The expression of P-gp in leukemia cells is associated with cross-resistance to protein N-glycosylation inhibitor tunicamycin

Abstract

Abstract. In P-gp-positive cell variants obtained from L1210 cells either by selection with vincristine (L1210/R) or by transfection with the human gene encoding P-gp (L1210/T), we have previously described cross-resistance to tunicamycin (TNM), a protein N-glycosylation inhibitor. Here we studied whether this cross-resistance also underlies P-gp-positive variants of human acute myeloid leukemia cells (AML) derived from SKM-1 and MOLM-13 cells (SKM-1/VCR, SKM-1/LEN, MOLM-13/ VCR) by selection with vincristine (VCR) and lenalidomide (LEN). While SKM-1/LEN cells were P-gp positive, no P-gp was detected in MOLM-13/LEN cells. P-gp-positive cells could be repeatedly passaged in medium containing TNM. In contrast, more than 90% of P-gp-negative cells were entering and progressing through cell death mechanisms after the third passage in medium containing TNM. Combined apoptosis/necrosis cell death was detected in L1210 cells after exposure to TNM. Passaging of P-gp-negative AML cells in medium containing TNM induced preferentially apoptosis. Damage to P-gp-negative cells induced with TNM was associated with arrest in the G1 phase of the cell cycle. P-gp-positive leukemia cells differed from P-gp-negative cells in the composition of plasma membrane glycoproteins, which we monitored with the aid of different lectins. The application of TNM to cells induced additional changes in membrane-linked glycosides.

Overexpression of GRP78/BiP in P-Glycoprotein-Positive L1210 Cells is Responsible for Altered Response of Cells to Tunicamycin as a Stressor of the Endoplasmic Reticulum

“…Therefore, qualitative and quantitative differences in protein N-glycosylation could occur in L1210 cells when P-gp is overexpressed. Consistent with this, we previously detected differences between P-gp-negative S and P-gp-positive R and T cells in the specific binding of several lectins with cell surface-located sugars [35,38,50,51,64,65]. Culture of S, R, and T cells in the presence of 0.1 µM tunicamycin did not affect the expression of this CNX ( Figure S5 in Supplementary Materials).…”

“…While R and T cells could be repeatedly cultured in medium containing sublethal concentrations of tunicamycin (0.1 µM), S cells did not endure these conditions well, and after the third passage, massive cell death was detected [33,35]. This result is also documented in the Supplementary Materials ( Figure S1).…”

“…Moreover, we used calcein retention assay to evaluate P-gp efflux activities and found that R and T cells possessed P-gp efflux activities but that S cells lacked this activity [33]. Both R and T cells were much less sensitive to P-gp substrates, such as VCR, doxorubicin, and mitoxantrone, than S cells [35]. All these features were periodically controlled in S, R, and T cells in our laboratory.…”

“…In this study, we aimed to explain why P-gp-positive R and T cells are less sensitive to the ER stressor tunicamycin [33,35,50,51]. After passaging R and T cells with tunicamycin, P-gp protein was detected only in unglycosylated [33,51] and ubiquitinated [50] molecular forms, and this transporter was localized in its typical position in the plasma membrane with sustained calcein-efflux activity.…”

“…Variants of mouse lymphocytic leukemia L1210 cells that overexpress P-gp due to either selection with vincristine (R) or transfection with a human gene encoding P-gp (T) are considerably less sensitive to repeated culturing in media containing sublethal concentrations of tunicamycin than parental L1210 cells (S) [33]. Similar behavior was observed when P-gp-negative and P-gp-positive variants of human acute myeloid leukemia (AML) cells were compared [35]. Only the unglycosylated 140 kDa P-gp form was present in R and T cells after culturing in medium containing tunicamycin, but interestingly, this form of P-gp was either integrated into the plasma membrane or had full efflux activity as measured by a calcein retention assay [33].…”