Type 1 vasoactive intestinal peptide receptor expression in PC3/AR cells is evidence of prostate epithelial differentiation.

BACKGROUND: Type 1 vasoactive intestinal peptide receptor (VIP1R) is expressed in many secretory epithelial cells. We investigated VIP1R expression as a marker of prostate secretory epithelial differentiation in normal and malignant prostate tissues and in PC-3 human prostate cancer cells either lacking or expressing a functional androgen receptor. METHODS: VIP1R mRNA in rat prostate was assessed by in situ hybridization. VIP1R mRNA in human prostate tissue was identified by Northern blot hybridization. VIP1R mRNA expression in human prostate cancer cell lines in the presence or absence of androgen was determined by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). Human prostate cell lines were treated with VIP, and changes in intracellular cAMP were measured by radioimmunoassay. RESULTS: VIP1R mRNA was expressed only in epithelial cells in normal rat prostate. VIP1R mRNA was present in both normal and malignant human prostate. Well-differentiated LNCaP cells expressed functional VIP receptors, while poorly differentiated PC-3 cells did not. PC-3 cells stably expressing the androgen receptor (PC3/AR) did express functional VIP receptors, but VIP1R mRNA levels were not androgen-regulated. CONCLUSIONS: VIP1R expression indicates epithelial differentiation in normal and malignant prostate. PC3/AR cells and LNCaP cells, both of which express VIP1R and prostate-specific antigen (PSA), are the only prostate cancer cell lines known to express these two markers of prostate epithelial differentiation.

Expression of rat thyrotropin-releasing hormone (TRH) gene in TRH-producing tissues of transgenic mice requires sequences located in exon 1.

TRH, an amidated tripeptide secreted by certain hypothalamic neurons, is a principal regulator of TSH secretion and thyroid hormone release. TRH is also produced by other neurons in the central nervous system, where it appears to function as a neuromodulator or neurotransmitter, and by certain endocrine cells, where it may act as an autocrine or paracrine factor. The genomic organization of the rat TRH (rTRH) gene is well understood; however, the domains of the rTRH gene that regulate expression are less well characterized. We observed that the region between -47 and +6 of the rTRH gene (relative to the transcription start site at +1) was active in CA-77 cells, a medullary thyroid carcinoma cell line model of TRH production, but was not active in transgenic mice. Inclusion of most of exon 1 (84 out of 103 bp; -47 to +84) increased promoter activity in CA-77 cells and was active in transgenic mice, principally in tissues that normally express the TRH gene. Further lengthening of the 5' end to -243, -547, or -776 retained this expression in TRH-producing tissues in transgenic mice, while further increasing activity in CA-77 cells. These results suggest that cis element(s) located within exon 1 are necessary for the expression of the rTRH gene in vivo.

Combined octreotide and insulin therapy in acromegaly.

OBJECTIVE: To describe an unusual treatment strategy for certain patients with both acromegaly and insulin-requiring diabetes. METHODS: We present a case history of a man with a pituitary adenoma and diabetes mellitus and chronicle his response to various treatment modalities. RESULTS: Acromegaly, a disease state with excess growth hormone (GH) and insulin-like growth factor-I, is associated with carbohydrate intolerance. Octreotide, a somatostatin analogue, is used in the treatment of acromegaly to lower GH levels. Despite effective lowering of GH levels, certain patients with acromegaly have persistent or even worsening carbohydrate intolerance and may require insulin therapy. Such a regimen would necessitate five or more injections per day. We describe a 51-year-old man who was diagnosed with a GH-producing pituitary adenoma in 1987. Despite transsphenoidal resection, frontal craniotomy, and radiation therapy, symptoms and increased levels of GH persisted. The patient was diagnosed with diabetes mellitus in August 1989 and treated with glipizide. Because of persistently increased GH levels, he was treated with octreotide. His glycohemoglobin level increased to 21% despite use of maximal doses of glipizide. The patient refused insulin therapy because of his objection to numerous daily injections. Despite adjustments in diet and exercise, glycemic control remained poor. As a trial, we thus attempted combining octreotide and regular insulin in the same syringe and administering the medications in a single subcutaneous injection. No precipitate formation was evident, and he had no adverse effects. Glucose control improved, and the glycohemoglobin level was lower but still elevated. GH levels remained at less than 5 ng/mL. His symptoms of acromegaly were unchanged, but his overall attitude and energy level improved. CONCLUSION: The efficacy of the individual components of this treatment was demonstrated in the combination of insulin and octreotide. The combined administration of insulin and octreotide has limited application in patients with acromegaly and insulin-requiring diabetes mellitus.

Vasoactive intestinal peptide stimulates prostate-specific antigen secretion by LNCaP prostate cancer cells.

The secretion of prostate-specific antigen (PSA) by prostate cancer provides an important tool in the diagnosis and management of this disorder. While androgens are required for PSA synthesis, the neuroendocrine regulation of PSA secretion is less understood. Human prostate is extensively innervated with vasoactive intestinal peptide (VIP)-containing neurons, while both normal and malignant prostate cells contain VIP receptors. Therefore, we investigated the effects of VIP on PSA secretion by LNCaP prostate cancer cells. We found that 1-4-h VIP treatment produces 60-100% increases in PSA secretion by LNCaP cells. Increases in PSA secretion were seen with as little as 10(-10) M VIP with maximum effects at 10(-7) M. The predominant acute effect of VIP was to increase the secretion of stored PSA without increasing PSA mRNA. VIP's effect on PSA secretion involved the production of intracellular cAMP since all doses of VIP which increased secretion were associated with increased cyclic AMP and since dibutyryl-cyclic AMP treatment increased secretion similarly to VIP. These results suggest that VIP regulates PSA secretion by prostate cancer cells and also suggest a role for VIP to regulate PSA secretion by normal prostate epithelial cells.

Androgenic up-regulation of androgen receptor cDNA expression in androgen-independent prostate cancer cells.

The expression of the androgen receptor (AR) gene is regulated by androgens. Although androgens down-regulate AR mRNA in most cell lines and tissues, including the prostate, up-regulation occurs in some tissues. Androgen-mediated reduction in AR mRNA is reproduced in COS1 cells and in the androgen-sensitive human prostate cancer cell line LNCaP when each expresses the AR cDNA. We have previously established that the AR cDNA contains the requisite sequences for this down-regulation. Here we shown that androgen promoted up-regulation of AR mRNA in two androgen-independent human prostate cancer cell lines, PC3 and DU145, when each was transfected with a human AR cDNA. This effect was due to the AR cDNA and not to the heterologous promoter driving AR expression. In addition to up-regulation of AR mRNA, androgen induced comparable increases in AR protein levels in PC3 cells stably expressing an AR cDNA (PC3/AR). Up-regulation of AR in PC3/AR cells was accompanied by failure of these cells to undergo desensitization or inactivation of AR following prolonged (96 h) androgen administration, whereas the same conditions resulted in desensitization of AR transactivation in LNCaP cells and in CVl cells that stably express the AR cDNA. Androgen treatment of PC3/AR cells resulted in induction of an androgen-regulated reporter gene (MMTV-CAT) as well as the native prostate-specific antigen gene, which is silent in untransfected PC3 but is androgen up-regulated in LNCaP and in the prostate. These results suggest that ectopic expression of AR in androgen-independent prostate cancer cell lines establishes both typical and atypical androgenic responses in a target gene-specific manner. Androgenic up-regulation of AR cDNA expression may be due to distinct signaling mechanisms that influence androgen action in androgen-independent prostate cancer cells.

Expression and processing of peptidylglycine alpha-amidating monooxygenase messenger RNA in rat prostate.

The prostate gland contains neuroendocrine cells and amidated neuroendocrine peptides whose presence has been related to aggressive forms of prostate cancer. The enzyme peptidylglycine alpha-amidating monooxygenase (PAM) is critical to the bio-synthesis of amidated peptides and is commonly present in neuroendocrine cells. By northern blot hybridization analysis, PAM mRNA was detected in similar quantities in dorsolateral and ventral prostates of 3-month-old and 13-month-old rats. Multiple forms of PAM mRNA were present whose size distribution was more similar to PAM mRNAs found in pituitary than atrium. Alternative splice sites in PAM mRNA were investigated by reverse-transcriptase polymerase chain reaction. Similar alternatively spliced forms of PAM mRNA were found in both prostate lobes, pituitary, and atrium. However, the distribution of forms in the prostate most resembled that of pituitary. Multiple forms of PAM mRNA are present in prostate and may serve as markers of neuroendocrine differentiation.

Neuroendocrine peptides stimulate adenyl cyclase in normal and malignant prostate cells.

Elevations of intracellular cAMP in human prostate cancer cells have been shown to increase invasiveness and to promote neuronal differentiation. Since neuroendocrine peptides capable of activating adenyl cyclase are present in prostatic nerves and epithelial neuroendocrine cells, we investigated normal and malignant human prostate cells for changes in intracellular cAMP in response to the prostatic peptides vasoactive intestinal peptide (VIP), calcitonin (CT), and calcitonin gene-related peptide (CGRP). Normal prostate epithelial cells and LNCaP prostate cancer cells exhibited, respectively, 6- and 30-fold increases in intracellular cAMP in response to VIP. ALVA-31 and PPC-1 prostate cancer cells demonstrated 20- to 200-fold increases in cAMP in response to CGRP, while normal epithelial cells and LNCaP cells exhibited smaller (2- to 6-fold) responses. Only DU-145 cells increased cAMP substantially in response to CT. VIP receptor mRNA was identified by Northern blot analysis only in those cells that responded to VIP. CT receptor mRNA was identified only in DU-145 cells by polymerase chain reaction and Southern blot analysis. These results suggest that VIP and possibly CGRP receptors are likely to be present in both normal and malignant prostate cells. VIP or CGRP may regulate secretion of proteases by normal or prostate cancer cells and may influence epithelial cell differentiation.

Neuroendocrine peptides in the prostate.

Circulating androgens are required for normal growth and maintenance of function of the prostate. However, the prostate also contains neuroendocrine peptides, found either in nerve terminals or in prostatic neuroendocrine cells, which are likely to regulate prostate growth or function. The neuronal peptides are likely to participate in the regulation of the synthesis and secretion of prostatic secretory products. While the function of the neuroendocrine cells is undefined, there is evidence for growth-regulating effects of several neuroendocrine cell peptides. Since neuroendocrine differentiation has been correlated with tumor grade and poor prognosis in prostate cancer, the peptide products of the neuroendocrine cells may influence cancer cell replication as well. Recent evidence in other tissues suggests that peptide hormone receptor second-messenger systems may interact with steroid receptors to modulate their actions. These findings raise the possibility that prostatic neuroendocrine peptides may modulate the response of prostate to androgens.

Identification of the human seminal TRH-like peptide pGlu-Phe-Pro-NH2 in normal human prostate.

Human and rat prostate contain thyrotropin-releasing hormone immunoreactivity (iTRH) including TRH and an uncharged TRH-like peptide. Recently the uncharged TRH-like peptide pGlu-Phe-Pro-NH2 was purified from human semen. To determine whether this peptide was of prostatic origin, human and rat prostate extracts were analyzed by ion-exchange chromatography and reversed-phase HPLC. The predominant uncharged iTRH comigrated exactly with synthetic pGlu-Phe-Pro-NH2 on HPLC and had identical affinity to pGlu-Phe-Pro-NH2 in a TRH radioimmunoassay. We conclude that prostate is a source of this peptide in humans and rats. This amidated TRH-like peptide may play a role in human reproductive physiology.

Expression of prostatic TRH-like peptides differs between species and between malignant and nonmalignant tissues.

Thyrotropin-releasing hormone-immunoreactive peptides (iTRH) were analyzed in normal rat and rabbit prostates and in human benign prostatic hyperplasia (BPH) and prostate cancer. Peptides were extracted from tissues, fractionated by anion and cation exchange chromatography, and analyzed by TRH radioimmunoassay. pGlu-Glu-Pro-NH2 predominated in rabbit, but accounted for only 10-15% of iTRH in rat and human BPH. Uncharged peptides predominated in rat and human prostate. Authentic TRH (pGlu-His-Pro-NH2) is not present in rabbit prostate, but may account for up to 25% of iTRH in rat and human prostate. iTRH was virtually absent in prostate cancer. These results demonstrate considerable heterogeneity in the expression of TRH-like peptides in the prostates of various animal species, and suggest decreased expression of these peptides in prostate cancer.

Hypercalcemia in association with acute renal failure and rhabdomyolysis. Case report and literature review.

The syndrome of hypercalcemia during the course of acute renal failure (usually associated with rhabdomyolysis) occurs most commonly in young men with very severe renal failure. Although fewer than 90 such patients have been reported, the prevalence of hypercalcemia in patients with rhabdomyolysis-associated renal failure averages 30%. Hypercalcemia occurs most commonly in the diuretic phase and resolves spontaneously. The mean duration of hypercalcemia is 14 days. The pathogenesis of this syndrome has not been clearly defined. In the rare instances where it has been measured, intact PTH is suppressed. In contrast, both elevated and suppressed values of plasma 1,25-dihydroxyvitamin D have been reported. The release of calcium from ectopic calcification in damaged muscle tissue provides a potential explanation for this syndrome. Therapy for the hypercalcemia should generally be conservative given its self-limited nature.

The thyrotrophin-releasing hormone (TRH)-like peptides in rat prostate are not formed by expression of the TRH gene but are suppressed by thyroid hormone.

Thyrotrophin-releasing hormone (TRH)-immunoreactive peptides were extracted from rat prostate and divided into two groups by mini-column cation exchange chromatography. The amounts of the peptides in each group were determined by radioimmunoassay with a TRH antiserum. The unretained peptides which lacked a basic group and the retained peptides which possessed a basic group were further purified by high-performance liquid chromatography. The unretained fraction was found to contain a series of TRH-immunoreactive peptides, one of which corresponded chromatographically to synthetic pGlu-Glu-Pro amide and another to pGlu-Phe-Pro amide. None of the TRH-immunoreactive peptides in either fraction exhibited the chromatographic behaviour of TRH. Additional evidence for the absence of TRH gene expression in the prostate was obtained by Northern blot analysis and by application of polymerase chain reaction amplification, which failed to reveal TRH mRNA. Furthermore the preproTRH-derived peptide, preproTRH(53-74), could not be detected by radioimmunoassay. The influence of thyroid status was investigated on the levels of the TRH-like peptides in the prostate. Adult rats were treated chronically with thyroxine (T4) or propylthiouracil (PTU) and the concentrations of the TRH-immunoreactive peptides were determined by chromatography and radioimmunoassay. Treatment with T4 caused the levels of the neutral and acidic TRH-like peptides to fall to approximately one-third of the levels in the controls. No significant difference from the controls was seen in the concentrations of the peptides in the prostates of rats rendered hypothyroid by administration of PTU. The results demonstrate that rat prostate contains TRH-immunoreactive peptides which are not derived from the TRH gene.(ABSTRACT TRUNCATED AT 250 WORDS)

Tetany induced on separate occasions by administration of potassium and magnesium in a patient with hungry-bone syndrome.

The case is described of a 68-year-old man whose therapy induced tetany during each of two consecutive hospital admissions. On each occasion the patient had marked hypocalcemia and hypomagnesemia, presumably as a result of the hungry-bone syndrome associated with diffuse prostatic osteoblastic metastases. During the February 1991 admission, marked hypokalemia was the principal initial concern. It seems likely that the tetany associated with the administration of KCl, without sufficient calcium, resulted from attenuation of the protection against hypocalcemia-enhanced neuromuscular excitability conferred by coexisting hypokalemia. The admission in March 1991 was prompted by the finding (without symptoms) of very low levels of both serum Mg and serum Ca. Tetany occurred during the infusion of MgSO4, without calcium. An acute decrement in plasma ionized Ca resulting from complexing of Ca with sulfate ions together with augmented urinary excretion of Ca were likely pathogenic factors.

Thyrotropin-releasing hormone gene expression in normal thyroid parafollicular cells.

The rat TRH gene encodes a 255-amino-acid precursor polypeptide, preproTRH, containing five copies of TRH and seven non-TRH peptides. Expression of this gene is well documented in the central nervous system, particularly in the hypothalamus. Thyroids also contain TRH immunoreactivity, but it is unknown whether this immunoreactivity results from expression of the TRH gene or from other genes encoding TRH-like products. Since the CA77 neoplastic parafollicular cell line expresses the TRH gene, we investigated whether TRH gene expression also occurs in normal thyroid parafollicular cells. Northern analysis of total thyroid RNA with a preproTRH-specific RNA probe identified a single hybridizing band the same size as authentic TRH mRNA found in hypothalamus and CA77 cells. Gel filtration analysis of thyroid extracts identified the same 7-kilodalton and 3-kilodalton species of immunoreactive preproTRH53-74 previously identified in hypothalamus and CA77 cells. Immunoreactive preproTRH115-151, not previously identified, was found in all three tissues. Part of this immunoreactivity comigrated with the synthetic preproTRH115-151 standard on gel filtration and reversed-phase HPLC. PreproTRH53-74 was localized to thyroid parafollicular cells by immunostaining. These findings demonstrate authentic TRH gene expression by normal rat thyroid parafollicular cells and establish the CA77 cell line as the only model system of a normal TRH-producing tissue. In addition to expanding the range of neuroendocrine peptides known to be produced by parafollicular cells, these results also suggest a potential paracrine regulatory role for TRH gene products within the thyroid.

Dexamethasone stimulates thyrotropin-releasing hormone production in a C cell line.

The hypothalamic peptide hormone TRH is also found in other tissues, including the thyroid. While TRH may be regulated by T3 in the hypothalamus, other regulators of TRH have not been identified and the regulation of TRH in nonhypothalamic tissues is unknown. We recently demonstrated the biosynthesis of TRH in the CA77 neoplastic thyroidal C cell line. We studied the regulation of TRH by dexamethasone in this cell line because glucocorticoids have been postulated to inhibit TSH secretion by decreasing TRH in the hypothalamus. Furthermore, TRH in the thyroid inhibits thyroid hormone release. Thus by regulating thyroidal TRH, glucocorticoids could also directly affect thyroid hormone secretion. Treatment of CA77 cells for 4 days with dexamethasone produced dose-dependent increases in both TRH mRNA and cellular and secreted TRH. Increases in TRH mRNA and peptide levels could be seen with 10(-9) M dexamethasone. A 4.8-fold increase in TRH mRNA and a 4-fold increase in secreted peptide were seen with 10(-7) M dexamethasone. Dexamethasone treatment did not increase beta-actin mRNA levels or cell growth. These results suggest that glucocorticoids may be physiological regulators of TRH in normal C cells. In addition to their inhibitory effects on TSH, glucocorticoids may decrease thyroid hormone levels by increasing thyroidal TRH. Since the glucocorticoid effects on C cell TRH are the converse of what is expected for hypothalamic TRH, glucocorticoid effects in these two tissues may be mediated by different regulators.

Glycosylation reduces the bioactivity of calcitonin.

The biological significance of peptide hormone glycosylation is uncertain. To examine the effect of Asn-linked glycosylation on calcitonin's bioactivity we purified glycosylated calcitonin from a transplantable rat medullary thyroid carcinoma. Glycosylated calcitonin constituted 2.3% of the total extracted immunoreactive calcitonin. The structure of this peptide differed from nonglycosylated calcitonin only by the oligosaccharide modification of asparagine 3. Affinity of glycosylated calcitonin for lentil lectin indicated that the oligosaccharide was a complex processed form. In a standard in vivo bioassay glycosylated calcitonin had a markedly reduced hypocalcemic activity compared to nonglycosylated calcitonin, an effect most likely due to the presence of the oligosaccharide.

Biosynthesis of calcitonin gene-related peptide and calcitonin by a human medullary thyroid carcinoma cell line.

cDNA analyses predict that the human calcitonin gene encodes the two precursor proteins of calcitonin and calcitonin gene-related peptide (CGRP). The mRNAs for the putative precursors of these peptides are derived from alternative processing of the primary transcript of this gene. The 75 amino-terminal residues of each preprohormone are predicted to be identical. We have developed the TT human medullary thyroid carcinoma cell line as a model to study human calcitonin gene expression. Mature calcitonin and CGRP are major secretory products of this cell line. Extracts of TT cells radiolabeled with amino acids for 1 h contained only one peak of either immunoprecipitable calcitonin or CGRP on reverse-phase high-pressure liquid chromatography. Each of these species had an Mr = 12,800 as estimated by gel filtration chromatography. The results of partial amino-terminal microsequencing of these two precursors were identical. Residues 2, 11, and 14 of each precursor were proline, and residues 7, 17, 24, 25, 26, and 29 were leucine. The unique alignment of the positions of these amino acids with the cDNA-predicted sequence for the common region of preprocalcitonin and preproCGRP suggests that the site of signal sequence cleavage occurs after residue 25 of both preprophormones. These studies represent the first identification of procalcitonin (116 amino acids) from a human cell line and of proCGRP (103 amino acids) from any tissue.

Squamous carcinoma model of humoral hypercalcemia of malignancy.

Squamous carcinomas are the most common cause of humoral hypercalcemia of malignancy (HHM) in humans. To develop an animal model of this syndrome, CD-1 female mice were painted with dimethylbenzanthracene, which produced cutaneous squamous carcinomas in the majority of those painted. Greater than 90% of tumor-bearing mice developed a syndrome of hypercalcemia, hypophosphatemia, hypercalciuria, elevated plasma 1,25-dihydroxyvitamin D, normal immunoreactive PTH, elevated urinary cAMP, and accelerated bone resorption compared to control mice. Tumor excision reversed the hypercalcemia and hypophosphatemia, and autopsies revealed no evidence of skeletal or other metastases. Dietary calcium restriction did not affect the hypercalcemia in tumor-bearing mice. Extracts of tumor tissue contained potent bioactivity paralleling that of bovine (b) PTH in a PTH-sensitive canine renal cortical adenylate cyclase assay. The activity was trypsin sensitive and partially inhibitable by Nle, Tyr bPTH amide. The activity coeluted with chymotrypsinogen (mol wt, 25,700) on Sephacryl S-200 chromatography, well ahead of bPTH. This is the first description of an animal squamous carcinoma that produces HHM. With the exception of elevated plasma 1,25-dihydroxyvitamin D levels, the syndrome precisely mimics that seen in human HHM. The presence of a biologically active protein larger than PTH in tumor extracts, similar to that extracted from human tumors, suggests a common mode of pathogenesis. This model should be useful in further studying the pathophysiology of HHM.