Karle J M, Cowan K H, Chisena C A, Cysyk R L
Mol Pharmacol. 1986 Aug;30(2):136-41.
Cultured wild-type MCF-7 human breast cancer cells and two MCF-7 sublines that overproduce enzymes of the de novo pyrimidine biosynthetic pathway were compared with regard to: rate of de novo biosynthesis of uracil nucleotides, sensitivity of the de novo and salvage pathways to the concentration of intracellular uracil nucleotides, and potential of exogenous uridine at concentrations equivalent to plasma levels to affect de novo pyrimidine biosynthesis. The PALAR MCF-7 subline, which is resistant to N-(phosphonacetyl)-L-aspartate and has 5.2 times the activity of the first de novo enzyme as the wild-type MCF-7 cells, synthesizes uracil nucleotides via the de novo pathway at a rate that is 5.8 times that of the wild type MCF-7 cells. The PYRR MCF-7 subline, which is resistant to pyrazofurin and has 15.1 times the activity of orotate phosphoribosyltransferase as the wild-type MCF-7 cells, synthesizes uracil nucleotides via the de novo pathway at a rate that is 1.4 times that of wild-type MCF-7 cells. These results are consistent with carbamyl phosphate synthetase being the rate-controlling step of de novo pyrimidine biosynthesis. In the presence of exogenous uridine at concentrations equivalent to that found in plasma (4.4-8.6 microM), the uracil nucleotide pool of wild-type MCF-7 cells was expanded by 20% and de novo synthesis was inhibited by 55%. Incubation of PALAR MCF-7 cells with uridine at concentrations between 7.3 and 16.8 microM caused a 40% increase in the uracil nucleotide pool and a 30% inhibition of de novo synthesis. De novo synthesis of uracil nucleotides in PYRR MCF-7 cells was not affected by a greater than 10-fold increase in the uracil nucleotide pool. Salvage of [14C] uridine was inhibited by an expanded uracil nucleotide pool in the wild-type and PYRR MCF-7 cells but was not inhibited in the PALAR MCF-7 cell line. These results demonstrate that, although the overproduced enzymes exhibit substrate affinities and specificities in cell-free preparations similar to those of the wild-type enzymes, in intact cells the resistant cell lines exhibit marked differences in the control of de novo and salvage pyrimidine biosynthetic pathways by intracellular uracil nucleotides.
将培养的野生型MCF-7人乳腺癌细胞以及两个过量产生嘧啶从头生物合成途径中酶的MCF-7亚系，在以下方面进行了比较：尿嘧啶核苷酸的从头生物合成速率、从头途径和补救途径对细胞内尿嘧啶核苷酸浓度的敏感性，以及相当于血浆水平浓度的外源性尿苷影响嘧啶从头生物合成的潜力。PALAR MCF-7亚系对N-(膦酰乙酰基)-L-天冬氨酸具有抗性，其第一种从头酶的活性是野生型MCF-7细胞的5.2倍，通过从头途径合成尿嘧啶核苷酸的速率是野生型MCF-7细胞的5.8倍。PYRR MCF-7亚系对吡唑呋林具有抗性，乳清酸磷酸核糖基转移酶的活性是野生型MCF-7细胞的15.1倍，通过从头途径合成尿嘧啶核苷酸的速率是野生型MCF-7细胞的1.4倍。这些结果与氨甲酰磷酸合成酶是嘧啶从头生物合成的速率控制步骤一致。在存在相当于血浆中浓度（4.4 - 8.6 microM）的外源性尿苷时，野生型MCF-7细胞的尿嘧啶核苷酸池扩大了20%，从头合成受到55%的抑制。用浓度在7.3至16.8 microM之间的尿苷孵育PALAR MCF-7细胞，导致尿嘧啶核苷酸池增加40%，从头合成受到3%的抑制。尿嘧啶核苷酸池增加超过10倍时，PYRR MCF-7细胞中尿嘧啶核苷酸的从头合成不受影响。野生型和PYRR MCF-7细胞中，尿嘧啶核苷酸池的扩大抑制了[14C]尿苷的补救，但在PALAR MCF-7细胞系中未受抑制。这些结果表明，尽管过量产生的酶在无细胞制剂中表现出与野生型酶相似的底物亲和力和特异性，但在完整细胞中，抗性细胞系在细胞内尿嘧啶核苷酸对嘧啶从头生物合成途径和补救途径的控制方面表现出显著差异。
