FTIR microspectroscopy of malignant fibroblasts transformed by mouse sarcoma virus

FTIR microspectroscopy of malignant fibroblasts

transformed by mouse sarcoma virus

Ahmad Salmana, Jagannathan Ramesha,1, Vitaly Erukhimovitchb,Marina Talyshinskyb, Shaul Mordechaia,*, Mahmoud Huleihelb,*

aDepartment of Physics, Ben Gurion University of the Negev, Beer-Sheva 84105, Israelb Institute for Applied Biosciences, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel

Received 21 May 2002; received in revised form 7 November 2002; accepted 29 November 2002

Abstract

Fourier transform infrared microspectroscopy (FTIR-MSP), which is based on the characteristic

molecular vibrational spectra of cells, was used to investigate spectral differences between normal

primary rabbit bone marrow (BM) cells and bone marrow cells transformed (BMT) by murine

sarcoma virus (MuSV). Primary cells, rather than cell lines, were used for this research because

primary cells are similar to normal tissue cells in most of their characteristics. Our results showed

dramatic changes in absorbance between the control cells and MuSV124-transformed cells. Various

biological markers, such as the phosphate level and the RNA/DNA obtained, based on the analysis

of the FTIR-MSP spectra, also displayed significant differences between the control and transformed

cells. Preliminary results suggested that the cluster analysis performed on the FTIR-MSP spectra

yielded 100% accuracy in classifying both types of cells.

D 2002 Elsevier Science B.V. All rights reserved.

Keywords: Fibroblast; MuSV; FTIR microspectroscopy; Cluster analysis

1. Introduction

Progress in deciphering the molecular mechanisms of carcinogenesis has been

enhanced substantially by the study of the action of viruses as one of the causatives of

0165-022X/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved.

doi:10.1016/S0165-022X(02)00182-3

* Corresponding authors. S. Mordechai is to be contacted at Tel.: +972-8-646-1749; fax: +972-8-647-2903.

M. Huleihel is to be contacted at Tel.: +972-8-646-1999; fax: +972-8-647-2970.

E-mail addresses: [email protected] (S. Mordechai), [email protected] (M. Huleihel).1 Present address: Advanced Biomedical Science and Technology Group, Life Sciences Division, Oak Ridge

National Laboratory, TN 37831-6101, USA.

www.elsevier.com/locate/jbbm

J. Biochem. Biophys. Methods 55 (2003) 141–153

cancer [1]. Viruses consist of either DNA or RNA as nucleic acid and have been well

studied in the last 20 years. Among them, retroviruses containing RNA are known to be

responsible for various types of malignancies in humans [2,3]. Cellular transformation by

retroviruses involves the insertion and activation of viral proto-oncogenes [4]. More than

70 different proto-oncogenes are documented in the literature [5]. Cervical cancer is the

second type of malignancy causing mortality in women in the World. The human

papiloma viruses (HPV) have been incriminated in the transformation of epithelia in

cervical cancer [6]. Powerful molecular techniques are available today to detect the virus

in the cells obtained from patients [7]. However, these methods are expensive and time-

consuming in nature.

Fourier transform infrared (FTIR) spectroscopy has been widely applied in biology

and medicine. FTIR has expanded our knowledge of the structure, conformation and

dynamics of various molecular components of the cell [8]. With the introduction of

microscopy in modern FTIR instrumentation, FTIR analysis of cells and tissues has

become a reality. Naumann’s group successfully achieved the classification of different

classes of bacteria using FTIR spectroscopy [9]. In recent years, there has been

tremendous interest in applying FTIR as a tool for the diagnosis of cancer. Successful

diagnoses of lung [10], breast [11], cervical [12] and prostate [13] cancers have been

reported. In addition to the in vitro studies on cells [14], our group has contributed to

the accurate diagnosis of colon cancer using FTIR microspectroscopy (FTIR-MSP)

[15].

There is enough evidence in the literature that viral infections, especially in cervical

cancer, could be detected using FTIR-MSP [16]. This is the first controlled study in

which the malignant transformation of fibroblasts in ex vivo was performed by

retroviral infection and detected by FTIR-MSP. Our initial results indicated that

retroviral infection could be successfully detected in fibroblasts by means of FTIR-

MSP.

2. Materials and methods

2.1. Cells and viruses

Primary rabbit cells obtained from the bone marrow (BM) of 1.5-kg rabbits were

grown at 37 jC in Roswell Park Memorial Institute (RPMI) medium, supplemented

with 10% newborn calf serum (NBCS) and antibiotics: penicillin, streptomycin and

neumycin. All the chemicals used were obtained from Biological Industries (Bet ha-

Emeq, Israel). Clone 124 of TB cells (mouse fibroblast cells), chronically releasing

Moloney MuSV-124, was used to prepare the appropriate virus stock. TB cells were

grown in a minimum amount of medium containing 2% serum for 24 h. Then, this

medium was spinned at 2000 rpm for 5 min in order to remove cells and cell debris

(Virus particles are released from the TB cells into the medium) and used for infection.

The normal rabbit bone marrow fibroblasts were designated as BM cells and those

transformed by murine sarcoma virus (MuSV) as bone marrow-transformed (BMT)

cells.

A. Salman et al. / J. Biochem. Biophys. Methods 55 (2003) 141–153142

2.2. Cell infection and determination of cell transformation

A monolayer of primary fibroblast rabbit cells grown in 9-cm2 tissue culture plates was

treated with 8 Ag/ml of polybrene (a cationic polymer required for neutralizing the

negative charge of the cell membrane) for 24 h before infection with the virus. Excess

polybrene was then removed and the cells were incubated at 37 jC for 2 h with the

infecting virus (MuSV-124) at various concentrations in RPMI medium containing 2% of

NBCS. The unabsorbed virus particles were removed, fresh medium containing 2% NBCS

was added, and the monolayers were incubated at 37 jC. Control cells were also treated

with polybrene using the same procedure as for the MuSV-transformed cells. After 2–3

days, these cell cultures were examined for the appearance of malignant transformed cells.

In addition, normal primary bone marrow cells and transformed cells were synchronized

separately at G1 phase by sodium butyrate treatment [17] and their spectra were tested

every 2 h of growth.

2.3. Sample preparation

Because ordinary glass slides exhibit strong absorbance in the wavelength range of

interest, zinc sellenide crystals, which are highly transparent to IR radiation, were used.

Normal cells from passages 3–5 or transformed cells (from a fully transformed cell

culture) were washed twice with saline and culled from the tissue culture plates after a 1-

min treatment with trypsin (0.25%). These cells were pelleted by centrifugation at 1000

rpm for 5 min. Each pellet was then washed twice with saline and resuspended in 100 Al ofsaline. The number of cells was counted with an hematocytometer, and all the tested

samples were pelleted again and resuspended in an appropriate volume of saline to give a

concentration of 1000 cells/Al. One drop of 1 Al from each sample was placed on a certain

area on the zinc sellenide crystal, air-dried for 4 h and examined then by FTIR microscopy.

The radius of such a 1-Al drop was found to be about 0.5 mm.

2.4. FTIR microspectroscopy (FTIR-MSP)

FTIR measurements were performed in transmission mode using the FTIR microscope

IRscope II, equipped with a mercury cadmium telluride (HgCdTe) (MCT) detector,

coupled to the FTIR spectrometer (BRUKER EQUINOX model 55 OPUS software).

This microscope is also equipped with a CCD camera, for the visible range of the

spectrum, and a fully computerized X–Y stage, allowing for the measurement of a large

number of spectra, which can be used for creating FTIR maps. The measured spectra cover

the wave number range 600–4000 cm� 1. During each measurement, the measured sites

were circular, measuring about 100 Am diameter at most. There are about 100 cells within

a circle of 100 Am. The spectra taken consisted of an average of 128 scans each to increase

the signal-to-noise ratio. Baseline corrections for all the spectra were done using the rubber

band baseline correction method, and they were normalized arbitrarily to 2 for the amide I

peak at 1643 cm� 1 after the baseline corrections of the entire spectrum. This normal-

ization, with respect to total protein content, nullifies the differences in thickness among

the various samples. Amide II and vector normalization have also been tested in the

A. Salman et al. / J. Biochem. Biophys. Methods 55 (2003) 141–153 143

present work. In the vector normalization, the sum of the square of the absorbances (600–

1800 cm� 1) is normalized to one. The data presented in this report was produced in seven

different experiments. For each cell type (BM or BMT), 10 different site measurements

were performed.

2.5. Cluster analysis

Cluster analysis is an unsupervised technique that examines the interpoint distances

between all the samples and represents the information in the form of a two-dimensional

plot, known as a dendrogram. The dendrogram presents the data from high-dimensional

row spaces in a form that facilitates the use of human pattern recognition abilities. To

generate a dendrogram, cluster analysis methods form clusters of samples based on their

nearness in row space. Cluster analysis was performed with good quality spectra, those

having a high signal-to-noise ratio (>1000). The ‘‘Ward minimum variance method,’’

provided in the OPUS software, was used for the cluster analysis. The total number of

spectra used in our analysis was 111 including normal and transformed samples.

3. Results

3.1. Cell characteristics

In the present study, primary cells (1–2 passages/culture) and malignant cells trans-

formed by retroviruses (MuSV) were used.

Primary rabbit BM fibroblasts, grown in plastic dishes in RPMI medium with 10%

NBCS, appear as flat cells under an inverted light microscope (Fig. 1a). These cells are

completely unable to grow in soft agar. When BM cells were infected by MuSV,

transformed cells, with a highly refractive shape, were detected growing randomly in a

Fig. 1. (a) Normal primary rabbit fibroblast cells (BM). (b) BM transformed by murine sarcoma virus (BMT).


criss-cross fashion (Fig. 1b). These BMT cells were able to grow on soft agar and to

produce large colonies within 5–10 days. BM cells replicated very slowly in culture and

could not survive high densities; most of them died after about 10 passages. In contrast,

the BMT cells grew rapidly and reached high densities in cell culture (Fig. 2).

When 5� 106 cells were injected subcutaneously to newborn mice, only malignant

cells were able to produce tumors 2 weeks after injection (data not shown). Primary cells

are not able to replicate and produce tumors in newborn mice.

3.2. FTIR microspectroscopy

FTIR-MSP spectra of the BM and BMT normalized to amide I are shown in Fig. 3a.

Baseline corrections were performed for all the spectra in the range between 600–4000

cm� 1. The absorbance in the BM was higher than in the BMT.

The absorbance changes were different at various regions of the spectrum. No major

spectral pattern changes were observed between the two types of cells. The absorbance

changes in the region between 2600 and 3200 cm� 1 (data not shown) were similar to the

lower wave number region. In comparison, the FTIR-MSP spectra of the NIH 3T3

fibroblast cell line (23) and its corresponding BMT (by the same virus reported in this

article) spectra are shown in Fig. 3b. The absorbance was higher for normal cells in both

cell types as compared to those transformed by the virus. The percentage of change in

absorbance between these two different sources can be better understood from the

difference spectra shown in Fig. 3c. The percentage of change was much higher for

BM-BMT than for the NIH-MuSV, indicating that the effects of the retroviral infection of

the fibroblasts varies for different sources.

In addition, our data showed that the cell cycle of the tested cells did not affect the

spectral differences observed between BM and BMT cells (data not shown). Both of these

cell cultures were synchronized and their spectra were examined every 2 h of growth. No

Fig. 2. Cell proliferation of BM and BMT.


Fig. 3. FTIR-microscopic spectra of: (a) Control cells (BM—solid line) and MuSV-transformed fibroblasts

(BMT—broken line) isolated from bone marrow of rabbits; (b) BM (solid line) and BMT (broken line) NIH 3T3

fibroblasts (Ref. [23]); and (c) Differential spectra generated by the subtraction of BMT spectra from the BM

ones. The spectra shown in Fig. 1a are the averages of seven different measurements. All the spectra were

baseline corrected and normalized to the amide I band at 1643 cm� 1.


significant differences were observed between the spectra of each kind of cells at the

various tested times.

To understand the effects of various methods of spectral processing, the FTIR-MSP

spectra of BM and BMT are presented again in Fig. 4a and b with two additional methods

of normalization, amide II (Fig. 4a) and vector (Fig. 4b) normalization.

Both types of normalization methods showed that absorbance for BM was higher than

for the BMT, except for in amide I band, where the absorbance changes were reversed.

Hence, it can be concluded that the quantitative changes observed in our study, in the

fingerprints regions (e.g., phosphate), were independent of the methods of spectral

manipulation.

The quantification of phosphate metabolites, which are composed of energy yielding

molecules and nucleic acids, provides a clue about the various states of the cell [18]. The

phosphate contents calculated by measuring the integrated area of phosphate symmetric

Fig. 4. (a) Amide II-normalized FTIR-microscopic spectra; (b) Vector-normalized FTIR-microscopic spectra for

BM (solid line) and BMT (broken line) isolated from the bone marrow of rabbits.


(980–1149 cm� 1) and phosphate asymmetric (1151–1350 cm� 1) bands for the control

cells and the BMT are given in Fig. 5. The phosphate content was dramatically higher in

the control cells relative to the BMT. It should be noted that the phosphate content (Fig. 5)

reported in this article for BM and BMT was measured with the spectra normalized to the

amide I peak. In addition, the variation among the BMTwas lower than that of the control

cells.

Ratio of amide I/II bands was reported to shed light on the change in the DNA content.

The ratio of amide I/II is unity for red blood cells (RBC), and deviation from unity is an

indication of DNA absorbance by infrared radiation from the cells [19]. The contribution

Fig. 6. The absorbance ratio of amide I (1643)/II (1544) for BM and BMT isolated from the bone marrow of

rabbits.

Fig. 5. Phosphate level as a biological marker derived from the FTIR spectra for BM and BMT isolated from the

bone marrow of rabbits. The phosphate content was calculated as the sum of the integrated absorbance of the

symmetric and asymmetric bands of the phosphate group. OPUS software was used for this purpose. The error

bars in Fig. 3–5 represent the standard error for each sample.


of the DNA due to the carbonyl group from the bases is quantified by the ratio of the

integrated area of amide I/II. Our results, shown in Fig. 6, indicated that the BMT had

higher DNA absorbance than the control cells. In 42% of the cases, the difference between

the control cells and the BMT was significantly high, in accordance with observations in

the literature.

Progress in the malignant transformation of the cell can be deduced from RNA/DNA

(intensity ratio at 1121/1020), which could be one of the biological markers. Earlier reports

[20] indicated that malignant cells had an increased RNA/DNA ratio as compared to the

control cells. The RNA/DNA for control cells and BMT is shown in the Fig. 7. Higher

RNA/DNAwas observed in the BMT than in the control cells. Our group obtained similar

results with a fibroblast cell line (balb/c) transformed by H-ras oncogene [14].

Various mathematical methods are applied for in biological and medical classification

[21,22]. The analysis of data leading to classification is the main purpose of the

abovementioned methods. Among the methods available today, cluster analysis is one

of the simplest and fastest procedures, which we adopted to classify the FTIR spectra of

the control cells and of the BMT. Cluster analysis was performed for different segments of

the spectra to obtain the best results. Table 1 shows the preliminary results of our cluster

analysis of the FTIR spectra for both the control cells and the BMT. The regions which

Fig. 7. The intensity ratio at 1121/1020 is presented as RNA/DNA for BM and BMT isolated from the bone

marrow of rabbits.

Table 1

Summary of cluster analysis resultsa

980–

1149

1151–

1350

1340–

1477

1475–

1593

1591–

1753

980–

1350

980–

1483

980–

1593

980–

1759

BM as BM 100% 85.5% 89% 96.4% 96.4% 100% 85.5% 100% 100%

BM as BMT 0% 14.5% 11% 3.6% 3.6% 0% 14.5% 0% 0%

BMT as BMT 100% 100% 98.2% 69.6% 87.5% 100% 100% 89.3% 87.5%

BMT as BM 0% 0% 1.8% 30.4% 12.5% 0% 0% 10.7% 12.5%

a All wavenumbers are in cm� 1.


correspond to the symmetric and asymmetric (980–1350 cm� 1) stretching vibrations of

phosphate were shown to classify both types of cells with 100% accuracy.

The symmetric region (980–1149 cm� 1) alone provided a 100% accurate classification

of both the control cells and the BMT. The region between 980 and 1759 cm� 1, covering

the phosphate, side-chain and protein bands, provided 100% classification for the control

cells, whereas only 87.5% was obtained for the BMT in this region. Other segments of the

spectra provided 70–100% classification accuracy. Our results showed that the phosphate

regions were the best for the classification of the two types of cells used in our study. The

dendrogram showing the results of a cluster analysis in the region between 980 and 1350

cm� 1 is given in Fig. 8. The normal (control cells) could be divided into two different

subclusters with populations of 42% and 58%. The BMT had a major and a minor cluster

with a distribution similar to that of the BM. Heterogeneity was higher for the BM than for

the BMT. The different growth stages might be responsible for the higher heterogeneity in

the BM which was reduced by retroviral infections.

4. Discussion

Our FTIR-MSP results on normal and MuSV-infected fibroblasts, isolated from the

bone marrow of rabbits, have suggested directions for the applications of advanced optical

Fig. 8. Dendrogram presentation of control cells (BM) and MuSV-transformed (BMT) fibroblasts isolated from

the bone marrow of rabbits. These results were obtained by a cluster analysis of 111 spectra in the region between

980 and 1350 cm� 1. For convenience sake, the averages of only seven clusters of BM and BMT are shown in this

figure.


technology in the detection of malignancies caused by retroviruses. Significant differences

in absorbance were observed between BM and BMT. Similar changes were observed in

cases where the same virus infected the NIH 3T3 cell line [23]. In addition, in our earlier

report on FTIR-MSP studies on H-ras-transfected fibroblast cells (balb/c), the absorbance

in the BM was higher than in the BMT in the entire region between 800 and 4000 cm� 1

[14]. The transformation of fibroblasts by MuSV124 is due to the mos oncogeneic product,

which is a Ser/Thr kinase [24]. NIH 3T3 fibroblast cells transformed by ras or mos

oncogenes have elevated glucose and the expression of glycolytic enzyme glyceraldehyde-

3-phosphate dehydrogenase (GADPH) [25]. This leads to an increased energy status

(phosphate content) of the cells. In addition, H-ras-transformed fibroblasts (NIH 3T3)

showed significant increase in cell volume when compared to control cells [26]. Hence, as

in the case of H-ras-transformed fibroblasts, the volume change occurring in retroviral

transformed cells may explain the observed absorbance changes in our studies. The

changes in volume found in BMT may surpass the effect of increased glucose levels

quantitatively.

The enhanced transcriptional activity observed in the BMT accounts for the higher

RNA/DNA ratio as compared to the BM. The higher amide I/II ratio for the BMT can be

understood as the effect of the mos oncogeneic product resulting in increased DNA

synthesis. Our studies are significant and bolster earlier reports by suggesting that the

susceptibility of skin fibroblasts to viral transformation by Kirsten sarcoma virus was

higher for familial cases of colorectal cancer patients [27].

General methods of viral detection include cell culture, antigen detection (fluores-

cence and ELISA), nucleic acid detection (PCR) and histological methods [28]. Among

the available methods, antigen detection methods are currently in use and nucleic acid

detection techniques are gaining recent attention. Nucleic acid detection techniques

have high sensitivity and their drawbacks are that they are time-consuming and

expensive [29]. The present study investigates the effect of retroviral infection in

fibroblasts using FTIR-MSP. Our results indicate that the FTIR spectra of BM and

BMT exhibited significant differences. The initial results indicated that the classification

of these two types of cells, using cluster analysis, was highly successful. The

development of any optical method in medicine is usually executed in several steps.

Initially, studies performed on a model system (as reported in this article) reveal the

feasibility and technical difficulties involved in the chosen approach. For example,

cervical cancer is the type of malignancy caused mainly by the human papiloma virus

(HPV). There are already reports in the literature [12] on FTIR-MSP analyses of

exfoliated cells (cervical scrapping). The extension of such FTIR-MSP studies to ex

vivo, and later to in vivo (FTIR-fiber optic evanescent wave spectroscopy), is most

promising toward the optical diagnoses of cervical cancer. In addition, it might be of

great interest to study skin cancers (sarcoma) and skin-related infections by a battery of

all the optical methods available today. In the light of our findings, it certainly seems

worthwhile to continue with the development of FTIR-MSP for the purpose of

quantifying the effects of viral infection in various types of cancers. In other words,

FTIR-MSP will be useful in the follow-up procedure of the abovementioned malig-

nancies and can be done routinely. The low cost and rapidity of this technique makes

this goal possible.


Acknowledgements

We gratefully acknowledge the Harry Stern Applied Research Grant Program and the

Israel Science Foundation (ISF grant number: 788/01) for their financial support.

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FTIR microspectroscopy of malignant fibroblasts transformed by mouse sarcoma virus

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