Formation Of Chromosome Abnormalities Research Articles

Talazoparib enhances resection at DSBs and renders HR-proficient cancer cells susceptible to Polθ inhibition

Background and purposeThe PARP inhibitor (PARPi), Talazoparib (BMN673), effectively and specifically radiosensitizes cancer cells. Radiosensitization is mediated by a shift in the repair of ionizing radiation (IR)-induced DNA double-strand breaks (DSBs) toward PARP1-independent, alternative end-joining (alt-EJ). DNA polymerase theta (Polθ) is a key component of this PARP1-independent alt-EJ pathway and we show here that its inhibition can further radiosensitize talazoparib-treated cells. The purpose of the present work is to explore mechanisms and dynamics underpinning enhanced talazoparib radiosensitization by Polθ inhibitors in HR-proficient cancer cells. Methods and MaterialsRadiosensitization to PARPis, talazoparib, olaparib, rucaparib and veliparib was assessed by clonogenic survival. Polθ-proficient and −deficient cells were treated with PARPis and/or with the Polθ inhibitors ART558 or novobiocin. The role of DNA end-resection was studied by down-regulating CtIP and MRE11 expression using siRNAs. DSB repair was assessed by scoring γH2AX foci. The formation of chromosomal abnormalities was assessed as evidence of alt-EJ function using G2-specific cytogenetic analysis. ResultsTalazoparib exerted pronounced radiosensitization that varied among the tested cancer cell lines; however, radiosensitization was undetectable in normal cells. Other commonly used PARPis, olaparib, veliparib, or rucaparib were ineffective radiosensitizers under our experimental conditions. Although genetic ablation or pharmacological inhibition of Polθ only mildly radiosensitized cancer cells, talazoparib-treated cells were markedly further radiosensitized. Mechanistically, talazoparib shunted DSBs to Polθ-dependent alt-EJ by enhancing DNA end-resection in a CtIP- and MRE11-dependent manner – an effect detectable at low, but not high IR doses. Chromosomal translocation analysis in talazoparib-treated cells exposed to Polθ inhibitors suggested that PARP1- and Polθ-dependent alt-EJ pathways may complement, but also back up each other. ConclusionWe propose that talazoparib promotes low-dose, CtIP/MRE11-dependent resection and increases the reliance of irradiated HR-proficient cancer cells, on Polθ-mediated alt-EJ. The combination of Polθ inhibitors with talazoparib suppresses this option and causes further radiosensitization. The results suggest that Polθ inhibition may be exploited to maximize talazoparib radiosensitization of HR-proficient tumors in the clinic.

Three-dimensional quantitative analysis of chromosomes in the oocytes of aging mice during meiosis I in vitro

The mechanism of senescence is very complicated and can involve formation of chromosome abnormalities and a decline in female fertility. In this study, 3-D visualization of fluorescently labeled chromosomes in oocytes from aging and pubertal mice during in vitro maturation was done with a two-photon laser scanning microscope. Differences between aging and pubertal groups at various maturation stages were analyzed quantitatively in terms of chromosomal morphology, shape, and spatial arrangement. Compared with the pubertal group, the chromosomal morphology of oocytes from aging mice changed: both the mean volume and the mean surface area of chromosomes increased by approximately 20% (P < 0.05) at prometaphase and metaphase of meiosis I (considered to be the weakly condensed folded form of the chromosomes). Furthermore, at these stages, the shape of the chromosomal array became rounder (roundness factor increased by approximately 10%; P < 0.001) and the adhesion among chromosomes became more severe (P < 0.001) at approximately the same stages. Additionally, trends over time for both chromosomal morphology and shape were quite distinct between oocytes from aging and pubertal mice. Interestingly, trends for mean distance were similar; therefore, aging did not seem to influence chromosome movement toward the metaphase plate. These morphologic results should be useful to study age-related degradation of oocyte quality and to interpret results derived from molecular biology.

Molecular Cytogenetic Delineation of Chromosomal Structure and Complexity

There has been an explosion of technology in the entire field of genetics, but especially in the sub discipline of cytogenetics. Today we can not only visualize chromosomes microscopically, we can utilize FISH to look at any specific region of interest within a chromosome regardless of size, and to use array technology to look for subtle gains or losses of chromosome material. New syndromes and small changes leading to abnormal phenotypes have been identified utilizing this new technology. It has always been believed in cytogenetics that the most logical approach is to suspect the simplest route in the formation of abnormalities. However based on many studies using both FISH analysis and array studies, we have begun to learn that many abnormalities are more complex than originally envisioned. Utilizing an approach combining classical cytogenetics, FISH and array analysis, we have studied numerous abnormalities that now have been shown to be more complex than originally thought. These include: "balanced" translocations that have been shown to have deleted material; rearrangements thought to involve 2 or 3 breaks which actually have up to 12 different breakpoints; deletion which appear to be simple but have material within the deleted region present; ring chromosomes which are fragmented so that they contain discontinuous regions of the normal chromosomes; and inversions which can be shown to be insertions. These findings teach us several aspects concerning the formation of chromosome abnormalities. First, it shows that there are not always simple mechanisms leading to abnormalities and by studying these we may eventually better understand the underlying causes of structural abnormalities. Second, these complex rearrangements have an impact on the phenotype as genes initially thought to be involved may in fact not be.

Elevated incidence of chromosomally chaotic embryos among frozen-thawed preimplantation embryos

Objective: The aim of the study was to evaluate the effect of cryopreservation on the formation of chromosomal abnormalities in human preimplantation embryos. Study design: The chromosomal constitutions of cleavage stage embryos ( n=61) were assessed using fluorescent in situ hybridisation (FISH) technique, applying probes for chromosomes 13, 16, 18, 21, X and Y. Study group embryos frozen at zygote or two-cell stage ( n=29) were cultured in vitro post-thawing until they reached four- to six-cell stage, after which their chromosomal constitutions were assessed. Control group embryos frozen at four- to six-cell stage ( n=32) were analysed immediately after thawing in order to exclude any post-thaw effect. The proportions of genetically normal and abnormal embryos were compared between study and control group. Results: The proportions of normal, aneuploid and mosaic embryos were similar in both groups. However, significantly ( P<0.05) higher proportion of chaotic embryos in study (24.1%) compared to control group (6.3%) was observed. Conclusion: The elevated level of chromosomally chaotic embryos among embryos that had undergone cellular division after thawing as compared to embryos analysed immediately after thawing indicates a potential negative impact of cryopreservation on the formation of chromosomal abnormalities in preimplantation embryos.

Twenty-six patients with hematologic disorders and X chromosome abnormalities: Frequent idic(X)(q13) chromosomes and Xq13 anomalies associated with pathologic ringed sideroblasts

In our experience, acquired structurally abnormal X chromosomes are found in about 1% of patients with hematologic disorders who have a chromosomally abnormal clone. Over a 10-year period, we identified 26 patients with hematologic disorders who had an acquired X chromosome abnormality. In 13 of these patients, the breakpoints were at Xq13, and in 13 the breakpoints were other than Xq13. Seven of the patients with Xq13 breakpoints had idic(X)(q13) chromosomes. The 13 patients with Xq13 breakpoints were all older females, and 12 had pathologic ringed sideroblasts with dysmyelopoietic syndromes or a history of these syndromes. Among the 13 patients with breakpoints other than Xq13, seven were male and six were female; only one of these patients had pathologic ringed sideroblasts, and all of these patients had a variety of hematologic disorders. Xq13 may contain genes that, when altered by the formation of chromosome abnormalities, may be associated with neoplastic disorders involving pathologic ringed sideroblasts.