Lymphoma caused by intestinal microbiota.

The intestinal microbiota and gut immune system must constantly communicate to maintain a balance between tolerance and activation: on the one hand, our immune system should protect us from pathogenic microbes and on the other hand, most of the millions of microbes in and on our body are innocuous symbionts and some can even be beneficial. Since there is such a close interaction between the immune system and the intestinal microbiota, it is not surprising that some lymphomas such as mucosal-associated lymphoid tissue (MALT) lymphoma have been shown to be caused by the presence of certain bacteria. Animal models played an important role in establishing causation and mechanism of bacteria-induced MALT lymphoma. In this review we discuss different ways that animal models have been applied to establish a link between the gut microbiota and lymphoma and how animal models have helped to elucidate mechanisms of microbiota-induced lymphoma. While there are not a plethora of studies demonstrating a connection between microbiota and lymphoma development, we believe that animal models are a system which can be exploited in the future to enhance our understanding of causation and improve prognosis and treatment of lymphoma.

Intestinal microbiome and lymphoma development.

The intestinal microbiota and gut immune system must communicate to maintain a balance between tolerance and activation. Our immune system protects us from pathogenic microbes at the same time that our bodies are host to trillions of microbes, symbionts, mutualists, and some that are essential to human health. Since there is such a close interaction between the immune system and the intestinal microbiota, it is not surprising that some lymphomas such as mucosal-associated lymphoid tissue lymphoma have been shown to be caused by the presence of certain bacteria. Animal models have played an important role in elucidating the causation and establishing the mechanism of bacteria-induced mucosal-associated lymphoid tissue lymphoma. In this review, we discuss different ways that animal models have been applied to investigate links between the gut microbiota and lymphoma and have helped to reveal the mechanisms of microbiota-induced lymphoma. Although there is a paucity of published studies demonstrating the interplay between the microbiota and lymphoma development, we believe that the connection is real and that it can be exploited in the future to enhance our understanding of causation and to improve the prognosis and treatment of lymphoma.

Intestinal bacteria modify lymphoma incidence and latency by affecting systemic inflammatory state, oxidative stress, and leukocyte genotoxicity.

Ataxia-telangiectasia is a genetic disorder associated with high incidence of B-cell lymphoma. Using an ataxia-telangiectasia mouse model, we compared lymphoma incidence in several isogenic mouse colonies harboring different bacterial communities, finding that intestinal microbiota are a major contributor to disease penetrance and latency, lifespan, molecular oxidative stress, and systemic leukocyte genotoxicity. High-throughput sequence analysis of rRNA genes identified mucosa-associated bacterial phylotypes that were colony-specific. Lactobacillus johnsonii, which was deficient in the more cancer-prone mouse colony, was causally tested for its capacity to confer reduced genotoxicity when restored by short-term oral transfer. This intervention decreased systemic genotoxicity, a response associated with reduced basal leukocytes and the cytokine-mediated inflammatory state, and mechanistically linked to the host cell biology of systemic genotoxicity. Our results suggest that intestinal microbiota are a potentially modifiable trait for translational intervention in individuals at risk for B-cell lymphoma, or for other diseases that are driven by genotoxicity or the molecular response to oxidative stress.

Effects of side-stream tobacco smoke and smoke extract on glutathione- and oxidative DNA damage repair-deficient mice and blood cells.

Cigarette smoke causes direct oxidative DNA damage as well as indirect damage through inflammation. Epidemiological studies show a strong relationship between secondhand smoke and cancer; however, the mechanisms of secondhand smoke-induced cancer are not well understood. Animal models with either (i) deficient oxidative DNA damage repair, or (ii) a decreased capacity to combat oxidative stress may help determine the pathways important in mitigating damage caused by smoke. In this study, we used mice lacking Ogg1 and Myh, both of which are involved in base excision repair by removing oxidatively damaged DNA bases. Gclm-deficient mice, which have decreased levels of glutathione (GSH), were used to look at the role of smoke-induced oxidative damage. Ex vivo experiments show significantly elevated levels of DNA single-strand breaks and chromosomal aberrations in peripheral blood lymphocytes from Ogg1(-/-)Myh(-/-) double knockout mice compared to wild type (WT) mice after 24h of exposure to cigarette smoke extract (CSE). The average γH2AX foci per cell was significantly elevated 3h after exposure to CSE in cells from Ogg1(-/-)Myh(-/-) double knockout mice compared to wildtype mice. In vivo we found that all mice had increased markers of DNA damage after exposure to side-stream tobacco smoke (SSTS). Ogg1(-/-)Myh(-/-) and Gclm(-/-) mice had altered levels of peripheral blood glutathione after SSTS exposure whereas wild type mice did not. This may be due to differential regulation of glutathione synthesis in the lung. We also found that Ogg1(-/-)Myh(-/-) mice had a decreased lifespan after oral gavage with benzo[a]pyrene compared to wildtype mice and sham-exposed Ogg1(-/-)Myh(-/-) mice. Our results are important in investigating the roles of oxidative stress and oxidative DNA damage repair in cigarette smoke-induced cancers and characterizing the role of genetic polymorphisms in smoke-related disease susceptibility.

Effects of 1 GeV/nucleon 56Fe particles on longevity, carcinogenesis and neuromotor ability in Atm-deficient mice.

As therapeutic uses of high-LET radiation become more prevalent and human space exploration continues to be a focus of NASA, it is important to understand the biological effects of high-LET radiation and the role of genetics in sensitivity to high-LET radiation. To study genetic susceptibility to radiation, we used mice deficient in Atm activity (AtmΔSRI). ATM is important in DNA repair, apoptosis and cell cycle regulation. Although homozygous mutations in ATM are rare, the prevalence of ATM heterozygosity is estimated to be 1% and results in an increased cancer risk. We found that the effects of 1 Gy 1 GeV/nucleon 56Fe particles on life span and tumorigenesis are genotype- and sex-specific. Significant effects of 1 Gy 1 GeV/nucleon 56Fe particles on incidence of non-cancer end points were seen; however, 2 Gy 1 GeV/nucleon 56Fe particles significantly affected neuromotor ability. Our results represent an extensive investigation into the late effects of high-LET radiation exposure in a sex- and genotype-dependent manner and provide a baseline for understanding the long-term risks of high-LET radiation.

Genomic instability in mice is greater in Fanconi anemia caused by deficiency of Fancd2 than Fancg.

Fanconi anemia (FA) results from mutations in the FANC genes and is characterized by bone marrow failure, birth defects, and a high incidence of cancer. FANCG is a part of the FA core complex that is responsible for monoubiquitination of FANCD2 and FANCI. The precise role of the FA pathway is not well understood, although it may be involved in homologous recombination (HR), nonhomologous end joining, and translesion synthesis (TLS). Fancd2(-/-) mice have a more severe phenotype than Fancg(-/-), and other FA core complex-deficient mice, although both Fancg and Fancd2 belong to the same FA pathway. We hypothesized that Fancd2 deficiency results in a more severe phenotype because Fancd2 also has a FA pathway-independent function in the maintenance of genomic integrity. To test this hypothesis, we determined the level of DNA damage and genomic instability in Fancd2(-/-), Fancg(-/-), and wild-type controls. Fancd2(-/-) mice displayed a higher magnitude of chromosomal breakage and micronucleus formation than the wild-type or Fancg(-/-) mice. Also, DNA strand breaks were increased in Fancd2(-/-) but not in Fancg(-/-) mice. In addition, Fancd2(-/-) mice displayed an elevated frequency of DNA deletions, resulting from HR at the endogenous p(un) locus. In contrast, in Fancg(-/-) mice, the frequency of DNA deletions was decreased. Thus, Fancd2 but not Fancg deficiency results in elevated chromosomal/DNA breakage and permanent genome rearrangements. This provides evidence that Fancd2 plays an additional role in the maintenance of genomic stability than Fancg, which might explain the higher predisposition to cancer seen in the Fancd2(-/-) mice.

Effects of human Werner helicase on intrachromosomal homologous recombination mediated DNA deletions in mice.

Werner syndrome (WS) is a rare genetic disorder characterized by accelerated aging and aging-related diseases including cancer. WS is caused by autosomal recessive mutations in the WRN gene, which is involved in genome maintenance although precise functions of WRN are not well understood. To further investigate the role of WRN, we used transgenic mice over-expressing a human helicase mutant WRN gene (hMW). We determined homologous recombination (HR) events leading to 70 kb deletions in the p(un) locus visualized as pigmented cells in the retinal pigment epithelium. hMW mice had an increased spontaneous frequency of DNA deletions compared to control mice, consistent with WRN involvement in HR suppression. In addition, 4-nitroquinoline 1-oxide (4-NQO), which can cause both oxidative stress and DNA adduct formation, significantly increased the frequency of DNA deletions in both control and hMW mice. In order to assess how oxidative stress may modulate this phenotype, we treated mice with the glutathione (GSH) synthesis inhibitor, buthionine sulfoximine (BSO). The frequency of DNA deletions increased significantly in control mice, but not in hMW littermates. To elucidate the cause of this discrepancy, we determined total GSH levels as a measure of anti-oxidative defense. BSO significantly decreased GSH levels in both hMW mice and control mice, while 4-NQO increased GSH levels in all mice. These findings suggest that the reduction of GSH by BSO or compensatory increase of GSH by 4-NQO had little impact on hMW mice in which HR repair is compromised. Therefore, oxidative stress impacts HR repair in hMW mice less than control mice and effects of the mutated gene may be exacerbated by direct DNA damage from 4-NQO. This mouse model of WS in conjunction with different DNA damaging agents may provide insight into mechanisms of genomic instability, DNA repair, and carcinogenesis.

Gene expression changes in human small airway epithelial cells exposed to Delta9-tetrahydrocannabinol.

Marijuana smoking is associated with inflammation, cellular atypia, and molecular dysregulation of the tracheobronchial epithelium. While marijuana smoke shares many components in common with tobacco, it also contains a high concentration of Delta9-tetrahydrocannabinol (THC). The potential contribution of THC to airway injury was assessed by exposing primary cultures of human small airway epithelial (SAE) cells to THC (0.1-10.0 microg/ml) for either 1 day or 7 days. THC induced a time- and concentration-dependent decrease in cell viability, ATP level, and mitochondrial membrane potential. Using a targeted gene expression array, we observed acute changes (24 h) in the expression of mRNA for caspase-8, catalase, Bax, early growth response-1, cytochrome P4501A1 (CYP1A1), metallothionein 1A, PLAB, and heat shock factor 1 (HSF1). After 7 days of exposure, decrease in expression of mRNA for heat shock proteins (HSPs) and the pro-apoptotic protein Bax was observed, while expression of GADD45A, IL-1A, CYP1A1, and PTGS-2 increased significantly. These findings suggest a contribution of THC to DNA damage, inflammation, and alterations in apoptosis. Treatment with selected prototypical toxicants, 2,3,7,8-tetrachlorodibenznzo-p-dioxin (TCDD) and carbonyl cyanide-p-(trifluoramethoxy)-phenyl hydrazone (FCCP), produced partially overlapping gene expression profiles suggesting some similarity in mechanism of action with THC. THC, delivered as a component of marijuana smoke, may induce a profile of gene expression that contributes to the pulmonary pathology associated with marijuana use.

A gene(s) for all-trans-retinoic acid-induced forelimb defects mapped and confirmed to murine chromosome 11.

All-trans-retinoic acid (RA) induces various anatomical limb dysmorphologies in mice dependent on the time of exposure. During early limb development, RA induces forelimb ectrodactyly (digital absence) with varying susceptibilities for different inbred mouse strains; C57BL/6N are highly susceptible while SWV are resistant. To isolate the genetic basis of this defect, a full-genome scan was performed in 406 backcross fetuses of F(1) males to C57BL/6N females. Fetuses were exposed via a maternal injection of 75 mg of RA per kilogram of body weight on gestational day 9.25. The genome-wide analysis revealed significant linkage to a chromosome 11 locus near D11Mit39 with a maximum LOD score of 9.0 and to a chromosome 4 locus near D4Mit170. An epistatic interaction was detected between loci on chromosome 11 (D11Mit39) and chromosome 18 (D18Mit64). Linkage to the chromosome 11 locus (D11Mit39) was confirmed in RA-treated backcross fetuses of F(1) females to C57BL/6N males. Loci associated with bone density/mass in both human and mouse were previously detected in the same region, suggesting a mechanistic linkage with bone homeostasis. The human syntenic region of this locus has been previously linked to Meckel syndrome; the phenotype includes postaxial polydactyly, an ectopic digital defect hypothesized to be induced by a common molecular pathway with ectrodactyly.