Role of Werner syndrome gene product helicase in carcinogenesis and in resistance to genotoxins by cancer cells.

Werner syndrome (WS) is an autosomal recessive genetic disorder causing premature aging, and WRN has been identified as the causative gene of WS. The product of the WRN gene (WRN) acts as a DNA helicase with exonuclease activity, and data have accumulated showing that the WRN gene strongly participates in carcinogenesis: (1) the normal WRN gene likely participates in the immortalization of B-lymphoblastoid cell lines through telomeric crisis caused by telomere shortening, (2) a much higher incidence of rare cancers occurs in WS patients than in other kinds of patients, and (3) levels of WRN expressed in virus-transformed cells and cancer cells are usually markedly up-regulated and are inversely correlated with the sensitivity of these cells against various genotoxins, including camptothecin. In this paper, we review the events that show a close correlation of the WRN gene and WRN with carcinogenesis and their underlying molecular mechanisms.

Steps involved in immortalization and tumorigenesis in human B-lymphoblastoid cell lines transformed by Epstein-Barr virus.

Epstein-Barr virus (EBV) is closely associated with the generation of various tumors, including Burkitt's lymphoma. Human resting B cells from peripheral blood are easily transformed by EBV to actively proliferating B-lymphoblastoid cell lines (LCLs). These LCLs with normal diploid karyotypes have been believed to be "immortal", without becoming tumorigenic. A series of recent studies, however, indicate that this initial, simple concept needs extensive reconsideration. Most LCLs from normal individuals are mortal because their telomeres shorten. Some LCLs are truly immortalized by developing strong telomerase activity and aneuploidy, accompanied by various other changes: down-regulation of p16/Rb; mutation of the p53 gene; modulation of apoptosis; and sensitivity to various chemical agents. Some post-immortal LCLs additionally develop the ability to form colonies in agarose and even become tumorigenic by developing the ability to grow in nude mice. The genetic background of LCLs markedly affects the frequency of immortalization. In summary, changes of B cells after infection by EBV are roughly divided into two steps: (a) transformation of B cells into LCLs caused by EBV proteins; and (b) immortalization and tumorigenesis of LCLs mainly regulated by the factors of host cells in cooperation with EBV proteins. The new concept as reviewed here is essential for the future study of tumorigenesis by EBV.

A Cell Kinetic and Cytological Study on the Asymmetric Cell Division of Thymic Lymphoblasts of the Embryonic Rat.

Cell division of thymus lymphoid cells from embrynonic and young rats was investigated cytologically on cell smears, focusing attention on asymmetric cell division. Some of thymic lymphoblasts displayed features implicating asymmetric cell division. At the telophase of such cells, two immature daughter cells looked dissimilar: one of them was smaller in size and possessed a more condensed nucleus, compared with the counterpart cell. Furthrmore, in most cases the cytoplasm of the smaller daughter cell was stained with Giemsa more deeply. It was suggested that the asymmetry of the nucleus emerges at anaphase and telophase probably due to some polarized situation of the cytoplasm. Asymmetrically-dividing cells were relatively frequently observed during the developmental period when large lymphoblasts actively transform into smaller lymphocytes :16% to 17% of whole dividing cells were under asymmetric cell division on days 16 and 17 of gestation, while less than 5% on day 19 or thereafter. In correlation with this observation, asymmetrically-dividing cells were more frequently observed among large lymphoblasts than among other smaller cell fractions. These results support the view that the asymmetric cell division may play some essential role in the transformation of large lymphoblasts into smaller lymphocytes.

DEVELOPMENT OF THE EMBRYONIC CHICKEN THYMUS III. UNEQUAL DIVISION OF LYMPHOID CELLS.

Cell division of thymus lymphoid cells from 11- to 17-day old embryonic chickens, as well as chickens just after hatch was investigated on cell smears stained with Giemsa. Unequally dividing cells were observed in the developmental stage of thymocytes. At the telophase of such cells, the cytoplasm of one of two future daughter cells was apparently larger in amount and was sometimes stained deeper than the cytoplasm of its counterpart. Unequal division was also observed in pro-, meta- and anaphase; sometimes a dividing cell had a large cytoplasmic process belonging to one hemisphere, suggesting that only one of the two daughter cells would receive the cytoplasmic process through cell division. The incidence of unequal division calculated by a rough estimation was around 10% of the total cell division between 11 and 13 days of embryonic development, and decreased progressively thereafter.

In vitro establishment of tumorigenic human B-lymphoblastoid cell lines transformed by Epstein-Barr virus.

We studied tumorigenic and phenotypic characteristics of pre- and postimmortal human B-lymphoblastoid cell lines (LCLs) transformed by Epstein-Barr virus (EBV): preimmortal LCLs showed low telomerase activity and a normal diploid karyotype while postimmortal LCLs showed much higher telomerase activity and maintained a clonal aneuploidic state. Among five postimmortal LCLs tested, LCLs N0005 and N6803 formed colonies in agar medium and showed marked aneuploidy, and N6803 was transplantable into nude mice indicating that it had a complete malignant phenotype, but all preimmortal LCLs and the remaining three postimmortal LCLs lacked these characteristics. The products of tumor suppresser genes, p16(INK4A) and pRb, were downregulated in these two LCLs, and the p53 gene was mutated in N0005 LCL. We believe these results showed for the first time that some postimmortal EBV-transformed LCLs can become tumorigenic, contrary to previous reports, and that these LCLs provide an in vitro model of tumorigenesis induced by EBV.

WRN gene and other genetic factors affecting immortalization of human B-lymphoblastoid cell lines transformed by Epstein-Barr virus.

The immortalization of human B-lymphoblastoid cell lines (LCL) transformed by Epstein-Barr virus (EBV) is accompanied by two major events: increase in telomerase activity and change in karyotype from normal diploid to aneuploidy. We investigated the effect of genetic factors on the incidence of immortalization by putting old and new data together to collect enough samples for statistical analysis. Among 50 LCL from normal individuals, 5 LCL (10.0%) were immortalized and the remaining 45 LCL were mortal. None of the 44 LCL (0%; P < 0.031 against normal individuals by chi square test) from patients having Werner syndrome (WS), a recessive genetic disorder showing premature aging, were immortalized. Among 11 LCL from a family with a tendency to have hereditary type 2 diabetes mellitus, 5 LCL (45.5%; P < 0.0040 against normal individuals, P < 0.00001 against WS patients) were immortalized. Duplicated measurements of the lifespan of 33 LCL showed a good coincidence (r=0.785) between the first and second estimations, indicating that each mortal LCL has a predetermined lifespan. These results strongly suggest that the normal WRN gene, the causative gene of WS, is essential for LCL to immortalize, and genetic factor(s) of a family having diabetes mellitus increases immortalization, implicating that host genetic factors affect immortalization of EBV and probably carcinogenesis by EBV.

Proteome analysis of Epstein-Barr virus-transformed B-lymphoblasts and the proteome database.

The proteome is the entire protein complement of the genome expressed in a particular cell, tissue, or organism at a given time under a specific set of environmental conditions. Proteomics is a combinatorial methodology to comprehensively analyze the proteome. The general protocol of the expression proteomics consists of advanced methods of high-resolution protein separation, high-quality image analysis and high-throughput protein identification. Although Epstein-Barr virus-transformed B-lymphoblastoid cell lines (LCLs) have long been believed to be immortalized, recent studies have provided ample evidence that a large proportion of LCLs have limited life spans due to shortening of telomeres, and that part of them are truly immortalized by developing strong telomerase activity to maintain telomeres. Differential proteome analysis of pre- and post-immortal LCLs would provide a powerful tool to analyze proteins participating in the process of immortalization. We focus in this review on cumulative data of proteomic information on pre- and post-immortal LCLs.

The dinucleotide microsatellite polymorphism of the IFNAR1 gene promoter correlates with responsiveness of hepatitis C patients to interferon.

We studied a possible correlation between the dinucleotide microsatellite polymorphism of the interferon alpha/beta receptor-1 gene (IFNAR1) and responsiveness of hepatitis C patients to interferon therapy. The length of GT-repeat (n of (GT)(n)) in the IFNAR1 promoter was determined in 157 patients, of whom 50 were responders and 107 were nonresponders to interferon. The genotypes 5/5 (i.e. homozygotes of (GT)(5)) and 5/14 (i.e. heterozygotes of (GT)(5) and (GT)(14)) were more frequently found in responders than in nonresponders: 80 versus 58%, P=0.008. Thus, determining (GT)(n) of the IFNAR1 promoter may help predict treatment outcome in patients treated with interferon alpha and/or beta. In addition, we sought for genetic polymorphism in the promoter region of the interferon alpha/beta receptor-2 gene (IFNAR2), and found single nucleotide polymorphisms (SNPs) at -134 and -75. But these SNPs did not show a clinical significance, as compared with the GT-repeat in IFNAR1, in the context of interferon responsiveness.

Chromosomal instability in B-lymphoblasotoid cell lines from Werner and Bloom syndrome patients.

Werner's syndrome (WS) and Bloom's syndrome (BS) are rare autosomal genetic diseases that predispose to cancer and are associated with genomic instability. To characterize the genomic instability of WS and BS, we analyzed and compared the cytogenetics of B-lymphoblastoid cell lines (LCLs) from WS and BS patients and healthy donors. Although, similar spontaneous frequencies of micronuclei (MN) and sister chromatid exchanges (SCE) were observed in LCLs from WS patients and healthy donors, they were much higher in BS-LCLs. We also examined the cells' cytotoxic and cytogenetic formation (MN) response to camptothecin (CAM), etoposide (ETO), 4-nitroquinoline 1-oxide (4NQO), and mitomycin C (MMC). Compared to healthy donor LCLs, BS-LCLs but not WS-LCLs tended to be resistant to cytotoxicity and sensitive to MN induction by 4NQO and MMC. Spectrum karyotyping analysis revealed that most WS- and BS-LCLs generated "variegated translocation mosaicism" at high frequencies during cell culture. These findings support the idea that the basis of genomic instability in WS is different from that in BS.

A cascade leading to premature aging phenotypes including abnormal tumor profiles in Werner syndrome (review).

This perspective review focused on the Werner syndrome (WS) by addressing the issue of how a single mutation in a WRN gene encoding WRN DNA helicase induces a wide range of premature aging phenotypes accompanied by an abnormal pattern of tumors. The key event caused by WRN gene mutation is the dysfunction of telomeres. Studies on normal aging have identified a molecular circuit in which the dysfunction of telomeres caused by cellular aging activates the TP53 gene. The resultant p53 suppresses cell growth and induces a shorter cellular lifespan, and also compromises mitochondrial biogenesis leading to the overproduction of reactive oxygen species (ROS) causing multiple aging phenotypes. As an analogy of the mechanism in natural aging, we described a hypothetical mechanism of premature aging in WS: telomere dysfunction induced by WRN mutation causes multiple premature aging phenotypes of WS, including shortened cellular lifespan and inflammation induced by ROS, such as diabetes mellitus. This model also explains the relatively late onset of the disorder, at approximately age 20. Telomere dysfunction in WS is closely correlated with abnormality in tumorigenesis. Thus, the majority of wide and complex pathological phenotypes of WS may be explained in a unified manner by the cascade beginning with telomere dysfunction initiated by WRN gene mutation.

Aging-associated inflammation in healthy Japanese individuals and patients with Werner syndrome.

Minor inflammation-driven aging (inflammaging) has been proposed to explain human aging mechanism. To study the inflammatory condition associated with normal human aging, highly sensitive CRP (hsCRP) was examined in the sera collected from 217 healthy Japanese individuals aged between 1 and 100years and 41 mutation-proven Japanese Werner syndrome (WS) patients. The serum hsCRP was assayed by ELISA. The serum hsCRP level increased significantly (p<0.001) with normal aging from both sexes. The serum hsCRP was significantly elevated in WS (mean±SE: 11.0±1.6µg/ml) compared with age-matched normal population (1.3±0.3µg/ml, p<0.001) and normal elderly population ages between 71 and 100years (4.2±0.7µg/ml, p<0.001). Both normal aging and WS were associated with minor inflammation that can be evaluated by serum hsCRP. WS may be a good candidate to study inflammaging.

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.

Hepatocyte-protective and anti-oxidant effects of rifampicin on human chronic hepatitis C and murine acute hepatocyte disorder.

Rifampicin (RFP) is a semisynthetic antibiotic derived from the rifamycins and is one of the most commonly used pharmaceutical compounds worldwide in the treatment of tuberculosis. We previously reported that low-dose and long-term oral administration of RFP to 6 hepatitis C virus-related liver cirrhosis patients who were at high risk for presenting with hepatocellular carcinoma (HCC) resulted in a marked suppression of the occurrence of HCC without showing an adverse effect. The underlying mechanism was found to be due to the anticancer effect based on the potent anti-angiogenic properties of RFP. The present study revealed that RFP has an additional hepatocyte-protective effect by lowering the release of hepatic enzymes alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in chronic hepatitis C patients. Experimentally, we were able to show that RFP had hepatocyte-protective effects in acute hepatocyte disorder models of mice and rats induced by concanavalin A and by D-galactosamine, respectively: RFP significantly prevented an increase in the levels of ALT, AST and lactate dehydrogenase in these animal models. In addition, we found that RFP had a strong anti-oxidant action which was approximately three times stronger than the action of silibinin, an anti-inflammatory agent of human hepatic stellate cells, implicating that the hepatocyte-protective effects of RFP are mediated by its anti-oxidant activity. These results reveal that oral administration of RFP exerts not only a prophylactic effect on the occurrence or recurrence of HCC for an extensive period of time, but also exerts hepatocyte-protective effects on both human chronic hepatitis C and acute hepatocyte disorder in rodent models, and the anti-oxidant activity of RFP is implicated to participate in the latter effects.

Increased chemotherapeutic activity of camptothecin in cancer cells by siRNA-induced silencing of WRN helicase.

Werner syndrome helicase (WRN) participates in a wide range of DNA activities, including replication, double-strand DNA break repair, telomere and retrovirus long terminal repeat maintenance. Mutations of the WRN gene cause Werner syndrome (WS), an autosomal recessive premature ageing disorder associated with various symptoms related to ageing. In this study, we investigated the siRNA that specifically down-regulates WRN expression. WRN silencing increased markedly the chemotherapeutic activity of camptothecin (CPT) on cancer cells in terms of the extent of efficacy and lowering effective drug dosage, accompanied by suppressing recovery from DNA damage caused by CPT. Here, we propose a potential combination therapy of WRN-siRNA and CPT, looking forward to minimizing the inevitable adverse effects associated with cancer chemotherapy.

Differential cytotoxicity of anticancer agents in pre- and post-immortal lymphoblastoid cell lines.

We studied the cytotoxic effect of various anticancer agents on lymphoblastoid cell lines transformed by Epstein-Barr virus. Post-immortal N0005 (post-N0005) is an immortalized cell line derived from pre-immortal N0005 (pre-N0005) accompanied by increased telomerase activity, short-telomere, abnormal karyotypes, mutation of p53 gene, down regulation of p16/Rb and the ability to grow in soft agar medium. Compared with pre-N0005 cells, post-N0005 cells were significantly (p<0.001 by the Student t test) more resistant to the killing activity of seven DNA-modifying agents: camptothecin, etoposide, bleomycin, fluorouracil, thioguanine, melphalan and actinomycin D. However, both pre-N0005 and post-N0005 cells showed similar levels of cytotoxicity against four DNA-non-modifying agents: colchicine, paclitaxel, vincristine and methotrexate. DNA-modifying and DNA-non-modifying agents are distinguished by their different sensitivities with pre-N0005 and post-N0005. Based on these results, we propose that pre-N0005 and post-N0005 cell lines be used as a new method to assess and screen anticancer agents.

Molecular biology of Werner syndrome.

Human RecQ helicases, including the Werner syndrome helicase, participate in maintaining the integrity of the genome by suppressing illegitimate recombination or by repair of local DNA structural damage. Deterioration or loss of RecQ helicase associated with aging or genetic disorder results in genomic instability in tissues and organs where certain RecQ helicases are needed to correct aberrant DNA during proliferation. Such genomic instability, if not corrected, causes increased apoptotic cell death that would result in reduction of cell numbers in some tissues, leading to the deterioration of tissues and organs, the phenotypes of aging. Besides being associated with aging, genomic instability increases the risk of the development of neoplasms, both benign and malignant. These neoplasms are produced if either a checkpoint system or apoptosis is not functioning appropriately. Malignant tumor cells would then be selected from the mixed population of neoplasms by acquiring phenotypes that permit rapid cell growth and a strong capability to maintain their genome, such as tumor cells having increased levels of RecQ helicase expression, as we have observed in various tumor cell lines. Further studies are needed to discover effective measures to control genomic instability and to manage malignant tumor cells.

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.