An integrated genomic approach identifies ARID1A as a candidate tumor-suppressor gene in breast cancer.

Tumor-suppressor genes (TSGs) have been classically defined as genes whose loss of function in tumor cells contributes to the formation and/or maintenance of the tumor phenotype. TSGs containing nonsense mutations may not be expressed because of nonsense-mediated RNA decay (NMD). We combined inhibition of the NMD process, which clears transcripts that contain nonsense mutations, with the application of high-density single-nucleotide polymorphism arrays analysis to discriminate allelic content in order to identify candidate TSGs in five breast cancer cell lines. We identified ARID1A as a target of NMD in the T47D breast cancer cell line, likely as a consequence of a mutation in exon-9, which introduces a premature stop codon at position Q944. ARID1A encodes a human homolog of yeast SWI1, which is an integral member of the hSWI/SNF complex, an ATP-dependent, chromatin-remodeling, multiple-subunit enzyme. Although we did not find any somatic mutations in 11 breast tumors, which show DNA copy-number loss at the 1p36 locus adjacent to ARID1A, we show that low ARID1A RNA or nuclear protein expression is associated with more aggressive breast cancer phenotypes, such as high tumor grade, in two independent cohorts of over 200 human breast cancer cases each. We also found that low ARID1A nuclear expression becomes more prevalent during the later stages of breast tumor progression. Finally, we found that ARID1A re-expression in the T47D cell line results in significant inhibition of colony formation in soft agar. These results suggest that ARID1A may be a candidate TSG in breast cancer.

Beta cell compensation for insulin resistance in Zucker fatty rats: increased lipolysis and fatty acid signalling.

AIMS/HYPOTHESIS: The aim of this study was to determine the role of fatty acid signalling in islet beta cell compensation for insulin resistance in the Zucker fatty fa/fa (ZF) rat, a genetic model of severe obesity, hyperlipidaemia and insulin resistance that does not develop diabetes. MATERIALS AND METHODS: NEFA augmentation of insulin secretion and fatty acid metabolism were studied in isolated islets from ZF and Zucker lean (ZL) control rats. RESULTS: Exogenous palmitate markedly potentiated glucose-stimulated insulin secretion (GSIS) in ZF islets, allowing robust secretion at physiological glucose levels (5-8 mmol/l). Exogenous palmitate also synergised with glucagon-like peptide-1 and the cyclic AMP-raising agent forskolin to enhance GSIS in ZF islets only. In assessing islet fatty acid metabolism, we found increased glucose-responsive palmitate esterification and lipolysis processes in ZF islets, suggestive of enhanced triglyceride-fatty acid cycling. Interruption of glucose-stimulated lipolysis by the lipase inhibitor Orlistat (tetrahydrolipstatin) blunted palmitate-augmented GSIS in ZF islets. Fatty acid oxidation was also higher at intermediate glucose levels in ZF islets and steatotic triglyceride accumulation was absent. CONCLUSIONS/INTERPRETATION: The results highlight the potential importance of NEFA and glucoincretin enhancement of insulin secretion in beta cell compensation for insulin resistance. We propose that coordinated glucose-responsive fatty acid esterification and lipolysis processes, suggestive of triglyceride-fatty acid cycling, play a role in the coupling mechanisms of glucose-induced insulin secretion as well as in beta cell compensation and the hypersecretion of insulin in obesity.

Derivatives of monoglycerides as apoptotic agents in T-cells.

Recently, lipids have received considerable attention for their potential to induce apoptosis when added exogenously to cells. In this study, we directly demonstrate that murine T-cells undergo rapid apoptosis following treatment with various forms of monoglycerides, which are a family of naturally occurring lipids consisting of a single fatty acid moiety attached to a glycerol backbone. The potency of these lipids varied depending on their chemical structure, whereas glycerol backbone or corresponding fatty acids alone were ineffective. Moreover, monoglyceride-mediated apoptosis was suppressed either by Bcl-2 overexpression, treatment with a broad inhibitor of caspases, or RNA and protein synthesis inhibitors. In addition, treatment of cells with derivatives of monoglycerides induced a calcium flux, which could be inhibited by both extracellular (EGTA) or intracellular (EGTA-AM) calcium chelators. To our knowledge, this is the first report demonstrating a role for derivatives of monoglycerides as inducers of apoptosis in mammalian cells.

Amine oxidase, spermine, and hyperthermia induce cytotoxicity in P-glycoprotein overexpressing multidrug resistant Chinese hamster ovary cells.

Multidrug resistance is a major obstacle for the successful use of chemotherapy. The multidrug resistance phenotype is often attributed to overexpression of P-glycoprotein, which is an energy-dependent drug efflux pump. We investigated a new strategy to overcome multidrug resistance, using purified bovine serum amine oxidase, which generates two major toxic products from the polyamine spermine. The cytotoxicity of the aldehyde(s) and H2O2, produced by the enzymatic oxidation of micromolar concentrations of spermine, was evaluated in multidrug resistant Chinese hamster ovary cells CHRC5 with overexpression of P-glycoprotein, using a clonogenic cell survival assay. We examined the ability of hyperthermia (42 degrees C), and inhibition of cellular detoxification systems, to sensitize multidrug resistant cells to spermine oxidation products. Severe depletion of intracellular glutathione was achieved using L-buthionine sulfoximine and inhibition of glutathione S-transferase by ethacrynic acid. CH(R)C5 cells showed no resistance to the toxic oxidation products of spermine, relative to drug-sensitive AuxB1 cells. Exogenous catalase protected cells against cytotoxicity of H2O2, but spermine-derived aldehyde(s) still caused some cytotoxicity. Hyperthermia (42 degrees C) enhanced cytotoxicity of spermine oxidation products. Cytotoxic responses in CH(R)C5 cells were compared to the drug-sensitive cells, to determine whether there are differential responses. CH(R)C5 cells were more sensitive to the cytotoxic effect of spermine oxidation products under more extreme conditions (higher temperature, higher spermine concentration, and longer exposure time). Glutathione depletion or glutathione S-transferase inhibition also led to enhanced cytotoxicity of spermine oxidation products in CH(R)C5 and AuxB1 cells. Our findings suggest that hyperthermia, combined with toxic oxidation products generated from spermine and amine oxidase, could be useful for eliminating drug-sensitive and multidrug resistant cells.

Correlation between glutathione and stimulation of the pentose phosphate cycle in situ in Chinese hamster ovary cells exposed to hydrogen peroxide.

The effect of glutathione on stimulation of pentose phosphate cycle activity during oxidative challenge was evaluated in intact Chinese hamster ovary cells in situ. Glutathione was depleted to varying levels with L-buthionine-[S,R] sulfoximine. The level of stimulation of pentose phosphate cycle activity by exogenous H2O2 (4 mumol/10(7) cells) was dependent on the time of pretreatment with L-buthionine-[S,R] sulfoximine and was proportional to the total glutathione concentration. This was not related to the amount of GSSG, since its level was exceedingly low under conditions where H2O2 stimulated pentose phosphate cycle activity. The amount of GSSG in cells increased after exposure to 10-fold higher concentrations of H2O2 under conditions where there was no stimulation of pentose phosphate cycle activity above the basal level. Paraquat caused stimulation of pentose phosphate cycle activity which was independent of L-buthionine-[S,R] sulfoximine pretreatment and of the glutathione content of cells. The stimulatory effects of both oxidants on pentose phosphate cycle activity appeared to be independent of glutathione reductase activity since they were unaffected in cells treated with 1,3-bis(2-chloroethyl)-1-nitrosourea. The inhibitory effect of L-buthionine-[S,R] sulfoximine on stimulation of pentose phosphate cycle activity by H2O2 did not appear to be due to the inhibitor itself, but rather to the overall level of glutathione. Glutathione could have a role in maintaining activity of the pentose phosphate cycle at a level which is appropriate for the severity of the oxidative challenge as well as for the capacity of the cellular antioxidant defenses.

Glucose, glutathione, and cellular response to spermine oxidation products.

Bovine serum amineoxidase (BSAO) oxidatively deaminates polyamines, which contain primary amine groups with formation of several toxic products, H2O2, and aldehyde(s). We evaluated the role of glucose metabolism via the pentose phosphate cycle and the level of intracellular glutathione on cytotoxicity induced by each of the toxic products in Chinese hamster ovary (CHO) cells. Glucose protected cells against cytotoxicity in the presence of BSAO at low spermine concentrations ( < 50 microM), where H2O2 was the only toxic species present. When catalase was present, cytotoxicity is attributed to spermine-derived aldehyde(s). Glucose did not protect cells against cytotoxicity induced by spermine-derived aldehyde(s), nor by the aldehyde acrolein. Hydrogen peroxide produced by spermine and BSAO stimulated pentose cycle activity, whereas the aldehyde(s) did not. Depletion of intracellular glutathione with L-buthionine sulfoximine (1 mM, 24 h) sensitized cells to the cytotoxic effects of both H2O2 and the aldehyde(s) produced by spermine and BSAO. The pentose cycle and the glutathione redox cycle have an important role in protection against H2O2 generated from spermine oxidation. Glutathione appears to have a role in protecting cells against cytotoxicity attributed to spermine-derived aldehyde(s), most likely by conjugation in a reaction catalyzed by glutathione S-transferase, whereas metabolism of glucose via the pentose cycle did not. The metabolism of both glucose and glutathione, affect the cellular response to H2O2 and aldehyde(s) derived from spermine, although different pathways are involved.

Heat enhancement of cytotoxicity induced by oxidation products of spermine in Chinese hamster ovary cells.

This study investigates the potential of using polyamines as thermosensitizers, in the presence of bovine serum amine oxidase (BSAO), as a new anticancer strategy. The effect of hyperthermia on cytotoxicity of spermine oxidized by purified bovine serum amine oxidase was investigated in Chinese hamster ovary cells. Several different spermine concentrations were employed in the presence of BSAO at 37 degrees and 42 degrees. Cytotoxicity was considerably enhanced at 42 degrees. Heat also increased the individual cytotoxicity of both exogenous H2O2 and the exogenous aldehyde acrolein. Thus, both of these species could contribute to the thermal enhancement of cytotoxicity caused by BSAO and spermine. The effect of temperature was especially marked in the presence of exogenous catalase. This cytotoxicity cannot be accounted for by H2O2 and was attributed to aldehyde(s). The involvement of aldehyde(s) in cytotoxicity at 42 degrees was also confirmed by the complete inhibition of cytotoxicity with both exogenous aldehyde dehydrogenase and exogenous catalase. A particularly interesting finding, in the presence of exogenous catalase, was that conditions of BSAO and spermine (< or = 50 microM) which were non-toxic at 37 degrees became cytotoxic at 42 degrees. This suggests that spermine-derived aldehyde(s), that were non-toxic at 37 degrees, contributed to cytotoxicity at 42 degrees and resemble thermosensitizers. The thermosensitizing activity of aldehyde(s) produced in the BSAO-catalysed oxidation of spermine has potential value for improving the therapeutic effects of hyperthermia and could be considered for future application in cancer therapy. Polyamines are present at elevated levels in tumour cells and have been considered as heat sensitizers. By delivering BSAO into tumour cells, toxic oxidation products of polyamines could be produced in situ for selective killing of tumour cells.

The role of glucose in cellular defences against cytotoxicity of hydrogen peroxide in Chinese hamster ovary cells.

The presence of glucose in the cellular environment influences the response of cells to hydrogen peroxide. This study examines the effect of glucose on clonogenic cell survival of Chinese hamster ovary cells exposed to micromolar concentrations of exogenous hydrogen peroxide. Exposure to hydrogen peroxide (20 mumol/10(7) cells) resulted in considerable cytotoxicity that was unaffected by the presence or absence of glucose. However, glucose protected the cells from killing induced by milder exposure (1 mumol/10(7) cells) to the oxidant causing a shift in the dose-response curve. This effect was considered in terms of glucose metabolism via the pentose phosphate cycle. A low nontoxic concentration of hydrogen peroxide (0.1 mumol/10(7) cells) markedly increased pentose phosphate cycle activity in normal cells. Clonogenic survival and activity of the cycle for cells depleted of total glutathione to about 8.6% of its initial value and/or with catalase activity reduced to about 10% of control levels were also determined. Neither of these modifications alone completely abolished the protective effect of glucose. Efficacy of glucose protection against cytotoxicity of hydrogen peroxide diminished in cells depleted of glutathione, and this was not accompanied by any detectable increase in pentose phosphate cycle activity above the control level. Cells depleted of catalase alone had a profile of survival and pentose phosphate cycle activity similar to that of control cells when exposed to hydrogen peroxide. Cells depleted of both glutathione and catalase were almost as sensitive to hydrogen peroxide as the cells incubated without glucose. They also did not express any detectable increase in pentose phosphate cycle activity. Survival of those cells, when exposed to hydrogen peroxide, was almost the same regardless if glucose was present or not. These results demonstrate an important role for the glutathione redox cycle, catalase, and the pentose phosphate cycle in protection against hydrogen peroxide in Chinese hamster ovary cells. They confirm the essential role of glucose and pentose phosphate cycle activity for the detoxification of hydrogen peroxide via the glutathione redox cycle. The data suggest that the ability of catalase to metabolise peroxide may also depend on metabolism of glucose via the pentose phosphate cycle. A clear understanding of the protective mechanisms in cells against hydrogen peroxide has many applications since this common reactive oxygen species is implicated in several pathophysiologies and in the action of certain chemotherapeutic drugs.

Aldehyde dehydrogenase and cytotoxicity of purified bovine serum amine oxidase and spermine in Chinese hamster ovary cells.

Bovine serum amine oxidase (EC 1.4.3.6) catalyses the oxidative deamination of polyamines giving rise to the corresponding aldehydes, ammonia, and hydrogen peroxide. It has been suggested that the dialdehyde produced during the oxidation of spermine subsequently undergoes spontaneous beta-elimination to form acrolein. Oxidation of the aldehydes by aldehyde dehydrogenase (EC 1.2.1.5) thus eliminates these reactive species and prevents the formation of acrolein. This work studies the role of each of the oxidation products of spermine in cytotoxicity induced by purified bovine serum amine oxidase. The inhibition patterns of NAD-dependent aldehyde dehydrogenase and catalase against cytotoxicity of bovine serum amine oxidase were determined in Chinese hamster ovary cells at 37 degrees C. Cytotoxicity caused by exogenous hydrogen peroxide, added directly (> 10 microM) or generated by glucose oxidase (0.5 U/mL), was completely inhibited by catalase. Cytotoxicity caused by bovine serum amine oxidase (5.7 x 10(-3) U/mL) and spermine (340 microM) was completely inhibited by catalase only during short incubation times after which time cytotoxicity occurred. This indicates that hydrogen peroxide was the only species contributing to cytotoxicity at this stage of the reaction. Aldehyde dehydrogenase alone caused partial inhibition of cytotoxicity, but only later in the reaction. Cytotoxicity was completely eliminated in the presence of both catalase and aldehyde dehydrogenase. Exogenous acrolein (> 50 microM) also caused cytotoxicity in Chinese hamster ovary cells. However, hydrogen peroxide was toxic to cells at lower concentrations and at shorter exposure times relative to aldehydes. These data show that both peroxide and aldehydes contribute to cytotoxicity of oxidation products of spermine.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytotoxicity and kinetic analysis of purified bovine serum amine oxidase in the presence of spermine in Chinese hamster ovary cells.

Bovine serum amine oxidase (BSAO, EC 1.4.3.6) catalyzes the oxidative deamination of polyamines giving rise to the corresponding aldehydes, ammonia and hydrogen peroxide (H2O2). This study demonstrates that amine oxidase (BSAO) purified from bovine serum and exogenous spermine caused cytotoxicity in Chinese hamster ovary (CHO) cells. Cytotoxicity occurred when cells were exposed to BSAO (0.0164-16.4 micrograms/ml) in the presence of spermine (1.9-340 microM). BSAO and spermine alone were not toxic at these concentrations. Cytotoxicity was dependent on the concentration of spermine and on the incubation time, and was also accelerated at 42 degrees C relative to 37 degrees C. Kinetic analysis of the enzymatic reaction, as a function of spermine concentration, showed Michaelis-Menten saturation kinetics. The apparent Vmax increased from 19.1 +/- 0.4 microM min-1 at 37 degrees C to 23.0 +/- 0.3 microM min-1 at 42 degrees C. The apparent Km decreased from 25.5 +/- 2.6 microM at 37 degrees C to 17.7 +/- 1.3 microM at 42 degrees C. Catalase inhibited cytotoxicity, suggesting that H2O2 was partially responsible for cytotoxicity. This work shows that the oxidation products of polyamines, rather than the polyamines themselves, are responsible for cytotoxicity in mammalian cells. The significance of this study is that amine oxidases could have therapeutic potential in cancer treatment regimens and a beneficial effect is likely when the enzyme is used together with clinical hyperthermia.

A model-free way of representing hyperthermia cell survival data.

We present a method of fitting curves to cell survival data that is free from all model assumptions, requiring only that the fitted curves be decreasing and reasonably smooth, where the degree of smoothness is determined from considerations of experimental error. The fitted curves are then differentiated to yield frequency distributions of cell killing times, which may be of value in defining subpopulations with different sensitivities to the cytotoxic agent under study. In addition, confidence intervals on the fitted curves and frequency distributions are obtained by Monte Carlo simulation. The results allow the objective and model-free assessment of the effects of various experimental interventions on cell survival.

Thermal adaptation in CHO cells at 40 degrees C: the influence of growth conditions and the role of heat shock proteins.

The kinetics of thermal adaptation at the nonlethal temperature of 40 degrees C was studied in CHO (Chinese hamster ovary) cells in vitro. Thermal resistance, demonstrated as an increase in mean 45 degrees C killing time or as an increase in the shoulder of the 45 degrees C survival curve, was fully developed by 2 h. Control cells in early logarithmic phase were more heat sensitive than those in stationary phase. Corresponding 45 degrees C killing time frequency distributions were unimodal with an increase in mean killing time from early logarithmic to stationary phase. Cells which were thermally adapted at 40 degrees C for 6 h had biphasic 45 degrees C killing time frequency distributions, and as cells progressed from early logarithmic to stationary phase the heat-sensitive subpopulation progressively declined. Exposure to 40 degrees C produced a 30% increase in total protein synthesis. Proteins with molecular weights 72, 89, and 109 kDa which correspond to those induced by lethal heat shock were synthesized at 40 degrees C, but there was no close temporal correlation between the development of heat resistance at 40 degrees C and synthesis of the heat shock proteins. Cycloheximide (100 micrograms/ml) reduced the mean 45 degrees C killing time but did not totally prevent the development of heat resistance at 40 degrees C.