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.

Reinitiation of DNA synthesis and cell division in senescent human fibroblasts by microinjection of anti-p53 antibodies.

In human fibroblasts, growth arrest at the end of the normal proliferative life span (induction of senescence) is dependent on the activity of the tumor suppressor protein p53. In contrast, once senescence has been established, it is generally accepted that reinitiation of DNA synthesis requires loss of multiple suppressor pathways, for example, by expression of Simian virus 40 (SV40) large T antigen, and that even this will not induce complete cell cycle traverse. Here we have used microinjection of monoclonal antibodies to the N terminus of p53, PAb1801 and DO-1, to reinvestigate the effect of blocking p53 function in senescent human fibroblasts. Unexpectedly, we found that both antibodies induce senescent cells to reenter S phase almost as efficiently as SV40, accompanied by a reversion to the "young" morphology. Furthermore, this is followed by completion of the cell division cycle, as shown by the appearance of mitoses, and by a four- to fivefold increase in cell number 9 days after injection. Immunofluorescence analysis showed that expression of the p53-inducible cyclin/kinase inhibitor p21sdi1/WAF1 was greatly diminished by targeting p53 with either PAb1801 or DO-1 but remained high and, moreover, still p53 dependent in cells expressing SV40 T antigen. As previously observed for induction, the maintenance of fibroblast senescence therefore appears to be critically dependent on functional p53. We suggest that the previous failure to observe this by using SV40 T-antigen mutants to target p53 was most probably due to incomplete abrogation of p53 function.

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.

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.

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.

Conditional immortalization of human thyroid epithelial cells: a tool for analysis of oncogene action.

To overcome the difficulty of assessing oncogene action in human epithelial cell types, such as thyroid, which have limited proliferative potential in culture, we have explored the use of temperature-sensitive (ts) mutants of simian virus 40 (SV40) early region to create conditionally immortalized epithelial cell lines. Normal primary cultures of human thyroid follicular cells were transfected with a plasmid containing the SV40 early region from mutant tsA58. Expanding epithelial colonies were observed after 2 to 3 months, all of which grew to greater than 200 population doublings without crisis. All showed tight temperature dependence for growth. After switch-up to the restrictive temperature (40.5 degrees C), no further increase in cell number was seen after 1 to 2 days. However, DNA synthesis declined much more slowly; the dissociation from cell division led to marked polyploidy. Viability was maintained for up to 2 weeks. Introduction of an inducible mutant ras gene into ts thyroid cells led, as expected, to morphological transformation at the permissive temperature when ras was induced. Interestingly, this was associated with a marked reduction in net growth rate. At the restrictive temperature, induction of mutant ras caused rapid cell death. These results demonstrate the utility of a ts SV40 mutant to permit the study of oncogene action in an otherwise nonproliferative target cell and reveal important differences in the interaction between ras and SV40 T in these epithelial cells compared with previously studied cell types.

Experimental induction of parathyroid adenomas in the rat.

Neonatal inbred Wistar albino rats were given either 5 or 10 microCi radioiodine (131I) within 24 hours of birth. After weaning, animals were placed on diets high, normal, or deficient in vitamin D3 (cholecalciferol) for periods up to 2 years. In animals aged 12 months and older, adenomas were found in 0 of 67 unirradiated controls, in 22 of 67 given 5 microCi 131I, and in 25 of 67 given to microCi 131I. The incidence of tumors in irradiated animals was highest (55%) in those on a low-vitamin D diet and lowest (20%) in those on a high-vitamin D diet. Plasma calcium levels were significantly increased by the high-vitamin D diet, but the low-vitamin D diet did not lead to any significant decrease as compared to the calcium levels of the normal vitamin D diet group. Small but significant calcium increases were found in tumor-bearing animals. These findings indicate that parathyroid tumors in the rat can be induced by radiation and that their incidence is strongly influenced by dietary vitamin D content. The possibility that metabolites of vitamin D3 may influence parathyroid growth and tumor formation directly is discussed.

Expression of mutant p21ras induces insulin-like growth factor 1 secretion in thyroid epithelial cells.

There is substantial evidence supporting the existence of insulin-like growth factor 1 (IGF-1) autocrine circuits in many tumor types, although the underlying inducing event has remained undefined. In order to address this matter, we have generated several immortalized human thyroid epithelial cell lines containing a zinc-inducible mutant H-ras gene and used these to investigate the relationship between expression of mutant p21ras and secretion of IGF-1. Induction of the transgene in the presence of zinc ions (Zn2+) was confirmed by Northern blot analysis and immunocytochemistry. IGF-1 levels in serum-free medium, stripped of binding proteins, were monitored using a sensitive radioimmunoassay. Expression of mutant p21ras in these cells, induced by Zn2+, resulted in an approximate 30-fold increase in the IGF-1 production rate, reaching a level exceeding that of human embryo fibroblasts. The data presented here suggest that an activating mutation of a ras oncogene may directly account for IGF-1 secretion in some human tumor cells.

Prolongation of fibroblast life span associated with epithelial rat tumor development.

We have examined the proliferative behavior in culture of stromal cells of fibroblast morphology taken from various stages in the development of experimentally induced epithelial tumors in the rat thyroid. In this model long-term stimulation by an elevated level of serum thyroid stimulating hormone leads first to hyperplasia, then to multiple benign, and finally malignant tumors. Fibroblasts from hyperplastic glands, despite having already undergone more divisions in vivo, consistently showed a longer life span than normal fibroblasts in culture, averaging 16 compared with six divisions before senescing. In the three tumor bearing glands studied, all cultures have undergone more than 40 divisions (in one case over 70) and none has yet shown signs of senescence. We conclude that the life span of thyroid fibroblasts observed in culture has been reset to a higher limit by growth stimulation in vivo and furthermore that this limit may be lost altogether in fibroblasts associated with epithelial tumors.

N-myc expression in neoplasia of human thyroid C-cells.

In view of the frequent reports of the increased expression of myc oncogenes in several neuroendocrine tumor types, we have investigated c- and N-myc expression in human medullary carcinoma, a malignant tumor derived from the neuroendocrine "C"-cell subpopulation of the thyroid gland. In situ nucleic acid hybridization was used to permit analysis not only of tumors but also of normal C-cells which form a tiny, scattered, minority of the thyroid epithelial cell population. N-myc expression was readily demonstrable in 6 of 21 tumor samples and c-myc in one case, whereas neither N- nor c-myc mRNA was ever detected in normal C-cells. We conclude that N-myc expression is a specific feature of C-cell tumors and is not merely a differentiation marker of their cell of origin. The data therefore strengthen the hypothesis that myc oncogene activation plays a role in neuroendocrine neoplasia.

A phenotypically and karyotypically stable human thyroid epithelial line conditionally immortalized by SV40 large T antigen.

Primary cultures of normal human neonatal thyroid follicular cells were transfected with a plasmid expressing a temperature-sensitive (tsA58) mutant of SV40 large T antigen. An epithelial cell line, designated B-thy-ts.1, was obtained which showed tight temperature-dependent growth. In sharp contrast to previous such lines, which were derived from adult thyroid, B-thy-ts.1 has retained a well-differentiated phenotype as reflected in its morphology and cytokeratin expression pattern. In addition to phenotypic stability the line also displays an unusually stable karyotype, lacking the usual clastogenic effects of SV40, which we speculate to result from a greater DNA repair capacity of its cell of origin. B-thy-ts.1 should be a particularly useful tool with which to study the effects of activated oncogenes on epithelial growth and differentiation.

Activated ras oncogenes in human thyroid cancers.

Human thyroid epithelial (follicular) cells give rise to two malignant tumors--"follicular" carcinomas, which metastasize almost exclusively via the bloodstream, and "papillary" carcinomas, which metastasize predominantly via lymphatics (Williams, E. D. In: W. Duncan (ed.), Recent Results in Cancer Research: Thyroid Cancer, pp. 47-55. Berlin: Springer-Verlag, 1980). We have investigated whether this contrast in biological behavior might be associated with different patterns of oncogene activation. DNA transfection analysis of five follicular and ten papillary cancers indeed showed a statistically significant difference in the pattern of genes responsible, activated ras oncogenes being found in 80% of follicular tumors but only 20% of papillary tumors. In addition, in follicular cancers we have found activation of all three ras oncogenes (H-ras, K-ras, and N-ras), the first time that this has been demonstrated in a primary human tumor type (as opposed to cell lines). We suggest therefore that ras activation may be an important determinant of metastatic capability in these epithelial cancers.

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.

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.

The proliferation of normal human fibroblasts is dependent upon negative regulation of p53 function by mdm2.

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.

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.

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.

Interaction between p53 and TGF beta 1 in control of epithelial cell proliferation.

Although loss of sensitivity to transforming growth factor beta (TGF beta) may be a key step in the escape of epithelial tumours from normal growth control, the intracellular signals determining responsiveness remain controversial, particularly the role of p53. We have investigated this question using thyroid epithelial lines as a model. We analysed (i) human thyroid cancer cell lines having either wild-type (wt) or mutant p53; (ii) rat thyroid lines derived by spontaneous immortalisation following introduction of mutant H-ras, which exhibit high levels of wt p53 but loss of p53-mediated cell-cycle control. Loss of response to TGF beta 1 was found in all human lines bearing mutant p53, and in the majority of the functionally equivalent rat lines, consistent with a role of wt p53 in mediating response. However, introduction of a dominant negative p53 mutant into TGF beta 1 responsive human lines containing wt p53 did not reduce responsiveness, demonstrating that p53 function is not necessary for TGF beta 1 response. On the other hand, expression of a temperature-sensitive (ts) p53 gene in a partially-responsive rat line demonstrated a highly significant modulation of TGF beta response, which fell from 65% inhibition of 3H-thymidine labelling index at 32.5 degrees C (wt p53 conformation) to only 14% at 37.5 degrees C (mutant conformation). The results suggest that p53 and TGF beta generate separate but interacting inhibitory signals, i.e. that p53 modulates but does not mediate TGF beta response. This conclusion explains previous conflicting data and is consistent with current models of cell cycle control by multiple inhibitors of cyclin-dependent kinases.

Radiation-associated and 'spontaneous' human thyroid carcinomas show a different pattern of ras oncogene mutation.

Activated ras oncogenes in experimentally induced rodent tumours have been demonstrated to show specific patterns of oncogene activation which depend on the inducing agent, with H-ras activation in nitrosomethylurea (NMU) induced tumours, and K-ras activation in tumours induced by ionising radiation. We report a study of 12 radiation-associated human thyroid tumours, using polymerase chain reaction (PCR) amplification of paraffin-embedded material and allele-specific hybridisation with mutant-specific probes for the 3 ras oncogenes. Compared to 68 'spontaneous' human thyroid tumours, the radiation-associated cases show the same overall prevalence of ras mutation. However there is a significantly higher rate of K-ras mutation in radiation-associated follicular carcinomas than in 'spontaneous' follicular carcinomas (60% compared to 6%, P less than 0.05), suggesting that radiation may preferentially activate K-ras in human as well as rodent tumours.