Infrared Characterization of Nuclei Isolated from Normal and Leukemic (B-CLL) Lymphocytes: Part III

E N Z O B E N E D E T T I , * M A R I A PIA P A L A T R E S I , P I E R G I O R G I O V E R G A M I N I , F E D E R I C O P A P I N E S C H I , M A R I A C R I S T I N A A N D R E U C C I , and G I U L I A N O S P R E M O L L A Dipartimento di Chimica e Chimica Industriale, Universitd di Pisa, Via Risorgimento, 35, 56100 Pisa, Italy (E.B., M.P.P., P. V.); and 1 ~ Clinica Medica, Universitd di Pisa, Via Roma, 55, 56100 Pisa, Italy (F.P., M.C.A., G.S.)

Nuclei extracted from lymphocytes coming from normal subjects and from patients affected by chronic lymphatic leukemia (B-CLL) were studied by means of infrared spectroscopy. Substantial spectral differ- ences were found above all in the region of the phosphate-group vibra- tions of DNA. The ratios of the integrated areas of the bands at 1080 and 1540 cm ', due to the symmetrical stretching vibrations of PO2 ~- groups and to proteic components, respectively, assume increasing val- ues, which are localized in quite separate ranges, for normal lympho- cytes, leukemic lymphocytes, normal nuclei, and leukemic nuclei. These values, indicating a different distribution of DNA and of proteic com- ponents between normal and leukemic cells and between normal and leukemic nuclei, may assume a remarkable significance in the preco- cious diagnosis of leukemia (B-CLL) and in following the course of the disease. In the case of leukemic nuclei, the ratio, in weight, of the two components reaches the value of 1:1.

Index Headings: Infrared spectroscopy; Leukemia.

INTRODUCTION

Developments in molecular biology have made it pos- sible to detect some specific mechanisms in the process of oncogenesis. Gene activation is always associated with extensive modifications in cellular assembly. In recent years, it has been possible to recognize that the process involves the structural order of chromatin. An under- standing of the mechanisms which lead to these alter- ations could represent the focal point in the interpre- tation of the modified genetic expression.

As far as leukemic cells are concerned, apparently no increase in the synthesis of DNA occurs. 1 Deoxyribo- nucleotide synthesis in normal and leukemic lympho- cytes does not show any significant differencesJ Fur- thermore, no difference has been found between DNA coming from leukemic and normal human cells? ,4

The study of nuclei is presently attracting a great deal of interest, since they represent the part of the cells where the most important processes occur, such as cel- lular mitosis, or alterations due to viruses, chemicals, or radiation) At the present time, differential thermic analysis and high-resolution analytical techniques make possible a global approach to the investigation of the initial stages of the transformation process and suggest new scientific and technological possibilities.

In previous experiments, infrared spectroscopy was used to study leukemic (B-CLL) and normal lympho- cytes, in order that indications about the distribution of the molecular components in the two kinds of cells could be obtained.

Received 16 April 1985. * Author to whom correspondence should be sent.

In the first step 6 of our research it was observed that the spectra of leukemic cells revealed absorptions at 965 and 530 cm i which were not present in those of normal cells. Furthermore, differences were found in the CH stretching region at the level of the methyl and meth- ylene groups. The relative intensities of the asymmet- rical stretching vibrations of the methyl groups (2960 cm -1) compared with those of the asymmetrical stretch- ing modes of the methylene groups (2925 cm -1) were more intense in leukemic cells. On this basis, it was sup- posed that more branched components were present in these lymphocytes. Furthermore, it was observed that the values of the ratios of the optical densities of the bands at 2925 and 2960 cm -1 were localized in different ranges, and that the spectra of the leukemic cells in the finger-print region were more constantly reproducible than those of normal cells. This fact could suggest that in the case of leukemic lymphocytes, unlike normal ones, certain components had to prevail over others.

In the second step of our experiments, 7 these cells were examined by means of Fourier transform infrared spectroscopy (FT-IR). The accuracy of the FT-IR spec- tra and the difference spectra allowed us to obtain fur- ther indications about the chemical and biological mod- ifications responsible for the spectral differences observed in the two series of lymphocytes.

The infrared spectra of leukemic lymphocytes com- pared with those of normal ones revealed, above all, remarkable differences in the region of the asymmetrical and symmetrical stretching vibrations of the phosphate groups of nucleic acids. Values of the ratio of the inte- grated areas of the bands at 1080 and 1540 cm -1 above 1.5 had to be considered as being made up of leukemic lymphocytes. It was suggested, in the light of further experiments, s that this ratio could be used as a method of precocious diagnosis of chronic lymphatic leukemia, especially in the cases of patients with lymphocytosis of doubtful interpretation.

In order to gain an insight into the molecular prop- erties of these kinds of cells, we carried out examinations on the various cellular components, such as the nuclei, membranes, and so on. This paper reports the results of experiments regarding the nuclei isolated from normal and leukemic (B-CLL) lymphocytes.

EXPERIMENTAL

The nuclei were isolated from normal and leukemic lymphocytes obtained by Boyum's method, 9 as reported in previous papers. 6,7

Volume 40, Number 1, 1 9 8 6 0003-7028/86/4001-003952.00[0 APPLIED SPECTROSCOPY 39 © 1986 Society for Applied Spectroscopy

FIG. 1. Infrared spectra of nuclei isolated from normal (N) and leu- kemic (L) lymphocytes.

In order to break down the whole cells, we used the detergent Triton X 100. To remove the cytoplasmatic residue, sucrose solutions were employed. 1° Finally, two rinsings with physiological solutions were necessary to eliminate completely the absorption bands of these chemicals. The nuclei thus obtained were dried on glass slides under a vacuum and removed in order that we might obtain, for the infrared measurements, KBr pel- lets containing 3 % of material.

All the infrared spectra were obtained by the use of a Model 180 Perkin-Elmer dispersive spectrophotometer. The difference spectrum between normal and leukemic nuclei (shown in Fig. 2) was obtained with the use of a Perkin-Elmer infrared data station interfaced with a Perkin-Elmer Model 283 spectrophotometer. We ob- tained the spectra of samples of the two kinds of cells in aqueous solutions, keeping the samples between KRS-5 windows.

RESULTS

The characterization of nuclei extracted from normal and leukemic lymphocytes (B-CLL) by means of in- frared spectroscopy represents a part of a wider program devoted to the gathering of indications about the whole cells and about their various components, at the molec- ular level, in order that an insight into the parameters

40 Volume 40, Number 1, 1986

L-N

N \

I L ] I J I

1300 900 crn-1 500

Fro. 2. Comparison between the spectra of two samples of normal (N) and leukemic (L) nuclei and difference spectrum obtained by the subtraction of the normal nuclei from the leukemic ones.

differentiating leukemic cells from normal ones can be gained.

The infrared spectra of five leukemic (B-CLL) and of three normal nuclei samples extracted from the corre- sponding lymphocytes are shown in Fig. 1. Like the spectra of the whole lymphocytes, they show remarkable differences which can only be explained by a different distribution of the molecular components or their struc- tural modifications. The most striking changes are ob- servable in the 1300-900 cm -1 region, where the vibra- tions of the phosphate groups of nucleic acids are localized. In this range the proteic components are prac- tically transparent. In the case of DNA, the bands at 1225 and 1080 cm -1 have been attributed to the PO~- asymmetrical and symmetrical stretching vibrations, re- spectively. The absorption at 965 cm -I is related to the O-P-O antisymmetrical stretching mode. The band at 1700 cm -1 is broader, since it is made up of more com- ponents than that of normal nuclei.

Figure 2 shows the spectra corresponding to the two kinds of nuclei in the region of the phosphate group vibrations, together with the difference spectrum that we obtained by subtracting the spectrum of a sample of normal nuclei from that of leukemic nuclei. This differ- ence spectrum assumes a particular significance when compared with the whole cells and their difference spec- tra, since the number of molecular components is in this case considerably reduced.

The association of the absorptions in the 1300-900 cm -I region with the vibrations of DNA phosphate groups is reasonable enough, especially when one considers the spectra obtained from normal lymphocytes (N), leuke- mic lymphocytes (L,, L2), leukemic nuclei (LN), and DNA in an aqueous solution: going from N towards L ,

w 0 Z <

O u')

<

e~ ~ j

17 II i I

I _ I

", K/ /

N

L1

LN

\ DNA

1 3 0 0 1200 1100 cm -1 1 0 0 0

Fro. 3. Infrared spectra of normal lymphocytes (N), leukemic lymphocytes (L~, L~), leukemic nuclei (LN), and DNA in aqueous solution.

L2, and LN samples, a progressively greater similarity with DNA can be seen, as shown in Fig. 3.

In order to verify the degree of similarity existing be- tween all the spectra corresponding to normal and leu- kemic lymphocytes and to normal and leukemic nuclei and that of DNA, we considered the trends of the ratios of the optical densities of the bands at 965 cm -1 and at 1080 cm -1 (Fig. 4). The values of this ratio seem to con- firm our previous hypothesis about a substantial in- crease of DNA through the four kinds of samples, reach- ing a maximum similarity in the case of the leukemic nuclei. Furthermore, the absorption at about 965 cm changes in frequency in the case of the four kinds of samples, as shown in Table I. Also in this case, a shift toward the DNA frequency is observed, again confirm- ing our above-mentioned results.

In a previous paper, 7 some ratios of the optical den- sities of bands showing modifications in frequency and intensity in the spectra of leukemic lymphocytes with respect to normal ones were considered. All the values of these ratios were localized in two quite separate ranges, even when the bands were produced mainly by vibra- tions of DNA itself (D 960 cm-~/D 530 cm -~, and D 1080 cm-1/D 1240 cm-0. This phenomenon could only be ex- plained by the admission that there had to be an addi- tional contribution in these absorptions due to other

molecular components or to modifications in the struc- ture of the DNA itself. Particular attention was paid to the ratios of the integrated areas of the bands at 1080 cm -1 and at 1540 cm -1 (1140-950 cm -1, 1590-1480 cm -1 base line points, respectively), the former due to vibra- tions of the P02- group of DNA, and the latter to the symmetrical vibration of the proteic components. We chose this kind of ratio in order to reduce the margin of experimental error with respect to the ratios of the op- tical densities. Furthermore, this ratio allowed us to compare in a semiquantitative way two different molec- ular species, that is to say, proteins and nucleic acids. In Fig. 5 the values of this ratio for the three normal and five leukemic nuclei samples are shown. These val- ues, localized in two different ranges, show an increase in the intensity of the bands of the symmetrical vibra- tions of PO2- groups with respect to the band of the proteic material. In the previous paper 7 we considered the density distribution of this ratio for leukemic and normal whole cells. At this point, it is interesting to com- pare the values of the ratios of these integrated areas for normal lymphocytes, leukemic lymphocytes, normal nuclei, and leukemic nuclei. The density distribution of these values is shown in Fig. 6. These values show that the integrated area of the band at 1080 cm -1 increases compared with the one at 1540 cm -~ (going from normal

APPLIED SPECTROSCOPY 41

D 960cm -1 AlO8Ocm- 1 D 1080cm 1

O,5-

0,4

0,3-

O,2 ̧

=DNA

L

T

2 4 6 SAMISLES FIa. 4. Values of the ratio D 960 cm 1/D 1080 cm -~ for leukemic nuclei (LN), for normal nuclei (NN), and for DNA. The ranges for these values are also reported for the normal lymphocytes (N) and leukemic lymphocytes (L).

lymphocytes, to leukemic lymphocytes, to normal nu- clei, to leukemic nuclei), and that the ranges of the four kinds of samples are quite separate.

Since infrared spectroscopy makes it possible to lo- calize absorptions in different ranges for DNA (1080 cm -l) and for proteic components (1540 cm-1), an at- tempt was made to correlate these values for whole cells and nuclei with the data obtained for these same ratios of integrated areas in the case of mixtures prepared by us with different compositions of DNA (salmon sperm) and a protein (albumin). On the basis of what is shown in Fig. 6, it is possible to predict a trend in the relative amounts of these two components in the whole cells and nuclei: this trend leads us to postulate for leukemic nu- clei a DNA/protein ratio of the order 1:1. Of course, this kind of approach has to be considered approximate

TABLE I. Shift of the O-P-O band for different samples.

Samples ~0-P.0 (=965 cm -~) Normal lymphocytes 971 Leukemic lymphocytes 963 Normal nuclei 963 Leukemic nuclei 962 DNA 962 RNA 966

A1540cm-1

5. f

J / 4 ¸

3 2 ,4 'SAMPLES

FIG. 5. Values of the ratios of the integrated areas of the bands at 1080 cm i and 1540 cm 1 for normal nuclei (NN) and for leukemic nuclei (LN).

since only one protein (albumin) and one nucleic acid (DNA) were employed.

CONCLUSIONS

The differences already observed in the spectra of normal and leukemic lymphocytes can be seen even bet- ter in the spectra of the corresponding nuclei. Going from normal to leukemic whole cells, and from normal to leukemic nuclei, the intensities of the bands due to the phosphate groups of DNA increase with respect to those attributed to the proteic components.

After examination of mixtures with different compo- sitions of DNA and a protein (albumin), a value of the order 1:1 for the DNA/protein ratio is proposed for leu- kemic nuclei. The spectra of leukemic nuclei in an aqueous solution and in the solid state (KBr matrix) are very similar to the spectra of form B and A of DNA, especially in the region of the phosphate-group vibra- tions.

The value of the ratio of the integrated areas of the bands at 1080 cm -1 and at 1540 cm 1 for the whole lym- phocytes and the nuclei corresponding to the two kinds of cells may be of remarkable significance in helping us to distinguish between normal and leukemic cells and thus arrive at a precocious diagnosis of chronic lym- phatic leukemia. Furthermore, we may also employ it to follow the progress of patients who are under treatment with drugs.

1. R. C. Gallo, Acta Haemat. 45, 136 (1971). 2. S. Fujioka, Acta Haemat. 45, 159 (1971). 3. G. Corneo, E. Ginelli, and E. Polli, Acta Haemat. 45, 167 (1971). 4. G. Zerbi, P. A. Bianchi, and E. Polli, Makromol. Chem. 60, 223

(1963). 5. M. M. Wintrobe, G. R. Lee, Dave R. Boggs, T. C. Bithell, J. Foers-

ter, J. W. Athens, and J. N. Lukens, Clinical Hematology (Lea and Febiger, Philadelphia, 1981), Chap. 60, p. 1471.

6. E. Benedetti, F. Papineschi, P. Vergamini, R. Consolini, and G. Spremolla, Leukemia Res. 8, 483 (1984).

42 Volume 40, Number 1, 1986

DNA Prot.

1,0.

0,8

0,6

0,4

i , I I i I i

O

Q

A 108Ocm -1 A 1540 cm -1

S A M P L E S

4

3

2

FIG. 6. Correlation between the density distribution of the values of the ratios of the integrated areas A 1080 cm-1/A 1540 cm 1 for normal lymphocytes [], leukemic lymphocytes D, normal nuclei @, leukemic nuclei [], and the relative amounts of DNA with respect to the proteic component (DNA:protein by weight).

7. E. Benedetti, M. P. Palatresi, P. Vergamini, F. Papineschi, and G. Spremolla, Leukemia Res. 9~ 1001 (1985).

8. G. Spremolla, F. Ambrogi, A. Azzar&, and E. Benedetti, "Diagnosi Precoce Della Leucemia Linfatica Cronica (LLC). Considerazioni su di un Nuovo Marker; Studio Mediante Spettroscopia Infraros- sa," Communication presented to the meeting of Soc. Ital. of Ema-

tology, Sez. Toscana, Florence, December 1, 1984, Haematologica (in press).

9. A. Boyum, Scand. J. Clin. Lab. Invest. 21~ (Suppl. 97), (1968). 10. F. Gabrielli, D. P. Aden, S. C. Carrel, C. yon Bahr, A. Rane, C. A.

Angeletti, and R. Hancock, Molecular and Cellular Biology 6S~ 57 (1985).

APPLIED SPECTROSCOPY 43