αCP1 Mediates Stabilization of hTERT mRNA by Autocrine Human Growth Hormone

B Starling Emerald,Yong Chen,Tao Zhu,Zhe Zhu,Kok-Onn Lee,Peter D Gluckman,Peter E Lobie

doi:10.1074/jbc.m600224200

Abstract

We herein demonstrate that autocrine human growth hormone production in human mammary carcinoma cells results in increased telomerase activity as a result of specific up-regulation of telomerase catalytic subunit (human telomerase reverse transcriptase (hTERT)) mRNA and protein. This increase in hTERT gene expression is not due to increased transcriptional activation of the hTERT promoter but is the result of increased stability of hTERT mRNA exerted by CU-rich cis-regulatory sequences present in the 3'-untranslated region of TERT mRNA. Autocrine human growth hormone up-regulates two poly(C)-binding proteins, alphaCP1 and alphaCP2, which bind to these cis-regulatory elements and stabilize hTERT mRNA. We have therefore demonstrated that post-transcriptional modulation of the level of hTERT mRNA is one mechanism for regulation of cellular telomerase activity.

Highlights

Chromosomal ends consist of small DNA repeats that are not duplicated faithfully during replication resulting in loss of DNA during each replication
Given the described oncogenic potential of autocrine hGH and that both the hGH receptor [20] and telomerase [21] are expressed highly in the human mammary stem cell population compared with differentiated mammary epithelial cells, we have examined the potential regulation of telomerase activity by autocrine hGH
Autocrine hGH Up-regulates Telomerase Activity in a JAK2dependent Manner—To examine the potential contribution of autocrine hGH to neoplastic progression of the human mammary epithelial cell and to determine the mechanism by which it results in oncogenic transformation, we examined the effect of autocrine hGH on telomerase activity in human mammary carcinoma cells

Summary

EXPERIMENTAL PROCEDURES

Cell Lines and Cell Culture—The MCF-7 cell line was obtained from ATCC. The MCF-7 cells were stably transfected either with wild type hGH gene (pMT-hGH) (designated MCFhGH) or with a translation-deficient hGH (designated MCFMUT) [55]. Sequences Used in Reverse Transcriptase-PCR—Extraction of total RNA and the semi-quantitative RT-PCR assay were performed as described previously [38]. The RT-PCR product of hTERT, ␤-actin, and luciferase were gel-purified and used as a template for generating the probes using a high prime DNA labeling kit (Roche Applied Science) according to the manufacturer’s protocol. RNA was extracted at the indicated times, and the endogenous hTERT and ␤-actin mRNA levels were analyzed by Northern blotting. Binding reactions were incubated at 22 °C for 30 min, after which 2 units of RNase T1 (Roche Applied Science) was added for 10 min, followed by the addition of heparin (final concentration, 5 mg/ml) (Sigma) for 10 min. 5 ϫ 104 MCF-7 cells were seeded into six-well plates and were cultured as above They were transiently transfected with 1 ␮g of the scrambled or the siRNA constructs and grown for an additional 18 h. The construct with the sequence 5Ј-TCGACAAGCTGGAGGAAGATA-3Ј was found to be effective and was used

RESULTS

Autocrine hGH Regulates the

DISCUSSION