Expression of Growth Hormone-Releasing Hormone and Its Receptor Splice Variants in a Cohort of Hungarian Pediatric Patients with Hematological and Oncological Disorders: A Pilot Study

Abstract

Hematological and oncological diseases are still among the leading causes of childhood mortality. Expression of growth hormone-releasing hormone (GHRH) and its receptors (GHRH-R) has been previously demonstrated in various human tumors, but very limited findings are available about the presence and potential function of GHRH-Rs in oncological and hematological disorders of children. In this study, we aimed to investigate the expression of mRNA for GHRH and splice variant 1 (SV) of GHRH-R in 15 pediatric hematological/oncological specimens by RT-PCR. The presence and binding characteristics of GHRH-R protein were also studied by Western blot and ligand competition assays. Of the fifteen specimens studied, eleven pediatric samples (73%) showed the expression of mRNA for GHRH. These eleven samples also expressed mRNA for GHRH receptor SV1. GHRH-R protein was found to be expressed in two benign tumor samples and five malignant tumors examined by Western blot. The presence of specific, high affinity binding sites on GHRH-R was demonstrated in all of the seven human pediatric solid tumor samples investigated. Our results show that the expression of GHRH and SV1 of GHRH-R in hemato-oncological diseases in children can pave the way for further investigation of GHRH-Rs as potential molecular targets for diagnosis and therapy.

Keywords: GHRH; GHRH receptor; hematological-oncological disorders in children; splice variant.

Figure 1

Hormonal activities of GHRH and its antagonistic analogs via indirect and direct pathways (also reviewed in detail in [5,6,7]).

Figure 2

Representative RT-PCR analysis of mRNA for the GHRH ligand in human pediatric tumor specimens. The PCR products were of the expected size of 150 bp for the GHRH-L; Lane M, molecular marker (50-bp DNA ladder); Lane N, no template control; Lane P, positive control (human pituitary tissue); Lane 1: RMS, Lane 2: ALL, Lane 3: CBN, Lane 4: HL.

Figure 3

Representative RT-PCR analysis of mRNA for SV1 of GHRH-R in human pediatric tumor specimens. The PCR products were of the expected size of 373 bp for the SV1 isoform of GHRH-R; Lane M, molecular marker (50-bp DNA ladder); Lane P, positive control (human pituitary tissue); Lane N, no template control; Lanes 1: RMS, Lane 2: TR, Lane 3: HL, Lane 4: FD.

Figure 4

Western blot analysis of GHRH-receptor protein in human pediatric hematological/oncological specimens. Protein lysates obtained from the samples were separated on 10% SDS-PAGE (A). The size of the proteins was defined using a molecular weight marker (Precision Plus Dual Color Protein Standard, Bio-Rad). HPRT (hypoxanthine phosphoribosyl transferase) protein was detected as a loading control to quantify the level of GHRH-R protein in each sample using Bio-Rad Image Lab 5.2.1 software (B). Lanes: 1: HL, 2 and 3: RMS, 4: TR, 5: BMH, 6: FD, 7: JM.