Pot1 OB-fold mutations unleash telomere instability to initiate tumorigenesis.

Chromosomal aberrations are a hallmark of human cancers, with complex cytogenetic rearrangements leading to genetic changes permissive for cancer initiation and progression. Protection of Telomere 1 (POT1) is an essential component of the shelterin complex and functions to maintain chromosome stability by repressing the activation of aberrant DNA damage and repair responses at telomeres. Sporadic and familial mutations in the oligosaccharide-oligonucleotide (OB) folds of POT1 have been identified in many human cancers, but the mechanism underlying how hPOT1 mutations initiate tumorigenesis has remained unclear. Here we show that the human POT1's OB-folds are essential for the protection of newly replicated telomeres. Oncogenic mutations in hPOT1 OB-fold fail to bind to single-stranded telomeric DNA, eliciting a DNA damage response at telomeres that promote inappropriate chromosome fusions via the mutagenic alternative non-homologous end joining (A-NHEJ) pathway. hPOT1 mutations also result in telomere elongation and the formation of transplantable hematopoietic malignancies. Strikingly, conditional deletion of both mPot1a and p53 in mouse mammary epithelium resulted in development of highly invasive breast carcinomas and the formation of whole chromosomes containing massive arrays of telomeric fusions indicative of multiple breakage-fusion-bridge cycles. Our results reveal that hPOT1 OB-folds are required to protect and prevent newly replicated telomeres from engaging in A-NHEJ mediated fusions that would otherwise promote genome instability to fuel tumorigenesis.

Predicting clinical outcome in feline oral squamous cell carcinoma: tumour initiating cells, telomeres and telomerase.

Feline oral squamous cell carcinoma (SCC) has very poor prognosis. Here, a retrospective pilot study was conducted on 20 feline oral SCC patients who underwent stereotactic radiation therapy (SRT), to evaluate: (1) the value of putative tumour initiating cell (TIC) markers of human head and neck SCC (CD44, Bmi-1); (2) telomere length (TL) specifically in putative TICs; and (3) tumour relative telomerase activity (TA). Significant inverse correlations were found between treatment outcomes and Bmi-1 expression, supporting the predictive value of Bmi-1 as a negative prognostic indicator. While TL exhibited a wide range of variability, particularly in very short fractions, many tumours possessed high levels of TA, which correlated with high levels of Bmi-1, Ki67 and EGFR. Taken together, our results imply that Bmi-1 and telomerase may represent novel therapeutic targets in feline oral SCC, as their inhibition - in combination with SRT - would be expected to have beneficial treatment outcome.

Understanding cancer development processes after HZE-particle exposure: roles of ROS, DNA damage repair and inflammation.

During space travel astronauts are exposed to a variety of radiations, including galactic cosmic rays composed of high-energy protons and high-energy charged (HZE) nuclei, and solar particle events containing low- to medium-energy protons. Risks from these exposures include carcinogenesis, central nervous system damage and degenerative tissue effects. Currently, career radiation limits are based on estimates of fatal cancer risks calculated using a model that incorporates human epidemiological data from exposed populations, estimates of relative biological effectiveness and dose-response data from relevant mammalian experimental models. A major goal of space radiation risk assessment is to link mechanistic data from biological studies at NASA Space Radiation Laboratory and other particle accelerators with risk models. Early phenotypes of HZE exposure, such as the induction of reactive oxygen species, DNA damage signaling and inflammation, are sensitive to HZE damage complexity. This review summarizes our current understanding of critical areas within the DNA damage and oxidative stress arena and provides insight into their mechanistic interdependence and their usefulness in accurately modeling cancer and other risks in astronauts exposed to space radiation. Our ultimate goals are to examine potential links and crosstalk between early response modules activated by charged particle exposure, to identify critical areas that require further research and to use these data to reduced uncertainties in modeling cancer risk for astronauts. A clearer understanding of the links between early mechanistic aspects of high-LET response and later surrogate cancer end points could reveal key nodes that can be therapeutically targeted to mitigate the health effects from charged particle exposures.

Breed, age and gender distribution of dogs with chronic hepatitis in the United Kingdom.

Standardised histological criteria are now available for the diagnosis of canine chronic hepatitis (CH). CH is common in dogs, but no studies have reported breed, age and gender distributions in the United Kingdom (UK). The objective of this study was to determine which breeds had an increased risk for developing CH in the UK and to report the age and gender distribution for those breeds. The databases of six veterinary histopathology laboratories were searched for cases with a histological diagnosis of CH according to standardised criteria. The breed, age and gender of dogs was recorded and compared to a control population to calculate the odds ratio and 95% confidence intervals for developing CH. A total of 551 cases of CH were identified, consisting of 61 breeds. Nineteen breeds were represented by five or more cases. Breeds with an increased risk for developing CH included the American cocker spaniel, Cairn terrier, Dalmatian, Dobermann pinscher, English cocker spaniel, English springer spaniel, Great Dane, Labrador retriever and Samoyed. The median age at diagnosis for all breeds with CH was 8 years (range 7 months to 16 years). Dalmatians, Dobermann pinschers and English springer spaniels with CH were significantly younger than Cairn terriers, English cocker spaniels and Labrador retrievers with CH. Females were over-represented when all cases were examined together. In conclusion, several breeds in the UK have an increased risk of CH, some of which have not been previously reported.

Impact of telomerase status on canine osteosarcoma patients.

BACKGROUND: We demonstrated previously that canine osteosarcoma (OSA) cell lines and samples from clinical patients are predominantly telomerase positive. In contrast, the majority of OSA samples from human patients appear to be telomerase negative, maintaining telomere length by an alternative lengthening of telomeres (ALT) mechanism. The purpose of the current study was to examine the telomerase status of a large number of OSA samples from dogs and determine if telomerase status can serve as a prognostic factor. HYPOTHESIS: The majority of clinical canine OSA appendicular lesions will be telomerase positive, and telomerase positivity will negatively impact disease outcome. ANIMALS: Sixty-seven dogs with appendicular OSA presenting to the Colorado State University Animal Cancer Center for treatment. METHODS: The Telomeric Repeat Amplification Protocol was performed on tissue samples from primary canine appendicular OSA to determine the presence of telomerase activity. Telomere restriction fragment (TRF) analysis was utilized to determine telomere length and detect ALT. Outcome data were obtained in a retrospective manner and correlated with telomerase status. RESULTS: Seventy-three percent of canine OSA samples were telomerase positive. Telomerase status did not have an impact on disease-free interval or survival time. Nine of 10 telomerase-negative samples examined were consistent with an ALT phenotype, based on TRF analysis. CONCLUSIONS AND CLINICAL IMPORTANCE: These results are consistent with the hypothesis that the majority of canine OSA are telomerase positive, suggesting that telomerase may be a valuable target for canine OSA therapy. Additionally, telomerase status does not appear to be a prognostic factor in canine OSA.

DNA-PKcs and ATM influence generation of ionizing radiation-induced bystander signals.

The phenomenon by which irradiated cells influence non-irradiated neighboring cells, referred to as the bystander effect (BSE), is not well understood in terms of the underlying pathways involved. We sought to enlighten connections between DNA damage repair and the BSE. Utilizing sister chromatid exchange (SCE) frequencies as a marker of the BSE, we performed cell transfer strategies that enabled us to distinguish between generation versus reception of a bystander signal. We find that DNA-dependent Protein Kinase catalytic subunit (DNA-PKcs) and Ataxia Telangectasia Mutated (ATM) are necessary for the generation of such a bystander signal in normal human cells following gamma (gamma)-ray exposure, but are not required for its reception. Importantly, we also show that directly irradiated human cells do not respond to receipt of a bystander signal, helping to explain why the BSE is a low-dose phenomenon. These studies provide the first evidence for a role of the DNA damage response proteins DNA-PKcs and ATM specifically in the generation of a bystander signal and intercellular signaling.

Telomeres and DNA double-strand breaks: ever the twain shall meet?

Telomeres were first recognized as a bona fide constituent of the chromosome based on their inability to rejoin with broken chromosome ends produced by radiation. Today, we recognize two essential and interrelated properties of telomeres. They circumvent the so-called end-replication problem faced by genomes composed of linear chromosomes, which erode from their termini with each successive cell division. Equally vital is the end-capping function that telomeres provide, which is necessary to deter chromosome ends from illicit recombination. This latter property is critical in facilitating the distinction between the naturally occurring DNA double-strand breaks (DSBs) found at chromosome ends (i.e., telomeres) and DSBs produced by exogenous agents. Here we discuss, in a brief historical narrative, key discoveries that led investigators to appreciate the unique properties of telomeres in protecting chromosome ends, and the consequences of telomere dysfunction, particularly as related to recombination involving radiation-induced DSBs.

Tradeoffs between oxygen and energy in tibial growth at high altitude.

Some studies of high altitude populations argue that stature reduction results from caloric, rather than hypoxic, stress. However, tradeoff models of oxygen and glucose metabolism predict that in hypoxemia, glucose metabolism will be downregulated. We used tradeoff assumptions in two hypotheses: First, that hypoxia targets leg segment growth differentially, and second, that proportions of leg segments partition the impact of high altitude into hypoxemic and energetic components. A group of 113 Han and Tibetan middle school children at 3100 m aged 8 to 11 were measured for segment anthropometries, skinfolds, vital capacity, blood oxygen saturation, and percent body fat. MANOVA showed that Tibetan children were significantly larger and fatter than Han children. Independent of ethnicity or caloric status, absolute and relative tibia length was significantly reduced in children with lower blood oxygen saturation. Height, chest circumference, sitting height, tibia length, and ankle diameter were greatest in fatter children, independent of ethnicity or blood oxygen. For children of either ethnicity with the lowest blood oxygen, size as well as proportion was impacted. These results support the tradeoff model. Caloric reserves and ethnicity independently affect total skeletal size. Oxygen saturation and ethnicity affect leg proportions. In hypoxemia, body fat has less impact on growth than when ample oxygen is present. Therefore, we should qualify the claim that size in high altitude populations stems from nutritional stress. The findings also suggest that decanalization may have different meanings and outcomes depending on which body segments contribute to the effect.

Telomerase activity in canine osteosarcoma.

Appendicular osteosarcoma (OSA) is the most common primary bone tumour in dogs, and the prognosis with standard of care therapy of amputation and adjunctive chemotherapy is generally poor, with median survival times of 1 year. The ability of neoplastic cells to maintain their telomere length, by either telomerase activity or alternate methods, is an important step in tumour development and malignancy. The purpose of this study was to determine the presence of telomerase activity in canine OSA. To evaluate the frequency of alternative lengthening of telomeres in canine OSA, we have used the telomeric repeat amplification protocol in five canine cell lines and in six samples taken from clinical patients at the time of amputation. Our results reveal the presence of telomerase activity in 100% of canine OSA cell lines and 83% of clinical samples evaluated. This is in contrast to human OSA where 25-40% expression levels of telomerase are reported. Importantly, our results not only suggest that canine OSA may serve as a good model for aggressive telomerase-positive forms of human OSA but also that antitelomerase therapy strategies for treatment of canine OSA may be more successful than in the treatment of majority of human patients with OSA.

Strand-specific fluorescence in situ hybridization: the CO-FISH family.

The ability to prepare single-stranded chromosomal target DNA allows innovative uses of FISH technology for studies of chromosome organization. Standard FISH methodologies require functionally single-stranded DNAs in order to facilitate hybridization between the probe and the complementary chromosomal target sequence. This usually involves denaturation of double-stranded probes to induce temporary separation of the DNA strands. Strand-specific FISH (CO-FISH; Chromosome Orientation-FISH) involves selective removal of newly replicated strands from DNA of metaphase chromosomes which results in single-stranded target DNA. When single-stranded probes are then hybridized to such targets, the resulting strand-specific hybridization is capable of revealing a level of information previously unattainable at the cytogenetic level. Mammalian telomeric DNA consists of tandem repeats of the (TTAGGG) sequence, oriented 5'-->3' towards the termini of all vertebrate chromosomes. Based on this conserved structural organization, CO-FISH with a telomere probe reveals the absolute 5'-->3' orientation of DNA sequences with respect to the pter-->qter direction of chromosomes. Development and various applications of CO-FISH will be discussed: detection of cryptic inversions, discrimination between telomeres produced by leading- versus lagging-strand synthesis, and replication timing of mammalian telomeres.

DNA and telomeres: beginnings and endings.

How a cell deals with its DNA ends is a question that returns us to the very beginnings of modern telomere biology. It is also a question we are still asking today because it is absolutely essential that a cell correctly distinguishes between natural chromosomal DNA ends and broken DNA ends, then processes each appropriately - preserving the one, rejoining the other. Effective end-capping of mammalian telomeres has a seemingly paradoxical requirement for proteins more commonly associated with DNA double strand break (DSB) repair. Ku70, Ku80, DNA-PKcs (the catalytic subunit of DNA-dependent protein kinase), Xrcc4 and Artemis all participate in DSB repair through nonhomologous end-joining (NHEJ). Somewhat surprisingly, mutations in any of these genes cause spontaneous chromosomal end-to-end fusions that maintain large blocks of telomeric sequence at the points of fusion, suggesting loss or failure of a critical terminal structure, rather than telomere shortening, is at fault. Nascent telomeres produced via leading-strand DNA synthesis are especially susceptible to these end-to-end fusions, suggesting a crucial difference in the postreplicative processing of telomeres that is linked to their mode of replication. Here we will examine the dual roles played by DNA repair proteins. Our review of this rapidly advancing field primarily will focus on mammalian cells, and cannot include even all of this. Despite these limitations, we hope the review will serve as a useful gateway to the literature, and will help to frame the major issues in this exciting and rapidly progressing field. Our apologies to those whose work we are unable to include.

A phase I/II study of oral uracil/tegafur (UFT), leucovorin and irinotecan in patients with advanced colorectal cancer.

BACKGROUND: The aim of this study was to determine the maximum tolerated dose (MTD), toxicity profile and response rate of the oral 5-fluorouracil prodrug UFT (tegafur/uracil) and leucovorin (LV) in combination with irinotecan in patients with advanced or metastatic colorectal cancer. PATIENTS AND METHODS: Patients with histologically proven advanced or metastatic colorectal adenocarcinoma received first-line chemotherapy comprising UFT 250 mg/m(2)/day and LV 90 mg/day given on days 1 to 14, with escalating doses of irinotecan (200-300 mg/m(2)) administered intravenously on day 1 of a three-weekly cycle. Eligibility criteria were standard. The MTD was defined as the dose at which >33% of six patients experienced a dose-limiting toxicity (DLT) during cycle 1. RESULTS: A total of 32 patients were studied. Initially, six patients were treated at each of the irinotecan dose levels (200, 250 and 300 mg/m(2)) combined with UFT 250 mg/m(2)/day and LV 90 mg/day. DLTs consisting of grade 3 or 4 diarrhoea and febrile neutropenia were observed in one of 20 patients at 250 mg/m(2) and three of six patients at the 300 mg/m(2) irinotecan dose level. Having defined the MTD, the 250 mg/m(2) dose level was established as the recommended dose (RD) and expanded to 20 patients in whom treatment was generally well tolerated. The overall response rate was 19%, with five patients having a partial response (PR) and 18 stable disease (SD) out of 32 response-evaluable patients. CONCLUSION: UFT and LV can be safely combined with irinotecan. The RDs for future studies are UFT 250 mg/m(2)/day and LV 90 mg/day given on days 1-14, with irinotecan 250 mg/m(2) administered on day 1, every 3 weeks. This combination is well tolerated and active. Further investigation of UFT and LV in combination with irinotecan is warranted in patients with colorectal cancer.

Strand-specific postreplicative processing of mammalian telomeres.

Telomeres are specialized nucleoprotein structures that stabilize the ends of linear eukaryotic chromosomes. In mammalian cells, abrogation of telomeric repeat binding factor TRF2 or DNA-dependent protein kinase (DNA-PK) activity causes end-to-end chromosomal fusion, thus establishing an essential role for these proteins in telomere function. Here we show that TRF2-mediated end-capping occurs after telomere replication. The postreplicative requirement for TRF2 and DNA-PKcs, the catalytic subunit of DNA-PK, is confined to only half of the telomeres, namely, those that were produced by leading-strand DNA synthesis. These results demonstrate a crucial difference in postreplicative processing of telomeres that is linked to their mode of replication.

Involvement of NADPH: cytochrome P450 reductase in the activation of indoloquinone EO9 to free radical and DNA damaging species.

Evidence suggests that DT-diaphorase is involved in the activation and mechanism of cytotoxicity of the investigational indoloquinone anticancer drug EO9 under aerobic conditions. Data also implicate a role for other enzymes including NADPH: cytochrome P450 reductase, especially in low DT-diaphorase tumour cells and under hypoxic conditions. Here, we used purified rat NADPH: cytochrome P450 reductase to provide additional evidence in support of a role for this enzyme in activation of EO9 to generate free radical and DNA-damaging species. Electron spin resonance spectrometry studies showed that NADPH: cytochrome P450 reductase reduced EO9 to a free radical species, including a drug radical (most likely the semiquinone) and reactive oxygen species. Plasmid DNA experiments showed that reduction of EO9 catalysed by NADPH: cytochrome P450 reductase results in single-strand breaks in DNA. The information obtained may contribute to the understanding of the molecular mechanism of DNA damage and cytotoxicity exerted by EO9 and may be useful in the design of future bioreductive drugs.

Ethanol and oxidative stress.

This article represents the proceedings of a workshop at the 2000 ISBRA Meeting in Yokohama, Japan. The chair was Albert Y. Sun. The presentations were (1) Ethanol-inducible cytochrome P-4502E1 in alcoholic liver disease, by Magnus Ingelman-Sundberg and Etienne Neve; (2) Regulation of NF-kappaB by ethanol, by H. Matsumoto, Y. Nishitani, Y. Minowa, and Y. Fukui; (3) Chronic ethanol consumption increases concentration of oxidized proteins in rat liver, by Shannon M. Bailey, Vinood B. Patel, and Carol C. Cunningham; (4) Antiphospholipids antibodies and oxidized modified low-density lipoprotein in chronic alcoholic patients, by Tomas Zima, Lenka Fialova, Ludmila Mikulikova, Ptr Popov, Ivan Malbohan, Marta Janebova, and Karel Nespor; and (5) Amelioration of ethanol-induced damage by polyphenols, by Albert Y. Sun and Grace Y. Sun.

Ethanol consumption and liver mitochondria function.

The mitochondrion is the subcellular organelle affected earliest during the development of alcoholic liver disease. As a result of chronic ethanol consumption mitochondrial protein synthesis is decreased significantly due to a depression in the functioning of the mitochondrial ribosome. This causes a significant decrease in the concentrations of the thirteen mitochondria gene products, all of which are components of the oxidative phosphorylation system. Consequently, there is a depression in the rate at which ATP is synthesized in hepatic mitochondria. In addition to this loss in function, hepatic mitochondria either acutely or chronically exposed to ethanol generate increased levels of reactive oxygen species (ROS). This elevation in ROS has been demonstrated in both isolated mitochondria and hepatocytes. The increase in mitochondrial ROS production accompanying acute ethanol exposure is due to mitochondrial associated reoxidation of NADH produced during ethanol and acetaldehyde metabolism. The elevation in ROS generation observed in mitochondria from chronic ethanol consumers is likely due to decreases in mitochondrial-derived electron transport components, which in turn results in higher levels of the semiquinone forms of flavin mononucleotide and ubiquinone. Both these semiquinones readily donate electrons to molecular oxygen to form superoxide.

Chronic ethanol consumption alters the glutathione/glutathione peroxidase-1 system and protein oxidation status in rat liver.

BACKGROUND: Alcohol-induced liver damage is associated with oxidative stress, which might be linked to disturbances in liver antioxidant defense mechanisms. The effect of chronic ethanol consumption on the mitochondrial and cytosolic glutathione/glutathione peroxidase-1 (GSHPx-1) system and oxidative modification of proteins was therefore studied in the rat. METHODS: Male Sprague-Dawley rats were fed liquid diets that provided 36% total calories as ethanol for at least 31 days. Pair-fed controls received isocaloric diets with ethanol calories substituted with maltose-dextrins. Mitochondrial and cytosolic fractions were prepared from livers and assayed for GSHPx-1 and glutathione reductase activities and total and oxidized concentrations of glutathione. Catalase activity was measured in the postmitochondrial supernatant. Levels of GSHPx-1, lactate dehydrogenase, and the beta subunit of the F1 portion of the ATP synthase protein were determined by western blot analysis. Concentrations of mitochondrial and cytosolic protein carbonyls were measured to assess ethanol-induced oxidation of proteins. RESULTS: Chronic ethanol consumption significantly decreased cytosolic and mitochondrial GSHPx-1 activities by 40% and 30%, respectively. Levels of GSHPx-1 protein in cytosol were unaffected by ethanol feeding, whereas there was a small decrease in GSHPx-1 protein levels in mitochondria isolated from ethanol-fed rats. Glutathione reductase activities were increased in both intracellular compartments and catalase activity was increased as a consequence of ethanol exposure. Cytosolic total glutathione was mildly decreased, whereas ethanol feeding increased mitochondrial levels of total glutathione. Chronic ethanol feeding significantly increased both cytosolic and mitochondrial concentrations of protein carbonyls by 30% and 60%, respectively. CONCLUSIONS: This study demonstrates that chronic ethanol-induced alterations in the glutathione/GSHPx-1 antioxidant system might promote oxidative modification of liver proteins, namely those of the mitochondrion, which could contribute to the adverse effects of ethanol on the liver.

A second-stage partogram.

OBJECTIVE: To describe a second-stage partogram based on a system of scoring the descent and position of the fetal head and to use this system for studying progress in the second stage of labor and predicting mode of delivery and obstetric outcome. METHODS: A prospective observational study of 1413 women at term with a singleton, cephalic presentation. The position and station of the fetal head were observed and scored at diagnosis of the second stage of labor, 1 hour later, and then at 30 minute intervals until delivery was achieved. The score at diagnosis of the second stage of labor was assessed for its ability to predict eventual mode of delivery and duration of labor. A normogram was defined for nulliparas and multiparas and was used to define normal and abnormal progress in the second stage, associated factors in the first stage of labor, and mode of delivery. RESULTS: Increasing total score at the start of the second stage of labor is associated with increasing chance of spontaneous vaginal delivery (odds ratio [OR] 1.68 for nulliparas, 1.59 for multiparas), decreasing chance of instrumental vaginal delivery (OR 0.67 for nulliparas, 0.64 for multiparas), and emergency cesarean delivery (OR 0.39 for nulliparas). Abnormal progress as defined by the normogram is associated with use of epidural anesthesia, induction of labor, augmentation, dystocia, and increased incidence of operative delivery. No significant difference is found between normal and abnormal second stages of labor in fetal outcome as determined by Apgar scores. CONCLUSION: The second-stage partogram offers an objective basis for management of the second stage of labor.

Antioxidants and gadolinium chloride attenuate hepatic parenchymal and endothelial cell injury induced by low flow ischemia and reperfusion in perfused rat livers.

The objective of this study was to determine whether Kupffer cells contribute to parenchymal and endothelial cell damage induced by ischemia-reperfusion in perfused rat livers. Parenchymal and endothelial cell injury were determined by measuring activities of lactate dehydrogenase (LDH) and purine nucleoside phosphorylase (PNP), respectively, in the effluent perfusate of livers subjected to 60 min of low flow ischemia followed by 30 min of reperfusion. Upon reperfusion, LDH and PNP activities increased significantly within the first 10 min of reperfusion and remained elevated over control values throughout the duration of reperfusion. Pretreatment with gadolinium chloride, an inhibitor of Kupffer cell function, significantly decreased LDH and PNP efflux during reperfusion by approximately 60% and 50%, respectively. When Kupffer cells were stimulated by vitamin A pretreatment, PNP efflux was doubled during reperfusion. Vitamin E pretreatment attenuated LDH and PNP release by approximately 70% during reperfusion compared to enzyme release in untreated livers. Moreover, the water-soluble antioxidants superoxide dismutase and desferrioxamine reduced reperfusion injury, whereas catalase had no effect on enzyme release. These results demonstrate that superoxide anions released from Kupffer cells are involved in oxidative damage to endothelial cells as well as hepatocytes during the early stages of hepatic reperfusion.