Genomic variation in captive deer mouse (Peromyscus maniculatus) populations.

BACKGROUND: Deer mice (genus Peromyscus) are the most common rodents in North America. Despite the availability of reference genomes for some species, a comprehensive database of polymorphisms, especially in those maintained as living stocks and distributed to academic investigators, is missing. In the present study we surveyed two populations of P. maniculatus that are maintained at the Peromyscus Genetic Stock Center (PGSC) for polymorphisms across their 2.5 × 109 bp genome. RESULTS: High density of variation was identified, corresponding to one SNP every 55 bp for the high altitude stock (SM2) or 207 bp for the low altitude stock (BW) using snpEff (v4.3). Indels were detected every 1157 bp for BW or 311 bp for SM2. The average Watterson estimator for the BW and SM2 populations is 248813.70388 and 869071.7671 respectively. Some differences in the distribution of missense, nonsense and silent mutations were identified between the stocks, as well as polymorphisms in genes associated with inflammation (NFATC2), hypoxia (HIF1a) and cholesterol metabolism (INSIG1) and may possess value in modeling pathology. CONCLUSIONS: This genomic resource, in combination with the availability of P. maniculatus from the PGSC, is expected to promote genetic and genomic studies with this animal model.

Core circadian protein CLOCK is a positive regulator of NF-κB-mediated transcription.

The circadian clock controls many physiological parameters including immune response to infectious agents, which is mediated by activation of the transcription factor NF-κB. It is widely accepted that circadian regulation is based on periodic changes in gene expression that are triggered by transcriptional activity of the CLOCK/BMAL1 complex. Through the use of a mouse model system we show that daily variations in the intensity of the NF-κB response to a variety of immunomodulators are mediated by core circadian protein CLOCK, which can up-regulate NF-κB-mediated transcription in the absence of BMAL1; moreover, BMAL1 counteracts the CLOCK-dependent increase in the activation of NF-κB-responsive genes. Consistent with its regulatory function, CLOCK is found in protein complexes with the p65 subunit of NF-κB, and its overexpression correlates with an increase in specific phosphorylated and acetylated transcriptionally active forms of p65. In addition, activation of NF-κB in response to immunostimuli in mouse embryonic fibroblasts and primary hepatocytes isolated from Clock-deficient mice is significantly reduced compared with WT cells, whereas Clock-Δ19 mutation, which reduces the transactivation capacity of CLOCK on E-box-containing circadian promoters, has no effect on the ability of CLOCK to up-regulate NF-κB-responsive promoters. These findings establish a molecular link between two essential determinants of the circadian and immune mechanisms, the transcription factors CLOCK and NF-κB, respectively.

Curaxins: anticancer compounds that simultaneously suppress NF-κB and activate p53 by targeting FACT.

Effective eradication of cancer requires treatment directed against multiple targets. The p53 and nuclear factor κB (NF-κB) pathways are dysregulated in nearly all tumors, making them attractive targets for therapeutic activation and inhibition, respectively. We have isolated and structurally optimized small molecules, curaxins, that simultaneously activate p53 and inhibit NF-κB without causing detectable genotoxicity. Curaxins demonstrated anticancer activity against all tested human tumor xenografts grown in mice. We report here that the effects of curaxins on p53 and NF-κB, as well as their toxicity to cancer cells, result from "chromatin trapping" of the FACT (facilitates chromatin transcription) complex. This FACT inaccessibility leads to phosphorylation of the p53 Ser(392) by casein kinase 2 and inhibition of NF-κB-dependent transcription, which requires FACT activity at the elongation stage. These results identify FACT as a prospective anticancer target enabling simultaneous modulation of several pathways frequently dysregulated in cancer without induction of DNA damage. Curaxins have the potential to be developed into effective and safe anticancer drugs.

Inhibition of encephalomyocarditis virus and poliovirus replication by quinacrine: implications for the design and discovery of novel antiviral drugs.

The 9-aminoacridine (9AA) derivative quinacrine (QC) has a long history of safe human use as an antiprotozoal and antirheumatic agent. QC intercalates into DNA and RNA and can inhibit DNA replication, RNA transcription, and protein synthesis. The extent of QC intercalation into RNA depends on the complexity of its secondary and tertiary structure. Internal ribosome entry sites (IRESs) that are required for initiation of translation of some viral and cellular mRNAs typically have complex structures. Recent work has shown that some intercalating drugs, including QC, are capable of inhibiting hepatitis C virus IRES-mediated translation in a cell-free system. Here, we show that QC suppresses translation directed by the encephalomyocarditis virus (EMCV) and poliovirus IRESs in a cell-free system and in virus-infected HeLa cells. In contrast, IRESs present in the mammalian p53 transcript that are predicted to have less-complex structures were not sensitive to QC. Inhibition of IRES-mediated translation by QC correlated with the affinity of binding between QC and the particular IRES. Expression of viral capsid proteins, replication of viral RNAs, and production of virus were all strongly inhibited by QC (and 9AA). These results suggest that QC and similar intercalating drugs could potentially be used for treatment of viral infections.

Anti-malaria drug blocks proteotoxic stress response: anti-cancer implications.

The number of physical conditions and chemical agents induce accumulation of misfolded proteins creating proteotoxic stress. This leads to activation of adaptive pro-survival pathway, known as heat shock response (HSR), resulting in expression of additional chaperones. Several cancer treatment approaches, such as proteasome inhibitor Bortezomib and hsp90 inhibitor geldanamycin, involve activation of proteotoxic stress. Low efficacy of these therapies is likely due to the protective effects of HSR induced in treated cells, making this pathway an attractive target for pharmacological suppression. We found that the anti-malaria drugs quinacrine (QC) and emetine prevented HSR in cancer cells, as judged by induction of hsp70 expression. As opposed to emetine, which inhibited general translation, QC did not affect protein synthesis, but rather suppressed inducible HSF1-dependent transcription of the hsp70 gene in a relatively selective manner. The treatment of tumor cells in vitro with a combination of non-toxic concentrations of QC and proteotoxic stress inducers resulted in rapid induction of apoptosis. The effect was similar if QC was substituted by siRNA against hsp70, suggesting that the HSR inhibitory activity of QC was responsible for cell sensitization to proteotoxic stress inducers. QC was also found to enhance the antitumor efficacy of proteotoxic stress inducers in vivo: combinatorial treatment with 17-DMAG + QC resulted in suppression of tumor growth in two mouse syngeneic models. These results reveal that QC is an inhibitor of HSF1-mediated HSR. As such, this compound has significant clinical potential as an adjuvant in therapeutic strategies aimed at exploiting the cytotoxic potential of proteotoxic stress.

Targeting transcription factor NFkappaB: comparative analysis of proteasome and IKK inhibitors.

Nuclear factorkappaB (NFkappaB) plays a critical role in cancer development and progression. Thus, the NFkappaB signaling pathway provides important targets for cancer chemoprevention and anticancer chemotherapy. The central steps in NFkappaB activation are phosphorylation and proteasome-dependent degradation of its inhibitory proteins termed IkappaBs. Consequently, the major pharmacological approaches to target NFkappaB include (1) repression of IkappaB kinases (IKKs) and (2) blocking the degradation of IkappaBs by proteasome inhibitors. We quantitatively compared the efficacy of various proteasome inhibitors (MG132, lactacystin and epoxomicin) and IKK inhibitors (BAY 11-7082 and PS1145) to block NFkappaB activity induced by TNFalpha or TPA and to sensitize LNCaP prostate carcinoma cells to apoptosis. Our studies revealed significant differences between these two classes of NFkappaB inhibitors. We found that proteasome inhibitors epoxomicin and MG132 attenuated NFkappaB induction much more effectively than the IKK inhibitors. Furthermore, in contrast to IKK inhibitors, all studied proteasome inhibitors specifically blocked TPA-induced generation de novo of NFkappaB p50 homodimers--(p50/p50). These results suggest that the proteasome plays a dominant role in TPA-induced formation of functional p50 homodimers, while IKK activity is less important for this process. Interestingly, profound attenuation of p50/p50 DNA-binding does not reduce the high potency of proteasome inhibitors to suppress NFkappaB-dependent transcription. Finally, proteasome inhibitors were much more effective in sensitizing LNCaP cells to TNFalpha-induced apoptosis compared to IKK inhibitors at the concentrations when both types of agents similarly attenuated NFkappaB activity. We conclude that this remarkable pro-apoptotic potential of proteasome inhibitors is partially mediated through NFkappaB-independent mechanism.

Quinacrine inhibits the epidermal dendritic cell migration initiating T cell-mediated skin inflammation.

Quinacrine (QC) is an anti-inflammatory drug that has been used for the treatment of malaria and rheumatoid diseases. The mechanism(s) underlying the anti-inflammatory activity of QC remains poorly understood. We recently reported the QC-mediated inhibition of the NF-kappaB pathway using an in vitro model. To test this potential mechanism in vivo, we used the contact hypersensitivity response (CHS) to chemical allergen sensitization and challenge in mice as a model of skin inflammation. The results indicated that QC treatment inhibited NF-kappaB activation in the skin during allergen sensitization. This inhibition was reflected by decreased mRNA expression and protein production of the NF-kappaB-dependent cytokines TNF-alpha and IL-1beta and the chemokine CCL21 in the skin. The decreases in these cytokines resulted in reduced migration of allergen-presenting dendritic cells from the skin into skin-draining lymph nodes and markedly decreased activation of effector CD8+ T cells for the CHS response to allergen challenge (inhibitory concentration 50% or IC50 was 55 mg/kg). These findings reveal a previously unrecognized mechanism of QC-mediated inhibition of inflammation.

Selenium compounds inhibit I kappa B kinase (IKK) and nuclear factor-kappa B (NF-kappa B) in prostate cancer cells.

Selenium compounds are potential chemopreventive agents for prostate cancer. There are several proposed mechanisms for their anticancer effect, including enhanced apoptosis of transformed cells. Because the transcription factor nuclear factor-kappa B (NF-kappa B) is often constitutively activated in tumors and is a key antiapoptotic factor in mammalian cells, we tested whether selenium inhibited NF-kappa B activity in prostate cancer cells. In our work, we used sodium selenite and a novel synthetic compound, methylseleninic acid (MSeA), that served as a precursor of the putative active monomethyl metabolite methylselenol. We found that both selenium forms inhibited cell growth and induced apoptosis in DU145 and JCA1 prostate carcinoma cells. Sodium selenite and MeSeA, at the concentrations that induced apoptosis, inhibited NF-kappa B DNA binding induced by tumor necrosis factor-alpha and lipopolysaccharide in DU145 and JCA1 prostate cells. Both compounds also inhibited kappa B. Luciferase reporter activity in prostate cells. A key to NF-kappa B regulation is the inhibitory kappa B (I kappa B) proteins that in response to diverse stimuli are rapidly phosphorylated by I kappa B kinase complex, ubiquitinated, and undergo degradation, releasing NF-kappa B factor. We showed that sodium selenite and MSeA inhibited I kappa B kinase activation and I kappa B-alpha phosphorylation and degradation induced by TNF-alpha and lipopolysaccharide in prostate cells. NF-kappa B blockage by I kappa B-alpha d.n. mutant resulted in the sensitization of prostate carcinoma cells to apoptosis induced by selenium compounds. These results suggest that selenium may target the NF-kappa B activation pathway to exert, at least in part, its cancer chemopreventive effect in prostate.

The role of IKK in constitutive activation of NF-kappaB transcription factor in prostate carcinoma cells.

Rel/NF-kappaB transcription factors are implicated in the control of cell proliferation, apoptosis and transformation. The key to NF-kappaB regulation is the inhibitory IkappaB proteins. During response to diverse stimuli, IkappaBs are rapidly phosphorylated by IkappaB kinases (IKKs), ubiquitinated and undergo degradation. We have investigated the expression and function of NF-kappaB, IkappaB inhibitors and IKKs in normal prostate epithelial cells and prostate carcinoma (PC) cell lines LNCaP, MDA PCa 2b, DU145, PC3, and JCA1. We found that NF-kappaB was constitutively activated in human androgen-independent PC cell lines DU145, PC3, JCA1 as well as androgen-independent CL2 cells derived from LNCaP. In spite of a strong difference in constitutive kappaB binding, Western blot analysis did not reveal any significant variance in the expression of p50, p65, IkappaBs, IKKalpha, and IKKbeta between primary prostate cells, androgen-dependent and androgen-independent PC cells. However, we found that in androgen-independent PC cells IkappaBalpha was heavily phosphorylated and displayed a faster turnover. Using an in vitro kinase assay we demonstrated constitutive activation of IKK in androgen-independent PC cell lines. Blockage of NF-kappaB activity in PC cells by dominant-negative IkappaBalpha resulted in increased constitutive and TNF-alpha-induced apoptosis. Our data suggest that increased IKK activation leads to the constitutive activation of NF-kappaB 'survival signaling' pathway in androgen-independent PC cells. This may be important for the support of their androgen-independent status and growth advantage.