Transforming growth factor beta1 stimulates contrasting responses in metastatic versus primary mouse prostate cancer-derived cell lines in vitro.

Tumor progression to the stage of metastasis may result in part from the selection of certain primary tumor cell clones which are phenotypically competent for survival, invasion, and growth at secondary sites. Selection for traits such as loss of growth inhibitory responses, acquisition of increased adhesiveness, increased local immunosuppression, and enhanced motility and collagenase activities likely contribute to cancer progression and may be regulated through the action of growth factors. The transforming growth factors (TGF-beta) family of growth factors has often been associated with these traits and tumor progression; therefore, elimination or subversion of TGF-beta-responsive pathways should be considered as a mechanistic framework for metastatic events. In this report, we have compared growth and extracellular matrix responses to TGF-beta in six metastatic and six primary tumor-derived cell lines in a mouse model of prostate cancer. We have found that tumor cell lines derived from focal pulmonary metastasis secreted relatively greater quantities of total TGF-betas, lost most or all TGF-beta1 growth inhibition, but responded to TGF-beta1 through induction of the type IV collagenase matrix metalloproteinase-9, whereas cell lines derived from tumors which proliferated at the primary site retained the growth inhibition but lacked collagenase activity. Synthesis of another extracellular matrix protein, plasminogen activator inhibitor 1, was stimulated by TGF-beta1 in both primary as well as metastatic tumors. These results suggest that acquisition of differential responses to the TGF-beta family could result in phenotypic traits which facilitate tumor metastasis from certain primary site clones.

Progression to androgen insensitivity in a novel in vitro mouse model for prostate cancer.

We have shown previously that the ras and myc oncogenes can induce poorly differentiated mouse prostate carcinomas in vivo with high frequency (greater than 90%) using inbred C57BL/6 mice in the mouse prostate reconstitution model system. To study the androgen sensitivity of these carcinomas, we have developed an in vitro model system which includes a cell line from normal urogenital sinus epithelium (CUGE) and cell lines from three ras + myc transformed mouse prostate carcinomas (RM-9, RM-1, and RM-2). CUGE cells, as well as all prostate carcinoma cell lines, were positive for cytokeratin 18 mRNA and immunoreactive to cytokeratin-specific antiserum. Two out of three of the early passage carcinoma cell lines were clonal with respect to Zipras/myc 9 retrovirus integration as determined by Southern blot analysis. Whereas significant mitogenic effects of testosterone (10 nM) were not seen in CUGE cells grown in serum-free medium, under similar conditions approx. 2-fold increases in cell number were seen in all low passage prostate carcinoma cell lines. Also, in the presence of growth inhibitory levels of suramin (50 micrograms/ml), testosterone was capable of significant growth stimulation in the carcinoma cell lines. With further propagation from low passage [20-25 population doublings (PD)] to high passage (75-100 PD), all carcinoma cell lines demonstrated increased and similar growth rate in the presence and absence of testosterone. These cell lines maintained stable androgen receptor numbers and binding kinetics during the transition from testosterone-responsive growth to reduced responsivity over multiple passages in culture (> 150 PD). Overall, our studies indicate that the capacity to bind testosterone is stably maintained through the transition of the androgen-sensitive to insensitive phenotype and raise the possibility that androgen sensitivity can persist throughout progression but is masked by the acquisition of autocrine pathways.

Development of a clinical protocol for hepatic gene transfer: lessons learned in preclinical studies.

Strategies for hepatic gene therapy have been proposed that involve isolation of primary hepatocytes and introduction of recombinant genes into these cells in culture, followed by autologous hepatocellular transplantation (HCT). Consideration of clinical applications requires data suggesting that HCT can be performed safely in human subjects in addition to data indicating that recombinant gene expression can reverse a disease process. This report describes preclinical studies that underlie a clinical trial of HCT in which hepatocytes would be labeled with a marker gene to facilitate assessment of engraftment in the recipient. Human hepatocytes were harvested from liver segments preserved in Belzar's solution and transduced with an amphotropic retroviral vector carrying a recombinant marker gene (neomycin phosphotransferase II). Human hepatocytes were recovered from monolayer culture, stained with the fluorescent dye 1,1'-dioctadecyl-3,3,3,3'-tetra-methylindo-carbocyanine perchlorate (DiI) and transplanted into severe combined immunodeficient mice by splenic injection. Engrafted hepatocytes were identified in the liver and spleen of severe combined immunodeficient mice but not immunocompetent controls. Two large animal models of HCT are described. In a dog model, neomycin phosphotransferase II-containing hepatocytes were identified in the liver 7 wk after transplantation. In a baboon model, autologous HCT with DiI-stained cells demonstrated that transplanted cells assume a normal morphology and constitute up to 5% of hepatocytes. These data demonstrate transduction and transplantation of human hepatocytes and the feasibility of HCT in large animals. On the basis of these studies, the proposed clinical trial for gene transfer and transplantation in human subjects has been approved by the National Institutes of Health and the Food and Drug Administration.(ABSTRACT TRUNCATED AT 250 WORDS)

The use of DiI-marked hepatocytes to demonstrate orthotopic, intrahepatic engraftment following hepatocellular transplantation.

A novel method is described for marking primary hepatocytes with the fluorescent dye DiI prior to hepatocellular transplantation and identifying these cells within the hepatic parenchyma of recipient animals by fluorescence microscopy and flow cytometry. Optimal conditions are described for marking cells with DiI in suspension or in monolayer cultures prior to transplantation. DiI is shown to be nontoxic to hepatocytes and not to be exchanged between adjacent cells in vitro. Histological analysis of transplanted tissues shows DiI staining of engrafted hepatocytes and phagocytotic cells (Kupffer cells). This analysis shows that hepatocytes engraft within the hepatic parenchyma and exhibit a histological appearance indistinguishable from normal by conventional hematoxylin and eosin staining. Many previous reports of hepatocellular transplantation have been limited by their inability to unequivocally identify transplanted cells within the liver. These data illustrate the importance of having specific markers for transplanted cells that engraft in an orthotopic location and assume a normal morphological appearance.

Expression of human alpha 1-antitrypsin in dogs after autologous transplantation of retroviral transduced hepatocytes.

The liver represents an excellent organ for gene therapy since many genetic disorders result from the deficiency of liver-specific gene products. We have previously demonstrated that transgenic mouse hepatocytes can be heterologously transplanted into congenic recipients where they survived indefinitely and continued to function as hepatocytes. Here we demonstrate the autologous transplantation of retrovirally transduced canine hepatocytes. At least 1 x 10(9) hepatocytes or 5% of the liver mass can be transplanted by the portal vasculature. In two animals we have transplanted hepatocytes transduced with a retroviral vector containing the human alpha 1-antitrypsin cDNA under transcriptional control of the cytomegalovirus promoter. Both animals had significant human alpha 1-antitrypsin in the serum for 1 month. Although the serum levels of human alpha 1-antitrypsin eventually fell due to inactivation of the cytomegalovirus promoter, PCR analysis demonstrated that a significant fraction of transduced hepatocytes migrated to the liver and continued to survive in vivo. The results suggest that gene therapy of hepatic deficiencies may be achieved by hepatocellular transplantation after genetic reconstitution with the use of promoters of cellular genes that are active in the normal liver.

Modulation of extracellular-matrix synthesized by cultured stromal cells from normal human breast tissue by epidermal growth factor.

A routine, reproducible procedure was developed for the preparation and characterization of stromal cells from normal human breast tissue obtained by reduction mammaplasty. Isolates (n = 15) all exhibited enhanced rates of proliferation, even in the presence of 20% fetal calf serum, when exposed to epidermal growth factor or transforming growth factor a (both 10(-8) M). Cellular responsiveness to these growth factors was consistent with expression of specific surface receptors for epidermal growth factor (approximately 10(4)/cell). In cultures, stromal cells elaborated an extensive, cross-linked, insoluble extracellular matrix which remained firmly associated with the plastic surface of tissue culture ware upon lysis of cells. The insoluble matrix material was analyzed using enzymatic digestion procedures following incorporation of radiolabelled precursors into macromolecular material prior to lysis and preparation. The relative proportion of glycoconjugate (glycopeptides and proteoglycans) and collagenous material present in matrix material was approximately 45% and approximately 55%, respectively, and this was modulated by inclusion of epidermal growth factor into culture medium to approximately 60% and approximately 40%, respectively. Under similar culture conditions stromal cells synthesized twice as much hyaluronate as was produced by control cultures. By use of specific antibody preparations we identified at least four species of glycopeptide present in stromal matrices (namely, fibronectin, laminin, tenascin, and thrombospondin) as well as three types of collagen (types I, III, and IV). The rapid and reproducible procedure for the preparation of radiolabelled insoluble matrix material from normal human breast tissue allows for the study of cellular interaction involving extracellular matrix turnover and degradation.

Endothelin messenger RNA and receptors are differentially expressed in cultured human breast epithelial and stromal cells.

Paracrine regulation is implicit in the biosynthesis and secretion of milk in the breast. An important determinant for this regulation in vivo is proximate cellular location as exemplified by stromal and epithelial cells in breast tissue. Cultured human breast epithelial cells exhibited low constitutive expression of mRNA for endothelin which was enhanced 20-fold after prolactin stimulation. Human breast stromal cells did not express measurable levels of endothelin mRNA under similar conditions. In a similar differential manner, the stimulated release of immunoreactive endothelin into medium overlay was observed only for breast epithelial and not stromal cells. Specific cell-surface receptors for endothelin and biochemical responsiveness to the peptide were observed only in the stromal cells.