Repair of DNA in Xeroderma Pigmentosum Conjunctiva David A. Newsome, MD; Kenneth H. Kraemer, MD;
Xeroderma pigmentosum (XP) is an autosomal recessive disease with tumor formation on sun-exposed areas of the skin and eyes. Cells from most XP patients are deficient in repairing DNA damaged by ultraviolet (UV) light as shown by a reduced rate of tritiated thymidine (3HTdR) incorporation during their DNA repair synthesis. We have studied such repair synthesis in conjunctival cells from an XP patient with a conjunctival epithelioma and from normal cadaver conjunctiva. Cultured conjunctival cells were irradiated with UV light and then incubated with 3HTdR. Autoradiograms were prepared and showed that UV radiation induced a considerably slower rate of DNA repair synthesis in the XP cells than in normal cells. Many of the ocular abnormalities of XP, including tumor formation, may be the result of this defective DNA repair process.
is autosomal recessive dis¬ ease with tumor formation on sun-ex¬ posed areas of the skin and eyes, in¬ cluding the cornea and conjunctiva.25 Epidermal cells,69 dermal fibro¬ blasts,11014 and peripheral blood lym¬ phocytes1·15 from most XP patients have been shown to be deficient in the ability to repair DNA damaged by ul¬ traviolet (UV) light. The present study provides evidence that normal conjunctival cells can repair UV-damaged DNA and that the rate of such repair is considerably greater than
Xeroderma pigmentosum (XP)1 a rare
Submitted for publication June 11, 1974. From the Laboratory of Vision Research, National Eye Institute, and the Dermatology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Md. Reprint requests to Massachusetts Eye and Ear Infirmary, 243 Charles St, Boston, MA 02114
(Dr. Newsome).
Jay
H.
Robbins, MD
conjunctival cells from an XP patient who had a conjunctival malig¬ nant neoplasm. that in
MATERIALS AND METHODS Conjunctival Cultures Xeroderma pigmentosum conjunctiva obtained from clinically uninvolved bulbar conjunctiva of a 16-year-old girl at the time of surgery for removal of a con¬ junctival intraepithelial epithelioma. This patient, designated as XP patient 10 of the National Institutes of Health series,1 had typical cutaneous and ocular manifesta¬ tions of XP, including conjunctivitis, kera¬ titis, and symblepharon. Her peripheral blood lymphocytes and dermal fibroblasts have previously been shown to have a DNA repair rate 10% to 25% of normal.1 Normal conjunctiva was excised from a cadaver eye of a 25-year-old woman who died of valvular heart disease. Cultures of this tis¬ sue were established 12 hours postmortem. The conjunctival cultures were estab¬ lished in the following manner: Using a was
dissecting stereomicroscope, epithelial explants with minimal adherent stroma were prepared. Stromal expiants, apparently free of epithelium, were also prepared. Each expiant was placed into a 60-mm plastic tissue culture dish with 3.0 ml of modified Ham F-12 medium16 and covered with a glass coverslip. All cultures were maintained at 37.5 C in a 5^C02:95%-air atmosphere with 100% humidity. When primary outgrowths of epithelial cells were to be studied for DNA repair, those epithe¬ lial expiants that adhered to the coverslips were used. For studies in which serially propagated conjunctival cells were to be used, the uni¬ formity of cell type in primary outgrowths from conjunctival expiants was judged by
phase-contrast microscopy. Primary out¬ growths of what appeared to be predomi¬ nantly epithelial cells were obtained after four to six days in vitro by removing the epithelial expiant from the cells that had become adherent to the culture dish before
Downloaded From: http://archopht.jamanetwork.com/ by a Oakland University User on 06/05/2015
readily apparent migration of contaminat¬ ing fibrocytes from the expiant had begun. Such primary outgrowths of epithelial cells and outgrowths containing predominantly fibrocytic cells from the stromal expiants were washed in CV*- and Mg**-free phos¬ phate-buffered saline solution (PBS),1' sus¬ pended by incubation for 20 minutes at 37.5 C in 1 ml of 0.25% trypsin solution with 10'* Methylenediaminetetracetic acid (EDTA) in the case of the epithelial cells and without EDTA for the fibroblasts. The cells were then cultured in the modified Ham F-12 medium.
UV Irradiation
Primary outgrowths and serially propa¬ gated cells growing on their coverslips washed twice with room-temperature PBS and then covered with a small volume of PBS. They were irradiated for 150 sec¬ onds with a 2,537-Angstrom germicidal lamp (General Electric No. G15T8) at an incident flux of approximately 1 erg/sq mm/sec as measured by an intensity me¬ ter (Blak-Ray J225 UV). The PBS was then removed, and 2 ml of medium 199 contain¬ ing bicarbonate, 20% human plasma, and 20 microcuries of 3HTdR (specific activity, 18 to 25 curies/mmol) were added. The cells were placed in a 5%-C02 atmosphere at 37 C for three hours. The cells were then washed twice with cold PBS, fixed in a glutaraldehyde fixative, and washed suc¬ cessively in PBS and in 70% ethanol. After air drying, the coverslips were attached to a microscope slide with the cells upward. were
Autoradiography microscope slides were dipped in au¬ toradiographic emulsion (Kodak NTB-3) melted at 42 C, allowed to dry at room tem¬ perature, and placed in lightproof boxes The
a desiccant at 4 C for seven or eight days. The emulsion was developed in devel¬ oper (Kodak D19) for three minutes and
with
washed in tap water for three minutes and fixer (Kodak) for three minutes, all at 15 C. After further washing in tap water, the
Fig 1 .—Non-irradiated, serially propagated conjunctival cells from XP patient. Only labeled cell is heavily labeled cell (arrow) in S-phase DNA synthesis (acid hematoxylin, original magnification 700).
slides were air dried and with acid hematoxylin.
lightly
stained
Fig 2.—Irradiated, serially propagated conjunctival cells from XP culture. Sparse but definite light labeling over nuclei of cells not in S-phase synthesis. (Arrow indicates S-phase cell.) Sparseness of this light labeling indicates slow rate of DNA repair in these XP cells (acid hematoxylin, original magnification 700). of Dryness explants epithelial cells shown in same
Opacification
Fig 3.
RESULTS
COMMENT
In the nonirradiated cells from both normal and XP conjunctiva, cells in S-phase semiconservative DNA syn¬ thesis had many grains over their nuclei due to 3HTdR incorporation in preparation for mitotic division (Fig 1, arrow). These cells are called "heav¬ ily labeled" cells. The other cells had no evidence of 3HTdR incorporation. They had over their nuclei only the very rare grains attributable to back¬ ground. However, virtually all the ir¬ radiated conjunctival cells from the XP patient (Fig 2) and from the nor¬ mal donor (Fig 3) incorporated 3HTdR into their nuclei as shown by the labeling over the cells not in S-
Xeroderma pigmentosum was de¬ scribed more than 100 years ago,22 and the eye manifestations were re¬ ported in detail 16 years later.23 While the cutaneous manifestations of XP are usually recognized clinically be¬ fore the ocular abnormalities, reports of series of cases have shown a greater than 70% prevalence of mod¬ ocular erate-to-severe complica¬ tions.1·24·25 The range of ocular abnormalities in XP is so extensive and the occur¬ rence so frequent that ocular involve¬ ment should be considered a major manifestation of this disease. The fol¬ lowing are abnormalities associated with XP:1
phase synthesis. Such labeled cells are referred to as "lightly labeled" cells to distinguish them from the heavily labeled S-phase cells (arrows). This light labeling is a measure of the re¬ pair synthesis occurring in the cells' UV-damaged DNA.1821 It is apparent that the lightly labeled XP cells in Fig 2 have many fewer grains over their nuclei than the lightly labeled cells from the normal donor (Fig 3). These results, therefore, indicate that the XP conjunctival cells have a lower rate of UV-induced DNA repair than the normal conjunctival cells. The rate of repair in cells of pri¬ mary XP explants was essentially similar to the rate shown in the se¬ rially propagated cells of Fig 2. Like¬ wise, the rate of repair in serially propagated normal conjunctival fi¬ broblasts or epithelial cells was essen¬ tially similar to that of the primary
Lids
Blepharitis Erythema, pigmentation, kératoses Atrophy leading to entropion, ectropion, loss of cilia, and loss of lower lid
Neoplasms Papillomas Epitheliomas of free border of lid
Basal and squamous cell carcinomas
Conjunctiva Conjunctivitis
with photophobia, lacrimation, edema
Pigmentation, telangiectasia Dryness Symblepharon Inflammatory nodules Neoplasms Intraepithelial epitheliomas Squamous cell carcinomas Cornea
Exposure
keratitis with edema, cellular invasion, vascularization
Downloaded From: http://archopht.jamanetwork.com/ by a Oakland University User on 06/05/2015
Ulcération and
scarring
Neoplasms Iris Iritis
Synechiae Atrophy Neoplasms Cells from most persons with XP are unable to repair UV-damaged DNA as rapidly as normal cells, as measured by the rate of UV-induced 3HTdR incorporation.1·6"15 Major photoproducts of UV irradiation of DNA are pyrimidine dimers, of which the thymine dimer predominates.1·2631 Such dimers distort the DNA strand.30 The damaged DNA is then normally repaired by a series of en¬ zymatic reactions as follows1·30: (1) endonucleolytic incision of the DNA strand near the dimer, (2) exonucleolytic excision of the dimer-containing segment, (3) insertion of new purines and pyrimidines into the gap so formed, and (4) joining of the newly inserted bases to the remain¬ ing intact DNA strand by DNA li¬
activity. Complementation test¬ ing with fused cells has shown the
gase
existence of at least four different mutations, each of which can cause decreased DNA repair in .1·32·33 The precise enzymatic defect has not yet been determined for any of these XP complementation groups. An etiological relationship between the high incidence of cutaneous ma¬ lignant neoplasms in XP and the de¬ fect in UV-induced DNA repair syn¬ thesis has been suggested.1·6,0 The incidence of primary malignant neo-
Fig 3.—Irradiated epithelial cell outgrowth from normal donor's primary epithelial con¬ junctival expiant. There are two heavily labeled cells in S-phase synthesis (arrows). Vir¬ tually all of remaining cells are lightly labeled, indicating that they had incorporated 3HTdR during their repair synthesis. From intensity of their light labeling in comparison with that of XP cells in Fig 2, it is apparent that normal donor's cells have considerably greater rate of DNA repair than XP cells (acid hematoxylin, original magnification 700).
plasms of the conjunctiva and cornea in the general population is quite small. However, several groups of XP patients have been reported with ex¬ amples of such primary cancer.125 Thus, XP patients clearly have an ab¬ normally high incidence of conjuncti¬
val and corneal tumors. Our demon¬ stration that normal conjunctival cells can repair UV-damaged DNA faster than XP conjunctival cells sug¬ gests that the higher incidence of these ocular tumors in XP patients may be due to the XP defect in DNA repair. In fact, the patient whose con¬ junctiva we used for this demonstra¬ tion had developed a malignant intraepithelial epithelioma of the
conjunctiva. Sun-exposed areas of her skin also showed the typical clinical manifesta¬ tions of XP, including tumor forma¬ tion, and her dermal fibroblasts and peripheral blood lymphocytes have previously been shown1 to have the same reduced DNA repair rate, which we have now shown in her conjuncti¬
val cells. Her inherited XP DNA re¬ pair defect is, therefore, probably present in all of the nucleated cells of her body, but only those tissues ex¬ posed to UV radiation have been in¬ volved in tumor formation. All the UV-exposed areas of the eyes of XP patients are subject to numerous ocular abnormalities. Many of these abnormalities may also be caused, in whole or in part, by the in¬ ability of these ocular tissues to re¬ pair their UV-damaged DNA as rap¬ idly as normal. In this regard, the
fundus, which is not exposed to UV radiation, is rarely, if ever, involved
patients. Whatever the exact relationship might be between faulty DNA repair and the ocular abnormal¬ ities in XP patients, it is apparent that all XP patients should wear UVimpenetrable glasses with appropri¬ in XP
ate side-shields to prevent any UV radiation from reaching their eyes and eyelids. The photomicrographs were prepared by Mr. Robert Petinga and Mr. Harry Schaefer.
References 1. Robbins JH, Kraemer KH, Lutzner MA, et al: Xeroderma pigmentosum: An inherited disease with sun sensitivity, multiple cutaneous neoplasms, and abnormal DNA repair. Ann Intern Med 80:221-248, 1974. 2. Lukasiewicz W: Ueber xeroderma pigmentosum (Kaposi). Arch Dermatol Syph 33:37\x=req-\ 67, 1895. 3. Ibrahim HA: Xeroderma pigmentosum. Bull Ophthalmol Soc Egypt 26:145-149, 1933. 4. Reese A, Wilber I: The eye manifestations of xeroderma pigmentosum. Am J Ophthalmol 26:901-911, 1943. 5. Mortada A: Incidence of lids, conjunctival and orbital malignant tumors in xeroderma pigmentosum in Egypt. Bull Ophthalmol Soc Egypt 61:231-236, 1968. 6. Epstein JH, Fukuyama K, Reed WB, et al: Defect in DNA synthesis in skin of patients with xeroderma pigmentosum demonstrated in vivo. Science 168:1477-1478, 1970. 7. Jung EG: New form of molecular defect in xeroderma pigmentosum. Nature 228:361-362, 1970. 8. Robbins JH, Levis WR, Miller AE: Xeroderma pigmentosum epidermal cells with normal UV-induced thymidine incorporation. J Invest Dermatol 59:402-408, 1972. 9. Robbins JH, Burk PG: Relationship of DNA repair to carcinogenesis in xeroderma pigmentosum. Cancer Res 33:929-935, 1973. 10. Cleaver JE: Defective repair replication of DNA in xeroderma pigmentosum. Nature 218:652-656, 1968. 11. Cleaver JE: DNA repair and radiation sensitivity in human (xeroderma pigmentosum)
Downloaded From: http://archopht.jamanetwork.com/ by a Oakland University User on 06/05/2015
cells. Int J Radiat Biol 18:557-565, 1970. 12. Bootsma D, Mulder MP, Pot B, et al: Different inherited levels of DNA repair replication in xeroderma pigmentosum cell strains after exposure to ultraviolet irradiation. Mutat Res 9:507-516, 1970. 13. Parrington JM, Delhanty JDA, Baden HP: Unscheduled DNA synthesis, UV-induced chromosome abberations and SV40 transformation in cultured cells from xeroderma pigmentosum. Ann Hum Genet 35:149-160, 1971. 14. Regan JD, Setlow RB, Ley RD: Normal and defective repair of damaged DNA in human cells: A sensitive assay utilizing the photolysis of bromodeoxyuridine. Proc Natl Acad Sci USA 68:708-712, 1971. 15. Burk PG, Lutzner MA, Clarke DD, et al: Ultraviolet-stimulated thymidine incorporation in xeroderma pigmentosum lymphocytes. J Lab Clin Med 77:759-767, 1971. 16. Coon HG, Weiss M: A quantitative comparison of formation of spontaneous and virusproduced viable hybrids. Proc Natl Acad Sci USA 62:852-859, 1969. 17. Dulbecco R, Vogt M: Plaque formation and isolation of pure lines with poliomyelitis viruses. J Exp Med 99:167-182, 1954. 18. Rasmussen RE, Painter RB: Radiationstimulated DNA synthesis in cultured mammalian cells. J Cell Biol 29:11-19, 1966. 19. Painter RB, Cleaver JE: Repair replication, unscheduled DNA synthesis, and the repair of mammalian DNA. Radiat Res 37:451-466, 1969. 20. Cleaver JE, Painter RB: Evidence for repair replication of HeLa cell DNA damaged by ultraviolet light. Biochim Biophys Acta 161:552\x=req-\ 554, 1968. 21. Djordjevic B, Tolmach LJ: Responses of synchronous populations of HeLa cells to ultraviolet irradiation at selected stages of the generation cycle. Radiat Res 32:327-346, 1967. 22. Hebra F, Kaposi M: On Diseases of the Skin Including the Exanthemata. London, New Sydenham Society, 1874, vol 3. 23. Archambault PJV: De la dermatose de Kaposi (xeroderma pigmentosum), thesis. Bordeaux, France, 1890. 24. Labib MA, Barrada A, Choukry I, et al: Three cases of xeroderma pigmentosum. Bull Ophthalmol Soc Egypt 54:51-57, 1961. 25. El-Hafnawi H, Mortada A: Ocular manifestations of xeroderma pigmentosum. Br J Dermatol 77:261-276, 1965. 26. Setlow RB: Molecular changes responsible for ultraviolet inactivation of the biological activity of DNA, in Pavan C (ed): Mammalian Cytogenetics and Related Problems in Radiobiology. New York, Pergamon Press Inc, 1964. 27. Deering RA: Ultraviolet radiation and nucleic acid. Sci Am 207:135-144, 1962. 28. Jagger J: Introduction to research, in Ultraviolet Photobiology. London, Prentice-Hall Inc, 1967. 29. Hanawalt PC, Haynes RH: The repair of DNA. Sci Am 216:36-43, 1967. 30. Watson J: Molecular Biology of the Gene, ed 2. New York, WA Benjamin Inc, 1970. 31. Setlow RB: The photochemistry, photobiology and repair of polynucleotides. Prog Neucleic Acid Res Mol Biol 8:257-295,1968. 32. De Weerd-Kastelein EA, Keijzer W, Bootsma D: Genetic heterogeneity of xeroderma pigmentosum demonstrated by somatic cell hybridization. Nature 238:80-83, 1972. 33. Kleijer WJ, De Weerd-Kastelein EA, Sluyter ML: UV-induced DNA repair synthesis in cells of patients with different forms of xeroderma pigmentosum and of heterozygotes. Mutat Res 20:417-428, 1973.
Xeroderma pigmentosum (XP) is an autosomal recessive disease with tumor formation on sun-exposed areas of the skin and eyes. Cells from most XP patients are deficient in repairing DNA damaged by ultraviolet (UV) light as shown by a reduced rate of tritiated thymidine (3HTdR) incorporation during their DNA repair synthesis. We have studied such repair synthesis in conjunctival cells from an XP patient with a conjunctival epithelioma and from normal cadaver conjunctiva. Cultured conjunctival cells were irradiated with UV light and then incubated with 3HTdR. Autoradiograms were prepared and showed that UV radiation induced a considerably slower rate of DNA repair synthesis in the XP cells than in normal cells. Many of the ocular abnormalities of XP, including tumor formation, may be the result of this defective DNA repair process.
is autosomal recessive dis¬ ease with tumor formation on sun-ex¬ posed areas of the skin and eyes, in¬ cluding the cornea and conjunctiva.25 Epidermal cells,69 dermal fibro¬ blasts,11014 and peripheral blood lym¬ phocytes1·15 from most XP patients have been shown to be deficient in the ability to repair DNA damaged by ul¬ traviolet (UV) light. The present study provides evidence that normal conjunctival cells can repair UV-damaged DNA and that the rate of such repair is considerably greater than
Xeroderma pigmentosum (XP)1 a rare
Submitted for publication June 11, 1974. From the Laboratory of Vision Research, National Eye Institute, and the Dermatology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Md. Reprint requests to Massachusetts Eye and Ear Infirmary, 243 Charles St, Boston, MA 02114
(Dr. Newsome).
Jay
H.
Robbins, MD
conjunctival cells from an XP patient who had a conjunctival malig¬ nant neoplasm. that in
MATERIALS AND METHODS Conjunctival Cultures Xeroderma pigmentosum conjunctiva obtained from clinically uninvolved bulbar conjunctiva of a 16-year-old girl at the time of surgery for removal of a con¬ junctival intraepithelial epithelioma. This patient, designated as XP patient 10 of the National Institutes of Health series,1 had typical cutaneous and ocular manifesta¬ tions of XP, including conjunctivitis, kera¬ titis, and symblepharon. Her peripheral blood lymphocytes and dermal fibroblasts have previously been shown to have a DNA repair rate 10% to 25% of normal.1 Normal conjunctiva was excised from a cadaver eye of a 25-year-old woman who died of valvular heart disease. Cultures of this tis¬ sue were established 12 hours postmortem. The conjunctival cultures were estab¬ lished in the following manner: Using a was
dissecting stereomicroscope, epithelial explants with minimal adherent stroma were prepared. Stromal expiants, apparently free of epithelium, were also prepared. Each expiant was placed into a 60-mm plastic tissue culture dish with 3.0 ml of modified Ham F-12 medium16 and covered with a glass coverslip. All cultures were maintained at 37.5 C in a 5^C02:95%-air atmosphere with 100% humidity. When primary outgrowths of epithelial cells were to be studied for DNA repair, those epithe¬ lial expiants that adhered to the coverslips were used. For studies in which serially propagated conjunctival cells were to be used, the uni¬ formity of cell type in primary outgrowths from conjunctival expiants was judged by
phase-contrast microscopy. Primary out¬ growths of what appeared to be predomi¬ nantly epithelial cells were obtained after four to six days in vitro by removing the epithelial expiant from the cells that had become adherent to the culture dish before
Downloaded From: http://archopht.jamanetwork.com/ by a Oakland University User on 06/05/2015
readily apparent migration of contaminat¬ ing fibrocytes from the expiant had begun. Such primary outgrowths of epithelial cells and outgrowths containing predominantly fibrocytic cells from the stromal expiants were washed in CV*- and Mg**-free phos¬ phate-buffered saline solution (PBS),1' sus¬ pended by incubation for 20 minutes at 37.5 C in 1 ml of 0.25% trypsin solution with 10'* Methylenediaminetetracetic acid (EDTA) in the case of the epithelial cells and without EDTA for the fibroblasts. The cells were then cultured in the modified Ham F-12 medium.
UV Irradiation
Primary outgrowths and serially propa¬ gated cells growing on their coverslips washed twice with room-temperature PBS and then covered with a small volume of PBS. They were irradiated for 150 sec¬ onds with a 2,537-Angstrom germicidal lamp (General Electric No. G15T8) at an incident flux of approximately 1 erg/sq mm/sec as measured by an intensity me¬ ter (Blak-Ray J225 UV). The PBS was then removed, and 2 ml of medium 199 contain¬ ing bicarbonate, 20% human plasma, and 20 microcuries of 3HTdR (specific activity, 18 to 25 curies/mmol) were added. The cells were placed in a 5%-C02 atmosphere at 37 C for three hours. The cells were then washed twice with cold PBS, fixed in a glutaraldehyde fixative, and washed suc¬ cessively in PBS and in 70% ethanol. After air drying, the coverslips were attached to a microscope slide with the cells upward. were
Autoradiography microscope slides were dipped in au¬ toradiographic emulsion (Kodak NTB-3) melted at 42 C, allowed to dry at room tem¬ perature, and placed in lightproof boxes The
a desiccant at 4 C for seven or eight days. The emulsion was developed in devel¬ oper (Kodak D19) for three minutes and
with
washed in tap water for three minutes and fixer (Kodak) for three minutes, all at 15 C. After further washing in tap water, the
Fig 1 .—Non-irradiated, serially propagated conjunctival cells from XP patient. Only labeled cell is heavily labeled cell (arrow) in S-phase DNA synthesis (acid hematoxylin, original magnification 700).
slides were air dried and with acid hematoxylin.
lightly
stained
Fig 2.—Irradiated, serially propagated conjunctival cells from XP culture. Sparse but definite light labeling over nuclei of cells not in S-phase synthesis. (Arrow indicates S-phase cell.) Sparseness of this light labeling indicates slow rate of DNA repair in these XP cells (acid hematoxylin, original magnification 700). of Dryness explants epithelial cells shown in same
Opacification
Fig 3.
RESULTS
COMMENT
In the nonirradiated cells from both normal and XP conjunctiva, cells in S-phase semiconservative DNA syn¬ thesis had many grains over their nuclei due to 3HTdR incorporation in preparation for mitotic division (Fig 1, arrow). These cells are called "heav¬ ily labeled" cells. The other cells had no evidence of 3HTdR incorporation. They had over their nuclei only the very rare grains attributable to back¬ ground. However, virtually all the ir¬ radiated conjunctival cells from the XP patient (Fig 2) and from the nor¬ mal donor (Fig 3) incorporated 3HTdR into their nuclei as shown by the labeling over the cells not in S-
Xeroderma pigmentosum was de¬ scribed more than 100 years ago,22 and the eye manifestations were re¬ ported in detail 16 years later.23 While the cutaneous manifestations of XP are usually recognized clinically be¬ fore the ocular abnormalities, reports of series of cases have shown a greater than 70% prevalence of mod¬ ocular erate-to-severe complica¬ tions.1·24·25 The range of ocular abnormalities in XP is so extensive and the occur¬ rence so frequent that ocular involve¬ ment should be considered a major manifestation of this disease. The fol¬ lowing are abnormalities associated with XP:1
phase synthesis. Such labeled cells are referred to as "lightly labeled" cells to distinguish them from the heavily labeled S-phase cells (arrows). This light labeling is a measure of the re¬ pair synthesis occurring in the cells' UV-damaged DNA.1821 It is apparent that the lightly labeled XP cells in Fig 2 have many fewer grains over their nuclei than the lightly labeled cells from the normal donor (Fig 3). These results, therefore, indicate that the XP conjunctival cells have a lower rate of UV-induced DNA repair than the normal conjunctival cells. The rate of repair in cells of pri¬ mary XP explants was essentially similar to the rate shown in the se¬ rially propagated cells of Fig 2. Like¬ wise, the rate of repair in serially propagated normal conjunctival fi¬ broblasts or epithelial cells was essen¬ tially similar to that of the primary
Lids
Blepharitis Erythema, pigmentation, kératoses Atrophy leading to entropion, ectropion, loss of cilia, and loss of lower lid
Neoplasms Papillomas Epitheliomas of free border of lid
Basal and squamous cell carcinomas
Conjunctiva Conjunctivitis
with photophobia, lacrimation, edema
Pigmentation, telangiectasia Dryness Symblepharon Inflammatory nodules Neoplasms Intraepithelial epitheliomas Squamous cell carcinomas Cornea
Exposure
keratitis with edema, cellular invasion, vascularization
Downloaded From: http://archopht.jamanetwork.com/ by a Oakland University User on 06/05/2015
Ulcération and
scarring
Neoplasms Iris Iritis
Synechiae Atrophy Neoplasms Cells from most persons with XP are unable to repair UV-damaged DNA as rapidly as normal cells, as measured by the rate of UV-induced 3HTdR incorporation.1·6"15 Major photoproducts of UV irradiation of DNA are pyrimidine dimers, of which the thymine dimer predominates.1·2631 Such dimers distort the DNA strand.30 The damaged DNA is then normally repaired by a series of en¬ zymatic reactions as follows1·30: (1) endonucleolytic incision of the DNA strand near the dimer, (2) exonucleolytic excision of the dimer-containing segment, (3) insertion of new purines and pyrimidines into the gap so formed, and (4) joining of the newly inserted bases to the remain¬ ing intact DNA strand by DNA li¬
activity. Complementation test¬ ing with fused cells has shown the
gase
existence of at least four different mutations, each of which can cause decreased DNA repair in .1·32·33 The precise enzymatic defect has not yet been determined for any of these XP complementation groups. An etiological relationship between the high incidence of cutaneous ma¬ lignant neoplasms in XP and the de¬ fect in UV-induced DNA repair syn¬ thesis has been suggested.1·6,0 The incidence of primary malignant neo-
Fig 3.—Irradiated epithelial cell outgrowth from normal donor's primary epithelial con¬ junctival expiant. There are two heavily labeled cells in S-phase synthesis (arrows). Vir¬ tually all of remaining cells are lightly labeled, indicating that they had incorporated 3HTdR during their repair synthesis. From intensity of their light labeling in comparison with that of XP cells in Fig 2, it is apparent that normal donor's cells have considerably greater rate of DNA repair than XP cells (acid hematoxylin, original magnification 700).
plasms of the conjunctiva and cornea in the general population is quite small. However, several groups of XP patients have been reported with ex¬ amples of such primary cancer.125 Thus, XP patients clearly have an ab¬ normally high incidence of conjuncti¬
val and corneal tumors. Our demon¬ stration that normal conjunctival cells can repair UV-damaged DNA faster than XP conjunctival cells sug¬ gests that the higher incidence of these ocular tumors in XP patients may be due to the XP defect in DNA repair. In fact, the patient whose con¬ junctiva we used for this demonstra¬ tion had developed a malignant intraepithelial epithelioma of the
conjunctiva. Sun-exposed areas of her skin also showed the typical clinical manifesta¬ tions of XP, including tumor forma¬ tion, and her dermal fibroblasts and peripheral blood lymphocytes have previously been shown1 to have the same reduced DNA repair rate, which we have now shown in her conjuncti¬
val cells. Her inherited XP DNA re¬ pair defect is, therefore, probably present in all of the nucleated cells of her body, but only those tissues ex¬ posed to UV radiation have been in¬ volved in tumor formation. All the UV-exposed areas of the eyes of XP patients are subject to numerous ocular abnormalities. Many of these abnormalities may also be caused, in whole or in part, by the in¬ ability of these ocular tissues to re¬ pair their UV-damaged DNA as rap¬ idly as normal. In this regard, the
fundus, which is not exposed to UV radiation, is rarely, if ever, involved
patients. Whatever the exact relationship might be between faulty DNA repair and the ocular abnormal¬ ities in XP patients, it is apparent that all XP patients should wear UVimpenetrable glasses with appropri¬ in XP
ate side-shields to prevent any UV radiation from reaching their eyes and eyelids. The photomicrographs were prepared by Mr. Robert Petinga and Mr. Harry Schaefer.
References 1. Robbins JH, Kraemer KH, Lutzner MA, et al: Xeroderma pigmentosum: An inherited disease with sun sensitivity, multiple cutaneous neoplasms, and abnormal DNA repair. Ann Intern Med 80:221-248, 1974. 2. Lukasiewicz W: Ueber xeroderma pigmentosum (Kaposi). Arch Dermatol Syph 33:37\x=req-\ 67, 1895. 3. Ibrahim HA: Xeroderma pigmentosum. Bull Ophthalmol Soc Egypt 26:145-149, 1933. 4. Reese A, Wilber I: The eye manifestations of xeroderma pigmentosum. Am J Ophthalmol 26:901-911, 1943. 5. Mortada A: Incidence of lids, conjunctival and orbital malignant tumors in xeroderma pigmentosum in Egypt. Bull Ophthalmol Soc Egypt 61:231-236, 1968. 6. Epstein JH, Fukuyama K, Reed WB, et al: Defect in DNA synthesis in skin of patients with xeroderma pigmentosum demonstrated in vivo. Science 168:1477-1478, 1970. 7. Jung EG: New form of molecular defect in xeroderma pigmentosum. Nature 228:361-362, 1970. 8. Robbins JH, Levis WR, Miller AE: Xeroderma pigmentosum epidermal cells with normal UV-induced thymidine incorporation. J Invest Dermatol 59:402-408, 1972. 9. Robbins JH, Burk PG: Relationship of DNA repair to carcinogenesis in xeroderma pigmentosum. Cancer Res 33:929-935, 1973. 10. Cleaver JE: Defective repair replication of DNA in xeroderma pigmentosum. Nature 218:652-656, 1968. 11. Cleaver JE: DNA repair and radiation sensitivity in human (xeroderma pigmentosum)
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