Telomerase activation and p53 mutations in urethane-induced A/J mouse lung tumor development

Abstract

The mouse telomerase holoenzyme, which synthesizes telomeric DNA de novo, is a ribonucleoprotein complex that includes the mouse telomerase RNA component (mTERC), mouse telomerase-associated protein (mTEP1) and mouse telomerase reverse transcriptase (mTERT). To determine the role of telomerase in urethane-induced lung tumorigenesis in A/J mice we examined telomerase activity and the expression of each telomerase subunit in 20 tumor samples, harvested at 16, 28, 40 and 50 weeks after urethane treatment. The telomeric repeat amplification protocol assay showed that statistically significant telomerase activation occurred both early and late in tumorigenesis. Semi-quantitative reverse transcription-polymerase chain reaction analysis revealed that mRNA expression levels of mTEP1 and mTERT were up-regulated during tumor progression, while mTERC expression was not significantly different between tumors and normal lung. We further examined mTEP1 protein expression in normal lung tissue and lung tumors; western blot analysis showed preferential expression of mTEP1 protein in lung tumors compared with normal lung and immunohistochemistry revealed that a majority of the adenoma cells were positively stained in the nucleus, whereas only a few of the adjacent normal alveolar cells were immunoreactive. In addition, we investigated DNAs of the 20 tumor samples by single strand conformation polymorphism and sequencing analyses to examine whether alterations of the p53 gene in exons 5-8 were associated with telomerase activity. Although we found one nonsense, two missense, two silent and one simultaneous double mutation at different codons in six late stage tumors, there was no apparent correlation between telomerase activity and p53 mutations. Collectively, these results suggest that mTEP1 as well as mTERT may be involved in the regulation of telomerase activity and that telomerase activation may contribute to lung tumorigenesis in A/J mice independently of p53 gene alterations.

Expression profiles of proliferative and antiapoptotic genes in sporadic and colitis‐related mouse colon cancer models

“…2003). Similar to the results obtained by Ohno et al. (2001) in urethane‐induced lung tumour in A/J mice, we found upregulation of mouse TERT mRNA in both models of CRC.…”

Long-termin vitroexpansion of human adipose-derived stem cells showed low risk of tumourigenicity

“…The p53 ‐specific mutation has been reported to be involved in telomerase activation (Ueda et al ., ). On the contrary, studies on mouse tumour lung development (Ohno et al ., ) and human breast cancer (Roos et al ., ) showed no correlation between telomerase activity and p53 mutations but it was associated with p53 protein accumulation (Roos et al ., ). In this study, telomerase activity in ASCs showed no significant changes in the relative telomerase activity (RTA) at P5, P10, P15 and P20.…”

Function of AP-1 in Transcription of the Telomerase Reverse Transcriptase Gene (TERT) in Human and Mouse Cells

“…Expression of hTERT and mTERT mRNAs was analyzed by reverse transcription (RT)-PCR and real-time RT-PCR as described previously (33,39). Briefly, total RNA was isolated from the cells using an RNeasy Mini Kit (QIAGEN) according to the manufacturer's protocol.…”

“…PCR used 30 s at 94°C, 30 s at 60°C, and 30 s 72°C for hTERT and 30 s at 94°C, 30 s at 55°C, and 30 s at 72°C for mTERT, with 28 cycles for conventional PCR and 40 cycles for real-time PCR. The efficiency of cDNA synthesis from each sample was estimated by PCR for human glyceraldehyde-3-phosphate dehydrogenase or mouse ␤-actin as described previously (33,39).…”