Identification of a G(2) arrest domain in the E1 wedge E4 protein of human papillomavirus type 16

Human papillomavirus type 16 (HPV16) is the most common cause of cervical carcinoma. Cervical cancer develops from low-grade lesions that support the productive stages of the virus life cycle. The 16E1 wedge E4 protein is abundantly expressed in such lesions and can be detected in cells supporting vegetative viral genome amplification. Using an inducible mammalian expression system, we have shown that 16E1 wedge E4 arrests HeLa cervical epithelial cells in G(2). 16E1 wedge E4 also caused a G(2) arrest in SiHa, Saos-2 and Saccharomyces pombe cells and, as with HeLa cells, was found in the cytoplasm. However, whereas 16E1 wedge E4 is found on the keratin networks in HeLa and SiHa cells, in Saos-2 and S. pombe cells that lack keratins, 16E1 wedge E4 had a punctate distribution. Mutagenesis studies revealed a proline-rich region between amino acids 17 and 45 of 16E1 wedge E4 to be important for arrest. This region, which we have termed the "arrest domain," contains a putative nuclear localization signal, a cyclin-binding motif, and a single cyclin-dependent kinase (Cdk) phosphorylation site. A single point mutation in the putative Cdk phosphorylation site (T23A) abolished 16E1 wedge E4-mediated G(2) arrest. Arrest did not involve proteins regulating the phosphorylation state of Cdc2 and does not appear to involve the activation of the DNA damage or incomplete replication checkpoint. G(2) arrest was also mediated by the E1 wedge E4 protein of HPV11, a low-risk mucosal HPV type that also causes cervical lesions. The E1 wedge E4 protein of HPV1, which is more distantly related to that of HPV16, did not cause G(2) arrest. We conclude that, like other papillomavirus proteins, 16E1 wedge E4 affects cell cycle progression and that it targets a conserved component of the cell cycle machinery.     

Role for Wee1 in inhibition of G2-to-M transition through the cooperation of distinct human papillomavirus type 1 E4 proteins

The infectious cycle of human papillomavirus type 1 (HPV1) is accompanied by abundant expression of the full-length E1;E4 protein (17-kDa) and smaller E4 polypeptides (16-, 11-, and 10-kDa) that arise by sequential loss of N-terminal E1;E4 sequences. HPV1 E4 inhibits G(2)-to-M transition of the cell cycle. Here, we show that HPV1 E4 proteins mediate inhibition of cell division by more than one mechanism. Cells arrested by coexpression of E1;E4 (E4-17K) and a truncated protein equivalent to the 16-kDa species (E4-16K) contain inactive cyclin B1-cdk1 complexes. Inactivation of cdk1 is through inhibitory Tyr(15) phosphorylation, with cells containing elevated levels of Wee1, the kinase responsible for inhibitory cdk1 phosphorylation. Consistent with these findings, overexpression of Wee1 enhanced the extent to which E4-17K/16K-expressing cells arrest in G(2), indicating that maintenance of Wee1 activity is necessary for inhibition of cell division induced by coexpression of the two E4 proteins. Moreover, we have determined that depletion of Wee1 by small interfering RNA (siRNA) alleviates the G(2) block imposed by E4-17K/16K. In contrast however, maintenance of Wee1 activity is not necessary for G(2)-to-M inhibition mediated by E4-16K alone, as overexpression or depletion of Wee1 does not influence the G(2) arrest function of E4-16K. Cells arrested by E4-16K expression contain low levels of active cyclin B1-cdk1 complexes. We hypothesize that differential expression of HPV1 E4 proteins during the viral life cycle determines the host cell cycle status. Different mechanisms of inhibition of G(2)-to-M transition reinforce the supposition that distinct E4 functions are important for HPV replication.     

Cooperation between different forms of the human papillomavirus type 1 E4 protein to block cell cycle progression and cellular DNA synthesis

Posttranslational modification-oligomerization, phosphorylation, and proteolytic cleavage-of the human papillomavirus (HPV) E4 protein occurs as the infected keratinocytes migrate up through the suprabasal wart layers. It has been postulated that these events modify E4 function during the virus life cycle. In HPV type 1 (HPV1)-induced warts, N-terminal sequences are progressively cleaved from the full-length E4 protein (E1(wedge)E4) of 17 kDa to produce a series of polypeptides of 16, 11 and 10 kDa. Here, we have shown that in human keratinocytes, a truncated protein (E4-16K), equivalent to the 16-kDa species, mediated a G(2) arrest in the cell cycle that was dependent on a threonine amino acid in a proline-rich domain of the protein. Reconstitution of cyclin B1 expression in E4-16K cells reversed the G(2) arrest. Expression of E4-16K also induced chromosomal rereplication, and this was associated with aberrant nuclear morphology. Perturbation of the mitotic cell cycle was a biological activity specific to the truncated protein. However, coexpression of the full-length E1(wedge)E4 protein and the truncated E4-16K protein inhibited normal cellular proliferation and cellular DNA rereplication but did not prevent cells from arresting in G(2). Our findings provide the first evidence to support the hypothesis that proteolytic cleavage of the E1(wedge)E4 protein modifies its function. Also, different forms of the HPV1 E4 protein cooperate to negatively influence keratinocyte proliferation. We predict that these distinct biological activities of E4 act to support efficient amplification of the viral genome in suprabasal keratinocytes.     

Non-malignant and tumor-derived cells differ in their requirement for p27Kip1 in transforming growth factor-beta-mediated G1 arrest

Transforming growth factor beta (TGF-beta) induces G(1) arrest in susceptible cells by multiple mechanisms that inhibit the G(1) cyclin-dependent kinases (Cdks), including Cdk2, Cdk4, and Cdk6. TGF-beta treatment of early passage finite lifespan human mammary epithelial cells (HMECs) led to an accumulation of p27(Kip1) in cyclin E1-Cdk2 complexes and kinase inhibition. The requirement for p27 in the G(1) arrest by TGF-beta was assessed by transfection of antisense p27 (ASp27) oligonucleotides into TGF-beta-treated HMECs. Despite a reduction in total and cyclin E-Cdk2 bound p27 after ASp27 transfection, HMECs remained arrested in the G(1) phase. Maintenance of the G(1) arrest was accompanied by increased association of the Cdk inhibitor p21(WAF-1/Cip-1) and the retinoblastoma family member p130(Rb2) in cyclin E1-Cdk2 complexes along with kinase inhibition. In contrast to the findings in HMECs, p27 was essential for G(1) arrest by TGF-beta in two tumor-derived lines. ASp27 transfection into two TGF-beta-responsive, cancer-derived lines was not associated with increased compensatory binding of p21 and p130 to cyclin E1-Cdk2, and these cell lines failed to maintain G(1) arrest despite the continued presence of TGF-beta. Progressive cell cycle deregulation leading to impaired checkpoint controls during malignant tumor progression may alter the role of p27 from a redundant to an essential inhibitor of G(1)-to-S phase progression.     

New alternative phosphorylation sites on the cyclin dependent kinase 1/cyclin a complex in p53-deficient human cells treated with etoposide: possible association with etoposide-induced apoptosis

Cell cycle arrest is a major cellular response to DNA damage preceding the decision to repair or die. Many malignant cells have non-functional p53 rendering them more “aggressive” in nature. Arrest in p53-negative cells occurs at the G2M cell cycle checkpoint. Failure of DNA damaged cells to arrest at G2 results in entry into mitosis and potential death through aberrant mitosis and/or apoptosis. The pivotal kinase regulating the G2M checkpoint is Cdk1/cyclin B whose activity is controlled by phosphorylation. The p53-negative myeloid leukemia cell lines K562 and HL-60 were used to determine Cdk1 phosphorylation status during etoposide treatment. Cdk1 tyrosine 15 phosphorylation was associated with G2M arrest, but not with cell death. Cdk1 tyrosine 15 phosphorylation also led to suppression of nuclear cyclin B-associated Cdk1 kinase activity. However cell death, associated with broader tyrosine phosphorylation of Cdk1 was not attributed to tyrosine 15 alone. This broader phosphoryl isoform of Cdk1 was associated with cyclin A and not cyclin B. Alternative phosphorylations sites were predicted as tyrosines 4, 99 and 237 by computer analysis. No similar pattern was found on Cdk2. These findings suggest novel Cdk1 phosphorylation sites, which appear to be associated with p53-independent cell death following etoposide treatment.     

Retinoic acid-mediated G1 arrest is associated with induction of p27(Kip1) and inhibition of cyclin-dependent kinase 3 in human lung squamous carcinoma CH27 cells

Retinoids are promising agents for the prevention and treatment of several human malignancies including lung cancer. In this study, the effect of retinoic acid (RA) on cell growth and the mechanism of growth modulation were examined in human lung squamous carcinoma CH27 cells. Here we report that RA mediated the dose- and time-dependent growth arrest in G1 phase, accompanied by the up-regulation of p27(Kip1) and the down-regulation of the cyclin-dependent kinase 3 (Cdk3) and p21(CIP1/Waf1) proteins. Furthermore, RA-induced growth arrest of CH27 cells was also associated with increased retinoic acid receptor beta (RARbeta) and reduced c-Myc expression. However, RA had no effect on the levels of cyclins A, D1, D3, E, or H, or on Cdk2, Cdk4, Cdk5, CDk6, Cdk7, p16(Ink4A), p15(Ink4B), p53, or pRb proteins in CH27 cells. Evaluation of the kinase activity of cyclin-Cdk complexes showed that RA increases p27(Kip1) expression in CH27 cells leading to markedly reduced cyclin A/Cdk2 kinase activity and slightly reduced cyclin E/Cdk2 kinase activity, with no effect on cyclin D/Cdk4 and cyclin D/Cdk6 activities. Moreover, coincident with the decrease in kinase activity was a drastic increase in cyclin A-bound p27(Kip1). These results suggest that increases in the levels of p27(Kip1) and its binding to cyclin A, as well as reduction of Cdk3 protein expression, are strong candidates for the cell cycle regulator that prevents the entry into the S phase in RA-treated CH27 cells, with prolongation of G1 phase and inhibition of DNA synthesis.     

Hyperoxia induces S-phase cell-cycle arrest and p21(Cip1/Waf1)-independent Cdk2 inhibition in human carcinoma T47D-H3 cells

Little is known about cell-cycle checkpoint activation by oxidative stress in mammalian cells. The effects of hyperoxia on cell-cycle progression were investigated in asynchronous human T47D-H3 cells, which contain mutated p53 and fail to arrest at G1/S in response to DNA damage. Hyperoxic exposure (95% O(2), 40-64 h) induced an S-phase arrest associated with acute inhibition of Cdk2 activity and DNA synthesis. In contrast, exit from G2/M was not inhibited in these cells. After 40 h of hyperoxia, these effects were partially reversible during recovery under normoxic conditions. The inhibition of Cdk2 activity was not due to degradation of Cdk2, cyclin E or A, nor impairment of Cdk2 complex formation with cyclin A or E and p21(Cip1). The loss of Cdk2 activity occurred in the absence of induction and recruitment of cdk inhibitor p21(Cip1) or p27(Kip1) in cyclin A/Cdk2 or cyclin E/Cdk2 complexes. In contrast, Cdk2 inhibition was associated with increased Cdk2-Tyr15 phosphorylation, increased E2F-1 recruitment, and decreased PCNA contents in Cdk2 complexes. The latter results indicate a p21(Cip1)/p27(Kip1)-independent mechanism of S-phase checkpoint activation in the hyperoxic T47D cell model investigated.     

Retinoic Acid-Mediated G1 Arrest Is Associated with Induction of p27 and Inhibition of Cyclin-Dependent Kinase 3 in Human Lung Squamous Carcinoma CH27 Cells

Retinoids are promising agents for the prevention and treatment of several human malignancies including lung cancer. In this study, the effect of retinoic acid (RA) on cell growth and the mechanism of growth modulation were examined in human lung squamous carcinoma CH27 cells. Here we report that RA mediated the dose- and time-dependent growth arrest in G1 phase, accompanied by the up-regulation of p27^Kip1 and the down-regulation of the cyclin-dependent kinase 3 (Cdk3) and p21^CIP1/Waf1 proteins. Furthermore, RA-induced growth arrest of CH27 cells was also associated with increased retinoic acid receptor β (RARβ) and reduced c-Myc expression. However, RA had no effect on the levels of cyclins A, D1, D3, E, or H, or on Cdk2, Cdk4, Cdk5, CDk6, Cdk7, p16^Ink4A, p15^Ink4B, p53, or pRb proteins in CH27 cells. Evaluation of the kinase activity of cyclin–Cdk complexes showed that RA increases p27^Kip1 expression in CH27 cells leading to markedly reduced cyclin A/Cdk2 kinase activity and slightly reduced cyclin E/Cdk2 kinase activity, with no effect on cyclin D/Cdk4 and cyclin D/Cdk6 activities. Moreover, coincident with the decrease in kinase activity was a drastic increase in cyclin A-bound p27^Kip1. These results suggest that increases in the levels of p27^Kip1 and its binding to cyclin A, as well as reduction of Cdk3 protein expression, are strong candidates for the cell cycle regulator that prevents the entry into the S phase in RA-treated CH27 cells, with prolongation of G1 phase and inhibition of DNA synthesis.     

Multifaceted regulation of cell cycle progression by estrogen: regulation of Cdk inhibitors and Cdc25A independent of cyclin D1-Cdk4 function

Estrogens induce proliferation of estrogen receptor (ER)-positive MCF-7 breast cancer cells by stimulating G(1)/S transition associated with increased cyclin D1 expression, activation of cyclin-dependent kinases (Cdks), and phosphorylation of the retinoblastoma protein (pRb). We have utilized blockade of cyclin D1-Cdk4 complex formation through adenovirus-mediated expression of p16(INK4a) to demonstrate that estrogen regulates Cdk inhibitor expression and expression of the Cdk-activating phosphatase Cdc25A independent of cyclin D1-Cdk4 function and cell cycle progression. Expression of p16(INK4a) inhibited G(1)/S transition induced in MCF-7 cells by 17-beta-estradiol (E(2)) with associated inhibition of both Cdk4- and Cdk2-associated kinase activities. Inhibition of Cdk2 activity was associated with delayed removal of Cdk-inhibitory activity in early G(1) and decreased cyclin A expression. Cdk-inhibitory activity and expression of both p21(Cip1) and p27(Kip1) was decreased, however, in both control and p16(INK4a)-expressing cells 20 h after estrogen treatment. Expression of Cdc25A mRNA and protein was induced by E(2) in control and p16(INK4a)-expressing MCF-7 cells; however, functional activity of Cdc25A was inhibited in cells expressing p16(INK4a). Inhibition of Cdc25A activity in p16(INK4a)-expressing cells was associated with depressed Cdk2 activity and was reversed in vivo and in vitro by active Cdk2. Transfection of MCF-7 cells with a dominant-negative Cdk2 construct inhibited the E(2)-dependent activation of ectopic Cdc25A. Supporting a role for Cdc25A in estrogen action, antisense CDC25A oligonucleotides inhibited estrogen-induced Cdk2 activation and DNA synthesis. In addition, inactive cyclin E-Cdk2 complexes from p16(INK4a)-expressing, estrogen-treated cells were activated in vitro by treatment with recombinant Cdc25A and in vivo in cells overexpressing Cdc25A. The results demonstrate that functional association of cyclin D1-Cdk4 complexes is required for Cdk2 activation in MCF-7 cells and that Cdk2 activity is, in turn, required for the in vivo activation of Cdc25A. These studies establish Cdc25A as a growth-promoting target of estrogen action and further indicate that estrogens independently regulate multiple components of the cell cycle machinery, including expression of p21(Cip1) and p27(Kip1).     

Human papillomavirus type 16 E6 promotes retinoblastoma protein phosphorylation and cell cycle progression

We show that E6 proteins from benign human papillomavirus type 1 (HPV1) and oncogenic HPV16 have the ability to alter the regulation of the G(1)/S transition of the cell cycle in primary human fibroblasts. Overexpression of both viral proteins induces cellular proliferation, retinoblastoma (pRb) phosphorylation, and accumulation of products of genes that are negatively regulated by pRb, such as p16(INK4a), CDC2, E2F-1, and cyclin A. Hyperphosphorylated forms of pRb are present in E6-expressing cells even in the presence of ectopic levels of p16(INK4a). The E6 proteins strongly increased the cyclin A/cyclin-dependent kinase 2 (CDK2) activity, which is involved in pRb phosphorylation. In addition, mRNA and protein levels of the CDK2 inhibitor p21(WAF1/CIP1) were strongly down-regulated in cells expressing E6 proteins. The down-regulation of the p21(WAF1/CIP1) gene appears to be independent of p53 inactivation, since HPV1 E6 and an HPV16 E6 mutant unable to target p53 were fully competent in decreasing p21(WAF1/CIP1) levels. E6 from HPV1 and HPV16 also enabled cells to overcome the G(1) arrest imposed by oncogenic ras. Immunofluorescence staining of cells coexpressing ras and E6 from either HPV16 or HPV1 revealed that antiproliferative (p16(INK4a)) and proliferative (Ki67) markers were coexpressed in the same cells. Together, these data underline a novel activity of E6 that is not mediated by inactivation of p53.     

Cyclin/CDK regulates the nucleocytoplasmic localization of the human papillomavirus E1 DNA helicase

Cyclin-dependent kinases (CDKs) play key roles in eukaryotic DNA replication and cell cycle progression. Phosphorylation of components of the preinitiation complex activates replication and prevents reinitiation. One mechanism is mediated by nuclear export of critical proteins. Human papillomavirus (HPV) DNA replication requires cellular machinery in addition to the viral replicative DNA helicase E1 and origin recognition protein E2. E1 phosphorylation by cyclin/CDK is critical for efficient viral DNA replication. We now show that E1 is phosphorylated by CDKs in vivo and that phosphorylation regulates its nucleocytoplasmic localization. We identified a conserved regulatory region for localization which contains a dominant leucine-rich nuclear export sequence (NES), the previously defined cyclin binding motif, three serine residues that are CDK substrates, and a putative bipartite nuclear localization sequence. We show that E1 is exported from the nucleus by a CRM1-dependent mechanism unless the NES is inactivated by CDK phosphorylation. Replication activities of E1 phosphorylation site mutations are reduced and correlate inversely with their increased cytoplasmic localization. Nuclear localization and replication activities of most of these mutations are enhanced or restored by mutations in the NES. Collectively, our data demonstrate that CDK phosphorylation controls E1 nuclear localization to support viral DNA amplification. Thus, HPV adopts and adapts the cellular regulatory mechanism to complete its reproductive program.     

Identification of an arginine-rich motif in human papillomavirus type 1 E1;E4 protein necessary for E4-mediated inhibition of cellular DNA synthesis in vitro and in cells

Productive infections by human papillomaviruses (HPVs) are restricted to nondividing, differentiated keratinocytes. HPV early proteins E6 and E7 deregulate cell cycle progression and activate the host cell DNA replication machinery in these cells, changes essential for virus synthesis. Productive virus replication is accompanied by abundant expression of the HPV E4 protein. Expression of HPV1 E4 in cells is known to activate cell cycle checkpoints, inhibiting G(2)-to-M transition of the cell cycle and also suppressing entry of cells into S phase. We report here that the HPV1 E4 protein, in the presence of a soluble form of the replication-licensing factor (RLF) Cdc6, inhibits initiation of cellular DNA replication in a mammalian cell-free DNA replication system. Chromatin-binding studies show that E4 blocks replication initiation in vitro by preventing loading of the RLFs Mcm2 and Mcm7 onto chromatin. HPV1 E4-mediated replication inhibition in vitro and suppression of entry of HPV1 E4-expressing cells into S phase are both abrogated upon alanine replacement of arginine 45 in the full-length E4 protein (E1;E4), implying that these two HPV1 E4 functions are linked. We hypothesize that HPV1 E4 inhibits competing host cell DNA synthesis in replication-activated suprabasal keratinocytes by suppressing licensing of cellular replication origins, thus modifying the phenotype of the infected cell in favor of viral genome amplification.     

Analysis of transient G1/S arrest in E1A+E1B-19kDa transformed cells after ionizing radiation

Expression of human adenovirus type 5 E1A oncogene in normal rodent cells leads to disruption of the G1/S cell cycle arrest realization in response to DNA damage. It has been shown here that rat embryo fibroblasts transformed by E1Aad5 oncogene in complementation with E1B-19 kDa gene realize the irradiation-induced transient G1/S arrest, which depends on selective suppression of CyclinE-Cdk2 activity despite functional inactivation of p21Waf1 inhibitor. Inhibitor p21Waf1 is not revealed in complexes with cyclins E and A in E1A + E1B-19 kDa transformants, however, it is not due to p21Waf1 interaction with E1A oncoproteins, because the E1A-p21Waf1 complex formation in E1A + cHa-ras transformants does not prevent the high level of CycIE, A-p21Waf1 association. In the case of p21Waf1 inactivation, the main way of cyclin-kinase activity regulation in E1A + E1B-19 kDa cells may be Cdk2 phosphorylation. However, irradiation of E1A + E1B-19 kDa transformed cells induces no changes in CAK (Cdk7-associated) kinase activity and in the protein level of Cdc25A phosphatase, which are responsible for activating Thr160 phosphoralation and Tyr15 dephosphorylation on Cdk2. Using phospho-Tyr15-Cdk2 specific antibodies, no increase of phosphorylation at Tyr15 position on immunoprecipitated Cdk2 was detected after irradiation. It seems likely that in the case of inactivated inhibitor p21Waf1 the transient G1/S block after irradiation in E1A + E1B-19 kDa transformants depends on suppression of Cycl-E-Cdk2 activity caused by inhibition of Thr160 Cdk2 phosphorylation, but his occurs with the involvement of other kinases rather than CAK.