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T J Slaga

Publications and source records attributed to T J Slaga.

At least 91 records · Page 5Linked to original sources
PubMed1989

Gap-junctional intercellular communication in epidermal cell lines from selected stages of SENCAR mouse skin carcinogenesis.

Homologous and heterologous gap-junctional intercellular communication (IC) was characterized in a panel of cell lines derived from selected stages of SENCAR mouse skin carcinogenesis. This panel included a "carcinogen-altered" cell line, 3PC, obtained from Ca2+-resistant primary adult keratinocytes after exposure to dimethylbenz(a)anthracene as well as cell lines obtained from early and late-stage papillomas and a squamous cell carcinoma (CA3/7) generated during standard in vivo initiation/promotion protocols (dimethylbenz(a)anthracene/12-O-tetradecanoyl-phorbol-13-acetate). Also studied was a cell line (B66BA) obtained from a metastatic lesion following benzo(a)pyrene-induced skin tumorigenesis. Intercellular communication was measured in low-calcium (0.05 mM) medium by quantitation of cell-cell transfer of microinjected fluorescent dye Lucifer Yellow CH. Homologous IC ability diminished progressively from 68 dye-coupled cells per injection for 3PC cultures, to between 21 and 54 dye-coupled cells per injection for three papilloma-derived cell lines, to six and three dye-coupled cells per injection for CA3/7 and B66BA cells, respectively. To test communication of these cells with their normal counterparts, heterologous IC was examined in cocultures with primary adult keratinocytes. Under the conditions used, normal cells established functional communication channels with each cell line tested, showing no selectivity. These results suggest that progressive loss of homologous but not heterologous IC capacity accompanies neoplastic development in mouse skin carcinogenesis.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1989

Induction of terminal differentiation-resistant epidermal cells in mouse skin and in papillomas by different initiators during two-stage carcinogenesis.

Carcinogen treatment of normal mouse epidermal cells causes some cells, if cultured under the appropriate conditions, to continue to proliferate instead of terminally differentiate, forming foci at 37 degrees C in medium with a calcium level above 0.1 mM. We have examined these Calcium (Ca)-resistant cells formed in the skin of SENCAR mice after treatment with the carcinogen initiator 7,12-dimethylbenz[a]anthracene (DMBA) followed by tumor promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA). Although in our previous studies TPA promotion initially increased the size but reduced the number of foci caused by the carcinogen initiator N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), TPA promotion of DMBA-treated mice increased the size but had no effect on the number of foci. Papillomas resulting from DMBA plus TPA treatment contained many rapidly growing Ca-resistant cells, corroborating our earlier results with MNNG. Permanent cell lines prepared from papilloma-derived foci formed squamous cell carcinomas in nude mice after relatively short periods in culture. These data provide further evidence that Ca-resistant cells may be papilloma (and perhaps carcinoma) precursors in vivo. In addition, since TPA tends to reduce the number of early Ca-resistant cells caused by MNNG but not by DMBA, this may at least partially explain why treatment with DMBA plus TPA is much more effective in producing papillomas in SENCAR mice than is treatment with MNNG plus TPA.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1989

Sequential trisomization of chromosomes 6 and 7 in mouse skin premalignant lesions.

Using a direct cytogenetic technique, we identified a nonrandom trisomy of chromosome 6 in 12 of 12 aneuploid mouse skin papillomas and in 10 of 11 squamous cell carcinomas induced by chemical carcinogenesis. The second most common abnormality observed was trisomy of chromosome 7 found in most dysplastic papillomas and in 10 of 11 carcinomas. The two trisomies were the only abnormalities found in all aneuploid papillomas and in several carcinomas. Mutation at codon 61 of the Ha-ras gene, which resides on chromosome 7, was also a common feature of the tumors sampled. Extensive homology exists between mouse chromosome 6 and human chromosome 7, the trisomy of which was recently suggested as a primary cytogenetic event in several human epithelial cancers. We propose a multistep model of tumor progression in which a sequence of specific nonrandom chromosomal abnormalities appear to be required for malignant transformation.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1988

Tumor progression in Sencar mouse skin as a function of initiator dose and promoter dose, duration, and type.

The influence of initiator dose and promoter dose, duration, and type on the progression of papillomas to carcinomas was examined in Sencar mice. A good dose-response relationship for promotion of papilloma formation by 12-O-tetradecanoylphorbol-13-acetate (TPA) [following initiation with 6.5 micrograms of 7,12-dimethylbenz(a)anthracene (DMBA)] was observed in the range of 0.125 to 2.0 micrograms/mouse. A maximal papilloma response was induced with 2 micrograms/mouse (24 papillomas/mouse). When adjusted for mortality, the carcinoma incidence after 60 wk of promotion was essentially the same (approximately 80%) for doses above 0.5 micrograms/mouse. In a related experiment, mice were given an initiation dose of either 2 or 20 micrograms of DMBA followed by applications of 2 micrograms of TPA for 3, 5, 7, or 60 wk. Papilloma formation was proportional to length of treatment, with a maximum of 29 papillomas/mouse (20-micrograms initiating dose of DMBA) and 10 papillomas/mouse (2-micrograms initiating dose of DMBA) occurring between 10 and 15 wk of promotion. In this experiment, the carcinoma incidence was clearly proportional to the duration of promoter treatment at the low initiation dose of DMBA. The carcinoma incidence, on the other hand, was similar (approximately 70%) in groups of mice given an initiation dose of 20 micrograms of DMBA and promotion treatment for greater than or equal to 5 wk. Thus, the initiator dose had a dramatic effect on the outcome of these experiments. Additional experiments were performed to compare tumor progression with the anthrone promoter, chrysarobin. At optimal promoting doses, chrysarobin treatment produced a maximum number of papillomas that was approximately 1/3 that produced by TPA (6.4 versus 17.0 papillomas per mouse, respectively). However, the carcinoma response was very similar in these two treatment groups, confirming previous work from this laboratory. In addition, chrysarobin treatment following 10 wk of TPA promotion did not enhance the progression of preexisting papillomas to carcinomas. The data presented in this paper are consistent with a model in which several types or stages of papillomas are initially produced during two-stage carcinogenesis in mouse skin with different probabilities of progressing to carcinomas. However, the data indicate that optimal doses of promoter and initiator exist and can influence interpretation of tumor progression studies in mouse skin.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1988

Quantitation of primary in vitro clonogenic keratinocytes from normal adult murine epidermis, following initiation, and during promotion of epidermal tumors.

A quantitative in vitro assay for clonogenic epidermal cells from adult mice has been designed to focus on the cells from normal epidermis that are potential targets for carcinogens. Dorsal epidermal cells were isolated by trypsinization from groups of three to six CD-1 female mice with yields of 1.31 +/- 6.84 X 10(6) (average of n = 7; SD) viable epidermal cells per square centimeter of skin. Suspensions of single epidermal cells were plated at clonal density onto irradiated Swiss 3T3 cells. The cultures were maintained in high calcium SPRD-105 medium designed to support concomitant proliferation and terminal differentiation of keratinocytes from normal as well as carcinogen-exposed mice. Two weeks later, the dishes were fixed and stained with rhodanile blue, and epidermal colonies were counted. The average number of colonies from normal epidermis was 45 +/- 8.5 (n = 11; SD) per 10(4) cells plated for mice 9 to 69 weeks of age. To determine the effects of initiation on the number of clonogenic epidermal cells, groups of mice 8 weeks of age were treated topically with 200 nmol of 7,12-dimethylbenz(a)anthracene or acetone alone. The number of colonies in both treatment groups remained within the control (untreated) range at all intervals from 7 to 61 weeks after initiation. In contrast, the number of clonogenic cells from control as well as initiated epidermis remained elevated at 1 month following multiple in vivo treatments of skin with 12-O-tetradecanoylphorbol-13-acetate (TPA). The increase in the number of clonogens was always greater from initiated epidermis treated with TPA than from control epidermis treated with TPA. These results suggest that an increase in the clonogenic population was a consequence of promotion rather than initiation and are in agreement with a concomitant carcinogenesis experiment confirming the apparent irreversibility of initiation, the veritable absence of tumors in the absence of promotion, and the similarity of the tumor responses regardless of the age of the animal at initiation or the length of the delay interval between initiation and promotion.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1988

Sequential development of aneuploidy, keratin modifications, and gamma-glutamyltransferase expression in mouse skin papillomas.

To elucidate the role and timing of expression of different premalignant and malignant markers in tumor promotion, we correlated alterations in keratin patterns and gamma-glutamyltransferase (GGT) expression with the chromosomal status of individual mouse skin papillomas. Papillomas were induced by 7,12-dimethylbenz[a]anthracene initiation and 12-O-tetradecanoylphorbol-13-acetate promotion. Individual tumors were randomly sampled at 20 and 35 weeks of promotion. Each tumor was cytogenetically analyzed and serial paraffin sections were used for GGT detection, immunoblotting, and immunohistochemistry studies. Monospecific antibodies elicited against keratins K1 (Mr 67,000) and K14 (Mr 55,000) were used to analyze keratin modifications. Most tumors at 20 weeks of promotion, although exhibiting aneuploidy, still presented high levels of the K1 differentiation-associated keratin. Later during promotion those tumors bearing the highest aneuploidy indexes were those that showed a marked decrease in or absence of the K1 protein. Furthermore, those same tumors with the highest levels of genomic alterations also exhibited foci of GGT activity. These results support the idea that the majority of papillomas under continuous promotion are progressing toward malignancy. Aneuploidy seems to precede detectable keratin modifications, and GGT activity appears to be the latest marker to be expressed.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1988

In vivo behavior of murine epidermal cell lines derived from initiated and noninitiated skin.

The in vivo behavior of cell cultures derived from normal and carcinogen-treated mouse epidermis was studied by implanting the cultures in a s.c. vascularized bed protected by a silicone chamber. Cells derived from normal adult mouse epidermis as well as cells derived from tumor-promoter-treated skin were unable to grow in these systems. Conversely, cell lines derived from skin initiated with single doses of N-methyl-N'-nitro-N-nitrosoguanidine or 9,10-dimethyl-1,2-benzanthracene proliferated in these chambers, reforming an epithelial structure. The type of structure in the chambers varied, ranging from formation of almost normal epithelia to atypical invasive behavior. The variable in vivo behavior among the different cell lines may be attributed to the initiation agent, the number of passages of the cultures, random genetic events, the strain of mouse, or a combination of these factors. Most of the cell types used in this study and all the cell lines that were able to grow in these chambers were selected for resistance to Ca-induced terminal differentiation. However, resistance to terminal differentiation according to the Ca2+ switch does not always correlate with the ability to grow in the chambers, since cell lines derived from spontaneous foci of resistance failed to grow in this system. These studies showed some of the possibilities of the SC silicone chambers to study the histogenic potential of cell lines derived from carcinogen-treated epidermis. This system also appears suitable to study the complex relationship between epidermal cells and specialized (dermal) stroma.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1988

Expression pattern of a gene for a secreted metalloproteinase during late stages of tumor progression.

We have reported that transin RNA, a 1.9-kb RNA coding for a novel, secreted proteinase, was overexpressed during the progression of benign mouse skin papillomas to malignant squamous cell carcinomas (SCCs) induced by a two-stage protocol (Proc Natl Acad Sci USA 83:9413, 1986). Recently a high degree of similarity has been demonstrated between rabbit stromelysin, a secreted metalloproteinase that degrades proteoglycans found in the basement membrane and the amino acid sequence predicted in rat transin cDNA. DNA sequencing of a mouse cDNA isolated from an SCC (initiated by 7,12-dimethylbenz[a]anthracene [DMBA] and promoted by 12-O-tetradecanoylphorbol-13-acetate [TPA]) showed greater than 85% nucleotide similarity and 90% amino acid similarity to the rat transin-1 cDNA nucleotide and predicted amino acid sequences. Using this mouse transin cDNA clone as a probe (labeled with 32P) we found enhanced levels of transin mRNA transcripts in SCCs induced by a protocol giving rise to metastatic tumors (repeated N-methyl-N-nitroso-N'-nitroguanidine [MNNG] treatments) compared with the level found in SCCs induced by a protocol that had a lower probability of giving rise to metastatic tumors (MNNG initiation followed by TPA promotion). A study of primary SCCs and metastatic lesions induced by repeated benzo[a]pyrene treatment showed that the levels of transin mRNA transcripts were reduced in the metastatic lesions in comparison to the primary tumors. Southern analysis of the DNA isolated from epidermis, papillomas, and SCCs indicated that neither transin gene amplification nor rearrangement accounted for increased levels of the transin mRNA transcripts. These data suggest a role for enhanced levels of transin production in the invasion and metastasis of chemically induced SCCs.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1988

Malignant progression of papilloma-derived keratinocytes: differential effects of the ras, neu, and p53 oncogenes.

The p117 keratinocyte cell line was derived in culture from chemically induced mouse papillomas. The benignly transformed nature of these cells was demonstrated by their ability to re-form benign papillomas when grafted back onto the animal. Retroviral vectors were used to introduce into the p117 cells three distinct oncogenes: v-Ha-ras, p53, and neu. All three oncogenes were able to induce tumorigenic conversion of the p117 keratinocytes when assayed by subcutaneous injection into nude mice. However, grafting the oncogene-transformed cells onto the back of the mouse revealed important differences in the ability of the three oncogenes to induce a fully malignant phenotype. While the ras-transformed papilloma cells formed aggressive carcinomas, p53 and neu transformation yielded an intermediate phenotype, with formation of large exophytic tumors, not yet invasive but with highly dysplastic features remarkably similar to those of in situ carcinomas. These findings establish a homologous, genetically modifiable cell system in which various stages of malignant transformation can be directly compared.

Animals↗
PubMed1988

Enhancement of mezerein-promoted papilloma formation by treatment with 12-O-tetradecanoylphorbol-13-acetate or mezerein prior to initiation.

The effects of promoter treatments prior to initiation on subsequent promotion by mezerein were examined in SENCAR mice. Groups of mice received two applications of various complete as well as first and second stage promoters given at various time intervals prior to initiation ranging from 3 days to 10 weeks. The mice were then initiated with 2 micrograms of 7,12-dimethylbenz[a]anthracene (DMBA) followed 2 weeks later by twice-weekly treatments with 2 micrograms of mezerein. The papilloma response in mice, receiving pretreatments with 2 micrograms of 12-O-tetradecanoylphorbol-13-acetate (TPA) either 3 days, 1, 2, 3 or 5 weeks before initiation, was similar to that seen when TPA was given after initiation during stage I of promotion followed by stage II of promotion with mezerein (4-5 papillomas per mouse in all groups). Surprisingly, pretreatment with the stage II promoter, mezerein (2 micrograms), either 2 or 5 weeks prior to initiation, also gave papilloma responses similar to that induced with the standard two-stage promotion protocol (4.7 and 6.4 papillomas per mouse, respectively). The papilloma response was less than that in the standard two-stage promotion protocol when pretreatments with the stage I promoter A23187 (80 micrograms/mouse) were given either 2 or 5 weeks before initiation (2.6 and 2.3 papillomas per mouse, respectively). However, a repeat experiment (currently in progress) with a higher dose of A23187 (160 micrograms/mouse) given 2 weeks prior to initiation indicates that it is more effective than the 80 micrograms dose. When the time interval between pretreatment and initiation was increased to 10 weeks, the papilloma response with TPA and A23187 pretreatment was reduced to below two papillomas per mouse and with mezerein pretreatment to below three papillomas per mouse, indicating the effect was reversible. Histological changes in epidermis of mice which received two applications of these compounds correlated with the tumor response. In this regard, treatment with two applications of TPA and mezerein resulted in an epidermal hyperplasia of similar magnitude (epidermal thickness of 53.5 +/- 1.5 and 50.0 +/- 1.1 microns, respectively). The hyperplasia produced by treatment with two applications of 80 micrograms A23187 (39.4 +/- 1.8 microns) was significantly less. The ability of pretreatments with benzoyl peroxide (20 mg) and chrysarobin (50 micrograms) to affect the subsequent promoting activity of mezerein was also examined.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1988

Cytogenetic profile of mouse skin tumors induced by the viral Harvey-ras gene.

Several investigators have postulated that the activation of the Harvey-ras (rasH) gene is the initiating event in mouse skin carcinogenesis. In support of this speculation, Roop et al. (Nature, 323, 822-824, 1986) have shown that papillomas developed from skin grafts of cultured keratinocytes in which the v-rasH gene had been introduced by a defective retroviral vector. Using the same technique we have analyzed the cytogenetic profile of primary mouse epidermal cultures bearing the activated ras gene and of the tumors generated by skin grafts of the same cells. The epidermal cultures infected with the replication-defective virus which incorporated the v-rasH gene into a packaging-defective Moloney murine leukemia virus did not show any detectable karyotypic abnormalities. These cells presented a diploid/tetraploid configuration similar to uninfected controls. Furthermore, papillomas produced by skin grafts of these cells also showed euploid chromosomal profiles. On the other hand, two carcinomas that arose spontaneously from pre-existing papillomas showed 80% aneuploid cells and aneuploid stem lines. It appears, from these results, that the activated rasH gene is able to generate benign lesions without the involvement of further gross genomic alterations, but that such alterations may be associated with malignant conversion.

Animals↗
PubMed1988

Effect of exogenous glutathione on tumor progression in the murine skin multistage carcinogenesis model.

Oxidative stress has been suggested to play an integral role in the cancer process. It may be particularly significant during tumor progression, where there is likely to be a large amount of free radicals generated by infiltrating inflammatory cells and dying tumor cells. In order to test this hypothesis, a variety of free radical scavengers and antioxidants were assessed for their ability to inhibit tumor progression. The murine skin multistage carcinogenesis model was used to generate papillomas, which are a population of putative precancerous lesions. Various test agents were applied topically to papillomas in order to determine if they would decrease the incidence of the malignant lesion, squamous cell carcinoma. The agents tested included: reduced glutathione (GSH), butylated hydroxyanisole, vitamin E, copper(II) (3,5-diisopropylsalicylate)2, sodium benzoate, N-acetyl cysteine and disulfiram. Under the conditions of our experiments, only GSH and disulfiram inhibited tumor progression to a significant degree. Additional studies indicated that GSH prevented cancer development in a dose-dependent manner. Another experiment demonstrated that when papillomas received repeated topical applications of diethylmaleate, a GSH-depleting agent, tumor progression was enhanced. Collectively these data suggest that sufficient glutathione levels may be important in preventing cancer formation.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1988

Interspecies comparisons of tissue DNA damage, repair, fixation, and replication.

The many anatomical, physiological, and biochemical differences among various mammalian species make it difficult to extrapolate carcinogenic potency data from animals to humans. The process is further complicated by the multistep origin of most malignant tumors in animals and humans due to the interaction of target cells with both endogenous and exogenous factors. Species differences in these aspects of carcinogenesis must also be considered when attempting to evaluate the carcinogenic risks of chemicals to humans. Cancer development in animals involves at least three distinct stages: initiation, promotion, and progression. Intra- and interspecies differences in susceptibility to carcinogenesis may be related to any one or a combination of these stages. Variation in species susceptibility to tumor initiation may result from differences in the abilities of various species to metabolize a potential carcinogen to an ultimate carcinogenic form and/or to detoxify the carcinogen. Most comparative studies among species have only revealed subtle differences in metabolism. DNA adducts from several activated carcinogens have been found to be the same in a number of tissues from various species, including humans. Capacity for DNA repair is apparently a critical factor in the initiation of carcinogenesis in target cells of different species but is less critical among mice that differ in susceptibility to two-stage carcinogenesis of the skin and liver. Susceptibility variations among stocks and strains to such carcinogenesis appear to be related to alterations in tumor promotion. Additional comparative studies are critically needed on all aspects of carcinogenesis to permit effective extrapolation of carcinogenic potency data from animals to humans.

Animals↗
PubMed1987

Events associated with mouse skin tumor promotion with respect to arachidonic acid metabolism: a comparison between SENCAR and NMRI mice.

NMRI and SENCAR, two stocks of mice commonly used in multistage skin carcinogenesis studies, were compared with respect to the effects of inhibitors of arachidonic acid metabolism for the following 12-O-tetra-decanoylphorbol-13-acetate (TPA)-elicited events: tumor promotion, DNA synthesis in vivo and in vitro, ornithine decarboxylase induction, and prostaglandin (PG) E2 synthesis. Previous work had shown that the cyclooxygenase inhibitor indomethacin enhanced TPA promotion in SENCAR mice. We report here that over the same dose range (50 to 200 micrograms) indomethacin caused a dose-dependent inhibition of promotion in NMRI mice. Significant reversal of this inhibition was achieved with concomitant application of 10 micrograms PGF2 alpha but not PGE2. DNA synthesis studies showed that low doses of indomethacin and flurbiprofen increased TPA-stimulated DNA synthesis in primary cultures from SENCAR mice; indomethacin suppressed this response in NMRI cultures. In vivo DNA synthesis studies showed the same pattern: indomethacin enhanced TPA-stimulated DNA synthesis in SENCAR mice but inhibited in NMRI mice. Other classes of inhibitors of arachidonate metabolism (i.e., the cyclooxygenase-lipoxygenase inhibitors 5,8,11,14-eicosatetraynoic acid and phenidone and the phospholipase A2 inhibitor dibromoacetophenone) had inhibitory activity in vitro and in vivo in both stocks of mice. Indomethacin was found to inhibit TPA-induced ornithine decarboxylase activity to the same extent in both mice. Indomethacin was also very effective in inhibiting TPA-induced PGE2 synthesis in both stocks of mice. 5,8,11,14-Eicosatetraynoic acid and phenidone were likewise suppressive in both stocks of mice. It is concluded that the NMRI and SENCAR mice respond similarly to TPA with respect to promotion, DNA synthesis, ornithine decarboxylase induction, and PG synthesis. The difference appears to be in the degree of involvement of the lipoxygenase pathway.

5,8,11,14-Eicosatetraynoic Acid↗