Elevated Levels of DNA Strand Breaks Induced by a Base Analog in the Human Cell Line with the P32T ITPA Variant
Figure 3
Accumulation of ITP in the P32T cell line. Areas of chromatogram corresponding to ITP and IMP are enlarged in boxes above the actual printout. Whole-cell lysates corresponding to 100βg of protein were deproteinized by acid extraction with 1βN HCl followed by alkali treatment with an equal volume of 1βN NaOH. The samples were centrifuged at 14,000βRPM for 10βmin. The volume of aqueous fraction (supernatant) was adjusted to 500βuL with HPLC-grade water. The samples were run on an Alltech hypersil BDS C18 5u column. The HP 1100 HPLC connected to an autosampler and a DAD detector (which was set at 262βnm) was used. Flow rate was kept constant at 1.1βmL/min, and a gradient flow was used: 0 to 5 minutes, 100% (a). 5β5β:β10 ramp up to 100% (b), 5β:β10β8β:β10, 100% (b), 8β:β10β8β:β20 ramp down to 100% (a). In the preliminary experiments we have determined the retention of pure 0.5βmM solutions of ITP (~2.6) and IMP (~5.2).
