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.

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.

Reduction of endogenous GRP78 levels improves secretion of a heterologous protein in CHO cells

1988 ◽  
Vol 8 (10) ◽  
pp. 4063-4070
Author(s):  
A J Dorner ◽  
M G Krane ◽  
R J Kaufman
Keyword(s):  
Endoplasmic Reticulum ◽  
Chinese Hamster Ovary ◽  
Heterologous Protein ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Glycosylation Site ◽  
Ovary Cells ◽  
Tissue Plasminogen ◽  
Stable Association

GRP78 is localized in the endoplasmic reticulum and associates with improperly folded or underglycosylated proteins. The role of GRP78 in secretion was studied in Chinese hamster ovary cells expressing a tissue plasminogen activator (tPA) variant which lacks potential N-linked glycosylation site sequences because of mutagenesis. The expression of variant tPA resulted in elevated levels of GRP78 and its stable association with tPA. The introduction of antisense GRP78 genes resulted in a two- to threefold reduction in GRP78 levels compared with those of the original cells. Cells with reduced levels of GRP78 secreted two- to threefold-higher levels of tPA activity. tPA expressed in these cells displayed reduced association with GRP78, and a greater proportion was processed to the mature form and secreted. These results demonstrate that reduction of GRP78 level can improve the secretion of an associated protein.

Sign in / Sign up

Export Citation Format

Share Document