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Biomedical subjects

Olga A Sedelnikova

Publications and source records attributed to Olga A Sedelnikova.

14 recordsLinked to original sources
PubMed2006

GammaH2AX in cancer cells: a potential biomarker for cancer diagnostics, prediction and recurrence.

Current advances in cancer biology have identified major pathways involved in tumorigenesis. The association of DNA damage with premalignant stages of tumor progression, genome instability and further oncogenic transformation opens the possibility of using common DNA damage markers for early cancer detection, prediction, prognosis, therapeutics and possibly for cancer prevention. Perhaps the most sensitive DNA damage marker is gammaH2AX formation in the chromatin flanking the free DNA double-stranded ends in double-strand breaks (DSBs) and eroded telomeres, both present during oncogenic transformation. Our group and others found elevated endogenous levels of in various human cancer cell lines, premalignant lesions and solid tumors. These data suggest that increased DNA damage is a general characteristic of cancer development. GammaH2AX-based assay can be applied to human biopsies, aspirates and, possibly, to mononuclear cells of the peripheral blood. We propose that detection of gammaH2AX could benefit for the early cancer screening and to ascertain the efficiency of clinical treatment involving chemo- and radiotherapeutic protocols.

Adolescent↗
PubMed2006

The phosphorylation status of PAS-B distinguishes HIF-1alpha from HIF-2alpha in NBS1 repression.

Hypoxia promotes genetic instability for tumor progression. Recent evidence indicates that the transcription factor HIF-1alpha impairs DNA mismatch repair, yet the role of HIF-1alpha isoform, HIF-2alpha, in tumor progression remains obscure. In pursuit of the involvement of HIF-alpha in chromosomal instability, we report here that HIF-1alpha, specifically its PAS-B, induces DNA double-strand breaks at least in part by repressing the expression of NBS1, a crucial DNA repair gene constituting the MRE11A-RAD50-NBS1 complex. Despite strong similarities between the two isoforms, HIF-2alpha fails to do so. We demonstrate that this functional distinction stems from phosphorylation of HIF-2alpha Thr-324 by protein kinase D1, which discriminates between subtle differences of the two PAS-B in amino-acid sequence, thereby precluding NBS1 repression. Hence, our findings delineate a molecular pathway that functionally distinguishes HIF-1alpha from HIF-2alpha, and arguing a unique role for HIF-1alpha in tumor progression by promoting genomic instability.

Animals↗
PubMed2006

Techniques for gamma-H2AX detection.

When a double-strand break (DSB) forms in DNA, many molecules of histone H2AX present in the chromatin flanking the break site are rapidly phosphorylated. The phosphorylated derivative of H2AX is named gamma-H2AX, and the phosphorylation site is a conserved serine four residues from the C-terminus, 139 in mammals and 129 in budding yeast. An antibody to gamma-H2AX reveals that the molecules form a gamma-focus at the DSB site. The gamma-focus increases in size rapidly for 10-30 min after formation, and remains until the break is repaired. Studies have revealed that small numbers of gamma-foci are present in cells even without the purposeful introduction of DNA DSBs. These cryptogenic foci increase in number during senescence in culture and aging in mice. This chapter presents techniques for revealing gamma-H2AX foci in cultured cells, in metaphase spreads from cultured cells, in tissues, and in yeast.

Animals↗
PubMed2005

Ionizing radiation induces DNA double-strand breaks in bystander primary human fibroblasts.

That irradiated cells affect their unirradiated 'bystander' neighbors is evidenced by reports of increased clonogenic mortality, genomic instability, and expression of DNA-repair genes in the bystander cell populations. The mechanisms underlying the bystander effect are obscure, but genomic instability suggests DNA double-strand breaks (DSBs) may be involved. Formation of DSBs induces the phosphorylation of the tumor suppressor protein, histone H2AX and this phosphorylated form, named gamma-H2AX, forms foci at DSB sites. Here we report that irradiation of target cells induces gamma-H2AX focus formation in bystander cell populations. The effect is manifested by increases in the fraction of cells in a population that contains multiple gamma-H2AX foci. After 18 h coculture with cells irradiated with 20 alpha-particles, the fraction of bystander cells with multiple foci increased 3.7-fold. Similar changes occurred in bystander populations mixed and grown with cells irradiated with gamma-rays, and in cultures containing media conditioned on gamma-irradiated cells. DNA DSB repair proteins accumulated at gamma-H2AX foci, indicating that they are sites of DNA DSB repair. Lindane, which blocks gap-junctions, prevented the bystander effect in mixing but not in media transfer protocols, while c-PTIO and aminoguanidine, which lower nitric oxide levels, prevented the bystander effect in both protocols. Thus, multiple mechanisms may be involved in transmitting bystander effects. These studies show that H2AX phosphorylation is an early step in the bystander effect and that the DNA DSBs underlying gamma-H2AX focus formation may be responsible for its downstream manifestations.

Bystander Effect↗
PubMed2005

DNA damage produced by 125I-triplex-forming oligonucleotides as a measure of their successful delivery into cell nuclei.

Decay of an Auger-electron-emitting radioisotope can knock out a targeted gene by producing DNA strand breaks within its sequence. For delivery of Auger emitters to genomic targets we used triplex-forming oligonucleotides (TFOs) that bind specifically to their target sequences by forming hydrogen bonds within the major groove of the target duplex. We named this approach antigene radiotherapy. In our previous studies, we demonstrated that (125)I-labeled TFOs targeted against the human MDR1 gene produced sequence-specific double strand breaks (DSBs) within this gene in live cultured cells. We also found that conjugation of TFO with nuclear localization signal peptide significantly increased the efficiency of targeting. To screen the wide variety of possible TFO modifications a sensitive and robust assay of DNA damage produced by such (125)I-TFOs would be highly desirable. Recently we showed a direct correspondence between the number of decays of (125)I incorporated into DNA as (125)I-UdR and the number of histone gamma-H2AX foci per cell revealed by staining with gamma-H2AX antibodies. The technique is 100-fold more sensitive than other DSB-detection methods, thus it is possible to detect as few as an average of 0.5 DSBs per cell in a population of cultured cells. Here we applied this method to evaluate the intracellular DNA damage produced by two (125)I-TFOs, the first targeted to the single-copy HPRT gene ((125)I-TFO-HPRT) and second to a multicopy repeated sequence (GA)(n) that occurs almost 7000 times in the human genome ((125)I-TFO-GA). DNA damage produced by (125)I-TFO was assessed by staining the cells with gamma-H2AX antibody followed by either direct counting gamma-H2AX foci or by measuring the gamma-H2AX signal using flow cytometry. Both methods produced quantitatively close results; (125)I-TFO-GA with multiple nuclear targets produced on average 1.93 times more gamma-H2AX foci per cell and generated 1.96 times increase in gamma-H2AX antibody staining signal than (125)I-TFO-HPRT with a single target. The gamma-H2AX-based assay requires considerably less time and effort than the direct measurement of DSB by Southern hybridization applied previously. Therefore, we believe that gamma-H2AX-based measurement of DNA damage could be useful for evaluation and cellular DNA accessibility by (125)I-labeled DNA targeting agents.

Base Sequence↗
PubMed2004

Senescing human cells and ageing mice accumulate DNA lesions with unrepairable double-strand breaks.

Humans and animals undergo ageing, and although their primary cells undergo cellular senescence in culture, the relationship between these two processes is unclear. Here we show that gamma-H2AX foci (gamma-foci), which reveal DNA double-strand breaks (DSBs), accumulate in senescing human cell cultures and in ageing mice. They colocalize with DSB repair factors, but not significantly with telomeres. These cryptogenic gamma-foci remain after repair of radiation-induced gamma-foci, suggesting that they may represent DNA lesions with unrepairable DSBs. Thus, we conclude that accumulation of unrepairable DSBs may have a causal role in mammalian ageing.

Aging↗
PubMed2003

H2AX haploinsufficiency modifies genomic stability and tumor susceptibility.

Histone H2AX becomes phosphorylated in chromatin domains flanking sites of DNA double-strand breakage associated with gamma-irradiation, meiotic recombination, DNA replication, and antigen receptor rearrangements. Here, we show that loss of a single H2AX allele compromises genomic integrity and enhances the susceptibility to cancer in the absence of p53. In comparison with heterozygotes, tumors arise earlier in the H2AX homozygous null background, and H2AX(-/-) p53(-/-) lymphomas harbor an increased frequency of clonal nonreciprocal translocations and amplifications. These include complex rearrangements that juxtapose the c-myc oncogene to antigen receptor loci. Restoration of the H2AX null allele with wild-type H2AX restores genomic stability and radiation resistance, but this effect is abolished by substitution of the conserved serine phosphorylation sites in H2AX with alanine or glutamic acid residues. Our results establish H2AX as genomic caretaker that requires the function of both gene alleles for optimal protection against tumorigenesis.

Animals↗
PubMed2003

Phosphorylation of histone H2AX and activation of Mre11, Rad50, and Nbs1 in response to replication-dependent DNA double-strand breaks induced by mammalian DNA topoisomerase I cleavage complexes.

DNA double-strand breaks originating from diverse causes in eukaryotic cells are accompanied by the formation of phosphorylated H2AX (gammaH2AX) foci. Here we show that gammaH2AX formation is also a cellular response to topoisomerase I cleavage complexes known to induce DNA double-strand breaks during replication. In HCT116 human carcinoma cells exposed to the topoisomerase I inhibitor camptothecin, the resulting gammaH2AX formation can be prevented with the phosphatidylinositol 3-OH kinase-related kinase inhibitor wortmannin; however, in contrast to ionizing radiation, only camptothecin-induced gammaH2AX formation can be prevented with the DNA replication inhibitor aphidicolin and enhanced with the checkpoint abrogator 7-hydroxystaurosporine. This gammaH2AX formation is suppressed in ATR (ataxia telangiectasia and Rad3-related) deficient cells and markedly decreased in DNA-dependent protein kinase-deficient cells but is not abrogated in ataxia telangiectasia cells, indicating that ATR and DNA-dependent protein kinase are the kinases primarily involved in gammaH2AX formation at the sites of replication-mediated DNA double-strand breaks. Mre11- and Nbs1-deficient cells are still able to form gammaH2AX. However, H2AX-/- mouse embryonic fibroblasts exposed to camptothecin fail to form Mre11, Rad50, and Nbs1 foci and are hypersensitive to camptothecin. These results demonstrate a conserved gammaH2AX response for double-strand breaks induced by replication fork collision. gammaH2AX foci are required for recruiting repair and checkpoint protein complexes to the replication break sites.

Animals↗
PubMed2003

Characteristics of gamma-H2AX foci at DNA double-strand breaks sites.

Phosphorylated H2AX (gamma-H2AX) is essential to the efficient recognition and (or) repair of DNA double strand breaks (DSBs), and many molecules, often thousands, of H2AX become rapidly phosphorylated at the site of each nascent DSB. An antibody to gamma-H2AX reveals that this highly amplified process generates nuclear foci. The phosphorylation site is a serine four residues from the C-terminus which has been evolutionarily conserved in organisms from giardia intestinalis to humans. Mice and yeast lacking the conserved serine residue demonstrate a variety of defects in DNA DSB processing. H2AX Delta/Delta mice are smaller, sensitive to ionizing radiation, defective in class switch recombination and spermatogenesis while cells from the mice demonstrate substantially increased numbers of genomic defects. gamma-H2AX foci formation is a sensitive biological dosimeter and presents new and exciting opportunities to understand important biological processes, human diseases, and individual variations in radiation sensitivity. These potentialities demonstrate the importance of understanding the parameters and functions of gamma-H2AX formation.

Amino Acid Sequence↗
PubMed2002

Genomic instability in mice lacking histone H2AX.

Higher order chromatin structure presents a barrier to the recognition and repair of DNA damage. Double-strand breaks (DSBs) induce histone H2AX phosphorylation, which is associated with the recruitment of repair factors to damaged DNA. To help clarify the physiological role of H2AX, we targeted H2AX in mice. Although H2AX is not essential for irradiation-induced cell-cycle checkpoints, H2AX-/- mice were radiation sensitive, growth retarded, and immune deficient, and mutant males were infertile. These pleiotropic phenotypes were associated with chromosomal instability, repair defects, and impaired recruitment of Nbs1, 53bp1, and Brca1, but not Rad51, to irradiation-induced foci. Thus, H2AX is critical for facilitating the assembly of specific DNA-repair complexes on damaged DNA.

Amino Acid Sequence↗
PubMed2002

Quantitative detection of (125)IdU-induced DNA double-strand breaks with gamma-H2AX antibody.

When mammalian cells are exposed to ionizing radiation and other agents that introduce DSBs into DNA, histone H2AX molecules in megabase chromatin regions adjacent to the breaks become phosphorylated within minutes on a specific serine residue. An antibody to this phosphoserine motif of human H2AX (gamma-H2AX) demonstrates that gamma-H2AX molecules appear in discrete nuclear foci. To establish the quantitative relationship between the number of these foci and the number of DSBs, we took advantage of the ability of (125)I, when incorporated into DNA, to generate one DNA DSB per radioactive disintegration. SF-268 and HT-1080 cell cultures were grown in the presence of (125)IdU and processed immunocytochemically to determine the number of gamma-H2AX foci. The numbers of (125)IdU disintegrations per cell were measured by exposing the same immunocytochemically processed samples to a radiation-sensitive screen with known standards. Under appropriate conditions, the data yielded a direct correlation between the number of (125)I decays and the number of foci per cell, consistent with the assumptions that each (125)I decay yields a DNA DSB and each DNA DSB yields a visible gamma-H2AX focus. Based on these findings, we conclude that gamma-H2AX antibody may form the basis of a sensitive quantitative method for the detection of DNA DSBs in eukaryotic cells.

Antibodies↗
PubMedDate not supplied

Assessment of DNA damage produced by 125I-triplex-forming oligonucleotides in cells.

PURPOSE: Triplex-forming oligodeoxyribonucleotides (TFOs) bind specifically to their target sequences by forming hydrogen bonds within the major groove of the target duplex. When labeled with Auger-electron-emitting radioisotopes, TFOs are able to damage the target gene in a process named antigene radiotherapy. We compared radiotoxicity and the amount of DNA damage produced within cultured cells by two 125I-labeled TFOs, one with a single target in the genome and another with multiple targets. MATERIALS AND METHODS: Radiotoxicity was measured by clonogenic assay while DNA damage was assessed by the number of histone gamma-H2AX foci formed at the sites of DNA double strand breaks (DSBs). RESULTS: The TFO with multiple nuclear targets was 1.7 fold more radiotoxic and produced on average 1.9 fold more gamma-H2AX foci per cell than the TFO with a single target. CONCLUSION: Since the two methods gave comparable results, measuring the number of gamma-H2AX foci per decay may be a useful procedure for the assessment of cytotoxic effects and the intranuclear localization of radionuclides when they produce DSBs.

Cell Line↗