Transforming growth factor-beta promotes invasion in tumorigenic but not in nontumorigenic human prostatic epithelial cells

Transforming growth factor-beta (TGF-beta) is a pleiotropic growth factor with actions that are dependent on circumstances, including dose, target cell type, and context. TGF-beta can elicit both growth-promoting and growth-suppressive activities. In normal tissues, TGF-beta generally acts to restrict growth and maintain differentiation. However, during tumorigenesis, changes in TGF-beta expression and cellular responses can promote tumorigenesis. The present study examines the effects of TGF-beta on the nontumorigenic human prostatic epithelial cell line BPH1 and on three derivative tumorigenic sublines BPH1(CAFTD)1, BPH1(CAFTD)3, and BPH1(CAFTD)5. The data show that TGF-beta has different effects on the nontumorigenic and tumorigenic cells. The nontumorigenic cells are growth inhibited by TGF-beta. In contrast, the tumorigenic sublines are not growth inhibited but instead undergo an epithelial to mesenchymal transformation (EMT) in response to TGF-beta. The tumorigenic lines show constitutively elevated levels of phosphorylated Akt, which modulates their response to TGF-beta by blocking Smad3 and p21 nuclear translocation. On TGF-beta stimulation of the tumorigenic sublines, the activated Akt allows the cell to escape cell cycle arrest. The phosphatidylinositol 3-kinase/Akt pathway is also involved in TGF-beta-induced EMT, defined here by induction of vimentin expression and enhanced cellular motility. In vivo, tumorigenic cells with constitutively active TGF-beta signaling show increased invasion with EMT, which express vimentin, located specifically at the invasive front of the tumor. These data indicate that following malignant transformation TGF-beta can play a direct role in promoting prostatic cancer and further that these responses are context specific in vivo.     

5alpha-dihydrotestosterone-binding proteins and androgen sensitivity in prostatic cancers of Copenhangen rats.

A line (R-3327-A) of androgen-insensitive squamous cell carcinomas of the prostate of Copenhagen rats has been derived from an established line (R-3327) of androgen-responsive tumors R-3327 A tummors have a growth rate of approximately 10 times that of R-3327. The response of R=3327 A to hormonal environment has been determined by measuring the growth rate of the tumor transplanted to males, females, and castrated males. No great differences were observed. The metabolism of testosterone by tumore samples was studied following s.c. injections of the labeled steroid. The results show very little 5alpha-reduction of testosterone compared with that obtained in the prostate gland itself, as well as in the androgen-sensitive R-3327 tumors. A comparison of the presence of 17beta-hydroxy-5alpha-androstan-3-one-binding proteins in cytoplasmic extracts from both lines of tumors shows that the receptor proteins are present only in the androgen-sensitive R-3327 and not in the androgen-insensitive R-3327 A. The levels of the receptor proteins in R-3327 tumors are higher in tumors borne by male than by female animals, and castration of males decreases the amount of 17beta-hydroxy-5alpha-androstan-3-one binding.     

TGF-beta 1 and skin carcinogenesis: antiproliferative effect in vitro and TGF-beta 1 mRNA expression during epidermal hyperproliferation and multistage tumorigenesis.

The role of transforming growth factor-beta 1 (TGF-beta 1) in multisage carcinogenesis in mouse skin was assessed by studying its growth inhibitory effects on nontumorigenic and tumorigenic keratinocytes and by examining its mRNA expression in vitro and during epidermal hyperproliferation and multistage carcinogenesis. While growth of primary basal keratinocytes was inhibited by TGF-beta 1 in doses as low as 0.1 ng/mL, the immortal keratinocyte line MCA/3D ("putatively initiated" cells) responded to TGF-beta 1 with slightly reduced sensitivity, and the papilloma-producing keratinocyte line 308 was considerably less sensitive. In contrast, the squamous carcinoma cell line Carc B was completely nonresponsive, and two other tumorigenic cell lines (PDV and PDVC57) were sensitive to growth inhibition by TGF-beta 1. Steady-state levels of TGF-beta 1 mRNA were high in all the malignant cell lines and in line 308 papilloma cells, but low in primary basal cells and in the nontumorigenic keratinocyte lines V2, Reb1, and MCA/3D. Our in vivo studies showed that tumor promoters, but not mitogenic or weak hyperplasiogenic agents, were able to induce transient expression of TGF-beta 1 mRNA in mouse epidermis. A constitutive overexpression of TGF-beta 1 mRNA was observed in malignant carcinomas but not in the benign premalignant lesions, indicating that overexpression may be associated with malignant progression.     

Growth and metastasis of human tumors in nude mice following tumor-cell inoculation into a vascularized polyurethane sponge matrix

Growth and metastasis of human tumor cells in immunodeficient nude mice were improved when tumor cells were inoculated within a vascularized artificial polyurethane sponge matrix. The sponges had been implanted 7-10 days earlier and were vascularized at the time of cell injection. All cell lines tested, including colon carcinoma-derived lines from primary tumors (HT29, PT3 and PT4) or from liver metastasis (LM3), and a metastatic variant from a melanoma (MeWo-Met) grew in a high percentage (78-94%) of the inoculated sponge grafts. When growth in sponge grafts is compared with growth at a subcutaneous site, the sponge matrix appears to increase tumorigenicity, at least for some cell lines. Regular formation of metastases was observed when cells had been injected into sponges. Most metastases were found in a second sponge graft implanted at a contralateral site, but some were also found at other s.c. sites. In vivo depletion of NK cells by pre-treatment with cyclophosphamide could not further enhance the formation of metastasis. Tumor cells from fresh surgical specimens could be propagated in sponge matrix grafts and subsequently established as cell lines in tissue culture.     

Isolation and characterization of propagable cell lines (HUNC) from the androgen-sensitive Dunning R3327H rat prostatic adenocarcinoma

The Dunning H rat prostate tumor (R3327H) is a widely used experimental model of human prostatic adenocarcinoma (CaP). The Dunning H tumor has been characterized as androgen-sensitive, androgen-receptor (AR) positive, prostate-specific antigen and prostatic acid phosphatase (PAP) positive. To date, the tumor has been maintained by serial passage in vivo because of the lack of an in vitro cell line that retains the characteristics of the in vivo tumor. The objective of the present study was to establish a propagable cell line from R3327H adenocarcinoma that maintained androgen sensitivity and expression of AR, PSA and PAP. Tissue harvested from an in vivo R3327H tumor was dissociated with collagenase and placed into Richter's improved media (with supplements). A cytokeratin-positive epithelial cell line (HUNC- E) and a vimentin-positive stromal cell line (HUNC-S) were generated from the primary culture, subcultured continuously for >300 days, and passaged >50 times. Survival of the HUNC-E cell line in vitro depended on several media supplements, including nicotinamide, insulin, transferrin, selenium and epidermal growth factor (EGF). HUNC-E cells expressed AR and produced PSA and PAP throughout the culture period, as confirmed by immunocytochemistry and Western blot analyses. Addition of 14 nM testosterone (T) or dihydrotestosterone (DHT) to HUNC-E cells, stimulated DNA synthesis as well as anchorage-independent growth and PSA production, which demonstrated the androgen-sensitive nature of the cells in vitro. When HUNC-E and HUNC-S cells were combined in a 3:1 ratio and introduced subcutaneously into syngeneic male hosts, tumors formed in 2/3 animals with an average latency of 7 months. RT-PCR and immunocytochemical characterization of the HUNC cell lines revealed that the cells expressed several growth factors and their cognate receptors, including HGF, TGF-alpha and the TGF-betas, indicating the establishment of potential autocrine loops in the neoplastic cells. The HUNC-E and HUNC-S CaP cell lines, which retain the characteristics of the epithelial and stromal components of the in vivo R3327H tumor, will allow a more thorough and informative molecular and biological analysis of prostatic adenocarcinoma.      

Elevated expression of phosphatidylserine in the outer membrane leaflet of human tumor cells and recognition by activated human blood monocytes

We determined whether the presence of phosphatidylserine (PS) in the outer membrane leaflet of human tumor cells correlated with their recognition by activated human monocytes. Three tumorigenic cell lines, A375 melanoma and A431 and Colo-16 carcinomas, and a normal human epidermal keratinocyte line (NHEK) were incubated with monocytes activated to the tumoricidal state by gamma-interferon and lipopolysaccharide. Activated human monocytes bound to and lysed all tumorigenic targets, while the nontumorigenic NHEK were neither bound nor killed. Semiquantitative analysis of PS in the outer leaflet of the cells revealed that the tumorigenic cells expressed 3-7-fold more PS than did the nontumorigenic NHEK. To determine whether enhanced PS expression on the tumor cells was responsible for their recognition by activated monocytes, NHEK were supplemented with exogenously supplied analogues of PS and phosphatidylcholine. PS-labeled NHEK but not phosphatidylcholine-labeled nor control NHEK bound to activated human monocytes. These results suggest a role for PS in monocyte recognition of tumor cells.     

Down modulation of fibronectin messenger RNA in metastasizing rat prostatic cancer cells revealed by differential hybridization analysis

To identify genes whose expression is down modulated in the process of metastasis, gene expression was analyzed in cell lines derived from Dunning R-3327 rat prostatic tumor sublines. A complementary DNA (cDNA) library from the anaplastic nonmetastasizing subline AT-1 was used for a differential hybridization analysis, using probes derived from mRNAs of the AT-1 and the metastasizing MAT-LyLu subline. In this way 14 cDNA clones were isolated representing 6 differentially expressed genes. The expression levels in a panel of tumor sublines measured with these cDNA clones were tested for correlation with the anaplastic non-metastasizing phenotype. One cDNA clone, designated pSE-1, whose expression was high in all tested sublines with that phenotype, appeared to represent the gene for fibronectin. To further investigate the down modulation of this gene, we studied its expression in AT-2 (anaplastic, nonmetastasizing tumor) and lines derived therefrom that exhibited a high metastatic potential after transfection with the v-Ha-ras oncogene. In the genetically manipulated metastasizing tumor sublines, fibronectin mRNA levels were approximately 4- to 8-fold lowered compared to the nonmetastasizing parental AT-2 line.     

Transient inflammatory response induced by apoptotic cells is an important mediator of melanoma cell engraftment and growth

Two murine melanoma cell lines, Tm1 and Tm5, were derived from a nontumorigenic lineage of pigmented murine melanocytes, melan-a. Both Tm1 and Tm5 are invariably tumorigenic in syngeneic mice when inoculated s.c. in doses higher than 10(4) cells; 10(3) or fewer cells rarely give rise to tumors. We demonstrate that subtumorigenic inocula of Tm1 or Tm5 cells (10(3)) as well as of a known murine melanoma cell line (B16F10) develop as vigorously growing tumor grafts only when coinoculated with apoptotic, but not necrotic cells. The presence of apoptotic cells correlates with a transient inflammatory infiltrate, composed mainly of neutrophils and macrophages. Kinin B1 receptor-deficient mice, which have impaired transmigration of neutrophils to inflamed tissues, had significant growth inhibition of subtumorigenic doses of melanoma cells coinjected with apoptotic cells. Using the same model, tumor take in athymic mice was similar to that seen in wild-type mice, suggesting that a T cell-dependent inflammatory response is not necessary to promote the survival and growth of subtumorigenic doses of melanoma cells. Taken together, our results describe how tumor engraftment and growth can be profoundly affected by microenvironmental alterations in response to the presence of apoptotic cells. Disrupting the delicate balance between apoptotic cells and leukocyte infiltration may provide potentially important insights for understanding and interfering with tumor cell viability during treatment with either gamma-radiation or apoptosis-inducing drugs.     

Heterogeneous expression of invasive and metastatic properties in a prostate tumor model

Cellular heterogeneity of neoplasia is well demonstrated in the Dunning R-3327 rat prostate adenocarcinoma. In this study, we measured the differential expression of invasive and metastatic properties of this prostate model by cloning from a heterogeneous parental cell line. Four cell clones were derived and characterized by morphological studies, E-cadherin expression, and invasive and metastatic potential. Three of the clones (clones 5A, 5C, and 5D) demonstrated a fibroblastic morphology and were anchored to the substrate by loose microvillous processes. The fourth clone (clone 5B) grew in tight clusters and displayed many closely spaced microvilli, long overlapping cytoplasmic regions with well-defined junctional complexes. The parental line (R3327-5) demonstrated a combination of both these growth patterns. E-cadherin expression was absent in clones 5A, 5C, and 5D and very prominent in clone 5B, when compared to the parental line. The absence of E-cadherin expression correlated with increased invasiveness, as measured in an in vitro invasion assay. Subcutaneous injections of clones 5A, 5C, and 5D yielded lung metastases and no primary tumors at the site of inoculation while clone 5B was tumorigenic and produced fewer lung metastases in vivo. These clones, therefore, provide a potential for studying a variety of molecules involved in prostate cancer invasion and metastasis, especially for the direct testing of the significance of E-cadherin expresssion in prostate cancer progression.     

Tumour seeding in peritoneal wound sites in relation to growth-factor expression in early granulation tissue

The purpose of these experiments was to identify growth factors produced during the formation of a peritoneal wound in relation to tumour cell seeding and stimulated growth in granulation tissue. Gelfoam(c) gelatin sponge was implanted in the mesenteric fan of nude mice to initiate the granulation process. Human HT29 colon carcinoma cells were inoculated intraperitoneally at various times after sponge implantation and tumour growth in granulation tissue was determined. RNA isolated from granulation tissue was used for polymerase chain reaction analysis of the expression of specific growth factors and receptors [vascular endothelial growth factor (VEGF), transforming growth factor-beta (TGF-beta) and lysophosphatic acid (LPA)], and for microarray analysis of differentially expressed genes in early vs. late granulation tissue. Inflammatory cells infiltrated the sponge within 1 day, followed by fibroblasts and the formation of an extracellular matrix. Tumour cell inoculation at 8 h to 3 days after sponge implantation resulted in extensive tumour formation in all cases. Inoculation at 10-28 days resulted in focal tumour growth in only 16-33% of the sponges. Low amounts of VEGF, TGF-beta(1-3), TGF-beta RIII and LPA receptors 1,2 were detected in early granulation tissue, with increased expression from day 10. Microarray analysis identified additional differentially expressed genes that may stimulate tumour take and growth in early granulation tissue.     

Spontaneous mutation rates of tumorigenic and nontumorigenic Chinese hamster embryo fibroblast cell lines

The genomic stability of a series of nontumorigenic, tumorigenic, and tumor-derived Chinese hamster embryo fibroblastic (CHEF) cell lines was compared by examining their rates of spontaneous mutation at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus, using thioguanine resistance for selection of mutants. The spontaneous mutation rates were 1.1 x 10(-6) mutations/cell/generation in the non-tumor-forming CHEF/18 cell line and 4.9 x 10(-6) in the tumorigenic CHEF/16 cells. Three tumorigenic and tumor-derived CHEF cell lines derived from CHEF/18 (J132 3-2 T3L, focus 2, focus 3) and two lines (16-2 Tuk 4 and 204 Bu50 Tuk 2) derived from CHEF/16 were chosen on the basis of their karyotypes, which demonstrated a considerable level of chromosomal rearrangement. Mutation rates of four of these five lines ranged from 1.2 x 10(-6) to 8.9 x 10(-6) mutations per cell per generation. Only the fifth line, 16-2 Tuk 4, showed a significantly elevated rate of mutation as compared with the nontumorigenic CHEF/18 cell line. Thus, we have found no simple correlation between spontaneous mutation rate and the malignant phenotype, and we conclude that mutation rate per se is not a sensitive index of malignancy. In addition, we have compared three methods of calculating mutation rate and find that they rank the cell lines in the same order, but each stresses a different aspect of the distribution and therefore produces different estimates of the mutation rate.     

Development and characterization of DP-153, a nontumorigenic prostatic cell line that undergoes malignant transformation by expression of dominant-negative transforming growth factor beta receptor type II

We have developed a nontumorigenic epithelial cell line, DP-153, from the dorsal prostate of a Lobund/Wistar rat treated with N-methyl-N-nitrosourea and testosterone propionate. DP-153 cells express cytokeratins 5 and 14, but not cytokeratin 18, consistent with a basal epithelial cell phenotype. Similar to the nontumorigenic NRP-152 prostatic cell line, DP-153 cells do not form tumors in athymic mice and retain many of the properties of normal prostatic cells. They express prostatic acid phosphatase and androgen receptors and require several mitogens (epidermal growth factor, insulin, dexamethasone, and cholera toxin) for sustained growth in culture under serum-containing conditions. DP-153 cells are also growth-stimulated by keratinocyte growth factor and basic fibroblast growth factor and growth-inhibited by all-trans-retinoic acid, 1,25-dihydroxyvitamin D(3), and transforming growth factor (TGF)-beta1. We demonstrate that expression of dominant-negative TGF-beta receptor type II by retroviral transduction of DP-153 cells leads to complete loss of TGF-beta1-induced growth inhibition. When transplanted s.c. in athymic mice, DP-153 cells expressing dominant-negative TGF-beta receptor type II form tumors as early as 4 weeks, in contrast to the vector control and parental cell line, which do not form tumors even 8 months after transplantation, supporting the observation that TGF-beta functions as a tumor suppressor in these cells. Our data further support that DP-153 is a suitable cell line for analysis of normal prostatic growth and carcinogenesis.     

Establishment and in vivo characterization of multidrug-resistant dunning R3327 rat prostate-carcinoma cell-lines

We describe the selection of 3 new multidrug-resistant cell lines derived from tumor cells of different metastatic phenotypes within the Dunning R3327 model of rat prostatic carcinoma. Cell lines of weak (AT2) and strong (AT3 and MAT-LyLu) metastatic behavior were cultured in vitro and challenged with doxorubicin at progressively increasing concentrations. Chemosensitivity was determined colorimetrically by release of precipitated formazan pigment (MTT assay). Expression of the multidrug-resistance glycoprotein (P-170) was monitored immunocytochemically and by Western blotting using monoclonal antibody C219. The behavior of the parental and resultant drug-resistant cells was assessed by their growth in syngeneic rats. Doxorubicin challenge of the initially drug-sensitive parental prostatic carcinoma cell lines resulted in the rapid development of multidrug resistance together with simultaneous expression of P-glycoprotein. While lung and lymph-node metastases developed in host animals inoculated with parental AT3 and MAT-LyLu cells, no metastases developed in the multidrug-resistant progeny of these cell lines. This study has shown that Dunning rat prostate-carcinoma cell lines, previously sensitive to different cytotoxic agents, rapidly become multidrug-resistant and express P-glycoprotein following exposure to doxorubicin. Further more, development of multidrug resistance is associated with a less aggressive tumor phenotype and loss of metastatic potential. Nevertheless, it is unlikely that the non-metastatic phenotype of Dunning rat prostatic carcinoma cells is solely associated with expression of P-glycoprotein. These new multidrug-resistant cell lines exhibiting an altered behavioral phenotype will provide a valuable mode with which to analyze the relationship between expression of P-glycoprotein and the metastatic phenotype of prostatic carcinoma cells.     

Isolation and characterization of a cloned cell line R3327H-G8-A1 derived from the dunning R3327H rat adenocarcinoma

A cloned cell line (R3327H-G8-A1) has been isolated from the Dunning R3327H adenocarcinoma. Light and electron microscopic studies showed that the cell line possessed features common to secretory epithelial cells. These cells, which grow in monolayer culture, produced s.c. hind flank tumors when inoculated inoculated into Copenhagen X Fischer F1 rats.l Chromosomal karyotype analysis confirmed that the cell line is distinctly that of the Rattus norvegicus genus and species. The cells specifically bind testosterone and dexamethasone with equilibrium dissociation constants (Kd) of 0.49 and 0.8 nM, respectively. The numbers of saturable binding sites per cell are 10,000 for testosterone and 60,000 for dexamethasone. The cells also have 5 alpha-reductase activity. These properties are characteristic of the prostate and of the Dunning tumor from which the cells are derived. Cell growth in vitro was stimulated by androgens and inhibited by glucocorticoids at concentrations of 10(-8) M. An int riguing finding was that estradiol and progestins dramatically stimulated growth in the apparent absence of receptors for these hormones. Finally, comparisons between the G8-A1 cells and the tumor induced by the G8-A1 clone and a second generation of cells from this G8-A1-induced tumor showed that the cloned cells retained their properties following passaging in the animal.     

A highly conserved vascular permeability factor secreted by a variety of human and rodent tumor cell lines

We have previously reported that rodent tumor cell lines secrete a potent vascular permeability factor with a molecular weight of 34,000-42,000 (Senger et al. Tumor cells secrete a vascular permeability factor that promotes accumulation of ascites fluid. Science (Wash. DC), 219: 983-985, 1983). This tumor-secreted vascular permeability factor (VPF) causes a rapid and completely reversible increase in microvascular permeability in the species (guinea pig or rat) from which the tumors were derived without causing mast cell degranulation or endothelial cell damage or exciting an inflammatory cell infiltrate. This VPF may be responsible, at least in part, for the increased permeability which is commonly displayed by solid and ascites tumor vessels. We have now examined 7 human tumor cell lines and have determined that 5 of them also secrete this same VPF. Antibody raised to guinea pig line 10 VPF neutralized more than 90% of the vascular permeability-increasing activity secreted by these 5 human tumor lines. Furthermore, VPFs from both guinea pig and human tumor sources bound to and were eluted similarly from immobilized heparin and comigrated identically on sodium dodecyl sulfate-polyacrylamide gels. Finally, 2 tumorigenic (in nude mice) human cell lines were found to secrete at least 14-fold more VPF than their directly matched, nontumorigenic counterparts, suggesting that elevated expression of this permeability factor may correlate with neoplastic transformation. These data suggest that a broad spectrum of tumor cells from several species, including humans, secretes a highly conserved molecule that enhances local vascular permeability and that this function may be important for tumor growth.     

Malignant transformation of human bladder epithelial cells by DNA transfection with the v-raf oncogene

Transfection of the v-raf oncogene into immortalized, nontumorigenic human bladder epithelial cells resulted in the isolation of two tumorigenic transformants. Both were identified as human and of the same origin as the parent cell line by human leukocyte antigen typing and Southern blot analysis. Both the primary tumorigenic transfectants and the cell lines established from the induced tumors expressed v-raf mRNA and v-raf protein. In both tumorigenic transformants the level of c-myc mRNA was enhanced compared with that of the parent cell line.     

Monoamine oxidase inhibitors l-deprenyl and clorgyline protect nonmalignant human cells from ionising radiation and chemotherapy toxicity

l-Deprenyl (R-(-)-deprenyl, selegiline) is an inhibitor of monoamine oxidase-B (MAO-B) that is known to protect nerve cells from a variety of chemical and physical insults. As apoptosis is a common mechanism of radiation-induced cell death, the effect of l-deprenyl on the survival of cultured cells and tissue explants was studied following exposure to gamma radiation. The results obtained were compared with the effects of the less-selective MAO-B inhibitor pargyline and the MAO-A inhibitor clorgyline. l-Deprenyl at a concentration of 10(-9) M protected the nontumorigenic cell line (HaCaT) and normal human urothelial explants from the effects of cobalt-60 gamma radiation, but did not protect tumorigenic human cell lines HaCaT-ras, HPV-transfected human keratinocytes (HPV-G cells), or PC3. Human bladder carcinoma explants were not protected. Clorgyline showed a smaller protective effect of normal cells, whereas pargyline had no effect. Radiation-induced delayed effects (genomic instability measured as delayed cell death) were prevented in normal cells by l-deprenyl but, interestingly, deprenyl appeared to increase the amount of delayed death in the tumorigenic cell lines. Studies using l-deprenyl prior to the exposure of nonmalignant cells to cisplatin showed that cell death due to this agent was also reduced. Treatment of cultures of nontumorigenic cells with l-deprenyl or clorgyline significantly increased the levels of the protein Bcl-2 following irradiation, but there was no such effect on the already-elevated levels of this protein in the tumour samples. Since the Bcl-2 has been shown to be an inhibitor of apoptosis or programmed cell death, this would imply that the protective effects of l-deprenyl and clorgyline involve activation of antiapoptotic pathways within the normal cell. This hypothesis is supported by data showing reduced levels of apoptosis in HaCAT cells and in normal bladder explant cultures following treatment with l-deprenyl.     

Tumor selective replication of Newcastle disease virus: association with defects of tumor cells in antiviral defence

To investigate tumor-selective viral replication, we compared several tumorigenic human cell lines to nontumorigenic human cells from the blood for the sensitivity to become infected by a recombinant lentogenic strain of Newcastle Disease Virus (NDV) with incorporated transgene EGFP (NDFL-EGFP). Although fluorescence signals in nontumorigenic cells were only weak or missing completely, a massive and long-lasting transgene-expression was observed in all tumor cell lines. The majority of tumor cells (50-95%) could be infected, and viral replication was associated with an increase in the cell surface density of viral antigens. To clarify the underlying mechanism of the observed difference in virus susceptibility we examined the kinetics of interferon-induced antiviral enzymes because NDV is a strong type-I interferon inducer. This analysis revealed several defects of tumor cells in their antiviral defence responses: They showed no response to UV-inactivated NDV, whereas nontumorigenic cells reacted with induction of high-levels of the antiviral enzymes PKR and MxA. Upon coincubation with live NDV, tumor cells showed a delayed response in the increased expression of the antiviral enzymes in comparison with PBMC. In nontumorigenic cells the replication cycle of NDV stopped after the production of positive-strand RNA, while tumor cells continued in the replication cycle and copied viral genomes 10-50 hr after infection. These results can explain the tumor selective replication behavior of this interesting antineoplastic virus.     

Inhibition of in vivo proliferation of androgen-independent prostate cancers by an antagonist of growth hormone-releasing hormone.

Tumour-inhibitory effects of a new antagonist of growth hormone-releasing hormone (GH-RH), MZ-4-71, were evaluated in nude mice bearing androgen-independent human prostate cancer cell lines DU-145 and PC-3 and in Copenhagen rats implanted with Dunning R-3327 AT-1 prostatic adenocarcinoma. After 6 weeks of therapy, the tumour volume in nude mice with DU-145 prostate cancers treated with 40 microg day(-1) MZ-4-71 was significantly decreased to 37 +/- 13 mm3 (P < 0.01) compared with controls that measured 194 +/- 35 mm3. A similar inhibition of tumour growth was obtained in nude mice bearing PC-3 cancers, in which the treatment with MZ-4-71 for 4 weeks diminished the tumour volume to 119 +/- 35 mm3 compared with 397 +/- 115 mm3 for control animals. Therapy with MZ-4-71 also significantly decreased weights of PC-3 and DU-145 tumours and increased tumour doubling time. Serum levels of GH and IGF-I were significantly decreased in animals treated with GH-RH antagonist. In PC-3 tumour tissue, the levels of IGF-I and IGF-II were reduced to non-detectable values after therapy with MZ-4-71. The growth of Dunning R-3327 AT-1 tumours in rats was also significantly inhibited after 3 weeks of treatment with 100 microg of MZ-4-71 day(-1) i.p. as shown by a reduction in tumour volume and weight (both P-values < 0.05). Specific high-affinity binding sites for IGF-I were found on the membranes of DU-145, PC-3 and Dunning R-3327 AT-1 tumours. Our results indicate that GH-RH antagonist MZ-4-71 suppresses growth of PC-3, DU-145 and Dunning AT-1 androgen-independent prostate cancers, through diminution of GH release and the resulting decrease in the secretion of hepatic IGF-I, or through mechanisms involving a lowering of tumour IGF-I levels and possibly an inhibition of tumour IGF-I and IGF-II production. GH-RH antagonists could be considered for further development for the therapy of prostate cancer, especially after the relapse.