DNA mismatch repair proficiency executing 5-fluorouracil cytotoxicity in colorectal cancer cells

Abstract

IntroductionThe fluoropyrimidine 5-fluorouracil (5-FU) is the cornerstone of chemotherapy and the standard of care for patients with advance staged colorectal cancer (CRC). In particular, 5-FU-based chemotherapy improves survival in patients with stage III colon cancer, 1-3 and in patients with stage II and III rectal cancer. 4 However, individual patient tumor response rates are still overall poor (20-30%), 5 and there is not an efficient method to determine which patient will have the best tumor response to 5-FU.5-FU is incorporated into the RNA of a cell, but also inhibits thymidylate synthetase (TS), depleting thymidine triphosphates (TTPs) available for DNA synthesis and enhancing the incorporation of UTP and FdUTP into a cell's DNA as substitutes for TTP. 6 Biochemically, we and others have demonstrated that DNA Methods: We constructed a 5FdU-containing heteroduplex plasmid (5FdU plasmid) and 5FdU-containing linear ds-DNA (5FdU linear DNA), and transfected these into MMR-proficient, hMLH1-/-and hMSH6 -/-cells. We observed cell growth characteristics of both transfectants for 5-FU-induced cytotoxicity.Results: MMR-proficient cells transfected with the 5FdU plasmid but not the 5FdU linear DNA showed reduced cell proliferation by MTS and clonogenic assays, and demonstrated cell morphological change consistent with apoptosis. In MMR-deficient cells, neither the 5FdU plasmid nor 5FdU linear DNA induced cell growth or morphological changes different from controls.Conclusion: 5FdU as heteroduplex DNA in plasmid but not linear form triggered cytotoxicity in a MMR-dependent manner. Thus 5-FU incorporated into DNA, separated from its effects on RNA, can be recognized by DNA MMR to trigger cell death.

Acidic tumor microenvironment downregulates hMLH1 but does not diminish 5-fluorouracil chemosensitivity

“…In experimental studies, hMutSα and hMutSβ participate in recognition of 5FU incorporated within DNA [23,24], which parallels MMR-directed 5FU cytotoxicity, with hMutSα > hMutSβ [25]. Although it has been demonstrated that some components of the tumor microenvironment such as hypoxia diminish DNA MMR repair activity [39–42], there is no data regarding DNA MMR binding or function for 5FU induced cytotoxicity in the typically acidic tumor microenvironment.…”

“…5FU is incorporated into DNA because it is available as a substrate after 5FU's action upon thymidylate synthetase (TS), causing depletion of 2′-deoxythymidine 5′-triphosphate (dTTP). Our demonstration that DNA-containing 5FU specifically triggers a DNA MMR-dependent cell death independent of its RNA actions suggests the importance of 5FU incorporated into DNA for its cytotoxicity [25]. …”

An active molecule from Pulsatilla chinensis, Pulsatilla saponin A, induces apoptosis and inhibits tumor growth of human colon cancer cells without or with 5-FU

“…Hypothetically, 5-FU may cause DNA deterioration, thus accelerating the rates of apoptosis in cancer cells (9,10). However, the effective rate of inducing cancer cell apoptosis for 5-FU was only ~30% with respect to colon cancer clinical chemotherapy.…”

GRG Profiles: John M. Carethers

“…We later showed that this had an effect on patient survival from colorectal cancer, as patients with a defective DNA mismatch repair system did not show a survival benefit with adjuvant 5-FU treatment [10]. We worked out the mechanism, showing that individual complexes from DNA mismatch repair had specific binding affinities for 5-FU incorporated into DNA [11, 12] and that isolating the DNA component of 5-FU (from the RNA component) proved that DNA mismatch repair could trigger cell death once it recognizes 5-FU [13]. Talented postdocs Akihiro Tajima, Moriya Iwaizumi, and Yasushi Hamaya [14] were key in figuring out this important process.…”