αCP1 Mediates Stabilization of hTERT mRNA by Autocrine Human Growth Hormone

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, ␣CP1 and ␣CP2, 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.Chromosomal ends consist of small DNA repeats that are not duplicated faithfully during replication resulting in loss of DNA during each replication. To faithfully replicate chromosomal ends, cells use a reverse transcriptase holoenzyme termed the telomerase complex (1). The template human telomerase RNA (hTR) 3 and the reverse transcriptase catalytic subunit (hTERT) are considered to be the major components of the human telomerase complex, because the expression of hTERT and hTR alone is able to restore the majority of telomerase activity (2). Telomerase activity is subject to stringent regulation and is regulated by multiple mechanisms such as hTERT mRNA transcription (3), splicing and maturation (4), protein phosphorylation and transport (5), and subcellular localization of each component of the telomerase complex (6). Telomerase activity is minimal in most somatic cells resulting in chromosome shortening after each proliferative cycle eventually resulting in crisis and cell death (7). However, telomerase activity is readily detected in 90% of all tumor cells (8), in all tissues during early development (9), and in tissues with continuous proliferation, such as the hematopoietic system or the epidermis (10, 11).mRNA turnover is one important mechanism by which gene expression is regulated (13). Specific interactions between sequences within the mRNA (cis-acting regulatory elements) and cellular RNA-binding proteins (trans-acting factors) regulate ribonuclease action and subsequent mRNA decay rates. The majority of these cis-acting elements are present in the 3Ј-untranslated region (3Ј-UTR) of the mRNA. cis-Regulatory elements such as AU-rich elements (12), Iron-responsive element (14), and CU-rich elements (15) within the 3Ј-UTR have been demonstrated to possess a role in mRNA stability. ␣-Globin mRNA poly(C)-rich segment-binding proteins (␣CPs), also referred to as poly(C)-binding proteins, and hnRNP K bind to CU-rich cis-regulatory elements (16). Two of the five members of poly(C)-binding proteins, ␣CP1 and ␣CP2, are highly homologous (16). Both ␣CP1 and ␣CP2 bind to pyrimidinerich elements in the 3Ј-UTR and stabilize a variety of mRNAs including ␣-globin and eryt...

Autocrine human GH promotes radioresistance in mammary and endometrial carcinoma cells

“…However, autocrine hGH increased mRNA levels of the breast cancer susceptibility gene, BRCA2, and the DNA repair protein involved in homologous recombination, X-ray repair cross complementing group-2 (XRCC2), in MDA-hGH cells compared with MDA-vec cells (Table 2). Consistent with our previous observations in MCF-7 cells (Emerald et al 2007), autocrine hGH also increased mRNA levels of TERT, the catalytic subunit of telomerase in MDA-MB-435S cells (Table 2). Functional antagonism of autocrine hGH reduces RL95-2 cell viability, total cell number, clonogenic survival and DNA damage after treatment with IR RL95-2 wild-type cells endogenously express and secrete hGH (Pandey et al 2008) and hence provide a suitable system for investigating the effect of functional antagonism of hGH on post-radiation cell survival.…”

“…Another mechanism contributing to autocrine hGH-mediated radioresistance may be through the upregulation of telomerase (Emerald et al 2007). Autocrine hGH has been found to increase hTERT gene and protein expression, leading to an increase in telomerase activity in MCF-7 cells (Emerald et al 2007). Consistent with these studies we observed that autocrine hGH increased hTERT mRNA expression in MDA-MB-435S cells (Table 2).…”

“…While telomere length is closely correlated with cancer cell radiosensitivity (Ayouaz et al 2008). Another mechanism contributing to autocrine hGH-mediated radioresistance may be through the upregulation of telomerase (Emerald et al 2007). Autocrine hGH has been found to increase hTERT gene and protein expression, leading to an increase in telomerase activity in MCF-7 cells (Emerald et al 2007).…”

Thymidine phosphorylase mRNA stability and protein levels are increased through ERK-mediated cytoplasmic accumulation of hnRNP K in nasopharyngeal carcinoma cells

“…Several studies have shown that CURE sequences function posttranscriptionally to regulate the mRNA stability of the CURE-containing mRNA (Levy et al, 1996;Czyzyk-Krzeska and Bendixen, 1999;Waggoner and Liebhaber, 2003;Wang et al, 2006;Emerald et al, 2007). To confirm that the CURE sequence in TP functions in its posttranscriptional regulation, we inserted three copies of wild-type and mutated TP CURE into the 3 0 -UTR of a firefly luciferase reporter gene, generating pMIR-CURE (CURE) and pMIRCUREmut (CUREmut), respectively ( Figure 2b; also see Supplementary materials and methods).…”

Inhibition of Human Tumor Cell Proliferation by the Telomerase Inhibitor TELIN

“…Six micrograms of total RNA was used for cDNA synthesis with d(T) 20 primer (Omniscript RT, Qiagen, Tokyo, Japan). Quantitative PCR was performed for hTERT and b-actin cDNA with the primer set according to the report by Emerald et al (2007). Appropriate PCR cycle numbers were determined as 32 and 18 for hTERT and b-actin, respectively, at which the amount of all the PCR products were plotted within a linear range.…”