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1.
Carcinogenesis ; 39(2): 202-213, 2018 02 09.
Artigo em Inglês | MEDLINE | ID: mdl-29106503

RESUMO

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.


Assuntos
Morte Celular/genética , Células Epiteliais/patologia , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas ras/genética , Linhagem Celular Tumoral , Células Cultivadas , Humanos , Neoplasias/genética , Neoplasias/patologia , Proteínas rac1 de Ligação ao GTP/metabolismo
2.
Cancer Sci ; 108(7): 1303-1309, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28440909

RESUMO

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.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Transformação Celular Neoplásica/metabolismo , Queratinócitos/virologia , Proteínas de Membrana/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Proteínas Repressoras/metabolismo , Neoplasias do Colo do Útero/virologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Western Blotting , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica/fisiologia , Xenoenxertos , Humanos , Queratinócitos/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Domínios PDZ/fisiologia , Infecções por Papillomavirus/complicações , Infecções por Papillomavirus/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias do Colo do Útero/patologia
3.
J Virol ; 89(9): 5040-59, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25717108

RESUMO

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.


Assuntos
Interações Hospedeiro-Patógeno , Papillomavirus Humano 16/imunologia , NF-kappa B/imunologia , NF-kappa B/metabolismo , Proteínas Oncogênicas Virais/imunologia , Proteínas Oncogênicas Virais/metabolismo , Replicação Viral , Células Cultivadas , Papillomavirus Humano 16/fisiologia , Humanos , Queratinócitos/virologia , Proteólise
4.
Carcinogenesis ; 35(8): 1840-6, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24858378

RESUMO

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.


Assuntos
Carcinoma Ductal Pancreático/patologia , Transformação Celular Neoplásica/patologia , Células Epiteliais/patologia , Regulação Neoplásica da Expressão Gênica , Oncogenes/fisiologia , Ductos Pancreáticos/patologia , Neoplasias Pancreáticas/patologia , Animais , Western Blotting , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Técnicas de Cultura de Células , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Células Cultivadas , Ciclina D1/genética , Quinase 4 Dependente de Ciclina/genética , Células Epiteliais/metabolismo , Feminino , Humanos , Técnicas Imunoenzimáticas , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Mutação/genética , Ductos Pancreáticos/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas p21(ras) , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Telomerase/genética , Proteína Supressora de Tumor p53/genética , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas ras/genética , Neoplasias Pancreáticas
5.
J Virol ; 86(6): 3276-83, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22238312

RESUMO

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.


Assuntos
Genoma Viral , Papillomavirus Humano 16/fisiologia , Proteínas Oncogênicas Virais/metabolismo , Infecções por Papillomavirus/virologia , Replicação Viral , Linhagem Celular , Replicação do DNA , Papillomavirus Humano 16/genética , Humanos , Proteínas Oncogênicas Virais/genética
6.
Carcinogenesis ; 33(4): 910-7, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22345164

RESUMO

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.


Assuntos
Transformação Celular Neoplásica , Papillomavirus Humano 16/fisiologia , Proteínas E7 de Papillomavirus/fisiologia , Proteínas Proto-Oncogênicas c-myc/fisiologia , Proteínas Proto-Oncogênicas p21(ras)/fisiologia , Células Cultivadas , Humanos
7.
Mol Cell Biol ; 27(10): 3732-42, 2007 May.
Artigo em Inglês | MEDLINE | ID: mdl-17353266

RESUMO

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.


Assuntos
Células Epiteliais/fisiologia , Regulação da Expressão Gênica , Receptor Notch1/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Sequência de Bases , Biomarcadores/metabolismo , Diferenciação Celular/fisiologia , Células Cultivadas , Dano ao DNA , Células Epiteliais/citologia , Perfilação da Expressão Gênica , Inativação Gênica , Humanos , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/metabolismo , RNA/química , RNA/metabolismo , Receptor Notch1/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transcrição Gênica , Proteína Supressora de Tumor p53/genética
8.
Sci Rep ; 10(1): 17503, 2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-33060611

RESUMO

Hepatocytes are an important tool for in vitro toxicology testing. In addition to primary cultures, a limited number of immortalized cell lines have been developed. We here describe a new cell line, designated as HepaMN, which has been established from a liver associated with biliary atresia. Hepatocytes were isolated from a liver of 4-year-old girl with biliary atresia and immortalized by inoculation with CSII-CMV-TERT, CSII-CMV-Tet-Off, CSII-TRE-Tight-cyclin D1 and CSII-TRE-Tight-CDK4R24C (mutant CDK4: an INK4a-resistant form of CDK4) lentiviruses at the multiplicity of infection of 3 to 10. HepaMN cells exhibited morphological homogeneity, displaying hepatocyte-like phenotypes. Phenotypic studies in vivo and in vitro revealed that HepaMN cells showed polarized and functional hepatocyte features along with a canalicular cell phenotype under defined conditions, and constitutively expressed albumin and carbamoyl phosphate synthetase I in addition to epithelial markers. Since HepaMN cells are immortal and subcloned, kinetics and expression profiles were independent of population doublings. HepaMN cells showed increased CYP3A4 expression after exposure to rifampicin, implying that their close resemblance to normal human hepatocytes makes them suitable for research applications including drug metabolism studies.


Assuntos
Atresia Biliar/metabolismo , Técnicas de Cultura de Células/métodos , Ciclina D1/metabolismo , Quinase 4 Dependente de Ciclina/metabolismo , Hepatócitos/citologia , Fígado Artificial , Telomerase/metabolismo , Linhagem Celular , Pré-Escolar , Análise Custo-Benefício , Citocromo P-450 CYP3A/metabolismo , Hepatócitos/efeitos dos fármacos , Humanos , Cinética , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fenótipo , Análise de Componente Principal , Medicina Regenerativa , Rifampina/farmacologia
9.
Mol Cell Biol ; 33(22): 4434-47, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24019071

RESUMO

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.


Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Queratinócitos/citologia , Receptor Notch1/metabolismo , Transdução de Sinais , Quinases Associadas a rho/metabolismo , Adulto , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Ativação Enzimática , Feminino , Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Queratinócitos/metabolismo , Receptor Notch1/genética
10.
Cancer Res ; 70(10): 4034-44, 2010 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-20442293

RESUMO

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.


Assuntos
Queratinócitos/patologia , Receptor Notch1/genética , Transativadores/genética , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/genética , Neoplasias do Colo do Útero/patologia , Animais , Northern Blotting , Western Blotting , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Feminino , Humanos , Queratinócitos/metabolismo , Luciferases/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor Notch1/antagonistas & inibidores , Receptor Notch1/metabolismo , Elementos de Resposta , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Deleção de Sequência , Transativadores/metabolismo , Fatores de Transcrição , Proteína Supressora de Tumor p53/genética , Proteínas Supressoras de Tumor/metabolismo , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/metabolismo
11.
Cancer Res ; 68(14): 5699-705, 2008 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-18632622

RESUMO

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.


Assuntos
Regulação Neoplásica da Expressão Gênica , Neoplasias do Colo do Útero/etiologia , Neoplasias do Colo do Útero/patologia , Animais , Linhagem Celular Tumoral , Transformação Celular Neoplásica , Feminino , Humanos , Queratinócitos/citologia , Camundongos , Camundongos Nus , Modelos Biológicos , Transplante de Neoplasias , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Receptor ErbB-2/metabolismo , Telomerase/metabolismo
12.
J Virol ; 81(3): 1379-89, 2007 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17121805

RESUMO

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.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Papillomaviridae/química , Ubiquitinas/metabolismo , Linhagem Celular , Proteínas de Ligação a DNA/química , Proteína 4 Homóloga a Disks-Large , Proteínas de Drosophila/química , Genes Supressores de Tumor , Células HeLa , Humanos , Proteínas Oncogênicas Virais/química , Ubiquitina-Proteína Ligases
13.
Cancer Sci ; 98(2): 147-54, 2007 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17233832

RESUMO

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.


Assuntos
Inibidor p16 de Quinase Dependente de Ciclina/genética , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Telomerase/metabolismo , Catálise , Proliferação de Células , Células Cultivadas , Senescência Celular , Metilação de DNA , Regulação para Baixo , Ativação Enzimática , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Humanos , Cariotipagem , Queratinócitos/metabolismo , Complexo Repressor Polycomb 1 , Regiões Promotoras Genéticas/genética , Interferência de RNA , Telomerase/genética , Regulação para Cima
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