MDM2 amplification and loss of heterozygosity at Rb and p53 genes: no simultaneous alterations in the oncogenesis of liposarcomas

PURPOSE: The present study aimed to investigate the status of alterations of the MDM2, Rb and p53 genes in a series of 45 liposarcomas. Furthermore, the possible correlation with histological and clinical parameters was studied. METHODS: MDM2 amplification was examined by non-radioactive Southern blot hybridization with a human MDM2 cDNA probe. Mutations in the p53 gene were screened by polymerase chain reaction/single-strand conformation polymorphism analysis and direct sequencing. To study loss of heterozygosity (LOH) at the tumor-suppressor genes Rb and p53, we used four polymorphic intragenic Rb markers (introns 1, 17, 20, and 25) and two p53 markers (intron 1 and exon 4). RESULTS: MDM2 amplification was found in 19 of 45 liposarcomas (42.2%). The frequency of LOH in Rb and p53 was nearly identical (22%). In 4 of 9 tumors (44.4%) with LOH, allelic loss was a concurrent event in both genes. Of 45 liposarcomas, 6 (13.3%) showed p53 mutations. Overall, alterations of the p53/MDM2/Rb pathway occurred in 30 of 45 liposarcomas (66.6%). In contrast to myxoid and pleomorphic variants, well-differentiated liposarcomas were characterized by a high frequency of MDM2 amplification, a lack of LOH of Rb and p53, and p53 mutations. CONCLUSIONS: Obviously MDM2 amplification and LOH at the Rh and p53 genes do not occur simultaneously in the oncogenesis of liposarcomas, as is the case for MDM2 amplification and p53 gene mutations (with one exception). We suggest that well-differentiated, myxoid and pleomorphic liposarcomas are characterized by a different pattern of molecular alterations.     

MDM2 protein overexpression promotes proliferation and survival of multiple myeloma cells

The murine double minute 2 (MDM2) protein facilitates G1 to S phase transition by activation of E2F-1 and can enhance cell survival by suppressing wild-type p53 (wtp53) function. In this study, we examined MDM2 expression and function in multiple myeloma (MM) cells. MDM2 is strongly and constitutively expressed in MM cell lines (ARH-77, RPMI 8226, and OCI-My5) and in the cells of plasma cell leukemia (PCL) patients, but is not expressed in normal bone marrow mononuclear cells (BM MNCs). Treatment of MM cells with MDM2 antisense, but not sense, nonsense, or scrambled, oligodeoxyribonucleotides (ODNs) decreased DNA synthesis and cell viability; it also induced G1 growth arrest, as evidenced by propidium iodide (PI) staining and induction of retinoblastoma protein (pRB) to E2F-1 binding. Moreover, inhibition of MDM2 using antisense ODNs also triggered MM cell apoptosis as evidenced by acridine orange-ethidium bromide staining. We next studied the association of MDM2 with wtp53 and/or mutant p53 (mtp53), E2F-1, CDK4, and p21. MDM2 constitutively binds to E2F-1 in all MM cells, to both wtp53 and mtp53, and to p21 in tumor cells lacking p53. These data suggest that MDM2 may enhance cell-cycle progression in MM cells both by activating E2F-1 and by downregulating cell-cycle inhibitory proteins (wtp53 and p21). Overexpression of MDM2 may therefore contribute to both growth and survival of MM cells, suggesting the potential utility of treatment strategies targeting MDM2 in MM.     

Survival of patients with glioblastoma multiforme is not influenced by altered expression of p16, p53, EGFR, MDM2 or Bcl-2 genes

Deregulated expression of one or more growth control genes including p16, p53, EGF receptor (EGFR), MDM2 or Bcl-2 may contribute to the treatment resistance phenotype of GBM and generally poor patient survival. Clinically, GBM have been divided into two major groups defined by (1) histologic progression from a low grade tumor ("progressive" or "secondary" GBM) contrasted with (2) those which show initial clinical presentation without a prior history ("de novo" or "primary" GBM). Using molecular genetic analysis for p53 gene mutations together with immunophenotyping for overexpression of EGFR, up to four GBM variants can be distinguished, including the p53+/EGFR- progressive or the p53-/EGFR+ de novo variant. We examined the survival of 80 adult patients diagnosed with astrocytic GBM stratified by age category (>40, 41-60 or 61-80) to determine whether alterations in any one given growth control gene or whether different genetic variants of GBM (progressive versus de novo) were associated with different survival outcomes. Survival testing using Kaplan-Meier plots for GBM patients with or without altered expression of p16, p53, EGFR, MDM2 or Bcl-2 showed no significant differences by age group or by gene expression indicating a lack of prognostic value for GBM. Also the clinical outcome among patients with GBM showed no significant differences within each age category for any GBM variant including the progressive and de novo GBM variants indicating similar biologic behavior despite different genotypes. Using a pairwise comparison, one-third of the GBM with normal p16 expression showed accumulation of MDM2 protein and this association approached statistical significance (0.01 < P < 0.05) using the Bonferroni procedure. These GBM may represent a variant in which the p19ARF/MDM2/p53 pathway may be deregulated rather than the p16/cyclin D-CDK4/Rb pathway.     

p53-independent role of MDM2 in TGF-beta1 resistance

Transforming growth factor-beta (TGF-beta) inhibits cell proliferation, and acquisition of TGF-beta resistance has been linked to tumorigenesis. A genetic screen was performed to identify complementary DNAs that abrogated TGF-beta sensitivity in mink lung epithelial cells. Ectopic expression of murine double minute 2 rescued TGF-beta-induced growth arrest in a p53-independent manner by interference with retinoblastoma susceptibility gene product (Rb)/E2F function. In human breast tumor cells, increased MDM2 expression levels correlated with TGF-beta resistance. Thus, MDM2 may confer TGF-beta resistance in a subset of tumors and may promote tumorigenesis by interference with two independent tumor suppressors, p53 and Rb.     

Human papillomavirus oncoproteins E6 and E7 independently abrogate the mitotic spindle checkpoint

The E6 and E7 genes of the high-risk human papillomavirus (HPV) types encode oncoproteins, and both act by interfering with the activity of cellular tumor suppressor proteins. E7 proteins act by associating with members of the retinoblastoma family, while E6 increases the turnover of p53. p53 has been implicated as a regulator of both the G1/S cell cycle checkpoint and the mitotic spindle checkpoint. When fibroblasts from p53 knockout mice are treated with the spindle inhibitor nocodazole, a rereplication of DNA occurs without transit through mitosis. We investigated whether E6 or E7 could induce a similar loss of mitotic checkpoint activity in human keratinocytes. Recombinant retroviruses expressing high-risk E6 alone, E7 alone, and E6 in combination with E7 were used to infect normal human foreskin keratinocytes (HFKs). Established cell lines were treated with nocodazole, stained with propidium iodide, and analyzed for DNA content by flow cytometry. Cells infected with high-risk E6 were found to continue to replicate DNA and accumulated an octaploid (8N) population. Surprisingly, expression of E7 alone was also able to bypass this checkpoint. Cells expressing E7 alone exhibited increased levels of p53, while those expressing E6 had significantly reduced levels. The p53 present in the E7 cells was active, as increased levels of p21 were observed. This suggested that E7 bypassed the mitotic checkpoint by a p53-independent mechanism. The levels of MDM2, a cellular oncoprotein also implicated in control of the mitotic checkpoint, were significantly elevated in the E7 cells compared to the normal HFKs. In E6-expressing cells, the levels of MDM2 were undetectable. It is possible that abrogation of Rb function by E7 or increased expression of MDM2 contributes to the loss of mitotic spindle checkpoint control in the E7 cells. These findings suggest mechanisms by which both HPV oncoproteins contribute to genomic instability at the mitotic checkpoint.     

Rapid and selective depletion of CD4+ T lymphocytes and preferential loss of memory cells on interaction of mononuclear cells with HIV-1 glycoprotein-expressing cells

Apigenin is a plant flavonoid that has been shown to significantly inhibit ultraviolet-induced mouse skin tumorigenesis when applied topically and may be an alternative sunscreen agent for humans. A long-term goal of our laboratory is to elucidate the molecular mechanism or mechanism by which apigenin inhibits skin tumorigenesis. In a previous publication, we characterized the mechanism by which apigenin induced G2/M arrest in keratinocytes. More recent studies in our laboratory have provided evidence that apigenin can induce G1 arrest in addition to arresting cells at G2/M. Here we describe the mechanism of the apigenin-induced G1 arrest in human diploid fibroblasts (HDF). Treatment of asynchronous HDF for 24 h with 10-50 microM apigenin resulted in dose-dependent cell-cycle arrest at both the G0/G1 and G2/M phases as measured by flow cytometry. The G0/G1 arrest was more clearly defined by using HDF that were synchronized in G0 and then released from quiescence by replating at subconfluent densities in medium containing 10-70 microM apigenin. The cells were analyzed for cell-cycle progression or cyclin D1 expression 24 h later. A dose of apigenin as low as 10 microM reduced the percentage of cells in S phase by 20% compared with control cultures treated with solvent alone. Western blot analysis of apigenin-treated HDF indicated that cyclin D1 was expressed at higher levels than in untreated cells, which signifies that they were arrested in G1 phase rather than in a G0 quiescent state. The G1 arrest was further studied by cyclin-dependent kinase 2 (cdk2) immune complex-kinase assays of apigenin-treated asynchronous HDF, which demonstrated a dose-dependent inhibition of cdk2 by apigenin. Inhibition of cdk2 kinase activity in apigenin-treated cells was associated with the accumulation of the hypophosphorylated form of the retinoblastoma (Rb) protein as measured by western blot analysis. The cdk inhibitor p21/WAF1 was also induced in a dose-dependent manner, with a 22-fold induction of p21/WAF1 in 70 microM apigenin-treated cells. In conclusion, apigenin treatment produced a G1 cell-cycle arrest by inhibiting cdk2 kinase activity and the phosphorylation of Rb and inducing the cdk inhibitor p21/WAF1, all of which may mediate its chemopreventive activities in vivo. To our knowledge this is the first report of a chemopreventive agent inducing p21/WAF1, a known downstream effector of the p53 tumor suppressor protein.     

Tumor suppressor gene alteration in adult acute lymphoblastic leukemia (ALL). Analysis of retinoblastoma (Rb) and p53 gene expression in lymphoblasts of patients with de novo, relapsed, or refractory ALL treated in Southwest Oncology Group studies

To examine the impact of inactivation of tumor suppressor genes on outcome in adult ALL, we compared two groups of patients registered to SWOG treatment protocols for loss of the Rb gene product and p53 overexpression: (1) 89 patients with de novo ALL, and (2) 26 patients with relapsed/refractory ALL. The groups were comparable with respect to age, sex, and race. Cell lysates (> or = 80% blasts) were analyzed by immunoblotting which enabled detection of Rb or p53 proteins in as little as 1 microg of lysate. Loss of Rb expression (pRbneg) was found in 54/85 (64%) de novo and 11/19 (58%) relapsed patients (P = 0.79). Overexpression of p53 (p53abn), indicative of p53 point mutations, was found in 16/75 (21%) de novo and 8/19 (42%) relapsed patients (P = 0.08). Using a nonisotopic RNase cleavage assay, p53 point mutations in exons 5-9 were confirmed in 14/23 (61%) p53abn specimens. For the de novo ALL group, patients with normal Rb protein had higher WBC and higher peripheral blast and lymphocyte counts. Otherwise neither abnormal Rb or p53 expression correlated with any of a large panel of clinical and laboratory variables including FAB class, blast lineage, expression of myeloid antigens or CD34, and presence of the Ph1 chromosome or BCR-ABL. Analyses of treatment outcomes demonstrated no significant impact of Rb or p53 status alone on CR rates, relapse-free or overall survival. An identical percentage (11%) of both de novo and relapsed/refractory patients had concurrent abnormalities of both Rb and p53 expression (pRbneg/p53abn). The survival curve of these patients suggests an increased rate of early death, but the number of patients in this group was small. Summarizing, (1) loss of Rb expression is common in adult ALL; (2) overexpression of p53 may be more frequent in relapsed/refractory than de novo adult ALL; and (3) although Rb or p53 alterations alone are not strong independent predictors of outcome, their concurrent expression may predict a poor response to therapy.     

A 60 kd MDM2 isoform is produced by caspase cleavage in non-apoptotic tumor cells

The MDM2 oncogene product is a regulator of the p53 tumor suppressor. MDM2 is cleaved by Caspase 3 (CPP32) during apoptosis after aspartic acid-361, generating a 60 kd fragment. Here we report that human tumor cell lines often express high levels of a 60 kd MDM2 isoform (p60) in the absence of apoptosis. We demonstrate that p60 is a product of caspase cleavage of full length MDM2 after residue 361. The protease that cleaves MDM2 in non-apoptotic cells appears to be distinct from the apoptosis-specific Caspase 3, since Caspase 3 substrate poly(ADP-ribose) polymerase (PARP) is not cleaved in cells producing p60. The p60 form of MDM2 is a significant fraction of the p53-bound MDM2 protein in certain tumor cells, suggesting that it functions in the regulation of p53. p60 is also detected in breast tumors overexpressing MDM2. These observations suggest that MDM2 is regulated by caspase processing in non-apoptotic cells, and may account for the MDM2 proteins of similar mobility seen in tumors and other cell lines.     

Time course comparison of cell-cycle protein expression following partial hepatectomy and WY14,643-induced hepatic cell proliferation in F344 rats

During recent years, there has been an extensive research focus in the area of cell-cycle control in eukaryotes and the relationship that exists between cell proliferation and cancer. The eukaryotic cell-cycle is governed by signal transduction pathways mediated by complexes of cyclin dependent kinases (CDK) and their partner cyclin proteins. This study was performed to identify differences in cell-cycle control protein expression following physical and chemical stimuli of hepatic cell growth. Protein levels of cell cycle mediators, cyclin dependent kinases (CDK 1,2,4,5), cyclin proteins (A,B,D1-D3 and E), proliferating cell nuclear antigen (PCNA), tumor suppressor proteins (p53 and Rb), and CDK inhibitory proteins (p16Ink4, p21Waf1 and p27Kip1) were examined in F344 rats following 70% partial hepatectomy or a single dose of WY14,643 over 96- and 48-h time courses, respectively. CDK1 (p34cdc2) and PCNA protein concentrations, quantified by ELISA, were significantly increased beginning at the 24-h time point and maximal at 48 h (6.9- and 3.7-fold for partial hepatectomy and 4.2- and 3.3-fold for WY14,643, respectively). Differential effects were observed with the G1 cell-cycle mediators CDK4, CDK5, and cyclin D3, p21Waf1 and p27Kip1 CDK inhibitory protein concentrations rose in accordance with the induction of DNA synthesis and histone H1 kinase activity. In addition, there were dramatic differences in p53 protein expression patterns following partial hepatectomy versus WY14,643 dosing. Because non-genotoxic hepatocarcinogens are known to induce cellular proliferation, data generated from this study may aid in elucidating the specific hepatocarcinogenic signal transduction pathways stimulated by non-genotoxic carcinogens.     

Implicit time topography of multifocal electroretinograms

The aim of this study was to investigate the expression of p53 and bcl2 proteins in a series of 107 non-small cell lung cancers (NSCLC), and to relate such protein expression to neovascularisation and the expression of vascular endothelial growth factor (VEGF). Moreover, we analysed the prognostic impact of these biological parameters on overall survival, both in univariate and multivariate analyses. An inverse association was found between bcl2 expression and microvessel count (MVC; P = 0.0004) and bcl2 and VEGF (P = 0.007). In contrast, a significant association was found between p53 expression and MVC (P = 0.03) and p53 and VEGF expression (P = 0.04). In univariate analysis, nodal status (P < 0.000001), MVC (P < 0.000001), bcl2 (P = 0.002), p53 (P = 0.03) and VEGF expression (P < 0.000001) significantly affected overall survival, but in multivariate analysis only MVC and VEGF expression retained their prognostic influence. Our results suggest that bcl2 and p53 possibly control the development of tumour angiogenesis in NSCLC, with putative mediation by VEGF. Moreover, the important influence of angiogenesis in the progression of NSCLC is further highlighted.     

Features of the lateralization of speech function in stutterers as a function of the sex of the subjects

BACKGROUND: To analyze the response to eradicative therapy and prognostic factors in 52 patients with primary gastrointestinal lymphoma (PGIL) diagnosed at a single institution in a 13 year period. PATIENTS AND METHODS: The main clinical, biological and evolutive data were recorded. Pathologic diagnosis of PGIL was made according to the Working Formulation. Clinical stage was determined by the Ann Arbor system modified by Mushoff. The results of therapy as well as the influence of such characteristics on complete remission (CR), disease-free survival (DFS) and overall survival (OS) were studied. RESULTS: Mean age of the series was 53 years (SD 15). Thirty patients were males. HIV infection preceded PGIL diagnosis in 10 cases. Seventeen had bad performance status (ECOG 2-4) and 30 B symptoms. The PGIL localization was gastric in 31 cases and 29 had a low grade malignant lymphoma. B phenotype was demonstrated in 98% and 22 patients presented advanced stages (IIE2-IV). Treatment was radical surgery followed by intensive chemotherapy in 32 cases, intensive chemotherapy alone in 17, and surgical resection in 3. CR was obtained in 34 patients and 6 of them relapsed. The projected DFS from CR at 9 years was 72% and OS was 26%. CR and survival were not influenced by PGIL localization and treatment type. The main unfavourable prognostic factors were advanced stage (CR and OS), B symptoms (DFS and OS) and advanced ECOG score (CR, DFS and OS). Previous HIV infection had an independent prognostic influence on both CR and OS. CONCLUSIONS: In patients with PGIL, the achievement of CR, DFS and survival have been independent of the type of eradicative treatment used. Performance status, B symptoms and clinical stage have been the main prognostic factors. HIV infection carried an independent prognostic significance.     

Banner year for U.S. longevity

Tumor-associated proteins are studied immunohistochemically in 86 medulloblastomas (MB). Three main variants of their coexpression (70% of all cases studied) are distinguished. The 1st variant (24 cases): expression of Rb and nm23 proteins and the lack of tenascin expression. The 2nd variant (25 cases): tenascin expression and lack of Rb and nm23 expression. The 3rd variant (12 cases): the expression of tenascin, Rb, p53 and/or bc12. Tenascin-positive MB much more frequently expressed oncoproteins p53, bc12, c-erb B-2 and p30/32, and had higher indices of "growth fraction". Tenascin immunoreactivity and that of other oncoproteins prevailed in MB with desmoplasia and in tumors with immunohistochemical features of neuronal differentiation.     

The role of p53, MDM2 and c-erb B-2 oncoproteins, epidermal growth factor receptor and proliferation markers in the prognosis of urinary bladder cancer

The immunohistological expression of p53 and MDM2 oncoproteins was examined in paraffin embedded tissue from 106 patients with transitional cell carcinoma of the urinary bladder and was related to various clinicopathological features, the expression of proliferation associated markers (proliferating cell nuclear antigen - PCNA - and Ki-67), c-erb B-2 oncoprotein and epidermal growth factor receptor (EGFR), as well as to survival. MDM2 immunoreactivity was seen in 38% of our cases, and in 14% was accompanied by p53 positive immunohistochemistry. The rate of p53 positivity was associated with grade, stage and papillary status, whereas MDM2 immunopositivity increased with grade and stage (Ta VS T1), and MDM2 labeling index (LI) with stage. MDM2 expression was related to p53 expression and less strongly to proliferation rate (Ki-67 LI). The simultaneous p53 and MDM2 expression was more frequently observed in higher grade and stage tumours. C-erb B-2, EGFR and proliferation marker expression increased with grade, stage and non-papillary configuration. In univariate analysis high grade, solid growth pattern, advanced T-category, cystectomy, EGFR and Ki-67 expression were linked to shorter overall survival but only Ki-67 LI, along with T-category and type of therapy, had independent prognostic value. C-erb B-2 expression and stage were the two independent predictors of disease-free survival and Ki-67 LI and EGFR LI the independent predictors of post-relapse survival. For patients with superficial tumors PCNA LI emerged as the single independent determinator of survival. p53 and MDM2 expression did not appear to have any significant impact on survival, although the simultaneous expression of p53 and MDM2 turned out to be a highly significant parameter of shortened overall survival in univariate analysis.     

SPECT brain perfusion abnormalities in mild or moderate traumatic brain injury

The accumulation of wild-type p53 protein results in two pathways, cell cycle G1 arrest by p21WAF1/CIP1/SDI1 and apoptosis inhibited by bcl2, which together carry out the tumor suppressor function. Since genetic alterations of p53 are frequently observed in gastric cancers, the expression of p21 and bcl2 may be altered in gastric carcinogenesis. We therefore analyzed normal mucosa, nondysplastic lesions, hyperplastic polyps, adenomas and carcinomas of the human stomach using immuno-histochemistry, polymerase chain reaction-single-strand conformation polymorphism and DNA sequencing. In normal gastric mucosa, the expression of p21, bcl2 and p53 was topographically restricted: a) p21 expression was limited to foveolar epithelial cells; b) bcl2 and p53 expression was confined to only a few regenerative epithelial cells of the mucous neck region. In chronic gastritis or intestinal metaplasia, topographic expression became more obvious. This topographic expression was altered in hyperplastic polyps and adenomas. Hyper-plastic polyp showed an increased p21 and p53 expression with no bcl2 expression. Where as bcl2 expression increased and extended up parabasal and superficial dysplastic epithelium, p21 expression increased and was limited to surface dysplastic epithelium. Weak p53 expression was in full thickness of dysplastic epithelium. p21 and bcl2 expression in adenoma was higher than in intestinal type of carcinoma. In carcinomas, this topography was abrogated, but p53 mutation (36%) was present. There was no relationship between p53, p21 and bcl2 expression. As a result, in normal gastric epithelial cells, there was a precisely ordered topographic pattern of p21, bcl2 and wild-type p53 expression that becomes disordered during neoplasia. These results suggest that altered cell cycle and apoptosis control by wild-type p53 and its mediators appears to be an early event in gastric carcinogenesis that may facilitate tumor progression.     

The expression of MDM2 and other p53-regulated proteins in the tissues of the developing rat

The expression of MDM2 was monitored in tissues taken from rats at different stages of development. Two major protein species of 90K and 130K were identified, although expression was not the same in all tissues. In most the level of the larger protein tended to decrease as the foetus developed, being relatively scarce after birth. The expression of the 90K product (usually considered to be the major MDM2 protein), however, increased as the animals developed, being most abundant in adult tissues, suggesting that the two proteins play quite different roles during development. p53 expression mirrored that of the smaller MDM2 protein in most, but not all, tissues. This is consistent with previous suggestions made on the basis of in vitro studies that expression of the latter protein is regulated by p53. In addition, the level of two other p53-regulated proteins, Bcl-2 and Bax, were examined. In most tissues expression of both proteins decreased as the foetus developed, being virtually undetectable in adults. These data strongly suggest that factors other than p53 exert the major influence over Bcl-2 and Bax expression, although levels of the 90K MDM2 protein are more likely to be regulated by p53. However, it was noted that the levels of MDM2 in the tissues of p53-null mice were comparable to those seen in the normal rat.     

Audit of ascertainment of deaths to children born in Cumbria, UK, 1950-89 through the NHS central register

Expression of mdm-2 mRNA was measured in 90 ovarian-cancer tissue specimens using the S1 nuclease assay, to investigate a possible association between MDM2 expression and prognosis. mdm-2 mRNA expression was an independent prognostic factor for patients with primary ovarian cancer, FIGO (International Federation of Gynecology and Obstetrics) stages III and IV (n = 57), who all received chemotherapy with carboplatin or cisplatin and cyclophosphamide. Median survival time for patients (FIGO stages III and IV) with no detectable expression of mdm-2 mRNA (n = 14) was 171 days, as compared with 839 days for patients (n = 43) with detectable mdm-2 mRNA (p = 0.0194; log-rank test). However, no association between mdm-2 mRNA expression and survival was observed for patients with FIGO stages I and II who did not receive chemotherapy. mdm-2 expression was not associated with FIGO stage, residual disease, histologic grade and type. Our results suggest that mdm-2, which is known to disrupt p53 function, sensitizes ovarian-cancer cells to cisplatin/cyclophosphamide, possibly by inhibition of p53-mediated G1 cell-cycle arrest and p53-stimulated nucleotide-excision repair.