PubMed · 2654318
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C M Huguley. 1989. Progress against leukemia.. https://pubmed.ncbi.nlm.nih.gov/2654318/
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Cleavage of cellular chromatin at internucleosomal sites is a characteristic change of DNA integrity in cells undergoing apoptosis. We have developed an assay for quantitation of internucleosomal DNA fragmentation in apoptotic cells. This technique involves purification of cellular DNA, dephosphorylation of the DNA ends, labeling of DNA with 32P at the 5'-end, gel electrophoresis through agarose, and quantitation of the radioactivity in DNA bands. This assay, which is about 1000- to 2000-fold more sensitive than visualization of DNA bands by ethidium staining, allows the detection of DNA fragments at picogram levels. A method for quantitatively determining the number of fragmented DNA strands is also described. Application of this new assay to evaluate the time course of internucleosomal DNA fragmentation was demonstrated in apoptotic cells induced by an anticancer nucleoside analogue.
Cellular metabolism studies had demonstrated previously that low cellular concentrations of 2',2'-difluorodeoxycytidine (dFdC) nucleotides are eliminated by deoxycytidylate deaminase (dCMPD), whereas dCMPD activity is inhibited at high cellular dFdC nucleotide levels (Heinemann et al., Cancer Res 52: 533-539, 1992). An assay for measuring dCMPD activity in intact human leukemia cells has now been developed to permit investigations of the interactions of dFdC nucleotides with dCMPD in intact cells in which the regulated nature of this enzyme was not disrupted. Using [14C]dCyd as the substrate, radioactivity that accumulated in dTTP was quantitated after high-pressure liquid chromotography by a radioactive flow detector. The assay was first characterized using either the dCMPD inhibitor tetrahydrodeoxyuridine (H4dUrd) which directly inhibits dCMPD, or thymidine and 5-fluoro-2'-deoxyuridine (FdUrd) which indirectly inhibit and activate dCMPD, respectively, by affecting the cellular dCTP:dTTP value. Measured by this in situ assay, there was a strong correlation between dCMPD activity and dCTP:dTTP levels. Consistent with previous studies using partially purified enzyme, incubation of cells with dFdC resulted in a concentration-dependent inhibition of dCMPD in situ. The mechanism of modulation of dCMPD by dFdC, however, was clearly different from that of thymidine and FdUrd. In addition to the effect of dFdC on cellular dCTP:dTTP, our findings also suggested an additional inhibitory mechanism, possibly a direct interaction between dCMPD and dFdC 5'-triphosphate. Thus, results obtained using this direct assay of dCMPD in intact cells support the hypothesis that dCMPD is inhibited by nucleotides of dFdC.
The anti-cancer drug 2',2'-difluoro-2'-deoxycytidine (dFdC) is internally incorporated into DNA in vitro. To determine the effects of this incorporation on DNA structure and function, the beta-cyanoethyl phosphoramidite of dFdC was synthesized and oligodeoxyribonucleotides containing dFdC were made using automated solid phase DNA synthesis techniques. Extension of the coupling time was required to achieve high coupling efficiency, suggesting a significant reduction in the rate of phosphotriester formation. Insertion of dFdC 5' into the recognition sequence of restriction enzymes HpaII and KpnI reduced the rate of cutting by 4% and 14% over 60 minutes. This reduction is similar to the effects seen with arabinofuranosylcytidine (ara-C) but small compared to the reductions caused by base analogues and phosphothioates. Insertion of dFdC into the BamHI recognition sequence, but not 5' to the cut site, did not alter the rate of cutting/recognition. The presence of a single dFdC reduced the Tm's of oligomers by 2-4 degrees C, depending on sequence and location. These results demonstrate that, once incorporated into DNA, dFdC does not greatly alter recognition between DNA and restriction enzymes; however, it does significantly alter duplex stability.