[Report based on fiscal 2010 diagnostic X-ray equipment questionnaire survey (conditions of radiography)].

This is the eighth investigation which has been carried out every 5 years since 1974 for the purpose of grasping the trend of X-ray devices and the radiographic condition. We gathered it up mainly on a radiographic condition, in this report. As for the chest radiography and double contrast gastrography, introduction of the flat panel detector (FPD) advanced in comparison with the last survey. Ratio of the imaging system at chest radiography was 65% for computed radiography (CR), 33% for FPD, 1% for screen/film (S/F), and 1% for others. The radiographic condition of FPD was current time product less than CR. Ratio of the imaging system at gastrography was 3% for CR, 48% for FPD, 34% for image intensifier-digital radiography (I.I.-DR), and 15% for S/F. The tube voltage and the exposure time were similar to the last survey time, but the tube current became lower. Through this survey, the change of the radiographic condition was seen in the radiography part where introduction of the FPD advanced. We think the continuous survey is necessary in future.

Involvement of WRN helicase in immortalization and tumorigenesis by the telomeric crisis pathway (Review).

The repeated replication of cells shortens telomeres, culminating in their instability, after which most cells cease to replicate and die. However, a small fraction of the cells become immortalized by maintaining telomeres with activated telomerase activity. It has been proposed that WRN helicase encoded by the WRN gene, the causative gene of Werner syndrome (WS), is required for immortalization by the telomeric crisis pathway (TCP) in a system that uses lymphoblastoid cell lines transformed by the Epstein-Barr virus. Taken together, these characteristics indicate that WRN helicase is also required for the immortalization of epithelial cells by TCP and consequent carcinogenesis, suggesting that the tumorigenesis of epithelial cells by TCP is suppressed in WS lacking the WRN helicase function. Notably, in WS the pathway of alternative lengthening of telomeres without activation of telomerase activity has been suggested to be involved in immortalization and tumorigenesis. This factor is consistent with the abundance of non-epithelial cancers in WS in that the ratio of epithelial to non-epithelial cancers is approximately 1:1 in WS patients compared to 10:1 in the general population. A hypothetical scheme showing the role of WRN helicase in immortalization by means of the supposed 'breakage-fusion-bridge cycle' of chromosomes at telomeric crisis is described.

Establishment of functional telomerase immortalized human hepatocytes and a hepatic stellate cell line for telomere-targeting anticancer drug development.

We previously reported that the telomere-targeting drug telomestatin induces apoptosis accompanied by G-tail reduction and dissociation of binding protein TRF2 from telomeres in cancer cell lines but not normal or human telomerase reverse transcriptase (hTERT)-immortalized cells. Because telomere-targeting drugs induce growth arrest in normal cells at higher doses, their development is dependent on the ability to predict toxicity before in vivo use, but no models for this are available. Here, we established two new cell lines, telomerase immortalized human fetal hepatocytes, Hc3716-hTERT, and telomerase immortalized hepatic stellate cells, NPC-hTERT. Examinations showed that Hc3716-hTERT maintained normal mammalian cell morphology, cell growth, albumin expression, and wild-type p53 responsiveness, whereas NPC-hTERT maintained hepatic stellate-like morphology, expression of hepatic stellate markers, alpha-smooth muscle actin, and secretion of type I collagen, an extracellular matrix protein. Given our finding that telomere G-tail length in Hc3716 cells was decreased in senescence and increased by hTERT infection, we next examined the effect of high-dose telomestatin-induced telomere dysfunction and G-tail shortening on cellular functions in Hc3716-hTERT cells. Interestingly, telomestatin decreased expression of cytochrome P450 (CYP) family members CYP3A3/4, CYP3A5, and CYP3A7, mRNA and induced albumin expression at both mRNA and protein levels. These gene expression responses to telomestatin were similar to those of the normal parental cell Hc3716. These established cell lines thus represent the first model for predicting the side-effects of telomere-targeting drugs in normal cells, and should be powerful tools in the development of these drugs.

MOV10 as a novel telomerase-associated protein.

A novel telomerase-associated protein was isolated from porcine testis. The 115-kDa protein, purified with telomerase activity, was molecular cloned using human cDNA library, and identified as MOV10. The expression levels of both MOV10 mRNA and MOV10 protein in cancer cells were 2-3 times higher than that of the normal cells, and MOV10 mRNA was highly expressed in human testis and ovary. The anti-MOV10 antibody precipitated the telomerase activity from cancer cell extracts, and inhibited the telomerase activity in vitro. Sf9-expressed MOV10 protein bound to G-rich strand of both single- and double-stranded telomere-sequenced DNA, but not to single C-rich strand. ChIP assay showed the binding of MOV10 to telomere region in vivo. These data suggest that MOV10 is involved in the progression of telomerase-catalyzing reaction via the interaction of telomerase protein and telomere DNA.

[Report based on fiscal 2005 diagnostic X-ray equipment questionnaire survey-conditions of radiography-].

In this report, we summarize the survey of diagnostic X-ray equipment conducted in 2005, focusing on exposure conditions. What we would like to point out in this report is how prevalent digital image receptor systems have become, including flat panel detectors (FPD), which did not exist in our last survey five years ago. FPD have become popular very quickly among the digital receptor systems used in general radiography. Twenty-three percent of all chest standing-position X-ray equipment now have FPD. Since FPD has become prevalent, exposure conditions are becoming somewhat different from screen/film systems (s/f) and computed radiography (CR). While the image receptor system is expected to shift to FPD, we would like to continue our survey to determine what exposure conditions will be like in the near future.

[Report based on fiscal 2005 diagnostic X-ray equipment questionnaire survey-A photography system-].

In accordance with a questionnaire survey of diagnostic X-ray equipment enforced in 2005, this report was focused mainly on a photography device. The paper questionnaire was mailed to 667 institutions, and the collection rate was 20.4%. Introductions of digital photographic devices are progressing and flat panel detectors (FPD) are spreading in addition to computed radiography (CR). Examination revealed that the spread rate of FPD was 14.4%. The inverter-type device occupied 73.4% in group of high-voltage-generation devices. We reason that the spread rate indicates changeover to an inverter-type device. Changes in photography equipment and photography devices will bring change in photographic technology. Such investigation will be necessary in the future.

Floating culture promotes the maintenance of hematopoietic stem cells.

In this study we examined the effect of the specific gravity of culture medium on the frequency of hematopoietic stem cell (HSC) maintenance. We used a newly developed high-specific-gravity media. Bone marrow cells were isolated and cultured, and HSC activity was evaluated. The number of hematopoietic progenitor/stem cells was markedly higher in the medium with high specific gravity. In high-specific-gravity media, cells did not precipitate, maintenance of HSCs was increased, and there was a concomitant accumulation of beta-catenin. This novel technique for maintaining HSC populations provides an important new tool for studies in regenerative medicine.

Telomerase activation induces elongation of the telomeric single-stranded overhang, but does not prevent chromosome aberrations in human vascular endothelial cells.

Chromosome aberrations such as loss of chromosome 13 were frequently observed in human endothelial cells from umbilical cord veins (HUVEC). A recent study showed that the length of telomeric single-stranded 3'-overhangs (G-tails) is more important as an essential structure for chromosome maintenance than the net telomere length in telomere t-loop formation. Here, we have examined G-tail length using G-tail telomere HPA in normal and hTERT-transduced HUVECs. We found that forced expression of hTERT in HUVEC induced G-tail as well as total telomere length elongation. G-tail length was well correlated with total telomere length. However, hTERT introduction did not prevent chromosome aberrations such as loss of chromosome 13. Normal characteristics such as morphology, up-regulation of vWF, and tube formation were observed in hTERT-HUVEC as in young normal HUVEC. These results show that chromosome aberrations in HUVEC are independent of telomere G-tail and total telomere attrition.

[Mechanism of cell proliferation--cell cycle, oncogenes, and senescence].

Cell proliferation is regulated through a transition between the G0 phase and cell cycle. We isolated a mammalian temperature-sensitive mutant cell line defective in the function from the G0 phase to cell cycle. Senescent human somatic cells fail to enter into the cell cycle from the G0 phase with stimulation by any growth factor. Telomere shortening was found to be a cause of cellular senescence, and reexpression of telomerase immortalized human somatic cells. Immortalized human somatic cells showed normal phenotypes and were useful not only for basic research but also for clinical and applied fields. The importance of p53 and p21 activation/induction i now well accepted in the signal transduction process from telomere shortening to growth arrest, but the precise mechanism is largely unknown as yet. We found that the MAP kinase cascade and histone acetylase have an important role in the signaling process to express p21. Tumor tissues and cells were found to have strong telomerase activity, while most normal somatic human tissues showed very weak or no activity. Telomerase activity was shown to be a good marker for early tumor diagnosis because significant telomerase activity was detected in very early tumors or even in some precancerous tissues compared with adjacent normal tissues. Telomere/telomerase is a candidate target for cancer chemotherapeutics, and an agent that abrogated telomere functions was found to kill tumor cells effectively by inducing apoptosis whereas it showed no effect on the viability of normal cells.

Mitogenic signalling and the p16INK4a-Rb pathway cooperate to enforce irreversible cellular senescence.

The p16(INK4a) cyclin-dependent kinase inhibitor has a key role in establishing stable G1 cell-cycle arrest through activating the retinoblastoma (Rb) tumour suppressor protein pRb in cellular senescence. Here, we show that the p16(INK4a) /Rb-pathway also cooperates with mitogenic signals to induce elevated intracellular levels of reactive oxygen species (ROS), thereby activating protein kinase Cdelta (PKCdelta) in human senescent cells. Importantly, once activated by ROS, PKCdelta promotes further generation of ROS, thus establishing a positive feedback loop to sustain ROS-PKCdelta signalling. Sustained activation of ROS-PKCdelta signalling irreversibly blocks cytokinesis, at least partly through reducing the level of WARTS (also known as LATS1), a mitotic exit network (MEN) kinase required for cytokinesis, in human senescent cells. This irreversible cytokinetic block is likely to act as a second barrier to cellular immortalization ensuring stable cell-cycle arrest in human senescent cells. These results uncover an unexpected role for the p16(INK4a)-Rb pathway and provide a new insight into how senescent cell-cycle arrest is enforced in human cells.

Microgravity potentiates stem cell proliferation while sustaining the capability of differentiation.

A three-dimensional (3D) clinostat is a device for generating multidirectional G force, resulting in an environment with an average of 10(3) G. Here we report that human mesenchymal stem cells (hMSCs) cultured in a 3D-clinostat (group CL) showed marked proliferation (13-fold in a week) compared with cells cultured under normal conditions of 1 G (group C) (4-fold in a week). Flow cytometry revealed a 6-fold increase in the number of hMSCs double-positive for CD44/CD29 or CD90/CD29 in group CL after 7 days in culture, compared with group C. Telomere length remained the same in cells from both groups during culturing. Group C cells showed increasing expression levels of type II collagen and aggrecan over the culture period, whereas group CL cells showed a decrease to undetectable levels. Pellets of hMSCs from each group were explanted into cartilagedefective mice. The transplants from group CL formed hyaline cartilage after 7 days, whereas the transplants from group C formed only noncartilage tissue containing a small number of cells. These results show that hMSCs cultured in a 3D-clinostat possess the strong proliferative characteristic of stem cells and retain their ability to differentiate into hyaline cartilage after transplantation. On the contrary, cells cultured in a 1-G environment do not maintain these features. Simulated microgravity may thus provide an environment to successfully expand stem cell populations in vitro without culture supplements that can adversely affect stem cell-derived transplantations. This method has significant potential for regenerative medicine and developmental biology.

Novel electrical stimulation sets the cultured myoblast contractile function to 'on'.

OBJECTIVE: In the present study, the effect of electrical stimulation was examined for the ability to induce morphological, physiological, and molecular biological effects on myoblasts during cell differentiation. METHODS: L6 rat myoblasts were electrically stimulated by newly developed methods on culture days 6, 8, 10 and 12. RESULTS: This electrical stimulation accelerated the appearance of myotubes, and subsequently produced spontaneously contracting muscle fibers. Measurement of membrane potential showed that the contracting cell had functional ion channels and gap junctional intercellular communication. In the electrically stimulated cells, an enhanced expression of MyoD family and M-cadherin was also observed. Expression of connexin 43 was increased and maintained at a high level in the electrically stimulated cells. CONCLUSION: This is the first demonstration of in vitro induction of myoblasts in spontaneously contractile muscle fibers by intermittent stimulation. This novel method for induction of myoblast differentiation represents an important advance in cell therapy.

G-tail telomere HPA: simple measurement of human single-stranded telomeric overhangs.

Accurate measurement of telomeric 3'-overhang (G-tail) lengths is essential for investigation of the biological effects of telomere dysfunction. G-tail telomere hybridization protection assay (Gt-telomere HPA) has the advantages of being simple to perform, accurate and highly sensitive for G tails as short as 20 nucleotides. Furthermore, Gt-telomere HPA is specific and quantitative for human G tails, and can be used to assay cell lysates as well as genomic DNA.

Establishment of cementoblast cell lines from rat cementum lining cells by transfection with temperature-sensitive simian virus-40 T-antigen gene.

Defining the regulatory mechanisms promoting differentiation and proliferation of cementoblasts has not been well understood, because of the lack of cell models in vitro. To establish an in vitro cell model for the cementoblasts, extracted rat molars obtained from 8-week-old rats were used. Cells lining the root surface (cemetoblasts) were obtained by an enzymatic digestion method, and immediately immortalized by transfection of thermolabile SV40 T-antigen gene. The transfected cementum lining cell clones, RCM-C3 and -C4, were maintained for more than 200 population doublings (PD), while the original cells stopped their growth at 60 PD. Thus, immortalized cell lines decreased expression of SV40 T-antigen and subsequently cell proliferation at non-permissive temperature (39 degrees C). Reverse-transcribed-polymerase chain reaction indicated expression of gene for type I collagen, alkaline phosphatase (ALP), osteopontin, and osteocalcin mRNA at both permissive (33 degrees C) and non-permissive (39 degrees C) temperatures. RCM-C4 expressed higher bone siaploprotein (BSP) mRNA than RCM-C3, and further RCM-C4 showed higher BSP mRNA at 39 degrees C than 33 degrees C. High ALP activity and mineralized nodule formation were observed at 39 degrees C in both cell lines. These findings suggested that the cell lines, RCM-C3 and -C4, are useful model for studying the regulatory mechanisms of differentiation and proliferation of cementoblasts.

G1P3, an interferon inducible gene 6-16, is expressed in gastric cancers and inhibits mitochondrial-mediated apoptosis in gastric cancer cell line TMK-1 cell.

Expression of an interferon inducible gene 6-16, G1P3, increases not only in type I interferon-treated cells but also in human senescent fibroblasts. However, the function of 6-16 protein is unknown. Here we report that 6-16 is 34 kDa glycosylated protein and localized at mitochondria. Interestingly, 6-16 is expressed at high levels in gastric cancer cell lines and tissues. One of exceptional gastric cancer cell line, TMK-1, which do not express detectable 6-16, is sensitive to apoptosis induced by cycloheximide (CHX), 5-fluorouracil (5-FU) and serum-deprivation. Ectopic expression of 6-16 gene restored the induction of apoptosis and inhibited caspase-3 activity in TMK-1 cells. Thus 6-16 protein has anti-apoptotic function through inhibiting caspas-3. This anti-apoptotic function is expressed through inhibition of the depolarization of mitochondrial membrane potential and release of cytochrome c. By two-hybrid screening, we found that 6-16 protein interacts with calcium and integrin binding protein, CIB/KIP/Calmyrin (CIB), which interacts with presenilin 2, a protein involved in Alzheimer's disease. These protein interactions possibly play a pivotal role in the regulation of apoptosis, for which further detailed analyses are need. These results overall indicate that 6-16 protein may have function as a cell survival protein by inhibiting mitochondrial-mediated apoptosis.

Establishment of immortalized clonal cells derived from periodontal ligament cells by induction of the hTERT gene.

Since the periodontal ligament (PDL) contains a heterogeneous cell population, it is challenging to identify all cell types within the tissue and to determine whether they function alone to produce tissue components or interact with other cell types. Further, it is difficult to isolate and expand single cell clones from PDL cells, as normal cells have a limited life span and are phenotypically unstable. In the present study, we inserted the human telomerase reverse transcriptase (hTERT) gene, which encodes the catalytic subunit of the telomerase holoenzyme, into normal human periodontal ligament (HPL) cells and successfully obtained single cell clones. Expression of the inserted gene and telomerase activity in each of the clones was confirmed. Unlike the original HPL cells, at the end of the study (day 120), clone populations continued to actively double without phenotypic alteration. Osteogenic characteristics were present in some but not all clones. In conclusion, immortalization of HPL cells was successfully accomplished by transduction with the hTERT gene. This is the first report of immortalization of different cell types derived from PDL.

Terminal telomere repeats are actually short in telomerase-negative immortal human cells.

Telomerase-negative immortal human cells maintained telomere length by a mechanism called alternative lengthening of telomeres (ALT mechanism). These cells (ALT cells) have two prominent characteristics of long telomere repeats at each chromosome end revealed by Southern blotting (terminal restriction fragments: TRF) and the presence extrachromosomal telomere repeat (ECTR) DNA. We report here that the TRF length of ALT cells revealed by the conventional unidirectional (UD) current or pulse-field (PF) current electrophoresis appeared to be over estimated. The TRF length determined by the pulse inverse-field (PIF) current electrophoresis (2-9 kbp depending upon cell lines) was much smaller than that (ca. 23 kbp) by UD or PF current electrophoresis. These results were in consistent with very weak telomere staining in situ at chromosome ends in ALT cells. When a mixture of HinfI-digested genomic DNA of human diploid fibroblasts and synthetic telomere repeat DNA with similar size of ECTR DNA was electrophoresed using a UD current, the apparent TRF size shifted to larger molecular weight, while the size shift did not occur by PIF current electrophoresis. These results together with other data indicate that the unusually long TRF of ALT cells determined by using conventional electrophoresis is an artifact produced by a complex formed by short TRF and short ECTR DNA.

A highly sensitive and quantitative telomerase activity assay with pancreatic juice is useful for diagnosis of pancreatic carcinoma without problems due to polymerase chain reaction inhibitors: analysis of 100 samples of pancreatic juice from consecutive patients.

BACKGROUND: Early detection of pancreatic carcinoma is difficult even with current diagnostic tools. Novel biomarkers and detection techniques are urgently needed. Telomerase activity is a promising diagnostic marker. However, the conventional telomeric repeat amplification protocol (TRAP) assay is not suitable for clinical application because of its complexity, time-consuming nature, and the effects of polymerase chain reaction (PCR) inhibitors in samples leading to difficulties in quantification. METHODS: The authors used a hybridization protection assay in combination with TRAP (TRAP/HPA) to investigate the effects of PCR inhibitors in pancreatic juice on quantification of telomerase activity. They analyzed 117 consecutive samples of pancreatic juice to determine the feasibility of TRAP/HPA for diagnosis of pancreatic carcinoma. RESULTS: The authors found that TRAP/HPA was 1000-fold more sensitive than the conventional TRAP assay, and that the effects of PCR inhibitors could be avoided by diluting samples. In a large analysis of pancreatic juice samples with TRAP/HPA, 17 samples were excluded from the final analysis because of insufficient follow-up periods or inadequate treatment of the samples. Relative telomerase activity (RTA) in samples from patients with pancreatic carcinoma was significantly higher in comparison to samples from patients with pancreatitis and 13 (61.9%) of 21 samples from patients with pancreatic carcinoma showed high RTA (> 4 U). Meanwhile, high RTAs were observed in 4 of 35 (11.4%) samples from patients with intraductal papillary mucinous tumor and in 1 of 40 samples (2.5%) fom patients without malignant disease. CONCLUSIONS: TRAP/HPA accurately evaluated weak telomerase activity in pancreatic juice samples without the problem due to PCR inhibitors. This large analysis of nonselected pancreatic juice samples suggested that TRAP/HPA is a promising approach for the diagnosis of pancreatic carcinoma.