Identification and characterization of the human testes-specific protease 50 gene promoter.

Testes-specific protease 50 (TSP50) has been identified as one of the testis-specific proteins that is expressed at high levels in approximately 92% of human breast cancer samples, making it an attractive molecular marker and a potential target for diagnosis and therapy. However, little is known about the transcriptional mechanisms controlling TSP50 gene expression. In the present study, we have characterized the 5' regulatory region of the TSP50 gene in order to understand the molecular mechanisms regulating its expression. Analysis with a series of deletions demonstrated that a 624-bp region was essential for the basal promoter activity of the TSP50 gene. Further analysis results indicated that the two fragment regions +231 to +251 and -22 to -8, especially the putative Sp1 binding site (+237 to +239) and the putative CCAAT/enhancer binding protein (C/EBP) binding site (-15 to -13), are more important for the basal transcription activity of the human TSP50 promoter. Overexpression of Sp1 and C/EBPbeta transcriptional factors upregulated the activities of the TSP50 promoter. Taken together, these results will help to better understand the role of the TSP50 gene in signal-dependent transcriptional regulation, and to develop new reagents for therapeutic downregulation of the TSP50 gene in human breast cancer.

Screening of single-chain variable fragments against TSP50 from a phage display antibody library and their expression as soluble proteins.

A novel gene, testes-specific protease 50 (TSP50), is abnormally activated and differentially expressed in most patients with breast cancer, suggesting it as a novel biomarker for this disease. The possibility that TSP50 may be an oncogene is presently under investigation. In this study, the single-chain variable fragments (scFvs) against TSP50 were panned from a phage display antibody library using TSP50-specific peptide, pep-50, as a target antigen. After 4 rounds of panning, 3 clones (A1, A11, and C8) from the library were verified to show strong binding affinities for TSP50 by enzyme-linked immunosorbent assay (ELISA) and to contain the variable region genes of the light and heavy chains of scFv antibodies but different complementary determining regions by sequencing. The genes of scFv-A1 and scFv-A11 were cloned into expression vector pPELB and successfully expressed as a soluble protein inEscherichia coli Rosetta. The yields of expressions were about 4.0 to 5.0 mg of protein from 1 L of culture. The expressed proteins were purified by a 2-step procedure consisting of ion-exchange chromatography, followed by immobilized metal affinity chromatography. The purified proteins were shown a single band at the position of 31 KDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Sandwich ELISA demonstrated that the expressed scFv proteins were able to specifically react with pep-50, laying a foundation for the investigation of the function of TSP50 in the development and treatment of breast cancer.

Localization and expression of TSP50 protein in human and rodent testes.

OBJECTIVES: To present our recent observations obtained from the continuing characterization of the TSP50 gene pertaining to its evolutionary importance and behavior in human testicular germ cell tumors. Previous studies have reported that expression of the human TSP50 gene is testes specific. Its product is similar to many serine proteases but possesses its own unique features. In addition, TSP50 is abnormally activated in most tested patients with breast cancer. METHODS: Testicular tissue from rats, mice, and humans was obtained through biopsy or orchiectomy. The expression of the TSP50 protein was determined using immunohistochemical staining and Western blotting techniques. RESULTS: The Western blot results showed that the polyclonal anti-human TSP50antibody reaction pattern in both rodent testes was the same as that observed in the human testes. In addition, the immunohistochemical staining patterns in the human, mouse, and rat testes were similar. We also discovered that expression of TSP50 was largely downregulated in all testicular germ cell tumors examined by immunohistochemical analysis. CONCLUSIONS: The results of our studies suggest that the TSP50 gene could be of evolutionary importance in mammalian reproduction. Unlike the results generated from patients with breast cancer, in whom upregulation of the TSP50 gene correlates with disease development, the TSP50 gene was downregulated in patients with seminoma. This information indicates that altered expression levels of the TSP50 gene in different microenvironments are associated with different or distinct types of human cancer.

Partially unspliced and fully spliced ELF3 mRNA, including a new Alu element in human breast cancer.

Using modified representational difference analysis, a DNA fragment (GC3) was isolated as a difference between a breast cancer and a normal cell line from the same patient. GC3 proved to be a fragment of intron 7 of the ELF3 gene, an ets family transcription factor, amplified in the breast cancer cell line. Using genomic walking technology, a new Alu (Alu(kwd)) was found downstream of GC3 in an antisense position between nt 8762 and nt 8763 within intron 8 of the ELF3 gene. This ELF3 intron fragment(GC3) was expressed in human breast cancer cell lines and four of six breast cancer tissues, but not in matched normal cell lines and tissues. Similarly, Alu(kwd) was also found in the same breast cancer cell lines and five of eight other breast cancer tissues, but not in matched normal cell lines and tissue. This was confirmed by RNase and DNase digestion analysis. Moreover, GC3 and Alu(kwd) were detected in both the nuclear and cytoplasmic RNA fractions of breast cancer cell lines. The finding of cytoplasmic intron retention was verified with northern blotting and the 5' and 3' rapid amplification cDNA ends procedure (5' and 3'RACE) to search for cDNA sequences in RNA from these cancer cell lines. Partially unspliced ELF3 mRNA and fully spliced ELF3 mRNA was found in the same breast cancer cell line. Partially unspliced ELF3 mRNA contained introns 4-7 without any nucleotide mutation at intron/exon splice junction borders. Fully spliced 1959 bp ELF3 mRNA showed a different 5'UTR from the published ELF3 mRNA, and was predicted to encode a 371 amino acid protein sharing 98% homology with the ELF3 protein sequence. This is the first report of intron retention of ELF3 as well as the pathological appearance of both spliced and unspliced cytoplasmic ELF3 mRNA in human breast cancer cells.

WTH3, a new member of the Rab6 gene family, and multidrug resistance.

The WTH3 gene was obtained by a DNA fragment isolated by the methylation-sensitive representational difference analysis technique due to its hypermethylation in the human multidrug resistant (MDR) breast cancer cell line MCF7/AdrR. The WTH3 gene product is 89% and 91% identical to the human Rab6 and Rab6c proteins, but possesses an elongated C-terminal region which contains 46 extra amino acids. Nevertheless, we consider the WTH3 gene a new member of the Rab6 gene family. Semi-quantitative reverse transcriptase-polymerase chain reaction results showed that WTH3 was 15 and 4 times downregulated in MCF7/AdrR and MES-SA/Dx5, a human MDR uterine sarcoma cell line, as compared to their non-MDR parental cell lines. Permanent expression of the WTH3 transgene in MDR cell lines increased to varying degrees their sensitivity to several anticancer drugs, which included doxorubicin, taxol, vinblastine, vincristine, and etoposide, as compared to the control sublines transfected with the empty vector. Flow cytometry and fluorescence microscope experiments suggest that the WTH3 transgene stimulated the host's uptake and retention of DOX. Our results imply that the WTH3 gene plays a role(s) in MDR phenotype development in vitro.

TSP50, a possible protease in human testes, is activated in breast cancer epithelial cells.

Initial studies have identified TSP50 as a human testes-specific gene that is demethylated in breast cancer. In this study, we will present new data related to the TSP50 gene. We have found that the TSP50 gene product shares a similar enzymatic structure with many serine proteases. However, the most critical catalytic site, serine, has been replaced by threonine. Western analysis revealed that in human testes, the TSP50 antibody detected two closely positioned protein bands whose estimated molecular masses were 37 kDa, whereas in a large portion of breast cancer tissues, but not normal control tissues, only one band was present. Immunohistochemistry assays found TSP50 proteins located in the spermatocytes of human testes, whereas in situ hybridization and immunohistochemistry confirmed that gene activation in breast tumors took place in malignant mammary epithelial cells. These results suggested that the normal function of the TSP50 gene was involved in spermatogenesis, whereas the up-regulation of TSP50 in many breast cancer patients not only indicated that it might be a novel biomarker for this disease but also encouraged us to further explore the possibility of whether it was an oncogene.

Lefty contributes to the remodeling of extracellular matrix by inhibition of connective tissue growth factor and collagen mRNA expression and increased proteolytic activity in a fibrosarcoma model.

Homeostasis of the extracellular matrix (ECM) of tissues is regulated by controlling deposition and degradation of ECM proteins. The breakdown of ECM is essential in blastocyst implantation and embryonic development, tissue morphogenesis, menstrual shedding, bone formation, tissue resorption after delivery, and tumor growth and invasion. TGF-beta family members are one of the classes of proteins that actively participate in the homeostasis of ECM. Here, we report on the effect of lefty, a novel member of the TGF-beta family, on the homeostasis of extracellular matrix in a fibrosarcoma model. Fibroblastic cells forced to express lefty by retroviral transduction lost their ability to deposit collagen in vivo. This event was associated with down-regulation of the steady-state level of connective tissue growth factor that induces collagen type I mRNA. In addition, lefty transduction significantly decreased collagen type I mRNA expression and simultaneously increased collagenolytic, gelatinolytic, elastolytic, and caseinolytic activities in vivo by the transduced fibroblasts. These findings provide a new insight on the actions of lefty and suggest that this cytokine plays an active role in remodeling of the extracellular matrix in vivo.