Reductase enzyme expression across the National Cancer Institute Tumor cell line panel: correlation with sensitivity to mitomycin C and EO9.
BACKGROUND
Many antitumor drugs require metabolic activation to exert their cytotoxic or cytostatic effects. The so-called bioreductive compounds, whose conversion into active antitumor agents is catalyzed by reductase enzymes, are examples of such drugs. The identification of specific enzymes involved in the activation of these compounds is important in understanding cellular factors that may influence drug antitumor activity.


OBJECTIVE
We measured expression levels of three different reductase enzymes-DT-diaphorase [NAD(P)H (i.e., reduced nicotinamide adenine dinucleotide, with or without phosphate): quinone oxidoreductase]; NADPH:cytochrome P-450 reductase; and NADH (i.e., reduced nicotinamide adenine dinucleotide): cytochrome-b5 reductase- in 69 cell lines (most of the National Cancer Institute [NCI] human tumor cell panel) to see if relationships could be established between the activities of these enzymes and cellular sensitivities to the bioreductive compounds mitomycin C and EO9.


METHODS
For all 69 cell lines, the activity of each enzyme was determined using cellular extracts and photometric assays involving the reduction of cytochrome c. Western blot analysis was used to measure the relative amount of DT-diaphorase protein in each extract, and coupled reverse transcription and polymerase chain reactions were employed to assess DT-diaphorase and NADPH:cytochrome P-450 reductase messenger RNA (mRNA) levels in a subset of the cell lines. The cytotoxic and/or cytostatic activities of mitomycin C and EO9 toward the cell lines were determined under aerobic conditions. Relationships between enzyme activity levels and drug sensitivities were assessed by use of the COMPARE program and Pearson correlation coefficients.


RESULTS
In general, DT-diaphorase activity levels were higher than those observed for the other two reductases across the entire cell line panel. Measured activities for all three enzymes varied among cell lines derived from the same tissue as well as between lines derived from different tissues; however, tissue-specific patterns of expression could be discerned. Differences in the activity levels of individual enzymes appeared to reflect differences in corresponding enzyme protein and/or mRNA levels. A relationship between enzyme activity and chemosensitivities to mitomycin C and EO9 was observed only for DT-diaphorase (Pearson correlation coefficient = .424 [two-sided P<.0005] for mitomycin C and .446 [two-sided P< or = to .0013] for EO9).


CONCLUSIONS
Reductase enzyme expression is heterogeneous across human tumor cell lines, and tissue-specific patterns of expression are apparent. DT-diaphorase activity levels correlate with sensitivities to mitomycin C and EO9, supporting a role for this enzyme in the bioactivation of these anticancer compounds.


CONCLUSIONS
Comparison of biochemical, molecular biological, and chemosensitivity data obtained from screening a large number of cell lines (e.g., the NCI tumor cell line panel) may facilitate investigation of factors influencing drug antitumor activity. The knowledge gained may be of value in the development of new anticancer agents or in the selection of patients to receive specific therapies.
Fitzsimmons
S A
SA
CRC Department of Medical Oncology, University of Glasgow, Scotland, U.K.
Workman
P
P
Grever
M
M
Paull
K
K
Camalier
R
R
Lewis
A D
AD
D000970:Antineoplastic Agents / Q000494:pharmacology*; D001388:Aziridines / Q000494:pharmacology*; D003579:Cytochrome Reductases / Q000378:metabolism*; D042966:Cytochrome-B(5) Reductase; D006801:Humans; D045563:Indolequinones; D007211:Indoles / Q000494:pharmacology*; D016685:Mitomycin / Q000494:pharmacology*; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000235:genetics / Q000378:metabolism*; D009251:NADPH-Ferrihemoprotein Reductase / Q000235:genetics / Q000378:metabolism*; D009369:Neoplasms / Q000188:drug therapy / Q000201:enzymology*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis; D014407:Tumor Cells, Cultured
D000970:Antineoplastic Agents; D001388:Aziridines; D045563:Indolequinones; D007211:Indoles; D012333:RNA, Messenger; D016685:Mitomycin; D003579:Cytochrome Reductases; D042966:Cytochrome-B(5) Reductase; D009251:NADPH-Ferrihemoprotein Reductase; D016660:NAD(P)H Dehydrogenase (Quinone); C060817:apaziquone