Doctoral Dissertations

Date of Award

8-1981

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Biomedical Sciences

Major Professor

T. J. Slaga

Committee Members

Susan Fischer, John Cook, Sankar Mitra

Abstract

The purpose of this study was to determine if the phorbol ester tumor promoter 12-0-tetradecanoylphorbol-13-acetate (TPA) induced any specific changes in mouse epidermal proteins using the high resolution technique of two-dimensional electrophoresis. To accomplish this goal of determining the specificity and possibly the stage in promotion with which these protein changes were associated, epidermal proteins were analyzed (1) after treatment of adult mouse epidermis with several weakly promoting hyperplasiogenic agents, (2) following treatment with TPA in combination with various inhibitors of tumor promotion, (3) in basal kerotinocytes isolated from adult epidermis following treatment with TPA or several weakly promoting agents, and (4) during an initiationpromotion experiment.

The more soluble protein components not crosslinked by disulfide bonds were removed from the epidermis using 4M urea. Following the urea extraction, the remaining protein consisting primarily of the epidermal keratins were solubilized using 2% sodium dodecylsulfate and 10mM dithiothreitol (SDS-DTT). The pH range (pH 4-6) of the isoelectric focusing dimension was chosen such that maximal separation of individual keratin polypeptides was achieved. The proteins were resolved further by separation on the basis of their molecular weights in the second dimension which usually consisted of 10% SDS-polyacrylamide gels. The parameters that were used for the identification and characterization of some of the epidermal proteins were extractability, isoelectric points, molecular weights, assembly into filaments in vitro, immunological crossreactivity, peptide maps, and amino acid composition.

Evidence was found which indicated that the potent tumor promoter TPA as well as the weakly promoting hyperplasiogenic agents, mezerein, ethylphenylpropiolate (EPP), and mechanical abrasion, induced similar modifications of epidermal proteins, particularly among the keratins. These keratin modifications progressed with time following treatment resulting in a keratin pattern which resembled that of newborn epidermis. The protein modifications induced by EPP, however, were quantitatively less than those observed with the other agents. This observation is supported by the findings of others which showed that many of the biochemical changes induced by EPP were quantitatively less when compared to those induced by TPA or mezerein. Significant keratin modifications were also observed in basal cells isolated from adult epidermis following treatment with TPA and the other weakly promoting hyperplasiogenic agents. Modification of the epidermal keratins, which represent the major differentiation product of the epidermis, could result in the alteration of the properties of the filaments formed from these proteins. Several other protein changes were observed among the more soluble proteins extracted with urea which were also prominent proteins in newborn epidermis. The induction of hyperplasia by strong or weak promoters appeared to result in shifts in the gene expression in adult epidermis to that of a newborn state, causing the reappearance of newborn proteins. These data indicated that many of the protein changes induced by TPA were associated with its hyperplasiogenic properties.

Further evidence which indicated that these protein changes were associated with hyperplasia and not necessarily related to complete promoting ability was obtained by studying the effects of various inhibitors of tumor promotion on the TPA induced protein modifications. The inhibitors which interfered with the induction of hyperplasia, fluocinolone acetonide (FA) and cycloheximide, were found to also interfere with the promoter-induced protein changes. Retinoic acid (RA) and tosylphenylalanylchloromethyl ketone (TPCK) which did not prevent the TPA induced hyperplasia did not significantly influence these protein changes.

The keratin polypeptide patterns of papillomas, carcinomas, and epidermis during an initiation-promotion experiment were studied to further understand the role that the keratin modifications play in two-stage carcinogenesis. Distinct keratin patterns were found to be associated with each of these tissues. The TPA-treated epidermis, initiated and noninitated, contained the same keratin modifications that were observed with one TPA treatment. These keratin changes were also features of all papillomas; however, papillomas could be distinguished from surrounding epidermis by the presence of considerable charge heterogeneity among the high molecular weight keratins. The most striking protein alterations observed in the carcinomas was a significant reduction in the high molecular weight keratins. In fact, the carcinoma keratin profiles were very similar to those patterns observed in the basal cells isolated from adult epidermis following a single treatment with a hyper-plasiogenic agent. This suggests that the program of keratin synthesis is fixed at the basal cell level of differentiation. Changes in keratin synthesis, therefore, may serve as a biochemical marker of malignant progression in mouse epidermis.

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