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Expansion of stem cell numbers without reduction in their regenerative potential is crucial for therapeutic applications. However, the repeated cell divisions and aging impair stem cell function. We found that Pot1a, a component of the shelterin that protects telomeres, involves the maintenance of hematopoietic stem cell (HSC) activity during aging. Pot1a maintained the self-renewal activity of HSCs through the prevention of DNA damage responses in HSCs and suppression of the production of reactive oxygen species. Furthermore, the exogenous Pot1a expanded the HSC number and rejuvenated aged HSCs function upon ex vivo culture. Consistent with these results, treatment with exogenous human POT1 protein maintains human HSC activity in culture. Collectively, these results show that Pot1a or POT1 sustains HSC activity and can be used to expand HSC numbers ex vivo.

Protein p53 is described as guardian of the genome contributing for the suppression of tumorigenesis. p53 is also known to be the critical regulator of cellular senescence. It is activated by various genomic stresses including telomere attrition, excessive mitogenic sig- naling and genotoxic agents(eg. reactive oxygen species), and mediates cellular senescence. Senescent cells develop growth arrest, associated with altered gene profiles. These changes induce chronic inflammation and organ dysfunction, promoting pathologies in age related disorders. In this review, we delineate the role of p53 induced cellular senescence in age related disorders, such as obesity/diabetes and heart failure.

Type 2 diabetes is a major threat to human lifespan, and is caused by tissue dysfunction in insulin sensitive tissues and insulin secreting pancreatic P cells. Prevalence of diabetes is known to increase with age. Tissue dysfunctions in diabetes may be caused in part by a convergence of cellular senescence in adipose tissues and pancreatic p cells. Cellular senescence occurs in response to various cellular stressors, such as telomere erosion, DNA damage and oxidative stress. Senescent adipocytes and pancreatic p cells adopt several unique characteristics including enzymatic activity of senescence associated p galactosidase(SA- f -GAL) and upregulation of cell cycle inhibitors such as p53 and p16. Senotherapies are therapies targeting senescent cells, and is a promising strategy to overcome diabetes.

Aging is characterized by a progressive decline in the efficiency of physiological function and by the increased susceptibility to disease. Recently, the prevalence of elderly hepatocel- lular carcinoma (HCC) has been increasing in Japan. Inflammation, DNA-damaging agents (reactive oxygen and nitrogen species), methylation/ demethylation of DNA, and shortening of the telomere will lead. to genetic changes in pro- liferating cells and hepatocarcinogenesis. Because, these factors and processes are interlinked, it is difficult to assert the main causes of hepatocarcinogenesis. In conclusion, aging has become one of the most important risk factors for HCC. The inves- tigation of the mechanism of hepatocarcinogenesis by aging would have potentially impor- tance that lead to the novel diagnostic system, treatment strategy and prevention for HCC in the elderly.

Kidney is an energy-consuming organ and its function deteriorates according as aging. Various mechanisms for cellular senescence and organ aging have an impact on the initiation and progression of CKD, which includes telomere attrition, genomic instability, stem cell exhaustion, mitochondrial dysfunction, impaired nutrient signaling, inactivation of Sirtuin or Klotho-mediated pathway and renin-angiotensin system activation. Interventions in these pathways have provided therapeutic strategy against CKD. On the other hand, renal dys- function, in turn, affects human aging process. Because of this close relationship between human aging process and CKD, aging phenotype, such as frailty can be used as a clinical marker of the prognosis of CKD as well as of other age-related diseases including cardio- vascular disease and type II diabetes. Amelioration of this aging phenotype can provide a novel therapeutic strategy against CKD for the purpose of providing CKD patients with suc- cessful aging.

We examined the role of molecules related to drug resistance, such as P-glycoprotein (P-gp) and telomerase (TERT), signaling molecules of STATs and FLT3 in leukemia pathogenesis in de novo acute myeloid leukemia (AML), and myelodysplastic syndrome in the phase of overt leukemia (MDS-OL). Subjects were 18 patients with de novo AML, in which expression of P-gp, TERT, STAT3, STAT5, and FLT3 was observed in 11, 14, 16, 18, and 14 of patients, respectively. Phosphorylation of STAT3, STAT5, and FLT3 in patients with de novo AML was observed in 10 out of 14, 14 out of 18, and 10 out of 14 patients, respectively. Phosphorylation of STAT5 was associated with expression of both P-gp and TERT, suggesting that STAT5 is one of the transcription factors for these genes. On the other hand, P-gp, TERT, STAT3, STAT5, and FLT3 were expressed in 3, 1, 1, 6, and 1 of the 7 patients with MDS-OL, respectively. While phosphorylation of STAT5 was observed in 4 out of 7 patients, phosphorylation of STAT3 or FLT3 was not detected in all cases examined. Telomere length varied from 2.7 kb to 6.0 kb in de novo AML, accompanied by an increased level of telomerase activity in 4 of 5 patients with de novo AML. In contrast, all MDS-OL cases showed a similar telomere length of 4-5 kb. These results indicate that consideration should be given to the differences of molecular mechanisms in the pathogenesis of de novo AML and MDS-OL for the treatment strategy of AML.

Telomerase, a ribonucleoprotein enzyme which can compensate for the erosion of telomeric repeats each cell division, is considered to be associated with cellular immortality. The genes encoding the major components of human telomerase, TERC for telomerase RNA component, TEP1 for a human homolog of p80, and TERT for catalytic subunit, have been cloned. In addition, TERF1 and TERF2, encoding telomeric repeat binding proteins, have been cloned and shown to be involved in telomere length regulation and the protection of chromosome end fusions, respectively. Although the activity level of telomerase is similar at each stage of the cell cycle in telomerase-positive cells, it is generally upregulated with cellular proliferation and repressed with withdrawal from cell cycle. Although biological mechanisms regulating telomerase activity and telomere length remain elucidated, telomerase activity can be detected in approximately 85% of human malignant neoplasms, and the telomerase is a promising and novel tumor marker for clinical diagnosis of cancer, as well as a novel target of anticancer therapy.

Telomerase is a ribonuclear protein that is detected in more than 90% of primary cancer tissues using a telomeric repeat amplification protocol (TRAP) assay, thus, telomerase is considered to be a novel marker for cancer. Telomerase activity is not detectable in somatic cells, except for hematopoietic cells and cryptic cells in the intestine and hair follicles, thus, quantitation of telomerase is important to delineate clinical implication of telomerase activity. We have developed semiquantitative fluorescence-based TRAP assay. Moreover, we also developed an in situ TRAP assay that detects telomerase activity at the cellular level. Thus, using these TRAP assays, we are able to detect telomerase activity in various kinds of extracts or cytological specimens and therefore these applications may have additive information in the early detection of cancer and monitoring disease condition.

Human papillomavirus type 16 is a strong ethiologic agent of tumor development in human mucous epithelia such as uterine cervix. The virus two early genes, E6 and E7, have been characterized to be oncogenes that transform various cells and cell lines in vitro. Extensive analyses of E6 and E7 proteins revealed that these two oncoproteins interacted with cellular tumor suppressors, p53 and pRb, respectively. Thus, oncogenic potentials of the E6/E7-genes could be explained in part by the consequence of crosstalk of E6/E7-proteins and the cell cycle regulators. Furthermore, telomerase activation that protects telomere erosion appeared to by necessary for HPV16-oncogenesis and was demonstrated in HPV16-immortalized human epithelial cell lines. This line of evidence now enabled us to describe, but not fully, a scheme of an earlier process of human carcinogenesis induced by this virus type.

The molecular genetics of mitochondria relevant to NIDDM is described, because the mutations in mitochondrial DNA cause diabetes. The non-Mendelian genetics, including maternal inheritance, heteroplasmy, stochastic segregation are characteristic of mitochondrial gene. Since aging causes rapid loss of mitochondrial function, which results in the retardation of insulin secretion via ATP-sensitive K-channel. This loss is not caused by the age-dependent mutation in the mitochondrial DNA, but by a nuclear aging, perhaps accompanied by the shortening of telomere. This was shown by the cybrid experiment. The aged mitochondria in cytoplasts are transferred to immortal rho = cells (cells devoid of mitochondrial DNA) and restored its oxidative and transcriptional activities (J. Biol. Chem. 269:6878, 1994). Thus, the control mechanism of transcription in mitochondria has been analyzed. The genes for mitochondrial transcription factor (mtTF1) an MRP-RNA have been sequenced and their regulatory elements are found (BBRC 194:544, 1993 etc.) The mutations in mitochondrial DNA that cause diabetes have been found, and from the stochastic segregation of the heteroplasmic mutated mitochondrial DNA, we could explain why the MELAS mutation is concentrated in some tissue (J. Neurol. Sci. 120:174, 1993). Although there have been many reports on the mitochondrial mutations found in diabetic patients, we have to be careful on polymorphism (J. Biol. Chem. in press).