Girish V. Shah

Muscarinic cholinergic receptors promote growth of human prostate cancer cells

Recent evidence suggests that muscarinic receptors induce mitogenesis in cells capable of undergoing cell proliferation. Human prostate gland is innervated by the autonomic nervous system and muscarinic receptors have been localized in the prostate gland.

Calcitonin is a prostate epithelium-derived growth stimulatory peptide

Locally secreted growth factors and neuropeptides may play an important role in sustaining the growth of hormone-independent prostate cancer. Our previous studies have shown that calcitonin-like immunoreactive peptide (CTI) is secreted by primary prostate cells in culture, and its secretion from malignant prostate cells is significantly higher than benign cells. Exogenously added calcitonin (CT) induces DNA synthesis in serum-starved prostate cancer LNCaP and PC-3M cells. Present studies extended these findings by cloning cDNAs for CT and CT receptor (CT-R) from prostate cancer cells and studying the expression of CT and CT-R mRNA in prostate cancer cell lines and primary prostate tumor specimens. The results have shown that PC-3 cells expressed CT, and not CT-R, mRNA, whereas CT-R, but not CT, mRNA was expressed by LNCaP cells. Conditioned media from PC-3 cells induced DNA synthesis of LNCaP cells, and this mitogenic response was abolished by anti-CT serum. Highly aggressive PC-3M cells co-expressed CT and CT-R mRNAs. CT also induced a twofold increase in DNA synthesis of primary prostate cells and anti-CT serum caused a 56% decline. In-situ hybridization histochemistry of archival prostate specimens has selectively localized CT and CT-R mRNA in basal epithelium of benign and low grade PC specimens, and these mRNAs were not detected in either luminal epithelium or stroma. In contrast, CT and CT-R mRNA were detected throughout the luminal epithelium of moderate and high-grade PC specimens. Most epithelial cells of low and moderately differentiated tumors expressed either CT or CT-R mRNA, suggesting that CT may serve as a paracrine factor. In contrast, CT and CT-R mRNAs were co-expressed by most tumor cells in advanced PC specimens. The cells expressing CT-R mRNA in primary tumors also co-expressed PCNA. These results, when combined with mitogenic actions of CT on primary prostate cells as well as PC cell lines, strongly support the role for CT in sustaining the growth of cancer cells.

Role of stimulatory guanine nucleotide binding protein (Gsα) in proliferation of PC‐3M prostate cancer cells

Previous studies have shown that calcitonin‐like immunoreactive substances are secreted by primary prostate cells. Furthermore, exogenously added calcitonin stimulates proliferation of androgen‐responsive LnCaP cells. To examine the possible effect of calcitonin on growth of invasive prostate cancer cells, we tested its effects on proliferation of PC‐3M cells. Calcitonin stimulated DNA synthesis of PC‐3M cells in a dose‐dependent fashion, and also stimulated adenylyl cyclase and protein kinase C activities. To further delineate the role of these signaling cascades in proliferation of PC‐3M prostate cancer cells, we selectively activated these pathways by transfecting cDNAs expressing constitutively active forms of either Gsα (Gsα‐QL) or Gqα (Gqα‐QL). cDNAs expressing wild‐type forms of G‐proteins (Gsα‐WT and Gqα‐WT) were used as vehicle controls. Gqα‐QL transfectants exhibited growth inhibition and terminal differentiation. Those expressing Gsα‐QL exhibited a dramatic increase in growth rate. Gsα‐QL transfectants displayed an almost 3‐fold increase in [3H]‐thymidine incorporation and over a 4‐fold increase in growth rate when compared with parental PC‐3M cells or those expressing wild‐type Gsα (Gsα‐WT). The growth‐promoting action of Gsα‐QL could not be mimicked by either 8‐bromo cAMP or forskolin. However, nifedipine, a calcium channel antagonist, potently and selectively inhibited DNA synthesis in Gsα‐QL transfectants. These results suggest that the growth‐promoting actions of Gsα on PC‐3M cells may be mediated by nifedipine‐sensitive proliferative events. Int. J. Cancer 91:46–54, 2001.

Calcitonin Inhibits Anterior Pituitary Cell Proliferation in the Adult Female Rats

Previous studies have shown that CT-like immunoreactive peptide(s) (pit-CT) is synthesized by the anterior pituitary (AP) gland, and exogenously added salmon(s) CT inhibits PRL release and PRL gene transcription in cultured AP cells. Anti-sCT serum, which immunoreacts with pit-CT, stimulates PRL secretion, suggesting pit-CT is a physiologically relevant PRL-inhibiting hormone. Using proliferating cell nuclear antigen (PCNA) staining and 5-bromo-2′deoxyuridine (BrdU) incorporation into newly replicated DNA, the effect of calcitonin (CT) on cellular proliferation in the rat anterior pituitary gland (AP) was examined. CT significantly attenuated PCNA-immunopositive as well as BrdU-positive AP cell populations in dispersed rat AP cells. A second series of experiments tested the effects of CT on AP cell proliferation in vivo. OVX + E2 rats were injected with 200 μg CT (iv), the rats killed at various time points, and the APs were processed for BrdU staining. CT inhibited BrdU incorporation at all time points u...

Calcitonin inhibits prolactin gene transcription in rat pituitary cells.

Our recent studies have shown that calcitonin (CT)-like immunoreactive peptide is synthesized and released from cultured rat anterior pituitary (AP) cells, and may serve as a paracrine inhibitor of PRL release. The present studies investigated effects of CT on basal and TRH-induced PRL mRNA levels in rat AP and rat pituitary tumor GH3 cells. CT attenuated steady-state PRL mRNA levels in a dose-dependent fashion in primary rat AP and GH3 cells. The kinetics of CT action suggests that 100nm CT caused a significant decline after 3 h, and the inhibition was sustained at least until the longest tested incubation period of 30 h. Results from nuclear run-on assays suggest that 100 nM CT decreased the rate of PRL gene transcription by 80% after 30 min of incubation. CT did not affect PRL mRNA levels in Ca2+-depleted GH3 cells but dramatically decreased them in Ca2+-repleted cells. Bay K 8644 induced increase in PRL mRNA levels of Ca2+-repleted GH3 cells and CT did not affect this increase. These results suggest that CT rapidly and selectively inhibits PRL gene transcription in primary AP and GH3 cells, and support a possibility that CT-induced attenuation of PRL mRNA may involve cytoplasmic Ca2+.

3′,5′ Cyclic Adenosine Monophosphate Mediates the Salmon Calcitonin-Induced Increase in Hypothalamic Tyrosine Hydroxylase Activity1

This study examined the effect of salmon calcitonin (sCT) on hypothalamic tyrosine hydroxylase (TH) activity and evaluated the cellular signaling mechanisms involved in the response. Fetal hypothalamic cells were cultured in a defined medium and treated with sCT and/or specific protein kinase inhibitors on day 14 in vitro. sCT (0.1–10 nm) increased both TH activity and cellular cAMP content in a concentration-dependent manner. sCT (10 nm) increased TH activity to 150–175% of control values and resulted in a 10-fold increase in cellular cAMP content. Both the C1a and C1b CT receptor isoforms were present in the cultures, as assessed by RT-PCR. Rp-adenosine 3′,5′-cyclic monophosphothioate (Rp-cAMPS), a cAMP antagonist, and H-8, a cyclic nucleotide kinase inhibitor, blocked the sCT-induced increase in TH activity, with complete abolition of the response observed at concentrations of 1 mm and 5 μm, respectively. sCT (10 nm) increased radiolabeled phosphate incorporation into TH protein to 169% of control valu...

Estrogen attenuates expression of calcitonin-like immunoreactivity in the anterior pituitary gland.

Estrogens increase prolactin (PRL) synthesis and release in rats and humans, whereas pituitary-derived calcitonin-like immunoreactive peptide (pit-CT) inhibits PRL gene expression and release. To test the hypothesis that estrogens stimulate lactotrophs by diminishing pit-CT expression, the present studies examined effects of ovariectomy (ovx) and estradiol (E2) administration on (1) pit-CT IR cell population; (2) pit-CT IR content and (3) release of pit-CT IR by cultured anterior pituitary (AP) cells. Ability of anti-calcitonin immunoglobulins (anti-CT IgG) to stimulate PRL release from cultured AP cells was also examined. The results suggest that ovariectomy induced a large increase in pit-CT IR cell population in the AP gland and E2-treatment dramatically reversed this increase. Similar changes were observed in pit-CT IR content of AP extracts. Cultured AP cells from ovx rats released significantly higher amounts of pit-CT IR, and anti-CT IgG induced a significant increase in basal PRL release. AP cells from E2-treated rats secreted lower amounts of pit-CT IR and this was associated with significantly higher PRL release. These results suggest that estrogens may stimulate lactotrophs, at least in part, by removing inhibitory influence of endogenous pit-CT.

Calcitonin inhibits prolactin promoter activity in rat pituitary GGH3 cells: evidence for involvement of p42/44 mitogen-activated protein kinase in calcitonin action.

Previous findings from our laboratory have shown that pituitary calcitonin-like peptide (pit-CT) is synthesized and released by gonadotrophs and inhibits prolactin (PRL) release, synthesis, and lactotroph proliferation. To investigate further the regulation of PRL gene transcription by CT, we examined the effect of CT on rat PRL (rPRL) promoter activity in rat pituitary GGH3 cells. GGH3 cells were transiently transfected with rPRL promoter-luciferase and control plasmids. Thirty-six hours later, the cells were treated with CT or other agents and their effect on luciferase activity was examined. The effect of CT and/or thyrotropin-releasing hormone (TRH) on p42/44 mitogen-activated protein kinase (MAPK) activity was also investigated. CT inhibited basal rPRL promoter activity in a dose-dependent fashion, with an approximate IC50 of 3 nM. The maximal inhibition occurred 1 h after the CT addition, and the peptide was equipotent in inhibiting −600 and −2500 rPRL promoter constructs. CT also inhibited TRH-, Bay K 8644-, and ionomycin-induced rPRL promoter activity. CT mimicked the actions of MEK inhibitors U0126 and PD 980089. However, CT could not inhibit rPRL promoter activity in GGH3 cells expressing constitutively active ERK1 or ERK2. CT markedly attenuated phospho-MAPK immunoreactivity in untreated as well as TRH-treated GGH3 cells. These results suggest that CT inhibits rPRL promoter activity by antagonizing Ca2+ and ERK1/2-mediated signaling events. They also demonstrate that CT is a potent inhibitor of early events associated with PRL gene activation and may play an important role in regulation of lactotroph function.