The use of premature chromosome condensation to study in interphase cells the influence of environmental factors on human genetic material

Abstract

Nowadays, there is a constantly increasing concern regarding the mutagenic and carcinogenic potential of a variety of harmful environmental factors to which humans are exposed in their natural and anthropogenic environments. These factors exert their hazardous potential in humans' personal (diet, smoking, pharmaceuticals, cosmetics) and occupational environments that constitute part of the anthropogenic environment. It is well known that genetic damage due to these factors has dramatic implications for human health. Since most of the environmental genotoxic factors induce arrest or delay in cell cycle progression, the conventional analysis of chromosomes at metaphase may underestimate their genotoxic potential. Premature chromosome condensation (PCC) induced either by means of cell fusion or specific chemicals, enables the microscopic visualization of interphase chromosomes whose morphology depends on the cell cycle stage, as well as the analysis of structural and numerical aberrations at the G 1 and G 2 phases of the cell cycle. The PCC method has been successfully used in problems involving cell cycle analysis, diagnosis, and prognosis of human leukemia, assessment of interphase chromosome malformations resulting from exposure to radiation or chemicals, as well as elucidation of the mechanisms underlying the conversion of DNA damage into chromosomal damage. In this report, particular emphasis is given to the advantages of the PCC methodology used as an alternative to conventional metaphase analysis in order to answer questions in the fields of radiobiology, biological dosimetry, toxicogenetics, clinical cytogenetics, and experimental therapeutics. KEYWORDS: premature chromosome condensation, cell fusion, calyculin-A, lymphocytes, DNA damage, genotoxicity, sister chromatid exchanges, chromosomal damage, chromosome aberrations, cell cycle delay, chemicals, ionizing radiation Hatzi et al.: Interphase genotoxicity assessment using PCC TheScientificWorldJOURNAL (2006TheScientificWorldJOURNAL ( ) 6, 1174TheScientificWorldJOURNAL ( -1190 INTRODUCTION Nowadays, there is a constantly increasing concern regarding the mutagenic and carcinogenic potential of a variety of harmful environmental agents to which humans are exposed in their natural and anthropogenic environments. Harmful environmental agents, exerting their effect on living organisms from the beginning of life, developed the appropriate evolutionary pressure that resulted in their defense mechanisms. Human technological development as well as human lifestyle introduced new series of harmful agents that, either alone or in combination, affect humans and all other organisms. These new agents exert their hazardous potential in humans' personal (diet, smoking, pharmaceuticals, cosmetics) and occupational environments that constitute part of the anthropogenic environment. The most important consequences of human exposure to environmental hazards are DNA damage induced either by direct or indirect binding to DNA. Direct-acting chemicals like DNA alkylating agents (e.g., mitomycin-C), crosslinking agents (e.g., methyl methanesulfonate), and oxygen radicals (e.g., hydrogen peroxide), bind covalently to DNA The exposure of cells to genotoxic factors may result in changes at the chromatid level, such as sister chromatid exchanges (SCEs), as well as in structural chromosomal alterations such as chromosomal aberrations Until now, the conventional cytogenetic analysis of chromosomal damage as a result of exposure to environmental chemicals is mainly based on the microscopic analysis of chromosomes in metaphase In this report, the advantages of the PCC methodologies used as an alternative to conventional metaphase analysis or in combination with other cytogenetic techniques are reviewed. Furthermore, the use of PCC in overcoming problems that could not be solved with the conventional metaphase analysis is Hatzi et al.: Interphase genotoxicity assessment using PCC TheScientificWorldJOURNAL (2006) 6, 1174-1190 1176 presented, and particular emphasis is given to the potential use of PCC to elucidate the mechanisms underlying conversion of DNA damage into chromosomal damage. Additional applications of the PCC methodologies in the fields of radiobiology, biological dosimetry, toxicogenetics, clinical cytogenetics, and experimental therapeutics are described, and future perspectives are discussed. PCC METHODOLOGIES PCC Induction Using Cell Fusion Central for the PCC assay is the fusion of interphase "test" cells with mitotic "inducer" cells that can be mediated either by incubation with Sendai virus As an alternative to the Sendai virus, the well-known fusogen polyethylene glycol (PEG) was applied to PCC-induction procedures Using the PCC assay by means of cell fusion, interphase cells that are either cycling, noncycling, or arrested can be visualized and analyzed. In the hybrids formed by cell fusion, the mitotic factors present in the donor mitotic cell dissolve the nucleus membrane and condense chromatin of the interphase "test" cell. More specifically, cells that have undergone PCC assume a morphology that is characteristic of the position of the interphase cell in the cell cycle: single chromatid per chromosome in G 1 phase, double chromatids per chromosome in G 2 , and pulverized chromosome regions in S phase Chemically Induced PCC Chemically induced PCCs can be obtained by the use of the chemical compounds such as calyculin-A. Calyculin-A was initially isolated from the marine sponge Discodermia calyx 1177 APPLICATIONS OF PCC METHODOLOGIES Biodosimetry and Biomonitoring of Exposure to Ionizing Radiation Exposure of cells to ionizing radiation has been shown to cause a wide variety of phenomena, the most prominent of which are the induction of mutations, the induction of transformation, cell cycle arrest, and cell death. As a result, the study of phenomena elicited by radiation are of particular importance to human health and, therefore, elucidation of the underlying biochemical mechanisms and cytogenetic effects of ionizing radiation are a high priority in radiation biology research. In the field of biological dosimetry, numerous methods capable of detecting radiation-induced changes at the molecular, cytogenetic, and cellular level have been used in order to obtain absorbed dose estimates For these reasons, the PCC methodology was proposed as an alternative biodosimetric tool by Pantelias and Maillie in 1984[50] and since then it has been extensively used to assess and evaluate the induction and repair of chromosome damage after in vivo or in vitro exposure of human cells to ionizing radiation Mechanisms Underlying Conversion of DNA Damage into Chromosomal Damage Visualizing cells with conventional analysis at metaphase can give information concerning only the residual damage after exposure to genotoxic environmental factors. The analysis of such interactions is based, therefore, only on those cells that proceed to mitosis. Therefore, it is difficult to elucidate the mechanism underlying the conversion of DNA damage into chromosomal damage. With the PCC methodologies, it is possible to gain valuable information not only to understand the biochemical mechanisms that affect the conversion of DNA damage into chromosomal damage, but also to determine possibly chromosomal radiosensitivity in G 2 phase, as well as variability in radiosensitivity at various stages of the cell cycle. The Hatzi et al.: Interphase genotoxicity assessment using PCC TheScientificWorldJOURNAL (2006) 6, 1174-1190 1181 onset and the efficiency of chromatin condensation-decondensation are important determinants of these processes. Data obtained so far demonstrate the important role of cdk1/cyclin-B complex and of the G 2 checkpoint control mechanism in affecting chromatin conformation changes and conversion of DNA damage into chromosomal damage. Using the PCC method, it was realized specifically that changes in chromatin conformation soon after irradiation, presumably as a result of histone-H1 phosphorylationdephosphorylation, strongly affected the conversion of DNA lesions into visual PCC fragments. The cdk1/cyclin-B complex was originally defined as the mitosis promoting factor (MPF), identified in mitotic frog eggs as a factor capable of inducing mitosis in G 2 -phase cells. Regulation of cdk1/cyclin-B complexes at multiple levels ensures the tight regulation of the timing of mitotic entry In early reports, G 0 human lymphocytes were irradiated and analyzed at various times after fusion with mitotic CHO cells, i.e., as chromatin condensation proceeded. The yield of fragments observed was directly related to the amount of chromosome condensation allowed to take place after irradiation and inversely related to the extent of chromosome condensation at the time of irradiation. From these experiments, it was concluded that changes in chromosome conformation interfered with repair processes of DNA damage. In contrast, resting chromosomes (as G 0 lymphocytes irradiated before fusion) showed efficient repair of chromosomal damage. These results supported the hypothesis that DNA damage is converted into cytogenetic lesions and becomes observable when chromatin conformation changes occur during the cell cycle G 2 checkpoint facilitates repair of chromosomal damage, and the hypothesis that G 2 -checkpoint defects during the G 2 -to M-phase transition can also affect G 2 -chromosomal sensitivity. This was tested using caffeine to abolish G 2 checkpoint by inhibiting ATM protein (Ataxia Telangiectasia mutated protein) Concerning the variability of radiosensitivity to ionizing radiation at various stages of the cell cycle, it is already known that middle to S phase and G 1 phase are resistant stages, while mitosis, G 1 /S, and G 2 /M transition are very sensitive to radiation Genotoxicity of Exposure to Chemical Agents It is well known that increased rates of cell proliferation can escalate the risk of malignancy following exposures to chemical agents 1183 The Uses of PCC in Clinical Cytogenetics and Experimental Therapeutics The fact that chromosomal analysis by means of the PCC method requires a small amount (0.5 ml or less) of sample (i.e., peripheral blood or bone marrow) makes it especially suitable for in vivo and in vitro studies, and also in clinical applications for diagnostic purposes. In particular, the ability to visualize the interphase chromosomes of bone marrow and blood cells using PCC by means of mitotic cell fusion with interphase cells has proved useful and accurate in the study of human acute leukemia Recently, calyculin-A-induced PCC has been combined with multicolor FISH [pq-COBRA-FISH (COmbined Binary RAtio labeling-fluorescence in situ hybridization)] for the cytogenetic analysis of cancer cell lines In the field of prenatal diagnosis, preliminary attempts have been made to combine calyculin-Ainduced PCC with GTG banding for fetus examination Furthermore, a major area of experimental therapeutic research centers on the function of anticancer drugs. In this field, the PCC methodology has been applied to study the effect of several anticancer drugs, such as BCNU, CCNU, Cis-acid, VM-26, adriamycin, and neocarzinostatin, whose mode of action depends on cell cycle arres