calcimycin and Prostatic-Neoplasms

Human DNAJC12 is a J domain-containing protein whose regulation, subcellular localization, and function are currently unknown. We show here that the abundance of DNAJC12 in human LNCaP prostate cancer cells is upregulated by the stress-inducing drug A23187 and by the stressregulated transcription factor AIbZIP/CREB3L4. The DNAJC12 gene encodes two isoforms, only one of which (isoform a) is expressed in these cells. Immunofluorescence studies showed that a recombinant DNAJC12 protein is diffusely distributed in the cytoplasm. To identify substrates of DNAJC12, we used an immunoaffinity-mass spectrometry approach in cells that express epitope-tagged DNAJC12. The list of potential DNAJC12-binding proteins that were identified in this screen includes several nucleotide-binding proteins. The most frequently identified partner of DNAJC12 in unstressed cells was Hsc70, a cognate Hsp70 chaperone, whereas in stressed cells, the ER chaperone BiP was frequently associated with DNAJC12. Immunoprecipitation experiments confirmed that the endogenous DNAJC12 and Hsc70 proteins interact in LNCaP cells. These results clarify the role of DNAJC12 in the regulation of Hsp70 function.

Topics: Basic-Leucine Zipper Transcription Factors; Calcimycin; Cell Line, Tumor; Cyclic AMP Response Element-Binding Protein; Endoplasmic Reticulum Stress; HSC70 Heat-Shock Proteins; Humans; Male; Nuclear Proteins; Prostatic Neoplasms; Protein Binding; Protein Transport; Proteins; Subcellular Fractions; Up-Regulation

Although inactivation of the androgen receptor (AR) by androgen-ablation or anti-androgen treatment has been frontline therapy for disseminated prostate cancer for over 60 years, it is not curative because castration-resistant prostate cancer cells retain AR activity. Therefore, curative strategy should include targeted elimination of AR protein. Since AR binds to calmodulin (CaM), and since CaM-binding proteins are targets of calpain (Cpn)-mediated proteolysis, we studied the role of CaM and Cpn in AR breakdown in prostate cancer cells. Whereas the treatment of prostate cancer cells individually with anti-CaM drug or calcimycin, which increases intracellular Ca(++) and activates Cpn, led to minimal AR breakdown, combined treatment led to a precipitous decrease in AR protein levels. This decrease in AR protein occurred without noticeable changes in AR mRNA levels, suggesting an increase in AR protein turnover rather than inhibition of AR mRNA expression. Thus, CaM inactivation seems to sensitize AR to Cpn-mediated breakdown in prostate cancer cells. Consistent with this possibility, purified recombinant human AR (rhAR) underwent proteolysis in the presence of purified Cpn, and the addition of purified CaM to the incubation blocked rhAR proteolysis. Together, these observations demonstrate that AR is a Cpn target and AR-bound CaM plays an important role in protecting AR from Cpn-mediated breakdown in prostate cancer cells. These observations raise an intriguing possibility that anti-CaM drugs in combination with Cpn-activating agents may offer a curative strategy for the treatment of prostate cancer, which relies on AR for growth and survival.

Topics: Antineoplastic Agents; Calcimycin; Calcium; Calcium-Calmodulin-Dependent Protein Kinases; Calmodulin; Calpain; Cell Line, Tumor; Dose-Response Relationship, Drug; Humans; Ionophores; Male; Prostatic Neoplasms; Protein Kinase Inhibitors; Protein Processing, Post-Translational; Receptors, Androgen; Recombinant Proteins; RNA Interference; RNA, Messenger; Sulfonamides; Time Factors; Transfection; Trifluoperazine

The androgen-regulated protein androgen-induced bZIP (AIbZIP) is a bZIP transcription factor that localizes to the membrane of the endoplasmic reticulum (ER). The physiological role of AIbZIP is unknown, but other ER-bound transcription factors such as ATF6 and SREBPs play a crucial role in the regulation of protein processing and lipid synthesis, respectively. In response to alterations in the intracellular milieu, ATF6 and SREBPs are processed to their transcriptionally active forms by regulated intramembrane proteolysis. In humans, AIbZIP mRNA is expressed in several organs including the pancreas, liver, and gonads, but it is especially abundant in prostate epithelial cells. We therefore used LNCaP human prostate cancer cells as a model to identify stimuli that lead to AIbZIP activation and define the transcriptional targets of AIbZIP. In LNCaP cells, AIbZIP was processed to its transcriptionally active form by drugs that deplete ER calcium stores (i.e., A23187 and caffeine), but it was unaffected by an inhibitor of protein glycosylation (tunicamycin). To identify AIbZIP-regulated genes, we generated LNCaP cell lines that conditionally express the processed form of AIbZIP and used Affymetrix microarrays to screen for AIbZIP-regulated transcripts. Selected genes (n = 48) were validated by Northern blot hybridization. The results reveal that the downstream targets of AIbZIP include genes that are implicated in protein processing (e.g., BAG3, DNAJC12, KDELR3). Strikingly, a large number of AIbZIP-regulated transcripts encode proteins that are involved in transcriptional regulation, small molecule transport, signal transduction, and metabolism. These results suggest that AIbZIP plays a novel role in cell homeostasis.

Topics: Adenocarcinoma; Amino Acid Sequence; Basic-Leucine Zipper Transcription Factors; Brefeldin A; Caffeine; Calcimycin; Calcium Signaling; Cell Line, Tumor; Cyclic AMP Response Element-Binding Protein; Endoplasmic Reticulum; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Golgi Apparatus; Homeostasis; Humans; Male; Molecular Sequence Data; Neoplasm Proteins; Nuclear Proteins; Prostatic Neoplasms; Protein Processing, Post-Translational; Recombinant Fusion Proteins; RNA, Small Interfering; Thapsigargin; Transcription, Genetic; Tunicamycin

Chemotherapy of prostate cancer targets androgen receptor (AR) by androgen ablation or antiandrogens, but unfortunately, it is not curative. Our attack on prostate cancer envisions the proteolytic elimination of AR, which requires a fuller understanding of AR turnover. We showed previously that calmodulin (CaM) binds to AR with important consequences for AR stability and function. To examine the involvement of Ca(2+)/CaM in the proteolytic breakdown of AR, we analyzed LNCaP cell extracts that bind to a CaM affinity column for the presence of low molecular weight forms of AR (intact AR size, approximately 114 kDa). Using an antibody directed against the NH(2)-terminal domain (ATD) of AR on Western blots, we identified approximately 76-kDa, approximately 50-kDa, and 34/31-kDa polypeptides in eluates of CaM affinity columns, suggesting the presence of CaM-binding sites within the 31/34-kDa ATD of AR. Under cell-free conditions in the presence of phenylmethylsulfonyl fluoride, AR underwent Ca(2+)-dependent degradation. AR degradation was inhibited by N-acetyl-leu-leu-norleu, an inhibitor of thiol proteases, suggesting the involvement of calpain. In intact cells, AR breakdown was accelerated by raising intracellular Ca(2+) using calcimycin, and increased AR breakdown was reversed with the cell-permeable Ca(2+) chelator bis-(O-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid tetra-(acetoxymethyl)-ester. In CaM affinity chromatography studies, the Ca(2+)-dependent protease calpain was bound to and eluted from the CaM-agarose column along with AR. Caspase-3, which plays a role in AR turnover under stress conditions, did not bind to the CaM column and was present in the proenzyme form. Similarly, AR immunoprecipitates prepared from whole-cell extracts of exponentially growing LNCaP cells contained both calpain and calpastatin. Nuclear levels of calpain and calpastatin (its endogenous inhibitor) changed in a reciprocal fashion as synchronized LNCaP cells progressed from G(1) to S phase. These reciprocal changes correlated with changes in AR level, which increased in late G(1) phase and decreased as S phase progressed. Taken together, these observations suggest potential involvement of AR-bound CaM in calcium-controlled, calpain-mediated breakdown of AR in prostate cancer cells.

Topics: Androgen Antagonists; Calcimycin; Calcium; Calmodulin; Calpain; Cell Line, Tumor; Cell Nucleus; Chelating Agents; Chromatography, Affinity; Cytoplasm; Humans; Ionophores; Isoleucine; Male; Prostatic Neoplasms; Receptors, Androgen; Signal Transduction; Ultraviolet Rays

Calreticulin has been identified previously as one of the androgen-response genes in the prostate. The role of calreticulin in androgen action was studied using androgen-sensitive LNCaP and androgen-insensitive PC-3 human prostate cancer cell lines. Calreticulin appears to be a primary androgen-response gene in cultured LNCaP cells because androgen induction of calreticulin mRNA resists protein synthesis inhibition. Calreticulin is a high capacity intracellular Ca2+ binding protein, suggesting that calreticulin expression is likely to be associated with the intracellular Ca2+ buffering capacity that could regulate the sensitivity to cytotoxic intracellular Ca2+ overload. As expected, androgen protects androgen-sensitive LNCaP but not androgen-insensitive PC-3 cells from cytotoxic intracellular Ca2+ overload induced by Ca2+ ionophore A23187. To provide evidence for the role of calreticulin in reducing cytotoxic effect of Ca2+ influx in prostatic cells, we have shown that calreticulin antisense oligonucleotide down-regulates calreticulin protein level and significantly increases the sensitivity to A23187-induced apoptosis in both LNCaP and PC-3 cells. Furthermore, calreticulin antisense oligonucleotide reverses the androgen-induced resistance to A23187 in LNCaP cells. The above observations collectively suggest that calreticulin mediates androgen regulation of the sensitivity to Ca2+ ionophore-induced apoptosis in LNCaP cells.

Topics: Androgens; Apoptosis; Calcimycin; Calcium; Calcium-Binding Proteins; Calreticulin; Down-Regulation; Endoplasmic Reticulum; Humans; Ionophores; Male; Oligonucleotides, Antisense; Prostatic Neoplasms; Ribonucleoproteins; Tumor Cells, Cultured

In androgen-responsive LNCaP human prostatic cancer cells, human TR3 orphan receptor, a member of the steroid receptor superfamily, can be rapidly induced by androgen. In contrast, ablation of androgen by castration can induce the expression of the TR3 orphan receptor gene in rat ventral prostate that has undergone apoptosis. This phenomenon prompted us to further analyze the potential role of human TR3 orphan receptor in prostate cancer cells in which apoptosis had been induced. Northern blot analysis shows that human TR3 orphan receptor expression can be induced rapidly after treatment of LNCaP and PC-3 prostate cancer cells with calcium ionophore or etoposide. Our data further demonstrate that a much higher concentration of etoposide was needed to kill the same number of cells in LNCaP and PC-3 cells transfected stably with antisense TR3 orphan receptor compared with that in control vector transfectants. Together, our data suggest that the human TR3 orphan receptor may play an important role in modulating drug-induced prostate apoptosis.

Topics: Androgens; Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Blotting, Northern; Calcimycin; Cell Survival; DNA-Binding Proteins; Etoposide; Humans; Male; Metribolone; Nuclear Receptor Subfamily 4, Group A, Member 1; Orchiectomy; Prostatic Neoplasms; Rats; Rats, Sprague-Dawley; Receptors, Cytoplasmic and Nuclear; Receptors, Steroid; Receptors, Thyroid Hormone; Transcription Factors; Transfection

Elevation of intracellular calcium levels in the presence of normal androgen levels has been implicated in apoptotic prostate cell death. Since the androgen receptor (AR) plays a critical role in the regulation of growth and differentiation of the prostate, it was of interest to determine whether Ca2+ would affect the expression of androgen receptor messenger RNA (mRNA) and protein, thus affecting the ability of androgens to control prostate function. AR-positive human prostate cancer cells, LNCaP, were incubated with either the calcium ionophore A23187 or the intracellular endoplasmic reticulum Ca(2+)-ATPase inhibitor thapsigargin. Subsequently, AR mRNA and protein levels were assessed by Northern and Western blot analysis. Both A23187 and thapsigargin were found to down-regulate steady state AR mRNA levels in a time- and dose-dependent manner. AR mRNA began to decrease after 6-8 h of incubation with 10(-6) M A23187 or 10(-7) M thapsigargin, reaching a nadir at 16 and 10 h of incubation, respectively. In contrast, control mRNA (glyceraldehyde 3-phosphate dehydrogenase) did not change significantly during the treatments with either A23187 or thapsigargin. AR protein levels were found to be decreased after 12 h of incubation with either 10(-6) M A23187 or 10(-7) M thapsigargin. The decrease in AR mRNA and protein seemed to precede apoptosis, since neither A23187 (24 h) nor thapsigargin (30 h) was found to alter cell morphology within the treatment time. Cycloheximide and actinomycin D were unable to change the calcium-mediated decrease in AR mRNA, ruling out the necessity for de novo protein synthesis or a change in mRNA stability. Moreover, the decrease in AR mRNA induced by calcium does not seem to involve protein kinase C- or calmodulin-dependent pathways, since inhibitors of these cellular components had no effect. Nuclear run-on assays demonstrated little or no effects of either A23187 or thapsigargin treatment on AR gene transcription (8 h and 10 h). In conclusion, these studies show that intracellular calcium seems to be a potent regulator of AR gene expression in LNCaP cells.

Topics: Alkaloids; Apoptosis; Blotting, Northern; Blotting, Western; Calcimycin; Calcium; Calcium-Transporting ATPases; Cell Line; Cell Nucleus; Dactinomycin; Dose-Response Relationship, Drug; Gene Expression; Humans; Kinetics; Male; Prostatic Neoplasms; Protein Kinase C; Receptors, Androgen; RNA, Messenger; RNA, Neoplasm; Staurosporine; Terpenes; Thapsigargin; Tumor Cells, Cultured

Prostate-specific antigen (PSA) is the most sensitive marker available for monitoring the progression of prostate cancer and response to therapy. In a previous study, we demonstrated tissue-specific expression of PSA glycoprotein and mRNA and its regulation through the androgen receptor. In this study, we examine the effects of protein kinase A (PKA) and protein kinase C (PKC) on the androgen regulation of PSA in a human adenocarcinoma cell line, LNCaP. Northern blot analysis demonstrated that forskolin, an activator of PKA, had no effect on the androgen regulation of PSA. However, the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA), a direct activator of PKC, showed a time- and dose-dependent repression of the androgen regulation of PSA glycoprotein and mRNA. The biologically inactive phorbol ester, 4 alpha-phorbol-12,13-didecanoate, had no effect. Staurosporine, a PKC inhibitor, blocked the TPA-mediated repression of the androgenic stimulation of PSA glycoprotein. In addition, the calcium ionophore, A23187, was able to simulate the actions of TPA, presumably through activation of PKC via calcium mobilization. In summary, the androgenic regulation of PSA protein and mRNA is repressed by tumor-promoting phorbol esters through the PKC pathway. This indicates that the effects of TPA may be secondary to repressed gene transcription or altered mRNA stability. In addition, this study emphasizes that the androgenic regulation of PSA is complex and may involve other extracellular transduction signals.

Topics: Alkaloids; Antigens, Neoplasm; Biomarkers, Tumor; Calcimycin; Colforsin; Dose-Response Relationship, Drug; Down-Regulation; Humans; Male; Nandrolone; Phorbols; Prostate-Specific Antigen; Prostatic Neoplasms; RNA, Messenger; Staurosporine; Testosterone Congeners; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

When human PC-3 cells derived from a metastatic prostatic adenocarcinoma were incubated for 15 min to 4 h with the in vitro inhibitor of eicosanoid biosynthesis, eicosatetraynoic acid (ETYA) at 10-80 microM, DNA synthesis was suppressed. No reduction in cellular viability occurred, as judged by exclusion of trypan blue or unaltered release of 51Cr-labeled proteins, and the inhibition was partially reversible. Indomethacin (to 12.5 micrograms/ml) did not inhibit DNA synthesis or alter the suppression of DNA synthesis by ETYA, suggesting a role for a lipoxygenase product in this effect. Addition of leukotriene B4 (LTB4) at 10(-8) M did not reverse the inhibition of DNA synthesis produced by ETYA, nor did arachidonic acid (10(-5) - 10(-9) M) incubated with control cells mimic the effect of that agent. 3H-arachidonic acid incubated with PC-3 cells was rapidly incorporated into phospholipids and this labeling was differentially inhibited by ETYA. Positive modulation of PC-3 cellular DNA synthesis by lipoxygenase products and inhibition of their synthesis by ETYA is one attractive hypothesis with which to account for these results. Other consequences of producing a selective deficiency of arachidonic acid in cellular membrane phospholipids and even the probable substitution of ETYA for arachidonic acid could also contribute to the inhibition of DNA synthesis by ETYA.

Topics: 5,8,11,14-Eicosatetraynoic Acid; Adenocarcinoma; Arachidonic Acid; Arachidonic Acids; Calcimycin; Cyclooxygenase Inhibitors; DNA; Fatty Acids, Unsaturated; Humans; Indomethacin; Leukotriene B4; Lipoxygenase Inhibitors; Male; Phospholipids; Prostatic Neoplasms; Tumor Cells, Cultured