RESUMO
Squamous cell carcinoma (SCC) immortality is associated with p53 and INK4A dysfunction, high levels of telomerase and loss of heterozygosity (LOH) of other chromosomes, including chromosome 4. To test for a functional cancer mortality gene on human chromosome 4 we introduced a complete or fragmented copy of the chromosome into SCC lines by microcell-mediated chromosome transfer (MMCT). Human chromosome 4 caused a delayed crisis, specifically in SCC lines with LOH on chromosome 4, but chromosomes 3, 6, 11 and 15 were without effect. The introduction of the telomerase reverse transcriptase into the target lines extended the average telomere terminal fragment length but did not affect the frequency of mortal hybrids following MMCT of chromosome 4. Furthermore, telomerase activity was still present in hybrids displaying the mortal phenotype. The MMCT of chromosomal fragments into BICR6 mapped the mortality gene to between the centromere and 4q23. Deletion analysis of the introduced chromosome in immortal segregants narrowed the candidate interval to 2.7 Mb spanning D4S423 and D4S1557. The results suggest the existence of a gene on human chromosome 4 whose dysfunction contributes to the continuous proliferation of SCC and that this gene operates independently from telomeres, p53 and INK4A.
Assuntos
Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Transformação Celular Neoplásica/genética , Cromossomos Humanos Par 4/genética , Perda de Heterozigosidade/genética , Animais , Sobrevivência Celular , Mapeamento Cromossômico , Coloração Cromossômica , Proteínas de Ligação a DNA , Humanos , Marcação In Situ das Extremidades Cortadas , Camundongos , Repetições de Microssatélites/genética , Fenótipo , Telomerase/antagonistas & inibidores , Telomerase/metabolismo , Telômero/metabolismo , Telômero/patologia , Células Tumorais CultivadasRESUMO
We show here that histone deacetylase inhibitors (HDACIs) sodium dibutyrate (SDB) and trichostatin A (TSA) induce a phenotype that has similarities to replicative senescence in human fibroblasts. There was no evidence that SDB accelerated a constitutive cell division counting mechanism as previously suggested because cells pretreated with SDB for three mean population doublings (MPDs) exhibited a similar overall proliferative life span to controls once SDB was withdrawn. SDB-treated cells upregulated the cell cycle inhibitors p21(WAF1) and p16(INK4A), but not p14(ARF), in the same sequential order as in senescence and the cells developed biochemical markers of senescence. However, the mechanism of senescence did not involve telomere dysfunction and there was no evidence for any posttranslational modification of p53. The expression of human papillomavirus (HPV) 16 E6 in human fibroblasts or targeted disruption of the p53 and p21(WAF) genes only weakly antagonized HDACI-induced senescence. However, expression of the E7 gene, which inhibits the function of pRb, cooperated with E6 to block SDB-induced senescence completely and human cells deficient in p16(INK4A) (but not p14(ARF)) were also resistant to SDB-induced senescence, suggesting that the p16(INK4A)/pRb pathway is the major mediator of HDACI-induced senescence in human cells. However, p53-/- mouse fibroblasts were resistant to HDACI-induced senescence, identifying p53 as the major pathway to senescence in this species.
Assuntos
Divisão Celular/fisiologia , Senescência Celular/fisiologia , Inibidores Enzimáticos/farmacologia , Fibroblastos/metabolismo , Inibidores de Histona Desacetilases , Animais , Ácido Butírico/farmacologia , Linhagem Celular , Inibidor p16 de Quinase Dependente de Ciclina/deficiência , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Diploide , Feto , Fibroblastos/enzimologia , Regulação da Expressão Gênica , Histona Desacetilases/efeitos dos fármacos , Histona Desacetilases/metabolismo , Humanos , Ácidos Hidroxâmicos/farmacologia , Cariotipagem , Camundongos , Papillomaviridae/metabolismo , Testes de Precipitina , Proteína do Retinoblastoma/genética , Proteína do Retinoblastoma/metabolismo , Transdução de Sinais , Pele/citologia , Especificidade da Espécie , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Regulação para CimaRESUMO
Mml loci have been identified as provirus integration sites among a subset of monocytic tumours induced by murine leukaemia virus (MuLV) infection of BALB/c and DBA/2 mice. These myeloid leukaemias contain a retrovirus integrated on chromosome 10 in proximity to the c-myb locus; however, c-myb expression was not altered. Detailed physical mapping enabled placement of the retroviral integration sites approximately 25 kb (Mml1), approximately 51 kb (Mml2), and approximately 70 kb (Mml3) upstream of the c-myb locus. Furthermore, the Fti1 (fit-1) locus, a common integration site in feline leukaemia virus-induced T cell lymphomas, was mapped upstream of Mml3. Sequence analysis of Mml1, Mml2 and Mml3 loci (39.6, 16.4 and 5.9 kb, respectively) in conjunction with the BLAST (basic local alignment search tool) homology searches against the expressed sequence tag (EST) database and the use of gene/exon prediction programs revealed potential coding sequences that were not confirmed by Northern analysis or RT-PCR. The sequences between c-myb and Fti1, which were shown to include two potential scaffold/matrix attachment regions (S/MARs), are most likely regulatory in nature. An extended search for transcribed sequences far upstream of Mml3 revealed five genes, four of which were expressed in multiple tissues in mice. These genes could not be linked to tumour formation by the virus but their homologous sequences were found on human chromosome 6, thus allowing extension of the syntenic region on mouse chromosome 10 to approximately 250 kb.