Genomic instability in neoplasia

Recent studies have demonstrated novel alterations of microsatellite DNA in tumor tissue. The alterations, termed microsatellite instability or replication error phenotype, have now been observed in tumors from patients with hereditary nonpolyposis colorectal cancer (HNPCC), the Muir-Torre syndrome (MTS) and in an increasing number of sporadic tumors. These observations, along with the use of genetic linkage analysis, have led to the identification of at least four genetic susceptibility loci for HNPCC, hMSH2, hMLH1, hPMS1 and hPMS2, each of which are involved in DNA mismatch repair. For those tumors demonstrating microsatellite instability, several different phenotypes may exist, the significance of which is currently unknown. Defective DNA mismatch repair may have important implications for the mechanism of tumorigenesis and the clinical behavior of tumors.     

Chromosome instability in Candida albicans

Candida albicans maintains genetic diversity by random chromosome alterations, and this diversity allows utilization of various nutrients. Although the alterations seem to occur spontaneously, their frequencies clearly depend on environmental factors. In addition, this microorganism survives in adverse environments, which cause lethality or inhibit growth, by altering specific chromosomes. A reversible loss or gain of one homolog of a specific chromosome in this diploid organism was found to be a prevalent means of adaptation. We found that loss of an entire chromosome is required because it carries multiple functionally redundant negative regulatory genes. The unusual mode of gene regulation in Candida albicans implies that genes in this organism are distributed nonrandomly over chromosomes.     

Chromosome instability and tumor lethality suppression in carcinogenesis

The maintenance and survival of each organism depends on its genome integrity. Alterations of essential genes, or aberrant chromosome number and structure lead to cell death. Paradoxically, cancer cells, especially in solid tumors, contain somatic gene mutations and are chromosome instability (CIN), suggesting a mechanism that cancer cells have acquired to suppress the lethal mutations and/or CIN. Herein we will discuss a tumor lethality suppression concept based on the studies of yeast genetic interactions and transgenic mice. During the early stages of the multistep process of tumorigenesis, incipient cancer cells probably have adopted genetic and epigenetic alterations to tolerate the lethal mutations of other genes that ensue, and to a larger extent CIN. In turn, CIN mediated massive gain and loss of genes provides a wider buffer for further genetic reshuffling, resulting in cancer cell heterogeneity, drug resistance and evasion of oncogene addiction, thus CIN may be both the effector and inducer of tumorigenesis. Accordingly, interfering with tumor lethality suppression could lead to cancer cell death or growth defects. Further validation of the tumor lethality suppression concept would help to elucidate the role of CIN in tumorigenesis, the relationship between CIN and somatic gene mutations, and would impact the design of anticancer drug development.     

Chromosome evolution in the cercopithecidae and its relationship to human fragile sites and neoplasia

Seven species of the family Cercopithecidae have been studied using highresolution banding techniques. Comparative studies allowed us to identify the main chromosomal reorganizations in this group, as well as to establish the phylogenetic relationships between species. Some of the regions involved in evolutionary rearrangements correspond to human fragile sites and/or chromosomal rearrangements related to neoplasia.     

Chromosome instability in Spodoptera frugiperda Sf-9 cell line

Homogeneous cell lines are essential in industry and research if reliable and reproducible data are to be obtained. The Spodoptera frugiperda (Sf-9) cell line routinely used for the production of recombinant proteins was found to be heterogeneous, containing a mixture of diploid and tetraploid cells. Using dilution-cloning techniques, diploid and tetraploid subpopulations were isolated from a Sf-9 parental cell line, and their cytogenetic state was monitored using Vinblastine to arrest cells in mitosis. Flow cytometry was used to obtain a snapshot of the predominant subpopulations present to verify the karyological results. The rate at which clonal populations digress into the heterogeneous state was found to be more rapid for the diploid subpopulation, with the emergence of tetraploid cells after only 11 passages, than for the tetraploid subpopulation, where diploid clones appeared after 18 passages. The chromosomes in both diploid and tetraploid subpopulations as well as the parental cell line were found to spontaneously fragment during growth and expansion processes, giving rise to variable chromosome numbers. DNA analysis of cell lines obtained from laboratories worldwide have shown that the Sf-9 cell line used for the production of many recombinant proteins is cytologically unstable, leading to varying degrees of polyploidal state depending on its culture history and supplier.     

Chromosome aberration frequencies and chromosome instability in mice after long-term exposure to low-dose-rate gamma-irradiation

Chronological changes of chromosome aberration rates related to accumulated doses in chronically exposed humans and animals at a low-dose-rate have not been well studied. C3H female specific pathogen-free mice (8 weeks of age) were chronically irradiated. Chromosome aberration rate in mouse splenocytes after long-term exposure to low-dose-rate (LDR) gamma-rays was serially determined by conventional Giemsa method. Incidence of dicentrics and centric rings increased almost linearly up to 8000 mGy following irradiation for about 400 days at a LDR of 20 mGy/day. Clear dose-rate effects were observed in the chromosome aberration frequencies between dose rates of 20 mGy/day and 200 Gy/day. Furthermore, the frequencies of complex aberrations increased as accumulated doses increased in LDR irradiation. This trend was also observed for the incidences of micronuclei and trisomies of chromosomes 5, 13 and 18 in splenocytes, detected by micronucleus assay and metaphase fluorescence in situ hybridization (FISH) method, respectively. Incidences of 2-4 micronuclei and trisomy increased in mouse splenocytes after irradiation of 8000 mGy at a LDR of 20 mGy/day. These complex chromosome aberrations and numerical chromosome aberrations seem to be induced indirectly after radiation exposure and thus the results indicate that continuous gamma-ray irradiation for 400 days at LDR of 20 mGy/day induced chromosomal instability in mice. These results are important to evaluate the biological effects of long-term exposure to LDR radiation in humans.     

Chromosome breakage-fusion-bridge-cycle and phenotypic instability in isochromosome lines of tomato

Summary Cultivated tomato lines with 7–9 copies of extra chromosomes, which were fully heterochromatic, were found to have normal growth and reproduction. Cytological evidence suggested that all extra chromosomes in the 24 plants investigated were derived from a single isochromosome of the short arm of chromosome 2. In spite of their common origin, the individual extra chromosomes within and between plants varied considerably with respect to size and morphology. The morphological variation of these extra chromosomes was found to result from chromosome-or chromatid breakage-fusion-bridge-cycle (BFBC). In order to verify whether BFBC could induce genomic shock and instability, the phenotypes of more than 10,000 seedlings from some of the progeny were examined. There was a 3-to 4-fold increase in the occurrence of the number of chlorophyll variegated sectors in the progeny of plants with BFBC when compared to the control variety.     

Chromosome instability in Hordeum vulgare x H. bulbosum hybrids

Chromosome number in Hordeum vulgare x H. bulbosum hybrids ranged between the haploid and diploid number but with peaks in frequency occurring at the 14 and 7 chromosome level. This was reflected in a gradual change from hybrid morphology to that of haploid H. vulgare. The rate of chromosome elimination differed significantly between hybrids, while within each hybrid, differences in mean chromosome number were recorded between and within individual tillers. An increase in temperature from 25–30° C caused a significant increase in the rate of elimination of H. bulbosum chromosomes.     

Modelling plasmid instability in batch and continuous fermentors

The probability of complete loss of plasmid material from plasmid bearing cells undergoing active growth has been modelled and incorporated into predictions for the dynamic concentrations of plasmid bearing cells in both batch and continuous flow, stirred tank bioreactors. The new model is based on an extension of the well-used model of Imanaka and Aiba [Ann. New York Acad. Sci. 369 (1981) 1] and is relatively easy to use compared to complex structured models. The new model predicts that both accelerating and decelerating rates of plasmid loss occur in both batch and continuous flow operation, and agrees well with data collected here and published earlier by others.     

Chromosome instability revealed in children of fathers irradiated during Chernobyl accident

Four children groups with and without thyroid pathology born to fathers exposed to ionizing irradiation in 1986 during Chernobyl accident as liquidators as well as residents of territory with radioactive contamination have been cytogenetically observed. The frequency and spectrum of chromosome aberrations in peripheral blood lymphocytes have been studied using two-termed cultivation (during 48 and 144 hours). Under the short-term incubation the observed groups did not significantly differ on the mean-group integral cytogenetic parameters which corresponded to age norm, but in progeny of parents from radionuclide contaminated territory the increased level of chromosome type exchanges has been revealed. In long-termed lymphocyte cultures of children with chronic thyroiditis the significantly increased cytogenetic effects of both chromatid (single fragments) and chromosome types (abnormal monocentrics, centric rings) have been established. The data received testified the reality of the transmissible chromosome instability phenomenon in progeny of irradiated parents and confirmed the possibility of its expression in consequent mitoses.     

Magnesium and neoplasia: from carcinogenesis to tumor growth and progression or treatment

Magnesium is involved in a wide range of biochemical reactions that are crucial to cell proliferation, differentiation, angiogenesis, and apoptosis. Changes in magnesium availability have been shown to influence biological responses of immuno-inflammatory cells. Equally plausible seems to be an involvement of magnesium in the multistep and interconnected processes that lead to tumor formation and development; however, the "how" and "when" of such an involvement remain to be defined. Here, we reviewed in vitro and in vivo data that indicated a role for magnesium in many biological and clinical aspects of cancer (from neoplastic transformation to tumor growth and progression or pharmacologic treatment). In adopting this approach we went through a full circle from molecular aspects to observational or epidemiological studies that could reconcile in a unifying picture the otherwise fragmentary or puzzling data currently available on the role of magnesium in cancer.     

Proteomic analysis of response to long-term continuous stress in roots of germinating soybean seeds

Germination is a complex process, highly dependent on various environmental factors, including temperature and water availability. Germinating soybean seeds are especially vulnerable to unfavorable environmental conditions and exposure to long-term abiotic stresses may result in diminishing much of the yield and most importantly – restrained germination. In the present study, a proteomic approach was employed to analyze influence of cold and osmotic stress on roots of germinated soybean (Glycine max, L.) seeds. Seeds were germinating under continuous conditions of cold stress (+10 °C/H₂O), osmotic stress (+25 °C/−0.2 MPa) as well as cold and osmotic stress combined (+10 °C/−0.2 MPa). Proteome maps established for control samples and stress-treated samples displayed 1272 CBB-stained spots. A total of 59 proteins, present in both control and stress-treated samples and showing significant differences in volume, were identified with LC/nanoESI-MS. Identified proteins divided into functional categories, revealed 9 proteins involved in plant defense, 8 proteins responsible for plant destination and storage and 10 proteins involved in various tracks of carbohydrate metabolism. Furthermore, a number of proteins were assigned to electron transport, range of metabolic pathways, secondary metabolism, protein synthesis, embryogenesis and development, signal transduction, cellular transport, translocation and storage. By analyzing differences in expression patterns, it was possible to trace the soybean response to long-term abiotic stress as well as to distinguish similarities and differences between response to cold and osmotic stress.     

Oscillatory growth movements of roots in weightlessness

The gravitropic curvature of lentil roots ( Lens culinaris L. cv. Verte du Puy), grown in near weightlessness and stimulated on a 1-g centrifuge for 5 to 60 min was followed by time lapse photography. The experiment was carried out in the frame of the IML 1 Mission of Spacelab. Due to the applied acceleration field, the tip of the roots bent and reoriented with respect to the acceleration vector. However, visual inspection of the data could indicate an oscillatory movement superimposed on the gravitropic reorientation.
We applied two signal processing techniques, fast Fourier transform (FFT) and maximum entropy spectral analysis (MESA), to provide quantitative data about the oscillatory movements of the lentil roots under gravity free conditions. In the case with very few data points in the time series the MESA method is superior to the conventional FFT. In the lentil root movements, the Fourier analysis could not extract and resolve the oscillatory signals present in the time series. The MESA approach revealed oscillations with periods around 35 and 50 min for the present lentil roots.
Circumnutations are, therefore, present in roots also in weightlessness.     

Plant roots: recycled auxin energizes patterning and growth

Recent studies show that, in plant roots, mutually dependent regulatory mechanisms operating at cell and tissue levels interact to generate a self-sustaining distribution of the hormone auxin which provides a framework for developmental patterning and growth.     

Analysis of growth components in Allium roots

Vacuoles were prepared from cultured parsley cells by polyamine-induced rupture of protoplasts. Acid-phosphatase activity, associated exclusively with the vacuoles, served for determination of vacuole yield in subsequent transport studies. Isolated vacuoles rapidly accumulated [2-¹⁴C]apigenin 7-O-(6-O-malonylglucoside) or 2-¹⁴C]-methyl D-6-O-malonylglucoside added at approximately 20 nM and 1.5 M concentration, respectively, to the incubation mixture. The accumulation was linear with time and strongly dependent on alkaline buffer conditions as well as on the age of the vacuole preparation. Subsequent addition of a malonic hemiester esterase did not relase the label from the vacuoles. Moreover, neither [2-¹⁴C]apigenin 7-O-glucoside or [2-¹⁴C]malonic acid accumulated in the vacuoles under any assay conditions, nor did such compounds or -methyl D-glucopyranoside, a malonic diester, and a succinic monoester inhibit transport of the acylated flavonoid. Transport was, however, inhibited by -methyl D-6-O-malonylglucopyranoside. Vacuoles which had been incubated for more than 40 min at pH 8.0 did not stain any more with neutral-red dye and concomitantly lost the previously accumulated acylated glucoside. Our data confirm that malonylglucoside uptake by parsley vacuoles involves selective transport sites. It is suggested that changes in the molecular symmetry of the malonylglucosides are responsible for vacuolar trapping of flavonoids in parsley.Abbreviation DEAE diethylamionethyl     

Chromosome instability in industrial strains of Saccharomyces cerevisiae batch cultivated under laboratory conditions

Industrial ethanol fermentation is a complex microbiological process to which yeast cells must adapt for survival. One of the mechanisms for adaptation is thought to involve chromosome rearrangements. We found that changes in chromosome banding patterns measured by pulsed-field gel electrophoresis can also be produced in laboratory media under simulated industrial conditions. Based on analysis of their generational variation, we found that these chromosome changes were specific to the genetic backgrounds of the initial strains. We conclude that chromosome rearrangements could be one of the factors involved in yeast cell adaptation to the industrial environment.     

Chromosome instability mutants of Saccharomyces cerevisiae that are defective in microtubule-mediated processes.

By using a multiply marked supernumerary chromosome III as an indicator, we isolated mutants of Saccharomyces cerevisiae that display increased rates of chromosome loss. In addition to mutations in the tubulin-encoding TUB genes, we found mutations in the CIN1, CIN2, and CIN4 genes. These genes have been defined independently by mutations causing benomyl supersensitivity and are distinct from other known yeast genes that affect chromosome segregation. Detailed phenotypic characterization of cin mutants revealed several other phenotypes similar to those of tub mutants. Null alleles of these genes caused cold sensitivity for viability. At 11 degrees C, cin mutants arrest at the mitosis stage of their cell cycle because of loss of most microtubule structure. cin1, cin2, and cin4 mutations also cause defects in two other microtubule-mediated processes, nuclear migration and nuclear fusion (karyogamy). Overproduction of the CIN1 gene product was found to cause the same phenotype as loss of function, supersensitivity to benomyl. Our findings suggest that the CIN1, CIN2, and CIN4 proteins contribute to microtubule stability either by regulating the activity of a yeast microtubule component or as structural components of microtubules.