An effect of cell-culture passage on ultraviolet-enhanced viral reactivation by mammalian cells

417

Mutation Research, 62 (1979) 417--423 © Elsevier/North-Holland Biomedical Press

AN E F F E C T OF CELL-CULTURE PASSAGE ON ULTRAVIOLETENHANCED V I R A L REACTIVATION BY MAMMALIAN CELLS

SHARON P. MOORE and THOMAS P. COOHILL

Departments of Biology and Physics, Western Kentucky University, Bowling Green, K Y 42101 (U.S.A.) (Received 13 December 1978) (Revision received 29 May 1979) (Accepted 14 June 1979)

Summary The ability of ultraviolet-irradiated African green m o n k e y kidney cells to reactivate UV-damaged herpes simplex virus was tested over a range of 57 passages (24--81). Several types of controls were employed to minimize daily variations. The results indicate that in this system cells demonstrate an increasing efficiency to reactivate UV
Cultured mammalian cells in vitro may change properties from those of their in vivo ancestors [15]. This is especially true with regard to a " p e r m a n e n t " cell line since these lines must undergo genetic changes in order to become established lines [9]. It is likely that changes in cells do n o t cease u p o n establishment of the line, b u t rather continue as cells undergo successive passages. Since changes due to passage level of cells may result in extraneous variables in experiments, many investigators a t t e m p t to minimize this effect by limiting use of cells to a narrow range of passages [1,3,6]. Passage level has been considered analogous to cell culture "age" [11]. During experiments on the wavelength dependence of host-cell capacity and ultraviolet-enhanced viral reactivation (UVR) in the CV-1 cell--herpes virus system [3,4], we noticed an effect which appeared to be passage-related. U V R involves the reactivation of UV-irradiated virus b y UV-irradiated mammalian cells. We chose these systems to test passage effects since U V R appears to be similar to Weigle reactivation in bacteria. Weigle reactivation has been linked to " S O S " functions including error-prone repair [8], mutagenesis [ 7,16,17], prophage induction [18] and photoreactiva-

418 tion [16]. The effect of cell-culture passage level on these systems is important not only as a basis for comparison of data obtained in different laboratories, but also to help elucidate factors involved in DNA repair. Materials and methods Cell cultures A TC-7 clone of African green m o n k e y kidney cells (CV-1 line) was obtained from Dr. G. Zamansky of the Harvard Medical School. These cells were maintained in a modification of Dulbecco's Modified Eagle's Medium [5]. Cells were incubated in closed flasks or in petri dishes in closed chambers at 37°C. Cells were obtained at passage 15 and frozen at passage 18. 6 additional passages were required to obtain sufficient cells for an experiment, thus experiments began at passage 24. Each passage represents approximately 3 population doublings. Viral assay: plaque-forming ability (pfa) We used a macroplaque strain of HSV-1 (herpes virus hominus) given to us by Dr. C.D. Lytle of the Bureau of Radiological Health. Details of the harvesting and assay procedures have been previously described [3]. To maximize viral reactivation, infection with virus was delayed until 48 h post-irradiation [2]. After infection, cell monolayers were incubated for 2 days in the case of unirradiated virus and 3 days in the case of irradiated virus to allow for visible plaque formation. Following incubation, the medium was removed from each plate and the plates were UV-irradiated overnight to inactivate remaining viable virions. The following day monolayers were stained with a 1% solution of crystal violet (Carolina Biological Co., Burlington, NC). Co n tro Is Several controls were employed to minimize the effects of daily variation. In some instances cells which extended over a wide passage range were irradiated on the same day. In other experiments cells at similar passages were irradiated concomitantly. Throughout the study cells were removed from the freezer n o t only at the lowest passage number [ t 8 ] but also at increasing passages up to 68. No daily variations were observed. Cells freshly removed from the freezer gave results similar to those of the same passage which had been in culture for several months. In addition, each author maintained cells and conducted experiments independently of each other and obtained the same results. UV-irradiation A General Electric (G8T5) 15-W germicidal bulb with a principal o u t p u t of 86% of its energy at 254 nm was used as the radiation source. This system has been described in a previous publication [ 5]. Cells or viruses were irradiated in open 60-ram plastic petri dishes. Prior to irradiation the growth medium was removed from the cells, the monolayers were rinsed twice in Dulbecco's phosphate-buffered saline (PBS) and overlaid with a final 2.0 ml of PBS which remained on the cells during irradiation. Cells were irradiated with different

419 exposures as shown in the results. Immediately following irradiation the PBS was replaced with growth medium and the cells were incubated for 48 h. Viruses were diluted in PBS and irradiated with an exposure of 400 J / m 2, which lowered the pfa of the virus to approximately 2% of the control (unirradiated) virus. This survival level varied somewhat from experiment to experiment. Cells were infected with an appropriate viral dilution yielding approximately 60 plaques on control (unirradiated) capacity experiments and approximately 30 plaques on control U V R experiments.

Chromosome counting Chromosomes were counted at selected passages. The procedure used was adapted from that of Pollack and Pfeiffer [14]. Due to limitations of our facility and the nature of this work, more extensive chromosome investigations were n o t possible. Results

53 experiments were conducted over a passage range of 24--81 from the origin of this clonal line. For purposes of visualization, representative experiments were averaged in Fig. 1 (A--D). The abscissa shows the exposure of radiation to the cells; the ordinate shows survival of virus (as measured by pfa). The upper curves (®) were obtained from irradiating cells but n o t virus (capacity). A slight decrease in capacity occurred in all experiments as exposure increased. The lower curves (A) obtained from irradiating both cells and virus show a rise in survival of pfa (UVR) even after the capacity curves begin to drop. Since U V R can reflect changes in capacity, the effect of capacity was compensated for by dividing the pfa of irradiated virus by the pfa of unirradiated virus on cells that received the same exposure of radiation [4]. Using this method, a reasonably straight line was obtained for each experiment (UVR factor), the slope of which indicates the extent of reactivation obtained. In each figure, the averaged U V R factor is drawn at the b o t t o m of the figure (G). The slope of each U V R factor was calculated over the region 0--30 J/m 2 by subtracting the logarithm of the control point from the logarithm of the point at 30 J / m 2 and dividing this difference by the difference in exposure. In some experiments, cells began to detach from the dishes at 40 J / m 2, and the U V R factors dropped at this point. Even in those experiments in which the U V R factor continued to rise at 40 J / m 2, the data analysis was still restricted to 0--30 J / m 2. The slope of each U V R factor is plotted in Fig. 2. The smaller open circles represent slopes of U V R factors from individual experiments; the larger circles are the average of groups of experiments within a range of 10 passages, with error bars representing standard deviation. The number next to each averaged point indicates the number of points considered in that average. Fig. 1A shows representative data averaged from 3 experiments using cells of passages 24a, 24b and 25. The 2 sets of cells at passage 24 represent cells removed from the freezer at different times, i.e. they are n o t sister populations. Error bars indicate the standard error resulting from averaging experiments. Fig. 1B (passages 32, 34a, 34b and 36), Fig. 1C (passages 45, 47 and 48) and Fig. 1D) (passages 67, 68 and 69) are plotted in a manner similar to Fig. 1A.

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Fig. 2. S l o p e o f all U V R f a c t o r curves p l o t t e d against passage n u m b e r . Smaller, lighter p o i n t s r e p r e s e n t individual e x p e r i m e n t s ; larger, heavier circles r e p r e s e n t averages o f groups o f e x p e r i m e n t s w i t h i n a range of 1 0 passages. The n u m b e r beside e a c h average p o i n t i n d i c a t e s the n u m b e r o f individual p o i n t s considered in that average.

The passages shown in Fig. 1A (24a, 24b and 25) are the lowest passage cells tested. The UVR curve rises only slightly, and the slope of the averaged UVR factor is 0.08. The low point at 40 J/m 2 in Fig. 1 may be explained by the fact that at this exposure both UVR and capacity fall off at approx, the same rate. Again, although 2 of these experiments use cells at the same passage, they are not sister populations, but rather, represent cells removed from the freezer at different intervals. 4 separate experiments were averaged in Fig. 1B (32, 34a, 34b and 36). In this instance, the averaged UVR factor for these passages resulted in a straight line. The average of passages 45, 47 and 48 shown in Fig. 1C exhibits an even greater rise in UVR than passages shown in Figs. 1A and l B . This figure also shows the least effect due to capacity. The greatest rise in UVR occurs in the region 0--20 J/m 2, but capacity remains approximately the same level over this region. The slope of the averaged UVR factors for these passages is 0.189. Data from experiments averaged for passages 67, 68 and 69 are shown in Fig. 1D. Here UVR rises only slightly and attains a shoulder at a relatively low exposure -- 15 J/m 2. The slope of the averaged UVR factor curve for these passages is 0.09. All of the slopes of the averaged UVR factor curves agree with the averaged points in Fig. 2. In Fig. 2, the abscissa shows the cell passage number of each experiment, and the ordinate shows the slope of the reactivation factor line obtained from each experiment. Slope values ranged from 0.042 (at passage 26) to 0.30 (at passage 68). When slopes were averaged within groups of 10 passages, the

422 TABLE 1 RESULTS

OF CHROMOSOME

COUNTS ON CV-1 CELLS AT SELECTED

PASSAGES (Clone TC-7)

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highest average value occurred in the passage 60 region. Results from chromosome counts are shown in Table 1. Due to technical difficulties, varying numbers of cells were suitable for counting at each passage. The mean number of chromosomes and the standard error were calculated at each passage (see Table 1). Discussion Throughout these experiments capacity changed very little. UVR, however, appears to be "passage-dependent." Although UVR is relatively low in lowpassage cells, it rises rapidly until the cells reach a " t u r n o v e r " passage (in this case approximately passage 60) then decreases. By analogy Finch and Hayflick [9] present a diagrammatic representation of the survival of a mortal cell strain which resembles the curve we obtained in Fig. 2 using an established line. However, their curve reached the " t u r n o v e r " point a few passages earlier than ours and indicated loss of cell viability while our cells remained viable. Chromosome counts showed no significant increase or decrease in number as passage increased. These data suggest that changes in UVR as passage increases are not due to chromosomal anomalies. At present we have no explanation for this phenomenon. Lytle [11] showed that in human cells host-cell reactivation changed little as passage number of the cells increased; however, the relationship between HCR and UVR is still unclear [11]. Painter et al. [13] working with a mortal human cell line (WI-38) found that " a g e " of the culture did not significantly affect the efficiency of excision repair until very close to the terminal stage of the culture. Lytle et al. [12] reported that excision repair is not necessary for UVR. Goldstein [10] using xeroderma pigmentosum and normal human cells found that "age" of the cells did not appear to impair DNA repair. From our results it appears that the efficiency of some factor(s) or mechnism(s) involved in UVR in the CV-1 cell line is passage-dependent. Acknowledgements The authors express appreciation to William E. Thompson for chromosome counting and L.E. Bockstahler, C.D. Lytle and K.B. Hellman for helpful discussion. All the reported work was supported by FDA contract No. 223-74-6067.

423

References l Black, P. H. ( 1 9 7 8 ) p e r s o n a l c o m m u n i c a t i o n . 2 B o c k s t a h l e r , L.E., C.D. L y t l e , J.E. S t a f f o r d a n d K.F. t t a y n e s , U V e n h a n c e d r e a c t i v a t i o n of a h u m a n virus: E f f e c t of d e l a y e d i n f e c t i o n , M u t a t i o n Res., 35 ( 1 9 7 6 ) 1 8 9 - - 1 9 8 . 3 Coohill, T.P., S.P. M o o r e a n d S. D r a k e , T h e w a v e l e n g t h d e p e n d e n c e of u l t r a v i o l e t i n a c t i v a t i o n of h o s t c a p a c i t y in a m a m m a l i a n cell-virus s y s t e m , P h o t o c h e m . P h o t o b i o l . , 26 ( 1 9 7 7 ) 3 8 7 - - 3 9 1 . 4 Coohill, T.P., L.C. J a m e s a n d S.P. Moore, T h e w a v e l e n g t h d e p e n d e n c e of u l t r a v i o l e t e n h a n c e d reactiv a t i o n in a m a m m a l i a n cell-virus s y s t e m , P h o t o c h e m . P h o t o b i o l . , 27 ( 1 9 7 8 ) 7 2 5 - - 7 3 0 . 5 Coohill, T.P., D.J. K n a u e r a n d D.G. F r y , T h e e f f e c t of c h a n g e s in cell g e o m e t r y on t h e sensitivity to u l t r a v i o l e t r a d i a t i o n of m a m m a l i a n cell c a p a c i t y , P h o t o c h e m . P h o t o b i o l . , ( 1 9 7 9 ) in press. 6 C o p p e y , J., G r o w t h of N e w c a s t l e disease a n d h e r p e s virus a n d i n t e r f e r o n p r o d u c t i o n in a m o n k e y - m o u s e h y b r i d line, J. Gen. Virol. 14 ( 1 9 7 2 ) 9 - - 1 4 , 7 D a s g u p t a , U.B., a n d W.C. S u m m e r s , U l t r a v i o l e t r e a c t i v a t i o n of h e r p e s s i m p l e x virus is m u t a g e n i c a n d i n d u c i b l e in m a m m a l i a n cells, Proc. Natl. A c a d . Sci. (U.S.A.), 75 ( 1 9 7 8 ) 2 3 7 8 - - 2 3 8 1 . 8 D e v o r e t , R., P. M o r e a u , A. G o z e , A. Bailane, A. Sarasin a n d Y. Moule, 7th Int. Congr. P h o t o b i o l . , R o m e , Italy, 1 9 7 6 . 9 Finch, C.D., and L. H a y f l i c k , H a n d b o o k of the Biology of Aging, Van N o s t r a n d / R e i n h o l d , N e w York, 1977. 10 G o l d s t e i n , S., T h e role of D N A r e p a i r in a g i n g of c u l t u r e d fibroblasts f r o m x e r o d e r m a p i g m e n t o s u m a n d n o r m a l s , Proc. Soc. Exp. Biol. Med., 137 ( 1 9 7 1 ) 7 3 0 - - 7 3 4 . 11 L y t l e , C.D., a n d S.G. Benane, H o s t cell r e a c t i v a t i o n in m a m m a l i a n cells, IV. Cell c u l t u r e c o n d i t i o n s a f f e c t i n g virus survival, Int. J. Radiat. Biol., 2 6 ( 2 ) ( 1 9 7 4 ) 1 3 3 - - 1 4 1 . 12 L y t l e , C.D., R.S. D a y III, K.B. H e l l m a n a n d L.E. B o c k s t a h l e r , I n f e c t i o n of U V - i r r a d i a t e d x e r o d e r m a p i g m e n t o s u m fibroblasts b y h e r p e s s i m p l e x virus: S t u d y of c a p a c i t y a n d Weigle r e a c t i v a t i o n , M u t a t i o n Res., 36 ( 1 9 7 6 ) 3 5 7 - - 3 6 4 . 13 Painter, R.B., J.M. Clarkson and B.R. Y o u n g , U l t r a v i o l e t i n d u c e d r e p a i r r e p l i c a t i o n in aging diploid h u m a n cells (WI-38), R a d i a t . Res., 56 ( 1 9 7 3 ) 5 6 0 - - 5 6 4 . 14 Pollack, R., a n d S. Pfeiffer, A n i m a l Cell Culture, Cold Spring H a r b o r L a b o r a t o r y , Cold Spring H a r b o r , NY, 19710 pp. 4 6 - - 4 9 . 15 T o o z e , J., T h e M o l e c u l a r Biology of T u m o r Viruses, Cold Spring H a r b o r L a b o r a t o r y , Cold Spring H a r b o r , NY 1 9 7 3 . 16 Weigle, J., I n d u c t i o n of m u t a g e n e s i s in a b a c t e r i a l virus, Proc. Natl. A c a d . Sci. (U.S.A.), 39 ( 1 9 5 3 ) 628--636. 17 Witkin, E.M., U l t r a v i o l e t m u t a g e n e s i s and i n d u c i b l e r e p a i r in Escherichia coli, Bacteriol, Rev., 4 0 ( 4 ) (1976) 869--907. 18 Witkin, E.M., T h e r a d i a t i o n sensitivity of Escherichia coli B: A h y p o t h e s i s relating f i l a m e n t f o r m a t i o n a n d p r o p h a g e i n d u c t i o n , Proc. Natl. A c a d . Sci. (U.S.A.), 57 ( 1 9 6 7 ) 1 2 7 5 - - 1 2 7 9 .