The WRN and MUS81 proteins limit cell death and genome instability following oncogene activation.

Oncogene-induced replication stress is recognized as the primary cause of accumulation of DNA damage and genome instability in precancerous cells. Although the molecular mechanisms responding to such type of replication perturbation are not fully characterized, it has been speculated that their dysfunction may enhance genome instability and accelerate tumor progression. Here, we show that the WRN protein, a member of the human RecQ helicases, is necessary to sustain replication fork progression in response to oncogene-induced replication stress. Loss of WRN affects cell cycle progression and results in enhanced accumulation of double-strand breaks and instability at common fragile sites in cells experiencing oncogene-induced replication stress. Moreover, we demonstrate that double-strand breaks, observed upon oncogene over-expression, depend on the MUS81 endonuclease, which represents a parallel pathway collaborating with WRN to prevent cell death. Overall, our findings give insights into the mechanisms protecting replication forks in cells experiencing oncogene-induced replication stress, and identify factors that, when mutated or dysfunctional, may enhance genome instability in precancerous cells. In addition, because concomitant depletion of WRN and MUS81 causes synthetic sickness in cells growing under oncogene-induced replication stress, our results support the possibility of targeting cancer cells with an impaired replication fork recovery pathway by a specific inactivation of the other parallel pathway.

The RAD9-RAD1-HUS1 (9.1.1) complex interacts with WRN and is crucial to regulate its response to replication fork stalling.

The WRN protein belongs to the RecQ family of DNA helicases and is implicated in replication fork restart, but how its function is regulated remains unknown. We show that WRN interacts with the 9.1.1 complex, one of the central factors of the replication checkpoint. This interaction is mediated by the binding of the RAD1 subunit to the N-terminal region of WRN and is instrumental for WRN relocalization in nuclear foci and its phosphorylation in response to replication arrest. We also find that ATR-dependent WRN phosphorylation depends on TopBP1, which is recruited by the 9.1.1 complex in response to replication arrest. Finally, we provide evidence for a cooperation between WRN and 9.1.1 complex in preventing accumulation of DNA breakage and maintaining genome integrity at naturally occurring replication fork stalling sites. Taken together, our data unveil a novel functional interplay between WRN helicase and the replication checkpoint, contributing to shed light into the molecular mechanism underlying the response to replication fork arrest.

Cassia angustifolia extract is not hepatotoxic in an in vitro and in vivo study.

BACKGROUND: Cassia angustifolia L. (senna) is traditionally used as a laxative. Its major components are sennosides that are responsible for the laxative effect. Senna is recommended for the short-term treatment of acute constipation. Nevertheless people use its preparations as self-medication, often for long periods, to treat chronic constipation thus exposing themselves to adverse reactions. Most reactions were associated with hepatotoxicity. AIMS: The present study was aimed to evaluate the toxicity of a C. angustifolia leafextract (standardized at 60% of sennosides) on rat liver cells and the long-term effects on liver functions, in Wistar rats. METHODS: Cytotoxicity was assessed in a buffalo normal rat liver cell line (BRL-3A) by the trypan blue assay and the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide reduction test. In vivo effects were observed after oral administration of the extract for 4 or 8 weeks at doses of 12 and 58 mg/kg/day. At the end of treatment, animals were sacrificed, the postmortem examination was performed and serum was used for biochemical analysis. Liver samples were used for histomorphological and immunohistochemical examination along with the determination of oxidative stress parameters. RESULTS AND CONCLUSION: In BRL-3A cells, the extract was cytotoxic at concentrations that appear largely higher than those attainable in humans. In Wistar rats, the extract did not induce any significant change in all of the parameters tested. In summary, the present study indicates a lack of hepatotoxicity of senna at doses higher than those generally used in humans.

Polycystic liver diseases.

Polycystic liver diseases (PCLDs) are genetic disorders with heterogeneous etiologies and a range of phenotypic presentations. PCLD exhibits both autosomal or recessive dominant pattern of inheritance and is characterized by the progressive development of multiple cysts, isolated or associated with polycystic kidney disease, that appear more extensive in women. Cholangiocytes have primary cilia, functionally important organelles (act as mechanosensors) that are involved in both normal developmental and pathological processes. The absence of polycystin-1, 2, and fibrocystin/polyductin, normally localized to primary cilia, represent a potential mechanism leading to cyst formation, associated with increased cell proliferation and apoptosis, enhanced fluid secretion, abnormal cell-matrix interactions, and alterations in cell polarity. Proliferative and secretive activities of cystic epithelium can be regulated by estrogens either directly or by synergizing growth factors including nerve growth factor, IGF1, FSH and VEGF. The abnormalities of primary cilia and the sensitivity to proliferative effects of estrogens and different growth factors in PCLD cystic epithelium provide the morpho-functional basis for future treatment targets, based on the possible modulation of the formation and progression of hepatic cysts.

New insights into liver stem cells.

Hepatic progenitor cells are bi-potential stem cells residing in human and animal livers that are able to differentiate towards the hepatocytic and the cholangiocytic lineages. In adult livers, hepatic progenitor cells are quiescent stem cells with a low proliferating rate, representing a reserve compartment that is activated only when the mature epithelial cells of the liver are continuously damaged or inhibited in their replication, or in cases of severe cell loss. Hepatic progenitor cell activation has been described in various acute and chronic liver diseases. Their niche is composed by numerous cells such as Hepatic Stellate Cells, endothelial cells, hepatocytes, cholangiocytes, Kupffer cells, pit cells and inflammatory cells. All these cells, numerous hormones and growth factors could interact and cross-talk with progenitor cells influencing their proliferative and differentiative processes. Hepatic progenitor cells and their niche could represent, in the near future, a target for therapeutic approaches to liver disease based on cell-specific drug delivery systems. Isolation and transplantation of hepatic progenitor cells could represent a new approach for therapy of end-stage chronic liver diseases, as they offer many advantages to transplantation of mature hepatocytes. The possibility of applying stem cell therapy to liver diseases will represent a major goal in this field.

Estrogens stimulate the proliferation of human cholangiocarcinoma by inducing the expression and secretion of vascular endothelial growth factor.

BACKGROUND: Estrogens may induce the proliferation of neoplastic cells by activating neo-angiogenesis. AIM: To evaluate the effect of estrogens on the expression of vascular endothelial growth factor (VEGF) and related receptors (VEGF-R) in human cholangiocarcinoma and the role played by VEGF in mediating the proliferative effects of estrogens. METHODS: Seven biopsies of intra-hepatic cholangiocarcinoma and the HuH-28 cell lines were investigated. Cell proliferation was measured by both PCNA Western blot and MTS proliferation assay. RESULTS: By immunohistochemistry, biopsies of human cholangiocarcinoma stained positively for VEGF-A and VEGF-C and related receptors. HuH-28 cells expressed VEGF-A, -C, and VEGFR-1, -2, -3 and, their protein level was enhanced by 17beta-estradiol in association with the stimulation of cell proliferation. 17beta-Estradiol-stimulated proliferation of HuH-28 cells was blocked by 70% by VEGF-TRAP, a receptor-based VEGF inhibitor. 17beta-Estradiol induced the secretion of VEGF in the supernatant of HuH-28 cells. The stimulatory effect of 17beta-estradiol on the protein expression of VEGF-A, VEGF-C and VEGFR-1, -2, -3 was blocked by antagonists of ER (Ici182,780) or insulin-like growth factor 1-receptor (alphaIR3). CONCLUSIONS: With the limitations of experiments performed in a cell line, our study indicates that VEGF plays a major role in mediating the proliferative effects of estrogens on human cholangiocarcinoma.

Effects of Cimicifuga racemosa extract on liver morphology and hepatic function indices.

UNLABELLED: Cimicifuga racemosa (black cohosh) is a herbaceous perennial plant, that has been traditionally used for a variety of ailments (dyspepsia, climacteric complaints, muscular rheumatisms, menstrual cramps). From laboratory and clinical studies, black cohosh seems to have a relatively good safety profile, even if a number of case reports of hepatotoxicity were a matter of recent concern. AIM: A number of case reports indicated that C. racemosa could induce hepatotoxicity. We evaluated the effects of black cohosh extract on liver morphology, and on levels of various hepatic function indices in rats. METHODS: Wistar rats received 300mg/kg/day of C. racemosa extract by gavage, for 30 days. Biochemical analysis of serum was conducted by an automated, random-access clinical chemistry analyzer. Liver samples were used for hystomorphological and immunohistochemical examination, for the detection of apoptosis (TUNEL assay), and for the determination of GSH level (spectrophotometrical analysis). RESULTS: C. racemosa extract does not affect liver morphology and hepatic function indices, in rats. CONCLUSIONS: On the basis of experimental data, the use of 300mg/kg/day of black cohosh appears quite safe in rats. Nevertheless, in humans the safety of C. racemosa should be further monitored, in terms of patient-related factors.

The logic and regulation of cell cycle exit and reentry.

Tissue repair and regeneration are very complex biological events, whose successful attainment requires far more than mere cell division. However, almost unavoidably they entail cell proliferation as a fundamental premise. Full regeneration or repair cannot be achieved without replacing cells lost to disease or injury, replacement that can only take place via proliferation of surviving cells. This review endeavors to outline the molecular bases of exit from and reentry into the cell cycle. In recent years, the decision to proliferate or not has been seen as mostly the concern of cyclins and cyclin-dependent kinases. This account tries to show that cell cycle inhibitors are as important as the positive regulators in the making of this decision. Finally, the authors wish to suggest that the molecular knowledge of the cell cycle can be harnessed to the benefit of many aspects of regenerative medicine.

Hepatic microcirculation and cholangiocyte physiopathology.

BACKGROUND: The peribiliary plexus (PBP) plays a fundamental role in supporting the functions of the biliary epithelium. After common bile duct ligation (BDL) progressive PBP proliferation is demonstrated. We have, recently, demonstrated that the biliary epithelium express Vascular Endothelial Growth Factor (VEGF), both subtype -A and -B and VEGF receptors. Taking in consideration the wide extension of PBP during BDL, aim of our study is to investigate the role of VEGF in stimulating angiogenesis and also in the modulation of epithelial cells proliferation. MATERIAL AND METHODS: Experimental studies were performed by evaluating the effects of: a) endogenous VEGF neutralization by chronic administration of anti VEGF-C antibody on cholangiocyte proliferation in BDL rats and; b) the hepatic artery ligation (HAL) immediately after BDL followed by treatment (7 days) with a recombinant of VEGF-A (administered through IP implanted minipumps) on cholangiocyte proliferative activities. RESULTS: Both administration of antiVEGF-C antibody and HAL decreases cholangiocyte proliferation. The decrease of cholangiocyte proliferation was associated with depressed VEGF-A protein expression. The administration of rVEGF-A to BDL, hepatic artery ligated rats prevented the decrease of cholangiocyte proliferation and VEGF-A expression as compared to BDL control rats. CONCLUSION: These data suggest that VEGF-C modulates the proliferative activities of cholangiocytes in experimental cholestasis and that circulating factors (i.e., VEGF) in the blood supply of the intra-hepatic biliary epithelium, play an important role in the balance between cholangiocyte proliferation/loss.

Alpha-SMA expression in hepatic stellate cells and quantitative analysis of hepatic fibrosis in cirrhosis and in recurrent chronic hepatitis after liver transplantation.

BACKGROUND: The alpha isotype of actin expressed by hepatic stellate cells reflects their activation to myofibroblast-like cell and has been directly related to experimental liver fibrogenesis, and indirectly to human fibrosis in chronic liver disease. AIMS: To evaluate the changes in distribution and percentage of alpha-smooth muscle actin-positive hepatic stellate cells and the correlation with the degree of the fibrosis in cirrhotic livers, as well as in patients with recurrent HCV chronic hepatitis after liver transplantation. METHODS: Human liver biopsies were divided in four groups: (1) normal livers obtained from cadaveric liver donors (n=35), (2) cirrhosis post-HBV hepatitis (n=11), (3) cirrhosis post-HCV hepatitis (n=10), and (4) post-transplant recurrent HCV chronic hepatitis (n=13). Samples were stained with anti-alpha-smooth muscle actin antibody by immunoperoxidase method and semi-quantitatively evaluated. Liver fibrosis was assessed from specimens stained with Masson's trichrome and quantified by computer image analysis. RESULTS: The percentage of alpha-smooth muscle actin-positive hepatic stellate cells was significantly higher in the HBV cirrhosis, HCV cirrhosis and post-transplant HCV recurrent hepatitis groups (36.1+/-15.2, 23.8+/-19.7 and 27.8+/-16.4%, respectively) compared to the liver donor group (2.9+/-4.0%). The alpha-smooth muscle actin-positive hepatic stellate cells to fibrous tissue ratio were significantly higher in the post-transplant recurrent HCV hepatitis group (2.36+/-1.12) compared to both the donor livers and the HCV cirrhosis groups (0.74+/-1.09 and 1.03+/-0.91, respectively). The alpha-smooth muscle actin-positive hepatic stellate cell percentage and fibrosis correlated positively in the post-transplant recurrent HCV hepatitis group and negatively in the HCV cirrhosis group. No difference in the immunohistochemical and morphometrical variables was found between the HCV cirrhosis and HBV cirrhosis groups. CONCLUSIONS: These results indirectly confirm that, in vivo, alpha-smooth muscle actin expression is a reliable marker of hepatic stellate cells activation which precedes fibrous tissue deposition even in the setting of recurrent HCV chronic hepatitis after liver transplantation, and it could be useful to identify the earliest stages of hepatic fibrosis and monitoring the efficacy of the therapy. In the presence of advanced cirrhosis other factors, rather than alpha-smooth muscle actin-positive hepatic stellate cells, may sustain fibrosis deposition.

Alfa and beta estrogen receptors and the biliary tree.

This manuscript summarizes recent data showing that estrogens and their receptors play an important role in modulating cholangiocyte proliferation. We have recently demonstrated that rat cholangiocytes express both estrogen receptors (ER)-alpha and -beta subtypes, while hepatocytes only express ER-alpha. ER and especially the ER-beta subtype, are overexpressed in cholangiocytes proliferating after bile duct ligation (BDL) in the rat, in association with enlarged bile duct mass and with enhanced estradiol serum levels. Cholangiocyte proliferation, during BDL, is impaired by estrogen antagonists (tamoxifen, ICI 182,780) which furthermore, induce the overexpression of Fas antigen and activate apoptosis of proliferating cholangiocytes. 17beta-estradiol stimulates, in vitro cholangiocyte proliferation, and this effect is individually blocked by tamoxifen or ICI 182,780. Cholangiocyte proliferation during BDL was associated with an enhanced protein expression of phosphorylated extracellular regulated kinases (ERK)1/2 which is, in contrast, negatively modulated by tamoxifen in association with its antiproliferative effect. This indicates a major involvement of the ERK system in the estrogen modulation of cholangiocyte proliferation.

Immunohistochemical features of bile duct epithelial cells in normal and experimental liver conditions.

It is well known that estrogen (E) modulates the processes of liver growth and regeneration. However, while estrogen receptors (Er) have been detected in hepatocytes, little is known on the occurrence of Er in cholangiocytes and the role of E on the physiopathology of the biliary epithelium. The purpose of this study was to investigate the occurrence of Er and their alpha or beta subtypes in cholangiocytes of normal and Bile Duct Ligated (BDL) rats and to evaluate the role and mechanisms of E in the modulation of cholangiocyte proliferation. In this study normal and BDL rats (utilized as experimental model of cholestasis) were used. Er alpha and beta subtypes, CK-19, PCNA and Fas were analysed by immunohistochemistry. The antiestrogens tamoxifen or ICI 182,780 were administered in the BDL group and the effects on cholangiocyte proliferation (bile duct mass) and apoptotic phenomenon (Tunel and Fas expression) were evaluated. Our results demonstrated that cholangiocytes express both Er-alpha and Er-beta subtypes, while hepatocytes only express Er-alpha. The increased percentage of cholangiocytes during BDL-induced proliferation was correlated with Er and PCNA expression and with enlarged Bile Duct Mass (BDM). Treatment of BDL rats with antiestrogens induced: i) inhibition of cholangiocyte proliferadon as indicated by the decreased BDM and PCNA expression; ii) over-expression of Fas antigen in cholangiocytes and induction of apoptosis (TUNEL) and iii) inhibition of cholangiocyte secretory activities. In condusion, our findings demonstrate that cholangiocytes express Er which are up-regulated during cholangiocyte proliferation. Inhibition of Er with antiestrogens blocks cholangiocyte proliferation and triggers apoptosis of Fas+ cholangiocytes suggesting a crucial role of estrogens in modulating cholangiocyte proliferation during bile duct obstruction.

Hypersensitivity to camptothecin in MSH2 deficient cells is correlated with a role for MSH2 protein in recombinational repair.

DNA mismatch repair (MMR) corrects DNA polymerase insertion errors that have escaped proofreading in order to avoid the accumulation of deleterious mutations. While the role of MMR in the correction of replication errors is well established, its involvement in the processing of DNA damage induced by chemical and physical agents is less clear. A role for some of the MMR proteins, such as MSH2, in the repair of double strand break (DSBs) through recombination has also been envisaged. Why MMR- deficient cells are sensitive to agents causing replication fork stalling and thus DSBs remains unclear. To verify a possible role of MSH2 in homologous recombinational repair, we have treated cells from knockout mice for the MSH2 gene and mouse colorectal carcinoma cells also defective for MSH2 with different doses of camptothecin, an agent known to interfere with DNA replication. In the absence of MSH2, we found a reduced survival rate accompanied by higher levels of chromosomal damage and SCE induction. Furthermore, MSH2(-/-) cells displayed an elevated spontaneous RAD51 focus-forming activity and a higher induction of RAD51 foci following camptothecin treatment. Thus, the absence of MSH2 could result in both spontaneous DNA damage and uncontrolled recombination events leading to the observed higher yield of chromosomal damage and the higher induction of RAD51 foci following CPT treatment. Therefore, our results suggest an involvement of MSH2 in the early events leading to correct RAD51 relocalization after the formation of DSBs specifically produced at the blocked replication fork.

Werner's syndrome protein is required for correct recovery after replication arrest and DNA damage induced in S-phase of cell cycle.

Werner's syndrome (WS) is a rare autosomal recessive disorder that arises as a consequence of mutations in a gene coding for a protein that is a member of RecQ family of DNA helicases, WRN. The cellular function of WRN is still unclear, but on the basis of the cellular phenotypes of WS and of RecQ yeast mutants, its possible role in controlling recombination and/or in maintenance of genomic integrity during S-phase has been envisaged. With the use of two drugs, camptothecin and hydroxyurea, which produce replication-associated DNA damage and/or inhibit replication fork progression, we find that WS cells have a slower rate of repair associated with DNA damage induced in the S-phase and a reduced induction of RAD51 foci. As a consequence, WS cells undergo apoptotic cell death more than normal cells, even if they arrest and resume DNA synthesis at an apparently normal rate. Furthermore, we report that WS cells show a higher background level of DNA strand breaks and an elevated spontaneous induction of RAD51 foci. Our findings support the hypothesis that WRN could be involved in the correct resolution of recombinational intermediates that arise from replication arrest due to either DNA damage or replication fork collapse.

Investigation of G2-phase chromosomal radiosensitivity in hereditary non-polyposis colorectal cancer cells.

PURPOSE: To investigate whether cells from hereditary nonpolyposis colorectal cancer (HNPCC) patients, a genetic condition characterized by constitutional mutations in DNA mismatch repair genes and associated with predisposition to colorectal carcinoma (CRC), could present a higher G2 chromosomal radiosensitivity. It is generally hypothesized that cancer predisposition in HNPCC is associated with the loss of the wild-type allele in somatic cells, resulting in defective DNA mismatch repair but, to date, no data on G2 radiosensitivity have been reported for HNPCC. MATERIALS AND METHODS: Lymphoblastoid cell lines derived from six HNPCC patients heterozygous for MLH1, one HNPCC patient carrying a mutant MSH2 allele and three healthy controls were treated with 50 cGy of X-rays and sampled at various harvesting times, monitoring cell-cycle progression by 5-bromo-2-deoxyuridine (BrdUrd) incorporation in order to analyse chromosomal damage in the homogeneous G2 population. RESULTS: There were no differences between lymphoblasts derived from patients in the frequency of G2 chromosomal aberrations induced by X-rays when compared with control cell lines. However, despite the absence of G2 radiosensitivity in HNPCC cells, lymphoblasts from patients heterozygous for MLH1 mutations showed a higher induction of chromatid exchanges. CONCLUSIONS: The observed possible incorrect rejoining of double-strand breaks in MLH1 heterozygotes would be an additional and important factor contributing to loss of heterozygosity in HNPCC patients.

Peyer's patches epithelium in the rat: a morphological, immunohistochemical, and morphometrical study.

The epithelial layer covering lymphoid follicles of Peyer's patches consists of cells with a different surface morphology. Some of these cells have been described as a distinct cytotype, the so-called M cells. In order to resolve the controversy on the specific morphological and biochemical markers of M cells, structural, ultrastructural, and morphometrical study of the epithelium covering the rat Peyer's patches were performed. Peyer's patches from healthy rats were processed for light microscopy, immunohistochemistry, in situ nick-end labeling (TUNEL), and scanning and transmission electron microscopy. A morphometric study was also performed to evaluate microvillus density, length, and number of lysosomes in different areas of the epithelium. Peyer's patches were covered by simple columnar/cubical dome epithelium (DE). Scarce goblet cells and a large number of enterocytes were observed. Ultrastructural observations revealed that the DE showed cells with different morphology. The density and length of microvilli and the lysosome number varied along the whole dome without significant differences. The DE cells characterized by short and disorganized microvilli appeared always in close spatial relationship with lymphocytes. In conclusion, the concept that distinct cell types (enterocytes and M cells) can be identified in the rat DE does not appear to be valid based on morphological criteria. It seems correct to consider that in rat Peyer's patches the presence of scarce goblet cells and a large number of enterocytes showing dynamic morphofunctional modifications is related to the functional state and/or to cell cycle.

Estrogens stimulate proliferation of intrahepatic biliary epithelium in rats.

BACKGROUND & AIMS: We investigated the expression of estrogen receptor (ER) alpha and beta subtypes in cholangiocytes of normal and bile duct-ligated (BDL) rats and evaluated the role and mechanisms of estrogens in the modulation of cholangiocyte proliferation. METHODS: ER-alpha and ER-beta were analyzed by immunohistochemistry, reverse-transcription polymerase chain reaction, and Western blotting in normal and BDL rats. The effects of the ER antagonists tamoxifen and ICI 182,780 on cholangiocyte proliferation were evaluated. RESULTS: Cholangiocytes expressed both ER-alpha and ER-beta subtypes, whereas hepatocytes expressed only ER-alpha. In association with a marked cholangiocyte proliferation and with enhanced estradiol serum levels, the immunoreactivity for ER-alpha involved a 3-fold higher percentage of cholangiocytes in 3-week BDL than in normal rats; immunoreactivity for ER-beta showed a 30-fold increase. Western blot analysis showed that during BDL, the total amount of ER-beta in cholangiocytes was markedly increased (5-fold), whereas that of ER-alpha decreased slightly (-25%). Treatment with tamoxifen or ICI 182,780 of 3-week BDL rats inhibited cholangiocyte proliferation and induced overexpression of Fas antigen and apoptosis in cholangiocytes. In vitro, 17 beta estradiol stimulated proliferation of cholangiocyte, an effect blocked to the same extent by tamoxifen or ICI 182,780. CONCLUSIONS: This study suggests that estrogens and their receptors play a role in the modulation of cholangiocyte proliferation.

Hepatic microvascular features in experimental cirrhosis: a structural and morphometrical study in CCl4-treated rats.

BACKGROUND/AIMS: In this study, a detailed morphometrical analysis of the hepatic microvasculature in the different zones of hepatic parenchyma was performed in normal and cirrhotic rat liver (CCl4-induced). The aims were to detect, in CCl4-induced cirrhosis, the real presence of the "capillarization" of hepatic sinusoids and to assess alterations of the sinusoid/parenchyma ratio within the nodule. METHODS: Cirrhosis was promoted by controlled intragastric CCl4 administration. Scanning electron microscopy of the vascular corrosion cast technique associated with light microscopy and transmission electron microscopy were used. RESULTS: Evidence of connective tissue in the space of Disse was found only in sinusoids located near portal tracts or large fibrotic areas, and this was also confirmed by laminin immunohistochemistry. In contrast, all the intranodular sinusoids lacked real basal membrane and connective fibers in the space of Disse and, displayed normal fenestrations. The parenchymal area, sinusoidal area, mean sinusoidal area, sinusoidal perimeter, hepatocyte area and the reciprocal ratios were all considered in the morphometrical analysis. The sinusoids were of uniform size in the periportal, periseptal and pericentral areas of the cirrhotic liver without the typical zonal differences of the normal liver. The areas occupied by sinusoids per unit of parenchyma and the sinusoid/hepatocyte interfaces disposable for metabolic exchanges were markedly smaller (p<0.01) in cirrhotic than normal liver. CONCLUSION: Our findings indicate that capillarization of hepatic sinusoids occurs only in very limited regions of the cirrhotic parenchyma, and thus this phenomenon does not have relevant functional consequences. Furthermore, the cirrhotic parenchyma appears not to be supplied by sinusoids and lacks features of zonation, which is a condition that could play a major role in the development and progression of liver failure.

Werner's syndrome cell lines are hypersensitive to camptothecin-induced chromosomal damage.

Werner's syndrome (WS) is a recessive human genetic disorder associated with an elevated incidence of many types of cancer. The WS gene product, WRNp, belongs to the RecQ family of DNA helicases and is required for the maintenance of genomic stability in human cells. A possible interaction between helicases and topoisomerases that could co-operate in many aspects of DNA metabolism such as progression of the replication forks, recombination and repair has been recently suggested. In addition, sgs1 gene product in yeast, homologous to WS gene, has been shown to physically interact with topoisomerase types I and II. Earlier data from our laboratory suggested that WRN helicase might play a role in a G2 recombinational pathway of double strand breaks (DSBs) repair, co-operating with topoisomerase II. In this work, the effect of the topoisomerase I inhibitor camptothecin in WS cells has been investigated at the chromosomal level. The data from the present work suggest that the inhibition of topoisomerase I activity by camptothecin results in a higher induction of chromosomal damage in WS cell lines in the G2-phase and in the S-phase of the cell cycle compared to normal cells, perhaps associated with the defects in DNA replication synthesis.

Evidence that camptothecin-induced aberrations in the G(2) phase of cell cycle of Chinese hamster ovary (CHO) cell lines is associated with transcription.

It is widely accepted that camptothecin (CPT) is an S-dependent genotoxin. In this study, we aimed to elucidate the 'puzzling' induction of chromosomal damage by CPT in the G(2) phase of CHO cells, where no DNA synthesis is expected, focusing the attention on the possible role of the ongoing RNA synthesis, supposed to cause the conversion of CPT-single stranded cleavage complexes spaced closely on opposite DNA strands into DNA double strand breaks (DSB's) by the action of traversing RNA polymerase.CHO AA8 and its parental mutant EM9 cell lines were pre-treated with alpha-amanitin, which prevents transcription to pre-m-RNA and challenged cells with CPT for the last hour in culture to evaluate whether G(2)-CPT-induced aberrations would have been reduced or abolished in the absence of RNA synthesis compared with G(2)-CPT treatment alone. The results obtained indicated a marked and significant reduction of aberration yields, to almost the control values (alpha-amanitin alone) when inhibition of RNA synthesis was substantial (3h total alpha-amanitin). Partial inhibition of RNA synthesis (2h total alpha-amanitin) slightly reduced the CPT-induced aberrations yield only at the high dose-level employed of CPT (20mM). This finding strongly supports the hypothesis that CPT-single stranded cleavages complexes spaced closely on opposite DNA strands are converted into DNA double strand breaks by the action of traversing RNA polymerase.