Development of a polyclonal antiserum for the detection of the isoforms of the receptors for human growth hormone-releasing hormone on tumors

Abstract

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.splice variant ͉ growth hormone-releasing hormone receptor ͉ polyclonal antibody ͉ non-Hodgkin's lymphoma A ntagonists of growth hormone-releasing hormone (GHRH) inhibit the growth of experimental human cancer cell lines xenografted into nude mice or cultured in vitro and are being developed for cancer therapy (1-4). To design still more potent antagonists, we have to fully understand their mechanism of action. GHRH antagonists suppress tumor growth through indirect and direct pathways. The indirect mechanism operates through a suppression of the growth hormone release from the pituitary and the resulting inhibition of the production of insulin-like growth factor I in the liver (1-11). However, the principal action of GHRH antagonists is probably exerted directly on tumors and appears to be mediated by specific receptors for GHRH antagonists on cancer cells (1-9, 11-13). Although the mRNA for GHRH and immunologically active GHRH were demonstrated in various human tumor cells, the mRNA for human pituitary GHRH receptor (GHRH-R) has not been detected on these tumor cells or any of the other cancer models (11,14,15).Because of the structural similarities between GHRH and vasoactive intestinal peptide (VIP), the receptors for VIP could be a target for the GHRH antagonists, but GHRH antagonists inhibit the proliferation of MiaPaCa-2 human pancreatic tumor cells, which do not express the receptors for VIP (13). Moreover, in LNCaP human prostatic carcinoma cells, which are positive for the VIP receptors, GHRH antagonists inhibit tumor growth more powerfully than the antagonists of VIP (1...

The expression of the pituitary growth hormone-releasing hormone receptor and its splice variants in normal and neoplastic human tissues

“…Interassay variation was Ͻ15%, and intraassay variation was Ͻ10%. We did not find a significant crossreaction with [Ala (25). No detectable crossreactivity was found with luteinizing hormone-releasing hormone, somatostatin, [Tyr-4]bombesin, and GHRH(1-29)NH 2 .…”

“…The synthesis of [Ala-28,41]pGHRH-R(23-45)-Tyr-Cys-NH 2 peptide hapten (designated pGHRH-R-hapten) was described in ref. 25. This peptide is an analog of segment 23-45 of the pGHRH-R protein in which two internal cysteine residues (Cys-28 and Cys-41) were replaced by alanine residues (26,27).…”

“…Optimal dilutions of the antibodies were determined in preliminary studies. The blots were probed at 4°C overnight with polyclonal antibodies to GHRH C-16 and V-17 (1:500) (Santa Cruz Biotechnology) and to SV1-SV2-SV4 (1:2,000) and pGHRH-R (1:2,000), with antisera raised and affinity-purified in our laboratory (25). The batch numbers for SV1-SV2-SV4 and pGHRH-R antisera were 2317͞7 and 2321͞5, respectively.…”

“…The detectability of pGHRH-R-hapten and the crossreaction with unrelated peptides were estimated (25,28) by using the JH-2321͞5 batch at the final dilution of 1:210,000. The standard curve of the RIA was set up in the range of 2 fmol per tube to 4,000 fmol per tube and showed the following characteristics: binding of the antiserum to the tracer (percentage of bound radioactivity to total radioactivity) was 41.2%.…”

Knocking down gene expression for growth hormone-releasing hormone inhibits proliferation of human cancer cell lines

“…The blots were then incubated at 41C overnight with an affinity purified goat polyclonal antibody against a peptide mapping near the N terminus of GHRH of human origin (1 : 425) (cat no. 10280, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and with rabbit antiserum to SV1 (1 : 2000) raised in our laboratory (Toller et al, 2004). The antiserum batch number for SV1 was JH 2317/5.…”