The molecular mechanisms of sensing nucleoside analogue-induced DNA damage

Nucleoside analogues are antimetabolites effective in the treatment of a wide variety of solid tumors and hematological malignancies. Upon being metabolized to their active triphosphate form, these agents are incorporated into DNA during replication or excision repair synthesis. Because DNA polymerases have a greatly decreased affinity for primers terminated by most nucleoside analogues, their incorporation causes stalling of replication forks. The molecular mechanisms that recognize blocked replication may contribute to drug resistance but have not yet been elucidated. Here, several molecules involved in sensing nucleoside analogue-induced stalled replication forks have been identified and examined for their contribution to drug resistance. ^ The phosphorylation of the DNA damage sensor, H2AX, was characterized in response to nucleoside analogues and found to be dependent on both time and drug concentration. This response was most evident in the S-phase fraction and was associated with an inhibition of DNA synthesis, S-phase accumulation, and activation of the S-phase checkpoint pathway (Chk1-Cdc25A-Cdk2). Exposure of the Chk1 inhibitor, 7-hydroxystaurosporine (UCN-01), to cultures previously treated with nucleoside analogues caused increased apoptosis, clonogenic death, and a further log-order increase in H2AX phosphorylation, suggesting enhanced DNA damage. Ataxia-telangiectasia mutated (ATM) has been identified as a key DNA damage signaling kinase for initiating cell cycle arrest, DNA repair, and apoptosis while the Mre11-Rad50-Nbs1 (MRN) complex is known for its functions in double-strand break repair. Activated ATM and the MRN complex formed distinct nuclear foci that colocalized with phosphorylated H2AX after inhibition of DNA synthesis by the nucleoside analogues, gemcitabine, ara-C, and troxacitabine. Since double-strand breaks were undetectable, this response was likely due to stalling of replication forks. A similar DNA damage response was observed in human lymphocytes after exposure to ionizing radiation and in acute myelogenous leukemia blasts during therapy with the ara-C prodrug, CP-4055. Deficiencies in ATM, Mre11, and Rad50 led to a two- to five-fold increase in gemcitabine sensitivity, suggesting that these molecules contribute to drug resistance. Based on these results, a model is proposed for the sensing of nucleoside analogue-induced stalled replication forks that includes H2AX, ATM, and the Mre11-Rad50-Nbs1 complex. ^

A methodologic study of pain assessment in Anglo and Hispanic children with cancer

There is growing clinical evidence that even young children experience pain and accompanying anxiety. Few instruments have been validated to assess pain characteristics in children. The study of related demographic, illness, psychologic and parental factors in children has also been limited. This study examines the reliability and validity of pain assessment tools in an outpatient pediatric cancer population. A total of 78 children from three to fifteen years of age were observed and interviewed about the pain of invasive procedures. The effect of cultural factors and the stress of acculturation were examined by comparing data from two cultural groups, Anglo and Hispanic.^ Spielberger State-Trait Anxiety Scales were administered to children and parents prior to an invasive procedure. The Procedure Behavioral Checklist (PBCL) was used for observation of the child's response during the procedure. The Children's Procedural Interview (CPI) which contains items on the PBCL and visual analogues (scales of faces indicating varying degrees of pain and anxiety) was administered following the procedure.^ Reliability coefficients for Anglos were.78 on the PBCL,.79 on the CPI and.85 on the visual analogue scales. For Hispanics, the reliability for the PBCL was.54, while the CPI had a reliability of.72 and the visual analogue scales,.87. Construct validity was demonstrated by high correlations between the PBCL and CPI scores for both ethnic groups (.66 for Anglos and.64 for Hispanics) and by the significant correlation of State anxiety scores with both PBCL and CPI scores. Age was inversely correlated with PBCL and CPI scores for both ethnic groups. Hispanic parents' anxiety scores were higher than Anglo parents, but were not highly correlated with their child's PBCL, CPI or State-Trait anxiety scores. Caregivers' ratings were correlated with the PBCL scores for Anglos but not for Hispanics.^ The findings of this study indicate that pain responses may be reliably assessed using both observational and self-report methods in children, though differences in Anglo and Hispanic cultures exist. Differences in pain symptomatology and assessment in the two cultural groups warrant further study. ^

Examination of synthetic retinoid -induced apoptosis in cutaneous squamous cell carcinomas: Mechanisms and implications for chemoprevention and chemotherapy with retinoids

Non-melanoma skin cancers, including basal cell carcinoma and squamous cell carcinoma (SCC), are the most common neoplasms in the United States with a lifetime risk nearly equal to all other types of cancer combined. Retinoids are naturally occurring and synthetic analogues of vitamin A that bind to nuclear retinoid receptors and modulate gene expression as a means of regulating cell proliferation and differentiation. Retinoids have been employed for many years in the treatment of various cutaneous lesions and for cancer chemoprevention and therapy. The primary drawback limiting the use of retinoids is their toxicity, which is also associated with receptor-gene interactions. In this study, the effects of the synthetic retinoids N-(4-hydroxyphenyl)retinamide (4HPR) and 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437) were examined in cutaneous keratinocytes. Four human cutaneous SCC cell lines were examined along with normal human epidermal keratinocyte (NHEK) cells from two donors. Sensitivity to 4HPR or CD437 alone or in combination with other agents was determined via growth inhibition, cell cycle distributions, or apoptosis induction. Both synthetic retinoids were able to promote apoptosis in SCC cells more effectively than the natural retinoid all-trans retinoic acid. Apoptosis could not be inhibited by nuclear retinoic acid receptor antagonists. In NHEK cells, 4HPR induced apoptosis while CD437 promoted G1 arrest. 4HPR acted as a prooxidant by generating reactive oxygen species (ROS) in SCC and NHEK cells. 4HPR-induced apoptosis in SCC cells could be inhibited or potentiated by manipulating cellular defenses against oxidative stress, indicating an essential role for ROS in 4HPR-induced apoptosis. CD437 promoted apoptosis in SCC cells in S and G2/M phases of the cell cycle within two hours of treatment, and this rapid induction could not be blocked with cycloheximide. This study shows: (1) 4HPR- and CD437-induced apoptosis do not directly involve a traditional retinoid pathway; (2) 4HPR can act as a prooxidant as a means of promoting apoptosis; (3) CD437 induces apoptosis in SCC cells independent of protein synthesis and is potentially less toxic to NHEK cells; and (4) 4HPR and CD437 operate under different mechanisms with respect to apoptosis induction and this may potentially enhance their therapeutic index in vivo. ^

A new modern analogue reference dataset and its application to the 430-kyr pollen record from Lake Biwa

This study presents a newly compiled dataset of modern pollen and climate data from 798 sites across Japan and the Russian Far East. This comprehensive reference dataset combined with the modern analogue technique (MAT) provides a powerful tool for pollen-based reconstruction of the Quaternary Northwest Pacific climate. Pollen-derived reconstruction of the modern climate at the reference pollen-sampling sites matches well with the estimated modern climate values (R2 values vary between 0.79 and 0.95, and RMSEP values vary between 5.8 and 9.7% of the modern climatic range for all nine tested variables). The successful testing of the method encourages its application to the fossil pollen records. We used a coarse-resolution pollen record from Lake Biwa to reconstruct glacial-interglacial climate dynamics in central Japan since ~438 kyr and compared it to the earlier reconstruction based on a less representative reference dataset. The current and earlier results consistently demonstrate that the coldest glacial intervals experienced pronounced cooling in winter and moderate cooling in summer, supporting the growth of cool mixed forest (COMX) where warm mixed forest (WAMX) predominates today. During the last glacial, maximum (~24 kyr BP) mean temperatures of the coldest (MTCO) and warmest (MTWA) month were about -13 °C (RMSEP = 2.34 °C) and 21 °C (RMSEP = 1.66 °C) respectively, and annual precipitation (PANN) was about 800 mm (RMSEP = 158.06 mm). During the thermal optimums of the interglacial intervals, the temperatures of the coldest and warmest month were above 0 °C and 25 °C respectively, leading to the reconstruction of WAMX and temperate conifer forest (TECO). Although both these vegetation types grow in the southern part of Japan today, WAMX requires warmer space. The presence of WAMX during marine isotope stages (MIS) 11 and 1, and its absence during MIS 9 and MIS 5 contradict the marine isotope and Antarctic ice records, suggesting that the latter two interglacials were the warmest of the last 800 kyr. The apparent contradiction allows at least three different explanations including low temporal resolution of the pollen record; different trends in CO2 concentrations during 'short' and 'long' interglacials; and regional climate variability and non-linear response of different regions to the global forcing. More definitive conclusions will be possible on the basis of forthcoming high-resolution pollen records from central Japan.

Chemical and isotopic compositions of rocks, ores, and hydrothermal minerals from the Rainbow and Logachev-2 hydrothermal fields

The aim of this paper is to analyze and compare mineralogy and geochemistry of copper-zinc sulfide ores from the Logachev-2 and Rainbow hydrothermal fields of the Mid-Atlantic Ridge (MAR) confined to serpentinite protrusions. It was found that Zn(Fe) and Cu, Fe(Zn) sulfides had been deposited in black smokers pipes almost simultaneously from intermittently flowing, nonequilibrium H2S-low solutions of different temperatures. Pb isotope composition confirmed that the deep oceanic crust had been a source of lead. The ores from the Rainbow field are 20-fold higher in Co than ores restricted to basalts and show a high ratio of Co/Ni=46. The ores from the Rainbow field are enriched in 34S isotope (aver. d34S=10 per mil) because of constant flow of cold sea water into the subsurface zone of the hydrothermal system. Ores from the Logachev-2 field are 8 times higher in gold compared to other MAR regions. Sulfide ores from the Rainbow and Logachev-2 fields have no analogues among MAR ore occurrences in terms of enrichment in valuable components (Zn, Cd, Co, and Au).

Reference datasets and pollen record from sediment core YAK, Yakumo, SW Hokkaido

This study reconstructs middle and late Holocene vegetation and climate dynamics in the Oshima Peninsula, SW Hokkaido, using the published method of biome reconstruction and modern analogue technique applied to the Yakumo pollen record (42°17'03''N, 140°15'34''E) spanning the last 5500 years. Two previously published matrices assigning Japanese plant/pollen taxa to the major vegetation types (biomes) are tested using a newly compiled dataset of 78 surface pollen spectra from Hokkaido. With both matrices showing strengths and weaknesses in reconstructing cool mixed and temperate deciduous forests of Hokkaido, the results suggest the necessity to consider the whole list of identified terrestrial pollen taxa for generating robust vegetation reconstructions for northern Japan. Applied to the fossil pollen data, both biome-reconstruction approaches demonstrate consistently that oak-dominated cool mixed forest spread in the study region between 5.5 and 3.6 cal ka BP and was subsequently replaced by beech-dominated temperate deciduous forest. The pollen-based climate reconstruction suggests this change in the vegetation composition was caused by a shift from cooler and drier than present climate to warmer and wetter, similar to modern conditions about 3.6 cal ka BP. Comparing the pollen-based reconstruction results with the published marine records from the NW Pacific, the reconstructed vegetation and climate dynamics can be satisfactorily explained by the greater role played by the warm Tsushima Current in the Sea of Japan and in the Tsugaru Strait during the middle and late Holocene. An increase in sea surface temperatures west and south of the study site would favour air temperature rise and moisture uptake and cause an increase in precipitation and snow accumulation in the western part of Hokkaido during the late Holocene.