Photochemistry and Photobiology Vol. 5 5 , No. 2, pp. 212-219, 1992
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CELL DENSITY DEPENDENCE OF ULTRAVIOLET LIGHT ENHANCED REACTIVATION OF Herpes simplex TYPE I AND THE LARGE PLAQUE EFFECT IN C3H/lOT+ MOUSE FIBROBLASTS J. G. MONTES’ and W. D. TAYLOR*~ ‘Department of Biophysics, University of Maryland School of Medicine, Baltimore, MD 21201, USA and *Department of Molecular and Cell Biology, The Pennsylvania State University, University Park, PA 16802, U S A (Received 11 March 1991; accepted 21 June 1991)
Abstract-43HIlOT4 mouse fibroblasts were grown to different cell densities either by plating at low density and allowing different growth periods, or by plating at a series of increasing densities and allowing the same growth period. These plates were UV irradiated at 7.5 J/mZor mock irradiated and 24 h later infected with UV-irradiated Herpes simprex type I virus which had been UV irradiated at 50 or 125 J/m2 or mock irradiated. The numbers and sizes of plaques were measured and these data used to calculate the extent of UV-enhanced host cell reactivation, the capacity enhancement, the large plaque effect (LPE) and the small plaque effect (SME). The influence of cell density on these phenomena was similar for both series of density experiments. Ultraviolet-enhanced host cell reactivation could be demonstrated only for cultures of lower density. The capacity of the cells for Herpes simplex type I virus decreased with cell density, but UV irradiated cells showed an increase in capacity with cell density. Plaque sizes decreased in all cases with cell density but the LPE and SPE were not significantly altered. The greatest variation in the above parameters occurred just as the cells were approaching confluence, where most host cell reactivation experiments are carried out. We conclude that the reproducibility of such experiments depends critically on cell density, a dependence which may be relevant to mechanistic interpretations of the UV-dependent phenomena.
cells (Lytle ef af., 1974) and the mouse fibroblast line C3HhOTf at confluence (Montes and Taylor, Enhanced host cell reactivation ( E R ) t of nuclear1986). The fact that it is possible to induce E R with replicating viruses inactivated with different DNAagents known to inhibit D N A synthesis, such as damaging agents occurs in various mammalian cell cycloheximide, hydroxyurea, caffeine, and bromolines (Bockstahler and Lytle, 1970; Lytle et af., deoxyuridine (Sarasin and Hanawalt, 1978; Fogel et 1974; Bockstahler et al., 1976; Lytle et af., 1978; af., 1979; Lytle and Goddard, 1979), has prompted Sarasin and Hanawalt, 1978; Fogel et af., 1979; the hypothesis that E R is induced by the delay of Jeeves and Rainbow, 1979a,b). In E R , survival of D N A synthesis, whether produced by agents affectinactivated virus is greater on cells that have been ing D;IA polymerases, or by damaged D N A itself pretreated with certain DNA-damaging agents than (Lytle and Goddard, 1979). The search for a model on untreated cells. This phenomenon has been likexplaining E R in general has been complicated ened to Weigle Reactivation, one of several “SOS” additionally by the finding that a biological agent, responses seen in bacteria (Witkin, 1976; Radman, human cytomegalovirus, can induce E R (Nishiyama 1980); it has been argued whether E R and “SOS” and Rapp, 1980). are caused by a common mechanism (Rossman and Enhanced host cell reactivation occurs in associKlein, 1985). Ultraviolet light is one DNA-altering ation with other phenomena, some of which may agent which has been given particular attention as be useful in explaining its nature. These include the an inducer of ER. small plaque effect (SPE), the large plaque effect Ultraviolet light-enhanced reactivation (UVER) (LPE), and an increase in the capacity of monoof Herpes simplex type I virus (HSV-1) occurs in layers to allow formation of viral plaques, herein most systems studied thus far, although notable referred to as capacity enhancement, or CE. In the exceptions exist, including human embryonic lung SPE plaque size is reduced by treatment of viruses with DNA-damaging agents before infection of cells; this phenomenon has been observed with Her*To whom correspondence should be addressed. tAbbreviutions: CE, capacity enhancement; DPBS, Dul- pes virus in various mammalian cell lines (Ross et becco’s phosphate buffeted saline; ER, enhanced reacti- al., 1971; Cameron et af., 1979; Fogel et af., 1979; vation; HSV-1, Herpes simplex virus, type 1; LPE, large Coohill et al., 1980). The LPE results in the proplaque effect; RF, reactivation factor; SEM, standard error of the mean; SPE, small plaque effect; UVER, duction of larger plaques on host cells pretreated with such diverse agents as prostaglandin (Harbour ultraviolet enhanced reactivation. INTRODUCTION
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J . G. MONTESand W. D. TAYLOR
et a f . , 1978), cycloheximide (Babich et a f . , 1981), and U V light (Babich et a f . , 1981; Montes and Taylor, 1986). Some time ago. we reported on the significant increase in plaque number (CE) which occurs when mouse embryo fibroblast cells (C3WlOTt) are treated with U V light shortly before inoculation with HSV-1 (Montes and Taylor, 1986). In that paper we concluded that absence of E R in a particular cell line did not necessarily preclude the presence of the LPE and the SPE. One of the obvious results of irradiating monolayers with U V light is the decline in the number of attached cells that can be attributed to cell death, thereby causing a reduction in cell density. This observation brings into question the direct o r indirect role of cell density in U V E R , as well as in the LPE and C E ; previous studies using HSV-1 on other types of cells indicate, for example, that U V E R depends on age of confluent cultures (Zurlo and Yager, 1984) and that U V E R may only be exhibited by cells undergoing DNA synthesis (Ryan and Rainbow, 1986). We therefore carried out experiments in which cell density was varied by either seeding density or culture age, in order to determine directly the possible role of cell density on these phenomena. A hypothetical explanation is proposed that may account for the failure to observe LJVER under certain conditions in C3H/lOTi and in perhaps other cell lines. MATERIALS AND METHODS
Virus stocks. Herpes simplex virus type 1. MP (Macroplaque strain), was used to infect cells. Stocks were prepared by infecting freshly-confluent monolayers of CV1-P (African green monkey kidney) cells. Inoculation with virus was performed in Dulbecco's phosphate-buffered saline (DPBS) (Dulbecco and Vogt, 1954) at a multiplicity of infection between 0.001 and 0.01 as calculated for the unirradiated virus. Culture medium and growth conditions for CVl-P are detailed elsewhere (Taylor er al., 1982; Montes and Taylor, 1986). Host cell culrure. C3HIlOTd mouse embryo fibroblasts were cultured as described previously (Montes and Taylor, 1986). Experiments were performed at passages 32-37. and cells were passaged every 8-12 days. Pretreatmenr irradiation of cells. Cell monolayers were irradiated as described previously (Montes and Taylor, 1986). Total dose to the cells was either 0 Jlm2 (mockirradiation) or 7.5 Jim'; the latter dose was selected because of optimum effects seen previously at that dose (Montes and Taylor. 1986). Irrudiarion of virrcs. Virus was irradiated in uncovered 100 mm tissue culture dishes under a GE G&T5 lamp as described (Montes and Taylor, 1986). Total dose to virus was either SO or 125 Jim'. Reucrivation experimenrs. The surviving fraction of irradiated virus was measured on unirradiated cells, S,,, and UV-irradiated cells, S,. The reactivation factor, RF. was calculated as: RF = S , / S , , , (Bockstahler er a / . . 1976). The effect of irradiation of cells on their capacity to support the growth of unirradiated virus (the CE) was measured by comparing the number of plaques on irradiated plates with the number on unirradiated plates. PQque number and size assays. Plaques were counted in monolayers stained and dried as described in our previous report (Montes and Taylor. 1986). All cultures, regardless
of condition or protocol, were terminated and stained exactly 4 days after inoculation with virus; from previous trials bracketing the range of conditions used in this study it was found that further increases in incubation time did not result in any significant changes in plaque number. Plaque size (area) was determined by projecting the image of the cellular layer of stained dishes onto a sheet of graph paper (Ross er a l . , 1971). Control of cell monolayer densities. In these experiments, one of two different approaches was used to attain the resulting densities: (A) seeding dishes at different cell densities on the same day and then allowing the same time (2, 3, or 4 days) before irradiation or mock-irradiation with UV light; or (B) seeding all dishes on the same day at 4.5 x 104 cells per dish (plating efficiency 32-38%; about 1.5-1.7 x 104 cells adhered) and beginning reactivation experiments at different times (4,5,6,7, or 8 days) thereafter. In approach (A) the actual number of cells that attached was not determined for seeding densities greater than 5 x 105/dish,although for lower densities the plating efficiency remained at about 32-38% (see Table 1). In both approaches, the pretreatment UV light doses were 0 or 7.5 J/m2. In the first approach. virus was inactivated with 125 Jim', whereas 50 J/mZwas used in the second. The rationale for using two different approaches to obtain different cell densities was based on the observation that monolayers attaining confluence immediately, by seeding at very high density, had final cell densities different from those that reached confluence starting from lower seeding densities (see Discussion). It was not known if cells that become quiescent via different routes of initial seeding density and of time allowed to reach confluence would possess properties of importance to the study. Cell densities were measured for both types of experiments at the time of irradiation of the cells and at the time of inoculation (UV-irradiation temporarily suppressed growth of cultures). All densities were found by detaching cells with 0.05% trypsin + 0.016% EDTA in DPBS and then counting the cells in suspension by using a Neubauer hemacytometer (American Optical, Buffalo, NY). RESULTS
Density studies: dishes seeded at different cell densities on the same day Cultures were started by seeding at different cell numbers per 60 mm dish (range: 1.6 x lo4 to 1.2 x 10" cells per dish; actual number of cells that attached was significantly less because of a 32-38% plating efficiency). In Figs. 1-3, panels (a), (b) and (c) correspond to experiments in which the cells were mock irradiated or irradiated with 7.5 Jlm2 of U V light 2 days (a), 3 days (b) or 4 days (c) after plating. The time allowed between seeding and pretreatment resulted in further differences in cell density. Table 1 provides data on population densities of cells seeded, as well as the numbers of cells attached to dishes at critical times during the experiments. It was seen that U V light resulted in decreases in density as measured 24 h after irradiation of the cells, with the effect being more prominent at lower densities. Seemingly paradoxical was the confluent or nearly confluent condition of some of the monolayers at densities lower than usual for confluence, arising from seeding at high density. Microscopic observation verified that the
Density dependence of enhanced reactivation
215
Table 1. Correlation of C3WlOTf cell density at time of UV-irradiation of cells and viral inoculation with number of cells initially seeded Number of cells x lO-Vdish
Day of irradiation of cells
On day of viral inoculation On day of seeding
On day of irradiation
2
0.40 f 0.04 0.75 ? 0.08 2.00 f 0.20
~
Unirradiated
Irradiated
0.97 f 0.14 2.20 f 0.24 3.30 f 0.18
2.90 f 0.25 4.10 ? 0.14 5.80 f 0.19
1.60 rt 0.17 2.60 ? 0.31 4.20 2 0.25
3
0.50 f 0.05 1.60 t 0.16 3.00 f 0.30 6.00 f 0.60 12.00 f 0.12
2.30 f 0.15 4.90 & 0.37 6.40 f 0.10 6.20 f 0.40 6.40 ? 0.26
6.0 t 0.26 10.00 t 0.90 11.00 ? 1.10 7.70 2 0.62 7.40 f 0.48
2.60 f 0.10 4.40 f 0.20 7.00 ? 0.48 6.80 ? 0.67 7.00 f 0.10
4
0.35 f 0.04 1.20 2 0.06 2.00 f 0.20 8.00 f 0.04 12.00 f 1.20
1.80 f 0.23 4.60 ? 0.48 6.90 f 0.68 7.00 2 0.74 6.30 t 0.33
4.70 f 0.41 8.10 2 0.38 15.00 ? 0.35 8.80 ? 0.46 6.50 f 0.75
1.80 0.12 6.20 f 0.27 8.10 2 0.41 7.20 2 0.36 5.80 rt 0.64
"
Cells were either UV-irradiated (7.5 J/m2) or mock-irradiated 1 day before inoculation with UV-irradiated (125 J/m2) or unirradiated HSV-1. Day = 0 corresponds to day of seeding. Data correspond to experiments of [Figs. l(a)] (in part), [l(b)], and [l(c)], and [Fig. 2(a)] (in part). Results are based on at least 4 independent hemacytometer counts from 1 sacrificed dish (or from seeding stock, for second column) per condition.
1 2 3 4 5 6 7 8 9 D l l l Z NUMBER OF CELLS SEEDED (CELLS 1 lO-'lDlsn)
Figure 1. Survival of HSV-1 on C3H/lOTI cells as a function of different seeding densities. Cells were mockirradiated (0)or pretreated with 7.5 J/m7 UV light (A); virus were inactivated with 125 J/m2 UV light. Inoculation with virus took place 24 h after pretreatment of cells. Surviving fraction for cells treated 2 days after seeding (performed at only 2 different population densities) (a), 3 days after seeding (performed at 5 population densities) (b), or 4 days after seeding (performed at 5 population densities) (c). (Each panel represents an experiment performed in duplicate. Standard deviation is displayed with error bars; points without error bars are not significantly different statistically from each other.)
lack of increase in cell density for cells seeded at the high densities of 6, 8, or 12 x lo5 per dish correlated with the fact that these higher starting densities ultimately resulted in cells that were larger than those found in confluent dishes plated in the normal way, 1.e. starting with about 5 x lo4 cells/ dish; in this way, contact inhibition could occur at a lower cell density. Clearly, the cell density at confluence is a function of the density at which the cells are plated. Virrrs survival. The survival of virus inactivated with 125 J/m2 UV light depends to a large extent on the number of cells seeded. Reference to Table 1 will indicate that at or near confluence survival was relatively low and within a narrow range of variation, regardless of the population density at which confluence finally occurred. Therefore, when 6 x lo5 cells per dish or more were used, the results were essentially the same, with surviving fractions of between 1and 2 x At lower seeding densities, however, survival was greater (up to more than 8x depending on seeding density and days after seeding that cells were infected), gradually declining from its highest point as the number of cells seeded increased. At the lower seeding densities (1.6 X 104 to about 3 X 105 cellddish) viral survival was almost always higher when the host cells had been pretreated with 7.5 J/m2 of UV light than in controls (i. e. UV-enhanced reactivation occurred according to the mathematically operational definition of enhanced reactivation, that is, surviving
J. G. MONTES and W.D. TAYLOR
216 1
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I 2 3 4 S 6 7 8 9 1 0 1 1 1 2 NUMBER O f CELLS SEEDED (CELLS n lO-’/DlSH)
Figure 2. Capacity of C3WlUTt cells for HSVl as a function of different seeding densities. All titers (i.e. plaque numbers) were normalized to maximum titer measured on unirradiated cells for each set. Cells were either mockirradiated (0)or exposed to 7.5 J/m2 UV light (A). Inoculation with unirradiated virus took place 24 h after treatment of cells. Relative capacity for cells that were treated 2 days after seeding (a), 3 days after seeding (b), or 4 days after seeding (c). (Statistical information provided as in Fig. 1 . ) fraction of irradiated virus on irradiated cells/surviving fraction of irradiated virus on unirradiated cells > unity); although the evidence for UVER was statistically significant for the very lowest seeding densities in Fig. 1 (calculations not shown), the RF never exceeded 1.5. At confluence or near confluence the RF was less than unity. The possible relevance of this observation to the mechanisms responsible for enhanced reactivation are considered below in the Discussion. Capacity enhancement. Maxima in plaque yields occurred (Fig. 2) for unirradiated cells when dishes were seeded at 1-2 x 10’ cells per dish and inoculated with unirradiated virus 2, 3 or 4 days after seeding (i.e. mock-irradiated 1, 2 or 3 days after seeding, respectively). At seeding densities below 6 x lo5 cells per dish (resulting in confluence or near-confluence 1 day later), plaque yields were lower on irradiated cells (7.5 J/mZ) than on corresponding unirradiated cells. A crossover of the plaque yield curves for treated and untreated cells occurred above 4 x los cells per dish. It is interesting that the maximum plaque yield obtained for pretreated cells was roughly equal to the maximum observed in control cells, even though the cell seeding densities corresponding to maximum plaque number were very different for the two plaque yield curves. Plaque sizes. Plaque size (Fig. 3) was greatest for cells seeded at the lowest densities, regardless of treatment to cells (7.5 J/mZ) or virus (125 J/mZ);
Figure 3. HSV-1 plaque size as a function of seeding density. Cells were mock-irradiated (0.0) or exposed to 7.5 J/mZ( A , A ) of UV light before inoculation with unirradiated virus (A,O) or UV-irradiated (125 J/mZ) virus (A,.) 24 h later. Plaque sizes were measured for cells seeded 3 days (a), or 4 days (b) before irradiation or mock-irradiation. (Error bars show standard deviation for duplicate experiments; points without error bars are not significantly different statistically from points immediately below or above them.) with increase in the number of cells seeded, plaque size declined, reaching essentially constant values for cultures infected 4 days [Fig. 3(a)], or 5 days after seeding [Fig. 3(b)]. Irradiation of virus generally resulted in smaller plaque sizes for both irradiated and unirradiated cells within the time allowed for plaque development; pretreatment of cells resulted in larger plaques for both irradiated and unirradiated virus. It is notable that the LPE remained constant at about 2 regardless of density at time of irradiation of the cells.
Density studies: dishes seeded at sanie initial density but treated on different subsequent days In these experiments cells were allowed to divide from a common starting population density (4.5 x lo4cells seeded, about 1.5 x lo4 successfully plated), the desired variation in population density being obtained by pretreatment on different days after seeding. The growth curve for the cells in these experiments is shown in Fig. 4. Thus, it is possible to match a value on the abscissa (days after seeding) in Figs. 5-7 to a corresponding cell density on the ordinate of Fig. 4. Survival of virus. It was evident (Fig. 5) that the survival of irradiated virus (50 J/m2) decreased with age of cultures, i.e. with increasing density (it should be noted that HSV-1 survival on C3H/lOTi monolayers is non-linear on a semi-log plot, and may in fact be multi-component; see Montes and Taylor, 1986). Only in cultures pretreated 4 days after seeding (i.e. inoculated 5 days after seeding) was the surviving fraction in pretreated cultures (7.5 J/mZ)
217
Density dependence of enhanced reactivation
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-
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0.1
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J W
u
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2 4 6 DAYS AFTER SEEDING
8
Figure 4. Growth curve for C3H/lOTt cells. Dishes were seeded with 4.5 X 104 cells each (= 1.5 X 104 successfully plated) on Day 0. Open circles indicate number of attached cellsldish at different times. Triangles indicate number of attached cellddish 24 h after exposure of cells to 7.5 J/mZ of UV light. (Shown are the average results from 3 experiments; error bars indicate standard error of the mean (SEM) for unirradiated cells; SEM = 6.4 lr: 4.6% for triangles.)
higher than in non-pretreated cultures, and then by a small margin; reactivation factors for older cultures were below unity, i.e. no evidence could be found for UVER in older cultures (Fig.5, right
t
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ot14
I
I
I JI I
I 6 1 DAYS AFTER SEEDING
8
Figure 5. Survival and reactivation factor for HSV-1 on C3WlOT4 cells as functions of time after seeding. Dishes were seeded with 4.5 x 104 cells each (= 1.5 x 104 successfully plated) on Day 0. Cells were mock-irradiated (0)or irradiated with 7.5 J/m2 ( A ) of UV light 24 h before inoculation with UV-irradiated (50 J/m2) virus on the days indicated. The corresponding reactivation factors are also given ( x ) (the dashed line was fit “by eye” and is suggestive only). (Each point represents the results of 3 separate experiments; error bars indicate SEM.)
W
a
LI
0 ’4
I I 1 5 6 1 DAYS AFTER SEEDING
I 1
e
Figure 6. Relative plaque number and capacity enhancement of HSV-1 in C3H/lOTi cells as functions of time after seeding. Dishes were seeded with 4.5 x 104 cells each (= 1.5 x 104 successfully plated) on Day 0. Cells were mock-irradiated (0)or exposed to 7.5 J/m2 ( A ) of UV light 24 h before inoculation with virus on the days indicated. All plaque numbers were normalized to the maximum number observed on unirradiated cells in order to obtain relative plaque numbers. Capacity enhancement ( x ) was calculated by dividing plaque number for irradiated cells by the number for unirradiated cells. (Each point represents the results of 3 separate experiments; SEM are shown with error bars.)
margin). It was seen that only at 4 days after seeding (cell density = 9.5 x lo5 cells/dish) were cultures still in exponential growth (Fig. 4). Capacity enhancement. It was found that in control cultures, relative capacity, calculated by dividing the average plaque number for each density by the average number of plaques observed on day 4, declined with age of cultures, with most obvious differences occurring between days 6 and 7 (Fig. 6). However, for pretreated cultures (7.5J/m*) a welldefined maximum occurred at 6 days. As in Fig. 2, the highest titer (i.e. plaque number) measured on irradiated monolayers was not significantly different from the maximum achieved on unirradiated monolayers. It is interesting that the CE (obtained by dividing plaque number for pretreated cultures by plaque number for non-pretreated controls) nonetheless vaned as a smooth sigmoidal function (Fig. 6, right margin). Plaque size. Plaque size declined with days after seeding (i.e.cell density) regardless of whether cells, virus or both, received radiation (Fig. 7). Pretreated cultures yielded larger plaques than control cultures, for both irradiated and unirradiated virus. The LPE was relatively constant for unirradiated virus, between 1.5 and 2.0; the LPE for irradiated virus, however, was considerably lower and not as constant. Smaller plaques were obtained with irradiated virus (SPE) on both control and irradiated cells.
J. G. MONTES and W. D. TAYLOR
218
r
m1
T
A
T\
DAYS AFTER SEEDING
Figure 7. Plaque size of HSV-1 on C3HIlOTI cells as a function of time after seeding. Dishes were seeded with 4.5 x 104 cells each (= 1.5 x 104 successfully plated) on Day 0. Cells to be inoculated with unirradiated virus were mock-irradiated (0)or exposed to 7.5 J/mz ( A ) of UV light on days indicated; cells to be inoculated with UVirradiated (50 Urn2) virus similarly were mock-irradiated (0)or exposed to 7.5 J/mZ (A) of UV light on days indicated. Inoculation took place 24 h after day indicated.
(Each point represents results of 3 separate experiments; error bars show SEM). These results are qualitatively similar to those found in the first study. DISCUSSION AND CONCLUSIONS
It is clear from these data that important parameters of UV radiation effects on the HSV-1C3WlOTI system depend strongly on the cell density, especially as confluence is approached. In these two series of experiments in which cell density was varied in two different ways, qualitatively similar results were obtained for the three kinds of measurements made. In the first kind (Figs. 1 and 5), the surviving fraction of irradiated virus declined with cell density on both irradiated and control cultures. The RF calculated from these data also declined from somewhat more than unity to less than unity. In the second kind of measurement, in which the capacity of cell cultures to support the growth of unirradiated virus was measured, opposite cell density effects were seen (Figs. 2 and 6) for control vs irradiated cultures. In control cultures the capacity declined with increasing density; in irradiated cultures the capacity increased with increasing cell density. This could arise if quiescent cells were less susceptible to viral infection and if in irradiated monolayers sufficient cells were killed to produce an apparent displacement to lower densities. In the third kind of measurement, of plaque sizes, the results of both series of experiments coincided; all plaque sizes declined with cell density. However, the SPE and LPE appeared to be relatively insensitive to cell density.
Comparing the 2 series of experiments, one can reach the conclusion that the growth state of the cells is the important parameter, rather than the absolute cell monolayer density. For example, the RF, often used as evidence for the possible induction of repair processes, depends strongly in this system on the cell density just as the cells reach confluence (see Fig. 4), which is where most studies are carried out. An interesting possibility that gives UVER (or perhaps other forms of ER) a novel explanation is that UV light applied to a confluent or nearly confluent culture may result in a dosedependent shift of the growth state of cells in the culture. In Figs. 1 and 5 it is clear that the virus surviving fraction decreases considerably at confluence whether cells are pre-irradiated or not. The result of UV pre-irradiation of the cells may be simply to break contact inhibition among them via cell death or damage, so that a new culture of effectively lower cell density is created. In effect, calculation of a RF (surviving fraction on irradiated cells divided by surviving fraction on unirradiated cells) may be equivalent to comparing the surviving fraction of virus for a confluent culture (i.e. lower survival) with that for a culture of lower density (i.e. higher survival), even though experimentally both cultures started at confluence. This explanation would suggest that whether UVER can be demonstrated may depend, at least partially, on the suscep tibility of a given cell type to the lethal effects of UV light and the UV dosing regimen used. Thus, it is important to control more precisely the growth state of cells in these kinds of experiments in order to obtain data that are both reproducible and more informative. REFERENCES
Babich, M.A.. T. P. Coohill, W. Snipes and W. D. Taylor (1981) The effect of metabolic inhibitors on the large plaque effect with Herpes simplex virus. Phorochem. Photobiol. 34, 197-201. Bockstahler, L. E. and D. C. Lytle (1970) Ultraviolet light enhanced reactivation of a mammalian virus. Biochem. Biophys. Res. Commun. 41, 184-189. Bockstahler, L. E., D. C. Lytle, J. E. Stafford and K. F. Haynes (1976) Ultraviolet enhanced reactivation of a human virus: effect of delayed infection. M u . Res. 35, 189-1 98. Cameron, K. R., L. M.Tomkins, R. P. Eglin, L. J. N. Ross, P. Wildy and W. C. Russell (1979) The effects of ultraviolet and ionizing radiation on herpes virus, SV40, and adenoviruses in relation to the small-plaque effect. Arch. Virol. 62, 31-40. Coohill, T. P., M.A. Babich, W. D. Taylor and W. Snipes (1980) Herpes simplex virus produced larger plaques when assayed in ultraviolet irradiated CV1 cells. Photochem. Photobiol. 32, 97-99. Dulbecco, R. and M.Vogt (1954) Plaque formation and isolation of pure lines with poliomyelitis viruses. 1. Exp. Med. 99, 167-182. Fogel, M.,K. Yamanischi and F. Rapp (1979) Enhancement of host cell reactivation of UV irradiated Herpes simplex virus by caffeine, hydroxyurea, and 5-bromodeoxyuridine. Inr. J. Cancer 23, 657-662.
Density dependence of enhanced reactivation Harbour, D. A., W. A. Blyth and T. J. Hill (1978) Prostaglandins enhance spread of Herpes simplex virus in cell cultures. J . Gen. Virol. 41, 87-95. Jeeves, W. P. and A. J. Rainbow (1979a) y-Ray enhanced reactivation of UV-irradiated adenovirus in normal human fibroblasts. Murat. Res. 60,33-41. Jeeves, W. P. and A. J. Rainbow (1979b) y-Ray enhanced reactivation of y-irradiated adenovirus in human cells. Biochem. Biophys. Res. Commun. 90, 567-574. Lytle, C. D., S. G. Benane and C. E. Bockstahler (1974) Ultraviolet-enhanced reactivation of herpes virus in human tumor cells. Photochem. Photobiol. 20, 91-94. Lytle, C. D., J. Coppey and W. D. Taylor (1978) Enhanced survival of ultraviolet-irradiated Herpes simplex virus in carcinogen-pretreated cells. Nature 272, 60-62. Lytle, C. D. and J. B. Goddard (1979) UV-enhanced virus reactivation in mammalian cells: effects of metabolic inhibitors. Photochem. Photobiol. 29, 959-962. Montes, J. G. and W. D. Taylor (1986) The effects of ultraviolet light on host cell reactivation and plaque size of Herpes simplex virus Type I in C3WlOTi mouse cells. Photochern. Photobiol. 43, 35-40. Nishiyama, Y. and F. Rapp (1980) Enhanced survival of ultraviolet-irradiated Herpes simplex virus in human cytomegalovirus-infected cells. Virology 100, 189-193. Radman, M. (1980) Is there SOS induction in mammalian
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cells? Photochern. Photobiol. 32, 823-830. Ross, L. J. N., P. Wildy and K. R. Cameron (1971) Formation of small plaques by herpes virus irradiated with ultraviolet light. Virology 45, 808-812. Rossman, T. G. and C. B. Klein (1985) Mammalian SOS system: a case of misplaced analogies. Cancer Invest. 3 , 175- 187. Ryan, D. K. G. and A. J. Rainbow (1986) Comparative studies of host-cell reactivation, cellular capacity and enhanced reactivation of herpes simplex virus in normal, xeroderma pigmentosum and Cockayne syndrome fibroblasts. Mu!. Res. 166, 99-111. Sarasin, A. R. and P. C. Hanawalt (1978) Carcinogens enhance survival of UV-irradiated simian virus 40 in treated monkey kidney cells: induction of a recovery pathway? Proc. Nut. Acad. Sci. U.S.A. 75, 344-350. Taylor, W. D., L. E. Bockstahler, J. Montes, M. A. Babich and C. D. Lytle (1982) Further evidence that ultraviolet radiation-enhanced reactivation of simian virus 40 in monkey kidney cells is not accompanied by mutagenesis. Mutat. Res. 105, 291-298. Witkin, E. M. (1976) Ultraviolet mutagenesis and inducible DNA repair in Escherichia coli. Bacteriol. Rev. 40,869-907. Zurlo, J. and J. D. Yager (1984) U.V.-enhanced reactivation of U.V.-irradiated herpes virus by primary cultures of rat hepatocytes. Carcinogenesis 5 , 495-500.
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CELL DENSITY DEPENDENCE OF ULTRAVIOLET LIGHT ENHANCED REACTIVATION OF Herpes simplex TYPE I AND THE LARGE PLAQUE EFFECT IN C3H/lOT+ MOUSE FIBROBLASTS J. G. MONTES’ and W. D. TAYLOR*~ ‘Department of Biophysics, University of Maryland School of Medicine, Baltimore, MD 21201, USA and *Department of Molecular and Cell Biology, The Pennsylvania State University, University Park, PA 16802, U S A (Received 11 March 1991; accepted 21 June 1991)
Abstract-43HIlOT4 mouse fibroblasts were grown to different cell densities either by plating at low density and allowing different growth periods, or by plating at a series of increasing densities and allowing the same growth period. These plates were UV irradiated at 7.5 J/mZor mock irradiated and 24 h later infected with UV-irradiated Herpes simprex type I virus which had been UV irradiated at 50 or 125 J/m2 or mock irradiated. The numbers and sizes of plaques were measured and these data used to calculate the extent of UV-enhanced host cell reactivation, the capacity enhancement, the large plaque effect (LPE) and the small plaque effect (SME). The influence of cell density on these phenomena was similar for both series of density experiments. Ultraviolet-enhanced host cell reactivation could be demonstrated only for cultures of lower density. The capacity of the cells for Herpes simplex type I virus decreased with cell density, but UV irradiated cells showed an increase in capacity with cell density. Plaque sizes decreased in all cases with cell density but the LPE and SPE were not significantly altered. The greatest variation in the above parameters occurred just as the cells were approaching confluence, where most host cell reactivation experiments are carried out. We conclude that the reproducibility of such experiments depends critically on cell density, a dependence which may be relevant to mechanistic interpretations of the UV-dependent phenomena.
cells (Lytle ef af., 1974) and the mouse fibroblast line C3HhOTf at confluence (Montes and Taylor, Enhanced host cell reactivation ( E R ) t of nuclear1986). The fact that it is possible to induce E R with replicating viruses inactivated with different DNAagents known to inhibit D N A synthesis, such as damaging agents occurs in various mammalian cell cycloheximide, hydroxyurea, caffeine, and bromolines (Bockstahler and Lytle, 1970; Lytle et af., deoxyuridine (Sarasin and Hanawalt, 1978; Fogel et 1974; Bockstahler et al., 1976; Lytle et af., 1978; af., 1979; Lytle and Goddard, 1979), has prompted Sarasin and Hanawalt, 1978; Fogel et af., 1979; the hypothesis that E R is induced by the delay of Jeeves and Rainbow, 1979a,b). In E R , survival of D N A synthesis, whether produced by agents affectinactivated virus is greater on cells that have been ing D;IA polymerases, or by damaged D N A itself pretreated with certain DNA-damaging agents than (Lytle and Goddard, 1979). The search for a model on untreated cells. This phenomenon has been likexplaining E R in general has been complicated ened to Weigle Reactivation, one of several “SOS” additionally by the finding that a biological agent, responses seen in bacteria (Witkin, 1976; Radman, human cytomegalovirus, can induce E R (Nishiyama 1980); it has been argued whether E R and “SOS” and Rapp, 1980). are caused by a common mechanism (Rossman and Enhanced host cell reactivation occurs in associKlein, 1985). Ultraviolet light is one DNA-altering ation with other phenomena, some of which may agent which has been given particular attention as be useful in explaining its nature. These include the an inducer of ER. small plaque effect (SPE), the large plaque effect Ultraviolet light-enhanced reactivation (UVER) (LPE), and an increase in the capacity of monoof Herpes simplex type I virus (HSV-1) occurs in layers to allow formation of viral plaques, herein most systems studied thus far, although notable referred to as capacity enhancement, or CE. In the exceptions exist, including human embryonic lung SPE plaque size is reduced by treatment of viruses with DNA-damaging agents before infection of cells; this phenomenon has been observed with Her*To whom correspondence should be addressed. tAbbreviutions: CE, capacity enhancement; DPBS, Dul- pes virus in various mammalian cell lines (Ross et becco’s phosphate buffeted saline; ER, enhanced reacti- al., 1971; Cameron et af., 1979; Fogel et af., 1979; vation; HSV-1, Herpes simplex virus, type 1; LPE, large Coohill et al., 1980). The LPE results in the proplaque effect; RF, reactivation factor; SEM, standard error of the mean; SPE, small plaque effect; UVER, duction of larger plaques on host cells pretreated with such diverse agents as prostaglandin (Harbour ultraviolet enhanced reactivation. INTRODUCTION
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J . G. MONTESand W. D. TAYLOR
et a f . , 1978), cycloheximide (Babich et a f . , 1981), and U V light (Babich et a f . , 1981; Montes and Taylor, 1986). Some time ago. we reported on the significant increase in plaque number (CE) which occurs when mouse embryo fibroblast cells (C3WlOTt) are treated with U V light shortly before inoculation with HSV-1 (Montes and Taylor, 1986). In that paper we concluded that absence of E R in a particular cell line did not necessarily preclude the presence of the LPE and the SPE. One of the obvious results of irradiating monolayers with U V light is the decline in the number of attached cells that can be attributed to cell death, thereby causing a reduction in cell density. This observation brings into question the direct o r indirect role of cell density in U V E R , as well as in the LPE and C E ; previous studies using HSV-1 on other types of cells indicate, for example, that U V E R depends on age of confluent cultures (Zurlo and Yager, 1984) and that U V E R may only be exhibited by cells undergoing DNA synthesis (Ryan and Rainbow, 1986). We therefore carried out experiments in which cell density was varied by either seeding density or culture age, in order to determine directly the possible role of cell density on these phenomena. A hypothetical explanation is proposed that may account for the failure to observe LJVER under certain conditions in C3H/lOTi and in perhaps other cell lines. MATERIALS AND METHODS
Virus stocks. Herpes simplex virus type 1. MP (Macroplaque strain), was used to infect cells. Stocks were prepared by infecting freshly-confluent monolayers of CV1-P (African green monkey kidney) cells. Inoculation with virus was performed in Dulbecco's phosphate-buffered saline (DPBS) (Dulbecco and Vogt, 1954) at a multiplicity of infection between 0.001 and 0.01 as calculated for the unirradiated virus. Culture medium and growth conditions for CVl-P are detailed elsewhere (Taylor er al., 1982; Montes and Taylor, 1986). Host cell culrure. C3HIlOTd mouse embryo fibroblasts were cultured as described previously (Montes and Taylor, 1986). Experiments were performed at passages 32-37. and cells were passaged every 8-12 days. Pretreatmenr irradiation of cells. Cell monolayers were irradiated as described previously (Montes and Taylor, 1986). Total dose to the cells was either 0 Jlm2 (mockirradiation) or 7.5 Jim'; the latter dose was selected because of optimum effects seen previously at that dose (Montes and Taylor. 1986). Irrudiarion of virrcs. Virus was irradiated in uncovered 100 mm tissue culture dishes under a GE G&T5 lamp as described (Montes and Taylor, 1986). Total dose to virus was either SO or 125 Jim'. Reucrivation experimenrs. The surviving fraction of irradiated virus was measured on unirradiated cells, S,,, and UV-irradiated cells, S,. The reactivation factor, RF. was calculated as: RF = S , / S , , , (Bockstahler er a / . . 1976). The effect of irradiation of cells on their capacity to support the growth of unirradiated virus (the CE) was measured by comparing the number of plaques on irradiated plates with the number on unirradiated plates. PQque number and size assays. Plaques were counted in monolayers stained and dried as described in our previous report (Montes and Taylor. 1986). All cultures, regardless
of condition or protocol, were terminated and stained exactly 4 days after inoculation with virus; from previous trials bracketing the range of conditions used in this study it was found that further increases in incubation time did not result in any significant changes in plaque number. Plaque size (area) was determined by projecting the image of the cellular layer of stained dishes onto a sheet of graph paper (Ross er a l . , 1971). Control of cell monolayer densities. In these experiments, one of two different approaches was used to attain the resulting densities: (A) seeding dishes at different cell densities on the same day and then allowing the same time (2, 3, or 4 days) before irradiation or mock-irradiation with UV light; or (B) seeding all dishes on the same day at 4.5 x 104 cells per dish (plating efficiency 32-38%; about 1.5-1.7 x 104 cells adhered) and beginning reactivation experiments at different times (4,5,6,7, or 8 days) thereafter. In approach (A) the actual number of cells that attached was not determined for seeding densities greater than 5 x 105/dish,although for lower densities the plating efficiency remained at about 32-38% (see Table 1). In both approaches, the pretreatment UV light doses were 0 or 7.5 J/m2. In the first approach. virus was inactivated with 125 Jim', whereas 50 J/mZwas used in the second. The rationale for using two different approaches to obtain different cell densities was based on the observation that monolayers attaining confluence immediately, by seeding at very high density, had final cell densities different from those that reached confluence starting from lower seeding densities (see Discussion). It was not known if cells that become quiescent via different routes of initial seeding density and of time allowed to reach confluence would possess properties of importance to the study. Cell densities were measured for both types of experiments at the time of irradiation of the cells and at the time of inoculation (UV-irradiation temporarily suppressed growth of cultures). All densities were found by detaching cells with 0.05% trypsin + 0.016% EDTA in DPBS and then counting the cells in suspension by using a Neubauer hemacytometer (American Optical, Buffalo, NY). RESULTS
Density studies: dishes seeded at different cell densities on the same day Cultures were started by seeding at different cell numbers per 60 mm dish (range: 1.6 x lo4 to 1.2 x 10" cells per dish; actual number of cells that attached was significantly less because of a 32-38% plating efficiency). In Figs. 1-3, panels (a), (b) and (c) correspond to experiments in which the cells were mock irradiated or irradiated with 7.5 Jlm2 of U V light 2 days (a), 3 days (b) or 4 days (c) after plating. The time allowed between seeding and pretreatment resulted in further differences in cell density. Table 1 provides data on population densities of cells seeded, as well as the numbers of cells attached to dishes at critical times during the experiments. It was seen that U V light resulted in decreases in density as measured 24 h after irradiation of the cells, with the effect being more prominent at lower densities. Seemingly paradoxical was the confluent or nearly confluent condition of some of the monolayers at densities lower than usual for confluence, arising from seeding at high density. Microscopic observation verified that the
Density dependence of enhanced reactivation
215
Table 1. Correlation of C3WlOTf cell density at time of UV-irradiation of cells and viral inoculation with number of cells initially seeded Number of cells x lO-Vdish
Day of irradiation of cells
On day of viral inoculation On day of seeding
On day of irradiation
2
0.40 f 0.04 0.75 ? 0.08 2.00 f 0.20
~
Unirradiated
Irradiated
0.97 f 0.14 2.20 f 0.24 3.30 f 0.18
2.90 f 0.25 4.10 ? 0.14 5.80 f 0.19
1.60 rt 0.17 2.60 ? 0.31 4.20 2 0.25
3
0.50 f 0.05 1.60 t 0.16 3.00 f 0.30 6.00 f 0.60 12.00 f 0.12
2.30 f 0.15 4.90 & 0.37 6.40 f 0.10 6.20 f 0.40 6.40 ? 0.26
6.0 t 0.26 10.00 t 0.90 11.00 ? 1.10 7.70 2 0.62 7.40 f 0.48
2.60 f 0.10 4.40 f 0.20 7.00 ? 0.48 6.80 ? 0.67 7.00 f 0.10
4
0.35 f 0.04 1.20 2 0.06 2.00 f 0.20 8.00 f 0.04 12.00 f 1.20
1.80 f 0.23 4.60 ? 0.48 6.90 f 0.68 7.00 2 0.74 6.30 t 0.33
4.70 f 0.41 8.10 2 0.38 15.00 ? 0.35 8.80 ? 0.46 6.50 f 0.75
1.80 0.12 6.20 f 0.27 8.10 2 0.41 7.20 2 0.36 5.80 rt 0.64
"
Cells were either UV-irradiated (7.5 J/m2) or mock-irradiated 1 day before inoculation with UV-irradiated (125 J/m2) or unirradiated HSV-1. Day = 0 corresponds to day of seeding. Data correspond to experiments of [Figs. l(a)] (in part), [l(b)], and [l(c)], and [Fig. 2(a)] (in part). Results are based on at least 4 independent hemacytometer counts from 1 sacrificed dish (or from seeding stock, for second column) per condition.
1 2 3 4 5 6 7 8 9 D l l l Z NUMBER OF CELLS SEEDED (CELLS 1 lO-'lDlsn)
Figure 1. Survival of HSV-1 on C3H/lOTI cells as a function of different seeding densities. Cells were mockirradiated (0)or pretreated with 7.5 J/m7 UV light (A); virus were inactivated with 125 J/m2 UV light. Inoculation with virus took place 24 h after pretreatment of cells. Surviving fraction for cells treated 2 days after seeding (performed at only 2 different population densities) (a), 3 days after seeding (performed at 5 population densities) (b), or 4 days after seeding (performed at 5 population densities) (c). (Each panel represents an experiment performed in duplicate. Standard deviation is displayed with error bars; points without error bars are not significantly different statistically from each other.)
lack of increase in cell density for cells seeded at the high densities of 6, 8, or 12 x lo5 per dish correlated with the fact that these higher starting densities ultimately resulted in cells that were larger than those found in confluent dishes plated in the normal way, 1.e. starting with about 5 x lo4 cells/ dish; in this way, contact inhibition could occur at a lower cell density. Clearly, the cell density at confluence is a function of the density at which the cells are plated. Virrrs survival. The survival of virus inactivated with 125 J/m2 UV light depends to a large extent on the number of cells seeded. Reference to Table 1 will indicate that at or near confluence survival was relatively low and within a narrow range of variation, regardless of the population density at which confluence finally occurred. Therefore, when 6 x lo5 cells per dish or more were used, the results were essentially the same, with surviving fractions of between 1and 2 x At lower seeding densities, however, survival was greater (up to more than 8x depending on seeding density and days after seeding that cells were infected), gradually declining from its highest point as the number of cells seeded increased. At the lower seeding densities (1.6 X 104 to about 3 X 105 cellddish) viral survival was almost always higher when the host cells had been pretreated with 7.5 J/m2 of UV light than in controls (i. e. UV-enhanced reactivation occurred according to the mathematically operational definition of enhanced reactivation, that is, surviving
J. G. MONTES and W.D. TAYLOR
216 1
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Figure 2. Capacity of C3WlUTt cells for HSVl as a function of different seeding densities. All titers (i.e. plaque numbers) were normalized to maximum titer measured on unirradiated cells for each set. Cells were either mockirradiated (0)or exposed to 7.5 J/m2 UV light (A). Inoculation with unirradiated virus took place 24 h after treatment of cells. Relative capacity for cells that were treated 2 days after seeding (a), 3 days after seeding (b), or 4 days after seeding (c). (Statistical information provided as in Fig. 1 . ) fraction of irradiated virus on irradiated cells/surviving fraction of irradiated virus on unirradiated cells > unity); although the evidence for UVER was statistically significant for the very lowest seeding densities in Fig. 1 (calculations not shown), the RF never exceeded 1.5. At confluence or near confluence the RF was less than unity. The possible relevance of this observation to the mechanisms responsible for enhanced reactivation are considered below in the Discussion. Capacity enhancement. Maxima in plaque yields occurred (Fig. 2) for unirradiated cells when dishes were seeded at 1-2 x 10’ cells per dish and inoculated with unirradiated virus 2, 3 or 4 days after seeding (i.e. mock-irradiated 1, 2 or 3 days after seeding, respectively). At seeding densities below 6 x lo5 cells per dish (resulting in confluence or near-confluence 1 day later), plaque yields were lower on irradiated cells (7.5 J/mZ) than on corresponding unirradiated cells. A crossover of the plaque yield curves for treated and untreated cells occurred above 4 x los cells per dish. It is interesting that the maximum plaque yield obtained for pretreated cells was roughly equal to the maximum observed in control cells, even though the cell seeding densities corresponding to maximum plaque number were very different for the two plaque yield curves. Plaque sizes. Plaque size (Fig. 3) was greatest for cells seeded at the lowest densities, regardless of treatment to cells (7.5 J/mZ) or virus (125 J/mZ);
Figure 3. HSV-1 plaque size as a function of seeding density. Cells were mock-irradiated (0.0) or exposed to 7.5 J/mZ( A , A ) of UV light before inoculation with unirradiated virus (A,O) or UV-irradiated (125 J/mZ) virus (A,.) 24 h later. Plaque sizes were measured for cells seeded 3 days (a), or 4 days (b) before irradiation or mock-irradiation. (Error bars show standard deviation for duplicate experiments; points without error bars are not significantly different statistically from points immediately below or above them.) with increase in the number of cells seeded, plaque size declined, reaching essentially constant values for cultures infected 4 days [Fig. 3(a)], or 5 days after seeding [Fig. 3(b)]. Irradiation of virus generally resulted in smaller plaque sizes for both irradiated and unirradiated cells within the time allowed for plaque development; pretreatment of cells resulted in larger plaques for both irradiated and unirradiated virus. It is notable that the LPE remained constant at about 2 regardless of density at time of irradiation of the cells.
Density studies: dishes seeded at sanie initial density but treated on different subsequent days In these experiments cells were allowed to divide from a common starting population density (4.5 x lo4cells seeded, about 1.5 x lo4 successfully plated), the desired variation in population density being obtained by pretreatment on different days after seeding. The growth curve for the cells in these experiments is shown in Fig. 4. Thus, it is possible to match a value on the abscissa (days after seeding) in Figs. 5-7 to a corresponding cell density on the ordinate of Fig. 4. Survival of virus. It was evident (Fig. 5) that the survival of irradiated virus (50 J/m2) decreased with age of cultures, i.e. with increasing density (it should be noted that HSV-1 survival on C3H/lOTi monolayers is non-linear on a semi-log plot, and may in fact be multi-component; see Montes and Taylor, 1986). Only in cultures pretreated 4 days after seeding (i.e. inoculated 5 days after seeding) was the surviving fraction in pretreated cultures (7.5 J/mZ)
217
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Figure 4. Growth curve for C3H/lOTt cells. Dishes were seeded with 4.5 X 104 cells each (= 1.5 X 104 successfully plated) on Day 0. Open circles indicate number of attached cellsldish at different times. Triangles indicate number of attached cellddish 24 h after exposure of cells to 7.5 J/mZ of UV light. (Shown are the average results from 3 experiments; error bars indicate standard error of the mean (SEM) for unirradiated cells; SEM = 6.4 lr: 4.6% for triangles.)
higher than in non-pretreated cultures, and then by a small margin; reactivation factors for older cultures were below unity, i.e. no evidence could be found for UVER in older cultures (Fig.5, right
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Figure 5. Survival and reactivation factor for HSV-1 on C3WlOT4 cells as functions of time after seeding. Dishes were seeded with 4.5 x 104 cells each (= 1.5 x 104 successfully plated) on Day 0. Cells were mock-irradiated (0)or irradiated with 7.5 J/m2 ( A ) of UV light 24 h before inoculation with UV-irradiated (50 J/m2) virus on the days indicated. The corresponding reactivation factors are also given ( x ) (the dashed line was fit “by eye” and is suggestive only). (Each point represents the results of 3 separate experiments; error bars indicate SEM.)
W
a
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I I 1 5 6 1 DAYS AFTER SEEDING
I 1
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Figure 6. Relative plaque number and capacity enhancement of HSV-1 in C3H/lOTi cells as functions of time after seeding. Dishes were seeded with 4.5 x 104 cells each (= 1.5 x 104 successfully plated) on Day 0. Cells were mock-irradiated (0)or exposed to 7.5 J/m2 ( A ) of UV light 24 h before inoculation with virus on the days indicated. All plaque numbers were normalized to the maximum number observed on unirradiated cells in order to obtain relative plaque numbers. Capacity enhancement ( x ) was calculated by dividing plaque number for irradiated cells by the number for unirradiated cells. (Each point represents the results of 3 separate experiments; SEM are shown with error bars.)
margin). It was seen that only at 4 days after seeding (cell density = 9.5 x lo5 cells/dish) were cultures still in exponential growth (Fig. 4). Capacity enhancement. It was found that in control cultures, relative capacity, calculated by dividing the average plaque number for each density by the average number of plaques observed on day 4, declined with age of cultures, with most obvious differences occurring between days 6 and 7 (Fig. 6). However, for pretreated cultures (7.5J/m*) a welldefined maximum occurred at 6 days. As in Fig. 2, the highest titer (i.e. plaque number) measured on irradiated monolayers was not significantly different from the maximum achieved on unirradiated monolayers. It is interesting that the CE (obtained by dividing plaque number for pretreated cultures by plaque number for non-pretreated controls) nonetheless vaned as a smooth sigmoidal function (Fig. 6, right margin). Plaque size. Plaque size declined with days after seeding (i.e.cell density) regardless of whether cells, virus or both, received radiation (Fig. 7). Pretreated cultures yielded larger plaques than control cultures, for both irradiated and unirradiated virus. The LPE was relatively constant for unirradiated virus, between 1.5 and 2.0; the LPE for irradiated virus, however, was considerably lower and not as constant. Smaller plaques were obtained with irradiated virus (SPE) on both control and irradiated cells.
J. G. MONTES and W. D. TAYLOR
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DAYS AFTER SEEDING
Figure 7. Plaque size of HSV-1 on C3HIlOTI cells as a function of time after seeding. Dishes were seeded with 4.5 x 104 cells each (= 1.5 x 104 successfully plated) on Day 0. Cells to be inoculated with unirradiated virus were mock-irradiated (0)or exposed to 7.5 J/mz ( A ) of UV light on days indicated; cells to be inoculated with UVirradiated (50 Urn2) virus similarly were mock-irradiated (0)or exposed to 7.5 J/mZ (A) of UV light on days indicated. Inoculation took place 24 h after day indicated.
(Each point represents results of 3 separate experiments; error bars show SEM). These results are qualitatively similar to those found in the first study. DISCUSSION AND CONCLUSIONS
It is clear from these data that important parameters of UV radiation effects on the HSV-1C3WlOTI system depend strongly on the cell density, especially as confluence is approached. In these two series of experiments in which cell density was varied in two different ways, qualitatively similar results were obtained for the three kinds of measurements made. In the first kind (Figs. 1 and 5), the surviving fraction of irradiated virus declined with cell density on both irradiated and control cultures. The RF calculated from these data also declined from somewhat more than unity to less than unity. In the second kind of measurement, in which the capacity of cell cultures to support the growth of unirradiated virus was measured, opposite cell density effects were seen (Figs. 2 and 6) for control vs irradiated cultures. In control cultures the capacity declined with increasing density; in irradiated cultures the capacity increased with increasing cell density. This could arise if quiescent cells were less susceptible to viral infection and if in irradiated monolayers sufficient cells were killed to produce an apparent displacement to lower densities. In the third kind of measurement, of plaque sizes, the results of both series of experiments coincided; all plaque sizes declined with cell density. However, the SPE and LPE appeared to be relatively insensitive to cell density.
Comparing the 2 series of experiments, one can reach the conclusion that the growth state of the cells is the important parameter, rather than the absolute cell monolayer density. For example, the RF, often used as evidence for the possible induction of repair processes, depends strongly in this system on the cell density just as the cells reach confluence (see Fig. 4), which is where most studies are carried out. An interesting possibility that gives UVER (or perhaps other forms of ER) a novel explanation is that UV light applied to a confluent or nearly confluent culture may result in a dosedependent shift of the growth state of cells in the culture. In Figs. 1 and 5 it is clear that the virus surviving fraction decreases considerably at confluence whether cells are pre-irradiated or not. The result of UV pre-irradiation of the cells may be simply to break contact inhibition among them via cell death or damage, so that a new culture of effectively lower cell density is created. In effect, calculation of a RF (surviving fraction on irradiated cells divided by surviving fraction on unirradiated cells) may be equivalent to comparing the surviving fraction of virus for a confluent culture (i.e. lower survival) with that for a culture of lower density (i.e. higher survival), even though experimentally both cultures started at confluence. This explanation would suggest that whether UVER can be demonstrated may depend, at least partially, on the suscep tibility of a given cell type to the lethal effects of UV light and the UV dosing regimen used. Thus, it is important to control more precisely the growth state of cells in these kinds of experiments in order to obtain data that are both reproducible and more informative. REFERENCES
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Density dependence of enhanced reactivation Harbour, D. A., W. A. Blyth and T. J. Hill (1978) Prostaglandins enhance spread of Herpes simplex virus in cell cultures. J . Gen. Virol. 41, 87-95. Jeeves, W. P. and A. J. Rainbow (1979a) y-Ray enhanced reactivation of UV-irradiated adenovirus in normal human fibroblasts. Murat. Res. 60,33-41. Jeeves, W. P. and A. J. Rainbow (1979b) y-Ray enhanced reactivation of y-irradiated adenovirus in human cells. Biochem. Biophys. Res. Commun. 90, 567-574. Lytle, C. D., S. G. Benane and C. E. Bockstahler (1974) Ultraviolet-enhanced reactivation of herpes virus in human tumor cells. Photochem. Photobiol. 20, 91-94. Lytle, C. D., J. Coppey and W. D. Taylor (1978) Enhanced survival of ultraviolet-irradiated Herpes simplex virus in carcinogen-pretreated cells. Nature 272, 60-62. Lytle, C. D. and J. B. Goddard (1979) UV-enhanced virus reactivation in mammalian cells: effects of metabolic inhibitors. Photochem. Photobiol. 29, 959-962. Montes, J. G. and W. D. Taylor (1986) The effects of ultraviolet light on host cell reactivation and plaque size of Herpes simplex virus Type I in C3WlOTi mouse cells. Photochern. Photobiol. 43, 35-40. Nishiyama, Y. and F. Rapp (1980) Enhanced survival of ultraviolet-irradiated Herpes simplex virus in human cytomegalovirus-infected cells. Virology 100, 189-193. Radman, M. (1980) Is there SOS induction in mammalian
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cells? Photochern. Photobiol. 32, 823-830. Ross, L. J. N., P. Wildy and K. R. Cameron (1971) Formation of small plaques by herpes virus irradiated with ultraviolet light. Virology 45, 808-812. Rossman, T. G. and C. B. Klein (1985) Mammalian SOS system: a case of misplaced analogies. Cancer Invest. 3 , 175- 187. Ryan, D. K. G. and A. J. Rainbow (1986) Comparative studies of host-cell reactivation, cellular capacity and enhanced reactivation of herpes simplex virus in normal, xeroderma pigmentosum and Cockayne syndrome fibroblasts. Mu!. Res. 166, 99-111. Sarasin, A. R. and P. C. Hanawalt (1978) Carcinogens enhance survival of UV-irradiated simian virus 40 in treated monkey kidney cells: induction of a recovery pathway? Proc. Nut. Acad. Sci. U.S.A. 75, 344-350. Taylor, W. D., L. E. Bockstahler, J. Montes, M. A. Babich and C. D. Lytle (1982) Further evidence that ultraviolet radiation-enhanced reactivation of simian virus 40 in monkey kidney cells is not accompanied by mutagenesis. Mutat. Res. 105, 291-298. Witkin, E. M. (1976) Ultraviolet mutagenesis and inducible DNA repair in Escherichia coli. Bacteriol. Rev. 40,869-907. Zurlo, J. and J. D. Yager (1984) U.V.-enhanced reactivation of U.V.-irradiated herpes virus by primary cultures of rat hepatocytes. Carcinogenesis 5 , 495-500.
