Studies on biological macromolecules

Experimental

Cell Research 42, 89-98 (1966)

89

STUDIES ON BIOLOGICAL II. EFFECT OF ACRIFLAVINE MACROMOLECULES S.

BOSE, Indian

MACROMOLECULES

EXPOSURE

ON THE SYNTHESIS

IN LIVER CELLS IN

B. P. GOTHOSKAR Cancer Research

OF

VITRO

and K. J. RANADIVE

Centre, Parel, Bombay,

India

Received August 21, 1965l

A

NUMBER of acridine dyes, particularly Acriflavine and Proflavine, have been shown to be mutagenic for viruses and bacteria [5, 12, 261. In the presence of light they inactivate phages due to their photodynamic action-dye mediated and light stimulated oxidation of nitrogenous bases of deoxyribonucleic acid, DNA [25, 281. Acriflavine has also been shown to be capable of modifying UV-induced mutagenesis in bacteria [S, 271. This modifying effect of the dye can be reversed by addition of the excess of Na-ribonucleate parameters [16] or Na-deoxyribonucleate [27]. A number of independent provide evidence that such acridine molecules combine with DNA [ 143 by intercalating between two otherwise sequential base pairs in the DNA molecules [15]. Such intercalation might interfere with the replication of DNA molecules and other biosynthetic processes involving their direct or indirect participation. Involvement of ribonucleic acid (RNA) molecules

in the biological

action of acridines

cannot be ruled out [ 151. Present investi-

gations were therefore started to evaluate the effect of acriflavine exposure on the synthesis of macomolecules in a mammalian cell system in uitro. The results are presented

in this communication.

MATERIALS

AND

METHODS

Cells.-Liver cells, originated from human tissue [6], were commercially obtained and serially.cultivated in vitro on Eagle’s basal medium [9] supplemented with 15 per cent human adult serum. Cells from passages 308 to 312 were used in these studies. For acriflavine exposure, 1.2 x lo5 cells per tube were inoculated in standard Leighton tubes containing coverslips and incubated at 37°C for 24 hr. Exposure to acriflavine (Riedel).-Stock solutions of acriflavine were prepared in iV saline at 25, 50, and 100 pg/ml concentrations. They were filtered through bac1 Revised version received December 13, 1965. Experimental

Cell Research 42

90

S. Bose, B. P. Gothoskar

and K. J. Ranadive

terial filter and stored in darkness. Medium from 24 hr incubated tubes was replaced with 0.9 ml of the fresh medium and 0.1 ml of the stock dye solution of the required concentration was added. The final dye concentration in each set of tubes was 2.5, 5.0, and 10.0 pg per ml of the medium. The dye treated tubes were covered with aluminium foil to prevent exposure to light and incubated at 37°C for 4, 2, 4 and 6 hr. After the required dye exposure the medium from the tubes was removed and the tubes were given two quick rinses with saline before adding I ml of the fresh medium. Exposure to the labeled precursors.--To study DNA, RNA, and protein synthesis, 3H-thymidine, 3H-uridine, and W-phenylalanine were used as precursors. The concentrations used were; 3H-thymidine, 0.05 ,UC per tube (specific activity 3 c/m&f); 3H-uridine, 0.1 ,UC per tube (specific activity 1.22 c/m&f); and W-phenylalanine, 1.0 ,UC per tube (specific activity 1 mc/mM). Contact time was 1 hr for 3H-thymidine, and 3 hr for both 3H-uridine and W-phenylalanine. Necessary enzyme and acid treatments were carried out and the cells fixed as previously reported [4]. After the treatments the coverslips were coated with Kodak AR 10 stripping film, following the technique of Doniach and Pelt [7]. Autoradiographic exposure for the tritiated precursors was 5 days and for 14C-phenylalanine it was 3 days. All the photographic and staining procedures were carried out as reported earlier [4]. For DNA synthesis, per cent labeled nuclei as well as grain distribution per 64 ,u2 (taken as unit area) were registered. For RNA and protein synthesis, cellwise and cell organellewise distribution of grains was registered. All the cells were chosen at random.

RESULTS

Fig. 1 shows the effect of acriflavine exposure on 3H-thymidine incorporation in liver cells. Table I shows the effect of the same on per cent labeled nuclei. The incorporation is found to be severely decreased by the dye ex-

EXPOSURE

Fig. Experimental

l.-%H-Thymidine Cell Research

incorporation 42

TIME

IN HOURS

in liver

cells after

acriflavine

exposure.

Studies on biological

macromolecules.

II

91

posure, even at 2.5 ,ug concentration. Percentage of labeled nuclei similar effect of the dye exposure, the rate of decrease being slower the first case. Fig. 2a, b, c and d show the effect of acriflavine exposure on and cell organellewise incorporation of 3H-uridine in liver cells. TABLE

I. Effect

Liver cells treated with the effect was evaluated

Exposure (W 0 (control) t

of acriflavine

different concentrations on the basis of the

time

(with standard 2.5 pg/ml

5 +

2 i: 4 Ik 6 -t

a About

exposure on 3H-thymidine in liver cells.

2000 cells were

39.4 8.9 38.5 8.4 36.4 7.8 24.5 7.0 16.0 3.8

scored

of acriflavine were percentage of labeled

cellwise The re-

incorporation

exposed to BH-thymidine, and nuclei in the cell populations.

Per cent labeled nuclei deviation)a in cells exposed 5.0 ,Ug/ml

show a than in

to the dye 10.0 pg/ml

37.7 8.4 31.8 f 7.7 22.0 Al 5.4 11.1 k 2.8

28.7 7.2 15.7 It 4.4 0

+

rt

for each value.

sponse is found to be different in various intracellular sites. RNA synthesis in the nucleolus and cytoplasm show an inhibition, clearly noticed in the cells exposed for 2 hr. On the other hand, 6 hr exposure of the cells to 2.5 pug of the dye causes only 40 per cent decrease in RNA synthesis in the non nucleolar region. Fig. 3a, b, c and d show the cellwise and cell organellewise incorporation .of l*C-phenylalanine in liver cells exposed to acriflavine. The incoroporation at all the sites is affected by the dye exposure, though the nucleolar and the cytoplasmic regions are comparatively more affected. Each value in the above figures is a mean of 81 samples and zero hour values plotted are those obtained in the controls. For values in Table I, about 2000 cells were scored in each set. Experimental

Cell Research

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S. Bose, B. P. Gothoskar

92

C,

NON-NUECLEOLAR

and K. J. Ranadive

REGION

;;;;Ps

0EXPOSVRE 0 24TlYE 6 8 2TIME 4IN 68 EXPOS HOU IN HOURS Fig.

2.--SH-Uridine

incorporation

in liver

cells after

acriflavine

exposure.

Figs. 4 a, b, 5 a, b, and 6 a, b are the photomicrographs of the cells showing the incorporation of the three precursors before and after the dye exposure. DISCUSSION

DNA synthesis The photodynamic action of the dye can be ruled out in these studies since the cells were kept in darkness as long as they were in contact with the dye. The results clearly indicate a very preferential action of the dye exposure on DNA synthesis. Even with 4 hr exposure to 2.5 ,ug of the dye, 3H-thymidine incorporation has come down to about 60 per cent of that in the control. The same exposure produces negligible effect on the incorporation of the other Experimental

Cell Research

42

Studies on biological

macromolecules.

d,

II

93

CYTOPl.ISM

t

Fig.

3.-*%-Phenylalanine

incorporation

in liver

cells after

acriflavine

exposure.

two precursors. 3H-thymidine incoroporation in the treated cells seems to be dose dependent and, even at the low dose used, decreases rapidly with the increasing exposure times. This early and rapid decrease evidently points out the involvement of DNA molecules and is in agreement with the suggestion of Lerman [15] that acridine intercalation of the DNA double helix might interfere with the replication of DNA molecules, perhaps by hindering the polymerization of fresh deoxynucleotides [3]. It should, however, be noted that even though exposure to the low dye concentration drastically affects DNA duplication as evidenced by reduction in the grain count per unit area, it has comparatively less influence on the transition of the cells from G, to S phase as see from Table I. 4 hr exposure, which decreases DNA duplication by about 40 per cent, has least effect on the per cent labeled Experimental

Cell

Research

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S. Bose, B. P. Gothoskar

94

nuclei. Thus the dye exposure more than its initiation.

and K. J. Ranadive

seems to affect DNA

duplication

phenomenon

RNA synthesis The intracellular pattern of RNA synthesis shows a heterogenous response to acriflavine exposure. 3H-uridine incorporation in the nucleolar and cytoplasmic regions is atfected earlier and to a greater extent than in the nonnucleolar (chromatin) region of the nucleus. Proflavine, chemically a close associate of acriflavine, has been reported to produce a similar effect; total inhibition of nucleolar RNA synthesis but only partial inhibition of chromatin RNA synthesis [24]. Recently Scholtissek [20] reported that chick tibroblasts showed residual RNA synthesis even in the presence of very high amounts of proflavine. He has not reported (i) the site at which this residual synthesis goes on, and (ii) the effect of the same dye treatment on DNA synthesis. Finding the base composition of this residual RNA fraction to be abnormal, he suggests that proflavine disturbs the template function of DNA molecules. Present studies on aciflavine clearly show that it is possible to discriminate between the two types of ternplating activities of the DNA molecules. The treatment which hinders the DNA duplication allows the RNA synthesis in the non-nucleolar region to escape unharmed. It is known that RNAs in different parts of the cell have distinctive base composition [lo, 111. Under certain conditions nucleolar and cytoplasmic RNA behaves differently from RNA in the chromatin region of the nucleus [18]. Actinomycin D [17], thioacetamide [23], and UV microbeam irradiation of the nucleolus [19] also cause a similar marked suppression of nucleolar and cytoplasmic RNA synthesis without affecting the chromatin RNA synthesis. The parallel decrease in the nucleolar and cytoplasmic region is not strange since nucleolar RNA is known to be a precursor of major part of the cytoplasmic RNA [21]. The nucleolus is known to contain various types of RNAs, extrinsic and intrinsic [22, 231. Present results show that short exFig. 4.-Photomicrograph of liver cells showing the effect of acriflavine exposure on 8H-thymidine incorporation. x 850. (a) Incorporation in the control cells; (b) incorporation after 6 hr exposure to 2.5 pg/ml dye concentration. Fig. 5.-Photomicrographs of liver cells showing the effect of acriflavine exposure on SH-uridine incorporation. x 850. (a) Incorporation in the control cells; (b) incorporation after 6 hr exposure to 2.5 ,ug/ml dye concentration. Note the remarkable inhibition in the nucleolus (arrow). Fig. 6.-Photomicrographs alanine incorporation. exposure to 2.5 yg/ml Experimental

of liver cells showing x 850. (a) Incorporation dye concentration.

Cell Research

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the effect of acriflavine exposure on 14C-phenylin the control cells; (a) incorporation after 6 hr

Studies on biological

macromolecules.

Fig.

4.

Fig.

5.

Fig.

6.

II

Experimental

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S. Bose, B. P. Gothoskar

96

and K. J. Ranadive

posures to the low dye concentration do not affect the DNA-dependent, chromatin RNA synthesis but cause a decrease in the RNA-dependent, intrinsic RNA synthesis. Lerman’s experiments [ 151 with synthetic polyribonucleotide solutions show that the addition of low amounts of acridine leads to the stabilisation of a double helix containing equivalent amounts of adenine and uracil. Probably intercalation of acriflavine molecules in the DNA-RNA hybrid (present in the chromatin region of the nucleus) prevents the dissociation of this RNA from the hybrid, which normally should have proceeded to the nucleolus to prime the synthesis of intrinsic RNA. Attention is drawn to the fact that 2 hr dye exposure at 2.5 ,ug concentration, which causes almost 75 per cent inhibition in DNA synthesis, leaves the RNA synthesis in the chromatin region almost unscathed. Apparently aciflavine intercalation interferes with the functioning of the DNA polymerase but not the DNA dependent RNA polymerase. Protein

synthesis

14C-phenylalanine incorporation studies reveal a picture logically to be expected from the pattern of RNA synthesis in the dye treated cells. The nucleolus has been shown as a source of ribosomes [a]. Since ribosomes are the main sites of protein synthesis in the cytoplasm [13], acriflavine exposure which disturbs the nucleolar and cytoplasmic RNA synthesis is bound to produce parallel repercussions in the protein synthesis. Figs. 3a-d show that 14C-phenylalanine incorporation is decreased at all sites in the dye treated cells, particularly in the nucleolar and cytoplasmic regions. It is interesting to note that protein synthesis in the non-nucleolar region is not as resistant to the dye treatment as the RNA synthesis in the same region. Short exposure to 2.5 ,ug of the dye elicits a differential response in the non-nucleolar region, where RNA synthesis is almost intact but protein synthesis is decreased. Allfrey [l] has shown the major role of nuclear ribosomes in the synthesis of nuclear proteins. If this synthesis is linked in some manner with the supply of some fresh RNA from the nucleolus, then the decreased protein synthesis in the non-nucleolar region, seen in the present case, may be a secondary phenomenon occurring as a result of the drastic inhibition of RNA synthesis in the nucleolai region. In conclusion it can be said that exposure of the liver cells to low concentration of acriflavine affects DNA synthesis, which rapidly decreases with the increasing exposure times, RNA synthesis shows a differential response. RNA synthesis in the non-nucleolar (chromatin) region of the nucleus seems to be resistant to the dye treatment which causes decreased RNA synthesis Experimental

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Studies on biological in the nucleolar and cytoplasmic at all intracellular sites.

macromolecules.

regions.

Protein

II

synthesis

97 shows

a decrease

SUMMARY With the help of 3H-thymidine, 3H-uridine, and l*C-phenylalanine, the pattern of synthesis of macromolecules in liver cells exposed to acriflavine was investigated. 1. DNA synthesis is the process primarily affected by the dye exposure. Even 3 hr exposure to 2.5 ,ug/ml of the dye causes nearly 40 per cent decrease in 3H-thymidine incorporation. The decrease seems to be dependent on the amount of the dye and the time of exposure. Short exposure to the low dye concentration does not significantly affect the percentage of labeled nuclei. 2. Intracellular RNA synthesis shows a heterogenous response. Nucleolar and cytoplasmic RNA synthesis decrease with increasing dye exposures, though not as rapidly as DNA synthesis. At 2.5 ,ug/ml of the dye concentration, RNA synthesis in the non-nucleolar region of the nucleus seems to be resistant to short exposures. 3. Protein synthesis responds to the dye treatment in a different manner by showing decreased l*C-phenylalanine incorporation at all the intracellular sites in the dye treated cells. Protein synthesis in the non-nucleolar region is not as resistant as the RNA synthesis in the same region. The authors gratefully acknowledge the cooperation of the colleagues at the Tissue Culture Laboratory of the centre. They also thank Mr S. P. Gothoskar, Joint Director, Bureau of Economics and Statistics, Maharashtra Government, for analysis of the data. REFERENCES 1. ALLFREY, 2. BIRNSTIEL,

V.

G., Expil Cell M. L., CHIPCHASE,

Res. M.

Suppl. 9, 183 (1963). I.

H.

and

HYDE,

B.

B.,

Biochim.

Biophys.

Acta

76, 454

(1963).

BOLLUM, F. J., Progr. Nucleic Acid Res. 1, 1 (1963). BOSE, S., COUTINHO, W. G. and RANADIVE, K. J., Znd. J. Expfl Biol. 2, 167 (1964). BRENNER, S., BARNETT, L., CRICK, F. H. C. and ORGEL, A., J. Mol. Biol. 3, 121 CHANG, R. S., Proc. Sot. Exptl Biot. Med. 87, 440 (1955). DONIACH, J. and PELC, S. FL, Brit. J. RadioI. 23, 184 (1950). DOUDNEY, C. O., WHITE, B. F. and BRUCE, B. J., Biochem. Biophys. Res. Commun. (1964). 9. EAGLE, H.,’ Science 122, 501 (1955). 10. EDSTROM, J. E., J. Biophys. Biochem. Cytot. 8, 47 (1960). 11. EDSTROM, J. E., GRAMPP, W. and SCHOR, N., ibid. 11, 549 (1961). 12. FREESE, E., Proc. Nat1 Acad. Sci. 45, 622 (1959). 13. HOAGLAND, M. B., KELLER, E. B. and ZAMECNIK, P. C., J. Biol. Chem. 218, 345 Biol. 3, 18 (1961). 14. LERMAN, L. S., .7. Mol.

3. 4. 5. 6. 7. 8.

7 - 661803

Experimental

Cell

(1961).

15, 70

(1956).

Research

42

98 15. 16. 17. 18. 19. 20. 21.

22. 23. 24. 25. 26. 27.

S. Bose, B. P. Gothoskar

and Ii. J. Ranadive

SvmD. Molecular Action of Mutagenic and Carcinogenic Agents. J. Cellular Coma. Phys;.ol. 64, Suppl. 1, 1 (1964). MCILWAIN, H., Biochem. J. 35, 1311 (1941). PERRY, R. P., Exptl Cell Res. 29, 400 (1963). PERRY, R. P., ERRERA, H., HELL, A. and DURWALD, H., J. Biophvs. Biochem. Cufol. 11. 1 . _ (i961). PERRY, R. I’., HELL, A. and ERRERA, ill., Biochim. Biophys. Acfa 49, 47 (1961). SCHOLTISSEK, C., Biochim. Biophys. Acta 103, 146 (1965). SIBATANI, A.. DE KLOET, S. R., ALLFREY, V. G. and MIRSKY. A. E.. Proc. Nat2 Acad. Sci, U.S. 48,471 (1962j. SIRLIN, J. L., Progr. Biophys. Biophys. Chem. 12, 25 (1962). SIRLIN, J. L., JACOB, J. and KATO, K. I., Expfl Cell Res. 27, 355 (1962). SIRLIN, J. L., TANDLER, C. J. and JACOB, J., Exptl Cell Res. 31, 611 (1963). WELSH, J. N. and ADAMS, M. H., J. Bacterial. 68,122 (1954). WITKIN, E. M., Cold Spring Harbour Symp. Quanf. Biol. 12, 256 (1947). -Symp. Recovery of Cells from Injury. J. CeUuZar Comp. Physiol. 58, Suppl. 1, l(1961). -~

28. YAMAMOTO,

Experimental

N.,

Cell

J. Bacferiol.

Research

42

75, 443 (1958).