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[Signal function of the reactive oxygen species in regulatory networks of the cell reaction to damaging effects: contribution of radiosensitivity and genome instability].

Reactive oxygen species (ROS) have a few possible effects, such as metabolic (participation in regulation of protein functions), damaging (oxidative damage to proteins, lipids and nucleic acids) and signal; the latter is reviewed in the article. Superoxide anion-radical (O2-.), hydroperoxide (HO2) and nitroxide (NO) are capable to act as signal substances in the cell regulatory network, which determines a mode of cell response to disturbance: proliferation pace, a course of differentiation or a start of the apoptosis program. A role of ROS in the reaction network is reviewed: importance of their content in a cell; ROS-bound signal pathways, which trigger the programs of cell reactions to stimuli; initiations of the regulatory network, which determine a content of ROS in a cell; ROS reactions with network components, which influence its functioning. A significance of the ROS-bound segment of the network, which realizes regulatory signals of the damage, in formation of radiobiological effect is estimated. The data obtained by the authors are submitted; the prospects of studing substances (such as phenozan etc.), which can actively influence redox processes, as means of modification of radiation-induced genome instability and prevention of oncogenic transformation are considered.

Animals↗

In utero exposure to diethylstilbestrol (DES) does not increase genomic instability in normal or neoplastic breast epithelium.

BACKGROUND: In 1992, the National Cancer Institute (NCI) established the Continuation of Follow-Up of DES-Exposed Cohorts to study the long-term health effects of exposure to diethylstilbestrol (DES). Genetic effects on human breast tissue have not been examined. The authors investigated whether breast tissue of women exposed in utero to DES might exhibit the genetic abnormalities that characterize other DES-associated tumors. METHODS: Subjects enrolled in the NCI Cohort were queried about breast biopsies or breast cancer diagnoses. Available tissue blocks were obtained for invasive cancers (IC), in situ cancers (CIS), or atypical hyperplasia (AH). Exposure status was blinded, lesions were microdissected, and their DNA was analyzed for microsatellite instability (MI) and loss of heterozygosity (LOH), or allele imbalance (AI), at 20 markers on 9 chromosome arms. RESULTS: From 31 subjects (22 exposed, 9 unexposed), 273 samples were analyzed (167 normal epithelium, 16 AH, 30 CIS, 60 IC). Exposed and unexposed subjects exhibited no differences in breast cancer risk factors or demographic characteristics, except for age at diagnosis (exposed vs. unexposed: 43.2 vs. 48.8 years of age, P = .02). The authors found that MI was rare and that AI was common, with frequencies consistent with previous reports. The global age-adjusted relative rate (RR) of AI was 1.3, 95% CI = 0.8-2.4. No statistically significant associations were observed after adjustment for risk factors or after stratification by histology or by chromosome arm. CONCLUSIONS: In utero DES exposure does not appear to significantly increase genomic instability in breast epithelium, as measured by MI and AI. Breast tissue may respond differently from that of the reproductive tract to in utero DES exposure. Consequences of in utero DES exposure on the breast may be mediated by proliferative effects of estrogen.

Adult↗

Radiation-induced genomic instability.

Quantitative assessment of the heritable somatic effects of ionizing radiation exposures has relied upon the assumption that radiation-induced lesions were 'fixed' in the DNA prior to the first postirradiation mitosis. Lesion conversion was thought to occur during the initial round of DNA replication or as a consequence of error-prone enzymatic processing of lesions. The standard experimental protocols for the assessment of a variety of radiation-induced endpoints (cell death, specific locus mutations, neoplastic transformation and chromosome aberrations) evaluate these various endpoints at a single snapshot in time. In contrast with the aforementioned approaches, some studies have specifically assessed radiation effects as a function of time following exposure. Evidence has accumulated in support of the hypothesis that radiation exposure induces a persistent destabilization of the genome. This instability has been observed as a delayed expression of lethal mutations, as an enhanced rate of accumulation of non-lethal heritable alterations, and as a progressive intraclonal chromosomal heterogeneity. The genetic controls and biochemical mechanisms underlying radiation-induced genomic instability have not yet been delineated. The aim is to integrate the accumulated evidence that suggests that radiation exposure has a persistent effect on the stability of the mammalian genome.

Animals↗

Monoclonality and surface lesion-specific microsatellite alterations in premalignant and malignant neoplasia of uterine cervix: a local field effect of genomic instability and clonal evolution.

Invasive squamous carcinoma of the uterine cervix (CC) arises from sequential progression of low-grade (L) and high-grade (H) squamous intraepithelial lesions (SILs). In clinical observations, these lesions are frequently found as synchronous multiple foci. The nature and evolutionary mechanism of these lesions are largely unknown. We have performed allelotyping of three 3p markers (at 3p14, 3p22-24, and 3p25) on 22 LSILs and 15 HSILs microdissected from patients with multiple (n = 21) or uniform (n = 6) cervical lesions. The results were analyzed together with our previous allelotyping of 57 deeply invasive CCs. Loss of heterozygosity at one of the three markers was observed in 23%, 27%, and 31 % of LSILs, HSILs, and CCs, respectively. Frequent and early allelic loss was noted (in 30% of LSILs and 50% of HSILs) at 3p14, which may harbor tumor suppressor genes involved in early stages of cervical carcinogenesis. A high frequency of microsatellite alteration (MA) was found in LSIL (41%) and HSIL (67%) but not in CC (5.3%). In particular, MA was more frequently found in low-grade lesions in association with invasive cancers (75%, 6/8) than in those associated with SILs (29%, 4/14) (P < 0.05). Together with the finding of a monoclonal origin of premalignant and malignant cervical lesions, the present results allow us to propose a model of local field effect of genomic instability that progressively affects the clonal evolution of SIL of uterine cervix.

Carcinoma in Situ↗

Loss of heterozygosity and genomic instability in synchronous endometrioid tumors of the ovary and endometrium.

BACKGROUND: The unknown etiology of endometrioid carcinomas of the ovary and the relatively high frequency of a concomitant carcinoma of the endometrium in these patients warrants study of such tumors. The aim of this study was to identify the genetic alterations involved in endometrioid ovarian cancer development, and to determine whether primary tumors of the endometrium and synchronous primary endometrioid tumors of the ovary could be distinguished based on differing patterns of genetic alterations. The distinction of metastatic carcinoma of the ovary from other synchronous primary tumors is often difficult but has important therapeutic and prognostic implications. METHODS: This study examined the genetic alterations at 28 polymorphic DNA markers in the DNA of tumors of 17 patients with endometrioid carcinoma of the ovary, including 5 nonmetastatic ovarian tumors, 5 ovarian tumors metastatic to the uterus, and 7 endometrioid ovarian tumors with a synchronous primary endometrial tumor. RESULTS: Chromosomes 17 and 22 were found to be the most common sites of loss of heterozygosity (LOH) in the 17 patients studied. Loss of heterozygosity on chromosome 17 was associated with advanced stage ovarian tumors. In 96% of LOH events in the metastatic tumors, LOH was observed in the primary tumor and in the metastatic site. Conversely, in four of seven synchronous tumors in which LOH was observed, LOH was confined to the ovarian tumor. Genomic instability was identified in two of seven patients with synchronously occurring tumors that did not demonstrate LOH. A positive family history was noted for these two patients. CONCLUSIONS: A lack of shared genetic alterations and in synchronously occurring endometrial and endometrioid ovarian tumors indicates independent developmental pathways for these tumors. Loss of heterozygosity on chromosome 17 in endometrioid ovarian carcinoma may indicate transition to a more aggressive tumor.

Carcinoma↗

V(D)J-mediated translocations in lymphoid neoplasms: a functional assessment of genomic instability by cryptic sites.

Most lymphoid malignancies are initiated by specific chromosomal translocations between immunoglobulin (Ig)/T cell receptor (TCR) gene segments and cellular proto-oncogenes. In many cases, illegitimate V(D)J recombination has been proposed to be involved in the translocation process, but this has never been functionally established. Using extra-chromosomal recombination assays, we determined the ability of several proto-oncogenes to target V(D)J recombination, and assessed the impact of their recombinogenic potential on translocation rates in vivo. Our data support the involvement of 2 distinct mechanisms: translocations involving LMO2, TAL2, and TAL1 in T cell acute lymphoblastic leukemia (T-ALL), are compatible with illegitimate V(D)J recombination between a TCR locus and a proto-oncogene locus bearing a fortuitous but functional recombination site (type 1); in contrast, translocations involving BCL1 and BCL2 in B cell non-Hodgkin's lymphomas (B-NHL), are compatible with a process in which only the IgH locus breaks are mediated by V(D)J recombination (type 2). Most importantly, we show that the t(11;14)(p13;q32) translocation involving LMO2 is present at strikingly high frequency in normal human thymus, and that the recombinogenic potential conferred by the LMO2 cryptic site is directly predictive of the in vivo level of translocation at that locus. These findings provide new insights into the regulation forces acting upon genomic instability in B and T cell tumorigenesis.

Adaptor Proteins, Signal Transducing↗

Bystander effects in radiation-induced genomic instability.

Exposure of GM10115 hamster-human hybrid cells to X-rays can result in the induction of chromosomal instability in the progeny of surviving cells. This instability manifests as the dynamic production of novel sub-populations of cells with unique cytogenetic rearrangements involving the "marker" human chromosome. We have used the comet assay to investigate whether there was an elevated level of endogenous DNA breaks in chromosomally unstable clones that could provide a source for the chromosomal rearrangements and thus account for the persistent instability observed. Our results indicate no significant difference in comet tail measurement between non-irradiated and radiation-induced chromosomally unstable clones. Using two-color fluorescence in situ hybridization we also investigated whether recombinational events involving the interstitial telomere repeat-like sequences in GM10115 cells were involved at frequencies higher than random processes would otherwise predict. Nine of 11 clones demonstrated a significantly higher than expected involvement of these interstitial telomere repeat-like sequences at the recombination junction between the human and hamster chromosomes. Since elevated levels of endogenous breaks were not detected in unstable clones we propose that epigenetic or bystander effects (BSEs) lead to the activation of recombinational pathways that perpetuate the unstable phenotype. Specifically, we expand upon the hypothesis that radiation induces conditions and/or factors that stimulate the production of reactive oxygen species (ROS). These reactive intermediates then contribute to a chronic pro-oxidant environment that cycles over multiple generations, promoting chromosomal recombination and other phenotypes associated with genomic instability.

Animals↗

Clonal chromosome aberrations and genomic instability in X-irradiated human T-lymphocyte cultures.

To study the effects of X-irradiation on clone forming ability and karyotypic abnormalities in human peripheral blood lymphocytes, cells were exposed to 3 Gy of X-rays in vitro and either individual T-cell clones or long-term T-cell cultures were established. The karyotypes were analyzed in G-banded chromosome preparations after proliferation for 9-34 days in vitro. T-cell clonal karyotype abnormalities were found in 24 of 37 (65%) irradiated and in two of 43 (5%) control clones. Balanced reciprocal translocations and deletions were the predominating types of clonal aberrations. Complex aberrations and unstable karyotypes were found in about half of the irradiated clones. Some of the T-cell clones demonstrated sequential change from normal to aberrant karyotype. Other clones seemed to develop multiple, heterogeneous chromosomal aberrations during growth in vitro. X-Irradiated T-cells grown in long-term T-cell culture displayed karyotype abnormalities in 60-80% of the cells, and the types of aberrations were similar to those found in the individual, irradiated T-cell clones. An increasing number of cells with the same abnormal karyotype was observed when the cultivation time was extended, indicating clonal proliferation. These results demonstrate that a surprisingly high proportion of T-cells with stable and often complex irradiation-induced chromosome aberrations are able to proliferate and form expanding cell clones in vitro. Furthermore, the results indicate that X-irradiation induces latent chromosome damage and genomic instability in human T-lymphocytes.

Adult↗

Genomic instability and catalase gene amplification induced by chronic exposure to oxidative stress.

Chronic exposure (>200 days) of HA1 fibroblasts to increasing concentrations of H2O2 or O2 results in the development of a stable oxidative stress-resistant phenotype characterized by increased cellular antioxidant levels, particularly catalase (D. R. Spitz et al, Arch. Biochem. Biophys., 279: 249-260, 1990; D. R. Spitz et al., Arch. Biochem. Biophys., 292: 221-227, 1992; S. J. Sullivan et al., Am. J. Physiol. (Lung Cell. Mol. Physiol.), 262: L748-L756, 1992). Acutely stressed cells failed to develop a stably resistant phenotype or increased catalase activity, suggesting that chronic exposure is required for the development of this phenotype. This study investigates the mechanism underlying increased catalase activity in the H2O2- and O2-resistant cell lines. In H2O2- and O2-resistant cells, catalase activity was found to be 20-30-fold higher than that in the parental HA1 cells and correlated with increased immunoreactive catalase protein and steady-state catalase mRNA levels. Resistant cell lines also demonstrated a 4-6-fold increase in catalase gene copy number by Southern blot analysis, which is indicative of gene amplification. Chromosome banding and in situ hybridization studies identified a single amplified catalase gene site located on a rearranged chromosome with banding similarities to Z-4 in the hamster fibroblast karyotype. Simultaneous in situ hybridization with a Z-4-specific adenine phosphoribosyltransferase (APRT) gene revealed that the amplified catalase genes were located proximate to APRT on the same chromosome in all resistant cells. In contrast, HA1 cells contained only single copies of the catalase gene that were not located on APRT-containing chromosomes, indicating that amplification is associated with a chromosomal rearrangement possibly involving Z-4. The fact that chronic exposure of HA1 cells to either HO2 or 95% O2 resulted in gene amplification suggests that gene amplification represents a generalized response to oxidative stress, contributing to the development of resistant phenotypes. These results support the hypothesis that chronic exposure to endogenous metabolic or exogenous environmental oxidative stress represents an important factor contributing to gene amplification and genomic instability.

Adenine Phosphoribosyltransferase↗

A mutation in separase causes genome instability and increased susceptibility to epithelial cancer.

Proper chromosome segregation is essential for maintenance of genomic integrity and instability resulting from failure of this process may contribute to cancer. Here, we demonstrate that a mutation in the mitotic regulator separase is responsible for the cell cycle defects seen in the zebrafish mutant, cease&desist (cds). Analysis of cds homozygous mutant embryos reveals high levels of polyploidy and aneuploidy, spindle defects, and a mitotic exit delay. Carcinogenesis studies demonstrated that cds heterozygous adults have a shift in tumor spectrum with an eightfold increase in the percentage of fish bearing epithelial tumors, indicating that separase is a tumor suppressor gene in vertebrates. These data strongly support a conserved cross-species role for mitotic checkpoint genes in genetic stability and epithelial carcinogenesis.

Animals↗

Increased genome instability in Escherichia coli lon mutants: relation to emergence of multiple-antibiotic-resistant (Mar) mutants caused by insertion sequence elements and large tandem genomic amplifications.

Thirteen spontaneous multiple-antibiotic-resistant (Mar) mutants of Escherichia coli AG100 were isolated on Luria-Bertani (LB) agar in the presence of tetracycline (4 microg/ml). The phenotype was linked to insertion sequence (IS) insertions in marR or acrR or unstable large tandem genomic amplifications which included acrAB and which were bordered by IS3 or IS5 sequences. Five different lon mutations, not related to the Mar phenotype, were also found in 12 of the 13 mutants. Under specific selective conditions, most drug-resistant mutants appearing late on the selective plates evolved from a subpopulation of AG100 with lon mutations. That the lon locus was involved in the evolution to low levels of multidrug resistance was supported by the following findings: (i) AG100 grown in LB broth had an important spontaneous subpopulation (about 3.7x10(-4)) of lon::IS186 mutants, (ii) new lon mutants appeared during the selection on antibiotic-containing agar plates, (iii) lon mutants could slowly grow in the presence of low amounts (about 2x MIC of the wild type) of chloramphenicol or tetracycline, and (iv) a lon mutation conferred a mutator phenotype which increased IS transposition and genome rearrangements. The association between lon mutations and mutations causing the Mar phenotype was dependent on the medium (LB versus MacConkey medium) and the antibiotic used for the selection. A previously reported unstable amplifiable high-level resistance observed after the prolonged growth of Mar mutants in a low concentration of tetracycline or chloramphenicol can be explained by genomic amplification.

Anti-Bacterial Agents↗

Human papillomaviruses and centrosome duplication errors: modeling the origins of genomic instability.

The majority of human cancers are genomically unstable, often with gains or losses of whole chromosomes. In high-risk human papillomavirus (HPV)-associated cervical neoplasia, the two HPV-encoded oncoproteins E6 and E7 have been implicated in mitotic infidelity by their ability to induce centrosome-related mitotic disturbances. However, the mechanisms by which HPV E6 and E7 subvert centrosome homeostasis are strikingly different. Whereas the E7 oncoprotein rapidly drives centrosome duplication errors in cells that appear phenotypically normal, expression of the HPV E6 oncoprotein results in an accumulation of supernumerary centrosomes in multinucleated cells. The primary centrosome duplication defect in HPV E7 expressing cells may be linked to the ability of E7 to disrupt regulatory nodes that govern both the host cell division cycle machinery and the initiation of centrosome duplication. Most importantly, the E7 oncoprotein has been shown to dysregulate cdk2 activity, a major determinant for the initiation of centrosome duplication. HPV-induced centrosome abnormalities, multipolar mitoses, and aneuploidy often occur at early stages during cervical carcinogenesis and increase with malignant conversion. These findings suggest that HPV oncoprotein-induced chromosomal instability increases the risk for genetic changes that may ultimately facilitate carcinogenic progression.

Cell Transformation, Neoplastic↗

Sequence rearrangements and genome instability. A possible step in carcinogenesis.

A substantial part of the mammalian genome is composed of sequences that do not contain structural genes. These sequences may constitute the major target for physical, chemical and biological DNA-damaging agents and can be involved in carcinogenesis. DNA-damaging agents contribute to the instability of the genome by introducing recombination-prone sites at DNA; these agents lead to extensive chromosomal lesions and rearrangements of genes and their regulatory sequences. Movable sequences that exist and operate in certain bacteria, yeast, and the fruit fly are responsible for sequence rearrangements and contribute to the majority of mutations. Their presence and role in higher animals is not well established. Extensive chromosomal rearrangements were identified in numerous malignancies in man and animals and definitely seem to represent a characteristic of malignancy. Vast chromosomal damage and sequence reshuffling may be of no less importance in the malignant transformation than the point mutation of a particular gene.

Animals↗

Suppression of genomic instability by SLX5 and SLX8 in Saccharomyces cerevisiae.

Replication forks can stall spontaneously at specific sites in the genome, and upon encountering DNA lesions resulting from chemical or radiation damage. In Saccharomyces cerevisiae proteins implicated in processing of stalled replication forks include those encoded by the SGS1, TOP3, MUS81, MMS4, SLX1, SLX4, SLX5/HEX3, and SLX8 genes. We tested the roles of these genes in suppressing gross chromosomal rearrangements (GCRs), which include translocations, large interstitial deletions, and loss of a chromosome arm with de novo telomere addition. We found that mus81, mms4, slx1, slx4, slx5, and slx8 mutants all have elevated levels of spontaneous GCRs, and that SLX5 and SLX8 are particularly critical suppressors of GCRs during normal cell cycle progression. In addition to increased GCRs, deletion of SLX5 or SLX8 resulted in increased relocalization of the DNA damage checkpoint protein Ddc2 and activation of the checkpoint kinase Rad53, indicating the accumulation of spontaneous DNA damage. Surprisingly, mutants in slx5 or slx8 were not sensitive to transient replication fork stalling induced by hydroxyurea, nor were they sensitive to replication dependent double-strand breaks induced by camptothecin. This suggested that Slx8 and Slx8 played limited roles in stabilizing, restarting, or resolving transiently stalled replication forks, but were critical for preventing the accumulation of DNA damage during normal cell cycle progression.

Cell Cycle Proteins↗

[Genomic instability associated with human LINE-1 rétrotransposition].

LINE-1 (L1) retrotransposon accounts for approximately 17 % of the human genome. Because of the great number of identical copies, L1 can be implicated in genomic rearrangements associated with events of homologous recombination between heterologous sites. Moreover, even if the vast majority of the L1 elements are inactive, some are still able to mobilize themselves by retrotransposition. Thus, L1 is regarded as an insertional mutagenic agent. Moreover, recent works have shown that active retrotransposons were able to mobilize other sequences to generate retro-pseudogenes or to amplify other repeated sequences. Finally, L1 has been associated recently with new genomic rearrangements generated upon insertions such as large genomic deletions. L1 then can be considered as a major factor that has affected and shaped the human genome through several mechanisms.

Genomic Instability↗

Telomere dysfunction in genome instability syndromes.

Telomeres are nucleoprotein complexes located at the end of eukaryotic chromosomes. They have essential roles in preventing terminal fusions, protecting chromosome ends from degradation, and in chromosome positioning in the nucleus. These terminal structures consist of a tandemly repeated DNA sequence (TTAGGG in vertebrates) that varies in length from 5 to 15 kb in humans. Several proteins are attached to this telomeric DNA, some of which are also involved in different DNA damage response pathways, including Ku80, Mre11, NBS and BLM, among others. Mutations in the genes encoding these proteins cause a number of rare genetic syndromes characterized by chromosome and/or genetic instability and cancer predisposition. Deletions or mutations in any of these genes may also cause a telomere defect resulting in accelerated telomere shortening, lack of end-capping function, and/or end-to-end chromosome fusions. This telomere phenotype is also known to promote chromosomal instability and carcinogenesis. Therefore, it is essential to understand the interplay between telomere biology and genome stability. This review is focused in the dual role of chromosome fragility proteins in telomere maintenance.

Animals↗