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

Characterization of the novel mutant A78T-HERG from a long QT syndrome type 2 patient: Instability of the mutant protein and stabilization by heat shock factor 1

Background:The human ether-a-go-go-related gene (HERG) encodes the α-subunit of rapidly activating delayed-rectifier potassium channels. Mutations in this gene cause long QT syndrome type 2 (LQT2). In most cases, mutations reduce the stability of the channel protein, which can be restored by heat shock (HS). Methods: We identified the novel mutant A78T-HERG in a patient with LQT2. The purpose of the current study was to characterize this mutant protein and test whether HS and heat shock factors (HSFs) could stabilize the mutant protein. A78T-HERG and wild-type HERG (WT-HERG) were expressed in HEK293 cells and analyzed by immunoblotting, immunoprecipitation, immunofluorescence, and whole-cell patch clamping. Results: When expressed in HEK293 cells, WT-HERG gave rise to immature and mature forms of the protein at 135 and 155 kDa, respectively. A78T-HERG gave rise only to the immature form, which was heavily ubiquitinated. The proteasome inhibitor MG132 increased the expression of immature A78T-HERG and increased both the immature and mature forms of WT-HERG. WT-HERG, but not A78T-HERG, was expressed on the plasma membrane. In whole-cell patch clamping experiments, depolarizing pulses evoked E4031-sensitive HERG channel currents in cells transfected with WT-HERG, but not in cells transfected with A78T-HERG. The A78V mutant, but not A78G mutant, remained in the immature form similarly to A78T. Maturation of the A78T-HERG protein was facilitated by HS, expression of HSF-1, or exposure to geranyl geranyl acetone. Conclusions: A78T-HERG was characterized by protein instability and reduced expression on the plasma membrane. The stability of the mutant was partially restored by HSF-1, indicating that HSF-1 is a target for the treatment for LQT2 caused by the A78T mutation in HERG

Study on the neovascularization of limb ischemia induced by pretreatment with fucoidan

【Background】Peripheral artery disease（ PAD） caused the poor prognosis of the patients. The life style intervention is necessary to protect patients from PAD. Fucoidan is the member of the polysaccharide originated from Mozuku sea weed, which is useful supplement for life style related disease. 【Purpose】We examined whether fucoidan could improve limb ischemic lesions in mice and rats. 【Method and Result】We created the ischemic limb model of mice and rats and evaluated their blood flow using the laser Doppler blood perfusion. We separated them into three groups, control group （without any treatment），post-treatment group （treated with fucoidan after ischemia for 4 weeks），and pre- and post-treatment group （treated with fucoidan before ischemia for 1 week and after ischemia for 4 weeks）．Fucoidan significantly improved the blood perfusion of ischemic limb in pre- and post-treatment group compared to those in control group. Neovascularization was remarkably increased in muscle of ischemic limb in pre- and post-treatment group compared to those in control group. Fucoidan did not improve either the blood perfusion or neovascularization in post-treatment group. 【Conclusion】Pretreatment with fucoidan through oral administration would be a promising supplement for protection from PAD

Cited4 is related to cardiogenic induction and maintenance of proliferation capacity of embryonic stem cell-derived cardiomyocytes during in vitro cardiogenesis.

Cardiac progenitor cells have a limited proliferative capacity. The CREB-binding protein/p300-interacting transactivator, with the Glu/Asp-rich carboxy-terminal domain (Cited) gene family, regulates gene transcription. Increased expression of the Cited4 gene in an adult mouse is associated with exercise-induced cardiomyocyte hypertrophy and proliferation. However, the expression patterns and functional roles of the Cited4 gene during cardiogenesis are largely unknown. Therefore, in the present study, we investigated the expression patterns and functional roles of the Cited4 gene during in vitro cardiogenesis. Using embryoid bodies formed from mouse embryonic stem cells, we evaluated the expression patterns of the Cited4 gene by quantitative reverse transcriptase-polymerase chain reaction. Cited4 gene expression levels increased and decreased during the early and late phases of cardiogenesis, respectively. Moreover, Cited4 gene levels were significantly high in the cardiac progenitor cell population. A functional assay of the Cited4 gene in cardiac progenitor cells using flow cytometry indicated that overexpression of the Cited4 gene significantly increased the cardiac progenitor cell population compared with the control and knockdown groups. A cell proliferation assay, with 5-ethynyl-2'-deoxyuridine incorporation and Ki67 expression during the late phase of cardiogenesis, indicated that the number of troponin T-positive embryonic stem cell-direived cardiomyocytes with proliferative capacity was significantly greater in the overexpression group than in the control and knockdown groups. Our study results suggest that the Cited4 gene is related to cardiac differentiation and maintenance of proliferation capacity of embryonic stem cell-derived cardiomyocytes during in vitro cardiogenesis. Therefore, manipulation of Cited4 gene expression may be of great interest for cardiac regeneration

The <i>Cited4</i> gene is expressed transiently during the early cardiac stage of <i>in vitro</i> cardiogenesis.

<p>(A-D) mRNA expression levels of lineage marker genes during <i>in vitro</i> cardiogenesis were measured by qRT-PCR and normalized to <i>β-actin</i> mRNA levels. <i>Rex1</i>, <i>Bra</i>, <i>Flk1</i>, and <i>Nkx2</i>.<i>5</i> genes were lineage markers for undifferentiated pluripotent stem cells, early primitive mesodermal cells, lateral mesodermal cells, and cardiac progenitor cells, respectively (N = 4). (E) The proportion of beating EBs, a classical <i>in vitro</i> cardiogenesis marker, was evaluated during <i>in vitro</i> cardiogenesis (N = 3, n = 240). (F) mRNA expression levels of the <i>Cited4</i> gene during <i>in vitro</i> cardiogenesis were measured by qRT-PCR and normalized to <i>β-actin</i> mRNA levels (N = 3). N: number of experiments; n: total number of EBs counted. *<i>P</i> < 0.05 vs. <i>Rex1</i> expression at day 4.5, <i>Bra</i> expression at day 3.5, <i>Flk1</i> expression at day 0, <i>Nkx2</i>.<i>5</i> expression at day 0, <i>Cited4</i> expression at day 0, and proportion of beaing EBs at day 5.0.</p

Sequence of the primers for qRT-PCR.

<p>Sequence of the primers for qRT-PCR.</p

The <i>Cited4</i> gene is expressed specifically in cardiac progenitor cell populations during <i>in vitro</i> cardiogenesis.

<p>(A) Time-dependent expression of GFP under the control of a Rex1-promoter was evaluated at day 0 of undifferentiation embryonic stem cells and day 3.5 in the EBs formed with the Rex1-ht7 cell line. Scale bar = 100 μm. (B) Time-dependent expression of GFP under the control of a Bra-promoter was evaluated at day 3.5, day 4.5, day 5.5, and day 7.5 in the EBs formed with the Bra-ht7 cell line. Scale bar = 100 μm. (C) To obtain the pluripotent embryonic stem cell population, the Rex1 and E-cadherin double positive cell population was isolated from the Rex1-ht7 cell line at day 0 before forming the EBs. (D) To obtain the primitive mesodermal cell population and the early lateral mesodermal cell population, the Bra-positive and Flk1-negative cell population and the Bra-negative and Flk1-positive cell population were isolated from the Bra-ht7 cell line at day 4.5 in the EBs, respectively. (E) To obtain the cardiac progenitor cell population, the Nkx2.5-positive and Flk1-negative cell population was isolated from the hcgp7 cell line at day 7.5 in the EBs. (F) The <i>Cited4</i> gene expressions in the isolated lineage marker-positive cell populations were evaluated by qRT-PCR and normalized to <i>β-actin</i> mRNA levels (N = 3). Values are presented as the mean ± SEM. N: number of experiments. *<i>P</i> < 0.05 vs. Rex1-positive embrynic stem cells.</p