Cellular senescence in naevi and immortalisation in melanoma: a role for p16?

Cellular senescence, the irreversible proliferative arrest seen in somatic cells after a limited number of divisions, is considered a crucial barrier to cancer, but direct evidence for this in vivo was lacking until recently. The best-known form of human cell senescence is attributed to telomere shortening and a DNA-damage response through p53 and p21. There is also a more rapid form of senescence, dependent on the p16-retinoblastoma pathway. p16 (CDKN2A) is a known melanoma susceptibility gene. Here, we use retrovirally mediated gene transfer to confirm that the normal form of senescence in cultured human melanocytes involves p16, since disruption of the p16/retinoblastoma pathway is required as well as telomerase activation for immortalisation. Expression (immunostaining) patterns of senescence mediators and markers in melanocytic lesions provide strong evidence that cell senescence occurs in benign melanocytic naevi (moles) in vivo and does not involve p53 or p21 upregulation, although p16 is widely expressed. In comparison, dysplastic naevi and early (radial growth-phase, RGP) melanomas show less p16 and some p53 and p21 immunostaining. All RGP melanomas expressed p21, suggesting areas of p53-mediated senescence, while most areas of advanced (vertical growth-phase) melanomas lacked both p16 and p21, implying escape from both forms of senescence (immortalisation). Moreover, nuclear p16 but not p21 expression can be induced in human melanocytes by oncogenic BRAF, as found in around 80% of naevi. We conclude that cell senescence can form a barrier to melanoma development. This also provides a potential explanation of why p16 is a melanoma suppressor gene.

p53 promotes adenoviral replication and increases late viral gene expression.

The tumor suppressor protein, p53, plays a critical role in viro-oncology. However, the role of p53 in adenoviral replication is still poorly understood. In this paper, we have explored further the effect of p53 on adenoviral replicative lysis. Using well-characterized cells expressing a functional p53 (A549, K1neo, RKO) and isogenic derivatives that do not (K1scx, RKOp53.13), we show that virus replication, late virus protein expression and both wtAd5 and ONYX-015 virus-induced cell death are impaired in cells deficient in functional p53. Conversely, by transfecting p53 into these and other cells (IIICF/c, HeLa), we increase late virus protein expression and virus yield. We also show, using reporter assays in IIICF/c, HeLa and K1scx cells, that p53 can cooperate with E1a to enhance transcription from the major late promoter of the virus. Late viral protein production is enhanced by exogenous p53. Taken together, our data suggest that functional p53 can promote the adenovirus (Ad) lytic cycle. These results have implications for the use of Ad mutants that are defective in p53 degradation, such as ONYX-015, as agents for the treatment of cancers.

Biological activity of activating thyroid-stimulating hormone receptor mutants depends on the cellular context.

Activating TSH receptor (TSHR) mutations are a major cause of toxic thyroid adenoma and familial hyperthyroidism, and more than 37 such mutations have been described. Previously their functional activity had been assessed in terms of cAMP and inositol phosphate production and predominantly in transiently transfected COS-7 (monkey embryonic kidney cells), a model that does not reflect effects on thyrocyte proliferation and function. Here we have performed a systematic comparison of wild-type and seven gain-of-function TSHR mutants, introduced into rat FRTL-5 and human thyrocytes, using retroviral vectors. Our results show that 1) biological potency of TSHR mutants in thyroid cells does not correlate with their cAMP levels in transfected COS cells, highlighting the importance of cellular context and level of expression when assessing biological effects of oncogenic mutations; 2) dissociation between stimulation of function and growth occurs with thyrocyte differentiated functions more readily stimulated than growth; 3) TSHR mutants show a similar order of potency in FRTL-5 cells and human thyrocytes; 4) mutants inducing the highest stimulation of adenylyl cyclase may paradoxically fail to induce proliferation; and 5) biological effects of cAMP activating TSHR mutants are attenuated by complex counterregulatory mechanisms at least at the level of phosphodiesterases and cAMP regulatory element modulator isoforms.

Telomerase activity and telomere length in thyroid neoplasia: biological and clinical implications.

Despite several recent studies, the biological status and clinical relevance of telomerase expression in tumours derived from the thyroid follicular cell remain controversial. This study has analysed a series of normal, benign, and malignant thyroid samples using two novel approaches: the use of purified epithelial cell fractions to eliminate false-positives due to telomerase-positive infiltrating lymphocytes; and the simultaneous measurement of telomere length to provide a clearer interpretation of telomere dynamics in thyroid neoplasia. The data obtained support the prediction that the epithelial component of non-neoplastic thyroid and of follicular adenomas is telomerase-negative, any positive results being explicable by lymphocyte infiltration. In contrast, many malignant tumours, both follicular and papillary, were telomerase-positive. However, serial dilution of extracts indicated a wide spectrum of activity in these cancers, possibly related to variation in the proportion of telomerase-positive cells. Furthermore, an unexpectedly high proportion were telomerase-negative, a finding which was not explicable by technical problems such as TRAP (telomeric repeat amplification protocol) assay sensitivity. Many of these apparently telomerase-negative tumours had abnormally long telomeres. Correlation of telomerase and telomere length data suggests that thyroid cancers fall into three biological groups: telomerase-positive lesions, consistent with the conventional model of telomere erosion followed by telomerase reactivation; telomerase-negative tumours, which maintain telomere length by a mechanism independent of telomerase; and telomerase-negative tumours which are still undergoing telomere erosion and may therefore be composed of mortal cancer cells. From a clinical standpoint, it is concluded that telomerase detection on unfractionated tissue, such as fine needle aspirates, is of no value as a marker of malignancy in follicular lesions, due to both low sensitivity and specificity.

Simian virus 40 detection in human mesothelioma: reliability and significance of the available molecular evidence.

Simian virus 40 was discovered as a contaminant of early poliovirus vaccines that were inadvertently administered to millions of people in Europe and the United States between 1955 and 1963. Although SV40 was proven to be oncogenic in rodents and capable of transforming human and animal cells in vitro, its role in human cancer could not be proven epidemiologically. The matter was forgotten until 1993 when SV40 was accidentally found to cause mesotheliomas in hamsters injected intra-cardially. Subsequently, DNA sequences associated with its powerful oncogenic principal, the large T antigen, were found with high frequency in human pleural mesothelioma using the polymerase chain reaction (PCR). Since then many laboratories have confirmed the human findings. However, a few laboratories have failed to reproduce these data and the authors of the studies have claimed that the detection of SV40 DNA may simply represent PCR contamination artefacts. The controversy raised by this viewpoint is reviewed in this article together with a critical appraisal of the reliability of the molecular techniques used to detect SV40 DNA, in order to evaluate the potential aetiopathogenic role of SV40 in human mesothelioma.

Transient ectopic expression of PTEN in thyroid cancer cell lines induces cell cycle arrest and cell type-dependent cell death.

The tumour suppressor gene PTEN/MMAC1/TEP1 has been implicated in a variety of human cancers and several inherited hamartoma tumour syndromes, including Cowden syndrome, which has a high risk of breast and thyroid cancer. We have previously reported that overexpression of PTEN in MCF-7 breast cancer cells induces cell cycle arrest and apoptosis. In this study, we analysed PTEN status at both the structural and expression levels and explored PTEN's growth-suppressive effects on thyroid. We found that 1 of 10 thyroid cancer lines [follicular thyroid carcinoma FTC-133] had hemizygous deletion and a splice variant IVS4--19G-->A in the remaining allele. Four lines, including FTC-133, express PTEN mRNA at low levels. In general, PTEN protein levels correlated with mRNA levels, except for NPA87, which has low levels of transcript and relatively high levels of PTEN protein. Transient expression of PTEN in seven thyroid cancer cell lines resulted in G(1) arrest in two well differentiated papillary thyroid cancer lines (PTCs) and both G(1) arrest and cell death in the remaining five lines, including three FTCs, one poorly differentiated PTC and one undifferentiated thyroid cancer. The level of phosphorylated Akt was inversely correlated with the endogenous level of PTEN protein and overexpression of PTEN-blocked Akt phosphorylation in all cells analysed. Our results suggest that downregulation of PTEN expression at the mRNA level plays a role in PTEN inactivation in thyroid cancer and PTEN exerts its tumour-suppressive effect on thyroid cancer through the inhibition of cell cycle progression alone or both cell cycle progression and cell death.

Evidence for a telomere-independent "clock" limiting RAS oncogene-driven proliferation of human thyroid epithelial cells.

An initiating role for RAS oncogene mutation in several epithelial cancers is supported by its high incidence in early-stage tumors and its ability to induce proliferation in the corresponding normal cells in vitro. Using retroviral transduction of thyroid epithelial cells as a model we ask here: (i) how mutant RAS can induce long-term proliferation in an epithelial cell in contrast to the premature senescence observed in fibroblasts; and (ii) what is the "clock" which eventually triggers spontaneous growth arrest even in epithelial clones generated by mutant RAS. The early response to RAS activation in thyroid epithelial cells showed two features not seen in fibroblasts: (i) a marked decrease in expression of the cyclin-dependent kinase inhibitor (CDKI) p27(kip1) and (ii) the absence of any induction of p21(waf1). When proliferation eventually ceased (after up to 20 population doublings) this occurred despite undiminished expression of mutant RAS and was tightly correlated with a return to the initial high level of p27(kip1) expression, together with the de novo appearance of p16(ink4a). Importantly, neither the CDKI changes nor the proliferative life span of RAS-induced epithelial clones was altered by induction of telomerase activity through forced expression of the catalytic subunit, hTERT, at levels sufficient to immortalize human fibroblasts. These data provide a basis for cell-type differences in sensitivity to RAS-induced proliferation which may explain the corresponding tumor-type specificity of RAS mutation. They also show for the first time in a primary human cell model that a telomere-independent mechanism can limit not only physiological but also oncogene-driven proliferation, pointing therefore to a tumour suppressor mechanism additional, or alternative, to the telomere clock.

Replicative senescence: mechanisms and implications for human cancer.

The proliferative lifespan of most normal human cells, even in ideal growth conditions, is limited by intrinsic inhibitory signals which induce cell cycle arrest after a preset number of cell divisions. This process of 'replicative senescence' is activated in many cell types by the progressive erosion of the specialised ends of chromosomes--telomeres--which act as a molecular 'clock'. Although many details are still to be elucidated, one major signal pathway linking telomere shortening to growth arrest operates via activation of the tumour suppressor gene (TSG) product, p53, which in turn induces the cell-cycle inhibitor p21WAF1, and at least in some cell types wild-type p53 function is an absolute requirement for normal senescence. Given the evidence that replicative senescence represents a natural obstacle to tumour progression, the need to escape p53-mediated senescence may therefore represent a major selection pressure for loss of p53 function in many human cancers.

PI-3-kinase is an essential anti-apoptotic effector in the proliferative response of primary human epithelial cells to mutant RAS.

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.

Posttranslational modifications of p53 in replicative senescence overlapping but distinct from those induced by DNA damage.

Replicative senescence in human fibroblasts is absolutely dependent on the function of the phosphoprotein p53 and correlates with activation of p53-dependent transcription. However, no evidence for posttranslational modification of p53 in senescence has been presented, raising the possibility that changes in transcriptional activity result from upregulation of a coactivator. Using a series of antibodies with phosphorylation-sensitive epitopes, we now show that senescence is associated with major changes at putative regulatory sites in the N and C termini of p53 consistent with increased phosphorylation at serine-15, threonine-18, and serine-376 and decreased phosphorylation at serine-392. Ionizing and UV radiation generated overlapping but distinct profiles of response, with increased serine-15 phosphorylation being the only common change. These results support a direct role for p53 in signaling replicative senescence and are consistent with the generation by telomere erosion of a signal which shares some but not all of the features of DNA double-strand breaks.

RAS oncogene activation induces proliferation in normal human thyroid epithelial cells without loss of differentiation.

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.

Activation of mitogen-activated protein kinase is necessary but not sufficient for proliferation of human thyroid epithelial cells induced by mutant Ras.

Given the high frequency of ras oncogene activation in several common human cancers, its signal pathways are an important target for novel therapy. For practical reasons, however, these have been studied mainly in the context of transformation of established fibroblast cell lines, whereas ras acts at an earlier stage in human tumorigenesis and predominantly on epithelial cells. Here we have developed a more directly relevant model - human primary thyroid epithelial cells - which are a major target of naturally-occurring Ras mutation, and in which expression of mutant Ras in culture induces clonal expansion without morphological transformation, closely reproducing the phenotype of the corresponding tumour in vivo. Transient or stable expression of mutant H-ras (by scrapeloading or retroviral infection) at levels which stimulated proliferation induced sustained activation and translocation of MAP kinase (MAPK) in these cells. Inhibition of the MAPK pathway at the level of MAPKK, by expression of a dominant-negative mutant or by the pharmacological inhibitor PD98059, efficiently blocked the proliferative response. Conversely, selective activation of MAPK by a constitutively-active MAPKK1 mutant failed to mimic the action of Ras and, although this was achievable with activated Raf, micro-injection of anti-ras antibodies showed that this still required endogenous wild-type Ras function. In contrast to recent results obtained with a rodent thyroid cell line (WRT), therefore, activation of the MAPK pathway is necessary, but not sufficient, for the proliferogenic action of mutant Ras on primary human thyroid cells. These data emphasize the unreliability of extrapolation from cell lines and establish the feasibility of using a more representative human epithelial model for Ras signalling studies.

Contrasting effects of activating mutations of GalphaS and the thyrotropin receptor on proliferation and differentiation of thyroid follicular cells.

The cyclic AMP pathway is a major regulator of thyrocyte function and proliferation and, predictably, its inappropriate activation is associated with a sub-set of human thyroid tumours. Activating mutations are, however, more common in the thyrotropin receptor (TSHR) than in its downstream transducer, Galphas. To investigate whether this reflects an inherent difference in their oncogenic potency, we compared the effects of retrovirally-transduced mutant (A623I) TSHR or (Q227L) Galphas (GSP), using the rat thyroid cell line FRTL5 and primary human thyrocytes. In FRTL5, expression of GSP or mutant (m) TSHR induced a 2 - 3-fold increase in basal levels of cAMP. This was associated with TSH-independent proliferation (assessed by both cell number and DNA synthesis) and function (as shown by increased expression of thyroglobulin (Tg) and the sodium/iodide symporter). In primary cultures, expression of mTSHR, but not GSP, consistently induced formation of colonies with epithelial morphology and thyroglobulin expression, capable of 10 - 15 population doublings (PD) compared to less than three in controls. Thus, while mTSHR and GSP exert similar effects in FRTL5, use of primary cultures reveals a major difference in their ability to induce sustained proliferation in normal human thyrocytes, and provides the first direct evidence that mTSHR is sufficient to initiate thyroid tumorigenesis.

p53-Dependent growth arrest and altered p53-immunoreactivity following metabolic labelling with 32P ortho-phosphate in human fibroblasts.

The tumour suppressor gene p53 plays a major role in the cellular response to DNA damage, mediating growth arrest and/or apoptosis. Phosphorylation of the protein occurs at numerous sites in vivo and is likely to be a major mechanism for modulation of its activity as a transcriptional transactivator. Not surprisingly, therefore, p53 has been intensively studied by 32P metabolic labelling. Here we show however, using normal human fibroblasts, that typical labelling conditions induce (i) a p53-dependent inhibition of DNA synthesis and (ii) an increase in the cellular content of p53 protein detectable by the phosphorylation-sensitive antibody DO-1 but not by antibody DO-12. These data demonstrate for the first time that 32P labelling is sufficient to induce a biologically-significant, p53-mediated cellular response and strongly suggest that it perturbs the phosphorylation state of p53 which it is being used to measure. This highlights the need to re-evaluate earlier data by non-radioactive approaches using phospho-specific antibodies.

Cellular senescence and cancer.

The proliferative lifespan of normal mammalian cells is limited by intrinsic controls, which desensitize the cell-cycle machinery to extrinsic stimulation after a given number of cell divisions. One underlying clock driving this process of 'replicative senescence' is the progressive erosion of chromosome telomeres, which occurs with each round of DNA replication. This appears to trigger growth inhibition via activation of the tumour suppressor gene (TSG) product, p53, and the consequent up-regulation of the cell-cycle inhibitor p21WAF1. Other inhibitory pathways are also activated (possibly by additional clocks), including the TSG p16INK4a and the less well-defined complementation group genes. Loss of one pathway can be compensated, after a limited extension of lifespan, by further up-regulation of the others, so that to escape mortality a developing tumour must overcome multiple 'proliferative lifespan barriers' (PLBs) by successive genetic events, each conferring a new wave of clonal expansion. This provides one explanation for the existence of multiple genetic abnormalities in human cancers; furthermore, the diversity in the nature and timing of these PLBs between different cell types may explain the variation in the spectrum of abnormalities observed between the corresponding cancers. Even if all senescence pathways are inactivated, immortalization can only be achieved if erosion of telomeres is halted, before their end-protecting function is lost. This usually requires either activation of telomerase during tumour development, if the cell of origin is telomerase-negative, or up-regulation if the normal cell already has some activity, but not enough to prevent erosion. In either case, cancers often maintain near-critical telomere lengths; hence pharmacological inhibition of telomerase remains an attractive approach to the selective killing of tumour cells.

[Molecular basis of tumors arising in thyroid follicular cells]. / Base moléculaire des tumeurs développées à partir des cellules thyroïdiennes folliculaires.

Tumours arising from the thyroid follicular cell have proven to be a very useful model for studying the molecular genetics of tumour development. This review summarises our current knowledge of the principal abnormalities of oncogenes and tumour suppressor genes associated with the major sub-types of thyroid tumour. The pattern which emerges demonstrates well how successive genetic abnormalities drive clonal progression. In addition, comparison of follicular and papillary sub-types provides a fascinating example of how the nature of the underlying genetic abnormality may determine the clinico-pathological behaviour of the resulting tumour. The potential impact of this molecular data on clinical management is also discussed.

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.