RESUMO
Human chromosome 4 was previously shown to elicit features of senescence when introduced into cell lines that map to complementation group B for senescence, including HeLa cells. Subsequently, a DNA segment encoding the pseudogene Mortality Factor 4 (MORF4) was shown to reproduce some of the effects of the intact chromosome 4 and was suggested to be a candidate mortality gene. We have identified multiple MORF4 alleles in several cell lines and tissues by sequencing and have failed to detect any cancer-specific mutations in three of the complementation group B lines (HeLa, T98G, and J82). Furthermore, MORF4 was heterozygous in these lines. These results question whether MORF4 is the chromosome 4 mortality gene. To map other candidate mortality gene(s) on this chromosome, we employed microcell-mediated monochromosome transfer to introduce either a complete copy, or defined fragments of the chromosome into HeLa cells. The introduced chromosome 4 fragments mapped the mortality gene to a region between the centromere and the marker D4S2975 (4q27), thus excluding MORF4, which maps to 4q33-q34.1. Analysis of microsatellite markers on the introduced chromosome in 59 immortal segregants identified a frequently deleted region, spanning the markers BIR0110 and D4S1557. This defines a new candidate interval of 130 kb at 4q22-q23.
Assuntos
Mapeamento Cromossômico , Cromossomos Humanos Par 4/genética , Fatores de Transcrição/genética , Alelos , Animais , Senescência Celular/genética , Coloração Cromossômica , Cromossomos Humanos Par 4/metabolismo , Células Clonais , Genes Supressores de Tumor , Teste de Complementação Genética , Genótipo , Células HeLa/metabolismo , Humanos , Perda de Heterozigosidade , Camundongos , Repetições de Microssatélites , Fenótipo , Polimorfismo Genético , Fatores de Transcrição/metabolismoRESUMO
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.
