RESUMO
We previously reported that transgenic mice produced with a transgene consisting of the SV40 T antigen and vasopressin without the 3'-flanking region exhibit brain tumors and lymphoma. In this study, transgenic mice were produced with the fusion gene containing the SV40 T antigen and the whole vasopressin gene with the 3'-flanking region. Six transgenic mice were generated, five which died after 2-6 weeks. The remaining founder mouse was investigated for fusion gene expression and tumor progression at the age of 6 weeks. Brain tumor cells were characterized for phenotypes and transgene expression. During in vitro cell cultures, the phenotypic appearances at 10, 20, and 30 passages were as a uniform monolayer with similar growth rates. The site of SV40 T antigen integration was in the A2 region of chromosome 11, and SV40 T antigen was expressed at the same level in cells of both earlier and later passages. Thirty passages were probably insufficient to reach crisis and immortalization. These cells enriched brain tumor cell compositions with astrocytes and neuronal cells.
Assuntos
Camundongos , Animais , Vasopressinas/genética , Transgenes/genética , Proteínas Recombinantes de Fusão/genética , Plasmídeos/genética , Camundongos Transgênicos , Camundongos Endogâmicos ICR , Hibridização in Situ Fluorescente/métodos , Técnicas Imunoenzimáticas , Expressão Gênica/genética , Proliferação de Células , Linhagem Celular Tumoral , Neoplasias Encefálicas/genética , Western Blotting , Antígenos Transformantes de Poliomavirus/genéticaRESUMO
SV40 large T antigen, a viral oncoprotein, is known to immortalize human diploid fibroblast by soaking up cellular RB and p53, but its frequency is extremely low. Additional genetic alteration is necessary for single-step immortalization. We attempted to find out what this alteration is by overexpressing cellular signal mediator genes; c-myc and cyclin D frequently amplified in many cancer cells. Overexpression of cyclin D did not affect the immortalization, but, overexpression of c-myc along with T antigen could immortalize normal human diploid fibroblast. Several cellular markers tested during immortalization process showed that p21, a cyclin-dependent kinase inhibitor and a marker of cellular senescence, disappeared in the life span-extended cells by T antigen and in the immortalized cells by c-myc. p21 was, however, elevated in the senescent cells and in the cells of crisis. Interestingly, p16 was upregulated whenever T antigen is overexpressed. Telomerase activity was also activated only in the immortalized cells. These results suggest that overexpression of c-myc contributes to immortalization of human diploid fibroblast by activating telomerase activity and suppressing p21 activity.
Assuntos
Humanos , Antígenos Transformantes de Poliomavirus/genética , Biomarcadores , Senescência Celular/genética , Transformação Celular Viral , Células Cultivadas , Ciclinas/metabolismo , Diploide , Fibroblastos/metabolismo , Genes myc/genética , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Vírus 40 dos Símios/genética , Telomerase/metabolismoRESUMO
SV40 large T antigen, a viral oncoprotein, is known to immortalize human diploid fibroblast by soaking up cellular RB and p53, but its frequency is extremely low. Additional genetic alteration is necessary for single-step immortalization. We attempted to find out what this alteration is by overexpressing cellular signal mediator genes; c-myc and cyclin D frequently amplified in many cancer cells. Overexpression of cyclin D did not affect the immortalization, but, overexpression of c-myc along with T antigen could immortalize normal human diploid fibroblast. Several cellular markers tested during immortalization process showed that p21, a cyclin-dependent kinase inhibitor and a marker of cellular senescence, disappeared in the life span-extended cells by T antigen and in the immortalized cells by c-myc. p21 was, however, elevated in the senescent cells and in the cells of crisis. Interestingly, p16 was upregulated whenever T antigen is overexpressed. Telomerase activity was also activated only in the immortalized cells. These results suggest that overexpression of c-myc contributes to immortalization of human diploid fibroblast by activating telomerase activity and suppressing p21 activity.
Assuntos
Humanos , Antígenos Transformantes de Poliomavirus/genética , Biomarcadores , Senescência Celular/genética , Transformação Celular Viral , Células Cultivadas , Ciclinas/metabolismo , Diploide , Fibroblastos/metabolismo , Genes myc/genética , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Vírus 40 dos Símios/genética , Telomerase/metabolismoRESUMO
Polyomavirus is a DNA tumor virus that induces a variety of tumors in mice. Its genome encodes three proteins, namely large T (LT), middle T (MT), and small T (ST) antigens, that have been implicated in cell transformation and tumorigenesis. LT is associated with cell immortalization, whereas MT plays an essential role in cell transformation by binding to and activating several cytoplasmic proteins that participate in growth factor-induced mitogenic signal transduction to the nucleus. The use of different MT mutants has led to the identification of MT-binding proteins as well as analysis of their importance during cell transformation. Studying the molecular mechanisms of cell transformation by MT has contributed to a better understanding of cell cycle regulation and growth control