Tài liệu Báo cáo khoa học: Infrared spectroscopy as a tool for discrimination between sensitive and

Infrared spectroscopy as a tool for discrimination between sensitive

and multiresistant K562 cells

Anthoula Gaigneaux, Jean-Marie Ruysschaert and Erik Goormaghtigh

Laboratory of Structure and Function of Biological Membranes, Free University of Brussels, Belgium

Fourier transform infrared spectroscopy was performed on

human leukemic daunorubicin-sensitive K562 cells and their

multiresistant counterpart derived by selection. Statistical

analysis, including variable reduction and linear discrimi￾nant analysis was performed on sensitive and multiresistant

cells spectra in order to establish a diagnostic tool for

multiresistant pattern. For each of the two methods of data

reduction tested [genetic algorithm or principal component

analysis (PCA)] discrimination between the two cell lines was

found to be possible. The best results, obtained with

PCA-reduction, showed an accuracy of 93% on a distinct

test set of spectra. These results demonstrate the efficiency of

Fourier transform infrared spectroscopy for classification.

Further analysis of the spectral differences indicated that

discrimination between resistant and sensitive cells was

based on variations in all cellular contents. Lipid and nucleic

acid decreased, relatively, while the protein content

increased.

Keywords: multiresistance; infrared spectroscopy; multivar￾iate statistics; K562; leukemia.

In recent years, infrared spectroscopy has been a powerful

tool for biodiagnostics [1]. A major advantage of infrared

spectroscopy over more classical techniques of investigation

is that neither staining of the samples nor chemical reagent

additions are necessary. Just a few minutes and a few lL of

a cell suspension are sufficient to obtain a spectrum

representative of all cell constituents.

This technique is based on absorption of infrared light by

the vibrational transitions in covalent bonds. Intensities

provide quantitative information, while frequencies give

qualitative information about the nature of these bonds,

their structure, and their molecular environment.

In complex systems such as cells, the main absorptions

arise from N–H, C¼O, C–H and P¼O bonds from the

proteins, lipids, and nucleic acids present in the cells. An

infrared spectrum of cells is the sum of all these contribu￾tions. A classical group frequency approach can be used to

interpret changes in one of the cell component, as previously

done on leukemic cell lines [2]. Another way to analyse

infrared spectra is to use the spectral signature to correlate

spectral patterns with biological properties. Rigas [3] proved

that IR spectroscopy was able to detect features of human

normal or malignant cultured colonocytes. Multivariate

statistics known as Ôpattern recognition techniquesÕ have

been used to classify spectra in intrinsic groups when they

are unsupervised (cluster analysis, or principal component

analysis). Naumann et al. [4] successfully used cluster

analysis to characterize hundreds of bacterial cell lines. The

same approach was also used to clearly distinguish between

normal and chronic lymphocytic leukemia cells [5]. Super￾vised multivariate methods, such as linear discriminant

analysis (LDA) or partial least squares regression are

powerful tools to build rules of discrimination that are used

later to identify new samples. This method was successfully

applied to skin tumours [6] and to lymph cells and tissues [7].

The multiresistant phenotype is an significant problem in

cancer chemotherapy. It is characterized by cell resistance to

multiple and structurally unrelated drugs [8]. It may be

expressed by cells selected for resistance to a single agent.

Many of these multiresistant cells differ from their sensitive

counterpart by overexpression of a membranous protein of

170 kDa, named P-glycoprotein (P-gp) [9]. Although the

sole presence of P-gp has proven in some cell lines to confer

multidrug resistance phenotype [10], previous studies have

shown that molecular changes in lipid and nucleic acid

fractions of the cells accompany P-gp overexpression

[11,12].

In this study, we worked with sensitive (K562/DNS) and

multiresistant (K562/DNR) human chronic myelogenous

leukemia K562 cells. First, we examined whether infrared

spectroscopy, associated with data reduction techniques and

multivariate statistics, is able to identify multidrug resistant

phenotype in these cells with a high accuracy. Second, we

tried to learn more about biological origin of the spectral

differences that exist between the K562-multiresistant cell

line and its sensitive counterpart.

MATERIALS AND METHODS

Cell culture

K562 is a human chronic myelogenous leukemia cell line. In

this study, two different K562 lines were used. The first cell

line (cell line A) has been described previously [13]. A second

Correspondence to G. Erik, Laboratory of Structure and Function of

Biological Membranes, Free University of Brussels, CP 206/2,

Boulevard du Triomphe, B-1050 Brussels, Belgium.

Fax: + 32 2 650 5382, Tel.: + 32 2 650 5386,

E-mail: [email protected]

Abbreviations: P-gp, P-glycoprotein; K562/DNS, sensitive K562 cells;

K562/DNR, daunorubicin resistant K562 cells; PCA, principal com￾ponent analysis; LDA, linear discriminant analysis; MDR, multidrug

resistant.

(Received 4 January 2002, accepted 21 January 2002)

Eur. J. Biochem. 269, 1968–1973 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02841.x