Lovastatin inhibits mesangial cell proliferation via p27Kip1

Mesangial cell proliferation is a key feature of glomerulonephritis. The hydroxymethylglutaryl-coenzyme A reductase inhibitor lovastatin is known to inhibit cell cycle progression. To determine the inhibitory mechanisms of mesangial cell proliferation by lovastatin, the cyclin-dependent kinase (CDK) activity, and expression of CDK inhibitor (p27Kip1, p21Cip1, and p16INK4) mRNA and protein were measured. Lovastatin inhibited phosphorylation of retinoblastoma protein and mesangial cell proliferation dose dependently. Lovastatin increased the p27Kip1 protein level but produced no changes in the abundance of the p27Kip1 mRNA level both in the presence and absence of mitogens. Treatment with lovastatin revealed the increment of both CDK2- and CDK4-bound-p27Kip1. The experiment using antisense oligonucleotide against p27Kip1 showed significant amelioration of lovastatin-induced cell cycle arrest. Lovastatin reduced both platelet-derived growth factor-stimulated CDK2 and CDK4 kinase activities. In conclusion, lovastatin inhibited mesangial proliferation via translational upregulation or impairment of p27Kip1 protein degradation. Lovastatin serves as a potential therapeutic approach to mesangial proliferative disease.     

Cytoplasmic displacement of cyclin E-cdk2 inhibitors p21Cip1 and p27Kip1 in anchorage-independent cells

Loss of attachment to an extracellular matrix substrate arrests the growth of untransformed cells in the G1 phase. This anchorage-dependent cell cycle arrest is linked to increased expression of the p21Cip1 (p21) and p27Kip1 (p27) cyclin-dependent kinase inhibitors. The result is a loss of cdk2-associated kinase activity, especially that of cyclin E-cdk2. The levels of p21 and p27 are also upregulated in unattached transformed cells, but cyclin E-cdk2 activity remains high, and the cells are able to grow in an anchorage-independent manner. Increased expression of cyclin E and cdk2 appears to be partially responsible for the maintenance of cyclin E-cdk2 activity in transformed cells. To explore further the regulation of cyclin E-cdk2 in transformed cells, we have analysed the subcellular distribution of cyclin-cdk complexes and their inhibitors in normal human fibroblasts, their transformed counterparts, and in various human tumor cell lines. In substrate-attached normal fibroblasts, cyclin E and cdk2 were exclusively in the nuclear fraction, associated with one another. When normal fibroblasts were detached and held in suspension, cyclin E-cdk2 complexes remained nuclear, but were now found associated with the p21 and p27 cdk inhibitors and lacked histone H1 phosphorylating activity. In contrast, the transformed fibroblasts and tumor cells, which are anchorage-independent, had more than half of their cyclin E, cdk2, p21 and p27 in the cytoplasmic fraction, both in attached and suspended cultures. The cytoplasmic p21 and p27 were bound to cyclin E-cdk2, as well as to complexes containing cyclin A and cyclin D. The nuclear cyclin E-cdk2 complexes from the transformed cells grown in suspension contained only low levels of p21 and p27 and had histone H1 kinase activity. Thus, at least three mechanisms contribute to keeping cyclin E-cdk2 complexes active in suspended anchorage-independent cells: cyclin E and cdk2 are upregulated, as reported previously, cdk inhibitors are sequestered away from the nucleus by cytoplasmic cyclin-cdk complexes, and the binding of the inhibitors to nuclear cyclin E-cdk2 complexes is impaired.     

Role of p27Kip1 and cyclin-dependent kinase 2 in the proliferation of non-small cell lung cancer

There is strong evidence that depression can have profound negative effects on the functional status, psychologic outlook, and possibly the medical outcome of cancer patients. Its presence often is taken for granted by both patients and physicians as being either inevitable or easily explained by the grim prognosis and complex treatment regimens experienced by many cancer patients, and thereby less deserving of an aggressive approach to diagnosis and treatment. This view is inappropriate, and hinders an effective approach to the problem. Effective treatment, similar to that provided for any other depressed patient, can enhance the cancer patient's overall approach to life and the disease, irrespective of the eventual medical outcome.     

The role of cell cycle regulatory proteins, cyclin D1, cyclin E, and p27 in thyroid carcinogenesis

The cell cycle is controlled in part by cyclin-dependent kinases (CDKs), which are activated by forming complexes with cyclins. CDKs phosphorylate certain substrates to facilitate the proliferating cells through the cell cycle. CDK inhibitors (CDKIs) such as p27 inhibit cyclin-CDK complexes and function as a negative cell cycle regulator. The overexpression of the positive regulators (cyclins) or the underexpression of the negative regulators including p27 has been seen in a variety of neoplasms, but their role and interaction in thyroid carcinogenesis is yet to be established. We studied the expression of cyclins D1 and E, and the CDKI, p27 by immunohistochemistry in 116 cases, including 59 cases of follicular variant of papillary carcinoma (FVPC) and 57 cases of follicular adenoma (FA). The positive staining was divided into four grades: 1+ if less than 10%, 2+ if 11% to 25%, 3+ if 26% to 50%, and 4+ if greater than 50% of the nuclei of tumor cells stained positively. Cyclin D1 expression was seen in 37 (63%) FVPC and 34 (60%) FA. Cyclin E-positive cells were seen in 51 (86%) FVPC and 47 (82%) FA. No significant differences in the grade of cyclins D1 (P = .261) and E (P = .284) staining was seen between FVPC and FA. Of the 59 FVPC, 53 (89%) showed p27-positive cells; of these, 33 were 1+, nine were 2+, seven were 3+ and only four were 4+ positive. Conversely, all 57 FA were p27 positive, 53 were 4+, and four were 3+ positive. This difference in the grade of p27 staining between FVPC and FA was statistically significant (P < .001). This study shows a significant underexpression of p27 in FVPC compared with FA, suggesting that a decrease in p27 expression plays a more important role than overexpression of cyclins D1 and E alone in thyroid carcinogenesis and that p27 immunostaining may be helpful in the diagnosis of FVPC.     

Prognostic significance of the cell cycle inhibitor p27Kip1 in chronic B-cell lymphocytic leukemia

B-cell chronic lymphocytic leukemia (B-CLL) is characterized by the accumulation of resting lymphocytes. The identification of p27(kip1), a cyclin-dependent kinase inhibitor that contributes to cell cycle arrest and represents a link between extracellular signals and cell cycle, prompted us to study p27 protein in the lymphocytes from 88 patients with B-CLL and 32 patients with other chronic B-lymphoproliferative disorders. The expression of p27 protein was higher in B-CLL samples with variations among them. In B-CLL, p27 levels were independent of absolute number of circulating lymphocytes, but strongly correlated with both lymphocyte and total tumor mass (TTM) doubling time. High p27 expression was associated with a poorer overall prognosis. In vitro, there was an increased spontaneous survival of B-CLL cells expressing high p27 levels. Interleukin-4 (IL-4) upregulated p27 levels in B-CLL cells, while fludarabine decreased p27 levels. Thus, our results indicate that p27 may be a valuable kinetic marker in B-CLL by providing instantaneous estimation of the disease doubling time. In addition, these results suggest that there is a link between p27 expression and the ability of CLL cells to undergo apoptosis.     

Butyrate-induced differentiation of Caco-2 cells is associated with apoptosis and early induction of p21Waf1/Cip1 and p27Kip1

BACKGROUND: Intestinal mucosal turnover is a process of proliferation, differentiation, and apoptosis; the mechanisms remain largely undefined. The purpose of our study was to (1) assess the relationship between apoptosis and enterocyte differentiation and (2) determine whether the cell-cycle inhibitors, p21Waf1/Cip1 and p27Kip1, or the apoptosis inhibitors, Bcl-2 and Bcl-XL, may be involved. METHODS: Gut-derived Caco-2 cells were treated with sodium butyrate. Apoptosis was assessed by Hoechst stain, DNA laddering, and annexin V assay; differentiation was determined by alkaline phosphatase and sucrase activity. RNA and protein were analyzed for expression of p21Waf1/Cip1, p27Kip1, and members of the Bcl-2 family. RESULTS: Treatment of Caco-2 cells with sodium butyrate resulted in the concomitant induction of both differentiation (increased alkaline phosphatase and sucrase activity) and apoptosis. Increased levels of p21Waf1/Cip1 and p27Kip1 mRNA and protein were detected at 24 hours, occurring before apoptosis or differentiation; decreased mRNA levels of Bcl-2 and Bcl-XL were noted at 24 hours. CONCLUSIONS: Differentiation and apoptosis occurred simultaneously in Caco-2 cells, suggesting that apoptosis may be linked to enterocyte differentiation. The induction of p21Waf1/Cip1 and p27Kip1 and the down-regulation of Bcl-2 and Bcl-XL further suggest a link between the cell-cycle mechanisms regulating enterocyte differentiation and apoptosis.     

A recombinant adenovirus expressing p27Kip1 induces cell cycle arrest and loss of cyclin-Cdk activity in human breast cancer cells

In order to elucidate the biochemical mechanisms by which the universal cyclin kinase inhibitor p27Kip1 regulates cell cycle progression in human breast cancer cells, a recombinant adenovirus expressing human p27 was constructed (Adp27). Upon infection of human breast cancer cells MDA-MB-231 and MCF-7 with Adp27, a high level of p27 expression was observed, and this resulted in a marked decrease in the proportion of cells in S-phase. In multiple cell lines, comparison of the cytotoxicity of Adp27 with another adenovirus vector expressing the related universal cyclin kinase inhibitor WAF1/Cip1 (AdWAF1), showed Adp27 to be markedly more (up to 56-fold) toxic than AdWAF1. DNA histograms showed Adp27 to cause a G1/S arrest at lower viral doses than AdWAF1. Analysis of cyclin dependent kinase activity following Adp27 infections showed decreased Cdk2 and cyclin B1-Cdc2 activity at lower viral doses when compared with AdWAF1. Adp27 is therefore potentially useful for studies of growth regulation and for gene therapy when growth inhibition is desired.     

Modulation of p27(Kip1) levels by the cyclin encoded by Kaposi's sarcoma-associated herpesvirus

DNA tumour viruses have evolved a number of mechanisms by which they deregulate normal cellular growth control. We have recently described the properties of a cyclin encoded by human herpesvirus 8 (also known as Kaposi's sarcoma-associated herpesvirus) which is able to resist the actions of p16(Ink4a), p21(Cip1) and p27(Kip1) cdk inhibitors. Here we investigate the mechanism involved in the subversion of a G1 blockade imposed by overexpression of p27(Kip1). We demonstrate that binding of K cyclin to cdk6 expands the substrate repertoire of this cdk to include a number of substrates phosphorylated by cyclin-cdk2 complexes but not cyclin D1-cdk6. Included amongst these substrates is p27(Kip1) which is phosphorylated on Thr187. Expression of K cyclin in mammalian cells leads to p27(Kip1) downregulation, this being consistent with previous studies indicating that phosphorylation of p27(Kip1) on Thr187 triggers its downregulation. K cyclin expression is not able to prevent a G1 arrest imposed by p27(Kip1) in which Thr187 is mutated to non-phosphorylatable Ala. These results imply that K cyclin is able to bypass a p27(Kip1)-imposed G1 arrest by facilitating phosphorylation and downregulation of p27(Kip1) to enable activation of endogenous cyclin-cdk2 complexes. The extension of the substrate repertoire of cdk6 by K cyclin is likely to contribute to the deregulation of cellular growth by this herpesvirus-encoded cyclin.     

Progression of LNCaP prostate tumor cells during androgen deprivation: hormone-independent growth, repression of proliferation by androgen, and role for p27Kip1 in androgen-induced cell cycle arrest

The molecular mechanism of androgen-independent growth of prostate cancer after androgen ablation was explored in LNCaP cells. An androgen-dependent clonal subline of the LNCaP human prostate carcinoma cell line, LNCaP 104-S, progressed to a slow growing stage (104-R1) and then to a faster growing stage (104-R2) during more than 2 yr of continuous culture in the absence of androgen. Androgen-induced proliferation of 104-S cells is inhibited by the antiandrogen Casodex, while proliferation of 104-R1 and 104-R2 cells is unaffected by Casodex. This indicates that proliferation of 104-R1 and 104-R2 cells is not supported by low levels of androgen in the culture medium. Compared with LNCaP 104-S cells, both 104-R1 and 104-R2 cells express higher basal levels of androgen receptor (AR), and proliferation of these two cell lines is paradoxically repressed by androgen. After continuous passage in androgen-containing medium, 104-R1 cells reverted back to an androgen-dependent phenotype. The mechanism of androgenic repression of 104-R1 and 104-R2 sublines was further evaluated by examining the role of critical regulatory factors involved in the control of cell cycle progression. At concentrations that repressed growth, androgen transiently induced the expression of the cyclin-dependent kinase (cdk) inhibitor p21waf1/cip1 in 104-R1 cells, while expression of the cdk inhibitor p27Kip1 was persistently induced by androgen in both 104-R1 and 104-R2 cells. Induced expression of murine p27Kip1 in 104-R2 cells resulted in G1 arrest. Specific immunoprecipitates of Cdk2 but not Cdk4 from androgen-treated 104-R1 cells contained both p21waf1/cip1 and p27Kip1. This observation was confirmed by in vitro assay of histone H1 and Rb (retinoblastoma protein) phosphorylation by the proteins associated with the immune complex. Furthermore, inhibition of Cdk2 activity correlated with the accumulation of p27Kip1 and not p21waf1/cip1. From these results we conclude that androgenic repression of LNCaP 104-R1 and 104-R2 cell proliferation is due to the induction of p27Kip1, which in turn inhibits Cdk2, a factor critical for cell cycle progression and proliferation.     

Accumulation of the cyclin-dependent kinase inhibitor p27/Kip1 and the timing of oligodendrocyte differentiation

Many types of vertebrate precursor cells divide a limited number of times before they stop and terminally differentiate. In no case is it known what causes them to stop dividing. We have been studying this problem in the proliferating precursor cells that give rise to postmitotic oligodendrocytes, the cells that make myelin in the central nervous system. We show here that two components of the cell cycle control system, cyclin D1 and the Cdc2 kinase, are present in the proliferating precursor cells but not in differentiated oligodendrocytes, suggesting that the control system is dismantled in the oligodendrocytes. More importantly, we show that the cyclin-dependent kinase (Cdk) inhibitor p27 progressively accumulates in the precursor cells as they proliferate and is present at high levels in oligodendrocytes. Our findings are consistent with the possibility that the accumulation of p27 is part of both the intrinsic counting mechanism that determines when precursor cell proliferation stops and differentiation begins and the effector mechanism that arrests the cell cycle when the counting mechanism indicates it is time. The recent findings of others that p27-deficient mice have an increased number of cells in all of the organs examined suggest that this function of p27 is not restricted to the oligodendrocyte cell lineage.     

c-Myc or cyclin D1 mimics estrogen effects on cyclin E-Cdk2 activation and cell cycle reentry

Estrogen-induced progression through G1 phase of the cell cycle is preceded by increased expression of the G1-phase regulatory proteins c-Myc and cyclin D1. To investigate the potential contribution of these proteins to estrogen action, we derived clonal MCF-7 breast cancer cell lines in which c-Myc or cyclin D1 was expressed under the control of the metal-inducible metallothionein promoter. Inducible expression of either c-Myc or cyclin D1 was sufficient for S-phase entry in cells previously arrested in G1 phase by pretreatment with ICI 182780, a potent estrogen antagonist. c-Myc expression was not accompanied by increased cyclin D1 expression or Cdk4 activation, nor was cyclin D1 induction accompanied by increases in c-Myc. Expression of c-Myc or cyclin D1 was sufficient to activate cyclin E-Cdk2 by promoting the formation of high-molecular-weight complexes lacking the cyclin-dependent kinase inhibitor p21, as has been described, following estrogen treatment. Interestingly, this was accompanied by an association between active cyclin E-Cdk2 complexes and hyperphosphorylated p130, identifying a previously undefined role for p130 in estrogen action. These data provide evidence for distinct c-Myc and cyclin D1 pathways in estrogen-induced mitogenesis which converge on or prior to the formation of active cyclin E-Cdk2-p130 complexes and loss of inactive cyclin E-Cdk2-p21 complexes, indicating a physiologically relevant role for the cyclin E binding motifs shared by p130 and p21.     

The cyclin-dependent kinase inhibitor p27(Kip1) induces N-terminal proteolytic cleavage of cyclin A

Progression through the cell cycle is regulated in part by the sequential activation and inactivation of cyclin-dependent kinases (CDKs). Many signals arrest the cell cycle through inhibition of CDKs by CDK inhibitors (CKIs). p27(Kip1) (p27) was first identified as a CKI that binds and inhibits cyclin A/CDK2 and cyclin E/CDK2 complexes in G1. Here we report that p27 has an additional property, the ability to induce a proteolytic activity that cleaves cyclin A, yielding a truncated cyclin A lacking the mitotic destruction box. Other CKIs (p15(Ink4b), p16(Ink4a), p21(Cip1), and p57(Kip2)) do not induce cleavage of cyclin A; other cyclins (cyclin B, D1, and E) are not cleaved by the p27-induced protease activity. The C-terminal half of p27, which is dispensable for its kinase inhibitory activity, is required to induce cleavage. Mechanistically, p27 does not appear to cause cleavage through direct interaction with cyclin/CDK complexes. Instead, it activates a latent protease that, once activated, does not require the continuing presence of p27. Mutation of cyclin A at R70 or R71, residues at or very close to the cleavage site, blocks cleavage. Noncleavable mutants are still recognized by the anaphase-promoting complex/cyclosome pathway responsible for ubiquitin-dependent proteolysis of mitotic cyclins, indicating that the p27-induced cleavage of cyclin A is part of a separate pathway. We refer to this protease as Tsap (pTwenty-seven- activated protease).     

Comparative effects of overexpression of p27Kip1 and p21Cip1/Waf1 on growth and differentiation in human colon carcinoma cells

Recent studies have shown that decreased expression of p27Kip1 is associated with high grade tumors and an unfavorable prognosis in several types of human cancer. To clarify the role of p27Kip1 in colon cancer, we have overexpressed this protein in the HT29 colon cancer cell line. The derivatives displayed an increase in the p27Kip1 protein in cyclin E/CDK2 immunoprecipitates and a decrease in cyclin E-associated kinase activity when compared to vector control clones, providing evidence that the overexpressed protein was functional. Clones with a high level of p27Kip1 displayed partial growth inhibition in monolayer culture and a decrease in plating efficiency, even though they expressed increased levels of the cyclin D1 protein. Using alkaline phosphatase expression as a marker, we found that the p27Kip1 overexpressor clones displayed a 2-3-fold increase in sensitivity to induction of differentiation by 2 mM sodium butyrate. In contrast to these results, derivatives of HT29 cells that stably overexpressed p21Cip1/Waf1 displayed decreased sensitivity to the induction of differentiation. These findings may explain why decreased levels of p27Kip1 in certain human cancers is associated with high grade (poorly differentiated) tumors, and suggest that strategies that increase the level of p27Kip1 may be useful in cancer therapy.     

Regulation of cyclin-dependent kinase 4 during adipogenesis involves switching of cyclin D subunits and concurrent binding of p18INK4c and p27Kip1

Terminal differentiation of many cell lineages involves an exit from the mitotic cycle and entry into, and maintenance of, a permanent state of G1 arrest. We found that during terminal differentiation of mouse 3T3-L1 preadipocytes, the level of cyclin-dependent kinase 4 (CDK4) remained constant, but the subunit composition of the CDK4 complex underwent a dynamic rearrangement. As 3T3-L1 cells differentiated, the levels of cyclin D1 and cyclin D1-CDK4 complexes declined to negligible levels. Meanwhile, cyclins D2 and D3 levels and their associations with CDK4 increased transiently and persistently, respectively, with cyclin D3 becoming the predominant cyclin partner of CDK4 in mature adipocytes. At least five CDK inhibitors are expressed during the differentiation program of 3T3-L1 cells. Both p15INK4b and p16INK4a continuously declined to undetectable levels immediately after differentiation induction. p21 was transiently expressed during the exit of 3T3-L1 cells from mitotic clonal expansion and then decreased to undetectable levels in mature adipocytes. The level of p27KiP1 and p27-CDK4 complexes remain high during differentiation and in mature adipocytes. Distinctly, there is a remarkable induction of p18INK4c mRNA and protein that was not seen in the closely related nondifferentiating 3T3-C2 cell line, suggesting that p18 induction in 3T3-L1 cells is related to cell differentiation, not cell cycle arrest. The pRb kinase activity of cyclin D3 and CDK4 was not detected in quiescent 3T3-L1 cells and was then induced as the cells entered the mitotic clonal expansion phase. Unexpectedly, cyclin D3 and CDK4 pRb kinase activity remained high after 3T3-L1 cells completed their mitotic division and was still readily detectable in mature adipocytes. Our study reveals an active regulation, rather than passive inhibition, of CDK4 activity during adipocyte differentiation. Two central features of this complex regulation are switching of activating cyclin D subunits and concurrent binding by the p18 and p27 CDK inhibitors.     

Differential expression of the p27Kip1 mRNA in IFN-sensitive and resistant cell lines

STUDY OBJECTIVES: In a previous study published by our group, six out of nine subjects with mild allergic asthma were shown to have an enhanced response to allergen challenge following a 1-h exposure in an 0.8-m3 exposure chamber (modified from a body plethysmograph) to an average of 120 parts per billion (ppb) ozone at rest. Other studies failed to confirm this effect. In the present study, using a similar design, we reexamined this effect using a larger group of asthmatics and a larger chamber allowing minimal fluctuations in ozone levels during exposures. DESIGN: Prospective, randomized single-blinded crossover study. SETTING: Pulmonary function laboratory equipped with an exposure chamber. SUBJECTS: Fifteen subjects had mild allergic asthma; 9 men and 6 women; the mean (SD) age was 32.5 (10) years; FEV1 was 3.4 (0.8) L; baseline methacholine provocation concentration causing a 20% fall in FEV1 was (PC20) 3.28 (4.1) mg/mL. INTERVENTIONS: Each participant was exposed, at rest, on 1 day to filtered air and on another day to ozone (mean level=120 ppb) in a larger exposure chamber than the one used in our first study with less variability in ozone level (110 to 130 vs 85 to 175 ppb) using a random, single-blinded design. After each exposure, the subject was challenged with allergen (nine with grass pollen extract and six with ragweed extract) and allergen PC15 was measured. RESULTS: Ozone preexposure did not affect allergen PC15 when compared with clean air preexposure (allergen PC15 dilution 1/114 vs 1/119, respectively). Ozone vs air preexposure resulted in an allergen PC15 that was lower in five subjects, higher in six, and unchanged (within one doubling dose) in four. CONCLUSIONS: At this low level with less variability and lower peaks than our previous study, ozone had no significant effect on airway allergen responsiveness.     

Mantle cell lymphomas lack expression of p27Kip1, a cyclin-dependent kinase inhibitor

The TCRs expressed on T lymphocytes recognize foreign peptides bound to MHC molecules. This reactivity is the basis of specific immune response to the foreign Ag. How such specificities are generated in the thymus is still being debated. Signals generated through TCR upon interaction with self MHC-peptide complexes are critical for maturation of the CD4+ helper and CD8+ cytotoxic subsets. We have observed maturation of CD4+ but not CD8+ T cells in Ly-6A.2 transgenic MHC null mice. Since there can be no interactions with MHC molecules in these mice, these CD4+ cells must express the T cell repertoire that exists before positive and negative selection. Interestingly, despite an absence of selection by MHC molecules, the CD4+ cells that mature recognize MHC molecules at a frequency as high as in CD4+ cells in normal mice. These results demonstrate that: 1) the germline sequences encoding TCRs are biased toward reactivity to MHC molecules; and 2) CD4+ cells as opposed to CD8+ cells have distinct lineage commitment signals. These results also suggest that signals originating from Ly-6 can promote or substitute for signals generated from TCR that are required for positive selection. Moreover, this animal model offers a system to study T cell development in the thymus that can provide insights into mechanisms of lineage commitment in developing T cells.     

Critical role for p27Kip1 in cell cycle arrest after androgen depletion in mouse mammary carcinoma cells (SC-3)

The molecular mechanisms underlying androgen-regulated cancer growth and the frequent development of refractoriness to endocrine therapy remain unknown. In this study functional and quantitative alterations in cell cycle regulators after androgen depletion were examined in androgen-dependent mouse mammary carcinoma cells (SC-3) as a model system to clarify the initial response of cancer cells to anti-androgen therapy. FACS analysis of SC-3 cells cultured with or without 10(-7) M testosterone revealed that suppression of cell growth after hormone withdrawal was due to GI arrest. Although cyclin D1/Cdk4 activity decreased along with a reduced level of cyclin D1 protein, this was a later event (48-72 h) than the G1 arrest (24 h). Taken together with the results that constitutive expression of cyclin D1 in SC-3 cells did not overcome the growth suppression following androgen depletion, the existence of an alternative pathway(s) causing G1 arrest was suggested. Cyclin E/Cdk2 and cyclin A/Cdk2 activities decreased significantly at 24 h without apparent changes in the amounts of Cdk2, cyclin E or cyclin A. Among various Cdk inhibitors (CKIs) examined, p27Kip1 was upregulated at both mRNA and protein levels at 24 h after androgen depletion. In addition, immunoprecipitation-Western analysis showed that the amount of p27Kip1 associated with Cdk2 complexes increased as early as 24 h. These results suggest that p27Kip1 CKI is a critical target in the initial response of cancer cells to androgen depletion and plays a key role in Cdk2 inactivation through association with the kinase complex, leading to cell cycle arrest.     

Inactivation of p27Kip1 by the viral E1A oncoprotein in TGFbeta-treated cells

The adenovirus oncoprotein E1A and the simian virus SV40 large T antigen can both reverse the strong growth-inhibitory effect of transforming growth factor(TGF)-beta on mink lung epithelial cells: exposure of TGF-beta causes these cells to arrest late in the G1 phase of the cell cycle (ref. 3). This arrest correlates with an increase in expression of the protein p15Ink4B (ref. 4), inactivation of the cyclin E/A-cdk2 complex by the inhibitory protein p27Kip1 (refs 5-7), and with the accumulation of unphosphorylated retinoblastoma protein. The rescue by E1A of cells from TGF-beta arrest is partly independent of its binding to retinoblastoma protein. Here we show that E1A directly affects the cyclin-dependent kinase inhibitor p27Kip1 in TGF-beta-treated cells by binding to it and blocking its inhibitory effect, thereby restoring the activity of the cyclin-cdk2 kinase complex. In this way, E1A can overcome the effect of TGF-beta and modulate the cell cycle. To our knowledge, E1A provides the first example of a viral oncoprotein that can disable a cellular protein whose function is to inhibit the activity of cyclin-dependent kinases.     

Neurotransmitter receptor activation triggers p27(Kip1 )and p21(CIP1) accumulation and G1 cell cycle arrest in oligodendrocyte progenitors

We examined the pathways that link neurotransmitter receptor activation and cell cycle arrest in oligodendrocyte progenitors. We had previously demonstrated that glutamate receptor activation inhibits oligodendrocyte progenitor proliferation and lineage progression. Here, using purified oligodendrocyte progenitors and cerebellar slice cultures, we show that norepinephrine and the beta-adrenergic receptor agonist isoproterenol also inhibited the proliferation, but in contrast to glutamate, isoproterenol stimulated progenitor lineage progression, as determined by O4 and O1 antibody staining. This antiproliferative effect was specifically attributable to a beta-adrenoceptor-mediated increase in cyclic adenosine monophosphate, since analogs of this cyclic nucleotide mimicked the effects of isoproterenol on oligodendrocyte progenitor proliferation, while alpha-adrenoceptor agonists were ineffective. Despite the opposite effects on lineage progression, both isoproterenol and the glutamate receptor agonist kainate caused accumulation of the cyclin-dependent kinase inhibitors p27(Kip1)and p21(CIP1), and G1 arrest. Studies with oligodendrocyte progenitor cells from INK4a-/- mice indicated that the G1 cyclin kinase inhibitor p16(INK4a) as well as p19(ARF)were not required for agonist-stimulated proliferation arrest. Our results demonstrate that beta-adrenergic and glutamatergic receptor activation inhibit oligodendrocyte progenitor proliferation through a mechanism that may involve p27(Kip1) and p21(CIP1); but while neurotransmitter-induced accumulation of p27(Kip1) is associated with cell cycle arrest, it does not by itself promote oligodendrocyte progenitor differentiation.     

Intestinal cell cycle regulations. Interactions of cyclin D1, Cdk4, and p21Cip1

OBJECTIVE: The p21Cip1 protein is a potent stoichiometric inhibitor of cyclin-dependent kinase activity, and p21Cip1 mRNA expression is localized to the nonproliferative compartment of the intestinal villus, suggesting an in vivo growth-inhibitory role in the gut. The authors determined whether nontransformed rat intestinal epithelial cells (IECs) underwent reversible cell cycle arrest by contact inhibition, and determined whether increases in the relative amount of p21 associated with cyclin D/Cdk4 protein complexes were associated with cell growth arrest. METHODS: Density arrest was achieved by prolonged culture IEC-6 in confluent conditions (5 or more days). Release from density arrest was achieved by detaching the cells from the culture plate and reseeding them at a 1:4 ratio. The DNA synthesis was estimated by [3H]-thymidine incorporation and expressed as mean plus or minus standard error of the mean (n = 4). Cyclin D1, Cdk4, and p21 mRNA and protein levels were determined by standard Northern and Western blot analyses, respectively. Cyclin D1, Cdk4, and p21 protein complex formation was analyzed by immunoprecipitating the complexes from cell lysates with an antibody to one of the constituents, followed by SDS polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis of the precipitated complexes using antibodies to the other proteins. The kinase activity of the immunoprecipitated Cdk4 was determined using recombinant Rb as substrate. RESULTS: The IEC-6[3H]-thymidine incorporation was decreased 7.5-fold from day 1 confluence to day 7 of confluence. Twenty-four hours after release from density arrest, there was a 43-fold increase in [3H]-thymidine incorporation. Cyclin D1 and Cdk4 mRNA levels remained relatively constant during contact inhibition, whereas immunoblotting showed that the levels of cyclin D1 and Cdk4 proteins decreased by 70.9% and 68.7%, respectively, comparing day 3 with day 9 during density arrest. The levels of cyclin D1 increased 5.8-fold and Cdk4 increased by 4.4-fold by 24 hours after reseeding the day 9 density-arrested cultures, coincident with the increase in DNA synthesis. The amount of p21 associated with the cyclin D1 and Cdk4 complex in the density-arrested cells was 170% of that observed in the reseeded, proliferating cells. More important, the p21::Cdk4 ratio was 6.4-fold higher in the density-arrested (quiescent) cells as compared with rapidly proliferating cells by 24 hours after release from growth arrest. Recovery of Cdk4-dependent kinase activity occurred by 4 hours after release from growth arrest, coincident with decreased binding of p21 to the complex. CONCLUSIONS: Intestinal epithelial cells in culture can undergo density-dependent growth arrest. This process involves downregulation of cyclin D1 and Cdk4 at the level of protein expression, whereas the mRNA levels remain relatively unchanged. Further, during contact inhibition, there is more p21 associated with cyclin D1/Cdk4, which further contributes to the inhibition of the kinase complex. The authors also have shown that the process of contact inhibition is reversible, which may explain partly the ability of the intestinal epithelium to increase proliferative activity in response to injury.