C-Myc-mediated Regulation of Telomerase Activity Is Disabled in Immortalized Cells

Rachid Drissi,Frederique Zindy,Martine F Roussel,John L Cleveland

doi:10.1074/jbc.m101899200

Rachid Drissi, Frederique Zindy + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m101899200

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Abstract

Myc overexpression is a hallmark of human cancer and promotes transformation by facilitating immortalization. This function has been linked to the ability of c-Myc to induce the expression of the catalytic subunit of telomerase, telomerase reverse transcriptase (TERT), as ectopic expression of TERT immortalizes some primary human cell types. c-Myc up-regulates telomerase activity in primary mouse embryonic fibroblasts (MEFs) and myeloid cells. Paradoxically, Myc overexpression also triggers the ARF-p53 apoptotic program, which is activated when MEFs undergo replicative crises following culture ex vivo. The rare immortal variants that arise from these cultures generally suffer mutations in p53 or delete Ink4a/ARF, and Myc greatly increases the frequency of these events. Alternative reading frame (ARF)- and p53-null MEFs have increased telomerase activity, as do variant immortal clones that bypass replicative crisis. Similarly, immortal murine NIH-3T3 fibroblasts and myeloid 32D.3 and FDC-P1.2 cells do not express ARF and have robust telomerase activity. However, Myc overexpression in these immortal cells results in remarkably discordant regulation of TERT and telomerase activity. Furthermore, in MEFs and 32D.3 cells TERT expression and telomerase activity are regulated independently of endogenous c-Myc. Thus, the regulation of TERT and telomerase activity is complex and is also regulated by factors other than Myc, ARF, or p53.

Highlights

Transformation of normal cells to cancer requires the bypass of several hurdles that monitor the ability of the cell to proliferate, differentiate, and survive
This function has been linked to the ability of c-Myc to induce the expression of the catalytic subunit of telomerase, telomerase reverse transcriptase (TERT), as ectopic expression of TERT immortalizes some primary human cell types. c-Myc up-regulates telomerase activity in primary mouse embryonic fibroblasts (MEFs) and myeloid cells
Activation of p53 in this scenario requires the p19ARF nucleolar tumor suppressor protein [5], which is expressed from an alternative reading frame of the INK4a locus [6], which encodes the p16Ink4a inhibitor of the cyclin D-dependent kinases 4 and 6 [7]

Summary

EXPERIMENTAL PROCEDURES

Cell Culture—MEFs derived from wild-type, ARF-, p53- and p21CIP1null day E13.5 embryos were explanted and cultured as described previously [4]. To ensure that parental and c-Myc-overexpressing myeloid cells were at similar phases of growth prior to analysis, the cells were sequentially passaged for two consecutive days at 0.3 ϫ 106 (FDC-P1.2) or at 0.5 ϫ 106 cells per ml (32D.3), and on the 3rd day they were assessed for levels of mTR and mTERT and telomerase activity. PCR amplification of the 160 base pairs of mTERT mRNA (telomerase reverse transcriptase) was performed with primers coined LT7 (5Ј-CACATTCCAGAAGAACAGG-3Ј) and LT8 (5ЈCAGATGGGCATGGCTAG-3Ј), based on sequence from GenBankTM accession number AF029235, for 38 cycles of denaturation (95 °C, 1 min), annealing (60 °C, 1 min), and extension (72 °C, 2 min). The membrane was incubated with a rabbit serum containing polyclonal antibody raised against the C-terminal peptide sequence (SRKLPGTTLTALEAAAPAL) corresponding to amino acids 1105–1124 from hTERT sequence (GenBankTM accession number NM_003219) This antibody recognizes both hTERT and mTERT proteins

RESULTS

Myc Regulation of TERT Is Context Specific

Findings

DISCUSSION