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

2001 ◽  
Vol 79 (2) ◽  
pp. 165-175 ◽  
Author(s):  
Stephanie Lord-Fontaine ◽  
Enzo Agostinelli ◽  
Ewa Przybytkowski ◽  
Diana A Averill-Bates
Keyword(s):  
Multidrug Resistance ◽  
Chinese Hamster Ovary ◽  
Amine Oxidase ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Multidrug Resistant ◽  
Oxidation Products ◽  
Glutathione S Transferase ◽  
Ovary Cells ◽  
P Glycoprotein

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°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. CHRC5 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°C) enhanced cytotoxicity of spermine oxidation products. Cytotoxic responses in CHRC5 cells were compared to the drug-sensitive cells, to determine whether there are differential responses. CHRC5 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 CHRC5 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.Key words: amine oxidase, spermine, multidrug resistance, P-glycoprotein, hyperthermia.

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

1994 ◽  
Vol 72 (1-2) ◽  
pp. 36-42 ◽  
Author(s):  
Diana A. Averill-Bates ◽  
Enzo Agostinelli ◽  
Ewa Przybytkowski ◽  
Bruno Mondovi
Keyword(s):  
Hydrogen Peroxide ◽  
Aldehyde Dehydrogenase ◽  
Chinese Hamster Ovary ◽  
Bovine Serum ◽  
Amine Oxidase ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Oxidation Products ◽  
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 β-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 °C. Cytotoxicity caused by exogenous hydrogen peroxide, added directly (> 10 μM) or generated by glucose oxidase (0.5 U/mL), was completely inhibited by catalase. Cytotoxicity caused by bovine serum amine oxidase (5.7 × 10−3 U/mL) and spermine (340 μM) 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 μM) 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. Aldehydes such as acrolein are responsible for cytotoxicity that cannot be accounted for by hydrogen peroxide.Key words: acrolein, hydrogen peroxide, catalase, polyamine.

scholarly journals Simultaneous expression of two P-glycoprotein genes in drug-sensitive Chinese hamster ovary cells.

1987 ◽  
Vol 7 (11) ◽  
pp. 4075-4081 ◽  
Author(s):  
J A Endicott ◽  
P F Juranka ◽  
F Sarangi ◽  
J H Gerlach ◽  
K L Deuchars ◽  
...  
Keyword(s):  
Chinese Hamster Ovary ◽  
Multigene Family ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Alternative Polyadenylation ◽  
Multidrug Resistant ◽  
Ovary Cell ◽  
Ovary Cells ◽  
Cdna Sequences ◽  
P Glycoprotein

Overexpression of P-glycoprotein is characteristic of multidrug-resistant cells. We analyzed four P-glycoprotein transcripts that are simultaneously expressed in a drug-sensitive Chinese hamster ovary cell line. We concluded that these transcripts are encoded by two distinct members of a P-glycoprotein multigene family, each of which has two alternative polyadenylation sites. A comparison of the two hamster sequences with the single reported human and mouse P-glycoprotein cDNA sequences demonstrates that P-glycoprotein is a highly conserved protein, that the hamster multigene family is undergoing concerted evolution, and that differences between gene family members are maintained across species. These conserved differences suggest that there may be functional differences between P-glycoprotein molecules.

Simultaneous expression of two P-glycoprotein genes in drug-sensitive Chinese hamster ovary cells

1987 ◽  
Vol 7 (11) ◽  
pp. 4075-4081
Author(s):  
J A Endicott ◽  
P F Juranka ◽  
F Sarangi ◽  
J H Gerlach ◽  
K L Deuchars ◽  
...  
Keyword(s):  
Chinese Hamster Ovary ◽  
Multigene Family ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Alternative Polyadenylation ◽  
Multidrug Resistant ◽  
Ovary Cell ◽  
Ovary Cells ◽  
Cdna Sequences ◽  
P Glycoprotein

Overexpression of P-glycoprotein is characteristic of multidrug-resistant cells. We analyzed four P-glycoprotein transcripts that are simultaneously expressed in a drug-sensitive Chinese hamster ovary cell line. We concluded that these transcripts are encoded by two distinct members of a P-glycoprotein multigene family, each of which has two alternative polyadenylation sites. A comparison of the two hamster sequences with the single reported human and mouse P-glycoprotein cDNA sequences demonstrates that P-glycoprotein is a highly conserved protein, that the hamster multigene family is undergoing concerted evolution, and that differences between gene family members are maintained across species. These conserved differences suggest that there may be functional differences between P-glycoprotein molecules.

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