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Cancer Research Program Garvan Institute of Medical Research St. Vincent’s Hospital Sydney, New South Wales 2010, Australia
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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50% at 9–18 h.
Similarly, cyclin E-associated kinase activity decreased by 60% at
12–24 h in the absence of significant changes in the abundance of
cyclin E and Cdk2. The CDK inhibitor p21 increased in mRNA and protein
abundance and was present at increased levels in cyclin D1 and cyclin E
complexes at times when their kinase activity was decreased. Increased
p21 protein abundance was observed in another antiprogestin-sensitive
cell line, BT 474, but not in two breast cancer cell lines insensitive
to antiprogestins. These data suggest increased p21 abundance and
concurrent inhibition of CDK activity as a mechanism for antiprogestin
induction of growth arrest. Antiprogestin effects on proliferation
were markedly reduced after ectopic expression of cyclin D1, indicating
that inhibition of cyclin D1 function is a critical element in
antiprogestin inhibition of proliferation. However, these data
also implicate regulation of cyclin E function in antiprogestin
regulation of cell cycle progression. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Studies using breast cancer cells in vitro and rodent mammary tumors in vivo suggest that the antitumor activity of antiprogestins is mediated by inhibition of proliferation (8). Like other steroid or retinoid receptor ligands, antiprogestins have cell cycle phase-specific effects on cell proliferation (9, 10). Antiprogestin treatment leads to accumulation of breast cancer cells in G1 phase at the expense of cells in S, G2, and M phases (11, 12, 13), but the molecular targets involved in mediating this effect have not been defined. However, recent studies implicate regulation of cyclin/cyclin-dependent kinase (CDK) activity, particularly cyclin D-associated kinase activity, in steroidal regulation of proliferation. Cyclin D1 appears to be necessary for the progesterone-dependent development and differentiation of the mammary gland, because cyclin D1-deficient mice fail to develop lobular alveoli during pregnancy (14, 15), and this phenotype is shared by progesterone receptor-deficient mice (16). Furthermore, antiestrogen and retinoid inhibition of breast cancer cell cycle progression is accompanied by decreased cyclin D1 function (17, 18, 19), whereas decreased cyclin D3 function contributes to glucocorticoid inhibition of lymphoma cell proliferation (20).
Sequential activation of cyclin/CDK enzyme complexes regulates progress through the cell cycle. These enzymes are regulated at multiple levels, providing a variety of possible means by which overall activity of the complex, and hence the rate of cell cycle progression, might be modulated. The catalytic activity of CDKs depends not only on cyclin association but also on appropriate phosphorylation of the CDK subunit (21). The CDK-activating kinase (CAK) is itself a cyclin/CDK complex (cyclin H/Cdk7) regulated by phosphorylation. However, regulation of cyclin abundance governs much of the regulation of CDK activity during cell cycle progression and is a frequent response to treatment either by mitogens or inhibitors of cell proliferation (22, 23). Alteration of cyclin abundance is sufficient to alter the rate of cell cycle progression because overexpression of cyclins D or E accelerates cells through G1 and, conversely, inhibition of their function by antibody microinjection prevents entry into S phase (23).
A further means of regulating cyclin/CDK function is provided by endogenous low molecular weight proteins that physically associate with the cyclins, CDKs, or their complexes and inhibit CDK activity (24). A growing family of such inhibitors, for which p16INK4 is the prototype, selectively targets Cdk4 and Cdk6 (24). A second family of inhibitors, including p21 (WAF1, Cip1, Sdi1) and p27 (Kip1), are active against a wider range of cyclin/CDK complexes (24). The balance between levels of inhibitor and cyclin/CDK complexes is thought to set a threshold for activation of the kinase. The mechanism for inhibition of CDK activity has not been defined, but p21 immunoprecipitates display kinase activity indicating that association per se is not sufficient for inactivation (25, 26). Increased p21 or p27 abundance accompanies inhibition of proliferation during quiescence, senescence, and differentiation and contributes to inhibition of growth by transforming growth factor-ß (24, 27).
The retinoblastoma tumor suppressor protein, pRB, is a critical substrate for the G1 CDKs. pRB is hypophosphorylated during early G1 and in this form is growth-inhibitory. The pRB hyperphosphorylation that relieves this growth inhibition is first apparent in late G1 phase and continues during the remainder of the cell cycle (28). Cells without functional pRB lose dependence on cyclin D1 for G1 progression but demonstrate an absolute requirement for cyclin D1 upon reintroduction of pRB (29, 30). These data provide compelling evidence that pRB is a critical physiological target for cyclin D1. However, it is likely that cyclin E/Cdk2 also phosphorylates pRB in vivo, perhaps contributing to the further phosphorylation of pRB as cells progress into S phase (28).
In breast cancer cells cyclin D1 is both necessary and sufficient for G1 phase progression (31, 32). Furthermore, cyclin D1 abundance is rate-limiting in these cells as well as in fibroblasts (32, 33, 34). Initial studies demonstrated that neither cyclin D1 nor Cdk4 mRNA decreased in abundance after antiprogestin treatment, despite inhibition of proliferation (12, 19). However, as outlined above, other mechanisms for regulation of cyclin function exist and, in view of increasing evidence for regulation of cyclin function after treatment with steroids, steroid antagonists, and retinoids, the possibility that antiprogestins might regulate cyclin function in T-47D human breast cancer cells was investigated. Both RU 486, which is the prototypic progestin antagonist but also has glucocorticoid antagonist activity, and ORG 31710, which is representative of newer progestin antagonists with little antiglucocorticoid activity, were used. These studies demonstrate that antiprogestins induce p21 and regulate G1 cyclin function and indicate that this is likely to account for their inhibition of breast cancer cell proliferation.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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). ORG 31710 was more potent than RU
486 such that the response to ORG 31710 was maximal at 1
nM, compared with 10 nM after RU 486 treatment
(Fig. 1A). The decrease in cell number was associated with a sustained
decrease in the proportion of cells in S phase over the same
concentration range, apparent within 1 day of treatment and at
concentrations of 
1 nM, maintained until the conclusion
of the experiment at 5 days (Fig. 1B). Maximally effective
concentrations of RU 486 (100 nM) or ORG 31710 (10
nM) were chosen on the basis of these data and used for
further experiments.
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). Although the time courses
for the two compounds were similar, ORG 31710 was slightly more
effective. The effects of both antiprogestins on S phase fraction
decreased between 18–24 h exposure but were thereafter maintained
(Fig. 1, B and C).
Since pRB is a physiological substrate for the G1 phase
CDKs, pRB phosphorylation in vivo was examined to determine
whether changes in CDK activity might accompany antiprogestin
treatment. In untreated exponentially proliferating cells pRB was
predominantly in the hyperphosphorylated form (ppRB, Fig. 2). After 9 h or more of antiprogestin treatment
the abundance of hypophosphorylated pRB increased, reaching a more than
2-fold increase after 18 h ORG 31710 treatment, and there was a
concomitant decrease in the abundance of ppRB (Fig. 2 and data not
shown). On average, the ppRB/pRB ratio was reduced to 50% of
control at 12–24 h (Fig. 2B).
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50%
after 9–18 h ORG 31710 treatment (Fig. 3A). This
decrease, while modest, was statistically significant at 9 and 12
h. Cyclin E-associated kinase activity was measured using histone H1
substrate and was decreased by 60% from 12–24 h ORG 31710
treatment (Fig. 3B). RU 486 treatment led to similar effects on kinase
activity (not shown). These data demonstrated a decrease in
G1 cyclin-associated kinase activity which paralleled
decreased pRB phosphorylation.
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), consistent with previous examination of cyclin D1
mRNA after RU486 treatment (12, 19). No change in the abundance of
cyclin E, Cdk4, or Cdk6 was detected (Fig. 4), and there was at most a
minor decrease in Cdk2 abundance (not shown). Since regulation of the
abundance of these cyclins and CDKs did not appear to contribute to
decreased kinase activity CDK inhibitor expression was next examined.
No change in the abundance of p27 was detected, but p21 levels
increased markedly after 12 h or more antiprogestin treatment
(Fig. 4B). Quantification of data from multiple experiments showed an
average 4- to 5-fold increase after ORG 31710 treatment and a slightly
smaller increase of >3-fold after RU 486 treatment (Fig. 4C). To
determine the basis for the increase in p21 abundance Northern analysis
was performed after ORG 31710 treatment. Within 6 h treatment p21
mRNA increased by 3-fold and the increase was maintained at 18
h (Fig. 5), indicating that antiprogestin regulation of
p21 mRNA levels is one cause of the increase in p21 protein levels.
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12 treatment (Fig. 6A), to an average
of 3-fold relative to control. Cyclin E immunoprecipitates contained
similar amounts of cyclin E and Cdk2 in control and
antiprogestin-treated cells, but the amount of coimmunoprecipitated p21
increased by 2- to 3.3-fold after 12–24 h ORG 31710 treatment (Fig. 6B), when the kinase activity was decreased (Fig. 3B). Overall, these
data are consistent with the interpretation that increased p21
abundance contributes to the reduction in cyclin-associated kinase
activity after antiprogestin treatment.
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). Similarly, there
was no effect on cyclin D1, p21, or p27 abundance, nor was there any
alteration in the amount of p21 coimmunoprecipitating with cyclin D1
(Fig. 7, B and C). In BT 474 no effect on S phase was observed after
15 h treatment but a significant, albeit small, effect was
observed after 30 h (Fig. 7A). This delay in action is likely a
reflection of the longer doubling time of these cells, 3 days
compared with 1–1.5 days for the other three cell lines. Concomitant
with the decrease in S phase fraction, there was an approximately
3-fold increase in p21 abundance but no change in the abundance of
cyclin D1 or p27 (Fig. 7B). Cyclin D1 immunoprecipitates indicated
increased p21 bound to cyclin D1 after ORG 31710 treatment of BT 474
(Fig. 7C). However, the level of p21 in cyclin D1 immunoprecipitates
from treated BT 474 cells was still markedly lower than that in
immunoprecipitates of a similar amount of cyclin D1 from T-47D cells.
These data indicate an association between antiprogestin induction of
p21 and inhibition of cell proliferation.
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), suggesting that the cyclin D1
complexes in control cells contained significant amounts of p21. To
assess the degree of saturation of the complexes, the effect of adding
recombinant GST-p21 fusion protein to a constant amount of cyclin D1
immunoprecipitate from exponentially proliferating cells was
determined. Recruitment of GST-p21 into the cyclin D1 complexes
increased in proportion to the amount of GST-p21 added until the total
p21 (the sum of the endogenous p21 and GST-p21) reached approximately 3
times the initial abundance (Fig. 8, B and C). However, increasing the
amount of added GST-p21 by 10-fold led to a relatively modest further
increase in the total p21 bound (Fig. 8, B and C). Thus the cyclin D1
complexes became saturated with p21 upon a 4-fold increase in
associated p21. These data indicate that the 3-fold relative
increase in cyclin D1-associated p21 after antiprogestin treatment is
likely to be sufficient to account for the observed decrease in kinase
activity.
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MTcycD1-3 (32, 35), were used to examine the effects of increased
cyclin D1 abundance on the response to antiprogestins. After zinc
treatment of these cells cyclin D1 protein abundance increases within
3 h and reaches maximum levels by 6–9 h (32). The resulting
acceleration of cells through G1 phase leads to an increase
in the proportion of cells in S phase, which is maximal at 15–24 h
(32). Since zinc induction of cyclin D1 and antiprogestin induction of
p21 occur over a similar time course, the cyclin D1/p21 ratio would be
expected to remain relatively constant after simultaneous treatment
with ZnSO4 and antiprogestin. The increase in cyclin D1
abundance after zinc treatment was unaffected by simultaneous treatment
with ORG 31710 (Fig. 9A). ORG 31710 treatment reduced
the S phase fraction relative to untreated control cells when cyclin D1
levels were increased by up to 2-fold (i.e. zinc
concentrations of 30 µM or below) (Fig. 9B). A 2.5-fold
increase in cyclin D1 abundance after treatment with 40
µM zinc prevented ORG 31710 treatment from decreasing the
S phase fraction relative to untreated control cells, while a further
increase in cyclin D1 abundance led to increased S phase despite the
presence of antiprogestin (Fig. 9B). In the same experimental design,
treatment of parental T-47D cells with up to 50 µM zinc
did not significantly increase either cyclin D1 abundance or S phase
fraction.
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MTcycD1-3 cells treated with zinc
together with antiprogestin was consistently lower than that of cells
treated with zinc in the absence of antiprogestin treatment, despite
equivalent cyclin D1 levels (Fig. 9, A and B). However, consistent with
previous data (32), cyclin D1 abundance and S phase fraction were
linearly related in both cases (Fig. 9C). Lines of best fit generated
by linear regression were essentially parallel, with slopes of 1.52 and
1.59 for control and ORG 31710, respectively (Fig. 9C), i.e.
in either the presence or absence of antiprogestin a given absolute
increase in cyclin D1 abundance led to the same absolute increase in S
phase. Thus, the effect of antiprogestin treatment was to increase the
amount of cyclin D1 necessary to achieve a particular S phase value, as
would be expected if the threshold for cyclin D1 function had been
increased as a result of p21 induction after antiprogestin
treatment. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Accumulation of underphosphorylated pRB was coincident with the decrease in S phase fraction. Both were first evident at 9 h and reached a minimum after 18 h ORG 31710 treatment. These responses were preceded slightly by decreased cyclin D1- and cyclin E-associated kinase activity, which reached a minimum by 12 h. Since pRB is a key substrate for G1 cyclin-associated kinases, these data suggest that decreased pRB phosphorylation results from decreased CDK activity and that it is the presence of growth-inhibitory, underphosphorylated, pRB that ultimately mediates G1 arrest. Investigation of possible mechanisms underlying the decrease in CDK activity revealed that the abundance of the CDK inhibitor p21 was increased by 3- to 4-fold after 12–24 h treatment, when decreased kinase activity was observed. Antiprogestin induction of p21 mRNA was also observed, suggesting that the increased protein abundance was, at least in part, a result of either increased transcription or stabilization of p21 mRNA. Induction of p21 was not observed in either a progesterone receptor-negative cell line (MDA-MB-231) or a progesterone receptor-positive MCF-7 variant that is insensitive to antiprogestins. However, it was observed in BT 474 cells accompanying an antiprogestin-induced decrease in S phase fraction, indicating a close correspondence between p21 induction and growth inhibition. Regulation of the abundance of cyclin D1, Cdk4, Cdk6, or p27 did not appear to contribute to the decreased kinase activity, nor did the relative Cdk4 abundance in cyclin D1 immunoprecipitates alter after antiprogestin treatment (our unpublished data). The latter observation argues against the induction of p16INK4 or a related CDK inhibitor after antiprogestin treatment because this would be expected to displace Cdk4 from the complexes (24). Furthermore, preliminary examination of cyclin D1 immunoprecipitates from 35S-methionine-labeled T-47D cells did not provide evidence for alterations in complex composition other than increased p21 association after antiprogestin treatment (our unpublished data). Similarly, decreases in the abundance of cyclin E or Cdk2 sufficient to account for the decrease in cyclin E-associated kinase activity were not observed, but the relative abundance of p21 increased in cyclin E immunoprecipitates at times when kinase activity was reduced.
In a variety of cell types, ectopic expression of p21 leads to arrest
in G1 phase (26, 40, 41), consistent with the hypothesis
that induction of p21 might be responsible for the G1 phase
arrest after antiprogestin treatment. Further experiments indicated
that the observed increase in p21 abundance was likely to be sufficient
to account for the decrease in cyclin D1-associated kinase activity.
Although addition of p21 to recombinant cyclin D1/Cdk4 complexes
inhibits kinase activity in a concentration-dependent fashion, the
complexes retain near-maximal kinase activity in the presence of p21
levels representing 25–50% of the level required to completely
inhibit kinase activity (42). Although the precise details of the p21
binding required for inhibition remain undefined, it is apparent that
kinase activity is likely to decrease as the complexes approach
saturation. In untreated T-47D cells a third of the total cellular p21
coimmunoprecipitated with cyclin D1 (Fig. 8), consistent with data from
other cell types (e.g. Ref.43). This observation suggested
that the cyclin D1 complexes contained p21 at a level near that
required for inhibition of kinase activity. Addition of recombinant
GST-p21 to cyclin D1 immunoprecipitates indicated that saturation of
the cyclin D1 complexes with p21 would occur after an approximately
4-fold increase in bound p21 (Fig. 8) and thus that the observed
3-fold increase in p21 coimmunoprecipitating with cyclin D1 was
likely to be sufficient to decrease cyclin D1-associated kinase
activity. The observation that the threshold for cyclin D1 function was
increased after antiprogestin treatment (Fig. 9) is consistent with
this interpretation since the abundance of CDK inhibitors is thought to
set this threshold (24). Although the degree of induction of p21 in BT
474 was similar to that in T-47D, there was more Cdk4 and less p21
coimmunoprecipitated with cyclin D1 in BT 474, even after antiprogestin
induction of p21 (Fig. 7C), suggesting a greater proportion of active
complexes. This could then contribute to the modest level of the
decrease in BT 474 S phase fraction after ORG 31710 treatment.
Because the activity of both cyclin D1 and cyclin E is required for progress into S phase (44, 45), inhibition of either could account for growth inhibition after antiprogestin treatment. However, despite the antiprogestin-induced decrease in both cyclin D1-associated and cyclin E-associated kinase activity, a 2.5-fold increase in cyclin D1 abundance alone prevented antiprogestin inhibition of cell cycle progression, apparently overriding effects on cyclin E-associated kinase activity. Thus, inhibition of cyclin D1-associated kinase activity appears to be a critical element in antiprogestin inhibition of cell cycle progression. An implication of this conclusion is that sensitivity to inhibition by antiprogestins may, in part, depend on the abundance of cyclin D1 and hence that the significant fraction, 30–50%, of breast cancers that overexpress cyclin D1 (31, 46, 47, 48, 49) may display altered sensitivity to antiprogestin therapy.
Occupancy of cyclin E complexes by p21 was not investigated in such
detail as that of cyclin D1 complexes, but the correspondence between
increased p21 binding and decreased kinase activity is consistent with
increased p21 abundance contributing to the inhibition of cyclin
E-associated kinase activity. However, it does not exclude other
mechanisms, including alterations in the level or activity of the
kinases and phosphatases controlling the level of Cdk2 phosphorylation
and hence its activity. Furthermore, there is increasing evidence for
growth-inhibitory effects of p21 not mediated via direct inhibition of
CDK activity but rather by interaction with other cell cycle-regulatory
pathways. For example, the activity of E2F, a transcription factor with
a central role in cell proliferation, is repressed by p21 (50, 51). A
possible mechanism is suggested by p21 disruption of the interaction
between Cdk2, E2F, and the pRB-related proteins p107 and p130 (50, 51, 52),
and this has been suggested to play a role in the inhibitory function
of p21 (51). In addition, p21 binds the proliferating cell nuclear
antigen (PCNA), blocking its ability to activate DNA polymerase 
,
and the PCNA-binding domain alone is capable of blocking cell cycle
progression (24). Since the PCNA-binding domain is a less effective
growth inhibitor than the CDK-inhibitory domain (24), it is unlikely
that inhibition of DNA replication by this mechanism makes a major
contribution to antiprogestin inhibition of proliferation. Finally, p21
has recently been shown to inhibit the activity of kinases other than
CDKs, i.e. the stress-activated protein kinases (also known
as the c-Jun amino-terminal kinases) and protein kinase CK2 (53, 54),
and it is conceivable that such inhibition also contributes to its
growth-inhibitory effects.
The effects of antiprogestin on breast cancer cells are not limited to inhibition of cell proliferation. Other responses including induction of differentiation and apoptosis are apparent after several days’ antiprogestin treatment (55, 56). Although both differentiation and apoptosis can occur in the absence of p21 induction (24), increased p21 has been associated with differentiation in a number of cellular systems both in vivo and in vitro (24) and with retinoid induction of apoptosis in breast cancer cells (57). Overexpression of p21 in human melanocytes led to morphological changes characteristic of differentiation and increased melanin production, in some cases followed by cell death (41), while overexpression in MCF-7 and T-47D breast cancer cells led to apoptosis (58). These data suggest that p21 induction could contribute to antiprogestin effects other than growth arrest alone.
In summary, the data presented in this manuscript suggest a model for the effects of antiprogestin treatment on proliferation in which decreased activity of both cyclin D1/CDK and cyclin E/CDK and consequent decreased pRB phosphorylation result in inhibition of entry into S phase. Decreased kinase activity does not result from regulation of cyclin abundance but induction of the CDK inhibitor p21 accompanies these changes and is thus a likely mediator of the effects of antiprogestins on cell proliferation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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MTcycD1-3 are a clonal derivative of T-47D expressing ectopic cyclin
D1 under the control of a metal-inducible truncated human
metallothionein IIA promoter lacking steroid-responsive sequences (32, 35). RPMI 1640 medium was supplemented with 10 µg/ml human insulin
(Actrapid, CSL-Novo, North Rocks, NSW, Australia), HEPES (20
mM), sodium bicarbonate (14 mM), and
L-glutamine (6 mM). Stock cultures were
maintained as previously described (36), in medium supplemented with
10% FCS and without antibiotics. Experiments used cells cultured in
medium supplemented with 5% FCS. RU 486 (generously provided by Dr J-P
Raynaud of Roussel-Uclaf, Romainville, France) and ORG 31710
(generously provided by Dr W. Schoonen, Organon, Oss, The Netherlands)
were dissolved in ethanol at 1000- or 2000-fold final concentration and
added to cells in exponential growth. Control cultures received ethanol
to the same final concentration. Data presented are representative of
at least two experiments. Cell cycle phase distribution was determined
by flow cytometry (59). Antiprogestin effects on cell number were measured using a colorimetric cell proliferation assay (CellTiter, Promega, Madison, WI). Cells (103) were seeded into 96-well plates, and the next day antiprogestin or vehicle was added to quadruplicate wells for each treatment. Plates were assayed at intervals and relative absorbances, i.e. relative cell numbers, were determined near the end of exponential growth for control cultures, after 6–7 days’ exposure to antiprogestin.
Recombinant p21
The coding region of p21 was amplified by PCR using pZL.WAF1
(60) as a template and p21N (TAC ATG GAT CCA TGT CAG AAC CGG CTG GGG A)
and p21C (AGA CTG AAT TCT TAG GGC TTC CTC TTG GAG A) as primers. The
resulting fragment was cloned into the
BamHI/EcoRI sites of the expression vector
pGEX-2t (Pharmacia, Uppsala, Sweden) to yield pGEX-p21. To prepare
GST-p21, Eschericia coli were transformed with pGEX-p21, and
expression of the protein was induced by incubation with 0.1
mM isopropylthioglycoside) (2.5 h, room temperature).
Frozen cell pellets were lysed by sonication at 4 C after resuspension
in PBS with protease inhibitors (1 mM
phenylmethylsulfonylfluoride, 0.5 M EDTA, 0.05% (vol/vol)
ß-mercaptoethanol, 10 µg/µl aprotonin, 10 µg/µl leupeptin).
After addition of 0.5% Triton X-100, the lysate was centifuged at 4 C.
The supernatant was then incubated with gentle rotation at 4 C for
1 h with 0.5 ml of a 50% (vol/vol) suspension of
glutathione-agarose (Sigma, St.Louis, MO). The resin was washed once
with ice-cold PBS/0.05% ß-mercaptoethanol/0.5% Triton and twice
with ice-cold PBS/0.05% ß-mercaptoethanol and finally resuspended in
0.5 ml ice-cold PBS/0.05% ß-mercaptoethanol/0.1% azide. The fusion
protein was then eluted with 50 mM Tris-Cl, pH 8.0, 15
mM reduced glutathionine, 0.05% ß-mercaptoethanol, and
the purity of fusion protein was assessed by PAGE followed by Coomassie
blue staining.
Cell Lysis, Western Blot Analysis, and Immunoprecipitation
Cells were lysed either as described below for cyclin
D1-associated kinase assays or as previously described, using lysis
buffer consisting of 50 mM HEPES (pH 7.5), 150
mM NaCl, 10% (vol/vol) glycerol, 1% Triton X-100, 1.5
mM MgCl2, 1 mM EGTA, 10 µg/ml
aprotinin, 10 µg/ml leupeptin, 1 mM PMSF, 200
µM sodium orthovanadate, 10 mM sodium
pyrophosphate, and 100 mM NaF (32, 61). Similar results
were obtained from Western blotting or immunoprecipitation using either
lysis technique.
Cell lysates were precleared by incubation with protein A-Sepharose
beads (Zymed, San Francisco, CA) (1 h, 4 C) then immunoprecipitated by
incubation (3 h, 4 C) with protein A-Sepharose beads that had been
conjugated with either an anti-cyclin E antibody (C-19, Santa Cruz
Biotechnology, Santa Cruz, CA) or rabbit polyclonal anti-cyclin D1
serum [raised against a human cyclin D1-GST fusion protein (35)]. In
some experiments the antibodies were chemically cross-linked to the
protein A-Sepharose beads by incubation in 5 mg/ml dimethyl
pimelimidate/0.2 M sodium tetraborate (pH 9.0) for 30 min
at room temperature, essentially as described (62). The
immunoprecipitated proteins were then washed as previously described
(35). In the experiments presented in Fig. 8, recombinant GST-p21 was
added to the immunoprecipitates, and the samples were incubated at 30 C
for 1 h with vortexing every 10 min. The beads were then washed
with 50 mM HEPES (pH 7.5), 1 mM dithiothreitol
before resuspension in SDS-PAGE sample buffer.
Samples of immunoprecipitated or total protein in SDS-PAGE sample buffer were heated to 95 C for 3 min, then separated by SDS-PAGE and transferred to nitrocellulose. Specific proteins were visualized by chemiluminescence (Dupont NEN, Boston, MA) after incubation (2–4 h at room temperature or overnight at 4 C) with the following primary antibodies: p21 antiserum kindly provided by Dr David Beach (Cold Spring Harbor, NY); cyclin E (HE12), Cdk2 (M2), Cdk4 (C-22), and Cdk6 (C-21) antibodies from Santa Cruz Biotechnology; cyclin D1 antibody (DCS6) from Novocastra, Newcastle-upon-Tyne, U.K.; pRB (14001A) antibodies from Pharmingen (San Diego, CA); p21 (C24420) and p27 (K25020) antibodies from Transduction Laboratories (Lexington, KY). Relative abundance was quantitated using a Molecular Dynamics (Sunnyvale, CA) densitometer and IP LabGel analysis software (Signal Analytics, Vienna, VA).
Kinase Assays
The histone H1 kinase activity of cyclin E immunoprecipitates
was measured as previously described for Cdk2 assays (35) using 10 µg
histone H1 as substrate. For cyclin D1-associated kinase assays, cells
were harvested and lysed as previously described using kinase lysis
buffer [50 mM HEPES (pH 7.5), 1 mM
dithiothreitol, 150 mM NaCl, 1 mM EDTA, 2.5
mM EGTA, 0.1% Tween-20, 10% glycerol, 10 mM
ß-glycerophosphate, 1 mM NaF, 0.1 mM sodium
orthovanadate, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 0.1
mM PMSF] (35, 63). Kinase activity of cyclin D1
immunoprecipitates of these lysates was measured using either a
pRB(379–928)-maltose binding protein fusion protein, GST-pRB(769–921)
fusion protein substrate (Santa Cruz) or GST-pRB(773–928) (64) as
previously described (35). After termination of kinase reactions,
samples were incubated at 90 C for 2 min in SDS sample buffer and
separated using 10% SDS-PAGE. Relative intensities were quantitated
using a Molecular Dynamics PhosphorImager Scanner (model 445 SI)
followed by analysis using IP LabGel analysis software (Signal
Analytics) or in some cases after exposure to x-ray film as decribed
above for Western analysis. The degree of background phosphorylation in
cyclin D1-associated kinase assays was estimated from parallel control
samples either immunoprecipitated using preimmune serum or assayed
after incubation of immunoprecipitates with an excess of GST-p21 (30
min, 30 C) and has been subtracted in the data presented in Fig. 3A.
RNA Isolation and Northern Analysis
Total RNA was extracted (using a guanidinium
isothiocyanate-cesium chloride procedure) and blotted as previously
described, using 20 µg total RNA/lane (46). The membranes were
hybridized overnight at 50 C in 50% (vol/vol) formamide, 2x SSPE (0.3
M NaCl, 20 mM NaH2PO4,
2 mM EDTA, pH 7.4), 1% (wt/vol) SDS, 0.5% (wt/vol) low
fat skim milk (Diploma, St Kilda, Victoria, Australia), 10% (wt/vol)
dextran sulfate (Mr 500,000), 200 µg/ml yeast RNA, 40
µg/ml polyadenylic acid (5'), 500 µg/ml salmon sperm DNA. The
2.1-kb p21 cDNA from pZL.WAF1 (60) was labeled with
[
-32P]dCTP (Amersham Australia, North Ryde, New South
Wales, Australia; specific activity 3000 Ci/mmol) to a specific
activity of approximately 1 x 109 cpm/µg DNA using
the Multiprime DNA labeling kit (Amersham Australia) then added to the
hybridization mix at a final concentration of 10 ng/ml. The
membranes were washed at a highest stringency of 0.2 x SSC (30
mM NaCl, 3 mM sodium citrate, pH 7.0), 1% SDS
at 65 C and exposed to Kodak X-OMAT film at -70 C. Equivalent RNA
loading was verified as previously described (46) by hybridizing
membranes with a [
-32P]ATP end-labeled oligonucleotide
complementary to 18S rRNA.
Statistical Analysis
Data pooled from multiple experiments were analyzed using
Statview II software (Abacus Concepts, Inc, Berkeley, CA). The
significance of differences from control was determined using a
one-tailed t test.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This study was supported by research grants from the National Health and Medical Research Council of Australia and the New South Wales State Cancer Council.
Received for publication September 16, 1996. Accepted for publication October 3, 1996.
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) or ORG 31710 (•) at the indicated
concentrations. Proliferation was assayed after 6–7 days’ treatment
using a colorimetric assay, and absorbance of the treated wells is
presented relative to that in vehicle-treated control wells, as
relative cell number. Data points indicate mean ± SEM
of values from two or three replicate experiments each performed in
quadruplicate or (points without error bars) mean of quadruplicates in
a single experiment. B, Exponentially proliferating T-47D human breast
cancer cells were treated with ethanol vehicle or the indicated
concentrations of RU 486 or ORG 31710. Individual flasks were harvested
and stained for DNA analysis by flow cytometry after 1 (
), 3 (
),
or 5 days (
) treatment. Data represent mean ± SEM
or range of results from two to four experiments. The S phase of
control cells was similar over the entire experiment and, therefore,
data from days 1, 3, and 5 have been pooled and are shown as mean
± SEM (C,
). C, Exponentially proliferating T-47D human
breast cancer cells were
treated with ethanol vehicle (x) RU 486 (100 nM,
