Search

The hormone 1alpha,25-dihydroxyvitamin D (1alpha,25(OH)(2)D) inhibits growth and induces differentiation of prostate cells. The enzyme responsible for 1alpha,25(OH)(2)D synthesis, 25-hydroxyvitamin D (25(OH)D)-1alpha-hydroxylase (1alpha-OHase), has been demonstrated in human prostate cells. We compared the levels of 1alpha-OHase activity in prostate cancer cell lines, LNCaP, DU145 and PC-3 and in primary cultures of normal, cancerous and benign prostatic hyperplasia (BPH) prostate cells. We observed a marked decrease in 1alpha-OHase activity in prostate cancer cells, including an undetectable level of activity in LNCaP cells. Transient or stable transfection of 1alpha-OHase cDNA into LNCaP cells increased 1alpha-OHase activity from undetectable to 4.95pmole/mg+/-0.69pmole/mg and 5.8pmole/mg+/-0.7pmole/mg protein per hour, respectively. In response to 25(OH)D, the prohormone of 1alpha,25(OH)(2)D, the transfected LNCaP cells showed a significant inhibition of 3H-thymidine incorporation (37%+/-6% and 56%+/-4% at 10(-8)M for transiently and stably transfected cells, respectively). These findings support an important autocrine role for 1alpha,25(OH)(2)D in the prostate and suggest that the re-introduction of the 1alpha-OHase gene to prostate cancer cells, in conjunction with the systemic administration of 25(OH)D, constitutes an endocrine form of gene therapy that may be less toxic than the systemic administration of 1alpha,25(OH)(2)D.

We have analyzed structural and functional features of the human glucocorticoid receptor (hGR) for their effects on receptor subcellular distribution. COS 1 cells transiently transfected with wild type and mutant hGR cDNAs were assessed immunocytochemically using well-characterized antipeptide antibodies to the hGR. The effect of administration of steroid hormones (and the antiglucocorticoid RU486) on receptor localization was evaluated. Unliganded wild type receptors expressed in COS 1 cells were predominately cytoplasmic. Addition of glucocorticoids or the glucocorticoid receptor antagonist, RU486, resulted in complete translocation of these receptors into the nucleus whereas non-glucocorticoid steroids or dibutyryl cAMP were not effective in promoting nuclear translocation. Thus, nuclear translocation was specific for steroids capable of high affinity binding to the hGR. To elucidate the potential role of receptor domains in receptor localization, COS 1 cells transiently transfected with various receptor cDNA mutants were analyzed in a similar manner. Translocation of an hGR deletion mutant lacking the majority of the amino terminus (deletion of amino acids 77-262) was identical to the wild type receptor despite the absence of a transactivation domain. Receptors in which the DNA binding domain was either partially or totally deleted showed an impaired capacity to undergo hormone-inducible nuclear translocation. Deletion of the hinge region of the hGR (which also contains part of the nuclear localization signal, NL1) resulted in receptor localization in the cytoplasm. Mutants in the ligand binding domain exhibited two localization phenotypes, exclusively nuclear or cytoplasmic. Receptor mutants truncated after amino acid 550 were found in the nucleus in the presence and absence of hormone consistent with the existence of nuclear localization inhibitory sequences in the ligand binding domain of the receptor. However, a linker insertion mutant (at amino acid 582) which results in a receptor deficient in ligand binding did not undergo nuclear translocation indicating that nuclear localization inhibitory sequences were intact in this mutant. The role of receptor phosphorylation on hormone induced nuclear translocation was also examined. Mouse glucocorticoid receptors which contained mutations of certain hormone inducible phosphorylation sites exhibited translocation properties similar to wild type mGR indicating that these phosphorylation sites on the receptor do not play a major role in hormone inducible nuclear translocation.