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.

Oncogenic Ras/ERK signaling activates CDCP1 to promote tumor invasion and metastasis.

UNLABELLED: Involvement of Ras in cancer initiation is known, but recent evidence indicates a role in cancer progression, including metastasis and invasion; however, the mechanism is still unknown. In this study, it was determined that human lung cancer cells with Ras mutations, among other popular mutations, showed significantly higher expression of CUB domain-containing protein 1 (CDCP1) than those without. Furthermore, activated Ras clearly induced CDCP1, whereas CDCP1 knockdown or inhibition of CDCP1 phosphorylation by Src-directed therapy abrogated anoikis resistance, migration, and invasion induced by activated-Ras. Activation of MMP2 and secretion of MMP9, in a model of Ras-induced invasion, was found to be regulated through induction of phosphorylated CDCP1. Thus, CDCP1 is required for the functional link between Ras and Src signaling during the multistage development of human malignant tumors, highlighting CDCP1 as a potent target for treatment in the broad spectrum of human cancers associated with these oncogenes. IMPLICATIONS: CDCP1 protein induced by oncogenic Ras/Erk signaling is essential for Ras-mediated metastatic potential of cancer cells.

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.

NRF2 mutation confers malignant potential and resistance to chemoradiation therapy in advanced esophageal squamous cancer.

Esophageal squamous cancer (ESC) is one of the most aggressive tumors of the gastrointestinal tract. A combination of chemotherapy and radiation therapy (CRT) has improved the clinical outcome, but the molecular background determining the effectiveness of therapy remains unknown. NRF2 is a master transcriptional regulator of stress adaptation, and gain of-function mutation of NRF2 in cancer confers resistance to stressors including anticancer therapy. Direct resequencing analysis revealed that Nrf2 gain-of-function mutation occurred recurrently (18/82, 22%) in advanced ESC tumors and ESC cell lines (3/10). The presence of Nrf2 mutation was associated with tumor recurrence and poor prognosis. Short hairpin RNA-mediated down-regulation of NRF2 in ESC cells that harbor only mutated Nrf2 allele revealed that themutant NRF2 conferred increased cell proliferation, attachment-independent survival, and resistance to 5-fluorouracil and γ-irradiation. Based on the Nrf2 mutation status, gene expression signatures associated with NRF2 mutation were extracted from ESC cell lines, and their potential utility for monitoring and prognosis was examined in a cohort of 33 pre-CRT cases of ESC. The molecular signatures of NRF2 mutation were significantly predictive and prognostic for CRT response. In conclusion, recurrent NRF2 mutation confers malignant potential and resistance to therapy in advanced ESC, resulting in a poorer outcome. Molecular signatures of NRF2 mutation can be applied as predictive markers of response to CRT, and efficient inhibition of aberrant NRF2 activation could be a promising approach in combination with CRT.

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.

[Mechanisms of high-risk human papillomavirus-induced cervical carcinogenesis].

Human papillomaviruses (HPVs) are believed to be the primary causal agents for development of pre-neoplastic and malignant lesions of the uterine cervix and high-risk types such as type 16 and 18 are associated with more than 90% of all cervical carcinomas. The E6 and E7 genes of HPVs are thought to play causative roles, since E6 promotes the degradation of p53 through its interaction with E6AP, an E3 ubiquitin ligase, whereas E7 binds to the retinoblastoma protein pRb and disrupts its complex formation with E2F transcription factors. Although prophylactic vaccines have become available, it is still necessary to clarify the mechanisms of HPV-induced carcinogenesis because of the widespread nature of HPV infection. In this article, the mechanisms of high-risk HPV E6 and E7-induced multistep carcinogenesis and recently identified functions of these oncoproteins are reviewed.

Involvement of human ORC and TRF2 in pre-replication complex assembly at telomeres.

The origin recognition complex (ORC) binds to replication origins to regulate the cell cycle-dependent assembly of pre-replication complexes (pre-RCs). We have found a novel link between pre-RC assembly regulation and telomere homeostasis in human cells. Biochemical analyses showed that human ORC binds to TRF2, a telomere sequence-binding protein that protects telomeres and functions in telomere length homeostasis, via the ORC1 subunit. Immunostaining further revealed that ORC and TRF2 partially co-localize in nuclei, whereas chromatin immunoprecipitation analyses confirmed that pre-RCs are assembled at telomeres in a cell cycle-dependent manner. Over-expression of TRF2 stimulated ORC and MCM binding to chromatin and RNAi-directed TRF2 silencing resulted in reduced ORC binding and pre-RC assembly at telomeres. As expected from previous reports, TRF2 silencing induced telomere elongation. Interestingly, ORC1 silencing by RNAi weakened the TRF2 binding as well as the pre-RC assembly at telomeres, suggesting that ORC and TRF2 interact with each other to achieve stable binding. Furthermore, ORC1 silencing also resulted in modest telomere elongation. These data suggest that ORC might be involved in telomere homeostasis in human cells.

An in vitro multistep carcinogenesis model for human cervical cancer.

Human papillomaviruses (HPV) are believed to be the primary causal agents for development of cervical cancer, and deregulated expression of two viral oncogenes E6 and E7 in basal cells, mostly by integration, is considered to be a critical event for disease progression. However, lines of evidence suggest that, besides expression of E6 and E7 genes, additional host genetic alterations are required for cancer development. To directly test this hypothesis, we first transduced HPV16 E6 and E7 with or without hTERT into several lines of normal human cervical keratinocytes (HCK) from independent donors and then searched for additional alterations required for carcinogenesis. Oncogenic Hras(G12V) (Hras) provided marked tumor forming ability in nude mice and ErbB2 or c-Myc (Myc) endowed weaker but significant tumor forming ability. Combined transduction of Myc and Hras to HCKs expressing E6 and E7 resulted in the creation of highly potent tumor-initiating cells. These results show that only one or two genetic changes occurring after deregulated expression of high-risk HPV oncogenes might be sufficient for development of cervical cancer.

Identification of novel human Cdt1-binding proteins by a proteomics approach: proteolytic regulation by APC/CCdh1.

In mammalian cells, Cdt1 activity is strictly controlled by multiple independent mechanisms, implying that it is central to the regulation of DNA replication during the cell cycle. In fact, unscheduled Cdt1 hyperfunction results in rereplication and/or chromosomal damage. Thus, it is important to understand its function and regulations precisely. We sought to comprehensively identify human Cdt1-binding proteins by a combination of Cdt1 affinity chromatography and liquid chromatography and tandem mass spectrometry analysis. Through this approach, we could newly identify 11 proteins, including subunits of anaphase-promoting complex/cyclosome (APC/C), SNF2H and WSTF, topoisomerase I and IIalpha, GRWD1/WDR28, nucleophosmin/nucleoplasmin, and importins. In vivo interactions of Cdt1 with APC/C(Cdh1), SNF2H, topoisomerase I and IIalpha, and GRWD1/WDR28 were confirmed by coimmunoprecipitation assays. A further focus on APC/C(Cdh1) indicated that this ubiquitin ligase controls the levels of Cdt1 during the cell cycle via three destruction boxes in the Cdt1 N-terminus. Notably, elimination of these destruction boxes resulted in induction of strong rereplication and chromosomal damage. Thus, in addition to SCF(Skp2) and cullin4-based ubiquitin ligases, APC/C(Cdh1) is a third ubiquitin ligase that plays a crucial role in proteolytic regulation of Cdt1 in mammalian cells.

CUB domain-containing protein 1 is a novel regulator of anoikis resistance in lung adenocarcinoma.

Malignant tumor cells frequently achieve resistance to anoikis, a form of apoptosis induced by detachment from the basement membrane, which results in the anchorage-independent growth of these cells. Although the involvement of Src family kinases (SFKs) in this alteration has been reported, little is known about the signaling pathways involved in the regulation of anoikis under the control of SFKs. In this study, we identified a membrane protein, CUB-domain-containing protein 1 (CDCP1), as an SFK-binding phosphoprotein associated with the anchorage independence of human lung adenocarcinoma. Using RNA interference suppression and overexpression of CDCP1 mutants in lung cancer cells, we found that tyrosine-phosphorylated CDCP1 is required to overcome anoikis in lung cancer cells. An apoptosis-related molecule, protein kinase Cdelta, was found to be phosphorylated by the CDCP1-SFK complex and was essential for anoikis resistance downstream of CDCP1. Loss of CDCP1 also inhibited the metastatic potential of the A549 cells in vivo. Our findings indicate that CDCP1 is a novel target for treating cancer-specific disorders, such as metastasis, by regulating anoikis in lung adenocarcinoma.

Basic mechanisms of high-risk human papillomavirus-induced carcinogenesis: roles of E6 and E7 proteins.

Human papillomaviruses (HPV) are believed to be the primary causal agents for development of pre-neoplastic and malignant lesions of the uterine cervix, and high-risk types such as type 16 and 18 are associated with more than 90% of all cervical carcinomas. The E6 and E7 genes of HPV are thought to play causative roles, since E6 promotes the degradation of p53 through its interaction with E6AP, an E3 ubiquitin ligase, whereas E7 binds to the retinoblastoma protein (pRb) and disrupts its complex formation with E2F transcription factors. Although prophylactic vaccines have become available, it is still necessary to clarify the mechanisms of HPV-induced carcinogenesis because of the widespread nature of HPV infection. Approximately 493,000 new cases of cervical cancer are diagnosed each year with approximately 274,000 mortalities due to invasive cervical cancer. In the present article, the mechanisms of HPV16 E6- and E7-induced multistep carcinogenesis and recently identified functions of these onco-proteins are reviewed.

Regulation of Notch1 gene expression by p53 in epithelial cells.

The E6 protein of cervical cancer-associated human papillomaviruses (HPVs) is known to suppress keratinocyte differentiation through unidentified mechanisms. Notch1 is a determinant of keratinocyte differentiation and functions as a tumor suppressor in mammalian epidermis. Here, we report that the Notch1 gene is a novel target of p53 and can be down-regulated by E6 through p53 degradation in normal human epithelial cells. Thus, inactivation of p53 by E6 or short-hairpin RNA (shRNA) resulted in reduced Notch1 expression at the transcription level, and a p53-responsive element could be identified in the Notch1 promoter. The expression of E6, p53 shRNA, or Notch1 shRNA suppressed both spontaneous keratinocyte differentiation in culture and its induction upon DNA damage. Furthermore, the induction of Notch1 and differentiation makers as well as thickening of the epidermal layer upon UV irradiation was observed in wild-type but not in p53-deficient mouse skin. Together, our findings not only demonstrate a novel link between p53 and Notch1 in keratinocyte differentiation upon genotoxic stress but also suggest a novel tumor suppressor mechanism of p53 in the development of squamous cell carcinomas, including HPV-induced tumors.

Efficient immortalization of primary human cells by p16INK4a-specific short hairpin RNA or Bmi-1, combined with introduction of hTERT.

Activation of telomerase is sufficient for immortalization of some types of human cells but additional factors may also be essential. It has been proposed that stress imposed by inadequate culture conditions induces senescence due to accumulation of p16(INK4a). Here, we present evidence that many human cell types undergo senescence by activation of the p16(INK4a)/Rb pathway, and that introduction of Bmi-1 can inhibit p16(INK4a) expression and extend the life span of human epithelial cells derived from skin, mammary gland and lung. Introduction of p16(INK4a)-specific short hairpin RNA, as well as Bmi-1, suppressed p16(INK4a) expression in human mammary epithelial cells without promoter methylation, and extended their life span. Subsequent introduction of hTERT, the telomerase catalytic subunit, into cells with low p16(INK4a) levels resulted in efficient immortalization of three cell types without crisis or growth arrest. The majority of the human mammary epithelial cells thus immortalized showed almost normal ploidy as judged by G-banding and spectral karyotyping analysis. Our data suggest that inhibition of p16(INK4a) and introduction of hTERT can immortalize many human cell types with little chromosomal instability.

E6AP-dependent degradation of DLG4/PSD95 by high-risk human papillomavirus type 18 E6 protein.

In most cervical cancers, DNAs of high-risk mucosotropic human papillomaviruses (HPVs), such as types 16 and 18, are maintained so as to express two viral proteins, E6 and E7, suggesting that they play important roles in carcinogenesis. The carboxy-terminal PDZ domain-binding motif of the E6 proteins is in fact essential for transformation of rodent cells and induction of hyperplasia in E6-transgenic mouse skin. To date, seven PDZ domain-containing proteins, including DLG1/hDLG, which is a human homologue of the Drosophila discs large tumor suppressor (Dlg), have been identified as targets of high-risk HPV E6 proteins. Here, we describe DLG4/PSD95, another human homologue of Dlg, as a novel E6 target. DLG4 was found to be expressed in normal human cells, including cervical keratinocytes, but only to a limited extent in both HPV-positive and HPV-negative cervical cancer cell lines. Expression of HPV18 E6 in HCK1T decreased DLG4 levels more strongly than did HPV16 E6, the carboxy-terminal motif of the proteins being critical for binding and degradation of DLG4 in vitro. DLG4 levels were restored by expression of either E6AP-specific short hairpin RNA or bovine papillomavirus type 1 E2 in HeLa but not CaSki or SiHa cells, reflecting downregulation of DLG4 mRNA as opposed to protein by an HPV-independent mechanism in HPV16-positive cancer lines. The tumorigenicity of CaSki cells was strongly inhibited by forced expression of DLG4, while growth in culture was not inhibited at all. These results suggest that DLG4 may function as a tumor suppressor in the development of HPV-associated cancers.

Deregulation of Cdt1 induces chromosomal damage without rereplication and leads to chromosomal instability.

The activity of human Cdt1 is negatively regulated by multiple mechanisms. This suggests that Cdt1 deregulation may have a deleterious effect. Indeed, it has been suggested that overexpression of Cdt1 can induce rereplication in cancer cells and that rereplication activates Ataxia-telangiectasia-mutated (ATM) kinase and/or ATM- and Rad3-related (ATR) kinase-dependent checkpoint pathways. In this report, we highlight a new and interesting aspect of Cdt1 deregulation: data from several different systems all strongly indicate that unregulated Cdt1 overexpression at pathophysiological levels can induce chromosomal damage other than rereplication in non-transformed cells. The most important finding in these studies is that deregulated Cdt1 induces chromosomal damage and activation of the ATM-Chk2 DNA damage checkpoint pathway even in quiescent cells. These Cdt1 activities are negatively regulated by cyclin A/Cdks, probably through modification by phosphorylation. Furthermore, we found that deregulated Cdt1 induces chromosomal instability in normal human cells. Since Cdt1 is overexpressed in cancer cells, this would be a new molecular mechanism leading to carcinogenesis.