This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Brooke, J. Articles by Christopherson, R. I. Search for Related Content PubMed PubMed Citation Articles by Brooke, J. Articles by Christopherson, R. I.

Cytotoxic Effects of Dihydroorotase Inhibitors upon Human CCRF-CEM Leukemia¹

Department of Biochemistry, University of Sydney, Sydney, New South Wales 2006, Australia

6-L-Thiodihydroorotate (TDHO) and 2-oxo-1,2,3,6-tetrahydropyrimidine-4,6-dicarboxylate (HDDP) are potent inhibitors of mammalian dihydroorotase in vitro (R. I. Christopherson, K. J. Schmalzl, E. Szabados, R. J. Goodridge, M. C. Harsanyi, M. E. Sant, E. M. Algar, J. E. Anderson, A. Armstrong, S. C. Sharma, W. A. Bubb, and S. D. Lyons, Biochemistry, 28: 463–470, 1989). Using human CCRF-CEM leukemia cells growing in culture, TDHO and HDDP as the free acids have 50% inhibitory concentration (IC₅₀) values of 32 µM and >1000 µM, respectively, whereas for TDHO methyl ester, the IC₅₀ value is 25 µM, and for HDDP dimethyl ester, the IC₅₀ value is 21 µM. These IC₅₀ values were not affected by addition of dihydroorotate, uridine, or deoxycytidine to the culture medium. TDHO methyl ester (25 µM) had only slight inhibitory effects upon the dihydroorotase reaction of de novo pyrimidine biosynthesis in growing leukemia cells, cells arrested in G₂ + M phases of the cell cycle. At 250 µM TDHO methyl ester, analysis of cell extracts by high-performance liquid chromatography showed that after 4 h carbamyl aspartate had accumulated from undetectable levels to 760 µM, whereas UTP decreased from 580 to 110 µM and CTP from 350 to 86 µM, indicating inhibition of dihydroorotase in growing leukemia cells. IMP accumulated from 63 to 350 µM, total guanylates increased while adenylates decreased, and the adenylate energy charge decreased from 0.91 to 0.69 after 4 h. The cellular concentration of 5-phosphoribosyl 1-pyrophosphate increased from 180 to 290 µM due to sparing from pyrimidine nucleotide biosynthesis resulting in complementary stimulation of the de novo purine pathway. HDDP dimethyl ester at concentrations of up to 250 µM had no discernable effect upon pyrimidine or purine nucleotide biosynthesis. At 25 µM HDDP-dimethyl ester, cells arrested in G₂ + M phases initially, with accumulation of cells in G₁/G₀ at later times. These data suggest that the primary mechanisms of growth inhibition for TDHO and HDDP involve inhibition of cell cycle progression from late G₂ or M phase to G₁ phase and that blockade of the pyrimidine pathway by TDHO is a secondary effect found at higher concentrations.

¹ This work was supported by Wellcome Australia Ltd., and Australian Research Council Grant A08415281.

² To whom requests for reprints should be addressed.

Received 5/14/90. Accepted 9/10/90.