Protection against induction of mouse skin ... - Carcinogenesis

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Carcinogenesis vol.18 no.3 pp.497–502, 1997

Protection against induction of mouse skin papillomas with low and high risk of conversion to malignancy by green tea polyphenols

Santosh K.Katiyar, Rajiv R.Mohan, Rajesh Agarwal and Hasan Mukhtar1 Department of Dermatology, Skin Diseases Research Center, University Hospitals of Cleveland, Case Western Reserve University, Cleveland, OH 44106-5028, USA 1To

whom correspondence should be addressed

We earlier showed that a polyphenolic fraction isolated from green tea (GTP) affords protection against tumor promotion and tumor progression in SENCAR mouse skin. The present study was designed to further evaluate the protective effect of GTP against the induction and subsequent progression of papillomas to squamous cell carcinomas (SCCs) in experimental protocols where papillomas were developed with a low or high probability of their malignant conversion. Topical application of GTP (6 mg/ animal) 30 min prior to that of 12-O-tetradecanoylphorbol13-acetate (TPA) either once a week for 5 weeks (high risk TPA protocol) or once a week for 20 weeks (low risk TPA protocol) or mezerein (MEZ) twice a week for 20 weeks (high risk MEZ protocol) in 7,12-dimethylbenz[a]anthracene (DMBA)-initiated mouse skin resulted in significant protection against skin tumor promotion in terms of tumor incidence (32–60%), multiplicity (49–63%) and tumor volume/mouse (73–90%) at the termination of the experiment at 20 weeks. In three separate malignant progression experiments when papilloma yield in DMBA-initiated and TPA or MEZ promoted low and high risk protocols was stabilized at 20 weeks, animals were divided into two subgroups. These animals were either topically treated twice weekly with acetone (0.2 ml/animal, spontaneous malignant conversion group) or with GTP (6 mg/animal in 0.2 ml acetone) for an additional period of 31 weeks. During these treatment regimens, all suspected carcinomas were recorded and each one was verified histopathologically either at the time when tumor-bearing mouse died/ moribund or at the termination of the experiment at 51 weeks. GTP resulted in significant protection against the malignant conversion of papillomas to SCC in all the protocols employed. At the termination of the experiment at 51 weeks, these protective effects were evident in terms of mice with carcinomas (35–41%), carcinomas per mouse (47–55%) and percent malignant conversion of papillomas to carcinomas (47–58%). The kinetics of malignant conversion suggest that a subset of papillomas formed in the early phase of tumor promotion in all the protocols had a higher probability of malignant conversion into SCCs because all the positive control groups (acetone treated) produced nearly the same number of carcinomas (33–38 in a group *Abbreviations: DMBA, 7,12-dimethylbenz[a]anthracene; TPA, 12-Otetradecanoylphorbol-13-acetate; GTP, polyphenolic fraction isolated from green tea; SSCs, squamous cell carcinomas; MEZ, mezerein; LR-TPA, low risk TPA; HR-TPA, high risk TPA; HR-MEZ, high risk MEZ; LC, Langerhans cells. © Oxford University Press

of 20 animals) at the end of the progression period. In the GTP-treated group of animals the number of carcinomas formed was less (14–20 in a group of 20 animals), which shows the ability of GTP to protect against the malignant conversion of papillomas of higher probability of malignant conversion to SCCs. The results of this study suggest that irrespective of the risk involved, GTP may be highly useful in affording protection against skin cancer risk.

Introduction Multistage models of carcinogenesis have been developed for various tissues and animal species for mechanistic studies. These animal models also play an important role as in vivo test systems in the identification of exogenous and/or endogenous agents playing a role in enhancement and prevention of various stages of carcinogenesis. In this context the initiation– promotion protocol of mouse skin carcinogenesis has been widely used both for studies on mechanisms involved in chemical carcinogenesis and for testing of carcinogenic or anti-carcinogenic agents (1–3). In the mouse skin the process of carcinoma formation has been subdivided into at least three operationally defined stages: initiation, promotion and malignant progression (2,3). Collective information from numerous studies using the initiation–promotion model of skin carcinogenesis suggests a sequence of events starting from an initiated cell, passing through a benign papilloma stage and finally leading to the development of carcinomas (4). Initiation can be brought about by a single topical application of a subcarcinogenic dose of a carcinogen, such as 7,12-dimethylbenz[a]anthracene (DMBA*). Initiation causes a permanent genetic alteration in some epidermal cells (initiated cells), which give rise to epidermal tumors only upon subsequent promotion. Promotion of the DMBA-initiated skin to generate benign tumors (papillomas) is accomplished by repeated topical applications of a promoting agent, such as the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Carcinoma formation apparently does not occur in initiated skin without promotion, indicating that the latter is a prerequisite for malignant progression, i.e. carcinomas usually develop from papillomas, but in some other skin tumor protocols many carcinomas do not first go through the papilloma stage (4,5). However, such a view is complicated by the observation of a substantial heterogeneity among papillomas (reviewed in 5). Many papillomas have been found to require continued exposure to TPA in order to prevent regression (6–9), whereas some regress even in the presence of the promoter (9). A certain percentage of papillomas has been shown to persist for the lifetime of the animals and a definite percentage of these persistent papillomas spontaneously progress into carcinomas (6,7,10). In addition, these so-called high risk papillomas have been shown to arise early on and to be more sensitive to mutagen-induced progression in comparison with the so-called low risk papil497

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lomas (9–11), indicating that they may result from different populations of initiated cells rather than develop from promoterdependent precursor papillomas. This situation becomes more complicated by the fact that the ratio of persistent to reversible papillomas is different in mouse strains of different susceptibility to promoters (9). Tea, a product of Camellia sinensis, is a popular beverage consumed world wide and contains several polyphenolic antioxidants. These antioxidants play an important role as inhibitors, at least in the initiation and promotion stages, of multistage carcinogenesis (12). In earlier studies, by using the multistage model of skin carcinogenesis, we and others have shown that a polyphenolic fraction isolated from green tea (GTP) possesses significant chemopreventive effects against TPA-induced skin tumor promotion in DMBA-initiated SENCAR mouse skin (13–15). In addition, we have shown that topical application of GTP also affords protection against spontaneous malignant conversion as well as against malignant conversion enhanced by the free radical-generating compound benzoyl peroxide and the carcinogenic agent 4-nitroquinoline N-oxide in SENCAR mice (16). To gain further knowledge about the anticarcinogenic effects of GTP, in the present study experimental protocols were adopted to elicit papillomas with a lower as well as higher probability of malignant conversion in mouse skin (17,18). Thus the aim of our study was to evaluate the chemopreventive efficacy of GTP among a heterogenous population of papillomas with variable risk of cancer induction. By using three different protocols of the mouse skin multistage chemical carcinogenesis model, the effect of GTP was assessed against tumor promotion as well as subsequent progression of papillomas to squamous cell carcinomas (SCC). Materials and methods Chemicals TPA and mezerein (MEZ) were purchased from Sigma Chemical Co. (St Louis, MO) and DMBA was obtained from Aldrich Chemical Co. (Milwaukee, WI). GTP was prepared from green tea leaves as described earlier and is a mixture of four epicatechin derivatives, namely (–)-epicatechin, (–)-epicatechin 3-gallate, (–)-epigallocatechin and (–)-epigallocatechin 3-gallate (19). All other chemicals were of the highest quality commercially available. Animals Six–seven week old female SENCAR mice, obtained from Harlan-Sprague Dawley (Indianapolis, IN), were acclimatized for 1 week before use and subjected to a 12 h light/12 h dark cycle, housed at 24 6 2°C and 50 6 10% relative humidity in a room with 12–15 cycles of air exchange/h and fed Purina chow diet and water ad libitum. Induction of papillomas in SENCAR mouse skin One week after their arrival, the dorsal skin of the mice was shaved using electric clippers and then Nair depilatory cream was applied. Only those mice which were in the resting phase of their hair cycle were used in this study. The details of tumor protocols employed are those described earlier (17,18). In brief, mice were divided into three groups termed Groups I, II and III (40 mice in each) and treated topically on the shaved area with a single application of DMBA (20 µg/mouse in 0.2 ml acetone). One week later, mice in all three groups were subdivided randomly into two subgroups, A and B. The animals in Group 1A were treated topically once a week with TPA (2 µg/mouse in 0.2 ml acetone) for 20 weeks, designated the ‘low risk TPA protocol’ (LRTPA). In this protocol the papillomas which develop are known to have a low probability of conversion to carcinomas (17,18). The mice in Group 1B were topically treated with GTP (6 mg/mouse in 0.2 ml acetone) 30 min before each TPA application as in Group 1A. The animals in Group IIA were topically treated once a week with TPA (2 µg/mouse in 0.2 ml acetone) for 5 weeks and then were left for an additional 15 weeks, designated the ‘high risk TPA protocol’ (HR-TPA). In this protocol the papillomas which develop have a high probability of conversion to carcinomas (17,18). The mice in Group IIB were topically treated with GTP (6 mg/mouse in 0.2 ml acetone)

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30 min before each TPA application as in Group IIA. The mice in Group IIIA were topically treated twice a week with MEZ (4 µg/mouse in 0.2 ml acetone) for 20 weeks, designated the ‘high-risk MEZ protocol’ (HR-MEZ). The papillomas which develop in this protocol have a high probability of conversion to carcinomas (18). The mice in Group IIIB were topically treated with GTP (6 mg/mouse in 0.2 ml acetone) 30 min before each MEZ application as in Group IIIA. All the treatments of TPA or MEZ, GTP1TPA or GTP1MEZ were topical on the same defined area of the dorsal skin of the mice which had previously been initiated with DMBA. Skin tumors .1 mm in diameter which persisted for ù2 weeks were recorded. The tumor incidence and multiplicity were determined weekly until week 20 in each group of animals. At the end of week 20, when papilloma yield was stabilized in each group with no additional tumor appearance, the volume of each tumor was determined. Malignant progression of papillomas to carcinomas in SENCAR mouse skin To study whether GTP can afford protection against malignant progression of the tumors having a low or high risk of probability of conversion into SCC in the multistage carcinogenesis model, we took 80 mice in three separate groups. In these mice, tumors were induced by the topical application of tumor promoters in DMBA-initiated SENCAR mice using the same protocols described for Groups IA, IIA and IIIA. In brief, Group I will develop papillomas with low probability of conversion into SCC, while Groups II and III will develop papillomas with a higher probability of conversion into SCC. When the tumor yield was stabilized at week 20, out of 80 mice in each group, 40 tumor-bearing mice which bore an almost identical number of papillomas were selected and the rest of the mice were removed from the study. No carcinomas were present on any mice selected for the progression study at the start of this experiment, i.e. at 20 weeks. These 40 mice were divided into two subgroups of 20 mice in each. In each of the three protocols, mice in the first subgroup were treated twice weekly with acetone (0.2 ml/ mouse), whereas in the second subgroup they were treated twice weekly with GTP (6 mg/mouse in 0.2 ml acetone). These treatments were continued up to the end of the experiment at week 51 (31 weeks after promoter treatment). Carcinomas formed on each mouse during these treatment protocols were counted grossly as downward invading lesions per week and each one was verified histopathologically at the time of termination of the experiment at week 51 as described earlier (17,18). In addition, when carcinoma-bearing mice were moribund, they were killed and all tumors were harvested for histological verification. The criteria for the diagnoses of various tumors were based on those described earlier (20). The percent malignant conversion of benign skin papillomas into SCCs was calculated by dividing the total number of carcinomas by the total number of papillomas (papillomas used in the study at the beginning of the progression experiment at 20 weeks) and multiplying by 100 (18,20). Histological evaluation Tumor tissue, from papilloma-bearing or suspected carcinoma-bearing mice, for histological evaluation was prepared using conventional paraffin sections and hematoxylin and eosin staining. Histology was performed for all the tumors at week 20, i.e. at the end of the tumor promotion experiment, and in week 51 of the progression experiment. SCCs were diagnosed microscopically as downward invading lesions. Statistical analysis The statistical significance of differences between positive controls and GTPtreated experimental groups in terms of papilloma and carcinoma incidence and multiplicity was evaluated by χ2 analysis and the Wilcoxon rank sum test.

Results Effect of GTP on tumor promotion In previous studies we and others (13–15) have shown that topical applications of GTP before each application of TPA affords substantial protection against tumor formation in DMBA-initiated SENCAR mouse skin in a dose-dependent manner. In the present study we modulated the development of the number of papillomas per mouse by adopting three different protocols (17) which have a high or low probability for conversion into carcinomas. Topical applications of GTP prior to each application of the tumor promoters in DMBAinitiated SENCAR mice resulted in a significant protection against tumor promotion in each of the low and high risk protocols employed. The protective effect of GTP was evident in terms of tumor incidence and tumor multiplicity as well as size of the tumor.

Protection by green tea against malignant conversion

Fig. 1. Protective effect of topical application of GTP against tumor promotion in SENCAR mice skin on the LR-TPA protocol (left panel), the HR-TPA protocol (middle panel) and the HR-MEZ protocol (right panel). The details of the treatments are provided in Materials and methods. The percentage of mice with tumors (top panels) and tumors per mouse (mean 6 SE, bottom panels) data are plotted as a function of the number of weeks on test.

As shown by the data in Figure 1 (left top panel), at the termination of the experiment at week 20, compared with 95% of animals with skin tumors in the non-GTP-treated group, 65% of animals exhibited the appearance of skin tumors in the GTP-treated group of animals (31% protection, P , 0.05, χ2 test). With regard to tumor multiplicity, at the end of the experiment at week 20, while the TPA-treated group of animals showed 10.0 6 1.2 papillomas/mouse, animals in the GTPtreated group showed only 5.1 6 0.4 papillomas/mouse (49% protection, P , 0.03, Wilcoxon rank sum test) (Figure 1, left bottom panel). Figure 1 also shows the kinetics of papilloma formation in groups of SENCAR mice receiving respectively TPA application for only 5 weeks (high risk of malignant conversion into SCC) or continued promotion with MEZ for up to 20 weeks (high risk of malignant conversion into SCC). Compared with the LR-TPA protocol showing 95% mice with papillomas, the HR-TPA and HR-MEZ protocols generated papillomas in only 50 and 85% of mice respectively at the end of week 20. However, pre-application of GTP to that of either TPA or MEZ resulted in 20 or 35% of mice with tumors respectively in the HR-TPA and HR-MEZ protocols, which indicated significant protection by GTP. In terms of tumor incidence the protection was 60% (P , 0.005, χ2 test). In terms of tumor multiplicity, a significant variation was observed among different protocols, as shown in Figure 1 (bottom panels). At 20 weeks, whereas in the LR-TPA protocol 10.0 6 1.2 tumors/ mouse were evident (Figure 1, left bottom panel), in the HRTPA and HR-MEZ protocols 1.2 6 0.3 and 3.3 6 0.4 papillomas/mouse (Figure 1, middle and right bottom panels) respectively were observed. At termination of the experiment, in comparison with non-GTP-treated animals, pre-application of GTP to that of the tumor promoters resulted in 42 and 63% inhibition (P , 0.01 and 0.005, Wilcoxon rank sum test) in tumor multiplicity in high risk-associated papillomas respectively in the TPA- and MEZ-treated groups of animals. When these tumor data were considered in terms of total tumor volume per mouse at the termination of the experiment

at 20 weeks, pre-application of GTP to that of the tumor promoters resulted in 74, 73 and 90% reductions (P , 0.0005, Student’s t-test) in size (mm3) in the LR-TPA, HR-TPA and HR-MEZ protocols, as shown in Figure 2 (top panel). Similarly, in terms of tumor volume per tumor, pre-application of GTP also resulted in 60, 58 and 74% reductions (P , 0.0005, Student’s t-test) in size of the developing papillomas in the LR-TPA, HR-TPA and HR-MEZ protocols (Figure 2, bottom panel). Effect of GTP on malignant progression One novel aspect of these studies was the modulation of the number of papillomas generated per mouse by adopting three different protocols as described in Materials and methods. Two high risk tumor protocols also allowed us to examine, under conditions of relatively low tumor burden, the protective effects of GTP against progression of papillomas to carcinomas in mouse skin. Similarly to anti-skin tumor promoting effects, GTP also afforded significant protection against the malignant conversion of chemically induced papillomas to SCC in all the employed protocols of this study. The protective effect of GTP was evident in terms of carcinoma incidence, carcinoma multiplicity and percent malignant conversion of papillomas to SCCs. The malignant conversion of papillomas to SCC in the LRTPA protocol is shown in Figure 3 (left panel). Treatment with GTP resulted in significant protection throughout the experiment and at the end of the experiment at 51 weeks as much as 41% protection (P , 0.01, χ2 analysis) in terms of percent mice with carcinomas (Figure 3, left top panel) was observed. In terms of carcinoma multiplicity (Figure 3, left bottom panel), GTP treatment resulted in 47% protection (P , 0.01, Wilcoxon rank sum test) in comparison with the acetone-treated control group (spontaneous malignant conversion group) of animals. Similarly to the LR-TPA protocol, treatment with GTP also resulted in significant protection against spontaneous conversion of papillomas to SCC throughout the experiment 499

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Fig. 2. Protective effect of topical application of GTP against tumor volume per mouse (top panel) and tumor volume per tumor (bottom panel) in all three protocols employed. The data shown (mean 6 SE) were obtained at the termination of the promotion studies at week 20 of the experiment.

on the HR-TPA protocol, as shown in Figure 3 (middle panel). At the end of the experiment at 51 weeks, treatment with GTP resulted in 40% protection (P , 0.01, χ2 analysis) in terms of the percentage of mice with carcinomas (Figure 3, middle top panel), while 49% (P , 0.03, Wilcoxon rank sum test) protection was observed in terms of number of carcinomas per mouse (Figure 3, middle bottom panel). As shown in Figure 3 (right panel), treatment with GTP also resulted in protection against malignant conversion of papillomas to SCC in the HR-MEZ protocol throughout the experiment in comparison with the acetone-treated positive control group (spontaneous malignant conversion group) of animals. At the end of the experiment at 51 weeks, GTP treatment resulted in 35% protection (P , 0.02, χ2 analysis) in terms of the percentage of mice with carcinomas (Figure 3, right top panel), while 55% protection (P , 0.01, Wilcoxon rank sum test) was evident in terms of number of carcinomas per mouse (Figure 3, right bottom panel). The percent malignant conversion of chemically induced papillomas was computed at different time intervals during the progression period in the acetone-treated spontaneous group as well as in the GTP-treated group, which clearly indicates that application of GTP afforded protection against malignant conversion of papillomas to SCC throughout the experimental period (data not shown). At the end of the experiment at 51 weeks, we summarized the percent malignant conversion in each of the groups. These data showed that the rate of percent malignant conversion of the non-malignant 500

tumors is in the order HR-TPA . HR-MEZ . LR-TPA (Figure 4). It is apparent from the data shown in Figure 4 that application of GTP afforded a significant reduction in the percent malignant conversion of papillomas into SCC in the LR-TPA, HR-TPA and HR-MEZ groups respectively (47, 48 and 58%, P , 0.03–0.01, Mantel–Haenszel χ2 test) at the termination of the experiment at 51 weeks. Remarkably, larger SCC developed in the HR-TPA group in comparison with the LR-TPA and HR-MEZ groups. This may be because of fewer numbers of tumors on the backs of each animal, which provided more space for carcinoma development. It is important to mention here that no additional tumors appeared in any mice between 20 and 51 weeks on test. Prevention kinetics of GTP against malignant conversion At the termination of the experiment at 51 weeks, the ultimate number of carcinomas formed in a group of 20 mice in the spontaneous malignant conversion subgroups of the LR-TPA, HR-TPA and HR-MEZ protocols ranged between 33 and 38. This shows that a subset of papillomas formed in the early phase is possibly highly charged with the probability of conversion into SCC. Continuous treatment with either TPA, as in the low risk tumor protocol (Figure 1, left panel), or MEZ, as in the high risk tumor protocol (Figure 1, right panel), although producing a greater number of papillomas per mouse, were not associated with a higher risk of malignant conversion. This information led to the suggestion that different subsets of papillomas exist with different probabilities of malignant conversion and that many papillomas are not associated with a risk of progression to SCC. This study also provides information that only limited treatment with TPA (5 weeks) in SENCAR mice is required to produce a similar number of SCC (35 carcinomas) compared with mice receiving continuous tumor promoter treatment, e.g. TPA or MEZ (38 and 33 carcinomas). An analysis of these data also reveals that the maximum percentage (87%) of papillomas was converted to SCC in the HR-TPA group, while 55 and 19% papillomas were converted to SCC respectively in the HR-MEZ and LRTPA groups. Additional analysis of the data showed that application of GTP afforded 47, 48 and 58% protection against spontaneous malignant conversion respectively in the LR-TPA, and HR-TPA and HR-MEZ groups of animals. Discussion The mouse skin model of multistage carcinogenesis provides an experimental framework to study basic mechanisms associated with the initiation, promotion and progression stages. This model has also proved useful in identifying cancer chemopreventive agents. In several studies we and others have shown the chemopreventive effect of GTP in the mouse skin chemical carcinogenesis model system (13–15,21). These preventive effects appear to be a consequence of anti-inflammatory and/or anti-oxidant properties of GTP (13,15,22–24). The administration of GTP or its components either topically or through drinking water to mice has been shown to reduce both the occurrence and the growth of tumors induced chemically or by UVB radiation (25,26). Katiyar et al. (16) have also shown that GTP protects against malignant conversion of chemically induced benign skin papillomas to SCC in SENCAR mice. In this study different tumor promotion protocols were employed to elicit papillomas with a low as well as high probability of malignant conversion to further test the efficacy

Protection by green tea against malignant conversion

Fig. 3. Protective effect of topical application of GTP against tumor progression in SENCAR mice skin on the LR-TPA protocol (left panel), the HR-TPA protocol (middle panel) and the HR-MEZ protocol (right panel). The details of the treatments are provided in Materials and methods. The percentage of mice with carcinoma (top panels) and carcinomas per mouse (mean 6 SE, bottom panels) data are plotted as a function of the number of weeks on test.

Fig. 4. Protective effect of topical application of GTP against percent malignant conversion of papillomas to SCCs in all three protocols employed. The data shown were analyzed at termination of the malignant progression experiment at 51 weeks.

of GTP against the development of papillomas and their subsequent conversion to SCC. Several laboratories have provided evidence for an alternative model whereby initiation– promotion regimens generate at least two major classes of papillomas; one with a high probability of progression and a second with a low probability of progression to SCC (4,6,18,27). The results of the present study indicate that in both cases, whether the tumors have low or high probability of malignant conversion, pre-application of GTP to that of the tumor promoters is highly effective in reducing the generation of papillomas, as well as their subsequent conversion to SCC. Mechanistic studies regarding the anti-tumor promoting effect of GTP have shown that topical application of GTP prior to that of tumor promoters onto mouse skin inhibits tumor promoter induction of ornithine decarboxylase and cyclooxygenase- and lipoxygenase-catalyzed metabolism of arachidonic acid (13). Skin application of GTP has also been shown to inhibit TPA-induced epidermal edema, hyperplasia and

enhancement of expression of the pro-inflammatory cytokine IL-1α (28). In the case of protection against malignant conversion by GTP, the mechanism, although not clear, may be related to its anti-oxidant properties. Earlier we showed that GTP and its individual epicatechin components inhibit spontaneous lipid peroxidation (23) in mouse epidermal microsomes, which may also be an important factor in reducing free radical generation in mouse skin and hence in inhibition of tumor promotion as well as conversion of papillomas to SCC. In our previous study we also showed that GTP inhibits malignant conversion enhanced by the free radical-generating compound benzoyl peroxide (16). It has been demonstrated that application of DMBA and TPA to mouse skin results in depletion of Langerhans cells (LC), an important event associated with the pathogenesis of skin cancer (29,30). This depletion of LC from the epidermis (29,30) may provide an appropriate microenvironment for the induction of tolerance (31–33), suggesting that this could be a phenomenon specific to chemical carcinogens. This could be important because it provides evidence that depletion of LC from the skin is a crucial part of the tumor promotion process (34). In our tumor promotion protocol we utilized DMBA as the initiator and TPA as the tumor promoter, both of which can cause suppression of the immune system through LC depletion in the skin. We have shown that GTP protects against UVB radiation-induced local and systemic suppression of the contact hypersensitivity response in C3H/HeN mice (35). GTP thus may protect against the induction of immune suppression caused by either DMBA or TPA and this may be the possible mechanism involved in protection against tumor promotion and subsequent progression of papillomas to SCCs. In conclusion, the data in this manuscript indicate that a much higher proportion of existing papillomas generated with high risk-associated standard initiation–promotion protocols using TPA or MEZ as the tumor promoters progress to SCCs in comparison with low risk-associated papillomas, and in both the cases GTP affords protection against malignant conversion of papillomas to SCCs. Thus our data suggest that irrespective of the risk involved in skin carcinogenesis, topical 501

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application of GTP affords significant protection against induction of papillomas with a low as well as high probability of conversion to malignancy. If this could be extrapolated to tumors in other body organs then our data would imply that GTP may be capable of affording protection against cancers with a wide range of tumor risk. Much additional work is required to strengthen this suggestion. Acknowledgements This work was supported by American Institute for Cancer Research grant 92B35 and Skin Diseases Research Center Core Grant P-30-AR-39750.

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