Albert D A, Gudas L J
J Cell Physiol. 1986 May;127(2):281-7. doi: 10.1002/jcp.1041270214.
Phosphonoformic acid (PFA) and its congener phosphonoacetic acid (PAA) are inhibitors of viral replication whose mechanism of action appears to be the inhibition of viral DNA polymerase. These drugs inhibit mammalian DNA polymerase to a lesser extent. We sought to characterize the effects of phonoformic acid on mammalian cells by examining mutants of S49 cells (a mouse T-lymphoma line), which were selected by virtue of their resistance to phosphonoformic acid. The 11 mutant lines that were resistant to growth inhibition by 3 mM PFA had a range of growth rates, cell cycle distribution abnormalities, and resistance to the inhibitory effects of thymidine, acycloguanosine (acyclovir), aphidicolin, deoxyadenosine, and novobiocin. Most mutant lines had pools of ribonucleoside triphosphates and deoxyribonucleoside triphosphates similar to those of wild-type S49 cells. However, one line (PFA 3-9) had a greatly elevated dCTP pool. When this mutant line was further characterized, no apparent defect in DNA polymerase alpha activity was seen, but an increased ribonucleotide reductase activity, as assayed by CDP reduction in permeabilized cells, was observed. The CDP reductase activity in the PFA 3-9 cells decreased to wild-type control levels, and the CDP reductase activity of wild-type cells was also greatly reduced when PFA (2-3 mM) was added to permeabilized cells during the enzyme assay. These results demonstrate that PFA can directly inhibit ribonucleotide reductase activity in permeabilized cells. In addition, when PFA was added to exponentially growing cultures of either wild-type or PFA 3-9 mutant cells, the drug caused an arrest in S phase of the cell cycle and a decrease in all four deoxyribonucleotide pools, with the most dramatic decrease in the dCTP pools. The reduction in the dCTP pool level could be reversed by addition of exogenous deoxycytidine, but this reversed PFA toxicity only marginally. These observations suggest that PFA is an inhibitor of mammalian ribonucleotide reductase and that partial resistance to PFA can be effected by mutation to increased CDP reductase activity resulting in a large dCTP pool. This mutation results in less than twofold resistance to PFA, suggesting that other sites of inhibition coexist.
膦甲酸（PFA）及其同类物膦乙酸（PAA）是病毒复制的抑制剂，其作用机制似乎是抑制病毒DNA聚合酶。这些药物对哺乳动物DNA聚合酶的抑制作用较小。我们试图通过研究S49细胞（一种小鼠T淋巴瘤细胞系）的突变体来表征膦甲酸对哺乳动物细胞的影响，这些突变体是根据它们对膦甲酸的抗性而筛选出来的。对3 mM PFA的生长抑制具有抗性的11个突变细胞系具有一系列的生长速率、细胞周期分布异常，并且对胸苷、阿昔洛韦（无环鸟苷）、阿非科林、脱氧腺苷和新生霉素的抑制作用具有抗性。大多数突变细胞系的核糖核苷三磷酸和脱氧核糖核苷三磷酸库与野生型S49细胞相似。然而，一个细胞系（PFA 3-9）的dCTP库大大升高。当对这个突变细胞系进行进一步表征时，未发现DNA聚合酶α活性有明显缺陷，但通过测定通透细胞中CDP的还原情况发现核糖核苷酸还原酶活性增加。PFA 3-9细胞中的CDP还原酶活性降至野生型对照水平，并且在酶测定过程中向通透细胞中加入PFA（2-3 mM）时，野生型细胞的CDP还原酶活性也大大降低。这些结果表明，PFA可以直接抑制通透细胞中的核糖核苷酸还原酶活性。此外，当将PFA添加到野生型或PFA 3-9突变细胞的指数生长培养物中时，该药物会导致细胞周期停滞在S期，并且所有四种脱氧核苷酸库均减少，其中dCTP库的减少最为显著。添加外源性脱氧胞苷可以逆转dCTP库水平的降低，但这仅略微逆转了PFA的毒性。这些观察结果表明，PFA是哺乳动物核糖核苷酸还原酶的抑制剂，并且通过突变增加CDP还原酶活性导致dCTP库增大可以实现对PFA的部分抗性。这种突变导致对PFA的抗性小于两倍，表明还存在其他抑制位点。
