Multistage Carcinogenesis In Mouse Skin Research Articles

Cloning and chromosomal localisation of the murine epidermal-type fatty acid binding protein gene (Fabpe).

We succeeded in cloning the gene encoding the murine epidermal-type fatty acid binding protein (E-FABP). To avoid the screening of pseudogenes, the presence of which was shown by PCR, we designed an intron-specific probe and screened a bacterial artificial chromosome library from mouse embryonic stem cells. One of the clones obtained was analysed by restriction with various enzymes and an 11-kb EcoRI fragment with the complete gene was subcloned. The gene revealed the canonical exon/intron FABP structure consisting of four exons (112, 173, 102 and 544bp, respectively) and three introns (2217, 327 and 546bp, respectively). The exon sequences were identical with the cDNA encoding mouse E-FABP (Krieg, P., Feil, S., Fürstenberger, G., Bowden, T.G., 1993. Tumor-specific overexpression of a novel keratinocyte lipid-binding protein. Identification and characterisation of a cloned sequence activated during multistage carcinogenesis in mouse skin. J. Biol. Chem. 268, 17362-17369). Of the 5' region, 2470bp were sequenced and searched for transcription factor binding sites. Putative responsive elements within the promoter region were identified that may be responsible for the wide expression observed for E-FABP in mouse tissues. The 11-kb EcoRI fragment was used to localise Fabpe on chromosome 3 in the region 3A1-3 by fluorescence in-situ hybridisation.

Changes in protein expression during multistage mouse skin carcinogenesis

To directly compare the expression patterns of different proteins known to be altered during mouse skin carcinogenesis, serial sections of normal and hyperplastic skin and tumors from various stages of 7,12-dimethylbenz[a]anthracene-initiated, 12-O-tetradecanoylphorbol-13-acetate-promoted female SENCAR mice were examined by immunohistochemistry. In untreated, normal mouse skin, keratin 1 (K1) and transforming growth factor-beta1 (TGFbeta1) were strongly expressed in the suprabasal layers, whereas integrin alpha6beta4 was expressed only in basal cells and only moderate staining for transforming growth factor-alpha (TGFalpha) was seen. In hyperplastic skin, TGFalpha expression became stronger, whereas expression of another epidermal growth factor (EGF) receptor ligand, heparin-binding EGF-like growth factor (HB-EGF), was strongly induced in all epidermal layers from no expression in normal skin. Likewise, the gap-junctional protein connexin 26 (Cx26) became highly expressed in the differentiated granular layers of hyperplastic skin relative to undetectable expression in normal skin. Expression of cyclin D1 in the proliferative cell compartment was seen in all benign and malignant tumors but not in hyperplastic skin. Beginning with very early papillomas (after 10 wk of promotion), expression of alpha6beta4 in suprabasal cells and small, focal staining for keratin 13 (K13) were seen in some tumors. Later (after 20-30 wk), focal areas of gamma-glutamyl transpeptidase (GGT) activity appeared in a few papillomas, whereas TGFbeta1 expression began to decrease. Cx26 and TGFalpha staining became patchier in some late-stage papillomas (30-40 wk), whereas suprabasal alpha6beta4, K13, and GGT expression progressively increased and K1 expression decreased. Finally, in squamous cell carcinomas (SCCs), there was an almost complete loss of K1 and a further decline in TGFalpha, HB-EGF, TGFbeta1, and Cx26 expression. On the other hand, almost all SCCs showed suprabasal staining for alpha6beta4 and widespread cyclin D1 and K13 expression, whereas only about half showed positive focal staining for GGT activity.

Changes in protein expression during multistage mouse skin carcinogenesis

Changes in protein expression during multistage mouse skin carcinogenesis

Atypical association of H1 and H2 histamine receptors with signal transduction pathways during multistage mouse skin carcinogenesis.

In the present work we studied the association of histamine receptors with second messengers during multistage carcinogenesis in Sencar mice skin. 96 Sencar female mouse, divided into six groups were used. Tumors appeared only in the 7, 12-dimethylbenz[a]anthracene-initiated and 12-O-tetradecanoylphorbol-13-acetate-promoted group. Control groups received only TPA, or acetone or no treatment at all. Periodically during the promotion period, cAMP and inositol phosphate production were measured after stimulation with H1 or H2 agonists in samples from all groups. In non-treated skin, H1 receptors were coupled to phosphatidylinositol hydrolysis and H2 receptors mediated cAMP production. Conversely, in tumors H2 receptors were associated with phosphatidylinositol hydrolysis and H1 mediated a rise in cAMP levels. The skin among tumors and the skin from all control groups maintained the same coupling as non-treated skin. An increase in mast cell number, with a homogeneous subepithelial distribution and marked phenotypic changes, was also observed in promoted skin. These findings indicate an atypical association of histamine receptors with second messengers that could be a critical feature for the postulated action of histamine in tumor growth.

Overexpression of insulin-like growth factor-1 induces hyperplasia, dermal abnormalities, and spontaneous tumor formation in transgenic mice.

Transgenic animals were developed to assess the role of insulin-like growth factor 1 (IGF-1) in skin growth, differentiation and organization, as well as its importance in tumor formation. Expression of a human IGF-1 cDNA was targeted to the interfollicular epidermis of transgenic mice using a human keratin 1 promoter construct (HK1). Transgenic animals (HK1.IGF-1 mice) could be identified at birth by early ear unfolding and excessive ear and skin growth compared to non-transgenic littermates. Further examination of the skin from these mice showed epidermal hyperplasia and hyperkeratosis, marked thickening of the dermis and hypodermis, and early hair follicle generation in newborns. The severity of this phenotype correlated with transgene expression both of which subsided with age. Adult HK1.IGF-1 mice developed spontaneous tumors following treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) alone and exhibited an exaggerated epidermal proliferative response following treatment with the tumor promoter compared to non transgenic littermates. Additionally, HK1.IGF-1 transgenic mice developed papillomas faster and in markedly greater numbers compared to non-transgenic littermates in standard initiation-promotion experiments. The data presented suggest an important role for IGF-1 in the process of multistage carcinogenesis in mouse skin.

Different expression of prostaglandin-H synthase isozymes and lipoxygenases during multistage carcinogenesis in mouse skin.

An increasing body of epidemiologic data suggests the prostaglandin-H synthase (PGHS) pathway to arachidonic acid metabolisrii to be involved in human colon carcinogenesis. In addition, PGHS inhibitors were shown to reduce the tumor incidence in rat models of colon carcinogenesis (1). In the initiation-promotion model of mouse skin carcinogenesis inhibitors of both the PGHS and lipoxygenase (LOX) pathways were found to exhibit potent anti-promoting activities (2–5).

Altered expression of insulin-like growth factor I and its receptor during multistage carcinogenesis in mouse skin.

We examined the possible role of insulin-like growth factor-I (IGF-I) and IGF-I receptor (IGF-Ir) during multistage carcinogenesis in mouse skin. For this purpose, the expression of both IGF-I and IGF-Ir was investigated in mouse skin during tumor promoter treatment and in primary papillomas and squamous cell carcinomas (SCCs) obtained from SENCAR mice treated with standard initiation-promotion regimens. IGF-I transcripts were not detectable or only weakly detectable in normal SENCAR mouse epidermis by northern or reverse transcription (RT)-polymerase chain reaction (PCR) analysis, respectively, whereas IGF-I transcripts (primarily a 7.0-kb transcript) were readily detected in RNA preparations from the dermis by both northern blot analysis and RT-PCR analysis. In contrast, IGF-Ir transcripts were observed in RNA samples from both epidermis and dermis of control SENCAR mice. Single and multiple topical treatments with 3.4 nmol of 12-O-tetradecanoylphorbol-13-acetate (TPA) had no effect on dermal or epidermal IGF-I and IGF-Ir mRNA levels. In contrast, the levels of IGF-I transcripts were elevated (2.5- to 15-fold) in a significant number of mouse skin tumors (71% of all tumors examined). Transcripts of 7.0, 2.5, and 1.3 kb were more consistently overexpressed in skin tumors compared with epidermis, whereas the two smaller transcripts were most consistently overexpressed compared with the dermis. The levels of an 11.0-kb IGF-Ir transcript were also elevated (2.5- to 8-fold) in some papillomas (20%) and SCCs (55%), but the percentage of tumors exhibiting this property (32% of all tumors examined) was lower than the percentage overexpressing IGF-I. These data suggest that altered expression of IGF-I and IGF-Ir may play a role in multistage carcinogenesis in the mouse skin model. The inability of TPA to induce elevated IGF-I or IGF-Ir expression suggests that these changes in skin tumors are coincident with tumor formation and not a direct result of altered epidermal proliferation per se. Altered expression of IGF-I in a high percentage of papillomas may indicate that IGF-I has an important role in the development of autonomous growth in these tumors. The higher percentage of SCCs with altered levels of IGF-Ir mRNA may indicate a role for these changes in the later stages (i.e., tumor progression) of carcinogenesis in this model system.

Protective effect of all trans retinoic acid against tumor promotion and progression in low- and high-risk protocols of mouse skin chemical carcinogenesis

In this study, we assessed the protective effects of all trans retinoic acid (RA) against skin tumor promotion and progression in three different protocols of mouse skin multistage carcinogenesis. Under these protocols, where papillomas on the skin of SENCAR mice were induced for their low- and high-probability of conversion to malignant carcinomas, pre-application of RA (10 mu g/mouse/application) to that of tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or mezerein (MEZ) in 7,12-dimethylbenz(a)anthracene (DMBA)-initiated mouse skin resulted in a highly significant protection against skin tumor promotion. In terms of tumor incidence, multiplicity and volume/mouse, at the termination of the experiment at 20 weeks, RA showed 35-80%, 67-83% and 70-98% protection, respectively. While the effect of RA was profound in all the protocols, maximum affectivity was evident in high-risk papilloma induction protocol where DMBA was used as tumor initiator and one week later TPA was applied once a week for five weeks. In tumor progression studies, at 20 weeks of standard low- and high-risk protocols, when papilloma yield is stabilized, animals which did not receive RA in different protocols were divided into two groups and topically treated twice weekly either with acetone or RA (10 mu g). These treatments were continued for an additional 29 weeks. During these treatments, all suspected carcinoma formation was recorded and carcinomas were verified histologically at the termination of the experiment. In each case, RA showed remarkable protective effects against percentage of mice with carcinomas (35-45% protection), number of carcinomas per mouse (50-63% protection) and the rate of malignant conversion (50-63% protection). The results of the present study clearly demonstrate that irrespective of the risk for skin carcinogenesis, RA is capable of affording protection against the induction of nonmalignant lesions and their subsequent conversion to malignancy.

Aberrant expression of gap junction proteins (connexins) is associated with tumor progression during multistage mouse skin carcinogenesis in vivo.

To elucidate what changes in the expression of gap junction proteins (connexins) occur at what stages during multistage mouse skin carcinogenesis in vivo, we immunohistochemically and morphometrically analyzed the expression of connexin 26 (Cx26) and connexin 43 (Cx43) in papillomas, well-, moderately- and poorly-differentiated squamous cell carcinomas, as well as in squamous cell carcinomas at invasion sites and those metastasized into lymph nodes in female CD-1 mice as a result of treatment with dimethylbenz[a]anthracene and 12-O-tetradecanoylphorbol-13-acetate. In papillomas, no clear reduction of the two connexins was observed; however, Cx26 and Cx43 were frequently co-localized in the same gap junction plaques, whereas the two kinds of Cxs were differentially expressed in normal and surrounding non-tumorous epidermis. In squamous cell carcinomas, the expression of both Cx26 and Cx43 significantly decreased compared with surrounding non-tumorous epidermis and papillomas. The Western blot analysis confirmed that both Cx26 and Cx43 proteins were reduced in squamous cell carcinomas compared with papillomas. Furthermore, the expression of Cx26 was reduced as cancer cells became morphologically less differentiated, while that of Cx43 did not change. Squamous cell carcinomas at invasive sites showed clear reduction of Cx26 and Cx43. In squamous cell carcinomas metastasized into lymph nodes, Cx26 was expressed, but few carcinoma cells expressed Cx43. The localization of E-cadherin on the plasma membrane between cancer cells was maintained even at invasive and metastatic sites. Our data suggest that quantitative and qualitative changes in connexin expression are associated with tumor progression, including the loss of differentiation, and invasion and metastasis, during multistage mouse skin carcinogenesis.

Altered expression of the epidermal growth factor receptor and transforming growth factor-alpha during multistage skin carcinogenesis in SENCAR mice.

In the study presented here, we examined the possible role of the transforming growth factor-alpha (TGF alpha)/epidermal growth factor receptor (EGFR) system during multistage carcinogenesis in mouse skin. In this regard, the expression (mRNA and protein) of both TGF alpha and EGFR was examined in primary papillomas and squamous cell carcinomas (SCCs) obtained from SENCAR mice treated with standard initiation-promotion regimens and compared with the levels of expression in normal epidermis. The level of a 4.8-kb TGF alpha transcript was elevated in 100% of the skin tumors examined (both papillomas and SCCs), including papillomas obtained 13 wk after the start of promotion, compared with normal epidermis. Immunohistochemical analyses detected elevated levels of TGF alpha protein in these skin tumors and in papillomas as early as 10 wk after the start of promotion. The levels of EGFR transcripts were also significantly elevated in most (90%) of the skin tumors examined, including again those harvested after 13 wk of promotion. Interestingly, multiple EGFR transcripts (10.5, 5.8, 2.8, and 1.8 kb) were detected in both papillomas and SCCs. The two smaller transcripts appeared to encode truncated versions of the EGFR, and the 1.8-kb transcript appeared to be unique to RNA samples isolated from skin tumors, based on comparative analyses of several normal tissues. As with TGF alpha, immunohistochemical analyses detected elevated levels of EGFR protein in these skin tumors (both papillomas and SCCs), including papillomas harvested as early as 10 wk after the start of promotion. Southern analyses of genomic DNAs for TGF alpha and EGFR failed to detect any cases of gene rearrangements or amplification as a possible explanation for the elevated levels of the transcripts of these two genes. These results support the hypothesis that a key step in the development of autonomous growth in mouse skin papillomas generated in SENCAR mice by an initiation-promotion regimen may involve alterations in the synthesis of TGF alpha and its cognate receptor.

7,12-Dimethylbenz [a] anthracene-Induced Mouse Keratinocyte Malignant Transformation Independent of Harvey ras Activation

Independent clones of mouse keratinocytes initiated in vitro gave rise to tumor phenotypes typical of mouse skin multistage carcinogenesis and histologically similar to human tumors of the skin, and head and neck. High-molecular-weight genomic DNAs isolated from two 7,12-dimethylbenz[a]anthracene (DMBA)-initiated murine epithelial carcinoma cell lines and one papilloma cell line were examined for transforming activity by transfection into NIH3T3 cells. DNAs from each of these cell lines resulted in the formation of foci morphologically unlike foci containing an activated c-Ha-ras oncogene. Following polymerase chain reaction amplification of the c-Ha-ras gene, Xba I restriction analysis and oligonucleotide differential hybridization did not detect 61st, 12th, or 13th codon mutations. Southern and Northern analysis confirmed that the normal c-Ha-ras gene was not activated by amplification or overexpression. These results provide evidence that 7,12-dimethylbenz[a]anthracene-induced malignant transformation of murine keratinocytes occurred independent of point mutations associated with c-Ha-ras activation. The absence of an activated c-Ha-ras oncogene in these cell lines distinguishes our model from other mouse models of carcinogenesis and may provide a model for functional genetic changes during initiation and progression of human epithelial cancers.

Tumor-specific overexpression of a novel keratinocyte lipid-binding protein. Identification and characterization of a cloned sequence activated during multistage carcinogenesis in mouse skin

Differential screening of cDNA libraries from chemically induced malignant mouse skin squamous cell carcinomas (SCCs) identified sequences, including one called mal1, that were up-regulated in their expression at both the benign papilloma and the malignant SCC stages during tumor development. The mal1 plasmid cDNA clone was used to screen lambda phage cDNA libraries made from chemically induced papillomas and SCCs. Two size classes (655 and 933 nucleotides excluding the poly(A) tail) of full-length cDNAs were isolated. The corresponding mRNAs differ in their 3'-untranslated region by 278 nucleotides as a result of utilizing two alternative polyadenylation signals. Both transcripts were expressed simultaneously, showing the same expression patterns, with the smaller one being the predominant species. Most tissues examined showed a weak expression of mal1 mRNA. High levels of mal1 transcripts could be detected in adipose and mammary tissues and tongue epithelia and predominantly in epidermis. The expression observed in epidermis was up-regulated dramatically during tumor formation. Computer-assisted sequence analysis revealed one open reading frame that encoded a protein of 135 amino acid residues with extensive homology to members of the lipid-binding protein family. Residues determining the proposed beta-clam structure of these proteins and the structure of the lipid-binding region were shown to be conserved in the mal1 gene. In vitro translation of mal1 RNA yielded a polypeptide of the predicted size of 15 kDa that was immunoprecipitable with an anti-rat liver fatty acid-binding protein antiserum. Based on the sequence analysis and antigenic properties of mal1, we conclude that it encodes a novel member of the lipid-binding protein family.

Inhibition of benzoyl peroxide-induced tumor promotion and progression by copper(II)(3,5-diisopropylsalicylate) 2

The ability of a biomimetic superoxide dismutase agent, copper(II)(3,5-diisopropylsalicylate) 2 (CuDIPS), to modulate benzoyl peroxide (BzPo)-induced tumor promotion and progression in mouse skin multistage carcinogenesis was evaluated. The results showed a significant inhibition of tumor incidence by CuDIPS pretreatment during promotion-progression. Different types of tumors were developed: papillomas, keratoacanthomas and squamous cell carcinomas. There was a significant increase in the keratoacanthomapapilloma ratio when the period of treatment with BzPo was prolonged, which was inhibited by CuDIPS pretreatment. CuDIPS induced a significant inhibition of malignant conversion. Our results suggest that reactive oxygen species could be important in BzPo-induced promotion and progression.

Tumour induction in mouse epidermal cells irradiated by hot particles.

We have shown elsewhere that highly non-uniform exposure to ionizing radiation from authentic Chernobyl-released and artificially-produced hot particles (fragments of nuclear fuel) transform fibroblastic 10T1/2 cells in vitro effectively. We have also shown that hot-particle exposure leads to mutation and overexpression of the tumour suppressor gene p53 (and some other growth-related genes) in mouse skin in vivo at a high frequency. In the present paper it is shown that hot-particles produced by irradiating natural uranium with slow neutrons, when implanted (immobilized) under the skin of hairless and nude mice, induce epidermal tumours in excess compared with the conventional non-threshold stochastic model of radiation-induced cancer. One explanation for the effectiveness of the hot-particle exposure, under the present assay conditions, is that the same cells in which specific radiation-induced DNA damage is most likely to occur, are forced into sustained mitotic activity in the chronic wound which develops around the radiation source (combined genotoxic and nongenotoxic effects). The results are consistent with a role for cell proliferation in multistage carcinogenesis in mouse skin.

Identification of a cloned sequence activated during multi-stage carcinogenesis in mouse skin

Differential screening of cDNA libraries made from chemically induced malignant mouse skin squamous cell carcinomas (SCC) identified three sequences, including one called mal2, that were upregulated in their expression at both the benign papilloma and malignant SCC stages. The mal2 plasmid cDNA clone (containing a 350 bp insert) was used to screen lambda phage cDNA libraries made from chemically induced SCCs. Two of the largest mal2-related cDNA inserts obtained from the phage libraries were sequenced. In addition a mal2-related genomic clone was obtained by hybridization probing of a mouse spleen genomic DNA library. The sequence of the genomic clone overlapped and was identical with both the mal2 plasmid and lambda cDNA clones. Identity was found between the mal2 cDNAs, the mal2 genomic sequence and the cDNA sequence for a mouse hyperproliferative keratin called K6. A synthetic oligonucleotide specific for the 3' untranslated region of the mal2 or keratin K6 gene was used in Northern analyses to demonstrate elevated steady-state levels of K6 keratin transcripts in SCCs induced by various protocols involving both chemical and ionizing radiation initiation of tumors as well as complete chemical and radiation carcinogenesis protocols. Metastatic lung lesions derived from SCCs generated by repeated doses of benzo[a]pyrene showed moderate levels of K6 keratin transcripts, whereas normal lung showed very low levels of K6 transcripts. The overexpression of the mal2 or keratin K6 gene in malignant SCCs was independent of the protocol, either chemical or radiation, that was used to induce the tumors.

Effects of antihistamines on phorbol ester tumor promotion and vascular permeability changes

Substantial evidence suggests that inflammation is an essential component of the phorbol ester tumor promotion stage of multistage carcinogenesis in mouse skin. In order to understand better the significance of this relationship, studies were directed at identifying the principal mediators of the vascular permeability component of inflammation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA). Antihistamines and inhibitors of arachidonic acid metabolism were compared with respect to their anti-inflammatory activity and the correlation of this parameter with their effect on tumor promotion. The H1 histamine receptor antagonist, diphenhydramine, suppressed TPA-induced vascular leakage by 25 and 50% at topical doses of 0.342 mumol (100 micrograms) and 0.856 mumol (250 micrograms) respectively. In initiated mice, these same doses inhibited papilloma development by 40 and 75%. Inhibition of tumors was also observed when diphenhydramine was given orally. The H2 antagonist, cimetidine, which could only be supplied orally, had little effect on either TPA-induced vascular permeability or promotion. The lipoxygenase inhibitor nordihydroguaiaretic acid also suppressed vascular permeability and has been reported to inhibit papilloma development. The cyclooxygenase inhibitor indomethacin, however, has no effect on TPA-induced vascular permeability. Collectively, these data suggest that the increased vascular leakage observed with TPA contributes to tumor development and that this event is mediated by both the H1 histamine receptors and one or more of the lipoxygenase products of arachidonic acid.

Effect of diterpene esters on actin cytoskeleton of SV40-transformed keratinocytes is not reproduced by diacylglycerols.

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) induces a disorganization of the actin cytoskeleton and its redistribution at the periphery of the cells in SV40-transformed human keratinocytes. This phenomenon is induced by other diterpene esters, such as 4-O-methyl TPA, 12-O-ethacrynylphorbol-13-acetate (EPA), 12-O-retinoylphorbol-13-acetate (RPA) and mezerein, which exert either convertogenic or promoting effects in skin tumor development in vivo. Two diacylglycerols: oleyl-acetyl glycerol (OAG) and dioctanoyl-glycerol (DOG) do not induce the disorganization of actin. Thus, the effects of these compounds, as they are found in SV40-transformed human keratinocytes, do not exhibit clear-cut correlations to either their protein kinase C activating abilities, or their effects on different stages of multistage carcinogenesis in mouse skin in vivo.

Molecular cloning of sequences activated during multi-stage carcinogenesis in mouse skin

Recombinant DNA techniques were employed to isolate sequences that were activated during multi-stage carcinogenesis in the skin of NMRI mice. Differential screening of 5000 cDNA clones made from poly(A)+ RNA of squamous cell carcinomas induced by 7,12-dimethylbenz[a]anthracene (DMBA) and the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) resulted in the identification of 35 cDNA clones displaying a different hybridization signal. Eight cDNA clones, three of which proved to be identical, were used as probes in transfer hybridization experiments. These cDNA clones, designated pmal-1 to pmal-6, showed a strong signal with carcinoma RNA but either a weak or no signal with RNA from normal epidermis. Clone pmal-2 contained repetitive sequences as shown by Southern analysis resulting in a smeared RNA hybridization signal in RNA blots whereas the other five clones corresponded to discrete size classes of mRNA. Studies on the expression pattern of mal-1,-2 and -3 related sequences revealed transcriptional activation already in the benign papilloma stage of multi-step carcinogenesis.

Effects of a biomimetic superoxide dismutase on complete and multistage carcinogenesis in mouse skin.

The effects of a selective detoxifier of the proximate oxygen radical, superoxide anion, on the induction of tumors in the skin of CD-1 mice by either the initiation-promotion regimen or the complete carcinogenesis process were investigated. The principle agent of interest, copper (II) (3,5-diisopropylsalicylate)2 (CuDIPS), is a low molecular weight, lipophilic copper coordination complex that catalytically disproportionates superoxide anion at a rate comparable to native Cu-Zn superoxide dismutase (SOD). The protocols used to elicit tumors were: (i) a single application of 0.2 mumol of 7,12-dimethylbenz[a]anthracene (DMBA) followed by twice-weekly applications of 12-O-tetradecanoylphorbol-13-acetate (TPA) in an initiation-promotion study, and (ii) either a single application of 3.6 mumol DMBA followed by no further treatment or weekly applications of 0.2 mumol DMBA in complete carcinogenesis protocols. Application of 2 mumol CuDIPS 15 min prior to the initiating dose of DMBA was without significant effect on tumor yield or incidence, whereas application prior to each dose of TPA substantially reduced tumor incidence and yield. This anti-promoting property of CuDIPS can be attributed to its SOD-mimetic activity in as much as the corresponding zinc coordination complex lacking in SOD activity, zinc (II) (3,5-diisopropylsalicylate)2, was non-inhibitory. Significant reductions in tumor yield were also observed when CuDIPS was applied prior to DMBA in either of the complete carcinogenesis protocols. Additionally, covalent binding of [3H]DMBA to epidermal DNA was markedly reduced by CuDIPS pre-treatment, suggesting that the anti-carcinogenic properties may reflect a perturbation in superoxide anion-dependent metabolic activation of DMBA. The induction by DMBA of ornithine decarboxylase activity, a biochemical marker of tumor promoter activity, was not affected by CuDIPS; however, induction of ornithine decarboxylase by TPA was potently blocked. Collectively, these effects of a biomimetic SOD further implicate reactive oxygen species at multiple stages in chemical carcinogenesis.

The "promoting" activity of methyl methanesulphonate in rat bladder carcinogenesis.

The carcinogenic activity of the alkylating agent methyl methanesulphonate (MMS) was investigated in the F344 rat bladder, both untreated and pretreated with a single threshold dose of N-methyl-N-nitrosourea (MNU). On its own, 6 doses of 2.5 mg MMS produced a 7% incidence of bladder cancer. After a single intravesical instillation of MNU, the same MMS treatment produced a bladder cancer incidence of 56%. This was significantly higher than the incidence (24%) observed after treatment with MNU alone, and greater than the sum of the lesions produced by either treatment alone. By reference to the mouse skin multistage carcinogenesis model, it is argued that MMS is a complete, albeit weak carcinogen with little initiating but powerful late-stage activity. Its promoting activity is most probably attributable to its potent mitogenic action and in this model it is analogous to a stage 2, rather than a stage 1 skin promoter.