Ras activity late in G1 phase required for p27kip1 downregulation, passage through the restriction point, and entry into S phase in growth factor-stimulated NIH 3T3 fibroblasts.

It is well documented that Ras functions as a molecular switch for reentry into the cell cycle at the border between G0 and G1 by transducing extracellular growth stimuli into early G1 mitogenic signals. In the present study, we investigated the role of Ras during the late stage of the G1 phase by using NIH 3T3 (M17) fibroblasts in which the expression of a dominant negative Ras mutant, p21(Ha-Ras[Asn17]), is induced in response to dexamethasone treatment. We found that delaying the expression of Ras(Asn17) until late in the G1 phase by introducing dexamethasone 3 h after the addition of epidermal growth factor (EGF) abolished the downregulation of the p27kip1 cyclin-dependent kinase (CDK) inhibitor which normally occurred during this period, with resultant suppression of cyclin Ds/CDK4 and cyclin E/CDK2 and G1 arrest. The immunodepletion of p27kip1 completely eliminated the CDK inhibitor activity from EGF-stimulated, dexamethasone-treated cell lysate. The failure of p27kip1 downregulation and G1 arrest was also observed in cells in which Ras(Asn17) was induced after growth stimulation with a phorbol ester or alpha-thrombin and was mimicked by the addition late in the G1 phase of inhibitors for phosphatidylinositol-3-kinase. Ras-mediated downregulation of p27kip1 involved both the suppression of synthesis and the stimulation of the degradation of the protein. Unlike the earlier expression of Ras(Asn17) at the border between G0 and G1, its delayed expression did not compromise the EGF-stimulated transient activation of extracellular signal-regulated kinases or inhibit the stimulated expression of a principal D-type cyclin, cyclin D1, until close to the border between G1 and S. We conclude that Ras plays temporally distinct, phase-specific roles throughout the G1 phase and that Ras function late in G1 is required for p27kip1 downregulation and passage through the restriction point, a prerequisite for entry into the S phase.     

Cyclic AMP-dependent phosphorylation of cyclin D3-bound CDK4 determines the passage through the cell cycle restriction point in thyroid epithelial cells

According to current concepts, the cell cycle commitment after restriction (R) point passage requires the sustained stimulation by mitogens of the synthesis of labile d-type cyclins, which associate with cyclin-dependent kinase (CDK) 4/6 to phosphorylate pRb family proteins and sequester the CDK inhibitor p27kip1. In primary cultures of dog thyroid epithelial cells, the cAMP-dependent cell cycle induced by a sustained stimulation by thyrotropin or forskolin differs from growth factor mitogenic pathways, as cAMP does not upregulate d-type cyclins but increases p27 levels. Instead, cAMP induces the assembly of required cyclin D3-CDK4 complexes, which associate with nuclear p27. In this study, the arrest of forskolin stimulation rapidly slowed down the entry of dog thyrocytes into S phase and the phosphorylation of pRb family proteins. The pRb kinase activity, but not the formation, of the cyclin D3-CDK4-p27 complex was strongly reduced. Using two-dimensional gel electrophoresis, a phosphorylated form of CDK4 was separated. It appeared in response to forskolin and was bound to both cyclin D3 and p27, presumably reflecting the activating Thr-172 phosphorylation of CDK4. Upon forskolin withdrawal or after cycloheximide addition, this CDK4 phosphoform unexpectedly persisted in p27 complexes devoid of cyclin D3 but it disappeared from the more labile cyclin D3 complexes. These data demonstrate that the assembly of the cyclin D3-CDK4-p27 holoenzyme and the subsequent phosphorylation and activation of CDK4 depend on distinct cAMP actions. This provides a first example of a crucial regulation of CDK4 phosphorylation by a mitogenic cascade and a novel mechanism of cell cycle control at the R point.     

Regulation of Exit from Quiescence by p27 and Cyclin D1-CDK4

The synthesis of cyclin D1 and its assembly with cyclin-dependent kinase 4 (CDK4) to form an active complex is a rate-limiting step in progression through the G₁ phase of the cell cycle. Using an activated allele of mitogen-activated protein kinase kinase 1 (MEK1), we show that this kinase plays a significant role in positively regulating the expression of cyclin D1. This was found both in quiescent serum-starved cells and in cells expressing dominant-negative Ras. Despite the observation that cyclin D1 is a target of MEK1, in cycling cells, activated MEK1, but not cyclin D1, is capable of overcoming a G₁ arrest induced by Ras inactivation. Either wild-type or catalytically inactive CDK4 cooperates with cyclin D1 in reversing the G₁ arrest induced by inhibition of Ras activity. In quiescent NIH 3T3 cells expressing either ectopic cyclin D1 or activated MEK1, cyclin D1 is able to efficiently associate with CDK4; however, the complex is inactive. A significant percentage of the cyclin D1-CDK4 complexes are associated with p27 in serum-starved activated MEK1 or cyclin D1 cell lines. Reduction of p27 levels by expression of antisense p27 allows for S-phase entry from quiescence in NIH 3T3 cells expressing ectopic cyclin D1, but not in parental cells.     

Parathyroid hormone-related protein induces G1 phase growth arrest of vascular smooth muscle cells

In this study, we investigated the mechanisms responsible for the growth-inhibitory action of parathyroid hormone-related protein (PTHRP) in A10 vascular smooth muscle cells (VSMC). Fluorescence-activated cell sorting analysis of serum-stimulated VSMC treated with PTHRP or dibutyryl-cAMP (DBcAMP) demonstrated an enrichment of cells in G1 and a reduction in the S phase. Measurement of DNA synthesis in platelet-derived growth factor-stimulated VSMC treated with DBcAMP revealed that cells became refractory to growth inhibition by 12-16 h, consistent with blockade in mid-G1. cAMP treatment blunted the serum-induced rise in cyclin D1 during cell cycle progression without altering levels of the cyclin-dependent kinase cdk4 or cyclin E and its associated kinase, cdk2. Exposure of cells to PTHRP or cAMP resulted in a reduction in retinoblastoma gene product (Rb) phosphorylation. Immunoblotting of extracts from cAMP-treated cells with antibodies to cdk inhibitors revealed a striking increase in p27(kip1) abundance coincident with the G1 block. Immunoprecipitation with an anti-cyclin D1 antibody of cell lysates prepared from cAMP-treated cells followed by immunoblotting with antisera to p27(kip1) disclosed a threefold increase in p27(kip1) associated with cyclin D1 compared with lysates treated with serum alone. We conclude that PTHRP, by increasing intracellular cAMP, induces VSMC cycle arrest in mid-G1. This occurs secondary to a suppression in cyclin D1 and induction of p27(kip1) expression, which in turn inhibits Rb phosphorylation.     

Nature of the Critical Labile Event That Controls RB Phosphorylation in the Cyclic AMP-Dependent Cell Cycle of Thyrocytes in Primary Culture

This study addresses the nature of the critical labile event that couples at restriction point mitogenic cascades with the autonomous part of the cell cycle. In primary cultures of dog thyroid epithelial cells, kinetic experiments indicate that a labile cAMP-dependent event positively controls a late G1 commitment to DNA replication and RB phosphorylation. As previously shown in this system, the cAMP-dependent mitogenic pathway differs from the most generally envisaged growth factor cascades as it stimulates the accumulation of p27(kip1) but not of cyclins D. Nevertheless, cyclin D3 and CDK4 activity are essential in the cAMP-dependent mitogenesis, and cAMP unmasks the DCS-22 epitope of cyclin D3 and induces the nuclear translocations and assembly of cyclin D3 and CDK4 in a complex that also contains p27(kip1). Unexpectedly, the washing out of forskolin rapidly arrested S phase entry and the accumulation of hyperphosphorylated RB, but did not reverse any of the above events associated with cyclin D3-CDK4 activation. This implies that even after induction of stable nuclear cyclin D3-CDK4 complexes, dog thyrocytes still depend on cAMP for RB phosphorylation and commitment to DNA synthesis, which suggests that a key labile event responsible for a last control of restriction point passage remains to be uncovered, in the cAMP-dependent cell cycle of dog thyrocytes and possibly other systems.     

Prolyl oligopeptidase inhibition-induced growth arrest of human gastric cancer cells

Prolyl oligopeptidase (POP) is a serine endopeptidase that hydrolyzes post-proline peptide bonds in peptides that are <30 amino acids in length. We recently reported that POP inhibition suppressed the growth of human neuroblastoma cells. The growth suppression was associated with pronounced G₀/G₁ cell cycle arrest and increased levels of the CDK inhibitor p27^kip1 and the tumor suppressor p53. In this study, we investigated the mechanism of POP inhibition-induced cell growth arrest using a human gastric cancer cell line, KATO III cells, which had a p53 gene deletion. POP specific inhibitors, 3-({4-[2-(E)-styrylphenoxy]butanoyl}-l-4-hydroxyprolyl)-thiazolidine (SUAM-14746) and benzyloxycarbonyl-thioprolyl-thioprolinal, or RNAi-mediated POP knockdown inhibited the growth of KATO III cells irrespective of their p53 status. SUAM-14746-induced growth inhibition was associated with G₀/G₁ cell cycle phase arrest and increased levels of p27^kip1 in the nuclei and the pRb2/p130 protein expression. Moreover, SUAM-14746-mediated cell cycle arrest of KATO III cells was associated with an increase in the quiescent G₀ state, defined by low level staining for the proliferation marker, Ki-67. These results indicate that POP may be a positive regulator of cell cycle progression by regulating the exit from and/or reentry into the cell cycle by KATO III cells.     

P27 expression is regulated by separate signaling pathways, downstream of Ras, in each cell cycle phase

The cyclin inhibitory protein p27Kip1 (p27) plays a vital role in regulating cell proliferation in response to the extracellular growth environment. Active proliferation requires the suppression of p27 levels throughout the cell cycle. Late in the cell cycle, p27 degradation requires phosphorylation of Thr 187 by cyclin dependent kinase 2, leading to recognition by the SCF ubiquitin ligase containing the Skp2 F-box protein. Suppression of p27 is also essential for cell proliferation early in the cell cycle, but this occurs independently of Skp2, whose expression is suppressed during G1 phase. In this study, we use a time lapse and quantitative imaging approach to study the connection between proliferative signaling and the degradation of p27 during each cell cycle period in actively cycling cells. Ras activity was required for the suppression of p27 levels throughout the cell cycle, but separate pathways downstream of Ras signaling were required in different cell cycle periods. For example, inhibitors of MEK and phosphatidylinositol-3-kinase induced p27 expression primarily in G1 phase, while inhibitors of AKT activity stimulated these levels primarily in S phase. Skp2 was expressed in a Ras-dependent manner at higher levels late in the cell cycle. Its ablation resulted in higher p27 levels primarily in G2 phase as expected. The fact that separate signaling pathways downstream of Ras function in each cell cycle phase to suppress p27 levels helps explain the vital connection between proliferative signaling, cell cycle control, and p27 expression.     

Regulation of p27Kip1 by intracellular iron levels

Enhanced intracellular iron levels are essential for proliferation of mammalian cells. If cells have entered S phase when iron is limiting, an adequate supply of deoxynucleotides cannot be maintained and the cells arrest with incompletely replicated DNA. In contrast, proliferating cells that are not in S phase, but have low iron pools, arrest in late G1. In this report the mechanism of iron-dependent G1 arrest in normal fibroblasts was investigated. Cells were synchronized in G0 by contact inhibition and serum deprivation. Addition of serum caused the cells to re-enter the cell cycle and enter S phase. However, if the cells were also treated with the iron chelator deferoxamine, S phase entry was blocked. This corresponded to elevated levels of the cyclin dependent kinase inhibitor p27^Kip1 and inhibition of CDK2 activity. Expression of other cell cycle regulatory proteins was not affected, including the induction of cyclins D1 and E. When the quiescent serum starved cells were supplemented with a readily usable form of iron in the absence of serum or any other growth factors, a significant population of the cells entered S phase. This was associated with downregulation of p27^Kip1 and increased CDK2 activity. Using an IPTG-responsive construct to artificially raise p27^Kip1 levels blocked the ability of iron supplementation to promote S phase entry. Thus it appears that p27^Kip1 is a mediator of G1 arrest in iron depleted Swiss 3T3 fibroblasts. We propose that this is part of an iron-sensitive checkpoint that functions to ensure that cells have sufficient iron pools to support DNA synthesis prior to entry into S phase.     

Follicle stimulating hormone-induced DNA synthesis in the granulosa cells of hamster preantral follicles involves activation of cyclin-dependent kinase-4 rather than cyclin d2 synthesis

Although cyclin D2 mRNA synthesis precedes gonadotropin-induced DNA synthesis in quiescent granulosa cells in culture, it is unclear whether a similar mechanism exists for the granulosa cells of growing preantral follicles in cyclic animals. The objective was to evaluate whether the synthesis of cyclin D2 protein was a prerequisite for FSH-induced DNA synthesis in the granulosa cells of intact preantral follicles of cyclic hamsters. Preantral follicles from cyclic hamsters were cultured in the presence or absence of FSH, and cell cycle parameters were examined. FSH stimulated cyclin-dependent kinase (CDK)-4 activity by 2 h and DNA synthesis by 4 h without altering the levels of cyclin D2 in the granulosa cells. The FSH effect was mimicked by epidermal growth factor administered in vivo. Although FSH increased the levels of cyclin D2 mRNA, it also stimulated the degradation of cyclin D2 as well as p27(Kip1) and p19(INK4) proteins. FSH activation of CDK4 was mediated by cAMP and ERK-1/2. In contrast to granulosa cells in intact follicles, FSH or cAMP significantly increased cyclin D2 protein levels in cultured granulosa cells but failed to induce DNA synthesis. Collectively, these data suggest that granulosa cells of preantral follicles, which are destined to enter the S phase during the estrous cycle, contain necessary amounts of cyclin D2 and other G1 phase components. FSH stimulation results in the formation and activation of the cyclin D2/CDK4 complex leading to DNA synthesis. This mechanism may be necessary for rapid movement of follicles from preantral to antral stages during the short duration of the murine estrous cycle.     

Inactivation of a Cdk2 inhibitor during interleukin 2-induced proliferation of human T lymphocytes.

Peripheral blood T lymphocytes require two sequential mitogenic signals to reenter the cell cycle from their natural, quiescent state. One signal is provided by stimulation of the T-cell antigen receptor, and this induces the synthesis of both cyclins and cyclin-dependent kinases (CDKs) that are necessary for progression through G1. Antigen receptor stimulation alone, however, is insufficient to promote activation of G1 cyclin-Cdk2 complexes. This is because quiescent lymphocytes contain an inhibitor of Cdk2 that binds directly to this kinase and prevents its activation by cyclins. The second mitogenic signal, which can be provided by the cytokine interleukin 2, leads to inactivation of this inhibitor, thereby allowing Cdk2 activation and progression into S phase. Enrichment of the Cdk2 inhibitor from G1 lymphocytes by cyclin-CDK affinity chromatography indicates that it may be p27Kip1. These observations show how sequentially acting mitogenic signals can combine to promote activation of cell cycle proteins and thereby cause cell proliferation to start. CDK inhibitors have been shown previously to be induced by signals that negatively regulate cell proliferation. Our new observations show that similar proteins are down-regulated by positively acting signals, such as interleukin 2. This finding suggests that both positive and negative growth signals converge on common targets which are regulators of G1 cyclin-CDK complexes. Inactivation of G1 cyclin-CDK inhibitors by mitogenic growth factors may be one biochemical pathway underlying cell cycle commitment at the restriction point in G1.     

Estrogens down-regulate p27Kip1 in breast cancer cells through Skp2 and through nuclear export mediated by the ERK pathway

The cyclin-dependent kinase (CDK) inhibitor p27Kip1 plays a key role in growth and development of the mammary epithelium and in breast cancer. p27Kip1 levels are regulated through ubiquitin/proteasome-mediated proteolysis, promoted by CDK2 and the F box protein Skp2 at the G1/S transition, and independent of Skp2 in mid-G1. We investigated the respective roles of Skp2 and subcellular localization of p27Kip1 in down-regulation of p27Kip1 induced in MCF-7 cells by estrogens. 17beta-Estradiol treatment increased Skp2 expression in MCF-7 cells; however, this increase was prevented by G1 blockade mediated by p16Ink4a or the CDK inhibitor roscovitine, whereas down-regulation of p27Kip1 was maintained. Exogenous Skp2 prevented growth arrest of MCF-7 cells by antiestrogen, coinciding with decreased p27Kip1 expression. Under conditions of G1 blockade, p27Kip1 was stabilized by inhibition of CRM1-dependent nuclear export with leptomycin B or by mutation of p27Kip1 (Ser10 --> Ala; S10A) interfering with CRM1/p27Kip1 interaction. Antisense Skp2 oligonucleotides and a dominant-interfering Cul-1(1-452) mutant prevented down-regulation of p27Kip1S10A, whereas Skp2 overexpression elicited its destruction in mitogen-deprived cells. Active mediators of the extracellular signal-regulated kinase (ERK) pathway including Raf-1caax induced cytoplasmic localization of p27Kip1 in antiestrogen-treated cells and prevented accumulation of p27Kip1 in these cells independent of Skp2 expression and coinciding with ERK activation. Genetic or chemical blockade of the ERK pathway prevented down-regulation and cytoplasmic localization of p27Kip1 in response to estrogen. Our studies indicate that estrogens elicit down-regulation of p27Kip1 in MCF-7 cells through Skp2-dependent and -independent mechanisms that depend upon subcellular localization of p27Kip1 and require the participation of mediators of the Ras/Raf-1/ERK signaling pathway.     

Involvement of p27Kip1 in the G1- and S/G2-phase lengthening mediated by glucocorticoids in normal human lymphocytes.

Glucocorticoids inhibit cell proliferation by inducing cell cycle lengthening. In this report, we have analyzed, in normal peripheral blood lymphocytes, the involvement of p27Kip1 in this slowing of proliferation. Following dexamethasone (DXM) treatment, p27Kip1 expression and regulation varied differently with the level of lymphocyte stimulation. In quiescent cells, DXM inhibited p27Kip1 protein expression by decreasing its rate of synthesis, whereas its half-life and mRNA steady state remained constant. In contrast, in stimulated lymphocytes, DXM increased p27Kip1 expression by enhancing its mRNA steady state. This increase is not only a consequence of the DXM-induced interleukin 2 inhibition: we also found an increase in p27Kip1 mRNA stability that was not observed in quiescent lymphocytes. Cyclin/cyclin-dependent kinase (CDK) complexes immunoprecipitated with p27Kip1 are differentially modified by DXM addition: (a) G1 kinasic complexes (cyclin D/CDK4 or CDK6) associated with p27Kip1 are strongly decreased by DXM, (b) S-phase complexes (CDK2/cyclin E and A) remained stable or increased, and (c) the association of p27Kip1 with the phosphorylated forms of CDK1 is increased by DXM. In addition, CDK2 kinase activity was decreased in DXM-treated cells: we suggest that p27Kip1 might participate in inhibiting its catalytic activity. These results indicated that, in normal lymphoid cells, p27Kip1 may be involved in DXM antiproliferative effects. The increase of p27Kip1 expression and a decrease in G1 mitogenic factors, together with the redistribution of p27Kip1 to S/G2-M regulatory complexes, may explain the lengthening of G1 and S/G2 after DXM treatment in lymphocytes.     

p27Kip1 inhibits the cell cycle through non-canonical G1/S phase-specific gatekeeper mechanism

The cyclin-dependent kinase (CDK) inhibitor p27Kip1 has been shown to regulate cellular proliferation via inhibition of CDK activities. It is now recognized that p27Kip1 can regulate cellular processes through non-canonical, CDK-independent mechanisms. We have developed an inducible p27Kip1 model in cultured cells to explore CDK-independent p27Kip1 regulation of biological processes. We present evidence that p27Kip1 can function in a CDK-independent manner to inhibit entry and/or progression of S phase. Even though this p27Kip1 mechanism is non-canonical it does requires the intact cyclin-binding motif in p27Kip1. We suggest a mechanism similar to that proposed in post-mitotic neural cells whereby p27Kip1 functions to coordinate growth arrest and apoptosis. Our hypothesis supports the concept that p27Kip1 is a gatekeeper for the entry and progression of S phase through interaction with specific protein(s) or via binding to specific DNA sequences in a CDK-independent manner.     

Transforming growth factor B1 stimulated DNA synthesis in the granulosa cells of preantral follicles: negative interaction with epidermal growth factor

EGF or TGFB1 alone stimulates but together attenuate granulosa cell DNA synthesis. Intact preantral follicles from hamsters were cultured with TGFB1, EGF, or both to reveal the mechanisms of such unique regulation. Follicular CCND2 (also known as cyclin D2), CDKN1B (also known as p27(kip1)), and the involvement of appropriate signaling intermediaries and kinases were examined. TGFB1, acting via SMAD2 and SMAD3, antagonized the degradation of CCND2 protein by blocking its phosphorylation. In contrast, TGFB1 supported CDKN1B degradation by involving MAPK14 (also known as p38 Map Kinase) and PKC, resulting in CDK4 activation and DNA synthesis. EGF via MAPK3/1 maintained functional levels of CCND2 through CCND2 synthesis as well as degradation. EGF and TGFB1 together inhibited CDK4 activation and DNA synthesis. EGF attenuated TGFB1 stimulated phosphorylation of SMAD3, TGFB1-induced activation of MAPK14 and PKC, and TGFB1 suppression of CCND2 degradation. In contrast, TGFB1 suppressed EGF-induced increase in CCND2 mRNA levels. The final outcome was CCND2 degradation without replenishment and decreased activities of MAPK14 and PKC leading to suppression of CDK4 activation. The results indicate that each growth factor involves a separate mechanism to maintain an effective level of CCND2 in granulosa cells for the activation of CDK4 and induction of DNA synthesis. However, their simultaneous action is inhibitory to follicular DNA synthesis because they counteract each other's activity by interfering at specific sites. Because both EGF and TGFB1 are present in granulosa cells, this mechanism may explain how their effects are temporally modulated for granulosa cell proliferation and folliculogenesis.     

Spy1 interacts with p27Kip1 to allow G1/S progression

Progression through the G1/S transition commits cells to synthesize DNA. Cyclin dependent kinase 2 (CDK2) is the major kinase that allows progression through G1/S phase and subsequent replication events. p27 is a CDK inhibitor (CKI) that binds to CDK2 to prevent premature activation of this kinase. Speedy (Spy1), a novel cell cycle regulatory protein, has been found to prematurely activate CDK2 when microinjected into Xenopus oocytes and when expressed in mammalian cells. To determine the mechanism underlying Spy1-induced proliferation in mammalian cell cycle regulation, we used human Spy1 as bait in a yeast two-hybrid screen to identify interacting proteins. One of the proteins isolated was p27; this novel interaction was confirmed both in vitro, using bacterially expressed and in vitro translated proteins, and in vivo, through the examination of endogenous and transfected proteins in mammalian cells. We demonstrate that Spy1 expression can overcome a p27-induced cell cycle arrest to allow for DNA synthesis and CDK2 histone H1 kinase activity. In addition, we utilized p27-null cells to demonstrate that the proliferative effect of Spy1 depends on the presence of endogenous p27. Our data suggest that Spy1 associates with p27 to promote cell cycle progression through the G1/S transition.     

Fbxw8 is involved in the proliferation of human choriocarcinoma JEG-3 cells

Fbxw8 is the F-box component of a SCF-like E3 ubiquitin ligase complex. Mice lacking Fbxw8 exhibit pathological defects in placenta and embryo similar to fetal growth retardation, suggesting a role of Fbxw8 in placentation. Proliferative capacity of trophoblast cells is very important in placental development. In this context, we revealed that Fbxw8 was expressed in four different human trophoblast cell lines. Silencing of Fbxw8 expression by siRNA inhibited the growth of choriocarcinoma JEG-3 cells. By Western blotting, cell cycle analysis, we showed that down-regulation of Fbxw8 by RNAi induced cell-growth arrest at G2/M phase through decreasing the levels of CDK1, CDK2, cyclin A and cyclin B1 and up-regulation of p27 at protein level. Conversely, over-expression of Fbxw8 led to the opposite effect. These results suggest that Fbxw8 plays an essential role in the proliferation of human trophoblast cells, especially JEG-3 cells, via G2/M phase transition in association with regulation of CDK1, CDK2, cyclin A, cyclin B1 and p27 expression.     

p27/Kip1 mediates retinoic acid-induced suppression of ovarian carcinoma cell growth

We have investigated the mechanisms by which all-trans retinoic acid (ATRA) causes growth inhibition of ovarian carcinoma cells. As a model, we have studied the CAOV3 cell line, which is sensitive to ATRA, and the SKOV3 cell line, which is resistant. We have found that treatment of CAOV3 cells with ATRA causes a 5-10 fold increase in the protein level of the cyclin dependent kinase inhibitor p27/Kip1. p27/Kip1 protein upregulation is important in ovarian carcinoma as primary tumors are frequently found lacking this protein. The increase in p27/Kip1 is detected by day 3 of ATRA treatment of CAOV3 cells, and is maximal by day 5. Messenger RNA levels of p27/Kip1 do not change in CAOV3 cells following ATRA treatment, however, we have shown that p27/Kip1 mRNA is more stable in ATRA treated CAOV3 cells. Conversely, the ATRA resistant cell line SKOV3 fails to show p27/Kip1 accumulation. Interestingly, the SCF component protein SKP2 appears to be decreased in CAOV3 cells treated with ATRA. We have also shown that the ATRA dependent increase in p27/kip1 protein in CAOV3 cells leads to a decrease in the kinase activity of cyclin dependent kinase 4 (CDK4) following ATRA treatment. Finally, we found that CAOV3 cells stably transfected with a p27/kip1antisense construct, which express lower levels of p27/kip1 following ATRA treatment, and have a higher CDK4 kinase activity are less sensitive to ATRA induced growth suppression. Taken together our data suggest ATRA-induced growth inhibition in CAOV3 ovarian carcinoma cells involves modulation of the CDK inhibitor p27/kip1.