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Determinants of trimetrexate lethality in human colon cancer cells
J L Grem1, D M Voeller, F Geoffroy
1National Cancer Institute-Navy Medical Oncology Branch, National Naval Medical Center, Bethesda, Maryland 20889-5105.
Abstract:
We examined the cytotoxicity and biochemical effects of the lipophilic antifol trimetrexate (TMQ) in two human colon carcinoma cell lines, SNU-C4 and NCI-H630, with different inherent sensitivity to TMQ. While a 24 h exposure to 0.1 microM TMQ inhibited cell growth by 50-60% in both cell lines, it did not reduce clonogenic survival. A 24 h exposure to 1 and 10 microM TMQ produced 42% and 50% lethality in C4 cells, but did not affect H630 cells. Dihydrofolate reductase (DHFR) and thymidylate synthase were quantitatively and qualitatively similar in both lines. During drug exposure, DHFR catalytic activity was inhibited by > or = 85% in both cell lines; in addition, the reduction in apparent free DHFR binding capacity (< or = 20% of control), depletion of dTTP, ATP and GTP pools and inhibition of [6-3H]deoxyuridine incorporation into DNA were similar in C4 and H630 cells. TMQ produced a more striking alteration of the pH step alkaline elution profile of newly synthesised DNA in C4 cells compared with 630 cells, however, indicating greater interference with DNA chain elongation or more extensive DNA damage. When TMQ was removed after a 24 h exposure to 0.1 microM, recovery of DHFR catalytic activity and apparent free DHFR binding sites was evident over the next 24-48 h in both cell lines. With 1 and 10 microM, however, persistent inhibition of DHFR was evident in C4 cells, whereas DHFR recovered in H630 cells. These data suggest that, although DHFR inhibition during TMQ exposure produced growth inhibition, DHFR catalytic activity 48 h after drug removal was a more accurate predictor of lethality in these two cell lines. Several factors appeared to influence the duration of DHFR inhibition after drug removal, including initial TMQ concentration, declining cytosolic TMQ levels after drug removal, the ability to acutely increase total DHFR content and the extent of TMQ-mediated DNA damage. The greater sensitivity of C4 cells to TMQ-associated lethality may be attributed to the greater extent of TMQ-mediated DNA damage and more prolonged duration of DHFR inhibition after drug exposure.
Insights
Trimetrexate (TMQ) exhibits differential cytotoxicity in colon cancer cells. Prolonged dihydrofolate reductase (DHFR) inhibition and DNA damage after TMQ removal predict cell lethality, not just initial growth inhibition.
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Research
Background:
- Trimetrexate (TMQ) is a lipophilic antifolate drug.
- Colon carcinoma cell lines SNU-C4 and NCI-H630 exhibit varying sensitivity to TMQ.
Purpose of the Study:
- To investigate the cytotoxicity and biochemical mechanisms of TMQ in two human colon carcinoma cell lines.
- To correlate biochemical effects with differential cell sensitivity to TMQ.
Main Methods:
- Exposure of SNU-C4 and NCI-H630 cells to varying concentrations of TMQ.
- Assays for cell growth, clonogenic survival, and DNA damage (pH step alkaline elution).
- Enzyme activity assays for dihydrofolate reductase (DHFR) and thymidylate synthase.
Main Results:
- TMQ inhibited cell growth similarly in both lines at low concentrations, but higher concentrations (1-10 microM) caused significant lethality only in SNU-C4 cells.
- Biochemical effects like DHFR inhibition and nucleotide depletion were initially similar, but TMQ caused more DNA damage in SNU-C4 cells.
- Persistent DHFR inhibition after drug removal correlated with lethality in SNU-C4 cells, while DHFR recovered in NCI-H630 cells.
Conclusions:
- DHFR inhibition during TMQ exposure causes growth inhibition, but prolonged DHFR inhibition and DNA damage after drug removal are better predictors of cell lethality.
- Differential sensitivity of colon cancer cells to TMQ is linked to the duration of DHFR inhibition and the extent of DNA damage post-exposure.