Various Methods of Analysis of Mdr-1/P-glycoprotein in Human Colon Cancer Cell Lines

Background: Multidrug resistance (MDR) mediated by high levels of mdr-1 (also known as PGY1)/P-glycoprotein (Pgp) has been studied in tissue culture systems; however, most tumor samples which express mdr-1/Pgp have much lower levels.

Purpose: We wanted to determine if levels seen clinically could be detected by commonly used methods and to determine if these levels conferred MDR reversible by Pgp antagonists.

Methods: We studied multi-drug-resistant cell lines and sublines with levels of mdr-1/Pgp expression comparable to those seen clinically. We evaluated the expression of mdr-1 RNA by Northern blot analysis, slot blot analysis, polymerase chain reaction (PCR) analysis, and in situ hybridization. We evaluated protein expression by immunofluorescence, immunohistochemistry, fluorescence-activated cell sorting, and immunoblotting analyses. Drug resistance and reversibility were determined by cell growth during continuous drug exposure.

Results: In most cases, the low level of mdr-1/Pgp present in these cell lines could be detected by each method, but the assays were at the limit of sensitivity for all methods except the PCR method. These low levels of mdr-1/Pgp are capable of conferring MDR, which can be antagonized by verapamil.

Conclusions: Levels of mdr-1/Pgp similar to those found in clinical samples can be detected by each of these methods, but the PCR method was the most sensitive and most reliably quantitative.

Implications: In vitro sensitization by the addition of verapamil in cell lines with these low levels of mdr-1/Pgp suggests that clinically detected levels may confer drug resistance in vivo.