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By imposing a limit on the proliferative lifespan of most somatic cells, telomere erosion represents an innate mechanism for tumor suppression 1 and may contribute to age-related disease 2 . A detailed understanding of the pathways that link shortened telomeres to replicative senescence has been severely hindered by the inability of current methods to analyze telomere dynamics in detail. Here we describe single telomere length analysis (STELA), a PCR-based approach that accurately measures the full spectrum of telomere lengths from individual chromosomes. STELA analysis of human XpYp telomeres in fibroblasts identifies several features of telomere biology. We observe bimodal distributions of telomeres in normal fibroblasts; these distributions result from inter-allelic differences of up to 6.5 kb, indicating that unexpectedly large-scale differences in zygotic telomere length are maintained throughout development. Most telomeres shorten in a gradual fashion consistent with simple losses through end replication, and the rates of erosion are independent of allele size. Superimposed on this are occasional, more substantial changes in length, which may be the consequence of additional mutational mechanisms. Notably, some alleles show almost complete loss of TTAGGG repeats at senescence.

Telomere shortening in normal human cells causes replicative senescence, a p53‐dependent growth arrest state, which is thought to represent an innate defence against tumour progression. However, although it has been postulated that critical telomere loss generates a ‘DNA damage’ signal, the signalling pathway(s) that alerts cells to short dysfunctional telomeres remains only partially defined. We show that senescence in human fibroblasts is associated with focal accumulation of γ‐H2AX and phosphorylation of Chk2, known mediators of the ataxia‐telangiectasia mutated regulated signalling pathway activated by DNA double‐strand breaks. Both these responses increased in cells grown beyond senescence through inactivation of p53 and pRb, indicating that they are driven by continued cell division and not a consequence of senescence. γ‐H2AX (though not Chk2) was shown to associate directly with telomeric DNA. Furthermore, inactivation of Chk2 in human fibroblasts led to a fall in p21 waf1 expression and an extension of proliferative lifespan, consistent with failure to activate p53. Thus, Chk2 forms an essential component of a common pathway signalling cell cycle arrest in response to both telomere erosion and DNA damage.

Hepatocyte Growth Factor (HGF) receptor, encoded by the protooncogene c-met, is overexpressed in many human tumours, including those of thyroid epithelium. The absence in most cases of any primary structural abnormality of the met gene suggests that overexpression is secondary to mutation of other gene(s). To test this hypothesis we investigated the effect on met expression of two activated oncogenes known to play a major role in thyroid oncogenesis, ras and ret. To minimize the possibility of unknown co-operating events, we introduced these genes directly into normal human thyrocytes in primary culture using amphotropic retroviral vectors and assessed met expression as early as possible in the resulting epithelial colonies. Double immunofluorescence revealed expression of met protein, strictly localized to cells expressing the mutant ras and ret vectors, expression in background normal cells being barely detectable. In contrast, colonies induced to proliferate at a comparable rate by a vector expressing SV40 T showed no increase in met expression. To permit quantitation by Western blotting we also extended these findings to a thyroid cell line (R18) containing a zinc-inducible mutant ras gene. Induction of the oncogene led to a fourfold increase in met protein expression. We conclude that overexpression of met is induced by activation of the ras or ret signalling pathway and not simply by deregulation of the cell cycle per se. The data suggest that the proliferative advantage conferred by these oncogenes may be in part due to the resulting sensitization of tumour epithelium to paracrine HGF secreted by stromal cells.

We report that a subject with Cockayne syndrome type A (CS3BE) was a compound heterozygote for mutations in CKN1 , the gene encoding the CSA protein (MIM 216400). CS3BE displayed a novel missense mutation (A160V) and a previously described nonsense mutation (E13X). Although residing between the second and third WD-40 repeats characteristic of the CSA protein, A160 is completely conserved in all species that possess a CKN1 homologue. We also describe a mutation in a previously uncharacterised xeroderma pigmentosum group C subject (XP8CA) in the XPC gene (MIM 278720). XP8CA was homozygous for a 2 bp TG deletion in codon 547 resulting in premature termination at codon 572. Immunoblotting of XP8CA extracts confirmed the absence of full-length XPC protein that was present in unaffected cell lines.

Loss of function of the tumour suppressor gene p53 is a key event in most human cancers. Although usually occurring through mutation, in some tumour types this appears to be achieved via an indirect mechanism involving inappropriate expression of a functional inhibitor, mdm2, which binds to the transactivation domain of p53. This interaction offers an ideal potential target for novel cancer therapies. However, therapeutic specificity may depend on the extent to which this p53-inhibitory action of mdm2 is also required by normal cells. Transgenic data have already established that mdm2 is needed to prevent embryonic lethality, but the situation in adult cells is still unclear. Here we show that micro-injection of normal human fibroblasts with an antibody directed against the p53-binding domain of mdm2 induces expression of p53-responsive genes, and furthermore results in p53-dependent growth arrest. We conclude that normal cell proliferation can be dependent on negative regulation of p53 by mdm2, a finding which raises an important note of caution for mdm2-directed cancer therapies.

Werner syndrome (WS) is a rare disorder inherited in an autosomal recessive manner and characterized by accelerated ageing 1 . WS fibroblasts display an accelerated rate of senescence in vitro 2 , which has been linked to this progeroid phenotype. The senescence of normal human fibroblasts is triggered by telomere shortening 3 , 4 , 5 , whereas the premature senescence of WS fibroblasts has been assumed 6 , 7 to reflect the accumulation of DNA damage. Here we show that forced expression of telomerase in WS confers extended cellular lifespan and probable immortality.

Replicative senescence is thought to be a significant barrier to human tumorigenesis, which in human fibroblasts, and many other cell types, can be overcome experimentally by combined loss of function of p53 and Rb 'pathways'. To avoid the confounding pleiotropic effects of HPVE7 frequently used in such studies, here we have employed retroviral vectors over-expressing CDK4 or CDK6 as a more representative model of naturally-occurring mutations targeting the Rb pathway. We show that these can extend fibroblast lifespan by approximately 10 population doublings, ending in a viable senescence-like state which contrasts with the apoptotic end-stage seen with E7. Compared with 'normal' senescence, this growth arrest was, in most cases, not accompanied by any further increase in p21(Waf1) levels but with up to a 19-fold increase in p16(Ink4a). Surprisingly however, this could not explain arrest, since expression of mutant CDK4 and/or CDK6, incapable of binding p16(Ink4a), did not confer any greater lifespan extension than the wild-type CDKs. Subsequent abrogation of p53 function by a second vector, encoding HPVE6, downregulated p21(Waf1) and conferred a second lifespan extension, ending in a crisis-like state, consistent with full escape from senescence. These data: (i) point to a back-up 'senescence' mechanism distinct from induction of p21(Waf1) or p16(Ink4a); and (ii) provide an in vitro model of clonal evolution through successive dysfunction of Rb and p53 pathways in a relevant human cell context.

In contrast to its growth-inhibitory effect on primary mesenchymal cells, RAS oncogene activation induces a proliferative phenotype in normal human thyroid epithelial cells in vitro, consistent with its putative role in tumour initiation. Using this model, we previously showed that activation of the MAP kinase (MAPK) pathway is necessary, but not sufficient for the proliferative response to mutant (V12) H-RAS. Here we extend this work to show that another major RAS effector-- phosphatidylinositol-3-kinase (PI-3-K)--while also insufficient alone, is able to synergize with MAPK activation to mimic the effect of mutant RAS, albeit at reduced efficiency. Furthermore we show that PI-3-K is an absolute requirement for the proliferative response to RAS in these cells, acting via suppression of RAS-induced apoptosis. These data extend our understanding of RAS signalling in a clinically-relevant cell context and point to the use of PI-3-K inhibitors as potential therapeutic agents for targetting human cancers induced by RAS mutation.

Current evidence suggests the papillary thyroid carcinoma oncogene (RET/PTC) generates papillary thyroid carcinomas in one genetic step. We tested a resulting prediction that RET/PTC expression in thyroid epithelium should be sufficient to cause the changes in nuclear morphology diagnostic of this tumor. Primary cultures of human thyroid epithelial cells were infected with a RET/PTC retroviral construct. Morphological scoring by two independent cytopathologists shows RET/PTC expression by immunohistochemistry to be highly associated ( p ≪ 0.0001) with an irregular nuclear contour and a euchromatic appearance compared with non-expressing cells in the same cultures. The altered nuclear morphology is not due to gene transfer or transformation per se as primary thyroid cell cultures infected with a retroviral H-RAS construct differ from RET/PTC-infected cells by showing round nuclear envelopes and coarser chromatin, as determined by the independent scoring of two cytopathologists ( p ≪ 0.0001). In addition, RET/PTC-transfected cells appear to disperse, whereas RAS-transfected cells grow as discrete colonies. The results provide additional support for the hypothesis that RET/PTC is sufficient to cause papillary thyroid carcinomas. A signaling pathway downstream of RET/PTC leads to restructuring of the nuclear envelope and chromatin, and the signal does not depend entirely, if at all, on a RAS pathway.

Neoplastic transformation of rodent thyroid epithelial cell lines by mutant RAS genes has been widely studied as an experimental model of oncogene-induced loss of tissue-specific differentiation. However, separate evidence strongly implicates RAS mutation as an early event in human thyroid tumour development at a stage prior to loss of differentiation. To resolve this controversy we examined the short- and long-term responses of normal human thyroid epithelial cells to mutant RAS introduced by micro-injection and retroviral transduction respectively. In both cases, expression of RAS at a level sufficient to induce rapid proliferation did not lead to loss of differentiation as shown by expression of cytokeratin 18, E-cadherin, thyroglobulin, TTF-1 and Pax-8 proteins. Indeed, RAS was able to prevent, and to reverse, the loss of thyroglobulin expression which occurs normally in TSH-deficient culture medium. These responses were partially mimicked by activation of RAF, a major RAS effector, indicating involvement of the MAP Kinase signal pathway. The striking contrast between the effect of mutant RAS on differentiation in primary human, compared to immortalized rodent, epithelial cultures is most likely explained by the influence of additional co-operating abnormalities in the latter, and highlights the need for caution in extrapolating from cell line data.