Transgenerational carcinogenesis: induction and transmission of genetic alterations and mechanisms of carcinogenesis

Parental exposure, i.e. germ cell exposure to radiation and chemicals, increased the incidence of tumors and malformations in the offspring, and the germ-line alterations that cause cancer are transmissible to further generations. However, tumor incidences were 100-fold higher than those of ordinary mouse mutations and there were apparent strain differences in the types of induced tumors. In human, higher risk of leukemia is reported in the children of fathers who had been exposed to radionuclides at the nuclear reprocessing plants or to diagnostic doses of radiation. However, these findings in mice and men have not been confirmed in the children of atomic bomb survivors in Hiroshima and Nagasaki. Another important finding was that germ-line exposure was very weakly tumorigenic by itself. However, the transmissible alterations caused persistent hypersensitivity to tumor induction in the offspring, e.g. enhanced by postnatal treatment with tumor promoting/carcinogenic agents. The above results suggest that transmissible alterations might be imprinted in germ cells for the future development of cancer by the postnatal environment. Many gene loci concerning immunological, biochemical and physiological function might be involved, and the cumulative changes in such genes may slightly elevate or enhance tumor incidences, although mutations of tumor suppressor genes such as p53 were also detected in some offspring and genomic instability may modify tumor occurrence in transgenerational manner. In fact, Gene Chip analysis showed suppression and/or over-expression of many functional genes rather than cancer-related genes in the preconceptionally irradiated cancer prone progeny.     

Genomic instability in human lymphocytes irradiated with individual charged particles: involvement of tumor necrosis factor alpha in irradiated cells but not bystander cells

Exposure to ionizing radiation can increase the risk of cancer, which is often characterized by genomic instability. In environmental exposures to high-LET radiation (e.g. 222Ra), it is unlikely that many cells will be traversed or that any cell will be traversed by more than one alpha particle, resulting in an in vivo bystander situation, potentially involving inflammation. Here primary human lymphocytes were irradiated with precise numbers of 3He2+ ions delivered to defined cell population fractions, to as low as a single cell being traversed, resembling in vivo conditions. Also, we assessed the contribution to genomic instability of the pro-inflammatory cytokine tumor necrosis factor alpha (TNFA). Genomic instability was significantly elevated in irradiated groups (> or = two-fold over controls) and was comparable whether cells were traversed by one or two 3He2+ ions. Interestingly, substantial heterogeneity in genomic instability between experiments was observed when only one cell was traversed. Genomic instability was significantly reduced (60%) in cultures in which all cells were irradiated in the presence of TNFA antibody, but not when fractions were irradiated under the same conditions, suggesting that TNFA may have a role in the initiation of genomic instability in irradiated cells but not bystander cells. These results have implications for low-dose exposure risks and cancer.     

Nontargeted effects of ionizing radiation: implications for low-dose exposures

The traditional thinking has been that the biological effects of ionizing radiation occur in irradiated cells as a consequence of the DNA damage they incur. This implies that: 1) biological effects occur only in irratiated cells, 2) radiation traversal through the nucleus of the cell is a prerequisite to produce a biological response, and 3) DNA is the target molecule in the cell. Evidence has been emerging, however, for non-DNA targeted effects of radiation; that is, effects including mutations, chromosomal aberrations, and changes in gene expression which occur in cells that in themselves receive no radiation exposure. Two of these phenomena will be described in this paper. The first is radiation-induced genomic instability whereby biological effects, including elevated frequencies of mutations and chromosomal aberrations, arise in the distant descendants of irradiated cells. The second phenomenon has been termed the "bystander effect", whereby in a mixed population of irradiated and nonirradiated cells, biological effects arise in those cells that receive no radiation exposure. The damage signals are transmitted from cell to cell through gap junction channels, and the genetic effects observed in bystander cells appear to result from an upregulation of oxidative stress. The possible influence of these non-targeted effects of radiation of the respounse to low-dose exposures is discussed.     

Role of induced genetic instability in the mutagenic effects of chemicals and radiation

Recent studies have demonstrated that cells exposed to ionizing radiation or alkylating agents can develop prolonged genetic instability. Induced genetic instability is manisested in multiple ways, including delayed reproductive death, an increased rate of point mutations, and an increased rate of chromosome rearrangements. In many respects these changes are similar to the genetic instability associated with cancer and some human genetic diseases. Therefore, as with cancer cells, multiple mechanisms may be involved, some occuring in the early stages and some in the later stages. The high percentage of cells that develop induced genetic instability after exposure to stress, and the prolonged period over which the instability occurs, indicates that the instability is not in response to residual damage in the DNA or mutations in specific genes. Instead, changes affecting most of the exposed cells, such as epigenetic alterations in gene expression or chain reactions of chromosome rearrangements, are a more likely explanation. Learning more about the mechanisms involved in this process is essential for understanding the consequences of exposure of cells to ionizing radiation or alkylating agents.     

Damaging and protective cell signalling in the untargeted effects of ionizing radiation

The major adverse consequences of radiation exposures are attributed to DNA damage in irradiated cells that has not been correctly restored by metabolic repair processes. However, the dogma that genetic alterations are restricted to directly irradiated cells has been challenged by observations in which effects of ionizing radiation arise in non-irradiated cells. These, so called, untargeted effects are demonstrated in cells that are the descendants of irradiated cells either directly or via media transfer (radiation-induced genomic instability) or in cells that have communicated with irradiated cells (radiation-induced bystander effects). Radiation-induced genomic instability is characterized by a number of delayed responses including chromosomal abnormalities, gene mutations and cell death. Bystander effects include increases or decreases in damage-inducible and stress-related proteins, increases or decreases in reactive oxygen and nitrogen species, cell death or cell proliferation, cell differentiation, radioadaptation, induction of mutations and chromosome aberrations and chromosomal instability. The phenotypic expression of untargeted effects and the potential consequences of these effects in tissues reflect a balance between the type of bystander signals produced and the responses of cell populations to such signals, both of which may be significantly influenced by cell type and genotype. Thus, in addition to targeted effects of damage induced directly in cells by irradiation, a variety of untargeted effects may also make important short-term and long-term contributions to determining overall outcome after radiation exposures.     

Increased sensitivity of subtelomeric regions to DNA double-strand breaks in a human cancer cell line

We previously reported that a single DNA double-strand break (DSB) near a telomere in mouse embryonic stem cells can result in chromosome instability. We have observed this same type of instability as a result of spontaneous telomere loss in human tumor cell lines, suggesting that a deficiency in the repair of DSBs near telomeres has a role in chromosome instability in human cancer. We have now investigated the frequency of the chromosome instability resulting from DSBs near telomeres in the EJ-30 human bladder carcinoma cell line to determine whether subtelomeric regions are sensitive to DSBs, as previously reported in yeast. These studies involved determining the frequency of large deletions, chromosome rearrangements, and chromosome instability resulting from I-SceI endonuclease-induced DSBs at interstitial and telomeric sites. As an internal control, we also analyzed the frequency of small deletions, which have been shown to be the most common type of mutation resulting from I-SceI-induced DSBs at interstitial sites. The results demonstrate that although the frequency of small deletions is similar at interstitial and telomeric DSBs, the frequency of large deletions and chromosome rearrangements is much greater at telomeric DSBs. DSB-induced chromosome rearrangements at telomeric sites also resulted in prolonged periods of chromosome instability. Telomeric regions in mammalian cells are therefore highly sensitive to DSBs, suggesting that spontaneous or ionizing radiation-induced DSBs at these locations may be responsible for many of the chromosome rearrangements that are associated with human cancer.     

Genetic instability and darwinian selection in tumours

Genetic instability has long been hypothesized to be a cardinal feature of cancer. Recent work has strengthened the proposal that mutational alterations conferring instability occur early during tumour formation. The ensuing genetic instability drives tumour progression by generating mutations in oncogenes and tumour-suppressor genes. These mutant genes provide cancer cells with a selective growth advantage, thereby leading to the clonal outgrowth of a tumour. Here, we discuss the role of genetic instability in tumour formation and outline future work necessary to substantiate the genetic instability hypothesis.     

Genetic instability and darwinian selection in tumours

Genetic instability has long been hypothesized to be a cardinal feature of cancer. Recent work has strengthened the proposal that mutational alterations conferring instability occur early during tumour formation. The ensuing genetic instability drives tumour progression by generating mutations in oncogenes and tumour-suppressor genes. These mutant genes provide cancer cells with a selective growth advantage, thereby leading to the clonal outgrowth of a tumour. Here, we discuss the role of genetic instability in tumour formation and outline future work necessary to substantiate the genetic instability hypothesis.     

Genetic instability and darwinian selection in tumours

Genetic instability has long been hypothesized to be a cardinal feature of cancer. Recent work has strengthened the proposal that mutational alterations conferring instability occur early during tumour formation. The ensuing genetic instability drives tumour progression by generating mutations in oncogenes and tumour-suppressor genes. These mutant genes provide cancer cells with a selective growth advantage, thereby leading to the clonal outgrowth of a tumour. Here, we discuss the role of genetic instability in tumour formation and outline future work necessary to substantiate the genetic instability hypothesis.     

Untargeted effects of ionizing radiation: implications for radiation pathology

The dogma that genetic alterations are restricted to directly irradiated cells has been challenged by observations in which effects of ionizing radiation, characteristically associated with the consequences of energy deposition in the cell nucleus, arise in non-irradiated cells. These, so called, untargeted effects are demonstrated in cells that have received damaging signals produced by irradiated cells (radiation-induced bystander effects) or that are the descendants of irradiated cells (radiation-induced genomic instability). Radiation-induced genomic instability is characterized by a number of delayed adverse responses including chromosomal abnormalities, gene mutations and cell death. Similar effects, as well as responses that may be regarded as protective, have been attributed to bystander mechanisms. Whilst the majority of studies to date have used in vitro systems, some adverse non-targeted effects have been demonstrated in vivo. However, at least for haemopoietic tissues, radiation-induced genomic instability in vivo may not necessarily be a reflection of genomically unstable cells. Rather the damage may reflect responses to ongoing production of damaging signals; i.e. bystander responses, but not in the sense used to describe the rapidly induced effects resulting from direct interaction of irradiated and non-irradiated cells. The findings are consistent with a delayed and long-lived tissue reaction to radiation injury characteristic of an inflammatory response with the potential for persisting bystander-mediated damage. An important implication of the findings is that contrary to conventional radiobiological dogma and interpretation of epidemiologically-based risk estimates, ionizing radiation may contribute to malignancy and particularly childhood leukaemia by promoting initiated cells rather than being the initiating agent. Untargeted mechanisms may also contribute to other pathological consequences.     

Progressive alteration of telomeric sequences at one end of a yeast linear plasmid and its possible association with reduced plasmid stability

After selection for migration into the nucleus, a cytoplasmic yeast linear plasmid bearing an inverted terminal repeat (ITRs) at each end replicates in Saccharomyces cerevisiae in a linear form, called pTLU, which carries host telomeric repeats (TG(1-3))(n) of about 300-350 bp added to the ITR ends. We previously showed that the nucleotide composition of the added telomeric sequences varied among individual pTLU isolates, while those on the two ends of any given pTLU were always identical. The telomeric sequences of pTLU remained unchanged over numbers of cell generations when cells were selected for expression of the plasmid-borne nuclear marker. We report here that progressive alterations in telomeric sequences can be detected in cells which are grown under non-selective conditions. Surprisingly, in any given molecule, the telomeric alterations occur exclusively on one side, either the left or the right end, while the sequence at the opposite end remained identical to the original, suggesting a difference in the mode of DNA replication between the plasmid ends. These alterations occur over a broad area extending from the termini of telomeres to nucleotides near the junction between the telomeric sequences and the pTLU-ITR, implying that the plasmid ends undergo successive rounds of extension and contraction. Clonal analysis under non-selective conditions indicated that the alterations in telomeric sequences are generally associated with extreme instability of the pTLU plasmid.     

Are chromosomal instabilities induced by exposure of cultured normal human cells to low- or high-LET radiation?

Radiation-induced genomic instability has been proposed as a very early, if not an initiating, step in radiation carcinogenesis. Numerous studies have established the occurrence of radiation-induced chromosomal instability in various cells of both human and rodent origin. In many of these studies, however, the cells were not "normal" initially, and in many cases they involved tumor-derived cell lines. The phenomenon clearly would be of even greater interest if it were shown to occur generally in cells that are normal at the outset, rather than cells that may have been "selected" because of a pre-existing susceptibility to induced instability. As a test of the generality of the phenomenon, we studied low-passage normal diploid human fibroblasts (AG1521A) to determine whether they are susceptible to the induction of chromosomal instability in the progeny of surviving cells after exposure in G(0) to low- and high-LET radiation. Cytogenetic assays for instability were performed on both mixed populations of cells and clones of cells surviving exposure. We found no evidence for the induction of such instability as a result of radiation exposure, though we observed a senescence-related chromosomal instability in the progeny of both irradiated and unirradiated cell populations.     

Transmission of genomic instability from a single irradiated human chromosome to the progeny of unirradiated cells

Ionizing radiation can induce chromosome instability that is transmitted over many generations after irradiation in the progeny of surviving cells, but it remains unclear why this instability can be transmitted to the progeny. To acquire knowledge about the transmissible nature of genomic instability, we transferred an irradiated human chromosome into unirradiated mouse recipient cells by microcell fusion and examined the stability of the transferred human chromosome in the microcell hybrids. The transferred chromosome was stable in all six microcell hybrids in which an unirradiated human chromosome had been introduced. In contrast, the transferred chromosome was unstable in four out of five microcell hybrids in which an irradiated human chromosome had been introduced. The aberrations included changes in the irradiated chromosome itself and rearrangements with recipient mouse chromosomes. Thus the present study demonstrates that genomic instability can be transmitted to the progeny of unirradiated cells by a chromosome exposed to ionizing radiation, implying that the instability is caused by the irradiated chromosome itself and also that the instability is induced by the nontargeted effect of radiation.     

A multistage theory of age-specific acceleration in human mortality

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.     

Telomeres, crisis and cancer

Eukaryotic chromosomes terminate in specialized nucleic acid-protein complexes known as telomeres. Disruption of telomere structure by erosion of telomeric DNA or loss of telomere binding protein function activates a signal transduction program that closely resembles the cellular responses generated upon DNA damage. Telomere dysfunction in turn induces a permanent proliferation arrest known as senescence. Senescence is postulated to perform a tumor suppressor function by limiting cellular proliferative capacity, thus imposing a barrier to cellular immortalization. Genetic or epigenetic silencing of components of the DNA damage pathway, allows cells to proliferate beyond senescence limits. However, these cells eventually reach a stage of extreme telomere dysfunction known as crisis that is characterized by cell death and the concomitant appearance of cytogenetic abnormalities. Telomeric crisis produces significant chromosomal instability, a hallmark of human cancer, and may thus be relevant to carcinogenesis by increasing the occurrence of genetic alterations that would favor neoplastic transformation. The following review examines the relationship of telomere function during crisis in accelerating chromosomal instability and cancer.     

Transgenerational transmission of radiation induced genomic instability

Stability of genome is one of the evolutionary important trait of cells. Various mutations (gene, chromosomal, genomic) as well as artificial manipulations with genomes (inbreeding, DNA transfection, introduction of Br-DU in DNA) cause the genetic instability. Ionizing radiation is known as the factor which induced instability of genome in late mitotic descendants of cells after in vitro and in vivo exposure. Radiation induced genetic instability can be transmitted through germline cells. On the cell level both types of radiation induced genomic instability are manifested in elevated frequency of mutations, chromosome aberrations, micronuclei, increased radiosensitivity, disappearance of adaptive response, changes in gene expression. In studies of 1970-1980 years clear evidences on the different morphological and functional injuries in tissues of irradiated organisms as well as in tissues of the progeny of exposed parents were obtained. On the organism level the instability of mitotic and of meiotic progeny of irradiated cells is resulted in increased risk of cancer and of other somatic diseases. It seems to be useful to review the earlier radiobiology literature where delayed and transgenerational effects of ionizing radiation on tissues and on organisms level were clearly shown in animals. For the estimation of pathogenic role of radiation induced genomic instability in humans, particularly in children of exposed parents the parallel study of the same human cohorts using clinical parameters and various characteristic of genomic instability seems to be very important.     

Bystander-mediated genomic instability after high LET radiation in murine primary haemopoietic stem cells

Communication between irradiated and unirradiated (bystander) cells can result in responses in unirradiated cells that are similar to responses in their irradiated counterparts. The purpose of the current experiment was to test the hypothesis that bystander responses will be similarly induced in primary murine stem cells under different cell culture conditions. The experimental systems used here, co-culture and media transfer, are similar in that they both restrict communication between irradiated and bystander cells to media borne factors, but are distinct in that with the media transfer technique, cells can only communicate after irradiation, and with co-culture, cells can communication before, during and after irradiation. In this set of parallel experiments, cell type, biological endpoint, and radiation quality and dose, were kept constant. In both experimental systems, clonogenic survival was significantly decreased in all groups, whether irradiated or bystander, suggesting a substantial contribution of bystander effects (BE) to cell killing. Genomic instability (GI) was induced under all radiation and bystander conditions in both experiments, including a situation where unirradiated cells were incubated with media that had been conditioned for 24h with irradiated cells. The appearance of delayed aberrations (genomic instability) 10-13 population doublings after irradiation was similar to the level of initial chromosomal damage, suggesting that the bystander factor is able to induce chromosomal alterations soon after irradiation. Whether these early alterations are related to those observed at later timepoints remains unknown. These results suggest that genomic instability may be significantly induced in a bystander cell population whether or not cells communicate during irradiation.     

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.     

The radiation bystander effect and its potential implications for human health

A long-held dogma in radiation biology has been that the biological effects of exposure to ionizing radiation occur as a result of damage in directly irradiated cells and that no effect would occur in neighboring unirradiated cells. This paradigm has been frequently challenged by reports of radiation effects in unirradiated or 'bystander' cells receiving signals from directly irradiated cells, an issue that may have substantial impact on radiation risk assessment and development of radiation-based therapies. Radiation-induced bystander effects have been shown in single-cell systems in vitro for an array of cancer relevant endpoints, and may trigger damage in more complex 3-D tissue systems. They may be mediated by soluble factors released by irradiated cells into the extracellular environment and/or by the passage of mediator molecules through gap-junction intercellular communication. To date, evidence that radiation-associated bystander or abscopal responses are effectual in vivo has been limited, but new data suggest that they may significantly affect tumor development in susceptible mouse models. Further understanding of how the signal/s is transmitted to unirradiated cells and tissues and how it provokes long-range and significant responses is crucial. By summarizing the existing evidence of radiation induced bystander-like effects in various systems with emphasis on in vivo findings, we will discuss the potential mechanisms involved in these observations and how effects in bystander cells contribute to uncertainties in assessing cancer risks associated with radiation exposure.