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Biochemistry and Biophysics |
Division of Cancer Biology Research, Sunnybrook Health and Women’s Science Centre, Toronto, Ontario M4N 3M5, Canada [C. S., J. S.], and Department of Urology, Stanford University, Stanford, California 94305-5118 [D. M. P.]
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Most previous studies investigating senescence have used fibroblasts. The high incidence of prostatic cancer in adult males and our limited understanding of prostatic oncogenesis motivated our study of prostatic epithelial cell senescence. Primary prostatic cancers frequently show telomerase activation (7, 8, 9, 10) , and it is possible to establish immortal lines from primary prostate cancers (11, 12, 13) . Thus, prostate cancer development may be associated with loss of the senescence checkpoint. An understanding of the molecular mechanisms whereby the senescence checkpoint is lost in cancers requires an assessment of this checkpoint in normal HPECs.3 In the present study, we investigated the mechanisms of cdk inhibition in HPEC senescence.
Transition from one phase of the cell cycle to the next requires the orderly activation and inactivation of a family of related cdks, cdk1–7 [reviewed by Sherr (14) and Morgan (15) ]. Cdks are activated by cyclin binding (14) and regulated by phosphorylation (16) . G1 phase to S phase progression requires phosphorylation of pRb, which is mediated primarily by cyclin D1-associated cdk4 or cdk6, and also by cyclin E-cdk2 (17 , 18) .
Two families of cdk inhibitors, the INK4 and KIP families, regulate cdk activity [reviewed by Sherr and Roberts (19 , 20) ]. The KIP family consists of three broadly acting inhibitors: p21CIP1, p27KIP1, and p57KIP2. KIP family members bind to and inhibit the cyclin-cdk complexes. Recent in vitro experiments demonstrated that a single KIP molecule is sufficient to inhibit cyclin/cdk kinase activity (21) . In contrast to the KIP inhibitors, members of the INK4 family (p15INK4B, p16INK4A, p18INK4C, and p19INK4D) bind specifically cdk4 and cdk6, with resulting loss of cyclin D binding and catalytic inactivation.
Cell culture models have identified a role for the cdk inhibitors in senescence. An increased expression of p21 and/or p16 at senescence has been identified in human and murine fibroblasts and melanocytes (22, 23, 24, 25, 26) . The induction of p21 in senescent fibroblasts led to the initial identification and cloning of this gene by Noda et al. (27) . The elevated expression of p16 and/or p21 at senescence is associated with their increased binding and inhibition of G1-S phase cdks. Elimination of p21 expression through homologous recombination extended the life span of human diploid fibroblasts in culture (28) . Thus, the loss of p21 expression, although itself not sufficient to abrogate senescence arrest, may represent a key step in the immortalization of cells of fibroblastic lineage. The loss of p21 and p16 expression in human cancers suggests that in vivo, these inhibitors may contribute to the senescence checkpoint and limit tumor development (29 , 30) .
In human prostatic tumors, although p16 is rarely mutated (31 , 32) , loss of expression occurs frequently through hypermethylation or deletion (29 , 33 , 34) . Furthermore, loss of p16 expression in human prostatic tumors may have prognostic implications (30) . Together, these results suggest that p16 may play an important tumor suppressor role in prostatic epithelial cells. In this study, we show that the progression toward HPEC senescence was associated with an increase in p16 levels. Novel phosphorylated forms of p16 showed increased association with the target cdk complexes, cdk4 and cdk6. In contrast to senescence in fibroblasts, neither the expression of p21 nor its association with target cdk complexes was increased. Phosphorylation of p16 may represent a novel regulatory pathway for p16 inhibitory activity.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-tocopherol, and 0.03 nM
all-trans retinoic acid (35)
. Cells that grew
out in primary culture were aliquoted and stored frozen in liquid
nitrogen. The epithelial nature of these cells was verified by
immunocytochemical staining for cytokeratins (35)
. To
verify the histology of origin, the prostatic specimens were inked
after dissection, fixed, and serially sectioned (36)
. The
histology of tissues immediately adjacent to and surrounding the
portion removed for culture was reviewed. Neither cancer nor benign
prostatic hyperplasia was present in the areas of tissue from which the
cell strains were derived.
Cells were serially passaged as follows: Cells were thawed and
inoculated into collagen-coated dishes containing MCDB 105 (Sigma, St.
Louis, MO) supplemented as described for PFMR-4A except with 10 rather
than 40 µg/ml pituitary extract. When 
50% confluent, a portion of
the cells was harvested for analysis of cell cycle regulators at
"passage 1." The remainder of the cells (10 or 20%) were
passaged after trypsinization into 40 dishes and again grown to 50%
confluency, at which time cells were again either harvested ("passage
2") or passaged. This process was repeated twice more (passages 3 and
4) until the cells ceased proliferation.
Flow Cytometric Analysis.
At different passages, cells were pulse labeled with 10
µM BrdUrd for 2 h. Cells were then harvested,
fixed with 70% ethanol, treated with 0.1 N HCl, and heated for 10 min
at 90°C to expose the labeled DNA. Cells were then stained with
anti-BrdUrd-conjugated FITC (Becton Dickinson, Bedford, MA) and
propidium iodide. Cell cycle analysis was carried out on a Becton
Dickinson FACScan, using Cell Quest software.
Antibodies.
Antibodies to pRb, cdk2, cyclins A and D1, and p21 were obtained from
PharMingen (San Diego, CA) or Santa Cruz Biotechnology (Santa Cruz,
CA). Cyclin E1-specific antibodies (mAbs E12 and E172; Refs. 37
, 38
) were from E. Lees and E. Harlow (Massachusetts General
Hospital, Boston, MA). Monoclonal PSTAIRE antibody
(39)
was a gift from S. Reed (The Scripps Research
Institute, La Jolla, CA), and cyclin D1 antibody, DCS-11, was
purchased from Neomarkers (Fremont, CA). Cyclin A mAb E67 was provided
by J. Gannon and T. Hunt (ICRF, London, United Kingdom).
Monoclonal p27 antibody was purchased from Transduction Labs
(Lexington, KY). Cdk4 and cdk6 polyclonal sera were provided by G.
Hannon and D. Beach (CSH Labs, Cold Spring Harbor, NY). The JC-6
monoclonal, provided by J. Koh and E. Harlow (Massachusetts General
Hospital; Ref. 40
), and p16 polyclonal antibody,
purchased from Santa Cruz, were used for immunoblotting of p16 in these
studies. ß-actin antibody was purchased from Sigma. Monoclonal p19
antibody was obtained from Neomarkers. Polyclonal p18 antibody was
kindly provided by Y. Xiong (University of North Carolina, Chapel Hill,
NC).
Immunoblotting.
Cells were lysed in ice-cold NP40 lysis buffer [0.1% NP40, 50
mM Tris (pH 7.5), 150 mM NaCl, 1 mM
phenylmethylsulfonyl fluoride, and 0.02 mg/ml each of aprotinin,
leupeptin, and pepstatin]. Lysates were sonicated and clarified
by centrifugation.
Protein was quantitated by Bradford analysis. Twenty or 50 µg of protein were loaded in each lane and resolved by SDS-PAGE. Transfer and blotting were as described (41) . Equal loading of the lanes was verified using a ß-actin antibody. For detection of cdk4-associated proteins by immunoprecipitation/Western analysis, cdk4 was immunoprecipitated from 200 µg of protein lysate, complexes were resolved and blotted, and the blot was reacted with cdk4, cyclin D1, p16, p21, or p27 antibodies. To verify the identity of associated proteins, control cyclin D1, p16, p21, and p27 immunoprecipitations were resolved along side the cdk4 immune complexes (data not shown). Similar methods were used to detect cdk6- and cyclin E1-associated proteins.
Protein Expression in Bacteria.
pRb substrate for cyclin D1-associated kinase assays was generated from
a pGEX vector containing the COOH terminus of pRb (amino acids
729–928) fused to glutathione S-transferase (kindly
provided by J. Zhao and E. Harlow, Massachusetts General Hospital).
Bacteria were lysed in PBS containing 1% Triton X-100, 1 mg/ml
lysozyme, and protease inhibitors, and lysates were clarified by
centrifugation at 15,000 rpm for 20 min. The pRb fragment was isolated
by incubating bacterial extract with glutathione beads for 1 h.
Beads were then washed repeatedly with PBS-1% Triton X-100 and then
with PBS alone. The pRb fragment was eluted in 20
mM glutathione in PBS (pH 7.8).
Kinase Assays.
For cdk4 kinase assays, cells were lysed in 50 mM HEPES,
150 mM NaCl, 1 mM EDTA (pH 8.0), 2.5
mM EGTA (pH 8.0), 10% glycerol, 10 mM
ß-glycerophosphate, 1 mM NaF, 0.1% Tween-20, 0.1
mM Na2VO4, 1
mM 4-(2-aminoethyl)benzenesulfonyl fluoride, 0.5
mM DTT and 1 mg/ml both leupeptin and aprotinin. Cdk4
kinase assays were performed following the method of LaBaer et
al. (42)
, using cdk4 antibody obtained from Santa
Cruz for immunoprecipitation and the COOH-terminal fragment of pRb as
substrate. Radioactivity was quantified using a Molecular Dynamics
PhosphorImager and ImageQuant software. Cyclin E1- and A-associated
kinase assays were performed as described (43)
. Histone H1
substrate was obtained from Boehringer Mannheim (Laval, Quebec).
Background levels of kinase activity were determined for each kinase
reaction by immunoprecipitating early passage cellular extract with
nonspecific mouse mAbs (Fig. 2
, Lane C).
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Two-Dimensional Gel Electrophoresis.
Isoeletric focusing of p16 was carried out by immunoprecipitating cdk6
complexes from 1 mg of protein cell extract. The immune complexes were
washed three times with NP40 lysis buffer and then once with 20
mM Tris (pH 7.5). The washed complexes were solubilized in
200 µl of rehydration buffer [8 M urea, 2% (w/v) CHAPS,
0.5% (v/v) pH 3–10 IPG (Immobiline Dry Strip Gels) buffer
(Amersham Pharmacia Biotech), and 18 mM DTT] and loaded
onto IPG strip holders containing pH 3–10 linear IPG strips (Amersham
Pharmacia Biotech). The gels were rehydrated with the sample solution
for 17 h at 20°C and then focused consecutively for 1 h at
500 V, 1 h at 1000 V, and 2 h at 8000 V in an IPGPHOR
Isoelectric Focusing System (Amersham Pharmacia Biotech). The focused
gel strips were incubated for 15 min at room temperature in
equilibration buffer [50 mM Tris (pH 8.8), 6 M
urea, 30% (v/v) glycerol, 2% (w/v) SDS, and 65 mM DTT]
prior to loading onto 17.5% SDS-PAGE gels. The electrophoresed gels
were then transferred to Immobilon, and p16 was detected by
immunoblotting.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). At early passage, 22% of asynchronously growing cells were in S
phase, 66% contained 2N DNA, and the remaining 12% of cells contained
4N DNA. Cellular proliferation ceased after six passages, which
corresponded to 30 population doublings at senescence. Cells were
considered to be senescent after they remained subconfluent for >1
month. Flow cytometry of senescent HPECs demonstrated a cell cycle
arrest with <1% of cells in S phase and 85% of the cells with 2N
DNA. The remaining 14% of cells had 4N DNA at senescence.
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. The activities of cyclin E1- and cyclin A-associated kinases and of
cdk4 decreased steadily as cells progressed from early passage to
senescence. Senescent cells showed no kinase activities above that in
nonspecific immune controls. Furthermore, analysis of cells of
increasing passage revealed a progressive loss of pRb phosphorylation,
indicative of cell cycle arrest in G1 (see Fig. 3A
). Because pRb is phosphorylated by cyclin E1- and
D1-associated kinases, loss of pRb phosphorylation provides further
evidence of inhibition of these cdks.
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). Western
analysis revealed no change in cyclin E1 or cyclin D1 levels during the
aging of prostatic epithelial cell populations. Cyclin A levels
gradually decreased, consistent with the gradual recruitment of cells
into G1 arrest at senescence. The decrease in
cyclin A levels was likely the consequence of decreased cyclin E1- and
cyclin D1-associated kinase activities, resulting in reduced
E2F-mediated transcription of cyclin A. The cdk4 and 6 levels remained
unchanged. The loss of cdk2 protein with increasing population
doublings was largely due to loss of the CAK-activated, Thr-160
phosphoform (faster migrating cdk2 band).
The levels of the KIP and INK inhibitors were also analyzed in
asynchronous prostatic epithelial cells at increasing population
doublings and at senescence. There was a steady decrease in the protein
levels of p21CIP1 and
p27KIP1. p18INK4C
expression was not detected (data not shown), and
p19INK4D levels remained unchanged, whereas
p16INK4A levels steadily increased, reaching
maximal levels of expression in senescent cells. In addition, longer
exposures of the p16 immunoblots revealed the presence of two
additional p16-reactive bands of decreased mobility on SDS-PAGE (Fig. 3B
). These slower mobility bands were detected using two
different anti-p16 antibodies (the JC-6 monoclonal and the Santa Cruz
polyclonal). The abundance of these two novel p16-related bands
increased with increasing population doublings.
KIP Binding to Cyclin E-cdk2 Did Not Increase in Senescent HPECs.
The composition of cyclin E1-cdk2 complexes showed no apparent change
during the progression of HPECs toward senescence (Fig. 4A
). The amount of cdk2 bound to cyclin E1 was not reduced.
Furthermore, the proportion of Thr-160 phosphorylated cdk2 bound to
cyclin E1 remained constant. The levels of cyclin E1-associated p21 and
p27 remained constant. Thus, the inhibition of cyclin E1-cdk2 activity
could not be attributed to increased KIP binding or to a lack of
activating phosphorylation at the Thr-160 residue of cdk2.
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). Although there was
no increase in the faster mobility band of p16 in cdk4 and cdk6
complexes (Fig. 4 and C
, short
exposure), longer exposures of the cdk-associated p16 immunoblots
(Fig. 4 and C
, long exposure) again
revealed the existence of two delayed mobility bands that cross-reacted
with p16-specific antibodies. The association of these two bands with
cdk4 and cdk6 complexes steadily increased with later cell passages.
Densitometry showed that association of the slower migrating forms of
p16 increased by 2-fold in cdk6 complexes and by 1.6-fold in cdk4
complexes between passage 2 and senescence. The accumulation of these
slower mobility p16 bands in cdk4 and cdk6 complexes was correlated
with a decrease in the binding of cyclin D1, p27, p21, and p19. None of
the anti-p16-reactive bands were cross-reactive with p18 or p19
antibodies.
Altered Phosphorylation of p16 in Senescent Prostatic Epithelial
Cells.
To determine whether the novel bands of delayed mobility were different
phosphoforms of p16, cdk6 complexes were immunoprecipitated from
senescent cellular extract (Fig. 5
, Lane 1) and treated with PAP (Fig. 5
, Lane 2);
immunoblots were reacted with p16 antibodies. PAP treatment of the
immunoprecipitates from senescent cells resulted in the loss of the
upper two p16 bands visible on long exposures and the formation of two
bands of increased mobility (data for senescent cells shown in Fig. 5
,
middle and bottom panels). Thus, the slower
mobility forms of p16, whose association with both cdk4 and cdk6
complexes increased in senescent HPECs, represented novel phosphoforms
of p16. These delayed mobility p16 phosphoforms were not detected, even
with prolonged exposure in the early passage HPECs. It is
notable that the dominant, faster mobility p16 band, detected in both
early passage and senescent cells, also shifted to two faster mobility
bands when the phosphatase reaction went to completion, indicating that
this dominant band also represents phosphorylated p16 (Fig. 5
). There
was no loss of any of the p16 reactive bands when PAP was preincubated
with phosphatase inhibitors (Fig. 5
, Lane 3). The
formation of two p16-reactive bands of increased mobility after PAP
treatment suggested that all of the cellular p16 is phosphorylated, but
the pattern of expression of the different phosphoforms differed
between early passage and senescent cells.
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). In early passage cells, the solitary band of p16 seen on
one-dimensional Western blots (Fig. 4
) resolved on 2D IEF as two forms
of p16, which focused to a pH between 5 and 6 (Fig. 6
, arrowhead
3). In senescent cells, in addition to the two forms of p16
observed in early passage cells, two additional p16 isoforms (Fig. 6
,
arrowheads 1 and 2) were present.
These two isoforms showed a delayed mobility on SDS-PAGE (the second
dimension) and focused just below pH 6.0. The relative mobilities of
isoforms 1 and 2 in the second dimension with respect to the
predominant forms of p16 was consistent with these being the two minor
p16 bands of delayed mobility that were observed in senescent cells in
one-dimensional Western blots in Fig. 4
.
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and indicate that all three p16 bands detected on
Western blotting represent p16 phosphoforms. The pattern differs
between early passage and senescence, with isoforms 1 and 2 increasing
as cells approach and enter senescence. |
DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The identification of key inhibitor(s) of cell cycle progression at senescence could potentially indicate a critical regulator whose expression or activity would need to be down-regulated for tumor progression to proceed. Investigations of senescence in fibroblasts suggest important roles for p21 and p16. Here we have demonstrated an increase in p16 and the appearance of novel phosphorylated forms of p16 that bind and inhibit cdk4 and cdk6 activity in senescent HPECs.
The prostatic epithelial cell cultures used in this study moved from a
proliferatively active state to a senescent state within six passages
or 30 population doublings. Senescent HPECs showed predominantly 2N
DNA. A small proportion of cells had 4N DNA at senescence. We and
others have observed an increase in tetraploidy as cultured cells
approach senescence4
(44)
. Thus, cells with 4N
DNA are most likely tetraploid cells arrested prior to S phase entrance
at the senescence checkpoint, as opposed to cells arrested at the
G2-M transition. The catalytic activity of the
cdks associated with the G1-S transition
underwent a steady decrease from early passage to senescence. The
steady loss of kinase activity implies that the entry of cells into
senescence does not occur in a synchronized manner. Rather, there
appears to be some heterogeneity with regard to the passage at which
epithelial cells enter senescence. Early studies of senescence
identified this phenomenon by the progressive reduction of tritiated
thymidine incorporation that occurred during the serial passaging of
fibroblasts (45)
. The established relationship between
telomere length and the proliferative capacity of cells suggests that
variations in telomere length within a population of cells may account
for the apparently stochastic manner whereby cells enter senescence
(46)
. The heterogeneity in telomere length may reflect
variations in the proliferation of epithelial stem cell populations
in vivo.
To investigate the cause of cdk inhibition in senescent HPECs, we examined the steady-state levels of G1-S-associated cell cycle regulators. Of the cyclins examined, only cyclin A diminished with increasing passage, accounting for the corresponding loss of cyclin A-associated kinase activity. The loss of cyclin A in senescent HPECs is likely a consequence of the cells arresting at a point within the cell cycle prior to cyclin A induction. Studies in human fibroblasts have shown that entrance into senescence is similarly associated with a loss of cyclin A (47) . However, in senescent fibroblasts there is a significant increase in the expression of cyclins D1 and E1 (48 , 49) . The levels of cdk4 and cdk6 were unaffected by increasing passage in HPECs, whereas loss of cdk2 was largely due to loss of the Thr-160 phosphorylated form of cdk2. Fibroblasts have a similar loss of the Thr-160 form of cdk2 during senescence (48 , 50) , and in addition, studies of fibroblast senescence have revealed a reduction in cdk4 levels (49) . Thus, it appears that the regulation of both cyclin and cdk expression differs between senescent fibroblasts and prostatic epithelial cells.
In contrast to reports in cells of fibroblastic and melanocytic lineage, the steady-state levels of both p21 and p27 proteins decreased as HPECs moved toward senescence (22 , 27 , 51) . Furthermore, there was no increase in the binding of p21 and p27 to cyclin E1-associated complexes in senescent HPEC extracts. These KIP molecules were also lost from cdk4 and cdk6 complexes during the progression toward senescence. These results contrast with previous findings in senescent human fibroblasts and keratinocytes, where p21 was shown to be induced at senescence and its binding to G1-S-associated cdks was increased (28 , 51 , 52) . Our findings in senescent HPECs and human mammary epithelial cells,4 indicate that p21 does not appear to mediate cdk inhibition in all senescent epithelial cells. The lack of p21 induction in senescent prostatic and mammary epithelial cells may represent a fundamental difference between these epithelial cell types and keratinocytes and fibroblasts at senescence.
Investigations of senescence in fibroblasts, lymphocytes, and uroepithelial cells have implicated p16 in the inhibition of cyclin D1-dependent kinases (22 , 26 , 53) . The high incidence of p16 inactivation in prostatic tumors suggests that p16 may play a role in arresting HPECs at senescence both in vivo and in vitro (29 , 33) . The present study and the recent report of Jarrard et al. (54) establish that there is an increase in p16 levels in senescent prostatic epithelial cells. Immortal HPEC derivatives, generated by the introduction of human papilloma virus E6 and/or E7, were associated with a loss of p16 or pRb expression (54) . We have identified two novel p16-related bands of delayed mobility that accumulate in senescent HPECs. Resolution on both one-dimensional gels and by 2D IEF after treatment with PAP demonstrates that the two novel p16 bands of delayed mobility observed in one-dimensional Western blots were p16 phosphoforms. In both early and late passage cell extracts, PAP treatment also shifted the most abundant (faster mobility) p16 band on the one-dimensional Western blots, suggesting that all detectable p16 is phosphorylated. The failure of p16 to resolve into a single band after prolonged PAP treatment may be a consequence of phosphorylation at sites on p16 that are not recognized efficiently by PAP, or alternatively, a fraction of the cellular p16 may undergo other posttranslational modifications, such as glycosylation, that serve to alter its gel mobility.
PAP treatment and 2D IEF confirmed the existence of different p16
phosphorylation patterns in early passage and senescent HPECs. The
dominant p16 band seen on immunoblots from both early passage and
senescent cdk6 immune complexes was composed of two p16 isoforms. Two
additional p16 isoforms of delayed mobility consistently appeared in
senescent cells, suggesting that p16 undergoes senescence-specific
posttranslational modifications. These senescent-specific isoforms
(Fig. 6
, arrowheads 1 and 2) of p16 focused at a
slightly higher pH, and thus represent p16 isoforms with a lower of
level of phosphorylation when compared with the two dominant isoforms
of p16 (Fig. 6
, arrowhead 3). After PAP treatment, all of
the p16 isoforms focused at a higher pH. These results are consistent
with the interpretation that all of the detected p16 is phosphorylated
but that novel phosphoforms appear in senescent cells.
The increased expression of p16 translated into an increased association of p16 with cdk4 and cdk6 complexes in senescent HPECs. In senescent HPECs, as in other forms of G1 arrest, the accumulation of this INK4 molecule in target kinases was associated with loss of cyclin D1 and KIP binding (55 , 56) . However, only the senescence-specific phosphoforms of p16 showed an increased binding to cdk complexes. The accumulation of these novel p16 phosphoforms in cdk4 and cdk6 complexes suggests a senescence-activated mechanism of posttranslational modification of p16 contributing to kinase inhibition and senescence arrest in HPECs.
Phosphorylation of p16 may represent an important mechanism of p16 regulation. p16 phosphorylation may regulate either the affinity for cdk4 and cdk6 and/or the localization of p16 within the cell. Phosphorylation of p27 functions to regulate the stability of the protein and its affinity for cdk complexes (57, 58, 59) . It is tempting to postulate that phosphorylation of specific sites on p16 in senescent HPECs facilitates the binding of p16 to target cdk complexes and contributes thereby to G1 arrest in senescence. The identification of these phosphorylation sites and the pathways that influence phosphorylation of p16 would aid in the understanding of the regulation of the INK family of inhibitors and elucidate further the pathways regulating p16 inhibitory activity.
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FOOTNOTES |
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1 J. Slingerland is a physician scientist
supported by Cancer Care Ontario and by career awards from the United
States Army Breast Cancer Program, and by the Burrough’s Welcome Fund.
This work was supported by grants from the United States Army Prostate
Cancer Research Program and the Canadian Breast Cancer Research
Initiative of the National Cancer Institute of Canada (to
J. M. S.) and from the United States Army Prostate Cancer
Program (to D. M. P.). 
2 To whom requests for reprints should be
addressed, at Division of Cancer Biology Research, Sunnybrook Health
and Women’s Science Centre, S-218, 2075 Bayview Avenue, Toronto, ON
M4N 3M5, Canada. Phone: (416) 480-6100, Ext. 3494; Fax:
(416) 480-5703; E-mail: joyce.slingerland{at}utoronto.ca
3 The abbreviations used are: HPEC, human
prostatic epithelial cell; cdk, cyclindependent kinase; pRb,
retinoblastoma protein; INK4, inhibitors of cdk4; KIP, kinase inhibitor
protein; BrdUrd, bromodeoxyuridine; mAb, monoclonal antibody; PAP,
potato acid phosphatase; 2D IEF, two-dimensional isoelectric
focusing.
4 C. Sandhu, J. Donovan, N. Bhattacharya, M.
Stampfer, P. Worland, and J. Slingerland. Loss of cdc25A contributes to
cyclin E1/cdk2 inhibition at senescence in human mammary epithelial
cells, submitted for publication.
Received 8/ 2/99. Accepted 3/20/00.
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