[Hypoxia increases chemotherapy resistance in nasopharyngeal carcinoma via inducing CDK6 deSUMOylation].

Objective: To understand the mechanism of chemotherapy resistance in nasopharyngeal carcinoma under hypoxic conditions through the perspective of protein SUMOylation modification. Methods: Cobalt chloride (CoCl(2)) was used to establish the hypoxic model of human nasopharyngeal carcinoma CNE1 cells. Then, the cell cycle was detected by flow cytometry, and the expression level of small ubiquitin-related modifier(SUMO) and cyclin-dependent kinase 6 (CDK6) proteins were detected by western blotting. MTT assay was used to determine the median lethal dose (IC(50)) of cancer cells against cisplatin, and enzyme-linked immunosorbent assay (ELISA) was used to determine lactate dehydrogenase (LDH) level. Results: The cell cycle of CNE1 induced by hypoxia was arrested in G0/G1 phase.The results of Western blot showed that the protein expression level of CDK6 in CNE1 cells was lower than that in the control group (0.83±0.25 vs. 0.43±0.21, t=14.67, P=0.003). The protein level of conjugated SUMO1 was significantly lower than that in the control group (2.69±0.48 vs. 1.38±0.31, t=17.22, P=0.001), while the level of free SUMO1 protein was significantly higher than that in the control group (2.01±0.43 vs. 2.60±0.59, t=15.45, P=0.002).The LC50 of CNE1 cells in the control group was significantly lower than that in the hypoxic group (29.44 µg/ml vs. 97.72 µg/ml, t=12.79, P=0.001). After CNE1 cells received 50 µg/ml cisplatin for 48 h, the LDH content in the supernatant of the control group was significantly higher than that in the hypoxic group ((541.49±64.59) ng/ml vs. (234.67±41.03) ng/ml, t=11.94, P=0.007)). The apoptosis rate of CNE1 cells in the control group was significantly higher than that in the hypoxic group ((76.64±5.37)% vs. (32.84±4.77) ng/ml, t=8.49, P=0.003)). Conclusion: Hypoxia can dissociate the covalent modification of CDK6 and SUMO1, inhibit cell cycle and increase the chemotherapy resistance of nasopharyngeal carcinoma.

[Spectomycin B1 induces VEGFR2 de-SUMO modification to inhibit angiogenesis in nasopharyngeal carcinoma].

Objective:To explore the new mechanism of spectomycin B1 in inhibiting angiogenesis of nasopharyngeal carcinoma and to provide a theoretical basis for targeted gene therapy of nasopharyngeal carcinoma. Method:Human nasopharyngeal carcinoma CNE1 cells were divided into two groups, the control group and spectomycin B1 group. Western blot was used to detect the expression levels of small ubiquitin-related modified protein（SUMO） 1 and vascular endothelial growth factor receptor 2（VEGFR2）. The angiogenesis assay was used to detect the angiogenic ability of CNE1 cells, and the apoptosis was detected by flow cytometry. The model of nasopharyngeal carcinoma-bearing mice was established, spectomycin B1 was administered, tumor volume and weight were measured, and protein expression of CD31 was detected by immunohistochemistry and microvessel density was compared. Result:Spectomycin B1 could reduce deSUMOylation of VEGFR2 protein by 4.05 times, significantly reduce the angiogenic ability of CNE1 cells, and increase the apoptosis rate by 20.68%. In the tumor-bearing mouse model, spectomycin B1 treatment could inhibit subcutaneous tumor growth rate and weight, and the blood vessel density decreased by 40.04%. Conclusion:Spectomycin B1 can inhibit neovascularization of nasopharyngeal carcinoma by inducing deSUMOylation of VEGFR2 protein.

The JaICA-genox oxidative stress profile--an overview on the profiling technique in the oxidative stress assessment and management.

It is widely accepted that oxidative stress (OS) is a major causative factor for many of the age-related dysfunctions and specific diseases. Since the oxidative stress state (OSS) of an individual depends on hereditary, dietary, and environmental factors, there is a large heterogeneity in the population that may be related to disease incidence and longevity. Hence there is a need to assess how well an individual is coping against OS. The Japan Institute for the Control of Aging (JaICA) and Genox have jointly developed a profiling technique to measure the "Oxidative Stress Profiles (JaICA-Genox OSP)" of individuals and laboratory test animals. The JaICA-Genox OSP consists of about 45 different assays measuring the levels of oxidative damage in lipids and nucleic acids, and the antioxidant defenses in the serum. In addition, several bio-markers for cardiovascular disease risk are also measured, and assays to measure specific age- and sex-related hormones in the serum and urine, and race elements in serum, urine, and drinking water are also undertaken. This overview discusses the designing of the JaICA-Genox OSP and its application in the testing of human subjects.

Expression of SV40 large T antigen stimulates reversion of a chromosomal gene duplication in human cells.

Transformation of human cells is characterized by altered cell morphology, frequent karyotypic abnormalities, reduced dependence on growth factors and substrate, and rare "immortalization"-clonal acquisition of unlimited proliferative potential. We previously reported a marked increase in DNA rearrangements, arising between two duplicated segments in a transfected plasmid substrate, for five immortal human cell lines relative to three normal fibroblast strains [Finn et al. (1989) Mol. Cell. Biol. 9, 4009-4017]. We have now assessed reversion of a 14-kilobase-pair duplication within the hypoxanthine phosphoribosyl transferase (HPRT) gene locus, in a fibroblast strain during its normal replicative lifespan and after stable transformation with SV40 large-T antigen. Revertants, selected under HPRT-dependent growth conditions immediately after purging preexisting HPRT+ cells, were confirmed as HPRT+ by hypoxanthine incorporation and 6-thioguanine sensitivity. Southern blot analyses indicate loss from most revertant clones of a restriction fragment representing the duplicated HPRT region, as predicted for homologous recombination between the 14-kilobase-pair repeats. Amplification of a subregion of HPRT mRNA implicated deletion of duplicated exons in 93% of revertant colonies. Reversion to HPRT+ was unaltered during the normal in vitro lifespan of these cells, but increased in 9 clones stably transformed with large-T antigen (mean = 3.8-fold; each P < 10(-5)). Stimulation of HPRT-reversion is abrogated in a variety of T-antigen mutants, and depends on continued induction of T antigen by glucocorticoid in two clones tested 10-30 doublings before replicative senescence. Since no immortal subclones arose from these clones, elevated reversion must precede immortalization. Increased DNA rearrangements, in cells expressing T-antigen, could facilitate the rare concurrence of multiple mutations necessary for immortalization.

Site-specific oxidation of histidine residues in glycated insulin mediated by Cu2+.

The site-specific oxidation of histidine residues in glycated insulin mediated by copper ions and the relationship between the oxidation sites and the steric conformation of insulin are discussed in this study. Glycated insulin was prepared by incubating native insulin with glucose in 67 mM sodium phosphate, pH 7.5, at 37 degrees C for 30 h. In the presence of micromolar concentrations of Cu2+, glycated insulin was oxidized and its fragmentation or aggregation was detected. Accompanying the fragmentation, new N-termini were generated. The residues in these N-termini were identified as alanine, proline, valine, leucine and isoleucine by comparing dansyl derivatives with standard dansyl-amino acid products. Furthermore, several oxidized products of glycated insulin were isolated using reverse-phase HPLC (P1-P3). From amino acid composition and sequence analyses, it was determined that His10 on the insulin B-chain was modified in each of these peptides, while His5 was also modified in P3. The difference in susceptibility of His10 and His5 to oxidative modification is considered to be due to easier coordination of Cu2+ with His10, which further forms a complex with the Amadori compound at B-chain Phe1 that is vicinal to His10 in the steric conformation of insulin. This complex may generate an active oxygen species, which induces the degradation of the imidazole ring at His10, leading to aggregation or fragmentation of insulin.

Selective oxidation of histidine residues in proteins or peptides through the copper(II)-catalysed autoxidation of glucosone.

Glucosone has been identified as the main intermediate sugar moiety product of the copper(II)-catalysed autoxidation of the Amadori compound [Kawakishi, Tsunehiro & Uchida (1991) Carbohydr. Res. 211, 167-171]. Oxidative fragmentation of the model protein, especially selective degradation of the histidine residue in protein or peptides mediated by the copper(II)-catalysed autoxidation of glucosone, is discussed in this paper. The oxidative damage to protein could be retarded by catalase (EC 1.11.1.16) and EDTA, while superoxide dismutase (EC 1.15.1.1) and hydroxyradical scavengers showed little effect. Through the process of the oxidative degradation of N-benzoylhistidine and other histidine-containing peptides, the oxidation of the imidazole ring in histidine caused by the glucosone-copper(II) system was the same as that by the ascorbate-copper(II) system. These facts suggest that the copper-catalysed autoxidation of glucosone could generate some active-oxygen species causing oxidative damage to protein similar to that caused by the ascorbate-copper(II) system.

Homologous recombination is elevated in some Werner-like syndromes but not during normal in vitro or in vivo senescence of mammalian cells.

Werner syndrome (WS) is a recessive genetic condition associated with markedly reduced replicative lifespans of cells in culture, high chromosomal instability in vivo and in vitro, and premature appearance of many characteristics of normal aging, including an increased incidence of cancer. We have monitored plasmid homologous recombination frequencies in diploid fibroblasts from 6 Werner or Werner-like syndrome patients, following transfection with a plasmid substrate containing 2 overlapping fragments of the TN5 Neor gene. Plasmid DNA recovered from these cells was then assayed for homologous recombination by (a) transformation of recA- bacteria to Ampr (indicating total viable plasmid) or Neor (indicating viable recombinant plasmid), and (b) by limited-cycle polymerase chain reaction (PCR) to co-amplify a recombinant fragment containing the overlap region, and a control region of the same plasmid, without bacterial transformation. Bacterial assay data indicated that recombination rates in 3 of the 6 WS strains were significantly elevated above normal controls; 4 of 6 appeared elevated by PCR assay. The highest-recombination WS strain showed evidence of reduced degradation of transfected plasmid DNA. For this small sample of WS strains, clinical severity of WS was not well correlated with recombination rate as determined by either assay (Pearson r = 0.78, not significant, for PCR assay); elevated recombination may, however, define a subset of WS at greatest risk for cancer and/or atherosclerosis. PCR assay of a hyperoxia-resistant HeLa cell line, displaying substantially increased chromosome breakage, indicated increased recombination between direct-repeat fragments. Nevertheless, elevated recombination in WS strains is unlikely to be secondary to impaired replicative capacity characteristic of WS cells, or to defective repair of chromosome damage which is increased in WS, since recombination in non-WS strains was unaffected by passage level or repeated UV irradiation.

Homologous plasmid recombination is elevated in immortally transformed cells.

The levels of intramolecular plasmid recombination, following transfection of a plasmid substrate for homologous recombination into normal and immortally transformed cells, have been examined by two independent assays. In the first assay, recovered plasmid was tested for DNA rearrangements which regenerate a functional neomycin resistance gene from two overlapping fragments. Following transformation of bacteria, frequencies of recombinationlike events were determined from the ratio of neomycin-resistant (recombinant) colonies to ampicillin-resistant colonies (indicating total plasmid recovery). Such events, yielding predominantly deletions between the directly repeated sequences, were substantially more frequent in five immortal cell lines than in any of three normal diploid cell strains tested. Effects of plasmid replication or interaction with T antigen and of bacterially mediated rejoining of linear molecules generated in mammalian cells were excluded by appropriate controls. The second assay used limited coamplification of a control segment of plasmid DNA, and of the predicted recombinant DNA region, primed by two sets of flanking oligonucleotides. Each amplified band was quantitated by reference to a near-linear standard curve generated concurrently, and recombination frequencies were determined from the ratio of recombinant/control DNA regions. The results confirmed that recombinant DNA structures were generated within human cells at direct repeats in the transfected plasmid and were markedly more abundant in an immortal cell line than in the diploid normal cells from which that line was derived.