Therapeutic implications of enhanced G(0)/G(1) checkpoint control induced by coculture of prostate cancer cells with osteoblasts

Osteoblastic metastases are common in lethal prostate cancer. Effective the rapy for bone metastases is lacking. Thus, developing an appropriate in vit ro screening system is critical to prioritize which of the newly developed agents should undergo additional expensive and time-consuming ill vivo eval uation in bone metastases animal models. In the past, such in vitro screeni ng evaluated the response of prostate cancer cells to chemotherapeutic agen ts in monoculture without the presence of osteoblasts. In such monoculture, prostate cancer cells have a high (i.e., >90%) proliferative growth fracti on. In contrast, the growth fraction (i.e., mean: 7.1 +/- 0.8%; median: 3.1 %) in 117 metastatic sites of prostate cancer obtained from 11 androgen abl ation failing patients at "warm" autopsy was found to be >10-fold lower. To better mimic the lower growth fraction observed clinically, LNCaP human pr ostate cancer cells were cocultured with membrane-separated hFOB human oste oblasts. Such coculturing significantly lowered the growth fraction of the LNCaP cells (i.e., from >90 to <30%) without enhancing their low rate (i.e. , <5%) of apoptosis. This lowering of the growth fraction was documented us ing flow cytometry, Ki-67 immunohistochemistry, and 5-bromo-2-deoxyuridine incorporation. Using RNase protection assays, it was documented that cocult ure with osteoblasts causes enhanced p53, p27, and p21 expression leading t o a decrease in the number of LNCaP cells entering the cell cycle (i.e., en hanced number of LNCaP cells in G(0)-G(1) and a decrease in S and G(2)-M an d thus the growth fraction). This osteoblast-induced enhanced G(0)-G(1) che ckpoint control affected the chemosensitivity of LNCaP cells. This was docu mented by coculturing LNCaP cells with hFOB cells to condition the medium f or 3 days to lower the growth fraction to <30% before exposing the LNCaP ce lls for 48 h to various concentrations of Taxol, doxorubicin, or thapsigarg in (TG). In standard high (i.e., >90%) growth fraction cultures (i.e., cult ures in the absence of osteoblast-conditioned medium), there was a dose-dep endent and significant (P < 0.05) increase in apoptosis of LNCaP cells expo sed to Taxol or doxorubicin. In contrast, even the highest dose of Taxol (1 <mu>M) did not enhance apoptosis of lower growth fraction LNCaP cells cult ured in osteoblast-conditioned medium. Similarly, only the highest concentr ation of doxorubicin (1 muM) enhanced apoptosis in lower growth fraction ce lls. In contrast, 100 nM TG induced high levels of apoptosis in both lower and high-growth fraction LNCaP cultures. These results demonstrate that the osteoblast/LNCaP coculture system is a better in vitro screen than monocul ture to identify proliferation-independent agents for the treatment of pros tate cancer bone metastases, and TG is such an agent.