Analysis of Interleukin-4-Induced Class Switch Recombination in Mouse Myeloma CH12F3-2 Cells.

Affinity maturation of B lymphocytes is a process that includes somatic hypermutation and class switch recombination. Class switch recombination is a fundamental factor of the human adaptive immunity. The perturbation of this process has an adverse effect on human health, and results in global chromosome rearrangements and cell transformation. Evaluation of the class switch recombination efficiency is an important component of laboratory diagnosis of immunotoxic components. Here we describe a method for testing the efficiency of the class switch recombination. Cultivation of mouse myeloma CH12F3-2 cell line with anti-CD40 antibodies, transforming growth factor beta, and recombinant interleukin-4 (IL-4) triggers a cascade of signal transduction network events that lead to switching the immunoglobulin isotypes from IgM to IgA. This chapter describes the methodology of class switch recombination assay for assessment of the effect of environmental pollutants in toxicological laboratory diagnostics.

Metformin Suppressed CXCL8 Expression and Cell Migration in HEK293/TLR4 Cell Line.

Chronic inflammation is associated with cancer. CXCL8 promotes tumor microenvironment construction through recruiting leukocytes and endothelial progenitor cells that are involved in angiogenesis. It also enhances tumor cell proliferation and migration. Metformin, type II diabetes medication, demonstrates anticancer properties via suppressing inflammation, tumor cell proliferation, angiogenesis, and metastasis. This study intended to address the role of metformin in regulation of CXCL8 expression and cell proliferation and migration. Our data indicated that metformin suppressed LPS-induced CXCL8 expression in a dose-dependent manner through inhibiting NF-κB, but not AP-1 and C/EBP, activities under the conditions we used. This inhibitory effect of metformin is achieved through dampening LPS-induced NF-κB nuclear translocation. Cell migration was inhibited by metformin under high dose (10 mM), but not cell proliferation.

N,N-Dimethylacetamide Significantly Attenuates LPS- and TNFα-Induced Proinflammatory Responses Via Inhibition of the Nuclear Factor Kappa B Pathway.

Previously, we have shown that N,N-dimethylacetamide (DMA) prevents inflammation-induced preterm birth in a murine model, inhibits LPS-induced increases in placental pro-inflammatory cytokines and up-regulates the anti-inflammatory cytokine Interleukin-10 (IL-10). However, DMA's mechanism of action remains to be elucidated. In the current study we investigate how DMA produces its anti-inflammatory effect. Using in vitro and ex vivo models, we show that DMA suppresses secretion of pro-inflammatory cytokines in lipopolysaccharide (LPS)-induced RAW 264.7 cells, TNFα-challenged JEG-3 cells and LPS-stimulated human placental explants. DMA significantly attenuated the secretion of TNFα, IL-6, IL-10, and granulocyte macrophage colony stimulating factor (GM-CSF) from LPS-stimulated RAW 264.7 cells, IL-6 secretion from TNFα-stimulated JEG-3 cells and TNFα, IL-6, IL-10, GM-CSF and Interleukin-8 (IL-8) from LPS-stimulated human placental explants. We further investigated if DMA's effect on cytokine expression involves the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and mitogen-activated protein kinase (MAPK) pathways. DMA (10 mM) significantly inhibited nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκBα) degradation in LPS-stimulated RAW 264.7 cells, but there was no significant change in the expression of phosphorylated or native forms of downstream proteins in the MAPK pathway. In addition, DMA significantly attenuated luciferase activity in cells co-transfected with NF-κB-Luc reporter plasmid, but not with AP-1-Luc or CEBP-Luc reporters. Overall, our findings suggest that the anti-inflammatory activity of DMA is mediated by inhibition of the NF-κB pathway via decreased IκBα degradation.

Rev1 is a base excision repair enzyme with 5'-deoxyribose phosphate lyase activity.

Rev1 is a member of the Y-family of DNA polymerases and is known for its deoxycytidyl transferase activity that incorporates dCMP into DNA and its ability to function as a scaffold factor for other Y-family polymerases in translesion bypass events. Rev1 also is involved in mutagenic processes during somatic hypermutation of immunoglobulin genes. In light of the mutation pattern consistent with dCMP insertion observed earlier in mouse fibroblast cells treated with a base excision repair-inducing agent, we questioned whether Rev1 could also be involved in base excision repair (BER). Here, we uncovered a weak 5'-deoxyribose phosphate (5'-dRP) lyase activity in mouse Rev1 and demonstrated the enzyme can mediate BER in vitro The full-length Rev1 protein and its catalytic core domain are similar in their ability to support BER in vitro The dRP lyase activity in both of these proteins was confirmed by NaBH4 reduction of the Schiff base intermediate and kinetics studies. Limited proteolysis, mass spectrometry and deletion analysis localized the dRP lyase active site to the C-terminal segment of Rev1's catalytic core domain. These results suggest that Rev1 could serve as a backup polymerase in BER and could potentially contribute to AID-initiated antibody diversification through this activity.

Bortezomib inhibits expression of TGF-ß1, IL-10, and CXCR4, resulting in decreased survival and migration of cutaneous T cell lymphoma cells.

Increased expression of the immunosuppressive cytokines, TGF-ß1 and IL-10, is a hallmark of the advanced stages of cutaneous T cell lymphoma (CTCL), where it has been associated with suppressed immunity, increased susceptibility to infections, and diminished antitumor responses. Yet, little is known about the transcriptional regulation of TGF-ß1 and IL-10 in CTCL, and about their function in regulating the CTCL cell responses. In this article, we show that TGF-ß1 and IL-10 expression in CTCL cells is regulated by NF-κB and suppressed by bortezomib (BZ), which has shown promising results in the treatment of CTCL. However, although the TGF-ß1 expression is IκBα dependent and is regulated by the canonical pathway, the IL-10 expression is IκBα independent, and its inhibition by BZ is associated with increased promoter recruitment of p52 that characterizes the noncanonical pathway. TGF-ß1 suppression decreases CTCL cell viability and increases apoptosis, and adding exogenous TGF-ß1 increases viability of BZ-treated CTCL cells, indicating TGF-ß1 prosurvival function in CTCL cells. In addition, TGF-ß1 suppression increases expression of the proinflammatory cytokines IL-8 and IL-17 in CTCL cells, suggesting that TGF-ß1 also regulates the IL-8 and IL-17 expression. Importantly, our results demonstrate that BZ inhibits expression of the chemokine receptor CXCR4 in CTCL cells, resulting in their decreased migration, and that the CTCL cell migration is mediated by TGF-ß1. These findings provide the first insights into the BZ-regulated TGF-ß1 and IL-10 expression in CTCL cells, and indicate that TGF-ß1 has a key role in regulating CTCL survival, inflammatory gene expression, and migration.

Evaluation of the adverse effect of low concentration of cadmium on interleukin-4 induced class switch recombination in Burkett's lymphoma Raji cell line.

Affinity maturation of B lymphocytes, a process that includes somatic hypermutation and class switch recombination, initiates global DNA rearrangements. The interruption of this process has an adverse effect on human health and results in immunodeficiency and autoimmune disease. Class switch recombination is a fundamental factor of the human adaptive immunity. Evaluation of the class switch recombination efficiency is an important component of laboratory diagnostic of immunotoxic components. Here, we describe a method for testing the efficiency of the class switch recombination. Cultivation of Raji Burkett's lymphoma cell line with anti-CD40 antibodies and recombinant interleukin-4 (IL-4) triggers a cascade of signal transduction network events that lead to switching the immunoglobulin isotopes from IgM to IgE. This chapter describes the methodology of class switch recombination assay for assessment of the effect of the environmental pollutants in toxicological laboratory diagnostics.

Analysis of LPS-induced, NFκB-dependent interleukin-8 transcription in kidney embryonic cell line expressing TLR4 using luciferase assay.

Gene expression is orchestrated by a complex network of signal transduction pathways that typically originate on cell surface receptors and culminate in DNA-binding transcription factors, which translocate to the nucleus and bind cis-regulatory elements in promoter regions of genes, thereby inducing de novo synthesis of the nascent RNA transcripts and their splicing. Gene expression arrays monitor abundance of the matured, spliced cDNA, which undergoes additional posttranscriptional modifications that greatly affect the half-life of the cDNA. Thus, the relative abundance of cDNA is not necessarily commensurable with the activity of promoters of the corresponding genes. In contrast, reporter gene assays provide valuable insight into the regulation of gene expression at the level of transcription and allow for discerning the contribution of individual transcription factors into changes in gene expression. Here, we describe a robust reporter gene assay method that is useful for exploration of transcription regulatory network, which regulates gene expression in response to inflammation. The method is exemplified by using the promoter region of the prototypic pro-inflammatory chemokine interleukin-8 (IL-8, CXCL8), which plays an important role in immune response as well as carcinogenesis. Using the luciferase reporter gene assay, we analyze the activation status of the IL-8 promoter in lipopolysaccharide (LPS)-stimulated human embryonic kidney cells.

Proteasome inhibition increases recruitment of IκB kinase ß (IKKß), S536P-p65, and transcription factor EGR1 to interleukin-8 (IL-8) promoter, resulting in increased IL-8 production in ovarian cancer cells.

Proinflammatory and pro-angiogenic chemokine interleukin-8 (IL-8, CXCL8) contributes to ovarian cancer progression through its induction of tumor cell proliferation, survival, angiogenesis, and metastasis. Proteasome inhibition by bortezomib, which has been used as a frontline therapy in multiple myeloma, has shown only limited effectiveness in ovarian cancer and other solid tumors. However, the responsible mechanisms remain elusive. Here, we show that proteasome inhibition dramatically increases the IL-8 expression and release in ovarian cancer cells. The responsible mechanism involves an increased nuclear accumulation of IκB kinase ß (IKKß) and an increased recruitment of the nuclear IKKß, p65-phosphorylated at Ser-536, and the transcription factor early growth response-1 (EGR-1) to the endogenous IL-8 promoter. Coimmunoprecipitation studies identified the nuclear EGR-1 associated with IKKß and with p65, with preferential binding to S536P-p65. Both IKKß activity and EGR-1 expression are required for the increased IL-8 expression induced by proteasome inhibition in ovarian cancer cells. Interestingly, in multiple myeloma cells the IL-8 release is not increased by bortezomib. Together, these data indicate that the increased IL-8 release may represent one of the underlying mechanisms responsible for the decreased effectiveness of proteasome inhibition in ovarian cancer treatment and identify IKKß and EGR-1 as potential new targets in ovarian cancer combination therapies.

Evidence for abasic site sugar phosphate-mediated cytotoxicity in alkylating agent treated Saccharomyces cerevisiae.

To better understand alkylating agent-induced cytotoxicity and the base lesion DNA repair process in Saccharomyces cerevisiae, we replaced the RAD27(FEN1) open reading frame (ORF) with the ORF of the bifunctional human repair enzyme DNA polymerase (Pol) ß. The aim was to probe the effect of removal of the incised abasic site 5'-sugar phosphate group (i.e., 5'-deoxyribose phosphate or 5'-dRP) in protection against methyl methanesulfonate (MMS)-induced cytotoxicity. In S. cerevisiae, Rad27(Fen1) was suggested to protect against MMS-induced cytotoxicity by excising multinucleotide flaps generated during repair. However, we proposed that the repair intermediate with a blocked 5'-end, i.e., 5'-dRP group, is the actual cytotoxic lesion. In providing a 5'-dRP group removal function mediated by dRP lyase activity of Pol ß, the effects of the 5'-dRP group were separated from those of the multinucleotide flap itself. Human Pol ß was expressed in S. cerevisiae, and this partially rescued the MMS hypersensitivity observed with rad27(fen1)-null cells. To explore this rescue effect, altered forms of Pol ß with site-directed eliminations of either the 5'-dRP lyase or polymerase activity were expressed in rad27(fen1)-null cells. The 5'-dRP lyase, but not the polymerase activity, conferred the resistance to MMS. These results suggest that after MMS exposure, the 5'-dRP group in the repair intermediate is cytotoxic and that Rad27(Fen1) protection against MMS in wild-type cells is due to elimination of the 5'-dRP group.

Rapid profiling of disease alleles using a tunable reporter of protein misfolding.

Many human diseases are caused by genetic mutations that decrease protein stability. Such mutations may not specifically affect an active site, but can alter protein folding, abundance, or localization. Here we describe a high-throughput cell-based stability assay, IDESA (intra-DHFR enzyme stability assay), where stability is coupled to cell proliferation in the model yeast, Saccharomyces cerevisiae. The assay requires no prior knowledge of a protein's structure or activity, allowing the assessment of stability of proteins that have unknown or difficult to characterize activities, and we demonstrate use with a range of disease-relevant targets, including human alanine:glyoxylate aminotransferase (AGT), superoxide dismutase (SOD-1), DJ-1, p53, and SMN1. The assay can be carried out on hundreds of disease alleles in parallel or used to identify stabilizing small molecules (pharmacological chaperones) for unstable alleles. As demonstration of the general utility of this assay, we analyze stability of disease alleles of AGT, deficiency of which results in the kidney stone disease, primary hyperoxaluria type I, identifying mutations that specifically affect the protein-active site chemistry.

Mutagenesis dependent upon the combination of activation-induced deaminase expression and a double-strand break.

We explored DNA metabolic events potentially relevant to somatic hypermutation (SHM) of immunoglobulin genes using a yeast model system. Double-strand break (DSB) formation has been discussed as a possible component of the SHM process during immunoglobulin gene maturation. Yet, possible mechanisms linking DSB formation with mutagenesis have not been well understood. In the present study, a linkage between mutagenesis in a reporter gene and a double-strand break at a distal site was examined as a function of activation-induced deaminase (AID) expression. Induction of the DSB was found to be associated with mutagenesis in a genomic marker gene located 7 kb upstream of the break site: mutagenesis was strongest with the combination of AID expression and DSB induction. The mutation spectrum of this DSB and AID-mediated mutagenesis was characteristic of replicative bypass of uracil in one strand and was dependent on expression of DNA polymerase delta (Polδ). These results in a yeast model system illustrate that the combination of DSB induction and AID expression could be associated with mutagenesis observed in SHM. Implications of these findings for SHM of immunoglobulin genes in human B cells are discussed.

HMGB1 is a cofactor in mammalian base excision repair.

Deoxyribose phosphate (dRP) removal by DNA polymerase beta (Pol beta) is a pivotal step in base excision repair (BER). To identify BER cofactors, especially those with dRP lyase activity, we used a Pol beta null cell extract and BER intermediate as bait for sodium borohydride crosslinking. Mass spectrometry identified the high-mobility group box 1 protein (HMGB1) as specifically interacting with the BER intermediate. Purified HMGB1 was found to have weak dRP lyase activity and to stimulate AP endonuclease and FEN1 activities on BER substrates. Coimmunoprecipitation experiments revealed interactions of HMGB1 with known BER enzymes, and GFP-tagged HMGB1 was found to accumulate at sites of oxidative DNA damage in living cells. HMGB1(-/-) mouse cells were slightly more resistant to MMS than wild-type cells, probably due to the production of fewer strand-break BER intermediates. The results suggest HMGB1 is a BER cofactor capable of modulating BER capacity in cells.

Down-regulation of DNA polymerase beta accompanies somatic hypermutation in human BL2 cell lines.

Somatic hypermutation (SHM) is a fundamental process in immunoglobulin gene maturation that results in increased affinity of antibodies toward antigens. In one hypothesis explaining SHM in human B cells, the process is initiated by enzymatic deamination of cytosine to uracil in the immunoglobulin gene V-region and this in turn triggers mutation-prone forms of uracil-DNA base excision repair (BER). Yet, an uncertainty with this model is that BER of uracil-DNA in mammalian cells is generally error-free, wherein DNA polymerase beta (pol beta) conducts gap-filling synthesis by insertion of bases according to Watson-Crick rules. To evaluate this inconsistency, we examined pol beta expression in various SHM proficient human BL2 cell line subclones. We report that expression of pol beta in SHM proficient cell lines was strongly down-regulated. In contrast, in other BL2 subclones, we found that SHM was deficient and that pol beta expression was much higher than in the SHM proficient subclones. We also found that overexpression of recombinant human pol beta in a SHM proficient subclone abrogated its capacity for SHM. These results suggest that down-regulation of the normal BER gap-filling DNA polymerase, pol beta, accompanies induced SHM in BL2 cells. This is consistent with the hypothesis that normal error-free BER must be silenced to make way for an error-prone BER process that may be required during somatic hypermutation.

DNA polymerase lambda protects mouse fibroblasts against oxidative DNA damage and is recruited to sites of DNA damage/repair.

DNA polymerase lambda (pol lambda) is a member of the X family of DNA polymerases that has been implicated in both base excision repair and non-homologous end joining through in vitro studies. However, to date, no phenotype has been associated with cells deficient in this DNA polymerase. Here we show that pol lambda null mouse fibroblasts are hypersensitive to oxidative DNA damaging agents, suggesting a role of pol lambda in protection of cells against the cytotoxic effects of oxidized DNA. Additionally, pol lambda co-immunoprecipitates with an oxidized base DNA glycosylase, single-strand-selective monofunctional uracil-DNA glycosylase (SMUG1), and localizes to oxidative DNA lesions in situ. From these data, we conclude that pol lambda protects cells against oxidative stress and suggest that it participates in oxidative DNA damage base excision repair.

REV1 mediated mutagenesis in base excision repair deficient mouse fibroblast.

The DNA polymerase beta (Pol beta) null background renders mouse embryonic fibroblast (MEF) cells base excision repair deficient and hyper-mutagenic upon treatment with the monofunctional alkylating agent, methyl methanesulfonate (MMS). This effect involves an increase in all types of base substitutions, with a modest predominance of G to A transitions. In the present study, we examined the hypothesis that the MMS-induced mutagenesis in the Pol beta null MEF system is due to a lesion bypass mechanism. We studied the effect of RNAi mediated down-regulation of the lesion bypass factor REV1. The steady-state level of REV1 protein was reduced by more than 95% using stable expression of a siRNA construct in a Pol beta null cell line. We found that REV1 expression is required for the MMS-induced mutagenesis phenotype of Pol beta null MEF cells. In contrast, cell survival after MMS treatment is not reduced in the absence of REV1.

Recombinogenic phenotype of human activation-induced cytosine deaminase.

Class switch recombination, gene conversion, and somatic hypermutation that diversify rearranged Ig genes to produce various classes of high affinity Abs are dependent on the enzyme activation-induced cytosine deaminase (AID). Evidence suggests that somatic hypermutation is due to error-prone DNA repair that is initiated by AID-mediated deamination of cytosine in DNA, whereas the mechanism by which AID controls recombination remains to be elucidated. In this study, using a yeast model system, we have observed AID-dependent recombination. Expression of human AID in wild-type yeast is mutagenic for G-C to A-T transitions, and as expected, this mutagenesis is increased upon inactivation of uracil-DNA glycosylase. AID expression also strongly induces intragenic mitotic recombination, but only in a strain possessing uracil-DNA glycosylase. Thus, the initial step of base excision repair is required for AID-dependent recombination and is a branch point for either hypermutagenesis or recombination.