Poly-ADP ribose polymerase (PARP) inhibitors have shown promise in the treatment of human malignancies characterized by deficiencies in the DNA damage repair proteins BRCA1 and BRCA2 and preclinical studies have demonstrated the potential effectiveness of PARP inhibitors in targeting ataxia-telangiectasia mutated (ATM)-deficient tumours. Here, we show that mantle cell lymphoma (MCL) cells deficient in both ATM and p53 are more sensitive to the PARP inhibitor olaparib than cells lacking ATM function alone. In ATM-deficient MCL cells, olaparib induced DNA-PK-dependent phosphorylation and stabilization of p53 as well as expression of p53-responsive cell cycle checkpoint regulators, and inhibition of DNA-PK reduced the toxicity of olaparib in ATM-deficient MCL cells. Thus, both DNA-PK and p53 regulate the response of ATM-deficient MCL cells to olaparib. In addition, small molecule inhibition of both ATM and PARP was cytotoxic in normal human fibroblasts with disruption of p53, implying that the combination of ATM and PARP inhibitors may have utility in targeting p53-deficient malignancies.

Thrombin-activable fibrinolysis inhibitor (TAFI) has recently been identified as a positive acute phase protein in mice, an observation that may have important implications for the interaction of the coagulation, fibrinolytic, and inflammatory systems. Activated TAFI (TAFIa) inhibits fibrinolysis by removing the carboxylterminal lysines from partially degraded fibrin that are important for maximally efficient plasminogen activation. In addition, TAFIa has been shown to be capable of removing the carboxyl-terminal arginine residues from the anaphylatoxins and bradykinin, thus implying a role for the TAFI pathway in the vascular responses to inflammation. In the current study, we investigated the ability of acute phase mediators to modulate human TAFI gene expression in cultured human hepatoma (HepG2) cells. Surprisingly, we found that treatment of HepG2 cells with a combination of interleukin (IL)-1␤ and IL-6 suppressed endogenous TAFI mRNA abundance in HepG2 cells (ϳ60% decrease), while treatment with IL-1␤ or IL-6 alone had no effect. Treatment with IL-1␤ and/or IL-6 had no effect on TAFI promoter activity as measured using a luciferase reporter plasmid containing the human TAFI 5 -flanking region, whereas treatment with IL-1␤ and IL-6 in combination, but not alone, decreased the stability of the endogenous TAFI mRNA. Treatment with the synthetic glucocorticoid dexamethasone resulted in a 2-fold increase of both TAFI mRNA levels and promoter activity. We identified a functional glucocorticoid response element (GRE) in the human TAFI promoter between nucleotides ؊92 and ؊78. The GRE was capable of binding the glucocorticoid receptor, as assessed by gel mobility shift assays, and mutation of this element markedly decreased the ability of the TAFI promoter to be activated by dexamethasone.Thrombin activable fibrinolysis inhibitor (TAFI) 1 was first identified in 1989 by two independent groups as a basic carboxypeptidase present in fresh serum that was distinct from the constitutive basic carboxypeptidase N (1, 2). By virtue of the intrinsic instability of this enzyme, whose activity disappeared within 2 h upon incubation at 37°C, Hendriks et al.(1) designated the novel activity "unstable" carboxypeptidase or carboxypeptidase U (1). Campbell and Okada (2) determined that the enzyme removed arginine residues from substrates more efficiently than lysines and therefore designated it carboxypeptidase R (2). In 1991, Eaton et al. (3) isolated a cDNA encoding the zymogen form of the enzyme and found that it was highly homologous to pancreatic procarboxypeptidase B. Bajzar et al. (4) independently isolated a protein on the basis of its ability to inhibit fibrinolysis in the setting of sustained activation of the coagulation cascade; on the basis of this property, they named the protein TAFI. Amino acid sequence analysis of TAFI revealed it to be identical to plasma procarboxypeptidase B and procarboxypeptidases U and R. TAFI can be activated by thrombin (4), plasmin (5), and thrombin in complex with thrombomodulin (6), wi...

The cold inducible RNA binding protein (CIRBP) responds to a wide array of cellular stresses, including short wavelength ultraviolet light (UVC), at the transcriptional and post-translational level. CIRBP can bind the 3'untranslated region of specific transcripts to stabilize them and facilitate their transport to ribosomes for translation. Here we used RNA interference and oligonucleotide microarrays to identify potential downstream targets of CIRBP induced in response to UVC. Twenty eight transcripts were statistically increased in response to UVC and these exhibited a typical UVC response. Only 5 of the 28 UVC-induced transcripts exhibited a CIRBP-dependent pattern of expression. Surprisingly, 3 of the 5 transcripts (IL1B, IL8 and TNFAIP6) encoded proteins important in inflammation with IL-1β apparently contributing to IL8 and TNFAIP6 expression in an autocrine fashion. UVC-induced IL1B expression could be inhibited by pharmacological inhibition of NFκB suggesting that CIRBP was affecting NF-κB signaling as opposed to IL1B mRNA stability directly. Bacterial lipopolysaccharide (LPS) was used as an activator of NF-κB to further study the potential link between CIRBP and NFκB. Transfection of siRNAs against CIRBP reduced the extent of the LPS-induced phosphorylation of IκBα, NF-κB DNA binding activity and IL-1β expression. The present work firmly establishes a novel link between CIRBP and NF-κB signaling in response to agents with diverse modes of action. These results have potential implications for disease states associated with inflammation.

Thrombin-activable fibrinolysis inhibitor (TAFI) is a plasma zymogen that acts as a molecular link between coagulation and fibrinolysis. Numerous single nucleotide polymorphisms (SNPs) have been identified in CPB2, the gene encoding TAFI, and are located in the 5-flanking region, in the coding sequences, and in the 3-untranslated region (UTR) of the CPB2 mRNA transcript. Associations between CPB2 SNPs and variation in plasma TAFI antigen concentrations have been described, but the identity of SNPs that are causally linked to this variation is not known. In the current study, we investigated the effect of the SNPs in the 5-flanking region on CPB2 promoter activity and SNPs in the 3-UTR on CPB2 mRNA stability. Whereas the 5-flanking region SNPs (with 2 exceptions) did not have a significant effect on promoter activity, either alone or in haplotypic combinations seen in the human population, all of the 3-UTR SNPs substantially affected mRNA stability. We speculate that these SNPs, in part, contribute to variation in plasma TAFI concentrations via modulation of CPB2 gene expression through an effect on mRNA stability. IntroductionThrombin-activable fibrinolysis inhibitor (TAFI), also known as procarboxypeptidase U or R or plasma procarboxypeptidase B, is a human plasma zymogen that may play a role in mediating the balance between blood coagulation and fibrinolysis. 1 Upon cleavage of TAFI by thrombin, 2 thrombin-thrombomodulin, 3 or plasmin, 4 an enzyme is formed (TAFIa) that possesses basic carboxypeptidase activity. TAFIa has been demonstrated to attenuate plasminogen activation, and thus fibinolysis, by removing from partially degraded fibrin the carboxyl-terminal lysine residues that mediate positive feedback in the fibrinolytic cascade. 5 In addition, TAFIa has been shown to remove the carboxyl-terminal arginine residues from bradykinin and the anaphylatoxins C3a and C5a, thereby implicating the TAFI pathway as a link between coagulation and inflammation. [6][7][8][9] Plasma concentrations of TAFI vary significantly in the human population. 10,11 The vast majority of individuals have TAFI antigen levels between 50% and 150% of the mean population value, 12,13 thereby ranging from approximately 100 to 200 nM. Importantly, these concentrations of TAFI fall below the K m for activation of TAFI by thrombin or thrombomodulin (1 M), 3 indicating that individuals with higher plasma TAFI concentrations would exhibit a higher rate of TAFIa production following a procoagulant stimulus. Indeed, variation in "functional" TAFI concentrations has been observed using a clot lysis assay of plasma samples. 14 Therefore, it is reasonable to consider the gene encoding TAFI (CPB2) as a candidate gene for thrombotic disorders. Indeed, elevated concentrations of TAFI have been shown to be a mild risk factor for first venous thrombosis 15 as well as recurrent venous thrombosis, 16 and to be more common in carriers of Factor V Leiden with venous thromboembolism than in asymptomatic carriers. 17 High functional TAFI concentrations hav...