The aim of our study was to evaluate if
p53 mutations, especially those in the L2/L3 domains of the
p53 gene, add prognostic information for node-positive and steroid receptor positive breast cancer ...patients. Two hundred and five tumour samples from a randomised clinical trial of 596 lymph node- and steroid receptor positive breast cancer patients were included. All patients had been randomly allocated to receive 20
mg of adjuvant tamoxifen (TAM) daily for 2 years or TAM plus one cycle of low-dose, short-term chemotherapy. For detection of
p53 mutations we used
in vitro amplification by polymerase chain reaction and consecutively performed temperature gradient gel electrophoresis (PCR-TGGE) and direct sequencing. We found
p53 mutations in 42/205 (20%) cases: 16/42 (38%)
p53 mutations occurred within the L2/L3 domains of the
p53 gene, and 26/42 (62%) outside the L2/L3 domains.
p53 mutation served as a statistically significant parameter in predicting disease-free survival in univariate (
P=0.02) and multivariate (
P=0.009) analysis. For overall survival, no significant differences were observed. Patients with tumours that had
p53 mutations within the L2/L3 domains of the gene showed no significant difference to those with mutations outside the L2/L3 domains for disease-free survival. For overall survival, mutations in the L2/L3 domains showed a marginally significant difference (
P=0.05) in multivariate analysis, but not in univariate analysis (
P=0.13). We conclude that mutation in the L2/L3 domains of the
p53 gene is not an independent prognostic indicator of disease outcome for patients suffering from breast cancer with lymph node metastases and positive steroid receptors.
Purpose: We aimed to determine the clinical role of the p53 family members p53 and p73 in the responsiveness to platinum-based chemotherapy
and survival in ovarian cancer, considering their ...cross-talk and the p53 polymorphism at codon 72.
Experimental Design: A detailed analysis of p53 and p73 in a series of 122 ovarian cancers was done. We used a functional yeast-based assay to determine the p53 mutational status. Red yeast colonies, indicating mutant p53 , were subsequently sequenced to determine the specific p53 alteration. p53 mutations were divided into two groups according to their previous characterization in the literature: those that efficiently
inhibit transcriptionally active TAp73 function and those that do not. A p53 polymorphism at codon 72 was determined in corresponding normal tissue or blood of ovarian cancer patients. Isoform-specific
p73 expression analysis using real-time reverse transcription-PCR has previously been done in the majority of ovarian cancers
included in this study. In a retrospective chart review, responsiveness to chemotherapy was assessed, and survival data with
long follow-up times were collected.
Results: Eighty of 122 (65.6%) of ovarian cancers harbored p53 mutations. p53 mutational status was an important determinant of responsiveness to platinum-based chemotherapy in all patients with a residual
tumor of <2 cm in diameter after initial surgery (wild-type versus mutant, P = 0.029). In addition, p53 mutational status was a strong prognosticator for recurrence-free and overall survival ( P < 0.0001 and P = 0.003, respectively) in univariate analyses. High expression levels of dominant-negative p73 isoforms (ΔNp73 and ΔN′p73) significantly correlated with chemotherapeutic failure ( P = 0.048) and with worse recurrence-free and overall survival in patients with p53 mutant cancers ( P = 0.048 and P = 0.005, respectively). Eight p53 mutations, present in 19 cases, were found that efficiently inhibit TAp73 (i.e., 175H, 220C, 245S, 245D, 248W, 248Q, 266E,
and 273H). Patients with p53 mutations that efficiently inhibit TAp73 function had a significantly shorter overall survival than patients with p53 mutations
of unknown effect on TAp73 ( P = 0.044). The p53 polymorphism at codon 72 had no influence on responsiveness to chemotherapy or survival.
Conclusion: We provide the first clinical evidence that dominant-negative p73 isoforms contribute to drug resistance in vivo , underscoring the importance of a p53-p73 cross-talk. NH 2 -terminally truncated p73 isoforms were of significant clinical effect by providing an additional unfavorable factor for response to platinum-based
chemotherapy and survival in p53 mutant ovarian cancers.
Abstract
Despite strong homology, the roles of TP53 and TP73 in tumorigenesis seem to be fundamentally different. In contrast to TP53, tumor-associated overexpression of TP73 in many different ...cancers, combined with virtual absence of inactivating mutations and lack of a cancer phenotype in the TP73 null mouse are inconsistent with a suppressor function but instead support an oncogenic function. The discovery of NH2-terminally truncated p73 isoforms, collectively called ΔTAp73, is now the focus of intense interest because they act as potent transdominant inihibitors of wild-type p53 and transactivation-competent TAp73. Therefore, establishing deregulated ΔTAp73 expression in tumors could be the crucial link to decipher which of the two opposing roles of this bipolar gene is the biologically relevant one. This study is the largest to date and encompasses 100 ovarian carcinomas with complete expression profile of all NH2-terminal isoforms, discriminating between TAp73 and ΔTAp73 (ΔNp73, ΔN′p73, Ex2p73, and Ex2/3p73) by isoform-specific real-time reverse transcription-PCR. We find that the set of NH2-terminal p73 isoforms distinguishes ovarian cancer patients from healthy controls and thus is a molecular marker for this diagnosis. Ovarian cancers strongly and almost universally overexpress ΔN′p73 compared with normal tissues (95% of cancers). About one-third of tumors also exhibit concomitant up-regulation of the antagonistic TAp73, whereas only a small subgroup of tumors overexpress ΔNp73. Thus, deregulation of the E2F1-responsive P1 promoter, rather than the alternate P2 promoter, is mainly responsible for the production of transdominant p53/TAp73 antagonists in ovarian cancer. Tumor stage, grade, presence of metastases, p53 status, and residual disease after resection are significant prognostic markers for overall and recurrence-free survival. A trend is found for better overall survival in patients with low expression of ΔN′p73/ΔNp73, compared with patients with high expression. A strong correlation between deregulated ΔTAp73 and p53 status exists. p53 wild-type cancers exhibit significantly higher deregulation of ΔN′p73, ΔNp73, and Ex2/3p73 than p53 mutant cancers. This data strongly supports the hypothesis that overexpression of transdominant p73 isoforms can function as epigenetic inhibitors of p53 in vivo, thereby alleviating selection pressure for p53 mutations in tumors.
In this study we examined whether low-density lipoprotein (LDL) receptor family members represent a link between blood flow characteristics and modified low-density lipoproteins involved in ...endothelial injury, a pivotal factor in atherogenesis. We demonstrated the expression of pro-atherogenic LDL receptor relative (LR11) for the first time in human coronary artery endothelial cells (HCAEC) in vitro and in vivo. Next, LR11 expression and regulation were explored in HCAEC cultured conventionally or on the inner surface of hollow fiber capillaries under exposure to shear stress for 10 days in the presence or absence of LDL. There was no LR11 expression under static conditions. When exposed to chronic low shear stress (2.5 dynes/cm²) transmembrane and soluble endothelial-LR11 were detected in high levels irrespective of the type of LDL added (carbamylated or native). In contrast, chronic high shear stress (25 dynes/cm²) inhibited the LR11-inducing effect of LDL such that transmembrane and soluble LR11 expression became non-detectable with native LDL. Carbamylated LDL significantly counteracted this atheroprotective effect of high shear stress as shown by lower, yet sustained expression of soluble and transmembrane LR11. Oxidised LDL showed similar effects compared to carbamylated LDL but caused significantly lower LR11 expression under chronic high shear stress. Medium from HCAEC under LR11-inducing conditions enhanced vascular smooth muscle cell migration, which was abrogated by the anti-LR11 antibody. Expression of LR11 depended entirely on p38MAPK phosphorylation. We conclude that coronary endothelial LR11 expression modulated by LDL and chronic shear stress contributes to atherogenesis. LR11 and p38MAPK are potential targets for prevention of atherosclerosis.
P-glycoprotein, encoded by the MDR-1 gene, confers multi-drug resistance against antineoplastic agents and is important for the transmembrane transition of various other common therapeutic drugs. ...Recently, a number of polymorphisms in the MDR-1 gene were identified and the T/T genotype at position 3435 in exon 26 was found to correlate with intestinal P-glycoprotein expression and bioavailability of digoxin after oral administration. We analysed the allelic frequencies at the polymorphic site C3435T in a group of patients with locally advanced breast cancer treated by preoperative chemotherapy to evaluate its predictive value. Sixty-eight patients diagnosed between 1998 and 2001 were treated by preoperative chemotherapy with anthracyclines or these agents combined with taxanes. From genomic DNA, a 106 bp fragment of MDR-1 exon 26 was amplified and the C3435T genotype was determined by Pyrosequencing methodology. A potential correlation with therapeutic response was calculated with Fisher's exact test. The overall clinical response rate (cCR and cPR) was 68% but only 7 patients (10.3%) achieved pathological complete response (pCR). Heterozygous C3435T occurred in 57% of the subjects and 22% of the analysed individuals possessed only T alleles. Statistical analysis revealed a significant correlation (p=0.029) between clinical complete response to preoperative chemotherapy and the T/T genotype. MDR-1 polymorphism C3435T in exon 26 may co-determine resistance to chemotherapy and provide useful information to individualize therapy.
DNA amplification seems to be particularly frequent in human breast tumours and has been associated with cancer evolution and aggressiveness. Recent data indicate that new events should be added to ...the list, such as the amplifications at chromosome 20q13 or the MDM2 gene. The present work aimed at determining the incidence and clinicopathological signification of these amplifications in a large series of breast and ovarian tumours. We tested 1371 breast and 179 ovarian tumours by Southern blotting and observed amplification of 20q13 in 5.4% breast and 2.8% ovarian carcinomas, whereas MDM2 was found amplified in 5.3% and 3.8% of breast and ovarian tumours respectively. MDM2 RNA expression levels were analysed in a subset of 57 breast tumours and overexpression was observed in 4/57 (7%) of the tumours. Elevated expression levels coincided with amplification of the gene. In breast cancer, 20q13 and MDM2 amplifications seem to define subsets of aggressive tumours. Indeed, 20q13 was correlated to axillary nodal involvement and occurred preferentially in younger patients (< 50 years). Furthermore, 20q13 correlated, as did MDM2 amplification, to aneuploidy. In parallel, we had also tested our tumour DNAs for amplification of CCND1, ERBB-2 and MYC, which made it possible to test for correlations with 20q13 or MDM2 amplifications. Whereas 20q13 showed a very strong correlation to CCND1 amplification, that of MDM2 was prevalent in MYC-amplified tumours. Interestingly, 20q13 and MDM2 amplifications showed some degree of correlation to each other, which may possibly be owing to the fact that both events occurred preferentially in aneuploid tumours. In ovarian cancer, no statistically significant correlation was observed. However, 20q13 amplification occurred preferentially in stage 3 tumours and MDM2 was correlated to ERBB-2 amplification. This may suggest that in ovarian tumours also, 20q13 and MDM2 amplifications occur in late or aggressive cancers.
Loss of heterozygosity on chromosomal band 8p22 is a common event in several epithelial tumors including ovarian carcinoma. So far, no clear evidence for a tumor suppressor gene (TSG) in this region ...has been found.
On the basis of publicly available expression data in ovarian tissues, the authors selected the eight most noteworthy genes from 8p22 (DLC1, N33, ZDHHC2, FLJ32642, PDGFRL, MTSG1, PCM1, and EFA6R) for a detailed expression analysis in 58 primary ovarian carcinoma tissues and in 38 ovarian cancer cell lines by using quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR). Expression data were correlated to various clinicopathologic characteristics and survival.
Two genes showed a significantly (P< 0.05) lower expression in grade 3 tumors compared with tumors of lower grade (N33) or compared with normal controls and tumors with lower grade (EFA6R). Expression of N33 and EFA6R seems to have an impact on survival, in particular when the combined expression of both genes was used as predictive factor (P< 0.003). In addition, N33 and EFA6R showed a complete loss of expression in several ovarian cancer cell lines. Three genes (FLJ32642, MTSG1, and PCM1) had a significantly (P< 0.001, P< 0.004, and P< 0.001) lower expression in primary ovarian carcinoma compared with controls (ovarian tissues and cysts).
Two to five new potential tumor suppressor or antagonizing gene candidates (N33 and EFA6R with impact on survival, and potentially FLJ32642, MTSG1, and PCM1) for ovarian carcinoma, were identified from the chromosomal band 8p22 and are promising candidates for further functional analysis in ovarian carcinoma.
Over the past few decades great interest has been focused on cell lines derived from tumors, because of their usability as models to understand the biology of cancer. At the same time, advanced ...technologies such as DNA-microarrays have been broadly used to study the expression level of thousands of genes in primary tumors or cancer cell lines in a single experiment. Results from microarray analysis approaches have provided valuable insights into the underlying biology and proven useful for tumor classification, prognostication and prediction. Our approach utilizes biclustering methods for the discovery of genes with coherent expression across a subset of conditions (cell lines of a tumor type). More specifically, we present a novel modification on Cheng & Church's algorithm that searches for differences across the studied conditions, but also enforces consistent intensity characteristics of each cluster within each condition. The application of this approach on a gynecologic panel of cell lines succeeds to derive discriminant groups of compact bi-clusters across four types of tumor cell lines. In this form, the proposed approach is proven efficient for the derivation of tumor-specific markers.
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