Altered spectra of hypermutation in DNA repair-deficient mice

Affinity maturation of the humoral immune response is based on the ability of immunoglobulin variable genes to undergo a process of rapid and extensive somatic mutation followed by antigenic selection for antibodies with higher affinity. While the behaviour of this somatic hypermutation phenomenon has been well characterized over the last 20 years, the molecular mechanism responsible for inserting mutations has remained shrouded. To better understand this mechanism, we studied the interplay between hypermutation and other DNA associated activities such as DNA repair. There was no effect on the frequency and pattern of hypermutation in mice deficient for nucleotide excision repair, base excision repair and ataxia-telangiectasia mutated gene repair of double strand breaks. However, variable genes from mice lacking some components of mismatch repair had an increased frequency of tandem mutations and had more mutations of G and C nucleotides. These results suggest that the DNA polymerase(s) involved in the hypermutation pathway produces a unique spectra of mutations, which is then altered by mismatch repair and antigenic selection. We, also describe the differential pattern of expression of some nuclear DNA polymerases in hypermutating versus non-hypermutating B lymphocytes. The rapidly dividing germinal centre B cells expressed DNA polymerases alpha, beta, delta, epsilon and zeta, whereas the resting non-germinal centre cells did not express polymerases alpha or epsilon at detectable levels, although they did express polymerases beta, delta and zeta. The lack of expression of polymerase epsilon in the non-germinal centre cells suggests that this enzyme has a critical role in chromosomal replication but does not participate in DNA repair in these cells.     

Relative sensitivities of repair-deficient mammalian cells for clonogenic survival after alpha-particle irradiation

The clonogenic survival of cells of the radiation-sensitive hamster cell lines irs1, irs2, irs3 and xrs5, representing different DNA repair pathways, was compared to that of their parent lines after alpha-particle irradiation. Measurements of nuclear area were made to calculate the probability of surviving a single alpha-particle traversal, the average number of lethal lesions per track and per unit dose, along with the "intrinsic radiosensitivity" of these cells, allowing for the potential of multiple lethal lesions per traversal. For all cell lines studied, alpha particles were found to be more biologically effective per unit absorbed dose than X rays at inducing cell inactivation. The repair-deficient cells showed an enhanced sensitivity to alpha particles compared to their parent line, but the degree of enhancement was less than for X rays. The reduction in additional sensitivity for alpha-particle irradiation was shown not to be due predominantly to differences in cell geometry limiting the probability of a cell nucleus being traversed. The results suggest that both the nonhomologous end-joining pathway and to a lesser extent the homologous recombination repair pathway play a role in successful repair of alpha-particle-induced damage, although a large proportion of damage is not repaired by either pathway.     

CTG repeat instability and size variation timing in DNA repair-deficient mice

Type 1 myotonic dystrophy is caused by the expansion of an unstable CTG repeat in the DMPK gene. We have investigated the molecular mechanisms underlying the CTG repeat instability by crossing transgenic mice carrying >300 unstable CTG repeats in their human chromatin environment with mice knockout for genes involved in various DNA repair pathways: Msh2 (mismatch repair), Rad52 and Rad54 (homologous recombination) and DNA-PKcs (non-homologous end-joining). Genes of the non-homologous end-joining and homologous recombination pathways did not seem to affect repeat instability. Only lack of Rad52 led to a slight decrease in expansion range. Unexpectedly, the absence of Msh2 did not result in stabilization of the CTG repeats in our model. Instead, it shifted the instability towards contractions rather than expansions, both in tissues and through generations. Furthermore, we carefully analyzed repeat transmissions with different Msh2 genotypes to determine the timing of intergenerational instability. We found that instability over generations depends not only on parental germinal instability, but also on a second event taking place after fertilization.     

Mutagenesis and carcinogenesis in nucleotide excision repair-deficient XPA knock out mice

Mice with a defect in the xeroderma pigmentosum group A (XPA) gene have a complete deficiency in nucleotide excision repair (NER). As such, these mice mimic the human XP phenotype in that they have a >1000-fold higher risk of developing UV-induced skin cancer. Besides being UV-sensitive, XPA^−/− mice also develop internal tumors when they are exposed to chemical carcinogens. To investigate the effect of a total NER deficiency on the induction of gene mutations and tumor development, we crossed XPA^−/− mice with transgenic lacZ/pUR288 mutation-indicator mice. The mice were treated with various agents and chemicals like UV-B, benzo[a]pyrene and 2-aceto-amino-fluorene. Gene mutation induction in several tumor target- and non-target tissues was determined in both the bacterial lacZ reporter gene and in the endogenous Hprt gene. Furthermore, alterations in the p53- and ras genes were determined in UV-induced skin tumors of XPA^−/− mice. In this work, we review these results and discuss the applicability and reliability of enhanced gene mutant frequencies as early indicators of tumorigenesis.     

Activation of the PI3K/Akt signal transduction pathway and increased levels of insulin receptor in protein repair-deficient mice

Protein L-isoaspartate (D-aspartate) O-methyltransferase is an enzyme that catalyses the repair of isoaspartyl damage in proteins. Mice lacking this enzyme (Pcmt1-/- mice) have a progressive increase in brain size compared with wild-type mice (Pcmt1+/+ mice), a phenotype that can be associated with alterations in the PI3K/Akt signal transduction pathway. Here we show that components of this pathway, including Akt, GSK3beta and PDK-1, are more highly phosphorylated in the brains of Pcmt1-/- mice, particularly in cells of the hippocampus, in comparison with Pcmt1+/+ mice. Examination of upstream elements of this pathway in the hippocampus revealed that Pcmt1-/- mice have increased activation of insulin-like growth factor-I (IGF-I) receptor and/or insulin receptor. Western blot analysis revealed an approximate 200% increase in insulin receptor protein levels and an approximate 50% increase in IGF-I receptor protein levels in the hippocampus of Pcmt1-/- mice. Higher levels of the insulin receptor protein were also found in other regions of the adult brain and in whole tissue extracts of brain, liver, heart and testes of both juvenile and adult Pcmt1-/- mice. There were no significant differences in plasma insulin levels for adult Pcmt1-/- mice during glucose tolerance tests. However, they did show higher peak levels of blood glucose, suggesting a mild impairment in glucose tolerance. We propose that Pcmt1-/- mice have altered regulation of the insulin pathway, possibly as a compensatory response to altered glucose uptake or metabolism or as an adaptive response to a general accumulation of isoaspartyl protein damage in the brain and other tissues.     

Diet-dependent shifts in ruminal butyrate-producing bacteria

The influence of a host's diet on Butyrivibrio and Pseudobutyrivibrio populations was investigated by competitive PCR. Specific primers were designed and competitive PCRs developed for both groups. Results (from 4 cows with different diets) suggested that high-fiber intake essentially increases the Butyrivibrio amounts in the rumen, whereas high-energy food additives lead to its suppression. The Pseudobutyrivibrio concentration also changed during the experiment but without any significant relation to the host's diet.     

Correction of liver dysfunction in DNA repair-deficient mice with an ERCC1 transgene

The ERCC1 gene is essential for the repair of UV-induced DNA damage. Unlike most genes in the nucleotide excision repair (NER) pathway, ERCC1 is also involved in recombinational repair. Perhaps for this reason, ERCC1 knockout mice are not a model for the human NER deficiency disorder, xeroderma pigmentosum. Instead, ERCC1 null mice are severely runted and die before weaning from liver failure with accelerated hepatocyte polyploidy that is more reminiscent of a premature ageing disorder. To permit study of the role of ERCC1 in other tissues we have corrected the liver ERCC1 deficiency with a transgene under the control of a liver-specific promoter. The transgene alleviated runting and extended the lifespan. The elevated level of oxidative DNA damage and premature liver polyploidy were reversed and liver function was corrected. A widespread mitochondrial dysfunction was identified and an essential role for ERCC1 in the kidney was also revealed with transgene-containing ERCC1-deficient animals going on to die of renal failure. The nuclei of kidney proximal tubule cells became polyploid in a similar way to the premature liver polyploidy observed in younger ERCC1-deficient animals. We believe that this is a response to the accumulation of endogenous DNA damage in these particularly susceptible tissues which cannot be repaired in ERCC1-deficient animals.     

UV light-induced DNA damage and tolerance for the survival of nucleotide excision repair-deficient human cells

DNA damage can cause cell death unless it is either repaired or tolerated. The precise contributions of repair and tolerance mechanisms to cell survival have not been previously evaluated. Here we have analyzed the cell killing effect of the two major UV light-induced DNA lesions, cyclobutane pyrimidine dimers (CPDs) and 6-4 pyrimidine-pyrimidone photoproducts (6-4PPs), in nucleotide excision repair-deficient human cells by expressing photolyase(s) for light-dependent photorepair of either or both lesions. Immediate repair of the less abundant 6-4PPs enhances the survival rate to a similar extent as the immediate repair of CPDs, indicating that a single 6-4PP lesion is severalfold more toxic than a CPD in the cells. Because UV light-induced DNA damage is not repaired at all in nucleotide excision repair-deficient cells, proliferation of these cells after UV light irradiation must be achieved by tolerance of the damage at replication. We found that RNA interference designed to suppress polymerase zeta activity made the cells more sensitive to UV light. This increase in sensitivity was prevented by photorepair of 6-4PPs but not by photorepair of CPDs, indicating that polymerase zeta is involved in the tolerance of 6-4PPs in human cells.     

Reduced hematopoietic reserves in DNA interstrand crosslink repair-deficient Ercc1-/- mice

The ERCC1-XPF heterodimer is a structure-specific endonuclease involved in both nucleotide excision repair and interstrand crosslink repair. Mice carrying a genetic defect in Ercc1 display symptoms suggestive of a progressive, segmental progeria, indicating that disruption of one or both of these DNA damage repair pathways accelerates aging. In the hematopoietic system, there are defined age-associated changes for which the cause is unknown. To determine if DNA repair is critical to prolonged hematopoietic function, hematopoiesis in Ercc1-/- mice was compared to that in young and old wild-type mice. Ercc1-/- mice (3-week-old) exhibited multilineage cytopenia and fatty replacement of bone marrow, similar to old wild-type mice. In addition, the proliferative reserves of hematopoietic progenitors and stress erythropoiesis were significantly reduced in Ercc1-/- mice compared to age-matched controls. These features were not seen in nucleotide excision repair-deficient Xpa-/- mice, but are characteristic of Fanconi anemia, a human cancer syndrome caused by defects in interstrand crosslink repair. These data support the hypothesis that spontaneous interstrand crosslink damage contributes to the functional decline of the hematopoietic system associated with aging.     

Effects of hexavalent chromium on the survival and cell cycle distribution of DNA repair-deficient S. cerevisiae

A broad spectrum of genetic damage results from exposure to hexavalent chromium. These lesions can result in DNA and RNA polymerase arrest, chromosomal aberrations, point mutations and deletions. Because of the complexity of Cr genotoxicity, the repair of Cr(VI)-induced DNA damage is poorly understood. Therefore, our aim was to investigate the sensitivities of DNA repair-deficient Saccharomyces cerevisiae strains to Cr(VI)-induced growth inhibition and lethality. Wild-type, translesion synthesis (rev3) and excision repair (apn1, ntg1, ntg2, rad1) mutants exhibited similar survival following Cr(VI) treatment (0-50mM) and underwent at least one population doubling within 2-4h post-treatment. The simultaneous loss of several excision repair genes (apn1 rad1 ntg1 ntg2) led to slower growth after Cr(VI) exposure (10mM) manifested as an initial delay in S phase progression. Higher concentrations of Cr(VI) (25mM) resulted in a prolonged transit through S phase in every strain tested. A G(2)/M arrest was evident within 1-2h after Cr(VI) treatment (10mM) in all strains and cells subsequently divided after this transient delay. In contrast to all other strains, only recombination-deficient (rad52, rad52 rev3) yeast were markedly hypersensitive towards Cr(VI) lethality. RAD52 mutant strains (rad52, rad52 rev3) also exhibited a significant delay (>6h) in the resumption of replication after Cr(VI) exposure which was related to the immediate and apparently terminal arrest of these yeast in G(2)/M after Cr(VI) treatment. These results, taken together with the recombinogenic effects of Cr(VI) in yeast containing a functional RAD52 gene, suggest that RAD52-mediated recombination is critical for the normal processing of lethal Cr-induced genetic lesions and exit from G(2) arrest. Furthermore, only the combined inactivation of multiple excision repair genes affects cell growth after Cr(VI) treatment.     

Diet-dependent cardiovascular lipid metabolism controlled by hepatic LXRalpha

The high-cholesterol/high-fat Western diet has abetted an epidemic of atherosclerotic cardiovascular disease, the leading cause of death in industrialized nations. Liver X receptors (LXRs) are oxysterol sensors that are required for normal cholesterol and triglyceride homeostasis, yet synthetic LXR agonists produce undesirable hypertriglyceridemia. Here we report a previously unrecognized role for hepatic LXRalpha in the links between diet, serum lipids, and atherosclerosis. A modest increase in hepatic LXRalpha worsened serum lipid profiles in LDL-receptor null mice fed normal chow but had the opposite effect on lipids and afforded strong protection against atherosclerosis on a Western diet. The beneficial effect of hepatic LXRalpha was abrogated by a synthetic LXR agonist, which activated SREBP-1c and its target genes. Thus, the interplay between diet and hepatic LXRalpha is a critical determinant of serum lipid profiles and cardiovascular risk, and selective modulation of LXR target genes in liver can ameliorate hyperlipidemia and cardiovascular disease.     

Expression of viral and virus-like elements in DNA repair-deficient/immunodeficient "wasted" mice

Wasted mice bear an autosomal recessive mutation (wst) that causes neurologic abnormalities, faulty DNA repair in lymphocytes, and immunodeficiency at mucosal sites. Recent work has suggested possible viral involvement in these manifestations by demonstrating abnormal viral gp70 expression that segregates with the wasted mutation. In the experiments reported here, we examined tissue-specific expression of AKR viral (AKV) sequences and virus-like 30S (VL30) elements in wasted and control mice. Our studies showed that AKV and VL30 RNA expression were two- to fivefold higher in spleen, brain, and thymus of mice bearing the wst allele than in those of control mice. (These tissues have all been shown to display functional or developmental abnormalities in wst/wst mice.) Expression of viral mRNA in Peyer's patches and mesenteric lymph nodes was similar in wasted and control animals. The majority of the VL30-hybridizing RNA in all tissues could be attributed to long-terminal-repeat rather than to main-body sequences. These differences in expression between wasted and control mice could not be attributed to differences in VL30 or AKV gene copy number. Our results demonstrate an association between altered expression of viral and virus-like sequences and the development of tissue-restricted abnormalities in wst/wst mice.     

Retinoblastoma protein activation of interleukin 8 expression inhibits tumor cell survival in nude mice.

Loss of retinoblastoma protein (Rb) has been implicated in the formation of a variety of human malignancies. Restoration of Rb expression in the cell lines representing these tumors eliminates or significantly reduces tumorigenicity in nude mice, but the mechanism for this Rb effect is unknown. Results from this study indicated that Rb expression reduced tumor cell survival in nude mice by dramatically enhancing interleukin 8 (IL-8) secretion. IL-8 secreted by the Rb-transformed cells attracted neutrophils in vitro and tumor-infiltrating neutrophils in vivo, which is consistent with the Rb-mediated tumor regression being dependent on IL-8. The apparent, contradictory roles of IL-8 as a protumorigenic and antitumorigenic cytokine are discussed.     

Diet-dependent cannibalism in the omnivorous phytoseiid mite Amblydromalus limonicus

Amblydromalus limonicus (Garman & McGregor) (Acari: Phytoseiidae) is a commercially available predator of key pests in protected crops, particularly of thrips and whiteflies. Basic information on the developmental and reproductive performance of the predator as a function of food is largely lacking. In the present study, development, reproduction and growth rates were determined for A. limonicus on four economically important pests: Western flower thrips, Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae), greenhouse whitefly, Trialeurodes vaporariorum (Westwood) (Hemiptera: Aleyrodidae), broad mite, Polyphagotarsonemus latus (Banks) (Acari: Tarsonemidae) and two-spotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae). The life history traits of females fed on these different target prey were compared with those of females offered Carpoglyphus lactis L. (Acari: Carpoglyphidae), which is the standard food source for mass-producing this predator. Additionally, three commercially available non-prey food sources with potential for use in the mass production or as supplementary food to sustain populations of the predator in the field were tested: the commercial pollen product Nutrimite (consisting of pollen of narrow-leaved cattail, Typha angustifolia L.), frozen eggs of the Mediterranean flour moth Ephestia kuehniella Zeller (Lepidoptera: Pyralidae) and frozen eggs of the Mediterranean fruit fly, Ceratitis capitata Wiedemann (Diptera: Tephritidae). Survival rates of immature A. limonicus were high (>94% survival) on all tested foods except on T. vaporariorum and T. urticae (76.0% and 17.1%, respectively). The fastest development was obtained when mites were fed on T. angustifolia, whereas the longest developmental times were obtained on T. urticae and T. vaporariorum. When females were offered P. latus, no reproduction was observed, despite a high prey consumption in both the juvenile and adult stages. The reproductive performance of A. limonicus fed on T. vaporariorum was significantly lower than that on F. occidentalis. Furthermore, no second generation could be obtained on a diet solely consisting of T. vaporariorum. Population growth rates were highest when A. limonicus was fed on Nutrimite, E. kuehniella or C. lactis, and exceeded those on a diet consisting of their natural prey, F. occidentalis. The phytoseiid showed cannibalistic behavior when maintained on E. kuehniella and C. capitata eggs and T. angustifolia pollen, with females consuming their own eggs. The rate of cannibalism was dependent on the food source offered, but always resulted in reduced population growth rates. This cannibalistic behavior should be taken into account when selecting food sources for mass rearing of A. limonicus or supporting its populations in the field.     

Diet-dependent body morphology and ontogenetic reaction norms in Eurasian perch

Individual morphology and performance are directly or indirectly under the influence of variation in resource levels. To study the effects of different resource conditions and their effects on morphology and ontogenetic reaction norms in young‐of‐the‐year (YOY) perch (Perca fluviatilis), we used three different approaches. First, we examined the morphological trajectories over early ontogeny in relation to lake‐specific resources in a field study. Second, one lake that lacked perch recruitment was stocked with perch eggs from a control lake in a whole‐lake experiment to study ontogenetic reaction norms. Third, we compared the development of YOY perch in the three lakes that mainly inhabited the littoral zone with YOY perch experimentally confined to enclosures in the pelagic zone of the lakes.
Overall body morphology of the YOY perch changed both as a function of size and as a function of diet. As perch increased in size they developed a deeper body morphology corresponding to an increased proportion of benthic macroinvertebrates in their diet. In pelagic enclosures where perch were constrained to feed mainly on zooplankton they had a more fusiform body morphology than perch in the lakes that fed on a mixture of zooplankton and macroinvertebrates. Similarly, the ontogenetic reaction norm of perch was related to the diet and lake‐specific zooplankton levels in the whole‐lake experiment.
In the pelagic enclosures, perch with high growth rates had a more fusiform body morphology than slow‐growing perch, whereas the opposite was found in the lakes, where perch included more macroinvertebrates in their diets. Perch in lakes with a higher proportion of macroinvertebrates in the diet also had deeper body morphology. The opposite morphology – growth rate relationship found between perch in the pelagic versus those using the whole lake suggest a morphological trade‐off between foraging on zooplankton and foraging on macroinvertebrates. Our results suggest that YOY perch show different ontogenetic reaction norms as a function of lake‐specific resource levels, which may allow YOY recruitment to later stages. Our results further suggest that diet‐related changes in morphology are a rapid process.     

Diet-dependent switch from circadian to hourglass-like operation of an insect photoperiodic clock

The results of resonance experiments gave evidence that circadian organization was involved in photoperiodic diapause induction of Pieris brassicae. However, giving the larvae an artificial diet instead of the host plant deeply altered the resonance effect and led to a response that could be accounted for by the operation of a noncircadian timer (hourglass). This important change, however, had no effect on the insect's ability to discriminate between short- and long-day 24-hr cycles. Thus, it is possible via the metabolism to switch the photoperiodic mechanism to a circadian or an hourglass-like mode of operation without impairing the photoperiodic time measurement.     

Bystander effects in repair-deficient cell lines

One of the current hypotheses concerning the role of bystander effects in biological systems is that they are protective because they terminate division in cells with collateral or possibly pre-existing DNA damage that is not properly repaired. Following the logic of this hypothesis led us to consider that cell lines that are repair deficient should have larger than usual bystander effects. To test this, several different "repair- deficient" cell lines were used for bystander experiments. Response was monitored by determining the cloning efficiency or, in the case of non-adherent cell lines, the cell number. The results show that the repair-deficient human cell lines and surviving progeny produced moderate to severe bystander- induced death effects in either autologous cells or a reporter cell line. Normal "repair-proficient" lines, which were matched as far as possible, have very much less severe or absent bystander-inducible effects on cloning efficiency. Cells of hamster cell lines derived from CHO-K1 cells did not produce similar severe effects. The results suggest that repair- deficient human cell lines, irrespective of the actual repair defect, may respond to the occurrence of DNA damage in the population by removing large numbers of cells from the proliferating pool.     

Unchanged survival rates of 14-3-3gamma knockout mice after inoculation with pathological prion protein

The diagnosis of sporadic Creutzfeldt-Jakob disease (CJD) is based on typical clinical findings and is supported by a positive 14-3-3 Western blot of cerebrospinal fluid. However, it is not clear whether 14-3-3 indicates general neuronal damage or is of pathophysiological relevance in CJD. The fact that the 14-3-3 isoform spectrum in cerebrospinal fluid does not correspond to that found in the brain points to a regulated process. To investigate a possible role of 14-3-3 proteins in transmissible spongiform diseases, we generated a 14-3-3gamma-deficient mutant mouse line by using a classical knockout strategy. The anatomy and cage behavior of the mutant mice were normal. Western blot analyses of brain homogenates revealed no changes in the protein expression of other 14-3-3 isoforms (epsilon, beta, zeta, and eta). Proteomic analyses of mouse brains by two-dimensional differential gel electrophoresis showed that several proteins, including growth hormone, 1-Cys peroxiredoxin, CCT-zeta, glucose-6-phosphate isomerase, GRP170 precursor, and alpha-SNAP, were differentially expressed. Mutant and wild-type mice were inoculated either intracerebrally or intraperitoneally with the Rocky Mountain Laboratory strain of scrapie, but no differences were detected in the postinoculation survival rates. These results indicate that 14-3-3gamma is unlikely to play a causal role in CJD and related diseases.