Therapeutic Implications of Enhanced G0/G1 Checkpoint Control Induced by Coculture of Prostate Cancer Cells with Osteoblasts
2001
Osteoblastic metastases are common in lethal prostate cancer. Effective
therapyfor bone metastases is lacking. Thus, developing an appropriate
in vitro screening system is critical to prioritize
which of the newly developed agents should undergo additional expensive
and time-consuming in vivo evaluation in bone metastases
animal models. In the past, such in vitro screening
evaluated the response of prostate cancer cells to chemotherapeutic
agents in monoculture without the presence of osteoblasts. In such
monoculture, prostate cancer cells have a high ( i.e., >
90%) proliferative growth fraction. 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
ablation failing patients at “warm” autopsy was found to be>
10-fold lower. To better mimic the lower growth fraction observed
clinically, LNCaP human prostate cancer cells were cocultured with
membrane-separated hFOB human osteoblasts. 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 using flow cytometry, Ki-67
immunohistochemistry, and 5-bromo-2-deoxyuridine incorporation. Using
RNase protection assays, it was documented that coculture with
osteoblasts causes enhanced p53, p27, and p21 expression leading to a
decrease in the number of LNCaP cells entering the cell cycle
( i.e., enhanced number of LNCaP cells in
G 0 -G 1 and a decrease in S and G 2 -M
and thus the growth fraction). This osteoblast-induced enhanced
G 0 -G 1 checkpoint control affected the
chemosensitivity of LNCaP cells. This was documented by coculturing
LNCaP cells with hFOB cells to condition the medium for 3 days to lower
the growth fraction to <30% before exposing the LNCaP cells for
48 h to various concentrations of Taxol, doxorubicin, or
thapsigargin (TG). In standard high ( i.e., >90%)
growth fraction cultures ( i.e., cultures in the absence
of osteoblast-conditioned medium), there was a dose-dependent and
significant ( P < 0.05) increase in
apoptosis of LNCaP cells exposed to Taxol or doxorubicin. In
contrast, even the highest dose of Taxol (1 μm) did not
enhance apoptosis of lower growth fraction LNCaP cells cultured in
osteoblast-conditioned medium. Similarly, only the highest
concentration of doxorubicin (1 μm) enhanced apoptosis in
lower growth fraction cells. 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 monoculture to
identify proliferation-independent agents for the treatment of prostate
cancer bone metastases, and TG is such an agent.
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