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Osteosarcoma is a highly metastatic form of bone cancer in adolescents and young adults that is resistant to existing treatments. Development of an effective therapy has been hindered by very limited understanding of the mechanisms of osteosarcomagenesis. Here, we used genetically engineered mice to investigate the effects of deleting the tumor suppressor Wwox selectively in either osteoblast progenitors or mature osteoblasts. Mice with conditional deletion of Wwox in preosteoblasts (Wwox Δosx1 ) displayed a severe inhibition of osteogenesis accompanied by p53 upregulation, effects that were not observed in mice lacking Wwox in mature osteoblasts. Deletion of p53 in Wwox Δosx1 mice rescued the osteogenic defect. In addition, the Wwox;p53 Δosx1 double knockout mice developed poorly differentiated osteosarcomas that resemble human osteosarcoma in histology, location, metastatic behavior, and gene expression. Strikingly, the development of osteosarcomas in these mice was greatly accelerated compared with mice lacking p53 only. In contrast, combined WWOX and p53 inactivation in mature osteoblasts did not accelerate osteosarcomagenesis compared with p53 inactivation alone. These findings provide evidence that a WWOX-p53 network regulates normal bone formation and that disruption of this network in osteoprogenitors results in accelerated osteosarcoma. The Wwox;p53 Δosx1 double knockout establishes a new osteosarcoma model with significant advancement over existing models. Cancer Res; 76(20); 6107-17. ©2016 AACR., Competing Interests: of Potential Conflicts of Interest. Non, (©2016 American Association for Cancer Research.)

ACE-inhibitors prevent the development of left ventricular hypertrophy (LVH). The tumor suppressor gene p53 up-regulates the cellular renin-angiotensin system, resulting in ANG II synthesis, which activates p53 creating a positive feedback loop. One hundred and fourteen rabbits were separated into groups A (control), B (sham-operated), C and D. In groups C and D, an aortic stenosis was performed, and in group D the animals were treated with enalapril. For p53 determination, LV specimens were examined by Western blot analysis and an immunohistochemical study was performed, except for samples from group D. In conclusion, LVH was significantly induced at 7 and 28 days after aortic stenosis with no difference between the two periods, while enalapril prevented hypertrophy in these two groups. p53 was transcriptionally activated and immunoreactively present after acute pressure overload as well as in the sham-operated group. Enalapril decreased the p53 expression at 180 min, 7 and 28 days following aortic stenosis.

The mechanisms of p53-dependent apoptosis involve a set of genes that possess the ability to modulate oxidative stress. One of them PIG3, is induced by p53 through a microsatellite in its promoter region. This microsatellite was found to acquire its full structure and p53-functional dependence only in Hominoidea (apes and humans) and has been proposed to represent an evolutionary adaptation of tumor suppressor mechanisms. Microsatellite instability and genetic constitution, comprising the presence of the low repetition allele (10 TGYCC repeats), at this locus have been hypothesized to provide an increased risk for cancer development. Therefore, in the present analysis we examined this polymorphism in two common human cancers, lung and breast and compared it with corresponding control cases. Furthermore, for lung cancer we employed two different ethnic groups, Greek and British. Analysis of this locus in this types of tumors showed: (1) a very low frequency of microsatellite instability and loss of heterozygosity (1.4% and 4%, respectively) in the examined carcinomas, (2) the homozygous presence of the 10 repeats allele only in the control cases, and (3) a non-significant increase of the most frequent allele (15 repeats) in the cancer groups as compared to control ones. The last two observations were found in both Greek and British populations. Taken together, these data do not support the notion that this PIG3 polymorphism is associated with an increased risk for cancer susceptibility. Larger studies including other types of cancer should also be performed.

Background: Several factors are currently employed for prognosis assessment and treatment determination in breast cancer. An array of molecular parameters, such as p53, Her2-neu (c-erbB 2) and Cathepsin-D, are also examined to improve clinical patient management. We have conducted a statistically powerful study of the prognostic value of conventional factors and of the investigational factors p53, Her2-neu and Cathepsin-D in patients with invasive breast carcinoma, in order to compare their significance. Our analysis was extended to determine the associations of p53 and Her2-neu with risk of death and relapse among patients with and without lymph node metastases., Materials and Methods: In a set of 125 primary breast tumors, p53 and Her2-neu expression were immunohistochemically evaluated. Cathepsin-D, estrogen and progesterone receptor concentrations were determined in cytosols by a standard immunoradiometric assay., Results: Over a mean of 62 months, 49 patients (39%) had a relapse and 29 patients (23%) died. Overexpression of p53, Her2-neu and Cathepsin-D was observed in 31%, 46% and 88% of cases, respectively. Overall survival was associated with histology (hazard ratio 0.04, 95% confidence interval: 0.01, 0.49 for lobular tumors) and stage (hazard ratio 5.94, 95% confidence interval: 1.30, 27.15 for stage III samples). Disease-free survival was also related to histology (hazard ratio 0.23, 95% confidence interval: 0.08, 0.73 for lobular tumors) and stage (hazard ratio 4.27, 95% confidence interval: 1.36, 13.36 for stage III tumors). Patients with both negative nodal status and Her2-neu overexpression tended to display an elevated risk of death., Conclusion: Our results support the prognostic power of tumor histology and stage and emphasize the need for further studies on the prognostic impact of p53. Her2-neu and Cathepsin-D in breast cancer. Additionally, our analysis indicates that deregulation of Her2-neu might characterize a subgroup of node-negative patients with poor prognosis who could benefit from an aggressive adjuvant therapy.

p53 is an oncosuppressor protein, which acts via transcriptional and non-transcriptional mechanisms. The transcriptional function of p53 is mediated by specific responsive elements. In the present study we found active responsive elements, specific for the p53 within the 5'flanking region and within the first intron of the gene encoding for the CD59 membrane inhibitor of reactive lysis, and within the first intron of the gene encoding for the CD58 membrane protein (LFA-3). The results suggest that p53 may enhance the transcription of both CD59 and CD58 and imply a novel role for p53 as a direct regulator of the immune response.

P53 is an oncosuppressor protein which acts via transcriptional and non-transcriptional mechanisms. The transcriptional function of p53 is mediated by specific responsive elements (RE). In the present study we found one perfect p53 RE located in the first intron of the gene encoding for CD58 (lymphocyte function Antigen-3/LFA-3) membrane protein. Additionally, we detected one perfect p53 RE within the promoter region and one imperfect p53 RE placed in the first intron of the gene encoding for CD59 (membrane inhibitor of reactive lysis/MIRL). The results of our investigation suggest that p53 may enhance the transcription of both CD59 and CD58 and imply a novel role for p53 as a direct regulator of the immune response., (Copyright 2002 Harcourt Publishers Ltd.)

The immunohistochemical expression of p53, p21, Rb, p16, cyclin D1, Ki67, cyclin A, cyclin B1, p27, bcl2, bax, and bak proteins and the apoptotic index (Al) were investigated in 20 normal thymuses (8 adults, 3 adolescents, 5 infants and 4 newborns). The expressions of Rb, Ki67, cyclin A and cyclin B1 were overlapping, being high in the cortex with a tendency for decreased expression toward the medulla. Apoptotic cells were mainly detected in the cortex and the corticomedullary junction, rarely being present in Hassall's corpuscles. The mean values of Ki67, cyclin A, and cyclin B1 expression in thymuses were 77.2%, 32.2% and 21.4% (newborns), 62.4%, 33.7% and 18.5% (infants), 56.9%, 23.4% and 18.9% (adolescents) and 38.7%, 21.7% and 14.6% (adults), respectively. The mean values of AI in thymuses from newborns, infants, adolescents and adults were 1.4%, 2.9%, 2.7% and 3.8%, respectively. This decrease in proliferation and increase in apoptosis may account for the process of thymic involution. P16 expression was widespread with most of Hassall's corpuscles being p16-positive. P16-positive cells and Hassall's corpuscles increased with the increase in age, in keeping with the suggested role of p16 in cellular senescence. P27 expression was undetectable in subcapsular thymocytes with a tendency for increased expression toward the medulla. The expressions of Ki67, cyclin A and cyclin B1 were inversly related with that of p27, consistent with previous evidence that p27 concentration is reduced when the cell-cycle progresses. P21 and much less frequently p53 proteins were mainly detected in a part of the subcapsular cortical epithelial cells. These findings suggest that a) in thymocytes, the apoptotic pathway is mostly p53-independent and the function of p21 as a negative regulator of the cell cycle must be redundant to other negative regulators, such as p16 and p27 which were abundantly detected in thymocytes and b) in some thymic epithelial cells, the p21 expression may be induced by p53, but in most of them seems to be p53-independent. Most of Hassall's corpuscles were p21-positive, consistent with previous evidence that these structures represent end stages of maturation of thymic medullary epithelium and that p21 protein is involved in the process of terminal differentiation. Cyclin D1 positivity was found in some macrophages. Bcl2 expression was mainly seen in medullary thymocytes, reflecting the surviving thymocytes in this region. The expressions of Bax and bak were more widespread in both the medulla and cortex, suggesting that these proteins play a broader role than bcl2 in the regulation of thymic apoptosis.

Background: Down-regulation or overexpression of the cyclin-dependent kinase inhibitor p27 have been observed in a range of malignancies, including lung cancer. To further elucidate the role of the molecule in tumor growth regulation, we evaluated p27 expression in a series of non-small cell lung carcinomas (NSCLCs), and examined its relation with histology, kinetic parameters, ploidy, and overall survival. We extended our investigation into the association of p27 levels with the presence of Ki-ras mutations, as well as with the expression status of p53 and pRb in tumor cells., Material and Methods: p27, p53, and pRb status were immunohistochemically evaluated in a total of 69 NSCLCs. In situ assays were employed to assess the kinetic parameters (Ki-67 immunohistochemistry for proliferation index, Tdt-mediated dUTP nick end labeling assay for apoptotic index). The ploidy status of the tumors was assessed after staining nuclei with the Feulgen procedure, and the presence of Ki-ras mutations was examined by restriction fragment length polymorphisms. All possible associations were assessed with a series of statistical methods., Results: Immunoreactivity for p27 was observed in the entire series of specimens, with the mean percentage of positive cells being 33%. Adenocarcinomas (AdCs) exhibited higher p27 levels compared to squamous cell carcinomas (SqCCs) (p < 0.01). An inverse correlation was established between p27 expression and proliferation index (PI) (r = -0.834, p < 0.01) but not with apoptotic index (AI), whereas aneuploid tumors were characterized by lower p27 levels than diploid ones (p < 0.01). No difference in p27 immunostaining was observed with regard to the presence of Ki-ras mutations, whereas aberrant p53 and/or pRb expression patterns were associated with p27 underexpression (p < 0.01 for p53 status, p < 0.05 regarding pRb levels, and p < 0.01 for a combined deregulation of both proteins). Two or more alterations in the p27/p53/pRb protein network (i.e., p27 levels lower than the estimated mean value, overexpressed p53, and/or aberrant pRb) were associated with increased PI and aneuploidy (p < 0.001 and p < 0.01, respectively). A powerful trend was found between p27 expression and overall survival (p = 0.066)., Conclusions: Our findings confirm the heterogeneity between AdCs and SqCCs, and are suggestive of an increased proliferative activity in NSCLCs underexpressing p27. Furthermore, our analysis supports the concept of p27 forming a functionally compact network with p53 and pRb, which is actively involved in the regulation of cellular proliferation and chromosomal stability.

Purpose: To evaluate the relationship between HER-2/neu overexpression and standard as well as investigational prognostic factors in Greek females with invasive breast carcinoma., Materials and Methods: Paraffin-embedded tumor sections from 128 consecutive primary breast cancer patients were screened for HER-2/neu oncoprotein (p185) overexpression by immunohistochemistry using the polyclonal antibody A 0485. The relationship between HER-2/neu staining and other established markers of prognosis were examined using both a univariate and a multivariate analysis., Results: HER-2/neu overexpression was detected in 46.1% of tumors. No statistical correlation was found between HER-2/neu and age, tumor diameter, histologic type, nodal status, tumor grade, stage, estrogen and progesterone receptor status or cathepsin-D. A significant correlation was noted between HER-2/neu and p53 overexpression in the total sample (p=0.014) as well as in node-positive patients (p=0.026). In those groups, HER-2/neu and p53 co-expression was found in 19.5% and 21.6% of cases, respectively. In node-negative patients, HER-2/neu overexpression did not correlate with any other marker., Conclusion: HER-2/neu overexpression seemed to correlate only with p53 oncoprotein.

Fifty-seven cases of T-cell lymphomas (TCL) including 5 lymphoblastic (T-LBL) and 52 peripheral TCL (PTCL) were analyzed by immunohistochemistry for the expression of p53, mdm2, p21, Rb, cyclin D1, cyclin A, cyclin B1, and Ki67/MIB1 proteins and 39/52 PTCL were also analyzed for the expression of p16 protein and for the presence of apoptotic cells by the TUNEL method. The aim was to search for abnormal immunoprofiles of p53 and Rb growth control pathways and to determine the proliferative activity and the apoptotic index of TCL. Abnormal overexpression of p53, p21 and mdm2, in comparison to normal lymph nodes, was found in 12/57, 10/57 and 2/57 cases of TCL, respectively. Abnormal loss of Rb and p16 expression was found in 1/57 and 2/39 cases, respectively, whereas abnormal overexpression of cyclin D1 was not detected in any of the 57 cases. Our data revealed entity-related p53/p21/mdm2 phenotypes. Indeed, most nodal and cutaneous CD30+ anaplastic large cell lymphomas (ALCL) showed concomitant overexpression of p53 and p21 proteins (7/8 cases), and mdm2 was overexpressed in 2 p53-positive nodal ALCL. In contrast, overexpression of p53 was found in 3/17 cases of nodal peripheral TCL unspecified (PTCL-UC) and 2/7 non-ALCL cutaneous pleomorphic TCL. Overexpression of p21 protein was detected in 2/3 p53-positive PTCL-UC and in 1/2 p53-positive non-ALCL cutaneous pleomorphic TCL. Finally, all the remaining 25 cases of TCL did not show p53 and p21 overexpression. Overall, the p53+/p21+ phenotype in 10/57 TCL suggests wild-type p53 capable of inducing p21 expression. The highest apoptotic index (AI) was found in ALCL and a positive correlation between apoptotic index and Ki67 index (p<0.001) was detected. Ki67, cyclin A and cyclin B1 expression was found in all 57 TCL and on the basis of the combined use of these 3 variables, 3 groups of proliferative activity could be determined: a) high in ALCL and T-LBL, b) low in mycosis fungoides (MF) and gammadelta hepatosplenic TCL, and c) intermediate in the remaining TCL entities. The proliferative activity in the 12 p53 overexpressing cases was higher in comparison to the 45 p53-negative cases. Ki67 expresion in more than 25% of tumour cells showed significant correlation with p53 overexpression (p<0.001). Rb expression tended to be parallel to Ki67, cyclin A and cyclin B1 expression in all but one case of nodal PTCL-UC which displayed loss of RB expression. Interestingly, this case was p53-negative, whereas the p53-positive cases were Rb-positive. These findings suggest that different pathogenetic routes may function in some TCL, involving either the p53 or, less frequently, the Rb pathways.

The aim was to investigate the combined immunoexpression of p53, p21, bcl-2, bax, Rb and Ki67 proteins in Hodgkin's lymphomas (HL) and correlate expression patterns with the histotype and the Epstein-Barr Virus (EBV) status. Paraffin-sections from 56 cases of HL (18 nodular sclerosis and 38 mixed cellularity) and from ten "reactive" lymph nodes were investigated. P53, p21, bcl-2, bax, Rb and Ki67 proteins were detected in Hodgkin and Reed-Sternberg (HRS) cells in 35/56, 56/56, 24/56, 23/56, 56/56 and 56/56 cases of HL, respectively. No correlation was found between the expression of each protein and the EBV status or the histotype of HL. Comparison between p53 and p21 staining revealed two patterns: a) p53+/p21+ (35 cases); and b) p53-/p21+ (21 cases). The pattern p53+/p21+ suggests wild type p53 protein able to induce the expression of p21 while the p53-/p21+ pattern suggests p53-independent p21 expression. These results are consistent with the interpretation that inactivating p53 gene mutations may be rare in HL. Comparison between bcl-2 and bax staining showed a statistically significant relationship (p<0.001) for coexpression (19 cases) or absence of expression of both proteins (28 cases) in HRS cells. In contrast, bax expression was observed in most lymphoid cells in all "reactive" lymph nodes. Since the proapoptotic bax protein may act as tumour suppressor it is possible that the absence of this protein in HRS cells in a substantial proportion of HL may confer growth advantage and play a role in their pathogenesis. This could suggest bax gene alterations in some HL since in other studies acute lymphoblastic leukaemia cell lines demonstrate bax gene mutations with loss of bax immunoexpression. Another possibility is that reduced bax expression may be due to post transcriptional regulation, as was described in lymphoma cell lines. Comparison between Rb and Ki67 staining disclosed two main deviations from the normal parallel relationship in reactive lymph nodes: a) 2 cases with low Rb and high Ki67 expression possibly reflecting loss of Rb expression due to chromosome loss or to other abnormalities in the structure or the expression of Rb gene; and b) 9 cases with high RB and low Ki67 possible reflecting an attempt of Rb protein in excess to induce cell cycle arrest. Taken together, our findings provide combined immunohistological evidence for deregulated expression of cell-cycle and apoptosis-related proteins, that may play a role in the pathogenesis of HL.

Background: Recent in vitro studies provide evidence that the cell cycle molecules pRb, p53 and MDM2 form a tightly regulated protein network. In this study, we examined the relationship of this protein network in a series of non-small cell lung carcinomas (NSCLCs), with the kinetic parameters, including proliferative activity or proliferation index (PI) and apoptotic index (AI), and ploidy status of the tumors., Material and Methods: A total of 87 NSCLCs were examined using immunohistochemical and molecular methods in order to estimate the status of the pRb-p53-MDM2 network. The kinetic parameters and the ploidy status of the tumors were assessed by in situ assays. The possible associations between alterations of the network, kinetic parameters and ploidy status of the carcinomas were assessed with a series of statistical methods., Results: Aberrant expression of pRb (Ab) and overexpression of p53 (P) and MDM2 (P) proteins were observed in 39%, 57%, and 68% of the carcinomas, respectively. The comprehensive analysis revealed that concurrent alterations in all three cell cycle regulatory molecules were the most frequent pattern, pRb(Ab)/p53(P)/MDM2(P); this "full abnormal" phenotype represented approximately 27% of the cases. This immunoprofile obtained the highest PI/AI value; whereas, the "normal" phenotype was the lowest one (p = 0.004). Furthermore, the pattern pRb(Ab)/p53(P)/MDM2(P) acquired the highest PI (p < 0.001) and lowest AI (p < 0.001) scores. Interestingly, the groups of carcinomas with impaired expression of one or two molecules attained PI/AI ratio values clustered in a narrow range placed in the middle of the scores exhibited by the "normal" and "full abnormal" phenotypes. These tumors had significantly lower AI, but similar PI values, compared with those noticed in the normal pattern. In addition, it was observed that the pRb(Ab)/p53(P)/MDM2(P) phenotype was also significantly associated with aneuploidy (p = 0.002) and a tendency was observed when the expression of two components was altered (p = 0.055)., Conclusions: Our findings suggest that simultaneous deregulation of all members of the pRb-p53-MDM2 network confers an additive effect on tumor growth. The apoptotic pathway seems to be more susceptible to its defects than the cell proliferation machinery. The findings of the ploidy analysis, which are in parallel with those regarding the proliferative activity and the apoptotic rate study, further support the concept that these molecules constitute a tightly regulated network participating in cell cycle control and chromosomal stability.

Wild-type (wt) p53 is a tumor-suppressor protein which acts via transcriptional and transcriptional-independent mechanisms. The transcriptional function of p53 is mediated by specific responsive elements (REs). The MDM2, WAF1/Cip1 and Bax genes possess p53REs and their activation by wt p53 induces cell cycle progression, arrest and programmed cell death (apoptosis), respectively. Mutations of the p53 gene are detected in more than 50% of the human malignant tumors. p53 mutants seem to have a more stable conformation and are suggested to exert dominant-negative inhibition of wt p53 in cells containing both wt and mutant (mt) alleles. However, recent studies show that certain mt p53 proteins posses a "gain of function" phenotype. In the present study, we examined the effects of the second most frequent p53 mutant R273H on the p53REs of the MDM2, WAF1/Cip1 and Bax genes in the H1299 non-small cell lung carcinoma cell line. Although mt p53 R273H alone was unable to bind and transactivate the corresponding p53REs, it enhanced the MDM2-p53RE mediated gene transcription of wt p53 (positive-dominant effect) and prevented the wt p53 transactivation of the p53REs of WAF1/Cip1 and Bax genes (negative-dominant effect). Our data suggest that in the appropriate environment, differential transcription of critical p53 target genes by certain p53 mutant proteins may illustrate another mechanism implicated in tumor development.

The p16-pRb and p53-MDM2 pathways represent vital cell cycle checkpoints. Recent studies provide evidence that these pathways are directly linked via MDM2-pRb interaction and p53 suppression of the RB1 gene. In the present study we investigated the alterations of this G1 phase protein network using immunohistochemical and molecular methods in a series of 68 non-small-cell lung carcinomas (NSCLCs) and correlated the findings with clinicopathological features and prognosis of the patients. Aberrant expression (Ab) of p16 and pRb was observed in 33 (49%) and 27 (40%) of the carcinomas, respectively. Analysis of the region that encodes for p16 by deletion mapping, a polymerase chain reaction (PCR)-based methylation assay and PCR single-strand conformation polymorphism (SSCP) analysis revealed that deletions and transcriptional silencing by methylation might represent the main mechanisms of CDKN2/p16ink4a inactivation in NSCLCs. The results of deletion mapping also suggest that other tumor suppressor genes may reside at the 9p21-22 region, which encodes for CDKN2/MTS1/p16ink4a, p14ARF, and MTS2/p15ink4b. In addition, microsatellite instability was observed with a frequency of 16% in the 9p21-22 chromosome area. Overexpression (P) of p53 and MDM2 proteins was found in 39 (58%) and 47 (70%) of the cases, respectively. A highly significant association was observed between p53 overexpression and p53 mutations (P = 0.006). Statistical analysis of the expression patterns of the biologically relevant molecules (p16/pRb, p53/MDM2, MDM2/pRb, and p53/pRb) showed coincident overexpression of p53 and MDM2 (P = 0.04) and that abnormal pRb was correlated with elevated levels of MDM2 (P = 0.013) and p53 (P = 0.01), respectively. We suggest that deregulated expression of these molecules may act synergistically. An important finding of the study was that multiple impairments (three and four molecules affected) of the p16/pRb/p53/MDM2 network occurred in a large proportion (43%) of the carcinomas. This finding in addition to the absence of correlation with clinical stage of the tumors suggests that multiple hits of this network may be a relatively early event in the development of a subset of NSCLCs. The relationship between the factors examined in the present study, clinicopathological features, and survival of the patients did not reveal any significant correlations with the exception of smoking, which was associated with microsatellite alterations (loss of heterozygosity and microsatellite instability) at the 9p21-22 locus (P = 0.04) and the immunophenotypes p53(P)/MDM2(P) (P = 0.04) and p16(Ab)/pRb(Ab)/p53(P)/MDM2(P) (P = 0.03), respectively. We suggest that in a subset of NSCLCs, simultaneous deregulation of the members of this network may represent one way of initiating the oncogenic procedure whereas in other NSCLC subgroups alternative pathways may play this role.

The present study represents a continuation of previous works in which we observed that lung carcinomas co-expressing MDM2 protein and p53 mutants (mt p53) exhibited more aggressive behaviour. In the above studies, we suggested a 'gain of function' mechanism of mt p53 proteins based on the fact that the MDM2 gene possesses a p53-responsive element (MDM2-p53RE). In this study, to prove our hypothesis, we selected 12 cases from a series of 51 bronchogenic carcinomas. In these 12 cases, we examined the ability of the expressed mt p53 to bind the MDM2-p53RE and correlated the findings with MDM2 expression. Furthermore, we constructed four of these p53 mutants and studied their transactivation properties by co-transfecting them with a reporter plasmid carrying MDM2-p53RE in the p53 null non-small-cell lung carcinoma cell line (NSCLC) H1299. We observed mutant p53 protein DNA-binding activity, which depended on the nature and the position of the amino acid substitution. The fact that the cases with DNA-binding activity were accompanied with MDM2 protein isoforms' overexpression is indicative of a 'gain of function' phenotype. This hypothesis was enforced by the findings of the transfection experiments, which revealed that certain p53 mutants enhanced the expression of the luciferase reporter gene either directly or indirectly via a dominant positive effect on the wild-type p53. In conclusion, this work is one first attempt to examine if the deregulation of the p53/MDM2 autoregulatory feedback loop is due to novel properties of certain p53 mutants in the specific environment of a subset of bronchogenic carcinomas.

Forty-one bronchogenic carcinomas were investigated for expression of MDM2 protein isoforms and their relationship to p53 protein levels and p53 gene alterations using molecular and immunohistochemical techniques. The findings were correlated with the pathological features of the carcinomas. MDM2 protein was overexpressed in 26 cases (63 percent). Western blot analysis with two monoclonal antibodies, 1B10 and IF2, revealed three MDM2 protein isoforms, p90, p57 and p76/74. p90 and p57 are capable of interacting with p53 protein, while p76/74 is not. Various patterns of MDM2 isoforms were seen. Although no correlation between the patterns and pathological features was observed, lymph node metastases were more frequent in the cases with MDM2 overexpression (P < 0.005). In 3 out of 17 specimens of normal lung tissue examined, there was a low level of expression of p90. Molecular analysis revealed that MDM2 overexpression was a consequence of increased transcription rather than MDM2 gene amplification. p53 protein was overexpressed in 21 cases (51 percent) and p53 gene alterations (mutations + allelic deletions) were detected in 23 patients (56 percent). A high degree of concordance (76 percent) between p53 mutations and p53 staining was noticed (P < 10(-5)). p53 gene alterations were significantly associated with lymph node disease (P < 0.01). MDM2 and p53 proteins were simultaneously detected in 21 cases (51 percent), of which 17 (42 percent) showed p53 and MDM2 overexpression. The latter group was positively correlated with p53 mutations (P < 0.05). A strong correlation between MDM2/p53 co-expression and lymph node metastases was observed (P < 0.001). The findings suggest that MDM2 overexpression is a common event in bronchogenic carcinoma. The selective expression of some MDM2 isoforms in neoplastic tissue and not in the surrounding normal areas underscores the pathological role of the various MDM2 products. Finally, the coexistence of MDM2 protein(s) and p53 aberrations (mutations and/or overexpression) in a subset of lung carcinomas may be indicative of a 'gain of function' phenotype, with more aggressive characteristics.

This study was undertaken in order to investigate the molecular nature of the p53 gene in 19 laryngeal squamous cell carcinomas and dysplasias. Moreover, we have examined the possible relationship between proliferating cell nuclear antigen (PCNA) expression and p53 protein detection status in 42 laryngeal premalignant and malignant lesions in which 14 of the 19 samples used in the molecular study were included. p53 gene analysis was performed with the single-strand conformation polymorphism technique. PCNA was stained with the peroxidase/antiperoxidase immunohistochemical method using the monoclonal antibody PC-10. Data from previous work concerning p53 expression was used. We found that 9 of 12 of the immunohistochemically p53 positive (+) cases had mutations in exons 5 or 6. In the remaining immunohistochemically p53(+) and p53 negative (-) specimens there was no indication of sequence alterations. Furthermore, we did not observe any deletions in the chromosomal region 17p31.1 which encodes exons 4-8 of the p53 gene. The PCNA labelling index (LI) increased progressively with p53 protein detection status (percentage of cells immunohistochemically positive for p53). The difference between the group with the higher percentage of p53(+) cells and the others was statistically significant. These data show that although there is a discrepancy between immunohistochemical demonstration of p53 and molecular analysis, a large proportion of the former harbours the mutant form of the protein. In addition, p53 overexpression is positively correlated with PCNA LI, a finding which accompanies tumour progression.

In order to evaluate the expression of p53 protein in 28 premalignant and 40 malignant squamous cell proliferations of the larynx and its relationship to tobacco consumption, human papillomavirus infection and differentiation grade of the lesions, p53 expression was examined by means of a microwave post-fixation immunohistochemical method using the PAb 240 and PAb 1801 monoclonal antibodies. HPV infection was assessed by non-isotopic in situ hybridization (NISH) and polymerase chain reaction (PCR). A large proportion of carcinomas (77.5%) and dysplasias (61%) expressed p53. No difference was found between differentiation grades of the lesions regarding p53 detection (P > 0.1), but moderate or intense p53 expression was more frequent in the carcinomas (P < 0.05). A statistical correlation was found between cigarette consumption and both p53 detection and p53 staining intensity (P < 0.05 in each case). HPV study revealed HPV 16 and 18 infection only in carcinomas. The frequency was 28% and the physical state of the virus as demonstrated by NISH was integration into the genome. We observed an inverse relationship between HPV infection and p53 expression (P = 0.006). Our findings suggest that p53 overexpression is a common and early event which increases in frequency with progression of laryngeal squamous cell carcinoma. The expression of p53 is influenced by tobacco and high-risk types of HPV.