Cutting Edge: RIP1 kinase activity is dispensable for normal development but is a key regulator of inflammation in SHARPIN-deficient mice.

RIP1 (RIPK1) kinase is a key regulator of TNF-induced NF-κB activation, apoptosis, and necroptosis through its kinase and scaffolding activities. Dissecting the balance of RIP1 kinase activity and scaffolding function in vivo during development and TNF-dependent inflammation has been hampered by the perinatal lethality of RIP1-deficient mice. In this study, we generated RIP1 kinase-dead (Ripk1(K45A)) mice and showed they are viable and healthy, indicating that the kinase activity of RIP1, but not its scaffolding function, is dispensable for viability and homeostasis. After validating that the Ripk1(K45A) mice were specifically protected against necroptotic stimuli in vitro and in vivo, we crossed them with SHARPIN-deficient cpdm mice, which develop severe skin and multiorgan inflammation that has been hypothesized to be mediated by TNF-dependent apoptosis and/or necroptosis. Remarkably, crossing Ripk1(K45A) mice with the cpdm strain protected against all cpdm-related pathology. Together, these data suggest that RIP1 kinase represents an attractive therapeutic target for TNF-driven inflammatory diseases.

C47T polymorphism in manganese superoxide dismutase (MnSOD), antioxidant intake and survival.

INTRODUCTION AND OBJECTIVE: Manganese superoxide dismutase (MnSOD), an enzyme that catalyzes superoxide radical quenching, is hypothesized to protect against premature aging. A C47T transition in the MnSOD gene may affect the enzyme's distribution to the mitochondrion, a site of high oxidative stress. We examined the association between this polymorphism and survival. METHODS: Individuals who donated a blood sample to the CLUE I and II campaigns in 1974 and 1989, respectively, and completed a food frequency questionnaire in 1989 (N=6151) were included in the analysis. Hazard ratios (HR) and 95% confidence intervals (CI) were calculated by Cox proportional hazards models. Mortality follow-up extended from 1989 to 2002. RESULTS: MnSOD genotype distributions were 27% CC (wildtype homozygotes), 50% CT (heterozygotes) and 23% TT (variant homozygotes). TT and CT genotypes compared to the CC genotype were not associated with all-cause or cardiovascular disease mortality. A slight, but non-statistically significant higher risk of cancer mortality was observed for the CT (HR=1.13, 95% CI: 0.86-1.49) and TT (HR=1.24, 95% CI: 0.90-1.70) genotypes compared to CC genotype (p-trend=0.19). CONCLUSION: We did not observe an association between the C47T polymorphism in the MnSOD gene and survival. These null associations were not modified by fruit and vegetable intake, cigarette smoking status, or body mass index.

MALT1 Protease Activity Is Required for Innate and Adaptive Immune Responses.

CARMA-BCL10-MALT1 signalosomes play important roles in antigen receptor signaling and other pathways. Previous studies have suggested that as part of this complex, MALT1 functions as both a scaffolding protein to activate NF-κB through recruitment of ubiquitin ligases, and as a protease to cleave and inactivate downstream inhibitory signaling proteins. However, our understanding of the relative importance of these two distinct MALT1 activities has been hampered by a lack of selective MALT1 protease inhibitors with suitable pharmacologic properties. To fully investigate the role of MALT1 protease activity, we generated mice homozygous for a protease-dead mutation in MALT1. We found that some, but not all, MALT1 functions in immune cells were dependent upon its protease activity. Protease-dead mice had defects in the generation of splenic marginal zone and peritoneal B1 B cells. CD4+ and CD8+ T cells displayed decreased T cell receptor-stimulated proliferation and IL-2 production while B cell receptor-stimulated proliferation was partially dependent on protease activity. In dendritic cells, stimulation of cytokine production through the Dectin-1, Dectin-2, and Mincle C-type lectin receptors was also found to be partially dependent upon protease activity. In vivo, protease-dead mice had reduced basal immunoglobulin levels, and showed defective responses to immunization with T-dependent and T-independent antigens. Surprisingly, despite these decreased responses, MALT1 protease-dead mice, but not MALT1 null mice, developed mixed inflammatory cell infiltrates in multiple organs, suggesting MALT1 protease activity plays a role in immune homeostasis. These findings highlight the importance of MALT1 protease activity in multiple immune cell types, and in integrating immune responses in vivo.

XPD polymorphism and risk of subsequent cancer in individuals with nonmelanoma skin cancer.

BACKGROUND: Individuals with nonmelanoma skin cancer (NMSC) are at increased risk of developing subsequent cancers. Genetic predisposition to reduced DNA repair capacity may be an underlying susceptibility factor explaining the excess risk of malignancies. To test this hypothesis, a cohort study was conducted to examine the association between XPD Lys751Gln polymorphism and risk of a second primary cancer in individuals with NMSC. METHODS: A subgroup of 481 individuals with a history of NMSC who participated in the CLUE II community-based cohort was followed for the development of a second primary cancer. Blood specimens donated in 1989 were genotyped for the XPD Lys751Gln polymorphism using the 5' nuclease assay. Cox proportional regression with delayed entry was used to calculate the incidence rate ratio (IRR) and 95% confidence interval (95% CI) for risk of developing a second primary cancer according to XPD genotype. All statistical tests were two sided. RESULTS: Eighty individuals developed a second primary cancer. The most frequent occurring cancers were of the prostate (18%), lung (15%), and breast (15%). Persons with at least one Gln allele had an increased risk of a second primary cancer compared with the reference Lys/Lys genotype (adjusted IRR 2.22, 95% CI 1.30-3.76). When the reference category was limited to never smokers with the Lys/Lys genotype, the risk of developing a second primary cancer associated with having at least one Gln allele was increased >3-fold in both never smokers (IRR 3.93, 95% CI 1.36-11.36) and ever smokers (IRR 6.14, 95% CI 2.17-17.37). CONCLUSION: These findings suggest that individuals with NMSC who have the variant XPD Gln allele are at increased risk of developing a second primary cancer.

C. elegans CEP-1/p53 and BEC-1 are involved in DNA repair.

p53 is a transcription factor that regulates the response to cellular stress. Mammalian p53 functions as a tumor suppressor. The C. elegans p53, cep-1, regulates DNA-damage induced germline cell death by activating the transcription of egl-1 and ced-13. We used the C. elegans model to investigate how, in the whole animal, different forms of DNA damage can induce p53-dependent versus p53-independent cell death and DNA repair. DNA damage was induced by ultraviolet type C (UVC) radiation, or 10-decarbamoyl mitomycin C (DMC, an agent known to induce mammalian p53-independent cell death). Wild-type or cep-1 loss-of-function mutant animals were assayed for germline cell death and DNA lesions. Wild-type animals displayed greater removal of UVC-lesions over time, whereas cep-1 mutant animals displayed increased UVC-lesion retention. The cep-1 mutation increased UVC-lesion retention directly correlated with a reduction of progeny viability. Consistent with DMC inducing p53-independent cell death in mammalian cells DMC induced a C. elegans p53-independent germline cell death pathway. To examine the influence of wild-type CEP-1 and DNA damage on C. elegans tumors we used glp-1(ar202gf)/Notch germline tumor mutants. UVC treatment of glp-1 mutant animals activated the CEP-1 target gene egl-1 and reduced tumor size. In cep-1(gk138);glp-1(ar202gf) animals, UVC treatment resulted in increased susceptibility to lesions and larger tumorous germlines. Interestingly, the partial knockdown of bec-1 in adults resulted in a CEP-1-dependent increase in germline cell death and an increase in DNA damage. These results strongly support cross-talk between BEC-1 and CEP-1 to protect the C. elegans genome.

8-Amino-adenosine activates p53-independent cell death of metastatic breast cancers.

8-Amino-adenosine (8-NH(2)-Ado) is a ribose sugar nucleoside analogue that reduces cellular ATP levels and inhibits mRNA synthesis. Estrogen receptor-negative (ER-) metastatic breast cancers often contain mutant p53; therefore, we asked if 8-NH(2)-Ado could kill breast cancer cells without activating the p53-pathway. Regardless of the breast cancer subtype tested or the p53 status of the cells, 8-NH(2)-Ado was more cytotoxic than either gemcitabine or etoposide. 8-NH(2)-Ado treatment inhibited cell proliferation, activated cell death, and did not activate transcription of the p53 target gene p21 or increase protein levels of either p53 or p21. This occurred in the estrogen receptor-positive (ER+) MCF-7 cells that express wild-type p53, the ER+ T47-D cells that express mutant p53, and the ER- MDA-MB-468 cells or MDA-MB-231 cells that both express mutant p53. 8-NH(2)-Ado induced apoptotic death of MCF-7 cells and apoptosis was not inhibited by knockdown of functional p53. Moreover, the pan-caspase inhibitor Z-VAD blocked the 8-NH(2)-Ado-induced MCF-7 cell death. Interestingly, 8-NH(2)-Ado caused the MDA-MB-231 cells to detach from the plate with only limited evidence of apoptotic cell death markers and the cell death was not inhibited by Z-VAD. Inhibition of MDA-MB-231 cell autophagy, by reduction of ATG7 or 3-methyladenine treatment, did not block this 8-NH(2)-Ado-mediated cytotoxicity. Importantly 8-NH(2)-Ado was highly cytotoxic to triple-negative breast cancer cells and worked through a pathway that did not require wild-type p53 for cytoxicity. Therefore, 8-NH(2)-Ado should be considered for the treatment of triple-negative breast cancers that are chemotherapy resistant.

C-reactive protein and colorectal adenoma in the CLUE II cohort.

OBJECTIVE: Circulating C-reactive protein concentration has been associated with colorectal cancer in some studies. Whether C-reactive protein is associated with earlier steps in the natural history of this cancer has not been published (aside from an abstract). Thus, we evaluated the association between plasma C-reactive protein concentration and development of colorectal adenoma in a nested case-control study. METHODS: Colorectal adenoma cases (n = 135) and matched controls (n = 269) who had a negative sigmoidoscopy or colonoscopy were identified between baseline in 1989 and 2000 from among participants in the CLUE II cohort of Washington County, Maryland. Cases were confirmed by medical record review. Controls were matched with cases on age, sex, race, date of blood draw, time since last meal, and type of endoscopy. The odds ratio (OR) of adenoma was estimated from conditional logistic regression models. RESULTS: C-reactive protein concentrations were similar between colorectal adenoma cases and controls (median C-reactive protein, 1.31 vs. 1.38 mg/l; p = 0.13). The OR of colorectal adenoma among those in the highest fourth (>2.95 mg/l) of C-reactive protein concentration compared with the lowest fourth (<0.65 mg/l) was 0.61 (95% confidence interval, 0.29-1.25; p for trend = 0.25). CONCLUSIONS: Pre-diagnostic plasma C-reactive protein concentration was not associated with an increased risk of colorectal adenoma. More work is needed to determine whether C-reactive protein is a valid marker of intra-colonic inflammation, and whether such inflammation contributes to the etiology of colorectal neoplasia.

Association among an ornithine decarboxylase polymorphism, androgen receptor gene (CAG) repeat length and prostate cancer risk.

PURPOSE: A single nucleotide substitution of guanine to adenine (A) at base +316 in the ornithine decarboxylase (ODC) gene may be associated with greater ODC expression and increased tumor growth. ODC is induced by androgens in human prostatic epithelial cells, presumably via transcriptional activation of androgen receptor (AR) and also by nicotine. A nested case-control study was done to examine the association between this ODC genotype and prostate cancer risk, and whether it varies by AR gene CAG repeat length and smoking. MATERIALS AND METHODS: A total of 164 cases were matched to 2 controls each from a community based cohort. ODC and AR genotyping was performed using a TaqMan (PE Applied Biosystems, Foster City, California) based assay and automated fragment analysis, respectively. Conditional logistic regression was used to estimate the OR and 95% CI. RESULTS: The presence of the ODC A allele was not significantly associated with an increased risk of prostate cancer (OR 1.33, 95% CI 0.90 to 1.96). However, men who inherited at least 1 ODC A alleles and less than 22 AR CAG repeats were at twice the risk of prostate cancer compared with those with 2 guanine alleles and 22 or greater AR CAG repeats (OR 2.09, 95% CI 1.23 to 3.57). Smoking was associated with prostate cancer only in men carrying a least 1 ODC A allele (p interaction = 0.02). CONCLUSIONS: The ODC A allele was not associated with a statistically significant increased risk of prostate cancer. However, this association may vary according to the number of CAG repeats in the AR receptor and smoking status.