Journal of Undergraduate Research
Volume 4, Issue 11 - August 2003
The Effect of p16INK4a on Differentiated and Undifferentiated Embryonic Stem Cells
Peter Piacenti
INTRODUCTION
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).
p16INK4a 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 attemted 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 p16INK4a). 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.
MATERIALS AND METHODS
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 (polyclonal 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. 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 p16 (Santa Cruz Biotechnology),
and developed with peroxidase labeled anti rabbit IgG using ECL (Amersham
Life Science). 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). Cells were fixed in 70% ethanol and were kept at –20Äc
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. Figure 1. The pTRE2hyg system that was used
to transfect ES cells After Transfecting ES cells with the vector containing
our p16 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. 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. 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. 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. 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. 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. Back to the Journal of Undergraduate
Research © University of Florida, Gainesville,
FL 32611; (352) 846-2032.
Transfection
Immunohistochemistry
Western Blot
Cells and media
Flow cytometry (FACS) analysis
RESULTS AND DISCUSSION
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.
REFERENCES
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