Supplementary MaterialsSupplementary Information. fluorophore conjugation on immobilized oligonucleotides. The four reactions gave a linear correlation coefficient >0.99 in the range of the concentration of dNTPs present in 106 cells, with little interference of cellular rNTPs. We present evidence indicating that data generated by this methodology is comparable to radioisotope-labeling data. Furthermore, the design and utilization of a strong microplate assay based on this technology evidenced the modulation of dNTPs in response to different chemotherapeutic brokers in malignancy cells. synthesis of dNTPs11. 5-FdU is usually metabolically converted into 5-fluoro-deoxyuridine monophosphate (5-FdUMP), which covalently modifies and inhibits thymidylate synthase, consequently blocking synthesis of dTTP32C34. A mismatch repair-deficient colon cancer cell collection, HCT116, was utilized for the drug treatments. Cells were exposed to Dox (1?M) for 1?hr ABT-199 (Venetoclax) and recovered in fresh medium for 8?hr. These conditions presumably allow for the modulation of dNTPs in response to the activation of DNA repair. Cells were also treated Akap7 with GEM (1?M) for 8?h or 5-FdU (2?M) for 6?h. After treatment, cells were harvested for Western blot analysis and methanol extraction for the microplate assay. Western blot analysis revealed that all the treatments caused an activation of the DNA damage checkpoint response, as indicated by the increase in phospho-CHK2 (pThr68). We found that both the expression levels of subunits of R1, R2, p53R2, thymidylate synthase (TS) and thymidine kinase 1(TK1) and the four dNTP pools remained unchanged in HCT116 cells after recovery from Dox exposure (Table?2). GEM treatment for 8?h clearly results in the depletion of dATP and ABT-199 (Venetoclax) dGTP pools while dTTP levels were increased probably due to upregulation of both TS and TK1 (Fig.?5). As for 5-FdU treatment for 6?h, we found that 90% of the dTTP pool was depleted accompanied by a 75% and 55% reduction in dCTP and dGTP, respectively, with no obvious switch in dATP levels. It was noted that levels of the R1 subunit of RNR diminished upon 5-FdU treatment (Fig.?5), which might contribute to the reduction of dCTP and dGTP (Table?2). Table 2 Effect of three anti-cancer brokers ABT-199 (Venetoclax) around the pools of four dNTPs in HCT116 cells.
dATP2.6??0.23.9??0.8Non detectable (0)1.9??0.6dTTP7.6??3.48.1??2.114.1??0.9 (1.9)Non detectable (0)dCTP1.2??0.92.0??1.60.9??0.6Non detectable (0)dGTP7.1??0.85.8??1.91.8??1.1 (0.3)3.2??0.3 (0.45) Open in a separate window Each data point represents the mean??SD determined from two indie experiments in duplicate. Figures in parentheses show fold changes relative to the control. and indicate the significant increase and decrease relative to the control. Dox, doxorubicin. GEM, gemcitabine. 5-FdU, 5-fluorodeoxyuridine Open in a separate window Physique 5 Western blot analysis of protein levels in response to different chemotherapeutic brokers. HCT116 ABT-199 (Venetoclax) cells were treated with doxorubicin (Dox, 1?M) for 1?hr, followed by recovery for 8?hr. In parallel, cells were treated with Gemcitabine (GEM, 1?M) and 5-fluoro-deoxyuridine (5-FdU, 2?M) for 8?hr and 6?hr, respectively. Cells were harvested for Western blot analysis. Conversation The levels of cellular dNTP pools provide important information indicating metabolic status for DNA synthesis. Information regarding alterations in dNTP pools in response to treatment with anti-cancer ABT-199 (Venetoclax) brokers in malignancy and immune cells is usually of paramount interest when developing precision medicine taking into account the context of molecular networks. Therefore, the availability of a convenient and versatile method for quantification of dNTPs is usually greatly sought after. In this study, we designed a method which combines enzymatic and click reactions to quantitate four dNTP pools and further developed a 96-well microplate assay to increase the assay capacity. By using this microplate assay, we compared the effect.