Questions and answers in the management of children with medulloblastoma over the time. How did we get here? A systematic review.

Introduction: Treatment of children with medulloblastoma (MB) includes surgery, radiation therapy (RT) and chemotherapy (CT). Several treatment protocols and clinical trials have been developed over the time to maximize survival and minimize side effects. Methods: We performed a systematic literature search in May 2023 using PubMed. We selected all clinical trials articles and multicenter studies focusing on MB. We excluded studies focusing exclusively on infants, adults, supratentorial PNETs or refractory/relapsed tumors, studies involving different tumors or different types of PNETs without differentiating survival, studies including <10 cases of MB, solely retrospective studies and those without reference to outcome and/or side effects after a defined treatment. Results: 1. The main poor-prognosis factors are: metastatic disease, anaplasia, MYC amplification, age younger than 36 months and some molecular subgroups. The postoperative residual tumor size is controversial.2. MB is a collection of diseases.3. MB is a curable disease at diagnosis, but survival is scarce upon relapse.4. Children should be treated by experienced neurosurgeons and in advanced centers.5. RT is an essential treatment for MB. It should be administered craniospinal, early and without interruptions.6. Craniospinal RT dose could be lowered in some low-risk patients, but these reductions should be done with caution to avoid relapses.7. Irradiation of the tumor area instead of the entire posterior fossa is safe enough.8. Hyperfractionated RT is not superior to conventional RT9. Both photon and proton RT are effective.10. CT increases survival, especially in high-risk patients.11. There are multiple drugs effective in MB. The combination of different drugs is appropriate management.12. CT should be administered after RT.13. The specific benefit of concomitant CT to RT is unknown.14. Intensified CT with stem cell rescue has no benefit compared to standard CT regimens.15. The efficacy of intraventricular/intrathecal CT is controversial.16. We should start to think about incorporating targeted therapies in front-line treatment.17. Survivors of MB still have significant side effects. Conclusion: Survival rates of MB improved greatly from 1940-1970, but since then the improvement has been smaller. We should consider introducing targeted therapy as front-line therapy.

High Diversity of Bradyrhizobial Species Fix Nitrogen with Woody Legume Spartocytisus supranubius in a High Mountain Ecosystem.

The symbiosis between rhizobia and legumes is of pivotal importance in nitrogen-poor ecosystems. Furthermore, as it is a specific process (most legumes only establish a symbiosis with certain rhizobia), it is of great interest to know which rhizobia are able to nodulate key legumes in a specific habitat. This study describes the diversity of the rhizobia that are able to nodulate the shrub legume Spartocytisus supranubius in the harsh environmental conditions of the high mountain ecosystem of Teide National Park (Tenerife). The diversity of microsymbionts nodulating S. supranubius was estimated from a phylogenetic analysis of root nodule bacteria isolated from soils at three selected locations in the park. The results showed that a high diversity of species of Bradyrhizobium and two symbiovars can nodulate this legume. Phylogenies of ribosomal and housekeeping genes showed these strains distributed into three main clusters and a few isolates on separate branches. These clusters consist of strains representing three new phylogenetic lineages of the genus Bradyrhizobium. Two of these lineages belong to the B. japonicum superclade, which we refer to as B. canariense-like and B. hipponense-like, as the type strains of these species are the closest species to our isolates. The third main group was clustered within the B. elkanii superclade and is referred to as B. algeriense-like as B. algeriense is its closest species. This is the first time that bradyrhizobia of the B. elkanii superclade have been reported for the canarian genista. Furthermore, our results suggest that these three main groups might belong to potential new species of the genus Bradyrhizobium. Analysis of the soil physicochemical properties of the three study sites showed some significant differences in several parameters, which, however, did not have a major influence on the distribution of bradyrhizobial genotypes at the different locations. The B. algeriense-like group had a more restrictive distribution pattern, while the other two lineages were detected in all of the soils. This suggests that the microsymbionts are well adapted to the harsh environmental conditions of Teide National Park.

When time is not of the essence: constraints to the carbon balance of bryophytes.

The data available so far indicate that the photosynthetic and relative growth rates of bryophytes are 10% of those reported for tracheophytes. By examining the existing literature and reanalysing data published in over 100 studies, this review examines the ecophysiological, biochemical, and structural reasons behind this phenomenon. The limiting Rubisco content and surface for gas exchange are the internal factors that can explain the low photosynthetic and growth rates of bryophytes. The role of the thicker cell walls of bryophytes in limiting CO2 diffusion is unclear, due to the current uncertainties regarding their porosity and permeability to CO2. From this review, it is also evident that, despite bryophytes having low photosynthetic rates, their positive carbon balance is tightly related to their capacity to deal with extreme conditions. Contributing factors include their capacity to deal with large daily temperature oscillations, and their capacity to delay the cessation of photosynthesis under water deficit (or to tolerate desiccation in extreme situations). Although further studies on bryophytes are needed before more solid conclusions can be drawn, it seems that their success relies on their remarkable tolerance to a highly variable environment, possibly at the expense of their maximum photosynthetic rate.

A synthetic mRNA cell reprogramming method using CYCLIN D1 promotes DNA repair, generating improved genetically stable human induced pluripotent stem cells.

A key challenge for clinical application of induced pluripotent stem cells (iPSC) to accurately model and treat human pathologies depends on developing a method to generate genetically stable cells to reduce long-term risks of cell transplant therapy. Here, we hypothesized that CYCLIN D1 repairs DNA by highly efficient homologous recombination (HR) during reprogramming to iPSC that reduces genetic instability and threat of neoplastic growth. We adopted a synthetic mRNA transfection method using clinically compatible conditions with CYCLIN D1 plus base factors (OCT3/4, SOX2, KLF4, LIN28) and compared with methods that use C-MYC. We demonstrate that CYCLIN D1 made iPSC have (a) lower multitelomeric signal, (b) reduced double-strand DNA breaks, (c) correct nuclear localization of RAD51 protein expression, and (d) reduced single-nucleotide polymorphism (SNP) changes per chromosome, compared with the classical reprogramming method using C-MYC. CYCLIN D1 iPSC have reduced teratoma Ki67 cell growth kinetics and derived neural stem cells successfully engraft in a hostile spinal cord injury (SCI) microenvironment with efficient survival, differentiation. We demonstrate that CYCLIN D1 promotes double-stranded DNA damage repair predominantly through HR during cell reprogramming to efficiently produce iPSC. CYCLIN D1 reduces general cell stress associated with significantly lower SIRT1 gene expression and can rescue Sirt1 null mouse cell reprogramming. In conclusion, we show synthetic mRNA transfection of CYCLIN D1 repairs DNA during reprogramming resulting in significantly improved genetically stable footprint in human iPSC, enabling a new cell reprogramming method for more accurate and reliable generation of human iPSC for disease modeling and future clinical applications.

Summit evergreen shrubs living at a semi-arid treeline: photoprotection systems activation in an open vs an understory site.

High-mountain-ecosystems in the Mediterranean-type climate are exceptional because of their outstanding biodiversity but also because of their characteristic drought stress in summer. Still, plant functioning in these habitats has been largely understudied. Here, morphological, photochemical, and biochemical traits were seasonally assessed in six shrubs characterized by contrasting morphological traits, in the Teide mountain in the Canary Islands. Two adjacent populations, the first located in an open site and the second in the understorey of Pinus canariensis treeline forest, were evaluated. We aimed at disentangling (1) the role of morphological and biochemical photoprotective strategies and of their seasonal plasticity to cope with changing environmental conditions in this semiarid ecosystem, (2) how the interspecific differences in biochemical photoprotection are related to leaf morphology and phenology and (3) how living in the understory of the treeline may affect those responses. Our results indicate that both morphological and biochemical traits (particularly leaf habit, morphology and carotenoids from the ß-branch) play an intricate role in photoprotection, and that a high interspecific variability exists. According to the down-regulation of photochemical activity and the upregulation of photoprotective molecules, species could be grouped into three types: (1) those more responsive to summer stress (e.g. Descurainia bourgeauana); (2) those more responsive to winter stress (e.g. Pterocephalus lasiospermus, Scrophularia glabrata and Adenocarpus viscosus); and (3) those showing rather constant behavior across seasons (e.g. Spartocytisus supranubius and Erysimum scoparium). In all the species, plants in the open site showed a marked seasonal physiological response in most of the studied parameters. Pinus canariensis canopy buffers environmental abiotic constrains. On a global change scenario, and provided further functional studies are needed, our results pinpoints heterogeneity in the sensitivity of these species against for instance late-frost or summer-heat/drought events, which could easily shift current species distribution in the coming years.

Effect of pyrolysis conditions on the total contents of polycyclic aromatic hydrocarbons in biochars produced from organic residues: Assessment of their hazard potential.

The interest of using biochar, the solid byproduct from organic waste pyrolysis, as soil conditioner is significantly increasing. Nevertheless, persistent organic pollutants, such as polycyclic aromatic hydrocarbons (PAHs), are formed during pyrolysis due to the incomplete combustion of organic matter. Consequently, these pollutants may enter the environment when biochar is incorporated into soil and cause adverse ecological effects. In this study, we examined the content of the 16 United States Environmental Protection Agency (USEPA) PAHs in biochars produced from rice husk, wood, wheat and sewage sludge residues using three different pyrolytic reactors and temperatures (400, 500 and 600 °C). The total concentration of PAHs (∑PAH) ranged from 799 to 6364 µg kg-1, being naphthalene, phenanthrene and anthracene the most abundant PAHs in all the biochars. The maximum amount of PAHs was observed for the rice husk biochar produced in the batch reactor at 400 °C, which decreased with increasing temperature. The ∑PAH value of the wood biochar produced via traditional kilns doubled compared with the wood biochar produced using the other pyrolytic reactors (5330 µg kg-1 in Kiln; 2737 µg kg-1 in batch and 1942 µg kg-1 in the rotary reactor). Looking for a more reliable risk assessment of the potential exposure of PAHs in biochar, the total toxic equivalent concentrations (TTEC) of the 14 produced biochars were calculated. When comparing the same feedstock and temperature, TTEC values indicated that the rotary reactor produced the safest biochars. In contrast, the biochars produced using the batch reactor at 400 and 500 °C have the greatest hazard potential. Our results provide valuable information on the potential risk of biochar application for human and animal health, as well as for the environment due to PAHs contamination.

Scientific evidence on perineal trauma during labor: Integrative review.

OBJECTIVE: To assess the scientific evidence for management and preservation of perineal integrity during the expulsive stage of labor. STUDY DESIGN: Integrative review that employed the Population, Intervention, Comparison, Outcome strategy to formulate the research question: Which perineal measure(s) is(are) effective in maintaining perineal integrity during labor? The search was performed in the databases MEDLINE, LILACS, BDENF and SciELO. The ten selected studies were analyzed based on their level of evidence and grade of recommendation. RESULTS: Four categories of measures were located: antenatal perineal care, perineal massage during the expulsive phase of labor, manual perineal support during the expulsive phase of labor and perineal hyaluronidase injection. CONCLUSION: Based on its level of evidence, perineal massage with lubricants performed by the women or their partners at the end of pregnancy may be recommended as a measure favorable for perineal protection.

Análise da sobrevida de pacientes com lesão renal aguda no pós-operatório de cirurgia cardíaca: revisão integrativa da literatura / Survival analysis of patients with acute kidney injury in the postoperative period of cardiac surgery: integrative literature review

Introdução: A lesão renal aguda (LRA) é uma importante complicação em cirurgias cardíacas. Pacientes que desenvolvem LRA implicam aumento do tempo de hospitalização,diminuição da qualidade de vida e da sobrevida. Objetivo: Avaliar a sobrevida de pacientes que desenvolvem LRA no pós-operatório de cirurgia cardíaca, bem como as variáveis associadasa ela. Método: Revisão integrativa da literatura realizada a partir da seleção de artigos publicados entre 2009 e 2014, indexados por Medline/PUBMED, nos idiomas português, inglês e espanhol. Foram identificados inicialmente 597 artigos com a utilização dos descritorescontrolados. Após aplicação dos critérios de inclusão e exclusão, 17 artigos foram selecionados para análise por dois pesquisadores independentes. Resultados: Os pacientesque desenvolvem LRA após cirurgia cardíaca podem apresentar fatores de risco pré, intra e pós-operatórios que interferem na taxa de sobrevida, e evoluem com uma sobrevida médiade cinco anos. Conclusão: A sobrevida foi gradativamente reduzida nos primeiros cinco anos após a cirurgia e os principais fatores apontados como preditores de mortalidade foram,entre outros, idade avançada, gravidade da disfunção renal e tipo de cirurgia.

Increased water use efficiency does not prevent growth decline of Pinus canariensis in a semi-arid treeline ecotone in Tenerife, Canary Islands (Spain).

KEY MESSAGE: Intrinsic water-use efficiency of Pinus canariensis (Sweet ex Spreng.) growing at a semi-arid treeline has increased during the past 37 years. Tree-ring width by contrast has declined, likely caused by reduced stomatal conductance due to increasing aridity. CONTEXT: Rising atmospheric CO2 concentration (Ca ) has been related to tree growth enhancement accompanied by increasing intrinsic water-use-efficiency (iWUE). Nevertheless, the extent of rising Ca on long-term changes in iWUE and growth has remained poorly understood to date in Mediterranean treeline ecosystems. AIMS: This study aimed to examine radial growth and physiological responses of P. canariensis in relation to rising Ca and increasing aridity at treeline in Tenerife, Canary Islands, Spain. METHODS: We evaluated temporal changes in secondary growth (tree-ring width; TRW) and tree ring stable C isotope signature for assessing iWUE from 1975 through 2011. RESULTS: Precipitation was the main factor controlling secondary growth. Over the last 36 years P. canariensis showed a decline in TRW at enhanced iWUE, likely caused by reduced stomatal conductance due to increasing aridity. CONCLUSION: Our results indicate that increasing aridity has overridden the potential CO2 fertilization on tree growth of P. canariensis at its upper distribution limit.

Generation of mice with longer and better preserved telomeres in the absence of genetic manipulations.

Although telomere length is genetically determined, mouse embryonic stem (ES) cells with telomeres of twice the normal size have been generated. Here, we use such ES cells with 'hyper-long' telomeres, which also express green fluorescent protein (GFP), to generate chimaeric mice containing cells with both hyper-long and normal telomeres. We show that chimaeric mice contain GFP-positive cells in all mouse tissues, display normal tissue histology and normal survival. Both hyper-long and normal telomeres shorten with age, but GFP-positive cells retain longer telomeres as mice age. Chimaeric mice with hyper-long telomeres also accumulate fewer cells with short telomeres and less DNA damage with age, and express lower levels of p53. In highly renewing compartments, such as the blood, cells with hyper-long telomeres are longitudinally maintained or enriched with age. We further show that wound-healing rates in the skin are increased in chimaeric mice. Our work demonstrates that mice with functional, longer and better preserved telomeres can be generated without the need for genetic manipulations, such as TERT overexpression.

ATRX driver mutation in a composite malignant pheochromocytoma.

Pheochromocytomas (PCCs) and paragangliomas (PGLs) are tumors arising from the adrenal medulla and sympathetic/parasympathetic paraganglia, respectively. Approximately 40% of PCCs/PGLs are due to germline mutations in one of 16 susceptibility genes, and a further 30% are due to somatic alterations in 5 main genes. Recently, somatic ATRX mutations have been found in succinate dehydrogenase (SDH)-associated hereditary PCCs/PGLs. In the present study we applied whole-exome sequencing to the germline and tumor DNA of a patient with metastatic composite PCC and no alterations in known PCC/PGL susceptibility genes. A somatic loss-of-function mutation affecting ATRX was identified in tumor DNA. Transcriptional profiling analysis classified the tumor within cluster 2 of PCCs/PGLs (without SDH gene mutations) and identified downregulation of genes involved in neuronal development and homeostasis (NLGN4, CD99 and CSF2RA) as well as upregulation of Drosha, an important gene involved in miRNA and rRNA processing. CpG island methylator phenotype typical of SDH gene-mutated tumors was ruled out, and SNP array data revealed a unique profile of gains and losses. Finally, we demonstrated the presence of alternative lengthening of telomeres in the tumor, probably associated with the failure of ATRX functions. In conclusion, somatic variants affecting ATRX may play a driver role in sporadic PCC/PGL.

Localization-dependent and -independent roles of SLX4 in regulating telomeres.

SLX4, a scaffold for structure-specific DNA repair nucleases, is important for several types of DNA repair. Many repair proteins bind to sites of DNA damage, resulting in subnuclear "foci," but SLX4 forms foci in human cells even without DNA damage. Using several approaches, we show that most, but not all, SLX4 foci localize to telomeres in a range of human cell lines irrespective of the mechanisms used to maintain telomere length. The SLX1 Holliday-junction-processing enzyme is recruited to telomeres by SLX4, and SLX4, in turn, is recruited by a motif that binds to the shelterin subunit TRF2 directly. We also show that TRF2-dependent recruitment of SLX4 prevents telomere damage. Furthermore, SLX4 prevents telomere lengthening and fragility in a manner that appears to be independent of telomere association. These findings reveal that SLX4 plays multiple roles in regulating telomere homeostasis.

Osteonecrosis mandibular asociada a bifosfonatos: Presentación de caso clínico / Bisphosphonate-associated jaw osteonecrosis: Clinical case presentation

El objetivo del trabajo es presentar la asociación de la osteonecrosis mandibular y el uso de bifosfonatos con fines terapéuticos. Los bifosfonatos son fármacos utilizados en el manejo de los desórdenes primarios y secundarios del hueso. Principalmente en la osteoporosis tanto local como general, enfermedades metabólicas óseas, calcificación de tejidos blandos y estados de hipercalcemia, entre otras. Asimismo también pueden actuar como antineoplásicos al inhibir la activación de proteínas vinculadas al cáncer. En los últimos años se ha incrementado su uso para la prevención de osteoporosis postmenopáusicas, gracias a que favorece el incremento en la densidad de minerales en huesos, lo que ha permitido la disminución fracturas. Se presenta un caso clínico de paciente femenina de 61 años de edad con el antecedente de cáncer de mama, ingesta de bifosfonato desde hace 3 años, la cual presentó exposición ósea espontánea, asintomática. Se muestra su manejo médico-quirúrgico y su evolución.

The aim of the present paper was to present the association between mandibular osteonecrosis and use of bisphosphonates for therapeutic purposes. Bisphosphonates are drugs used for the treatment of primary and secondary bone disorders. They are mainly used for local and general osteoporosis treatment, metabolic bone diseases, soft tissue calcification as well as hypercalcemia occurrence, among others. They can also act as antineoplastic agents through the inhibition of activation of cancer-linked proteins. In recent years, bisphosphonates have been used to prevent post-menopausae osteoporosis, since they enhance mineral density increase in bone and therefore contribute to a decrease in fractures. The clinical case here presented is that of a 61 year old female patient with previous history of breast cancer and ongoing 3 year bisphosphonate intake. The patient presented asymptomatic, spontaneous bone exposition. Surgical and medical handlings of the case are presented, as well as its evolution.

Telomerase gene therapy in adult and old mice delays aging and increases longevity without increasing cancer.

A major goal in aging research is to improve health during aging. In the case of mice, genetic manipulations that shorten or lengthen telomeres result, respectively, in decreased or increased longevity. Based on this, we have tested the effects of a telomerase gene therapy in adult (1 year of age) and old (2 years of age) mice. Treatment of 1- and 2-year old mice with an adeno associated virus (AAV) of wide tropism expressing mouse TERT had remarkable beneficial effects on health and fitness, including insulin sensitivity, osteoporosis, neuromuscular coordination and several molecular biomarkers of aging. Importantly, telomerase-treated mice did not develop more cancer than their control littermates, suggesting that the known tumorigenic activity of telomerase is severely decreased when expressed in adult or old organisms using AAV vectors. Finally, telomerase-treated mice, both at 1-year and at 2-year of age, had an increase in median lifespan of 24 and 13%, respectively. These beneficial effects were not observed with a catalytically inactive TERT, demonstrating that they require telomerase activity. Together, these results constitute a proof-of-principle of a role of TERT in delaying physiological aging and extending longevity in normal mice through a telomerase-based treatment, and demonstrate the feasibility of anti-aging gene therapy.

Association of circulating visfatin concentrations with insulin resistance and low-grade inflammation after dietary energy restriction in Spanish obese non-diabetic women: role of body composition changes.

BACKGROUND AND AIMS: To assess the influence of body composition changes on circulating serum visfatin after following 12 weeks of energy restricted diet intervention. We also examined the possible role of visfatin in glucose metabolism and in obesity-associated low-grade inflammation. METHODS AND RESULTS: A total of 78 obese (BMI 34.0 ± 2.8 kg/m²) women aged 36.7±7 y volunteered to participate in the study. We measured by DXA body fat mass (FM) and lean mass (LM). Fasting serum visfatin, glucose, insulin, adiponectin, leptin, IL-1ß, IL-6, IL-8, TNF-α and CRP concentrations were analyzed before and after the intervention and HOMA and QUIKI indexes were calculated. Mean weight loss 7.7 ± 3.0 kg and HOMA decreased in 24 ± 35%. Serum visfatin concentration change was negatively associated with LM difference (P < 0.05), whereas no significant relationship was observed with FM changes after energy restricted diet intervention. Changes in circulating serum visfatin levels were significantly and inversely associated with HOMA-IR (P < 0.01) and positively with QUICKI index (P < 0.02) after energy restricted diet intervention, regardless of achieved body weight loss. We did not find any significant association between changes in visfatin levels and IL-1ß, IL-6, IL-8, TNF-α and CRP levels after dietary intervention (all P > 0.2). CONCLUSION: Circulating visfatin concentration is associated with sensitivity improvement achieved after energy restricted diet intervention induced weight loss. Furthermore, LM changes could be an influencing factor on visfatin concentrations and consequently, on the improvement of insulin sensitivity after weight loss in obese non-diabetic women. Our findings did not provide any evidence for a role of visfatin increase on low-grade inflammation after weight loss.

Mammalian Rap1 controls telomere function and gene expression through binding to telomeric and extratelomeric sites.

Rap1 is a component of the shelterin complex at mammalian telomeres, but its in vivo role in telomere biology has remained largely unknown to date. Here we show that Rap1 deficiency is dispensable for telomere capping but leads to increased telomere recombination and fragility. We generated cells and mice deleted for Rap1; mice with Rap1 deletion in stratified epithelia were viable but had shorter telomeres and developed skin hyperpigmentation in adulthood. By performing chromatin immunoprecipitation coupled with ultrahigh-throughput sequencing, we found that Rap1 binds to both telomeres and to extratelomeric sites through the (TTAGGG)(2) consensus motif. Extratelomeric Rap1-binding sites were enriched at subtelomeric regions, in agreement with preferential deregulation of subtelomeric genes in Rap1-deficient cells. More than 70% of extratelomeric Rap1-binding sites were in the vicinity of genes, and 31% of the genes deregulated in Rap1-null cells contained Rap1-binding sites, suggesting a role for Rap1 in transcriptional control. These findings place a telomere protein at the interface between telomere function and transcriptional regulation.

TPP1 is required for TERT recruitment, telomere elongation during nuclear reprogramming, and normal skin development in mice.

The TPP1/ACD protein (hereafter TPP1) is a component of the shelterin complex at mammalian telomeres. Here we find that Tpp1-deficient mouse embryonic fibroblasts (MEFs) show increased chromosomal instability including sister chromatid fusions and chromosomes with multitelomeric signals related to telomere fragility. Tpp1 deletion decreases both TERT (the telomerase catalytic subunit) binding to telomeres in MEFs and telomerase function at chromosome ends in vivo. Abrogation of Tpp1 abolished net telomere elongation in the context of nuclear reprogramming of MEFs into induced pluripotent stem cells, whereas Tpp1 deletion in stratified epithelia of Tpp1(Delta/Delta)K5-Cre mice resulted in perinatal death, severe skin hyperpigmentation, and impaired hair follicle morphogenesis. p53 deficiency rescues skin hyperpigmentation and hair growth in these mice, indicating that p53 restricts proliferation of Tpp1-deficient cells. These results suggest a telomere-capping model where TPP1 protects telomere integrity and regulates telomerase recruitment to telomeres, thereby preventing early occurrence of degenerative pathologies.

ATR suppresses telomere fragility and recombination but is dispensable for elongation of short telomeres by telomerase.

Telomere shortening caused by incomplete DNA replication is balanced by telomerase-mediated telomere extension, with evidence indicating that the shortest telomeres are preferred substrates in primary cells. Critically short telomeres are detected by the cellular DNA damage response (DDR) system. In budding yeast, the important DDR kinase Tel1 (homologue of ATM [ataxia telangiectasia mutated]) is vital for telomerase recruitment to short telomeres, but mammalian ATM is dispensable for this function. We asked whether closely related ATR (ATM and Rad3 related) kinase, which is important for preventing replicative stress and chromosomal breakage at common fragile sites, might instead fulfill this role. The newly created ATR-deficient Seckel mouse strain was used to examine the function of ATR in telomerase recruitment and telomere function. Telomeres were recently found to resemble fragile sites, and we show in this study that ATR has an important role in the suppression of telomere fragility and recombination. We also find that wild-type ATR levels are important to protect short telomeres from chromosomal fusions but do not appear essential for telomerase recruitment to short telomeres in primary mouse embryonic fibroblasts from the ATR-deficient Seckel mouse model. These results reveal a previously unnoticed role for mammalian ATR in telomere protection and stability.

Zidovudine induces S-phase arrest and cell cycle gene expression changes in human cells.

Antiretroviral therapy for the human immunodeficiency virus-1 (HIV-1) typically includes two nucleoside reverse transcriptase inhibitors (NRTIs). 3'-Azido-3'-deoxythymidine (AZT, Zidovudine) plus 2'-deoxy-3'-thiacytidine (3TC, Lamivudine) is a combination that is used frequently. The NRTIs are mutagenic nucleoside analogs that become incorporated into DNA and terminate replication. We therefore hypothesized that exposure to this class of drug may alter cell cycle parameters. We used flow cytometry to examine the cell cycle in human epithelioid carcinoma (HeLa) cells exposed to AZT and 3TC alone, as well as a series of AZT/3TC dose combinations: (A) 125.0 microM AZT/12.5 microM 3TC; (B) 250.0 microM AZT/25.0 microM 3TC; and (C) 500 microM AZT/50 microM 3TC. At 24 h, at all doses, there was a good cell viability (>/=68%), and incorporation of AZT into nuclear DNA. Using flow cytometry, a dose-related increase in the percentage of cells in S phase, from 9.5% with no drug, to 36.0% with dose C, was observed in cells exposed for 24 h (P = 0.001, ANOVA). A concomitant decrease in the percentage of cells in G(1) phase, from 82.6% with no drug to 58.5% with dose C, was observed in cells exposed for 24 h (P = 0.017, ANOVA). A similar S phase arrest was seen in cells exposed to 125, 250 and 500 microM AZT alone, but there was no S phase alteration with 50 microM 3TC alone, suggesting that AZT is responsible for the accumulation of cells in S phase. To elucidate the accumulation of cells in S phase and explore the cell cycle gene expression changes induced by AZT and 3TC, we used c-DNA microarray, Cell Cycle Super Array and real-time PCR. There was a strong upregulation of the DNA damage-inducible transcript 3 (DDIT3 or GADD153) in NRTI-exposed cells. In addition, AZT induced an upregulation of cyclin D1 accompanied by a downregulation of the cyclin D1-associated inhibitors P18 and P57, and the G(1)-S check point gene P21, the net effect of which would be to foster a cell progression into S phase. Cyclin A2 was down-regulated in cells exposed to AZT, suggesting a block in S-G(2)-M progression that would also be consistent with the accumulation of cells in S phase. Overall, the study demonstrates that AZT, but not 3TC, causes an arrest of cells in S phase with a consistent alteration in the expression of several cell cycle genes.