Downregulation of hPMC2 imparts chemotherapeutic sensitivity to alkylating agents in breast cancer cells

Triple negative breast cancer cell lines have been reported to be resistant to the cyotoxic effects of temozolomide (TMZ). We have shown previously that a novel protein, human homolog of Xenopus gene which Prevents Mitotic Catastrophe (hPMC2) has a role in the repair of estrogen-induced abasic sites. Our present study provides evidence that downregulation of hPMC2 in MDA-MB-231 and MDA-MB-468 breast cancer cells treated with temozolomide (TMZ) decreases cell survival. This increased sensitivity to TMZ is associated with an increase in number of apurinic/apyrimidinic (AP) sites in the DNA. We also show that treatment with another alkylating agent, BCNU, results in an increase in AP sites and decrease in cell survival. Quantification of western blot analyses and immunofluorescence experiments reveal that treatment of hPMC2 downregulated cells with TMZ results in an increase in γ-H2AX levels, suggesting an increase in double strand DNA breaks. The enhancement of DNA double strand breaks in TMZ treated cells upon downregulation of hPCM2 is also revealed by the comet assay. Overall, we provide evidence that downregulation of hPMC2 in breast cancer cells increases cytotoxicity of alkylating agents, representing a novel mechanism of treatment for breast cancer. Our data thus has important clinical implications in the management of breast cancer and brings forth potentially new therapeutic strategies.     

Induction of DNA double-strand breaks in human gingival fibroblasts by eluates from titanium dioxide modified glass ionomer cements

Objectives

(1) To investigate the genotoxicity of a glass ionomer cement (GIC) and GIC incorporated with titanium dioxide nanopoarticle (TiO₂NPs) and with microparticle (TiO₂MPs) on DNA double-strand breaks of human gingival fibroblast cells (HGFs). (2) To compare the genotoxic differences between GIC and two modified cements.

X-ray induced DNA double-strand breaks in coronary CT angiography: comparison of sequential, low-pitch helical and high-pitch helical data acquisition

Background

Aim of this study was to compare DNA double-strand breaks (DSBs) in blood lymphocytes of patients undergoing high-pitch helical, low-pitch helical and sequential coronary CT angiography.

Methods and results

66 patients were examined with various scan protocols and modes (low-pitch helical scan: 100–120 kV, 320–438 mAs/rot, pitch 0.18–0.39, with or without ECG-pulsing, n = 35; prospectively ECG-triggered high-pitch helical scan: 100–120 kV, 320–456 mAs/rotation, pitch 3.2–3.4, n = 19; prospectively ECG-triggered sequential scan: 100–120 kV, 150–300 mAs or 320–370 mAs/rotation, n = 12) either using a 64-slice or 128-slice dual-source CT or a 128-slice single source CT scanner. Blood samples were obtained before and 30 min after CT and DSBs were analyzed in isolated lymphocytes using γ-H2AX immunofluorescence microscopy.A significant increase of DSBs was measurable 30 min after CTA (range 0.01–0.71/cell). CT induced DSBs showed a significant correlation with the estimated effective dose (ρ = 0.90, p < 0.00001). Both prospectively ECG-triggered sequential (0.10 DSBs/cell, 176 mGy cm, p < 0.00001) and high-pitch helical scan protocols (0.03 DSBs/cell, 109 mGy cm, p < 0.00001) led to a significant reduction of median DLP and DSB levels compared to low-pitch helical scans (0.34 DSBs/cell, 828 mGy cm). A reduction of the tube voltage resulted in significantly lower whereas additional calcium scoring resulted in elevated DLP and DNA damages (p < 0.05 each).

Conclusion

In coronary CTA, data acquisition protocols have a significant influence on the X-ray induced DSB levels. Using γ-H2AX immunofluorescence microscopy different scan modes in different CT generations can be compared concerning their biological impact.     

Sperm selection during ICSI treatments reduces single- but not double-strand DNA break values compared to the semen sample

PurposeTo detect a possible bias in sperm DNA fragmentation (SDF) testing when performed on semen samples or on those few spermatozoa selected for Intracytoplasmic Sperm Injection (ICSI) treatments.MethodsA multimethodological analysis of Single- and Double-Strand DNA Breaks (SSB and DSB, respectively) was performed through the Neutral Comet, the Alkaline Comet, the Sperm Chromatin Dispersion (SCD) and the Terminal deoxynucleotidyl transferase dUTP Nick End Labelling (TUNEL) assays. SDF was evaluated in (i) semen samples from 23 infertile patients (not achieving pregnancy or suffering recurrent miscarriage); (ii) samples after a Swim-up and (iii) spermatozoa microselected for ICSI (ICSI-S).ResultsThe analysis of 3217 ICSI-S revealed a significant reduction of SSB values compared to the Ejaculate and the Swim-up samples. On the contrary, DSB values were not reduced after any sperm selection method. The No-pregnancy group presented poorer semen parameters and higher SSB values. The Recurrent miscarriage group presented better semen parameters but also higher DSB values.ConclusionThe analysis of SDF on semen samples may not be fully representative of those few spermatozoa selected for ICSI. Since oxidative stress impairs sperm motility and causes SSB, selecting a motile sperm may intrinsically imply choosing a sperm not affected by this damage. DSB have an enzymatic origin which does not affect motility, making it difficult to select a sperm without this damage. Therefore, ICSI treatments could be effective in patients presenting high SSB values. Patients presenting high DSB values should expect bad ICSI results if this damage is not reduced through other specific methods.     

玻璃体切割手术相关医源性视网膜裂孔的临床观察

目的分析玻璃体切割手术过程中医源性视网膜裂孔(iatrogenic retinal breaks,氉IRB)的发生率、危险因素及预防措施。方法回顾分析129例133眼玻璃体手术病例,记录眼别、术前诊断、玻璃体后脱离状态、晶状体状态,分析术中IRB的数目、分布及预防措施。结果 133眼中12眼术中出现13个IRB,总体发生率为9.0%。其中,黄斑裂孔或黄斑前膜33眼中3眼(9.1%)出现IRB,增生性糖尿病视网膜病变52眼中6眼(11.5%)出现IRB,玻璃体积血40眼中3眼(7.5%)出现IRB。8眼(6.0%)出现巩膜切口相关IRB,5眼(3.8%)出现其他区域IRB,其中1眼同时存在巩膜切口相关IRB及其他区域IRB。术中制作玻璃体后脱离组的IRB发生率(12.3%)高于术前存在玻璃体后脱离组(6.6%),但差异无统计学意义(P=0.256);有晶状体组的IRB发生率(10.5%)高于术中联合超声乳化组(4.5%)及人工晶状体或无晶状体组(8.0%),但差异均无统计学意义(均为P>0.05)。术后发生视网膜脱离4眼(3.0%),所有视网膜脱离眼经二次手术后视网膜均达到解剖复位。结论各种疾病行玻璃体切割手术均有发生IRB的可能,没有证据表明术中制作玻璃体后脱离及晶状体状态与IRB的发生具有明显相关性。为避免IRB可能引起的玻璃体术后视网膜脱离,术中仔细排查与处理是非常重要的。     

不同周期的S-180V细胞DNA辐射损伤修复的比较

根据小鼠腹水瘤S-180 Ⅴ细胞的生长特点,设计了获取G_1、S、M期同步化细胞的方法,并用碱洗脱法对同步化细胞DNA单链断裂程度与重接修复速度进行了比较。观察到0～50Gyγ线照射范围内,不同周期细胞DNA单链断裂损伤无明显差别。30Gy照射后,不同周期细胞的断裂的DNA的修复速度不同,其中以S期重接修复速度最慢。     

Detection of DNA single-strand breaks in lymphocytes of smokers

Summary In a controlled study, ten male volunteers (five smokers and five nonsmokers) were subjected to different smoking conditions and compared to five non-smokers, not exposed to cigarette smoke. During the 4 days of the study, nonsmoking periods were strictly controlled. On the first day the ten subjects were sham exposed. On the second day the five smokers smoked 24 cigarettes in 8 h, while the five nonsmokers were exposed to the environmental tobacco smoke. After another day of sham exposure the smoke exposure was repeated under the same conditions. Blood was drawn before and after exposure and DNA single-strand breaks (SSBs) were analyzed in lymphocytes immediately (1 h) after isolation of cells and after 4 h incubation at 37°C, using a modified assay based on the nick translation reaction. Base levels of unscheduled DNA synthesis (UDS) and UDS levels were determined after 1 h incubation with methyl methanesulfonate. Duplicate analysis using the same method was performed in a second laboratory after transportation of blood samples at 0°C on a train from Munich to Hamburg. Tobacco smoke exposure of the subjects increased COHb and plasma cotinine levels. SSBs could be detected in all probands with some inter-individual day-to-day and morning-to-evening variations. In four of five active smokers, SSB increases were found after smoking. In nonsmokers exposed to tobacco smoke no exposure-related variation in SSB levels could be detected. In lymphocytes which were incubated in culture medium (DME/H) for 4 h at 37°C, SSBs correlated significantly with the SSBs of fresh (NT1) samples but the SSB level was lower in almost all cases and the effect of smoking was not as pronounced as in the NT1 samples. Larger interindividual variations and higher values in general were detected after 8–9h of transportation. Therefore, we recommend immediate determination of SSBs as soon as possible after blood sampling. We conclude that the modified nick translation assay is sensitive enough to detect SSBs caused by an in vivo genotoxic exposure when possible interindividual differences are considered in the study design and could therefore be used in biological monitoring of exposures at the workplace.     

DNA strand breaks, cytochrome P-450-dependent monooxygenase system activity and levels of chlorinated biphenyl congeners in the pyloric caeca of the seastar (Asterias rubens) from the North Sea

Seastars (Asterias rubens L.) were collecte d at sampling locations in different areas along transects radiating into the southern North Sea, representing areas impacted by contaminants to different degrees. Strand breakage in DNA isolated from tissue of the pyloric caeca was measured by the alkaline unwinding assay, modified to allow for the isolation of highly intact DNA. The interpretation of the results is based on the incidence of DNA strand breaks (expressed as the double:total DNA ratio or F value, indicating the degree of double-strandedness). The cytochrome P-450 concentration and the benzo [a] pyrene (BaP) hydroxylase activity were measured in microsomal fractions of the pyloric caeca. The chlorinated biphenyl (CB) congeners in tissue samples from the digestive gland were determined by temperature-programmed gas-chromatography, with a CPSil8 capillary column as the stationary phase, hydrogen as the carrier gas and 63Ni-electron capture detection. Areas where seastars showed different DNA integrity could be described. The highest integrity (0.75 < F < 0.85) was found in the off-shore reference sites of the Dogger Bank and Southern Bight. The mean F value in seastars from most sampling locations varied between 55 and 75%. The lowest DNA integrity (0.35 < F < 0.55) was found in specimens obtained from sampling locations near the river Rhine delta, along the Dutch coast and at two expected uncontaminated offshore areas. The BaP hydroxylase activity was relatively high near the mouth of the rivers Rhine and Scheldt, but also at a supposedly clean site near Dogger Bank. The concentration of CB congeners in the pyloric caeca of seastars decreased along transects radiating into the southern North Sea from the coastal areas of the Netherlands; the highest concentrations were in the nearby coastal areas and the lowest were in the open sea sampling locations. The data indicate that there might be a relationship between pollution from the Rivers Rhine, Meuse and Scheldt and the incidence of DNA strand breaks and/or BaP hydroxylase activity     

In vitro damage of isolated DNA from two brain tumor cell lines induced by a water-soluble antitumor nitrosourea

We report the DNA sites damaged by the antitumor drug, nimustine hydrochloride (ACNU), in highly reiterated DNA sequences of rat glioma cells. A reiterated fragment of 370 base pairs (bp), obtained after Hind III restriction endonuclease digestion of rat glioma C6 or 9L cells DNA, was divided into 167 and 203 bp by subsequent Hae III enzyme reaction. The reaction of end-labelled 167 and 203 bp fragments with ACNU resulted in scission breaks corresponding to the locations of guanine. Moreover, ACNU and subsequent piperidine hydrolysis produced more frequent breaks of the phosphodiester bonds at the guanine positions, thus forming alkali-labile sites. These results indicate that the preferred site of DNA strand scission induced by ACNU is at guanine positions.