Tumor cells purine synthesis

Methotrexate is a folic acid analogue. Its mechanism of action is based on the inhibition of dihydrofolate reductase. Inhibition of dihydrofolate reductase leads to depletion of the tetrahydrofolate cofactors that are required for the synthesis of purines and thymidylate (see Fig. 2). Enzymes that are required for purine and thymidylate synthesis are also directly inhibited by the polyglutamates of methotrexate which accumulate with dihydrofolate reductase inhibition. The mechanisms that can cause resistance include decreased transport of methotrexate into the tumor cells, a decreased affinity of the antifolate for dihydrofolate reductase, increased concentrations of intracellular dihydrofolate reductase and decreased thymidylate synthetase activity. 

Methotexate is an antineoplastic folic add analogue that blocks the conversion of dihydrofolate (FHj) to tetrahydro-folate (FH4) by binding to dihydrofolate reductase (DHFR) enzyme. Folate is essential for the normal synthesis of purines and pyrimidines, and therefore DNA and RNA. In order for folate to function as a cofactor, it must be reduced to FH by DHFR. Methotrexate binds to DHFR, prevents the conversion of FH2 to FH4, and, consequently, inhibits purine and pyrimidine synthesis. The antimetabolites are considered cell cycle specific, with most activity for cells in the S (synthesis) phase. With high-dose methotrexate, leucovorin rescue is often used to prevent severe toxicity to normal body tissues. Leucovorin (folinic acid) is a reduced form of folate (similar to FH ) that does not require the use of DHFR. Leucovorin is transported into healthy cells and is utilized for DNA and RNA synthesis. Tumor cells tend to have impaired transport mechanisms and usually cannot use leucovorin. Leucovorin is usually started within 24 to 36 hours of high-dose methotrexate administration and continues until methotrexate serum levels are below nontoxic levels (0.1 to 0.05 mol/L). 

Purine [adenosine] nucleoside antimetabolite active triphosphate form can prevent elongation of DNA chain also inhibits ribonucleotide reductase, depleting intracellular deoxynucleotides and impairing DNA synthesis Antipyrimidine antimetabolite inhibits DNA polymerase with inhibition of DNA strand elongation and replication activated in tumor cells to triphosphate form competes with conversion of cytidine to deoxycytidine nucleotides, further blocking polymerization of DNA leads to production of short DNA strands cell-cycle specific (S phase) acts only on proliferating cells... 

Fig. 4-14). Both 6-mercaptopurine and 6-thioguanine are not active purine inhibitors until they are converted to their respective nucleotides by hypoxanthine-guanine phosphoribo-syl transferase enzymes in the cell. Since this is an intrinsically destructive process to the cell, it is referred to as a lethal synthesis or salvage pathway. Tumor cells or normal cells that have very low levels of this enzyme are resistant to these two agents. Absence of this enzymatic function is not lethal to the cell since the synthesis of purines in the cell can occur by different pathways. 

Mechanisms of action and resistance Methotrexate is a substrate for and inhibitor of dihydrofolate reductase. This action leads to a decrease in the synthesis of thymidylate, purine nucleotides, and amino acids and thus interferes with nucleic acid and protein metabolism. The formation of polyglutamate derivatives of methotrexate appears to be important for cytotoxic actions. Tumor cell resishince mechanisms include decreased drug accumulation, changes in the drug sensitivity or activity of dihydrofolate reductase, and decreased formation of polyglutamates. 

Pentostatin (Fig. 42.25) is a ring-expanded purine ribonuoleoside that inhibits adenosine deaminase and is used in the treatment of hairy cell leukemia. The elevated levels of deoxyadenosine triphosphate that result from inhibition of this degradative enzyme inhibit the action of ribonucleotide reductase (the enzyme that converts ribose diphosphate to deoxyribose diphosphate), thus halting DNA synthesis within the tumor cell. 

Under physiological conditions the pathway of purine biosynthesis de novo is believed to be irreversible. Reversibility of some reactions can, however, be demonstrated under some conditions of incubation in vitro. If Ehrlich ascites tumor cells are incubated with formate- C in the absence of glucose, for example, the 2-position of the purine ring may contain 8 to 10 times as much C as does the 8-position (SO), although these two positions should be equally labeled if net synthesis only had occurred. Apparently inosinate can be reversibly converted to phosphoribosyl amino-imidazole carboxamide, which in reforming inosinate incorporates radioactive formate. 

PP-Ribose-P concentrations are much lower in Ehrlich ascites tumor cells in vivo than in vitro, and both PP-ribose-P levels and the rate of purine biosynthesis de novo are increased following the injection of glucose (SS). Both PP-ribose-P concentrations and rates of de novo s3mthesis are increased markedly in human skin fibroblasts which are deficient in hypo-xanthine-guanine phosphoribosyltransferase (3S), and this is probably related to the extraordinary rate of purine synthesis in vivo in patients with this enzyme deficiency (34). PP-Ribose-P levels are also elevated in some patients with gout and accelerated purine biosynthesis de novo (35,36). 

Glutamine is present in abundance in some tissues, such as brain, but is scarce in many tumor cells and is a rate-limiting factor in some cells, both in vivo and in vitro it may even be limiting in liver under certain conditions (17, 37). Similarly, glycine and aspartate can be rate-limiting for purine synthesis in some cells (17,38,39). As discussed above, aspartate can some-... 

For example, intact Ehrlich ascites tumor cells, or extracts therefrom, transfer the ribosyl group of uridine to hypoxanthine and thereby catalyze the net synthesis of inosine this reaction depends upon the coupled actions of uridine phosphorylase and purine nucleoside phosphorylase (89). Similar ribosyl transfers have been demonstrated with bacterial cells and extracts. Krenitsky has studied the kinetics of exchange between uracil-2- C and nonisotopic uridine catalyzed by highly purified uridine phosphorylase (30) ... 

LE PAGE,G.A. and M.JONES Purinethiols as feedback inhibitors of purine synthesis in ascites tumor cells. Cancer Res. 2, 642 (1961)... 

T There are several distinct types of inhibitors of nucleotide biosynthesis, each type acting at different points in the pathways to purine or pyrimidine nucleotides. All these inhibitors are very toxic to cells, especially rapidly growing cells, such as those of tumors or bacteria, because interruption of the supply of nucleotides seriously limits the cell s capacity to synthesize the nucleic acids necessary for protein synthesis and cell replication. In some cases, the toxic effect of such inhibitors makes them useful in cancer chemotherapy or in the treatment of bacterial infections. However, some of these agents can also damage the rapidly replicating cells of the intestinal tract and bone marrow. This danger imposes limits on the doses that can be used safely. 

Since folic acid is critical to the formation of purines, antagonists of folic acid metabolism are used as chemotherapy drugs to inhibit nucleic acid synthesis and cell growth. Rapidly dividing cells, such as those found in cancer and tumors, are more susceptible to these antagonists. 

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