Nitrogen mustard up-regulates Bcl-2 and GSH and increases NTP and PCr in HT-29 colon cancer cells.
We hypothesized that unexplained increases in nucleoside triphosphates (NTP) observed by 31P magnetic resonance spectroscopy (MRS) after treatment 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 HT-29 cells with 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 31P-detectable high-energy phosphates in these extracts were determined and correlated with changes in bcl-2 protein levels, cell viability, cell cycle, apoptosis and total cellular glutathione (GSH) (an important defence against DNA damage from alkylating agents). After HN2 administration, bcl-2 protein levels in the HT-29 cell line rose at 2 h. Cell viability declined to 25% within 18 h, but apoptosis measured using fluorescence techniques remained in the 1-4% range. Increased cell division was noted at 4 h. Two high-energy interconvertible phosphates, NTP (P < or = 0.006) and phosphocreatine (PCr) (P < or = 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 31P MRS phenomenon: increased NTP after chemotherapy.
Boddie
A W
AW
Department of Surgical Oncology, University of Illinois at Chicago, 60612, USA.
Constantinou
A
A
Williams
C
C
Reed
A
A
D000255:Adenosine Triphosphate / Q000378:metabolism; D000641:Ammonia / Q000494:pharmacology; D018906:Antineoplastic Agents, Alkylating / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D002453:Cell Cycle / Q000187:drug effects; D002851:Chromatography, High Pressure Liquid; D003110:Colonic Neoplasms / Q000378:metabolism / Q000473:pathology; D003570:Cytidine Triphosphate / Q000378:metabolism; D019254:Genes, bcl-2 / Q000187:drug effects*; D005978:Glutathione / Q000187:drug effects*; D006160:Guanosine Triphosphate / Q000378:metabolism; D019073:HT29 Cells / Q000187:drug effects; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008466:Mechlorethamine / Q000494:pharmacology*; D010725:Phosphocreatine / Q000378:metabolism*; D011685:Purine Nucleotides / Q000378:metabolism*; D015854:Up-Regulation; D014544:Uridine Triphosphate / Q000378:metabolism
D018906:Antineoplastic Agents, Alkylating; D011685:Purine Nucleotides; D010725:Phosphocreatine; D008466:Mechlorethamine; D003570:Cytidine Triphosphate; D000641:Ammonia; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D005978:Glutathione; D014544:Uridine Triphosphate