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Nitrogen mustard up-regulates Bcl-2 and GSH and increases NTP and PCr in HT-29 colon cancer cells
A W Boddie1, A Constantinou, C Williams
1Department of Surgical Oncology, University of Illinois at Chicago, 60612, USA.
Abstract:
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.
Insights
Chemotherapy using nitrogen mustard (HN2) increases nucleoside triphosphates (NTP) and phosphocreatine (PCr) in HT-29 cells. This is linked to bcl-2 gene and glutathione up-regulation, aiding DNA damage repair.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Unexplained increases in nucleoside triphosphates (NTP) are observed via 31P magnetic resonance spectroscopy (MRS) after DNA-damaging chemotherapy.
- These increases are hypothesized to relate to chemotherapy-induced bcl-2 gene up-regulation and DNA repair processes.
Purpose of the Study:
- To investigate the relationship between chemotherapy-induced nucleoside triphosphates (NTP) increases and bcl-2 gene expression and DNA damage repair mechanisms.
- To correlate changes in high-energy phosphates with bcl-2 protein levels, cell viability, cell cycle, apoptosis, and glutathione (GSH) levels.
Main Methods:
- HT-29 cells were treated with nitrogen mustard (HN2).
- Sequential perchloric acid extractions were performed over 0-18 hours.
- Absolute changes in 31P-detectable high-energy phosphates were determined using an internal standard and correlated with biological markers.
Main Results:
- Nitrogen mustard (HN2) administration led to increased bcl-2 protein and glutathione (GSH) levels within 2 hours.
- Nucleoside triphosphates (NTP) and phosphocreatine (PCr) significantly increased concurrently with bcl-2 and GSH.
- Cell viability decreased to 25% by 18 hours, while apoptosis remained low (1-4%).
Conclusions:
- This study demonstrates that bcl-2 and glutathione are up-regulated by HN2 treatment in HT-29 cells.
- The findings link the up-regulation of bcl-2 and glutathione to the previously unexplained increase in NTP observed via 31P MRS after chemotherapy.
- The results suggest a role for these protective mechanisms in cellular response to DNA-damaging agents.