No abstract

Activation of tumor-stromal interactions is considered to play a critical role in the promotion of tumorigenesis. To discover new therapeutic targets for hormone-refractory prostate tumor growth under androgen ablation therapy, androgen-sensitive LNCaP cells and the derived sublines, E9 (androgen-low-sensitive), and AIDL (androgen-insensitive), were recombined with androgendependent embryonic rat urogenital sinus mesenchyme (UGM). Tumors of E9CUGM and AIDLCUGM were approximately three times as large as those of LNCaPCUGM. Tumors grown in castrated hosts exhibited reduced growth as compared with those in intact hosts. However, in castrated hosts, E9CUGM and AIDLCUGM tumors were still approximately twice as large as those of LNCaPCUGM. Cell proliferation in tumors of E9CUGM and AIDLCUGM grown in castrated host, was significantly higher than that in tumors of LNCaPCUGM. In vitro, expression of fibroblast growth factor (FGF)-2 and IGF-I, but not FGF-7 mRNA, was significantly reduced in UGM under androgen starvation. In cell culture, E9 cells were responsive to FGF-2 and FGF-7 stimulation, while AIDL responded to FGF-7 and IGF-1. Expression of FGFR1 and FGFR2 was considerably higher in E9 than those in LNCaP, similarly expression of FGFR2 and IGF-IR were elevated in AIDL. These data suggest that activation of prostate cancer cell growth through growth factor receptor expression may result in the activity of otherwise androgenindependent stromal growth factor signals such as FGF-7 under conditions of androgen ablation.

Zinc exhibits inhibitory effects on apoptosis, and a deficiency in this metal generally causes this type of cell death to occur. In the present study, we found that exposure to zinc results in necrosis of prostate carcinoma cells. When zinc acetate was added to LNCaP or PC-3 cells in monolayer culture, they began to detach from the culture dishes, and viability was lost after 4Ϫ8 h. Most of the cell death was found to be due to necrosis as determined by double staining with fluorescein-isothiocyanate-labeled annexin V and ethidium bromide, and by detection of hypodiploid cells. Associated with the induction of necrosis was an increase in low molecular-mass proteins, identified by HPLC analysis to be thymosin β10, parathymosin and GAGE in LNCaP cells, and thymosin β4, parathymosin and metallothionein in PC-3. The time course of the increase of thymosin β10 in LNCaP cells and thymosin β4 in PC-3 cells was consistent with that of appearance of cell detachment and dead cells. These results indicate that zinc can induce necrosis and suggest that production of proteins including β-thymosins is involved in induction of processes leading to cell detachment.

Zinc levels in the prostate have been reported to be associated with the development and progression of malignant prostate cells. To investigate the reason why the zinc content decreases during the progression of prostate cancer to an androgen-independent state, we compared the expression levels of metallothionein and zinc transporters between androgen-responsive LNCaP cells and its androgen-independent subline, AIDL cells. AIDL cells showed lower zinc levels than LNCaP cells and comparable levels of androgen receptor expression to LNCaP cells, consistent with some clinical aspects of androgen-independent prostatic cancer. AIDL cells exhibited a lower expression of zinc transporter 1 (ZnT1) and higher expression of ZnT3 than LNCaP cells. The content of metallothionein, which is a major zinc-binding protein, was significantly lower in AIDL cells than in LNCaP cells. Furthermore, the expression of ZnT3 mRNA was decreased by incubating LNCaP cells in medium containing hormone-stripped fetal calf serum and increased by addition of synthetic androgen R1881 to the medium, whereas the intracellular zinc levels were not affected under these conditions. These findings suggest that factors such as ZnT1 and metallothioneins other than ZnT3 are associated with the low intracellular zinc content in AIDL cells.

Prostate cancer is a heterogeneous disease with varying degrees of androgen sensitivity. In this study, we performed a limiting dilution of human prostate LNCaP cells, and isolated two sublines, LNCaP-E9 and LNCaP-G4, with differential hormonesensitivity. Two LNCaP sublines were obtained by the limiting dilution method. The growth of E9 cells was decreased in the presence of androgens, while that of androgen-treated G4 cells was biphasic. Although the androgen receptor expression level in E9 cells was similar to that seen in G4 cells, the expression of PSA mRNA and protein was significantly lower in the E9 cells. Moreover, the androgen-based stimulation of PSA mRNA expression was less sensitive in E9 cells than G4 cells. Intracellular zinc level did not differ between E9 and G4 cells, but ZnT3 mRNA expression was significantly higher in the E9 cells. When the cells were grafted at the subrenal capsule, the number of CD31-positive vessels with a lumen was approximately 2.5 times higher than that in G4 tumors. LNCaP-E9 cells show lower androgen sensitivity than LNCaP-G4 cells. E9 and G4 cells would be helpful for understanding the biology of hormone-refractory prostate cancer.