Cancer Letters, 47 (1989) 53-56

Elsevier Scientific Publishers Ireland Ltd.

In vitro interaction between two antineoplastic drugs phopurinum and interferon cy2

J.R. Lazutka, G. Slapsyte, S. Jarmalaite and R. K. Lekevicius

Ecological Genetics Laboratory, Vilnius University, 21 Ciurlionis St., 232009 Vilnius (Lithuanian S.S.R.)

(Received 29 November 1988) (Accepted 11 April 1989)

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Summary

Induction of sister chromatid exchanges (SC&) and chromosome aberrations (CAs) by two antineoplastic drugs - phopurinum (Z-dimethylamino-6-diethyleneiminophosph- amido-7-methylpurine) and recombinant human interferon a2 (rHulFN-a,) was studied in human lymphocytes in oitro. Phopurinum was found to cause a significant increase of both SCEs and CAs in lymphocytes, while rHulFN-a, induced only SCEs. Combined treatment with these two drugs reduced SCE and CA levels as compared with those induced by phopurinum alone. The maximal extent of reduction, however, was observed at interme- diate doses of phopurinum.

Keywords: chromosome aberrations; anti- neoplastic drugs.

Introduction

Human interferon (IFN) is known to possess potent antineoplastic properties [4,6,12], though its mechanism of action remains largely obscure. Clinical studies concerning th.e effect of IFN in malignant diseases as an antineoplas- tic agent have been described in current litera- ture [8,11]. On the other hand, there was only

Correspondence to: J.R. Lazutka

Published and Printed in Ireland

limited information about the combined use of IFN with anticancer drugs [ 1,4,5], and no attempts to study mutagenic effects of such combined treatment have been made. It has been reported, however, that IFN may reduce the frequency of SCEs, CAs and point muta- tions induced under in vitro conditions by X- rays [19], UV light [17] and carcinogenic compounds such as 4-nitroquinoline-l-oxide 1191.

The scope of our study sought to examine the cytogenetic effects of combined treatment of human lymphocytes in vitro with rHuIFN-a, and the chemotherapeutic drug phopurinum, which is widely used in the U.S.S.R. in the clinical treatment of acute leukosis, chronic myeloleukosis, cutaneous reticulosis and reti- noblastoma [2]. The data obtained may be helpful for clinical use of the combination of IFN with other cytostatic drugs.

Materials and methods

Phopurinum (lot no. 40979) was kindly supplied by Professor V.A. Chernov (All- Union S. Ordzhonikidze Research Institute of Chemical Pharmacology, Moscow, U.S.S.R.). rHuIFN-a, was a generous gift from the Fermentas Scientific Production Amalga- mation (Vilnius, Lithuanian S. S .R .) , which was obtained in lyophylised form, containing human serum albumin as a stabilizer. 5-Bromo- 2’-deoxyuridine (BUdR) was obtained from

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Fluka AG (Switzerland). Bacto phytohemag- Statistical analysis was carried out using glutinin P (PHA) was purchased from Difco Student’s t-test. In order to stabilize dispersion Laboratories (U.S.A.); colchicine was from E. and normalize distribution, calculations were Merck AG (F.R.G.). All other reagents were of performed on the basis of following transfor- analytical grade. mations: (a) for SCE analysis

The study was conducted with peripheral blood lymphocytes obtained by venipuncture from a healthy female volunteer, 20 years of age. Cell cultivation, chromosome preparation and fluorescence-plus-Giemsa (FPG) staining of sister chromatids were carried out by stand- ard methods [ 131.

Y = (4X + 1.5p.5

where X is a number of SCEs per 46 chromo- somes in each analysed cell (this formula is analogous with that proposed by Wulf et al. [ 181); (b) for CA analysis

The treatment design used was elaborated by Zasukhina et al. [19]. Briefly, both drugs were dissolved in Eagle’s MEM immediately before use. rHuIFN-a, (50 IRU/ml) was introduced during the initiation of cell culture. Cells were exposed to phopurinum at the con- centrations indicated below for 4 h (at 20-24 h of incubation) after which cells were rinsed twice with cold (4OC) Hank’s BSS and then re- incubated in pre-warmed (37OC) fresh Eagle’s MEM, supplemented with 20% bovine serum, 100 pg/ml streptomycin, 100 IU/ml penicillin and 0.15% PHA. BUdR (10 pg/ml) was added at 24 h of incubation. The incuba- tion of cultures was then continued in the dark and 1.5 pg/ml colchicine was present for 3 h before harvesting at 72 h post-initiation.

q = 2 arcsin (p)“.5

where p is a relative frequency of CAs.

Results and discussion

The treatment of human lymphocyte cul- tures with phopurinum resulted in a genotoxic effect as measured by both the SCE (Table 1) and CA tests (Fig. 1). These results are in agreement with the well-documented genotox- icity of other alkylating drugs [7]. Treatment of cultures with rHuIFN-a, was found to cause a slight, but statistically significant increase in the baseline SCE levels. The pretreatment of human lymphocytes with rHuIFN-a, was

Table 1. Induction by phopurinum of sister chromatid exchanges in rHuIFN-a,-treated and untreated human blood cultures in vitro.

Concent- ration of phopurinum,

PM

Treatment with rHuIFN-a,, 50 IRU/ml

SCE/46 chromo- somesa

Transformed criterior?

Mean k S.E. Phopurinum- % induced Reduction parts k S.E.

0 - 9.97 6.33 f 0.13 - - 0 + 12.02 7.07 f 0.15’ - -

5.0 20.31 8.99 + 0.16 2.66 + 0.21 - 5.0 + 14.83 7.64 + 0.15 0.57 + 0.21c 78.6

15.0 35.93 11.94 * 0.18 5.61 k 0.22 - 15.0 + 31.45 11.19 + 0.18 4.12 & 0.22 26.6 25.0 - 44.28 13.28 f 0.15 6.95 f 0.20 - 25.0 + 45.63 13.44 f 0.14 6.37 f 0.2@ 8.3

“Eighty second-division metaphases were analysed for each point. bY = (4X + 1.5)05, where X is a number of SCEs per 46 chromosomes in each analysed cell. 'P< 0.05 as compared with rHuIFN-a,-untreated cultures.

d 0 5

4

r’

2‘5 ,A - 4M

Concentration

Fig. 1. Histogram showing number of chromosome aberrations per 100 metaphases induced by phopurinum in rHuIFN-cr, treated (filled columns) and untreated (open columns) human blood cultures in vitro. Two

hundred first-division metaphases were scored for each point. The bars indicate S.E. of the mean. *Statistically significant (P < 0.05) differences from rHulFN-a,-treated culture.

found to significantly decrease the frequency of SCEs induced by phopurinum. This decrease was inversely proportional to the phopurinum dosage (Table 1). A statistically significant decrease in the frequency of CAs (both chromosome- and chromatid-type) was observed only at a phopurinum dose of 15 PM (Fig. 1). These observations are Jn con- cordance with those of Zasukhina et al. [19] where natural human leukocyte IFN exhibited maximal anticlastogenic effect in human lym- phocytes exposed to fast neutrons at a dose of 2 Gy. At the same time, the anticlastogenic effect was minimal when doses of 1 or 4 Gy had been used. Thus, they also found that the effect of IFN was minimal at high and low doses of the mutagen.

Several mechanisms may be proposed to explain the anticlastogenic action of interferon. IFN may display or induce enzymatic activities

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participating in DNA-repair mechanisms in IFN-treated cells, as has been suggested by Zasukhina and co-workers [9,19,20] and Suzuki and co-workers [16,17]. IFN may also selectively inhibit histone synthesis in IFN- treated cells [3]. Such inhibition may retard maturation of the chromatin to give higher order structures. In turn, these changes in chromatin structure may facilitate DNA-repair. Finally, IFN pretreatment may lower the threshold for the heat-shock response [lo]. Heat-shock prior mutagenic treatment, how- ever, has been shown to significantly reduce the rates of clastogen-induced CAs [14]. This heat-shock effect is comparable to the clasto- genie adaptation which is observed after condi- tioning with low mutagen doses prior to challenge with high mutagen doses. Due to the lowered threshold for heat-shock response after IFN treatment, heat-shock genes may be acti- vated at the usual conditions and reaction resembling clastogenic adaptation may occur. Detailed biochemical studies, however, are needed for the elucidation of mechanisms proposed.

Our observations may have clinical relev- ance. Phopurinum has been reported to induce CAs is lymphocytes of ovarian carci- noma patients in vivo [15]. The average CA rate during the course of therapy was as large as 23.25 f 0.61%, it reduced up to 14.80 -+ 2.24% 3-4 days after the cessation of the clastogenic influence and to 8.59 & 1.49% 6 -45 days after. Serum peak concentrations after single injection of phopurinum (40-120 mg) were 6.8-20.5 FM. These concentra- tions in vivo are very similar to those used in our experiments in vitro. The present experi- mental observations together with several other studies [9,15- 17,19,20] suggest that IFN may have significant use in clinical application not only as an antineoplastic agent but also as a prophylactic agent, which may reduce the genotoxic effects caused by other cytostatic drugs. This is important because secondary neoplasias which arise after onco- therapy may be due to genotoxic properties of the applied cytostatics [7]. IFN may thus be used several days after the main course of

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chemotherapy when CA levels sensitive to modifying action of IFN will be reached (in our case it may be upon the 4th day after the ces- sation of treatment with phopurinum). The application of these experimental results to the treatment of human cancer patients, however, remains to be established.

Acknowledgements

The authors are grateful to Professor V.A. Chernov and to Professor A. Janulaitis for the supplying of phopurinum and interferon, respectively. Many thanks are also due to Dr. A. Zygas for revision of the English text.

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