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Oncogene 1998 Jun 11;16(23):3069-82
Department of Radiation Oncology, Long Island Jewish Medical Center, The Long Island Campus for the Albert Einstein College of Medicine, New Hyde Park, New York 11040, USA.
In addition to breast and ovarian cancer in women, recent evidence suggests that germ-line mutations of the breast cancer susceptibility gene-1 (BRCA1) also confer an increased life-time risk for prostate cancer in male probands. However, it is not known if and how BRCA1 functions in prostate cancer. We stably expressed wild-type (wt) and tumor-associated mutant BRCA1 transgenes in DU-145, a human prostate cancer cell line with low endogenous expression of BRCA1. As compared with parental cells and vector transfected clones, wtBRCA1 clones exhibited: (1) a slightly decreased proliferation rate (doubling time = 25 h as compared with 22 h for control cells); (2) a (3-6)-fold increase in sensitivity to chemotherapy drugs (adriamycin, camptothecin, and taxol); (3) increased susceptibility to drug-induced apoptosis; (4) reduced repair of single-strand DNA strand breaks; and (5) alterations in expression of key cellular regulatory proteins (including BRCA2, p300, Mdm-2, p21(WAF1/CIP1), Bcl-2 and Bax). Clones transfected with the 5677insA breast cancer-associated mutant BRCA1 (insBRCA1) displayed a similar phenotype to wtBRCA1 clones, except that insBRCA1 clones had a significantly decreased proliferation rate (doubling time = 42 h). On the other hand, cells transfected with with 185delAG mutant BRCA1 showed no obvious phenotype as compared with parental or vector transfected cells. These findings suggest that BRCA1 may function as a human prostate tumor suppressor by virtue of its ability to modulate proliferation and various components of the cellular damage response. They also suggest several potential target gene products for a BRCA1 prostate tumor suppressor function.
PMID: 9662340, UI: 98324717
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Cancer Res 1998 Jun 1;58(11):2282-7
The 4th Department, Osaka Bioscience Institute, Suita, Japan.
For cancer gene therapy, it is of primary importance to develop a system to sufficiently and selectively express therapeutic genes in cancer cells. In this study, we showed that an approximately 5.3-kb promoter region of the prostate-specific antigen (PSA) gene can replicate the endogenous expression pattern, although its expression is very weak. We then developed a novel two-step transcriptional activation system in which the PSA promoter drives an artificial transcriptional activator, GAL4-VP16 fusion protein, and it in turn activates transgene expressions under the control of GAL4-responsive elements. By using this system, transgene expressions can be greatly augmented while maintaining prostate-specific expression. Finally, we applied this system to drive an expanded polyglutamine, a potent proapoptotic molecule, to induce apoptosis selectively in PSA-positive prostate cancer cells. This novel system would provide an ideal approach for cancer gene therapy applicable not only to prostate cancer but to other cancers as well.
PMID: 9622059, UI: 98283378
Mol Endocrinol 1997 Nov;11(12):1766-81
Department of Medicine, University of Alabama at Birmingham 35294, USA.
The epidermal growth factor (EGF) system has been thought to play an important role in normal mammary development and carcinogenesis. To study the role of the EGF receptor (EGFR) in mammary development, we developed a transgenic mouse model in which a C-terminal truncated mouse EGFR (EGFR-TR) was expressed in the mouse mammary epithelium under the control of the mouse mammary tumor virus long terminal repeat. The EGFR-TR lacks most of the cytoplasmic domain of the receptor, including the entire protein tyrosine kinase domain. In cultured cells, we show that it acts in a dominant negative manner in EGF-signaled EGFR autophosphorylation. Several lines of mice were characterized and shown to express the transgene at the mRNA and protein levels not only in the mammary gland but also in the salivary glands, epididymis, and prostate. In postpubertal virgin female mice, the expression of the EGFR-TR in the mammary glands was greater than the expression of the endogenous wild type EGFR. In these virgin mice, inhibition in mammary ductal development and a decrease of mammary epithelial DNA synthesis were observed beginning at 5-6 weeks. The inhibition of duct development was most apparent by 15-16 weeks, resulting in a significant defect in ductal branching and outgrowth and an apparent overall decrease in the size of the mammary glands. However, during pregnancy, expression of the endogenous wild type EGFR was markedly increased relative to the EGFR-TR and, at this stage, normal presecretory alveoli developed from the hypoplastic duct tree. Postpartum, normal lactation occurred. Despite EGFR-TR expression in other tissues, no morphological abnormalities were observed. This model demonstrates that the EGFR-TR behaves as a dominant negative regulator of the EGFR system in vivo and that the EGFR system plays an important role in mammary ductal development.
PMID: 9369445, UI: 98034378
Prostate 1997 Nov 1;33(3):166-76
Urologic Oncology Research Laboratory, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA.
BACKGROUND: Studies of genes that may inhibit growth or induce death of cells are facilitated greatly by tightly controlled expression of those genes. A promising system for control of transgene expression over a wide range is the tetracycline-repressible transactivator (tTA) system developed by Gossen and Bujard [Proc Natl Acad Sci USA 1992;89:5547-5551]. We investigated the effectiveness of this system in three well-established human prostate cancer cell lines. METHODS: LNCaP, PC-3, and Tsu-Pr1 cells were transfected with a vector coding for the tTA protein and/or a luciferase reporter vector, and luciferase activity was measured in the presence and absence of tetracycline or the tTA protein. RESULTS: In the absence of tetracycline, the tTA system yielded high levels of luciferase activity in all three cell lines. Background luciferase activity in the presence of tetracycline was nearly undetectable in LNCaP cells, moderate in Tsu-Pr1 cells, and more than 20-fold higher in PC-3 than in Tsu-Pr1 cells. Similar background activity was observed in Tsu-Pr1 and PC-3 cells, even in the absence of the transactivator protein. CONCLUSIONS: The tTA system should be useful for stable transfection of cytotoxic transgenes in LNCaP cells and for control of transgene expression over a wide range in Tsu-Pr1 and PC-3 cells.
PMID: 9365544, UI: 98032248
Semin Cancer Biol 1997 Feb;8(1):3-9
Department of Oncology, Royal Pastgraduate Medical School, Hammersmith Hospital, London, UK.
Over the last decade gene therapy has emerged as probably the sexiest research topic in biology. Whilst few people would attach the same adjective to clinical studies on prostate cancer, the rise in mortality from this very common tumour coupled to the deficiencies of current therapies have kindled research on this malignancy. This article covers the overlap between these disciplines and describes laboratory and clinical experiments that investigate genetic manipulation for the management of prostate cancer.
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PMID: 9299576, UI: 97445671
Am J Pathol 1996 Oct;149(4):1177-91
Department of Medical Pathology, University of California at Davis 95616, USA.
Male transgenic mice expressing the polyomavirus middle T (PyV-MT) gene exhibited growth and developmental abnormalities in prostatic and other urogenital epithelium. Expression of PyV-MT was directed to these tissues by a novel, androgen-inducible expression vector based on the rat C3(1) gene. Epithelial growth disturbances (hyperplasia, dysplasia, and invasive carcinoma) were observed in the ventral and dorsal prostate, coagulating gland, epididymis, and vas deferens. The abnormalities were characterized by histological disorganization, nuclear pleomorphism, increased mitoses, and abnormal DNA content. Transgene transcription was detected in affected tissues, indicating that the C3(1)-based vector targeted androgen-sensitive urogenital tissues, especially the prostate. These results demonstrated that expression of a gene, the protein of which is known to interact with cellular proteins involved in signal transduction, dramatically disrupted urogenital growth and development.
PMID: 8863667, UI: 97017054
Toxicol Pathol 1996 Jul-Aug;24(4):502-4
Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030, USA.
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PMID: 8864193, UI: 97017576
Cancer Gene Ther 1996 Jul-Aug;3(4):230-7
UAB School of Medicine, Birmingham, Alabama, USA.
Achieving limited recombinant viral replication may provide a means of amplifying viral-mediated gene transfer in vivo. We have previously shown that cotransduction of an E1-defective adenovirus with a plasmid containing the deleted E1 functions into prostate carcinoma cells resulted in E1-defective virus production by those cells. The studies described here have extended these findings to more firmly establish the capacity of the trans complementation approach to achieve amplification of recombinant viral transgene expression. The recombinant virus used for all the studies was AdCMV-luc which contained a luciferase expression cassette; the replication-enabling plasmid, pE1, encoded the E1 functions deleted from AdCMV-luc. Quantitative in vitro studies with the HeLa cell line showed that for each plaque forming unit of AdCMV-luc originally exposed to the cells, 0.54 x 10(3) new replication-defective viruses were detected in supernatants and lysates over the following 4 days. Multiple cell lines were shown to support new virus production following cotransduction of AdCMV-luc and pE1. Small numbers of replication-competent viruses were detected in the lysates and supernatants from the cotransduced cells such that for every 10(5) replication-defective viruses approximately two replication-competent viruses were produced. Tumor nodules produced by engrafting a mixture of AdCMV-luc/pE1-cotransduced HeLa cells with uninfected HeLa cells yielded much higher levels of luciferase expression than control tumors containing mixtures of cells infected with AdCMV-luc alone. In total, these results demonstrate new virus production by cells receiving a replication-defective adenovirus and a replication-enabling plasmid are capable of amplifying recombinant viral transgene expression in vivo.
PMID: 8853547, UI: 97006253
DNA Cell Biol 1992 Jun;11(5):345-58
Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas 75235.
To define the regulatory strategy for the transcriptional control of the kallikrein multigene family, we analyzed the expression of several kallikrein/SV40 T-antigen (TAg) fusion genes in transgenic mice and rats. Kallikrein family members are normally expressed at a high level in the submandibular gland and are expressed in a wide range of tissues that vary among individual family members. A total of 1.7 kb of proximal 5'-flanking DNA from the tissue kallikrein gene (rKlk1) was sufficient to confer much of the correct tissue-specific pattern on a TAg reporter gene. TAg mRNA was detectable in tissues that normally express rKlk1 and TAg-induced tumors arose in brain and pancreas. However, absolute levels of transgene mRNA were very low relative to the expression of the normal endogenous tissue kallikrein gene. In particular, expression in the salivary glands, normally very high for endogenous rKlk1, was either low or absent. An intact rKlk1 transgene with extensive flanking DNA (4.5 kb 5' and 4.7 kb 3') and complete intragenic (4 kb) sequences was expressed similarly to the fusion transgene, demonstrating that regulatory elements necessary for comprehensively correct expression are not contained within these additional gene regions. Two additional kallikrein/SV40 fusion transgenes were derived from other family members, one from the rKlk2 gene, which encodes tonin, and another from the rKlk8 gene, which encodes a prostate kallikrein. Whereas the endogenous rKlk2 and rKlk8 genes normally are expressed at high levels in rat salivary glands, they were not expressed in the salivary glands as transgenes. The results for these transgenes of three different family members indicate that control elements that direct the particular nonsalivary gland expression pattern characteristic of each family member may be present within the proximal 5'-flanking region of each gene, whereas regulatory sequences necessary for normal levels of expression in these tissues and for maximal salivary gland expression are not. We propose that the gene-associated regulatory sequences are complemented by a dominant control region that imposes salivary gland expression on the extended kallikrein family locus.
PMID: 1605858, UI: 92297161
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