Centrosome and microtubule instability in aging Drosophila cells.

Several cytoskeletal changes are associated with aging which includes alterations in muscle structure leading to muscular atrophy, and weakening of the microtubule network which affects cellular secretion and maintenance of cell shape. Weakening of the microtubule network during meiosis in aging oocytes can result in aneuploidy or trisomic zygotes with increasing maternal age. Imbalances of cytoskeletal organization can lead to disease such as Alzheimer's, muscular disorders, and cancer. Because many cytoskeletal diseases are related to age we investigated the effects of aging on microtubule organization in cell cultures of the Drosophila cell model system (Schneider S-1 and Kc23 cell lines). This cell model is increasingly being used as an alternative system to mammalian cell cultures. Drosophila cells are amenable to genetic manipulations and can be used to identify and manipulate genes which are involved in the aging processes. Immunofluorescence, scanning, and transmission electron microscopy were employed for the analysis of microtubule organizing centers (centrosomes) and microtubules at various times after subculturing cells in fresh medium. Our results reveal that centrosomes and the microtubule network becomes significantly affected in aging cells after 5 days of subculture. At 5-14 days of subculture, 1% abnormal out of 3% mitoses were noted which were clearly distinguishable from freshly subcultured control cells in which 3% of cells undergo normal mitosis with bipolar configurations. Microtubules are also affected in the midbody during cell division. The midbody in aging cells becomes up to 10 times longer when compared with midbodies in freshly subcultured cells. During interphase, microtubules are often disrupted and disorganized, which may indicate improper function related to transport of cell organelles along microtubules. These results are likely to help explain some cytoskeletal disorders and diseases related to aging.     

Apoptosis and genomic instability

Genomic instability is intrinsically linked to significant alterations in apoptosis control. Chromosomal and microsatellite instability can cause the inactivation of pro-apoptotic pathways. In addition, the inhibition of apoptosis itself can be permissive for the survival and ongoing division of cells that have failed to repair DNA double-strand breaks, experience telomere dysfunction or are in an abnormal polyploid state. Furthermore, DNA-repair proteins can regulate apoptosis. So, genomic instability and apoptosis are intimately linked phenomena, with important implications for the pathophysiology of cancer.     

Centrosome overduplication and mitotic instability in PKD2 transgenic lines

Polycystin-2 (PC-2), a protein encoded by PKD2 and involved in autosomal dominant polycystic kidney disease (ADPKD), is a non-selective cationic channel recently implicated in the function of primary cilia. We recently constructed a new animal model in the form of a transgenic mouse with a BAC-containing human PKD2 inserted in its genome. Two transgenic mouse lines overexpressing human PKD2 showed mitotic instability. Fibroblasts from these transgenic mouse lines have abnormal chromosomal numbers. These lines also have supernumerary centrosomes. PC-2 overexpression is associated with mitotic instability and centrosome overduplication. PC-2 therefore seems to play a role in centrosome duplication, and this hypothesis is being evaluated in other models.     

The consequences of structural genomic alterations in humans: genomic disorders, genomic instability and cancer

Over the last decade or so, sophisticated technological advances in array-based genomics have firmly established the contribution of structural alterations in the human genome to a variety of complex developmental disorders, and also to diseases such as cancer. In fact, multiple 'novel' disorders have been identified as a direct consequence of these advances. Our understanding of the molecular events leading to the generation of these structural alterations is also expanding. Many of the models proposed to explain these complex rearrangements involve DNA breakage and the coordinated action of DNA replication, repair and recombination machinery. Here, and within the context of Genomic Disorders, we will briefly overview the principal models currently invoked to explain these chromosomal rearrangements, including Non-Allelic Homologous Recombination (NAHR), Fork Stalling Template Switching (FoSTeS), Microhomology Mediated Break-Induced Repair (MMBIR) and Breakage-fusion-bridge cycle (BFB). We will also discuss an unanticipated consequence of certain copy number variations (CNVs) whereby the CNVs potentially compromise fundamental processes controlling genomic stability including DNA replication and the DNA damage response. We will illustrate these using specific examples including Genomic Disorders (DiGeorge/Veleocardiofacial syndrome, HSA21 segmental aneuploidy and rec (3) syndrome) and cell-based model systems. Finally, we will review some of the recent exciting developments surrounding specific CNVs and their contribution to cancer development as well as the latest model for cancer genome rearrangement; 'chromothripsis'.     

Matrix metalloproteinase-induced genomic instability

Increased expression of matrix metalloproteinases (MMPs) is associated with nearly every tumor type. Although many studies have shown that MMPs can promote malignancy, recent evidence has revealed that MMPs can play a causative role also in the earliest stages of cancer development. A complex story is now emerging in which MMPs not only compromise cell-cell and cell-substratum adhesion processes that impact genomic surveillance mechanisms but also act directly on molecules at the cell surface to stimulate physiological processes that cause genetic alterations. Delineating the mechanisms involved in these processes and identifying how they are coordinated in vivo could aid identification of the crucial contribution of MMPs to tumorigenesis.     

The death-inducing effect and genomic instability

Exposure to ionizing radiation can induce a heritable change in the unirradiated progeny of irradiated cells. This non-targeted effect of ionizing radiation manifests as genomic instability, and although there is some debate as to the role of genomic instability in the carcinogenic process, it is thought by some to be an early step in radiation carcinogenesis. Although the mechanism of induction of genomic instability is not clearly understood, evidence suggests that secreted factors from irradiated cells may be involved. We have previously identified another non-targeted effect of ionizing radiation, the death-inducing effect. Exposure of unirradiated GM10115 cells to medium from chromosomally unstable clones was generally found to be cytotoxic. However, occasionally cells will survive in medium from unstable clones and can be clonally expanded. The absolute yield of survivors is independent of the initial number of cells plated when cell densities reached 5,000 or more cells/dish. After cytogenetic analysis of the surviving colonies, we found chromosomal instability in three of 40 clones analyzed, while some clones exhibited increased micronucleus frequency and HPRT mutation frequency. These data suggest that our chromosomally unstable GM10115 cells secrete factors that are cytotoxic to the majority of stable, parental cells but are also capable of inducing a heritable change in some of the survivors that can manifest as delayed genomic instability. These results suggest a mechanism whereby instability can be perpetuated through the influences of potentially cytotoxic factors produced by genomically unstable clones.     

Telomere higher-order structure and genomic instability

Telomeres, nucleoprotein complexes at the end of eukaryotic chromosomes, have vital roles in chromosome integrity. Telomere chromatin structure is both intricate and dynamic allowing for a variety of responses to several stimuli. A critical determinant in telomere structure is the G-strand overhang. Facilitated by telomeric proteins, the G-strand overhang stabilizes telomere higher-order assemblies most likely by adopting unusual DNA structures. These structures influence activities that occur at the chromosome end. Dysfunctional telomeres induce signals resulting in cell growth arrest or death. To overcome telomere dysfunction, cancer cells activate the DNA polymerase, telomerase. The presence of telomerase at the telomere may establish a particular telomeric state. If the chromosome ends of cancer and normal cells exist in different states, cancer-specific telomere structures would offer a unique chemotherapeutic target.     

Cancer models,genomic instability and somatic cellular Darwinian evolution

   

The biology of cancer is critically reviewed and evidence adduced that its development can be modelled as a somatic cellular Darwinian evolutionary process. The evidence for involvement of genomic instability (GI) is also reviewed. A variety of quasi-mechanistic models of carcinogenesis are reviewed, all based on this somatic Darwinian evolutionary hypothesis; in particular, the multi-stage model of Armitage and Doll (Br. J. Cancer 1954:8;1-12), the two-mutation model of Moolgavkar, Venzon, and Knudson (MVK) (Math. Biosci. 1979:47;55-77), the generalized MVK model of Little (Biometrics 1995:51;1278-1291) and various generalizations of these incorporating effects of GI (Little and Wright Math. Biosci. 2003:183;111-134; Little et al. J. Theoret. Biol. 2008:254;229-238).     

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.     

Zebrafish genomic instability mutants and cancer susceptibility

Somatic loss of tumor suppressor gene function comprising the second hit of Knudson's two-hit hypothesis is important in human cancer. A genetic screen was performed in zebrafish (Danio rerio) to find mutations that cause genomic instability (gin), as scored by Streisinger's mosaic-eye assay that models this second hit. The assay, based on a visible test for loss of wild-type gene function at a single locus, golden, is representative of genomewide events. Twelve ENU-induced genomic instability (gin) mutations were isolated. Most mutations showed weak dominance in heterozygotes and all showed a stronger phenotype in homozygotes. Trans-heterozygosity for 7 of these mutations showed greatly enhanced instability. A variety of spontaneous tumors were found in heterozygous adults from all gin lines, consistent with the expectation that genomic instability (mutator) mutations can accelerate carcinogenesis. The incidence of spontaneous cancer at 30-34 months was increased 9.6-fold in heterozygotes for the mutant with the strongest phenotype, gin-10. Tumors were seen in skin, colon, kidney, liver, pancreas, ovary, testis, and neuronal tissues, with multiple tumors in some fish. The study of these mutants will add to our understanding of the mechanisms of somatic loss of gene function and how those mechanisms contribute to cancer susceptibility.     

Strong association between cancer and genomic instability

After a first wave of radiation-induced chromosomal aberrations, a second wave appears 20–30 cell generations after radiation exposure and persists thereafter. This late effect is usually termed “genomic instability”. A better term is “increased genomic instability”. This effect has been observed in many cell systems in vitro and in vivo for quite a number of biological endpoints. The radiation-induced increase in genomic instability is apparently a general phenomenon. In the development of cancer, several mutations are involved. With increasing genomic instability, the probability for further mutations is enhanced. Several studies show that genomic instability is increased not only in the cancer cells but also in “normal” cells of cancer patients e.g. peripheral lymphocytes. This has for example been shown in uranium miners with bronchial carcinomas, but also in untreated head and neck cancer patients. The association between cancer and genomic instability is also found in individuals with a genetic predisposition for increased radiosensitivity. Several such syndromes have been found. In all cases, an increased genomic instability, cancer proneness and increased radiosensitivity coincide. In these syndromes, deficiencies in certain DNA-repair pathways occur as well as deregulations of the cell cycle. Especially, mutations are seen in genes encoding proteins, which are involved in the G₁/S-phase checkpoint. Genomic instability apparently promotes cancer development. In this context, it is interesting that hypoxia, increased genomic instability and cancer are also associated. All these processes are energy dependent. Some strong evidence exists that the structure and length of telomeres is connected to the development of genomic instability.     

R-loop-dependent replication and genomic instability in bacteria

DNA replication, the faithful copying of genetic material, must be tightly regulated to produce daughter cells with intact copies of the chromosome(s). This regulated replication is initiated by binding of specific proteins at replication origins, such as DnaA to oriC in bacteria. However, unregulated replication can sometimes be initiated at other sites, which can threaten genomic stability. One of the first systems of unregulated replication to be described is the one activated in Escherichia coli mutants lacking RNase HI (rnhA). In fact, rnhA mutants can replicate their chromosomes in a DnaA- and oriC-independent process. Because this replication occurs in cells lacking RNase HI, it is proposed that RNA from R-loops is used as a DNA polymerase primer. Replication from R-loops has recently attracted increased attention due to the advent of DNA:RNA hybrid immunoprecipitation coupled with high-throughput DNA sequencing that revealed the high prevalence of R-loop formation in many organisms, and the demonstration that R-loops can severely threaten genomic stability. Although R-loops have been linked to genomic instability mostly via replication stress, evidence of their toxic effects via unregulated replication has also been presented. Replication from R-loops may also beneficially trigger stress-induced mutagenesis (SIM) that assists bacterial adaptation to stress. Here, we describe the cis- and trans-acting elements involved in R-loop-dependent replication in bacteria, with an emphasis on new data obtained with type 1A topoisomerase mutants and new available technologies. Furthermore, we discuss about the mechanism(s) by which R-loops can reshape the genome with both negative and positive outcomes.     

Interrelationships amongst radiation-induced genomic instability, bystander effects, and the adaptive response

Over the past two decades, our understanding of radiation biology has undergone a fundamental shift in paradigms away from deterministic "hit-effect" relationships and towards complex ongoing "cellular responses". These responses include now familiar, but still poorly understood, phenomena associated with radiation exposure such as bystander effects, genomic instability, and adaptive responses. All three have been observed at very low doses, and at time points far removed from the initial radiation exposure, and are extremely relevant for linear extrapolation to low doses; the adaptive response is particularly relevant when exposure is spread over a period of time. These are precisely the circumstances that are most relevant to understanding cancer risk associated with environmental and occupational radiation exposures. This review will provide a synthesis of the known, and proposed, interrelationships amongst low-dose cellular responses to radiation. It also will examine the potential importance of non-targeted cellular responses to ionizing radiation in setting acceptable exposure limits especially to low-LET radiations.