Investigations of telomeres and telomerase following ionizing radiation exposure

2014 Spring.Telomeres are critical structures located at the termini of eukaryotic chromosomes that regulate the replicative lifespan of human cells. Telomeres shorten with cell division, a process that eventually leads to telomere based growth arrest and cellular senescence. Telomere length is maintained through the activity of the reverse transcriptase, telomerase that functions in embryonic and adult stem cells to elongate telomeres and prolong replicative lifespan. Telomerase is repressed in the vast majority of human somatic cells, and its reactivation is a critical early step in carcinogenesis. Thus, telomerase and telomere maintenance are critical factors in the processes of carcinogenesis, tumor maintenance, and tumor recurrence following interventional therapy. Ionizing radiation (IR) has long been acknowledged as both a potent carcinogen and an effective agent in the treatment of cancer. To investigate the role telomeres and telomerase play in the cellular response to IR exposure, we tracked telomerase activity and telomere length in a panel of cancer and immortalized non-cancer cell lines following both acute and low dose rate (LDR) exposures to γ-rays. We observed elevations of telomerase activity in cancer, but not non-cancer, cell lines following acute exposures to IR. Further, telomere length was significantly reduced in both cancer and non-cancer cells post-acute IR exposure. Taken together, these studies suggest telomerase activity is playing a role in accelerated tumor repopulation following radiation therapy and that the associated telomere loss may be contributing to genomic instability. As IR induced enrichment of cancer stem cells (CSC) in established cancer cell lines was recently suggested to play a role in accelerated tumor repopulation following radiation therapy, we investigated a potential role for telomerase in the IR induced enrichment of CSCs. Consistent with previous reports, we detected a significant enrichment of putative breast CSCs in MCF-7 mammary carcinoma cells at 5 days post exposure, and demonstrate significant enrichment of putative CSCs in the non-tumorigenic MCF-10a, WTK1, and LCL15044 cell lines. Further small molecule inhibition of telomerase activity was able to effectively block CSC enrichment in both MCF-7 and MCF-10a cells. Together, these results suggest that telomerase inhibition is a significant player in the IR induced enrichment of putative CSCs in both cancer and non-cancer mammary epithelial cells. Further, this process seems to be driven by non-canonical roles of telomerase

The Role of the Core Non-Homologous End Joining Factors in Carcinogenesis and Cancer

DNA double-strand breaks (DSBs) are deleterious DNA lesions that if left unrepaired or are misrepaired, potentially result in chromosomal aberrations, known drivers of carcinogenesis. Pathways that direct the repair of DSBs are traditionally believed to be guardians of the genome as they protect cells from genomic instability. The prominent DSB repair pathway in human cells is the non-homologous end joining (NHEJ) pathway, which mediates template-independent re-ligation of the broken DNA molecule and is active in all phases of the cell cycle. Its role as a guardian of the genome is supported by the fact that defects in NHEJ lead to increased sensitivity to agents that induce DSBs and an increased frequency of chromosomal aberrations. Conversely, evidence from tumors and tumor cell lines has emerged that NHEJ also promotes chromosomal aberrations and genomic instability, particularly in cells that have a defect in one of the other DSB repair pathways. Collectively, the data present a conundrum: how can a single pathway both suppress and promote carcinogenesis? In this review, we will examine NHEJ’s role as both a guardian and a disruptor of the genome and explain how underlying genetic context not only dictates whether NHEJ promotes or suppresses carcinogenesis, but also how it alters the response of tumors to conventional therapeutics

Telomeres and Telomerase in the Radiation Response: implications for instability, reprogramming, and carcinogenesis

Telomeres are nucleoprotein complexes comprised of tandem arrays of repetitive DNA sequence that serve to protect chromosomal termini from inappropriate degradation, as well as to prevent these natural DNA ends from being recognized as broken DNA (double-strand breaks; DSBs) and triggering of inappropriate DNA damage responses. Preservation of telomere length requires telomerase, the specialized reverse transcriptase capable of maintaining telomere length via template-mediated addition of telomeric repeats onto the ends of newly synthesized chromosomes. Loss of either end-capping function or telomere length maintenance has been associated with genomic instability or senescence in a variety of settings; therefore telomeres and telomerase have well-established connections to cancer and aging. It has long been recognized that oxidative stress promotes shortening of telomeres, and that telomerase activity is a radiation-inducible function. However, the effects of ionizing radiation (IR) exposure on telomeres per se are much less well understood and appreciated. To gain a deeper understanding of the roles telomeres and telomerase play in the response of human cells to ionizing radiations of different qualities, we tracked changes in telomeric end-capping function, telomere length, and telomerase activity in panels of mammary epithelial and hematopoietic cell lines exposed to low linear energy transfer (LET) gamma(γ)-rays or high LET high charge, high energy (HZE) particles, delivered either acutely or at low dose rates (LDR). In addition to demonstrating that dysfunctional telomeres contribute to IR-induced mutation frequencies and genome instability, we reveal non-canonical roles for telomerase, in that telomerase activity was required for IR-induced enrichment of mammary epithelial putative stem/progenitor cell populations, a finding also suggestive of cellular reprogramming. Taken together, the results reported here establish the critical importance of telomeres and telomerase in the radiation response and as such, have compelling implications not only for accelerated tumor repopulation following radiation therapy, but for carcinogenic potential following low dose exposures as well, including those of relevance to spaceflight-associated galactic cosmic radiations