Accumulation Of DNA Mutations Research Articles

Evolution of iPSC disease models

Human induced pluripotent stem cells (iPSCs) hold unprecedented potential to model human development and genetic disorders due to their capability to self-renew and differentiate into any somatic cell type. In the year of 2011, a number of iPSC disease models have been successfully developed for studying the mechanism of human diseases as well as establishing platforms for drug screening and testing (Batista et al., 2011; Brennand et al., 2011; Devine et al., 2011; Itzhaki et al., 2011; Koch et al., 2011; Liu et al., 2011a; Mazzulli et al., 2011; Nguyen et al., 2011; Pasca et al., 2011; Quarto et al., 2011; Tiscornia et al., 2011; Wu and Hochedlinger, 2011; Yazawa et al., 2011; Zhang et al., 2011; Zhu et al., 2011). One key hypothesis of current iPSC disease models is based on the presumption that wild type and diseased iPSCs are equal to their embryonic stem cell (ESC) counterparts. However, this hypothesis seems to be challenged by several recent findings on the striking differences between ESCs and iPSCs. At the genomic level, the reprogramming process tends to cause the accumulation of DNA mutations as well as other chromosomal abnormalities related to cancer pathways (Panopoulos et al., 2011). Whereas there is no relevant in vivo data indicating that these mutations are indeed linked to tumorigenesis, the genetic aberrances accumulated during reprogramming probably interfere with the cellular parameters in either iPSCs or their differentiated derivatives. If specific diseases, especially those associated with genomic instability, facilitate the accumulation of more genetic mutations during reprogramming, this could result in wrong explanation of the phenotypes of iPSC disease models. In fact, the evaluation on how aspects of specific diseases affect reprogramming-associated mutations has not been reported. Additionally, at the epigenomic level, the residual epigenetic memories of iPSCs from their original cellular environment likely represent another barrier to being a true phenocopy of their ESC counterparts. In support of this, mouse iPSCs, but not ESCs, show immune rejection upon transplantation (Zhao et al., 2011), suggesting that subtle epigenetic differences might cause substantially different cell identities. To date, it is not clear whether disease-associated epigenetic memories exist in patient-specific iPSCs. Only a few groups examined the successful resetting of abnormal epigenetic marks during reprogramming, such as histone and DNA methylation, in their iPSC disease models (Marchetto et al., 2010; Liu et al., 2011a). At the differentiation level, a number of reports have shown that iPSCs behave differently from their ESC counterparts (Buchholz et al., 2009; Bock et al., 2011; Kim et al., 2011), although researchers still don’t know how this difference affects recapitulation of disease phenotypes in vitro. It should also be noted that certain diseased somatic cellular environments might contribute to the defective reprogramming with a higher possibility. For example, cellular defects in Fanconi anemia patient fibroblasts resulted in a complete blockage of iPSC generation (Raya et al., 2009). Hence, to avoid misinterpretation of results, it seems essential to first evaluate whether generated patient iPSCs are completely reset to a patient ESC-like status. Along this line, thorough examination of various cellular parameters in patient-specific iPSCs could be a critical step before employment of any iPSC disease model in mechanistic studies or drug testing. As complementary approaches, the relevant assays with overexpression of a mutant (e.g. for dominant mutation) or knock-down of an endogenous protein (e.g. for recessive mutation) in disease-related cell types should be included to verify the specific aspects of disease phenotypes. Furthermore, the lack of appropriate control iPSC lines constructs another important experimental limitation for the use of patient-derived iPSCs. In fact, the “wild type” or “healthy” iPSC lines currently used as controls are derived from “phenotypically normal” populations, which nevertheless carry various genetic and epigenetic polymorphisms. The major concern with respect to these epigenetic and genetic variations is that they may cause inconsistent phenotypic

TRAPping telomerase within the intestinal stem cell niche

Adult intestinal stem cells (ISCs) reside at the crypt base, where they continuously proliferate to maintain homeostasis of the intestinal epithelium. As a result of a lifetime of cell division, these LGR5+ ISCs face the combined risk of genomic mutations and telomere attrition. In this issue of EMBOJ , [Schepers et al (2011)][1] examine whether ISCs employ telomerase expression and asymmetric chromosome segregation to protect their genome. [1]: #ref-9

No Longitudinal Mitochondrial DNA Sequence Changes in HIV-infected Individuals With and Without Lipoatrophy

The potential for mitochondrial (mt) DNA mutation accumulation during antiretroviral therapy (ART), and preferential accumulation in patients with lipoatrophy compared with control participants, remains controversial. We sequenced the entire mitochondrial genome, both before ART and after ART exposure, in 29 human immunodeficiency virus (HIV)-infected Swiss HIV Cohort Study participants initiating a first-line thymidine analogue-containing ART regimen. No accumulation of mtDNA mutations or deletions was detected in 13 participants who developed lipoatrophy or in 16 control participants after significant and comparable ART exposure (median duration, 3.3 and 3.7 years, respectively). In HIV-infected persons, the development of lipoatrophy is unlikely to be associated with accumulation of mtDNA mutations detectable in peripheral blood.

An age-dependent feedback control model of calcium dynamics in yeast cells

The functional decline of selected proteins or organelles leads to aging at the intracellular level. Identification of these proteins or organelles is usually challenging to traditional single-factor approaches since these factors are inter-connected via feedback or feedforward controls. Establishing a feedback control model to simulate the interactions of multiple factors is an insightful approach to guide the search for proteins involved in aging. However, there are only a few mathematical models describing the age-dependent accumulation of DNA mutations, which are directly or indirectly induced by deterioration of the intracellular environment including alteration of calcium homeostasis, a contributor of aging. Thus, based on Cui and Kaandorp's model, we develop an age-dependent mathematical model for the calcium homeostasis in budding yeast Saccharomyces cerevisiae. Our model contains cell cycle-dependent aging factors and can qualitatively reproduce calcium shocks and calcium accumulations in cells observed in experiments. Using this model, we predict calcium oscillations in wild type, pmc1 Delta, and pmr1 Delta cells. This prediction suggests that Pmr1p plays a major role in regulating cytosolic calcium. Combining the model with our experimental lifespan data, we predict an upper-limit of cytosolic calcium tolerance for cell survival. This prediction indicates that, for aged cells (>35 generations), no pmr1 Delta can tolerate the cytosolic calcium concentration of 0.1 microM while a very small fraction (1%) of aged wild type cells (>50 generations) can tolerate a high cytosolic calcium concentration of 0.5 microM.

Selective mtDNA mutation accumulation results in β-cell apoptosis and diabetes development

To test the hypothesis that somatic mitochondrial (mt)DNA mutation accumulation predisposes mice to beta-cell loss and diabetes development, transgenic mice expressing a proofreading-deficient mtDNA polymerase-gamma under the control of the rat insulin-1 promoter were generated. At 6 wk of age, mtDNA mutations reached 0.01% (1.05 mutations/10,000 bp) in islets isolated from transgenic mice. This mutational burden is associated with impaired glucose tolerance and a diabetes prevalence of 52% in male transgenic mice. Female transgenic mice maintain slightly elevated fasting glucose levels, mild glucose intolerance, and a diabetes prevalence of 14%. Diabetes in transgenic animals is associated with insulin insufficiency that results from a significant reduction in beta-cell mass. Importantly, apoptosis of beta-cells is increased 7-fold in female and 11-fold in male transgenic mice compared with littermate controls. These results are consistent with a causative role of somatic mtDNA mutation accumulation in the loss of beta-cell mass and diabetes development.

RAD51 and XRCC3 polymorphism frequency and risk of myelodysplastic syndromes

To the Editor: Single nucleotide polymorphisms in DNA repair genes are known to influence cancer risk. One common SNP in the 5'UTR of the RAD51 gene, “135G>C” (rs1801320), increases promoter activity, thus altering gene transcription and protein expression. Overexpression of RAD51 impairs initiation of HR.(1,2) Likewise, a C>T alteration in exon 8 of the XRCC3 gene (rs861539) changes the amino acid sequence (p.T241M, NP_005423). Cells with the XRCC3 T241M allele have nuclear abnormalities and low rates of spontaneous apoptosis, possibly promoting their malignant transformation, but the specific mechanism by with the SNP causes this behavior is unclear.(3,4) Myelodysplastic syndromes (MDS) are characterized by ineffective hematopoesis, recurrent clonal chromosomal abnormalities, and a predilection to progress to acute myeloid leukemia (AML).(5) Disease progression along the MDS-AML continuum is believed to be a consequence of stepwise accumulation of DNA mutations which infers a defect in DNA repair.(6) Seedhouse and colleagues showed that patients possessing both the RAD51 135G>C or XRCC3 T241M variants are at a 3.77-fold increase in risk of developing AML compared to controls.(7) Because 20-30% of patients with MDS develop AML, similar underlying pathophysiologic mechanisms may be present. We hypothesized that the presence of RAD51 135G>C or XRCC3 T241M would impair DSB repair and lead to genetic instability, consequently increasing the risk of developing MDS and the likelihood of having a karyotypic abnormality. We analyzed genomic DNA extracted from blood mononuclear cells from 292 consenting patients diagnosed with MDS using FAB criteria; 90 well-characterized DNA samples obtained from Coriell Institute panel NDPT024 (Camden, NJ) served as controls. Average age of the MDS patients was 70.9 versus 66.9 years in the control group. The MDS patients were 69.9% men versus 50% men in the controls. MDS subtypes are as noted in Table 1. Allele frequencies for RAD51 135G>C and XRCC3 T241M in all disease and controls were compared using the χ 2 test or Fisher's exact test as appropriate. Univariate logistic regression models were constructed to assess the relationship between each polymorphism and specific disease subtypes or IPSS stratification(8). The models were adjusted for age, gender, and

Diabetes Mellitus as a Risk Factor for Cancer: Stress or Viral Etiology?

Diabetes mellitus (DM) is a metabolic disorder. Currently over 230 million patients demonstrate already an epidemic scale of the disease. It is a lifelong progressive disease with a high mortality worldwide: every 10 seconds one patient dies on DM-related consequences. Whereas cardio-vascular complications are well-known for DM, it is relatively new consideration that diabetic patients are highly predisposed to cancer. Particularities of molecular pathomechanisms of cancer in diabetes are currently largely unclear. Disturbed glucose/insulin homeostasis is DM-specific stress factor resulting in increased production of Reactive Oxygen Species (ROS) and oxidative damage to chromosomal and mitochondrial DNA frequently observed in diabetic patients. Long-term accumulation of DNA mutations is well-acknowledged as triggering cancer. DNA-repair is highly energy consuming process which provokes increased mitochondrial activity. Particularly dangerous is a provoked activity of damaged mitochondria which leads to the "vicious circle" lowing energy supply and potentiating ROS production. Mitochondrial dysfunction is the well-acknowledged risk factor for neuro/degenerative diseases--one of possible pathomechanisms for various complications developed secondary to diabetes. At the same time, mitochondrial dysfunction might be implicated in pathomechanisms of diabetes-provoked cancer. There is a growing body of evidence that DM predisposes to almost all cancer types with some particular preferences. Frequently suffering from compromised immune response, diabetic patients is high-risk group for infectious disorders including viral infections. In its turn, viral infections are known to be implicated in cancer pathology. This review considers both stress and viral infections as possible etiology of cancer in diabetes.

The continuing search for cancer-causing somatic mutations

It is known that cancer is caused by an accumulation of mutations in DNA. Many genes have been associated with tumour progression either through germline or somatic mutations, but mutations in these genes by no means account for all instances of the disease. The availability of the completed human genome sequence and reduced costs of sequencing have allowed large-scale screens to uncover genes that are somatically mutated in cancer. In this issue, Chanock and colleagues present a screen of 91 breast cancers for somatic variants in a set of 21 genes.

Syndromes with genetic instability: Model diseases for (skin) cancerogenesis

Tumorigenesis is a multi-step process in which exogenous and endogenous mutational events lead to accumulation of DNA mutations resulting in loss of cellular growth control. During the last decades knowledge about the molecular mechanisms of DNA mutations and the cellular systems that counteract the degeneration of the genome has increased rapidly. This expansion was driven in part by clinical and molecular investigations of syndromes with genetic in-stability. These syndromes are typically associated with an increased predisposition for tumor formation. They include nucleotide excision repair defective syndromes, mismatch repair defective syndromes, and the chromosome break-age syndromes. These diseases mirror vividly the phenotypic consequences of the failure of important cellular systems for the maintenance of the genomic integrity. They significantly contribute to our understanding of the molecular mechanisms of (skin) carcinogenesis and for the development of preventive and therapeutic anticancer strategies.

Syndrome mit genetischer Instabilität: Modellerkrankungen für die (Haut‐) Kanzerogenese

Tumorigenesis is a multi-step process in which exogenous and endogenous mutational events lead to accumulation of DNA mutations resulting in loss of cellular growth control. During the last decades knowledge about the molecular mechanisms of DNA mutations and the cellular systems that counteract the degeneration of the genome has increased rapidly. This expansion was driven in part by clinical and molecular investigations of syndromes with genetic instability. These syndromes are typically associated with an increased predisposition for tumor formation.They include nucleotide excision repair defective syndromes, mismatch repair defective syndromes, and the chromosome breakage syndromes. These diseases mirror vividly the phenotypic consequences of the failure of important cellular systems for the maintenance of the genomic integrity. They significantly contribute to our understanding of the molecular mechanisms of (skin) carcinogenesis and for the development of preventive and therapeutic anticancer strategies.

Genomic instability in head and neck cancer patients.

In the normal host, there are a variety of cellular systems that ensure the accurate replication and repair of DNA. Recent evidence suggests that there are individual variations in the ability to preserve the genome. Certain individuals have defects in these checkpoints and have an inherent genomic instability. They are susceptible to the accumulation of DNA damage and are prone to carcinogenesis. This article examines the role of genomic instability in the development of head and neck cancer. Patients with either the chromosomal instability syndromes or the Li-Fraumeni syndrome have marked defects in either DNA repair or apoptosis. These patients are prone to have head and neck neoplasms develop. Head and neck cancer patients also have a diminished ability to repair DNA damage compared with the "normal" population. Abnormalities have been identified in mutagen sensitivity, the expression of DNA mismatch repair enzymes, the expression of p53, and telomerase activity when head and neck cancer patients are compared with controls. Subpopulations exist who have increased genomic instability. These individuals are at an increased risk for the accumulation of DNA mutations and the development of head and neck cancer. More research is needed to identify specific mechanisms of genomic instability and to further define the importance of this phenomenon.

Impact of age on hypermutation of immunoglobulin variable genes in humans.

Chronological aging is associated with an accumulation of DNA mutations that results in cancer formation. The effect of aging on spontaneous mutations in humans is difficult to study because mutations are infrequent in the overall genome and tumors are relatively rare. In contrast, somatic mutations in immunoglobulin variable genes are abundant and can be studied in peripheral blood lymphocytes. To determine if aging alters the frequency and pattern of hypermutation, we sequenced 331 cDNA clones with rearranged V(H)6 genes and compared 452 mutations from young humans to 570 mutations from old humans. There were more mutated clones in the young population compared to the old population. Among the mutated clones, the frequency, location, and types of substitutions were similar between the young and the old groups. However, the ratio of replacement-to-silent mutations was much higher in the complementarity-determining regions of heavy chains from old people, which indicates that their B cells had been selected by antigen. Among individuals, there was variability in the frequency of tandem mutations, which we have observed in mice defective for the PMS2 mismatch repair protein. Microsatellite variability in DNA, which is caused by impaired mismatch repair, was then measured, and there was a strong correlation between the frequency of tandem mutations and microsatellite alterations. The data suggest that individuals vary in their mismatch repair capacity, which can affect the mutational spectra in their antibodies.

Sunlight and Skin Cancer.

It is well known that chronic exposure to ultraviolet (UV) radiation present in sunlight is responsible for the induction of most nonmelanoma skin cancer (NMSC) in humans. Wavelengths in the UV-B (290–320 nm) region of the solar spectrum are absorbed into the skin, producing erythema, burns, and eventually skin cancer. NMSC is the most common type of human cancer. Recent surveys indicate that around one million new cases of skin cancer are diagnosed each year in the United States, about 70% of which result from repeated exposure of the skin to sunlight. Laboratory studies have shown that UV-B region of the solar spectrum is responsible for this effect. The first step in UV skin carcinogenesis involves the induction of DNA damage. Occasional mistakes during the repair of this damage leads to the incorporation of wrong bases into the genetic material. The DNA damage that is left unrepaired may also disrupt cellular processes by obstructing the DNA and RNA synthesizing machineries and introduce wrong bases into the DNA. These types of mistakes often result in mutation leading to loss or inappropriate expression of affected genes. Recent studies indicate that genetic alterations in the p53 tumor suppressor gene play an important role in the development of skin cancer. The p53 protein is also involved in programmed cell death (apoptosis), and it has been proposed that p53 serves as a “guardian of the genome” by aiding DNA repair or causing elimination of cells with excessive DNA damage. Unrepaired photoproducts in the p53 gene are transformed into mutations thereby initiating the process of carcinogenesis. Following repeated exposures to UV, keratinocytes carrying p53 mutations acquire a growth advantage by virtue of their increased resistance to apoptosis. Recent studies have shown that UV-B damaged keratinocytes (sunburn cells) are eliminated by Fas/Fas-ligand interaction and that this pathway is dysregulated during UV skin carcinogenesis resulting in accumulation of p53 mutations in DNA damaged keratinocytes. These results demonstrate a link between p53 pathway and Fas/Fas-ligand pathway and that dysregulation of both pathways can lead to the pathogenesis of UV-induced skin cancer. Several studies have shown that UV induces unique types of p53 mutations in skin cancers at a high frequency that are not commonly found in other types of human cancer. Analogous to human skin cancers, skin cancers induced in laboratory mice by UV radiation also display UV signature p53 mutations at a high frequency. More interestingly, p53 mutations are also present in sun-exposed skin and it can serve as an indicator of prior solar exposure in humans. It has been shown that p53 mutations in mouse skin arise as early as one week of chronic UV-irradiation and the frequency of p53 mutations reach a maximum at 4–8 week of UV exposure. These results suggest that p53 mutations arise well before skin cancer development and that they can serve as a surrogate early biological endpoint in skin cancer prevention studies. In fact, it has been shown that application of SPF-15 sunscreens to mouse skin before each UV-irradiation protect mice against induction of p53 mutations as well as skin cancer development. In addition to the p53 gene, another tumor suppressor gene known as patched (ptc) has been implicated in nevoid basal cell carcinoma syndrome (NBCCS). NBCCS, also called basal cell nevus syndrome or Gorlin's syndrome, is a rare autosomal dominant disorder characterized by multiple BCCs that appear at a young age on sun-exposed areas of the skin. Studies of NBCCS patients have shown that they have both germ-line and somatic mutations in the ptc gene. Somatic ptc mutations have been found in BCCs from otherwise normal individuals suggesting that genetic alterations in the ptc gene may also play a role in the development of BCC. In summary, recent advances have aided in the understanding of the mechanisms by which UV radiation induces skin cancer. Continued efforts should result in a greater understanding of the genetic mechanisms involved in the function of tumor suppressor and oncogenes that are now known, as well as genes yet to be discovered. The efforts of research in skin cancer may help to increase overall awareness of the harmful effects of UV exposure and result in better methods of skin cancer prevention and treatment.

Hepatitis B Virus X Protein Interferes with Cellular DNA Repair

The hepatitis B virus X protein (HBx) is a broadly acting transactivator implicated in the development of liver cancer. Recently, HBx has been reported to interact with several different cellular proteins, including our report of its binding to XAP-1, the human homolog of the simian repair protein UVDDB. In the present study, several HBx mutants were used to localize the minimal domain of HBx required for binding to XAP-1/UVDDB to amino acids 55 to 101. The normal function of XAP-1/UVDDB is thought to involve binding to damaged DNA, the first step in nucleotide excision repair (NER); therefore, we hypothesized that this interaction may affect the cell's capacity to correct lesions in the genome. When tested in two independent assays that measure NER (unscheduled DNA synthesis and host cell reactivation), the expression of HBx significantly inhibited the ability of cells to repair damaged DNA. Under the assay conditions, HBx was expressed at a level similar to that previously observed during natural viral infection and was able to transactivate several target reporter genes. These results are consistent with a model in which HBx acts as a cofactor in hepatocarcinogenesis by preventing the cell from efficiently repairing damaged DNA, thus leading to an accumulation of DNA mutations and, eventually, cancer. An adverse effect on cellular DNA repair processes suggests a new mechanism by which a tumor-associated virus might contribute to carcinogenesis.

Comparative analysis of DNA mutations in lacI transgenic mice with age

Transgenic mice have been developed recently that contain copies of the well-defined mutagenesis reporter gene, lacI, in an integrated bacteriophage-based shuttle vector. The lacI gene, which is present in all cells of the mouse, can be excised specifically from isolated genomic DNA and efficiently packaged into bacteriophage particles after the addition of packaging extracts. Mutations of the lacI gene are easily detected by the derepression of beta-galactosidase resulting in blue plaques in the presence of X-gal. Originally developed as a short-term in vivo mutagenesis assay to screen genotoxic agents, we have used this system to measure naturally occurring DNA mutations as a function of chronological age. There was a linear increase in the presence of phenotypic lacI mutants from birth to 24 months (r = 0.731, P < 0.001), such that 24-month-old mice have accumulated approximately fourfold more mutants than newborn pups. Molecular analysis of these spontaneously arising DNA mutations showed them to result predominantly from base substitutions (80.6-98.5%) that were equally distributed between transitions and transversions. However, lacI mutations in animals > 3 months of age demonstrated a higher percentage of mutations (12-19.4% vs. 1.2%) resulting from discriminable size changes (> 20 bp) than was observed for mice 1-2 months old. Sequence analysis of mutations resulting from a > 20 bp size change revealed them to be due to a duplication of adjacent lacI sequence. These results indicate that there is a gradual accumulation of DNA mutations with age and that the types of mutations also are influenced by the age of the animal.