Isolation and sequencing of cDNAs for splice variants of growth hormone-releasing hormone receptors from human cancers (original) (raw)
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Proceedings of The National Academy of Sciences, 2005
Various attempts to detect human pituitary growth hormonereleasing hormone receptor (pGHRH-R) in neoplastic extrapituitary tissues have thus far failed. Recently, four splice variants (SVs) of GHRH-R have been described, of which SV1 has the highest structural homology to pGHRH-R and likely plays a role in tumor growth. The aim of this study was to reinvestigate whether human tumors and normal human extrapituitary tissues express the pGHRH-R and to corroborate our previous findings on its SVs. Thus, we developed a real-time PCR method for the detection of the mRNA for the pGHRH-R, its SVs, and the GHRH peptide. Using real-time PCR, Western blotting, and radioligand-binding assays, we detected the mRNA for pGHRH-R and pGHRH-R protein in various human cancer cell lines grown in nude mice and in surgical specimens of human lung cancers. The expression of mRNA for SVs of pGHRH-R and GHRH was likewise found in xenografts of human non-Hodgkin's lymphomas, pancreatic cancer, glioblastoma, smallcell lung carcinomas, and in human nonmalignant prostate, liver, lung, kidney, and pituitary. Western blots showed that these normal and malignant human tissues contain SV1 protein and immunoreactive GHRH. Our results demonstrate that some normal human tissues and tumors express mRNA and protein for the pGHRH-R and its splice variants. These findings confirm and extend the concept that GHRH and its receptors play an important role in the pathophysiology of human cancers.
Breast Cancer Research and Treatment, 2003
Antagonists of GHRH inhibit the growth of various human tumors, including prostate cancer, but the tumoral receptors mediating the antiproliferative effect of GHRH antagonists have not been clearly identified. Recently, we demonstrated that human cancer cell lines express splice variants (SVs) of receptors for GHRH, of which SV1 exhibits the greatest similarity to the pituitary GHRH receptors. In this study we investigated the expression of GHRH and SVs of GHRH receptor and the binding characteristics of the GHRH receptor isoform in 20 surgical specimens of organ-confined and locally advanced human prostatic adenocarcinomas. The mRNA expression of GHRH and SVs of GHRH receptor was investigated by RT-PCR. The affinity and density of receptors for GHRH were determined by ligand competition assays based on bind-ing of 125 I-labeled GHRH antagonist JV-1-42 to tumor membranes. Twelve of 20 tumors (60%) exhibited specific, high affinity binding for JV-1-42, with a mean dissociation constant (K d ) of 0.81 nmol/liter and a mean maximal binding capacity of 185.2 fmol/mg membrane protein. The mRNA of SV1 was detected in 13 of 20 (65%) prostate cancer specimens and was consistent with the presence of GHRH binding. RT-PCR analyses also revealed the expression of mRNA for GHRH in 13 of 15 (86%) prostatic carcinoma specimens examined. The presence of GHRH and its tumoral receptor SVs in prostate cancers suggests the possible existence of an autocrine mitogenic loop. The antitumor effects of GHRH antagonists in prostate cancer could be exerted in part by interference with this local GHRH system. (J Clin Endocrinol Metab 87: 4707-4714, 2002) Abbreviations: B max , Maximal binding capacity; hGHRH, human GHRH; PACAP, pituitary adenylate cyclase-activating polypeptide; SCLC, small cell lung cancer; SV, splice variant; VIP, vasoactive intestinal peptide.
Proceedings of the National Academy of Sciences, 2004
Antagonists of growth hormone-releasing hormone (GHRH) inhibit the growth of various human cancers by multiple mechanisms, which include direct effects on tumor cells through the splice variants (SV) of the GHRH receptor. Our findings suggest that the tumoral protein encoded by SV 1 (SV 1) is a likely functional receptor. The aim of this study was to develop a polyclonal antiserum against a polypeptide analog of segment 1-25 of the putative SV 1 receptor protein. Rabbits were immunized with [Ala-23]SV 1 (1-25)-Tyr-26-Cys-27-NH2 as a hapten, conjugated to BSA or keyhole limpet hemocyanin. The antisera thus generated were evaluated by RIA for binding to the radiolabeled hapten. The specificity and sensitivity of the antisera were studied on xenografts of RL and HT human non-Hodgkin's lymphomas. The sera raised against keyhole limpet hemocyanin-SV 1 hapten, showed binding values of 50 -75% at a 1:56,000 dilution. In Western blot analyses, the purified polyclonal antibody recognized a specific signal with a molecular mass of Ϸ40 kDa in RL and HT lymphomas. This band corresponds to the estimated molecular mass of the GHRH receptor isoform encoded by SV 1. RT-PCR and ligand binding studies also revealed the expression of SV 1 and the presence of high-affinity binding sites for GHRH on RL and HT tumors. Because the antiserum developed recognizes the tumoral GHRH receptor protein encoded by SV 1, it should be of value in various investigations.
European Journal of Endocrinology, 2004
Objective: GHRH is secreted by the hypothalamus and, upon binding to specific GHRH receptors in the pituitary, stimulates growth hormone (GH) production and release from the pituitary. In addition to this neuroendocrine action, accumulated evidence implies additional roles for GHRH in carcinogenesis in non-pituitary tissues. In vitro and in vivo studies have shown that splice variant 1 (SV1) of the GHRH receptor, which is widely expressed in non-pituitary tissues and cancers, can mediate the proliferative effects of GHRH. The aim of the present study was to investigate the operation of an autocrine stimulatory loop between GHRH and SV1 in primary breast tumors. Design: Fifty-three primary breast tumors were evaluated for GHRH and SV1 expression. Methods: Expression of GHRH and SV1 was assessed by immunohistochemistry using anti-GHRH SV95 and anti-SV1 2317/5 polyclonal antibodies. Results: About 40% of the specimens tested express GHRH and/or SV1 (approx. 25% each), while in 35% of these positive specimens co-expression of these antigens was detected (P , 0.01). Furthermore, a correlation of GHRH, but not SV1, expression was detected in lobular compared with ductal carcinomas. Conclusions: These results constitute the first demonstration for the expression of GHRH and SV1 in primary breast cancers, and provide evidence for the operation of an autocrine stimulatory loop between GHRH and SV1 in primary cancers. Our findings indicate that GHRH analogs could have diagnostic and therapeutic applications for the management of breast cancer.
Background: Hypothalamic GH-releasing hormone (GHRH) regulates GH release from the pituitary, but an ectopic production of GHRH has been detected in various non-hypothalamic tissues, especially cancers. Objective: To investigate whether endometrial tumors produce GHRH. Methods: Twenty-four endometrioid, three serous papillary (SP), three mixed type endometrioid/ serous papillary adenocarcinomas and one malignant mixed Mü llerian tumor (MMMT) were assessed for GHRH immunoreactivity by the polyclonal anti-rabbit antibody SV95 and for the expression of GHRH mRNA by in situ hybridization using an oligonucleotide probe. Results: Increased GHRH immunoreactivity was detected in 15 out of 24 (63%) of the endometrioid tumors, including two out of three (66%) of the mixed type endometrioid/serous adenocarcinomas but not in the three SP or the MMMT tumor. Cytoplasmic staining was detected in all positive cases, while in three of them strong nuclear localization of GHRH was also revealed. In situ hybridization indicated the presence of GHRH mRNA in six cases, all characterized as positive for GHRH immunoreactivity. Conclusion: GHRH is expressed in a subset of endometrial tumors, of the endometrioid type in particular. A paracrine/autocrine role for GHRH in the development of the disease should be considered.
Peptides, 2002
The expression of mRNA for GHRH and splice variants (SVs) of GHRH receptors in LNCaP, MDA-PCa-2b and PC-3 human prostate cancers grown in nude mice was investigated by RT-PCR. The expression of mRNA for GHRH was detected in LNCaP and PC-3, but not in MDA-PCa-2b prostatic carcinoma. RT-PCR analyses of mRNA isolated from LNCaP, MDA-PCa-2b and PC-3 cancers, revealed the presence of 720 and 566 bp products, corresponding to SV(1) and SV(2) isoforms of GHRH receptors. In PC-3 tumor membranes a radiolabeled GHRH antagonist [125I]-JV-1-42 was bound to one class of high-affinity binding sites (K(d)=1.81+/-0.47 nM) and maximum binding capacity of 332.7+/-27.8 fmol/mg membrane protein. The in vivo uptake of [125I]-JV-1-42 was observed in all xenografts of human prostate cancer, the tracer accumulation being the highest in PC-3 tumors. These results indicate that GHRH and SVs of its receptors, different from those found in the pituitary, are present in experimental human prostate cancers and m...
Absence of Constitutively Activating Mutations in the GHRH Receptor in GH-Producing Pituitary Tumors
The Journal of Clinical Endocrinology & Metabolism, 2001
The molecular events leading to the development of GH-producing pituitary tumors remain largely unknown. We hypothesized that activating mutations of the GHRH receptor might occur in a subset of GH-producing pituitary tumors. Genomic DNA samples from 54 GH-producing pituitary tumor tissues were screened for mutations of the GHRH receptor. Eleven homozygous or heterozygous nucleotide substitutions
Regulatory Peptides, 2002
Splice variants (SV) of receptors for growth hormone-releasing hormone (GHRH) have been found in several human cancer cell lines. GHRH antagonists inhibit growth of various human cancers, including osteosarcomas and Ewing's sarcoma, xenografted into nude mice or cultured in vitro and their antiproliferative action could be mediated, in part, through these SV of GHRH receptors. In this study, we found mRNA for the SV 1 isoform of GHRH receptors in human osteosarcoma line MNNG/HOS and SK-ES-1 Ewing's sarcoma line. We also detected mRNA for GHRH, which is apparently translated into the GHRH peptide and secreted by the cells, as shown by the presence of GHRH-like immunoreactivity in the conditioned media of cell cultures. In proliferation studies in vitro, the growth of SK-ES-1 and MNNG/ HOS cells was dose-dependently inhibited by GHRH antagonist JV-1-38 and an antiserum against human GHRH. Our study indicates the presence of an autocrine stimulatory loop based on GHRH and SV 1 of GHRH receptors in human sarcomas. The direct antiproliferative effects of GHRH antagonists on malignant bone tumors appear to be exerted through the SV 1 of GHRH receptors on tumoral cells.
The Journal of Clinical Endocrinology & Metabolism, 1998
A novel G 11-protein-coupled receptor specific for synthetic GHreleasing peptides (GHRPs) has recently been cloned and sequenced. Two forms exist, types 1a and 1b, the latter of which is biologically inactive. Using RT-PCR, we looked for the presence in tumorous pituitary cells of messenger ribonucleic acid (mRNA) for this novel GH secretagogue receptor (GHS-R). Both subtypes of GHS-R mRNA were detected in all six human pituitary somatotropinomas removed from patients with acromegaly. In culture, four of the tumors exhibited strong responses to GHRP-2 in terms of both phosphatidylinositol (PI) hydrolysis and GH secretion, but two were resistant. There was no apparent difference in the type 1a and type 1b expression pattern, as judged by RT-PCR, between responsive and nonresponsive tumors. Similarly, the rat pituitary tumor cell line, GH 3 , was found to express GHS-R mRNA, although these cells also did not respond to GHRPs. RT-PCR failed to detect GHS-R mRNA in eight functionless human pituitary tumors. In contrast, prolactinomas were found to express the receptor and, in culture, significant stimulation of PRL secretion and PI hydrolysis occurred in two of three tumors tested. These results demonstrate that tumorous somatotrophs express the GHS-R gene and that the occasionally observed nonresponsiveness of somatotropinomas to GHRPs is not due to the absence of the biologically active type 1a receptor. Additionally, human pituitary prolactinomas also express GHS-R and are able to respond to GHRPs in terms of PI hydrolysis and PRL secretion. In contrast, GHS-R gene expression does not appear to be associated with human functionless pituitary tumors.
Molecular medicine (Cambridge, Mass.)
Hypothalamic growth hormone (GH)-releasing hormone (GHRH) regulates the release of GH from the pituitary gland. The receptors for GHRH (GHRH-R) are expressed predominantly in the pituitary. Recent evidence demonstrates that splice variants of the GHRH receptor are also expressed in several nonpituitary tissues, both normal and tumoral, as well as in cancer cell lines. The aim of this study was to investigate the expression of the splice variant 1 (SV-1) of GHRH-R in colorectal cancer (CRC). Seventy patients who underwent partial colectomy for CRC were enrolled in the study. Immunohistochemical expression of SV-1 was studied in paraffin-embedded sections of patient tumor tissue. A cytoplasmic supranuclear expression of SV-1 was observed in CRC as well as in the normal colon mucosa. Tumor grade and pathological stage were negatively correlated with expression of SV-1 (P = 0.012 and P = 0.013, respectively). CRCs metastatic to the liver showed a lower expression of SV-1 than did primar...