Biological activity of 2-phenylethanol and its derivatives

Archly fiir die gesamte Virusforschung 40, 205 9 by Springer-Verlag 1973 214 (1973) Bioloflieal Activity of 2-Phenylethanol and Its Derivatives V I I I . I n f l u e n c e on H e r p e s v i r u s D N A - S y n t h e s i s in R a b b i t K i d n e y Cells By W. E. G. M(YLLER~D. FALKE, B. HEICKE, and R. K. Z A ~ With technical assistance by R. BEYE~ Institut ftir Physiologische Chemie and Institut for Medizinische Mikrobiologie, der Johannes-Gutenberg-Universit/~t, Mainz, Federal Republic of Germany With 3 Figures Received July 13, 1972 Summary The biosynthesis of herpesvirus DNA in rabbit kidney cells is inhibited to 50% by P E A (2-Phenylethanol) at 0.65 mg PEA/ml. The inhibition of cellular DNA synthesis in uninfected cells by P E A is about twice as sensitive as that of viral DNA synthesis. The cellular DNA-dependent DNA polymerase is inhibited in a non-competitive way. The 50% inhibitory concentration amounts to 0.8 mg PEA/ml. In contrast the herpesvirus induced DNA-dependent DNA polymerase is 10-fold more resistant towards PEA. I t is assumed that, contrary to the synthesis of cellular DNA, the P E A induced inhibition of viral DNA synthesis is not caused by an inhibition of the virusinduced DNA-dependent DNA polymerase. 1. Introduction LILLY et al. (18) reported that 2-phenylethanol (PEA) exerts an inhibitory effect on the growth of gram-negative microorganisms. P E A was shown subsequently to be able to inhibit also proliferation in bacteria (1), moulds (17), yeast (4), plants (22) and mammalian cells (survey: 24). Results from previous studies have demonstrated the inhibition of DNA synthesis as well as production of RNA virus in bacteria (15, 23) and animal cells (27, 3) by PEA. In two publications RomMA~ (27) and MAASS et al. (19) previously studied P E A inhibition of multiplication in herpesvirus. ROIZMAN (27) postulates on the basis of his results, a block in the virus DNA synthesis b y this drug. Direct evidence for his hypothesis, however, is not yet available. 206 W . E . G . MOLLER, D. FALKE, B. I-IEIGKE,and R. K. ZA~t~: We a p p r o a c h e d t h e p r o b l e m b y d e t e r m i n a t i o n of t h e differential influence of P E A on cellular a n d on viral D N A synthesis, b y c o m p a r i n g t h e i n h i b i t o r y conc en t rat i o n s blocking D N A synthesis in the i n t a c t biologieM m o d el i.e. m a m m a l i a n cells w i t h the isolated e n z y m e system. 2. Materials and Methods 2.1. S o u r c e of M a t e r i a l s The following materials were purchased: Unlabeled deoxyribonueleoside triphosphate (dATP, dGTP, dCTP and dTTP) from Boehringer Mannheim, Tutzing; 3I-IdATP (spee. activity 13.5 Ci/mmole) and Thymidine-aH (spec. activity 24 Ci/mmole) from The I~adioehemical Centre, Amersham; ribonuelease (pancreas; 3000 units/mg, EC number : 2.7.7.16) from Worthington Biochemical Corp., Freehold ; cesium chloride from E. Merck, D a r m s t a d t ; insta-gel from Packard Instruments, Ztirich; penicillin and streptomycin from Bayer-Farbenfabriken, Leverkusen and moronal from v. Heyden, Mtinchen. Herring-DNA, isolated according to ZAHN et al. (28) was a gift from H. Mack, Illertissen (Bayern). 2.2. R a b b i t Kidney Cells Rabbit kidney cells were obtained from the kidneys of 6-- 12 days old rabbits by trypsinization. These primary cells were cultivated in I-Ianks's solution containing 10% calf serum, 0.50/o lactalbumine-hydrolysate and bicarbonate as well as antibiotics (penicillin: 100 units/ml, streptomycin: 0.1 mg/ml and moronal: 25 units/ml). After 4 or 5 days the cells were used in the experiments. 2.3. I n f e c t i o n of C e l l s w i t h I - I e r p e s v i r u s The cells were infected with the IES-strain of herpesvirus hominis (6) after changing the culture fluid. Eagle's basal medium (BME) with nonessential amino acids was used throughout the experiments. 2 hours after infection the nonadsorbed virus was washed away. Further incubation in BME was for different times. 2.4. L a b e l l i n g of C e l l u l a r a n d V i r a l D N A The labelling was effected with 10 BCi of thymidine per Petri dish, holding 10 ml of BME. At the end of the experiment, the cells were scraped off, washed two times and frozen at - - 2 0 ~ and kept until extraction of the DNA. F o r each experiment 3 Fetri dishes containing about 6 • 106 cells were used. 2.5. Extraction of D N A DNA from uninfected and virus-infected cells was extracted by the method of KIRBY (12). The D N A fraction was further freed of R N A by treatment with 20 Bg/ml ribonuelease (pancreas) for 120 minutes at 37 ~ C. The D N A finally was precipitated by the addition of ethanol (50% final concentration). The precipitate was dissolved in 0.015 • Tris-I-IC1 (pH 8.0). The yield of the extracted D N A amounted to about 20~o of the total. 2.6. I s o l a t i o n of C e l l u l a r D N A - D e p e n d e n t DNA Polymerase For preparation of crude extracts from rabbit kidney cells a modified method of KEII~ et al. (10) was used. All steps were carried out at 0 - - 4 ~ C. Five g of the sediment of isolated rabbit kidney ceils are washed three times with physiological saline and subsequently suspended in 5 ml buffer A (20 mM Tris-I-IC1, p i t 7.5, 1 m ~ ED TA , 5 m.'~ 2-mereaptoethanol). Influence of 2-Phenylcthanol on Herpesvirus DNA-Synthesis 207 The cells are lysed by rapid freezing and thawing three times. The lysate is centrifuged at 100,000 • g for 60 minutes and the supernatant which contains the DNA-de* pendent D N A polymcrase (EC number : 2.7.7.7), is decanted. In some assays this supernatant is dialysed against buffer B (20 mM Tris-tIC1, p H 7.5, 1 mM ED TA , 5 mM 2-mercaptoethanol, 4 mM MgSO4 and 150 m ~ KC1). The enzyme preparation (1 mg protein/ml) was free from DNA. 2.7. I s o l a t i o n of V i r u s - I n d u c e d DNA Dependent DNA Polymerase As described for control cells, cell batches 11 hours after infection were lysed and centrifuged. The supernatant contains cellular DNA-depcndcnt D N A polymerase as well as the virus induced enzyme (EC number: 2.7.7.7). The cellular DNA-dcpendent D N A polymerase can be inactivated completely by heating the enzyme preparation at 50 ~ C for 10 minutes (10). In this preparation (0.8 mg protein/ml) no D N A contamination was detectable. 2.8. P o l y m e r a s e A s s a y The assays for the determination of DNA-dependent D N A polymerase activities were based on the measurement of the conversion of nucleoside triphosphate into acid-insoluble form. The reaction mixture of 0.08 ml eontained: 0.1 m• att-dATP (4 • I06 counts/minute per ~xmole), 0.1 mM dGTP, dCTP and dTTP, 20 m ~ Tris-I-IC1 (pI-I 7.5), 8 mM MgCI~, I mM 2-mereaptocthanol and different amounts of native and heat denatured DNA, respectively. The reaction was started by addition of 0.02 ml enzyme preparation. After incubation times of 0 and 60 minutes in most cases at 37 ~ C, 20 ~1 were withdrawn, applied to a filter disc (Whatman No. 1) and processed as described by BOLL~M (2). 2.9. A n a l y t i c a l M e t h o d s D N A was determined by the method of KISSA~E et al. (13), protein by the biuret reagent of GORNALL et al. (7). Preparative eentrifugation was performed at 20~ in a 40 rotor of the Spineo model L for 90 hours at 30,000 r . p . m . The initial density of the CsC1 was adjusted to 1.700 g/cma; CsC1 was dissolved in 15 mM Tris-ItC1 (pI-I 8.0). Gradients were analysed by collecting 37 fractions by displacement with saturated CsC1 solution. The optical density was measured with a Zeiss PMQ I I spectrophotometer. For determination of radioactivity samples of 50 ~1 were dissolved in 10 ml instagel and counted in a scintillation counter (TriCarb Packard Inst., La Grange). 2.10. E v a l u a t i o n of D i f f e r e n t i a l I n h i b i t i o n of C e l l u l a r D N A and Herpesvirus DNA Synthesis D N A was extracted from virus infected rabbit kidney cells. The two D N A species (cellular and viral DNA) were separated by CsC1 density gradient (see there). The total radioactivity of the different D N A peaks obtained was calculated using the areas of the Gaussian curves (14). The ratio specific activity of cellular D N A to specific activity of viral D N A can indicate the differential inhibition of DNA-synthesis in case a particular substance had been added. 3. Results 3.1. I n f l u e n c e of P E A Rabbit on DNA-Synthesis Kidney Cells in Intact 3.1.1. Inhibition o / S y n t h e s i s in Unin[eeted Rabbit K i d n e y Cells Table 1 shows the inhibition of D N A - s y n t h e s i s b y P E A in u n i n f e c t e d cells expressed b y a decrease in specific a c t i v i t y ( 3 H - t h y m i d i n e i n c o r p o r a t i o n per m g D N A e x t r a c t e d from cells). I t is e v i d e n t t h a t the D N A synthesis of u n i n f e c t e d cells is a l r e a d y reduced to 41~ b y 0.55 mg P E A / m l . 208 W.E.G. I~IJLLEI%, D . FALKE, B. HEICKE, a n d R . K . Z a ~ s : Y1 u ,oool T 3,0007!-1'60 i 2,000" Woo. ! / ~ to. 1 /' /d,'~ ,,.I I; ~,,,, ~,': \',. '~ i ':..--,. ~11