Effects of the isoflavone 4',5,7-trihydroxyisoflavone (genistein) on psoralen plus ultraviolet A radiation (PUVA)-induced photodamage

Eileen Q. Shyong1, Yuhun Lu1, Alison Lazinsky1, Rao N. Saladi1,2, Robert G. Phelps2, Lisa M. Austin1, Mark Lebwohl1 and Huachen Wei1,3

1 Department of Dermatology and
2 Department of Dermatopathology, Mount Sinai School of Medicine, 1425 Madison Avenue, New York, NY 10029, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Long-term psoralen plus ultraviolet A radiation (PUVA) therapy is associated with an increased risk of squamous cell carcinoma and malignant melanoma. Genistein (4',5,7-trihydroxyisoflavone), a major isoflavone in soybeans and a specific inhibitor of protein tyrosine kinase, has been shown to inhibit UVB induced skin carcinogenesis in hairless mice. For this study we examined the protective effects of topical genistein on PUVA-induced photodamage. In two separate experiments, genistein in a dimethyl sulfoxide/acetone (1:9) solution was applied to SKH-1 female mice 1 h post 8-methoxy-psoralen dosing and 1 h prior to UVA irradiation. Application of genistein significantly decreased PUVA-induced skin thickening, and greatly diminished cutaneous erythema and ulceration in a dose-dependent manner. Histological examination showed that PUVA treatment of mouse skin induced dramatic inflammatory changes throughout the epidermis; topical genistein prevented these changes without noticeable adverse effects. Cells containing cleaved poly(ADP-ribose) polymerase (PARP) and active caspase-3 were significantly increased in PUVA-treated skin (P < 0.05 and P < 0.0001, respectively) as compared with unexposed control skin. Topical genistein completely inhibited cleavage of PARP and caspase-3. Proliferating cell nuclear antigen (PCNA) positive cells were observed in suprabasal areas of the epidermis and were significantly decreased in PUVA-treated skin compared with both control samples and samples treated with PUVA plus topical genistein (P < 0.005). These results indicate that genistein protects the skin from PUVA-induced photodamage.

Abbreviations: DMSO, dimethyl sulfoxide; genistein, 4',5,7-trihydroxyisoflavone; 8-MOP, 8-methoxy-psoralen; PARP, poly(ADP-ribose) polymerase; PBS, phosphate-buffered saline; PCNA, proliferating cell nuclear antigen; PUVA, psoralen plus ultraviolet A radiation; UVA, ultraviolet A radiation


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Psoralen plus ultraviolet A (320–400 nm) (PUVA) therapy is an effective and widely used form of treatment of several dermatoses, including psoriasis, mycosis fungoides and vitiligo (reviewed in ref. 1). Studies have shown that PUVA not only has potent mutagenic and carcinogenic effects (24), but also has immunosuppressive effects that can allow tumor development or change immune function in both immunosuppressed and healthy individuals (5,6). Long-term PUVA therapy causes a significant increase in the risk for the development of skin cancers, particularly squamous cell carcinoma and malignant melanoma (reviewed in refs 1, 7 and 8). Recent studies are showing that PUVA-induced DNA damage in these patient populations is a factor for increasing cancer risk (9,10). Our goal in this study was to identify a substance that can reduce the risk of cancer in these patient populations.

Epidemiological studies have revealed that the incidence of and deaths from certain human cancers are significantly higher in Western populations than in Asian populations. One major difference between these populations is that the Asians consume 20–50 times more soy-based foods than Americans (1114). Soybean based diets have been shown to inhibit radiation- and chemical-induced tumors of the mammary, skin and liver (1517). The primary bioactive components of soy are isoflavones, which are hypothesized to be responsible for soybean's anticarcinogenic effects.

Genistein (4',5,7-trihydroxyisoflavone) is the most abundant of the soy isoflavones. It is a potent and specific inhibitor of tyrosine phosphorylation of the epidermal growth factor receptor; this phosphorylation event plays an inductive role in cell proliferation and transformation (18). Genistein significantly inhibits chemical carcinogen-induced reactive oxygen species, oxidative DNA damage and proto-oncogene expression, namely c-fos and c-jun (1923). Recently, it was demonstrated that genistein inhibits UVB-induced skin carcinogenesis in hairless mice and UVB-induced erythema in human skin (24,25). Thus, the focus of this study was to determine if genistein could also inhibit (i) PUVA-induced changes in cell populations expressing the proliferating cell nuclear antigen (PCNA) and (ii) PUVA-induced photodamage as determined through the presence of cleaved caspase-3 and its substrate, poly(ADP-ribose) polymerase (PARP), both of which play key roles in the cell's response to UV-induced DNA damage (2628).


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chemical reagents
Genistein, dimethyl sulfoxide (DMSO), phosphate-buffered saline (PBS) were obtained from Alexis Biochemical (San Diego, CA), Sigma (St Louis, MO) and Fisher Scientific (Fairlawn, NJ), respectively. 8-Methoxy-psoralen (8-MOP) (Sigma) was initially dissolved in Tween 20 obtained from Fisher Scientific and then diluted with distilled water.

Animal treatment and PUVA therapy
Female SKH-1 mice, 6–7 weeks old, were purchased from Biological Testing (NCI, Frederick) and kept at the Mount Sinai Animal Facility under standard conditions (12 h light/12 h dark cycle, humidity at 50 ± 15%, temperature 22 ± 2°C and 12 air changes/h) for 1 week prior to experimentation. Mice were split into six groups of four mice each: (i) no treatment; (ii) ultraviolet A (UVA) only; (iii) 8-MOP only; (iv) PUVA only; (v) PUVA + 0.5 nmol/cm2 topical genistein; (vi) PUVA + 2 nmol/cm2 topical genistein. The Daavlin Phototherapy Unit was used to irradiate mice. It consisted of eight UVA lamps (F24T12BL-UVA-HO; Voltare, Fairfield, CT) with the predominant emitting peak at 320–360 nm. For UV exposure, four mice were placed under eight UVA lamps. The distance between UV lamps to mouse dorsal skin and the surface of the UVA detector was 40 cm. The dose of UVA was quantified and controlled by a UVA SEE038 detector (Daavlin, Bryan, OH). Applicable mice were fed 8-MOP via gastric gavage. One hour after 8-MOP administration (10 mg/kg) and 1 h prior to UVA irradiation (25 kJ/m2), genistein (0.5 or 2 nmol/cm2) was applied topically to the backs of each mouse in a DMSO/acetone (1:9) solution. A DMSO/acetone (1:9) solution without genistein was applied to mice treated only with PUVA. Optimal 8-MOP dosing and UVA irradiation were determined in preliminary mouse studies. Skin thickness measurements were taken 24, 48 and 72 h after treatment as described later. Photographs were taken at each of these time points as well as 1 week post-treatment. Each experiment was repeated three times with four mice per group. For immunohistochemical studies, 8-MOP dosing was increased to 20 mg/kg. Skin samples were taken 24 h post-PUVA treatment.

Histological and immunohistochemical staining
The expression of PCNA, cleaved PARP and cleaved (active) caspase-3 were demonstrated using the avidin–biotin complex immunoperoxidase method and the following antibodies: PCNA FL-261 (dilution 1:200) (Santa Cruz Biotechnology, Santa Cruz, CA), cleaved PARP antibody D214 (dilution 1:100) (Cell Signaling Technology, Beverly, MA) recognizing the large cleaved fragment of PARP (89 kDa) and anti-active caspase-3 monoclonal antibody C92-605 (dilution 1:1000) (PharMingen, San Diego, CA).

Skin samples of the dorsal skin (~2x2 cm) of each animal were obtained, fixed in formalin then embedded in paraffin, serially cut into 4 µm sections and mounted on poly-L-lysine (Sigma)-coated glass slides. Samples were stained with hematoxylin and eosin for light microscopic examination. Additional samples were deparaffinized in xylene, dehydrated in graded ethanol and then treated with 1% hydrogen peroxide for 20 min to block endogenous peroxidases. Treating the sections in a 1% non-fat milk blocking solution for 30 min blocked non-specific binding. The sections were incubated with PCNA, PARP or caspase-3 antibodies overnight at room temperature and then blocked with 10% normal serum specific for each secondary antibody in PBS for 40 min. (Prior to incubation with PCNA, the sections were heated in 10 mM citric acid monohydrate, pH 6.0, in a microwave oven.) Sections were then treated with biotinylated anti-mouse (dilution 1:200) (PARP) or anti-rabbit immunoglobulin (dilution 1:200) (caspase-3, PCNA) for 30–60 min and the avidin–biotin complex (30 min) (all from Vector Laboratories, Burlingame, CA). The color was developed with 3-amino-9-ethylcarbazole and the samples were lightly counterstained with methyl green (both from Vector Laboratories). The nucleus stains brown in a positive case and greenish-blue in a negative case. Mouse skin treated with both benzo[a]pyrene (100 µg) and 50 kJ/m2 UVA were used as positive controls for all antibodies. Sections incubated with mouse or rabbit IgG were used as negative controls. Labeling indices of cleaved PARP, active caspase-3 and PCNA were calculated from the number of positively stained cells/100 cells using NIH Image Analysis 1.62. Skin thickness measurements were made from hematoxylin and eosin stained paraffin embedded tissue also using this program.

Statistical analysis
The data representing skin-fold thickness and positively stained cells were analyzed by two sample Student's t-test assuming equal variances. All data are given as the mean ± SD.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Effects of genistein on PUVA-induced cutaneous changes
To evaluate if genistein could inhibit PUVA-induced cutaneous changes, genistein was topically applied to the backs of each applicable mouse after 8-MOP administration and prior to UVA irradiation. Application of genistein greatly diminished cutaneous erythema and ulceration in a dose-dependent manner (Figure 1 A–DGo). As expected, mice treated with UVA or 8-MOP alone showed no cutaneous changes. PUVA treatment significantly increased epidermal thickness as compared with unexposed control skin (p < 0.001). Two nmol/cm2 of genistein significantly decreased PUVA-induced skin thickening and edema (p < 0.0001) (Figure 2Go). Histological examination of mouse skin treated with PUVA alone displayed dramatic inflammatory changes as evidenced by epidermal acanthosis, dermal neutrophilic/lymphocytic infiltration, and prominent hydropic degeneration with extensive dyskeratotic and atypical keratinocytes throughout the epidermis. Genistein treatment inhibited these PUVA-induced inflammatory changes in a dose-dependent manner (Figure 1E–HGo). Mouse skin treated with UVA or 8-MOP alone showed no histological changes as compared to unexposed control skin (data not shown).



View larger version (47K):
[in this window]
[in a new window]
 
Fig. 1. Topical genistein inhibits PUVA-induced skin erythema and histological changes. (A–H) All applicable mice were given 10 mg/kg 8-methoxy-psoralen (8-MOP) and/or 25 kJ/m2 UVA. Genistein (0.5 or 2 nmol/cm2) in a dimethyl sulfoxide/acetone (1:9) solution was applied to SKH-1 female mice 1 h post 8-MOP dosing and 1 h prior to UVA irradiation. (A and E) Unexposed control; (B and F) psoralen plus ultraviolet A radiation (PUVA) only; (C and G) PUVA + 0.5 nmol/cm2 genistein and (D and H) PUVA plus 2 nmol/cm2 genistein. Each pictured mouse and stained section is representative of the other mice within the group. Gross pictures were taken 1 week post treatment. Biopsies for hematoxylin and eosin {sigma}taining were collected 72 h post treatment from the area indicated by the arrow heads. Scale bar, 12 µm

 


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 2. Topical genistein inhibits psoralen plus ultraviolet A radiation (PUVA)-induced skin thickening in a dose-dependent manner. All applicable mice were given 10 mg/kg 8-methoxy-psoralen (8-MOP) and/or 25 kJ/m2 UVA. Genistein (GST) (0.5 or 2 nmol/cm2) in a dimethyl sulfoxide/acetone (1:9) solution was applied to SKH-1 female mice 1 h post 8-MOP dosing and 1 h prior to UVA irradiation. Tissue samples of unexposed control, UVA only, 8-MOP only, PUVA only, PUVA + 0.5 nmol/cm2 genistein and PUVA + 2 nmol/cm2 genistein were taken 72 h post treatment. The results are expressed as the mean ± SD. Statistical analysis of the difference in skin thickness was conducted using the two sample Student's t-test of equal variance. *, P < 0.001 for PUVA versus unexposed control, 8-MOP only, and UVA only. +, P < 0.01 for PUVA versus PUVA + 0.5 nmol/cm2 genistein and ++, P < 0.0001 for PUVA versus PUVA + 2 nmol/cm2 genistein.

 
Effects of genistein on PUVA-induced cleaved PARP and active caspase-3 expression
In this study, we evaluated the effect of genistein on PUVA-induced DNA damage. Twenty-four hours after a single PUVA treatment, detection of cleaved PARP and active caspase-3 significantly increased by 26.1 ± 3.7%/100 cells (P < 0.05) and 34.7 ± 2.0%/100 cells (P < 0.0001), respectively, as compared with unexposed control skin. Expression of cleaved PARP and active caspase-3 was completely inhibited in skin treated with topical genistein (Figure 3A–FGo). 8-MOP or UVA only treated mice showed no positive cells for any of these proteins (data not shown).



View larger version (99K):
[in this window]
[in a new window]
 
Fig. 3. The effect of genistein on psoralen plus ultraviolet A radiation (PUVA)-induced apoptotic and proliferating pathway. (A–I) All applicable mice were given 20 mg/kg 8-methoxy-psoralen (8-MOP) and/or 25 kJ/m2 UVA. Genistein (2 nmol/cm2) in a dimethyl sulfoxide/acetone (1:9) solution was applied to SKH-1 female mice 1 h post 8-MOP dosing and 1 h prior to UVA irradiation. Samples were collected 24 h after treatment. Data represented here are from a typical experiment. Scale bar, 12 µm. (A–C) Active caspase-3 and (D–F) cleaved PARP immunohistochemical staining of formalin-fixed, paraffin-embedded tissue of (A and D) unexposed control; (B and E) PUVA only and (C and F) PUVA + 2 nmol/cm2 genistein-treated mice. Cells positive for active caspase-3 (P < 0.0001) and cleaved PARP (p < 0.05) were significantly increased as compared with unexposed control mice and mice treated with PUVA + 2 nmol/cm2 genistein. (G–I) PCNA immunohistochemical staining of formalin-fixed, paraffin-embedded tissue of (G) unexposed control; (H) PUVA only and (I) PUVA + 2 nmol/cm2 genistein-treated mice. Compared with unexposed control mice, PUVA-treated mice showed a significant decrease in PCNA positive cells (p < 0.005). The number of PCNA positive cells between (G) and (I) was not significantly different.

 
Effects of genistein on PCNA expression
The expression of PCNA, a marker for cell proliferation, was found in 29.3 ± 1.6%/100 cells cells in PUVA-treated skin as compared with 46.4 ± 5.4%/100 cells in unexposed skin (P < 0.005) (Figure 3G–IGo). The number of positively stained cells in skin treated with genistein plus PUVA, 40.7 ± 4.6%/100 cells, was not significantly different from the number of positively stained cells in unexposed, UVA only, 42.3 ± 0.41%/100 cells, or 8-MOP only treated skin, 39.5 ± 4.1%/100 cells (all P > 0.05). Immunostaining for PCNA in unexposed control, UVA only, 8-MOP only, PUVA plus genistein-treated skin showed the majority of positive cells within the basal layer of the epidermis. In addition to staining in the basal layer, PUVA-treated skin also showed positive cells in the suprabasal area.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
This study shows that both 0.5 and 2 nmol/cm2 genistein significantly decreased PUVA-induced skin thickening and edema, and greatly diminished cutaneous erythema and ulceration in a dose-dependent manner. Genistein also inhibited PUVA-induced inflammatory changes (Figure 1Go). Although one may question whether the effects of genistein are due to a `sunscreen effect', this can be ruled out because based on studies conducted in our laboratory in 10 cm2 Petri dishes, genistein has a maximum absorption of 262 nm and only begins blocking (inhibition of UVA penetration by 20% or greater) UVA radiation at a concentration of 400 nmol. At levels above 400 nmol, genistein precipitated out of solution.

PUVA treatment of skin clearly induces the type of DNA damage that triggers apoptotic biochemical pathways. PUVA sunburned SKH-1 mice had erythematous, thickened skin containing lower numbers of cycling cells and signs of apoptotic cells. Apoptosis is characterized by changes in cellular morphology and serial cleavage of molecules along the biochemical apoptotic pathway that lead to cell death. Caspase-3, which is synthesized as an inactive proenzyme and activated when its bioactive component is cleaved from the main molecule, plays a central role along the apoptotic pathway (29). The caspase-3 cleavage fragment then cleaves PARP (3032). PARP is a chromatin-associated nuclear enzyme that catalyzes the poly ADP-ribosylation of a variety of nuclear proteins. The catalytic ability of PARP is closely associated with the presence of strand breaks in DNA and has been shown to play a pivotal role in DNA damage repair and the execution of programmed cell death (reviewed in refs 33 and 34). Thus, activation of caspase-3 and the subsequent cleavage of PARP prevent replication of damaged DNA templates. Our results show that the topical application of genistein inhibited the expression of active caspase-3 and cleaved PARP induced by PUVA treatment. This suggests that genistein inhibits the initial PUVA-induced event, DNA damage, thus subsequently blocking the need for apoptosis to occur. This is supported by our previous publication where we demonstrated that genistein significantly inhibited PUVA-induced formation of 8-hydroxy-2'-deoxyguanosine and DNA fragmentation in mammalian cells (35). Thus, blocking active caspase-3 and cleaved PARP expression indirectly reflects a protective effect of genistein on PUVA-induced DNA damage. To our knowledge, this is the first time that these biomarkers have been studied in relation to PUVA and/or genistein. Our hypothesis for the site of action of genistein is detailed in Figure 4Go.



View larger version (35K):
[in this window]
[in a new window]
 
Fig. 4. Hypothetical model for the site of action of genistein. Psoralen plus ultraviolet A radiation induces DNA strand breaks, initiating the early apoptotic pathway that includes caspase-3 cleavage with subsequent poly(ADP-ribose) polymerase (PARP) cleavage. The catalytic activity of PARP is closely associated with the presence of strand breaks in DNA and has been shown to play a pivotal role in DNA damage repair. Since genistein inhibits caspase-3 cleavage and PARP cleavage, we hypothesize that genistein acts to inhibit the initial DNA strand breaks by an as of yet unidentified mechanism.

 
In this study we also show that the overall expression of PCNA in PUVA-treated skin samples (29. 3 ± 1.6%/100 cells) was decreased as compared with unexposed skin (46.4 ± 5.4%)/100 cells or PUVA plus 2 nmol/cm2 genistein-treated skin (40.7 ± 4.6%)/100 cells. PCNA is a well-known marker for cellular hyperproliferation (36,37). It is required for DNA replication and excision repair of DNA adducts or photoproducts (36,38). Although one would expect an increased percentage of PCNA positive cells since the cellular proliferation rate is enhanced by inducers of chronic inflammation (39), it is possible that the DNA damage induced by PUVA was so acute and extensive that it inhibited cell proliferation. The distribution of PCNA positive cells was also very different in PUVA-treated skin as compared with all other tested treatments. Specifically, positive immunostained cells were observed only in the basal layer of control samples. This would be expected because of the presence of proliferating stem cells within the basal layer of normal epidermis. PUVA-treated skin showed positive cells in suprabasal areas of the epidermis while skin treated with PUVA plus genistein showed normal staining patterns comparable with control skin. Because cellular proliferation rate is a factor that plays a role in the progression of intraepithelial neoplasia (39), these findings suggest that genistein may inhibit PUVA-induced carcinogenesis.

In summary, our results indicate that genistein may serve as a preventive agent against PUVA-induced photodamage, and thus, possibly PUVA-induced carcinogenesis. Clinical trials on the photoprotective ability of genistein are currently underway to determine the protective effects of genistein on PUVA-treated patients.


    Notes
 
3 To whom correspondence should be addressedEmail: huachen_wei{at}mountsinai.org Back


    Acknowledgments
 
This work was supported by research grants awarded to Dr. T.Huachen Wei from the National Caner Institute (RO1CA85360), the American Institute for Cancer Research (A017REN), and the Dermatology Foundation, as well as the Sigma Xi Grant in Aid of Research awarded to Eileen Shyong.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. Momtaz,K. and Fitzpatrick,T.B. (1998) The benefits and risks of long-term PUVA photochemotherapy. Dermatol. Clin., 16, 227–234.[ISI][Medline]
  2. Gunther,E.J., Yeasky,T.M., Gasparro,F.P. and Glazer,P.M. (1995) Mutagenesis by 8-methoxypsoralen and 5-methylangelicin photoadducts in mouse fibroblasts: mutations at cross-linkable sites induced by offoadducts as well as cross-links. Cancer Res., 55, 1283–1288.[Abstract]
  3. Yang,S.-C., Lin,J.-G., Chiou,C.-C., Chen,L.-Y. and Yang,J.-L. (1994) Mutation specificity of 8-methoxypsoralen plus two doses of UVA irradiation in the hprt gene in diploid human fibroblasts. Carcinogenesis, 15, 201–207.[Abstract]
  4. Kripke,M.L., Morison,W.L. and Parrish,J.A. (1982) Induction and transplantation of murine skin cancers induced by methox salen plus ultraviolet (320–400 nm) radiation. J. Natl Cancer Inst., 68, 685–690.[ISI][Medline]
  5. Morison,W.L., Wimberly,J., Parrish,J.A. and Bloch,K.J. (1983) Abnormal lymphocyte function following long-term PUVA therapy for psoriasis. Br. J. Dermatol., 18, 445–450.
  6. Moscicki,R.A., Morison,W.L., Parrish,J.A., Bloch,K.J. and Colvin,R.B. (1982) Reduction of the function of circulating helper-inducer T-cell identified by monoclonal antibodies in psoriatic patients treated with long-term 8-MOP/ultraviolet-A radiation (PUVA). J. Invest. Dermatol., 79, 205–208.[Abstract]
  7. Wang,S.Q., Setlow,R., Berwick,M., Polsky,D., Marghoob,A.A., Kopf,A.W. and Bart,R.S. (2001) Ultraviolet A and melanoma: a review. J. Am. Acad. Dermatol., 44, 837–846.[ISI][Medline]
  8. Wolff,K. (1988) Side-effects of psoralen photochemotherapy (PUVA). Br. J. Dermatol., 122 (Suppl. 36), 117–125.
  9. Nataraj,A.J., Black,H.S. and Ananthaswamy,H.N. (1996) Signature p53 mutation at DNA cross-linking sites in 8-methoxypsoralen and ultraviolet A (PUVA)-induced murine skin cancers. Proc. Natl Acad. Sci. USA, 93, 7961–7965.[Abstract/Free Full Text]
  10. Liu,Z., Lu,Y., Lebwohl,M. and Wei,H. (1999) PUVA (8-methoxy-psoralen plus ultraviolet A) induces the formation of 8-hydroxy-2'-deoxyguanosine and DNA fragmentation in calf thymus DNA and human epidermoid carcinoma cells. Free Radic. Biol. Med., 27, 127–133.[ISI][Medline]
  11. Setchell,K.D.R., Borriello,S.P., Kirk,D.N. and Axelson,M. (1984) Nonsteroidal estrogens of dietary origin: Possible role in hormone-dependent disease. Am. J. Clin. Nutr., 40, 569–578.[Abstract]
  12. Shimizu,H., Ross,R.K., Bernstein,L., Yatani,R., Henerson,B.E. and Mack,T.M. (1991) Cancers of the prostate and breast among Japanese and white immigrants in Los Angeles County. Br. J. Cancer, 63, 963–966.[ISI][Medline]
  13. Tominga,S. (1985) Cancer incidence in Japanese in Japan, Hawaii, and western United States. Natl Cancer Inst. Monogr., 69, 83–92.
  14. Messina,M. and Barnes,S. (1991) The role of soy products in reducing risk of cancer. J. Natl Cancer Inst., 83, 541–546.[ISI][Medline]
  15. Troll,W., Klassen,A. and Janoff,A. (1970) Tumorigenesis in mouse skin: Inhibition by synthetic inhibitors of proteases. Science, 169, 1211–1213.[ISI][Medline]
  16. Troll,W., Weisner,R., Shellabarger,C.J., Holtzman,S. and Stone,J.P. (1980) Soybean diet lowers breast tumor incidence in irradiated rats. Carcinogenesis, 1, 469–472.[Abstract]
  17. Becher,F.F. (1981) Inhibition of spontaneous hepatocarcinogenesis in C3H/HEN mice by Edipro A and isolated soy protein. Carcinogenesis, 2, 1213–1214.[ISI][Medline]
  18. Akiyama,T., Ishida,J., Nakagawa,S., Ogawara,H., Watanabe,S., Itoh,N., Shibuya,M. and Fukami,Y. (1987) Genistein, a specific inhibitor of tyrosine-specific protein kinases. J. Biol. Chem., 262, 5592–5595.[Abstract/Free Full Text]
  19. Wei,H., Wei,L., Frenkel,K., Bowen,R. and Barnes,S. (1993) Inhibition of tumor promoter-induced hydrogen peroxide production in vitro and in vivo by genistein. Nutr. Cancer, 2, 1–12.
  20. Wei,H., Barnes,S. and Wang,Y. (1996) The inhibitory effect of genistein on a tumor promoter c-fos and c-jun expression in mouse skin. Oncol. Rep., 3, 125–128.[ISI]
  21. Wei,H., Bowen,R., Barnes,S., Cai,Q. and Wang,Y. (1995) Antioxidant and anticarcinogenic properties of the soybean isoflavone genistein. Proc. Soc. Exp. Biol. Med., 208, 124–130.[Abstract]
  22. Wang,Y., Yaping,E., Zhang,X., Lebwohl,M., DeLeo,V. and Wei,H. (1998) Inhibition of ultraviolet B (UVB)-induced c-fos and c-jun expression in vivo by a protein tyrosine kinase inhibitor genistein. Carcinogenesis, 19, 649–654.[Abstract]
  23. Wei,H., Bowen,R., Zhang,X. and Lebwohl,M. (1998) Isoflavone genistein inhibits the initiation and promotion of two-stage skin carcinogenesis in mice. Carcinogenesis, 19, 1509–1514.[Abstract]
  24. Wei,H. (1998) Photoprotective action of isoflavone genistein: Models, mechanisms, and relevance to clinical dermatology. J. Am. Acad. Dermatol., 39, 271–272.[ISI][Medline]
  25. Wei,H., Spencer,J., Gelfand,J., Phelps,R. and Lebwohl,M. (2001) The isoflavone genistein, a new agent in dermatology? Cosmet. Dermatol., 14, 13–19.
  26. Kim,J.W., Won,J., Sohn,S. and Joe,C.O. (2000) DNA-binding activity of the N-terminal cleavage product of poly(ADP-ribose) polymerase is required for UV mediated apoptosis. J. Cell. Sci., 113, 955–961.[Abstract/Free Full Text]
  27. Fernet,M., Ponette,V., Deniaud-Alexandre,E., Menissier de Murcia,J., De Murcia,G., Giocanti,N., Megnin-Chanet,F. and Favaudon,V. (2000) Poly(ADP-ribose) polymerase, a major determinant of early cell response to ionizing radiation. Int. J. Radiat. Biol., 76, 1621–1629.[ISI][Medline]
  28. Chan,W.-H. and Yu,J.-S. (2000) Inhibition of UV irradiation-induced oxidative stress and apoptotic biochemical changes in human epidermal carcinoma A431 cells by genistein. J. Cell. Biochem., 78, 73–84.[ISI][Medline]
  29. Thornberry,N.A. and Lazebnik,Y. (1998) Caspases: enemies within. Science, 281, 1312–1316.[Abstract/Free Full Text]
  30. Stadelmann,C. and Lassmann,H. (2000) Detection of apoptosis in tissue sections. Cell Tissue Res., 301, 19–31.[ISI][Medline]
  31. Cryns,V. and Yuan,J. (1998) Proteases to die for. Genes Dev., 125, 1551–1570.
  32. Tewari,M., Quan,L.T., O'Rourke,K., Desnoyers,S., Zeng,Z., Beidler,D.R., Poirier,G.G., Salvesen,G.S. and Dixit,V.M. (1995) Yama/CPP32 beta, a mammalian homolog of CED-3, is a CrmA-inhibitable protease that cleaves the death substrate poly(ADP-ribose) polymerase. Cell, 81, 801–809.[ISI][Medline]
  33. Bürkle,A. (2001) Poly(APD-ribosyl)ation, a DNA damage-driven protein modification and regulator of genomic instability. Cancer Lett., 163, 1–5.[ISI][Medline]
  34. Herceg,Z. and Wang,Z.Q. (2001) Functions of poly(ADP-ribose) polymerase (PARP) in DNA repair, genomic integrity and cell death. Mutat. Res., 477, 97–110.[ISI][Medline]
  35. Liu,Z., Lu,Y., Lebwohl,M. and Wei,H. (1999) PUVA (8-methoxy-psoralen plus ultraviolet A) induces the formation of 8-hydroxy-2'-deoxyguanosine and DNA fragmentation in calf thymus DNA and human epidermoid carcinoma cells. Free Radic. Biol. Med., 27,127–133.[ISI][Medline]
  36. Shivji,K.K., Kenny,M.K. and Wood,R.D. (1992) Proliferating cell nuclear antigen is required for DNA excision repair. Cell, 69, 367–374.[ISI][Medline]
  37. Yang,G., Wang,Z.Y., Kim,S., Liao,J., Seril,D.N., Chen,X., Smith,T.J. and Yang,C.S. (1997) Characterization of early pulmonary hyperproliferation and tumor progression and their inhibition by black tea in a 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced lung tumorigenesis model with A/J mice. Cancer Res., 57, 1889–1894.[Abstract]
  38. Celis,J.E. and Madson,P. (1986) Increased nuclear cyclin/PCNA antigen staining of non S-phase transformed human amnion cells engaged in nucleotide excision DNA repair. FEBS Lett., 209, 277–283.[ISI][Medline]
  39. Boone,C.W., Keloff,G.J. and Steele,V.E. (1992) Natural history of intraepithelial neoplasia in humans with implications for cancer chemoprevention strategy. Cancer Res., 52, 1651–1659.[Abstract]
Received July 6, 2001; revised September 7, 2001; accepted October 9, 2001.