- Email: [email protected]
Topical enzyme therapy for skin diseases?
COMMENTARY
Topical enzyme therapy for skin diseases? Kenneth H. Kraemer, MD,a and John J. DiGiovanna, MDa,b Bethesda, Maryland, and Providence, Rhode Island
A
study recently appeared in Lancet1 that describes an exciting new advance in the treatment of skin disease. Patients with xeroderma pigmentosum were treated for 1 year with a topical liposome lotion containing a bacterial DNA repair enzyme. The authors found a 68% reduction in new actinic keratoses and a 30% reduction in new basal cell carcinomas. This study is relevant to the prevention of skin cancer, provides evidence of the role of new DNA damage in human skin carcinogenesis, and serves as a model for topical enzyme therapy for human skin diseases. Prevention of skin cancer in patients with xeroderma pigmentosum The study by Yarosh et al1 is an important breakthrough in the treatment of patients with xeroderma pigmentosum, particularly in the prevention of both premalignant lesions and skin cancers. Patients with xeroderma pigmentosum have a 1000-fold increase in skin cancer and a 50-year reduction in age at onset, with the first skin cancer typically occurring in children younger than 10 years.2-4 The mainstay of therapy is surgery, and children with xeroderma pigmentosum may have dozens to hundreds of surgical treatments. Preventing the development of skin cancer affords the patients decreased morbidity (pain, disfigurement, recovery time, utilization of resources) and, potentially, decreased mortality. To date, the main forms of skin cancer prevention in xeroderma pigmentosum are sun avoidance, wearing ultraviolet-
From Basic Research Laboratory, Center for Cancer Research, National Cancer Institute, Bethesdaa; and the Division of Dermatopharmacology, Department of Dermatology, Brown Medical School, Providence.b The opinions expressed are those of the authors and do not necessarily represent those of the US Government. The authors have no conflict of interest to disclose. Reprint requests: Kenneth H. Kraemer, MD, Basic Research Laboratory, National Cancer Institute, Bldg 37, Room 3E24, Bethesda, MD 20892. E-mail: [email protected]. J Am Acad Dermatol 2002;46:463-6. 16/1/119102 doi:10.1067/mjd.2002.119102
blocking clothing, and use of sunscreens. Our oral retinoid study5 showed isotretinoin to be effective in preventing new skin cancers but at the cost of substantial toxicity. The new study1 shows the efficacy of topical T4 endonuclease V enzyme. Although less effective than retinoids in terms of the extent of reduction in the frequency of new basal cell carcinomas, this new treatment has minimal toxicity. Antibodies to the bacterial protein were not detected in this small group of patients, but long-term followup will be needed to be sure of its safety. However, at this early stage of investigation, the topical enzyme approach appears to be safer, with less risk of systemic toxicity. Although its greatest benefit appears to be in younger patients, topical application of T4 endonuclease V is an exciting advance in treatment for these severely affected children and adults. New DNA damage and skin carcinogenesis In addition, the observation that application of a DNA repair enzyme over a period of only 1 year can reduce skin cancer is groundbreaking. It redefines and expands the role of DNA damage in the pathophysiology of skin cancer. If T4 endonuclease V is acting as a DNA repair enzyme, this observation implies that DNA damage continues to play an active role in carcinogenesis in addition to its role in inducing mutations that occurred years earlier (Fig 1), which emphasizes the continued need for use of sunscreens and other forms of sun protection to prevent DNA damage. There is a great body of evidence supporting the role of slow accumulation of DNA damage (mutations) over years to decades as an important factor in the process of skin cancer development6 (Fig 1). Our current notion is that sunlight-induced cutaneous carcinogenesis begins with ultraviolet-induced DNA damage in the form of the cyclobutane pyrimidine dimer and other photoproducts. Within an individual cell, unrepaired DNA damage may lead to death of the cell. In other situations, the cell can survive, and the change may result in an altered DNA sequence—usually a cytosine → thymine mutation that produces tumor initiation. Subsequent replication results in a clone of altered cells containing 463
464 Kraemer and DiGiovanna
J AM ACAD DERMATOL MARCH 2002
Fig 1. Multistep theory of sunlight-induced skin carcinogenesis. Sun exposure results in DNA damage. Unrepaired DNA damage may cause mutations in the tumor suppressor gene P53 (P531, P532, …), which results in initiated cells. Additional sun exposure acts as a promoter, permitting mutated cells to proliferate in normal-appearing skin. In a small fraction of cells additional DNA damage is associated with a precancerous state, such as an actinic keratosis (AK). A small fraction of these cells eventually become cancerous, resulting in squamous cell carcinomas (SCC). This entire process is modified by additional cellular processes that involve sun exposure and/or DNA damage including DNA repair, eicosanoid and proteinase production, cytosine activation, immune suppression, apoptosis, and mutations of other tumor suppressor genes, such as PATCHED in basal cell carcinomas (BCC). (Modified from Kraemer KH. Proc Natl Acad Sci U S A 1997;94:11-4.)
these mutations. The following stages—tumor promotion and progression—usually take many years. The common clinical correlate in skin cancer is best understood in squamous cell carcinoma (Fig 1). As squamous cell carcinoma develops, P53 mutations commonly occur early in the course of carcinogenesis as frequent mutations in premalignant actinic keratoses. Over many years some of these precancerous lesions undergo tumor progression to develop into squamous cell carcinoma.7 T4 endonuclease V attacks ultraviolet-induced cyclobutane pyrimidine dimer but does not affect the resulting cytosine → thymine mutations. The fact that the frequency of basal cell carcinomas and actinic keratoses fell in patients with xeroderma pigmentosum with only 1 year of topical therapy is a remarkable surprise. It indicates that recent DNA damage (as opposed to the slow accumulation of DNA mutations) is also playing a major role in ultraviolet-induced skin carcinogenesis. Yarosh et al1 make the reasonable suggestion that DNA damage–dependent immune mechanisms may be important in this process, although apoptosis, cytokine activation, and eicosanoid or proteinase production may also be involved (Fig 1).
Actions of T4 endonuclease V and photoreactivating enzyme T4 endonuclease V is a bacterial DNA repair enzyme that recognizes one class of ultravioletinduced DNA damage, the cyclobutane pyrimidine dimer, a covalent linking of adjacent pyrimidines thymine or cytosine (Fig 2). T4 endonuclease V makes two cuts at the site of a cyclobutane pyrimidine dimer: one cut separates the thymine or cytosine base from the deoxyribose sugar and the other cuts the DNA backbone between the adjacent pyrimidines. The resulting product must then be processed by the cell’s nucleotide excision repair system to remove the lesion and restore the original DNA sequence (Fig 2). In contrast to the bacterial system, the human nucleotide excision repair system ordinarily functions in a different manner to repair DNA damage. A series of proteins work together to recognize the DNA damage, unwind the DNA in the region of the damage, and excise the lesion within an approximately 30 nucleotide fragment (these cuts are made at a distance from the photoproducts). The resulting gap is then filled in by copying the undamaged strand of DNA (Fig 2). Xeroderma pigmentosum cells lack one of the components of the
J AM ACAD DERMATOL VOLUME 46, NUMBER 3
nucleotide excision repair system (each of the 7 xeroderma pigmentosum complementation groups has a defect in a different nucleotide excision repair protein8) and are thus unable to make the required incisions in the DNA. T4 endonuclease V bypasses this defect by making an incision in the DNA at a different site than the location used by the normal nucleotide excision repair system (Fig 2). In 1975, introduction of T4 endonuclease V into cultured xeroderma pigmentosum cells was shown to increase DNA repair.9 The study by Yarosh et al1 demonstrates that this treatment also has beneficial effects in patients with xeroderma pigmentosum. A few words of caution are in order. The size of the repaired region in cells expressing T4 endonuclease V is smaller than the size in repair-proficient normal cells10,11 (Fig 2). In addition, repair-proficient cells preferentially repair active genes. The observation that repair-deficient hamster cells expressing T4 endonuclease V do not show the repair localized to active genes suggests that the bacterial enzyme will not fully correct repair deficiencies.12 Thus T4 endonuclease V does not actually restore the normal nucleotide excision repair pathway and only acts on cyclobutane pyrimidine dimers and not other classes of photoproducts (Fig 2) (such as the 6-4 pyrimidine-pyrimidone photoproduct), which also may be important for ultraviolet mutagenesis.13-15 Longterm follow-up will be needed to determine whether the tumors that do occur despite T4 endonuclease V treatment behave in the same manner as the usual tumors. In addition, the small size of the study makes the contribution of individual patients relatively large so that when one placebo-treated patient dropped out after 9 months, the beneficial effect on actinic keratoses seemed to disappear. Topical application of another DNA repair enzyme, photolyase or photoreactivating enzyme (from the bacterium Anacystis nidulans) in a liposome formulation, was recently shown to reverse ultraviolet B (UVB)–induced cyclobutane pyrimidine dimer and UVB-induced immune suppression and to inhibit UVB-induced erythema.16 This enzyme differs from T4 endonuclease V in its mode of action. In the presence of visible light, photoreactivating enzyme results in direct reversal of cyclobutane pyrimidine dimer (Fig 2). This DNA repair action does not involve use of the nucleotide excision repair system and thus may have a different spectrum of biologic activity and toxicity than T4 endonuclease V. Topical enzyme therapy The demonstration that liposomes may be used for delivery of biologically active therapeutic molecules into skin cells is a major dimension of this arti-
Kraemer and DiGiovanna 465
Fig 2. Repair of ultraviolet-induced DNA damage by bacterial T4 endonuclease V (T4 endo) in comparison to the mammalian nucleotide excision repair (NER) system and bacterial photoreactivating enzyme (PRE). Ultraviolet induces formation of cyclobutane pyrimidine dimers (CPD) of thymine (T) and cytosine (C) and other DNA photoproducts such as the T-C 6-4 photoproduct (6-4 PP). T4 endo makes an incision (scissors) between the sugar and base of a cyclobutane pyrimidine dimer and another incision in the DNA backbone. The cell’s nucleotide excision repair system (orange) then removes the altered lesion, creating a gap that is filled in using the other DNA strand as a template. T4 endo is unable to repair 6-4 PP (dashed pink line and blue X) and other nondimer photoproducts. In repair-proficient cells, the nucleotide excision repair system makes a pair of incisions flanking cyclobutane pyrimidine dimer or 6-4 PP. A gap is created that is filled in using the other DNA strand as a template. Cells from patients with xeroderma pigmentosum are unable to perform the incision steps of nucleotide excision repair (dashed orange line and blue X) but can process DNA that is incised by T4 endo. Photoreactivating enzyme in the presence of visible light can directly reverse cyclobutane pyrimidine dimer, restoring the normal undamaged sequence.
cle. Genetic studies are identifying the mutations that underlie a variety of dermatologic diseases, and this knowledge is, for the first time, revealing their basic pathogenesis. These include the identification of mutations in transglutaminase 1 (protein-crosslinking enzyme) in lamellar ichthyosis,17,18 keratin 1
466 Kraemer and DiGiovanna
or 10 (structural protein) in epidermolytic hyperkeratosis,19-21 APT2A2 (calcium pump) in Darier disease,22,23 and connexin 31 (gap junction protein) in erythrokeratodermia variabilis.24 These insights have resulted in a glamorous race toward genetic intervention with a goal of correcting the underlying mutations in the DNA. Unfortunately, this commitment of resources has generally been disappointing, with elusive results. The successful demonstration of therapeutic benefit from the delivery of active enzyme protein into the skin (an alternative to gene therapy) can be a model for other diseases. Although patients with diabetes have used injections of insulin molecules for many years, this method is not suitable for many other proteins. The success of topical T4 endonuclease V liposomes suggests that the use of protein therapy may have an untapped potential for treatment of skin diseases. Theoretically, proteins can be delivered directly to the diseased skin cells, the amount of material can be precisely controlled, and, importantly, the treatment can be stopped in case of adverse events. There are substantial technical problems to be overcome relating to the differences between bacterial and mammalian enzymes. In addition to the use of liposomes, other skin protein delivery approaches are currently being investigated.25 Creative development and application of this approach to other disorders may be translated into useful therapies for many diseases affecting the general population. Indeed, topical T4 endonuclease V in liposomes may have beneficial effects in the large number of repair-proficient normal persons who are at high risk for skin cancer. Topical enzyme therapy of skin diseases may soon be a reality. REFERENCES 1. Yarosh D, Klein J, O’Connor A, Hawk J, Rafal E, Wolf P. Effect of topically applied T4 endonuclease V in liposomes on skin cancer in xeroderma pigmentosum: a randomised study. Xeroderma Pigmentosum Study Group. Lancet 2001;357:926-9. 2. Kraemer KH, Lee MM, Scotto J. DNA repair protects against cutaneous and internal neoplasia: evidence from xeroderma pigmentosum. Carcinogenesis 1984;5:511-4. 3. Kraemer KH, Lee MM, Scotto J. Xeroderma pigmentosum: cutaneous, ocular, and neurologic abnormalities in 830 published cases. Arch Dermatol 1987;123:241-50. 4. Kraemer KH, Lee M-M, Andrews AD, Lambert WC. The role of sunlight and DNA repair in melanoma and nonmelanoma skin cancer: the xeroderma pigmentosum paradigm. Arch Dermatol 1994;130:1018-21. 5. Kraemer KH, DiGiovanna JJ, Moshell AN, Tarone RE, Peck GL. Prevention of skin cancer in xeroderma pigmentosum with the use of oral isotretinoin. N Engl J Med 1988;318:1633-7. 6. Yuspa SH, Dlugosz AA, Cheng CK, Denning MF, Tennenbaum T, Glick AB, et al. Role of oncogenes and tumor suppressor genes in multistage carcinogenesis. J Invest Dermatol 1994;103(Suppl): 90S-5S. 7. Kraemer KH. Sunlight and skin cancer: another link revealed. Proc Natl Acad Sci U S A 1997;94:11-4.
J AM ACAD DERMATOL MARCH 2002
8. Van Steeg H, Kraemer KH. Xeroderma pigmentosum and the role of UV-induced DNA damage in skin cancer. Mol Med Today 1999;5:86-94. 9. Tanaka K, Sekiguchi M, Okada Y. Restoration of ultravioletinduced unscheduled DNA synthesis of xeroderma pigmentosum cells by the concomitant treatment with bacteriophage T4 endonuclease V and HVJ (Sendai virus). Proc Natl Acad Sci U S A 1975;72:4071-5. 10. Kusewitt DF, Ley RD, Henderson EE. Enhanced pyrimidine dimer removal in repair-proficient murine fibroblasts transformed with the denV gene of bacteriophage T4. Mutat Res 1991;255:1-9. 11. Kusewitt DF, Budge CL, Ley RD. Enhanced pyrimidine dimer repair in cultured murine epithelial cells transfected with the denV gene of bacteriophage T4. J Invest Dermatol 1994;102: 485-9. 12. Valerie K, de Riel JK, Henderson EE. Restoration of DNA repair in UV-sensitive Chinese hamster ovary cell by the denV gene from bacteriophage T4. Basic Life Sci 1986;38:319-26. 13. Protic-Sabljic M, Kraemer KH. Reduced repair of non-dimer photoproducts in a gene transfected into xeroderma pigmentosum cells. Photochem Photobiol 1986;43:509-13. 14. Brash DE, Seetharam S, Kraemer KH, Seidman MM, Bredberg A. Photoproduct frequency is not the major determinant of UV base substitution hot spots or cold spots in human cells. Proc Natl Acad Sci U S A 1987;84:3782-6. 15. Kraemer KH, Seetharam S, Protic-Sabljic M, Brash DE, Bredberg A, Seidman MM. Defective DNA repair and mutagenesis by dimer and non-dimer photoproducts in xeroderma pigmentosum measured with plasmid vectors. In: Friedberg E, Hanawalt P, editors. Mechanisms and consequences of DNA damage processing. UCLA Symposia on Molecular and Cellular Biology, New Series, Vol. 83. New York: Alan R Liss; 1988. p. 325-35. 16. Stege H, Roza L, Vink AA, Grewe M, Ruzicka T, Grether-Beck S, et al. Enzyme plus light therapy to repair DNA damage in ultraviolet-B-irradiated human skin. Proc Natl Acad Sci U S A 2000; 97:1790-5. 17. Russell LJ, DiGiovanna JJ, Rogers GR, Steinert PM, Hashem N, Compton JG, et al. Mutations in the gene for transglutaminase 1 in autosomal recessive lamellar ichthyosis. Nat Genet 1995;9:279-83. 18. Huber M, Rettler I, Bernasconi K, Frenk E, Lavrijsen SP, Ponec M, et al. Mutations of keratinocyte transglutaminase in lamellar ichthyosis. Science 1995;267:525-8. 19. Compton JG, DiGiovanna JJ, Santucci SK, Kearns KS, Amos CI, Abangan DL, et al. Linkage of epidermolytic hyperkeratosis to the type II keratin gene cluster on chromosome 12q. Nat Genet 1992;1:301-5. 20. Chipev CC, Korge BP, Markova N, Bale SJ, DiGiovanna JJ, Compton JG, et al. A leucine——proline mutation in the H1 subdomain of keratin 1 causes epidermolytic hyperkeratosis. Cell 1992;70:821-8. 21. Bale SJ, Compton JG, DiGiovanna JJ. Epidermolytic hyperkeratosis. Semin Dermatol 1993;12:202-9. 22. Sakuntabhai A, Ruiz-Perez V, Carter S, Jacobsen N, Burge S, Monk S, et al. Mutations in ATP2A2, encoding a Ca2+ pump, cause Darier disease. Nat Genet 1999;21:271-7. 23. Ringpfeil F, Raus A, DiGiovanna JJ, Korge B, Harth W, Mazzanti C, et al. Darier disease: novel mutations in ATP2A2 and genotypephenotype correlation. Exp Dermatol 2001;10:19-27. 24. Richard G, Smith LE, Bailey RA, Itin P, Hohl D, Epstein EH Jr, et al. Mutations in the human connexin gene GJB3 cause erythrokeratodermia variabilis. Nat Genet 1998;20:366-9. 25. Robbins PB, Rothbard JB, Sheu SM, Oliver SF, Goodnough JB, Wender PA, et al. Molecular transporters facilitate topical protein transduction into the skin [abstract]. J Invest Dermatol 2001;117:549.
Topical enzyme therapy for skin diseases? Kenneth H. Kraemer, MD,a and John J. DiGiovanna, MDa,b Bethesda, Maryland, and Providence, Rhode Island
A
study recently appeared in Lancet1 that describes an exciting new advance in the treatment of skin disease. Patients with xeroderma pigmentosum were treated for 1 year with a topical liposome lotion containing a bacterial DNA repair enzyme. The authors found a 68% reduction in new actinic keratoses and a 30% reduction in new basal cell carcinomas. This study is relevant to the prevention of skin cancer, provides evidence of the role of new DNA damage in human skin carcinogenesis, and serves as a model for topical enzyme therapy for human skin diseases. Prevention of skin cancer in patients with xeroderma pigmentosum The study by Yarosh et al1 is an important breakthrough in the treatment of patients with xeroderma pigmentosum, particularly in the prevention of both premalignant lesions and skin cancers. Patients with xeroderma pigmentosum have a 1000-fold increase in skin cancer and a 50-year reduction in age at onset, with the first skin cancer typically occurring in children younger than 10 years.2-4 The mainstay of therapy is surgery, and children with xeroderma pigmentosum may have dozens to hundreds of surgical treatments. Preventing the development of skin cancer affords the patients decreased morbidity (pain, disfigurement, recovery time, utilization of resources) and, potentially, decreased mortality. To date, the main forms of skin cancer prevention in xeroderma pigmentosum are sun avoidance, wearing ultraviolet-
From Basic Research Laboratory, Center for Cancer Research, National Cancer Institute, Bethesdaa; and the Division of Dermatopharmacology, Department of Dermatology, Brown Medical School, Providence.b The opinions expressed are those of the authors and do not necessarily represent those of the US Government. The authors have no conflict of interest to disclose. Reprint requests: Kenneth H. Kraemer, MD, Basic Research Laboratory, National Cancer Institute, Bldg 37, Room 3E24, Bethesda, MD 20892. E-mail: [email protected]. J Am Acad Dermatol 2002;46:463-6. 16/1/119102 doi:10.1067/mjd.2002.119102
blocking clothing, and use of sunscreens. Our oral retinoid study5 showed isotretinoin to be effective in preventing new skin cancers but at the cost of substantial toxicity. The new study1 shows the efficacy of topical T4 endonuclease V enzyme. Although less effective than retinoids in terms of the extent of reduction in the frequency of new basal cell carcinomas, this new treatment has minimal toxicity. Antibodies to the bacterial protein were not detected in this small group of patients, but long-term followup will be needed to be sure of its safety. However, at this early stage of investigation, the topical enzyme approach appears to be safer, with less risk of systemic toxicity. Although its greatest benefit appears to be in younger patients, topical application of T4 endonuclease V is an exciting advance in treatment for these severely affected children and adults. New DNA damage and skin carcinogenesis In addition, the observation that application of a DNA repair enzyme over a period of only 1 year can reduce skin cancer is groundbreaking. It redefines and expands the role of DNA damage in the pathophysiology of skin cancer. If T4 endonuclease V is acting as a DNA repair enzyme, this observation implies that DNA damage continues to play an active role in carcinogenesis in addition to its role in inducing mutations that occurred years earlier (Fig 1), which emphasizes the continued need for use of sunscreens and other forms of sun protection to prevent DNA damage. There is a great body of evidence supporting the role of slow accumulation of DNA damage (mutations) over years to decades as an important factor in the process of skin cancer development6 (Fig 1). Our current notion is that sunlight-induced cutaneous carcinogenesis begins with ultraviolet-induced DNA damage in the form of the cyclobutane pyrimidine dimer and other photoproducts. Within an individual cell, unrepaired DNA damage may lead to death of the cell. In other situations, the cell can survive, and the change may result in an altered DNA sequence—usually a cytosine → thymine mutation that produces tumor initiation. Subsequent replication results in a clone of altered cells containing 463
464 Kraemer and DiGiovanna
J AM ACAD DERMATOL MARCH 2002
Fig 1. Multistep theory of sunlight-induced skin carcinogenesis. Sun exposure results in DNA damage. Unrepaired DNA damage may cause mutations in the tumor suppressor gene P53 (P531, P532, …), which results in initiated cells. Additional sun exposure acts as a promoter, permitting mutated cells to proliferate in normal-appearing skin. In a small fraction of cells additional DNA damage is associated with a precancerous state, such as an actinic keratosis (AK). A small fraction of these cells eventually become cancerous, resulting in squamous cell carcinomas (SCC). This entire process is modified by additional cellular processes that involve sun exposure and/or DNA damage including DNA repair, eicosanoid and proteinase production, cytosine activation, immune suppression, apoptosis, and mutations of other tumor suppressor genes, such as PATCHED in basal cell carcinomas (BCC). (Modified from Kraemer KH. Proc Natl Acad Sci U S A 1997;94:11-4.)
these mutations. The following stages—tumor promotion and progression—usually take many years. The common clinical correlate in skin cancer is best understood in squamous cell carcinoma (Fig 1). As squamous cell carcinoma develops, P53 mutations commonly occur early in the course of carcinogenesis as frequent mutations in premalignant actinic keratoses. Over many years some of these precancerous lesions undergo tumor progression to develop into squamous cell carcinoma.7 T4 endonuclease V attacks ultraviolet-induced cyclobutane pyrimidine dimer but does not affect the resulting cytosine → thymine mutations. The fact that the frequency of basal cell carcinomas and actinic keratoses fell in patients with xeroderma pigmentosum with only 1 year of topical therapy is a remarkable surprise. It indicates that recent DNA damage (as opposed to the slow accumulation of DNA mutations) is also playing a major role in ultraviolet-induced skin carcinogenesis. Yarosh et al1 make the reasonable suggestion that DNA damage–dependent immune mechanisms may be important in this process, although apoptosis, cytokine activation, and eicosanoid or proteinase production may also be involved (Fig 1).
Actions of T4 endonuclease V and photoreactivating enzyme T4 endonuclease V is a bacterial DNA repair enzyme that recognizes one class of ultravioletinduced DNA damage, the cyclobutane pyrimidine dimer, a covalent linking of adjacent pyrimidines thymine or cytosine (Fig 2). T4 endonuclease V makes two cuts at the site of a cyclobutane pyrimidine dimer: one cut separates the thymine or cytosine base from the deoxyribose sugar and the other cuts the DNA backbone between the adjacent pyrimidines. The resulting product must then be processed by the cell’s nucleotide excision repair system to remove the lesion and restore the original DNA sequence (Fig 2). In contrast to the bacterial system, the human nucleotide excision repair system ordinarily functions in a different manner to repair DNA damage. A series of proteins work together to recognize the DNA damage, unwind the DNA in the region of the damage, and excise the lesion within an approximately 30 nucleotide fragment (these cuts are made at a distance from the photoproducts). The resulting gap is then filled in by copying the undamaged strand of DNA (Fig 2). Xeroderma pigmentosum cells lack one of the components of the
J AM ACAD DERMATOL VOLUME 46, NUMBER 3
nucleotide excision repair system (each of the 7 xeroderma pigmentosum complementation groups has a defect in a different nucleotide excision repair protein8) and are thus unable to make the required incisions in the DNA. T4 endonuclease V bypasses this defect by making an incision in the DNA at a different site than the location used by the normal nucleotide excision repair system (Fig 2). In 1975, introduction of T4 endonuclease V into cultured xeroderma pigmentosum cells was shown to increase DNA repair.9 The study by Yarosh et al1 demonstrates that this treatment also has beneficial effects in patients with xeroderma pigmentosum. A few words of caution are in order. The size of the repaired region in cells expressing T4 endonuclease V is smaller than the size in repair-proficient normal cells10,11 (Fig 2). In addition, repair-proficient cells preferentially repair active genes. The observation that repair-deficient hamster cells expressing T4 endonuclease V do not show the repair localized to active genes suggests that the bacterial enzyme will not fully correct repair deficiencies.12 Thus T4 endonuclease V does not actually restore the normal nucleotide excision repair pathway and only acts on cyclobutane pyrimidine dimers and not other classes of photoproducts (Fig 2) (such as the 6-4 pyrimidine-pyrimidone photoproduct), which also may be important for ultraviolet mutagenesis.13-15 Longterm follow-up will be needed to determine whether the tumors that do occur despite T4 endonuclease V treatment behave in the same manner as the usual tumors. In addition, the small size of the study makes the contribution of individual patients relatively large so that when one placebo-treated patient dropped out after 9 months, the beneficial effect on actinic keratoses seemed to disappear. Topical application of another DNA repair enzyme, photolyase or photoreactivating enzyme (from the bacterium Anacystis nidulans) in a liposome formulation, was recently shown to reverse ultraviolet B (UVB)–induced cyclobutane pyrimidine dimer and UVB-induced immune suppression and to inhibit UVB-induced erythema.16 This enzyme differs from T4 endonuclease V in its mode of action. In the presence of visible light, photoreactivating enzyme results in direct reversal of cyclobutane pyrimidine dimer (Fig 2). This DNA repair action does not involve use of the nucleotide excision repair system and thus may have a different spectrum of biologic activity and toxicity than T4 endonuclease V. Topical enzyme therapy The demonstration that liposomes may be used for delivery of biologically active therapeutic molecules into skin cells is a major dimension of this arti-
Kraemer and DiGiovanna 465
Fig 2. Repair of ultraviolet-induced DNA damage by bacterial T4 endonuclease V (T4 endo) in comparison to the mammalian nucleotide excision repair (NER) system and bacterial photoreactivating enzyme (PRE). Ultraviolet induces formation of cyclobutane pyrimidine dimers (CPD) of thymine (T) and cytosine (C) and other DNA photoproducts such as the T-C 6-4 photoproduct (6-4 PP). T4 endo makes an incision (scissors) between the sugar and base of a cyclobutane pyrimidine dimer and another incision in the DNA backbone. The cell’s nucleotide excision repair system (orange) then removes the altered lesion, creating a gap that is filled in using the other DNA strand as a template. T4 endo is unable to repair 6-4 PP (dashed pink line and blue X) and other nondimer photoproducts. In repair-proficient cells, the nucleotide excision repair system makes a pair of incisions flanking cyclobutane pyrimidine dimer or 6-4 PP. A gap is created that is filled in using the other DNA strand as a template. Cells from patients with xeroderma pigmentosum are unable to perform the incision steps of nucleotide excision repair (dashed orange line and blue X) but can process DNA that is incised by T4 endo. Photoreactivating enzyme in the presence of visible light can directly reverse cyclobutane pyrimidine dimer, restoring the normal undamaged sequence.
cle. Genetic studies are identifying the mutations that underlie a variety of dermatologic diseases, and this knowledge is, for the first time, revealing their basic pathogenesis. These include the identification of mutations in transglutaminase 1 (protein-crosslinking enzyme) in lamellar ichthyosis,17,18 keratin 1
466 Kraemer and DiGiovanna
or 10 (structural protein) in epidermolytic hyperkeratosis,19-21 APT2A2 (calcium pump) in Darier disease,22,23 and connexin 31 (gap junction protein) in erythrokeratodermia variabilis.24 These insights have resulted in a glamorous race toward genetic intervention with a goal of correcting the underlying mutations in the DNA. Unfortunately, this commitment of resources has generally been disappointing, with elusive results. The successful demonstration of therapeutic benefit from the delivery of active enzyme protein into the skin (an alternative to gene therapy) can be a model for other diseases. Although patients with diabetes have used injections of insulin molecules for many years, this method is not suitable for many other proteins. The success of topical T4 endonuclease V liposomes suggests that the use of protein therapy may have an untapped potential for treatment of skin diseases. Theoretically, proteins can be delivered directly to the diseased skin cells, the amount of material can be precisely controlled, and, importantly, the treatment can be stopped in case of adverse events. There are substantial technical problems to be overcome relating to the differences between bacterial and mammalian enzymes. In addition to the use of liposomes, other skin protein delivery approaches are currently being investigated.25 Creative development and application of this approach to other disorders may be translated into useful therapies for many diseases affecting the general population. Indeed, topical T4 endonuclease V in liposomes may have beneficial effects in the large number of repair-proficient normal persons who are at high risk for skin cancer. Topical enzyme therapy of skin diseases may soon be a reality. REFERENCES 1. Yarosh D, Klein J, O’Connor A, Hawk J, Rafal E, Wolf P. Effect of topically applied T4 endonuclease V in liposomes on skin cancer in xeroderma pigmentosum: a randomised study. Xeroderma Pigmentosum Study Group. Lancet 2001;357:926-9. 2. Kraemer KH, Lee MM, Scotto J. DNA repair protects against cutaneous and internal neoplasia: evidence from xeroderma pigmentosum. Carcinogenesis 1984;5:511-4. 3. Kraemer KH, Lee MM, Scotto J. Xeroderma pigmentosum: cutaneous, ocular, and neurologic abnormalities in 830 published cases. Arch Dermatol 1987;123:241-50. 4. Kraemer KH, Lee M-M, Andrews AD, Lambert WC. The role of sunlight and DNA repair in melanoma and nonmelanoma skin cancer: the xeroderma pigmentosum paradigm. Arch Dermatol 1994;130:1018-21. 5. Kraemer KH, DiGiovanna JJ, Moshell AN, Tarone RE, Peck GL. Prevention of skin cancer in xeroderma pigmentosum with the use of oral isotretinoin. N Engl J Med 1988;318:1633-7. 6. Yuspa SH, Dlugosz AA, Cheng CK, Denning MF, Tennenbaum T, Glick AB, et al. Role of oncogenes and tumor suppressor genes in multistage carcinogenesis. J Invest Dermatol 1994;103(Suppl): 90S-5S. 7. Kraemer KH. Sunlight and skin cancer: another link revealed. Proc Natl Acad Sci U S A 1997;94:11-4.
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