TNT . J . RADIAT . BIOL .,
1979,
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36,
NO .
6, 6 2 1 -629
Decreased repair of gamma-irradiated adenovirus in Xeroderma pigmentosum fibroblasts A . J . RAINBOW and M . HOWES
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Departments of Radiology and Biology, McMaster University, Hamilton, Ontario, L8S 4J9 Canada (Received 15 November 1978 ; accepted 3 May 1979)
The ability of gamma-irradiated adenovirus to produce viral structural antigens (Vag) was examined in several normal and Xeroderma pigmentosum (XP) fibroblast strains . The fibroblast cultures were infected with either irradiated or nonirradiated adenovirus and at 48 hours after infection, cells were examined for the presence of Vag using immunofluorescent staining . Survival of Vag synthesis for gamma-irradiated adenovirus had a D 37 value of 47 ± 4 x 10 4 rad following the infection of seven normal fibroblast strains . The survival of this viral function was found to be significantly less following infection of the XP strains . D 37 values for Vag synthesis expressed as a percentage of that obtained on normal strains were obtained for a representative strain from each of the XP complementation groups : group A, 57 per cent ; group B, 61 per cent ; group C, 61 per cent, group D, 59 per cent ; group E, 73 per cent ; and variant, 75 per cent . These results indicate that XP cells have a reduced repair capacity for some type of gamma-ray-induced DNA damage .
1.
Introduction Xeroderma pigmentosum (XP) is one of a group of autosomal recessive syndromes in man, which is characterized by chromosomal instability and increased incidence of cancer (German 1972) . The XP homozygote is extremely sensitive to ultra-violet (U .V .) radiation and eventually develops various forms of skin tumours (Reed, Landing, Sugarman, Cleaver and Melnyk 1969, Robbins, Kraemer, Lutzner, Festoff and Coon 1974) . Cell lines developed from skin biopsies of these individuals are defective in their ability to repair damage induced by U .V . radiation (Cleaver 1968, 1969) . Most XP strains show some defect in their ability to perform excision repair of U .V .-induced cyclobutane pyrimidine dimers (Setlow, Regan, German and Carrier 1969, Cleaver and Trosko 1970) whereas variant XP strains show normal excision but some defect in the post-replication repair of U .V.-induced DNA damage (Lehmann, Kirk-Bell, Arlett, Paterson, Lohman, de Weerd-Kastelein and Bootsma 1975) . However, at the present time, the precise step at which the repair pathways are affected remains unclear . Another report (Sutherland, Rice and Wagner 1975) has shown photoreactivating enzyme to be present in normal human cells and at reduced levels in XP cells . Cell hybridization studies have shown that the mutations leading to the XP phenotype fall into at least six groups : A,B,C,D,E and the variant (Robbins et al . 1974, de Weerd-Kastelein, Keijzer and Bootsma 1972, Bootsma, de WeerdKastelein, Kleyer and Keyzez 1975, Kraemer, de Weerd-Kastelein, Robbins, Keijzer, Barret, Petinga and Bootsma 1975) . This suggests that a number of different mutations are involved in XP . C, Taylor & Francis Ltd 0020-7616/79/3606 0621 $0200 1979 ^
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Several reports show that XP cells are deficient in the repair of DNA lesions induced by certain chemical mutagens and carcinogens (Cleaver 1973, Stich, San and Kawzoe 1973, Regan and Setlow 1973) . Another report (Setlow, Faulcan and Regan 1976) shows that some of the DNA damage induced by gamma rays under anoxic conditions is poorly repaired in XP as compared to normal human cells . The ability of U .V .-irradiated virus to form plaques as well as to express other viral functions has been successfully applied by a number of investigators in the detection of a defective repair mechanism for U .V .-induced DNA damage in fibroblasts from patients with XP (Day 1974, Day 1975 a, Stich, Stich and Lam 1974, Abrahams and Eb 1976, Aaronson and Lytle 1970) . Studies employing U .V .irradiated adenovirus and SV 40 were particularly sensitive in expressing this XP defect and were capable of detecting a repair deficiency in the XP variant strains (Day 1975 a, Abrahams and Eb 1976) . A reduced survival of plaque formation for nitrous acid-treated adenovirus has also been found for XP as compared to normal cell strains (Day 1975 b) . In this report, we have examined the ability of gamma-irradiated adenovirus type 2 (Ad 2) to form viral structural antigens (Vag) in several normal and XP fibroblast strains . The cell strains examined included seven normal strains, a representative strain from each of the five XP complementation groups and an XP variant strain shown to be defective in a post-replication repair mechanism (Lehmann et al . 1975) . A reduced survival of Vag formation for gamma-irradiated Ad 2 was found in all the XP strains examined as compared to normal strains . 2. Materials and methods 2 .1 . Cells and virus Stock monolayer cultures of diploid human fibroblasts were grown in screw-cap bottles (Falcon plastics) and placed in a CO 2 incubator at 37°C and 90-100 per cent humidity . The growth medium was Eagle's a-minimal essential medium (a-MEM) supplemented with 10 per cent foetal calf serum together with antibiotics . Normal strains RE, Al, A2 and A8 were kindly supplied by Dr . Samuel Goldstein, Departments of Medicine and Biochemistry, McMaster University, Hamilton, Ontario, Canada . The normal strain GM 964 was obtained from the Human Genetic Mutant Cell Repository, Cambden, Mass ., U .S .A . Normal strains CRL 1119, CRL 1220 and CRL, 1229 as well as all XP strains were obtained from the American Type Culture Collection, Rockville, Md, U .S .A . Cultures were generally confluent by 7-9 days following subculture with a split ratio of 1 :3 . The preparation of Ad 2 has been described (Rainbow and Mak 1970) . Stock virus generally containing around 10 12 particles/ml was suspended in TBS (Winocour 1963) plus 20 per cent glycerol and stored at -70 °C. 2 .2 . Irradiation
The method of gamma-irradiation was essentially the same as that described previously (Rainbow 1974) . Samples of 1 ml of stock Ad 2 were kept at dry-ice temperature (-75 °C) during irradiation at a dose rate of 1 . 6Mrad/h using a 60 Co source . The method of U .V. irradiation has also been described (Rainbow and Mak 1973) . Stock virus was diluted 2-3-fold in a-MEM and 1 ml of viral suspension was irradiated in a 35 mm diameter Petri dish (Falcon Plastics), kept on ice, with constant
Repair of y-irradiated adenovirus in XP
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swirling during the irradiation . Under these conditions the dose rate was about 6 J/m 2 /2 as determined using a J-225 short-wave U .V . meter (Ultraviolet Products, San Gabriel, California) .
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2.3 . Experimental procedure Nonirradiated and irradiated suspensions of Ad 2 were assayed for their ability to form viral structural antigens in human fibroblasts . Ad 2 is a double-stranded DNA virus which replicates in the nucleus of a susceptible host cell forming large quantities of Vag (Marusyk, Norrby and Marusyk 1972) which can be readily detected by immunofluorescent staining . For the experiments, fibroblasts at a subculture passage number of between 10 and 20 were seeded as monolayers into 8-well-chamber slides (Lak Tek Products, Naperville, Illinois) . The cell monolayers generally reached confluency 24 hours after seeding at which time they were infected with either irradiated or nonirradiated Ad 2 . Three serial dilutions of the virus were used to infect each slide . Duplicate wells were used for each viral dilution with the two additional wells serving as uninfected controls . Following viral adsorption for 90 min, infected cells were incubated in growth medium . At 48 h after infection, the monolayers were fixed in a cold acetone-ethanol mixture (1 : 1), and incubated in the presence of rabbit Ad 2 antiserum for 30 min at 37°C, and then incubated for the same time with fluoresceinconjugated anti-rabbit globulin . For each slide, the number of fluorescing centres was counted in duplicate wells at three serial dilutions of the virus, and the data points fitted to a straight line using least-squares analysis . The slope of the line was then used as a quantitative measure of Vag formation . 3 . Results 3 .1 . Gamma irradiation of adenovirus 2 Typical results from the infection of normal and XP fibroblasts with gammairradiated and nonirradiated virus are shown in figure 1 . It can be seen that the
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Relative Viral Input Multiplicity
Figure 1 . The frequency of Vag-positive cells following adenovirus infection of three normal and two Xeroderma pigmentosum cell strains : The dilution series for nonirradiated and irradiated virus were prepared separately . Each point is the average of two chambers and the total number of cells in each well was around 4 x 10 ° . Virus was adsorbed for 90 min and samples were taken 48 h post-infection and scored for Vagpositive cells (A) unirradiated, and (B) gamma-irradiated virus (2 Mrad) . 0 XP 10BE ; • XP 11BE ; 0 A2 ; 0 CRL 1220 ; A CRL 1229 .
A . J. Rainbow and M . Howes
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6 24
1
2
3
1
2
3
Dose
Mrads
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2
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Figure 2 . Survival of Vag formation for gamma-irradiated Ad 2 in normal and Xeroderma pigmentosum fibroblast strains . Virus was adsorbed for 90 min and cells examined at 48 h after infection . The frequency of Vag-positive cells was determined in duplicate at three serial dilutions for each dose to the virus . The data points were fitted to a straight line using least-squares analysis in order to obtain each survival point . A : Experiment 1 XP 10 BE ; A XP 12BE ; 0 CRL 1119 ; Q Al . B: Experiment 2 0 XP LOBE ; V XP 11 BE ; El CRL 1119 ; A CRL 1220 ; 0 A2 ; V CRL 1229 . C : Experiment 3 .E XP 5BE ; 0 XP 2RO ; A XP 4BE ; 0 CRL 1119 ; 0 A2 ; O RE .
D37 ± S .E . (104 rad) Experiment 1 Normal strains A1 Az RE CRL 1119 CRL 1220 CRL 1229 Meant XP XP XP XP XP XP XP
strains 12BE (A)$ 11 BE (B) LOBE (C) 5BE (D) 2R0 (E) 4BE (variant)
Experiment 2
Experiment 3
50±5
52±2 67+9 54±2
50±9
73±7
61 . 5
46±4 43+6 51+6 47 . 5
57 . 7
35+1 (57 per cent)§ 37±1 (60 per cent)
29 ± 1 (61 percent) 29±1 (61 per cent) 34±1 (59 per cent) 42±2 (73 per cent) 43+3 (75 per cent)
t Mean D 37 value for normals used in that experiment . $ Complementation group designation . § Percent HCR value : D 37 expressed as a percentage of that obtained for the mean value of normal strains used (a measure of the repair capacity of the XP strain) . Table 1 .
D 37 values for Vag expression of gamma-irradiated adenovirus in several Xeroderma pigmentosum cell strains .
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frequency of Vag- positive cells was similar for both the three normal and two XP strains, when infected with nonirradiated virus (figure 1(A)) . However, the frequency of Vag-positive cells was considerably reduced in the XP strains as compared to normals following infection with gamma-irradiated virus (figure 1 (B)) . Similar results were obtained for all the XP strains examined . Figure 2 shows survival data for three experiments including a representative strain from each of the five XP-complementation groups, the XP variant and a number of normal strains . Survival points for each strain were fitted to a straight line using least-squares analysis to obtain the D 37 values and the standard error as shown in table 1 . These experiments have been carried out twice or more for each XP strain with at least two normals and another XP strain in each experiment, with similar results .
3 .2. Normal human strains Figure 1 and table 2 give an indication of the variation in survival of Vag formation for gamma-irradiated Ad 2 in normal strains for different experiments . D 37 values for five different experiments using the normal strain A2 showed a mean . of 51 x 10 4 rad with a standard deviation of the mean of 13 x 10 4 rad . D 37 values obtained from pooled data for seven different normal strains are shown in table 2 . Differences between strains were not significant and resulted from differences due to variation between experiments rather than a true difference between strains . The mean D 31 value for the seven strains was 47 x 10 4 rad with a standard deviation of the mean of 4 x 10 4 rad . 3 .3 . XP fibroblast strains Figure 2 shows that for each experiment the survival of Vag formation for gamma-irradiated adenovirus was significantly less in the XP than in the normal strains . For each experiment the D 37 for Ad 2 Vag survival in the XP strain was expressed as a percentage of the mean value obtained in the normal strains . These values are presented in table 1 . It can be seen that infection of the XP strains from complementation groups A, B, C and D resulted in a similar survival of Vag formation which was about 60 percent of that observed for the normal strains . Infection of the XP strain from complementation group E and the XP variant strain resulted in a somewhat higher survival of Vag formation (about 74 per cent of that of the normal strains) . Cell strain RE (2)l A i (2) A2 (5) As (1) CRL 1119 (4) CRL 1220 (2) CRL 1220(2)
±S .E . (104 rad)
D371
54±6 44±6 48±5 43±6 50±5 44±7 46±6
f Mean D 37 ±S .D . =47±4 x 10 4 rad . I Indicates the number of experiments from which the data were pooled . Table 2 . D 37 values for Vag expression of gamma-irradiated adenovirus in normal human fibroblast strains .
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3 .4 . U . V. irradiation of Ad 2 Survival curves for Vag production of U .V . -irradiated adenovirus are shown for comparison in figure 3 . It can be seen that the reduction in Vag survival for an XP strain as compared to normal was greater for U .V .-irradiated than for gammairradiated virus . Using the initial portions of the curves, D 37 values for Ad 2 Vag survival in the two XP strains as compared to normal were 6 per cent for the group D strain (XP 5BE) and 62 per cent for the variant strain (XP 4BE) . A similar reduction in the survival of plaque formation for U .V .-irradiated adenovirus has been reported for these XP strains as compared to normal strains (Day 1974) .
4
8
12
16
Ultraviolet Dose J/M 2 x 10-2
Figure 3 .
Survival of Vag formation for U .V .-irradiated Ad 2 in normal and Xeroderma pigmentosum fibroblast strains . Pooled results for two experiments : at least two normals and one Xeroderma pigmentosum cell strain were used in each experiment . Virus was adsorbed for 90 min and cells examined at 48 h after infection . The frequency of Vagpositive cells was determined in duplicate at three serial dilutions for each dose to the virus . The data points were fitted to a straight line using least-squares analysis in order to obtain the survival points for each experiment . / XP SBE ; A XP 4BE; O RE ; El CRL 1119 ; Q A2 ; Q GM 964 .
4.
Discussion
The survival of Vag formation for gamma-irradiated Ad 2 yielded a mean D 37 value of 47±4 x 10 4 rad for the normal human fibroblast strains . This is in close agreement with the survival of plaque and inclusion-body formation of gammairradiated adenovirus in human KB cells which yielded D 37 values of 46 x 10 4 and 54x 104 rad respectively, for similar conditions of irradiation to the virus (Rainbow and Mak 1972) . The survival of Vag formation for gamma-irradiated Ad 2 was found to be significantly less in the XP strains than in the normal strains . These results indicate a reduced host cell reactivation (HCR) of Vag for gamma-irradiated Ad 2 in the XP strains and suggest a reduced repair capacity for some type of gamma-ray induced DNA damage for XP . Gamma irradiation of adenovirus, under the frozen conditions described here, results in several different types of DNA lesion in the viral DNA,
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Repair of y-irradiated adenovirus in XP
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including strand breakage (Rainbow and Mak 1972) and alkaline labile bonds (Rainbow 1977) . Gamma-ray-induced alkaline labile bonds are thought to reflect some type of base damage in the viral DNA . It has been shown that gamma-ray-induced single-strand breaks in adenovirus DNA can be repaired by a host-mediated mechanism following the infection of human cells (Rainbow 1974) . Since XP cells have normal levels of single-strand break repair for cellular DNA (Cleaver 1969) it is unlikely that the reduced HCR reported here reflects a defect in the repair of this type of lesion . Setlow and co-workers (Setlow et al . 1976) have shown that certain types of DNA base damage induced by gamma-rays under anoxic conditions are poorly repaired in XP cells . The reduced HCR for gamma-irradiated Ad 2 in XP reported in the present study may be a reflection of this reduced capacity for the repair of gamma-ray induced DNA base damage . The values of HCR for Vag formation by gamma-irradiated adenovirus were similar in strains XP 12 BE (group A), XP 11 BE (group B), XP 10BE (group C) and XP 5BE (group D) (about 60 per cent of those observed in normal strains) . Strains XP 2RE (group E) and XP 4BE (variant) showed somewhat higher HCR values (about 74 per cent of those obtained in normal strains) . Group E (XP 2RE) and variant strains also show higher HCR values than strains from groups A, B, C, and D for plaque formation of U .V .-irradiated adenovirus (Day 1974) and SV40 (Abrahams and Eb 1976). The survival of Vag formation was considerably less for U .V .-irradiated Ad 2 than for gamma-irradiated Ad 2 when comparing results for the same XP strain with those for the normal strains . XP 5BE (group D) and XP 4BE (variant) gave values of 6 per cent and 62 per cent for Vag formation of U .V .-irradiated Ad 2 . Values of 3 . 6 per cent and 69 per cent have been reported by Day for the same XP strains using adenovirus plaque survival (Day 1974) . A small reduction in the HCR of plaque formation for X-irradiated herpes virus has been reported for one XP strain as compared to normal (Lytle, Aaronson and Harvey 1972) . We have calculated the HCR value in this instance to be about 90 per cent of that obtained for the normal strain . This is higher than the HCR values obtained in this report for gamma-irradiated adenovirus, and considerably higher than the 30 per cent HCR values obtained for U .V .-irradiated herpes virus in XP cells as compared to normal cells (Lytle et al. 1972) . Presumably these differences are a reflection of the gamma-irradiation conditions and the assay system used . U .V.irradiated herpes virus also shows a higher HCR value than U .V .-irradiated adenovirus in XP strains as compared to normal strains (Day 1974, Lytle et al . 1972) . The insensitivity of HCR for herpes virus as compared to adenovirus in the detection of cellular repair defects may reflect a greater independence of host-cell functions for the larger herpes virus genome . The results of this study clearly demonstrate a reduced repair capacity for gamma-ray damaged DNA in XP . However, it is not known whether the reduced repair capacity for gamma-ray damaged DNA is a reflection of the same cellular defect which results in a reduced repair capacity for U .V.-damaged DNA in XP . It is possible that in addition to their known deficiencies for U .V .-Induced DNA damage, XP cells also possess a specific deficiency for the repair of gamma-ray-induced DNA lesions .
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Acknowledgments We would like to thank Dr . Frank Graham for his helpful discussions and suggestions concerning this work . This work has been supported by the National Cancer Institute of Canada . L'aptitude de l'adenovirus, ayant ete soumis a l'irradiation-y, a produire des antigenes viraux structuraux (Vag), a ete examinee clans des cellules humaines, provenant de plusieurs individus normaux et de certains atteints de Xeroderma Pigmentosum (XP) . Les cultures de fibroblastes furent infectees d'adenovirus soit irradie, soit non-irradie, et 48 heures apres inoculation, les deux types de cellules furent examines afin de verifier la presence on l'absence de Vag a l'aide d'une technique d'immunofluorescence . A la suite de ('inoculation de sept cultures normales d'origines differentes, la D 37 pour la synthese de Vag par l'adenovirus irradie aux rayons 7, etait de 47±4 x 10 4 rad . La survivance de cette fonction virale apres inoculation des cultures XP, se montrait inferieure d'une maniere significative . Pour une culture representative de chaque groupe de complementation XP, si l'on exprime la D 37 sous forme de pourcentage de la D 37 obtenue pour les cultures normales, l'on obtient : un groupe A, 57 pour cent, un groupe B, 61 pour cent, un groupe C, 61 pour cent, un groupe D, 59 pour cent, un groupe E, 73 pour cent ; et le XP variant, 75 pour cent . Ces resultats indiquent que les cellules provenant des individus atteints de XP, possedent une aptitude reduite a la restauration des dommages de I'ADN dus a 1'irradiation y .
Die Fahigkeit der Gamma bestrahlten Adenoviren, Antigene von Virusstruktur (Vag) zu produzieren, wurde in mehreren normalen and Xeroderma Pigmentosum (XP) Fibroblasttypen untersucht . Die Fibroblastkulturen wurden mit bestrahlten oder unbestrahlten Adenoviren infiziert, and achtundvierzig Stunden nach der Infizierung wurden die Zellen mittels immunofluoreszierender Farbung auf das Vorhandensein von Vag untersucht . Der D37 Wert der Uberlebensrate der Vag-Synthese bei Gamma bestrahlten Adenoviren betrug 47±4 x 10 4 rad nach Infizierung von sieben normalen Fibroblasttypen . Es wurde festgestellt, dal3 die Uberlebensrate dieser Virusfunktion nach Infizierung der XP Typen wesentlich geringer war . Die D 37 Werte fur die Vag-Synthese, ausgedruckt als Prozentsatz des bei normalen Typen ermittelten Prozentsatzes, wurden fur einen reprasentativen Typ von jeder der XP Komplementationsgruppen gefunden : Gruppe A, 57 Prozent ; Gruppe B, 61 Prozent ; Gruppe C, 61 Prozent ; Gruppe D, 59 Prozent ; Gruppe E, 73 Prozent ; and Varianten, 75 Prozent . Diese Ergebnisse zeigen, dal3 die XP Zellen bei einigen durch Gamma-Bestrahlung bewirkten DNS-Schaden ein reduziertes Reparaturvermogen besitzen . References AARONSON, S . A ., and LYTE, C . D ., 1970, Nature, Lond ., 228, 359 . ABRAHAMS, P . J ., and VAN DER EB, A . J ., 1976, Mutation Res ., 35, 13 . BOOTSMA, D ., DEWEERD-KASTELEIN, E . A ., KLEYER, W ., and KEYZEL, W ., 1975, Molecular Mechanisms for Repair of DNA, edited by P . C . Hanawalt and R . B . Setlow (New York : Plenum Press), p . 725 . CLEAVER, J . E ., 1968, Nature Lond ., 218, 652 ; 1969, Proc . natn . Acad . Sci. U.S . A ., 63, 428 ; 1973, Cancer Res ., 33, 362 . CLEAVER, J . E ., and TROSKO, S . E ., 1970, Photochem . Photobiol., 11, 547 . DAY, R . S ., III . 1974, Cancer Res ., 34, 1965 ; 1975 a, Nature, Lond ., 253, 748 ; 1975 b, Mutation Res ., 27, 407 . GERMAN, J ., 1972, Medical Genetics, edited by A . Steinberg and A . Beam (New York : Grime and Stratton), p . 61 . KRAEMER, K . H ., DEWEERD-KASTELEIN, E . A ., ROBBINS, J . H ., KEIJZER, W ., BARRET, S . F ., PETINGA, R . A ., and BOOTSMA, D ., 1975, Mutation Res ., 33, 327 . LEHMANN, A . R ., KIRK-BELL, S ., ARLETT, C . F ., PATERSON, M . C ., LOHMAN, P ., DEWEERDKASTELEIN, E . A ., and BOOTSIIA, D ., 1975, Proc . natn . Acad. Sci. U .S . A ., 72, 219 . LYTLE, C . D ., AARONSON, S . A ., and HARVEY, E ., 1972, Int . J. Radiat . Biol ., 22, 159 .
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Repair of 7-irradiated adenovirus in XP
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RAINBOW, A . J ., and MAK, S ., 1970, J . Virol ., 5,188 ; 1972, Radiat . Res ., 50,319 ; 1973, Int. J . Radiat . Biol ., 24, 59 . RAINBOW, A . J ., 1974, Radiat . Res ., 60, 155 ; 1977, Photochem . Photobiol ., 25, 457 . REED, W . B ., LANDING, B ., SUGARMAN, G ., CLEAVER, J . E ., and MELNYK, J ., 1969, J. Am . med . Assn ., 207, 2073 . REGAN, J . D ., and SETLOW, R . B ., 1973, Chemical Mutagens : Principles and Methods for Their Detection, Vol . 3 edited by A . Hollaender (New York : Plenum Press), p . 151 . ROBBINS, J . H ., KRAEMER, K . H ., LUTZNER, M . A ., FESTOFF, B . W ., and COON, H . G ., 1974, Ann . intern . Med ., 80, 221 . SETLOW, R . B ., REGAN, J . D ., GERMAN, J ., and CARRIER, W . L ., 1969, Proc . natn . Acad. Sci . U .S . A ., 64, 1035 . SETLOW, R . B ., FAULCON, F . M ., and REGAN, J . D ., 1976, Int. J. Radiat . Biol ., 29, 125 . STICH, H . F ., SAN, R . H . C ., and KAWZOE, Y ., 1973, Mutation Res ., 17, 127 . STICH, H . F ., STICH, W ., and LAM, P ., 1974, Nature, Lond ., 250, 599 . SUTHERLAND, B . M ., RICE, M ., and WAGNER, E . K ., 1975, Proc . natn . Acad . Sci . U.S . A ., 72, 103 . DEWEERD-KASTELEIN, E . A ., KEIJZER, W ., and BOOTSMA, D ., 1972, Nature, New Biol ., 238, 80 . WINOCOUR, E ., 1963, Virology, 19, 158 . Note added in proof We have recently obtained results for the XP25RO strain from complementation group A, the XP2BE strain from complementation group C, and the XP230S strain from complementation group F (Takebe, Fujiwara, Sasaki, Sato, Kozuka, Nikaido, Ishizaki, Arase and Ikenaga 1978) . XP25RO was obtained from the Human Genetic Mutant Cell Repository, Cambden, Mass ., U .S .A ., XP2BE was obtained from the American Type Culture Collection, Rockville, Md ., U .S .A ., and XP230S was a generous gift from Dr . Mituo Ikenaga, Department of Fundamental Radiology, Faculty of Medicine, Osaka University, Osaka, Japan . At least two survival curve experiments have been performed with each of these strains for both UV- and gammairradiated Ad 2 . HCR values for Vag formation of gamma irradiated Ad 2 in these strains as compared to those obtained on normal strains were about 65 per cent for XP25RO, 61 per cent for XP2BE and 77 per cent for XP230S . HCR values obtained for UV-irradiated Ad 2 in these strains were about 6 per cent for XP25RO, 14 per cent for XP2BE, and 22 per cent for XP230S . Reference TAKEBE, H ., FUJIWARA, Y ., SASAKI, M . S ., SATO, Y ., KOZUKA, T ., NIKAIDO, 0 ., ISHIZAKI, K ., ARASE, S ., and IKENAGA, M ., 1978, DNA Repair Mechanisms, edited by P . C . Hanawalt, E . C . Friedberg and C . F . Fox (New York: Academic Press), p . 617 .
1979,
VOL .
36,
NO .
6, 6 2 1 -629
Decreased repair of gamma-irradiated adenovirus in Xeroderma pigmentosum fibroblasts A . J . RAINBOW and M . HOWES
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Departments of Radiology and Biology, McMaster University, Hamilton, Ontario, L8S 4J9 Canada (Received 15 November 1978 ; accepted 3 May 1979)
The ability of gamma-irradiated adenovirus to produce viral structural antigens (Vag) was examined in several normal and Xeroderma pigmentosum (XP) fibroblast strains . The fibroblast cultures were infected with either irradiated or nonirradiated adenovirus and at 48 hours after infection, cells were examined for the presence of Vag using immunofluorescent staining . Survival of Vag synthesis for gamma-irradiated adenovirus had a D 37 value of 47 ± 4 x 10 4 rad following the infection of seven normal fibroblast strains . The survival of this viral function was found to be significantly less following infection of the XP strains . D 37 values for Vag synthesis expressed as a percentage of that obtained on normal strains were obtained for a representative strain from each of the XP complementation groups : group A, 57 per cent ; group B, 61 per cent ; group C, 61 per cent, group D, 59 per cent ; group E, 73 per cent ; and variant, 75 per cent . These results indicate that XP cells have a reduced repair capacity for some type of gamma-ray-induced DNA damage .
1.
Introduction Xeroderma pigmentosum (XP) is one of a group of autosomal recessive syndromes in man, which is characterized by chromosomal instability and increased incidence of cancer (German 1972) . The XP homozygote is extremely sensitive to ultra-violet (U .V .) radiation and eventually develops various forms of skin tumours (Reed, Landing, Sugarman, Cleaver and Melnyk 1969, Robbins, Kraemer, Lutzner, Festoff and Coon 1974) . Cell lines developed from skin biopsies of these individuals are defective in their ability to repair damage induced by U .V . radiation (Cleaver 1968, 1969) . Most XP strains show some defect in their ability to perform excision repair of U .V .-induced cyclobutane pyrimidine dimers (Setlow, Regan, German and Carrier 1969, Cleaver and Trosko 1970) whereas variant XP strains show normal excision but some defect in the post-replication repair of U .V.-induced DNA damage (Lehmann, Kirk-Bell, Arlett, Paterson, Lohman, de Weerd-Kastelein and Bootsma 1975) . However, at the present time, the precise step at which the repair pathways are affected remains unclear . Another report (Sutherland, Rice and Wagner 1975) has shown photoreactivating enzyme to be present in normal human cells and at reduced levels in XP cells . Cell hybridization studies have shown that the mutations leading to the XP phenotype fall into at least six groups : A,B,C,D,E and the variant (Robbins et al . 1974, de Weerd-Kastelein, Keijzer and Bootsma 1972, Bootsma, de WeerdKastelein, Kleyer and Keyzez 1975, Kraemer, de Weerd-Kastelein, Robbins, Keijzer, Barret, Petinga and Bootsma 1975) . This suggests that a number of different mutations are involved in XP . C, Taylor & Francis Ltd 0020-7616/79/3606 0621 $0200 1979 ^
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Several reports show that XP cells are deficient in the repair of DNA lesions induced by certain chemical mutagens and carcinogens (Cleaver 1973, Stich, San and Kawzoe 1973, Regan and Setlow 1973) . Another report (Setlow, Faulcan and Regan 1976) shows that some of the DNA damage induced by gamma rays under anoxic conditions is poorly repaired in XP as compared to normal human cells . The ability of U .V .-irradiated virus to form plaques as well as to express other viral functions has been successfully applied by a number of investigators in the detection of a defective repair mechanism for U .V .-induced DNA damage in fibroblasts from patients with XP (Day 1974, Day 1975 a, Stich, Stich and Lam 1974, Abrahams and Eb 1976, Aaronson and Lytle 1970) . Studies employing U .V .irradiated adenovirus and SV 40 were particularly sensitive in expressing this XP defect and were capable of detecting a repair deficiency in the XP variant strains (Day 1975 a, Abrahams and Eb 1976) . A reduced survival of plaque formation for nitrous acid-treated adenovirus has also been found for XP as compared to normal cell strains (Day 1975 b) . In this report, we have examined the ability of gamma-irradiated adenovirus type 2 (Ad 2) to form viral structural antigens (Vag) in several normal and XP fibroblast strains . The cell strains examined included seven normal strains, a representative strain from each of the five XP complementation groups and an XP variant strain shown to be defective in a post-replication repair mechanism (Lehmann et al . 1975) . A reduced survival of Vag formation for gamma-irradiated Ad 2 was found in all the XP strains examined as compared to normal strains . 2. Materials and methods 2 .1 . Cells and virus Stock monolayer cultures of diploid human fibroblasts were grown in screw-cap bottles (Falcon plastics) and placed in a CO 2 incubator at 37°C and 90-100 per cent humidity . The growth medium was Eagle's a-minimal essential medium (a-MEM) supplemented with 10 per cent foetal calf serum together with antibiotics . Normal strains RE, Al, A2 and A8 were kindly supplied by Dr . Samuel Goldstein, Departments of Medicine and Biochemistry, McMaster University, Hamilton, Ontario, Canada . The normal strain GM 964 was obtained from the Human Genetic Mutant Cell Repository, Cambden, Mass ., U .S .A . Normal strains CRL 1119, CRL 1220 and CRL, 1229 as well as all XP strains were obtained from the American Type Culture Collection, Rockville, Md, U .S .A . Cultures were generally confluent by 7-9 days following subculture with a split ratio of 1 :3 . The preparation of Ad 2 has been described (Rainbow and Mak 1970) . Stock virus generally containing around 10 12 particles/ml was suspended in TBS (Winocour 1963) plus 20 per cent glycerol and stored at -70 °C. 2 .2 . Irradiation
The method of gamma-irradiation was essentially the same as that described previously (Rainbow 1974) . Samples of 1 ml of stock Ad 2 were kept at dry-ice temperature (-75 °C) during irradiation at a dose rate of 1 . 6Mrad/h using a 60 Co source . The method of U .V. irradiation has also been described (Rainbow and Mak 1973) . Stock virus was diluted 2-3-fold in a-MEM and 1 ml of viral suspension was irradiated in a 35 mm diameter Petri dish (Falcon Plastics), kept on ice, with constant
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swirling during the irradiation . Under these conditions the dose rate was about 6 J/m 2 /2 as determined using a J-225 short-wave U .V . meter (Ultraviolet Products, San Gabriel, California) .
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2.3 . Experimental procedure Nonirradiated and irradiated suspensions of Ad 2 were assayed for their ability to form viral structural antigens in human fibroblasts . Ad 2 is a double-stranded DNA virus which replicates in the nucleus of a susceptible host cell forming large quantities of Vag (Marusyk, Norrby and Marusyk 1972) which can be readily detected by immunofluorescent staining . For the experiments, fibroblasts at a subculture passage number of between 10 and 20 were seeded as monolayers into 8-well-chamber slides (Lak Tek Products, Naperville, Illinois) . The cell monolayers generally reached confluency 24 hours after seeding at which time they were infected with either irradiated or nonirradiated Ad 2 . Three serial dilutions of the virus were used to infect each slide . Duplicate wells were used for each viral dilution with the two additional wells serving as uninfected controls . Following viral adsorption for 90 min, infected cells were incubated in growth medium . At 48 h after infection, the monolayers were fixed in a cold acetone-ethanol mixture (1 : 1), and incubated in the presence of rabbit Ad 2 antiserum for 30 min at 37°C, and then incubated for the same time with fluoresceinconjugated anti-rabbit globulin . For each slide, the number of fluorescing centres was counted in duplicate wells at three serial dilutions of the virus, and the data points fitted to a straight line using least-squares analysis . The slope of the line was then used as a quantitative measure of Vag formation . 3 . Results 3 .1 . Gamma irradiation of adenovirus 2 Typical results from the infection of normal and XP fibroblasts with gammairradiated and nonirradiated virus are shown in figure 1 . It can be seen that the
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Figure 1 . The frequency of Vag-positive cells following adenovirus infection of three normal and two Xeroderma pigmentosum cell strains : The dilution series for nonirradiated and irradiated virus were prepared separately . Each point is the average of two chambers and the total number of cells in each well was around 4 x 10 ° . Virus was adsorbed for 90 min and samples were taken 48 h post-infection and scored for Vagpositive cells (A) unirradiated, and (B) gamma-irradiated virus (2 Mrad) . 0 XP 10BE ; • XP 11BE ; 0 A2 ; 0 CRL 1220 ; A CRL 1229 .
A . J. Rainbow and M . Howes
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Figure 2 . Survival of Vag formation for gamma-irradiated Ad 2 in normal and Xeroderma pigmentosum fibroblast strains . Virus was adsorbed for 90 min and cells examined at 48 h after infection . The frequency of Vag-positive cells was determined in duplicate at three serial dilutions for each dose to the virus . The data points were fitted to a straight line using least-squares analysis in order to obtain each survival point . A : Experiment 1 XP 10 BE ; A XP 12BE ; 0 CRL 1119 ; Q Al . B: Experiment 2 0 XP LOBE ; V XP 11 BE ; El CRL 1119 ; A CRL 1220 ; 0 A2 ; V CRL 1229 . C : Experiment 3 .E XP 5BE ; 0 XP 2RO ; A XP 4BE ; 0 CRL 1119 ; 0 A2 ; O RE .
D37 ± S .E . (104 rad) Experiment 1 Normal strains A1 Az RE CRL 1119 CRL 1220 CRL 1229 Meant XP XP XP XP XP XP XP
strains 12BE (A)$ 11 BE (B) LOBE (C) 5BE (D) 2R0 (E) 4BE (variant)
Experiment 2
Experiment 3
50±5
52±2 67+9 54±2
50±9
73±7
61 . 5
46±4 43+6 51+6 47 . 5
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35+1 (57 per cent)§ 37±1 (60 per cent)
29 ± 1 (61 percent) 29±1 (61 per cent) 34±1 (59 per cent) 42±2 (73 per cent) 43+3 (75 per cent)
t Mean D 37 value for normals used in that experiment . $ Complementation group designation . § Percent HCR value : D 37 expressed as a percentage of that obtained for the mean value of normal strains used (a measure of the repair capacity of the XP strain) . Table 1 .
D 37 values for Vag expression of gamma-irradiated adenovirus in several Xeroderma pigmentosum cell strains .
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frequency of Vag- positive cells was similar for both the three normal and two XP strains, when infected with nonirradiated virus (figure 1(A)) . However, the frequency of Vag-positive cells was considerably reduced in the XP strains as compared to normals following infection with gamma-irradiated virus (figure 1 (B)) . Similar results were obtained for all the XP strains examined . Figure 2 shows survival data for three experiments including a representative strain from each of the five XP-complementation groups, the XP variant and a number of normal strains . Survival points for each strain were fitted to a straight line using least-squares analysis to obtain the D 37 values and the standard error as shown in table 1 . These experiments have been carried out twice or more for each XP strain with at least two normals and another XP strain in each experiment, with similar results .
3 .2. Normal human strains Figure 1 and table 2 give an indication of the variation in survival of Vag formation for gamma-irradiated Ad 2 in normal strains for different experiments . D 37 values for five different experiments using the normal strain A2 showed a mean . of 51 x 10 4 rad with a standard deviation of the mean of 13 x 10 4 rad . D 37 values obtained from pooled data for seven different normal strains are shown in table 2 . Differences between strains were not significant and resulted from differences due to variation between experiments rather than a true difference between strains . The mean D 31 value for the seven strains was 47 x 10 4 rad with a standard deviation of the mean of 4 x 10 4 rad . 3 .3 . XP fibroblast strains Figure 2 shows that for each experiment the survival of Vag formation for gamma-irradiated adenovirus was significantly less in the XP than in the normal strains . For each experiment the D 37 for Ad 2 Vag survival in the XP strain was expressed as a percentage of the mean value obtained in the normal strains . These values are presented in table 1 . It can be seen that infection of the XP strains from complementation groups A, B, C and D resulted in a similar survival of Vag formation which was about 60 percent of that observed for the normal strains . Infection of the XP strain from complementation group E and the XP variant strain resulted in a somewhat higher survival of Vag formation (about 74 per cent of that of the normal strains) . Cell strain RE (2)l A i (2) A2 (5) As (1) CRL 1119 (4) CRL 1220 (2) CRL 1220(2)
±S .E . (104 rad)
D371
54±6 44±6 48±5 43±6 50±5 44±7 46±6
f Mean D 37 ±S .D . =47±4 x 10 4 rad . I Indicates the number of experiments from which the data were pooled . Table 2 . D 37 values for Vag expression of gamma-irradiated adenovirus in normal human fibroblast strains .
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3 .4 . U . V. irradiation of Ad 2 Survival curves for Vag production of U .V . -irradiated adenovirus are shown for comparison in figure 3 . It can be seen that the reduction in Vag survival for an XP strain as compared to normal was greater for U .V .-irradiated than for gammairradiated virus . Using the initial portions of the curves, D 37 values for Ad 2 Vag survival in the two XP strains as compared to normal were 6 per cent for the group D strain (XP 5BE) and 62 per cent for the variant strain (XP 4BE) . A similar reduction in the survival of plaque formation for U .V .-irradiated adenovirus has been reported for these XP strains as compared to normal strains (Day 1974) .
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Figure 3 .
Survival of Vag formation for U .V .-irradiated Ad 2 in normal and Xeroderma pigmentosum fibroblast strains . Pooled results for two experiments : at least two normals and one Xeroderma pigmentosum cell strain were used in each experiment . Virus was adsorbed for 90 min and cells examined at 48 h after infection . The frequency of Vagpositive cells was determined in duplicate at three serial dilutions for each dose to the virus . The data points were fitted to a straight line using least-squares analysis in order to obtain the survival points for each experiment . / XP SBE ; A XP 4BE; O RE ; El CRL 1119 ; Q A2 ; Q GM 964 .
4.
Discussion
The survival of Vag formation for gamma-irradiated Ad 2 yielded a mean D 37 value of 47±4 x 10 4 rad for the normal human fibroblast strains . This is in close agreement with the survival of plaque and inclusion-body formation of gammairradiated adenovirus in human KB cells which yielded D 37 values of 46 x 10 4 and 54x 104 rad respectively, for similar conditions of irradiation to the virus (Rainbow and Mak 1972) . The survival of Vag formation for gamma-irradiated Ad 2 was found to be significantly less in the XP strains than in the normal strains . These results indicate a reduced host cell reactivation (HCR) of Vag for gamma-irradiated Ad 2 in the XP strains and suggest a reduced repair capacity for some type of gamma-ray induced DNA damage for XP . Gamma irradiation of adenovirus, under the frozen conditions described here, results in several different types of DNA lesion in the viral DNA,
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including strand breakage (Rainbow and Mak 1972) and alkaline labile bonds (Rainbow 1977) . Gamma-ray-induced alkaline labile bonds are thought to reflect some type of base damage in the viral DNA . It has been shown that gamma-ray-induced single-strand breaks in adenovirus DNA can be repaired by a host-mediated mechanism following the infection of human cells (Rainbow 1974) . Since XP cells have normal levels of single-strand break repair for cellular DNA (Cleaver 1969) it is unlikely that the reduced HCR reported here reflects a defect in the repair of this type of lesion . Setlow and co-workers (Setlow et al . 1976) have shown that certain types of DNA base damage induced by gamma-rays under anoxic conditions are poorly repaired in XP cells . The reduced HCR for gamma-irradiated Ad 2 in XP reported in the present study may be a reflection of this reduced capacity for the repair of gamma-ray induced DNA base damage . The values of HCR for Vag formation by gamma-irradiated adenovirus were similar in strains XP 12 BE (group A), XP 11 BE (group B), XP 10BE (group C) and XP 5BE (group D) (about 60 per cent of those observed in normal strains) . Strains XP 2RE (group E) and XP 4BE (variant) showed somewhat higher HCR values (about 74 per cent of those obtained in normal strains) . Group E (XP 2RE) and variant strains also show higher HCR values than strains from groups A, B, C, and D for plaque formation of U .V .-irradiated adenovirus (Day 1974) and SV40 (Abrahams and Eb 1976). The survival of Vag formation was considerably less for U .V .-irradiated Ad 2 than for gamma-irradiated Ad 2 when comparing results for the same XP strain with those for the normal strains . XP 5BE (group D) and XP 4BE (variant) gave values of 6 per cent and 62 per cent for Vag formation of U .V .-irradiated Ad 2 . Values of 3 . 6 per cent and 69 per cent have been reported by Day for the same XP strains using adenovirus plaque survival (Day 1974) . A small reduction in the HCR of plaque formation for X-irradiated herpes virus has been reported for one XP strain as compared to normal (Lytle, Aaronson and Harvey 1972) . We have calculated the HCR value in this instance to be about 90 per cent of that obtained for the normal strain . This is higher than the HCR values obtained in this report for gamma-irradiated adenovirus, and considerably higher than the 30 per cent HCR values obtained for U .V .-irradiated herpes virus in XP cells as compared to normal cells (Lytle et al. 1972) . Presumably these differences are a reflection of the gamma-irradiation conditions and the assay system used . U .V.irradiated herpes virus also shows a higher HCR value than U .V .-irradiated adenovirus in XP strains as compared to normal strains (Day 1974, Lytle et al . 1972) . The insensitivity of HCR for herpes virus as compared to adenovirus in the detection of cellular repair defects may reflect a greater independence of host-cell functions for the larger herpes virus genome . The results of this study clearly demonstrate a reduced repair capacity for gamma-ray damaged DNA in XP . However, it is not known whether the reduced repair capacity for gamma-ray damaged DNA is a reflection of the same cellular defect which results in a reduced repair capacity for U .V.-damaged DNA in XP . It is possible that in addition to their known deficiencies for U .V .-Induced DNA damage, XP cells also possess a specific deficiency for the repair of gamma-ray-induced DNA lesions .
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Acknowledgments We would like to thank Dr . Frank Graham for his helpful discussions and suggestions concerning this work . This work has been supported by the National Cancer Institute of Canada . L'aptitude de l'adenovirus, ayant ete soumis a l'irradiation-y, a produire des antigenes viraux structuraux (Vag), a ete examinee clans des cellules humaines, provenant de plusieurs individus normaux et de certains atteints de Xeroderma Pigmentosum (XP) . Les cultures de fibroblastes furent infectees d'adenovirus soit irradie, soit non-irradie, et 48 heures apres inoculation, les deux types de cellules furent examines afin de verifier la presence on l'absence de Vag a l'aide d'une technique d'immunofluorescence . A la suite de ('inoculation de sept cultures normales d'origines differentes, la D 37 pour la synthese de Vag par l'adenovirus irradie aux rayons 7, etait de 47±4 x 10 4 rad . La survivance de cette fonction virale apres inoculation des cultures XP, se montrait inferieure d'une maniere significative . Pour une culture representative de chaque groupe de complementation XP, si l'on exprime la D 37 sous forme de pourcentage de la D 37 obtenue pour les cultures normales, l'on obtient : un groupe A, 57 pour cent, un groupe B, 61 pour cent, un groupe C, 61 pour cent, un groupe D, 59 pour cent, un groupe E, 73 pour cent ; et le XP variant, 75 pour cent . Ces resultats indiquent que les cellules provenant des individus atteints de XP, possedent une aptitude reduite a la restauration des dommages de I'ADN dus a 1'irradiation y .
Die Fahigkeit der Gamma bestrahlten Adenoviren, Antigene von Virusstruktur (Vag) zu produzieren, wurde in mehreren normalen and Xeroderma Pigmentosum (XP) Fibroblasttypen untersucht . Die Fibroblastkulturen wurden mit bestrahlten oder unbestrahlten Adenoviren infiziert, and achtundvierzig Stunden nach der Infizierung wurden die Zellen mittels immunofluoreszierender Farbung auf das Vorhandensein von Vag untersucht . Der D37 Wert der Uberlebensrate der Vag-Synthese bei Gamma bestrahlten Adenoviren betrug 47±4 x 10 4 rad nach Infizierung von sieben normalen Fibroblasttypen . Es wurde festgestellt, dal3 die Uberlebensrate dieser Virusfunktion nach Infizierung der XP Typen wesentlich geringer war . Die D 37 Werte fur die Vag-Synthese, ausgedruckt als Prozentsatz des bei normalen Typen ermittelten Prozentsatzes, wurden fur einen reprasentativen Typ von jeder der XP Komplementationsgruppen gefunden : Gruppe A, 57 Prozent ; Gruppe B, 61 Prozent ; Gruppe C, 61 Prozent ; Gruppe D, 59 Prozent ; Gruppe E, 73 Prozent ; and Varianten, 75 Prozent . Diese Ergebnisse zeigen, dal3 die XP Zellen bei einigen durch Gamma-Bestrahlung bewirkten DNS-Schaden ein reduziertes Reparaturvermogen besitzen . References AARONSON, S . A ., and LYTE, C . D ., 1970, Nature, Lond ., 228, 359 . ABRAHAMS, P . J ., and VAN DER EB, A . J ., 1976, Mutation Res ., 35, 13 . BOOTSMA, D ., DEWEERD-KASTELEIN, E . A ., KLEYER, W ., and KEYZEL, W ., 1975, Molecular Mechanisms for Repair of DNA, edited by P . C . Hanawalt and R . B . Setlow (New York : Plenum Press), p . 725 . CLEAVER, J . E ., 1968, Nature Lond ., 218, 652 ; 1969, Proc . natn . Acad . Sci. U.S . A ., 63, 428 ; 1973, Cancer Res ., 33, 362 . CLEAVER, J . E ., and TROSKO, S . E ., 1970, Photochem . Photobiol., 11, 547 . DAY, R . S ., III . 1974, Cancer Res ., 34, 1965 ; 1975 a, Nature, Lond ., 253, 748 ; 1975 b, Mutation Res ., 27, 407 . GERMAN, J ., 1972, Medical Genetics, edited by A . Steinberg and A . Beam (New York : Grime and Stratton), p . 61 . KRAEMER, K . H ., DEWEERD-KASTELEIN, E . A ., ROBBINS, J . H ., KEIJZER, W ., BARRET, S . F ., PETINGA, R . A ., and BOOTSMA, D ., 1975, Mutation Res ., 33, 327 . LEHMANN, A . R ., KIRK-BELL, S ., ARLETT, C . F ., PATERSON, M . C ., LOHMAN, P ., DEWEERDKASTELEIN, E . A ., and BOOTSIIA, D ., 1975, Proc . natn . Acad. Sci. U .S . A ., 72, 219 . LYTLE, C . D ., AARONSON, S . A ., and HARVEY, E ., 1972, Int . J. Radiat . Biol ., 22, 159 .
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RAINBOW, A . J ., and MAK, S ., 1970, J . Virol ., 5,188 ; 1972, Radiat . Res ., 50,319 ; 1973, Int. J . Radiat . Biol ., 24, 59 . RAINBOW, A . J ., 1974, Radiat . Res ., 60, 155 ; 1977, Photochem . Photobiol ., 25, 457 . REED, W . B ., LANDING, B ., SUGARMAN, G ., CLEAVER, J . E ., and MELNYK, J ., 1969, J. Am . med . Assn ., 207, 2073 . REGAN, J . D ., and SETLOW, R . B ., 1973, Chemical Mutagens : Principles and Methods for Their Detection, Vol . 3 edited by A . Hollaender (New York : Plenum Press), p . 151 . ROBBINS, J . H ., KRAEMER, K . H ., LUTZNER, M . A ., FESTOFF, B . W ., and COON, H . G ., 1974, Ann . intern . Med ., 80, 221 . SETLOW, R . B ., REGAN, J . D ., GERMAN, J ., and CARRIER, W . L ., 1969, Proc . natn . Acad. Sci . U .S . A ., 64, 1035 . SETLOW, R . B ., FAULCON, F . M ., and REGAN, J . D ., 1976, Int. J. Radiat . Biol ., 29, 125 . STICH, H . F ., SAN, R . H . C ., and KAWZOE, Y ., 1973, Mutation Res ., 17, 127 . STICH, H . F ., STICH, W ., and LAM, P ., 1974, Nature, Lond ., 250, 599 . SUTHERLAND, B . M ., RICE, M ., and WAGNER, E . K ., 1975, Proc . natn . Acad . Sci . U.S . A ., 72, 103 . DEWEERD-KASTELEIN, E . A ., KEIJZER, W ., and BOOTSMA, D ., 1972, Nature, New Biol ., 238, 80 . WINOCOUR, E ., 1963, Virology, 19, 158 . Note added in proof We have recently obtained results for the XP25RO strain from complementation group A, the XP2BE strain from complementation group C, and the XP230S strain from complementation group F (Takebe, Fujiwara, Sasaki, Sato, Kozuka, Nikaido, Ishizaki, Arase and Ikenaga 1978) . XP25RO was obtained from the Human Genetic Mutant Cell Repository, Cambden, Mass ., U .S .A ., XP2BE was obtained from the American Type Culture Collection, Rockville, Md ., U .S .A ., and XP230S was a generous gift from Dr . Mituo Ikenaga, Department of Fundamental Radiology, Faculty of Medicine, Osaka University, Osaka, Japan . At least two survival curve experiments have been performed with each of these strains for both UV- and gammairradiated Ad 2 . HCR values for Vag formation of gamma irradiated Ad 2 in these strains as compared to those obtained on normal strains were about 65 per cent for XP25RO, 61 per cent for XP2BE and 77 per cent for XP230S . HCR values obtained for UV-irradiated Ad 2 in these strains were about 6 per cent for XP25RO, 14 per cent for XP2BE, and 22 per cent for XP230S . Reference TAKEBE, H ., FUJIWARA, Y ., SASAKI, M . S ., SATO, Y ., KOZUKA, T ., NIKAIDO, 0 ., ISHIZAKI, K ., ARASE, S ., and IKENAGA, M ., 1978, DNA Repair Mechanisms, edited by P . C . Hanawalt, E . C . Friedberg and C . F . Fox (New York: Academic Press), p . 617 .
