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PubMed2004

Imaging genome abnormalities in cancer research.

Increasing attention is focusing on chromosomal and genome structure in cancer research due to the fact that genomic instability plays a principal role in cancer initiation, progression and response to chemotherapeutic agents. The integrity of the genome (including structural, behavioral and functional aspects) of normal and cancer cells can be monitored with direct visualization by using a variety of cutting edge molecular cytogenetic technologies that are now available in the field of cancer research. Examples are presented in this review by grouping these methodologies into four categories visualizing different yet closely related major levels of genome structures. An integrated discussion is also presented on several ongoing projects involving the illustration of mitotic and meiotic chromatin loops; the identification of defective mitotic figures (DMF), a new type of chromosomal aberration capable of monitoring condensation defects in cancer; the establishment of a method that uses Non-Clonal Chromosomal Aberrations (NCCAs) as an index to monitor genomic instability; and the characterization of apoptosis related chromosomal fragmentations caused by drug treatments.

Journal Article↗
PubMed2004

The three-dimensional organization of telomeres in the nucleus of mammalian cells.

BACKGROUND: The observation of multiple genetic markers in situ by optical microscopy and their relevance to the study of three-dimensional (3D) chromosomal organization in the nucleus have been greatly developed in the last decade. These methods are important in cancer research because cancer is characterized by multiple alterations that affect the modulation of gene expression and the stability of the genome. It is, therefore, essential to analyze the 3D genome organization of the interphase nucleus in both normal and cancer cells. RESULTS: We describe a novel approach to study the distribution of all telomeres inside the nucleus of mammalian cells throughout the cell cycle. It is based on 3D telomere fluorescence in situ hybridization followed by quantitative analysis that determines the telomeres' distribution in the nucleus throughout the cell cycle. This method enables us to determine, for the first time, that telomere organization is cell-cycle dependent, with assembly of telomeres into a telomeric disk in the G2 phase. In tumor cells, the 3D telomere organization is distorted and aggregates are formed. CONCLUSIONS: The results emphasize a non-random and dynamic 3D nuclear telomeric organization and its importance to genomic stability. Based on our findings, it appears possible to examine telomeric aggregates suggestive of genomic instability in individual interphase nuclei and tissues without the need to examine metaphases. Such new avenues of monitoring genomic instability could potentially impact on cancer biology, genetics, diagnostic innovations and surveillance of treatment response in medicine.

Animals↗
PubMed2002

Induction of gene amplification as a gain-of-function phenotype of mutant p53 proteins.

Gene amplification accompanies tumor progression and is involved in the development of drug resistance. Previously, we reported (A. Albor et al., Cancer Res. 58: 2091-2094, 1998) that mutant p53 proteins conserve the capacity to interact with and activate topoisomerase I, and that this could be a mechanism for induction of genomic instability by mutant p53 proteins. To test this hypothesis, the effect of exogenous mutant p53 protein expression on genomic instability in human p53-/- Saos-2 cells was measured by the frequency of formation of N-(phosphoacetyl)-L-aspartate (PALA)-resistant (PALA(R)) colonies, mediated by gene amplification. Interaction of exogenous mutant p53 and topoisomerase I was confirmed by immunoprecipitation. Growth under continuous expression of mutant p53 proteins for 16-17 population doublings increased the frequency of appearance of PALA(R) colonies after subsequent exposure to PALA. Subtoxic concentrations of camptothecin (which stabilizes topoisomerase I cleavage complexes, mediating nonhomologous recombination) produced a dose-dependent increase in PALA(R) colonies, and combining expression of mutant p53 with exposure to camptothecin produced a greater than additive increase in PALA(R) colony formation. These results indicate that mutant p53 proteins promote gene amplification independently of their capacity to inactivate the wild-type p53 protein, and suggest that this effect is dependent on interaction of mutant p53 with topoisomerase I. Additional studies are needed to assess the potential of targeting mutant p53 interaction with topoisomerase I for the reduction of drug resistance development during chemotherapy.

Aspartic Acid↗
PubMed2003

Immortal, telomerase-negative cell lines derived from a Li-Fraumeni syndrome patient exhibit telomere length variability and chromosomal and minisatellite instabilities.

Five immortal cell lines derived from a Li-Fraumeni syndrome patient (MDAH 087) with a germline mutant p53 allele were characterized with respect to telomere length and genomic instability. The remaining wild-type p53 allele is lost in the cell lines. Telomerase activity was undetectable in all immortal cell lines. Five subclones of each cell line and five re-subclones of each of the subclones also showed undetectable telomerase activity. All five immortal cell lines exhibited variability in the mean length of terminal restriction fragments (TRFs). Subclones of each cell line, and re-subclones of the subclones also showed TRF variability, indicating that the variability is owing to clonal heterogeneity. Chromosome aberrations were observed at high frequencies in these cell lines including the subclones and re-subclones, and the principal types of aberrations were breaks, double minute chromosomes and dicentric chromosomes. In addition, minisatellite instability detected by DNA fingerprints was observed in the immortal cell lines. However, all of the cell lines were negative for microsatellite instability. As minisatellite sequences are considered recombinogenic in mammalian cells, these results suggest that recombination rates can be increased in these cell lines. Tumor-derived human cell lines, HT1080 cells and HeLa cells that also lack p53 function, exhibited little genomic instability involving chromosomal and minisatellite instabilities, indicating that chromosomal and minisatellite instabilities observed in the immortal cell lines lacking telomerase activity could not result from loss of p53 function.

Cell Line, Transformed↗
PubMed1998

Instability of dinucleotide repeats in Hodgkin's disease.

Tumorigenesis has been shown to proceed through a series of genetic alterations involving protooncogenes and tumor suppressor genes. However, the investigation of genomic instability of microsatellites has disclosed a new mechanism for human carcinogenesis, which is involved not only in hereditary nonpolyposis colon cancer (HNPCC) but in a number of other malignancies as well. To determine whether microsatellite instability is involved in Hodgkin's disease, we screened 16 such tumors using 7 microsatellite marker loci on 6 chromosome arms 4, 5, 9p, 9q, 11, 14, and 17. Using the polymerase chain reaction method, DNA samples from the tumors and from normal peripheral blood leukocytes from each patient were compared for the allelic pattern produced at each locus. Five cases of genomic instability were identified, suggesting that this mechanism is relevant to the pathogenesis of HD.

Chromosomes, Human↗
PubMed2002

Involvement of DNA mismatch repair in folate deficiency-induced apoptosis small star, filled.

Folate is a critical factor for DNA metabolism and its deficiency is associated with a number of human diseases and cancers. Although it has been shown that folate deficiency induces genomic instability and apoptotic cell death, the underlying mechanism is largely unknown. Given the role of mismatch repair in maintaining genomic integrity, mismatch repair was tested for its involvement in folate deficiency-induced genomic instability and cell death. Cells proficient in mismatch repair were highly sensitive to folate deficiency compared with cells defective in either hMutSalpha or hMutLalpha. Since wild-type cells but not mutant cells underwent apoptosis upon extensive folate depletion, the apoptotic response is dependent on a functional mismatch repair system. Our data also indicate that p53 is required for the folate depletion-induced apoptosis. In vitro biochemical studies demonstrated that hMutSalpha specifically recognized DNA damage induced by folate deficiency, suggesting a direct participation of mismatch repair proteins in mediating the apoptotic response. We conclude that while the mismatch repair-dependent apoptosis is necessary to protect damaged cells from tumorigenesis, it may damage a whole tissue or organ, as seen in patients with megaloblastic anemia, during extensive folate deficiency.

Journal Article↗
PubMed2002

Age-related mutation accumulation at a lacZ reporter locus in normal and tumor tissues of Trp53-deficient mice.

Increased genomic instability has been found associated with cancer and aging. The p53 tumor suppressor protein is a major determinant of genomic instability as a regulator of cell cycle control and apoptosis in response to DNA damage. To investigate the rate of age-related mutation accumulation in the absence of p53, we crossed Trp53 null mice with transgenic mice harboring a lacZ mutational target gene. In the hybrid animals, lacZ mutation frequencies at early age (i.e. at about 2 months) were found to be the same as in the control lacZ animals. However, up until about 6 months, when the Trp53-knockout mice usually die from cancer, mutations were found to accumulate with age in the spleen, and to a lesser extent in the liver, at a more rapid rate than in the control Trp53(+/+) or Trp53(+/-), lacZ hybrid mice. Treatment of 2-3-month-old Trp53(-/-), lacZ hybrid mice with the powerful mutagen ethyl nitrosourea (ENU) resulted in a higher number of mutations induced in the liver but not in the spleen, as compared to the Trp53(+/+), lacZ mice. These results suggest that p53 is not an important determinant of gene mutation induction, either spontaneously during development or after treatment with a mutagen. The accelerated age-related accumulation of mutations in normal spleen and liver could be explained by the defect in apoptosis, which would prevent severely damaged cells from being eliminated.

Aging↗
PubMed1992

Flow-cytometric and histological progression to malignancy in Barrett's esophagus: prospective endoscopic surveillance of a cohort.

To determine whether or not flow-cytometric evidence of aneuploidy and increased G2/tetraploid fractions predispose to neoplastic progression in Barrett's esophagus, 62 patients with Barrett's esophagus were evaluated prospectively for a mean interval of 34 months. Nine of 13 patients who showed aneuploid or increased G2/tetraploid populations in their initial flow-cytometric analysis developed high-grade dysplasia or adenocarcinoma during follow-up; none of the 49 patients without these abnormalities progressed to high-grade dysplasia or cancer (P less than 0.0001). Neoplastic progression was characterized by progressive flow-cytometric and histological abnormalities. Patients who progressed to high-grade dysplasia and carcinoma frequently developed multiple aneuploid populations of cells that were detectable flow-cytometrically. Similarly, patients appeared to progress through a phenotypic sequence that could be recognized histologically by the successive appearance of Barrett's metaplasia negative for dysplasia, abnormalities in the indefinite/low-grade dysplasia range, high-grade dysplasia, and eventually adenocarcinoma. These and prior results suggest that neoplastic progression in Barrett's esophagus occurs in a subset of patients who have an acquired genomic instability that generates abnormal clones of cells, some of which have aneuploid or increased G2/tetraploid DNA contents. With continued genomic instability, multiple aneuploid subclones may evolve, one of which may ultimately acquire the ability to invade and become an early carcinoma. The combination of histology and flow cytometry can be used to identify a subset of patients with Barrett's esophagus who merit more frequent endoscopic surveillance for the early detection of high-grade dysplasia or carcinoma.

Aneuploidy↗
PubMed2002

Application of inter-simple sequence repeat PCR to mouse models: assessment of genetic alterations in carcinogenesis.

Genomic instability is believed to play a significant role in cancer development by facilitating tumor progression and tumor heterogeneity. Inter-simple sequence repeat (inter-SSR) PCR has been proved to be a fast and reproducible technique for quantitation of genomic instability (amplifications, deletions, translocations, and insertions) in human sporadic tumors. However, the use of inter-SSR PCR in animal models of cancer has never been described. This new technique has been adapted in our laboratory for the analysis of spontaneous and induced mouse tumors. We established the best PCR conditions for each microsatellite-anchored primer and critically evaluated the reproducibility of the band patterns. We also studied the variation of the fingerprints between and within various inbred mouse strains, including wild-derived lines. Tumor-specific alterations were detected as gains, losses, or intensity changes in bands when compared with matched normal DNA. We quantitated the extent of alterations by dividing the number of altered bands in the tumor by the total number of bands in normal DNA (instability index). By means of inter-SSR PCR, we successfully analyzed genomic alterations in various mouse tumors, including spontaneous thymic lymphomas developed in Msh2 knockout mice as well as chemically induced squamous cell carcinomas and thymic lymphomas. Instability index values ranged between 0 and 9%, the highest levels observed in N-methyl-N-nitrosourea-induced thymic lymphomas generated in Trp53 (p53) nullizygote (-/-) mice. We report here, for the first time, the use of inter-SSR PCR to detect somatic mutations in mouse tumoral DNA, including laser-capture microdissected, methanol-fixed tissues. These PCR-based fingerprints provide a novel approach to assessing the number and onset of mutational events in mouse tumors and will help to understand better the mechanisms of carcinogenesis in mouse models.

Animals↗
PubMed2005

Implementation of accurate and fast DNA cytometry by confocal microscopy in 3D.

BACKGROUND: DNA cytometry is a powerful method for measuring genomic instability. Standard approaches that measure DNA content of isolated cells may induce selection bias and do not allow interpretation of genomic instability in the context of the tissue. Confocal Laser Scanning Microscopy (CLSM) provides the opportunity to perform 3D DNA content measurements on intact cells in thick histological sections. Because the technique is technically challenging and time consuming, only a small number of usually manually selected nuclei were analyzed in different studies, not allowing wide clinical evaluation. The aim of this study was to describe the conditions for accurate and fast 3D CLSM cytometry with a minimum of user interaction to arrive at sufficient throughput for pilot clinical applications. METHODS: Nuclear DNA was stained in 14 microm thick tissue sections of normal liver and adrenal stained with either YOYO-1 iodide or TO-PRO-3 iodide. Different pre-treatment strategies were evaluated: boiling in citrate buffer (pH 6.0) followed by RNase application for 1 or 18 hours, or hydrolysis. The image stacks obtained with CLSM at microscope magnifications of x40 or x100 were analyzed off-line using in-house developed software for semi-automated 3D fluorescence quantitation. To avoid sectioned nuclei, the top and bottom of the stacks were identified from ZX and YZ projections. As a measure of histogram quality, the coefficient of variation (CV) of the diploid peak was assessed. RESULTS: The lowest CV (10.3%) was achieved with a protocol without boiling, with 1 hour RNase treatment and TO-PRO-3 iodide staining, and a final image recording at x60 or x100 magnifications. A sample size of 300 nuclei was generally achievable. By filtering the set of automatically segmented nuclei based on volume, size and shape, followed by interactive removal of the few remaining faulty objects, a single measurement was completely analyzed in approximately 3 hours. CONCLUSIONS: The described methodology allows to obtain a largely unbiased sample of nuclei in thick tissue sections using 3D DNA cytometry by confocal laser scanning microscopy within an acceptable time frame for pilot clinical applications, and with a CV small enough to resolve smaller near diploid stemlines. This provides a suitable method for 3D DNA ploidy assessment of selected rare cells based on morphologic characteristics and of clinical samples that are too small to prepare adequate cell suspensions.

Adrenal Glands↗
PubMed2001

Lack of detectable transmissible chromosomal instability after in vivo or in vitro exposure of mouse bone marrow cells to 224Ra alpha particles.

Several studies over recent years have highlighted the possibility that radiation can induce transmissible genomic instability. Most of these involve in vitro irradiation and usually in vitro culture. Here it is reported that the short-half-life bone-seeking alpha-particle emitter (224)Ra did not induce excess transmissible chromosomal instability in CBA/H mouse bone marrow cells in a 100-day period after in vivo or in vitro exposure. Similarly, no excess transmissible chromosomal instability could be detected after in vivo whole-body X irradiation. It was noted, however, that short-term culture of murine bone marrow cells elevated yields of aberrations, as did transplantation of untreated marrow into radiation-ablated hosts. These findings emphasize the sensitivity of murine hemopoietic tissue to experimental manipulation and reinforce the importance of appropriate concurrent control experiments in any investigation of transmissible genomic instability.

Alpha Particles↗
PubMed2002

Endothelial cells maintain a reduced redox environment even as mitochondrial function declines.

Human umbilical vein endothelial cells (HUVECs) are an endothelial model of replicative senescence. Oxidative stress, possibly due to dysfunctional mitochondria, is believed to play a key role in replicative senescence and atherosclerosis, an age-related vascular disease. In this study, we determined the effect of cell division on genomic instability, mitochondrial function, and redox status in HUVECs that were able to replicate for approximately 60 cumulative population doublings (CPD). After 20 CPD, the nuclear genome deteriorated and the protein content of the cell population increased. This indicated an increase in cell size, which was accompanied by an increase in oxygen consumption, ATP production, and mitochondrial genome copy number and approximately 10% increase in mitochondrial mass. The antioxidant capacity increased, as seen by an increase in reduced glutathione, glutathione peroxidase, GSSG reductase, and glucose-6-phosphate dehydrogenase. However, by CPD 52, the latter two enzymes decreased, as well as the ratio of mitochondrial-to-nuclear genome copies, the mitochondrial mass, and the oxygen consumption per milligram of protein. Our results signify that HUVECs maintain a highly reducing (GSH) environment as they replicate despite genomic instability and loss of mitochondrial function.

Cells, Cultured↗
PubMed2003

Current status of the molecular genetics of human prostatic adenocarcinomas.

Molecular genetic mechanisms involved in the progression of prostate cancer are not well understood due to extensive tumor heterogeneity and lack of suitable models. New methods such as fluorescence in-situ hybridization (FISH), comparative genomic hybridization (CGH) and microsatellite analysis have documented losses or gains on various chromosomes. Altered chromosomal regions have been associated with the activation of oncogenes and the inactivation of tumor suppressor genes or defects in mismatch repair (MMR) genes. It is suggested that increased genomic instability is associated with decreased androgen-responsive and progressive behavior of human prostate tumors, but it remains unclear whether this genomic instability is causing the progression of cancer or is the consequence of cancer. Extended studies on hereditary prostate cancer have identified 7 prostate cancer susceptibility loci on several chromosomes, but no specific gene has been confined for a large proportion of susceptibility. In this review we summarize the ongoing molecular genetic events associated with the sporadic and hereditary prostate cancer development and progression.

Adenocarcinoma↗
PubMed2024

Loss of Fbxo45 in AT2 cells leads to insufficient histone supply and initiates lung adenocarcinoma.

Dysregulation of histone supply is implicated in various cancers, including lung adenocarcinoma (LUAD), although the underlying mechanisms remain poorly understood. Here, we demonstrate that knockout of Fbxo45 in mouse alveolar epithelial type 2 (AT2) cells leads to spontaneous LUAD. Our findings reveal that FBXO45 is a novel cell-cycle-regulated protein that is degraded upon phosphorylation by CDK1 during the S/G2 phase. During the S phase or DNA damage repair, FBXO45 binds to UPF1 and recruits the phosphatase PPP6C, thereby inhibiting UPF1 phosphorylation. This process is crucial for preventing the degradation of replication-dependent (RD) histone mRNAs and ensuring an adequate histone supply. In the absence of FBXO45, the impaired interaction between PPP6C and UPF1 results in sustained hyperphosphorylation of UPF1 throughout the cell cycle, leading to an insufficient histone supply, chromatin relaxation, genomic instability, and an increased rate of gene mutations, ultimately culminating in malignant transformation. Notably, analysis of clinical LUAD specimens confirms a positive correlation between the loss of FBXO45 and genomic instability, which is consistent with our findings in the mouse model. These results highlight the critical role of FBXO45 as a genomic guardian in coordinating histone supply and DNA replication, providing valuable insights into potential therapeutic targets and strategies for the treatment of LUAD.

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↗
PubMed1991

Evidence for increased in vitro recombination with insertion of human hepatitis B virus DNA.

Chromosomal translocation, deletion, and inversion/duplication directly linked to hepatitis B virus (HBV) DNA integration occur frequently in host DNA of human hepatocellular carcinomas. To test the possible recombinogenic effect of HBV DNA, we have utilized an in vitro recombination assay. Fragments containing the region spanning DR1, which is believed to be the origin of viral replication and a preferred site in the viral genome for integration, increased the recombination events reproducibly in the presence of extracts from actively dividing cells (e.g., hepatocellular carcinoma) but not resting cells (e.g., normal liver). Moreover, in these extracts we have found a protein(s) that specifically binds to these HBV DNA fragments. These results support the notion that in some instances integrated HBV DNAs cause further genomic instability, possibly involving specific cellular protein(s). The fact that extracts from nondividing, normal liver did not increase recombination events suggests that genomic instability depends upon active cellular growth, a feature more commonly found subsequent to HBV-induced hepatocellular injury than in healthy liver. Our results offer an explanation for the high incidence of liver cancer that accompanies chronic hepatitis and add HBV to the list of agents that can cause genetic recombination.

Animals↗
PubMed2001

A cytogenetic analysis of the long-term effect of uranium mining on peripheral lymphocytes using the micronucleus-centromere assay.

PURPOSE: To assess the long-term effect of radiation exposure of uranium miners on a cytogenetic endpoint: micronuclei (Mn) with and without a centromere. MATERIALS AND METHODS: Mn were scored using the cytochalasin-B technique. It is known that Mn can comprise acentric fragments or/and whole chromosomes. Mn containing whole chromosomes were identified by means of fluorescence in situ hybridization (FISH) with a centromere-specific probe. The frequency and percentage of Mn were analysed with centromeres (MnC+) in lymphocytes of healthy donors and uranium miners with large radiation exposures several decades ago employed by the Wismut AG in the former German Democratic Republic. The miners were subdivided into those with and those without bronchial carcinoma. RESULTS: It was shown previously that the relative frequency of MnC+ decreased with dose; this means that the number of Mn originating from acentric fragments increases. In the study presented here, no statistically significant difference in the overall Mn frequency was seen between the analysed groups. The fraction of MnC+, however, was highest in lymphocytes of healthy male donors (mean: 74.6%) followed by healthy miners (mean: 62.1%) and those suffering from cancer (mean: 55.8%). CONCLUSION: The results indicate the occurrence of a genomic instability in lymphocytes of miners, especially those with cancer. It appears that the low percentage of MnC+ may be a marker of genomic instability and cancer predisposition.

Adult↗
PubMed2005

Modeling premature aging syndromes with the telomerase knockout mouse.

Understanding the molecular basis of the aging process is a daunting problem, given the complex genetic and physiological changes that underlie human aging and the lack of genetically amenable primate model systems. However, analysis of mouse models exhibiting aging phenotypes reminiscent of those observed in elderly humans has enhanced our understanding of the biological mechanisms underlying mammalian aging. In particular, these mouse models have brought to light the importance of the DNA damage pathway during the aging process. Increased genomic instability is associated with accelerated cellular decline and manifestation of premature aging phenotypes in mice. Here I discuss how one form of genomic instability, initiated by critically short telomeres in the telomerase knockout mouse, perturb normal mammalian aging. Insights into the molecular pathways of the aging process may offer unprecedented opportunities to delay the deleterious effects of the aging process.

Aging, Premature↗