Mario Chevrette

Screen for genes differentially expressed during regeneration of the zebrafish caudal fin

The zebrafish caudal fin constitutes an important model for studying the molecular basis of tissue regeneration. The cascade of genes induced after amputation or injury, leading to restoration of the lost fin structures, include those responsible for wound healing, blastema formation, tissue outgrowth, and patterning. We carried out a systematic study to identify genes that are up‐regulated during “initiation” (1 day) and “outgrowth and differentiation” (4 days) of fin regeneration by using two complementary methods, suppression subtraction hybridization (SSH) and differential display reverse transcriptase polymerase chain reaction (DDRT‐PCR). We obtained 298 distinct genes/sequences from SSH libraries and 24 distinct genes/sequences by DDRT‐PCR. We determined the expression of 54 of these genes using in situ hybridization. In parallel, gene expression analyses were done in zebrafish embryos and early larvae. The information gathered from the present study provides resources for further investigations into the molecular mechanisms of fin development and regeneration. Developmental Dynamics 231:527–541, 2004.

Links between Fer tyrosine kinase expression levels and prostate cell proliferation.

In our cloning strategy to identify tyrosine kinases implicated in the regulation of prostate growth, the dog fer cDNA was obtained and shown to be highly homologous to known fer cDNAs. Using a polyclonal Fer antibody directed against a C-terminal peptide, we studied its associations with cortactin, beta-catenin and p120Cas in human prostate carcinoma PC-3 cells. In contrast to previous reports, no interactions were observed. To assess its functional role, fer cDNA constructs were transfected in PC-3 cells. Antisense clones exhibiting a marked diminution of Fer expression had a reduced growth rate (doubling time of 29 vs. 42 h) and were unable to form colonies in soft agar. In agreement with these results, Fer protein expression was linked to human prostatic proliferative diseases, with enhanced levels in extracts from cancer tissues as compared to those from normal and hyperplastic ones, and was also expressed in the human prostate carcinoma cell lines DU145 and LNCaP. In the dog model, Fer expression was up-regulated in dividing versus resting prostate epithelial cells in vitro, and also in vivo when basal cell hyperplasia and metaplasia was induced by estrogen after castration. Minimal effects were observed when renewing the luminal epithelium with androgens. Taken together, these results show that Fer expression is associated with prostate cell proliferation and enhanced in prostate cancer.

Down-Regulation of CD9 Expression during Prostate Carcinoma Progression Is Associated with CD9 mRNA Modifications

Purpose: Cluster-of-differentiation antigen 9 (CD9) protein, a member of the tetraspanin family, has been implicated in carcinogenesis of various human tumors. Although decreased expression of the CD82 tetraspanin protein, a close CD9 relative, is associated with prostate cancer progression, CD9 expression has not been analyzed in this malignancy. Experimental Design: CD9 expression in human prostatic adenocarcinoma was analyzed by immunohistochemistry on 167 primary tumors and 88 lymph node or bone metastases. CD9 cDNA was sequenced from two human prostate cancer cell lines, prostatic adenocarcinoma, high-grade prostatic intraepithelial neoplasia (PIN), and normal prostatic tissues. Results: Although CD9 was detected in the epithelium of normal prostatic tissues, reduced or loss of CD9 expression within neoplastic cells was observed in 24% of 107 clinically localized primary adenocarcinomas, 85% of 60 clinically advanced primary adenocarcinomas, 85% of 65 lymph node metastases, and 65% of 23 bone metastases. Difference in CD9 expression between clinically localized and advanced diseases was highly significant (P < 1 × 10−7). Whereas there was no alteration of CD9 cDNA in normal tissues, all PC-3–derived cell lines, one PIN, and four prostatic adenocarcinomas harbored deletions in their CD9 cDNAs. Recurring CD9 point mutations were also found in PC-3M-LN4 cells, one PIN, and seven prostatic adenocarcinomas. Conclusions: CD9 expression is significantly reduced and even lost during prostate cancer progression. Moreover, deletions and mutations of the CD9 mRNA may be associated with loss of protein expression observed in tumor cells. Our data suggest that CD9 inactivation may play an important role in prostate cancer progression.

Characterization of the 3p12.3-pcen region associated with tumor suppression in a novel ovarian cancer cell line model genetically modified by chromosome 3 fragment transfer

The genetic analysis of nontumorigenic radiation hybrids generated by transfer of chromosome 3 fragments into the tumorigenic OV‐90 ovarian cancer cell line identified the 3p12.3‐pcen region as a candidate tumor suppressor gene (TSG) locus. In the present study, polymorphic microsatellite repeat analysis of the hybrids further defined the 3p12.3‐pcen interval to a 16.1 Mb common region containing 12 known or hypothetical genes: 3ptel‐ROBO2‐ROBO1‐GBE1‐CADM2‐VGLL3‐CHMP2B‐POU1F1‐HTR1F‐CGGBP1‐ZNF654‐C3orf38‐EPHA3‐3pcen. Seven of these genes, ROBO1, GBE1, VGLL3, CHMP2B, CGGBP1, ZNF654, and C3orf38, exhibited gene expression in the hybrids, placing them as top TSG candidates for further analysis. The expression of all but one (VGLL3) of these genes was also detected in the parental OV‐90 cell line. Mutations were not identified in a comparative sequence analysis of the predicted protein coding regions of these candidates in OV‐90 and donor normal chromosome 3 contig. However, the nondeleterious sequence variants identified in the transcribed regions distinguished parent of origin alleles for ROBO1, VGLL3, CHMP2B, and CGGBP1 and cDNA sequencing of the hybrids revealed biallelic expression of these genes. Interestingly, underexpression of VGLL3 and ZNF654 were observed in malignant ovarian tumor samples as compared with primary cultures of normal ovarian surface epithelial cells or benign ovarian tumors, and this occurred regardless of allelic content of 3p12.3‐pcen. The results taken together suggest that dysregulation of VGLL3 and/or ZNF654 expression may have affected pathways important in ovarian tumorigenesis which was offset by the transfer of chromosome 3 fragments in OV‐90, a cell line hemizygous for 3p. Mol. Carcinog.

VGLL3 expression is associated with a tumor suppressor phenotype in epithelial ovarian cancer

Previous studies have implicated vestigial like 3 (VGLL3), a chromosome 3p12.3 gene that encodes a putative transcription co‐factor, as a candidate tumor suppressor gene (TSG) in high‐grade serous ovarian carcinomas (HGSC), the most common type of epithelial ovarian cancer. A complementation analysis based on microcell‐mediated chromosome transfer (MMCT) using a centric fragment of chromosome 3 (der3p12‐q12.1) into the OV‐90 ovarian cancer cell line haploinsufficient for 3p and lacking VGLL3 expression was performed to assess the effect on tumorigenic potential and growth characteristics. Genetic characterization of the derived MMCT hybrids revealed that only the hybrid that contained an intact VGLL3 locus exhibited alterations of tumorigenic potential in a nude mouse xenograft model and various in vitro growth characteristics. Only stable OV‐90 transfectant clones expressing low levels of VGLL3 were derived. These clones exhibited an altered cytoplasmic morphology characterized by numerous single membrane bound multivesicular‐bodies (MVB) that were not attributed to autophagy. Overexpression of VGLL3 in OV‐90 was achieved using a lentivirus‐based tetracycline inducible gene expression system, which also resulted in MVB formation in the infected cell population. Though there was no significant differences in various in vitro and in vivo growth characteristics in a comparison of VGLL3‐expressing clones with empty vector transfectant controls, loss of VGLL3 expression was observed in tumors derived from mouse xenograft models. VGLL3 gene and protein expression was significantly reduced in HGSC samples (>98%, p < 0.05) relative to either normal ovarian surface epithelial cells or epithelial cells of the fallopian tube, possible tissues of origin of HGSC. Also, there appeared to be to be more cases with higher staining levels in stromal tissue component from HGSC cases that had a prolonged disease‐free survival. The results taken together suggest that VGLL3 is involved in tumor suppressor pathways, a feature that is characterized by the absence of VGLL3 expression in HGSC samples.

Analysis of the conservation of synteny between Fugu and human chromosome 12

BackgroundThe pufferfish Fugu rubripes (Fugu) with its compact genome is increasingly recognized as an important vertebrate model for comparative genomic studies. In particular, large regions of conserved synteny between human and Fugu genomes indicate its utility to identify disease-causing genes. The human chromosome 12p12 is frequently deleted in various hematological malignancies and solid tumors, but the actual tumor suppressor gene remains unidentified.ResultsWe investigated approximately 200 kb of the genomic region surrounding the ETV6 locus in Fugu (fETV6) in order to find conserved functional features, such as genes or regulatory regions, that could give insight into the nature of the genes targeted by deletions in human cancer cells. Seven genes were identified near the fETV6 locus. We found that the synteny with human chromosome 12 was conserved, but extensive genomic rearrangements occurred between the Fugu and human ETV6 loci.ConclusionThis comparative analysis led to the identification of previously uncharacterized genes in the human genome and some potentially important regulatory sequences as well. This is a good indication that the analysis of the compact Fugu genome will be valuable to identify functional features that have been conserved throughout the evolution of vertebrates.

Chromosome 18 suppresses tumorigenic properties of human prostate cancer cells

Although prostate cancer is still the most diagnosed cancer in men, most genes implicated in its progression are yet to be identified. Chromosome abnormalities have been detected in human prostate tumors, many of them associated with prostate cancer progression. Indeed, alterations (including deletions or amplifications) of more than 15 human chromosomes have been reported in prostate cancer. We hypothesized that transferring normal human chromosomes into human prostate cancer cells would interfere with their tumorigenic and/or metastatic properties. We used microcell‐mediated chromosome transfer to introduce human chromosomes 10, 12, 17, and 18 into highly tumorigenic (PC‐3M‐Pro4) and highly metastatic (PC‐3M‐LN4) PC‐3‐derived cell lines. We tested the in vitro and in vivo properties of these hybrids. Introducing chromosome 18 into the PC‐3M‐LN4 prostate cancer cell line greatly reduced its tumorigenic phenotype. We observed retarded growth in soft agar, decreased invasiveness through Matrigel, and delayed tumor growth into nude mice, both subcutaneously and orthotopically. This phenotype is associated with a marker in the 18q21 region. Combined with the loss of human chromosome 18 regions often seen in patients with advanced prostate cancer, our results show that chromosome 18 encodes one or more tumor‐suppressor genes whose inactivation contributes to prostate cancer progression.

HUMAN CHROMOSOME 3 MEDIATES GROWTH ARREST AND SUPPRESSION OF APOPTOSIS IN MICROCELL HYBRIDS

Chemotherapeutic treatment of tumor cells leads either to tumor cell death (usually by apoptosis) or to the formation of drug-resistant subpopulations. Known mechanisms of cancer cell drug resistance include gene amplification and increased expression of drug transporters. On the other hand, normal cells survive many forms of chemotherapy with minimal damage probably because of their capacity for growth arrest and stringent control of apoptosis. Microcell hybrids between B78 (murine melanoma) and HSF5 (normal human fibroblasts) were analyzed to identify a new human chromosomal region involved in the promotion of drug-induced growth arrest and suppression of apoptosis. In these hybrids, the presence of human chromosome 3 was strongly associated with suppression of apoptosis via G1 and G2 growth arrest during exposure to the antimetabolite N-phosphonoacetyl-L-aspartate (PALA), suggesting that a gene(s) on chromosome 3 serves an antiproliferative role in a drug-responsive growth arrest pathway.

Somatic Molecular Genetics of Prostate Cancer

Despite progress in diagnosis and treatment, prostate cancer is still one of the most frequent lethal diseases in men in Western countries. Today, an increasing number of prostate cancers are detected through elevated serum prostate-specific antigen (PSA) levels. PSA detection is very sensitive. Determining free versus total PSA serum level has enabled the achievement of better specificity (Balk et al. 2003), however, this tool remains imperfect. Many patients undergo unnecessary diagnostic procedures, experiencing physiological and psychological stress. Similarly, current techniques such as imaging and biopsies are not optimal thus, hopes are high for the discovery of new molecular markers for prostate cancer. Studying prostate cancer progression may reveal new insights into the molecular mechanisms of cancer development to help improve prevention, provide better tools for diagnosis, as well as for prognosis and treatment.