Histone deacetylase inhibitors induce a senescence-like state in human cells by a p16-dependent mechanism that is independent of a mitotic clock.

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for three mean population doublings (MPDs) exhibited a similar overall proliferative life span to controls once SDB was withdrawn.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.

Telomerase alone extends the replicative life span of human skeletal muscle cells without compromising genomic stability.

Continuous cycles of muscle fiber necrosis and regeneration are characteristic of the muscular dystrophies, and in some cases this leads to premature replicative senescence of myoblasts in vitro. The molecular mechanism of senescence in human myoblasts is poorly understood but there is evidence to suggest that telomeric attrition may be one of the ways by which this is achieved. We report here, for the first time, the extension of normal human skeletal muscle cell replicative life span by the reconstitution of telomerase activity. The telomerase-expressing cells show no features of transformation in vitro and have stable genomes with diploid karyotypes, do not express exceptionally high levels of c-myc and have wild-type, unmethylated CDKN2A genes. In vivo, they regenerate to repair muscle injury in immunosuppressed RAG-1 mice. This work suggests that telomerase expression to repair short telomeres may aid the expansion of diploid human muscle cells and consequently attempts at gene therapy for muscle diseases.

High levels of telomere dysfunction bestow a selective disadvantage during the progression of human oral squamous cell carcinoma.

Human epithelial cells experience multiple barriers to cellular immortality in culture (mortality mechanisms 0, 1, and 2). Mortality mechanism 2 (M2) is termed crisis and involves telomere dysfunction due to lack of telomerase. However, proliferating normal keratinocytes in vivo can express telomerase, so it is unclear whether human squamous cell carcinomas (SCCs), which usually have high telomerase levels, develop from preexisting telomerase-positive precursors or by the activation of telomerase in telomerase-deficient somatic cells. We show that 6 of 29 oral SCCs show characteristics of M2 crisis in vivo, as indicated by a high anaphase bridge index (ABI), which is a good correlate of telomere dysfunction, and that 25 of 29 tumors possess some anaphase bridges. ABIs in excess of 0.2 in the primary tumor showed a decrease in the corresponding lymph node metastases. This suggests that high levels of telomere dysfunction (>0.2) and, by inference, M2 crisis bestow a selective disadvantage on SCCs during progression stages of the disease. Supporting this, SCCs with high levels of telomere dysfunction grow poorly in culture, and the ectopic expression of telomerase corrects this, together with other features of M2 crisis. Our data suggest that a substantial proportion of oral SCCs in vivo ultimately arise from telomerase-deficient keratinocytes rather than putative telomerase-proficient cells in the undifferentiated parts of the epithelium. Furthermore, the presence of significant levels of telomere dysfunction in a high proportion of SCCs at diagnosis but not in the normal epithelium implies that the therapeutic inhibition of telomerase should selectively compromise the growth of such tumors.

A mortality gene(s) for the human adenocarcinoma line HeLa maps to a 130-kb region of human chromosome 4q22-q23.

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.

Functional evidence for a squamous cell carcinoma mortality gene(s) on human chromosome 4.

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.