APVV-14-0334

Principal investigator: Zdena Sulová

Duration: July 2015 – June 2018
Coordinating institution: Institute of Molecular Physiology and Genetics, SAS, Bratislava

Annotation:

Project is focused on hypothesis that P-glycoprotein besides its generally accepted role as multidrug efflux pump for large groups of lipophilic substances also play additional role as regulatory protein influencing programmed cell death progression. Several lines of evidences indicated, that this regulatory action of P-gp is independent on P-gp transport activity. Therefore, P-gp when express in cells, could depress cell sensitivity also to substances which cannot be effluxed by its transport activity. We studied this trend on mice leukemia cells L1210 in which the P-gp expression was induced either by selection for resistance with vincristine, or by transfection with gene encoding full length P-gp. We found that P-gp overexpressing cells are resistant also to endoplasmic reticulum stressors like tunicamycin (inhibitor of N-glycosylation in endoplasmic reticulum), thapsigargin (inhibitor of Ca (2+)-ATPase from endoplasmic reticulum); but also to cisplatin (chemotherapeutic that is not substrate for P-gp), and lastly to lectin Concanavalin A. However P-gp positive cells were more sensitive than their P-gp negative counterparts to another lectin from wheat germ. These results are associated with massive remodalation of cell surface saccharides in cells with upregulated P-gp. Presence of P-gp in cells seems to induce strong alteration in cell regulatory pathways including either protein phosphorylation and glycosylation or regulation of apoptosis progression. These issues we plane to study by described P-gp cell models, or by using the human leukemia cells, in which the P-gp overexpresion was induced by adaptation with chemotherapeutics like vincristine, mitoxantrone or  azacytidine.

Keywords:

P-glycoprotein, leukemia, multidrug ressitance

Objectives:

Aims of this project are subjected to the detailed study of changes in the important cellular regulatory pathways, connected with the presence of P-gp in the leukemia cell line L1210, SKM 1 and MOLM 13 etc. which we have detected in the past. We will focus particularly on the changes: 1. In a quality control processes of proteins newly synthesized in the endoplasmic reticulum; 2. in the metabolism of carbohydrates; 3. in the onset and progression of apoptosis induced by cytotoxic agents that are not substrates of P-gp (5-azacytidine, cisplatin, lenalidomide, ceramides).

To achieve these aims, we will solve following stages:

• Monitoring of plasma membrane proteins glycosylation by interaction of viable cells with lectins having specificity for various carbohydrates. Comparison of changes in expression/glycosylation of membrane proteins associated with expression of P-gp in murine and human leukemia cells. Monitoring of protein glycosylation by microarray analysis using lectins.
• Study of ubiquitination and subsequent degradation of proteins in P-gp positive and negative cells cultivated in the presence of substances inducing stress of endoplasmic reticulum (tunicamycin – inhibitor of N-glycosylation and thapsigargin – inhibitor of Ca2+-ATPase of endoplasmic reticulum).
• Study of the changes in the onset of apoptosis after induction with compounds that are not substrates of P-gp in leukemia cells expressing and non-expressing P-gp. Monitoring the levels of pro- and anti-apoptotic proteins. Testing the effect of ceramides with different carbon chains in relations with activities of glucosylceramide synthase.
• 4. Observation of protein phosphorylation using radioactive phosphate in the presence of kinase inhibitors. Comparison of differences in the effect of substances on the protein phosphorylation in P-gp positive and negative human and mouse leukemia cells.
• Study of nuclear receptors for retinoids and rexinoids involvement to P-gp expression in human leukemia cells SKM 1 and MOLM 13. Study of involvement of appropriate miRNAs in the regulation of the expression and function of P-gp.
• Study of co-expression of regulatory and functional proteins with P-gp in leukemia cells.

Publications: