Resistance Mechanisms Of Methotrexate Enantiomers Resistant BALL-1, CCRF-CEM Cell Lines

Objective To establish methotrexate (MTX) enantiomers resistant human leukemiaBALL-1and CCRF-CEM cell lines, observe their biological characters and analyzethe relevant genes expression in epidermal growthfactor receptor (EGFR) signalpathway among different enantiomers resistant cell lines. Methods Using the methodof increasing concentrations combined with continuous low-dose induction to builddifferent enantiomers of MTX resistant cell lines. To observe BALL-1andCCRF-CEM cells in cell morphology and activity differences by Fluorescencemicroscopy. Detect resistance index of resistant cell lines and the parental cell line byMTT. The cell growth curve was determined by cell counting. The migration forcedifferences between D-(-)-MTX resistant cell lines with L-(+)-MTX resistant celllines by Transwell. The cell cycle distribution was analyzed by flow cytometryassayand RT-PCR was used to detect the expression of mRNA in, TGF and PI3K genes.Results1. Using the method of increasing concentrations combined with continuouslow-dose induction to build BALL-1and CCRF-CEM cell lines of different MTXenantiomers resistant. MTT results showed the resistance index ofL-(+)-MTX/BALL-1and L-(+)-MTX/CCRF-CEM is2.89and3.73. The resistanceindex of D-(-)-MTX/BALL-1and D-(-)-MTX/CCRF-CEM is9.65and10.72.2. Thecells BALL-1and CCRF-CEM added L-(+)-MTX that deaths and apoptosis rate wassignificantly higher than added D-(-)-MTX.3. The migration force of D-(-)-MTXresistant cell lines was higher than L-(+)-MTX resistant cell lines.4. The cell growthcurve flow cytometry results showed that compared with parental cell lines (BALL-1,CCRF-CEM) proliferation rate of resistant cell lines (L-type and D-type) weresignificant slower (P<0.05), the cells at S phase were reduced (P<0.05) whileincreased at G0/G1phase (P<0.05).5. EGFR was expressed in BALL-1cell-groups(BALL-1, L-MTX and D-MTX resistant cell lines), there was a significant differencein the EGFR mRNA gene (F=641.41, P=0.000) and there was statical difference between L-type and D-type MTX resistance cells (P=0.000). In BALL-1cell-groupsthere was a significant difference in K-Ras mRNA gene (F=673.086，P=0.000) andthere was statical difference between L-type and D-type MTX resistance cells (P=0.004). EGFR and KRas mRNA gene were expressed in CCRF-CEM cell-groups(EGFR: F=151.323, P=0.000, K-Ras: F=330.894, P=0.000). There was staticaldifference between L-type and D-type MTX resistance cells (EGFRmRNA P=0.000，K-RasmRNA P=0.003). TGF mRNA were expressed in the two cell-groups, but nosignificant difference were observed (P>0.05). Both two cell-groups didn’t expressPI3K mRNA gene. Conclusion The proliferation rate in L-(+)-MTX resistant celllines were slower than D-(-)-MTX. The migration force of D-(-)-MTX resistant celllines was higher than L-(+)-MTX resistant cell lines. The expression of EGFR andK-Ras genes presented a significant difference between L-(+)-MTX and D-(-)-MTXresistant cell lines.