ABSTRACT
Two different cell lines sharing the multidrug resistance (MDR) phenotype were investigated for 8 months by means of Fourier transform IR spectroscopy on cell smears. We studied (a) a human leukemic doxorubicin-sensitive K562 cell line, from which a doxorubicin-resistant K562 cell subline was subsequently derived; (b) a Chinese hamster LR73 drug-sensitive line, subsequently transfected with the expression plasmid pDREX4 containing the mdr1 gene, to produce a multidrug-resistant LR73 subline (MDR-LR73). The sensitivity of Fourier transform IR spectroscopy has allowed differentiation between sensitive and MDR phenotypes among the above lines, even in double blind studies. The MDR phenotype is characterized by three combined features in spectra: (a) a decrease in the intensities of the amide I and II bands; (b) a shoulder on the high wave numbers slope of the amide I bands; (c) a shift toward the high wave numbers of the amide II bands. Furthermore, computational treatment of Fourier transform IR spectra (deconvolution and Gaussian curve-fitting techniques), has evidenced, in MDR-K562 and MDR-LR73 cell sublines, a conformational change involving the same protein in both sublines. It is hypothesized that the protein implicated in the conformational change may be related to the MDR phenotype.
Subject(s)
Doxorubicin/metabolism , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/metabolism , Animals , Cell Line , Cricetinae , Drug Resistance , Humans , Phenotype , Spectrophotometry, Infrared , Tumor Cells, CulturedABSTRACT
Binding of free fatty acids (FFA) to human serum albumin (HSA) was studied by Fourier transform infrared (FT-IR) spectroscopy. Six volunteers ran a marathon race in order to induce changes in their basal plasma FFA levels. Three volunteers were well-trained and three untrained. The non-deconvolved HSA spectra show two types of spectra: the first one includes the untrained runners, who, after the run, display noticeable changes in the absorbance intensity of the amide I band, which also shifts to higher frequencies. The second one includes the well-trained subjects who exhibit few changes after the race. Examination of the deconvolved spectra of HSA shows two types of spectra, as well: the first class displays important decreases in the absorbance of the component assigned to alpha-helix after the race, together with a gradual growth of the two components that can be assigned to turns. The second class, which includes the well-trained subjects, exhibits few changes after the run. These data are interpreted in terms of conformational changes due to a less ordered alpha-helix state of HSA after FFA binding, while the emergence of components assigned to turns may reflect the exposure of several histidine residues to the solvent (which are buried in HSA when FFA/HSA less than 4). Our data are consistent with the Karush model of FFA to HSA binding.
Subject(s)
Fatty Acids, Nonesterified/chemistry , Physical Exertion , Serum Albumin/chemistry , Adult , Fatty Acids, Nonesterified/metabolism , Fourier Analysis , Humans , Physical Fitness , Protein Binding , Protein Conformation , Serum Albumin/metabolism , Spectrophotometry, InfraredABSTRACT
The multidrug resistance (MDR) phenotype has been investigated by means of Fourier transform infrared spectroscopy (FT-IR/S) on cell smears. We investigated K562 cell lines (sensitive and doxorubicin-resistant, the latter being MDR too) and primary cultures of rat hepatocytes (HEP). HEP displayed elevated levels of P-glycoprotein (P-gp) with time in 2-4 day-old culture, thus developing in the same time a MDR phenotype. No functional P-gp activity could be detected in HEP at day 1 after cell seeding. Given the sensitivity of FT-IR/S and using computational treatment of FT-IR data, we found that spectra of MDR-K562 and HEP from day 2 to day 4 displayed close protein conformational changes involving beta-sheets. These changes might be in close relationship with the MDR-phenotype and P-gp overexpression.