Androgen-dependent cell cycle arrest and apoptotic death in PC-3 prostatic cell cultures expressing a full-length human androgen receptor.
To assess the function of androgen receptor in androgen-independent prostate cancer cells, human PC-3 prostate carcinoma cells, which lack androgen receptor (AR) expression, were transfected with a full length human AR cDNA sequence inserted into an episomal expression vector system. Several clonal lines of transfected cells expressing varying levels of a 110 kDa AR, as determined by immunoblotting and ligand binding assay, were isolated. The expressed ectopic receptors displayed nuclear binding following androgen treatment and mediated androgen inducibility of a mouse mammary tumor virus (MMTV)-luciferase reporter gene construct in a dose-dependent manner. 5 alpha-dihydrotestosterone (DHT) activation of luciferase activity was blocked by the AR antagonist hydroxyflutamide, and was promoter-specific based on the inability of the hormone-insensitive RSV promoter to respond to DHT. Treatment of AR-expressing PC-3 cells with physiological levels of DHT for 3 days resulted in paradoxical inhibition of cell growth. The growth-inhibitory effect was observed in clonal lines expressing low, moderate and high levels of AR, indicating that it was not the result of AR overexpression. To determine whether AR-expressing PC-3 cells had become androgen dependent, albeit with slowed growth, the effect of 1.0 nM DHT on the growth of two clonal lines expressing low and moderate receptor levels (PC-3(AR)13 and PC-3(AR)2, respectively) was examined on over an 18 day period. DHT removed after 3, 6, or 9 days and replaced with steroid-free medium. Surprisingly, after 6 days of DHT treatment, the number of PC-3(AR)2 cells began to decrease such that all cells were dead by 15 days after initiation of DHT treatment. A similar effect was observed in PC-3(AR)13 cells, but required a longer initial period of DHT exposure. PC-3(AR)2 cells were rescued from cell death if DHT was withdrawn 3 days but not 6 or 9 days after initiation of DHT treatment. As determined by DNA cell cycle analysis, the proportion of cells in the G1 phase was enhanced by DHT treatment, accompanied by a decrease in cells in the S and G2M phase of the cell cycle. After 6 days of DHT treatment, the proportion of cells in G1 decreased which was accompanied by an increase in cells in a subG1 population consistent with apoptosis. DNA fragmentation in PC-3(AR)2 cells after 3 or 6 days of DHT treatment was demonstrated by agarose gel electrophoresis, further indicating the cell death was apoptotic. Removal of DHT from PC-3(AR)2 cultures after 3 days, but not after 6 or 9 days, was followed by a large shift in cells from G1 to S and G2M. These data suggest that DHT blocks the progression of AR transfected PC-3 cells through the cell cycle, resulting in growth inhibition and apoptosis.
Heisler
L E
LE
Division of Reproductive Science, Toronto Hospital Research Institute, Ontario, Canada.
Evangelou
A
A
Lew
A M
AM
Trachtenberg
J
J
Elsholtz
H P
HP
Brown
T J
TJ
D000728:Androgens / Q000494:pharmacology*; D017209:Apoptosis; D002453:Cell Cycle / Q000187:drug effects*; D002455:Cell Division / Q000187:drug effects; D002467:Cell Nucleus / Q000378:metabolism; D013196:Dihydrotestosterone / Q000494:pharmacology; D015870:Gene Expression; D006801:Humans; D008156:Luciferases / Q000235:genetics; D008297:Male; D008324:Mammary Tumor Virus, Mouse / Q000235:genetics; D011401:Promoter Regions, Genetic; D011471:Prostatic Neoplasms / Q000473:pathology*; D011944:Receptors, Androgen / Q000235:genetics / Q000502:physiology*; D011993:Recombinant Fusion Proteins; D014162:Transfection; D014407:Tumor Cells, Cultured
D000728:Androgens; D011944:Receptors, Androgen; D011993:Recombinant Fusion Proteins; D013196:Dihydrotestosterone; D008156:Luciferases