NITROGEN-MUSTARD UP-REGULATES BCL-2 AND GSH AND INCREASES NTP AND PCRIN HT-29 COLON-CANCER CELLS

We hypothesized that unexplained increases in nucleoside triphosphates (NTP) observed by P-31 magnetic resonance spectroscopy (MRS) after tr eatment of tumours by DNA-damaging agents were related to chemotherapy -induced up-regulation of the bcl-2 gene and DNA damage prevention and repair processes. To test this hypothesis, we treated MT-29 cells wit h 10(-4) M nitrogen mustard (HN2) and performed sequential perchloric acid extractions in replicate over 0-18 h. By reference to an internal standard (methylene diphosphonic acid), absolute changes in P-31-dete ctable high-energy phosphates in these extracts were determined and co rrelated with changes in bcl-2 protein levels, cell viability, cell cy cle, apoptosis and total cellular glutathione (GSH) (an important defe nce against DNA damage from alkylating agents). After HN2 administrati on, bcl-2 protein levels in the MT-29 cell line rose at 2 h. Cell viab ility declined to 25% within 18 h, but apoptosis measured using fluore scence techniques remained in the 1-4% range. Increased cell division was noted at 4 h. Two high-energy interconvertible phosphates, NTP (P less than or equal to 0.006) and phosphocreatine (PCr) (P less than or equal to 0.0002), increased at 2 h concurrently with increased levels of bcl-2 protein and glutathione. This study demonstrates that bcl-2 and glutathione are up-regulated by HN2 and links this to a previously unexplained P-31 MRS phenomenon: increased NTP after chemotherapy.