Development of Resistance to Endoplasmic Reticulum Stress-Inducing Agents in Mouse Leukemic L1210 Cells

Abstract

Four new variants of L1210 cells resistant to endoplasmic reticulum (ER) stressors, tunicamycin (STun), thapsigargin (SThap), bortezomib (SBor), and MG-132 (SMG-132), were developed via an 18-month periodic cultivation in culture medium with a gradual increase in substance concentration. Multidrug resistance was generated for STun (to tunicamycin, bortezomib and MG-132), SThap (to tunicamycin, thapsigargin and MG-132), SBor (to bortezomib and MG-132), and SMG-132 (to bortezomib and MG-132). These cells were compared to the original L1210 cells and another two variants, which expressed P-gp due to induction with vincristine or transfection with the gene encoding P-gp, in terms of the following properties: sensitivity to either vincristine or the ER stressors listed above, proliferative activity, expression of resistance markers and proteins involved in the ER stress response, and proteasome activity. The resistance of the new cell variants to ER stressors was accompanied by a decreased proliferation rate and increased proteasome activity. The most consistent change in protein expression was the elevation of GRP78/BiP at the mRNA and protein levels in all resistant variants of L1210 cells. In conclusion, the mechanisms of resistance to these stressors have certain common features, but there are also specific differences.

Insight into Bortezomib Focusing on Its Efficacy against P-gp-Positive MDR Leukemia Cells

“…BOR inhibited S, R, and T cell proliferation with IC 50 values of 6.0752 ± 1.7656, 4.7708 ± 0.8911, and 3.6738 ± 0.5412 (in nM), respectively ( Figure S2 in the supplementary files ). This result is consistent with previously published data [ 25 ]. Because there were weakly significant differences between the IC 50 values of the S cells and the R or T cells, it can be concluded that BOR affects S, R, and T cells with approximately equal efficacy.…”

“…Cutting AMC from the substrate and plotting the BOR effect as a function of time for 90 min revealed a straight line ( Figure S7A in the Supplementary files ), and similarly, a straight line was obtained when plotting the AADG effect measured for 75 min ( Figure S7B in the supplementary files ), both indicating a zero-order reaction that is typical of enzyme catalysis. In previous work, we observed similar behavior when measuring proteasome activity in L1210 cell variants [ 25 ]. This behavior makes it possible to determine the initial reaction rate of the proteasomal cleavage of a substrate, which is constant throughout the measurement, as the slope of the respective line.…”

“…We also observed an increase in the expression of the Cdk1 , Cdk2 , Cdk4, and Cdk6 genes in the R and T cells compared with the S cells. Increased expression of CDKs and their activating cyclins is probably critical for the accelerated proliferation of the R and T cells compared to the S cells, which has been previously described [ 25 ]. Increased expression of the genes encoding CDK4 and CDK6 and their activator, cyclin D1, as well as CDK2, with activating cyclin E1 in P-gp-positive R and T cells ( Figure 3 ), accelerates the shift of CC from the G0/G1 to S phase.…”

“…Since we showed that P-gp-positive R and T cells are resistant to thapsigargin [ 21 ] and tunicamycin [ 19 , 20 ], which cannot be explained by the transport activity of P-gp, we decided to further investigate the resistance of cells to ER stressors. For this purpose, we prepared variants of L1210 cells selected for resistance by all four ER stressors described above [ 25 ]. We found that the main molecular feature of these variants is the overexpression of the molecular chaperone GRP78/BiP, which is the central regulator of ER stress.…”

“…Interestingly, overexpression of this chaperone was found in the R and T cells, but not the S cells, and this property appears to be critical for the resistance of R and T cells to tunicamycin [ 13 ]. In contrast to the reduced sensitivity of R and T cells to thapsigargin and tunicamycin, the response of these P-gp-positive cells to BOR was similar to that of the P-gp-negative S cells [ 25 ]. These results are consistent with the finding that MDR proteins (P-gp, multidrug resistance protein-1 and both breast and lung cancer resistance proteins) did not affect the cytotoxicity of BOR [ 26 ].…”