Proliferating cell nuclear antigen and p21 are components of multiple cell cycle kinase complexes.

We have recently shown that two proteins, proliferating cell nuclear antigen (PCNA) and p21, are associated with cyclin D. Here we show that PCNA and p21 are common components of a wide variety of cyclin/cyclin-dependent kinase complexes in nontransformed cells. These include kinase complexes containing cyclin A, cyclin B, and cyclin D, associated either with CDC2, CDK2, CDK4, or CDK5. We show that PCNA and p21 form separate quaternary complex with each cyclin/CDK and that these quaternary complexes contain a substantial, if not major, fraction of the cell cycle kinases in asynchronously growing cells. These results suggest that PCNA and p21 may perform a common function for all these kinases.     

Human papillomavirus type 16 E1 E4-induced G2 arrest is associated with cytoplasmic retention of active Cdk1/cyclin B1 complexes

Human papillomavirus type 16 (HPV16) can cause cervical cancer. Expression of the viral E1 E4 protein is lost during malignant progression, but in premalignant lesions, E1 E4 is abundant in cells supporting viral DNA amplification. Expression of 16E1 E4 in cell culture causes G2 cell cycle arrest. Here we show that unlike many other G2 arrest mechanisms, 16E1 E4 does not inhibit the kinase activity of the Cdk1/cyclin B1 complex. Instead, 16E1 E4 uses a novel mechanism in which it sequesters Cdk1/cyclin B1 onto the cytokeratin network. This prevents the accumulation of active Cdk1/cyclin B1 complexes in the nucleus and hence prevents mitosis. A mutant 16E1 E4 (T22A, T23A) which does not bind cyclin B1 or alter its intracellular location fails to induce G2 arrest. The significance of these results is highlighted by the observation that in lesions induced by HPV16, there is evidence for Cdk1/cyclin B1 activity on the keratins of 16E1 E4-expressing cells. We hypothesize that E1 E4-induced G2 arrest may play a role in creating an environment optimal for viral DNA replication and that loss of E1 E4 expression may contribute to malignant progression.     

Cyclin E and SV40 small T antigen cooperate to bypass quiescence and contribute to transformation by activating CDK2 in human fibroblasts

Cyclin E overexpression is observed in multiple human tumors and linked to poor prognosis. We have previously shown that ectopic expression of cyclin E is sufficient to induce mitogen-independent cell cycle entry in a variety of tumor/immortal cell lines. Here we have investigated the rate-limiting step leading to cell cycle entry in quiescent normal human fibroblasts (NHF) ectopically expressing cyclin E. We found that in serum-starved NHF, cyclin E forms inactive complexes with CDK2 and fails to induce DNA synthesis. Coexpression of SV40 small t antigen (st), but not other tested oncogenes, efficiently induces mitogen-independent CDK2 phosphorylation on Thr-160, CDK2 activation, and DNA synthesis. Additionally, in contact-inhibited NHF ectopically expressing cyclin E, st induces cell cycle entry, continued proliferation, and foci formation. Coexpression of cyclin E and st also bypasses G(0)/G(1) arrests induced by CDK inhibitors. Although CDK2 is dispensable for G(0)/G(1) cell cycle entry and normal proliferation in mammals, CDK2 activity is an essential rate-limiting step in NHF with deregulated cyclin E expression and altered PP2A activity, which endows primary cells with transformed features. Consequently, CDK2 could be targeted therapeutically in tumors that involve these alterations. These data also suggest that alterations prior to cyclin E deregulation facilitate proliferation of tumor cells by bypassing mitogenic requirements and negative regulation by adjacent cells.     

Direct activation of cyclin-dependent kinase 2 by human papillomavirus E7

Addition of human papillomavirus (HPV) E7 CDK2/cyclin A or CDK2/cyclin E, purified from either insect cells or bacteria, dramatically upregulates histone H1 kinase activity. Activation is substrate specific, with a smaller effect noted for retinoblastoma protein (Rb). The CDK2 stimulatory activity is equivalent in high-risk (HPV type 16 [HPV16] and HPV31) and low-risk (HPV6b) E7. Mutational analyses of HPV16 E7 indicate that the major activity resides in amino acids 9 to 38, spanning CR1 and CR2, and does not require casein kinase II or Rb-binding domain functions. Synthetic peptides spanning HPV16 amino acid residues 9 to 38 also activate CDK2. Peptides containing this sequence that carry biotin on the carboxy terminus, as well as a photoactivated cross-linking group (benzophenone), also activate the complex and covalently associate with the CDK2/cyclin A complex in a specific manner requiring UV. Cross-linking studies that use protein monomers detect association of the E7 peptides with cyclin A but not CDK2. Together, our results indicate a novel mechanism whereby E7 promotes HPV replication by directly altering CDK2 activity and substrate specificity.     

Indole-3-carbinol (I3C) inhibits cyclin-dependent kinase-2 function in human breast cancer cells by regulating the size distribution, associated cyclin E forms, and subcellular localization of the CDK2 protein complex

Indole-3-carbinol (I3C), a dietary compound found in cruciferous vegetables, induces a robust inhibition of CDK2 specific kinase activity as part of a G1 cell cycle arrest of human breast cancer cells. Treatment with I3C causes a significant shift in the size distribution of the CDK2 protein complex from an enzymatically active 90 kDa complex to a larger 200 kDa complex with significantly reduced kinase activity. Co-immunoprecipitations revealed an increased association of both a 50 kDa cyclin E and a 75 kDa cyclin E immunoreactive protein with the CDK2 protein complex under I3C-treated conditions, whereas the 90 kDa CDK2 protein complexes detected in proliferating control cells contain the lower molecular mass forms of cyclin E. I3C treatment caused no change in the level of CDK2 inhibitors (p21, p27) or in the inhibitory phosphorylation states of CDK2. The effects of I3C are specific for this indole and not a consequence of the cell cycle arrest because treatment of MCF-7 breast cancer cells with either the I3C dimerization product DIM or the anti-estrogen tamoxifen induced a G1 cell cycle arrest with no changes in the associated cyclin E or subcellular localization of the CDK2 protein complex. Taken together, our results have uncovered a unique effect of I3C on cell cycle control in which the inhibition of CDK2 kinase activity is accompanied by selective alterations in cyclin E composition, size distribution, and subcellular localization of the CDK2 protein complex.     

Activation of cyclin-dependent kinase 2 by full length and low molecular weight forms of cyclin E in breast cancer cells

Cyclin E, a positive regulator of the cell cycle, controls the transition of cells from G(1) to S phase. Deregulation of the G(1)-S checkpoint contributes to uncontrolled cell division, a hallmark of cancer. We have reported previously that cyclin E is overexpressed in breast cancer and such overexpression is usually accompanied by the appearance of low molecular weight isoforms of cyclin E protein, which are not present in normal cells. Furthermore, we have shown that the expression of cyclin E low molecular weight isoforms can be used as a reliable prognostic marker for breast cancer to predict patient outcome. In this study we examined the role of cyclin E in directly activating cyclin-dependent kinase (CDK) 2. For this purpose, a series of N-terminal deleted forms of cyclin E corresponding to the low molecular weight forms detected only in cancer cells were translated in vitro and mixed with cell extracts. These tumor-specific N-terminal deleted forms of cyclin E are able to activate CDK2. Addition of cyclin E into both normal and tumor cell extracts was shown to increase the levels of CDK2 activity, along with an increase in the amount of phosphorylated CDK2. The increase in CDK2 activity was because of cyclin E binding to endogenous CDK2 in complex with endogenous cyclin E, cyclin A, or unbound CDK2. The increase in CDK2 phosphorylation was through a pathway involving cyclin-activating kinase, but addition of cyclin E to an extract containing unphosphorylated CDK2 can still lead to increase in CDK2 activity. Our data suggest that the ability of high levels of full-length and low molecular weight forms of cyclin E to activate CDK2 may be one mechanism that leads to the constitutive activation of cyclin E.CDK2 complexes leading to G(1)/S deregulation and tumor progression.     

HeLa cells are phenotypically limiting in cyclin E/CDK2 for efficient human papillomavirus DNA replication

Human papillomaviral (HPV) origin-containing plasmids replicate efficiently in human 293 cells or cell extracts in the presence of HPV origin-recognition protein E2 and replication initiation protein E1, whereas cervical carcinoma-derived, HPV-18-positive HeLa cells or cell extracts support HPV DNA replication poorly. We recently showed that HPV-11 E1 interacts with cyclin/cyclin-dependent kinase (cdk) complexes through an RXL motif and is a substrate for these kinases. E1 mutations in this motif or in candidate cdk phosphorylation sites are impaired in replication, suggesting a role for cdks in HPV replication. We now demonstrate that one limiting activity in HeLa cells is cyclin E/CDK2. Purified cyclin E/CDK2 or cyclin E/CDK3 complex, but not other cdks, partially complemented HeLa cell extracts. Cyclin E/CDK2 expression vectors also enhanced transient HPV replication in HeLa cells. HeLa cell-derived HPV-18 E1 protein is truncated at the carboxyl terminus but can associate with cyclin E/CDK2. This truncated E1 was replication-incompetent and inhibited cell-free HPV replication. These results indicate that HeLa cells are phenotypically limiting in cyclin E/CDK2 for efficient HPV replication, most likely due to sequestration by the endogenous, defective HPV-18 E1 protein. Further analyses of the regulation of HPV E1 and HPV replication by cyclin E may shed light on the roles of cyclin E/CDK2 in cellular DNA replication.     

Mammalian cell cycles without cyclin E-CDK2

The family of mammalian E-type cyclins is composed of two proteins, termed cyclin E1 and E2. These two cyclins are widely expressed in proliferating cells. E-cyclins bind and activate cyclin dependent kinase CDK2. Cyclin E-CDK2 complexes were believed to play critical function in driving cell cycle progression of normal, nontransformed cells and of cancer cells. Several recent reports challenge this notion.     

Mammalian Cell Cycles without Cyclin E-CDK2

The family of mammalian E-type cyclins is composed of two proteins, termed cyclin E1 and E2. These two cyclins are widely expressed in proliferating cells. E-cyclins bind and activate cyclin dependent kinase CDK2. Cyclin E-CDK2 complexes were believed to play critical function in driving cell cycle progression of normal, nontransformed cells and of cancer cells. Several recent reports challenge this notion.     

D type cyclins associate with multiple protein kinases and the DNA replication and repair factor PCNA.

Human cyclin D1 has been associated with a wide variety of proliferative diseases but its biochemical role is unknown. In diploid fibroblasts we find that cyclin D1 is complexed with many other cellular proteins. Among them are protein kinase catalytic subunits CDK2, CDK4 (previously called PSK-J3), and CDK5 (also called PSSALRE). In addition, polypeptides of 21 kd and 36 kd are identified in association with cyclin D1. We show that the 36 kd protein is the proliferating cell nuclear antigen, PCNA. Cyclin D3 also associates with multiple protein kinases, p21 and PCNA. It is proposed that there exists a quaternary complex of D cyclin, CDK, PCNA, and p21 and that many combinatorial variations (cyclin D1, D3, CDK2, 4, and 5) may assemble in vivo. These findings link a human putative G1 cyclin that is associated with oncogenesis with a well-characterized DNA replication and repair factor.     

Adeno-associated virus type 2 increases proteosome-dependent degradation of p21WAF1 in a human papillomavirus type 31b-positive cervical carcinoma line

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 onco-suppression 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 p21(WAF1) protein in a proteosome-dependent manner. Downstream consequences of lowering p21(WAF1) levels included a proportional loss of cyclin E/CDK2 complexes bound to p21(WAF1). The loss of stable p21(WAF1)/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 G(1)/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 p21(WAF1) protein levels, reminiscent of a block in G(1) phase progression. We propose that by down regulating p21(WAF1), AAV2 initiates cell cycle activities leading to enhanced G(1)/S phase-like conditions which may be favorable for AAV2-specific functions and may lead to downstream interference with HPV-associated cervical cancer progression.     

Progesterone inhibits the estrogen-induced phosphoinositide 3-kinase-->AKT-->GSK-3beta-->cyclin D1-->pRB pathway to block uterine epithelial cell proliferation

The mammalian cell cycle is regulated by the cyclin/cyclin-dependent kinase (CDK) phosphorylation of the retinoblastoma (pRB) family of proteins. Cyclin D1 with its CDK4/6 partners initiates the cell cycle and acts as the link between extracellular signals and the cell cycle machinery. Estradiol-17beta (E2) stimulates uterine epithelial cell proliferation, a process that is completely inhibited by pretreatment with progesterone (P4). Previously, we identified cyclin D1 localization as a key point of regulation in these cells with E2 causing its nuclear accumulation and P4 retaining it in the cytoplasm with the resultant inhibition of pRB phosphorylation. Here we show that E2 stimulates phosphoinositide 3-kinase to activate phosphokinase B/AKT to effect an inhibitory phosphorylation of glycogen synthase kinase (GSK-3beta). This pathway is suppressed by P4. Inhibition of the GSK-3beta activity in P4-treated uteri by the specific inhibitor, LiCl, reversed the nuclear accumulation of cyclin D1 and in doing so, caused pRB phosphorylation and the induction of downstream genes, proliferating cell nuclear antigen and Ki67. Conversely, inhibition of phosphoinositide 3 kinase by LY294002 or Wortmanin reversed the E2-induced GSK-3beta Ser9 inhibitory phosphorylation and blocked nuclear accumulation of cyclin D1. These data show the reciprocal actions of E2 and P4 on the phosphoinositide 3-kinase through to the GSK-3beta pathway that in turn regulates cyclin D1 localization and cell cycle progression. These data reveal a novel signaling pathway that links E2 and P4 action to growth factor-mediated signaling in the uterus.     

Intracellular localization of the cyclin-dependent kinase inhibitor p21<Superscript>CDKN1A</Superscript>-GFP fusion protein during cell cycle arrest

The cyclin-dependent kinase (CDK) inhibitor p21^CDKN1A is known to induce cell cycle arrest by inhibiting CDK activity and by interfering with DNA replication through binding to proliferating cell nuclear antigen. Although the molecular mechanisms have been elucidated, the temporal dynamics, as well as the intracellular sites of the activity of p21 bound to cyclin/CDK complexes during cell cycle arrest, have not been fully investigated. In this study we have induced the expression of p21^CDKN1A fused to green fluorescent protein (GFP) in HeLa cells, in order to visualize the intracellular localization of the inhibitor during the cell cycle arrest. We show that p21-GFP is preferentially expressed in association with cyclin E in cells arrested in G1 phase, and with cyclin A more than with cyclin B1 in cells arrested in the G2/M compartment. In addition, we show for the first time that p21-GFP colocalizes with cyclin E in the nucleolus of HeLa cells during the G1 phase arrest.O. Cazzalini and P. Perucca contributed equally to this work     

Cyclin D2 compensates for the loss of cyclin D1 in estrogen-induced mouse uterine epithelial cell proliferation

The cell cycle-regulatory protein, cyclin D1, is the sensor that connects the intracellular cell cycle machinery to external signals. Given this central role in the control of cell proliferation, it was surprising that mice lacking the cyclin D1 gene were viable and fertile. Fertility requires 17beta-estradiol (E2)-induced uterine luminal epithelial cell proliferation. In these cells E2 causes the translocation of cyclin D1/cyclin-dependent kinase 4 (CDK4) from the cytoplasm into the nucleus with the consequent phosphorylation of the retinoblastoma protein. In cyclin D1 null mice, E2 also induces retinoblastoma protein phosphorylation and DNA synthesis in a normal manner. CDK4 activity was slightly reduced in the D1 null mice compared with wild-type mice. This CDK4 activity was due to complexes of cyclin D2/CDK4. Cyclin D2 was translocated into the nucleus in response to E2 in the cyclin D1-/- mice to a much greater degree than in wild-type mice. This cyclin D2/CDK4 complex was also able to bind p27kip1 in cyclin D1-/- uterine luminal epithelial cells, allowing for the activation of CDK2. Our data show that in vivo cyclin D2 can completely compensate for the loss of cyclin D1 and reinforces the conclusions that cyclin Ds are the central regulatory point in the proliferative responses of epithelial cells to estrogens.     

HMGB1 mediates IFN-γ-induced cell proliferation in MMC cells through regulation of cyclin D1/CDK4/p16 pathway

Previous studies have revealed the elevated serum levels of High-mobility group box-1(HMGB1) and the interferon-γ (IFN-γ)-induced proliferation of renal mesangial cells in patients or experimental animals with systemic lupus erythematosus (SLE). However, it is still not elucidated whether HMGB1 involves in the pathogenesis of lupus nephritis (LN) and mediates IFN-γ-induced mesangial cell proliferation. Therefore, in the present study we demonstrated HMGB1 mRNA and protein levels were increased in the glomeruli of LN patients and BXSB mice. HMGB1 increased the proliferation index of mouse mesangial cells (MMC) that was accompanied with the up-regulation of cyclin D1, CDK4 and the down-regulation of p16, subsequently promoting the transition from the G0/G1 to S stage. Inhibition of HMGB1 by a specific short hairpin RNA vector prevented cyclin D1/CDK4/p16 up-regulation and attenuated IFN-γ-induced MMC cell proliferation and PCNA (proliferating cell nuclear antigen, PCNA) expression. These findings indicate that HMGB1 mediates IFN-γ-induced cell proliferation in MMC cells through regulation of cyclin D1/CDK4/p16 pathway and promoting the cell cycle transition from G1 to S stage.     

Overexpression of cyclin E/CDK2 complexes overcomes FGF-induced cell cycle arrest in the presence of hypophosphorylated Rb proteins

FGF signaling inhibits chondrocyte proliferation and requires the function of the p107 and p130 members of the Rb protein family to execute growth arrest. p107 dephosphorylation plays a critical role in the chondrocyte response to FGF, as overexpression of cyclin D1/CDK4 complexes (the major p107 kinase) in rat chondrosarcoma (RCS) cells overcomes FGF-induced p107 dephosphorylation and growth arrest. In cells overexpressing cyclin D1/CDK4, FGF-induced downregulation of cyclin E/CDK2 activity was absent. To examine the role of cyclin E/CDK2 complexes in mediating FGF-induced growth arrest, this kinase was overexpressed in RCS cells. FGF-induced dephosphorylation of either p107 or p130 was not prevented by overexpressing cyclin E/CDK2 complexes. Unexpectedly, however, FGF-treated cells exhibited sustained proliferation even in the presence of hypophosphorylated p107 and p130. Both pocket proteins were able to form repressive complexes with E2F4 and E2F5 but these repressors were not translocated into the nucleus and therefore were unable to occupy their respective target DNA sites. Overexpressed cyclin E/CDK2 molecules were stably associated with p107 and p130 in FGF-treated cells in the context of E2F repressive complexes. Taken together, our data suggest a novel mechanism by which cyclin E/CDK2 complexes can promote cell cycle progression in the presence of dephosphorylated Rb proteins and provide a novel insight into the key Retinoblastoma/E2F/cyclin E pathway. Our data also highlight the importance of E2F4/p130 complexes for FGF-mediated growth arrest in chondrocytes.     

Overexpression of cyclin E/CDK2 complexes overcomes FGF-induced cell cycle arrest in the presence of hypophosphorylated Rb proteins

FGF signaling inhibits chondrocyte proliferation and requires the function of the p107 and p130 members of the Rb protein family to execute growth arrest. p107 dephosphorylation plays a critical role in the chondrocyte response to FGF, as overexpression of cyclin D1/CDK4 complexes (the major p107 kinase) in rat chondrosarcoma (RCS) cells overcomes FGF-induced p107 dephosphorylation and growth arrest. In cells overexpressing cyclin D1/CDK4, FGF-induced downregulation of cyclin E/CDK2 activity was absent. To examine the role of cyclin E/CDK2 complexes in mediating FGF-induced growth arrest, this kinase was overexpressed in RCS cells. FGF-induced dephosphorylation of either p107 or p130 was not prevented by overexpressing cyclin E/CDK2 complexes. Unexpectedly, however, FGF-treated cells exhibited sustained proliferation even in the presence of hypophosphorylated p107 and p130. Both pocket proteins were able to form repressive complexes with E2F4 and E2F5 but these repressors were not translocated into the nucleus and therefore were unable to occupy their respective target DNA sites. Overexpressed cyclin E/CDK2 molecules were stably associated with p107 and p130 in FGF-treated cells in the context of E2F repressive complexes. Taken together, our data suggest a novel mechanism by which cyclin E/CDK2 complexes can promote cell cycle progression in the presence of dephosphorylated Rb proteins and provide a novel insight into the key Retinoblastoma/E2F/cyclin E pathway. Our data also highlight the importance of E2F4/p130 complexes for FGF-mediated growth arrest in chondrocytes.