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 (SV1) 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 SV1 receptor
protein. Rabbits were immunized with [Ala-23]SV1 (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-SV1
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 approximately 40 kDa in RL and HT
lymphomas. This band corresponds to the estimated molecular mass of the
GHRH receptor isoform encoded by SV1. RT-PCR and
ligand binding studies also revealed the expression of SV1 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 SV1, it should be of value in various investigations.