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Separation of resistance to antitumor diarylsulfonylurea agents from collateral sensitivity to mitochondrial toxins
1Department of Molecular Pharmacology, St. Jude Children's Research Hospital, Memphis, Tennessee, 38105-2794, USA.
Abstract:
Compared with parental GC3/c1 human colon adenocarcinoma cells, which are diarylsulfonylurea (DSU)-sensitive cells, the DSU-resistant clone LYC5 demonstrates 4.2-, 12.8-, and 5.3- fold increase in sensitivity to the mitochondrial toxins rotenone, antimycin, and oligomycin, respectively. Studies with hybrids formed by fusion of parental GC3/c1 cells with LYC5 cells have indicated that resistance to antitumor DSUs and collateral sensitivity to mitochondrial toxins are recessive and therefore potentially linked. To examine this, we transfected a cDNA library from GC3/c1 cells, constructed in pcDNA3, into LYC5 cells. G418-resistant colonies were selected and further selected in a single step for resistance to rotenone (100 nm). Individual colonies (designated T5LR) were expanded and tested for sensitivity to mitochondrial toxins, antitumor DSU agents (LY195779 and LY186391) that demonstrate a 45-50-fold differential potency against GC3/c1, LYC5 cells, and the antimitotic agent vincristine. Results demonstrate that resistance to mitochondrial toxins rotenone, antimycin, and oligomycin can be transferred without conferring a DSU-sensitive phenotype. Furthermore, in T5LR clones, resistance to mitochondrial toxins was not associated with increased resistance to vincristine or increased P-glycoprotein expression, supporting the contention that resistance to these agents is independent of P-glycoprotein. Southern blot analysis of T5LR clones demonstrated unique integration sites for the neomycin phosphotransferase gene into genomic DNA in clones 4 and 9, indicating independent derivation. Analysis of clones 4, 6, and 9 with use of polymerase chain reaction demonstrated a cDNA insert of approximately 1.0 kilobase.
Insights
Drug resistance in colon cancer cells can be transferred independently of sensitivity to diarylsulfonylureas (DSUs). This study shows mitochondrial toxin resistance can be isolated from DSU sensitivity in colon adenocarcinoma cells.
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Research
Background:
- Diarylsulfonylureas (DSUs) are antitumor agents with varying efficacy.
- Colon adenocarcinoma cell lines exhibit differential sensitivity to DSUs and mitochondrial toxins.
- Understanding drug resistance mechanisms is crucial for cancer therapy.
Purpose of the Study:
- To investigate the genetic basis of drug resistance in colon cancer.
- To determine if resistance to mitochondrial toxins is linked to DSU sensitivity.
- To identify genes responsible for altered drug sensitivity.
Main Methods:
- Transfection of a cDNA library into DSU-resistant colon cancer cells (LYC5).
- Selection of resistant clones for rotenone and other mitochondrial toxins.
- Analysis of drug sensitivity (DSU, vincristine) and P-glycoprotein expression in transfected cells.
- Southern blot and PCR analysis to confirm gene integration and insert size.
Main Results:
- Resistance to mitochondrial toxins (rotenone, antimycin, oligomycin) was successfully transferred to LYC5 cells.
- Transfected cells (T5LR clones) did not acquire sensitivity to DSUs.
- Mitochondrial toxin resistance was independent of vincristine resistance and P-glycoprotein expression.
- Southern blot and PCR confirmed unique gene integration events.
Conclusions:
- Resistance to mitochondrial toxins can be conferred independently of DSU sensitivity in colon cancer cells.
- The genetic determinants of mitochondrial toxin resistance are distinct from those conferring DSU sensitivity.
- These findings suggest potential therapeutic strategies targeting specific resistance mechanisms.