Cognitive performance in survivors of breast cancer and markers of biological aging.

BACKGROUND: Biological aging pathways accelerated by cancer treatments may be a mechanism for cognitive impairment in cancer survivors. The goal of the current study was to examine whether indicators of biological aging, namely elevated levels of DNA damage, reduced telomerase enzymatic activity, and shorter peripheral blood mononuclear cell (PBMC) telomere length (TL) would be related to cognitive function in a cohort of survivors of breast cancer. METHODS: The authors evaluated a cross-sectional sample of 94 women aged 36 to 69 years who were treated for early-stage breast cancer 3 to 6 years previously. Leukocyte DNA damage, PBMC telomerase enzymatic activity, PBMC TL, and the inflammatory marker soluble tumor necrosis factor receptor II (sTNF-RII) were determined from blood samples. Cognitive function was assessed using a neuropsychological test battery and self-report. Linear regression models examined the relationship between biological aging predictors and cognitive outcomes. RESULTS: Both higher DNA damage and lower telomerase were found to be statistically significantly related to lower executive function scores adjusting for age, body mass index, race, years from treatment, and intelligence score (standardized coefficients [B], -0.23 and 0.30; all P values <.05). In addition, lower telomerase activity was associated with worse attention and motor speed scores (B values, 0.30 and 0.24; P <.05). sTNF-RII and TL were found to be unrelated to any of the neurocognitive domains. CONCLUSIONS: The results of the current study suggest a significant association between measures of biological aging and objective measures of cognitive performance in survivors of breast cancer. Future prospective studies are needed to confirm a causal role of biological aging as a driver of declines in cognitive function after cancer treatment.

Ionizing radiation induces microhomology-mediated end joining in trans in yeast and mammalian cells.

DNA double-strand breaks repaired through nonhomologous end joining require no extended sequence homology as a template for the repair. A subset of end-joining events, termed microhomology-mediated end joining, occur between a few base pairs of homology, and such pathways have been implicated in different human cancers and genetic diseases. Here we investigated the effect of exposure of yeast and mammalian cells to ionizing radiation on the frequency and mechanism of rejoining of transfected unirradiated linear plasmid DNA. Cells were exposed to gamma radiation prior to plasmid transfection; subsequently the rejoined plasmids were recovered and the junction sequences were analyzed. In irradiated yeast cells, 68% of recovered plasmids contained microhomologies, compared to only 30% from unirradiated cells. Among them 57% of events used>or=4 bp of microhomology compared to only 11% from unirradiated cells. In irradiated mammalian cells, 54% of plasmids used>or=4 bp of microhomology compared to none from unirradiated cells. We conclude that exposure of yeast and mammalian cells to radiation prior to plasmid transfection enhances the frequency of microhomology-mediated end-joining events in trans. If such events occur within genomic locations, they may be involved in the generation of large deletions and other chromosomal aberrations that occur in cancer cells.

Student Satisfaction and Learning Outcomes in Asynchronous Online Lecture Videos.

Our study identified online lecture video styles that improved student engagement and satisfaction, while maintaining high learning outcomes in online education. We presented different lecture video styles with standardized material to students and then measured learning outcomes and satisfaction with a survey and summative assessment. We created an iterative qualitative coding scheme, "coding online asynchronous lectures" (COAL), to analyze open-ended student survey responses. Our results reveal that multimedia learning can be satisfying and effective. Students have strong preferences for certain video styles despite their equal learning outcomes, with the Learning Glass style receiving the highest satisfaction ratings. Video styles that were described as impersonal and unfamiliar were rated poorly, while those that were described as personal and engaging and evoked positive affective responses were rated highly. The students in our study rated lecture video styles that aligned with Mayer's multimedia learning principles as highly satisfying, indicating that student feedback can be a valuable resource for course designers to consider as they design their own online courses. Finally, we provide guidelines for creating engaging, effective, and satisfying asynchronous lecture videos to support establishment of best practices in online instruction.

Impaired DNA double strand break repair in cells from Nijmegen breakage syndrome patients.

Nijmegen breakage syndrome, caused by mutations in the NBS1 gene, is an autosomal recessive chromosomal instability disorder characterized by cancer predisposition. Cells isolated from Nijmegen breakage syndrome patients display increased levels of spontaneous chromosome aberrations and sensitivity to ionizing radiation. Here, we have investigated DNA double strand break repair pathways of homologous recombination, including single strand annealing, and non-homologous end-joining in Nijmegen breakage syndrome patient cells. We used recently developed GFP-YFP-based plasmid substrates to measure the efficiency of DNA double strand break repair. Both single strand annealing and non-homologous end-joining processes were markedly impaired in NBS1-deficient cells, and repair proficiency was restored upon re-introduction of full length NBS1 cDNA. Despite the observed defects in the repair efficiency, no apparent differences in homologous recombination or non-homologous end-joining effector proteins RAD51, KU70, KU86, or DNA-PK(CS) were observed. Furthermore, comparative analysis of junction sequences of plasmids recovered from NBS1-deficient and NBS1-complemented cells revealed increased dependence on microhomology-mediated end-joining DNA repair process in NBS1-complemented cells.

Adaptive response to gamma radiation in mammalian cells proficient and deficient in components of nucleotide excision repair.

Cells preconditioned with low doses of low-linear energy transfer (LET) ionizing radiation become more resistant to later challenges of radiation. The mechanism(s) by which cells adaptively respond to radiation remains unclear, although it has been suggested that DNA repair induced by low doses of radiation increases cellular radioresistance. Recent gene expression profiles have consistently indicated that proteins involved in the nucleotide excision repair pathway are up-regulated after exposure to ionizing radiation. Here we test the role of the nucleotide excision repair pathway for adaptive response to gamma radiation in vitro. Wild-type CHO cells exhibited both greater survival and fewer HPRT mutations when preconditioned with a low dose of gamma rays before exposure to a later challenging dose. Cells mutated for ERCC1, ERCC3, ERCC4 or ERCC5 did not express either adaptive response to radiation; cells mutated for ERCC2 expressed a survival adaptive response but no mutation adaptive response. These results suggest that some components of the nucleotide excision repair pathway are required for phenotypic low-dose induction of resistance to gamma radiation in mammalian cells.

Biomarkers of aging associated with past treatments in breast cancer survivors.

Radiation and chemotherapy are effective treatments for cancer, but are also toxic to healthy cells. Little is known about whether prior exposure to these treatments is related to markers of cellular aging years later in breast cancer survivors. We examined whether past exposure to chemotherapy and/or radiation treatment was associated with DNA damage, telomerase activity, and telomere length 3-6 years after completion of primary treatments in breast cancer survivors (stage 0-IIIA breast cancer at diagnosis). We also examined the relationship of these cellular aging markers with plasma levels of Interleukin (IL)-6, soluble TNF-receptor-II (sTNF-RII), and C-reactive protein (CRP). Ninety-four women (36.4-69.5 years; 80% white) were evaluated. Analyses adjusting for age, race, BMI, and years from last treatment found that women who had prior exposure to chemotherapy and/or radiation compared to women who had previously received surgery alone were more likely to have higher levels of DNA damage (P = .02) and lower telomerase activity (P = .02), but did not have differences in telomere length. More DNA damage and lower telomerase were each associated with higher levels of sTNF-RII (P's < .05). We found that exposure to chemotherapy and/or radiation 3-6 years prior was associated with markers of cellular aging, including higher DNA damage and lower telomerase activity, in post-treatment breast cancer survivors. Furthermore, these measures were associated with elevated inflammatory activation, as indexed by sTNF-RII. Given that these differences were observed many years after the treatment, the findings suggest a long lasting effect of chemotherapy and/or radiation exposure.

Point-source irradiation with eccentric rotation causes inhomogeneous dose distribution.

Radioactive point sources are regularly used for irradiating cell culture and other biological materials. Eccentric rotation is often used to minimize dose disparities that arise from irradiating samples that span a distance from the point source. Rotation provides a great improvement in dose homogeneity compared to inert irradiation yet still presents an obvious shortcoming for exposures in which the sample completes only partial rotation or fractional rotation. In such cases, certain areas of the sample have a closer average distance to the radiation source than other areas within the same sample. This obstacle can be partially overcome by adjusting rotation speed so the sample traverses a full rotation (or multiple thereof) throughout the total irradiation time. Here we investigate the effects of irradiation with eccentric rotation on dose homogeneity. We show that due to the inverse square law that governs dose, even exposures with full rotation result in inhomogeneous dose distributions. This dose inhomogeneity can be substantial, especially for large samples and small source- sample distances. We observed a 33% difference in survival across 100-mm dishes and a 400% difference for 150-mm dishes. The dose inhomogeneity inherent to eccentric rotation increases the actual average dose delivered across the sample compared to that delivered at sample center. We offer a table of correction factors that account for this dose increase and correct the dose delivered at center to the actual average dose delivered across the entire sample.

siRNA targeting NBS1 or XIAP increases radiation sensitivity of human cancer cells independent of TP53 status.

NBS1 is essential for the repair of radiation-induced DNA double-strand breaks (DSBs) in yeast and higher vertebrate cells. In this study, we examined whether suppressed NBS1 expression by small interference RNA (siRNA) could enhance radiation sensitivity in cancer cells with different TP53 status. We used human non-small cell lung cancer cells differing in TP53 gene status (H1299/wtp53 cells bearing wild-type TP53 or H1299/mp53 cells bearing mutant TP53). A DNA cassette expressing siRNA targeted for the NBS1 gene was transfected into those cell lines, and radiation sensitivity was examined with a colony-forming assay. Cellular levels of NBS1 and other proteins were analyzed using Western blotting. We found that the radiation sensitivity of H1299/wtp53 and H1299/mp53 cells was enhanced by transfection of the DNA cassette. In the NBS1-siRNA-transfected cells, we observed decreased constitutive expression of NBS1 protein and decreased radiation-induced accumulation of phosphorylated NBS1 protein. In addition, radiation-induced expression of the transcription factor NF-kappaB (NFKB) and XIAP (X-chromosome-linked inhibitor of apoptosis protein) was suppressed by NBS1-siRNA. Enhanced X-ray sensitivity after NBS1-siRNA transfection was achieved in TP53 wild-type cells and sensitivity was even more pronounced in TP53 mutant cells. The transfection of siRNA targeted for XIAP also enhanced X-ray sensitivity even more for TP53 mutant cells compared to TP53 wild-type cells. Our data suggest that the sensitization to radiation results from NBS1-siRNA-mediated suppression of DNA repair and/ or X-ray-induced cell survival signaling pathways through NFKB and XIAP. siRNA targeting appears to be a novel radiation-sensitizing agent, particularly in human TP53 mutant cancer cells.

Insulin-like growth factor I differentially regulates the expression of HIRF1/hCAF1 and BTG1 genes in human MCF-7 breast cancer cells.

Differential display PCR analysis (DD-PCR) was used to identify novel genes that respond to IGF-I treatment in human MCF-7 breast cancer cells. Fifty-three cDNAs showed alterations in their mRNA levels in IGF-I treated cells. One of these genes showed a significant increase in the mRNA level in IGF-I treated cells in comparison to non-treated cells. We named this gene HIRF1 (human IGF-I regulated factor 1). Nucleotide blast analysis revealed that this gene has a 100% sequence identity with the sequence for BTG1 (B-cell translocation gene) binding factor 1 (human CCR4-associated factor 1 gene, hCAF1). By alignment of cloned HIRF1 cDNA and genomic DNA 8p21.3-p22 sequence, we were able to determine the exon-intron structure of the cloned HIRF1 gene on chromosome 8. Northern blot and real-time PCR analysis showed that BTG1 and c-fos reached their maximal expression fairly early within 10 min to 1 h, and decreased to basal levels after 3 h of IGF-I treatment. HIRF1/hCAF1 expression reached maximal stimulation after 3 h of IGF-I treatment and then gradually decreased to basal level. HIRF1 and BTG1 mRNA was inhibited by inhibitors of the cell signaling pathways, PI3/Akt kinase and MAPK kinases (ERK1/2 and p38). In summary, cloned HIRF1/hCAF1 is coregulated with BTG1 in response to IGF-I. The regulation of these genes as early response genes may have an important role in differentiation, growth and proliferation of breast cancer cells.

Heat-induced phosphorylation of NBS1 in human skin fibroblast cells.

NBS1 is known to be involved in DNA damage-induced cellular responses after exposure to ionizing radiation (IR). Phosphorylation of NBS1 contributes to cell-cycle checkpoints. The aim of this study was to determine whether heat exposure induces or stimulates cellular responses mediated by the phosphorylation of NBS1 in human skin fibroblast cell lines. The results of immunofluorescent staining and Western blot analysis showed that NBS1 proteins are phosphorylated after exposure to heat in the nucleus of a normal skin fibroblast cell line (82-6 cells). This suggests that the NBS1-mediated signal transduction could be induced by heat. We further examined whether a deficiency in the NBS1 protein modifies heat sensitivity in human skin fibroblast cell lines. A skin fibroblast cell line (Gmtert), derived from a Nijmegen breakage syndrome (NBS) patient containing mutant NBS1, showed higher sensitivity to heat than the same cell line transfected with the wild-type copy of the NBS1 gene. We also showed that transfection of a DNA cassette expressing small interference RNA (siRNA) targeted to NBS1 into 82-6 cells enhanced cell sensitivity to heat. These results suggest that NBS1 is involved in cellular responses to DNA damage which is induced by heat exposure as well as by radiation exposure in human skin fibroblast cells.