Flavokawain A is the predominant chalcone from kava extract. We have assessed the mechanisms of flavokawain A's action on cell cycle regulation. In a p53 wild-type, low-grade, and papillary bladder cancer cell line (RT4), flavokawain A increased p21/WAF1 and p27/KIP1, which resulted in a decrease in cyclin-dependent kinase-2 (CDK2) kinase activity and subsequent G1 arrest. The increase of p21/WAF1 protein corresponded to an increased mRNA level, whereas p27/KIP1 accumulation was associated with the down-regulation of SKP2 and then increased the stability of the p27/KIP1 protein. The accumulation of p21/WAF1 and p27/KIP1 was independent of cell cycle position and thus not a result of the cell cycle arrest. In contrast, flavokawain A induced a G2-M arrest in six p53 mutant-type, high-grade bladder cancer cell lines (T24, UMUC3, TCCSUP, 5637, HT1376, and HT1197). Flavokawain A significantly reduced the expression of CDK1-inhibitory kinases, Myt1 and Wee1, and caused cyclin B1 protein accumulation leading to CDK1 activation in T24 cells. Suppression of p53 expression by small interfering RNA in RT4 cells restored Cdc25C expression and down-regulated p21/WAF1 expression, which allowed Cdc25C and CDK1 activation and then led to a G2-M arrest and an enhanced growth-inhibitory effect by flavokawain A. Consistently, flavokawain A also caused a pronounced CDK1 activation and G2-M arrest in p53 knockout but not in p53 wild-type HCT116 cells. This selectivity of flavokawain A for inducing a G2-M arrest in p53-defective cells deserves further investigation as a new mechanism for the prevention and treatment of bladder cancer.
Adeno-associated virus type 2 (AAV2) seropositivity is negatively correlated with the development of human papillomavirus (HPV)-associated cervical cancer. We have begun analysis of the molecular mechanisms underlying AAV2-mediated oncosuppression through cell cycle regulation in HPV-infected keratinocytes isolated from a low-grade cervical lesion. AAV2 superinfection of HPV type 31b (HPV31b)-positive cells at early times postinfection resulted in degradation of the cyclin-dependent kinase (CDK) inhibitor p21WAF1 protein in a proteosome-dependent manner. Downstream consequences of lowering p21WAF1 levels included a proportional loss of cyclin E/CDK2 complexes bound to p21WAF1. The loss of stable p21WAF1/cyclin E/CDK2 complexes coincided with an increase in CDK2-associated kinase activity and cyclin E levels. Both events have the potential to enhance the G1/S transition point mediated by active cyclin E/CDK2 complexes. Concurrently, cyclin A and E2F levels were decreased, conditions reminiscent of delayed entrance into the S phase of the cell cycle. On the other hand, infection of primary human foreskin keratinocytes with AAV2 resulted in upregulation of p21WAF1 protein levels, reminiscent of a block in G1 phase progression. We propose that by downregulating p21WAF1, AAV2 initiates cell cycle activities leading to enhanced G1/S phase-like conditions which may be favorable for AAV2-specific functions and may lead to downstream interference with HPV-associated cervical cancer progression.
The tumor suppressor p16INK4a inhibits cyclin-dependent kinases 4 and 6. This activates the retinoblastoma protein (pRB) and, through incompletely understood events, arrests the cell division cycle. To permit biochemical analysis of the arrest, we generated U2-OS osteogenic sarcoma cell clones in which p16 transcription could be induced. In these clones, binding of p16 to cdk4 and cdk6 abrogated binding of cyclin D1, p27KIP1, and p21WAF1/CIP1. Concomitantly, the total cellular level of p21 increased severalfold via a posttranscriptional mechanism. Most cyclin E-cdk2 complexes associated with p21 and became inactive, expression of cyclin A was curtailed, and DNA synthesis was strongly inhibited. Induction of p21 alone, in a sibling clone, to the level observed during p16 induction substantially reproduced these effects. Overexpression of either cyclin E or A prevented p16 from mediating arrest. We then extended these studies to HCT 116 colorectal carcinoma cells and a p21-null clone derived by homologous recombination. In the parental cells, p16 expression also augmented total cellular and cdk2-bound p21. Moreover, p16 strongly inhibited DNA synthesis in the parental cells but not in the p21-null derivative. These findings indicate that p21-mediated inhibition of cdk2 contributes to the cell cycle arrest imposed by p16 and is a potential point of cooperation between the p16/pRB and p14ARF/p53 tumor suppressor pathways.
p21WAF/CIP1/SDI1 is a recently identified gene expressed in cells harboring wild-type but not mutant p53 gene. It encodes a nuclear protein of 21 kD which inhibits cyclin-dependent kinase activity. Constitutive p21WAF1/CIP1/SDI1 mRNA expression was detected in neoplastic cells from patients with various hematological malignancies as well as in normal bone marrow mononuclear cells and in myeloid and lymphoid cell lines independent of their p53 status. Induced differentiation of the p53-deficient promyelocytic HL-60 cells along the monocytic lineage by phorbol ester or 1a,25 dihydroxyvitamin D3 resulted in a marked increase of both p21WAF1/CIP1/SDI1 mRNA and protein expression due to enhanced mRNA stability. Differentiation towards the granulocytic lineage by all-trans retinoic acid or dimethylsulfoxide failed to produce this effect. p21WAF1/CIP1/SDI1 is an immediate early gene since its upregulation occurred independently of de novo protein synthesis. The induction of p21WAF1/CIP1/SDI1 expression and its regulation in p53-deficient differentiating leukemic cells support the idea of an additional, p53-independent role of p21WAF1/CIP1/SDI1 in human hematopoiesis.
The mammalian CIP/KIP family of cyclin-dependent kinase (CDK) inhibitors (CKIs) comprises three proteins – p21Cip1/WAF1, p27Kip1, and p57Kip2 – that bind and inhibit cyclin–CDK complexes, which are key regulators of the cell cycle. CIP/KIP CKIs have additional independent functions in regulating transcription, apoptosis and actin cytoskeletal dynamics. These divergent functions are performed in distinct cellular compartments and contribute to the seemingly contradictory observation that the CKIs can both suppress and promote cancer. Multiple ubiquitin ligases (E3s) direct the proteasome-mediated degradation of p21, p27 and p57. This review analyzes recent data highlighting our current understanding of how distinct E3 pathways regulate subpopulations of the CKIs to control their diverse functions.
We are employing recent advances in the understanding of the cell cycle to study the inverse relationship between proliferation and neuronal differentiation. Nerve growth factor and aphidicolin, an inhibitor of DNA polymerases, synergistically induce neuronal differentiation of SH-SY5Y neuroblastoma cells and the expression of p21WAF1, an inhibitor of cyclin-dependent kinases. The differentiated cells continue to express p21WAF1, even after removal of aphidicolin from the culture medium. The p21WAF1 protein coimmunoprecipitates with cyclin E and inhibits cyclin E-associated protein kinase activity. Each of three antisense oligonucleotides complementary to p21WAF1 mRNA partially blocks expression of p21WAF1 and promotes programmed cell death. These data indicate that p21WAF1 expression is required for survival of these differentiating neuroblastoma cells. Thus, the problem of neuronal differentiation can now be understood in the context of negative regulators of the cell cycle.
Normal cell proliferation is closely regulated by proteins called cyclins. One of these, cyclin D1, in combination with its corresponding cyclin-dependent kinase (cdk), is essential for G1/S phase transition. Cyclin/cdk complexes are generally inhibited by cyclin-dependent kinase inhibitors(ckis), some of which are induced by wild-type p53. The aims of this study were: to investigate levels of cyclin D1 expression in transitional cell carcinoma (TCC) of the bladder; to correlate these results with data concerning the expression of p53, waf1, pRb and Ki67; and to determine whether cyclin D1 expression could predict clinical outcome. Paraffin-sections from 150 newly diagnosed bladder tumours (Ta/T1 = 97; T2–T4 = 53) were stained for cyclin D1 using immunohistochemistry and a cyclin D1 index assigned. These results were correlated with data relating to the expression of p53 and waf1 by the same tumours. A representative subset of 54 tumours (Ta/T1 = 28; T2–T4 = 26) was also stained for Ki67 and 55 were stained for pRb. The clinical course of each patient was recorded and multivariate analyses of risk factors for tumour recurrence, stage progression and overall survival were performed. Positive staining for cyclin D1 was found in 83% of tumours. The staining pattern varied between tumours with nuclear, cytoplasmic or a combination of the two evident in different tumours. 89% of Ta/T1 and 74% of T2–T4 tumours showed nuclear staining with or without cytoplasmic staining. The median value for cyclin D1 staining was significantly higher in Ta/T1 tumours (41%) compared with T2–T4 tumours (8%, P< 0.005) with 26% of muscle-invasive tumours demonstrating absent staining. In addition, the median value for cyclin D1 staining was significantly higher in G1/G2 tumours (43%) compared with G3 tumours (14%, P< 0.005). There was a significant positive correlation between expression of cyclin D1 and waf1 expression (P< 0.0001) as well as pRb expression but not between cyclin D1 expression and expression of p53. Ki67 expression was significantly associated with increasing tumour stage (P< 0.005) and histological grade (P< 0.05) but did not correlate with cyclin D1 expression. A cyclin D1 index ≥ 8% was associated with significantly better survival in those patients with muscle-invasive disease (T2–T4). In addition, there was a significantly higher progression rate for those patients with Ta/T1 disease whose tumours demonstrated cytoplasmic cyclin D1 staining. These results indicate that cyclin D1 expression is significantly higher in low-stage, well differentiated bladder tumours and strongly correlates with waf1 expression. In a multivariate analysis, cyclin D1 expression is an independent prognostic indicator of survival in those patients with muscle-invasive disease. © 2001 Cancer Research Campaign http://www.bjcancer.com
cyclin D1; bladder; carcinoma; immunohistochemistry; survival
The irreversible G1 arrest in senescent human diploid fibroblasts is probably caused by inactivation of the G1 cyclin–cyclin-dependent kinase (Cdk) complexes responsible for phosphorylation of the retinoblastoma protein (pRb). We show that the Cdk inhibitor p21Sdi1,Cip1,Waf1, which accumulates progressively in aging cells, binds to and inactivates all cyclin E-Cdk2 complexes in senescent cells, whereas in young cells only p21-free Cdk2 complexes are active. Furthermore, the senescent-cell-cycle arrest occurs prior to the accumulation of the Cdk4-Cdk6 inhibitor p16Ink4a, suggesting that p21 may be sufficient for this event. Accordingly, cyclin D1-associated phosphorylation of pRb at Ser-780 is lacking even in newly senescent fibroblasts that have a low amount of p16. Instead, the cyclin D1-Cdk4 and cyclin D1-Cdk6 complexes in these cells are associated with an increased amount of p21, suggesting that p21 may be responsible for inactivation of both cyclin E- and cyclin D1-associated kinase activity at the early stage of senescence. Moreover, even in the late stage of senescence when p16 is high, cyclin D1-Cdk4 complexes are persistent, albeit reduced by ≤50% compared to young cells. We also provide new evidence that p21 may play a role in inactivation of the DNA replication factor proliferating cell nuclear antigen during early senescence. Finally, because p16 accumulates in parallel with the increases in senescence-associated β-Gal activity and cell volume that characterize the senescent phenotype, we suggest that p16 upregulation may be part of a differentiation program that is turned on in senescent cells. Since p21 decreases after senescence is achieved, this upregulation of p16 may be essential for maintenance of the senescent-cell-cycle arrest.
HTLV-1 Tax can induce senescence by up-regulating the levels of cyclin-dependent kinase inhibitors p21CIP1/WAF1 and p27KIP1. Tax increases p27KIP1 protein stability by activating the anaphase promoting complex/cyclosome (APC/C) precociously, causing degradation of Skp2 and inactivation of SCFSkp2, the E3 ligase that targets p27KIP1. The rate of p21CIP1/WAF1 protein turnover, however, is unaffected by Tax. Rather, the mRNA of p21CIP1/WAF1 is greatly up-regulated. Here we show that Tax increases p21 mRNA expression by transcriptional activation and mRNA stabilization. Transcriptional activation of p21CIP1/WAF1 by Tax occurs in a p53-independent manner and requires two tumor growth factor-β-inducible Sp1 binding sites in the -84 to -60 region of the p21CIP1/WAF1 promoter. Tax binds Sp1 directly, and the CBP/p300-binding activity of Tax is required for p21CIP1/WAF1 trans-activation. Tax also increases the stability of p21CIP1/WAF1 transcript. Several Tax mutants trans-activated the p21 promoter, but were attenuated in stabilizing p21CIP1/WAF1 mRNA, and were less proficient in increasing p21CIP1/WAF1 expression. The possible involvement of Tax-mediated APC/C activation in p21CIP1/WAF1 mRNA stabilization is discussed.
The presence of two families of seven distinct mammalian cyclin-dependent kinase (CDK) inhibitor genes is thought to mediate the complexity of connecting a variety of cellular processes to the cell cycle control pathway. The distinct pattern of tissue expression of CDK inhibitor genes suggests that they may function as tumor suppressors with different tissue specificities. To test this hypothesis, we have characterized two strains of double mutant mice lacking either p18INK4c and p27KIP1 or p18INK4c and p21CIP1/WAF1. Loss of both p18 and p27 function resulted in the spontaneous development by 3 months of age of at least eight different types of hyperplastic tissues and/or tumors in the pituitary, adrenals, thyroid, parathyroid, testes, pancreas, duodenum, and stomach. Six of these hyperplastic tissues and tumors were in endocrine organs, and several types of tumors routinely developed within the same animal, a phenotype reminiscent of that seen in combined human multiple endocrine neoplasia syndromes. The p18-p21 double null mice, on the other hand, developed pituitary adenomas, multifocal gastric neuroendocrine hyperplasia, and lung bronchioalveolar tumors later in life. G1 CDK2 and CDK4 kinase activities were increased in both normal and neoplastic tissues derived from mice lacking individual CDK inhibitors and were synergistically stimulated by the simultaneous loss of two CDK inhibitors. This indicates that an increase in G1 CDK kinase activity is a critical step during but is not sufficient for tumor growth. Our results suggest that functional collaborations between distinct CDK inhibitor genes are tissue specific and confer yet another level of regulation in cell growth control and tumor suppression.
p21Cip1/WAF1 has cell cycle inhibitory activity by binding to and inhibiting both cyclin/Cdk kinases and proliferating cell nuclear antigen. Here we show that p21Cip1/WAF1 is induced in the cytoplasm during the course of differentiation of chick retinal precursor cells and N1E-115 cells. Ectopic expression of p21Cip1/WAF1 lacking the nuclear localization signal in N1E-115 cells and NIH3T3 cells affects the formation of actin structures, characteristic of inactivation of Rho. p21Cip1/WAF1 forms a complex with Rho-kinase and inhibits its activity in vitro and in vivo. Neurite outgrowth and branching from the hippocampal neurons are promoted if p21Cip1/WAF1 is expressed abundantly in the cytoplasm. These results suggest that cytoplasmic p21Cip1/WAF1 may contribute to the developmental process of the newborn neurons that extend axons and dendrites into target regions.
p21Cip1/WAF1; Rho-kinase; neurite outgrowth; differentiation; Rho
p16INK4a and p21WAF1, two major cyclin-dependent kinase inhibitors, are the products of two tumor suppressor genes that play important roles in various cellular metabolic pathways. p21WAF1 is up-regulated in response to different DNA damaging agents. While the activation of p21WAF1 is p53-dependent following γ-rays, the effect of ultraviolet (UV) light on p21WAF1 protein level is still unclear. In the present report, we show that the level of the p21WAF1 protein augments in response to low UVC fluences in different mammalian cells. This up-regulation is mediated through the stabilization of p21WAF1 mRNA in a p16INK4a-dependent manner in both human and mouse cells. Furthermore, using p16-siRNA treated human skin fibroblast; we have shown that p16 controls the UV-dependent cytoplasmic accumulation of the mRNA binding HuR protein. In addition, HuR immunoprecipitations showed that UV-dependent binding of HuR to p21 mRNA is p16-related. This suggests that p16 induces p21 by enabling the relocalization of HuR from the nucleus to the cytoplasm. Accordingly, we have also shown that p16 is necessary for efficient UV-dependent p53 up-regulation, which also requires HuR. These results indicate that, in addition to its role in cell proliferation, p16INK4a is also an important regulator of the cellular response to UV damage.
p21WAF1/CIP1 is a universal cyclin-dependent kinase inhibitor. To investigate the role of p21WAF1/CIP1 in proliferation of human liver cancer cells, we examined the expression of p53, p21WAF1/CIP1, cdk2 and cdk4 expression in two human liver cancer cell lines (HepG2 and PLC/PRF/5 cells). The effects of p21WAF1/CIP1 on [3H]thymidine incorporation and cdks were also examined in these cells. HepG2 cells expressed all these proteins with lower level of cdk4. PLC/PRF/5 cells expressed the other proteins except p21WAF1/CIP1. Transfection of p21WAF1/CIP1 gene inhibited [3H]thymidine incorporation of both cells with different extent. Although the transfection of p21WAF1/CIP1 did not affect cdk2 and cdk4 expression, it did reduce cdk2 kinase activity by 20%. These results suggest that: (a) p21WAF1/CIP1 involved in DNA synthesis of human liver cancer cells; (b) p21WAF1/CIP1 could be a target gene for the treatment of human hepatocellular carcinoma.
British Journal of Cancer (2002) 86, 625–629. DOI: 10.1038/sj/bjc/6600099 www.bjcancer.com
© 2002 Cancer Research UK
p21WAF1/CIP1; [3H]thymidine incorporation; liver cancer
Gastric carcinoma is characterised by numerous genetic and epigenetic alterations that influence cell cycle progression, apoptosis and DNA repair. These alterations include down‐regulation of the cyclin‐dependent kinase (CDK) inhibitors p21WAF1/CIP1 and p27Kip1, and mutations of the tumour suppressor protein p53 and the cell adhesion molecule E‐cadherin. Combined evaluation of the prognostic significance of these alterations has not been reported in Mexican Mestizo patients.
To evaluate p21WAF1/CIP1, p27Kip1, p53 and E‐cadherin protein expression, including mutant E‐cadherin variants with deletion of exon 8 (del 8) or 9 (del 9), in gastric cancer from Mexican patients.
Immunohistochemistry for the above‐mentioned markers, including mutation‐specific E‐cadherin antibodies, was carried out in 69 gastric carcinomas; expression levels were correlated with histotype, tumour stage and prognosis.
Expression of p21WAF1/CIP1 alone or in combination with p27Kip1 or in the absence of p53 was associated with favourable prognosis. Staining of del 8 and del 9 E‐cadherin was found exclusively in patients negative for p53 and positive for p21WAF1/CIP1, suggesting that the p21WAF1/CIP1 regulatory function of p53 was intact.
Combined evaluation of the prognostic significance of cell cycle regulators and E‐cadherin should be performed. Even though patients negative for p53 and positive for p21WAF1/CIP1 have a favourable prognosis, it may have a negative influence on prognosis if they acquire in addition E‐cadherin mutations which have been shown previously to be associated with poor survival.
; p53; E‐cadherin; gastric cancer
The transcriptional coactivators p300 and CREB binding protein (CBP) are important regulators of the cell cycle, differentiation, and tumorigenesis. Both p300 and CBP are targeted by viral oncoproteins, are mutated in certain forms of cancer, are phosphorylated in a cell cycle-dependent manner, interact with transcription factors such as p53 and E2F, and can be found complexed with cyclinE-Cdk2 in vivo. Moreover, p300-deficient cells show defects in proliferation. Here we demonstrate that transcriptional activation by both p300 and CBP is stimulated by coexpression of the cyclin-dependent kinase inhibitor p21WAF/CIP1. Significantly this stimulation is independent of both the inherent histone acetyltransferase (HAT) activity of p300 and CBP and of the previously reported carboxyl-terminal binding site for cyclinE-Cdk2. Rather, we describe a previously uncharacterized transcriptional repression domain (CRD1) within p300. p300 transactivation is stimulated through derepression of CRD1 by p21. Significantly p21 regulation of CRD1 is dependent on the nature of the core promoter. We suggest that CRD1 provides a novel mechanism through which p300 and CBP can switch activities between the promoters of genes that stimulate growth and those that enhance cell cycle arrest.
p27Kip1 (p27) acts as a tumor suppressor by inhibiting cyclin–cyclin-dependent kinase (cyclin-CDK) activity. However, mice expressing a form of p27 that is unable to bind or inhibit cyclin-CDK complexes (p27CK–) have increased incidence of tumor development as compared with wild-type and p27–/– mice, revealing an oncogenic role for p27. Here, we identified a phenotype of multinucleation and polyploidy in p27CK– mice not present in p27–/– animals, suggesting a role for p27 in G2/M that is independent of cyclin-CDK regulation. Further analysis revealed that p27CK– expression caused a cytokinesis and abscission defect in mouse embryonic fibroblasts. We identified the Rho effector citron kinase (citron-K) as a p27-interacting protein in vitro and in vivo and found that p27 and citron-K colocalized at the contractile ring and mid-body during telophase and cytokinesis. Moreover, overexpression of the minimal p27-binding domain of citron-K was sufficient to rescue the phenotype caused by p27CK–. Conversely, expression of a mutant p27CK– unable to bind citron-K did not induce multinucleation. Finally, by binding to citron-K, p27 prevented the interaction of citron-K with its activator RhoA. Taken together, these data suggest a role for p27 during cytokinesis via the regulation of citron-K activity.
The Krüppel-like transcription factors (KLFs) are important regulators of cell proliferation and differentiation in several different organ systems. The mouse Klf7 gene is strongly active in postmitotic neuroblasts of the developing nervous system, and the corresponding protein stimulates transcription of the cyclin-dependent kinase inhibitor p21waf/cip gene. Here we report that loss of KLF7 activity in mice leads to neonatal lethality and a complex phenotype which is associated with deficits in neurite outgrowth and axonal misprojection at selected anatomical locations of the nervous system. Affected axon pathways include those of the olfactory and visual systems, the cerebral cortex, and the hippocampus. In situ hybridizations and immunoblots correlated loss of KLF7 activity in the olfactory epithelium with significant downregulation of the p21waf/cip and p27kip1 genes. Cotransfection experiments extended the last finding by documenting KLF7's ability to transactivate a reporter gene construct driven by the proximal promoter of p27kip1. Consistent with emerging evidence for a role of Cip/Kip proteins in cytoskeletal dynamics, we also documented p21waf/cip and p27kip1 accumulation in the cytoplasm of differentiating olfactory sensory neurons. KLF7 activity might therefore control neuronal morphogenesis in part by optimizing the levels of molecules that promote axon outgrowth.
Our studies examined the effects of p27kip1 and p21cip1 on the assembly and activity of cyclin D3-cdk4 complexes and determined the composition of the cyclin D3 pool in cells containing and lacking these cyclin-dependent kinase inhibitors. We found that catalytically active cyclin D3-cdk4 complexes were present in fibroblasts derived from p27kip1-p21cip1-null mice and that immunodepletion of extracts of wild-type cells with antibody to p27kip1 and/or p21cip1 removed cyclin D3 protein but not cyclin D3-associated activity. Similar results were observed in experiments assaying cyclin D1-cdk4 activity. Data obtained using mixed cell extracts demonstrated that p27kip1 interacted with cyclin D3-cdk4 complexes in vitro and that this interaction was paralleled by a loss of cyclin D3-cdk4 activity. In p27kip1-p21cip1-deficient cells, the cyclin D3 pool consisted primarily of cyclin D3 monomers, whereas in wild-type cells, the majority of cyclin D3 molecules were complexed to cdk4 and either p27kip1 or p21cip1 or were monomeric. We conclude that neither p27kip1 nor p21cip1 is required for the formation of cyclin D3-cdk4 complexes and that cyclin D3-cdk4 complexes containing p27kip1 or p21cip1 are inactive. We suggest that only a minor portion of the total cyclin D3 pool accounts for all of the cyclin D3-cdk4 activity in the cell regardless of whether the cell contains p27kip1 and p21cip1.
Herpesvirus saimiri contains an open reading frame called eclf2 with homology to the cellular type D cyclins. We now show that the eclf2 gene product is a novel virus-encoded cyclin (v-cyclin). The protein encoded by the v-cyclin gene of this oncogenic herpesvirus was found to have an apparent molecular size of 29 kDa in transformed cells. v-Cyclin protein was found to be associated with cdk6, a cellular cyclin-dependent kinase known to interact with cellular type D cyclins. cdk6/v-cyclin complexes strongly phosphorylated Rb fusion protein and histone H1 as substrates in vitro. Mutational analyses showed that highly conserved amino acids in the cyclin box of v-cyclin were important for association with cdk6 and for activation of cdk6 kinase activity. Thus, v-cyclin resembles cellular type D cyclins in primary sequence, in its association with cdk6, by its ability to activate protein kinase activity, and by the presence of functional cyclin box sequences. v-Cyclin exhibited a selective preference for association with cdk6 over other cyclin-dependent kinases and a high level of kinase activation. The properties of v-cyclin suggest a likely role in oncogenic transformation by this T-lymphotropic herpesvirus.
Members of the protein kinase C (PKC) family of signal transduction molecules have been widely implicated in regulation of cell growth and differentiation, although the underlying molecular mechanisms involved remain poorly defined. Using combined in vitro and in vivo intestinal epithelial model systems, we demonstrate that PKC signaling can trigger a coordinated program of molecular events leading to cell cycle withdrawal into G0. PKC activation in the IEC-18 intestinal crypt cell line resulted in rapid downregulation of D-type cyclins and differential induction of p21waf1/cip1 and p27kip1, thus targeting all of the major G1/S cyclin-dependent kinase complexes. These events were associated with coordinated alterations in expression and phosphorylation of the pocket proteins p107, pRb, and p130 that drive cells to exit the cell cycle into G0 as indicated by concomitant downregulation of the DNA licensing factor cdc6. Manipulation of PKC isozyme levels in IEC-18 cells demonstrated that PKCα alone can trigger hallmark events of cell cycle withdrawal in intestinal epithelial cells. Notably, analysis of the developmental control of cell cycle regulatory molecules along the crypt–villus axis revealed that PKCα activation is appropriately positioned within intestinal crypts to trigger this program of cell cycle exit–specific events in situ. Together, these data point to PKCα as a key regulator of cell cycle withdrawal in the intestinal epithelium.
protein kinase C; cell cycle; intestinal mucosa; pocket proteins; cyclin-dependent kinase regulation
Histone deacetylase inhibitors (HDACi) induce growth arrest and differentiation, particularly in the colon where they are potential chemotherapeutic agents. A key mediator of HDACi action is the cyclin dependent kinase (CDK) inhibitor p21waf1. HDACi treatment of colonic cells promotes the formation of an ATM/ZBP-89/p300 complex on p21waf1 proximal promoter, and this multi-molecular complex plays an important role in HDACi induction of p21waf1 expression in vitro and mucosal protection in vivo. Here we found that ZBP-89 is phosphorylated by ATM kinase in vitro and in vivo. Disruption of the ATM phosphorylation motif 202SQ within the zinc finger domain of ZBP-89 attenuated its ability to enhance p21waf1 activation by butyrate. Moreover, disruption of the ATM phosphorylation site abrogated the ability of ZBP-89 to potentiate butyrate induction of endogenous p21waf1 expression. These results demonstrate that ATM phosphorylation of ZBP-89 contributes to HDACi induction of p21waf1 gene expression.
histone deacetylase inhibitor; trichostatin A; acetylation; phosphorylation; ZNF148
The retinoblastoma (pRB) family of proteins includes three proteins known to suppress growth of mammalian cells. Previously we had found that growth suppression by two of these proteins, p107 and p130, could result from the inhibition of associated cyclin-dependent kinases (cdks). One important unresolved issue, however, is the mechanism through which inhibition occurs. Here we present in vivo and in vitro evidence to suggest that p107 is a bona fide inhibitor of both cyclin A-cdk2 and cyclin E-cdk2 that exhibits an inhibitory constant (Ki) comparable to that of the cdk inhibitor p21/WAF1. In contrast, pRB is unable to inhibit cdks. Further reminiscent of p21, a second cyclin-binding site was mapped to the amino-terminal portions of p107 and p130. This amino-terminal domain is capable of inhibiting cyclin-cdk2 complexes, although it is not a potent substrate for these kinases. In contrast, a carboxy-terminal fragment of p107 that contains the previously identified cyclin-binding domain serves as an excellent kinase substrate although it is unable to inhibit either kinase. Clustered point mutations suggest that the amino-terminal domain is functionally important for cyclin binding and growth suppression. Moreover, peptides spanning the cyclin-binding region are capable of interfering with p107 binding to cyclin-cdk2 complexes and kinase inhibition. Our ability to distinguish between p107 and p130 as inhibitors rather than simple substrates suggests that these proteins may represent true inhibitors of cdks.
The Tax oncoprotein of human T-cell leukemia virus type 1 (HTLV-1) is leukemogenic in transgenic mice and induces permanent T-cell growth in vitro. It is found in active CDK holoenzyme complexes from adult T-cell leukemia-derived cultures and stimulates the G1- to-S phase transition by activating the cyclin-dependent kinase (CDK) CDK4. The Tax protein directly and specifically interacts with CDK4 and cyclin D2 and binding is required for enhanced CDK4 kinase activity. The protein-protein contact between Tax and the components of the cyclin D/CDK complexes increases the association of CDK4 and its positive regulatory subunit cyclin D and renders the complex resistant to p21CIP inhibition. Tax mutants affecting the N-terminus cannot bind cyclin D and CDK4.
To analyze, whether the N-terminus of Tax is capable of CDK4-binding, in vitro binding -, pull down -, and mammalian two-hybrid analyses were performed. These experiments revealed that a segment of 40 amino acids is sufficient to interact with CDK4 and cyclin D2. To define a Tax-binding domain and analyze how Tax influences the kinase activity, a series of CDK4 deletion mutants was tested. Different assays revealed two regions which upon deletion consistently result in reduced binding activity. These were isolated and subjected to mammalian two-hybrid analysis to test their potential to interact with the Tax N-terminus. These experiments concurrently revealed binding at the N- and C-terminus of CDK4. The N-terminal segment contains the PSTAIRE helix, which is known to control the access of substrate to the active cleft of CDK4 and thus the kinase activity.
Since the N- and C-terminus of CDK4 are neighboring in the predicted three-dimensional protein structure, it is conceivable that they comprise a single binding domain, which interacts with the Tax N-terminus.
We have used microinjection and time-lapse video microscopy to study the role of cyclin A in mitosis. We have injected purified, active cyclin A/cyclin-dependent kinase 2 (CDK2) into synchronized cells at specific points in the cell cycle and assayed its effect on cell division. We find that cyclin A/CDK2 will drive G2 phase cells into mitosis within 30 min of microinjection, up to 4 h before control cells enter mitosis. Often this premature mitosis is abnormal; the chromosomes do not completely condense and daughter cells fuse. Remarkably, microinjecting cyclin A/CDK2 into S phase cells has no effect on progress through the following G2 phase or mitosis. In complementary experiments we have microinjected the amino terminus of p21Cip1/Waf1/Sdi1 (p21N) into cells to inhibit cyclin A/CDK2 activity. We find that p21N will prevent S phase or G2 phase cells from entering mitosis, and will cause early prophase cells to return to interphase. These results suggest that cyclin A/CDK2 is a rate-limiting component required for entry into mitosis, and for progress through mitosis until late prophase. They also suggest that cyclin A/CDK2 may be the target of the recently described prophase checkpoint.
cyclin; CDK; mitosis; p21; GFP
Cyclin-dependent kinases (cdk's) have recently been suggested to regulate human immunodeficiency virus type 1 (HIV-1) transcription. Previously, we have shown that expression of one cdk inhibitor, p21/Waf1, is abrogated in HIV-1 latently infected cells. Based on this result, we investigated the transcription of HIV-1 in the presence of chemical drugs that specifically inhibited cdk activity and functionally mimicked p21/Waf1 activity. HIV-1 production in virally integrated lymphocytic and monocytic cell lines, such as ACH2, 8E5, and U1, as well as activated peripheral blood mononuclear cells infected with syncytium-inducing (SI) or non-syncytium-inducing (NSI) HIV-1 strains, were all inhibited by Roscovitine, a purine derivative that reversibly competes for the ATP binding site present in cdk's. The decrease in viral progeny in the HIV-1-infected cells was correlated with a decrease in the transcription of HIV-1 RNAs in cells treated with Roscovitine and not with the non-cdk general cell cycle inhibitors, such as hydroxyurea (G1/S blocker) or nocodazole (M-phase blocker). Cyclin A- and E-associated histone H1 kinases, as well as cdk 7 and 9 activities, were all inhibited in the presence of Roscovitine. The 50% inhibitory concentration of Roscovitine on cdk's 9 and 7 was determined to be ∼0.6 μM. Roscovitine could selectively sensitize HIV-1-infected cells to apoptosis at concentrations that did not impede the growth and proliferation of uninfected cells. Apoptosis induced by Roscovitine was found in both latent and activated infected cells, as evident by Annexin V staining and the cleavage of the PARP protein by caspase-3. More importantly, contrary to many apoptosis-inducing agents, where the apoptosis of HIV-1-infected cells accompanies production and release of infectious HIV-1 viral particles, Roscovitine treatment selectively killed HIV-1-infected cells without virion release. Collectively, our data suggest that cdk's are required for efficient HIV-1 transcription and, therefore, we propose specific cdk inhibitors as potential antiviral agents in the treatment of AIDS.