Mechanisms of cell death associated with death-inducing factors from genomically unstable cell lines

We recently described a unique non-targeted effect of ionizing radiation whereby growth medium from two clones of GM10115 cells exhibiting radiation-induced chromosomal instability was cytotoxic to parental GM10115 cells. We termed this the death-inducing effect (DIE). The goal of the present study was to determine how DIE killed cells. Our hypothesis was that DIE medium contained either a secreted factor(s) from unstable clones or products from dead/dying cells that were cytotoxic to parental cells. First, we investigated the apoptotic characteristics of our unstable clones by Annexin V binding and TUNEL assays. Both the parental GM10115 cells and cells from the unstable clone LS12 had a low background (approximately 2%) level of apoptosis. The unstable Fe-10-3 clone showed a high spontaneous level of apoptosis, indicating major differences in the spontaneously occurring levels of apoptosis. We then analyzed how medium from these unstable clones killed cells by investigating the induction of DNA breaks, micronucleus formation and apoptosis induction in cells exposed to medium from unstable clones. Medium from unstable clones was capable of eliciting DNA double-strand breaks and increased apoptosis. Increased micronucleus frequencies were also observed in cells exposed to medium from either unstable clone, indicating a role of mitotis-linked cell death in DIE. These data suggest that DIE most likely results from cytotoxic factors secreted into the culture medium that can cause DNA double-strand breaks in recipient cells. These breaks can then lead to mitotis-linked cell death, as measured by micronuclei, or apoptosis, which accounts for the DIE.     

Long-lasting genomic instability following arsenite exposure in mammalian cells: the role of reactive oxygen species

Previously, we reported that the progeny of mammalian cells, which has been exposed to sodium arsenite for two cell cycles, exhibited chromosomal instability and concurrent DNA hypomethylation, when they were subsequently investigated after two months of subculturing (about 120 cell generations) in arsenite-free medium. In this work, we continued our investigations of the long-lasting arsenite-induced genomic instability by analyzing additional endpoints at several time points during the cell expanded growth. In addition to the progressive increase of aneuploid cells, we also noted micronucleated and multinucleated cells that continued to accumulate up to the 50th cell generation, as well as dicentric chromosomes and/or telomeric associations and other complex chromosome rearrangements that began to appear much later, at the 90th cell generation following arsenite exposure. The increasing genomic instability was further characterized by an increased frequency of spontaneous mutations. Furthermore, the long-lasting genomic instability was related to elevated levels of reactive oxygen species (ROS), which at the 50th cell generation appeared higher than in stable parental cells. To gain additional insight into the continuing genomic instability, we examined several individual clones isolated at different time points from the growing cell population. Chromosomally and morphologically unstable cell clones, the number of which increased with the expanded growth, were also present at early phases of growth without arsenite. All genomically unstable clones exhibited higher ROS levels than untreated cells suggesting that oxidative stress is an important factor for the progression of genomic instability induced by arsenite.     

LINE-1 hypomethylation is associated with radiation-induced genomic instability in industrial radiographers

Global DNA hypomethylation is proposed as a potential biomarker for cancer risk associated with genomic instability, which is an important factor in radiation-induced cancer. However, the associations among radiation exposure, changes in DNA methylation, and carcinogenesis are unclear. The aims of this study were (1) to examine whether low-level occupational radiation exposure induces genomic DNA hypomethylation; and (2) to determine the relationships between radiation exposure, genomic DNA hypomethylation and radiation-induced genomic instability (RIGI) in industrial radiographers. Genomic DNA methylation levels were measured in blood DNA from 40 radiographers and 28 controls using the LINE-1 pyrosequencing assay and the luminometric methylation assay. Further, the micronucleus-centromere assay was performed to measure aneuploidy of chromosomes 1 and 4 as a marker of delayed RIGI. Genomic DNA methylation levels were significantly lower in radiographers than those in controls. LINE-1 hypomethylation was not significantly correlated with recent 1-year, recent 3-year, or total cumulative radiation doses in radiographers; however, LINE-1 hypomethylation significantly correlated with the cumulative radiation dose without recent 3-year exposure data (D3dose, r = −0.39, P < 0.05). In addition, LINE-1 hypomethylation was a significant contributor to aneuploidy frequency by D3dose (F (2, 34) = 13.85, P < 0.001), in which a total of 45% of the variance in aneuploidy frequency was explained. Our results provide suggestive evidence regarding the delayed effects of low-dose occupational radiation exposure in radiographers and its association with LINE-1 hypomethylation; however, additional studies using more subjects are needed to fully understand the relationship between genomic DNA hypomethylation and RIGI. Environ. Mol. Mutagen. 60: 174–184, 2019. © 2018 Wiley Periodicals, Inc.     

TP53-dependent chromosome instability is associated with transient reductions in telomere length in immortal telomerase-positive cell lines

Telomere shortening in telomerase-negative somatic cells leads to the activation of the TP53 protein and the elimination of potentially unstable cells. We examined the effect of TP53 gene expression on both telomere metabolism and chromosome stability in immortal, telomerase-positive cell lines. Telomere length, telomerase activity, and chromosome instability were measured in multiple clones isolated from three related human B-lymphoblast cell lines that vary in TP53 expression; TK6 cells express wild-type TP53, WTK1 cells overexpress a mutant form of TP53, and NH32 cells express no TP53 protein. Clonal variations in both telomere length and chromosome stability were observed, and shorter telomeres were associated with higher levels of chromosome instability. The shortest telomeres were found in WTK1- and NH32-derived cells, and these cells had 5- to 10-fold higher levels of chromosome instability. The primary marker of instability was the presence of dicentric chromosomes. Aneuploidy and other stable chromosome alterations were also found in clones showing high levels of dicentrics. Polyploidy was found only in WTK1-derived cells. Both telomere length and chromosome instability fluctuated in the different cell populations with time in culture, presumably as unstable cells and cells with short telomeres were eliminated from the growing population. Our results suggest that transient reductions in telomere lengths may be common in immortal cell lines and that these alterations in telomere metabolism can have a profound effect on chromosome stability.     

Characteristics of chromosome instability in the human lymphoblast cell line WTK1

The characteristics of spontaneous and radiation-induced chromosome instability were determined in each of 50 individual clones isolated from control populations of human lymphoblasts (WTK1), as well as from populations of these cells previously exposed to two different types of ionizing radiation, Fe-56 and Cs-137. The types of chromosome instability did not appear to change in clones surviving radiation exposure. Aneuploidy, polyploidy, chromosome dicentrics and translocations, and chromatid breaks and gaps were found in both control and irradiated clones. The primary effect of radiation exposure was to increase the number of cells within any one clone that had chromosome alterations. Chromosome instability was associated with telomere shortening and elevated levels of apoptosis. The results suggest that the proximal cause of chromosome instability is telomere shortening.     

Differential induction and activation of NF-kappaB transcription complexes in radiation-induced chromosomally unstable cell lines

Radiation-induced genomic instability is a delayed effect of ionizing radiation that may contribute to radiation carcinogenesis. Prior microarray studies investigating gene expression changes in genomically unstable cell lines isolated after radiation exposure uncovered the differential expression of the NF-kappaB p105 mRNA. In this study, the functionality of the NF-kappaB pathway was examined to determine its role in regulating gene expression changes after oxidative stress in chromosomally stable and unstable human-hamster hybrid clones. Basal DNA-binding activity assays showed no significant differences between the clones; however, further experiments established differences in NF-kappaB induction in three chromosomally unstable clones after acute hydrogen peroxide treatment. A second assay was used to confirm this differential activity in the chromosomally unstable clones by studying reporter gene activation after treatment with hydrogen peroxide. Yet an initial upstream analysis of the pathway revealed no significant increase in the level of IkappaBalpha inhibitor protein in the unstable clones. Downstream tests analyzing the induction of the antiapoptotic target protein Bcl-2 found variable induction among the stable and unstable clones. These differences did not translate to a reduction in clonogenic survival after acute exposure to oxidative stress, as the irradiated but chromosomally stable clone displayed the most sensitivity. Due to its role in regulating a diverse set of cellular functions, including responses to oxidative stress, alterations in the NF-kappaB pathway in chromosomally unstable clones may regulate the differential physiology of a subset of chromosomally unstable clones and could contribute to the perpetuation of the phenotype. However, a specific role for defective induction and activation of this pathway remains unidentified.     

Influence of cellular sequences on instability of plasmid integration sites in human cells

To learn more about mechanisms of genome instability in human cells, I investigated DNA sequences that promote high rates of recombination by analyzing rare unstable plasmid integration sites in simian virus 40-transformed human fibroblasts. Previous studies had hypothesized that rearrangement or loss of integrated sequences could be attributed to adjacent cellular DNA. Consistent with this interpretation, a cloned fragment containing both the integrated plasmid and 2.0 kb of adjacent cell DNA from one such unstable integration site in the cell line LM205 demonstrated a much higher incidence of rearrangements when integrated into other chromosome locations than did the original plasmid. To further test this hypothesis, portions of cellular DNA from this region were integrated in duplicate in other locations to determine their ability to promote restriction-fragment-length polymorphism, an indicator of high rates of homologous recombination. Although two types of instability were observed, neither could be attributed solely to the cell sequences being tested in the plasmid. The first type of instability was a transient deletion or amplification of the plasmid DNA soon after integration, which appeared to be a general phenomenon often associated with any type of newly integrated sequence. A second type of instability continued indefinitely for many cell generations, as did that observed in cell line LM205. Because this was rare (one of 78 clones tested), it could not be attributed solely to cell sequences contained within the plasmid. However, the rearrangements in this cell clone occurred exclusively within the cell DNA adjacent to the integration site, again suggesting a role forcis-acting cell sequences in this process. The inability to identify specific cell sequences responsible for instability may therefore indicate that a complex combination of sequences is involved, possibly within both the plasmid and cell DNA.     

Loss of chromosome 14 increases the radiosensitivity of CGL1 human hybrid cells but lowers their susceptibility to radiation-induced neoplastic transformation

Loss of active tumor suppressor alleles on fibroblast chromosomes 11 and 14 are involved in radiation-induced neoplastic transformation of human hybrid CGL1 cells. Loss of either chromosome 11 or 14 alone is not sufficient for neoplastic transformation. To gain insight into the potential functions of these tumor suppressor loci, we have investigated the effects of chromosome 11 or 14 loss on radiation-induced neoplastic transformation. We recently demonstrated that loss of chromosome 11 increases the susceptibility to X-ray induced cell killing, neoplastic transformation and the expression of delayed death. The data suggested that one possible function of the chromosome 11 tumor suppressor gene may be to help maintain genome stability after radiation damage. We postulated that if the chromosome 14 allele is functioning in a similar manner, then the loss of chromosome 14 may also make the hybrid cells more susceptible to radiation-induced cell killing and neoplastic transformation. A hybrid cell line which has lost one copy of chromosome 14 was isolated and designated CON3(-14). CON3(-14) cells were more sensitive to X-ray-induced cell killing when compared with parental CGL1 cells. However, the susceptibility to radiation-induced neoplastic transformation was significantly reduced (by a factor of two) compared with the parental CGL1 cells. The expression of delayed death in the progeny of the irradiated CON3(-14) cells, growing in transformation flasks, was similar to CGL1 cells during the 21 day assay period. Taken together, the data indicate that loss of chromosome 14 alone increased the X-ray sensitivity of the hybrid cells but reduced their susceptibility to radiation-induced neoplastic transformation. These data suggest that the tumor suppressor alleles on chromosomes 11 and 14 may be functionally distinct in terms of their regulation of genomic instability and neoplastic transformation after radiation exposure.     

Stably transfected common fragile site sequences exhibit instability at ectopic sites

Common fragile sites (CFSs) are loci that are especially prone to forming gaps and breaks on metaphase chromosomes under conditions of replication stress. Although much has been learned about the cellular responses to gaps and breaks at CFSs, less is known about what makes these sites inherently unstable. CFS sequences are highly conserved in mammalian evolution and contain a number of sequence motifs that are hypothesized to contribute to their instability. To examine the role of CFS sequences in chromosome breakage, we stably transfected two BACs containing FRA3B sequences and two nonCFS control BACs containing similar sequence content into HCT116 cells and isolated cell clones with BACs integrated at ectopic sites. Integrated BACs were present at just a few to several hundred contiguous copies. Cell clones containing integrated FRA3B BACs showed a significant, three to sevenfold increase in aphidicolin-induced gaps and breaks at the integration site as compared to control BACs. Furthermore, many FRA3B integration sites displayed additional chromosome rearrangements associated with CFS instability. Clones were examined for replication timing and it was found that the integrated FRA3B sequences were not dependent on late replication for their fragility. This is the first direct evidence in human cells that introduction of CFS sequences into ectopic nonfragile loci is sufficient to recapitulate the instability found at CFSs. These data support the hypothesis that sequences at CFSs are inherently unstable, and are a major factor in the formation of replication stress induced gaps and breaks at CFSs.     

Human fibroblasts transfected with an ATM antisense vector respond abnormally to ionizing radiation.

ATM, the gene mutated in ataxia-telangiectasia (A-T), mediates multiple cellular responses to DNA damage. A-T homozygotes have a high risk of cancer and exhibit spontaneous chromosomal instability, and cultured A-T cells react abnormally to ionizing radiation. We have developed an ATM antisense vector that confers an A-T phenotype on normal cells. An episomal antisense vector was created that contained a 1.3 kb segment of the ATM cDNA, and was transfected into normal human fibroblasts. Intracellular levels of ATM protein were typically reduced 10-fold in antisense-expressing (GM639-46alpha) clones. GM639-46alpha clones exhibited the low threshold for radiation-induced apoptosis, low clonogenic survival, and cell cycle defects normally seen in A-T cells. Transfection with the corresponding ATM sense strand vector had no effect on the behavior of normal cells, and neither vector affected the behavior of A-T cells. Our results demonstrate that interference with ATM gene expression recreates the A-T phenotype in normal cells, and provide functional evidence linking the ATM gene to cellular DNA damage responses. The ATM antisense vector should prove a useful tool for studying ATM function in a variety of normal, mutant, and malignant cell lines.     

Trans-acting factors in chromosomal instability

The hypothesis that trans-acting factors affect chromosome stability was explored using human X Chinese hamster somatic cell hybrids. Two types of hybrids were examined. In either case, the human parent consisted of human diploid fibroblasts, the chromosomes of which tended to be lost from the hybrid cell. Comparisons were made between hybrid clones in which the hamster parent had a very stable karyotype (line CHO) and clones from a hamster parent with an unusual ongoing unstable karyotype (line CHX). Chinese hamster-human hybrid cell clones were expanded, and metaphase spreads were analyzed with an in situ hybridization procedure that uses biotin-labeled human genomic DNA as probe. Analyses of chromosome numbers and interspecies translocations were made after 20, 60, and 100 population doublings. Throughout the experiments, the generation of human-hamster-translocated chromosomes was more frequent in the hybrid cells with the CHX background. In addition, these cells also generated human acentric fragments, which were rare in cells with the CHO background. These results favor explanations for the instability of the CHX line that involve ongoing production of a diffusible clastogenic factor.     

Cytogenetic heterogeneity: its role in tumor evolution

There is ample evidence that cytogenetic heterogeneity characterizes human solid tumors, despite the opposing influences of clonal origin and selection for tumor-specific chromosome aberrations. Different chromosome patterns are found within individual tumors and among phenotypically similar tumors. Some tumor cell populations contain mixtures of diploid and cytogenetically aberrant cells; others display multiple aberrant clones. The extent and biological significance of chromosomal heterogeneity is contrasted between examples of leukemias and of selected solid tumors (mainly of breast and central nervous system origin). Increasing degrees of chromosomal aberration appear correlated with increasingly malignant biological properties of tumors. Genic and chromosomal instability are potential sources for genetic diversity within all tumors. However, variations in local selective forces and differential survival within an expanding solid lesion may contribute to maintenance of a mixed cell population within the primary tumor. In turn, the resulting heterogeneity may permit selection and increase of aberrant cells that are responsible for tumor progression and metastasis.     

Variable levels of chromosomal instability and mitotic spindle checkpoint defects in breast cancer

Cytogenetic analyses have revealed that many aneuploid breast cancers have cell-to-cell variations of chromosome copy numbers, suggesting that these neoplasms have instability of chromosome numbers. To directly test for possible chromosomal instability in this disease, we used fluorescent in situ hybridization to monitor copy numbers of multiple chromosomes in cultures of replicating breast cancer-derived cell lines and nonmalignant breast epithelial cells. While most (7 of 9) breast cancer cell lines tested are highly unstable with regard to chromosome copy numbers, others (2 of 9 cell lines) have a moderate level of instability that is higher than the "background" level of normal mammary epithelial cells and MCF-10A cells, but significantly less than that seen in the highly unstable breast cancer cell lines. To evaluate the potential role of a defective mitotic spindle checkpoint as a cause of this chromosomal instability, we used flow cytometry to monitor the response of cells to nocodazole-induced mitotic spindle damage. All cell lines with high levels of chromosomal instability have defective mitotic spindle checkpoints, whereas the cell lines with moderate levels of chromosomal instability (and the stable normal mammary cells and MCF10A cells) arrest in G(2) when challenged with nocodazole. Notably, the extent of mitotic spindle checkpoint deficiency and chromosome numerical instability in these cells is unrelated to the presence or absence of p53 mutations. Our results provide direct evidence for chromosomal instability in breast cancer and show that this instability occurs at variable levels among cells from different cancers, perhaps reflecting different functional classes of chromosomal instability. High levels of chromosomal instability are likely related to defective mitotic checkpoints but not to p53 mutations.     

Effect of TH-lines and clones on the growth and differentiation of B cell clones in microculture

Antibody isotype expression by B cell clones was analyzed using in vitro microcultures containing low numbers of hapten-gelatin-enriched B cells and higher numbers of hemocyanin-specific helper T cell lines or clones. Twenty-eight to sixty-three percent of clones grown in microculture with haptenated hemocyanin and T cells from established lines expressed IgG and/or IgA isotypes in random mixtures, almost always accompanied by IgM. Helper T cells from hemocyanin-specific clones also supported the expression of non-IgM isotypes by the B cell clones, suggesting that a single specificity of T cell can provide sufficient growth and differentiation factors for the display of isotype switching. A positive correlation between the antibody output of clones and the expression of non-IgM isotypes indicated that the switching process may be associated with cell division. Although memory B cells that give clones expressing IgG and/or IgA in the absence of IgM are also enriched on haptenated gelatin, they are not stimulable under conditions of this microculture assay.     

Functional analysis of lung tumor suppressor activity at 3p21.3

The early and frequent occurrence of deletions at 3p21.3 in lung cancer has led to the consideration of this chromosomal region as a lung cancer (LUCA) critical region with tumor suppressor activity. We covered this 19 genes-containing region with overlapping P1 artificial chromosomes (PACs), in which genes are likely accompanied by their own promoters or other regulatory sequences. With these PACs we transfected cells from a small cell lung cancer (SCLC) cell line which readily caused tumors in nude mice. Per PAC we selected two cell clones with a low number of PAC copies integrated at a single genomic site. The selected clones were s.c. injected into nude mice to investigate whether the integrated genes suppressed the tumor-inducing capacity of the original SCLC cell line. We could demonstrate PAC-specific gene expression in the transfected cells. All of the PAC integration sites were different. It appeared that introduction of a PAC or even an empty PAC vector causes some chromosomal instability, which in principle may either promote or inhibit cell growth. However, both cell clones with integration of the same PAC from the centromeric part of the LUCA region in different genomic sites were the sole pair of clones that caused smaller tumors than did the original SCLC cell line. This suggests that rather than the induced chromosomal instability, the DNA sequence of that PAC, which in addition to two protein-encoding genes contains at least one potential miRNA gene, is responsible for the tumor suppressor activity.     

Establishment of stable human T-T hybridomas

Human T-T hybridomas potentially provide an invaluable resource for a variety of immunoregulatory molecules that modulate the immune response. To date, success in this technology, using human cell populations, has been hampered by several problems associated with proliferative and functional instability of the hybrid cells. These forms of instability are the result of a multifactorial process, with 1 parameter of importance being the chromosome number of the malignant parent cell line used for fusion. The present studies describe the production of a stable human T-T hybridoma generated by fusing a near diploid (modal chromosome number of 48) aminopterin-sensitive T cell line, CEM TG E11, and lectin-stimulated human peripheral blood lymphocytes. The rapidly growing hybrid cells have been clonally selected for the production of a B cell growth factor. Hybridization was documented by the presence of HLA phenotypes reflecting the combined antigens of the fusion partners. Fusions with 4 other partners besides CEM TG E11, where the majority of the cells had modal chromosome numbers ranging from 78 to 94, were proliferatively unstable. To date, hybrid cells derived from the CEM TG E11 fusion have been doubling approximately every 48 h for greater than 12 months, and selected clones constitutively produce B cell growth factor.     

Antigenic heterogeneity of a human melanoma tumor detected by autologous CTL clones

Peripheral blood lymphocytes from a melanoma patient were stimulated with autologous melanoma cells in mixed lymphocyte tumor cultures (MLTC). After three restimulations, the lytic activity of the responder cells directed against the autologous melanoma cells was higher than that against K-562 and autologous Epstein-Barr virus-transformed B cell line (EBV-B) cells. From these MLTC-responder cells, we derived specific cytolytic T cell (CTL) clones that lysed the autologous melanoma cells and did not lyse K-562 or autologous EBV-B cells. Autologous melanoma clones were found that were resistant to some or all of these CTL clones. The autologous CTL clones recognized at least two different antigens (A, B) on the melanoma cells and three types of melanoma clones could be distinguished (A+B+, A+B-, A-B-). This antigenic heterogeneity of melanoma clones was confirmed by testing the CTL clones in cold target competition and also in antigen-dependent CTL proliferation assays performed with very small numbers of stimulator cells. The data further indicated an instability of the expression of a melanoma-associated antigen in the course of a long culture period. Among the melanoma clones that expressed antigen A, one was found to stimulate the proliferation of anti-A CTL clones much more effectively than the others. This represents a new type of heterogeneity among tumor cells which may be of significance for the elicitation of an autologous anti-tumoral immune response.     

Abnormal nuclear shape in solid tumors reflects mitotic instability

Abnormalities in nuclear morphology are frequently observed in malignant tissues but the mechanisms behind these phenomena are still poorly understood. In this study, the relation between abnormal nuclear shape and chromosomal instability was explored in short-term tumor cell cultures. Mitotically unstable ring and dicentric chromosomes were identified by fluorescence in situ hybridization at metaphase and subsequently localized in interphase nuclei from five malignant soft tissue tumors. The vast majority (71 to 86%) of nuclear blebs, chromatin strings, and micronuclei contained material from the unstable chromosomes, whereas few (<11%) were positive for stable chromosomes. Nuclear morphology was also evaluated in fibroblasts and an osteosarcoma cell line exposed to irradiation. A linear correlation was found between the frequency of abnormalities in nuclear shape, on one hand, and cells with unstable chromosomes (r = 0.87) and anaphase bridge configurations (r = 0.98), on the other hand. The relation between nuclear shape and karyotypic pattern was investigated further in cultures from 58 tumors of bone, soft tissue, and epithelium. Blebs, strings, and micronuclei were significantly more frequent in tumors that contained rings, dicentrics, or telomeric associations than in those exhibiting only stable aberrations (P: < 0.001) and a positive correlation (r = 0.78) was found between the frequency of such nuclear abnormalities and the intratumor heterogeneity of structural chromosome aberrations. These results indicate that the formation of nuclear blebs, chromatin strings, and micronuclei in malignant tissues is closely related to the breakage-fusion-bridge type of mitotic disturbances. Abnormalities in nuclear shape may thus primarily be regarded as an indicator of genetic instability and intratumor heterogeneity, independent of cytogenetic complexity and the grade of malignancy.     

Deficiency in DNA mismatch repair increases the rate of telomere shortening in normal human cells

DNA mismatch repair (MMR) is essential for genome stability and inheritance of a mutated MMR gene, most frequently MSH2 or MLH1, results in cancer predisposition known as Lynch syndrome or hereditary nonpolyposis colorectal cancer (HNPCC). Tumors that arise through MMR deficiency show instability at simple tandem repeat loci (STRs) throughout the genome, known as microsatellite instability (MSI). The STR instability is dominated by errors that accumulate during replication in the absence of effective MMR. In this study we show that there is a high level of instability within telomeric DNA with a tendency toward deletions in tumor-derived MMR defective cell lines. We downregulated MSH2 expression in a normal fibroblast cell line and isolated four clones, with differing levels of MSH2 depletion. The telomere-shortening rate was measured at the Xp/Yp, 12q, and 17p telomeres in the MSH2 depleted and three control clones. Interestingly the mean telomere-shortening rate in the clones with MSH2 depletion was significantly greater than in the control clones. This is the first demonstration that MSH2 deficiency alone can lead to accelerated telomere shortening in normal human cells.