Increased activity and expression of NAD(P)H:quinone acceptor oxidoreductase in confluent cell cultures and within multicellular spheroids.
NAD(P)H:quinone acceptor oxidoreductase (NQO1, EC 1.6.99.2) is an enzyme that is believed to play a central role in the bioreductive activation of several compounds, particularly quinones. The results of this study demonstrate that the activity of NQO1 is significantly elevated (2.5-fold) in HT-29 human colon cells that are in the plateau phase of the growth curve as opposed to cells in the exponential phase. Analysis of gene expression using semiquantitative reverse transcription-polymerase chain reaction and Northern blot analysis demonstrates that the increased enzyme activity is associated with increased NQO1 mRNA levels. Sequential trypsinization of layers of cells from HT-29 multicellular spheroids and analysis of gene expression by reverse transcription-polymerase chain reaction demonstrate that NQO1 expression is elevated in cells close to the necrotic center. Maximum expression occurs at a depth of 90-110 microns, with reduced expression as the distance toward both the surface and the necrotic center decreases. HT-29 spheroids were significantly more responsive than monolayers (concentration producing 50% inhibition, 124.6 and 364 nM, respectively) to the experimental drug, 2,5-dimethyl-3,6 diaziridinyl-1,4 benzoquinone. While the environmental stimulus responsible for causing elevated NQO1 expression has not been identified, the fact that NQO1 expression is influenced by microenvironmental conditions will have important implications for those drugs that are activated by NQO1.
School of Pharmacy, University of Southern California, Los Angeles 90033.
R M
RM
Phillips
A
A
de la Cruz
R D
RD
Traver
N W
NW
Gibson
D001388:Aziridines / Q000494:pharmacology; D001483:Base Sequence; D016227:Benzoquinones / Q000494:pharmacology; D002455:Cell Division; D003110:Colonic Neoplasms / Q000201:enzymology* / Q000473:pathology; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D011808:Quinone Reductases / Q000096:biosynthesis* / Q000235:genetics; D014407:Tumor Cells, Cultured
D001388:Aziridines; D016227:Benzoquinones; C073715:2,5-dimethyl-3,6-diaziridinyl-1,4-benzoquinone; C041831:NADH dehydrogenase (quinone); D011808:Quinone Reductases