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Purpose and Experimental Design: Despite the therapeutic utility of progestin in invasive and preinvasive endometrial neoplasias, the molecular mechanisms through which it exerts inhibitory effects on endometrial epithelial growth are largely unknown. The aim of the study was to clarify the molecular mechanisms of progestin action to endometrial epithelial cells using originally established in vitro and in vivo treatment models for immortalized and transformed endometrial epithelial cell lines that express progesterone receptor., Results: In this model, progestin effectively inhibited the cell growth, inducing G0/G1 arrest rather than apoptosis without p21/WAF-1 induction. Using DNA microarray analysis, we identified 24 genes whose expression increased more than 10-fold on progestin treatment. Of these genes, we paid special attention to forkhead box transcription factor FOXO1, known as a key gene for endometrial decidualization. Progestin markedly induced FOXO1 gene expression mainly in the nuclei in vitro and in vivo. This induction was not due to the canonical activation of FOXO1 via protein dephosphorylation but due to FOXO1 promoter activation and mRNA induction. siRNA inhibition of FOXO1 significantly attenuated the effects of progestin to inhibit endometrial epithelial cell growth. Disrupting Akt activity by the introduction of the dominant negative form of Akt increased nuclear FOXO1 accumulation and enhanced the effect of progestin., Conclusion: These findings suggest that FOXO1 is a direct target of progestin, implicating novel molecular mechanisms of progestin to eradicate endometrial neoplasia., (©2010 AACR.)

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.

Objective: Dysregulated expression of disintegrin-metalloprotease proteins [a disintegrin and metalloproteases (ADAMs) and ADAMs with thrombospondin motif (ADAMTSs)] has been reported in many types of cancers and is believed to play an important role in cancer formation and metastasis. However, little is known about the expression of ADAMs and ADAMTSs in the development of human cervical cancer., Methods: Reverse transcriptase polymerase chain reaction and immunoblotting were performed to assess the expression of several disintegrin-metalloproteases and tissue inhibitors of metalloproteinases (TIMPs) in squamous-type cervical cancer cells and oncogenically modified keratinocytes (immortalized human cervical keratinocytes transduced with human papilloma virus-16 E6/E7 proteins with or without oncogenes). Immunohistochemistry of ADAM-9, ADAM-10 and TIMP-3 was performed on 31 primary human cervical tissue specimens of preinvasive and invasive cervical carcinoma., Results: mRNA levels of ADAM-9, ADAM-10, ADAM-12, TIMP-2 and TIMP-3 were upregulated as cervical cells progressed from dysplastic to malignant lesions compared to normal cervical cells. These results were corroborated at the protein level by Western blot analysis and immunohistochemistry., Conclusion: The expression of disintegrin-metalloproteases and their endogenous regulators was dysregulated during cervical carcinogenesis. The aberrant expression of ADAMs might contribute to the pathogenesis of cervical cancer formation and progression., (Copyright © 2011 S. Karger AG, Basel.)

Epithelial-mesenchymal transition (EMT) is a critical phenotypic alteration of cancer cells that triggers invasion and metastasis. Lung cancer cells often show mesenchymal phenotypes; however, a causative genetic alteration for the induction of EMT in lung cancer cells remains unknown. Recent studies have shown that the LKB1 gene is mutated in up to one-third of lung adenocarcinomas. Therefore, to pursue the possible involvement of LKB1 inactivation in the induction of EMT in lung carcinogenesis, we generated immortalized lung epithelial cells and lung adenocarcinoma cells with stable or transient LKB1 knockdown. LKB1 knockdown increased cell motility and invasiveness, and induced the expression of several mesenchymal marker proteins accompanied by the expression of ZEB1, a transcriptional repressor for E-cadherin and an EMT inducer. In agreement with the recent findings, expression of miR-200a/c was inversely correlated with that of ZEB1 in LKB1 knockdown clones with mesenchymal phenotype. Furthermore, transient knockdown of LKB1 induced ZEB1 mRNA and increased cell motility, and this motility was suppressed by ZEB1 repression. These results strongly indicate that LKB1 inactivation triggers EMT in lung cancer cells through the induction of ZEB1., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

14-3-3 proteins control various cellular processes, including cell cycle progression and DNA damage checkpoint. At the DNA damage checkpoint, some subtypes of 14-3-3 (beta and zeta isoforms in mammalian cells and Rad24 in fission yeast) bind to Ser345-phosphorylated Chk1 and promote its nuclear retention. Here, we report that 14-3-3gamma forms a complex with Chk1 phosphorylated at Ser296, but not at ATR sites (Ser317 and Ser345). Ser296 phosphorylation is catalysed by Chk1 itself after Chk1 phosphorylation by ATR, and then ATR sites are rapidly dephosphorylated on Ser296-phosphorylated Chk1. Although Ser345 phosphorylation is observed at nuclear DNA damage foci, it occurs more diffusely in the nucleus. The replacement of endogenous Chk1 with Chk1 mutated at Ser296 to Ala induces premature mitotic entry after ultraviolet irradiation, suggesting the importance of Ser296 phosphorylation in the DNA damage response. Although Ser296 phosphorylation induces the only marginal change in Chk1 catalytic activity, 14-3-3gamma mediates the interaction between Chk1 and Cdc25A. This ternary complex formation has an essential function in Cdc25A phosphorylation and degradation to block premature mitotic entry after DNA damage.

Human papillomavirus (HPV) infection is a causative event for the development of uterine cervical carcinoma. Human papillomavirus (HPV) 16, 18, and 33 DNA has been also detected frequently in lung adenocarcinomas (AdCs) in East Asian countries; however, its prevalence in Japan remains unclear. We therefore screened for HPV 16/18/33 DNA in 297 lung AdCs in a Japanese population by multiplex PCR with type-specific primers. As reported previously, HPV 16 DNA was detected in two cervical cancer cell lines, CaSki and SiHa, while HPV 18 DNA was detected in HeLa cells, and 0.1-1.0 copies of HPV-DNA per cell were detectable by this method. However, with this method, none of the 297 lung AdCs showed positive signals for HPV 16/18/33 DNA, indicating that HPV-DNA is not or is very rarely integrated in lung AdC genomes in the Japanese. Furthermore, none of the lung AdCs showed positive signals by nested PCR with HPV 16/18 type-specific primers. Therefore, we further attempted to detect HPV 16/18/33 DNA in 91 lung cancer cell lines, including 40 AdC cell lines. Among them, 30 have been established in Japan and the remaining 61 in the USA. No HPV signals were obtained in any of the 91 cell lines by either multiplex or nested PCR, while the p53 gene was mutated in 81 of them including 35 of the 40 AdC cell lines. These results indicate that HPV 16/18/33 infection does not play a major role in the development of lung AdC in Japan nor in the USA.

The transcutaneous communication system (TCS) is one of the key technologies for monitoring and controling artificial hearts and other artificial organs in the body. In this study, we have developed a new TCS that uses the human body as a conductive medium. Having no energy conversion from electric currents into electromagnetic waves and light provides energy-saving data transmission with a simple electrical circuit. Each unit of the TCS mainly consists of two electrodes, an amplitude shift keying (ASK) modulator and an ASK demodulator (carrier frequency: 4 and 10 MHz). A resonant frequency of an L-C tank circuit including the capacitance component of the body is tuned into each carrier frequency in order to apply the data current effectively into the body. Performance of the TCS was evaluated by a communication test on the surface of a human body. The TCS was able to transmit 3,315 bytes of data bi-directionally at a transmission rate of 115 kbps from a left wrist to a right forearm, to an abdomen and to a left calf without communication error. The power consumption of each TCS unit was 125 mW with an ASK modulated current of 7 mA (RMS). While further study is required to secure its safety, the TCS promises to be a next-generation transcutaneous communication device.

We reported previously that the forced expression of the chemokine BRAK/CXCL14 in head and neck squamous cell carcinoma cells decreased the rate of tumor formation and size of tumor xenografts in athymic nude mice and SCID mice. In order to clarify the expression of BRAK/CXCL14 affected either the settlement of carcinoma cells in host tissues in vivo or proliferation of the colonized carcinoma cells or both, we prepared oral floor carcinoma-derived HSC-2 cells in which BRAK/CXCL14 expression was induced upon doxycycline treatment. Then 30 nude mice were separated into 3 groups composed of 10 mice per group: Group I, the control, in which the engineered cells were directly xenografted onto the back of the mice; Group II, the cells were xenografted and then the mice were treated with doxycycline; and Group III, the cells were pretreated with doxycycline during culture, and the host mice were also treated with the drug before and after xenografting. The effects of BRAK/CXCL14 expression were examined by measuring the tumor size. The order of the size of tumor xenografts was Group I > II > III, even though the growth rate of the engineered cells was the same whether or not the cells were cultured in the presence of the drug. In addition, the size of tumors was significantly down-regulated after xenografting the doxycycline-pretreated cells in Group III. These data indicate that BRAK/CXCL14 expression in oral floor carcinoma cells reduced both the rate of settlement and the proliferation of the cells in vivo after settlement of the 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., ((c)2010 AACR.)

Several tumor viruses, such as human T-cell leukemia virus (HTLV), human papilloma virus (HPV), human adenovirus, have high-oncogenic and low-oncogenic subtypes, and such subtype-specific oncogenesis is associated with the PDZ-domain binding motif (PBM) in their transforming proteins. HTLV-1, the causative agent of adult T-cell leukemia, encodes Tax1 with PBM as a transforming protein. The Tax1 PBM was substituted with those from other oncoviruses, and the transforming activity was examined. Tax1 mutants with PBM from either HPV-16 E6 or adenovirus type 9 E4ORF1 are fully active in the transformation of a mouse T-cell line from interleukin-2-dependent growth into independent growth. Interestingly, one such Tax1 PBM mutant had an extra amino acid insertion derived from E6 between PBM and the rest of Tax1, thus suggesting that the amino acid sequences of the peptides between PBM and the rest of Tax1 and the numbers only slightly affect the function of PBM in the transformation. Tax1 and Tax1 PBM mutants interacted with tumor suppressors Dlg1 and Scribble with PDZ-domains. Unlike E6, Tax1 PBM mutants as well as Tax1 did not or minimally induced the degradations of Dlg1 and Scribble, but instead induced their subcellular translocation from the detergent-soluble fraction into the insoluble fraction, thus suggesting that the inactivation mechanism of these tumor suppressor proteins is distinct. The present results suggest that PBMs of high-risk oncoviruses have a common function(s) required for these three tumor viruses to transform cells, which is likely associated with the subtype-specific oncogenesis of these tumor viruses.

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.

Proteins of avian egg albumin have been suggested to play various biological roles during the development of chick embryo to confer protection. Recently, we have shown that ovotransferrin (OTf), the second major protein in egg albumin, undergoes thiol-linked autocleavage at distinct sites upon reduction. This study explores the physiological significance of OTf autocleavage by examining the effect of the reduced autocleaved OTf (termed rac-OTf) on modulation of cell proliferation, lethality, and apoptosis in two human cancer cell lines, colon cancer (HCT-116) and breast cancer (MCF-7). The rac-OTf was prepared by reduction of OTf with a non-thiol reductant (TCEP), to avoid reductive alkylation and produce highly soluble fragments. Unlike OTf, rac-OTf remarkably inhibited the proliferation of cancerous MCF-7 and HCT-116 cells in a dose-dependent manner, with the greatest effect on HCT-116, but had no effect on normal human mammary epithelial cells (HMEC). Cytofluorometric and trypan blue exclusion analyses indicated that rac-OTf exhibits cytotoxicity to HCT-116 in a dose-dependent fashion. The cytotoxic mechanism of rac-OTf against cancer cells was found to be induction of apoptosis as judged by changes in cell morphology, annexin-V binding, collapse of mitochondrial membrane potential, and caspase-9 and -6 activation, indicating the involvement of the mitochondrial pathway. This finding is the first to describe the reduction-dependent autocleaved OTf as an anticancer molecule, providing insights into a novel physiological function of OTf, suggesting its therapeutic potential in the treatment of human cancers and health benefit in nutraceuticals.

Chk1, one of the critical transducers in DNA damage/replication checkpoints, prevents entry into mitosis through inhibition of Cdk1 activity. However, it has remained unclear how this inhibition is cancelled at the G(2)/M transition. We reported recently that Chk1 is phosphorylated at Ser(286) and Ser(301) by Cdk1 during mitosis. Here, we show that mitotic Chk1 phosphorylation is accompanied by Chk1 translocation from the nucleus to the cytoplasm in prophase. This translocation advanced in accordance with prophase progression and was regulated by Crm-1-dependent nuclear export. Exogenous Chk1 mutated at Ser(286) and Ser(301) to Ala (S286A/S301A) was observed mainly in the nuclei of prophase cells, although such nuclear accumulation was hardly observed in wild-type Chk1. Induction of S286A/S301A resulted in the delay of mitotic entry. Biochemical analyses using immunoprecipitated cyclin B(1)-Cdk1 complexes revealed S286A/S301A expression to block the adequate activation of Cdk1. In support of this, S286A/S301A expression retained Wee1 at higher levels and Cdk1-induced phosphorylation of cyclin B(1) and vimentin at lower levels. A kinase-dead version of S286A/S301A also localized predominantly in the nucleus but lost the ability to delay mitotic entry. These results indicate that Chk1 phosphorylation by Cdk1 participates in cytoplasmic sequestration of Chk1 activity, which releases Cdk1 inhibition in the nucleus and promotes mitotic entry.

Human papillomaviruses (HPV) are the main etiological factor for cervical carcinoma. HPV-16 is the most prevalent high-risk HPV-genotype found in HPV-associated cancers. We studied the effect of HPV-16 E7 oncoprotein on cadherin-mediated cell adhesion. The expression of E7 strongly suppressed the cadherin-mediated cell adhesion in the rat fibroblast cell line 3Y1. This suppression was associated with the decreased expression of N-cadherin at the transcriptional level. The treatment of 3Y1 cells that express E7 (E7-3Y1) with MEK inhibitor recovered the cadherin-mediated cell adhesion together with the accumulation of N-cadherin at the cell-cell contact site. Moreover, the suppression of c-Jun, which is the element of AP-1 transcriptional factor, leads to the recovery of N-cadherin expression and cadherin-mediated cell adhesion in E7-3Y1 cells. Taken together, our results demonstrate that E7 regulates cadherin-mediated cell adhesion through the modulation of cadherin expression via the MEK-ERK and AP-1 signaling pathway.

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.

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.

Over the last two decades, since the initial discovery of human papillomavirus (HPV) type 16 and 18 DNAs in cervical cancers by Dr. Harald zur Hausen (winner of the Nobel Prize in Physiology or Medicine, 2008), the HPVs have been well characterised as causative agents for cervical cancer. Viral DNA from a specific group of HPVs can be detected in at least 90% of all cervical cancers and two viral genes, E6 and E7, are invariably expressed in HPV-positive cervical cancer cells. Their gene products are known to inactivate the major tumour suppressors, p53 and retinoblastoma protein (pRB), respectively. In addition, one function of E6 is to activate telomerase, and E6 and E7 cooperate to effectively immortalise human primary epithelial cells. Though expression of E6 and E7 is itself not sufficient for cancer development, it seems to be either directly or indirectly involved in every stage of multi-step carcinogenesis. Epidemiological and biological studies suggest the potential efficacy of prophylactic vaccines to prevent genital HPV infection as an anti-cancer strategy. However, given the widespread nature of HPV infection and unresolved issues about the duration and type specificity of the currently available HPV vaccines, it is crucial that molecular details of the natural history of HPV infection as well as the biological activities of the viral oncoproteins be elucidated in order to provide the basis for development of new therapeutic strategies against HPV-associated malignancies. This review highlights novel functions of E6 and E7 as well as the molecular mechanisms of HPV-induced carcinogenesis., (Copyright (c) 2009 John Wiley & Sons, Ltd.)

Investigation into the function of human trophoblasts has been largely restricted by a lack of suitable cell models. We aimed to produce normal human trophoblast cell lines with a long lifespan and to provide an ideal in vitro cell model. Primary human trophoblast cells were derived from a placenta that had undergone elective abortion at the 7th week of gestation. The cells were immortalized by infection with retroviral expression vectors containing the type 16 human papillomaviruses E6 and E7 in combination with human telomerase reverse transcriptase (hTERT). Characterization of the cell line was performed by immunocytochemistry using a panel of antibodies, Western blotting, real-time RT-PCR, an invasion assay, gelatin zymography, karyotype analysis and a nude mouse assay. Gene expression profiles under hypoxia (1% O2, 1 h) and subsequent reoxygenation (20% O2, 6 h) were analyzed using cDNA microarray. Immunocytochemistry revealed an extravillous trophoblastic phenotype by positive staining for hCGbeta, cytokeratin 7, HLA-G and CD9. A transwell insert invasion assay showed the invasiveness of this cell line and gelatin zymography detected the secretion of MMP-2 and MMP-9. Karyotype analysis exhibited an almost normal chromosomal number which ranged from 46 to 48 and the cells showed no tumorigenecity in a nude mouse assay. Forty-three genes showing reversible up- or down-regulation during hypoxia were detected using an oligonucleotide array. This newly immortalized cell line, HChEpC1b, is a useful model for the study of extravillous trophoblast function.

Mesenchymal stromal cells (MSC) are promising options to cellular therapy to several clinical disorders, mainly because of its ability to immunomodulate and differentiate into different cell types. Even though MSC can be isolated from different sources, a major challenge to understanding the biological effects is that the primary cells undergo replicative senescence after a limited number of cell divisions in culture, requiring time-consuming and technically challenging approaches to get a sufficient cell number for clinical applications. Therefore, a new isolation, characterization, and expansion is necessary every time, which increases the variability and is time-consuming. Immortalization is a strategy that can overcome these challenges. Therefore, here, we review the different methodologies available to cellular immortalization, and discuss the literature regarding MSC immortalization and the broader biological consequences that extend beyond the mere increase in proliferation potential., (© 2023. The Author(s) under exclusive licence to Japan Human Cell Society.)

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.

Epidermodysplasia verruciformis (EV) is a genodermatosis characterized by widespread and persistent cutaneous lesions caused by beta-papillomaviruses. Rare cases of acquired EV-like eruption associated with beta-papillomavirus infection have been reported in immunosuppressed patients. We report a case of acquired EV-like eruption in an immunosuppressed patient with adult T-cell leukemia. The cutaneous lesions clinically resembled pityriasis versicolor and exhibited the typical histological features of EV, but in some areas of the same biopsy specimen characteristic homogeneous intracytoplasmic inclusion bodies were observed. Although beta-papillomavirus was not detected by highly sensitive polymerase chain reaction, a putative novel type of gamma-papillomavirus was identified. This is the first documentation of an association between EV-like eruption and gamma-papillomavirus infection., ((c) 2009 S. Karger AG, Basel.)

Despite the initiation of tumor arises from tumorigenic transformation signaling in cancer cells, cancer cell survival, invasion, and metastasis also require a dynamic and reciprocal association with extracellular signaling from tumor microenvironment (TME). Primary cilia are the antenna-like structure that mediate signaling sensation and transduction in different tissues and cells. Recent studies have started to uncover that the heterogeneous ciliation in cancer cells and cells from the TME in tumor growth impels asymmetric paracellular signaling in the TME, indicating the essential functions of primary cilia in homeostasis maintenance of both cancer cells and the TME. In this review, we discussed recent advances in the structure and assembly of primary cilia, and the role of primary cilia in tumor and TME formation, as well as the therapeutic potentials that target ciliary dynamics and signaling from the cells in different tumors and the TME., (© 2023 The Authors. Journal of Cellular Physiology published by Wiley Periodicals LLC.)

Stem cells derived from dental/odontogenic tissue have the property of multiple differentiation and are prospective in tooth regenerative medicine and cellular and molecular studies. However, in the face of cellular senescence soon in vitro, the proliferation ability of the cells is limited, so studies are hindered to some extent. Fortunately, immortalization strategies are expected to solve the above issues. Cellular immortalization is that cells are immortalized by introducing oncogenes, human telomerase reverse transcriptase genes (hTERT), or miscellaneous immortalization genes to get unlimited proliferation. At present, a variety of immortalized stem cells from dental/odontogenic tissue has been successfully generated, such as dental pulp stem cells (DPSCs), periodontal ligament cells (PDLs), stem cells from human exfoliated deciduous teeth (SHEDs), dental papilla cells (DPCs), and tooth germ mesenchymal cells (TGMCs). This review summarized establishment and applications of immortalized stem cells from dental/odontogenic tissues and then discussed the advantages and challenges of immortalization., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

We previously reported Chk1 to be phosphorylated at Ser286 and Ser301 by cyclin-dependent kinase (Cdk) 1 during mitosis [T. Shiromizu et al., Genes Cells 11 (2006) 477-485]. Here, we demonstrated that Chk1-Ser286 and -Ser301 phosphorylation also occurs in hydroxyurea (HU)-treated or ultraviolet (UV)-irradiated cells. Unlike the mitosis case, however, Chk1 was phosphorylated not only at Ser286 and Ser301 but also at Ser317 and Ser345 in the checkpoint response. Treatment with Cdk inhibitors diminished Chk1 phosphorylation at Ser286 and Ser301 but not at Ser317 and Ser345 with the latter. In vitro analyses revealed Ser286 and Ser301 on Chk1 to serve as two major phosphorylation sites for Cdk2. Immunoprecipitation analyses further demonstrated that Ser286/Ser301 and Ser317/Ser345 phosphorylation occur in the same Chk1 molecule during the checkpoint response. In addition, Ser286/Ser301 phosphorylation by Cdk2 was observed in Chk1 mutated to Ala at Ser317 and Ser345 (S317A/S345A), as well as Ser317/Ser345 phosphorylation by ATR was in S286A/S301A. Therefore, Chk1 phosphorylation in the checkpoint response is regulated not only by ATR but also by Cdk2.

Over the last two decades since discovery of human papillomavirus (HPV) type 16 and 18 DNAs in cervical cancers by Dr. Harald zur Hausen, HPVs have been well characterized as causative agents for cervical cancer. Viral DNA from a specific group of HPVs can be detected in at least 90% of all cervical cancers and two viral genes, E6 and E7, are invariably expressed in HPV-positive cervical cancer cells. Their gene products are known to inactivate the major tumor suppressors, p53 and pRB, respectively. In addition, one function of E6 is to activate telomerase, and E6 and E7 cooperate to effectively immortalize human primary epithelial cells. Though expression of E6 and E7 is itself not sufficient for cancer development, it seems to be either directly or indirectly involved in every stage of multi-step carcinogenesis. Indeed, it has been shown that only one or two genetic alterations in addition to expression of E6 and E7 are experimentally sufficient to confer tumorigenicity to normal human cervical keratinocytes. Epidemiological and biological studies suggest the potential efficacy of prophylactic vaccines to prevent genital HPV infection as an anti-cancer strategy. However, given the widespread nature of HPV infection and unresolved issues about the duration and type specificity of the currently available HPV vaccines, it is crucial that molecular details of the natural history of HPV infection as well as the biological activities of the viral oncoproteins be elucidated in order to provide the basis for development of new therapeutic strategies against HPV-associated malignancies. This review highlights the novel functions of E6 and E7 as well as the molecular mechanisms of HPV-induced carcinogenesis.

The keratin intermediate filament network is abundant in epithelial cells, but its function in the establishment and maintenance of cell polarity is unclear. Here, we show that Albatross complexes with Par3 to regulate formation of the apical junctional complex (AJC) and maintain lateral membrane identity. In nonpolarized epithelial cells, Albatross localizes with keratin filaments, whereas in polarized epithelial cells, Albatross is primarily localized in the vicinity of the AJC. Knockdown of Albatross in polarized cells causes a disappearance of key components of the AJC at cell-cell borders and keratin filament reorganization. Lateral proteins E-cadherin and desmoglein 2 were mislocalized even on the apical side. Although Albatross promotes localization of Par3 to the AJC, Par3 and ezrin are still retained at the apical surface in Albatross knockdown cells, which retain intact microvilli. Analysis of keratin-deficient epithelial cells revealed that keratins are required to stabilize the Albatross protein, thus promoting the formation of AJC. We propose that keratins and the keratin-binding protein Albatross are important for epithelial cell polarization.

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.

For future cell-based therapies for liver diseases, the shortage of cell sources must be resolved. Immortalized human hepatocytes are expected to be among the new sources. In addition to telomerase activation by the introduction of human telomerase reverse transcriptase (hTERT), inactivation of the p16/RB pathway and/or p53 by E6/E7 of human papillomavirus type 16 (HPV16) has been shown to be useful for efficient immortalization of several human cell types. Here we report the immortalization of human hepatocytes by the introduction of HPV16 E6/E7 and hTERT. Human adult hepatocytes were lentivirally transduced with HPV16 E6/E7 and hTERT. Two human immortalized hepatocyte cell lines were established and were named HHE6E7T-1 and HHE6E7T-2. Those cells proliferated in culture beyond 200 population doublings (PDs). Albumin synthesis and expression of liver-enriched genes were confirmed, but gradually decreased as passages progressed. Karyotype analysis showed that HHE6E7T-1 cells remained near diploid but that HHE6E7T-2 cells showed severe aneuploidy at 150 PDs. Subcutaneous injection of these cells into severe combined immunodeficiency (SCID) mice did not induce tumor development. Intrasplenic transplantation of dedifferentiated HHE6E7T-1 cells over 200 PDs significantly improved the survival of acetaminophen-induced acute liver failure SCID mice. In conclusion, we successfully established immortalized human hepatocytes that retain the characteristics of differentiated hepatocytes. We also showed the reduction of hepatocyte-specific functions in long-term culture. However, the results of intrasplenic transplantation to SCID mice with acetaminophen-induced acute liver failure showed the possibility of HHE6E7T-1 serving as a cell source for hepatocyte transplantation.

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.

Stem cell therapy can help repair damaged heart tissue. Yet many of the suitable cells currently identified for human use are difficult to obtain and involve invasive procedures. In our search for novel stem cells with a higher cardiomyogenic potential than those available from bone marrow, we discovered that potent cardiac precursor-like cells can be harvested from human menstrual blood. This represents a new, noninvasive, and potent source of cardiac stem cell therapeutic material. We demonstrate that menstrual blood-derived mesenchymal cells (MMCs) began beating spontaneously after induction, exhibiting cardiomyocyte-specific action potentials. Cardiac troponin-I-positive cardiomyocytes accounted for 27%-32% of the MMCs in vitro. The MMCs proliferated, on average, 28 generations without affecting cardiomyogenic transdifferentiation ability, and expressed mRNA of GATA-4 before cardiomyogenic induction. Hypothesizing that the majority of cardiomyogenic cells in MMCs originated from detached uterine endometrial glands, we established monoclonal endometrial gland-derived mesenchymal cells (EMCs), 76%-97% of which transdifferentiated into cardiac cells in vitro. Both EMCs and MMCs were positive for CD29, CD105 and negative for CD34, CD45. EMCs engrafted onto a recipient's heart using a novel 3-dimensional EMC cell sheet manipulation transdifferentiated into cardiac tissue layer in vivo. Transplanted MMCs also significantly restored impaired cardiac function, decreasing the myocardial infarction (MI) area in the nude rat model, with tissue of MMC-derived cardiomyocytes observed in the MI area in vivo. Thus, MMCs appear to be a potential novel, easily accessible source of material for cardiac stem cell-based therapy.

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.

Here, we identified human myogenic progenitor cells coexpressing Pax7, a marker of muscle satellite cells and bone-specific alkaline phosphatase, a marker of osteoblasts, in regenerating muscle. To determine whether human myogenic progenitor cells are able to act as osteoprogenitor cells, we cultured both primary and immortalized progenitor cells derived from the healthy muscle of a nondystrophic woman. The undifferentiated myogenic progenitors spontaneously expressed two osteoblast-specific proteins, bone-specific alkaline phosphatase and Runx2, and were able to undergo terminal osteogenic differentiation without exposure to an exogenous inductive agent such as bone morphogenetic proteins. They also expressed the muscle lineage-specific proteins Pax7 and MyoD, and lost their osteogenic characteristics in association with terminal muscle differentiation. Both myoblastic and osteoblastic properties are thus simultaneously expressed in the human myogenic cell lineage prior to commitment to muscle differentiation. In addition, C3 transferase, a specific inhibitor of Rho GTPase, blocked myogenic but not osteogenic differentiation of human myogenic progenitor cells. These data suggest that human myogenic progenitor cells retain the capacity to act as osteoprogenitor cells that form ectopic bone spontaneously, and that Rho signaling is involved in a critical switch between myogenesis and osteogenesis in the human myogenic cell lineage.

The periodontal ligament (PDL) is situated between the tooth root and alveolar bone, thereby supporting the tooth, and is composed of collagen and elastic system fibers. Marfan syndrome type I (MFS1, MIM #154700) is caused by mutations in FBN1 encoding fibrillin-1, which is a major microfibrillar protein of elastic system fibers. MFS1 is characterized by tall stature, aortic/mitral valve prolapse, and ectopia lentis and is occasionally accompanied by severe periodontitis. Since little is known about the biological functions of elastic system fibers in PDLs and the pathogenesis of the periodontitis in MFS1, PDL cells were isolated from an MFS1 patient with a heterozygous missense mutation in a calcium-binding epidermal-growth-factor-like domain of FBN1. Isolated PDL cells were immortalized by transducing a retrovirus carrying genes for the human Polycomb group protein, Bmi-1, and human telomerase reverse transcriptase. Immortalized PDL cells from the MFS1 patient (termed M-HPL1) and those of a healthy volunteer (termed HPDL2) both expressed various PDL-related genes. The growth and attachment of M-HPL1 and HPDL2 to hydroxyapatite particles were comparable. However, when M-HPL1 were transplanted with hydroxyapatite particles into immunodeficient mice, disorganized cell alignment and irregular microfibril assembly were noted. The activation of the signaling of transforming grwoth factor-beta (TGF-beta) is thought to cause the pathogenesis for lung and cardiovascular abnormalities in MFS1. Interestingly, M-HPL1 shows a higher level of activated TGF-beta than HPDL2. Thus, M-HPL1 represent a powerful tool for clarifying the biological roles of elastic system fibers in PDL and the pathogenesis of periodontitis in MFS1. Our findings also suggest that FBN1 regulates cell alignment and microfibril assembly in PDLs.

Cellular senescence was originally described as a phenomenon observed in cultured human cells. Accumulating lines of evidence now indicate that the same processes also take place in vivo, suggesting important implications for tumor development. Telomere shortening is the most well-established cause of cellular senescence that can be induced by many other intrinsic and extrinsic factors. The retinoblastoma susceptibility gene product is a convergent target that is downstream of these factors. p53, p38MAPK and cyclin-dependent kinase inhibitors p16INK4a (p16) and p21CIP1 (p21) are key mediators. As most stresses that induce cellular senescence are also known causes of cancer, a common strategy might be applied to the development of cancer chemopreventive agents and anti-ageing drugs.

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.

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.

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.

Duchenne muscular dystrophy (DMD), the most common lethal genetic disorder in children, is an X-linked recessive muscle disease characterized by the absence of dystrophin at the sarcolemma of muscle fibers. We examined a putative endometrial progenitor obtained from endometrial tissue samples to determine whether these cells repair muscular degeneration in a murine mdx model of DMD. Implanted cells conferred human dystrophin in degenerated muscle of immunodeficient mdx mice. We then examined menstrual blood-derived cells to determine whether primarily cultured nontransformed cells also repair dystrophied muscle. In vivo transfer of menstrual blood-derived cells into dystrophic muscles of immunodeficient mdx mice restored sarcolemmal expression of dystrophin. Labeling of implanted cells with enhanced green fluorescent protein and differential staining of human and murine nuclei suggest that human dystrophin expression is due to cell fusion between host myocytes and implanted cells. In vitro analysis revealed that endometrial progenitor cells and menstrual blood-derived cells can efficiently transdifferentiate into myoblasts/myocytes, fuse to C2C12 murine myoblasts by in vitro coculturing, and start to express dystrophin after fusion. These results demonstrate that the endometrial progenitor cells and menstrual blood-derived cells can transfer dystrophin into dystrophied myocytes through cell fusion and transdifferentiation in vitro and in vivo.

Tissue stem cells may serve as progenitors for malignant tumors derived from the same tissue. Here, we report the establishment of immortalized human mesenchymal stem cells (ihMSC) and tested the feasibility of using ihMSC as presarcomatous cells. Immortalization was achieved by introducing the genes for human telomerase reverse transcriptase and Bmi1. ihMSC retained the potential for multi-directional differentiation of the original MSC. To transform ihMSC, we introduced an oncogenic H-ras(Val12) gene, and established the cell line ihMSC-ras. ihMSC-ras had the phenotype of fully transformed cells and retained adipogenic and chondrogenic, but not osteogenic, potential. Interestingly, ihMSC-ras demonstrated morphological features of autophagy, and inhibition of the ERK pathway suppressed the production of autophagosomes, indicating that ras/ERK signaling is responsible for the induction of autophagy. Thus ihMSC will serve as a material with which to analyze the tumorigenic and differentiation-modifying effects of candidate oncogenes involved in the development of sarcomas.

About 50% of cervical cancers are associated with human papillomavirus type 16 (HPV-16), and since the HPV-16 E6 and E7 oncoproteins are constitutively expressed in the tumor cells, they are attractive targets for cytotoxic T lymphocyte (CTL)-mediated immunotherapy. Nevertheless, only a limited number of HPV-16 E6 epitopes have been identified to date. Using reverse immunological methods, we have generated a CTL clone against the HPV-16 E6(49-57) epitope restricted by HLA-A*2402, which is the most common allele in Japan and relatively frequent worldwide, capable of lysing 293T cells transduced with HLA-A*2402 and HPV-16 E6. Although it was unable to recognize the SiHa cervical cancer cell line positive for HPV-16 and HLA-A*2402, the cells became susceptible to lysis when transduced with E6-E7 genes, which was unexpectedly offset by pretreatment with interferon (IFN)-gamma alone. Interestingly, however, combined pretreatment with a proteasome inhibitor, bortezomib and IFN-gamma fully restored CTL-mediated lysis of the original SiHa cells. Furthermore, such intervention of 2 of 4 other cervical cancer cell lines expressing HPV-16 E6 and HLA-A*2402 was found to induce IFN-gamma production by specific CTLs. Tetramer analysis further revealed that induction of E6(49-57)-specific T cells was possible in 5 of 7 patients with HPV-16-positive high grade cervical intraepithelial neoplasia or cervical cancer by in vitro stimulation with E6(49-57) peptide. Thus, these findings together indicate that E6(49-57) is a candidate epitope for immunotherapy and immunological monitoring of such patients.

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.

In current and past practice, murine or primate embryonic stem (ES) cells are usually cultured on live nurse cells for growth that keeps the cells in an undifferentiated state. It is troublesome, however, to prepare nurse cells for each cell culture and it is difficult to completely remove the nurse cells when they are transferred. In this study, mouse and monkey ES cells were therefore grown on chemically fixed mouse embryonic fibroblast (MEF) or human amniotic epithelial (HAE) cells. MEF cells were fixed by incubation in a glutaraldehyde or formaldehyde solution. HAE cells were immortalized by transfection of hTERT and chemically fixed with the same reagents. When mouse ES cells were cultured on these chemically fixed cells, the mouse ES cells grew well and expressed alkaline phosphatase, SSEA-1, and Oct-3/4 as their markers, indicating their undifferentiated state. The monkey ES cells also grew well and expressed alkaline phosphatase, SSEA-4, and Oct-4 as their markers, indicating their undifferentiated state. Freeze-drying HAE or MEF cells did not change their ability to support the undifferentiated growth of ES cells. Additionally, the chemically fixed cells could be utilized repeatedly in the culture of ES cells. These results demonstrate that chemically fixed nurse cells are useful for the maintenance of ES cells in an undifferentiated state in culture.