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1 INHIBITORY EFFECT OF BROMODE OXYURIDINE ON LONG-TERM CELL PROLIFERATION By LINDSAY HARRIS LEVKOFF A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2008
2 2008 Lindsay Harris Levkoff
3 To my mom for her unconditional love, support, and friendship!
4 ACKNOWLEDGMENTS I would first like to thank my mentor, Dr. Er ic Laywell, for his continued optimism and motivation. His encouragement and perseverance will not be forgotten. Dr. Laywells office door was always open for scientific di scussions and his willingness to meet regularly exemplifies his dedication. I would also like to thank all of the members of the Laywell and Steindler labs for their support. The assistance and mentorship from the postdoctoral associates, Greg Marshall and Heather Ross, was critical. Finally, I would espe cially like to thank my fellow Laywell Lab graduate student, Meryem Demir, for her cama raderie during our years as graduate students. I am also grateful to my committee members, Drs. Lucia Notterpek, Edward Scott, and Wolfgang Streit, for their helpful suggestions, fee dback, and expertise. I would also like to thank our collaborators, Drs. Dennis Steindler, Wo lfgang Streit, and Brent Reynolds for providing invaluable materials and sound advice for my project. I want to thank my mother, Marsha; and my husband, Stephen, for their unconditional love and support. Their friendship, encouragement, and humor were critical in quelling the stress of graduate school. I'd also like to thank Emma a nd Mr. Butters, the best tw o dogs in the world, for providing the perfect distraction; their limitless cuteness, hilarious antics, and free snuggles helped me survive the last few years. Lastly, I am grateful to the Beas tie Boys for creating the perfect soundtrack to endure the long days of monotonous research tasks, to Big Lous Pizzeria for providing the perfect escape for lab lunches, and to the Starbucks Double Shot on Ice for providing the caffeine-induced insp iration and motivation for the countless hours of dissertation writing.
5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF FIGURES................................................................................................................ .........8 ABSTRACT....................................................................................................................... ............10 CHAPTER 1 INTRODUCTION................................................................................................................. .12 Background..................................................................................................................... ........12 Mutagenecity and Toxicity.....................................................................................................1 3 Proliferation Label............................................................................................................ ......15 Neurogenesis................................................................................................................... ........16 Cancer......................................................................................................................... ............18 Radiosensitization............................................................................................................. ......19 Senescence..................................................................................................................... .........20 Cancer Stem Cells.............................................................................................................. .....23 Connecting Neural Stem Cells and Gliomas..........................................................................26 Concluding remarks............................................................................................................. ...27 2 MATERIALS AND METHODS...........................................................................................28 Neurosphere Culture............................................................................................................ ...28 Neurosphere Quantification and Measurement......................................................................28 Immunolabeling................................................................................................................. .....29 Senescence-Associated -Galactosidase Labeling.................................................................29 Cell Culture................................................................................................................... ..........30 In Vitro Drug Treatment and Quantif ication of Prolif erative Activity...................................30 Subcutaneous Tumors and In Vivo BrdU Administration.....................................................31 Flow Cytometry................................................................................................................. .....32 Cell cycle analysis (Propidium Iodide)...........................................................................32 Annexin-V...................................................................................................................... .32 JC-1........................................................................................................................... .......33 Western Blots and Densitometry............................................................................................33 TUNEL Assay.................................................................................................................... ....34 Telomere Assays................................................................................................................ .....34 Telomere Length.............................................................................................................34 Telomerase activity.........................................................................................................35 Statistics..................................................................................................................... .............35 3 BRDU INDUCES A SENESCENT-LIKE STATE IN NEURAL STEM AND PROGENITOR CELLS..........................................................................................................36
6 BrdU Incorporation into Neurosphere-Forming Cells (NFC) Results in the Alteration of Morphology and Growth Rate in Subsequent Neurospheres (NS).....................................39 In Vivo BrdU Administration Reduces Neurosphere Yield...................................................39 In Vivo BrdU Administration In creases the Population of SA Gal(+) Cells........................40 BrdU Inhibits Proliferation of the Puta tive Cancer Stem Cell of the Brain...........................40 Discussion..................................................................................................................... ..........42 4 BRDU INHIBITS PROLIFERATION OF CANCER CELLS IN VITRO AND IN VIVO........................................................................................................................... ...........52 BrdU Administration Reduces Cancer Cell Population Expansion over Time......................53 Anti-Proliferation Follows Even Brie f, Low-Dose BrdU Administration.............................54 A Second Pulse of BrdU Exacerba tes Proliferation Suppression...........................................56 BrdU-Mediated Anti-Proliferation is not Blocked by the Addition of Cytosine or Thymidine...................................................................................................................... .....57 Halogenated Pyrimidines Suppress Prolifer ation More Robustly than Current AntiCancer Nucleosides............................................................................................................. 58 Length of BrdU Exposure is Directly Rela ted to the Percentage of Labeled Cells................59 BrdU Administration Slows Glio ma Tumor Progression In Vivo.........................................60 Discussion..................................................................................................................... ..........61 5 CHARACTERIZING THE MECHANISM OF ACTION FOR THE ANTIPROLIFERATIVE EFFECT OF BRDU................................................................................74 Single-Pulse BrdU Does Not Result in Increased DNA Damage or Apoptosis.....................75 BrdU Alters the Cell-Cycle Profile.........................................................................................78 BrdU-Incorporating Cells Do Not Influen ce the Proliferation of Neighboring Cells............81 Discussion..................................................................................................................... ..........83 6 CONCLUSIONS AND SIGNIFICANCE..............................................................................97 LIST OF REFERENCES............................................................................................................. 101 BIOGRAPHICAL SKETCH.......................................................................................................110
7 LIST OF TABLES Table page 5-1 Reported statuses of prominent senescen ce-related markers for all cell lines tested........96
8 LIST OF FIGURES Figure page 3-1 Variations in neurosphere morphol ogy following Bromodeoxyuridine administration in vitro ............................................................................................................................... .46 3-2 Bromodeoxyuridine suppresses the grow th rate of primary neurospheres........................47 3-3 In vivo Bromodeoxyuridine administration reduces subsequent neurosphere yield.........48 3-4 In vivo Bromodeoxyuridine administra tion does not influence subsequent neurosphere size............................................................................................................... ..49 3-5 In vivo Bromodeoxyuridine administration increases the populat ion of senescent cells.......................................................................................................................... ..........50 3-6 Bromodeoxyuridine induces a progressive dose-responsive suppression of cancer stem cell population expansion..........................................................................................51 4-1 Bromodeoxyuridine induces a progressive dose-responsive suppression of cancer cell line population expansion...........................................................................................63 4-2 Eventual recovery following singlepulse Bromodeoxyuridine administration................64 4-3 Proliferation suppression is co mmon among all cancer cells examined............................65 4-4 Proliferation suppression is inde pendent of Bromodeoxyuridine retention......................66 4-5 Transient, low-dose Bromodeoxyur idine suppresses expansion rate................................67 4-6 Bromodeoxyuridine exposure time as short as 1 minute leads to suppressed expansion and delayed labeling of nuclear DNA..............................................................68 4-7 Second pulse of Bromodeoxyuridin e exacerbates expansion suppression........................69 4-8 Bromodeoxyuridine-mediated proliferati on suppression is not antagonized by excess cytidine....................................................................................................................... ........70 4-9 Bromodeoxyuridine-mediated proliferat ion suppression is matched by similar halogenated pyrimidines and surpasses current anti-cancer nucleoside analogs...............71 4-10 Exposure time corresponds with an in creased percentage of Bromodeoxyuridinelabeled cells.................................................................................................................. ......72 4-11 Bromodeoxyuridine administration slows tu mor progression in a syngeneic in vivo glioma model................................................................................................................... ..73 5-1 Bromodeoxyuridine does not lead to increased H2A.X immunoreactivity.....................85
9 5-2 Expansion suppression is not due to Bromodeoxyuridine-mediated photolysis...............86 5-3 Bromodeoxyuridine does not perturb mitochondrial membrane physiology....................87 5-4 Bromodeoxyuridine does not induce cleav age of caspase-3 in treated cells.....................88 5-5 Bromodeoxyuridine treatment causes variab le, cell line-specific Annexin V assay results........................................................................................................................ .........89 5-6 Bromodeoxyuridine induces a negligible incr ease in late-stage apoptotic cell death.......90 5-7 Bromodeoxyuridine alters the cell cycle profile of treated cells.......................................91 5-8 Phosphorylation of pRb is reduced in some cell types after Bromodeoxyuridine exposure, while total pRb and p21 remain unchanged......................................................92 5-9 Bromodeoxyuridine induces an increase in SA Gal activity............................................93 5-10 Bromodeoxyuridine has varying effects on telomerase expression but does not alter telomere length................................................................................................................ ...94 5-11 Bromodeoxyuridine-treated cells do not inhibit expansion of untreated cells..................95
10 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy INHIBITORY EFFECT OF BROMODE OXYURIDINE ON LONG-TERM CELL PROLIFERATION By Lindsay Harris Levkoff December 2008 Chair: Eric D. Laywell Major: Medical SciencesMolecular Cell Biology The thymidine analog bromodeoxyuridine (Brd U) can be incorporated into newlysynthesized DNA and is commonly used to birthda te proliferative cells. It has been suggested that BrdU alters the stability of DNA thereby increasing the risk of sister-chromatid exchanges, mutations, and double-strand breaks. However, most of these effects are found only when BrdU incorporation is combined with secondary stressors Early toxicity studies showed that BrdU can induce chromosomal breakage and in crease the sensitivity of treate d cells to ionizing radiation, and this radiosensitizing effect has continued to be pursued as an adjunctive therapy in the treatment of a variety of cancers. BrdU readily crosses the blood-brain barrie r, and has been combined with conventional chemotherapy and radi ation treatment in seve ral clinical trials. While the clinical benefits of including BrdU as a radiosensitizer have been disappointing showing, at best, modest improvements for some outcome measurementsit is possible that other therapeutic effects of BrdU were not appreciated, either because of interference by the other treatment modalities in these studies, or because fi ner analytical resolution is required to discern them. Despite its extensive history there is no accep ted consensus mechanism of action for BrdU. Surprisingly little attention has been focused on examining the influence that BrdU alone may
11 exert on cellular function. We re port a novel anti-proliferative e ffect of BrdU on populations of stem and cancer cells that is inde pendent of its role in radiosens itization. A single, brief in vitro exposure to BrdU induces a profound and sustained re duction in the proliferat ion rate of all cells examined. Cells do not die but variably upregulate some senescence-associated proteins as they accumulate in the G1 phase of the cell cycle. Br dU also impairs the proliferative capacity of primary tumor-initiating human glioma cells and may, therefore, represent a means of targeting cancer stem cells. Finally, conservative in vivo BrdU regimens (in the absence of any other treatment) significantly suppress tumor progression in a highly a ggressive, syngeneic rat glioma model. Results suggest that BrdU may have an im portant role as an adju nctive therapeutic for a wide variety of cancers based upon new insights into its effect as a negative regulator of cell cycle progression.
12 CHAPTER 1 INTRODUCTION Background Bromodeoxyuridine (BrdU) is a thymidine anal og that was introduced in the 1950s as a mutagen to target rapidly-divi ding cancer cells (Hakala, 1959; Hakala, 1962; Djordjevic and Szybalski, 1960), but is now used ubiquitously to birthdate dividi ng cells. While there are myriad reports dealing with the consequences of BrdU incorporation into DNA chains (Littlefield and Gould, 1959; Stellwagon and Tomkins, 1971; D unn and Smith, 1957; Terzaghi et al., 1962), the variations in dosage and exposure time in these studies make it difficult to compare individual results. Perhaps because of this, and because inco rporating cells appear to maintain relatively normal functionat least in th e short-term (Cameron and McKay, 2001)BrdU is generally thought to substitute relatively benignly for thym idine. Recent work, though, suggests that BrdU may play a role in premature senescence induction in a wide variety of ce lls (Michishita et al., 1999; Suzuki et al., 2001). Despite its extensive history there is no accep ted consensus mechanism of action for BrdU. It has been suggested that BrdU alters the stability of DNA there by increasing the ri sk of sisterchromatid exchanges, mutations, and doublestrand breaks (reviewed in Taupin, 2006). However, most of these effects are found only when BrdU incorporation is combined with secondary stressors. Early toxicity studies show ed that BrdU can induce chromosomal breakage and increase the sensitivity of tr eated cells to ionizing radiatio n (Djordjevic and Szybalski, 1960; Hsu and Somers, 1961; Erickson and Szybalski, 1963), and this radiosensitizing effect has continued to be pursued as an adjunctive therapy in the treatmen t of a variety of cancers. BrdU readily crosses the blood-brain barrier, and ha s been combined with conventional chemotherapy and radiation treatment in severa l clinical trials (Kinsella et al., 1984; Phillips et al., 1991;
13 Robertson et al., 1997a; Robertson et al., 1997b; Groves et al., 1999; Prados et al., 2004). While the clinical benefits of includi ng BrdU as a radiosensitizer have been disappointing showing, at best, modest improvements for some outcome meas urementsit is possible that other therapeutic effects of BrdU were not appreciated, either because of interferen ce by the other treatment modalities in these studies, or because finer anal ytical resolution is required to discern them. Understanding the mechanism by which BrdU af fects cells would allow scientists to maximize the efficacy of using BrdU in combin ation therapy. However, surprisingly little attention has been focused on examining the in fluence that BrdU alone may exert on cellular function. BrdU has been studied for over five decad es but the results are dispersed over various subfields of research. Compiling all of the BrdU -related research is further complicated by the lack of a standard reference for this co mpound (Bromodeoxyuridine, 5-Bromodeoxyuridine, 5Bromo-2-deoxyuridine, BrdU, Br dUrd, BUdR, etc.). Revisiting the extensive literature may provide key clues for elucidating the effector mechanism of BrdU. Mutagenecity and Toxicity While most of the recent scientific literature pertaining to BrdU focuses on its use as a proliferative label, one should not ignore the fact that this synthetic thymidine analog was originally designed in the 1950s to perturb DNA synt hesis in cancer cells. Within that decade scientists began characterizing how cells were affected by BrdU expos ure. Hakala (1959) reported that once BrdU was incorporated in place of thymine in the DNA it creates an irreversible abnormality which prevents further s ynthesis of DNA. Shortly thereafter, Littlefield and Gould (1960) showed that when cells were incubated for 5 to 7 days with increasing concentrations of BrdU, an in creasing amount of the analog was incorporated into DNA, and growth (as measured by cell number, total cell protein, and single cell pl ating efficiency) was progressively inhibited. Interestingl y, they believed that the toxic effect of BrdU was related to
14 an interference with the function of DNA (i.e., transfer of information required for protein synthesis). It has been over five decades since scientis ts began reporting the effects of BrdU and yet the mechanism of action remains unknown. The integration of a bromine atom into the DNA alters its stability, increasing the risk of sister-chromatid exchanges, mutations, DNA doublestrand breaks, and lengthens the cell cycle of incorporating ce lls (reviewed in Taupin, 2006). One of the earliest explanations (based on experi ments with prokaryotic systems) is that BrdUinduced mutations occur as the result of occasional mispairing of the analog during DNA replication. BrdU has ambiguous pairing properties due to the bromine atom, and it can shift from a keto form (which pairs with adenine) to an enol form (which pairs guanine) thereby causing a mispair (Peng et al., 2001 ). Hopkins & Goodman (1980) argue that the effects of BrdU mutagenesis are actually two-fold, involving the perturbation of normal deoxyribonucleoside triphosphate pool sizes (Meuth and Green, 1974) and base-pairi ng ambiguity. BrdUTP is an inhibitor of ribonucleotide diphospha te reductase. This inhibition ma inly affects the reduction of cytidine disphosphate (CDP) to deoxycytidine di phosphate (dCDP), and high levels of dTTP, dGTP, and dATP are generally synergistic with BrdUTP in this inhibition (Meuth and Green, 1974; Moore and Hurlbert, 1966). Therefore, high BrdUTP concentrations will prevent the formation of the cytosine substrate for DNA synt hesis, dCTP. With a decrease in dCTP pools, BrdUTP becomes increasingly competitive fo r sites opposite template guanines during DNA replication. Davidson et al. (1988) elabor ated on this idea by sugges ting that the BrdU-related mutagenesis results from a sequence of events: (1) there is an inducti on of a high BrdUTP/dCTP molar ratio (due to the inhibi tion by BrdUTP of the ribonucleotid e reductase catalyzed reduction
15 of CDP to dCDP); (2) then BrdU mispairs or misincorporates opposite a gu anine residue (due to the excess of BrdU and the lack of dCTP ); and (3) the resulting BrdU-induced G-C A-T transitions are driven by nuc leotide pool perturbation. Many early studies tried to combat the theo retical nucleoside pool imbalance by adding exogenous deoxycytidine to BrdU-t reated cultures. The belief was that the mutagenicity, toxicity, and even the effects on differentiati on associated with BrdU treatment might be thwarted if the presence of dC TP causes BrdUTP to compete less effectively for sites opposite guanine on DNA (Davidson and Kaufman, 1977; Davidson and Kaufman, 1978; Hopkins and Goodman, 1980; Kaufman and Davidson, 1979; Meuth and Gree n, 1974). Unfortunately, the results from such experiments are conflicting; while some results suggest a rescue effect, others report no difference. Proliferation Label The ability to label dividing cells and quan tify DNA synthesis is fundamental to the study of cell biology, specifically can cer biology. Understanding the ex act kinetics of the S-phase could maximize the efficacy of cell cycle-specific chemotherapeutic drugs that selectively kill tumor cells. Initially, scientists used techniques such as tritia ted thymidine with autoradiography to measure the percentage of S-phase cells and/or label mitotic cells. The analysis of cell cycle was greatly improved by the introduction of flow cytometry because this technique allowed for the precise measurement of DNA content of a statistically significan t population of cells. However, flow cytometers are not well suited for conventional autoradiographic measurements (e.g., tritiated thymidine) thus necess itating a new method (Leif et al., 2004). Over four decades ago, the collaboration be tween Robert Leif, Howard Gratzner, and Albert Castro resulted in the production of a good affinity-purified polyc lonal antibody to BrdU. The creation of this antibody a llowed scientists to label cells with BrdU and subsequently
16 determine S-phase populations using flow cytome try. To improve specificity and affinity, a mouse monoclonal antibody to BrdU was produced. The use of the BrdU antibody has facilitated cell cycle studies, and the knowledge of S-phase content and timing continues to be useful for the creation of better cancer therapies (Leif et al., 2004). While cancer was the original focus for utilizi ng BrdU as a prolifera tive label, it is now commonly associated with stem cell research. Br dU labeling represents a relatively easy method for tracking stem and progenitor ce ll proliferation in various regene rative tissues. In fact, BrdU labeling is the most commonly us ed technique for studying adult neurogenesis in situ (Taupin, 2006). Neurogenesis The adult brain was once thought to be relativel y static, lacking the plasticity and genesis of the developing brain. This dogma persisted fo r nearly a century desp ite numerous reports suggesting the birth of new neurons in the adult brain. The first suggestion of adult neurogenesis came from early studies with [3H]-thymidine autoradiography th at showed neurogenesis in the adult rodent brain. These resu lts were further supported by studies conducted with electron microscopy that reported autoradiographic and ultr astructural evidence of new neurons in adult rats as well as high levels of neurogenesis in the avian brain (as revi ewed in Gross, 2000). Despite continuing evidence for neurogenesis, the dogma persisted. It was not until the 1990s, when advances in techniques to study neurogene sis were discovered that the longstanding notion that no new neurons were born in the adult brain was finally put to rest. Interestingly, the introduction of the synthetic thymidine analog BrdU was a major milestone in neurogenesis research because it allowed for the stereological es timation of the total number of cells as well as demonstration that the new cells express markers of specific cell types (Gross, 2000).
17 It is now well established that neurog enesis occurs throughout adulthood in the mammalian brain (Gage, 2000). Ther e are two main regions in th e adult mammalian brain that undergo neurogenesis, the dentate gyrus of the hi ppocampus and the subventr icular zone (Taupin and Gage, 2002). Being able to label proliferatin g cells is requisite for studying neurogenesis. BrdU immunohistochemistry has been instrumental for studying the development of the nervous system and to confirm that neurogenesis does oc cur in the adult brain. The benefits of BrdU immunohistochemistry include fast er processing, utilization of a monoclonal antibody rather than a radiolabeled substance, and its usability wi th relatively thick tissues (Gratzner, 1982; West et al., 1991). However, knowing th at BrdU affects incorporati ng cells, it is important to distinguish cell proliferation and neurogenesi s from other events (e.g., DNA synthesis, DNA repair). BrdU can be administered by various methods including intracerebrov entricular (i.c.v.), intraperitoneal (i.p.) and intrave nous (i.v.) injection, or orally (in drinking water), for studying neurogenesis. While the mechanism of BrdU transport into the brain remains unknown, in vitro experiments suggest that BrdU uses the same nuc leoside transporter as thymidine (Lynch et al. 1977). Thymidine, and likely BrdU as well, crosse s the blood-brain barrie r through a facilitative low affinity, high-capacity carrier-mediated nucle oside transport system (Taupin, 2006; Thomas nee William and Segal, 1996; Thomas et al. 199 7; Spector and Berlinger, 1982). Hayes and Nowakowski (2000) suggest that BrdU is only ava ilable for labeling cells in the adult brain for approximately two hours following systemic injection. However, with all of the benef its of BrdU labeling, one must not forget that BrdU can be toxic. It is known that high doses and/or multip le exposures of BrdU can trigger cell death in embryonic and neonatal development and, when give n to pregnant rats, a ffect the litter size,
18 body weight and mortality of the offspring. These BrdU regimens also reduce the size of the cerebellum and produce defects in proliferation, migration, and patterning of the cerebellum in adult progenies (Sekerkova et al., 2004). This is in contrast to th e fact that single-injection lowdose BrdU does not appear toxic to the devel opment of certain brai n regions (Miller and Nowakowski, 1988; Takahashi et al., 1995). While th ere is no report of toxicity using BrdU to study adult neurogenesis, it is cert ainly plausible that even low dos es of BrdU are likely to have toxic effects on newly generated neuronal cells in the adult brain particularly when multiple doses are administered (Sekerkova et al., 2004). Even with its exte nsive history of mutagenicity and toxicity, BrdU remains the most comm only used method for studying neurogenesis. Cancer The focus of BrdU in cancer biology is generall y related to its role as a proliferative label and radiosensitizer. However, there have been reports of BrdU induci ng differentiation in a variety of cancers including ne uroblastomas and small cell lung carcinomas. Chen et al. (2007) show that BrdU treatment causes morphological cha nges in non-adherent cancer cells such that a new adherent subpopulation forms. Additionall y, this adherent subpopulation shows reduced colony-forming capacity (both in size and numbe r). Finally, when the BrdU-treated adherent subpopulation was tested for its ability to form tumors in vivo the authors found a reduction in tumor development. Interestingly, it appears that the differentiation events can take place in the absence of DNA synthesis, suggesti ng that BrdU need not be incorporated into DNA to alter the phenotype of the cell (Schubert and Jacob, 1970). Early experiments using BrdU suggest that BrdU weakens the DNA thereby increasing the risk of sister-chromatid exchanges, muta tions, and DNA double-strand breaks. Understanding the potential of those events in possibly weak ening and/or killing cancer cells, scientists combined BrdU treatment with radiation to determine if their combination had a synergistic
19 effect. The results showed that BrdU radiosens itizes cells, making them more susceptible to the damage induced by radiation. This discovery held great promise in improving the efficacy of radiation as a cancer therapy and continues to be studied in vari ous clinical trials. Radiosensitization Combining radiation therapy with radiosensiti zing drugs has been a focus for oncologists for many decades. The idea is that the drugs increas e the sensitivity of treated cells to radiation thereby improving treatment. However, there has b een limited success with the countless clinical trials looking at the combination of chemi cal/biological agents and radiation. Steele and Peckham (1979; 1988) categorized these types of c linical trials according to the nature of how the radiation and drug(s) inter act: spatial cooperation, toxicity independence, protection of normal tissue, and enhancement of tumor res ponse (Coleman and Mitchell, 1999). One of the biggest hurdles in achieving success is the lack of preclinical studies that would elucidate the optimal timing and dosing regimen for the various agents. Additionally, proper analysis of biological tissue during the treatments would gr eatly enhance the quality of these studies. Eisbruch et al. (1999) utilized data from preclinical stud ies to explore a unique schedule of BrdU delivery to cervical cancer patients receiving radiation treatment. By collecting multiple biopsies of tumor and normal tissue the inve stigators were able to obtain substantial results regarding the incorporation of BrdU into normal and tumor tissu es that supported the rationale of their study (Coleman and Mitchell, 1999). Cells exposed to halogenated thymidine analogs are more sensitive to ultraviolet light and radiation than untreated cells. The incorporation of the haloge nated deoxyuridine, BrdU, is known to increase the radi osensitivity of a variet y of organisms that include certain bacteria, bacteriophages, and mammalian cells (Greer an d Zamenhof, 1957; Kozinski and Szybalski, 1959; Djordjevic and Szybalski, 1960). Compared to other thymidine analogs, the molecular
20 structure of BrdU most closel y resembles that of thymidine and was therefore targeted for clinical use. Over the last three decades there have been myriad clinical trials designed around the use of BrdU as a radiosensitizer. These studie s were designed to find the most effective mode of administration and dosing re gimen for maximizing the radios ensitization effect of BrdU without producing toxic side effects. Scientists and clinicians deliv ered BrdU via intravenous infu sion or intra-arterial infusion at doses ranging from 25 mg/kg/day to 1000 mg/m2/day for various time periods. Some of the reported side effects included varying levels of anorexia, fatigue, ipsilateral forehead dermatitis, blepharitis, iritis, and nail ri dging. However, the most common t oxicities occurred in the bone marrow and skin (e.g., myelosuppression and macul opapular skin rash) (Kinsella et al., 1984; Greenberg et al., 1988). Many of the trials were able to minimize these toxicities to acceptable levels upon finding ideal dosing regimens, and conc luded that BrdU was ab le to significantly enhance radiotherapy in malignant brain cancer, pancreatic cancer, hepa tobiliary tumors, and colorectal liver metastases (K insella et al., 1984; Greenberg et al., 1988; Matsutani et al., 1988; Hegarty et al., 1990; Robertson et al., 1997a; Robertson et al., 1997b). BrdU was also combined with radiation and chemotherapy in a series of Phase II and III clinical trials that failed to show any positive effect of adding BrdU to the therapy. While the clinical benefits of in cluding BrdU as a radiosensitizer have been mixed showing, at best, modest improvements for some outcome measurem entsit is possible that other therapeutic effects of BrdU were not appreciated, either because of interferen ce by the other treatment modalities in these studies, or because finer anal ytical resolution is required to discern them. Senescence Senescence was originally described by Hayflic k and colleagues (1961) in studies showing that normal human fibroblasts have a finite ability to proliferate. Cellular senescence is confined
21 to mitotic cells which can stall for long intervals in a reversibly arrested state. These cells remain viable awaiting the appropriate signals to resume proliferati on. Interestingly, senescent cells undergo alterations in gene expression and often become resistant to apoptosis. A hallmark of senescence is the inability to progress through the cell cycle. To achieve this, senescent cells maintain a delicate balance of expressing cell cycle inhibitors (e.g., p21 and p16) and repressing genes that encode proteins that stimulate cel l cycle progression (e.g., cyclins, c-Fos, PCNA). Most of the observations regarding senescen ce have been made using cultured cells; however, the importance of senescence in vivo has gained support over the last decade. A major limitation in studying senescence is th e fact that the markers used to identify senescent cells are not exclusive to this state. Despite this limitati on in identifying senescent cells, the significance of this state is highlighted in two main areas of research, cancer and aging. Populations of senescent cells have been found in renewable tiss ues in various species as well as at sites of chronic age-related pa thology and benign dysplastic or pren eoplastic lesions (reviewed in Campisi and dAdda di Fagagna, 2007). Senescent cells may contribute to the aging process by altering the expression of tumorsuppressive (i.e., pro-agi ng) proteins that suppress stem ce ll proliferation and tissue regeneration, and by upregulating genes that encode extracellu lar-matrix-degrading enzymes, inflammatory cytokines and growth fact ors that influence the local environment. These secreted factors can actually stimulate the growth and angiogenic ac tivity of nearby premali gnant cells (Campisi, 2005; Campisi and dAdda di Fagagna, 2007). Th is relationship between mitotically inactive senescent cells and highly repli cative cancer cells seems paradoxical However, this may explain the rise in cancer developm ent in aging organisms.
22 Cellular senescence probably suppresses tumori genesis by limiting cell proliferation. This is supported by the presence of sene scent cells in preneoplastic le sions where they are presumed to be halting the progression to malignancy. However, senescence may not be permanent and the reversal of this state may allow damaged, stresse d, or oncogene-expressing cells to proliferate. Many of the stimuli that induce senescence are potentially oncogenic a nd cancer cells must acquire mutations that allow them to avoid sene scence. Interestingly, this does not necessarily result in malignant transformation. The loss of th e senescence response appears to be critical, albeit insufficient, in the development of cancer (reviewed in Campisi and dAdda di Fagagna, 2007). There is no defining marker for senescent cells. However, senescence is typically characterized by a flat, enlarged cell shape, the induction of senescence markers such as senescence-associated beta-galactosidase (SA Gal), and resistance to ap optosis (Cristofalo and Pignolo, 1993; Dimri et al., 1995; Wang, 1997). While the initiating events leading to senescence and the resulting molecular signature are not well understood, the following potential causes have been evaluated: degradation and/or dysfunctional telomeres, DNA damage, chromatin perturbation, activation of oncoge nes, and stress (reviewed in Ca mpisi and dAdda di Fagagna, 2007). The Ayusawa group has shown that BrdU induces an immediate senescence-like phenotype in any type of mammalian cell. In fact, th ey suggest that this effect may occur in all eukaryotic cells as the effect seems to carry over into thymidine-auxotrophic yeast as well (2002). Their results show that BrdU induces flat and enlarged cell shap e, characteristic of senescent cells, and senescence-a ssociated beta-galactosidase in mammalian cells regardless of cell type or species. Interest ingly, human cell lines lacking functional p21, p16, or p53 behaved
23 similarly. This is particularly surprising cons idering the well-documented relationship between alterations in the expression of these proteins and the induction of se nescence. Additionally, while telomerase activity was suppressed in pos itive cell lines, accelerate d telomere shortening was not observed in tumor cell lines. These re sults suggest the BrdU activates a common senescence pathway present in both mortal and immortal mammalian cells. However, it is unclear whether this BrdU-induced senescence is permanent and the au thors fail to define mechanism for this change. Masterson and ODea (2007) report that Br dU induces a DNA damage response that involves the activation of Chk1/2 and p53, subs equent cell cycle inhibition, and phenotypic changes consistent with senescence. Additiona lly, BrdU causes alteratio ns in the expression profiles of many of the quintessential senescence markers (e.g., p21, p27, SA Gal staining). Interestingly, despite inducing a DNA damage res ponse, the authors believe that the downstream effects of BrdU influence cell cycle dynamics, diffe rentiation, and senescen ce, but do not initiate the apoptotic cascade. Similarly, Peng et al. (20 01) showed that continuous exposure to 20M BrdU for 48 hours or more markedly inhibited the growth of cancer ce lls irrespective of p53 status, and that the growth suppression is mainly due to cell cycle arrest rather than cell death. Cancer Stem Cells The theory that cancer might arise from a rare population of cells that display stem cell characteristics was postulated over 150 years ag o and various studies have been conducted to isolate this proposed subpopulation. However, the ma jority of these studies have been limited to in vitro assays that measure proliferation rather than true self-renewal. Advances in stem cell biology and animal models now allo w scientists to more directly measure self-renewal, a central tenet in defining stem cell populations (reviewed in Wicha et al., 2006).
24 Stem cells and cancer cells share several importa nt properties that incl ude (a) capacity for self-renewal, (b) ability to differentiate, (c) ac tive telomerase expression, (d) activation of antiapoptotic pathways, (e) increased membrane tran sporter activity, and (f) the ability to migrate and metastasize (Wicha et al., 2006). Stem cells can undergo either symme tric (production of two identical stem cell progeny) or asymmetric (production of one exact copy and one daughter cell that undergoes differentiation) division. The symmetric division s produce the cells that are responsible for tumor infiltration and metastasis while asymmetric divisions produce the cells that comprise the tumor bulk. The regulation of self-renewal is critical for development and repair and is believed to be infl uenced by environmental signals pres ent in the stem cell niche. If the proper inhibition cues are not available, st em cells will continue to proliferate. This deregulation may represent an early stage in car cinogenesis, and is supported by the fact that signaling pathways involved in stem cell se lf-renewal (e.g., Wnt, Notch, and Hedgehog) are implicated in various human cancers as well. Alte rations in these pathways have been shown in colon, pancreatic, gastric, prostate, skin, cer vical, blood, and breast car cinomas (reviewed in Wicha et al., 2006). While the focus remains on the rare stem cell population, it is certainly plausible that the progenitor cells may also be responsible for tumo rigenesis. Whether these cells transform and/or de-differentiate, it is likely th at they acquire the capacity fo r self-renewal. Certain oncogenic mutations might deactivate the control seque nces involved in diffe rentiation, cell-cycle inhibition, or cell death. Wicha et al. (2006) propose that both types of cells are important because self-renewal drives tumorigenesis whereas differentiation (albeit aberrant in tumors) contributes to tumor phenotypic he terogeneity. The maintenance of these states are controlled by paracrine signaling pathways, negative feedback loops that limit the response to mitogenic
25 signals, and pathways that suppr ess activation of the different iation program. This suppression may be a result of translational repression or epigenetic changes that lead to repression of differentiation-associated genes (Clarke a nd Fuller, 2006). Ultimately, the mechanism of transformation remains unknown and the identifi cation of the intracellular and extracellular events that support the cancer stem cells is critical. Normal stem cells are programmed to survive a nd are inherently resistant to apoptosis. The assumption that cancer stem cells share this charac teristic has therapeutic relevance because this resistance to apoptosis may explain the inefficien cy of cytotoxic agents and radiation therapy in cancer treatment. This apoptosis resistance may be the result of alterations in cell cycle kinetics, DNA replication and repair, asynch ronous DNA synthesis, and/or th e expression patterns of antiapoptotic and transporter proteins. Further char acterization of these ch anges will allow for new targeted therapies. However, the similarity between normal and cancer stem cells poses a challenge can a therapy efficien tly target cancer stem cells wit hout affecting normal stem cells? Promising results from recent studies involving signaling pathway and enzyme inhibitors support the feasibility of selectively targeting the cancer stem cell population (Wicha et al., 2006). It is estimated that only a very small fract ion of cancer cells (< 1%) is clonogenic. Hamburger & Salmon (1977) found that only 1/1000 to 1/5000 cells from tumors of the lung and ovary formed colonies in agar. While scientis ts have found cancer cells that express markers commonly associated with stem cells (e.g., CD133, nestin, Oct3/4), ther e is still no unique marker for the cancer stem cell. In fact, it is likel y that the putative cancer stem cells within each type of tumor will possess a unique combination of cell surface markers. Cells possessing specific cell surface marker phenotypes have been isolated from blood, brea st, and brain tumors,
26 and have been shown to generate tumors in secondary hosts (e.g., NOD/SCID mice) that are nearly identical to the parent neop lasm (reviewed in Huang et al., 2007). There is great heterogeneity within tumors and the ability to identify and isolate the putative cancer stem cell will be critical for targ eting these cells. It is difficult to design a therapeutic that will effectivel y treat a heterogeneous population of cells. This may explain why most current therapies can induce tu mor shrinkage yet fail to actually cure cancer and prevent recurrence. An agent designed specif ically to kill the cancer stem cell could be more effective. However, it is likely that a treatm ent that is toxic to cancer stem cells will also affect the normal stem cell populations. Maintenance of normal tissue re quires a balance between se lf-renewal and differentiation within the stem cell pools. A small number of stem cells are responsible for producing a large population of differentiated progeny. However, the stem cells are not the only cells that can proliferate; the transit-amplif ying progenitor cells may undergo a few mitotic divisions before entering a fully differentiated, post-mitotic state. Stem cell replication is regulated by various internal and external stimuli but the alterations that cause a normal stem cell to become a cancer stem cell remain unknown. There are two ways that cancer stem cells may arise from normal stem cells. First, mutations may disrupt th e regulation of replica tion thereby transforming the cell. Alternatively, mutations may allow the transit-amplifying progenitor cells to continue proliferating without entering a pos t-mitotic differentiated state. In either case, a population of self-renewing cells originates that is resistant to differentiation and susceptible to additional mutations (Clarke and Fuller, 2006). Connecting Neural Stem Cells and Gliomas Alterations in cellular functions such as pr oliferation, apoptosis, and tissue invasion can lead to cancer. Neural stem and progenitor cel ls are regulated by the same pathways that
27 influence many brain tumors. Therefore, it is reasonable to assume that these neurogenic populations of cells are potentially susceptible to transformation. In support of this theory is the fact that, in animal models, highly-proliferativ e regions of the brain are more sensitive to chemical or viral oncogenesis than are areas with a low pr oportion of proliferating cells. Additionally, many gliomas ar e found near germinal regions, such as the subventricular zone in humans, and frequently express markers that ar e associated with progenitor cells. While the precise mechanism of transformation is unknown, recent experiments have shown that changing the expression profiles of certain signal transduction proteins cause the formation of brain tumors in rodents (reviewed in Sanai et al., 2005). Alternatively, thes e cells may arise from a dedifferentiation event occurring within the organ, allowing this subpopulation to adopt a stem celllike phenotype. Concluding remarks Conclusions drawn from the l iterature review suggest that the incorporat ion of BrdU probably carries consequences for cellular viab ility. While the mechanism(s) for the varied effects of BrdU remain unknown, it is possible that BrdU functions in a cell type-specific manner. Aside from the early BrdU studies, ve ry little research has been conducted on the consequences of incorporating BrdU in the abse nce of secondary stressors. Considering BrdU is the most commonly employed method for labeling pro liferative cells, it is im portant to determine whether the incorporatio n of this thymidine analog has c onsequences for these populations.
28 CHAPTER 2 MATERIALS AND METHODS Neurosphere Culture Primary neurospheres (NS) were derived fr om neonatal C57BL/6 mice (P4 to P8). In vivo studies were conducted using young C57BL/6 mi ce (~P21). Animals were housed at the University of Floridas Department of Animal Care Services, and all procedures were in compliance with the regulations of the Institutional Animal Care and Use Committee. NS were generated as described (Laywell et al., 2002). Briefly, brains were removed from euthanized animals, incubated in Trypsin, and dissociated into a single-cell slurry with a series of decreasing bore glass pipettes. The slurry was plated overn ight in growth media (DMEM/F12, 5% FBS, 10 ng/l bFGF and EGF). In order to isolate NFC, the slurry suspension was aspirated, pelleted by centrifugation and incubated in Tr ypsin for 2 minutes. Cells were gently triturated, washed and resuspended. NFC were then plated in non-adhere nt flasks at clonal density (10,000 cells/cm2) in growth medium. The typical dose ranges for Br dU administration (based on the literature) are 10-50M in vitro and 50-300mg/kg in vivo Neurosphere Quantification and Measurement NS were assessed for frequency and size as a measure of NFC number and proliferative capacity, respectively. NS frequency was dete rmined by counting random triplicate 50 l aliquots using a 10X objective. Total NS number was extrapolated to total culture volume for each field counted. NS size was determined by m easuring the diameter of ten randomly chosen spheres in each well (triplicat e conditions) using calibrated meas uring probes associated with digital image capture software (Spot Advanced).
29 Immunolabeling For cleaved caspase-3 and H2A.X immunolabeling cells were grown to ~75% confluency on polyornithine-coated glass coverslips. The me dia was removed and the cells were fixed by incubation in 4% paraformaldehyde in PBS at room temperature for 15 minutes then washed with PBS for 5 minutes. Cells were prepared fo r immunocytochemistry by first blocking at room temperature for 1 hour in PBS plus 0.01% Trit on X-100 (PBSt) containi ng 10% FBS. Primary antibodies were then ap plied to the cells for one hour in PBSt + 10% FBS with moderate agitation at 37oC. The antibodies used were either Cleaved Caspase-3 (1:400; #9661S; Cell Signaling Technology) or H2A.X phospho-S139 (1:200; ab2893; Abcam). Residual primary antibody was removed by three 5 minute washes with PBS and secondary antibodies were applied at room temperature for one hour in PBSt + 10% FBS. Residua l secondary antibodies were removed by three 5 minute washes in PBS. The cover-slips were placed onto glass slides and Vectashield mounting medium plus DAPI (Burlingame, CA: H-1200) was applied immediately prior to cover-slipping. Cells were processed for BrdU immunolabeling as previously described (Laywell et al., 2005). Briefly, cells were incubated for 2 hours in a 1:1 ratio of 2xSSC:formamide at 65oC. After a wash in 2xSSC the cells were then incubated for 30 min. at 37oC in 2N HCL. Finally, the cells were equilibrated at room temperature for 10 mi nutes in borate buffer, followed by standard indirect immunofluorescence dete ction of BrdU with a rat an ti-BrdU antibody (#ab6326, Abcam, Cambridge, MA). Senescence-Associated -Galactosidase Labeling SA Gal activity was detected as described (D imri et al., 1995). Briefly, tissue samples were collected from the neurogenic brain regions of animals receiving either BrdU (100mg/kg) or saline (0.9%) injections (13x/24h). The samples were incuba ted overnight at 37C in buffer
30 containing 1 mg/ml 5 bromo-4-chloro-3-indolyl BDgalactoside (X-Gal), 40 mM sodium citrate pH 6.0, 5% dimethylformamide, 5% potassium ferrocyanide, 5 mM ferricyanide, 150 mM sodium chloride and 2 mM magnesium chloride. Tissue samples were mounted on slides, viewed with a compound microscope, and scored for SA Gal label as indicated by blue/green reactivation product ove r the cell soma. SA Gal(+) cells were quantified for all brain regions (tissue samples were in triplicate). Cell Culture Cell lines were obtained from American T ype Culture Collection (www.atcc.org): H9, human cutaneous T-cell lymphoma (#HTB176); MG-63, human osteosarcoma (#CRL-1427); Saos-2, human osteosarcoma (#HTB-85); TT, human thyroid tumor (#CRL-1803); BJ, human fibroblasts (#CRL-2522); and RG2, rat glioma (# CRL-2433). Primary human glioma cells were generated from a surgical resection. Experime nts were performed in triplicate cultures maintained in a 37o C humidified chamber containing 5% CO2. In Vitro Drug Treatment and Quantifi cation of Proliferative Activity All reagents were purchased from Sigma (S t. Louis, MO) unless otherwise noted: BrdU (#B9285); BrdU (#B23151 from Molecular Probes, Eugene, OR); 5-ch loro-2-deoxyuridine (#C6891); 5-iodo-2-deoxyuridine (#I7125); 5-aza-2-deoxycytid ine (#A3656); 5-fluorouracil (#F6627); thymidine (#T1895); cy tidine (#C4654). Exposure times ranged from 1 min. to 24 hours, after which the medium was aspirated and replaced with fresh medium without analogs. Control and treated cultures received th e same number of medium changes. Cultures were initially plated at 2000 cells/cm2, and were quantified with a Z2 Coulter Counter (Beckman Coulter, Fullerton, CA) at vari ous intervals after the removal of BrdU (range = 1 min. to several weeks). Ne urosphere cultures of primar y human glioma cells were established and maintained as de scribed (Piccirillo et al., 2006).
31 Subcutaneous Tumors and In Vivo BrdU Administration A bolus of either untreated or pre-treated RG2 gl ioma cells (1x106 cells in 250L of PBS) was injected subcutaneously betw een the scapulae of anesthetized adult male Fisher 344 rats as previously described (Mariani et al., 2006). Pre-treated RG2 cells were treated with 50M BrdU for 24 hours prior to implantation. Tumors were measured every other day in two dimensions with digital calipers, and tumor volume was calculated [( /6) x W2 x L (W = shortest dimension and L = longest dimension)]. The experiment al endpoint was defined as a tumor volume > 3000mm3. At endpoint euthanasia was performed by tr anscardial perfusion with 200 mL of 4% paraformaldehyde in PBS under deep sodium pentobarbital anesthes ia (150 mg/kg, i.p.). BrdU administration, i.p. Untreated RG2 cells were implanted into 10 anim als as described above. The BrdU regimen was initiated when palpable tumors had reached a volume of 200mm3. Half of the animals received 3 i.p. injections of BrdU (300mg/kg) per day for 2 da ys, while the other half served as controls and received an equal number and volum e of sterile saline injections. BrdU administration, oral Again, untreated RG2 cells were implanted s ubcutaneously into 20 animals as described. Immediately after implantation, half of the animals were provided with drinking water containing BrdU (0.8mg/mL), a nd half received normal drinki ng water. All animals were provided with freshly prepared water (either with or wit hout BrdU) each day for 7 days, ad libitum On the eighth day after implantation, all animals were placed on normal drinking water for the duration of the experiment. A dose of 300mg/kg corresponds to a clinical dose of 1800mg/m2. The rats received three of these doses per day for two days thus receiving a total of 10,800 mg/m2. The drinking water dose (based on the standard 20mL per da y consumption by adult rats) is 640 mg/m2/day for
32 seven days, or a total of 4,480 mg/m2. By way of comparison, previous clinical trials (e.g. Kinsella et al., 1984) that included BrdU as a radiosensitizer as part of a multimodal therapy treated patients with 350mg/m2 for continuous 12 hour infusions every day for 14 days, or 4900mg/m2 total. Thus, the treatment range in our study is generally in accord with previous human clinical applications since, even though our injected BrdU was th eoretically about twice what humans received, it is known that BrdU is active in plasma only for about 2 hours. Therefore, the continuous infusion employed in the human trials likely resulted in more widespread BrdU incorporation than our injection paradigm. Flow Cytometry Cell cycle analysis (Propidium Iodide) Cells were fixed overnight in 70% ethanol a nd then incubated for 1 hour at 4C in PBS containing 50g/ml each of propidium iodide (#P-4170; Sigma-Aldrich) and RNase A (#R6513; Sigma-Aldrich). Samples were processed with a FACSCalibur flow cytome ter (BD Biosciences, San Jose, CA). Data were analyzed with Flow Jo Flow Cytometry Analysis Software (Ashland, OR). Annexin-V Cells were harvested at various time point s following BrdU administration (50M) to assess Annexin-V staining using the Vybran t Apoptosis Assay Kit #9 (V35113, Molecular Probes). Briefly, cell pellets were obtained by centrifugation and resuspended at 1 x 106 cells/ml in 1X Annexin Binding Buffer (ABB). Annexin V (APC) and SYTOX green stain were added to the cell suspension and incubated at 37oC, 5% CO2 for 15 minutes. The cell suspension was diluted with 1X ABB, gently mixed, and anal yzed by flow cytometry (530/660nm). Populations were separated based on high and low le vels of red and green fluorescence.
33 JC-1 The JC-1 assay was performed to determin e if BrdU exposure causes any changes in mitochondrial membrane potential. Control and Br dU-treated cells were suspended in warm medium at 1 x 106 cells/ml. The positive control sample wa s treated with CCCP and incubated at 37oC for 5 minutes. All groups received 2M JC-1 and were incubated at 37oC, 5% CO2 for 30 minutes. Cells were washed once and resuspended in 500l of PBS. Samples were then analyzed on a flow cytometer with 488nm excitation using th e appropriate emission filter for Alexa Fluor 488 dye and R-phycoerythrin. Cells we re gated to exclude debris and standard compensation was performed using the CCCP-treated sample. Western Blots and Densitometry Proteins were isolated from adherent cell lin es (RG2 and BJ) by incubation with RIPA extraction buffer (50 mM Tris -HCl, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 1% sodium dodecyl sulfate) contai ning protease inhibito r cocktail (Calbiochem, San Diego, CA) on flasks over an ice bath for 5 min followed by cell scraping. Extracted protein was quantified against a bovine serum albumin (BSA) standard using a DC protein assay reagent kit (Bio-Rad, Hercules, California). Equal protein was loaded onto Bis-Tris 4-12% density gradient gels and electrophoresis was performed using NuPAGE ME S [12(N-morpholino) ethane sulfonic acid] reducing buffer system (Invitrogen) for 50 minutes at 200 V. Proteins were transferred onto nitrocellulose membranes for 1-1.5 hour(s) at 30 V. Non-specific binding was blocked with 5% bovine milk in Tris buffered saline plus 0.05% Tween 20. Membranes were incubated in blocking buffer at room temperature for 1 hour pr ior to being probed with the following primary antibodies: Phospho-specific (Ser249,Thr252) anti-retinoblastoma (1:500, PC640, Oncogene Research Products), p21 (1:500, ab7960, Abcam) anti-human retinoblas toma protein (1:200, #554136, BD Pharmingen) and anti-actin (1:2000, A-4700, Sigma). Membranes were incubated
34 in primary antibody at 4oC overnight and then probed with an appropriate secondary antibody for one hour at room temperature. Finally, immunopositive proteins were detected by autoradiography using ECL reagents (GE Healthca re Life Sciences, UK) and densitometry was quantified with Image J software. TUNEL Assay Apoptotic cells from control and BrdU-tre ated cultures were visualized using a fluorimetric terminal deoxynucle otidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay (DeadEnd Fluoremetric TUNEL System G3250; Promega) according to the manufacturer's recommendations. This assay measures the fragm ented DNA of apoptotic cells by incorporating fluorescein-labeled dUTP at the 3 ends of DNA strands. Cells were counterstained with Vectashield + DAPI (H1200; Vector), and cell de ath was quantified by co unting the number of TUNEL+ nuclei on each of three coverslips at five predeter mined stage coordinates. The criterion for apoptotic cells wa s intentionally libera l to avoid undercounting (i.e., nuclei with even the faintest evidence of fluores cein label were considered positive). Telomere Assays Telomere Length To determine if the effects of BrdU are relate d to changes in telomere length we performed a TeloTAGGG assay (#2209136; Roche). Briefl y, genomic DNA was isolated (#11814770001; Roche) and digested with Hinf1 and Rsa1 enzy mes. Following digestion, the DNA fragments are separated by gel electro phoresis and transferred to a nylon membrane by Southern blotting. The blotted DNA fragments were hybridized to a di goxigenin(DIG)-labeled probe specific for telomeric repeats and incubated with a DIG-spec ific antibody covalently coupled to alkaline phosphatase. Finally, the immobilized telomere pr obe was visualized by virtue of alkaline phosphatase metabolizing CDPStar a highly sensitive chemiluminescence substrate.
35 Telomerase activity The TRAPeze Elisa kit (#S7750; Chemicon) a ssay was employed to determine levels of telomerase activity in our control and BrdU-treated cells. Briefly, the sample cells telomerase adds a number of telomeric repeats (GGTTAG) ont o the 3 end of the biotinylated Telomerase Substrate oligonucleotide (b-TS) and the extende d products are then amplified by the polymerase chain reaction (PCR). The extension/amplification was performed with biotinylated primer and DNP-labeled dCTP. Thus, the TRAP products are tagged with biotin and DNP residues and the labeled products can be immobili zed onto streptavidin-coated mi crotiter plates via biotinstreptavidin interaction, and then detected by anti-DNP an tibody conjugated to horseradish peroxidase (HRP). The amount of TRAP products was determined by means of the HRP activity using substrate TMB and subsequent color development. Statistics All analyses were performed with Graphpad InStat & Prism 4 (San Diego, CA). Twogroup comparisons of cell counts were perfor med with the Students T-test. Multiple group comparisons were performed with either a one-w ay or two-way analysis of variance (ANOVA). Tumor progression data is expressed as Kaplan-Meier survival curves. For the compound statistical model (Figure 4-3) the ratio is estimat ed using mean (exp group)/mean (control group) for each cancer line/ti me point combination. The standard deviation was computed according to Cochrans equation 6.4 (Cochran, William G. 1977 Sampling Techniques 3rd Ed., Wiley, New York). Under the null hypothesis that the ratio is 1, the test statistic is a z-statistic, which ha s the standard normal distribution if the null is true. An asterisk (*) indicates significance using the Bonferroni correction for multiple tests.
36 CHAPTER 3 BRDU INDUCES A SENESCENT-LIKE STAT E IN NEURAL STEM AND PROGENITOR CELLS Stem cells are generally characterized by func tional criteria that include the ability to proliferate, self-renew over an extended period of time, and generate progeny that can differentiate into the primary cell types of the tissue from which it is obtained (Reynolds & Rietze, 2005). To assess these char acteristics scientists must de sign and employ proper assays. In the case of the neural stem cell, the neurosphere assay (NSA) has emerged as the assay of choice for the detection and expansion of neural stem and progenitor cells. Neural stem cells can be isolated from neurogenic regions of brain tissue (e.g., subventricu lar zone, hippocampus) that has been mechanically dissociated and plated on nonadherent culture dishes in appropriate media containing growth factors. In time, free-floating colonies of cells called neurospheres will appear. Neurospheres can then be dissociated into a single cell susp ension and replated on culture dishes. Each series of growth and dissociation is referred to as a passage. To assess the differentiation potential of the cells within the neuros phere, one must plate the cells in an adherent culture condition and w ithdraw serum and growth factors. Generally the cells at the periphery of the neur osphere will migrate and different iate into three main cell types (astrocytes, oligodendrocytes, and neurons) while the cells at the cente r remain undifferentiated. Because the NSA allows for the expansion of bot h stem and non-stem cell populations, one must be cautious in the interpretation of the NSA becau se it does not provide a reliable or accurate readout of stem cell frequency (Reynolds & Riet ze, 2005). However, this assay continues to be the standard for studying the biology of this pa rticular population of stem cells. The NSA is critical in determining how various exogenous f actors influence the proliferation kinetics and differentiation potential of neur al stem and progenitor cells.
37 The halogenated thymidine analog 5-bromo-2deoxyuridine (BrdU) can incorporate into DNA during S-phase of the cell cycle and can therefor e be used to detect events including cell division, DNA repair and cell cycl e re-entry. BrdU is commonly us ed to quantitate proliferative index or to birthdate, iden tify and track cycling endogenous and transplanted neural stem/progenitor cells. However, quantitative comparison of indepe ndent studies of neurogenesis is difficult because BrdU does not exclusively la bel dividing cells and there is no standardized dosing schedule (reviewed in Taupin, 2006). In a ddition to these technical issues, some data suggest detrimental downstream functional effects of BrdU incorporation. Exposure to BrdU has been shown to be toxic to precursor and matu re neuronal cells and has also been shown to modulate the growth and differe ntiation of cultured neurobla stoma cells (Bannigan, 1985; Caldwell et al., 2005; Nagao et al., 1998; Schubert and Jacob, 1970). Finally, BrdU administration results in the upregulation of se veral markers of senes cence in a variety of primary and transformed mammalian cells (Michi shita et al., 1999; Su zuki et al., 2001). Senescence was originally described by Ha yflick and Moorehead (1961) in studies showing that normal human fibroblasts have a fini te ability to proliferate. These cells remain viable and bioactive but the culture in its entirety experiences an ir reversible loss of the ability to divide. Senescence is characterized most typically by a flat, enlarg ed cell shape, the induction of senescence markers such as senescence-associated beta-galactosidase (SA Gal), and resistance to apoptosis (Cristofalo and Pignolo, 1993; Dimri et al., 1995;Wa ng, 1997). Senescence can be induced by a variety of stimuli and is currently understood to be stim ulus-, cell-typeand species-specific to varying degrees (reviewed in Campisi and dA dda di Fagagna, 2007). Further, the initiating events leading to senescence, the resulting molecula r signature, and the sequence of altered gene/protein expression is not completely understood. Im portantly, despite its ubiquitous
38 use in stem cell biology, the im plications of BrdU incorporat ion on the long-term function of multipotent stem and progenitor cells has not been systematically investigated. Our studies were designed to test the hypothesis that BrdU induces senescence in ne ural stem and progenitor cells. We show that BrdU exerts a strong anti-prolife rative effect on cultured murine stem/progenitor cells. Reduced proliferation is concurrent with the onset of a senescent phenotype in BrdUtreated cells that alte rs cell morphology, differentiation potenti al and susceptibility to apoptosis (unpublished observation). We show altered proliferation of neurosphere forming cells (NFC) isolated from animals administered an experimentally relevant BrdU treatment regimen. Together, these data uncover a novel and uncharacterized effect of BrdU admi nistration on stem/progenitor cells that has profound implications for the interpretation of re sults obtained with this thymidine analog. Specifically, long-term in vivo BrdU administration may alter th e biology of stem and precursor cell pools, severely limiting downs tream applications. Conversely, the reliable i nduction of a senescent-like phenotype in e xpandable, multipotent neural stem cells may represent a novel platform for molecular studies designed to address questions regarding multiple aspects of neurogenesis and aging. Finally, we examined the effect of BrdU ad ministration on primary cancer cells with stemlike properties using the NSA. Putative cancer st em cells derived from primary human gliomas were treated with BrdU and th e rate of population doubling was co mpared to that of untreated controls. A single pulse of experimentally re levant BrdU induces a reliable and dramatic reduction in the proliferation of clonally expanded progeny over numerous population doublings. This result suggests the potential of BrdU as a potent therapeutic, targeting cancer stem cells and thereby preventing metastasis.
39 BrdU Incorporation into Neurosphere-Forming Cells (NFC) Results in the Alteration of Morphology and Growth Rate in Subsequent Neurospheres (NS) NFC from neonatal C57BL/6 mice (P4 to P8) we re plated at clonal density, treated with BrdU (50M) and propagated in non-adherent conditions until multicellular neurospheres were apparent. In cultures not receivi ng BrdU treatment, NS appear as cell clusters with a smooth and well-defined border (Figure 3-1A ). NS derived from NFC ex posed to BrdU, however, are substantially smaller than control NS, and usua lly have an uneven border (Figure 3-1B-D). Our observations led us to quantify NS prolifera tion dynamics by measuring both the number and diameter of spheres derived from control and Br dU-treated NFC. These parameters reflect the number of surviving NFC and the amount of pro liferation within each NS, respectively. Single NFC were treated with a single pulse of BrdU (0, 10, 50, 100 M), plated at clonal density and allowed to form NS for 7-10 days. Subsequent analysis reveals no cons istent or significant differences in NS number among groups (data no t shown). However, there is a significant, doseresponsive decline in NS diameter (F igure 3-2). These data show that in vitro administration of BrdU does not inhibit NS formation, but impairs the growth of resulting NS. In Vivo BrdU Administration Reduces Neurosphere Yield In order to determine if in vivo BrdU administration alters the growth potential of NFC progeny, we administered BrdU [100 mg/kg] or normal sa line [0.9%] to young C57BL/6 mice (~P21) via intraperitoneal in jections three times during a twenty-four hour period. Two hours following the final injection, animal s were sacrificed and tissue ha rvested for culture of primary NS as well as histology. NS were then pl ated, counted and measured as in the in vitro treatment experiments. NFC obtained from BrdU-treated anim als yield fewer primary NS (Figure 3-3) with no significant difference in neuros phere diameter (Figure 3-4) wh en compared to those obtained from control animals. When cultures are qualitativel y considered, it is clear that a wide variation
40 in NS diameter exists in BrdU-treated culture s. Rather than a population of uniformly smaller NS, there appear to be both larger spheres that resemble those of contro l cultures, and irregular small spheres that resemble in vitro -treated NS. These results conf irm an effect on NS growth following in vivo BrdU administration. In Vivo BrdU Administration Increases the Population of SA Gal(+) Cells We have shown that BrdU administration result s in suppressed proliferation of neural stem and progenitor cells. Interestingly, this does not appear to be the result of cell death (unpublished observation). The alteration in prol iferation kinetics suggests the possibility that BrdU-treated cells are either in a senescentor quiescent-lik e state. Since we did not find any evidence of recovery in treated cells, we argue that the cells are more likely to be in a senescent state. While there is no single definitive marker of senes cence, senescence-associated beta-galactosidase (SA Gal) labeling is the most commonly used method for identifying senescent cells. Animals were treated with BrdU as above and histological samples were collected from the neurogenic regions of the adult brai n. The tissue was stained with SA Gal and positive cells were quantified for the various brain regions. There is a baseline amount of senescent cells in the control samples. However, a dose-response can be seen in the BrdU-treated groups where samples from animals receiving three injections show an increase in SA Gal(+) cells compared to animals receiving saline injections or only one BrdU injection (Figure 3-5). Similar results were found in the lateral subventricular zone, dors al and ventral blades, wh ile the hilus contained significantly more senescent cells overall. BrdU Inhibits Proliferation of the Pu tative Cancer Stem Cell of the Brain The hierarchical model of cancer suggests that a rare subset of cells within the tumor have significant proliferation capacity and the ability to generate new tumors. In this model the remainder of the tumor cells represents different iating or terminally differentiated cells. Brain
41 tumor stem cells have been iden tified and isolated from glioblastomas, medulloblastomas, and ependymomas. These cells share the cardinal charact eristics of stem cells and also possess the ability to initiate cancer upon orthotopic implantation (Reya et al., 2001; Vescovi et al, 2006). While the mechanism responsible for the ge neration of cancer stem cells remains unknown, many believe that disruption of the regulatory m echanisms that control self-renewal are likely involved (Reya et al., 2001). The actual criteria for identif ying brain tumor stem cells re mains nebulous; however, to date, all of the reports that de scribe the isolation and characte rization of putative brain tumor stem cells have used the neurosphere assay to help confirm the existence of this specific population of cells (Vescovi et al., 2006). The ability to culture brain tumor stem cells in vitro provides both a model system for testing known ther apeutic agents and a platform for identifying specific antigenic and molecular markers th at might target the tumor-initiating cell. We examined the effect of BrdU administ ration on primary cancer cells with stem-like properties using the neurosphere assay (Reynol ds and Rietze, 2005; Marshall et al., 2007). Neural stem-like cells capable of forming NS are present in primary gliomas (Ignatova et al., 2002), and represent tumor-i nitiating cells in serial transplantation paradi gms (Piccirillo et al., 2006; Vescovi et al., 2006). NS derived from pr imary human gliomas and treated with a single pulse of either 5 or 10M BrdU show a dosedependent reduction in the rate of population doubling as compared to untreated control cells (Fi gure 3-6). Cancer stem cells seem particularly susceptible to the anti-proliferativ e effect of BrdU such that a single pulse of BrdU reliably and dramatically slows the prolif eration of clonally expanded pr ogeny over numerous population doublings.
42 Discussion We have demonstrated that ne ural stem and progenitor cells exhibit a dramatic inhibition of proliferative capacity after si ngle-pulse BrdU-administration. In vitro BrdU-treated neurosphere-forming cells (NFC) produce smalle r primary neurospheres that also show an impaired capacity for generating secondary neurospheres. These results have profound implications for the interpretation of experime ntal outcomes based upon BrdU incorporation, and may limit downstream functional assessment of la beled stem/progenitor cells and their progeny since incorporating cells may have radically altered biology. Likewise, quantitative long-term in vitro and in vivo labeling paradigms can be expected to underestimate stem/progenitor cells and their progeny due to the proliferation suppression following BrdU administration. In vivo administration of BrdU leads to a reduction in the number of primary NS subsequently obtained from the neural stem ce ll niche. Because BrdU is known to have a negative effect on repair mechanisms, we can specula te that incorporating cells are less likely to survive the stressful dissociation procedure wh ich combines both enzymatic and mechanical methods for separating cells. Under these circumst ances a percentage of incorporating cells may not survive long enough to produce id entifiable NS, thus reducing th e total yield. The fact that we see a dramatic effect in the neurosphere as say, a surrogate test of stem/progenitor cell presence, suggests that relative ly modest BrdU regimens may be a viable method of inducing senescence either in experimental paradigms of aging-associated changes in neural stem cell function and neurogenesis, or in the clinical setting as a potential antineoplastic approach. Our present data support recent findings sugges ting perturbed proliferation following BrdU exposure. Michishita and colleagues (1999) showed that BrdU leads to inc onsistent responses of important cell cycle regulatory proteins known to affect senescence and cancer pathways. Slowed proliferation resulting from BrdU inco rporation has been desc ribed in a number of
43 cancer cell lines (Minagawa et al., 2005), as well as in thymidine auxotrophic yeast (Fujii et al., 2002), suggesting that the effect may be universal among eukaryotic cells. We have also found that all tested primary and cancer cell lines are susceptible to Br dU-induced senescence, regardless of species, telomerase status, or stat us of cell cycle proteins such as p16, p21, and p53 (Levkoff et al., 2008). While BrdU is now most frequently used for birthdating and tracking proliferative cells, it was initially introduced as a mutagen to target rapidly-dividing cancer cells (Djordjevic and Szybalski, 1960; Hakala, 1959; Hakala, 1962). More recently it has been shown that BrdU may not affect all cells in the same way, and is selec tively toxic for neural progenitor cells at doses as low as 1M (Caldwell et al., 2005), which is well below the dose typically used in in vitro labeling paradigms. However, because most in corporating cells seem to maintain normal function (Cameron and McKay, 2001), at least in the short term and in the absence of secondary stressors, BrdU is generally regarded as a benign substitute for thymidine. It may seem surprising that the relationshi p between BrdU and senescence is only now becoming appreciated given its long history and ubiquitous use; howev er, this is likely due to the initially subtle, yet progressive, nature of senescence in BrdU incorporating cells. Multiple rounds of replication are required for dramatic ef fects on proliferation rate to become manifest given the tools typically used to assess normal cellular func tion. Thus, quantification of BrdUtreated cells shortly after exposur e will not reveal the large dive rgence from normal control cells that is seen with longer postincorporation intervals. Adu lt hippocampal neurogenesis, for instance, in which newly-generate d granule neurons do not continue dividing after genesis in the subgranular zone would not be expe cted (again, in the short-term) to show overt perturbation as a result of BrdU administration.
44 However, such neurogenesis may eventually b ecome impaired over time if the cell cycle kinetics of enough stem/progenitor cells is nega tively affected by BrdU incorporation. While there is strong evidence for se nescence induction by BrdU, the mechanism of this induction remains enigmatic. Prior microarray studies have identified a number of senescence-associated genes that are upregulated in response to BrdU administration (Suzuki et al., 2001), but their potential causative role has not been establishe d. More recently, chromatin unpacking, regulated by BrdU incorporation into scaffold/nuclear matrix attachment region sequences, has been proposed as an initiating event to senescence indu ction (Satou et al., 200 4; Suzuki, et al., 2002). BrdU incorporation leads to a delayed but progressive inducti on of senescence in neural stem/progenitor cells that manifests over multiple rounds of replication, and is accompanied by perturbed differentiation of neural progeny. This effect is likely to be common to all stem cell pools, and emphasizes the need for caution when interpreting results based on long-term BrdU tracking over multiple rounds of replication. The reliable induction of senescence in stem/progenitor cells in vitro and in vivo may yield a novel platform for molecular studies designed to address multiple aspects of aging, a nd may also represent a th erapeutic approach to slow the growth of cancer cells. Cancer stem cells seem particularly susceptible to the anti-proliferative effect of BrdU such that a single pulse of BrdU relia bly and dramatically slows the pr oliferation of clonally expanded progeny over numerous population doublings. This re sult strongly suggests that BrdU may be a potent therapeutic, targeting cancer stem cells and potentially slowing the regrowth of de-bulked primary tumors and/or the metastatic spread of secondary tumors. The wide penetrance of the anti-proliferative effect, combined with the ab ility for rapid transport across the blood-brain barrier makes BrdU an attractiv e candidate against all types of cancer. However, these same
45 attributes also make it likely that indigenous stem cell pools will be adversely affected. Therefore, potential therapeutic BrdU dosing regime ns will need to be carefully tested to avoid a permanent depletion of the stem cells and longterm progenitors needed for maintaining tissue homeostasis.
46 Figure 3-1. Variations in neurosphere morphology following BrdU administration in vitro Representative photomicrographs showing di fferences in size and border structure between control neurospheres (A) and neurospheres treated with 10, 50, or 100M BrdU (B-D).
47 Figure 3-2. BrdU suppresses the growth rate of primary neurospheres. There is a significant dose-responsive reduction in NS diameter in BrdU-treated (10, 50 or 100 M) NS generated from neonates.(Control v. 10M p<0.05; Control v. 50,100M p<0.01). Data analyzed with one-way ANOVA with the Tukey-Kramer post-hoc analysis; Error bars represent standard deviation.
48 Figure 3-3. In vivo BrdU admi nistration reduces subsequent neurosphere yield. C57/BL6 pups (P21) were given either one ( 1x) or three (3x) i.p. inje ctions of 100 mg/kg BrdU or 0.9% sterile saline over 24 hours and sacrif iced 2 hours after th e final injection. BrdU-treated animals yield significantly fewer neurospheres than control animals (Control v. 1x, p<0.05; Control v. 3x, p<0.01). Data analyzed with one-way ANOVA with Tukey-Kramer post hoc test; Error bars represent standard deviation.
49 Figure 3-4. In vivo BrdU admi nistration does not influence s ubsequent neurosphere size. C57/BL6 pups (P21) were given either one ( 1x) or three (3x) i. p. injections of 100 mg/kg BrdU or 0.9% sterile saline over 24 hours and sacrificed 2 hours after the final injection. Average neurosphere diameter was calculated for the control and experimental groups. There was no significan t difference in neurosphere size between BrdU-treated and control animals. Data analyzed with one-w ay ANOVA with TukeyKramer post hoc test; Error bars represent standard deviation.
50 Figure 3-5. In vivo BrdU admini stration increases the population of senescent cells. C57/BL6 pups (P21) were given either one (1x) or th ree (3x) i.p. injecti ons of 100 mg/kg BrdU or 0.9% sterile saline over 24 hours and sacrificed 2 hours after the final injection. SA Gal staining was performed on the neuroge nic tissues. A dose-response can be seen in the BrdU-treated groups where samp les from 3x animals show an increase in SA Gal(+) cells compared to the control or 1x animals. Error bars represent standard deviation.
51 Figure 3-6. BrdU induces a progressive, dos e-responsive suppression of cancer stem cell population expansion. Tumor-initiating cancer stem cells isolated from a primary human glioma were treated with a single pulse of 5 or 10M BrdU and grown in neurosphere cultures. Neurospheres were passaged, quantified, and replated every 47 days. Both doses severely suppress can cer stem cell population expansion. Data are represented as mean +/standard deviation.
52 CHAPTER 4 BRDU INHIBITS PROLIFERATION OF CA NCER CELLS IN VITRO AND IN VIVO The halogenated thymidine analog 5-bromo-2deoxyuridine (BrdU) can incorporate into DNA during S-phase of the cell cycle and can therefor e be used to detect events including cell division, DNA repair and cell cycl e re-entry. BrdU is commonly us ed to quantitate proliferative index, or to birthdate, identif y and track cycling endogenous a nd transplanted cells. In the previous chapter we reported a ltered proliferation of neural st em and progenitor cells following BrdU administration in vitro or in vivo The reduction in proliferation appears to be indicative of a senescent-like state rather than the result of cell death. Administering BrdU in vivo led to an increase in senescent (SA Gal+) cells in neurogenic regions of the murine brain. Finally, we were able to recapitulate the an ti-proliferative results from the primary neural stem/progenitor cells in a population of cancer stem cells. Thes e data uncover a novel and uncharacterized effect of BrdU administration on stem/p rogenitor cells that has profound implications for both the stem cell and cancer fields. In addition to altering the biology of stem and precursor cell pools, BrdU administration may exert its anti-prolifer ative effect on cancer cells as well. In this chapter we show that a single, brief exposure to BrdU leads to a reduction in the proliferation rate of all cell lines examined. This anti-proliferative effect follows even brief, lowdose BrdU administration. However, an increase in exposure time and/or a second pulse of BrdU exacerbates the proliferation suppression. Additionally, BrdU cannot be out-competed by the presence of exogenous cytidine or thymidine, su ggesting that cells may actually preferentially incorporate the analog. The anti-p roliferative effect of BrdU is mirrored in closely related thymidine analogs and appears to be more r obust than current anti -cancer analogs. Most importantly, we show that a brief oral or systemic regimen of BrdU leads to significantly delayed tumor progression in a highly aggres sive syngeneic rat model of glioma. Our results suggest that
53 BrdU possesses therapeutic potential as an anti-cancer agent that is independent of its role as a radiosensitizer, and that BrdU should be re-assessed as an ad junctive therapeuti c modality based on a new understanding of its anti-proliferative effect. BrdU Administration Reduces Cancer Cell Population Expansion over Time RG2 rat glioma cells were tr eated once with 0, 1, 10, or 50 M BrdU for 24 hours and cumulative growth curves were obtained over 18 days (Figure 4-1A). C ontrol and treated cells were quantified and replated at equal densities on Days 5, 12, and 18 post-treatment. At all time points the BrdU-treated cells de monstrate a statistically signifi cant dose-responsive decrease in cell numbers that becomes more pronounced with increasing rounds of replication (data are expressed as population doublings over time, statistical analysis was performed on total cell counts). To more finely analyze the temporal effect of BrdU on cell number we repeated this experiment with non-adherent H9 human lymphom a cells, quantifying at 1, 2, or 4 days after exposure (Figure 4-1B). These re sults show that there is a delay of about 48 hours before statistically significant differences in cell num ber are detected. The majority of cell lines examined fail to display any sign of recovery. Ho wever, when we followed H9 cells that were treated for 24 hours with 50M BrdU and quantif ied periodically over two months, we found the initial, dramatic reduction in the rate of proliferation was followe d by a gradual recovery to near normal levels over the course of 61 population doublings (Figure 4-2). The inhibitory effect of BrdU on proliferation is common to all mammalian cells that we have tested. Figure 4-3 is a gra phical representation of BrdU-induced reduction in expansion rate for a number of cell lines. Expansion is expressed as percent of control, an d all treated cells show a dramatic, sustained, and statistically significant reduction in the rate of expansion as compared to matched, untreated controls. Since our paradi gm includes only a single pulse of BrdU, and since treated cells are affected for numerous population doubli ngs, we reasoned that impaired
54 proliferation, while requiring init ial BrdU incorporation into cellu lar DNA, is maintained even as the amount of retained BrdU decreases due to dilution with each round of cell division. We exposed MG63 human osteosarcoma cells to 50 M BrdU for 18 hours and assessed BrdU immunolabeling over 2 weeks. At 24 hours after trea tment, greater than 95% of cells are BrdU+, and the labeling is characteristically spread ove r the entire nucleus (Figure 4-4A). By day 6 (Figure 4-4B) most cells are sti ll decorated, but the pattern is pa tchy and less intense. On day 11 (Figure 4-4C) only about half of all cells st ill express immunodetectable amounts of BrdU, and the patchy pattern is more pronounced. Finally, by day 13 (Figure 4-4D) only occasional cells are labeled, and these typically s how only a single focal point with in the nucleus. At later times nuclear BrdU is not detected (data not shown) These data demonstrat e that proliferation suppression does not depend on the continue d presence of BrdU within the DNA. Anti-Proliferation Follows Even Brie f, Low-Dose BrdU Administration To determine the lowest effective dose of BrdU for slowing expansion we treated RG2 cells with 0.01, 0.1, 1.0, or 10 M BrdU for 18 hours and quantifie d after 8 days. A dose response is again apparent, with 10 M eliciting a str onger effect than 1.0M. Ho wever, doses lower than 1.0M fail to alter expansion (Figure 4-5A). Identic al results were also obtained with BJ cells (data not shown). Anti-BrdU la beling reveals that only doses of 1M or higher result in immunodetectable levels of BrdU within the cell nuclei. At 24 hours post-treatment BrdU is seen in more than 95% of BJ cells treated with 1.0 or 10 M BrdU, while cells treated with 0.1 M BrdU or lower fail to demonstr ate any immunolabeling (Figure 45B). This finding demonstrates that BrdU must incorporate into cellular DNA at immunodetectable levels in order to elicit the anti-proliferation effect. BrdU is metabolized through dehalogenation and the resulting ur acil residue can be excised from the DNA by the uracil glycosylase re pair system (Hume and Saffill, 1986; Kriss et
55 al., 1963). Various studies report that BrdU is available for labeling anywhere from 2-6 hours following systemic injection in vivo However, the exact half-life of BrdU in culture remains unknown. We next reduced the length of BrdU e xposure in order to determine the shortest effective treatment to induce impa ired proliferation. In this expe riment RG2 cells were treated with 10M BrdU for 1, 5, 10, or 60 minutes, and th e cells were quantifie d 1 week later. All exposure periods result in statistically significan t reductions in proliferation as compared to control, and all exposure times are statistically indistinguishable from one another in terms of efficacy (Figure 4-6A). Similar results were obta ined with H9 human lymphoma cells exposed to 1M BrdU for 5, 10, 20, 30, 60, or 180 minutes. Again, all exposure times were equally effective at eliciting the anti -proliferative effect. Since we have shown that BrdU incorporat ion into DNA is required to elicit the antiproliferative effect, and 60 seconds is far too sh ort an exposure time to hit all asynchronous cells in S-phase, we further analyzed how such a tr ansient BrdU pulse can produce such a profound effect on subsequent replicati on rate. We exposed both RG2 ra t glioma cells and MG63 human osteosarcoma cells to 10M BrdU for 60 sec onds (60) during their l og-phase of expansion. After the 60 exposure, cells were either fi xed immediately and immuno stained for BrdU or replenished with normal medium and fixed 24 hours later for immunolabeling. As expected, none of the cells fixed immediately after the 60 pul se exhibit the typical nuclear staining pattern associated with BrdU incorporation. In marked contrast, however, nearly all of the cells fixed and stained 24 hours after the 60 pulse show an apparently normal distribution of BrdU characteristic of incorporating cells (Figure 46B). Finer analysis at higher magnification shows that about 30% of the cells fixe d immediately after the 60 puls e do show some punctate nuclear labeling that may be located at re plication forks (Figure 4-6B inse t), whereas greater than 95% of
56 the cells fixed 24 hours after the pulse show dramatically more robust immunolabeling (Figure 46B). This finding suggests that, even within 60 s econds of application, Br dU can cross the cell membrane and perhaps enter the nucleus. Furtherm ore, BrdU that immediately enters the cell must somehow be sequestered in such a way that, during subsequent replication, it can be utilized by the cell for DNA synthesi s. For instance, if BrdU is im mediately transported into the cytoplasm and remains soluble there, then as ce llular division proceeds, it is reasonable to presume that this sequestered BrdU can then be added to replicating DNA chains, thus accounting for the dramatic increase in BrdU imm unolabeling seen after 24 hours. That fact that free BrdU sequestered within the cel l is not detected in the immedi ately fixed cells is likely due to BrdU being washed out of the cell during the immunolabeling protocol, which requires both harsh denaturation to form single-strande d DNA and permeabilization to allow antibody penetration into the nucleus. A Second Pulse of BrdU Exacerbates Proliferation Suppression BrdU is metabolized quickly both in vitro and in vivo The degradation of this analog generally consists of a two-step process that includes dehaloge nation followed by the excision of the uracil base by a uracil glycosylase repair system. The window of opportunity for active BrdU to be incorporated into cellular DNA may be limited and, theref ore, administering only one BrdU pulse may be insufficient to elicit the maximum benefit. It is importa nt to find the correct balance in drug administration that maximizes efficacy without induci ng toxicity. Delivering multiple BrdU pulses may further weaken cells that have already incorporated BrdU in the initial hit while also improving the likelihood of incorporat ion into cells that may not have been cycling during the initial pulse.
57 Our previous results demonstrate the impressi ve anti-proliferative effect of single-pulse BrdU. To determine if a second hit of BrdU would further suppress pr oliferation we treated RG2 cells with a 5 hour pulse of 10M BrdU (treated). A subset of treated cells received a second 10uM pulse three weeks foll owing the first pulse (re-treate d). Cells were quantified and compared to control values at four weeks follo wing the initial pulse (Fig ure 4-7). Treated cells show significantly suppressed expansion as compar ed to untreated controls (p<0.001), and retreated cells expand significantly sl ower than treated cells (p<0.001). BrdU-Mediated Anti-Proliferation is not Bl ocked by the Addition of Cytosine or Thymidine. While BrdU normally pairs with adenosine during DNA replication, it is also known to frequently mispair with guanine (Ashman a nd Davidson, 1981; Kaufma n and Davidson, 1978; Meuth and Green, 1974). While the exact cause of BrdU mispairing is unknown, it may be due to perturbation of normal deoxyri bonucleoside triphosphate pool sizes (Meuth and Green, 1974). BrdU-triphosphate (BrdUTP) is an inhibitor of ribonucleosid e diphosphate reductase, which ultimately leads to a deficiency in the conve rsion of cytidine diphosphate to deoxycytidine diphosphate (Meuth and Green, 1974; Moore and Hulbert, 1966). High BrdUTP concentrations, therefore, may prevent the formation of dCTP s ubstrate for DNA synthesis. With a decrease in dCTP pools, BrdUTP becomes increasingly compet itive for sites opposite template guanines, an effect that can be mitigated by the addition of excess deoxycytidine (Davidson and Kaufman, 1978). To test whether the anti-pro liferative effect of BrdU re sults from mispairing due to a deoxycytidineless state, we followed the expans ion rates of cells treated with equimolar BrdU, thymidine, or cytidine, both alone and in combination (Figure 4-8) The reduced cellular expansion produced by 50M BrdU is not abroga ted by the co-administration of equimolar
58 thymidine or cytidine, nor by a combination of thymidine and cytidine. Furthermore, neither thymidine nor cytidine, alone or in combination, significantly re duces proliferation rate as compared to untreated controls cells. Finally, the anti-proliferativ e effect of 50M BrdU is not diminished even when co-administered with up to 250M cytidine (data not shown). These results suggest that anti-proliferation arises neither from BrdU out-c ompeting cytidine during DNA synthesis, nor as a result of simply alte ring the intracellular milie u by the addition of excess nucleotides. Halogenated Pyrimidines Suppress Proliferati on More Robustly than Current Anti-Cancer Nucleosides To determine if halogenated pyrimidines st ructurally similar to BrdU also perturb proliferation, we analyzed the effect of 5-chloro-2-deoxyuridine (CldU) and 5-iodo-2deoxyuridine (IdU) on H9 cell expansion for thr ee consecutive weeks af ter an 18 hour exposure to 1, 10, or 50M of each thymidine analog. All three analogs produce a st atistically significant dose-responsive reduction in prol iferation that is remarkably similar in degree (Figure 4-9A). Furthermore, this anti-prolifer ation is non-synergistic, since co mbinatorial administration does not strengthen the effect (data not shown). In the same experiment we compared expans ion among cells treated with either BrdU or one of two anti-cancer nucleoside s, 5-fluorouracil (5-FU) or 5aza-2-deoxycytidine (AZA). The chemotherapy agent 5-FU acts in several ways, but principally as a thymidylate synthase inhibitor. Interrupting the action of this enzyme blocks synthesis of the pyrimidine thymidine. 5FU is transformed inside the cell into different cytotoxic metabolites which are then incorporated into DNA and RNA, finally inducing cell cycle arrest and apoptosis by inhibiting the cell's ability to synthesize DNA. It is an S-phase specific drug and only act ive during certain cell cycles. AZA belongs to a class of cytosine anal ogues which were devel oped as inhibitors of
59 DNA methylation and have shown clinical efficacy in myelodysplastic syndromes (MDS) and acute myelogenous leukemia (Kiziltepe et al., 20 07). Despite the widely accepted demethylating activity of AZA, the exact basis of its clinical efficacy and its cytotoxic mechanism still remain unclear. To compare the efficacy of BrdU to 5-FU a nd AZA, H9 cells were administered either BrdU, 5-FU (FU), or AZA (A) at 0, 1, 10, or 50M for 18 hours and cells were quantified weekly for three consecutive weeks (Figure 49B). At one week AZA-treated cells are not significantly different from cont rols, whereas both BrdUand 5-FU -treated cells are reduced by 50-60%. At two weeks AZA-treated cells still matc h control levels, while the BrdU and 5-FU groups have dropped to about 10% of control. At week three the Br dU-treated cells are maintained near 10% of control level while the 5-FU treated cells reco ver to about 35%. AZA showed substantial variability, w ith some treated cells demonstrating a statistically significant reduction to about 80% of control, but this reduction is not maintain ed over time (data not shown). These results suggest th at the suppressive effect of single-pulse BrdU on cancer cell expansion is more effective than AZ A, and more persistent than 5-FU. Length of BrdU Exposure is Directly Rela ted to the Percentage of Labeled Cells The efficacy of BrdU treatment depends on widespread penetrance. To determine how well BrdU is incorporated in cancer cells in vitro we compared BrdU labeling in cells treated with BrdU (1, 5, 10 or 50 M) for 1, 6, or 24 hours. Fi gure 4-10 shows that the exposure time is more important than dose. The number of labeled cell s nearly doubles from one hour to six hours of exposure while the differences between doses are in significant. These results are consistent with the idea that longer exposure times allow for capturing more cycling cells in an asynchronous population. Interestingly, while the different doses do not alter the number of labeled cells, the
60 intensity of the BrdU labeling is directly related to dose; where cells treated with higher doses of BrdU display brighter st aining (data not shown). BrdU Administration Slows Glioma Tumor Progression In Vivo Glioblastoma multiforme (GBM) is the most common primary (i.e., non-metastatic) brain tumor of humans. Despite advances in cytoreduc tive and cytotoxic therap ies, the prognosis for this neoplasm remains dismal, with a median su rvival time of approximately 12 months (Akman et al., 2002; Basso et al., 2002; Nied er et al., 2000). This has foster ed an intense in terest in the search for alternative therapeuti c modalities that may prove to be more effective or that may augment standard surgical, radi ological or chemotherapeutic treatments for these neoplasms (Mariani et al., 2007). We chose a syngeneic, invasive, non-immunoge nic rat glioma mode l (Aas et al., 1995; Barth, 1998; Ko et al., 1980) to test whether the proliferation suppres sion of BrdU has a meaningful in vivo correlate. The RG2 glioma model has been posited as the equivalent of human GBM, and these tumors are refractory to therapeutic modalities, rendering them nearly impossible to treat efficiently or cure. Their in vasive pattern of growth and uniform lethality following an innoculum of relatively few cells make them a particularly attractive model to test new therapeutic modalities (Barth, 1998). First, we injected a bolus of RG2 cells -either untreated or pre-treated in v itro for 24 hours with 50M BrdUsubcutaneously into Fisher 344 rats and followed tumor progression. All animals ev entually develop tumors but the survival time (defined as 3000mm3 tumor volume) is dramatically dela yed in animals receiving pre-treated cells (Figure 4-11A). This result clearly demons trates that prolifera tion suppression induced by in vitro application of BrdU is maintained in the in vivo environment. In a second set of experiments we tested whether tumor progression can be slowed by in vivo administration of BrdU. Animals were inocul ated with nave RG2 cells and then treated
61 with either i.p. or oral BrdU. After implantation, one group of animals received 6 i.p. injections of BrdU (300mg/kg) over 2 days (Figure 4-11B ), while a second group was placed on ad libitum drinking water containing 0.8mg/m L BrdU for 1 week (Figure 4-11C ). In both cases there is a statistically significant increase in survival in th e animals receiving BrdU, i ndicating that BrdU is effective at slowing the growth of cancer cells in vivo even when it is administered after tumor initiation. Even a modest increase in survival time following a conservative dosing regimen is highly encouraging when one considers th e refractory nature of these tumors. Discussion Proliferation was suppressed in all of the cells that we exam ined and prior studies have also demonstrated the ubiquitous susceptibility of mammalian cells to BrdU (Michishita et al., 1999). There is even evidence that BrdU slow s replication in thym idine-auxotrophic yeast (unpublished observation, Fujii et al., 2002), su ggesting that BrdU-mediated proliferation suppression in all eukaryotic cells may be affected through a common yet still undefined mechanism. The delayed in vivo tumor progression in the extremely aggressive RG2 model is the most important aspect of our study. Despite advances in cytoreductive and cy totoxic therapies, the prognosis for GBM remains dismal, with a median survival time of approximately 12 months (Akman et al., 2002; Basso et al., 2002; Nieder et al., 2000). There is intense interest in finding alternative therapeutic modalities that may prove to be more effective or that may augment standard surgical, radiol ogical or chemotherapeutic treatments for these neoplasms (Mariani et al., 2007). The RG2 glioma model has been posited as the equivalent of human GBM. The fact that brief administration times used in our study result in statistically si gnificant delays in the growth of nave tumor cells raises the possibili ty that BrdU alone may be capable of producing biologically significant therapeu tic gains under optimized dosing schedules. In addition, the
62 dramatically delayed progression of BrdU pre-treated cells (tr eated prior to implantation) suggests that BrdU may prove effective against secondary metastatic tumor formation. Future studies will be directed at examining the homi ng and invasiveness of Br dU-incorporating cells and their progeny.
63 Figure 4-1. BrdU induces a pr ogressive, dose-responsive suppr ession of cancer cell line population expansion. (A) RG2 rat glioma ce lls treated for 24 hours with 1, 10, or 50M BrdU show a dose-responsive reducti on in the rate of population doubling over 18 days following removal of BrdU. Cells from all groups were replated at equal density at 120 & 288 hours to prevent overgro wth of the culture vessels. At all time points, BrdU-treated cells lag significantl y behind controls, rega rdless of dose (oneway ANOVA, Tukey-Kramer post-hoc test of significance was performed on the basis of total cell counts at each time poi nt; p<0.001, N=3 for all groups). (B) Finer temporal analysis reveals that significantly slowed expansion is a pparent as early as 48 hours post-BrdU. H9 human lymphoma ce lls treated for 24 hours with 1, 5, 10, or 50M BrdU are not significantly differe nt from control at 24 hours post-BrdU exposure, yet by 48 hours all treated groups lag significantly behind control, and the degree of lag is dose-responsive (one-way ANOVA, Bonferroni post-hoc test of significance; p<0.001, N=3 for all groups). Data are represented as mean +/standard deviation.
64 Figure 4-2. Eventual recovery following single-pulse BrdU admini stration. H9 cells treated for 24 hours with 50M BrdU and followed for 61 population doublings (approximately 1x1016 times expansion) show a gradual recove ry in expansion rate to near control levels.
65 Figure 4-3. Proliferation suppres sion is common among all cancer cells examined. Our standard treatment paradigm of 50M BrdU fo r 24 hours causes a reliable suppression of expansion in various cell lines. The top pa nel shows the graphical representation of expansion by BrdU-treated cells (shown as percent control) over a range of 0 14 population doublings after removal of BrdU; Statistical analysis of this model is presented in the table (below).
66 Figure 4-4. Proliferation s uppression is independent of BrdU retention. MG63 human osteosarcoma cells were exposed to a single, 18 hour pulse of 50M BrdU and assessed over time for BrdU retention. At 24 hours post-exposure (A) greater than 95% of all cells show substantial BrdU im munoreactivity (green) within the nucleus. The amount and intensity of BrdU label progr essively declines at 6 (B), 11 (C), and 13 (D) days post-exposure. Cell nuclei ar e counterstained with propidium iodide (red).
67 Figure 4-5. Transient, low-dose BrdU suppresses expansion rate (A) RG2 cells were treated with a single 18 hour pulse of 0.01, 0.1, 1.0, or 10M BrdU and quantified 8 days later. Cells receiving 0.01 or 0.1M doses were not significantly different from control at 8 days, whereas expansio n of both the 1.0 and 10M groups was significantly suppressed (one -way ANOVA with a Tukey-Kramer post-hoc test of significance; p<0.001. N=3 for each group.). (B) BrdU doses that fail to suppress expansion also fail to immunolabel treat ed cells. Both the 1M and 10M groups demonstrate BrdU immunolabeling (green) of nearly all cells 24 hours after BrdU exposure, whereas the 0.1M group does not contain immunodetectable BrdU. All nuclei are counterstained with propidium iodide (red). Sc ale bar = 20m. Data are represented as mean +/standard deviation.
68 Figure 4-6. BrdU exposure times as short as 1 minute leads to suppressed expansion and delayed labeling of nuclear DNA. (A) RG2 rat glioma cells were exposed to 10M BrdU for 1, 5, 10, or 60 minutes and quantified after 1 week. All exposure times result in statistically suppressed expansion as compared to control, and there are no significant differences among the treated groups. (B) RG 2 cells treated for 1 minute with 10M BrdU were fixed and immunostained either immediately after rem oval of BrdU (left panel), or 24 hours after removal of BrdU (right panel). Cell s fixed immediately show little or no BrdU immunoreactivity (gre en), except for a fraction of cells that display subtle nuclear labeli ng that is apparent only at 100x magnification (inset). In contrast, greater than 95% of the cells fixed 24 hours after the 1 minute pulse show the typical pattern of nuclear immunost aining. All nuclei are counterstained with propidium iodide (red). Scale bar = 20M.
69 Figure 4-7. A second pulse of BrdU exacerbates expansion suppression. RG2 rat glioma cells received one 5 hour pulse of 10M BrdU (treat ed). A subset of treated cells received a second 10uM pulse three weeks following the first pulse (re-treated). Cells were quantified and compared to control values at four weeks follow ing the initial pulse. Treated cells show significantly suppressed expansion as compared to untreated controls (p<0.001), and re-treated cells expa nd significantly slower than treated cells (p<0.001). One-way ANOVA with Tukey-Kramer post-hoc test of significance. N=3 for all groups.
70 Figure 4-8. BrdU-mediated proliferation suppr ession is not antagonize d by excess cytidine. 50M BrdU, deoxycytidine (dCyd), and deoxythy midine (dThy) were applied to H9 human lymphoma cells in factorial combin ations for 24 hours, and cellular expansion was assessed 1 week later. All of the gr oups receiving BrdU showed statistically indistinguishable reductions in expansion as compared to control (p<0.001). In contrast, groups receiving dCyd, dThy, or dCyd+dThy demonstrated expansion equivalent to control levels. One-way ANO VA with a Tukey-Kramer post-hoc test of significance. N=3 for each group. Error ba rs represent standard deviation.
71 Figure 4-9. BrdU-mediated proliferation s uppression is matched by similar halogenated pyrimidines and surpasses current anticancer nucleoside anal ogs. (A) BrdU (B), CldU (C), and IdU (I) were compared for the ability to suppress expansion of H9 human lymphoma cells. Each analog wa s administered for 18 hours at 1, 10, or 50M and cells were quantified weekly fo r three weeks (weekl y counts correspond to a left-to-right progression of color-coded tr iplicates of bars). At all doses and time points the three halogenated pyrimidines produce remarkably similar, statistically significant reductions in cell number as comp ared to untreated controls. (B) In the same experiment, the therapeutic anti-cance r nucleoside analogs 5-fluorouracil (FU) and 5-azacytidine (A) were also examined for their ability to suppress expansion of H9 cells in the same paradigm. 50M FU is slightly more effective than 50M BrdU at week one, but by week two 50M BrdU is substantially better at suppressing expansion. At week three 50M BrdU s uppression has not changed, while 50M FU suppression has started to rec over toward control. At a ll other doses and time points BrdU is as effective as or more effectiv e than FU at suppressi ng expansion, and BrdU suppression persists longer. 5-azacytidine s hows variable and transient suppression of expansion to near 80% of control levels Counts were analyzed with a two-way ANOVA followed by a Tukey-Kramer post-hoc test of significance. N=3 for each group. Data are represented as mean +/standard deviation.
72 Figure 4-10. Exposure time corresponds with an increased percentage of BrdU-labeled cells. RG2 cells were treated with 0, 5, 10, or 50 M BrdU for 1, 6, or 24 hours. The percentage of BrdU(+) cells was quantifie d in control and experimental groups for each dose and exposure time. There is a signi ficant increase in the percentage of BrdU(+) cells exposed for 6 or 24 hours compared to 1 hour (p<0.001); however, there were no significant di fferences between doses. Count s were analyzed with a two-way ANOVA followed by a Tukey-Kramer pos t-hoc test of significance. N=3 for each group. Data are represented as mean +/standard deviation.
73 Figure 4-11. BrdU administration slows tumor pr ogression in a syngeneic in vivo glioma model. RG2 rat glioma cells, either untreated or pre-treated with 50M BrdU, were injected subcutaneously in Fisher 344 rats. Tu mor progression was monitored by taking measurements every other day and calc ulating tumor volume. Animals were euthanized when the tumor volume r eached the critical end point (3000mm3). (A) Animals that received the RG2 glioma ce lls that had been pre-treated with 50M BrdU for 24 hours show a significant dela y in tumor progression when compared to the control animals (p = 0.002). (B) Animals th at received subcutaneous injections of untreated RG2 cells but were administered BrdU by i.p. injections (300mg/kg x 3 / 2days) also show significant delays in tu mor progression (p = 0.02). Additionally, (C) animals that received subcutaneous inj ections of untreated RG2 cells and were administered BrdU in their drinking water (0.8mg/ml for 7d) show a significant delay in tumor progression (p = 0.04). Kaplan-Meier survival curves (Chi-square test with associated p-value). N=10 for each group.
74 CHAPTER 5 CHARACTERIZING THE MECHANISM OF AC TION FOR THE ANTI-PROLIFERATIVE EFFECT OF BRDU Despite its extensive history there is no accep ted consensus mechanism of action for BrdU. It has been suggested that BrdU alters the stability of DNA there by increasing the ri sk of sisterchromatid exchanges, mutations, and doublestrand breaks (reviewed in Taupin, 2006). However, most of these effects are found only when BrdU incorporation is combined with secondary stressors. Early toxicity studies show ed that BrdU can induce chromosomal breakage and increase the sensitivity of tr eated cells to ionizing radiatio n (Djordjevic and Szybalski, 1960; Erickson and Szybalski, 1963; Hsu and Somers, 1961), and this radiosensitizing effect has continued to be pursued as an ad junctive therapy in the treatment of a variety of cancers. BrdU readily crosses the blood-brain barrier, and ha s been combined with conventional chemotherapy and radiation treatment in several clinical tr ials (Kinsella et al., 1984, Phillips et al., 1991, Robertson et al., 1997a, Roberts on et al., 1997b, Groves et al., 1999, Prados et al., 2004). While the clinical benefits of includi ng BrdU as a radiosensitizer have been disappointing showing, at best, modest improvements for some outcome meas urementsit is possible that other therapeutic effects of BrdU were not appreciated, either because of interferen ce by the other treatment modalities in these studies, or because finer anal ytical resolution is required to discern them. Surprisingly little attention has been focused on examining the influence that BrdU alone may exert on cellular function. In the present study we show that a single, brief exposure to BrdU leads to a progressive and sustained impair ment of cell-cycle progression in all examined cancer cells in vitro Treated cells do not die, but gradually accumulate in the G1 phase of the cell cycle while showing a variable upregulation of some senesc ence-associated proteins. DNA damage is one of the primary causes for the ch anges we see in the cell cycle kinetics and subsequent senescent phenotype in BrdU-treated cells. Interestingly, BrdU treatment does not
75 appear to cause DNA damage. It is known that senescent cells can influence their local environment through the secretion of various factors, altering gr owth signals for neighboring cells. However, BrdU-treated cells do not promot e or inhibit the prolifer ation of untreated cocultured cells. Single-Pulse BrdU Does Not Result in Increased DNA Damage or Apoptosis Reports associating BrdU with DNA damage generally involve the use of a secondary stressor such as irradiation, rende ring it difficult to discern whethe r BrdU alone has an effect. It is plausible that BrdU exposure induces DNA da mage which can cause stress in other cellular components, leading to amplification of apoptos is (Lin et al., 2007). H2A.X, a member of the histone H2A family, is synthesized in the G1/S phases and is involved in chromatin organization. In response to DNA damage, H2A.X is phosphorylated at the Serine 139 re sidue and referred to as H2A.X. We labeled control a nd BrdU-treated cells with H2A.X at various time points following BrdU administration to determine if DNA damage, specifically double-strand breaks (DSB), is induced. We found with BJ and MG63 cells that there are no consistent differences in the number of H2A.X positive cells between treated and control groups during the first 4 days after exposure to BrdU. At no time did the percentage of positive cells in either control or treated groups exceed 2% of the total popul ation, and in some instances th e percentage was higher in the controls (Figure 5-1). Previous studies reveal the sensitivity of Br dU-substituted DNA to visible light irradiation. This combination results in DNA lesions, specifically sister-chromatid exchanges. It is suggested that the debromination of 5-brom ouracil after visible light irradiat ion results in the production of uracil residues (Maldonado et al ., 1985; Hutchinson and Kohnlein, 1980). To determine if the anti-proliferative effect of BrdU is related to photosensitivity, we ex amined increased BrdUmediated sensitivity to photolysis, as describe d by Michishita and colleagues (2002). Treated
76 cells that were protected from ambient light for five days still show profound expansion suppression, demonstrating that DNA damage due to irradiation does not account for the observed effect (Figure 5-2). The relationship between cellu lar metabolism and cell cycle control is not well understood. However, it has been reported that energy deprivation can prevent passage through the G1-S cell cycle checkpoint (Mandal et al ., 2005). While BrdU can inco rporate into mitochondrial DNA (Davis and Clayton, 1996), litt le is known about how it affect s mitochondrial health and/or function. Mitochondrial membrane potential is a key indicator of cellular viab ility and is critical for ATP production. Additionally, the collapse of the electrochemical gradient across the mitochondrial membrane is an early event in the apoptotic cascade. We compared the mitochondrial membrane potential of treated and control MG63 cells at 1 and 7 days after a 24 hour pulse of 50 M BrdU. These results fail to reveal differences in mitochondrial membrane potential between control and BrdU-treated cells (Figure 5-3). Identical results were obtained with both H9 and BJ cells (data not shown). Activation of cleaved caspase 3 is another earl y event in cellular apoptosis. We examined BJ and MG63 cells for caspase 3 expression over 4 days after a 24 hour pulse of BrdU (Figure 54). The percentage of cells expressing caspase in all groups was very low, never exceeding 1% of the total. Additionally, there were no consistent differences between treated cells and matched controls in either group. We also examined Annexin-V binding which reveals the loss of plasma membrane asymmetry that allows phosphatidylserine, normally located in the inner layer, to be exposed on the cell surface. Such loss of as ymmetry is thought to be asso ciated with cells that will eventually execute an apoptotic program. H9, Saos-2, and BJ cells were examined after a single
77 24 hour exposure to BrdU (50M). In additi on, a dose response study was carried out with MG63 cells. The results with Annexin-V are highly variable and somewhat confusing (Figure 55). There is a dose responsive increase in the percentage of MG63 cells labeled with Annexin-V with greater than 60% of a ll cells either dead or Anne xin-V(+) after exposure to 50 M BrdU for 24 hours (Figure 5-5A). The results with the other cell lines are highly va riable (Figure 5-5B). Treated H9 cells seem to show a slight increas e in the Annexin-V(+) po pulation, while there is no increase in treated Saos-2 cells. BJ cells also fail to reveal differenc es in Annexin-V levels between treated and control groups, but the base level of expression is around 50% even in the untreated controls. Because the Annexin-V results are so dramatically different from the results obtained with other markers of apoptosis, and becau se there is large intracell line variability in both baseline Annexin-V expression and degree of change after BrdU exposure, we believe that these results do not accurately reflect cell death in our culture paradigm. This is supported by reports in the literature demonstrating that A nnexin-V labeling can be tr ansient and reversible (Hammill et al., 1999), and that Annexin-V labe ling can increase even without eventual cell death (Holder et al., 2006) Finally, in order to assess la te-stage apoptosis, we examined cells with the Terminal Uridine Deoxynucleotidyl Transferase dUTP ni ck end labeling (TUNEL) assay which detects DNA fragmentation characteristic of apoptotic cells. As with the H2A.X labeling, we found only negligible increases in TUNEL(+) trea ted cells that cannot account for the profound reduction in expansion rate, since TUNEL+ cells never accounted fo r more than 0.5% of the total population (Figure 5-6). Thus, three methods for detecting apoptosis i ndicate that the level of cell death in BrdU-treated cultures is very low and not significantly different fr om the control level.
78 BrdU Alters the Cell-Cycle Profile The cell cycle is regulated by the sequential ac tivation and inactivation of cyclin-dependent kinases (CDKs), through the periodic synthesis an d destruction of cyclin s. Understanding cell cycle control is a major focus of cancer resear ch because it provides information on both the process of tumorigenesis as well as potential therapeutic target s. During the first gap phase (G1) a cell prepares for DNA replication and determines its fate. Mitogenic and growth inhibitory signals are integrated and the cell uses this inform ation to make the ultimate decision to proceed, pause, or exit the cell cycle (Johnson and Wa lker, 1999). The Restriction Point within G1 represents a critical checkpoint in determining the viability of the cell. Events like DNA damage signal changes in the molecular network at the Rest riction Point that influence the cell to either pause for repair and/or (permanently) exit the cell cycle. Since BrdU leads to a reduced cellular expans ion over time that is not the result of increased cell death, we hypothesize th at there must be an alterati on in the cell cy cle profile of treated cells. We treated async hronous BJ fibroblasts with 50 M BrdU for 24 hours and compared their cell cycle profile to control cells after one we ek. As expected, there is a statistically significant reduction in the proportion of BrdU treated ce lls in S-phase that is offset by an increase in the frac tion of treated cells in G0/G1 (Figure 5-7). Finer analysis with similarly treated RG2 cells reveals that as early as 6 hours after BrdU administration there is a statistically significant reduction in the proportion of cells in S-phase in treated gr oups (Figure 5-7). The ratio of treated to control cells in S-phase varies but at all times over 1 week post-exposure there are fewer BrdU-treated cells in S-phase H9 cells treated for 24 hours with 50 M BrdU also show a reduction in the proporti on of cells in S-phase by 24 hours post-BrdU exposure, and this reduction remains relatively stable over the ne xt two days (data not shown). These findings
79 demonstrate that BrdU exposure leads to a rapid alteration in cell cycle di stribution that precedes a detectable delay in expansion rate as measured by total cell quantification. The increase in the population of BrdU-treated cells in G1 suggests the possibility that these cells are either exiting th e cell cycle or are unable to trav erse the Restriction Point. The control of cellular proliferation is tightly regul ated by various intrinsic and extrinsic factors. However, the retinoblastoma prot ein (pRb) is recognized as a gua rdian of the Restriction Point and cell cycle progress (Weinberg, 1995). To te st the effect of BrdU on pRb phosphorylation, RG2 and BJ cells were administer ed BrdU (50M) at 0 hours and cell lysates were collected at various post-administration time points (1 hour, 6 hours, 96 hours, and 7 days) for Western blot analysis. At 24 hours, the BrdU-containing media in the 96 hour and 7 day culture flasks was replaced with BrdU-free media. The level of phosphorylated pRb in BrdU-treated RG2 cells is negligible at 6 hours post-administration and ther e is no detectable expression at 96 hours or 7 days (Figure 5-8 A&C). Additionally, phosphorylated pRb expression is no longer detectable in BrdU-treated BJ cells by 96 hours post-administr ation (Figure 5-8 B&D). The time at which the level of phosphorylated pRb d eclines corresponds with the G1 accumulation (see Figure 5-7). The decrease in expression level appears to be phosphorylation-speci fic as the levels of total pRb are comparable between control and BrdU-treated cel ls, particularly in the BJ cells, at these time points (Figure 5-8 E&F). The two most commonly mutated genes in cancer are p53 and p16. These two prominent proteins can greatly influence cell cycle kinetics and can function dependently or independently of each other. Both the INK4 (p15, p16, p18, p19) and Cip/Kip (p21, p27, p57) families of cyclin-dependent kinase inhibitors (CDKIs) regulate cell progression through G1. Interestingly, the majority of the cell lines we employ have abnormal expression patterns for many of the
80 primary markers related to G1 arrest (see Table 5-1), making it di fficult to assign the effect of BrdU to any of the prominent cell cycle and/or senescence pathways. However, the expression profile of a key cell cycle protei n, p21, has been reported as normal in both the RG2 and BJ cell lines. The induction of p21 can prevent cell cycle progressi on by inhibiting a variety of cyclin/CDK complexes and/or by inhibiting DNA synthesis through PCNA binding (Johnson and Walker, 1999). Interestingly, the level of p21 does not change following BrdU treatment (Figure 5-8 E&F). This result is not necessarily surprisi ng knowing that both H9 and Saos-2 cells do not express p21 yet are still sensitive to BrdU treatment. The slowed expansion and altered cell cycle profile of BrdU-treated cells resembles a senescent-like phenotype and there is evidence that halogenated pyrimidines can induce senescence in a variety of cell t ypes (Michishita et al., 1999; Suz uki et al., 2001; Michishita et al., 2002; Minagawa et al., 2005) However, the expression le vels of known senescenceassociated proteins are not consistently altere d by BrdU exposure. For instance, senescenceassociated -galactosidase (SA Gal) activity (Dimri et al., 1995) is upregulated in RG2 cells 24 hours after exposure to 10M BrdU (Figure 59). In contrast, ther e is no detectable SA Gal activity in severely suppressed MG63 cells (data not shown). Similar ambiguous results were obtained in relation to telomerase activity and telomere mainte nance. Telomere erosion during cellular replication has been shown to activate DNA damage signaling pathways that can inhibit subsequent cell cycle progression and induce senescence (de Lange, 2006). To examine BrdUmediated perturbation of telomerase activity as a mechanism of slowed cell cycle progression, we performed Telomeric Repeat Amplification Pr otocol (TRAP) analysis on control and BrdUtreated RG2 cells. TRAP analysis performed 24 and 48 hours after treatment reveals strongly reduced telomerase activity (Figure 5-10A). Ag ain, though, this reduction is variable and cell-
81 line specific, as MG63 cells fail to demonstrate a reduction in telomerase activity, even three weeks post-exposure (Figure 5-10A). Furthermore, the telomerase-negative BJ and Saos-2 cell lines also demonstrates BrdU-m ediated proliferati on suppression, suggesti ng that telomerase activity is not directly involved in BrdU-mediated anti-prolife ration, but may itself be reduced in telomerase-positive cells subsequent to cell cycle alterations. Under some circumstances telomeres can be maintained by a telomerase-independent mechanism referred to as Alternative Lengtheni ng of Telomeres, or ALT (Dunham et al., 2000). Since telomere length can be maintained in the absence of telomerase activity, and since BrdU induces slowed proliferation in cells regardless of telomerase ac tivity, we looked for evidence of telomere shortening through terminal restriction fr agment (TRF) analysis in H9 cells, but failed to detect differences in average telomere length between treated and contro l cells (Figure 5-10B). BrdU-Incorporating Cells Do Not Influen ce the Proliferation of Neighboring Cells Senescent cells possess an unusual phenotype that maintains metabolic activity while preventing either cell division or cell death. This level of activ ity allows senescent cells to secrete various factors that influence the microenvironment and neighboring cells. Inducing senescence in cancer cells has been the focus of many groups looking for novel cancer therapies. However, it remains unknown exactly how senes cent cells may affect nearby cancer cells. Senescent fibroblasts have been shown to stim ulate the proliferation of co-cultured cells, specifically neoplastic cells. Inte restingly, the presence of senes cent cancer cells also increases the proliferation of co-cultured cells in vitro though less significantly than that seen with senescent fibroblasts. This bysta nder effect is believed to be a result of secreted factors including extracellular matrix pr oteins, growth factors and cytoki nes. However, senescent cells also show different secretion patterns and expres sion levels of these factors as compared to
82 actively replicating cells (reviewed in Ewald et al., 2008). These differences may allow for the specific targeting of senescent cancer cells. The idea that senescent cells can influence neighboring cells is well supported in vitro. The downstream effects caused by secreted factor s from senescent cells likely depend on the recipient cell type. However, the in vivo correlate does not appear as strong. Ewald et al. (2008) show that the transplantation of senescent cancer cells failed to increase the establishment, growth, or proliferation of nonsenescent cancer cells in a xenogr aft model. They argue that proliferative bystander e ffect of senescent cancer cells are negligible and support further development of senescence induction therapy. We have shown that BrdU administration leads to a senescent-like st ate in various cancer cell lines. However, it remains unclear how BrdU-t reated cells may influence neighboring cells, particularly untreated cells. Our data thus far indicate that BrdU must be incorporated into a cellular DNA to alter proliferation rate. An al ternative hypothesis that could account for the suppression of population doubling time is the suppr ession of cell cycle progression by a fraction of cells that incorporate BrdU. For example, if BrdU-incorporating cel ls secrete a growthinhibitory factor, then the prol iferation kinetics of a population of cells might be suppressed by a minority of BrdU-incorporating cells. We co-cultured BrdU-treated (1M, 3 hour expo sure time) and control (untreated) H9 cells in varying combinations to dete rmine if BrdU-treated cells aff ect the untreated cells. Control cells, BrdU-treated cells, a 1:1 control:treated mi xture, a 9:1 control:treat ed mixture, and a 1:9 control:treated mixture were compared after 1 w eek (Figure 5-11). As exp ected, expansion of the BrdU-treated cells is dramatically impaired, representing only 18% of the control value. Interestingly, there is a near linear relationship among the followi ng control:treated mixtures: the
83 9:1 ratio consists of 90% control cells, and thei r expansion is 92% of control; the 1:1 mixture falls near the midway between treated and contro l, at 62% of the control value; and the 1:9 mixture, containing 95% treated cells shows exactly 95% of the anti-proliferative effect with expansion that is 23% of contro l. These data strongly demonstr ate that the anti-proliferative effect of BrdU is intrinsic to only those cells exposed directly, and th ere is no proliferation suppression by treated cells. BrdU does not appear to cause a bys tander effect on the untreated co-cultured cells; therefore, the presence of BrdU-t reated cells neither prom otes nor inhibits the proliferation activity of neighboring cells. Discussion BrdU has a long history as a potential anti-cancer drug, and it is known that at high doses and in combination with secondary stressors, such as ionizing radiation, BrdU can have lethal consequences for incorporating cells. The pres ent findings are surprisi ng in that our BrdU regimen is exceedingly mild, even by current experimental pulse-chase birthdating paradigms in which cells incorporate BrdU and continue to function in an appare ntly normal manner. Furthermore, the suppressed prolif eration that is described occurs in the absence of secondary insults that would stress the ce lls ability to maintain homeo stasis or undergo DNA repair. A role for BrdU in senescence-induction of ma mmalian cells has recently been described in the gerontology field (see Minagawa et al., 2005) but this role is not yet widely appreciated by the larger scientific communit y, and has not previously been a pplied as an approach to slow tumor progression. Emerging in vivo studies underscore the importa nce of cellular senescence in altering the growth properties of tumors in humans and rodents (Campisi, 1995; Liu and Hornsby, 2007), and we show here that cancer cell s treated with single-pu lse BrdU show some signs consistent with senescence. Both the al tered cell-cycle profile and the upregulation of SA Gal are common manifestations of a senescent phenotype; however, SA Gal upregulation
84 varies widely by cell line, with some showing no enzyme activity even during severe suppression of proliferation rate, and ther e is evidence suggesting that SA Gal is not necessarily a selective marker of senescence (Severino et al., 2000). While the control of cellular proliferation is tightly regulated by various intrinsic and extrinsic factors, pRb is generally regarded as the master regulato r of the Restriction Point and is now implicated in cellular senescence. Rb is be lieved to control a senescence-initiating pathway that may synergize with, but is distinct from telomere loss (Thomas et al., 2003; Weinberg, 1995). Our result, showing perturbed pro liferation that corre sponds with hypo-/unphosphorylated pRb yet appears to be independent of telomere main tenance, supports this theory. The post-administration times at which the levels of phosphorylated pRb become undetectable in BrdU-treated RG2 and BJ cells al most perfectly correspond to the accumulation of these cells in G1. However, results showing similar prolifer ation suppression in pRb null Saos-2 human osteosarcoma cells (data not show n) again indicate that pRb is not required for BrdU effect. It seems, then, that BrdU exposure does not lead to classically defined senescence but rather to a generalized slowing of proliferation and does not, in our experimental paradigm, lead to crisis or subsequent cell death. Furthermore, the battery of cell lines we have examined all show similar proliferation suppression following Br dU exposure, yet differ widely in the status of quintessential senescence markers (see Table 5-1). This variability makes it difficult to define a universal mechanism of action and suggests that these molecular players, while possibly involved in a cell type dependent manner, are ne ither required nor causative for the suppressive effect of BrdU.
85 Figure 5-1. BrdU does not lead to increased H2A.X immunoreactivity. (A) BrdU-treated (B) and matched control (C) BJ or MG63 immunostained for H2A.X at 1, 6, 24, 72, and 96 hours after a single 24 hour pulse of Br dU, and positive cells are expressed as the percentage of the total ce ll population. While there is a trend toward greater H2A.X expression in both control and treated cells with increasing time in culture, there are no consistent differences between matched co ntrol and treated samples in either cell type. (B) Representative photomic rographs showing examples of H2A.X(+) and H2A.X(-) MG63 cells 96 hours after a single 24 hour pulse of 50 M BrdU. The panel on the left shows DAPI staining of two nuclei (pseudocolored magenta). The middle panel shows H2AX immunolabeling (green) of the same field of view. The right-hand panel is an overlay of two panels. Characteristic H2AX(+) foci are present in the upper nucleus, indicati ng double-strand DNA breaks. Scale bar = 10 M.
86 Figure 5-2. Expansion suppressi on is not due to BrdU-mediate d photolysis. Since BrdU has been shown to increase the sensitivity of cells to irradiati on, including light, we exposed MG63 human osteosarcoma cells to 50M BrdU for 24 hours and cultured them under light protection for 5 days. Even under these conditions BrdU-treated cells expanded at a significantly slower ra te than controls (unpaired t-test, Welchcorrected, p<0.0001). N=3 for each group; Erro r bars represent standard deviation.
87 Figure 5-3. BrdU does not perturb mito chondrial membrane physiology. MG63 human osteosarcoma cells received a 24 hour pul se of 50M BrdU and mitochondrial membrane physiology was assessed via the JC-1 potentiometric dye. Mitochondrial membrane depolarization is indicated by a decrease in the red/green fluorescence intensity ratio. (A) Contro l MG63 cells were treated with CCCP (a mitochondrial membrane disrupter) as a positive contro l for depolarization. At both 24 hours (B&C) and 7 days (D&E) post-BrdU exposure contro l and treated cells display equivalent membrane potentials.
88 Figure 5-4. BrdU does not induce cleavage of caspase-3 in trea ted cells. Cleaved caspase-3 was assessed in control (C) and BrdU-treated (B) BJ and MG 63 cells at 1, 6, 24, 72, and 96 hours after a single 24 hour exposure to 50 M BrdU. In none of the groups does the percentage of caspase 3+ cells exceed 1% of the total popul ation, and there are no consistent differences between treated and control groups in either cell line.
89 Figure 5-5. BrdU treatment cause s variable, cell line-specific Annexin V assay results. (A) Annexin-V labeling (purple) shows a dose-responsive increase in MG63 cells exposed for 24 hours to 1, 10, or 50 M BrdU. In addition, there is a slight increase in dead cells (green) with increasing concen tration of BrdU. (B) Annexin-V labeling was assessed in control (C) a nd treated (B) H9, Saos-2, and BJ cells at various times after a single 24 hour exposure to 50 M BrdU. H9, but not Saos-2 or BJ cells show an increase in Annexin-V label (purple) by treated cells. Additionally, untreated control cells show wide vari ability in the constitutive level of Annexin-V labeling, with approximately 50% of control BJ cells labeled positive.
90 Figure 5-6. BrdU induces a negligible increase in late-stage apoptotic cell death. Apoptosis was assessed with the TUNEL assay in RG2 rat gl ioma cells that received a 24 hour pulse of either 10 or 50 M BrdU. At all time points fo llowing BrdU exposure treated groups show significant increases in TUNEL+ cells as compared to control. However, even the highest rate of apoptosis represen ts less than 0.5% of the total cell number.
91 Figure 5-7. BrdU alters the cell cycle profile of treated cells. Cell cycle kinetics of asynchronous control and treated cells were assessed via flow cytometry (propidium iodide staining) after a 24 hour pulse of 50M Br dU. 144 hours after exposure, treated BJ cells (pie charts) show a substantial increase in the percentage of cells in G1/ G2 with a corresponding reduction in S-phase. The tabl e on the right shows the results of finer temporal analysis with H9 and RG2 cells Even as early as 6 hours after BrdU exposure there is a reduction in the percentage of cells in S-phase that persists over time.
92 Figure 5-8. Phosphorylation of pRb is reduced in some cell types following BrdU exposure, while total pRb and p21 remain unchanged. (A&B) RG2 and BJ cells were exposed to a single 24 hour pulse of 50 M BrdU and analyzed for phosphorylated pRb (Ser249,Thr252) by Western blot analysis at 1, 6, 96 hours, and 7 days postadministration. (C&D) Densitometric anal ysis was performed to quantitate the changes in phosphorylated pRb protein leve ls in RG2 and BJ samples normalized to actin. There is a dramatic reduction in phospho-Rb beginning at 6 hours in the RG2 cells, and at 96 hours in the BJ cells. (E&F) Similarly treated RG2 and BJ cells were analyzed for total Rb and p21 protein expres sion by Western blot analysis. Total Rb decreases slightly in RG2 cells at one week post-administration, but is not altered in BJ cells. In neither cell type is p21 expression altered as a result of BrdU exposure. (amount of protein loaded/well: (A&B ) RG2 1h, 6h: 17g; RG2 96h, 7d: 20g; BJ 1h, 6h: 15g; BJ 96h: 17g; (C &D) RG2: 15g; BJ: 17g).
93 Figure 5-9. BrdU induces an increase in SA Gal activity. RG2 rat glioma cells treated with 50M BrdU for 24 hours show an in crease in the percentage of SA Gal+ cells within 24 hours. Error bars represent standard deviation.
94 Figure 5-10. BrdU has varying effects on telo merase expression but doe s not alter telomere length. (A) RG2 rat glioma and MG63 human osteosarcoma cells were assayed for telomerase expression via TRAP analysis after a 24 hour pulse of 50M BrdU. RG2 cells show a dramatic and statisticall y significant reduction (p<0.001 at 24 and 48 hours) in telomerase activity within 24 hours of BrdU e xposure, while telomerase in treated MG63 cells does not vary from contro l levels even at 7 or 21 days post-BrdU when proliferation is severely suppre ssed. (B) TRF was performed on H9 human lymphoma cells 7 days following a 24 hour pulse of 50uM BrdU. There is no discernible difference in telomere length be tween the control and BrdU-treated cells.
95 Figure 5-11. The BrdU-treated cells do not inhibit expansion of untreated cells. Control H9 human lymphoma cells were mixed in vary ing ratios with H9 cells that had been exposed 1 week earlier to 1M BrdU for 3 hour s. Cells were quantified after 6 days of co-culture. The results show that there is a near-linear reduc tion in expansion as the ratio of BrdU-exposed cells increases. The 9\1 mixture (90% untreated cells) is reduced from control (10\0 ratio) by approxi mately 10%. Likewise, the 1\9 ratio (90% treated cells) shows approximately 10% greater expansion th an the 0\10 (100% treated cells) ratio. The equal mixture of control-to-treat ed cells (5\5) expanded at 62% of the control rate. These findings show that treated cells do not exert a suppressive effect on the expansion of untreated cells. One-way ANOVA with Student-Newman-Keuls post-hoc test of significance. All columns are significantly different with p<0.001, except 1\9 vs. 0\ 10 where p<0.05; N=3 for all groups.
96 Table 5-1. Reported statuses of prominent senescence-related markers for all cell lines tested Cell line Description p53 P16 p21 pRb Telomerase H9 Human lymphoma Mutant -/Not expressed Normal Positive RG2 Rat glioma -/-/Normal n/a Positive MG63 Human osteosarcoma -/-/Normal Normal Positive BJ Human immortalized fibroblasts Normal Normal Normal Normal Negative Saos-2 Human osteosarcoma -/Normal Not expressed -/Negative TT Human thyroid cancer Mutant n/a n/a n/a Positive
97 CHAPTER 6 CONCLUSIONS AND SIGNIFICANCE BrdU has a long history as a potential anti-cancer drug, and it is known that at high doses and in combination with secondary stressors, such as ionizing radiation, BrdU can have lethal consequences for incorporating cells. The pres ent findings are surprisi ng in that the BrdU regimen is exceedingly mild, even by current experimental pulse-chase birthdating paradigms in which cells incorporate BrdU and continue to function in an appare ntly normal manner. Furthermore, the suppressed proliferation we descri be occurs in the absence of secondary insults that would stress the cells ability to ma intain homeostasis or undergo DNA repair. A role for BrdU in senescence-induction of ma mmalian cells has recently been described in the gerontology field, but this role is not yet widely appreci ated by the larger scientific community, and has not previously been applie d as an approach to slow tumor progression. Emerging in vivo studies underscore the importance of cellu lar senescence in altering the growth properties of tumors in humans and rodents, a nd we show here that can cer cells treated with single-pulse BrdU show some si gns consistent with senescence Both the altered cell-cycle profile and the upregulation of SA Gal are common manifestations of a senescent phenotype; however, SA Gal upregulation varies wide ly by cell line, with some showing no enzyme activity even during severe suppression of proliferation rate, and there is evidence suggesting that SA Gal is not necessarily a selective marker of se nescence. It seems, then, that BrdU exposure does not lead to classically defined senescen ce but rather to a ge neralized slowing of proliferation and does not, in our experimental pa radigm, lead to crisis or subsequent cell death. Furthermore, the battery of cell lines we ha ve examined all show similar proliferation suppression following BrdU exposure, yet differ widely in the stat us of quintessential senescence markers. This variability makes it difficult to define a universal mechanism of action and
98 suggests that these molecular players, while possi bly involved in a cell type dependent manner, are neither required nor causative for the suppressive effect of BrdU. The cell cycle data indicate that BrdU-treated cells accumulate in G1 suggesting that these cells ma y either exit the cell cycle or are unable to traverse the Restriction Point. Wh ile the control of cellular proliferation is tightly regulated by various intrinsic a nd extrinsic factors, pRb is gene rally regarded as the master regulator of the Restriction Point and is now impli cated in cellular senescence. Rb is believed to control a senescence-initiating pa thway that may synergize with, but is distinct from, telomere loss. Results showing perturbed proliferati on that corresponds with hypo-/un-phosphorylated pRb yet appears to be independent of telome re maintenance support this theory. The postadministration times at which the levels of phosphorylated pRb become undetectable in BrdUtreated RG2 and BJ cells almost perfectly corre spond to the accumulation of these cells in G1. However, a result showing similar prolifer ation suppression in pRb null Saos-2 human osteosarcoma cells (data not show n) again indicates that pRb is not required for BrdU effect. Proliferation was suppressed in all of the ex amined cells and prior studies have also demonstrated the ubiquitous susceptibility of ma mmalian cells to BrdU. There is even evidence that BrdU slows replication in thymidine-auxo trophic yeast, suggesti ng that BrdU-mediated proliferation suppression in all eukaryotic ce lls may be affected through a common yet still undefined mechanism. Cancer stem cells seem part icularly susceptible to the anti-proliferative effect of BrdU. The neurosphere-forming assay allo ws us to study the behavior of stem cell-like tumor-initiating cells, and a si ngle pulse of BrdU reliably and dramatically slows the proliferation of clonally expanded progeny over numerous population doublings. This result strongly suggests that BrdU may be a potent therapeutic, targeting cancer stem cells and potentially slowing the regrowth of de-bulked primary tumors and/ or the metastatic spread of
99 secondary tumors. The wide penetr ance of the anti-prolif erative effect, combin ed with the ability for rapid transport across the blood-brain barrie r makes BrdU an attractiv e candidate against all types of cancer. However, these same attributes also make it likely th at indigenous stem cell pools will be adversely affected. Therefore, potential therapeutic BrdU dosing regimens will need to be carefully tested to avoid a perman ent depletion of the stem cells and long-term progenitors needed for maintaining tissue homeostasis. Delayed in vivo tumor progression in the extremely aggressive RG2 model is the most important aspect of our study. Glioblastoma mu ltiforme (GBM) is the most common primary (i.e., non-metastatic) brain tumor of humans. Desp ite advances in cytoreductive and cytotoxic therapies, the prognosis for this neoplasm remains dismal, with a median survival time of approximately 12 months. This has fostered an in tense interest in the search for alternative therapeutic modalities that may prove to be mo re effective or that may augment standard surgical, radiological or chemot herapeutic treatments for these neoplasms. The RG2 glioma model has been posited as the equivalent of human GBM. The fact that brief administration times used in this study result in statistically sign ificant delays in the growth of nave tumor cells raises the possibility that BrdU alone may be capable of prod ucing biologically significant therapeutic gains under optimized dosing schedu les. In addition, the dramatically delayed progression of BrdU pre-treated ce lls (treated prior to implanta tion) suggests that BrdU may prove effective against secondary metastatic tumo r formation. Future studies will be directed at examining the homing and invasiveness of Br dU-incorporating cells and their progeny. In re-assessing BrdU as a potential anti-cancer drug it is important to ask why the clinical trials of BrdU as a radiosensitizer were relativ ely ineffective at extending survival. Even though these previous studies were concerned only w ith the extent to which BrdU augmented the
100 standard chemical and radiation th erapies, one would expect that the anti-proliferative effect of BrdU should still have been evident. Based upon the present findings I can offer reasonable speculation as to why results from these earlier trials were so ambiguous. First, these clinical studies were performed solely on the basis of in vitro evidence of BrdU ra diosensitization; thus, the study designs did not have the benefit of in vivo models that may have revealed dosing schedules and drug interaction e ffects that either optimize or attenuate BrdU incorporation. Second, in all of the clinical trials BrdU was added to an existing, multimodal therapy that involved both chemotherapeutics and fractionated irradi ation. Under these circumstances it isnt clear to what extent BrdU was systemically avai lable during stages of active tumor cell division. Related to this is the question of dose and treatment length optim ization; without the benefit of animal models it simply was not possible to de termine sufficient dosing schedules to maximize therapeutic outcome. Even the present results with in vivo BrdU administration do not necessarily represent an optimized treatment para digm, as I was intentionally conservative in my approach, and was able to elicit a therapeutic effect while staying well below side effect limitations. It remains for future studies to extend these results to define the most effective treatment regimen from a co st/benefit perspective.
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110 BIOGRAPHICAL SKETCH Lindsay Harris Levkoff was born and raised in Sarasota, Florida. She graduated from Pine View School in June 1999. Lindsay attended th e University of Florida from 1999 to 2001 and then transferred to the Univers ity of Colorado, Boulder, where sh e graduated with her Bachelor of Arts degree in kinesiology and applied phys iology in May 2003. During college, Lindsay was awarded a Howard Hughes Medical Institute und ergraduate research opportunity program (UROP) award to conduct research in the laboratory of Dr. Linda Watkins. Lindsays research in behavioral neuroscience culminated in multiple pres entations at scientific conferences as well as authorship on two publicati ons (February 2005 issue of Behavioral Neuroscience and December 2006 issue of Pain ). In 2004, Lindsay enrolled in the Inte rdisciplinary Program in Biomedical Sciences (IDP) at the University of Florida Co llege of Medicine and re ceived the Presidential Fellowship and Grinter Scholarship upon admiss ion. She began her doctoral study under the guidance of Dr. Eric Laywell, in the Depa rtment of Anatomy and Cell Biology. Lindsays graduate research characterizing the anti-prolife rative effect of BrdU on highly proliferative cell populations was published in the August 2008 issue of Neoplasia Upon completion of her Ph.D. in December 2008, Lindsay plans to pursue a car eer dedicated to the study of cancer biology.