Thymineless death

The cell requires each of the four deoxyribonucleotides (dNTPsj to replicate and repair its DNA. This is a trivial statement. More interesting is to say that the cell needs a balanced supply of the four NTPs to replicate and repair its DNA properly. The importance of the regulation of the NTP pools becomes apparent from disturbances in cell function when a bias in the normal balance occurs. Two extensively studied cases, leading to cell death, are the effects of severe dTTP deprivation ( thymineless death) and certain immune diseases accompanied by the accumulation of dATP and dGTP. 

Fluorouracil (5-FU) is inactive in its parent form and requires activation via a complex series of enzymatic reactions to ribosyl and deoxyribosyl nucleotide metabolites. One of these metabolites, 5-fluoro-2 -deoxyuridine-5 -monophosphate (FdUMP), forms a covalently ternary complex with the enzyme thymidylate synthase and the reduced folate 5,10-methylenetetrahydrofolate, a reaction critical for the de novo synthesis of thymidylate. This results in inhibition of DNA synthesis through "thymineless death." 5-FU is converted to 5-fluorouridine-5 -triphosphate (FUTP), which is then incorporated into RNA, where it interferes with RNA processing and mRNA translation. 5-FU is also converted to 5-fluorodeoxyuridine-5 -triphosphate (FdUTP), which can be incorporated into cellular DNA, resulting in inhibition of DNA synthesis and function. Thus, the cytotoxicity of 5-FU is thought to be the result of combined effects on both DNA- and RNA-mediated events. 

Flucytosine (FQ and 5-fluorouraci (5-FU) are bioactivated to 5-fluorodeoxyuridine monophosphate (5-FdUMP), which inhibits thymidylate synthetase —> "thymineless death" of fungal cells (FC) or neoplastic cells (5-FU). 

Which of the following is a pro-drug that causes thymineless death of cells ... 

Answer C. All of the drugs listed are antimetabolites used in cancer chemotherapy or as immunosuppressants. The 5-fluorouracil is bioactivated to 5-fluorodeoxyuridine monophosphate (5-FdUMP), a substrate for and inhibitor of thymidylate synthase. When used in drug regimens for treatment of cancer, 5-FU causes thymineless death of cells. 

Mechanisms Fluorouracil is biotransformed to 5-fluoro-2 -deoxyuridine-5 -monophos-phate (5-FdUMP). which inhibits thymidylate synthase and leads to thymineless death of cells. Tumor cell resistance mechanisms include decreased activation of 5-FU, increased thymidylate S)mthase activity, and reduced drug sensitivity of this enzyme. 

Fluorouracil (5-FU) undergoes metabolism to form 5-fluoro-2 -deoxyuridine 5 -phosphate (5-dUMP). This metabolite forms a covalently bound ternary complex with thymidylate synthase and its coenzyme A-methylenetetrahydrofolate. The synthesis of thymine nucleotides is blocked, and a thymineless death of cells results. The answer is (E). 

Looked at simply, dTMP is produced via Cs-methylation of deoxyuridine monophosphate (dUMP). The rate-limiting enzyme of the dTMP synthetic pathway is the sulfhydryl-containing thymidylate synthase, with 5,10-methylenetetrahydrofolate (5,10-methylene-THF) serving as the methyl-donating cofactor. All dTMP synthesis inhibitors will inhibit thymidylate synthase either directly or indirectly, and this will result in a thymineless death in actively dividing cells. Without dTMP and its deoxythmidine triphosphate metabolite, DNAwill fragment, and the cell will die. 

By the thymineless death method a situation is created in which the parental cells die and the mutant cells do not grow but are able to stay alive in the selective environment. Since mammalian cells utilize thymidine instead of thymine, a state of thymidine starvation is created by exposing cells to a folic acid antagonist, such as aminopterin. Under these conditions, the prototrophs continue to grow until death, while the auxotrophs do not grow but remain alive and can thus be rescued. This method has been successfully applied to isolation of glutamine-requiring mutant lines from HeLa cells (De Mars and Hooper, 1960) and Chinese hamster cells (Chu et aLy 196%). 

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