Hiromi Tanaka

Factors impacting human telomere homeostasis and age-related disease.

Loss of telomere length homeostasis has been linked to age-related disease especially cancer. In this review, we discuss two major causes of telomere dysfunction that potentially lead to tumorigenesis: replicative aging and environmental assaults. Aging has long been recognized as a source for telomere dysfunction through increasing numbers of cell divisions in the absence of sufficient telomerase activity. However, environmental assaults that cause telomere dysfunction are only beginning to be identified and recognized. Environmental stressors that influence telomere length may be physical or induced by psychological situations like stress. Knowledge of all factors, including genetic and environmental forces, that moderate telomere length will be critical for understanding basic mechanisms of human telomere maintenance during development and aging as well as for disease prevention and treatment strategies.

Shared environmental factors associated with telomere length maintenance in elderly male twins

During aging, chromosome ends, or telomeres, gradually erode or shorten with each somatic cell division. Loss of telomere length homeostasis has been linked to age‐related disease. Remarkably, specific environmental assaults, both physical and psychological, have been shown to correlate with shortened telomeres. However, the extent that genetic and/or environmental factors may influence telomere length during later stages of lifespan is not known. Telomere length was measured in 686 male US World War II and Korean War veteran monozygotic (MZ) and dizygotic (DZ) twins (including 181 MZ and 125 DZ complete pairs) with a mean age of 77.5 years (range 73–85 years). During the entire process of telomere length measurement, participant age and twin status were completely blinded. White blood cell mean telomere length shortened in this elderly population by 71 base pairs per year (P < 0.0001). We observed no evidence of heritable effects in this elderly population on telomere length maintenance, but rather find that telomere length was largely associated with shared environmental factors (P < 0.0001). Additionally, we found that individuals with hypertension and cardiovascular disease had significantly shorter telomeres (P = 0.0025 and 0.002, respectively). Our results emphasize that shared environmental factors can have a primary impact on telomere length maintenance in elderly humans.

Telomere fusions in early human breast carcinoma

Several lines of evidence suggest that defects in telomere maintenance play a significant role in the initiation of genomic instability during carcinogenesis. Although the general concept of defective telomere maintenance initiating genomic instability has been acknowledged, there remains a critical gap in the direct evidence of telomere dysfunction in human solid tumors. To address this topic, we devised a multiplex PCR-based assay, termed TAR (telomere-associated repeat) fusion PCR, to detect and analyze chromosome end-to-end associations (telomere fusions) within human breast tumor tissue. Using TAR fusion PCR, we found that human breast lesions, but not normal breast tissues from healthy volunteers, contained telomere fusions. Telomere fusions were detected at similar frequencies during early ductal carcinoma in situ and in the later invasive ductal carcinoma stage. Our results provide direct evidence that telomere fusions are present in human breast tumor tissue and suggest that telomere dysfunction may be an important component of the genomic instability observed in this cancer. Development of this robust method that allows identification of these genetic aberrations (telomere fusions) is anticipated to be a valuable tool for dissecting mechanisms of telomere dysfunction.

Localization of an hTERT repressor region on human chromosome 3p21.3 using chromosome engineering

Telomerase is a ribonucleoprotein enzyme that synthesizes telomeric DNA. The reactivation of telomerase activity by aberrant upregulation/expression of its catalytic subunit hTERT is a major pathway in human tumorigenesis. However, regulatory mechanisms that control hTERT expression are largely unknown. Previously, we and others have demonstrated that the introduction of human chromosome 3, via microcell-mediated chromosome transfer (MMCT), repressed transcription of the hTERT gene. These results suggested that human chromosome 3 contains a regulatory factor(s) involved in the repression of hTERT. To further localize this putative hTERT repressor(s), we have developed a unique experimental approach by introducing various truncated chromosome 3 regions produced by a novel chromosomal engineering technology into the renal cell carcinoma cell line (RCC23 cells). These cells autonomously express ectopic hTERT (exohTERT) promoted by a retroviral LTR promoter in order to permit cellular division after repression of endogenous hTERT. We found a telomerase repressor region located within a 7-Mb interval on chromosome 3p21.3. These results provide important information regarding hTERT regulation and a unique method to identify hTERT repressor elements.

The Presence of Telomere Fusion in Sporadic Colon Cancer Independently of Disease Stage, TP53/KRAS Mutation Status, Mean Telomere Length, and Telomerase Activity

Defects in telomere maintenance can result in telomere fusions that likely play a causative role in carcinogenesis by promoting genomic instability. However, this proposition remains to be fully understood in human colon carcinogenesis. In the present study, the temporal sequence of telomere dysfunction dynamics was delineated by analyzing telomere fusion, telomere length, telomerase activity, hotspot mutations in KRAS or BRAF, and TP53 of tissue samples obtained from 18 colon cancer patients. Our results revealed that both the deficiency of p53 and the shortening of mean telomere length were not necessary for producing telomere fusions in colon tissue. In five cases, telomere fusion was observed even in tissue adjacent to cancerous lesions, suggesting that genomic instability is initiated in pathologically non-cancerous lesions. The extent of mean telomere attrition increased with lymph node invasiveness of tumors, implying that mean telomere shortening correlates with colon cancer progression. Telomerase activity was relatively higher in most cancer tissues containing mutation(s) in KRAS or BRAF and/or TP53 compared to those without these hotspot mutations, suggesting that telomerase could become fully active at the late stage of colon cancer development. Interestingly, the majority of telomere fusion junctions in colon cancer appeared to be a chromatid-type containing chromosome 7q or 12q. In sum, this meticulous correlative study not only highlights the concept that telomere fusion is present in the early stages of cancer regardless of TP53/KRAS mutation status, mean telomere length, and telomerase activity, but also provides additional insights targeting key telomere fusion junctions which may have significant implications for colon cancer diagnoses.

Evidence for inactivation of distinct telomerase repressor genes in different types of human cancers.

Telomerase activation, a critical event in human carcinogenesis, may result from defects in telomerase‐repressing mechanisms. Data from microcell‐mediated chromosome transfer (MMCT) suggests the presence of telomerase repressor genes that become inactivated during carcinogenic processes. The transfer of a normal human chromosome 3 represses telomerase activity of both human renal cell carcinoma (RCC) and breast carcinoma (BC) cells. For a genetic complementation analysis of telomerase repression, 2 RCC cell lines (KC12 and RCC23) and a BC cell line (21NT) were used to make somatic cell hybrids. All of the self‐hybrids (KC12×KC12 and 21NT×21NT) and hybrids from 2 RCC cell lines (KC12×RCC23) expressed the telomerase activity similarly to their parental cells, excluding the possibility of a ploidy‐associated change in telomerase activity and suggesting the same genetic defect shared by the 2 RCC cell lines. In contrast, the fusion of BC and RCC cells (21NT×KC12 and 21NT×RCC23) produced a significant number of telomerase‐negative hybrids, suggesting that the RCC and BC cells have different defects in the telomerase repression, which are functionally corrected through genetic complementation in the hybrids. This notion was supported by the mapping of the RCC telomerase repressor gene to a 5.7‐Mb region on 3p21, which is different from the candidate region for the BC telomerase repressor gene on the same chromosomal band. These findings provide direct evidence for inactivation of distinct telomerase repressor genes in different types of human cancers and may have implications in the tissue‐specific regulation of telomerase during human development and carcinogenesis.

Altered expression of telomere-associated genes in leukocytes among BRCA1 and BRCA2 carriers

Telomere dysfunction resulting from telomere shortening and deregulation of shelterin components has been linked to the pathogenesis of age‐related disorders, including cancer. Recent evidence suggests that BRCA1/2 (BRCA1 and BRCA2) tumor suppressor gene products play an important role in telomere maintenance. Although telomere shortening has been reported in BRCA1/2 carriers, the direct effects of BRCA1/2 haploinsufficiency on telomere maintenance and predisposition to cancer development are not completely understood. In this study, we assessed the telomere‐associated and telomere‐proximal gene expression profiles in peripheral blood leukocytes from patients with a BRCA1 or BRCA2 mutation, compared to samples from sporadic and familial breast cancer individuals. We found that 25 genes, including TINF2 gene (a negative regulator of telomere length), were significantly differentially expressed in BRCA1 carriers. Leukocyte telomere length analysis revealed that BRCA1/2 carriers had relatively shorter telomeres than healthy controls. Further, affected BRCA1/2 carriers were well differentiated from unaffected BRCA1/2 carriers by the expression of telomere‐proximal genes. Our results link BRCA1/2 haploinsufficiency to changes in telomere length, telomere‐associated as well as telomere‐proximal gene expression. Thus, this work supports the effect of BRCA1/2 haploinsufficiency in the biology underlying telomere dysfunction in cancer development. Future studies evaluating these findings will require a large study population.