Nickel (Ni) compounds are potent carcinogens and can induce malignant transformation of rodent and human cells. To uncover the molecular mechanisms of nickel sulfide (NiS)-induced cell transformation, we investigated epigenetic alterations in a set of DNA repair genes. The silencing of the O(6)-methylguanine DNA methyltransferase (MGMT) gene locus and upregulation of DNA methyltransferase 1 (DNMT1) expression was specifically detected in NiS-transformed human bronchial epithelial (16HBE) cells. In addition, we noted epigenetic alterations including DNA hypermethylation, reduced histone H4 acetylation and a decrease in the ratio of Lys-9 acetylated/methylated histone H3 at the MGMT CpG island in NiS-transformed 16HBE cells. Meanwhile, we identified concurrent binding of methyl-CpG-binding protein 2, methylated DNA-binding domain protein 2 and DNMT1 to the CpG island of the MGMT promoter, demonstrating that these components collaborate to maintain MGMT methylation in NiS-transformed cells. Moreover, depletion of DNMT1 by introduction of a small hairpin RNA construct into NiS-transformed cells resulted in a 30% inhibition of cell proliferation and led to increased MGMT gene expression by reversion of the epigenetic modifications at the MGMT promoter region. MGMT suppression and hypermethylation at the CpG island of the MGMT promoter occurred 6 days after NiS treatment, indicating that epigenetic modifications of MGMT might be an early event in tumorigenesis. Taken together, these observations demonstrate that epigenetic silencing of MGMT is associated with DNA hypermethylation, histone modifications and DNMT1 upregulation, which contribute to NiS-induced malignant transformation.
BackgroundG protein coupled receptors (GPCRs) represent the largest family of membrane proteins in the human genome and the richest source of targets for the pharmaceutical industry. A major limitation to characterizing GPCRs has been the difficulty in developing high-level heterologous expression systems that are cost effective. Reasons for these difficulties include inefficient transport and insertion in the plasma membrane and cytotoxicity. Additionally, GPCR purification requires detergents, which have a negative effect on receptor yields and stability.ResultsHere we report a detergent-free cell-free protein expression-based method to obtain pharmacologically active GPCRs in about 2 hours. Our strategy relies on the co-translational insertion of modified GPCRs into nanometer-sized planar membranes. As a model we employed an engineered β2-adrenergic receptor in which the third intracellular loop has been replaced with T4 lysozyme (β2AR -T4L). We demonstrated that nanolipoprotein particles (NLPs) are necessary for expression of active β2AR -T4L in cell-free systems. The binding specificity of the NLP- β2AR-T4L complex has been determined by competitive assays. Our results demonstrate that β2AR-T4L synthesized in vitro depends on similar oxidative conditions as those required by an in vivo-expressed receptor.ConclusionsAlthough the activation of β2AR-T4L requires the insertion of the T4 lysozyme sequence and the yield of that active protein limited, our results conceptually prove that cell-free protein expression could be used as a fast approach to express these valuable and notoriously difficult-to-express proteins.
1. The present study investigated the therapeutic effects of both single and combination treatment with Yunke (technetium-99 conjugated with methylene diphosphonate; (99)Tc-MDP) and colloidal chromic phosphate (32)P (phosphonium-32) in rats with adjuvant arthritis (AA). 2. Rats were randomly allocated to one of five groups: (i) normal control group (sham operated and treated with normal saline); (ii) AA control group (arthritis induced with adjuvant and treated with normal saline); (iii) (32)P colloid group (arthritis induced with adjuvant and treated with a single intra-articular injection of colloidal chromic phosphate phosphonium-32 (0.02 mCi) and i.p. injections of normal saline every other day); (iv) Yunke group (arthritis induced with adjuvant and treated with i.p. Yunke (2.5 x 10(-3) microg/kg) every other day and single intra-articular injection of normal saline); and (v) combination group (arthritis induced with adjuvant and treated with a combination of both therapies). 3. The left-to-right diameter (LRD) of the left hind ankle, serum levels of tumour necrosis factor (TNF) and interleukin (IL)-1b and histological sections of the ankle joints were examined at different time points. 4. The LRD of the left hind ankle was smaller for the combination group compared with (32)P colloid alone at Week 4 (7.11 +/- 0.28 vs 7.57 +/- 0.24 mm, respectively; P < 0.001). The combination treatment was more effective than (32)P colloid alone in decreasing serum TNF (1.614 +/- 0.368 vs 1.977 +/- 0.255 ng/mL, respectively; P = 0.002 for Week 4) and IL-1b (0.271 +/- 0.027 vs 0.308 +/- 0.020 ng/mL, respectively for Week 4; 0.209 +/- 0.023 vs 0.255 +/- 0.016 ng/mL, respectively for Week 6; both P = 0.001). Histologically, the combination group exhibited less synovium proliferation compared with Yunke treatment alone and decreased inflammatory cell infiltration compared with (32)P colloid alone. 5. In conclusion, the combination of Yunke and (32)P colloid is more effective in the treatment of AA in rats compared with Yunke or (32)P colloid alone.
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