Novel regulation of MHC class II function in B cells

The presence of post-translational regulation of MHC class II (MHC II) under physiological conditions has been demonstrated recently in dendritic cells (DCs) that potently function as antigen-presenting cells (APCs). Here, we report that MARCH-I, an E3 ubiquitin ligase, plays a pivotal role in the post-translational regulation of MHC II in B cells. MARCH-I expression was particularly high in B cells, and the forced expression of MARCH-I induced the ubiquitination of MHC II. In B cells from MARCH-I-deficient mice (MARCH-I KO), the half-life of surface MHC II was prolonged and the ubiquitinated form of MHC II completely disappeared. In addition, MARCH-I-deficient B cells highly expressed exogenous antigen-loaded MHC II on their surface and showed high ability to present exogenous antigens. These results suggest that the function of MHC II in B cells is regulated through ubiquitination by MARCH-I.     

Co-exposure to benzo[a]pyrene and UVA induces phosphorylation of histone H2AX

Phosphorylation of histone H2AX (termed gamma-H2AX) was recently identified as an early event after induction of DNA double strand breaks (DSBs). We have previously shown that co-exposure to benzo[a]pyrene (BaP), a wide-spread environmental carcinogen, and ultraviolet A (UVA), a major component of solar UV radiation, induced DSBs in mammalian cells. In the present study, we examined whether co-exposure to BaP and UVA generates gamma-H2AX in CHO-K1 cells. Single treatment with BaP (10(-9)-10(-7)M) or UVA ( approximately 2.4 J/cm(2)) did not result in gamma-H2AX, however, co-exposure drastically induced foci of gamma-H2AX in a dose-dependent manner. gamma-H2AX could be detected even at very low concentration of BaP (10(-9)M) plus UVA (0.6J/cm(2)), which did not change cell survival rates. NaN(3) effectively inhibited the formation of gamma-H2AX induced by co-exposure, indicating the contribution of singlet oxygen. This is the first evidence that co-exposure to BaP and UVA induced DSBs, involving gamma-H2AX.     

Benzo[a]pyrene exposed to solar-simulated light inhibits apoptosis and augments carcinogenicity

Polycyclic aromatic hydrocarbons (PAHs) such as benzo[a]pyrene (BaP) are widespread environmental pollutants and several lines of experimental evidence have suggested a role in carcinogenesis. PAHs in the environment are exposed to sunlight and photomodified PAHs have been detected in contaminated sediment and air particulate matter; however, the carcinogenicity of photomodified PAHs is not well understood. In this study, we found that solar-simulated light-irradiated BaP (LBaP) inhibited apoptosis, leading to cancer. LBaP suppressed apoptosis induced by cell detachment and serum depletion in a dose and light-irradiated time-dependent manner. The antiapoptotic effect was related to the production of reactive oxygen species from degraded BaP. The cells that survived apoptosis by LBaP treatment were transformed having the ability to form colonies in soft agar and tumors in nude mice. These capabilities were specific to LBaP, not BaP itself. The results suggested that the carcinogenicity of PAHs may be attributable not only to the genetic damage induced by their metabolites, but also to the antiapoptotic effects of oxidative products on exposure to sunlight.     

Hydrodynamic-based delivery of an interleukin-22-Ig fusion gene ameliorates experimental autoimmune myocarditis in rats

IL-22 is one of several cytokines with limited homology to IL-10. However, the biological activities of IL-22 are mostly unknown. The purpose of this study was to evaluate the effect of IL-22 on rat experimental autoimmune myocarditis (EAM) and elucidate an aspect of the biological activities of IL-22. Rats were immunized on day 0; IL-22-Ig-treated rats were injected with pCAGGS-IL-22-Ig and control rats with pCAGGS-Ig using hydrodynamics-based gene delivery on day 1 or day 6. IL-22-Ig gene therapy administered on day 1 or day 6 after immunization was effective in controlling EAM as monitored by the heart weight to body weight ratio, and the myocarditis area in rats was sacrificed on day 17. Examination of the expression of IL-22-related genes in purified cells from EAM hearts suggested that IL-22-Ig acting target cells were noncardiomyocytic (NC) noninflammatory cells such as fibroblasts, smooth muscle cells, and endothelial cells. Therefore, we examined the effect of rIL-22 or serum containing IL-22-Ig on the expression of immune-relevant genes in IL-1-stimulated NC cells cultured from EAM hearts. Results showed that the expression of immunologic molecules (PGE synthase, cyclooxygenase-2, MIP-2, MCP-1, IL-6, and cytokine-induced neutrophil chemoattractant-2) in IL-1-stimulated NC cells was significantly decreased by rIL-22 or serum containing IL-22-Ig. EAM was suppressed by hydrodynamics-based delivery of plasmid DNA encoding IL-22-Ig, and the reason for this effectiveness may be that IL-22 suppressed gene expression of PG synthases, IL-6, and chemokines in activated NC noninflammatory cells.     

Hydrogen peroxide is critical for UV-induced apoptosis inhibition

Apoptosis is an important cell death system that deletes damaged and mutated cells, preventing the induction of cancer. We previously have reported that UV irradiation inhibited the apoptosis induced by serum starvation and cell detachment. This phenomenon is suitable for clarifying the relationship between cancer and the dysregulation of apoptosis by UV irradiation. Here, we have studied the factors responsible for this inhibition of apoptosis, focusing on reactive oxygen species (ROS) and DNA damage. Treatment with xanthine oxidase in the presence of hypoxanthine, which is known to produce superoxide anion (O2*-) and hydrogen peroxide (H2O2), inhibited the induction of apoptosis. The xanthine oxidase-induced anti-apoptotic effect was suppressed in the presence of an H2O2-eliminating enzyme, catalase, but not in the presence of an O2*--eliminating enzyme, superoxide dismutase. Treatment with H2O2 itself significantly inhibited the induction of apoptosis. Furthermore, the effect of the inhibition of cell death by UVB irradiation and by H2O2 treatment decreased in H2O2-resistant cells. Although both UVB and H2O2 are known to induce DNA damage, other DNA damaging agents, like gamma-irradiation and treatment with cisplatin and bleomycin, showed no inhibition of apoptosis. These findings suggested that H2O2 was essential to the inhibition of apoptosis, in which DNA damage had no role.     

l-Leucine induces growth arrest and persistent ERK activation in glioma cells

Glioma is the most common type of brain tumor, and has the worst prognosis in human malignancy. Experimental evidence suggests that the use of high concentrations of various amino acids may perturb neoplastic cell growth. Thus, the aim of this study was to investigate whether essential amino acids can alter the growth and proliferation of glioma cells. Studies were performed using C6 rat glioma cell lines. High concentration of L-leucine induced growth arrest of glioma cell lines. Terminal transferase uridyl nick end labeling assay and cell cycle analysis showed that the effect of L-leucine on glioma cells growth was not cytotoxic, but rather cytostatic. Additionally, the extracellular signal-regulated protein kinase was activated in L-leucine-treated glioma cells, and inhibition of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1 (MEK) enhanced the effect of L-leucine on glioma cell growth. These data suggest that high concentration L-leucine combined with inhibition of MEK is a potential strategy for glioma cell growth arrest.     

Photomechanical Wave-Driven Delivery of siRNAs Targeting Intermediate Filament Proteins Promotes Functional Recovery after Spinal Cord Injury in Rats

The formation of glial scars after spinal cord injury (SCI) is one of the factors inhibiting axonal regeneration. Glial scars are mainly composed of reactive astrocytes overexpressing intermediate filament (IF) proteins such as glial fibrillary acidic protein (GFAP) and vimentin. In the current study, we delivered small interfering RNAs (siRNAs) targeting these IF proteins to SCI model rats using photomechanical waves (PMWs), and examined the restoration of motor function in the rats. PMWs are generated by irradiating a light-absorbing material with 532-nm nanosecond laser pulses from a Q-switched Nd:YAG laser. PMWs can site-selectively increase the permeability of the cell membrane for molecular delivery. Rat spinal cord was injured using a weight-drop device and the siRNA(s) solutions were intrathecally injected into the vicinity of the exposed SCI, to which PMWs were applied. We first confirmed the substantial uptake of fluorescence-labeled siRNA by deep glial cells; then we delivered siRNAs targeting GFAP and vimentin into the lesion. The treatment led to a significant improvement in locomotive function from five days post-injury in rats that underwent PMW-mediated siRNA delivery. This was attributable to the moderate silencing of the IF proteins and the subsequent decrease in the cavity area in the injured spinal tissue.