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.     

Comparison of bax, waf1, and IMP dehydrogenase regulation in response to wild-type p53 expression under normal growth conditions

Recently, we demonstrated that downregulation of inosine-5'-monophosphate dehydrogenase (IMPD; IMP:NAD oxidoreductase, EC 1.2.1.14), the rate-limiting enzyme for guanine nucleotide biosynthesis, is required for p53-dependent growth suppression. These studies were performed with cell lines derived from immortal, nontumorigenic fibroblasts that express wild-type p53 conditionally by virtue of a metal-responsive promoter. Here, the p53-dependent properties of the original "p53-inducible" fibroblasts are presented in detail and compared to related properties of epithelial cells that also express wild-type p53 conditionally, but by virtue of a temperature-responsive promoter. Both types of p53-inducible cells were designed to approximate normal physiologic relationships between the host cell and the regulated p53 protein. Together, they were used to investigate expression relationships between IMPD and other p53-responsive genes proposed as mediators of p53-dependent growth suppression. In both types of cells, IMPD activity, protein, and mRNA were consistently coordinately reduced in response to p53 expression. In contrast, mRNAs for waf1, bax, and mdm2 showed disparate patterns of expression, being induced in one conditional cell type, but not the other. This distinction in regulation pattern suggests that under normal growth conditions, unlike IMPD downregulation, bax and waf1 induction is not a rate-determining event for p53-dependent growth suppression.     

Stable reintroduction of wild-type P53 (MTmp53ts) causes the induction of apoptosis and neuroendocrine-like differentiation in human ductal pancreatic carcinoma cells

Pancreatic ductal adenocarcinoma is one of the major causes of cancer mortality in the industrialized world, having among the poorest prognosis of any malignancy. Mutations or alterations in the p53 tumor suppressor gene/protein are observed in 50-70% of these cancers, yet little information is available regarding the phenotypic effects of restoration of wild-type (wt) p53 function in pancreatic ductal carcinoma cells. The consequences of stable reintroduction of wt p53 on apoptosis and differentiation was examined in a poorly differentiated pancreatic carcinoma cell line (Panc-1), possessing only mutant (mt) p53 (codon 273 mutation). Cells were transfected with a temperature-sensitive mouse p53val135 (tsp53) vector under additional control of a genetically-modified metallothionein promoter. This tsp53 has a 'mt' phenotype at 37.5 degrees C, and a 'wt' phenotype at 32.5 degrees C and the presence of 100 microM ZnCl2. Stable expression of wt p53 caused upregulation of the p21/WAF1 gene, and G1 growth arrest as shown by flow cytometry and BrdU labeling. Additionally, apoptosis was induced 8-12 post-induction in the majority of the cells (60-70%), as demonstrated by morphological changes, in situ TdT labeling and internucleosomal laddering. However, a subpopulation (30%) of the transfectants survived this apoptotic fate. Unlike the epithelial parental Panc-1 cells, these cells exhibited the appearance of a neuroendocrine-like phenotype with extensive branch-like processes, and marked cytoplasmic and cytoskeletal immunostaining for tau-2, synaptophysin, and chromogranin A. These studies suggest that stable and regulated expression of wt p53 can have multiple phenotypic consequences (apoptosis and altered differentiation to a neuroendocrine-like phenotype) in poorly-differentiated pancreatic carcinoma cells.     

Expression of wild-type p53 increases etoposide cytotoxicity in M1 myeloid leukemia cells by facilitated G2 to M transition: implications for gene therapy

We have evaluated the role of p53 in the induction of cell death by the DNA topoisomerase II inhibitor etoposide in M1 myeloid leukemia cells. Three different clones of M1 cells were used: S6, which lacks p53; Phe-132, which expresses mutant p53 constitutively; and LTR-13, which expresses mutant protein at 37 degrees C and wild-type p53 at 32 degrees C. As described previously, LTR-13 cells undergo rapid apoptosis upon induction of wild-type p53 at 32 degrees C. Multiparameter flow cytometric analysis showed that etoposide treatment (0.5 microg/ml) of all three cell lines at 37 degrees C is associated with a block in the G2 phase of the cell cycle, whereas the cells preferentially die out of the next S phase. Induction of wild-type p53 in LTR-13 cells is associated with a loss of cells in late S and G2-M phase, and the cells die out of the early S phase. Interestingly, the simultaneous induction of apoptosis by both pathways (wild-type p53 and etoposide) leads to suppression of the etoposide-induced G2 block. To determine the effect of p53 on the G2 to M transition, LTR-13 cells were incubated with etoposide for 24 h at 37 degrees C and then either maintained for an additional 12 h at 37 degrees C or shifted to 32 degrees C to activate wild-type p53. The expression of wild-type p53 resulted in an increase in mitosis-specific phosphorylation, as determined by the MPM-2 antibody as well as the formation of mitotic spindles. This was associated with an important augmentation of the cytotoxic effect of etoposide. In contrast, a similar temperature shift of Phe-132 cells, which express mutant p53, had no effect on either immunostaining with MPM-2 or the cytotoxicity. Taken together, our results indicate that wild-type p53 can override the etoposide-induced G2 block in at least some cell types. These data propose a new role for p53 in the cell death induced by chemotherapeutic agents and may have important implications for gene therapy.     

Dexamethasone-mediated protection from drug cytotoxicity: association with p21WAF1/CIP1 protein accumulation?

Dexamethasone (DEX)-mediated inhibition of drug-induced, but not CD95 ligand-induced, apoptosis in malignant glioma cells correlates with wild-type p53 status. Here, we examined mechanisms underlying DEX-mediated protection from apoptosis. DEX did not induce p53 expression in two p53 wild-type cell lines (U87MG, LN-229) and did not alter drug-induced p53 accumulation. Forced expression of temperature-sensitive p53val135 in mutant conformation failed to prevent accumulation of endogenous wild-type p53 but acted in a transdominant negative manner to inhibit p53-mediated, camptothecin-induced p21WAF1/CIP1 expression. p53val135-transfected cells retained responsiveness to DEX at restrictive temperature, suggesting that p53 activity is not required for cytoprotection. Forced expression of wild-type p53val135 abrogated the protective effect of DEX, suggesting redundant cytoprotective effects of DEX and p53. Indeed, DEX induced moderate accumulation of p21WAF1/CIP1 in U87MG, LN-229 and p53 mutant LN-18 cells, but not in p53 mutant LN-308 or T98G cells. LN-18 is also the p53 mutant cell line with the best cytoprotective response to DEX. p21WAF1/CIP1 accumulation occurred in the absence of changes in p21WAF1/CIP1 mRNA expression. Wild-type p53 was not required for this DEX effect since DEX induced p21WAF1/CIP1 accumulation in p53val135-transfected LN-229 cells, too. DEX failed to induce p21WAF1/CIP1 expression or cytoprotection in untransformed rat astrocytes. The same lack of modulation of p21WAF1/CIP1 expression and drug toxicity was observed in p21(+/+), p21(+/-) and p21(-/-) human colon carcinoma cells. Paradoxically, while only p21(+/+) and p21(+/-) mouse embryonic fibroblasts showed enhance p21WAF1/CIP1 levels after exposure to DEX, only p21(-/-) fibroblasts were protected from drug toxicity by DEX. The present study links DEX-mediated protection from cancer chemotherapy to a p53-independent pathway of regulating p21WAF1/CIP1 expression in glioma cells but this effect appears to cell type-specific.     

Effect of ramipril on heart rate variability in digitalis-treated patients with chronic heart failure

Bilharzial-related bladder carcinoma (BBC) is the most common malignant neoplasm in Egypt, also occurring with a high incidence in other regions of the Middle East and East Africa. The clinical and pathological features of BBC are different than those described for the conventional transitional cell carcinoma of the bladder, including the high incidence of squamous cell carcinoma reported in BBC and the fact that over 90% of BBC cases at presentation are advanced-stage tumors (P3 and P4). This study was conducted to better define the phenotypic alterations associated with BBC affecting the p53 cell cycle control pathway, including altered patterns of expression of downstream effector proteins such as mdm2 and p21/WAF1. A well-characterized cohort of 125 patients affected with bilharzial-related bladder tumors was studied. Tumors were classified as squamous carcinomas (n = 68), transitional cell carcinomas (n = 55), or adenocarcinomas (n = 2). The products encoded by TP53, mdm2, and p21/WAF1 genes were analyzed by immunohistochemistry. Furthermore, the patterns of expression of these molecules were correlated with the Ki67 proliferative index. In addition, the microanatomical distribution of programmed cell death was assessed in a subset of tumors, using the so-called terminal deoxynucleotidyl transferase-mediated nick end labeling method. p53 nuclear overexpression was identified in 25 (20%) of 125 cases. Nuclear overexpression of mdm2 was detected in 74 (59.2%) of 125 cases. There was a statistically significant association between coexpression of both p53 and mdm2 and detection of lymph node metastases (P = 0.04). p21/WAF1 expression was detected in 87 (72%) of 121 evaluable cases. A high Ki67 proliferative index was observed in 99 (86%) of 115 evaluable cases. There was a statistically significant association between high Ki67 proliferative index and mdm2-positive phenotype (P = 0.005) and deep muscle invasion (P3b; P = 0.026) as well as lymph node metastases (P = 0.039). Apoptosis was observed in terminally differentiated tumor cells identified in the superficial layers of well-differentiated squamous carcinoma or exfoliating cells in transitional lesions. However, only rare apoptotic tumor cells were found in basal or suprabasal layers as well as in the invasive elements of the neoplasms studied. These results suggest that the frequency of p53 nuclear overexpression in BBC is lower than that reported for conventional transitional cell carcinoma. Nevertheless, tumors with p53 alterations have a greater propensity to progress. The prominent number of cases displaying an mdm2-positive phenotype suggests that this may be an early incident in BBC and should be regarded as a potential oncogenic phenomenon. This is supported by the significant correlation between high Ki67 proliferative index and mdm2 overexpression. The association of an aggressive clinical course with the coexpression of both p53 and mdm2 products might be viewed as a cooperative effect that develops in tumor progression.     

Calcium and S100B regulation of p53-dependent cell growth arrest and apoptosis

In glial C6 cells constitutively expressing wild-type p53, synthesis of the calcium-binding protein S100B is associated with cell density-dependent inhibition of growth and apoptosis in response to UV irradiation. A functional interaction between S100B and p53 was first demonstrated in p53-negative mouse embryo fibroblasts (MEF cells) by sequential transfection with the S100B and the temperature-sensitive p53Val135 genes. We show that in MEF cells expressing a low level of p53Val135, S100B cooperates with p53Val135 in triggering calcium-dependent cell growth arrest and cell death in response to UV irradiation at the nonpermissive temperature (37.5 degreesC). Calcium-dependent growth arrest of MEF cells expressing S100B correlates with specific nuclear accumulation of the wild-type p53Val135 conformational species. S100B modulation of wild-type p53Val135 nuclear translocation and functions was confirmed with the rat embryo fibroblast (REF) cell line clone 6, which is transformed by oncogenic Ha-ras and overexpression of p53Val135. Ectopic expression of S100B in clone 6 cells restores contact inhibition of growth at 37.5 degreesC, which also correlates with nuclear accumulation of the wild-type p53Val135 conformational species. Moreover, a calcium ionophore mediates a reversible G1 arrest in S100B-expressing REF (S100B-REF) cells at 37.5 degreesC that is phenotypically indistinguishable from p53-mediated G1 arrest at the permissive temperature (32 degreesC). S100B-REF cells proceeding from G1 underwent apoptosis in response to UV irradiation. Our data support a model in which calcium signaling and S100B cooperate with the p53 pathways of cell growth inhibition and apoptosis.     

The mummified Hodgkin cell: cell death in Hodgkin's disease

This study attempts to define more clearly the morphology and ultrastructure of mummified Hodgkin cells, to determine their incidence in the different histological subtypes of Hodgkin's disease (HD), and to correlate these data with the expression of p53, bcl-2, mdm2, and p21/WAF1. Forty-five cases of primary HD were examined at light and electron microscopic level. DNA strand breaks were detected by the in situ end-labelling (ISEL) and the TdT-mediated dUTP-digoxigenin nick end-labelling (TUNEL) technique. Mummified Hodgkin cells display morphological features that differ from those of classical apoptosis. In contrast to apoptotic cells, mummified Hodgkin and Reed-Sternberg (HRS) cells do not react in the ISEL or TUNEL procedures and maintain the expression of antigens such as CD30 and CD15. The morphology of mummified tissue cells could be simulated by CD95-mediated induction of apoptosis in the Hodgkin cell line HDLM2 if internucleosomal DNA fragmentation was inhibited by zinc ions. The highest incidence of mummified cells was found in the nodular sclerosis and mixed cellularity subtypes, whereas the lowest frequency was observed in nodular paragranuloma. The frequency was independent of p53, bcl-2, p21, and mdm2 expression. p21 and mdm2 immunoreactivity of HRS cells was correlated with p53 status. HRS cells in nodular paragranuloma were virtually negative for p21/WAF1 or bcl-2. Classical apoptotic cells reacting in the TUNEL and ISEL procedures are found in all subtypes of HD and are derived from the non-neoplastic cellular background. In conclusion, mummified Hodgkin cells display features of apoptosis lacking the internucleosomal DNA fragmentation. The pattern of the p53-transactivated genes mdm2 and p21/WAF1 suggests that inactivating mutations of p53 are rare in HD.     

Mdm2 association with p53 targets its ubiquitination

Key to p53 ability to mediate its multiple cellular functions lies in its stability. In the present study we have elucidated the mechanism by which Mdm2 regulates p53 degradation. Using in vitro and in vivo ubiquitination assays we demonstrate that Mdm2 association with p53 targets p53 ubiquitination. Exposure of cells to UV-irradiation inhibits this targeting. Mdm2 which is deficient in p53 binding failed to target p53 ubiquitination, suggesting that the association is essential for Mdm2 targeting ability. While mdm2-p53 complex is found in non-stressed cells, the amount of p53-bound mdm2 is decreased after UV-irradiation, further pointing to the relationship between mdm2 binding and p53 level. Similar to Swiss 3T3 cells, the dissociation of mdm2-p53 complex was also found in UV-treated Scid cells, lacking functional DNA-PK, suggesting that DNA-PK is not sufficient for dissociating mdm2 from p53. Together our studies point to the role of Mdm2, as one of p53-associated proteins, in targeting p53 ubiquitination.     

HIDA kinetics in children

We have investigated the immunohistochemical expression of p53, mdm2, p21/waf1 and bcl-2 proteins in 31 thymic epithelial tumours comprising five medullary thymomas (MDT), four mixed thymomas (MT), 12 cortical thymomas (CT), eight predominately cortical thymomas (PCT) and two well-differentiated thymic carcinomas (WDTC). We have found p53, mdm2, p21 and bcl-2 protein expression in 25/31, 8/31, 5/31 and 10/31 thymic epithelial tumours, respectively. Coexpression of p53 and mdm2 proteins was found in eight cases (three CT, four PCT and one WDTC). Five of them were also p21 positive and three p21 negative. Discordant p53+/mdm2-/p21- protein expression was found in 19 cases (three MDT, three MT, nine CT, three PCT and one WDTC). Mdm2 and p21 proteins were not expressed in the absence of p53 protein. Coexpression of bcl-2 and p53 proteins was found in seven cases (three MDT, three MT and one WDTC). Eighteen cases were p53+/bcl-2- (10 CT, seven PCT and one WDTC) and three cases (two MDT and one MT) were bcl-2+/p53-. Our findings indicate that in thymomas, p53 expression is more frequently associated with cortical histotypes while bcl-2 expression is strongly associated with medullary and mixed histotypes. In addition, there is an inverse correlation between p53 and bcl-2 protein expression in thymomas. Coexpression of p53/mdm2/p21 proteins may reflect thymomas with wild-type (wt), p53 gene since mdm2 and p21 proteins are inducible by wt p53 gene. However, in view of previous findings that p53 mutation is an early event in thymomas, the possibility of p53 gene mutation with p53-independent mdm2 and p21 expression should be considered in these cases. Discordant p53+/mdm2-/p21- protein expression may represent thymomas with p53 gene mutations unable to activate expression of mdm2 and p21 proteins.     

p53 and mdm2 are expressed independently during cellular proliferation

We analysed p53 expression during proliferation of serum stimulated Swiss mouse 3T3 cells and of concanavalin A stimulated mouse spleen lymphocytes and correlated it to rate of DNA synthesis and to expression of PCNA. We also analysed mdm2 gene expression, as rising p53 levels during proliferation might require MDM2 protein expression to functionally antagonize p53 mediated growth inhibition. p53 protein synthesis closely paralleled DNA synthesis and PCNA expression, suggesting a direct involvement of p53 in cellular DNA synthesis. mdm2 expression in 3T3 cells could not be correlated with p53 expression and DNA synthesis and was not detected at all in stimulated lymphocytes. We conclude that p53 and mdm2 expression during proliferation are not functionally related and that mdm2 expression is not required for proliferation.