Human thyroid cancer cells as a source of iso-genic, iso-phenotypic cell lines with or without functional p53.

Differentiated thyroid carcinomas (in contrast to the rarer anaplastic form) are unusual among human cancers in displaying a remarkably low frequency of p53 mutation and appear to retain wild-type (wt) p53 function as assessed by the response of derived cell lines to DNA damage. Using one such cell line, K1, we have tested the effect of experimental abrogation of p53 function by generating matched sub-clones stably expressing either a neo control gene, a dominant-negative mutant p53 (143ala) or human papilloma virus protein HPV16 E6. Loss of p53 function in the latter two groups was confirmed by abolition of p53-dependent 'stress' responses including induction of the cyclin/CDK inhibitor p21WAF1 and G1/S arrest following DNA-damage. In contrast, no change was detected in the phenotype of 'unstressed' clones, with respect to any of the following parameters: proliferation rate in monolayer, serum-dependence for proliferation or survival, tumorigenicity, cellular morphology, or tissue-specific differentiation markers. The K1 line therefore represents a 'neutral' background with respect to p53 function, permitting the derivation of functionally p53 + or - clones which are not only iso-genic but also iso-phenotypic. Such a panel should be an ideal tool with which to test the p53-dependence of cellular stress responses, particularly the sensitivity to potential therapeutic agents, free from the confounding additional phenotypic differences which usually accompany loss of p53 function. The results also further support the hypothesis that p53 mutation alone is not sufficient to drive progression of thyroid cancer to the aggressive anaplastic form.

Control of replicative life span in human cells: barriers to clonal expansion intermediate between M1 senescence and M2 crisis.

The accumulation of genetic abnormalities in a developing tumor is driven, at least in part, by the need to overcome inherent restraints on the replicative life span of human cells, two of which-senescence (M1) and crisis (M2)-have been well characterized. Here we describe additional barriers to clonal expansion (Mint) intermediate between M1 and M2, revealed by abrogation of tumor-suppressor gene (TSG) pathways by individual human papillomavirus type 16 (HPV16) proteins. In human fibroblasts, abrogation of p53 function by HPVE6 allowed escape from M1, followed up to 20 population doublings (PD) later by a second viable proliferation arrest state, MintE6, closely resembling M1. This occurred despite abrogation of p21(WAF1) induction but was associated with and potentially mediated by a further approximately 3-fold increase in p16(INK4a) expression compared to its level at M1. Expression of HPVE7, which targets pRb (and p21(WAF1)), also permitted clonal expansion, but this was limited predominantly by increasing cell death, resulting in a MintE7 phenotype similar to M2 but occurring after fewer PD. This was associated with, and at least partly due to, an increase in nuclear p53 content and activity, not seen in younger cells expressing E7. In a different cell type, thyroid epithelium, E7 also allowed clonal expansion terminating in a similar state to MintE7 in fibroblasts. In contrast, however, there was no evidence for a p53-regulated pathway; E6 was without effect, and the increases in p21(WAF1) expression at M1 and MintE7 were p53 independent. These data provide a model for clonal evolution by successive TSG inactivation and suggest that cell type diversity in life span regulation may determine the pattern of gene mutation in the corresponding tumors.

Dissociation of telomere dynamics from telomerase activity in human thyroid cancer cells.

Prevention of telomere erosion through acquisition of telomerase activity is thought to be an essential mechanism in most human cancer cells for avoidance of cellular senescence and crisis. It has been generally assumed that once telomerase has been activated, no further telomere shortening should ensue. We show here, however, that a much more complex pattern of telomere dynamics can exist in telomerase-positive immortal cancer cells. Using a panel of subclones derived from a human thyroid cancer cell line, K1E7, we found that some clones show persistent decline in mean telomere restriction fragment (TRF) length by up to 2 kb over 450 population doublings (pd), despite sustained high telomerase activity (as assessed by the in vitro "TRAP" assay). TRF length subsequently stabilized at around 5 kb, but with no corresponding increase in telomerase activity. One clone showed an even more unexpected biphasic time course, with the mean TRF length initially increasing by 1.5 kb over 90 pd, before "plateauing" and then returning over a similar period to its original value, again without any correlation to TRAP activity. Such dissociations between telomere dynamics and telomerase activity support the existence of additional controls on telomere length in the intact cell. Our observations are consistent with current negative-feedback models of telomere length regulation by telomere binding proteins and these cell lines should prove useful experimental tools for their further evaluation.

S phase cell-cycle arrest following DNA damage is independent of the p53/p21(WAF1) signalling pathway.

It is now likely that the cyclin-kinase inhibitor, p21(WAF1/SD11), is a key effector of p53-mediated cell-cycle arrest at the G(1)/S checkpoint following DNA damage. More recently, however, in vitro data has suggested that this pathway may also mediate the acute inhibition of DNA synthesis seen in cells already in S phase. Here we address this question in an intact cell system using normal human diploid fibroblasts in which p53 function is manipulated by expression of a dominant-negative mutant (ala(143)) introduced by a retroviral vector. Induction of DNA strand breaks in normal control fibroblasts by exposure to bleomycin led as expected to G(1)/S cell cycle arrest, induction of p2l(WAF1) and a rapid reduction in the rate of DNA synthesis in cells already in S phase. Stable expression of mutant p53 abrogated the G(1)/S (but not the G(2)/M) cell cycle checkpoint and abolished the induction of p21(WAF1), but had no significant effect on the inhibition of DNA replication in S phase nuclei. We conclude that, despite the in vitro evidence for inhibitory activity on PCNA/polymerase delta, p21(WAF1) induction does not appear to be essential for the acute inhibition of DNA synthesis in the intact cell following strand-break damage in S phase.

Mutant p53 rescues human diploid cells from senescence without inhibiting the induction of SDI1/WAF1.

Although the cyclin-dependent kinase inhibitor p21SDI1 (WAF1/CIP1) has been proposed as the mediator of p53-induced cell cycle arrest following DNA damage, several stimuli now appear to induce SDI1 independent of p53 function. We have examined the behavior of p53 and SDI1 in an isogeneic model by manipulating p53 status in normal diploid human fibroblasts using an amphotropic retroviral vector. Following DNA strand break damage induced by bleomycin, both SDI1 induction and G1-S cell cycle arrest are p53 dependent, consistent with SDI1 being the key mediator. In contrast, in cellular senescence (and following UV irradiation), induction of SDI1 occurs independent of p53 function yet growth arrest is still p53 dependent. We conclude (a) that redundant pathways exist for induction of SDI1, but that (b) SDI1, while perhaps necessary, is not sufficient for inhibition of cell cycle progression, requiring the cooperation of an additional factor (possibly another cyclin-dependent kinase inhibitor) whose expression, at least in the case of senescence, is strictly p53 dependent.

Evasion of p53-mediated growth control occurs by three alternative mechanisms in transformed thyroid epithelial cells.

Using the thyroid as a model of multistep epithelial tumorigenesis, we have used representative cell lines to correlate the degree of malignant transformation with the functional status of p53 and the integrity of cell-cycle check-points. Three distinct phenotypes were observed: Type I lines, derived from poorly-differentiated human thyroid cancers, expressed high levels of mutant p53 protein; Type II, also poorly-differentiated but derived from rat, showed over-expression of wild-type (wt) p53 with marked cell-cell heterogeneity: Type III, from well-differentiated human cancers, contained uniformly low levels of wt p53. All cell lines containing wt p53 retained a near-normal induction of p53 by DNA damage. However, the ability to undergo growth arrest differed strikingly. Whereas Type I and II lines had lost both G2/M and G1/S check points, Type III cells retained both. In Type III cells, as in diploid human fibroblasts, mutant p53 expression specifically abrogated G1/S check-point function with no other change in phenotype. These data demonstrate 3 mechanisms for evasion of p53 growth control: (i) direct mutation (ii) indirect inactivation, or (iii) 'avoidance' of activation, most probably due to failure to reach a critical threshold of DNA damage.