Keiko Hiyama

Telomere and telomerase in stem cells

Telomeres, guanine-rich tandem DNA repeats of the chromosomal end, provide chromosomal stability, and cellular replication causes their loss. In somatic cells, the activity of telomerase, a reverse transcriptase that can elongate telomeric repeats, is usually diminished after birth so that the telomere length is gradually shortened with cell divisions, and triggers cellular senescence. In embryonic stem cells, telomerase is activated and maintains telomere length and cellular immortality; however, the level of telomerase activity is low or absent in the majority of stem cells regardless of their proliferative capacity. Thus, even in stem cells, except for embryonal stem cells and cancer stem cells, telomere shortening occurs during replicative ageing, possibly at a slower rate than that in normal somatic cells. Recently, the importance of telomere maintenance in human stem cells has been highlighted by studies on dyskeratosis congenital, which is a genetic disorder in the human telomerase component. The regulation of telomere length and telomerase activity is a complex and dynamic process that is tightly linked to cell cycle regulation in human stem cells. Here we review the role of telomeres and telomerase in the function and capacity of the human stem cells.

Human mismatch repair gene, MLH1, is transcriptionally repressed by the hypoxia-inducible transcription factors, DEC1 and DEC2

Tumor hypoxia has been reported to cause a functional loss in DNA mismatch repair (MMR) system as a result of downregulation of MMR genes, although the precise molecular mechanisms remain unclear. In this study, we focused on the downregulation of a key MMR gene, MLH1, and demonstrated that hypoxia-inducible transcription repressors, differentiated embryo chondrocytes (DEC1 and 2), participated in its transcriptional regulation via their bindings to E-box-like motif(s) in MLH1 promoter region. In all cancer cell lines examined, hypoxia increased expression of DEC1 and 2, known as hypoxia-inducible genes, but decreased MLH1 expression in an exposure time-dependent manner at both the mRNA and protein levels. Co-transfection reporter assay revealed that DEC1 and, to greater extent, DEC2 as well as hypoxia-repressed MLH1 promoter activity. We further found that the action was remarkably inhibited by trichostatin A, and identified a possible DEC-response element in the MLH1 promoter. In vitro electrophoretic gel mobility shift and chromatin immunoprecipitation assays demonstrated that DEC1 or 2 directly bounds to the suggested element, and transient transfection assay revealed that overexpression of DEC2 repressed endogenous MLH1 expression in the cells. Hypoxia-induced DEC may impair MMR function through repression of MLH1 expression, possibly via the histone deacethylase-mediated mechanism in cancer cells.

Activation of the hypoxia-inducible factor-1 in overloaded temporomandibular joint, and induction of osteoclastogenesis

Vascular endothelial growth factor (Vegf) was previously shown to be expressed specifically in the condylar cartilage of temporomandibular joint-osteoarthritis (TMJ-OA) model rats. Here we demonstrate for the first time that hypoxia-inducible factor-1alpha (Hif-1alpha) is activated in mature chondrocytes of temporomandibular joint-osteoarthritis (TMJ-OA) model rat by mechanical overload, and that activated Hif-1 in chondrocytes can induce osteoclastogenesis via repression of osteoprotegerin (Opg) expression. In rat TMJs, degeneration of the condylar cartilage became prominent in proportion to the duration of overloading. Hif-1alpha expression was observed specifically in mature and hypertrophic chondrocytes, and Hif-1alpha-positivity, level of Vegf expression, and tartrate-resistant acid phosphatase (TRAP)-positive cell numbers all increased in the same manner. When ATDC5 cells induced differentiation by insulin were cultured under hypoxia, Hif-1alpha induction was observed in mature stage, but not in immature stage. Inductions of Hif-1-target genes showed a similar expression pattern. In addition, expression of Opg decreased in hypoxia, and Hif-1alpha played a role, in part, in its regulation.

Telomeres and Telomerase in Cancer

Telomerase can compensate for telomere shortening and helps prevent cellular senescence in eukaryotic cells. In humans, only specific germline cells and the vast majority of cancer cells have sufficient activity for indefinite proliferation. Lymphocytes and stem/progenitor cells in self-renewal tissues have weak activity for extension of their lifespan, but they still undergo replicative senescence. In contrast, most somatic cells do not have telomerase activity and display a finite replicative lifespan. Heterozygousmutations in either of principal telomerase components, TERT or TERC, cause telomere dysfunction and unexpectedly early senescence to stem cells of renewal tissues. Thus, restoration of telomere function in regenerative medicine via telomerase expression and inhibition of telomerase as an anticancer strategy is a double-edged sword of telomeres and telomerase in clinical medicine.

Detection of CpG island hypermethylation of caspase-8 in neuroblastoma using an oligonucleotide array.

The caspase‐8 gene (CASP8) is frequently inactivated in unfavorable neuroblastomas through DNA methylation. The present study utilized oligoarrays to evaluate the methylation status of a CpG island located between exons 2 and 3 of caspase 8 in neuroblastomas.

Genetic variations in detoxification enzymes and HIF‐1α in Japanese patients with COPD

Introduction:  Genetic factors contribute as major determinants in the pathophysiological mechanisms of chronic obstructive pulmonary disease (COPD). Therefore, identification of candidate genes and various gene polymorphisms have improved our understanding of COPD.

Telomerase activation without shortening of telomeric 3′‐overhang is a poor prognostic factor in human colorectal cancer

Our previous report demonstrated a good correlation between high telomerase activity of cancer tissues and a poor prognosis of patients with colorectal cancers, except for several cases. To elucidate the additional factors that contribute to patient prognosis, the correlation among the expression levels of telomere binding proteins (TBP), the lengths of telomeres, the lengths of telomere 3′‐overhang (3′‐OH) and telomerase activity in 106 paired colorectal cancer and corresponding noncancerous mucosa (NCM) specimens were examined. The expression levels of eight TBP genes (TRF1, TRF2, TIN2, TANK1, TANK2, POT1, RAP1 and TPP1) were analyzed. Among the 106 cases, 35 cases had shortened telomeres (<7 kb), 15 had shortened 3′‐OH (3′‐OH length ratio of cancer/NCM <0.5) and 88 were classified as telomerase‐activated cancers (activity ratio of cancer/NCM >2). Comparison between NCM and cancer in each case showed that all TBP except for POT1 were downregulated in cancers. A survival analysis using a Cox proportional hazard model showed that the survival rate of the telomerase‐activated cases with shortened 3′‐OH and that of telomerase‐inactivated cases were significantly better than that of telomerase‐activated cases without 3′‐OH shortening, that is, restored or maintained 3′‐OH (P = 0.018). In the telomerase‐activated cancers, the length of 3′‐OH was significantly correlated with the expression levels of POT1. Elongation of telomeric overhang by telomerase, which might be regulated by POT1, may contribute to the increase of malignant potential in colorectal cancers. (Cancer Sci 2011; 102: 330–335)

EMP3 as a candidate tumor suppressor gene for solid tumors

Background: Epithelial membrane protein 3 (EMP3), was recently reported to be a tumor suppressor gene for several solid tumors, and is drawing attention as a novel prognostic marker, since its expression level or hypermethylation of the promoter region is associated with clinical prognosis in neuroblastoma and esophageal cancer. However, some controversial data were also observed in gliomas and breast cancers, and there seems to be more than deletion/hypermethylation to its silencing mechanisms. Objective: To clarify the discrepancies in the biological behavior of EMP3 among the different organ-derived malignancies or histologies and validate the potential of EMP3 as a tumor suppressor for solid tumors. Methods: Literature dealing with EMP3 in the PubMed database was reviewed. Results/conclusions: EMP3 is a novel tumor suppressor gene in some kinds of malignancies, but not all, at the step of cellular immortalization rather than carcinogenesis. It may become a potent prognostic marker and a therapeutic target in such tumors.

Telomere and Telomerase for the Regulation of Stem Cells

Telomeres, guanine-rich tandem DNA repeats of chromosomal ends, provide chromosomal stability, and cellular replication causes their loss. In somatic cells, the activity of telomerase, a reverse transcriptase that can elongate telomeric repeats, is usually diminished after birth so that the telomere length is gradually shortened with cell divisions and triggers cellular senescence. In embryonic stem cells, telomerase is activated and maintains telomere length and cellular immortality. On the other hand, in adult stem cells, the level of telomerase activity is low and insufficient to maintain telomere length. Thus, even in stem cells, except for embryonic stem cells and cancer stem cells, telomere shortening occurs during replicative aging, possibly at a slower rate than that in normal somatic cells. In the past few years, the importance of telomere maintenance in human stem cells has been highlighted by the studies on dyskeratosis congenita, aplastic anemia, and idiopathic pulmonary fibrosis, a part of which are genetic disorders in the human telomerase component and are characterized by premature loss of tissue regeneration with stem cell dysfunction. The regulation of telomere length and telomerase activity is a complex and dynamic process that is tightly linked to cell cycle regulation in human stem cells. Here we review the role of telomeres and telomerase in human stem cells.

Exploration of the genes responsible for unlimited proliferation of immortalized lung fibroblasts

Regulation mechanism of lung fibroblast proliferation remains unknown. To elucidate the key molecules in it, the authors here established mortal and immortal nontransformed lung fibroblast cell line/strains with elongated life span by telomerase reversetranscriptase gene transfection. Comparing the expression profiles of them, 51 genes were explored to be the candidates responsible for regulation of cellular proliferation of lung fibroblasts. This set of fibrobrast strains of same origin with different proliferative capacities may become useful model cells for research on lung fibroblast growth regulation and the candidate genes explored in this study may provide biomarkers or therapeutic targets of pulmonary fibrosis.