The Wavelength Dependence of Ultraviolet Enhanced ... · of CV-l cells irradiated at 265 nm to...

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Western Kentucky University TopSCHOLAR® Masters eses & Specialist Projects Graduate School 4-1978 e Wavelength Dependence of Ultraviolet Enhanced Reactivation in A Mammalian Cell-Virus System Leslie C. James Follow this and additional works at: hp://digitalcommons.wku.edu/theses Part of the Biology Commons , Immunology and Infectious Disease Commons , and the Virus Diseases Commons is esis is brought to you for free and open access by TopSCHOLAR®. It has been accepted for inclusion in Masters eses & Specialist Projects by an authorized administrator of TopSCHOLAR®. For more information, please contact [email protected]. Recommended Citation James, Leslie C., "e Wavelength Dependence of Ultraviolet Enhanced Reactivation in A Mammalian Cell-Virus System" (1978). Masters eses & Specialist Projects. Paper 1705. hp://digitalcommons.wku.edu/theses/1705

Transcript of The Wavelength Dependence of Ultraviolet Enhanced ... · of CV-l cells irradiated at 265 nm to...

Page 1: The Wavelength Dependence of Ultraviolet Enhanced ... · of CV-l cells irradiated at 265 nm to reactivate irradiated (500 J m-2) HSV-l The virus reactivation curves for cells irradiated

Western Kentucky UniversityTopSCHOLAR®

Masters Theses & Specialist Projects Graduate School

4-1978

The Wavelength Dependence of UltravioletEnhanced Reactivation in A Mammalian Cell-VirusSystemLeslie C. James

Follow this and additional works at: http://digitalcommons.wku.edu/theses

Part of the Biology Commons, Immunology and Infectious Disease Commons, and the VirusDiseases Commons

This Thesis is brought to you for free and open access by TopSCHOLAR®. It has been accepted for inclusion in Masters Theses & Specialist Projects byan authorized administrator of TopSCHOLAR®. For more information, please contact [email protected].

Recommended CitationJames, Leslie C., "The Wavelength Dependence of Ultraviolet Enhanced Reactivation in A Mammalian Cell-Virus System" (1978).Masters Theses & Specialist Projects. Paper 1705.http://digitalcommons.wku.edu/theses/1705

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THE WAVELENGTH DEPENDENCE OF ULTRAVIOLET ENHANCED

REACTIVATION IN A MAMMALIAN CELL-VIRUS SYSTEM

A Thesis

Presented to

the Faculty of the Department of Biology

Western Kentucky University

Bowling Green, Kentucky

In Partial Fulfillment

of the Requfrements f or the Degree

Master of Science

by

Leslie C. James

April, 1978

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AUTHORIZATION FOR USE OF THESIS

ermission is hereby

I'V1 granted to the Western Kentucky University Library to lAJ. make , or allow to be made photocopies, microfilm or other

copies of this thesis fo r appropriate research or scholarly purposes.

O reserved to the author for the making of any copies of this the sis except for brief sections for re search or scholarly purposes .

'" (I;{ ..... ,a-

7 '-.,.- { Signed ./ t , (....... (;, , ...... /

Date .. j b ~ /-1 <; ~, f

Please place an " X" in the appropriate box.

Thi s Corm will be Ciled with the original of the thesis and will control future use of the thesis.

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-"~J l . ..

Dean Of the

THE WAVELENGTH DEPENDENCE OF ULTRAVIOLET ENHANCED REACTIVATION IN A MAMMALIAN CELL-VIRUS SYSTEM

Graduat . COllege

Recommended --_-f:0::!-0C7'/_7~ / 7,...::d,--/ __ (Date)

~~ 7?~tf2 - Director of Thesis _

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ACKNm~LEDGMENTS

I would like to t hank Dr. Fernando Morgado and Dr.

Larry Elliott for serving on my committee and for their

gu idance during my graduate program.

I would a lso like to t hank the excellent student

worker staff for their competence and cheerfulness in

performing the routine tasks which were necessary for the

completion of this project .

Special thanks go to my fellow workers in the Bio­

physics - Virology lab . Mr. Bobby Cobb, Mr. Richard Detsch

and Mr . Ed Ryan for always being available for irradiations,

and Mr . Daniel Knaue r and Mr. Dennis Fry for assisting in

experiments all deserve special consideration .

Very special thanks go to Ms. Sharon Moore for her

daily assistance in this research pro j ect , and also r

the typing of this manuscript .

Lastly, I would like to thank Dr . Thomas P. Coohill

for t eaching me how to do research , and for encouraging

and supporting me during al l of my research and academic

endeavors.

iii

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ACKNOWLEDGMENTS

LIST OF TABLES .

LIST OF FIGURES

TABLE OF CONTENTS

INTRODUCTION AND LITERATURE REVIEW

MATE RIALS AND METHODS

RESULTS

DISCUSSION

BIBLIOGRAPHY

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LIST OF TABLES

Table 1 . Fluence rate ranges for monochromatic expOsures to cells and the slope of the averages RER responses .. . ..

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Figur e 1 .

Figur e 2.

Figure 3 .

Figu re 4.

Figure 5.

Figure 6 .

LIST OF FIGURES

The effect of 265 nm radiation on the capacity of CV-l cells to SUpport the growth of either unirradiated ((), ~ ) or irradiated (500 J m- 2 ) ( .. , A ) HSV- l . . . . . . . . . . . . .

The effect of 235 nm radiation on cell capaci ty for unirrad~ated ( () or irr ad i ated (500 J m- ) ( .. ) virus . .

The effect of 302 nm radiation On cell capaci ty for unirradiated ( () or irradiated (500 J m- 2 ) virus .. .. .

The reactivation curves for the ability of CV- l cells irradiated at 265 nm to reactivate irradiated (500 J m-2) HSV-l

The virus reactivation curves for cells irradiated at 235 nm . . . . . . .

The virus reactivation curves for cells irradiated at 302 nm . . . . . . . . .

Figure 7 - 11. The average reactivation factors for RER of HSV- l by CV-l cells at different wavelengths . . . .

Figure 12. An action Spectrum for RER plotted as the slope (quantum corrected) of the lines from Figures 7 t h rough 11. An absorption spectrum for DNA is included (Jagger , 1967) ...... . . . ... .

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THE WAVELENGTH DEPENDENCE OF ULTRAVIOLET ENHANCED

REACTIVATION IN A ~~LIAN CELL-VIRUS SYSTEM

Leslie C. James April , 1978 2 7 pages

Di r ected by: Dr . Thomas P . Coohill, Dr . Larry Elliott , Dr . Fernando Morgado

Department of Biology Western Kentucky University

The effect of ultraviolet (UV) radiation in t he wavelength

region 230 nm to 30 2 nm on the ability of an irradiated

mammalian cell to reactivate UV-irradiated mammalian virus

was tested . An action spectrum for radiation enh anced reacti-

po i nts to a combined nucleic acid-protein target for UV

vation (RER) is presented . The shape of the action spectrum

biological events involved in RER does not a llow the identi-

of the results of others involving the biochemical and photo-

radiation effects on this cellular parameter. An analysis

to this effect . Studies involving an analogous phenomenon

fication of the macromolecule which is the major contL b uto r

involve similar mechanisms .

in bacteria (Weigle reactivation) imply that RER and NR may

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INTRODUCTION AND LITERATURE REVIEW

Today our environment contains many artificial light

sources. Increasingly, interest has focused on t h e effects

of these sources on biological cells and mol~cules . The wave­

length s which impart the most damage to cells are those in

the ultraviolet (UV) region. Therefore, UV-light is an

important environmental agent , and can be used as an analytical

tool to modify selective biological events. Several authors

have reported on the effects of ultraviolet light on several

mammalian cell parameters, for example : DNA replication

(I-Ihitfield, et al., 1961; Powell, 1962; Cleaver, 1965;

Rasmussen and Painter, 1966; Cleaver, 1967), mutation (Rao

and Puck , 1970; Trosko and Chu, 1971), chromosomal damage

(Chu, 1965) and cell death (Todd, et a l , 1968) . If the

effect under investigation is studied at a sufficient number

of wavelengths, an action spectrum of e f fect as a function of

wavelength can be plotted . A comparison of this spectrum a nd

the absorption spectra of important biological molecules can

help identify the target molecu le (or molecules) responsible

for the studied response (Jagger, 1967).

An action spectrum has recently been published for the

effect of UV-light on mammalian host cell capacity (Coohill,

et al ., 1977). Cellular capacity is defined by Benzer and

Jacob (1953) as the ability of a cell to support the growth

of a particular virus. This cellular function is sensitive

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to UV-radiation, irradiation of the cell Usually decreasing

its capacity to suppo rt viral growth (Anderson , 1948; Powell,

1 959; Bockstahler and Lytle, 1970; COppey, 1971). If the

virus was also UV- damaged and then allowed to infect UV-

irradiated cells, the expected result would be a furthe r

decrease in capacity , but this is not always the case.

I~hile investigating other phenomena in the Escherichia

coli-phage l a mbda system, Weigle (1953) made a surprising -observation: the survival of UV- irradiated phage lambda

increased if the host cells (or phage - bacterium complexes)

were also irradiated . If the bacteria were irradiated before

phage adsorption , the viral reactivating ability of the cells

was retained for several days . Also , there was a higher

proportion of mutants among reactiva ted phages than among non -

reactivated phages . Among these mutants , no reversions to

Dulbecco, 195 3) . the parent type were observed (Weigle, 1953; I~eigle and

Since the discovery of Weigle reacti vation I~R has been

demonstrated in several phage - bacterial systems (Rupert and

Harm , 1966). an analogous phenomenon has been more reCently

reported in mammalian cells (Bock stahler and Lytle, 1970;

Hellman , ~ ~., 1974; Lytle, ~ al. , 1974; Lytle and Benane ,

1975; BOCkstahler, ~ al ., 1976; Hellman , ~ al . , 1976; Lytle,

~ al . , 1976; Lytle, 1977) involving the reactivation of UV-

irradiated mammalian viruses grown in irradiated mammalian

cells . The term "radiation enhanced reactivation " (RER) has

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been recommended to describe this phenomenon in mammalian

cells since the molecular eVents that produce this reaction

may not be identical to those that produce Weigle reactivation in bacteria (Lytle, 1977).

3

No single theory has been accepted to eXplain the mechanisms

of WR and HER, but many researChers are in agreement that a Uv­

inducible repair process is responSible for both phenomena

(Devoret, 1973; Sedgewick, 1975; Lytle, 1977). This inducible

repair process was termed SOS repa ir by Radman (1974), the

term "SOS" implYing that DNA damage initiates its expression.

This mechanism is conSidered to be error-prone because its

inducement generates a large number of mutants. The induci_

bility of SOS repair indicates that protein synthesis is

required for its presence. Lytle (1972, 1977) has Shown that

HER is absent in mammalian cells treated With . ' otein syntheSis

inhibitors, implYin g the role of a protein in e nhanced reacti_

Vation. In add~tion , a delay of several days between cell

irt"adiatjon and vira l inoculation USually increases the

amount of HER observed in many mrunrnalian cells (BoCkstahler,

~ ~., 1976). These data indicated that the mec hanism of

enhanced reactivation is ind ucible and long-lived.

WR (dnu possibly also HER) has been interpreted by Lytle,

~ al. (1976) to be the result of the cell's response to DNA

d a mage which remains after eXcision repair, or to that damag",

Which remains in the absence of eXcision repair. Excision

repair is the mechanism which is responsible for the remova l

of Pyrimidine dimers . Pyrimidine dimers are the major cause

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of damage to cells due to UV-light. RER and ~IR are both

independent of excision repair. The UVR genes controllinq

excision repair in E. coli are not required for expression of

WR (Witkin, 1974). A similar finding has been made in the

exci sion repair deficient cells of xeroderma pigmentosum

patients in that the absence of excision repair does not in­

hibit the cells' expression of RER (Lytle, et al., 1976).

4

Finally , RER is present only for nuclear replicating

viruses . This suggests that the c e llular mechanisms responsible

for reactivation are located in the cell nucleus (Bockstahler

and Lytle , 1977).

The implications of the phenomena RER and IvR to the

study of mutation, virus activation , and carcinogenesis have

been recently stated by Devoret, et al . (1976) and Witkin

(1976). In fact, they have suggested that all three phenomena

are controlled by the same, or a closely related, mechanism

which is induced by UV-light .

In an attempt to elucidate the mech anism involved in

radiation enhanced reactivation in mammalian cells , experiments

were conducted involving the UV-wavelength (230 nm to 302 nm)

dependence of the ability of irradiated monkey kidney cells

to enhance the survival of UV-irradiated herpes simplex virus.

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MATERIALS AND METHODS

Cell Cultures

A permanent line of African green monkey kidney cells

(CV- l) was obtained from Dr . L . E . Bockstahler of the Bureau

of Radiological Health, Rockville, Maryland 20852. These

cells were maintained in a growth medium consisting of lX

Minimum Essential Medium (Eagle's) with the following

components added per liter: 10 % fetal calf serum (Reheis

Chemical Co., Kankakee, Illinois 60901) 0 . 25 g L-glutamine,

16 ml of SOX amino acids, 8 ml of 100 X vitamins, 80,000 units

penicillin, 0 . 08 g streptomycin (International Scientific

Industries , Cary , Illinois 60013), 100,000 units mycostatin

(E . R. Squibb, Princeton , New Jersey 08450) and buffered to

pH 7.5 with 0.3 g sodium bicarbonate. Cel ls were incubated

in closed flasks or in petri dishu i n clo sed ( a pproximately

5 % CO2 ) chambers at 37oC.

Virus Assay: Plaque Forming Ability (pfa)

A macroplaque strain of HSV-l ( Herpes hominus) was

obtained from Dr. C. D. Lytle of he Bureau of Radiological

Health , Rockville , Marylan d 20852. The virus was inoculated

into confluent monolayers of CV-l cells, harvested , and kept

in vials at -40o C until use . For virus assay freshly con­

flue~t monolayers of cells were inoculated with an appropriate

viral titre (approximately 100 plaque forming units/plate)

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in a maintenance medium consisting of IX MEM supplemented with

0.26 gil L-glutamine and 2% fe tal calf serum and kept at 370

c

for 90 min. The inoculum was then removed and 4 ml o f growth

medium Containing 0.25 % immune serum globulin (Armour Pharm­

aceutica l, Kankakee , Illinois 60901) was added to prevent non ­

cellular viral transfer . After inoculation with virus, cell

monolayers were incubated for two days in the case of unirrad ­

iated virus and three days in the c a se of irradiated virus to

allow for visible plaque formation (Ross, ~ al. , 1971) .

Following incubation, cells were stained by adding to the

growth medium one drop of a 3% aqueous solution of crystal

violet in 20% ethyl alcohol and 0 . 8% ammonium Oxalate (Carolina

Biological Co., Burlington, North Carolina 27215). The resulting

solution was poured off after 10 min and plaques were counted.

Monochromatic Exposures

To obtain sUfficient exposure rates , a 2 . 5 kW high

presSure mercury - xenon lamp was used (929B Hanovia Lamp, Newark,

New Jersey 07105). This source was air cooled to remove ozone,

and infrared radiation was partially removed from the beam by

means of a circulating water filter. Spectral separation was

achieved by means of two 25 cm grating rnonochromators coupled

in tandem (GM 250, Schoeffel Inst. , Westwood , New Jersey 07675) .

Half band widths were measured at several wavelengths and varied

from 1 . 5 to 6.0 nm depending on the slit width. The Spectral

purity of the monochromator was further checked by plaCing a

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7

series of cut-off filters into the exit beam . In this study,

only wavelengths corresponding to individual lines of the

mercury spectrum were used, and these were fOund to be Spectral_

ly pure. EXPOsure rates were measured at different times

during an experiment by plaCing a calibrated UV-sensitive

photodiode (Cal- Uv , United Detector Technology, (UDT) , Inc. ,

Santa Monica , California 90405) in the sample position and

measuring the photocurrent produced by the radiation beam with

a Keithley 610B electrometer (Keithley Inst., Cleveland, Ohio

44101). Cells or Viruses were irradiated in horizontally

oriented petri diShes. The UV beam was deflected downWard

onto the diShes by means of a 45 - degree front - surfaced mirror .

DiShes were rotated at 0.5 rps to compensate for POSsible

inhomogeneities in the radiation beam . EXPosure times Varied from 1 to 120 sec .

2 EXPOsure rates varied from 0.08 to 24 W/m .

Before exposure to radiation , the growth medium was

removed from confluent monolayers of cells i p lastic petri

diShes by Suction . The mono layers we re then rinsed twicp in

Dulbecco ' s phOSphate buffered saline (PBS) (Dulbecco and Vogt ,

1954) to remove UV-absorbing components in the medium. The cell

monolayers were kept mOist during irradiation by the presence

of 2 ml of PBS . Virus was also irradiated, as a SUspenSion,

in PBS . Virus was irradiated at 40 c and cells were irradiated

at room temperature (22 ! IOC) . During any experiment ten

monolayers were assayed at each fluence , and experiments were

r 2peate6 at least three times for each wavelength. Additional

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B

experiments were run at peak wavelengths to ins u re the accuracy

of t he peak response level .

Immediately following irradiation of the virus , the viral

suspension was diluted into maintenance medium and inoculat ed

onto the cells . Cells were incubated and stained as described

above.

All viral irradiations were at 265 nm at a fluence of

500 J/m2 . This fluence lowered the pfa of the virus to approx­

imately 1% of a control (unirradiated) virus , but this survival

varie d som~what from expe riment to experiment. Cells were

irradiated a t different wavele ngths as shown in t he results

(Table 1) .

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TABLE 1

FLUENCE RATE RANGES FOR MONOCHROMATIC EXPOSURES TO CELLS AND THE SLOPE OF THE AVERAGED RER RESPONSES

Fluence Wavelength rate re~ge RER RER Slope (in mn) (1'1 m ) Slope (Ouantum corrected)

230 0 .08 - 0 . 30 0.041 0.053 235 0.50 - 2.00 0.055 0.071 240 1.10 - 3.90 0.071 0 . 089 245 1. 30 - 5 .10 0.074 0.091 248 1.25 - 5.40 0 . 114 0.139 254 0 . 45 - 1.45 0.146 0.174 260 0.05 - 0.20 0.210 0.244 265 0.30 - 1.10 0.206 0.235 270 0.60 - 2 .00 0 . 220 0.246 275 0.75 - 2.60 0.190 0 .209 280 0 . 90 - 3.70 0 .152 0 . 164 289 1.05 - 3.50 0.060 0.063 297 6 . 00 - 19.00 0.011 0 . 011 302 8 . 00 - 24 .00 0.004 0.004

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RESULTS

Figure 1 represents the data obtained from two separate

experiments involving the effect of UV-radiation at 265 nm

on cellular capacity for the growth of unirradiated and

irradiated virus. The abscissa indicates the fluence of

radiation; the ordinate is the fractional survival of cellular

capacity compared with unirradiated c ontrols. Since each mono­

layer contained 20-100 plaques, each data point corresponds

to 200 to 1000 plaques. The upper curves obtained from ir­

radiating cells but not viruses show a loss of capacity as

fluence is increased. The lower curves obtained from irrad­

iating both cells and viruses show an increase in pfa at low

fluences fOllowed by a decrease at higher fluences. Figures

2 and 3 represent data obtained from experiments conducted

at two additional wavelengths (235 nm an r J 2 nm , respective ly).

In figure 2 are indicated results for a wa vel e ngth (235 nm)

which incurs less damage than 265 nm. At 235 nm, no decrease

i n the capacity to host unirradiated virus (upper curve ) is

seen at the fluence which at 265 nm (Figure 1 ) decreases the

capacity to 10 % of a control. Correspondingly , the reactivation

sh0wn at 235 nm (lower curve) is less than that shown at 265

nm. Figure 3 represents a wavelength (302 nm) which is also less

damaging to the cells than is 265 nm, a larger fluence is

required to lower cellular capacity to support virus growth .

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Figure 1. 'I'he effect of 265 nm radiation On the capacity of CV-I cells to support the growth of either_

2 unirradiated (O' ~ ) or irradiated ( 500 J m ) (. ,A) HSV-1.

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265 I'Im

>- 1.0 ~

(.) 0.5 ~

~ (.)

C) Z

0.1 Z -~ .05 ~ w a::

lL... 0 .01 Z 0 .005 ~ 0 ~ a:: lL...

0 10 20 30 40

FLUEI\CE IN ~l

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Fiqure 2. Tb effect of 235 na radiation on c II capact J tor un1rradiated ( 0 ) or irr d1 ted (~OO J .- ) ( . ) virus.

Page 23: The Wavelength Dependence of Ultraviolet Enhanced ... · of CV-l cells irradiated at 265 nm to reactivate irradiated (500 J m-2) HSV-l The virus reactivation curves for cells irradiated

>-...... 0 235nm

~ ~I.O (') 0.5 z z -<l ~ W a:: 0.1 LL

° .05 z 0 t-O <l .01 c:: LL

-

0 10 20 30 40 50 60 FLUENCE IN J/m2

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13

Figure 3. The effect of 302 ~ radi for un rradiatad ( 0 ) or ( . ) viru •.

tion 0 cell capac ty irr dta ad (500 J .-2)

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>-..... 302" ... 0 <l: 1.0 a.. <l: 0 0.5 C) Z

Z

<l: 0.1 ~ W cr .05 lJ... 0

Z 0

.0' ..... 0 <1 .005 0::: lJ...

o 25 t() 5 1 125 FLUENCE It, J/ ,.,.2

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14

The l ower cur ve repr esents th radiation ennanc d reactivat on

da ta for this wavelength. As in figure 2, the reactivation

apparent for l02 nm i. las. than that tor 265 n..

Experiments were co plated at an additional eleven wave­

length. and yielded siailar data. Por purpo ... of .i~licity,

only thr e important wavelength. are illuatra ed, one at the

peak response level and one at each end ot th wavelength.

tested. The shape of these r diatlon enhanced r ct~Yat 01'1

curves can vary from experi8ent to exper t.

To ttempt to arrive at an action _peet or thb effect with such a variat on in re.ponse uld be difficult. Howeva , it analys • • lia1ted !nth foll ng ay.,

tha probl m of choo.ing a c rta1n lev 1 of r c ivat.ion i.

alleviated:

1. The pta of rr d at d virus

divided by the pfa of unirr diat v rla on cell. tha the a tlu no. Thi. ratio i. ref rc toa.thee

factor (Bockat. hler, !! al., 1916, Caille _p

1972 / Radl!lan and Devor t, U7l). •

2. Only that port~on of the re.,pclnl,. that 1nYOl

e

t.1Yat.~

Det .,

increaaa in th abov ra 10 ia us

of r activation. to d t rain th a·:>uftt

l. Each RER experi t. as done on the • d Y capacity ex rl nt for unirr diat. d v ru p ss g nu r c U •.

Onc t a l1naita re illpO d, th n a 1 n on 8 mi-log paper w • oPt ined or e cn a r n . '" in,

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three important wavelength. were chosen to illustrate data.

Piqure 4 represents typical data analyz d a. above for

several experiments at 265 nm. Each line in f qure • rep­

resent. data from one experiment at 265 n.. Slope. re

calculated for each of these line. and an average slope value

was obtained for the wavelength. The lines n figure 5 rep­

resent experiments at 235 nm, and the 1 nes n figure 6 rep­

resent experiments at 302 nm. Calculated slopes are a1ao

presented for these wavelengths.

In order to obtain one repre ntative line for ea

wavelength, the data point. re then nOrrMlized to t cor-

responding data point. on th 1 1 ne (~.i. line E of

figure .). Th s valu ., calculat, d for all a lengt • were th n replotted in figures 7 tbrouq,h 11. The val of

the slope of this final line was eons d red to

value for th wav length.

In fi9ures 7 through 11 are ah nth effecta 0 uv-

radi tion of 14 different avel "9thS on r dtat on • ..la4n

reactivation. The alop of th re ctiv tion f ctor

for each w velength and 18 found lhted in T le l.

since photochemical ch nges occurring n bioloq c 1

Ho_vee,

are du to photon ab.orption, not j ust to the en rqy of x­

posure, th slope valu s w re quantum corr cted ( ) (J r,

1967). The OC values re also l i at d 1n Tab1 1.

15

In figure 12 is sho the corrected ct on SDec-.:naa

for RER plott d a alope veraua w n9th. An orpt on

apectrUlll for A is includ d for CO/IIDalr1.0n (J 99 r, 1 61) .

Page 28: The Wavelength Dependence of Ultraviolet Enhanced ... · of CV-l cells irradiated at 265 nm to reactivate irradiated (500 J m-2) HSV-l The virus reactivation curves for cells irradiated

16

Figure 4. The reactivation curv • tor the 1 ty of CV-l cell. irr,diat d at 2 5 na to re ctiv t. rr dia (500 J. I HSV-1.

Page 29: The Wavelength Dependence of Ultraviolet Enhanced ... · of CV-l cells irradiated at 265 nm to reactivate irradiated (500 J m-2) HSV-l The virus reactivation curves for cells irradiated

-

265nm

01

~ .06

::! c

z 0 f-

~ .01 -f-~ .006 w ..... Q:

o 5 10 15 FUDa:

Page 30: The Wavelength Dependence of Ultraviolet Enhanced ... · of CV-l cells irradiated at 265 nm to reactivate irradiated (500 J m-2) HSV-l The virus reactivation curves for cells irradiated

17

Figur 5. The viru. react vation curvea for cella read ted at 235 nJII.

Page 31: The Wavelength Dependence of Ultraviolet Enhanced ... · of CV-l cells irradiated at 265 nm to reactivate irradiated (500 J m-2) HSV-l The virus reactivation curves for cells irradiated

z o I-

~

.04

.01

~ .001

o

o a

10 20 .30 Fl.JJEta II

~QPfS ...... . ~ . . • • II - -•

Page 32: The Wavelength Dependence of Ultraviolet Enhanced ... · of CV-l cells irradiated at 265 nm to reactivate irradiated (500 J m-2) HSV-l The virus reactivation curves for cells irradiated

18

igure 6. The virus r act vat ion curve. to e.U. lrrad a at 302 nJD.

Page 33: The Wavelength Dependence of Ultraviolet Enhanced ... · of CV-l cells irradiated at 265 nm to reactivate irradiated (500 J m-2) HSV-l The virus reactivation curves for cells irradiated

a: o 0 .1 I-

~ LL .05

z o I­<l > t5 .01 <l W a:

.005

302"fI'\

8

o 500 1000 1500 2000 2500 FLUE NC IN J"/ rtlt

Page 34: The Wavelength Dependence of Ultraviolet Enhanced ... · of CV-l cells irradiated at 265 nm to reactivate irradiated (500 J m-2) HSV-l The virus reactivation curves for cells irradiated

19

Figures 7 - 11. The averaged r activation factor. for KEX of HSV-1 by CV-l cell. at different av 1 Dgtb ••

Page 35: The Wavelength Dependence of Ultraviolet Enhanced ... · of CV-l cells irradiated at 265 nm to reactivate irradiated (500 J m-2) HSV-l The virus reactivation curves for cells irradiated
Page 36: The Wavelength Dependence of Ultraviolet Enhanced ... · of CV-l cells irradiated at 265 nm to reactivate irradiated (500 J m-2) HSV-l The virus reactivation curves for cells irradiated

-

~ .05

~ u.:

z 0

DI -~ t- ~ 'ci W Q:

o

f,;0PEs HO~7.& • •

10 20 30

FlLEta

, , Uo

40 50

J/rtf

2.54

240

~

60

Page 37: The Wavelength Dependence of Ultraviolet Enhanced ... · of CV-l cells irradiated at 265 nm to reactivate irradiated (500 J m-2) HSV-l The virus reactivation curves for cells irradiated

Q: .05

E u II

z 0 .01 -

260 I-

~ -I-U <f W

- ·220 a · .20 a::

• t1'O - .217

o 5 10 15

FLL£NCE:

Page 38: The Wavelength Dependence of Ultraviolet Enhanced ... · of CV-l cells irradiated at 265 nm to reactivate irradiated (500 J m-2) HSV-l The virus reactivation curves for cells irradiated

cr f2 ~ z 0 ~ <l > ~ U <l W cr

0.1

.01

. 005

280

SL~ . 2,..-. • - .1iD e •

o 10 20 JO 40 50

FLUEf\CE I. ~'

Page 39: The Wavelength Dependence of Ultraviolet Enhanced ... · of CV-l cells irradiated at 265 nm to reactivate irradiated (500 J m-2) HSV-l The virus reactivation curves for cells irradiated

cr .05 ~ ()

i1.

z 0 I- .01 et >

&OfEs -I- .005 Si w cr

o 250 500 750 1000 &SQ)

FU.£ E ~.

Page 40: The Wavelength Dependence of Ultraviolet Enhanced ... · of CV-l cells irradiated at 265 nm to reactivate irradiated (500 J m-2) HSV-l The virus reactivation curves for cells irradiated

20

Figure 12. An ct on apectrua tor • plotted •• .10 (quantWII corrected) of the 1 n • fro. I'i ure 7 throug 11. An orptiQn. for • included (3 99 r. 1967).

Page 41: The Wavelength Dependence of Ultraviolet Enhanced ... · of CV-l cells irradiated at 265 nm to reactivate irradiated (500 J m-2) HSV-l The virus reactivation curves for cells irradiated

.3

.2

~ 0 ~

.I

IlCTION SPECTRLM

-DNA~I

/

I /

/ /

/'

230 240 250 260 270 200 290 300

A IN nm

Page 42: The Wavelength Dependence of Ultraviolet Enhanced ... · of CV-l cells irradiated at 265 nm to reactivate irradiated (500 J m-2) HSV-l The virus reactivation curves for cells irradiated

OISCUSSIO

An analysis of the action spectrua pre. nt.ed in figure

12 leads to several conclusions. Kajor protions of the •

action spectrum are similar in shape to the published action

spectrum tor cell capacity to support unirradiat.d virus 9th

in the s cell-virus syste (coon!1l,!! al., 1917 , . Since

mammalian cell c pacity is considered to be a A depend nt

function (Lytle and Benane, 1975 ; Coppey and Nocentini, 1 7',

Coohill, !! al., 1977) it s re sanAble to conclude that

is, a least partially, also 0 A dependent. ver, the ~.

action spectrUlll has its peal< n the ran fro. 2'0 to 275 llA.

Although a pea at 2 0 na indicates the lnyolv ll t ot ftucleic

acid ( A or AI, a peak &bov this va l ue ( 2 na) lip! es

protein involv nt (Ja9g r, 19 7). Th r fore, the ct Oft

spectrum pres nted her s 0 th 1nvol nt ot be n 0 1 c

oid and protein. This interpretation s able

one modification. Proteins also in 0

w v.lengths below 250 na , but this a c ion

rb _r trua

show an incr ase in aD orption in thiS r gion. In

the action .pectrua below 2 0 n .

rapidly than doe. the abaorption

s em to indicate th invol nl of

• i n val

etr or

not

molecules) in the RE r spons at thes berter a ths .

Whether this effect is dir ct (an in ract on) or t

(shielding of the t rg t molecule)

tion.

ai s fur r e r

Page 43: The Wavelength Dependence of Ultraviolet Enhanced ... · of CV-l cells irradiated at 265 nm to reactivate irradiated (500 J m-2) HSV-l The virus reactivation curves for cells irradiated

In bacteria, WR is clos ly aSSOCiated with prOphage

induction and mutagenesis. Witkin (1976) and Devoret (1976)

have Suggested that a COImIOn inducible regulatory aignal aay

Control all three pheno na. That all of the proc sa a re­

quire poa -irradiation protein syntheaia is corollary evi

22

of induoibility (Witkin, 1976). n (1974, 197 ) hal e 1-

Oped thi a concept further into a ·805· 1ndue ble eeror-prone

repair hypothesis. He haa also lhown that SOS-induc1ng treat­

ments allow DNA replication of the bacteriophag .XI7C to

prOceed p st (or around) pyriJUdine , penanal

communication). In addition, HR i. photoreact Vahle in e­

teria (Heigle , 1953) POinting to pyriJlidine d1 ra al a lor

cause of this r aponae. WR in bacteria ia not pendent Oft

exciSion r p ir (Radman and Devoret, 1'711. Pinally, sin

WR is vident in Single str ad aa 11 a do Ie r'ande'd

phages (Ono nd Shiaazu, 19 6/ T s n d Oz 1, 1 '0) •

not likely that a reco in conal type 0 r pair ia

for its e xpression.

Results of similar ex ri n a using .... ~l n cella

be n done. Lytle,!!!!. (1 16 ) have I

ia not necessary for RER. Thi, r ault,

relults of Hellman, !!!l. (197) ho

tha xc I on r

th

t t s decreased in the pr enc of eaf in, could be int rpret

luggesting that post-replication ~ pair aay be n

RER (Nilsson and Leh n , 1'75). Th r suIt, of Lytle (1 71)

with Kilh cat virul (a I ngl st vi) r ail r

to thoa ntioned &bov for I nqle b et r1opftag.1

r

Page 44: The Wavelength Dependence of Ultraviolet Enhanced ... · of CV-l cells irradiated at 265 nm to reactivate irradiated (500 J m-2) HSV-l The virus reactivation curves for cells irradiated

2 3

This resul t may mean t h at an inducible error-prone .override.

repai r sys tem ( Radman , 1974 and 1976) is common to both systems.

Hence , the results inVOlvinq RER in m 11an cells agree

in principle with the results Obtained with WR in bacteria with

o ne e xception; Lytle and Benane (1975) have reported that

Photoreactivating light treatment of potoroo cells has I ttle

Or no effect on delayed RER. This latter reSUlt L.plies th t

py r imidine dimers are not essential for RER, at least in marSupial cells.

In summary, th actiOn spectrum for RER in aa-=a11an cells

can be interpreted as evidence for involv t of both nUCleic

acid and protein in this pheno non. Corollary data of others

on the b iochemical events involved in this response 0 not all

one to distinquish betw neither DOlecuie as the .. ,or t r t

for UV-radiation effects on RER.

Page 45: The Wavelength Dependence of Ultraviolet Enhanced ... · of CV-l cells irradiated at 265 nm to reactivate irradiated (500 J m-2) HSV-l The virus reactivation curves for cells irradiated

BIBLIOGRAPHY

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BOCkstabler, L. E. and C. D. Lytle. (19 10 ) . Ultr violet light enhanced reactivation of a I i n v i rua. Biochem. Biophys. Rea. Co 41 : 18C-189.

BOCkstabler , L. E., C. D. Ly Ie, J. E. Stafford and X. E. Haynes. (1916). Ultraviolet enhanCed reactivation of a human virus: effect of elaYed infection. utat ..... 35:189-198.

BOckstabler, L. E. and C. D. Lytle. (1977). diation nh reactivation of nuclear r pI eating "~ali.n virua a. Photochem. Photobio1. 25:477-482.

Caillet-Fauquet, P. and H. Defai s. (1972 ) . UV r c ~y t on of phage 1 in a pol A Mutant of E. COIL. utat ..... 1 5,

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copper' J . (1971). n ultraviolet

cella. ture Repair of interteron ayn betic c c ty irr diated no 1 nd hybrid ....... U (London ) 234:14-15.

d

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25

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d L. 1:. r P1 StUdy ot c 3 .257-26 4 .

at Iller.

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26

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27

Whitfield, J. P., R. H. Rixon and T. Yaudale. (19 1). £ feets of ultraviole light on ultiplication and eoxyribonucl 1c acid synthesis in cultures of L strain .au e c lIs. Exp. Cell Res. 22:450- 454.

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