The Role of IgM Antibodies in T Cell Lymphoma Protection in a Novel Model Resembling Anaplastic Large Cell Lymphoma.

MRL/lpr mice typically succumb to immune complex-mediated nephritis within the first year of life. However, MRL/lpr mice that only secrete IgM Abs because of activation-induced deaminase deficiency (AID-/-MRL/lpr mice) experienced a dramatic increase in survival. Further crossing of these mice to those incapable of making secretory IgM (µS mice) generated mice lacking any secreted Abs but with normal B cell receptors. Both strains revealed no kidney pathology, yet Ab-deficient mice still experienced high mortality. In this article, we report Ab-deficient MRL/lpr mice progressed to high-grade T cell lymphoma that can be reversed with injection of autoreactive IgM Abs or following adoptive transfer of IgM-secreting MRL/lpr B cells. Anti-nuclear Abs, particularly anti-dsDNA IgM Abs, exhibited tumor-killing activities against a murine T cell lymphoma cell line. Passive transfers of autoreactive IgM Abs into p53-deficient mice increased survival by delaying onset of T cell lymphoma. The lymphoma originated from a double-negative aberrant T cell population seen in MRL/lpr mice and most closely resembled human anaplastic large cell lymphoma. Combined, these results strongly implicate autoreactive IgM Abs in protection against T cell lymphoma.

Mitochondrial dysfunction and DNA damage accompany enhanced levels of formaldehyde in cultured primary human fibroblasts.

Formaldehyde (FA) is a simple biological aldehyde that is produced inside cells by several processes such as demethylation of DNA and proteins, amino acid metabolism, lipid peroxidation and one carbon metabolism (1-C). Although accumulation of excess FA in cells is known to be cytotoxic, it is unknown if an increase in FA level might be associated with mitochondrial dysfunction. We choose to use primary human fibroblasts cells in culture (foreskin, FSK) as a physiological model to gain insight into whether an increase in the level of FA might affect cellular physiology, especially with regard to the mitochondrial compartment. FSK cells were exposed to increasing concentrations of FA, and different cellular parameters were studied. Elevation in intracellular FA level was achieved and was found to be cytotoxic by virtue of both apoptosis and necrosis and was accompanied by both G2/M arrest and reduction in the time spent in S phase. A gene expression assessment by microarray analysis revealed FA affected FSK cells by altering expression of many genes including genes involved in mitochondrial function and electron transport. We were surprised to observe increased DNA double-strand breaks (DSBs) in mitochondria after exposure to FA, as revealed by accumulation of γH2A.X and 53BP1 at mitochondrial DNA foci. This was associated with mitochondrial structural rearrangements, loss of mitochondrial membrane potential and activation of mitophagy. Collectively, these results indicate that an increase in the cellular level of FA can trigger mitochondrial DNA double-strand breaks and dysfunction.

Oxidative DNA Damage Modulates DNA Methylation Pattern in Human Breast Cancer 1 (BRCA1) Gene via the Crosstalk between DNA Polymerase ß and a de novo DNA Methyltransferase.

DNA damage and base excision repair (BER) are actively involved in the modulation of DNA methylation and demethylation. However, the underlying molecular mechanisms remain unclear. In this study, we seek to understand the mechanisms by exploring the effects of oxidative DNA damage on the DNA methylation pattern of the tumor suppressor breast cancer 1 (BRCA1) gene in the human embryonic kidney (HEK) HEK293H cells. We found that oxidative DNA damage simultaneously induced DNA demethylation and generation of new methylation sites at the CpGs located at the promoter and transcribed regions of the gene ranging from -189 to +27 in human cells. We demonstrated that DNA damage-induced demethylation was mediated by nucleotide misincorporation by DNA polymerase ß (pol ß). Surprisingly, we found that the generation of new DNA methylation sites was mediated by coordination between pol ß and the de novo DNA methyltransferase, DNA methyltransferase 3b (DNMT3b), through the interaction between the two enzymes in the promoter and encoding regions of the BRCA1 gene. Our study provides the first evidence that oxidative DNA damage can cause dynamic changes in DNA methylation in the BRCA1 gene through the crosstalk between BER and de novo DNA methylation.

Role of the oxidized form of XRCC1 in protection against extreme oxidative stress.

The multi-domain protein XRCC1 is without catalytic activity, but can interact with a number of known repair proteins. The interaction between the N-terminal domain (NTD) of XRCC1 and DNA polymerase ß (pol ß) is critical for recruitment of pol ß to sites of DNA damage and repair. Crystallographic and NMR approaches have identified oxidized and reduced forms of the XRCC1 NTD, and the corresponding forms of XRCC1 have been identified in cultured mouse fibroblast cells. Both forms of NTD interact with pol ß, but the interaction is much stronger with the oxidized form. The potential for formation of the C12-C20 oxidized conformation can be removed by alanine substitution at C12 (C12A) leading to stabilized reduced XRCC1 with a lower pol ß binding affinity. Here, we compare cells expressing C12A XRCC1 (XRE8) with those expressing wild-type XRCC1 (XC5). Reduced C12A XRCC1 is detected at sites of micro-irradiation DNA damage, but provides slower recruitment of pol ß. Expression of reduced XRCC1 does not affect sensitivity to MMS or H2O2. In contrast, further oxidative stress imposed by glutathione depletion results in increased sensitization of reduced XRCC1-expressing cells to H2O2 compared with wild-type XRCC1-expressing cells. There is no indication of enhanced H2O2-generated free radicals or DNA strand breaks in XRE8 cells. However, elevated cellular PAR is found following H2O2 exposure, suggesting BER deficiency of H2O2-induced damage in the C12A expressing cells.

Altered pattern of immunoglobulin hypermutation in mice deficient in Slip-GC protein.

We recently identified a novel germinal center GTPase, SLIP-GC, that localizes to replication factories in B cells and that, when reduced, induces DNA breaks in lymphoma B cell lines in an activation-induced deaminase (AID)-dependent manner. Herein, we generated mice deficient in SLIP-GC and examined the impact of SLIP-GC deficiency in immunoglobulin hypermutation and class switch recombination, both AID-dependent mechanisms. SLIP-GC-deficient mice experienced a substantial increase in mutations at G:C base pairs at the region downstream of JH4 in the immunoglobulin heavy chain locus. This change was reflected in the overall mutation frequency, and it was associated with an increase in transitions from G:C base pairs, a hallmark of AID-mediated deamination during replication. In addition, G:C transitions at non-immunoglobulin loci also increased in these mice. Given the intracellular localization of SLIP-GC to sites of replicating DNA, these results suggest that SLIP-GC protects replicating DNA from AID-mediated deamination of cytosines in both strands.

Macrophages, but not T and B lymphocytes, are critical for subepidermal blister formation in experimental bullous pemphigoid: macrophage-mediated neutrophil infiltration depends on mast cell activation.

Bullous pemphigoid (BP) is a subepidermal blistering disease associated with autoantibodies against two hemidesmosomal proteins, BP180 and BP230. Numerous inflammatory cells infiltrate the upper dermis in BP. We have previously shown by passive transfer studies that Abs to the ectodomain of murine BP180 are capable of triggering blisters in mice that closely mimic human BP. Experimental BP depends on complement activation and neutrophil infiltration. In the present study, we investigated the relative contribution of neutrophils, mast cells (MCs), macrophages (Mphi), and lymphocytes and their functional relationship in the immunopathogenesis of this disease model by using mice deficient in these cells. Wild-type, T cell-deficient, and T and B cell-deficient mice injected intradermally with pathogenic anti-murine BP180 IgG exhibited extensive subepidermal blisters. In contrast, mice deficient in neutrophils, MCs, and Mphi were resistant to experimental BP. MCs play a major role in neutrophil recruitment into the dermis. Furthermore, Mphi-mediated neutrophil infiltration depends on MC activation/degranulation.