The Effect of p16INK4a on Differentiated and Undifferentiated Embryonic Stem Cells

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The Effect of p16INK4a on Differentiated and Undifferentiated Embryonic Stem Cells
Piacenti, Peter
Terada, Naohiro ( Mentor )
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Gainesville, Fla.
University of Florida
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The Effect of p16INK4a on Differentiated and Undifferentiated Embryonic
Stem Cells

Peter Piacenti


Embryonic stem (ES) cells are cells which are isolated from the blastocyst stage embryo. They are found in the

inner cell mass, and can be maintained in an undifferentiated state under optimal conditions with the use of

leukemia inhibitory factor (LIF). These cells have the ability to differentiate into cells of all three germ layers. ES

cells also demonstrate the ability to continue proliferating indefinitely. These facts position stem cells to be

of immesurable value to modern medical research.

The life of each cell can be divided into cycles each containing four phases. In order for a cell to move from one

stage in the cycle to the next, permission is required from proteins called cyclins. There exists one cyclin for

each major stage of the cell cycle. If permission is given, the cyclin will bind to cyclin dependent kinases and

activate them, thereby driving the cell cycle. Another relevant term here is the restriction point or the "R point,"

the stage at which a cell is committed to replication. Cdk4/6-Cyclin D complexes have been shown to be

the regulators of the drive through the R point, which occurs in the G1 phase of the cell cycle (Ortega et al., 2001).

pl6INK4a is part of the INK4 family of proteins, whose function is to inhibit the activity of cdk4-cyclin D and

cdk6-cyclin D complexes. More accurately, the presence of p16INK4a directly inhibits the activity of Cdk4/6 by

altering the binding site of D-type cyclins and thereby reducing its affinity for ATP (Ortega et al., 2001). By

reducing the number of Cdk4/6-cyclin D complexes, p16INK4a inhibits the transition from G1 to S phase by

preventing the passage through the R point. It has been found that if p16INK4a is overexpressed in somatic

cells, senescence occurs and the cells eventually die. A 1995 paper examined the effect of p16INK4a on ES cells.

The experiment concluded that the overexpression of p16INK4a has no effect on the cell cycle of undifferentiated

ES cells, but that when LIF was removed and the cells differentiated, the distribution of cell cycle phase

became skewed such that a much larger proportion of the cells were in the G1 phase of the cycle (Savatier, et

al., 1995). It may be the case that the findings are inconclusive with regards to the effect of p16INK4a on ES cells.

We identify three points within this experiment that we have improved upon. First, the previous

experiment overexpressed p16INK4a only transiently. Second, the previous experiment attempted to compare

two groups of cells, one that was expressing p16INK4a and one that was not. The system employed allows

for probabilistic inconsistencies regarding the purity of the samples being examined. Third, the ratio of the

sample sizes was far from 1:1. The ratio of transfected ES cells to those that were not successfully transfected was

In fact the ratio of cells that were overexpressing p16INK4a to those that were not was .01:1, (i.e. approximately

1% of the ES cells were successfully transfected with pl6INK4a). We compared two cell cultures, one in which

the gene for p16INK4a is turned on by the tetracycline inducible system, to another of very similar size

and development in which the gene for p16INK4a is turned off. We believe that the comparable sample sizes and

the elimination of probabilistic dependence will allow for more accurate and convincing findings.


p16 construct

cDNA of mouse p16 was amplified from cDNAs of differentiated mouse ES cells using the primers

(5'- tcgaattcgcagcatggagtccgct-3', 5'- gctctagattagctctgctcttggg-3') and sequenced to confirm that

there was no PCR error in amino acid sequence.

This fragment was inserted into the multicloning sites of pTRE-hyg2 (Clontech).


pTRE-hyg2-p16 plasmid was transfected into TET-off Parental ES cell line** using Fugene6 (Roche).

For approximately two weeks selection with hygromycin, we got several positive clones containing

this plasmid confirmed by PCR and immunostaining.


After washing with PBS several times, cells were fixed in 4% formaldehyde. They were washed with

PBS several times followed by treatment of 0.5% triton/PBS for 10 minutes.

The samples were incubated with 0.5% BSA.PBS for one hour and incubated with the first

antibody polyclonall antibody against p16, Santa Cruz Biotechnology). After washing with PBS

several times, the cells were incubated with secondary antibody (Cy3, JacksonImmunoResearch) for

one hour, followed by washing with PBS for one hour, and then analysed with fluorescent microscope.

Western Blot

Cultured cells were harvested in RIPA buffer with protease inhibitors. Heated samples

were electrophoresed under reducing conditions and transferred into nitrocellulose membrane.

The membrane was probed with rabbit polyclonal antibody against pl6 (Santa Cruz Biotechnology),

and developed with peroxidase labeled anti rabbit IgG using ECL (Amersham Life Science).

Cells and media

TET-off ES cell line was a kind gift from Dr. Era (Era et al., 2002).

These cells were cultured on gelatin-coated dishes in Knock-out DMEM (Invitrogen) supplemented

with 10% knockout serum replacement (Invitrogen), 1% FCS (Wisent), 0.1mM 2mercaptoethanol,

1% non-essential aminoacid solution, L-glutamine, penicillin, streptomycin, and 1000 U/ml

LIF (Chemicon). For control of gene expression, we used doxcycline (DOX). With the use of this

system, we cultured cells in the presence of DOX, which would prevent them from expressing p16.

Upon the removal of DOX, the cells would begin overexpressing our gene. For differentiation, cells

were cultured in knockout-DMEM supplemented 20% FCS without LIF, or cultured without LIF in

the presence of retinoic acid (10-6M).

Flow cytometry (FACS) analysis

Cells were fixed in 70% ethanol and were kept at -20Ac until use. On the analysis day, cells were

stained with PI and analysed for cell cycle.


We first isolated the p16 cDNA from mouse ES cells. We ligated the p16 into the system shown in

Figure 1 called the pTRE2hyg vector.


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Figure 1. The pTRE2hyg system that was used to transfect ES cells

After Transfecting ES cells with the vector containing our pl6 gene, we added hygromycin to the cells

to determine which colonies contained our vector. Out of the 16 clones that we identified, we
performed PCR and found that eleven were positive for the insertion of the p16 sequence, as
illustrated in Figure 2.

Figure 2. Gel electrophoresis shows eleven clones with a strong band indicating the presence of

the p16INK4a PCR product.

a) Dox +

b) Dox-

Figure 3. a) clone number 4 is shown here in the presence of DOX. There is no expression of p16 in
the presence of DOX, shown by the absence of the red stain to the cells. b) the overexpression of p16
by clone number 7 is shown here after the removal of DOX.

c) Dox+

d) Dox-

Figure 3. c) Clone 7 showed no expression of p16 in the presence of DOX. d) Clone 7 showed
expression of p16 after the removal of DOX.

Next, we performed immunohistochemistry to identify which of the eleven clones had no

leaky expression in the presence of DOX, and those that also had overexpression of p16 in the absence

of DOX. Four clones out of the eleven fit the above description, and two are shown below in Figure 3.

We performed western blotting analysis with three of the clones that had positive results

from immunohistochemistry to confirm the overexpression of p16. Figure 4 revealed that clones 4 and

7 had some leaky expression in the presence of DOX, but clone 15 showed no leaky expression.

c+ c- 4+ 4- 7+ 7- 15+ 15-

Positive control: -actin
c+ c- 4+ 4- 7+ 7- 15+ 15-


Figure 4. Western blotting analysis was used to confirm the results from immunohistochemistry. After

2 days off DOX, the bands between DOX+ and DOX- are markedly different. The ES cells

are overexpressing p16 and continue to proliferate without senescence contrary to many expectations.


.x. % .J .',

Figure 5. a) FACS analysis for the positive control in the presence of DOX. b) positive control in

the absence of DOX. c) Clone 15 in the presence of DOX, showing no expression of p16. d) Clone 15 in

the absence of DOX, showing overexpression of p16, but maintaining an almost identical

spread regarding cell cycle phase as that of the control. The other three clones which

were overexpressing p16 showed a very similar pattern indicating no significant difference between

the spread of cell cycle phase between p16 expressing ES cells and those that are not expressing p16.

Finally, the FACS analysis was conducted to check the cell cycles of these clones (Fig. 5). We

compared the positive control and these clones and found that there is little or no difference in

the distribution of cell cycle phases among the clones. A more detailed account of the FACS

analysis result is located in Figure 6. As you can see in Figure 6, our result was markedly different

than the result obtained by Savatier et al. As of now, we have not yet checked the kinase activity

of CDK4/6, which is the main target of p16. We expect the kinase activity to be suppressed by p16 due

to the result we obtained from immunohistochemistry. Nonetheless, our next step will be to confirm

the virility of p16 by checking the kinase activity of CDK4/6.


C__ntml + Contml - 4+ 4- 7+ 7-
t.te-ff 16 days 16 days 16 days
Gam 1 22.57% 23.97% 22.44% ~5.88% 23.52% 21.82%
1 60.72% 60.01% 58.13% 56.35% 54. 11% 56.96%
GU/M. 16.70% 16.02% 19.4-3% 17.77% 22.37% 21.22%
G2m 1 i 2.01 2I.0 1 1.98 1.98 1.99 2.0


Contml+ Control - 15 + 15 - 16+ 16 -
tat-off 42 days 42 days 42 days
G0G 1 15.21% 18.78% 22.61% 2'5.51% 23.48% 23.64%
S 48.02% 52.61% 57.12% 57.34% 58.95% 57.63%
GmM. 36.77% 28.61% 20.27% 17.15% 17.57% 18.73%
Gu 1 !2.02 2.0 1 1.97 1.97 1.98 1.97

Control+ Control- 4+ 4- 7+ 7- 15+ 15-
tet off 25days 32days 27days 21 days
GO/G1 1 6.95% 1 8.09% 20.55% 23.09% 1 5.97% 1 8.13% 18.15% 18.82%
S 59.01% 58.33% 59.64% 56.90% 58.85% 57.90% 55.74% 56.64%
G2/M 24.04% 23.59% 1 9.81% 20.01% 25.18% 23.98% 26.11% 24.54%
G2/G1 1.9 1.91 1.92 1.91 1.92 1.93 1.91 1.93

Control+ Control- 4+ 4- 7+ 7- 15+ 15-
tet off days days days days
GO/G1 53.28% 53.65% 53.18% 54.93% 58.58% 61.36% 48.60% 47.03%
S 30.59% 28.49% 29.23% 28.24% 22.22% 20.71% 31.74% 32.85%
G2/M 16.13% 17.86% 17.59% 16.83% 19.19% 17.92% 19.66% 20.12%
G2/G1 1.96 1.93 1.94 1.94 1.98 1.97 1.94 1.94

Figure 6a-d. Throughout the culture period, there is no observed significant difference in the

distribution of cell cycle phase between our control cells and ES cells overexpressing p16. To further

our study, we removed LIF and allowed the cells to differentiate (table d). After 3 days culture

without LIF, the differentiated cells still showed no difference in cell cycle phase.


Era, Takumi, Wong, Stephane, Witte, Owen N. Analysis of Bcr-Abl Function Using an In Vitro
Embryonic Stem Cell Differentiation System. Methods Mol Biol. 2002; 185: 83-95.

Ortega, Sagrario, Malumbres, Marcos, Barbacid, Mariano. Cyclin D-dependent kinases, INK4
inhibitors and cancer. Biochimica et Biophysica Acta 1602. 2002: 73-87.

Savatier, P., Lapillonne, H., Grunsven, LA van, Rudkin, BB, Samarut, J. Withdrawal of
differentiation inhibitory activity/leukemia inhibitory factor up-regulates D-type cyclins and
cyclin-dependent kinase inhibitors in mouse embryonic stem cells. Oncogene 12 (1995) 309-322.


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