Decrease in telomerase activity in U-87MG human glioblastomas after treatment with an antagonist of growth hormone-releasing hormone (telomerase repeat amplification assayytelomerase catalytic subunit regulationytumor regressionydown-regulation of telomerase gene)

Antagonists of growth hormone-releasing hormone (GH-RH) inhibit the growth of various tumors through mechanisms that involve the suppression of the insulin-like growth factor I andyor insulin-like growth factor II levels or secretion. In the present study, we tested the hypothesis that the tumor inhibition is associated with a decrease in telomerase activity because telomerase is considered obligatory for continued tumor growth. Nude mice bearing xenografts of U-87MG human glioblastomas were treated with GH-RH antagonist MZ-5-156. Telomerase activity was assessed by the telomerase repeat amplification protocol. Treatment with MZ-5-156 reduced levels of telomerase activity as compared with controls. When U-87 glioblastomas, H-69 small cell lung carcinomas, H-23 non-small cell lung carcinomas, and MDA-MB-468 breast carcinoma cells were cultured in vitro, addition of 3 mM MZ-5-156 also inhibited telomerase activity. Reverse transcription–PCR analysis revealed that in U-87MG glioblastomas, the expression of the hTRT gene encoding for the telomerase catalytic subunit was significantly decreased by MZ-5-156, whereas the levels of mRNA for hTR and TP1, which encode for the telomerase RNA and telomerase-associated protein, respectively, were unaffected. The repression of the telomerase activity was not accompanied by a significant decrease of mRNA level for the c-myc protooncogene that regulates telomerase. Our findings suggest that tumor inhibition induced by the GH-RH antagonists in U-87MG glioblastomas is associated with the downregulation of the hTRT gene, resulting in a decrease in telomerase activity. Further studies are needed to establish whether GH-RH antagonists produce telomerase inhibition in other tumors. Growth hormone-releasing hormone (GH-RH) is secreted by the hypothalamus and through specific GH-RH receptors in the anterior pituitary stimulates the secretion of the GH (1). In addition to its physiological role in regulating GH release, GH-RH may play a role in the development of some neoplasms (2, 3). It has been demonstrated that GH-RH antagonists MZ-4-71 and MZ-5-156, synthesized in our laboratory, inhibit the proliferation of various experimental human and animal tumors in vivo and in vitro (4–10). Thus, GH-RH antagonists suppress growth of androgen-independent human DU-145 and PC-3 and rat Dunning R-3327-AT-1 prostate cancers (5, 7), H-69 small cell lung carcinomas (SCLC) and H-157 non-SCLC (6), CAKI-1 renal adenocarcinomas (8), SK-ES-1 and MNNGHOS osteosarcomas (9), and other cancers (4). GH-RH antagonists appear to inhibit the growth of cancers through indirect or direct pathways. The indirect mechanism operate through a suppression of the GH release from the pituitary and the resulting inhibition of the hepatic production of insulin-like growth factor (IGF) I (4, 7). In addition, a significant reduction in concentrations of IGF-I andyor IGF-II produced in osteosarcomas, renal cancers, and prostatic tumors as well as in non-SCLCs after treatment of nude mice with MZ-4-71 or MZ-5-156, points to a likely direct effect of GH-RH antagonists on tumors (5–9). A strong suppression of IGF-II mRNA expression in DU-145 tumors after treatment with MZ-5-156 supports this concept (7). In vitro studies also demonstrate that GH-RH antagonists cause a direct inhibition of growth, IGF-II production, and expression of IGF-II mRNA in human cancer cell lines (V. Csernus and A.V.S., unpublished work). IGF-I and IGF-II are involved in the regulation of normal and malignant growth through endocrine, paracrine, or autocrine mechanisms (4, 11–14). Telomerase is a ribonucleoprotein that functions as a specific DNA polymerase that is involved in the maintenance of telomeres, specialized structures at the ends of the eukaryotic chromosomes, by replacing the loss of telomeric DNA that occurs at each cell division (15–18). Telomerase maintains telomere length and chromosome stability, which are required for cellular immortality and subsequent malignant transformations (19–21). A striking association exists between telomerase activity and malignancy: 85–90% of the primary human tumors express this activity, as compared with approximately 24% of benign tumors, whereas it is absent in most normal somatic tissues (22–24). Three components of the telomerase ribonucleoprotein complex have been identified: the telomerase catalytic subunit (hTRT) (24–27), the telomerase RNA (hTR) (16), and telomerase-associated protein (TP1) (28, 29). Among them, hTRT is considered the rate-limiting determinant of the telomerase activity because a correlation was found between telomerase activity and hTRT mRNA, but not hTR and TP1 mRNA levels (25, 30). In addition, the introduction of the hTRT gene in telomerase-negative normal human fibroblasts restores telomerase activity and extends their life span (20). The mechanism for the reactivation of telomerase in tumor cells, which is a necessary event for the acquisition of cellular immortality, remains poorly understood, but an important role for the c-myc oncogene has been recognized (31, 32). In an attempt to investigate further the mechanism of antitumor action of MZ-5-156, we evaluated whether this analog affects the levels of telomerase activity in human The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. © 1999 by The National Academy of Sciences 0027-8424y99y96226-6$2.00y0 PNAS is available online at www.pnas.org. Abbreviations: GH, growth hormone; GH-RH, GH-releasing hormone; hGAPDH, human glyceraldehyde-3-phosphate dehydrogenase; IGF, insulin-like growth factor; SCLC, small cell lung carcinoma; TRAP, telomerase repeat amplification protocol; hTRT, telomerase catalytic subunit; hTR, telomerase RNA; TP1, telomerase-associated protein; RT, reverse transcription. *To whom reprint requests should be addressed at: Veterans Affairs Medical Center, 1601 Perdido Street, New Orleans, LA 70112-1262.