Cis-acting transmission of genomic instability

Genomic instability is a highly pleiotropic phenotype, which may reflect a variety of underlying mechanisms. Destabilization has been shown in some cases to involve mutational alteration or inactivation of trans-acting cellular factors, for example, p53 or mismatch repair functions. However, aspects of instability are not well explained by mutational inactivation of trans-acting factors, and other epigenetic and cis-acting mechanisms have recently been proposed. The trans and cis models result in divergent predictions for the distribution of instability-associated genetic alterations within the genome, and for the inheritance of genomic instability among sibling sub-clones of unstable parents. These predictions have been tested in this study primarily by tracking the karyotypic distribution of chromosomal rearrangements in clones and sub-clones exhibiting radiation-induced genomic instability; inheritance of mutator phenotypes was also analyzed. The results indicate that genomic instability is unevenly transmitted to sibling sub-clones, that chromosomal rearrangements within unstable clones are non-randomly distributed throughout the karyotype, and that the majority of chromosomal rearrangements associated with instability affect trisomic chromosomal segments. Observations of instability in trisomic regions suggests that in addition to promoting further alterations in chromosomal number, aneuploidy can affect the recovery of structural rearrangements. In summary, these findings cannot be fully explained by invoking a homogeneously distributed factor acting in trans, but do provide support for previous suggestions that genomic instability may in part be driven by a cis-acting mechanism.     

Chromosomal Evolution in the Genus Brachyscome (Asteraceae, Astereae): Statistical Tests Regarding Correlation Between Changes in Karyotype and Habit Using Phylogenetic Information

Brachyscome and 8 taxa of its allied genera, Australian Astereae. Statistical tests regarding correlations between changes in chromosome number, total chromosome length, mean chromosome length, karyotypic asymmetry and chromosome length heterogeneity and changes in habit were performed based on the matK molecular phylogenetic tree. The reductions in chromosome number and total chromosome length, and the increases in mean chromosome length, chromosome length heterogeneity and karyotypic asymmetry were found to be correlated with the change in habit from perennial to annual. A reduction in total chromosome length is favored to shorten the mitotic cell cycle and to produce smaller cells conducive to more rapid development of smaller annuals under the time-limited environment. Stepwise dysploidal reductions in chromosome number were achieved through the translocation of large chromosome segments onto other chromosomes, followed by the loss of a centromere, resulting in one fewer linkage group and one fewer haploid chromosome. The correlations between the dysploidal reduction in chromosome number and the increases in mean chromosome length, length heterogeneity and asymmetry in karyotype can be attributed to this mode of chromosomal change. These changes occurred independently in several different lineages in Brachyscome. Received 27 May 1998/ Accepted in revised form 18 January 1999     

Association of MAD2 expression with mitotic checkpoint competence in hepatoma cells

Chromosomal instability (CIN) refers to high rates of chromosomal gains and losses and is a major cause of genomic instability of cells. It is thought that CIN caused by loss of mitotic checkpoint contributes to carcinogenesis. In this study, we evaluated the competence of mitotic checkpoint in hepatoma cells and investigated the cause of mitotic checkpoint defects. We found that 6 (54.5%) of the 11 hepatoma cell lines were defective in mitotic checkpoint control as monitored by mitotic indices and flow-cytometric analysis after treatment with microtubule toxins. Interestingly, all 6 hepatoma cell lines with defective mitotic checkpoint showed significant underexpression of mitotic arrest deficient 2 (MAD2), a key mitotic checkpoint protein. The level of MAD2 underexpression was significantly associated with defective mitotic checkpoint response (p<0.001). In addition, no mutations were found in the coding sequences of MAD2 in all 11 hepatoma cell lines. Our findings suggest that MAD2 deficiency may cause a mitotic checkpoint defect in hepatoma cells.     

Stochastic cancer progression driven by non-clonal chromosome aberrations

Cancer research has previously focused on the identification of specific genes and pathways responsible for cancer initiation and progression based on the prevailing viewpoint that cancer is caused by a stepwise accumulation of genetic aberrations. This viewpoint, however, is not consistent with the clinical finding that tumors display high levels of genetic heterogeneity and distinctive karyotypes. We show that chromosomal instability primarily generates stochastic karyotypic changes leading to the random progression of cancer. This was accomplished by tracing karyotypic patterns of individual cells that contained either defective genes responsible for genome integrity or were challenged by onco-proteins or carcinogens that destabilized the genome. Analysis included the tracing of patterns of karyotypic evolution during different stages of cellular immortalization. This study revealed that non-clonal chromosomal aberrations (NCCAs) (both aneuploidy and structural aberrations) and not recurrent clonal chromosomal aberrations (CCAs) are directly linked to genomic instability and karyotypic evolution. Discovery of "transitional CCAs" during in vitro immortalization clearly demonstrates that karyotypic evolution in solid tumors is not a continuous process. NCCAs and their dynamic interplay with CCAs create infinite genomic combinations leading to clonal diversity necessary for cancer cell evolution. The karyotypic chaos observed within the cell crisis stage prior to establishment of the immortalization further supports the ultimate importance of genetic aberrations at the karyotypic or genome level. Therefore, genomic instability generated NCCAs are a key driving force in cancer progression. The dynamic relationship between NCCAs and CCAs provides a mechanism underlying chromosomal based cancer evolution and could have broad clinical applications.     

Molecular cytogenetic analysis of Haemulidae fish (Perciformes): Evidence of evolutionary conservation

Several fish species belonging to the family Haemulidae present a karyotype consisting of 48 acrocentric chromosomes (FN = 48), and apparently similar chromosomal microstructure, especially in genus Haemulon, representing a striking example of intrafamiliar chromosomal conservation. In this study, a more detailed cytogenetic analysis of the species Conodon nobilis and Pomadasys corvinaeformis was performed using C-banding, Ag-NOR, DAPI/CMA₃ staining, in situ digestion by distinct endonucleases and double-FISH to map the 18S and 5S ribosomal genes. Both species showed a similar karyotypic macrostructure with 2n = 48 acrocentric chromosomes and active ribosomal sites at interstitial position on long arms of chromosomal pair 18 and 24 in P. corvinaeformis and C. nobilis, respectively. These sites were the only CMA₃⁺/DAPI^-regions in the karyotype. Digestion with restriction enzymes revealed a low number of digestion sites in the heterochromatic segments of both species. The data indicate some degree of interspecific evolutionary diversification At the microstructural level, incorporated in a general pattern of extensive karyotypic conservatism. Thus, the interspecific reproductive isolation leading to phyletic diversification apparently occurred without the contribution of conspicuous karyotypic changes.     

Clonal analysis of delayed karyotypic abnormalities and gene mutations in radiation-induced genetic instability.

Many tumors exhibit extensive chromosomal instability, but karyotypic alterations will be significant in carcinogenesis only by influencing specific oncogenes or tumor suppressor loci within the affected chromosomal segments. In this investigation, the specificity of chromosomal rearrangements attributable to radiation-induced genomic instability is detailed, and a qualitative and quantitative correspondence with mutagenesis is demonstrated. Chromosomal abnormalities preferentially occurred near the site of prior rearrangements, resulting in complex abnormalities, or near the centromere, resulting in deletion or translocation of the entire chromosome arm, but no case of an interstitial chromosomal deletion was observed. Evidence for chromosomal instability in the progeny of irradiated cells also included clonal karyotypic heterogeneity. The persistence of instability was demonstrated for at least 80 generations by elevated mutation rates at the heterozygous, autosomal marker locus tk. Among those TK- mutants that showed a loss of heterozygosity, a statistically significant increase in mutation rate was observed only for those in which the loss of heterozygosity encompasses the telomeric region. This mutational specificity corresponds with the prevalence of terminal deletions, additions, and translocations, and the absence of interstitial deletions, in karyotypic analysis. Surprisingly, the elevated rate of TK- mutations is also partially attributable to intragenic base substitutions and small deletions, and DNA sequence analysis of some of these mutations is presented. Complex chromosomal abnormalities appear to be the most significant indicators of a high rate of persistent genetic instability which correlates with increased rates of both intragenic and chromosomal-scale mutations at tk.     

CENP-A overexpression promotes aneuploidy with karyotypic heterogeneity

Chromosomal instability (CIN) is a hallmark of many cancers. Restricting the localization of centromeric histone H3 variant CENP-A to centromeres prevents CIN. CENP-A overexpression (OE) and mislocalization have been observed in cancers and correlate with poor prognosis; however, the molecular consequences of CENP-A OE on CIN and aneuploidy have not been defined. Here, we show that CENP-A OE leads to its mislocalization and CIN with lagging chromosomes and micronuclei in pseudodiploid DLD1 cells and xenograft mouse model. CIN is due to reduced localization of proteins to the kinetochore, resulting in defects in kinetochore integrity and unstable kinetochore–microtubule attachments. CENP-A OE contributes to reduced expression of cell adhesion genes and higher invasion of DLD1 cells. We show that CENP-A OE contributes to aneuploidy with karyotypic heterogeneity in human cells and xenograft mouse model. In summary, our results provide a molecular link between CENP-A OE and aneuploidy, and suggest that karyotypic heterogeneity may contribute to the aggressive phenotype of CENP-A–overexpressing cancers.     

Quantitative analysis of Akt phosphorylation and activity in response to EGF and insulin treatment

The protein kinase Akt is a critical regulator of cell function and its overexpression and activation have been functionally linked to numerous pathologies such as cancer. Previous reports regarding the mechanism-regulating Akt's activation have revealed two phosphorylation events, at threonine 308 (T308) and serine 473 (S473), as necessary for the full activation of the kinase in response to insulin. For this reason and because of the availability of phospho-specific antibodies to both T308 and S473, many studies that focus on Akt's role in governing cell function rely on the measurement of these two sites to understand changes in kinase activity. Recent evidence, however, suggests the involvement of other phosphorylation sites; for example, in Src-transformed and epidermal growth factor (EGF)-treated cells, tyrosine phosphorylation has been found important for full kinase activation. In this study, we probed the quantitative reliability of using S473 and/or T308 phosphorylation as surrogates for Akt kinase activity across diverse treatment conditions. We performed quantitative Western blots and kinase activity assays on lysates generated during a 2h time course from two cell lines treated with either EGF or insulin. From the resulting approximately 250 quantitative measurements of phosphorylation and activity, we found that both T308 and S473 phosphorylation accurately captured quantitative changes in EGF-stimulated cells, but not in insulin-stimulated cells. Moreover, in all but one condition studied, we found a tight correlation between the onset of phosphorylation and dephosphorylation for both sites, despite the fact that they do not share common kinase- or phosphatase-mediated regulation. In sum, using a quantitative approach to study Akt activation identified ligand-dependent limits for the use of T308 or S473 as proxies for kinase activity and suggests the coregulation of Akt phosphorylation and dephosphorylation.     

Chronic oxidative DNA damage due to DNA repair defects causes chromosomal instability in Saccharomyces cerevisiae

Oxidative DNA damage is likely to be involved in the etiology of cancer and is thought to accelerate tumorigenesis via increased mutation rates. However, the majority of malignant cells acquire a specific type of genomic instability characterized by large-scale genomic rearrangements, referred to as chromosomal instability (CIN). The molecular mechanisms underlying CIN are not entirely understood. We utilized Saccharomyces cerevisiae as a model system to delineate the relationship between genotoxic stress and CIN. It was found that elevated levels of chronic, unrepaired oxidative DNA damage caused chromosomal aberrations at remarkably high frequencies under both selective and nonselective growth conditions. In this system, exceeding the cellular capacity to appropriately manage oxidative DNA damage resulted in a “gain-of-CIN” phenotype and led to profound karyotypic instability. These results illustrate a novel mechanism for genome destabilization that is likely to be relevant to human carcinogenesis.     

Non-random distribution of instability-associated chromosomal rearrangement breakpoints in human lymphoblastoid cells

Genomic instability is observed in tumors and in a large fraction of the progeny surviving irradiation. One of the best-characterized phenotypic manifestations of genomic instability is delayed chromosome aberrations. Our working hypothesis for the current study was that if genomic instability is in part attributable to cis mechanisms, we should observe a non-random distribution of chromosomes or sites involved in instability-associated rearrangements, regardless of radiation quality, dose, or trans factor expression. We report here the karyotypic examination of 296 instability-associated chromosomal rearrangement breaksites (IACRB) from 118 unstable TK6 human B lymphoblast, and isogenic derivative, clones. When we tested whether IACRB were distributed across the chromosomes based on target size, a significant non-random distribution was evident (p < 0.00001), and three IACRB hotspots (chromosomes 11, 12, and 22) and one IACRB coldspot (chromosome 2) were identified. Statistical analysis at the chromosomal band-level identified four IACRB hotspots accounting for 20% of all instability-associated breaks, two of which account for over 14% of all IACRB. Further, analysis of independent clones provided evidence within 14 individual clones of IACRB clustering at the chromosomal band level, suggesting a predisposition for further breaks after an initial break at some chromosomal bands. All of these events, independently, or when taken together, were highly unlikely to have occurred by chance (p < 0.000001). These IACRB band-level cluster hotspots were observed independent of radiation quality, dose, or cellular p53 status. The non-random distribution of instability-associated chromosomal rearrangements described here significantly differs from the distribution that was observed in a first-division post-irradiation metaphase analysis (p = 0.0004). Taken together, these results suggest that genomic instability may be in part driven by chromosomal cis mechanisms.     

Detection of chromosomal instability in alpha-irradiated and bystander human fibroblasts

There is increasing evidence biological responses to ionizing radiation are not confined to those cells that are directly hit, but may be seen in the progeny at subsequent generations (genomic instability) and in non-irradiated neighbors of irradiated cells (bystander effects). These so called non-targeted phenomena would have significant contributions to radiation-induced carcinogenesis, especially at low doses where only a limited number of cells in a population are directed hit. Here we present data using a co-culturing protocol examining chromosomal instability in alpha-irradiated and bystander human fibroblasts BJ1-htert. At the first cell division following exposure to 0.1 and 1Gy alpha-particles, irradiated populations demonstrated a dose dependent increase in chromosome-type aberrations. At this time bystander BJ1-htert populations demonstrated elevated chromatid-type aberrations when compared to controls. Irradiated and bystander populations were also analyzed for chromosomal aberrations as a function of time post-irradiation. When considered over 25 doublings, all irradiated and bystander populations had significantly higher frequencies of chromatid aberrations when compared to controls (2-3-fold over controls) and were not dependent on dose. The results presented here support the link between the radiation-induced phenomena of genomic instability and the bystander effect.     

Targeting Cancer Cells by Exploiting Karyotypic Complexity and Chromosomal Instability

Multiple karyotypic abnormalities and chromosomal instability are particular hallmarks of many cancers that are relatively resistant to long term control by current chemotherapeutic agents. We have asked whether these same hallmarks, karyotypic complexity and instability, can be used as determinants for the screening of potential anticancer compounds. Using a panel of well characterized cancer cell lines we have been able to identify specific groups of chemical compounds that are more cytotoxic toward the relatively more karyotypically complex and unstable panel members. Thus, we delineate an approach for the identification of “lead compounds” for anticancer drug discovery complementary to approaches that are focused at the outset on a given gene or pathway.     

Functional heterogeneity of non-small lung adenocarcinoma cell sub-populations

The morphological and functional heterogeneity of solid tumour cells can be observed in cancer cell lines cultured in vitro. We have combined analyses of microclones developed from single cells with micropore transmigration assays to demonstrate the co-existence of cellular subsets differing in morphology and motile activity, as well as Cx43 (connexin 43) and N-cadherin expression within lung carcinoma A549 populations. 'Fibroblastoid' cells, characterized by high motility, polarized morphology and plasmalemmal localization of Cx43, displayed the strongest aptitude for transmigration through narrow obstacles. Due to high mitotic activity, they maintain the whole population but can also give rise to a sub-population of quiescent and immobile 'epithelioid' cells. Our observations indicate that phenotypic transitions between the fibroblastoid and epithelioid phenotype account for the heterogeneity of metastable A549 cell populations.     

Chromosomal instability and cellular hypersensitivity to X-radiation of cultured fibroblasts derived from porokeratosis patients'' skin

Porokeratosis is a rare genetic skin disorder known to be associated with a propensity to develop skin cancer. To further elucidate the previously reported cytogenetic and cellular abnormalities, we studied karyotypic changes and the sensitivity to X-ray irradiation of cultured fibroblasts derived from skin lesions and normal-appearing skin of 3 patients with porokeratosis. Cultured fibroblasts from normal-appearing skin of 9 controls were similarly examined. Porokeratosis subjects had a greater number of cells with chromosomal abnormalities than controls. Two porokeratosis strains which were derived from the normal-appearing skin of a patient had a noticeable clone of abnormal cells. Porokeratosis fibroblasts were hypersensitive to the lethal effects of X-radiation. This hypersensitivity was common to both the lesion-derived strains and the ones derived from normal-appearing skin. The 2 strains with clonal abnormal cells were also similarly hypersensitive to X-radiation. These results suggest that chromosomal instability is strongly related to porokeratosis and that X-ray hypersensitivity is an inherent abnormality in cultured fibroblasts of porokeratosis patients.     

Effect of immobilized laminin on karyotypic variability in two karyotypically different variants of the indian muntjac skin fibroblast cell line

The numerical and structural karyotypic variability has been investigated in MTs of the markerless cell line of Indian muntjac skin fibroblasts, as well as in its karyotypic variant MT^D cultivated on a laminin 2/4 coated surface. In the MT cell line preincubated in serum-free medium for 2.5 and 1.0 h, then cultivated on a laminin-coated surface in serum-containing medium for one, two, and three days, the character of cell distribution for the chromosome number has changed. These changes involve a significant decrease in the frequency of cells with modal numbers of chromosomes and an increase in frequency of cells with lower chromosome numbers. Some new additional structural variants of the karyotype (SVK) appeared. The observed alterations seem to be due to disturbances of the chromosome segregation and the establishment of a new advantageous balanced karyotypic structure. In the karyotypic variant MT^D differing from MT by an increased number of dicentrics (telomeric associations) cultivated under the same conditions, the character of cell distribution for the chromosome number did not change. In the MT cell line, the frequency of chromosomal aberrations did not change relative to control variants. In the karyotypic variant MT^D under the same conditions, the frequency of chromosomal aberrations significantly increased after three days mainly due to the formation of dicentrics. These results confirm the conclusion that, like aneuploidy, the formation of dicentrics in markerless cell lines appears to be the way in which the cell population adapts to unfavorable environmental factors. Possible reasons for differences in the character of the numerical and structural karyotypic variability between the MT cell line and its karyotypic variant MT^D are discussed.     

High yields of isochromatid breaks and successive formation of chromosome exchanges may lead to reproductive cell death following high-LET irradiation

To clarify the relationship between cell death and chromosomal aberrations following exposure to heavy-charged ion particles beams, exponentially growing Human Salivary Gland Tumor cells (HSG cells) were irradiated with various kinds of high energy heavy ions; 13 keV/μm carbon ions as a low-LET charged particle radiation source, 120 keV/μm carbon ions and 440 keV/μm iron ions as high-LET charged particle radiation sources. X-rays (200 kVp) were used as a reference. Reproductive cell death was evaluated by clonogenic assays, and the chromatid aberrations in G2/M phase and their repairing kinetics were analyzed by the calyculin A induced premature chromosome condensation (PCC) method. High-LET heavy-ion beams introduced much more severe and un-repairable chromatid breaks and isochromatid breaks in HSG cells than low-LET irradiation. In addition, the continuous increase of exchange aberrations after irradiation occurred in the high-LET irradiated cells. The cell death, initial production of isochromatid breaks and subsequent formation of chromosome exchange seemed to be depend similarly on LET with a maximum RBE peak around 100–200 keV/μm of LET value. Conversely, un-rejoined isochromatid breaks or chromatid breaks/gaps seemed to be less effective in reproductive cell death. These results suggest that the continuous yield of chromosome exchange aberrations induced by high-LET ionizing particles is a possible reason for the high RBE for cell death following high-LET irradiation, alongside other chromosomal aberrations additively or synergistically.     

Cytological characterization of transgenic soybean

 Some of the transgenic soybean [Glycine max (L.) Merr.] plants produced by bombarding embryogenic suspension cultures with DNA-coated particles exhibit morphological aberrations, including stunted plant growth, leathery dark green leaves and partialto-total seed sterility. In general, cultures from two Asgrow soybean lines (A2242, A2872) that were maintained for 8 months or longer produced primary transformants with reduced fertility. Cytological examination (mitotic pro-metaphase to metaphase chromosomes) of cells of suspension cultures, of roots from germinating somatic embryos, and of plants (R₀ and R₁) derived from A2242, revealed, besides diploidy (2n=40), various chromosomal aberrations such as deletions, duplications, trisomics and tetraploidy. Diploid transgenic plants with a normal karyotype from A2242 generally exhibited good fertility. No chromosomal abnormalities were observed in A2872-derived plants. However, plants regenerated from relatively old cultures of A2872 (more than 1 year in culture) showed a range of phenotypic abnormalities although they all contained 2n=40 chromosomes. These results indicate that soybean genotypes differ in their susceptibility to chromosomal instability induced by tissue culture. Therefore, chromosome analysis of cell cultures and the plants derived from them can help eliminate chromosomally and genetically abnormal material from gene-transfer experiments. Received: 6 June 1997/Accepted: 9 October 1997