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

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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)

Passage n u m b e r

N u m b e r o f cells c o u n t e d

Mean number of chromosomes

24 27 42 47 53 72

22 9 16 21 15 25

89.4 88.0 93.0 84.0 91.7 90.7

-+ 8.2 ± 8.6 + 3.9 + 9.0 -+ 4 . 8 +- 4 . 8

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 .

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...
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