400
Biochimica et Biophysica Acta, 4 7 9 ( 1 9 7 7 ) 4 0 0 - - 4 1 0 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press
BBA 99058
TYPICAL XERODERMA PIGMENTOSUM COMPLEMENTATION GROUP A FIBROBLASTS HAVE DETECTABLE ULTRAVIOLET LIGHT-INDUCED UNSCHEDULED DNA SYNTHESIS
R O B E R T A. P E T I N G A a, A L A N D. A N D R E W S a, R O B E R T E. T A R O N E b a n d J A Y H. R O B B I N S a,*
a Dermatology Branch and b Biometry Branch, National Cancer Institute, National Institutes o f Health, Bethesda, Md. 20014 (U.S.A.) (Received June 22nd, 1977)
Summary Ultraviolet-induced nuclear uptake of tritiated thymidine [3H]dThd demonstrable by autoradiography in non-synthesis phases of the cell cycle is known as unscheduled DNA synthesis and reflects repair replication of ultraviolet-damaged DNA. We have reported t h a t the rate of any such unscheduled DNA synthesis in typical group A xeroderma pigmentosum fibroblasts, if present, is less than 2% of the normal rate. We have now performed experiments to determine whether these fibroblasts have any unscheduled DNA synthesis. Fibroblast coverslip cultures of four xeroderma pigmentosum group A strains were prepared. Irradiated (254 nm ultraviolet light) and unirradiated cultures from each strain were incubated with [3H]dThd at 37°C, and autoradiograms were prepared using NTB-3 emulsion. A nuclear grain c o u n t was made of 100 consecutive nuclei of non-S-phase irradiated and unirradiated cells. A slide background grain c o u n t was simultaneously made from an acellular area adjacent to each cell analyzed. When a strain's irradiated and unirradiated autoradiograms having similar slide background grain count averages were compared, the nuclear grain c o u n t average of the irradiated cells was always higher than t h a t of the unirradiated cells. This ultraviolet-induced increase in the mean nuclear grain count ranged from 0.4 to 1.3% of t h a t given by normal non-xeroderma pigmentosum fibroblasts and was n o t reduced by 10 -2 M h y d r o x y u r e a . Planimetric studies showed that the ultraviolet-induced increase in nuclear grain count is n o t due to an increased nuclear area in irradiated cells. We conclude that these typical group A xeroderma pigmentosum strains perform very low, but detectable, ultraviolet-induced unscheduled DNA synthesis which probably reflects repair replication. We cannot, however, determine if there are significantly different * To whom correspondence should be addressed.
401
rates of ultraviolet-induced unscheduled DNA synthesis among these ultraviolet strains.
Introduction Xeroderma pigmentosum is an autosomal recessive human disease in which the patients have a marked sensitivity to the ultraviolet radiation of sunlight [1]. At an early age the patients develop severe actinic skin damage including pigmentation abnormalities and cutaneous malignancies [1]. All xeroderma pigmentosum patients have defective repair of ultraviolet-induced damage of their DNA [2], and most of the xeroderma pigmentosum patients have abnormally slow excision repair of ultraviolet-induced pyrimidine dimers. These excision~leficient patients are known [3] to comprise at least five complementation groups which are designated groups A, B, C, D and E. All [1,3] but one [4] of the patients in complementation group A are so deficient in excision repair that no such repair has ever been claimed to have been detected [ 1--4]. We have reported [1,2] that if these typical group A xeroderma pigmentosum patients have any excision repair, as reflected by ultraviolet-induced unscheduled DNA synthesis, such unscheduled DNA synthesis must be less than 2% of the normal rate of such repair. We now report that we have been able to detect ultraviolet-induced unscheduled DNA synthesis in these typical group A cells and that such unscheduled DNA synthesis occurs at 0.4--1.3% of the normal rate and probably reflects the repair replication of DNA excision repair. Materials and Methods
Fibroblast strains and culture methods. The five fibroblast strains used were obtained from the American Type Culture Collection (ATCC), Rockville, Md., U.S.A. One strain was from a "normal" 3-year-old b o y who had no known diseases (ATCC culture designation CRL 1141). The other four strains were from excision
Biochimica et Biophysica Acta, 4 7 9 ( 1 9 7 7 ) 4 0 0 - - 4 1 0 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press
BBA 99058
TYPICAL XERODERMA PIGMENTOSUM COMPLEMENTATION GROUP A FIBROBLASTS HAVE DETECTABLE ULTRAVIOLET LIGHT-INDUCED UNSCHEDULED DNA SYNTHESIS
R O B E R T A. P E T I N G A a, A L A N D. A N D R E W S a, R O B E R T E. T A R O N E b a n d J A Y H. R O B B I N S a,*
a Dermatology Branch and b Biometry Branch, National Cancer Institute, National Institutes o f Health, Bethesda, Md. 20014 (U.S.A.) (Received June 22nd, 1977)
Summary Ultraviolet-induced nuclear uptake of tritiated thymidine [3H]dThd demonstrable by autoradiography in non-synthesis phases of the cell cycle is known as unscheduled DNA synthesis and reflects repair replication of ultraviolet-damaged DNA. We have reported t h a t the rate of any such unscheduled DNA synthesis in typical group A xeroderma pigmentosum fibroblasts, if present, is less than 2% of the normal rate. We have now performed experiments to determine whether these fibroblasts have any unscheduled DNA synthesis. Fibroblast coverslip cultures of four xeroderma pigmentosum group A strains were prepared. Irradiated (254 nm ultraviolet light) and unirradiated cultures from each strain were incubated with [3H]dThd at 37°C, and autoradiograms were prepared using NTB-3 emulsion. A nuclear grain c o u n t was made of 100 consecutive nuclei of non-S-phase irradiated and unirradiated cells. A slide background grain c o u n t was simultaneously made from an acellular area adjacent to each cell analyzed. When a strain's irradiated and unirradiated autoradiograms having similar slide background grain count averages were compared, the nuclear grain c o u n t average of the irradiated cells was always higher than t h a t of the unirradiated cells. This ultraviolet-induced increase in the mean nuclear grain count ranged from 0.4 to 1.3% of t h a t given by normal non-xeroderma pigmentosum fibroblasts and was n o t reduced by 10 -2 M h y d r o x y u r e a . Planimetric studies showed that the ultraviolet-induced increase in nuclear grain count is n o t due to an increased nuclear area in irradiated cells. We conclude that these typical group A xeroderma pigmentosum strains perform very low, but detectable, ultraviolet-induced unscheduled DNA synthesis which probably reflects repair replication. We cannot, however, determine if there are significantly different * To whom correspondence should be addressed.
401
rates of ultraviolet-induced unscheduled DNA synthesis among these ultraviolet strains.
Introduction Xeroderma pigmentosum is an autosomal recessive human disease in which the patients have a marked sensitivity to the ultraviolet radiation of sunlight [1]. At an early age the patients develop severe actinic skin damage including pigmentation abnormalities and cutaneous malignancies [1]. All xeroderma pigmentosum patients have defective repair of ultraviolet-induced damage of their DNA [2], and most of the xeroderma pigmentosum patients have abnormally slow excision repair of ultraviolet-induced pyrimidine dimers. These excision~leficient patients are known [3] to comprise at least five complementation groups which are designated groups A, B, C, D and E. All [1,3] but one [4] of the patients in complementation group A are so deficient in excision repair that no such repair has ever been claimed to have been detected [ 1--4]. We have reported [1,2] that if these typical group A xeroderma pigmentosum patients have any excision repair, as reflected by ultraviolet-induced unscheduled DNA synthesis, such unscheduled DNA synthesis must be less than 2% of the normal rate of such repair. We now report that we have been able to detect ultraviolet-induced unscheduled DNA synthesis in these typical group A cells and that such unscheduled DNA synthesis occurs at 0.4--1.3% of the normal rate and probably reflects the repair replication of DNA excision repair. Materials and Methods
Fibroblast strains and culture methods. The five fibroblast strains used were obtained from the American Type Culture Collection (ATCC), Rockville, Md., U.S.A. One strain was from a "normal" 3-year-old b o y who had no known diseases (ATCC culture designation CRL 1141). The other four strains were from excision
