An in vitro carcinogenesis model for cervical cancer harboring episomal form of HPV16.

Deregulated expression of viral E6 and E7 genes often caused by viral genome integration of high-risk human papillomaviruses (HR-HPVs) into host DNA and additional host genetic alterations are thought to be required for the development of cervical cancer. However, approximately 15% of invasive cervical cancer specimens contain only episomal HPV genomes. In this study, we investigated the tumorigenic potential of human cervical keratinocytes harboring only the episomal form of HPV16 (HCK1T/16epi). We found that the HPV16 episomal form is sufficient for promoting cell proliferation and colony formation of parental HCK1T cells. Ectopic expression of host oncogenes, MYC and PIK3CAE545K, enhanced clonogenic growth of both early- and late-passage HCK1T/16epi cells, but conferred tumor-initiating ability only to late-passage HCK1T/16epi cells. Interestingly, the expression levels of E6 and E7 were rather lower in late-passage than in early-passage cells. Moreover, additional introduction of a constitutively active MEK1 (MEK1DD) and/or KRASG12V into HCK1T/16epi cells resulted in generation of highly potent tumor-initiating cells. Thus an in vitro model for progression of cervical neoplasia with episomal HPV16 was established. In the model, constitutively active mutation of PIK3CA, PIK3CAE545K, and overexpression of MYC, in the cells with episomal HPV16 genome were not sufficient, but an additional event such as activation of the RAS-MEK pathway was required for progression to tumorigenicity.

Human Papillomavirus 16 E6 Suppresses Transporter Associated with Antigen-Processing Complex in Human Tongue Keratinocyte Cells by Activating Lymphotoxin Pathway.

Infection by high-risk human papillomaviruses (hrHPVs), including HPV type 16 (HPV16), is a major risk factor for oral squamous cell carcinomas (OSCCs). However, the pathogenic mechanism by which hrHPVs promote oral carcinogenesis remains to be elucidated. Here, we demonstrated that the suppression of a transporter associated with the antigen-processing complex (TAPs; TAP1 and TAP2), which is a key molecule in the transportation of viral antigenic peptides into MHC class-I cells, is affected by the E6 protein of HPV16. Mechanistically, HPV-mediated immune evasion is principally mediated via the signal-transduction network of a lymphotoxin (LT) pathway, in particular LTα1ß2 and LTßR. Our analysis of transcriptomic data from an HNSCC cohort from the Cancer Genome Atlas (TCGA) indicated that expression of TAP genes, particularly TAP2, was downregulated in HPV-infected cases. We further demonstrated that LTα1ß2 and LTßR were upregulated, which was negatively correlated with TAP1 and TAP2 expression in HPV-positive clinical OSCC samples. Taken together, our findings imply that HPV16 E6 regulates the machinery of the antigenic peptide-loading system and helps to clarify the role of oncogenic viruses in the context of oral carcinoma.

An Ex-Vivo Culture System of Ovarian Cancer Faithfully Recapitulating the Pathological Features of Primary Tumors.

The success rate of establishing human cancer cell lines is not satisfactory and the established cell lines often do not preserve the molecular and histological features of the original tissues. In this study, we developed a novel culture method which can support proliferation of almost all primary epithelial ovarian cancer cells, as well as primary normal human oviductal epithelial cells. Cancer cells from fresh or frozen specimens were enriched by the anti-EpCAM antibody-conjugated magnetic beads, plated on Matrigel-coated plate and cultivated under the optimized culture conditions. Seventeen newly established ovarian cancer cell lines, which included all four major histotypes of ovarian cancer, were confirmed to express histotype-specific markers in vitro. Some of the cell lines from all the four histotypes, except mucinous type, generated tumors in immune-deficient mice and the xenograft tumor tissues recapitulated the corresponding original tissues faithfully. Furthermore, with poorly tumorigenic cell lines including mucinous type, we developed a novel xenograft model which could reconstruct the original tissue architecture through forced expression of a set of oncogenes followed by its silencing. With combination of the novel culture method and cell-derived xenograft system, virtually every epithelial ovarian cancer can be reconstituted in mice in a timely fashion.

Induction of non-apoptotic programmed cell death by oncogenic RAS in human epithelial cells and its suppression by MYC overexpression.

Oncogenic mutations of RAS genes, found in about 30% of human cancers, are considered to play important roles in cancer development. However, oncogenic RAS can also induce senescence in mouse and human normal fibroblasts. In some cell lines, oncogenic RAS has been reported to induce non-apoptotic programed cell death (PCD). Here, we investigated effects of oncogenic RAS expression in several types of normal human epithelial cells. Oncogenic RAS but not wild-type RAS stimulated macropinocytosis with accumulation of large-phase lucent vacuoles in the cytoplasm, subsequently leading to cell death which was indistinguishable from a recently proposed new type of PCD, methuosis. A RAC1 inhibitor suppressed accumulation of macropinosomes and overexpression of MYC attenuated oncogenic RAS-induced such accumulation, cell cycle arrest and cell death. MYC suppression or rapamycin treatment in some cancer cell lines harbouring oncogenic mutations in RAS genes induced cell death with accumulation of macropinosomes. These results suggest that this type of non-apoptotic PCD is a tumour-suppressing mechanism acting against oncogenic RAS mutations in normal human epithelial cells, which can be overcome by MYC overexpression, raising the possibility that its induction might be a novel approach to treatment of RAS-mutated human cancers.

Roles of the PDZ-binding motif of HPV 16 E6 protein in oncogenic transformation of human cervical keratinocytes.

The high-risk human papillomavirus E6 proteins have been shown to interact with and lead to degradation of PDZ-domain-containing proteins through its carboxy-terminal motif. This PDZ-binding motif plays important roles in transformation of cultured cells and carcinogenesis of E6-transgenic mice. However, its biological effects on the natural host cells have not been elucidated. We have examined its roles in an in vitro carcinogenesis model for cervical cancer, in which E6 and E7 together with activated HRAS (HRASG12V ) can induce tumorigenic transformation of normal human cervical keratinocytes. In this model, E6Δ151 mutant, which is defective in binding to PDZ domains, almost lost tumorigenic ability, whereas E6SAT mutant, which is defective in p53 degradation showed activity close to wild-type E6. Interestingly, we found decreased expression of PAR3 in E6-expressing cells independently of E6AP, which has not been previously recognized. Therefore, we knocked down several PDZ-domain containing proteins including PAR3 in human cervical keratinocytes expressing E7, HRASG12V and E6Δ151 to examine whether depletion of these proteins can restore the tumorigenic ability. Single knockdown of SCRIB, MAGI1 or PAR3 significantly but partially restored the tumorigenic ability. The combinatorial knockdown of SCRIB and MAGI1 cooperatively restored the tumorigenic ability, and additional depletion of PAR3 further enhanced the tumorigenic ability surpassing that induced by wild-type E6. These data highlight the importance of the carboxy-terminal motif of the E6 protein and downregulation of PAR3 in tumorigenic transformation of human cervical keratinocytes.

Human Papillomavirus 16 E6 Upregulates APOBEC3B via the TEAD Transcription Factor.

The cytidine deaminase APOBEC3B (A3B) underlies the genetic heterogeneity of several human cancers, including cervical cancer, which is caused by human papillomavirus (HPV) infection. We previously identified a region within the A3B promoter that is activated by the viral protein HPV16 E6 in human keratinocytes. Here, we discovered three sites recognized by the TEAD family of transcription factors within this region of the A3B promoter. Reporter assays in HEK293 cells showed that exogenously expressed TEAD4 induced A3B promoter activation through binding to these sites. Normal immortalized human keratinocytes expressing E6 (NIKS-E6) displayed increased levels of TEAD1/4 protein compared to parental NIKS. A series of E6 mutants revealed that E6-mediated degradation of p53 was important for increasing TEAD4 levels. Knockdown of TEADs in NIKS-E6 significantly reduced A3B mRNA levels, whereas ectopic expression of TEAD4 in NIKS increased A3B mRNA levels. Finally, chromatin immunoprecipitation assays demonstrated increased levels of TEAD4 binding to the A3B promoter in NIKS-E6 compared to NIKS. Collectively, these results indicate that E6 induces upregulation of A3B through increased levels of TEADs, highlighting the importance of the TEAD-A3B axis in carcinogenesis.IMPORTANCE The expression of APOBEC3B (A3B), a cellular DNA cytidine deaminase, is upregulated in various human cancers and leaves characteristic, signature mutations in cancer genomes, suggesting that it plays a prominent role in carcinogenesis. Viral oncoproteins encoded by human papillomavirus (HPV) and polyomavirus have been reported to induce A3B expression, implying the involvement of A3B upregulation in virus-associated carcinogenesis. However, the molecular mechanisms causing A3B upregulation remain unclear. Here, we demonstrate that exogenous expression of the cellular transcription factor TEAD activates the A3B promoter. Further, the HPV oncoprotein E6 increases the levels of endogenous TEAD1/4 protein, thereby leading to A3B upregulation. Since increased levels of TEAD4 are frequently observed in many cancers, an understanding of the direct link between TEAD and A3B upregulation is of broad oncological interest.

Cdt1-binding protein GRWD1 is a novel histone-binding protein that facilitates MCM loading through its influence on chromatin architecture.

Efficient pre-replication complex (pre-RC) formation on chromatin templates is crucial for the maintenance of genome integrity. However, the regulation of chromatin dynamics during this process has remained elusive. We found that a conserved protein, GRWD1 (glutamate-rich WD40 repeat containing 1), binds to two representative replication origins specifically during G1 phase in a CDC6- and Cdt1-dependent manner, and that depletion of GRWD1 reduces loading of MCM but not CDC6 and Cdt1. Furthermore, chromatin immunoprecipitation coupled with high-throughput sequencing (Seq) revealed significant genome-wide co-localization of GRWD1 with CDC6. We found that GRWD1 has histone-binding activity. To investigate the effect of GRWD1 on chromatin architecture, we used formaldehyde-assisted isolation of regulatory elements (FAIRE)-seq or FAIRE-quantitative PCR analyses, and the results suggest that GRWD1 regulates chromatin openness at specific chromatin locations. Taken together, these findings suggest that GRWD1 may be a novel histone-binding protein that regulates chromatin dynamics and MCM loading at replication origins.

Activation of NF-κB by human papillomavirus 16 E1 limits E1-dependent viral replication through degradation of E1.

UNLABELLED: NF-κB is a family of transcription factors that regulate gene expression involved in many processes, such as the inflammatory response and cancer progression. Little is known about associations of NF-κB with the human papillomavirus (HPV) life cycle. We have developed a tissue culture system to conditionally induce E1-dependent replication of the human papillomavirus 16 (HPV16) genome in human cervical keratinocytes and found that expression of HPV16 E1, a viral helicase, results in reduction of IκBα and subsequent activation of NF-κB in a manner dependent on helicase activity. Exogenous expression of a degradation-resistant mutant of IκBα, which inhibits the activation of NF-κB, enhanced E1-dependent replication of the viral genome. Wortmannin, a broad inhibitor of phosphoinositide 3-kinases (PI3Ks), and, to a lesser extent, VE-822, an ATR kinase inhibitor, but not KU55933, an ATM kinase inhibitor, suppressed the activation of NF-κB and augmented E1-dependent replication of the HPV16 genome. Interestingly, the enhancement of E1-dependent replication of the viral genome was associated with increased stability of E1 in the presence of wortmannin as well as the IκBα mutant. Collectively, we propose that expression of E1 induces NF-κB activation at least in part through the ATR-dependent DNA damage response and that NF-κB in turn limits E1-dependent replication of HPV16 through degradation of E1, so that E1 and NF-κB may constitute a negative feedback loop. IMPORTANCE: A major risk factor in human papillomavirus (HPV)-associated cancers is persistent infection with high-risk HPVs. To eradicate viruses from infected tissue, it is important to understand molecular mechanisms underlying the establishment and maintenance of persistent infection. In this study, we obtained evidence that human papillomavirus 16 (HPV16) E1, a viral DNA helicase essential for amplification of the viral genomes, induces NF-κB activation and that this limits E1-dependent genome replication of HPV16. These results suggest that NF-κB mediates a negative feedback loop to regulate HPV replication and that this feedback loop could be associated with control of the viral copy numbers. We could thus show for the first time that NF-κB activity is involved in the establishment and maintenance of persistent HPV infection.

A human cancer xenograft model utilizing normal pancreatic duct epithelial cells conditionally transformed with defined oncogenes.

Pancreatic ductal adenocarcinomas (PDACs) are considered to arise through neoplastic transformation of human pancreatic duct epithelial cells (HPDECs). In order to evaluate the biological significance of genetic and epigenetic alterations in PDACs, we isolated primary HPDECs and established an in vitro carcinogenesis model. Firstly, lentivirus-mediated transduction of KRAS(G12V), MYC and human papillomavirus 16 (HPV16) E6/E7 under the control of a tetracyclin-inducible promoter efficiently immortalized and transformed primary HPDECs, which gave rise to adenocarcinomas subcutaneously in an immune-deficient mouse xenograft model, depending on expression of the four genes. The tumors regressed promptly upon shutting-off the oncogenes, and the remaining tissues showed histological features corresponding to normal ductal structures with simple columnar epithelium. Reexpression of the oncogenes resulted in development of multiple PDACs through pancreatic intraepithelial neoplasia-like structures. We also succeeded in efficient immortalization of primary HPDECs with transduction of mutant CDK4, cyclin D1 and TERT. The cells maintained a normal diploid status and formed duct-like structures in a three-dimensional culture. In combination with p53 silencing, KRAS(G12V) alone was sufficient to fully transform the immortalized HPDECs, and MYC markedly accelerated the development of tumors. Our PDAC model supports critical roles of KRAS mutations, inactivation of the p53 and p16-pRB pathways, active telomerase and MYC expression in pancreatic carcinogenesis and thus recapitulates many features of human PDAC development. The present system with reversible control of oncogene expression enabled de novo development of PDAC from quasinormal human tissues preformed subcutaneously in mice and might be applicable to carcinogenesis models in many organ sites.

ATM regulates Cdt1 stability during the unperturbed S phase to prevent re-replication.

Ataxia-telangiectasia mutated (ATM) plays crucial roles in DNA damage responses, especially with regard to DNA double-strand breaks (DSBs). However, it appears that ATM can be activated not only by DSB, but also by some changes in chromatin architecture, suggesting potential ATM function in cell cycle control. Here, we found that ATM is involved in timely degradation of Cdt1, a critical replication licensing factor, during the unperturbed S phase. At least in certain cell types, degradation of p27(Kip1) was also impaired by ATM inhibition. The novel ATM function for Cdt1 regulation was dependent on its kinase activity and NBS1. Indeed, we found that ATM is moderately phosphorylated at Ser1981 during the S phase. ATM silencing induced partial reduction in levels of Skp2, a component of SCF(Skp2) ubiquitin ligase that controls Cdt1 degradation. Furthermore, Skp2 silencing resulted in Cdt1 stabilization like ATM inhibition. In addition, as reported previously, ATM silencing partially prevented Akt phosphorylation at Ser473, indicative of its activation, and Akt inhibition led to modest stabilization of Cdt1. Therefore, the ATM-Akt-SCF(Skp2) pathway may partly contribute to the novel ATM function. Finally, ATM inhibition rendered cells hypersensitive to induction of re-replication, indicating importance for maintenance of genome stability.

Noncanonical NOTCH signaling limits self-renewal of human epithelial and induced pluripotent stem cells through ROCK activation.

NOTCH plays essential roles in cell fate specification during embryonic development and in adult tissue maintenance. In keratinocytes, it is a key inducer of differentiation. ROCK, an effector of the small GTPase Rho, is also implicated in keratinocyte differentiation, and its inhibition efficiently potentiates immortalization of human keratinocytes and greatly improves survival of dissociated human pluripotent stem cells. However, the molecular basis for ROCK activation is not fully established in these contexts. Here we provide evidence that intracellular forms of NOTCH1 trigger the immediate activation of ROCK1 independent of its transcriptional activity, promoting differentiation and resulting in decreased clonogenicity of normal human keratinocytes. Knockdown of NOTCH1 abrogated ROCK1 activation and conferred sustained clonogenicity upon differentiation stimuli. Treatment with a ROCK inhibitor, Y-27632, or ROCK1 silencing substantially rescued the growth defect induced by activated NOTCH1. Furthermore, we revealed that impaired self-renewal of human induced pluripotent stem cells upon dissociation is, at least in part, attributable to NOTCH-dependent ROCK activation. Thus, the present study unveils a novel NOTCH-ROCK pathway critical for cellular differentiation and loss of self-renewal capacity in a subset of immature cells.

Heterocomplex formation by Arp4 and ß-actin is involved in the integrity of the Brg1 chromatin remodeling complex.

Although nuclear actin and Arps (actin-related proteins) are often identified as components of multi-protein chromatin-modifying enzyme complexes, such as chromatin remodeling and histone acetyltransferase (HAT) complexes, their molecular functions still remain largely elusive. Here, we investigated the role of human Arp4 (BAF53, also known as actin-like protein 6A) in Brg1-containing chromatin remodeling complexes. Depletion of Arp4 by RNA interference impaired the integrity of these complexes and accelerated the degradation of Brg1, indicating a crucial role in their maintenance, at least in certain human cell lines. We further found that Arp4 can form a heterocomplex with ß-actin. Based on structural similarities between conventional actin and Arp4, and the assumption that actin-Arp4 binding might mimic actin-actin binding, we introduced a series of mutations in Arp4 that might be expected to impair its interaction with ß-actin. Some of them indeed caused reduced binding to ß-actin. Interestingly, such mutant Arp4 proteins also showed reduced incorporation into Brg1 complexes, and their interaction with Myc-associated complexes as well as Tip60 HAT complexes were also impaired. Based on these findings, we propose that ß-actin-Arp4 complex formation might be a crucial feature in some chromatin-modifying enzyme complexes, such as the Brg1 complex.

A critical role of MYC for transformation of human cells by HPV16 E6E7 and oncogenic HRAS.

Human papillomaviruses (HPVs) are the primary causal agents for development of cervical cancer, and deregulated expression of two viral oncogenes E6 and E7 is considered to contribute to disease initiation. Recently, we have demonstrated that transduction of oncogenic HRAS (HRAS(G12V)) and MYC together with HPV16 E6E7 is sufficient for tumorigenic transformation of normal human cervical keratinocytes (HCKs). Here, we show that transduction of HRAS(G12V) on the background of E6E7 expression causes accumulation of MYC protein and tumorigenic transformation of not only normal HCKs but also other normal primary human cells, including tongue keratinocytes and bronchial epithelial cells as well as hTERT-immortalized foreskin fibroblasts. Subcutaneous transplantation of as few as 200 HCKs expressing E6E7 and HRAS(G12V) resulted in tumor formation within 2 months. Dissecting RAS signaling pathways, constitutively active forms of AKT1 or MEK1 did not result in tumor formation with E6E7, but tumorigenic transformation was induced with addition of MYC. Increased MYC expression endowed resistance to calcium- and serum-induced terminal differentiation and activated the mammalian target of rapamycin (mTOR) pathway. An mTOR inhibitor (Rapamycin) and MYC inhibition a level not affecting proliferation in culture both markedly suppressed tumor formation by HCKs expressing E6E7 and HRAS(G12V). These results suggest that a single mutation of HRAS could be oncogenic in the background of deregulated expression of E6E7 and MYC plays a critical role in cooperation with the RAS signaling pathways in tumorigenesis. Thus inhibition of MYC and/or the downstream mTOR pathway could be a therapeutic strategy not only for the MYC-altered but also RAS-activated cancers.

The E1 protein of human papillomavirus type 16 is dispensable for maintenance replication of the viral genome.

Papillomavirus genomes are thought to be amplified to about 100 copies per cell soon after infection, maintained constant at this level in basal cells, and amplified for viral production upon keratinocyte differentiation. To determine the requirement for E1 in viral DNA replication at different stages, an E1-defective mutant of the human papillomavirus 16 (HPV16) genome featuring a translation termination mutation in the E1 gene was used. The ability of the mutant HPV16 genome to replicate as nuclear episomes was monitored with or without exogenous expression of E1. Unlike the wild-type genome, the E1-defective HPV16 genome became established in human keratinocytes only as episomes in the presence of exogenous E1 expression. Once established, it could replicate with the same efficiency as the wild-type genome, even after the exogenous E1 was removed. However, upon calcium-induced keratinocyte differentiation, once again amplification was dependent on exogenous E1. These results demonstrate that the E1 protein is dispensable for maintenance replication but not for initial and productive replication of HPV16.

An in vitro multistep carcinogenesis model for both HPV-positive and -negative human oral squamous cell carcinomas.

Oral squamous cell carcinomas (OSCCs) are considered to arise from human oral keratinocytes. DNAs of human papillomaviruses (HPVs), predominantly types 16 and 18, etiological agents of cervical cancer, have been detected in approximately 25% of OSCCs. In accordance with the established role of E6 and E7 in inactivating p53 and pRB, respectively, mutations of p53 and inactivation of p16(INK4a) are frequently observed in HPV-negative OSCCs. In addition, other alterations such as overexpression of epidermal growth factor receptor (EGFR) are often observed in both HPV-positive and -negative OSCCs. However, causal-relationships between accumulation of these abnormalities and multi-step carcinogenesis are not fully understood. To elucidate underlying processes, we transduced either HPV16 E6/E7 or mutant CDK4 (CDK4(R24C)), cyclin D1 and human telomerase reverse transcriptase (TERT) into primary human tongue keratinocytes (HTK), and obtained immortal cell populations, HTK-16E6E7 and HTK-K4DT. Additional transduction of oncogenic HRAS or EGFR together with MYC into the HTK-16E6E7 and dominant-negative p53 expressing HTK-K4DT resulted in anchorage-independent growth and subcutaneous tumor formation in nude mice. These results indicate that either HRAS mutation or activation of EGFR in cooperation with MYC overexpression play critical roles in transformation of HTKs on a background of inactivation of the pRB and p53 pathways and telomerase activation. This in vitro model system recapitulating the development of OSCCs should facilitate further studies of mechanisms of carcinogenesis in the oral cavity.

Chromatin remodeler sucrose nonfermenting 2 homolog (SNF2H) is recruited onto DNA replication origins through interaction with Cdc10 protein-dependent transcript 1 (Cdt1) and promotes pre-replication complex formation.

From late mitosis to the G(1) phase of the cell cycle, ORC, CDC6, and Cdt1 form the machinery necessary to load MCM2-7 complexes onto DNA. Here, we show that SNF2H, a member of the ATP-dependent chromatin-remodeling complex, is recruited onto DNA replication origins in human cells in a Cdt1-dependent manner and positively regulates MCM loading. SNF2H physically interacted with Cdt1. ChIP assays indicated that SNF2H associates with replication origins specifically during the G(1) phase. Binding of SNF2H at origins was decreased by Cdt1 silencing and, conversely, enhanced by Cdt1 overexpression. Furthermore, SNF2H silencing prevented MCM loading at origins and moderately inhibited S phase progression. Although neither SNF2H overexpression nor SNF2H silencing appeared to impact rereplication induced by Cdt1 overexpression, Cdt1-induced checkpoint activation was inhibited by SNF2H silencing. Collectively, these data suggest that SNF2H may promote MCM loading at DNA replication origins via interaction with Cdt1 in human cells. Because efficient loading of excess MCM complexes is thought to be required for cells to tolerate replication stress, Cdt1- and SNF2H-mediated promotion of MCM loading may be biologically relevant for the regulation of DNA replication.

DeltaNp63alpha repression of the Notch1 gene supports the proliferative capacity of normal human keratinocytes and cervical cancer cells.

The p53 family member p63 is a master regulator of epithelial development. One of its isoforms, DeltaNp63alpha, is predominantly expressed in the basal cells of stratified epithelia and plays a fundamental role in control of regenerative potential and epithelial integrity. In contrast to p53, p63 is rarely mutated in human cancers, but it is frequently overexpressed in squamous cell carcinomas (SCC). However, its functional relevance to tumorigenesis remains largely unclear. We previously identified the Notch1 gene as a novel transcriptional target of p53. Here, we show that DeltaNp63alpha functions as a transcriptional repressor of the Notch1 gene through the p53-responsive element. Knockdown of p63 caused upregulation of Notch1 expression and marked reduction in proliferation and clonogenicity of both normal human keratinocytes and cervical cancer cell lines overexpressing DeltaNp63alpha. Concomitant silencing of Notch1 significantly rescued this phenotype, indicating the growth defect induced by p63 deficiency to be, at least in part, attributable to Notch1 function. Conversely, overexpression of DeltaNp63alpha decreased basal levels of Notch1, increased proliferative potential of normal human keratinocytes, and inhibited both p53-dependent and p53-independent induction of Notch1 and differentiation markers upon genotoxic stress and serum exposure, respectively. These results suggest that DeltaNp63alpha maintains the self-renewing capacity of normal human keratinocytes and cervical cancer cells partly through transcriptional repression of the Notch1 gene and imply a novel pathogenetical significance of frequently observed overexpression of DeltaNp63alpha together with p53 inactivation in SCCs.

CDC6 interaction with ATR regulates activation of a replication checkpoint in higher eukaryotic cells.

CDC6, a replication licensing protein, is partially exported to the cytoplasm in human cells through phosphorylation by Cdk during S phase, but a significant proportion remains in the nucleus. We report here that human CDC6 physically interacts with ATR, a crucial checkpoint kinase, in a manner that is stimulated by phosphorylation by Cdk. CDC6 silencing by siRNAs affected ATR-dependent inhibition of mitotic entry elicited by modest replication stress. Whereas a Cdk-phosphorylation-mimicking CDC6 mutant could rescue the checkpoint defect by CDC6 silencing, a phosphorylation-deficient mutant could not. Furthermore, we found that the CDC6-ATR interaction is conserved in Xenopus. We show that the presence of Xenopus CDC6 during S phase is essential for Xenopus ATR to bind to chromatin in response to replication inhibition. In addition, when human CDC6 amino acid fragment 180-220, which can bind to both human and Xenopus ATR, was added to Xenopus egg extracts after assembly of the pre-replication complex, Xenopus Chk1 phosphorylation was significantly reduced without lowering replication, probably through a sequestration of CDC6-mediated ATR-chromatin interaction. Thus, CDC6 might regulate replication-checkpoint activation through the interaction with ATR in higher eukaryotic cells.

Redundant and differential regulation of multiple licensing factors ensures prevention of re-replication in normal human cells.

When human cells enter S-phase, overlapping differential inhibitory mechanisms downregulate the replication licensing factors ORC1, CDC6 and Cdt1. Such regulation prevents re-replication so that deregulation of any individual factor alone would not be expected to induce overt re-replication. However, this has been challenged by the fact that overexpression of Cdt1 or Cdt1+CDC6 causes re-replication in some cancer cell lines. We thought it important to analyze licensing regulations in human non-cancerous cells that are resistant to Cdt1-induced re-replication and examined whether simultaneous deregulation of these licensing factors induces re-replication in two such cell lines, including human fibroblasts immortalized by telomerase. Individual overexpression of either Cdt1, ORC1 or CDC6 induced no detectable re-replication. However, with Cdt1+ORC1 or Cdt1+CDC6, some re-replication was detectable and coexpression of Cdt1+ORC1+CDC6 synergistically acted to give strong re-replication with increased mini-chromosome maintenance (MCM) loading. Coexpression of ORC1+CDC6 was without effect. These results suggest that, although Cdt1 regulation is the key step, differential regulation of multiple licensing factors ensures prevention of re-replication in normal human cells. Our findings also show for the first time the importance of ORC1 regulation for prevention of re-replication.

Oncogenic transformation of human ovarian surface epithelial cells with defined cellular oncogenes.

Ovarian surface epithelium (OSE) is considered to give rise to epithelial ovarian carcinomas (EOCs). To elucidate early processes contributing to the development of EOCs from the OSE, two batches of primary human OSE cells were transduced with non-viral human genes (mutant Cdk4, cyclinD1 and hTERT) so as to efficiently establish normal diploid OSE cells without chromosomal instability. Then defined genetic alterations frequently observed in EOCs were transduced into the OSE cells. A combination of p53 inactivation and oncogenic Kras transduction did not confer tumor-forming ability in immunodeficient mice, though additional transduction of Akt or combined transduction of c-myc with bcl-2 did result in tumor formation. In the latter case, tumors demonstrated phenotypes reminiscent of human EOCs, including cytokeratin expression, a highly aggressive phenotype, metastatic behavior and formation of ascites. These results indicate that inactivation of p53 and activation of the Ras pathway play critical roles in ovarian carcinogenesis in co-operation with the Akt or c-myc pathways. This first in vitro model system faithfully recapitulating the development of EOCs using normal human OSE cells should greatly facilitate further studies of EOCs.