RESUMO
The CDKN2A locus is frequently inactivated in urothelial cell carcinoma (UCC), yet how this alteration contributes to bladder tumorigenesis is not known. Although most UCC express telomerase, inactivation of the p16/Rb pathway is generally required for in vitro immortalisation. This and the involvement of p16 in senescence of normal human urothelial cells (NHUC) suggest that CDKN2A deletion may aid bypass of senescence and allow immortalisation. CDKN2A encodes p16 and p14ARF and therefore inactivation of this locus can disrupt both the Rb and p53 tumour suppressor pathways. Retrovirus-mediated transduction was used to specifically modulate the p16/Rb and/or p53 tumour suppressor pathways in NHUC and to express human telomerase reverse transcriptase (hTERT). Expression of hTERT bypassed Rb and p53 pathway-dependent barriers to proliferation and immortalised NHUC. TERT-NHUC had normal karyotypes, were non-tumorigenic and unexpectedly retained CDKN2A. Thus, the phenotypic significance of inactivation of CDKN2A in UCC may not be solely related to bypass of senescence. Phenotypic assays in human urothelial cells have relied on cell strains derived from invasive tumours or NHUC immortalised by expression of SV40-large T. The production of genetically normal but immortal NHUC lines now provides a valuable platform for experiments to examine the timing and combination of events necessary for UCC tumorigenesis.
Assuntos
Transformação Celular Neoplásica/genética , Proteínas de Ligação a DNA/genética , Genes do Retinoblastoma , Genes p16 , Telomerase/genética , Urotélio/patologia , Sequência de Bases , Células Cultivadas , Primers do DNA , Humanos , Urotélio/citologia , Urotélio/metabolismoRESUMO
The melanoma-astrocytoma syndrome is characterized by a dual predisposition to melanoma and neural system tumours, commonly astrocytoma. Germline deletions of the region on 9p21 containing the CDKN2A and CDKN2B genes and CDKN2A exon 1beta have been reported in kindreds, implicating contiguous tumour suppressor gene deletion as a cause of this syndrome. We describe a family characterized by multiple melanoma and neural cell tumours segregating with a germline deletion of the p14(ARF)-specific exon 1beta of the CDKN2A gene. This deletion does not affect the coding or minimal promoter sequences of either the CDKN2A or CDKN2B genes. Our results are consistent with either: (i) loss of p14(ARF) function being the critical abnormality associated with this syndrome, rather than contiguous loss of both the CDKN2A and CDKN2B genes as suggested previously; or (ii) disruption of expression of p16 by mechanisms as yet unknown.
Assuntos
Astrocitoma/genética , Neoplasias Encefálicas/genética , Deleção de Genes , Genes p16/genética , Mutação em Linhagem Germinativa , Melanoma/genética , Proteínas/genética , Sequência de Bases , Mapeamento Cromossômico , Cromossomos Humanos Par 9 , Cosmídeos , Éxons , Saúde da Família , Feminino , Humanos , Hibridização in Situ Fluorescente , Masculino , Repetições de Microssatélites , Modelos Genéticos , Dados de Sequência Molecular , Linhagem , Fenótipo , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Mapeamento por Restrição , Proteína Supressora de Tumor p14ARFRESUMO
The tumour suppressor gene PTEN/MMAC1, which is mutated or homozygously deleted in glioma, breast and prostate cancer, is mapped to a region of 10q which shows loss of heterozygosity (LOH) in bladder cancer. We screened 123 bladder tumours for LOH in the region of PTEN. In 53 informative muscle invasive tumours (> or = pT2), allele loss was detected in 13 (24.5%) and allelic imbalance in four tumours (overall frequency 32%). LOH was found in four of 60 (6.6%) informative, non-invasive tumours (pTa/pT1). We screened 63 muscle invasive tumours for PTEN mutations by single-strand conformation polymorphism (SSCP) analysis and for homozygous deletion by duplex quantitative polymerase chain reaction (PCR). Two homozygous deletions were identified but no mutations. Of 15 bladder tumour cell lines analysed, three showed homozygous deletion of all or part of the PTEN gene, but none had mutations detectable by SSCP analysis. Our results indicate that PTEN is involved in the development of some bladder tumours. The low frequency of mutation of the retained allele in tumours with 10q23 LOH suggests that there may be another predominant mechanism of inactivation of the second allele, for example small intragenic deletions, that hemizygosity may be sufficient for phenotypic effect, or that there is another target gene at 10q23.
Assuntos
Carcinoma de Células de Transição/genética , Mutação , Monoéster Fosfórico Hidrolases/genética , Proteínas Supressoras de Tumor , Neoplasias da Bexiga Urinária/genética , Carcinoma de Células de Transição/sangue , Carcinoma de Células de Transição/enzimologia , Cromossomos Humanos Par 10 , DNA de Neoplasias/análise , DNA de Neoplasias/genética , Deleção de Genes , Genes Supressores de Tumor , Homozigoto , Humanos , Perda de Heterozigosidade , PTEN Fosfo-Hidrolase , Polimorfismo Conformacional de Fita Simples , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais Cultivadas , Neoplasias da Bexiga Urinária/sangue , Neoplasias da Bexiga Urinária/enzimologiaRESUMO
Deletions involving chromosome 9 occur in more than 50% of human bladder cancers of all grades and stages. Most involve loss of the whole chromosome or of an entire chromosome arm but some small deletions are found which can be used to define critical regions which may contain tumour suppressor genes. We have localized such a critical region of deletion at 9q34 between the markers D9S149 and D9S66, an interval which contains the Tuberous Sclerosis gene TSC1. Single strand conformation polymorphism (SSCP) and sequence analysis of TSC1 in bladder tumours and cell lines with 9q34 loss of heterozygosity (LOH) has identified five mutations in retained TSC1 alleles. Our results support the hypothesis that TSC1 can act as a bladder tumour suppressor gene.