Carclnogenesis vol.12 no.7 pp.1355-1357, 1991

SHORT COMMUNICATION

Effect of tumor necrosis factor on cell proliferation kinetics and sister chromatid exchange frequency in human lymphocytes

The testing of significance for differences between two RI values may be based (8) on statistics

Juozas R.Lazutka and Sonata Rudaitiene Ecological Genetics Laboratory, Vilnius University, 21 Ciurlionis St., 232009 Vilnius, Lithuania

Tumor necrosis factor a (TNF*) is an important cytokine released by activated macrophages. It was discovered in tumor-bearing mice as an endotoxin-induced serum factor which was able to elicit hemorrhagic necrosis of tumors (1). This factor is cytotoxic for several, but not all, tumor cell lines in vitro and causes necrosis of certain tumors in vivo (2). For certain types of normal as well as malignant cells, TNF exhibits mitogenic or growth stimulatory activity (3,4). TNF-induced cytotoxicity has been associated with DNA fragmentation (5). This DNA fragmentation has been suggested to involve topoisomerase II-like activities (6). Two biological end points measured in the present study after treatment in vitro of human lymphocytes with TNF were replication index (RI) and sister chromatid exchanges (SCEs). RI shows the average number of times cells have divided in the medium containing 5-bromo-2'-deoxyuridine (BrdUrd) (7). It can be calculated (8) from:

RI = E in,/N

(1)

where n, is the number of /'th division cells in the culture, k

i = — 1, . . . ,k, where k = A and N = E n,-. The number of divisions completed by a cell can be determined by analysing the distributions of cell cycle specific chromosome staining patterns (7) obtained at metaphase by the differential staining of sister chromatids (9). Variation of RI is given by: k

k

E i2n,{N-ni)-2 E imn?in

i" -

(2)

i

•Abbreviations: TNF, tumor necrosis factor; RI, replication index; SCE, sister chromatid exchange; BrdUrd, 5-bromo-2'-deoxyuridine; PHA, phytohemagghmnin.

© Oxford University Press

(RI

(3)

which refer to a table of critical values for a standard distribu-

tion N(0,\). Although the mechanisms and significance of SCEs are not fully understood, SCE test results have shown a good correlation with mutagenicity and carcinogenicity data (10). SCE frequency is enhanced when DNA damage is induced (11) or cellular metabolism is disturbed (12). Some evidence also exists that mammalian topoisomerases may play a role in the formation of SCEs (13,14). Thus, the present study was undertaken to examine whether TNF is able to induce SCE in human lymphocytes in vitro or to change cell proliferation kinetics. To evaluate this, we have used recombinant human TNF a (produced by the Institute of Applied Enzymology, Lithuania) with a sp. act. of 5 x 107 U/mg protein. A unit of TNF was defined as the amount of protein causing cell death of 50% mouse L-929 cells in the standard TNF assay in vitro (1). The study was conducted with peripheral blood lymphocytes obtained by venipuncture from two healthy female volunteers (23 and 31 years old). Heparinized whole blood was diluted in the ratio 1:15 by RPMI-1640 medium (Vektor, USSR) supplemented with 12% heat-inactivated fetal bovine serum (Institute of Biochemistry, Lithuania), 100 U/ml penicillin, 100 /xg/ml streptomycin, 10 /ig/ml BrdUrd (Aldrich, USA) and 0.15% Bacto PHA 'P' (Difco, USA). TNF (10, 50, 100, 500, 1000 and 5000 U/ml) was introduced at the initiation of culture and was present during the entire cultivation. Cells were cultured for 72 h at 37°C in complete darkness, and colchicine (Aldrich, USA) was present for the last 3 h at a final concentration of 0.5 /tg/ml. After hypotonic treatment (0.075 M KC1 for 30 min) and fixation (methanol:glacial acetic acid, 3:1, three times for 20 min), flame-dried slides were prepared. Differential staining of sister chromatids was carried out by a modified fluorescence/Giemsa technique (9). Briefly, the slides were stained for 10 min with 10 /ig/ml of Hoechst 33258 (Riedel de Haen, FRG) dissolved in 0.07 M Soerensen's buffer (pH 6.8), rinsed and mounted with citrate buffer (pH 8.5). Then the slides were covered with cover slips and exposed to UV light (400 W mercury lamp at a distance of 15 cm) for 6—7 min. Slides were then rinsed and stained for 3 min with azure II-eozin K solution (0.025 and 0.01 % respectively; Reachim, USSR) prepared in phosphate buffer (pH 6.8). All slides were coded, and 25 second mitotic division cells with 4 4 - 4 6 chromosomes were scored from each of two replicate cultures (for a total of 50 cells from each donor and treatment combination) for SCEs. About 100 cells of the first, second and third mitotic division from each replicate culture (no less than 200 cells from each donor and treatment combination) were scored to determine the RI. Since there were no marked differences in response to TNF treatment between the two donors (data not shown), all data were pooled

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Recombinant human tumor necrosis factor a (TNF) was found to cause a significant increase in cell proliferation rates and sister chromatid exchange (SCE) frequency in phytohemagglutinin-stimulated human lymphocytes in vitro. The cells were treated for the entire cultivation time with 10, 50, 100, 500, 1000 and 5000 U/ml TNF. Cell proliferation rates, as measured by the replication index, increased significantly (P < 0.01) in a concentration-independent manner. The maximal extent of SCE induction (15.18 ± 0.57 versus 10.26 ± 0.45 SCEs/cell in control, P < 0.001) was observed with 50 U/ml TNF.

J.R.Lazutka and S.Rudaftient

together. Statistical analysis was carried out using Student's f-test for SCEs and the Z-test (see above) for RI. The treatment of human lymphocyte cultures with TNF resulted in a dose-independent significant increase (P < 0.O1) in RI (Figure 1). At the same time, the mitotic activity remained unchanged in all cultures (data not shown) and was ~ 5%. Thus, TNF stimulated the rate of cell cycle progression in human lymphocyte cultures. The means for the SCE frequency are given in Figure 2. The SCE frequency was significantly increased in lymphocyte cultures treated with 10-1000 U/ml TNF (P < 0.05, Student's f-test). At 5000 U/ml TNF, the increase in SCE frequency was not significant {P > 0.05). The highest SCE frequencies were observed at low (10-100 U/ml) concentrations of TNF, with the peak response occurring at a TNF concentration of 50 U/ml. The dose-response curve may be described by a parabolic equation (4)

where yj is the number of SCEs/cell observed in lymphocyte culture after treatment with the jth dose of TNF, dj,

I 1-

1

6

5

S

o

woo TNF (U/ml)

Fig. 1. Effect of recombinant human TNF on RI values in cultured human lymphocytes. Values are means of pooled data from two independent experiments and expressed as means ± SE. All determinations were made in duplicate (— 400 cells were scored per experimental point).

Acknowledgements We thank Dr A.Bumelis of the Institute of Applied Enzymology, Vilnius, Lithuania for kindly supplying the recombinant human TNF.

References

TNF (U/ml) Fig. 2. Effect of recombinant human TNF on SCE frequency in cultured human lymphocytes. Values are mean of pooled data from two independent experiments and are expressed as mean ± SE. All determinations were made in duplicate (100 cells were scored per experimental point).

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1. Carswell.E.A., Old.L.J., Kassel.R.L., Green.S., Fiore.N. and WUliams.B. (1975) An endotoxin-induccd serum factor that causes necrosis of tumors. Proc. Nail. Acad. Sd. USA, 72, 3666-3670. 2. Old.L.J. (1985) Tumor necrosis factor (TNF). Science. 230, 630-632. 3.Sugarman,B.J., Aggarwal.B.B., Hass,P.E., Figari.I.S., PalladinorIr.,M.A. and Shepard,H.M. (1985) Recombinant human tumor necrosis factor-or: effects on proliferation of normal and transformed cells in vitro. Science, 230, 943-945. 4. Kamijo.R., Takeda.K., Nagumo,M. and Konno.K. (1989) Suppression of TNF-stimulated proliferation of diploid fibroblasts and TNF-induced cytotoxicity against transformed fibroblasts by TGF-(3. Biochem. Biophys. Res. Common., 158, 155-162. 5. Elias.L., Moore,P.B. and Rose.S.M. (1988) Tumor necrosis factor induced DNA fragmentation in HL-60 cells. Biochem. Biophys. Res. Commun., 157, 963-969. 6. Coffman.F.D., Green,L.M., Godwin.A. and Ware,C.F. (1989) Cytotoxicity mediated by tumor necrosis factor in variant subclones of the ME-180 cervical

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yj = 8.3480 + 10.5748JC; - 5.2638*? + 0.7085.^

j = 0, . . ., 6 and Xj = logl0(dj). Since log10(0) is undefined, the value of XQ was set below xx by an amount that was equal to the average spacing between consecutive values of xy This amount was calculated according to the formula proposed by Margolin et al. (15). This dose response for SCE induction by TNF may suggest that the SCE-inducing properties of TNF do not relate to its ability to form DNA adducts, since the dose-response relationship for SCE-inducing agents directly interacting with DNA has been previously reported to be linear (16). As mentioned above, TNF is cytotoxic for several malignant cell lines (2) and this cytotoxicity is often related to TNF-induced DNA fragmentation (5). DNA fragmentation induced by TNF was not blocked by inhibitors of protein or RNA synthesis nor by inhibitors of free radical mediated pathways and has been suggested to involve topoisomerase El-like activities (5). Furthermore, TNF has been reported to enhance cytotoxicity and DNA fragmentation produced by teniposide and etoposide (6), specific inhibitors of DNA topoisomerase II which act by preventing the DNA rejoining reaction. Our preliminary studies with novobiocin, an inhibitor of the DNA cleavage reaction mediated by topoisomerase II (17), showed a dramatic ability to decrease TNF-induced SCE frequency, suggesting that SCE induction by TNF is mediated at least in part by topoisomerase II. Similarly, novobiocin has been reported to reduce SCE frequency induced by m-AMSA (18), another inhibitor of the topoisomerase IImediated DNA rejoining reaction (19). To clarify the possible mechanisms involved in the SCE induction by TNF, further experiments are needed. Effects of TNF on lymphocyte proliferation rates are in agreement with previously published results about the in vitro stimulation of untransformed cell growth by TNF (3,20,21). However, the mechanism involved in the enhancement of lymphocyte proliferation by TNF seems to be different from that involved in SCE induction, since dose-response relationships were different for both end points measured. In conclusion, it should be noted that other cytokines such as human interferon-a2 and interleukin-2 have been reported to induce SCEs in lymphocytes in vitro (22-24). Thus, SCEinducing ability seems to be a common feature for all cytokines so far investigated cytogenetically. Elucidation of mechanisms involved in SCE induction by these polypeptides will be of great interest and importance.

Effect of tumor necrosis factor

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