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.

Characterization of a family of glycoproteins associated with the bile canalicular membrane of normal hepatocytes but not expressed by two transplantable rat hepatocellular carcinomas.

Xenoantisera were used to investigate the expression of normal cell-surface components on transplantable rat hepatocellular carcinoma (253 and 311c) cells. Cell-surface components, immunoprecipitated from non-ionic detergent extracts of 125I-labeled hepatocytes by xenoantisera raised against rat hepatocytes, 253 cells, or 311c cells, were analyzed by 2-dimensional polyacrylamide gel electrophoresis. Comparison of the gel patterns revealed that anti-hepatocyte antiserum was reactive with a component having an apparent molecular weight of 105,000 (gp105) that was not recognized by xenoantisera against 253 or 311c cells. This component was isolated by affinity adsorption to Sepharose-conjugated wheat germ agglutinin, reconstituted in liposomes, and used to immunize a rabbit. The resulting antiserum, anti-gp105, was reactive with a family of glycoproteins that had an apparent molecular weight of 105,000 but differed in isoelectric points. Immunohistochemical techniques revealed that gp105 was localized in the bile canalicular domain of the hepatocyte plasma membrane. Immunodepletion analysis, blocking of immunoprecipitation by extracts of tumor cells, and labeling of cells by immunohistochemical techniques indicated that gp105 was not expressed at the surface of 253 or 311c cells. These studies suggest that the altered expression of gp105 on 253 and 311c hepatocellular carcinoma cells may be associated with the abnormal tissue architecture and growth patterns exhibited by these transplantable tumors.

Polyacrylamide gel electrophoresis and immunoblotting of proteins extracted from paraffin-embedded tissue sections.

We show in this communication that polyacrylamide gel electrophoresis (PAGE) and immunoblotting of proteins can be performed using one to two 5-7 micron paraffin sections of tissues fixed in non-cross-linking fixatives (acetone, alcohol, or modified Carnoy's solution). Proteins for study were extracted from paraffin sections of mouse foot pad and liver. The presence of unaltered keratin polypeptides in tissues fixed with either acetone or alcohol was demonstrated in gels stained with Coomassie brilliant blue. The preservation of their antigenic determinants was demonstrated with immunoblotting. Furthermore, the immunoreactivity of soluble proteins, such as albumin, remained unaltered in immunoblots obtained from paraffin-embedded mouse liver sections. These data indicate that tissues embedded and stored in paraffin are useful for the above-mentioned biochemical and immunological studies and may therefore be an important technique for diagnostic pathology or retrospective studies.

Rapid low molecular weight polyethylene glycol embedding protocol for immunocytochemistry.

We describe an alternative polyethylene glycol (PEG) embedding procedure which utilizes PEG 200 for dehydration and PEG 600 for infiltration and embedding of perfusion-fixed rat liver. PEG 600 has a melting point of 22 degrees C, enabling infiltration of fixed tissue to be performed at room temperature. Sections (2 microM) cut in a cryostat at -20 degrees C and immobilized in agarose were readily labeled by immunoperoxidase protocols with monoclonal antibodies to hepatocyte membrane antigens. Subsequent examination by light microscopy or by electron microscopy after re-embedding in resin and ultra-thin sectioning showed excellent preservation of morphology, with minimal impairment of antigenicity.

Induction of short-term markers of tumor promotion by organic peroxides.

Experiments from different laboratories have shown that benzoyl peroxide (BzPo) and other organic peroxides are effective tumor promoters in the mouse skin two-stage carcinogenesis system. In the present paper we have studied the short-term effect of six other organic peroxides, which have not been previously assayed as skin tumor promoters. These compounds were chosen for their molecular diversity, the type of radical predicted to be formed, solubility and availability. The parameters evaluated in this study include a series of short-term markers of tumor promotion, hyperplasia, induction of dark basal keratinocytes and induction of ornithine decarboxylase activity. After single applications the biological activity of the compounds was: m-chloroperoxybenzoic acid greater than di-m-methylbenzoyl peroxide greater than dicumyl peroxide greater than O,O-t-butyl-O-(2-ethylhexyl)mono-peroxycarbonate greater than benzoyl peroxide greater than di-m-chlorobenzoyl peroxide greater than di-t-butyl peroxide greater than t-butyl hydroperoxide. After multiple applications, the order of activity of the compounds was: dicumyl peroxide greater than di-m-methyl-benzoyl peroxide greater than O,O-t-butyl-O-(2-ethylhexyl)monoperoxy carbonate greater than m-chloroperoxybenzoic acid greater than di-m-chlorobenzoyl peroxide greater than t-butyl hydroperoxide greater than benzoyl peroxide greater than di-t-butyl peroxide. The difference of activity among the different compounds did not seem to correlate directly with the chemical stability of the compound; it is more likely that the activity depends on different factors such as percutaneous absorption, metabolism, and the rate of free radical formation in vivo. The data presented here further support the association between free radicals and tumor promotion since all of the compounds, with the exception of one, were active in inducing the short-term markers of tumor promotion. It will also establish conditions for future tumor experiments.

Structural differences in envelope glycoproteins associated with rat leukaemia virus produced by Novikoff hepatocellular carcinoma and spontaneously transformed Wistar rat embryo cells.

Immunochemical and immunocytochemical techniques have been used to characterize viral glycoproteins and endogenous rat leukaemia viruses (RaLV) produced both by Novikoff hepatocellular carcinoma cells and spontaneously transformed Wistar rat embryo cells (WRC). Results from immunocytochemical analysis demonstrated that RaLV produced by Novikoff and WRC cells could be distinguished by their unique patterns of reactivity with xenoantisera raised against virus particles or viral glycoproteins. This differential labelling was unexpected since all the antisera tested had been shown by immunoprecipitation and immunodepletion analysis to be reactive with viral glycoproteins expressed on the cell surface. Since no significant differences in cell surface-associated viral glycoproteins and those shed from the cell surface were detected by pulse iodination analysis, it was concluded that the apparent discrepancy between immunoferritin labelling and immunoprecipitation analysis resulted from differences in antigen accessibility on intact virions caused by structural differences in the viral glycoproteins expressed on Novikoff and WRC cells. This conclusion was supported by results from ferritin-lectin labelling, affinity chromatography and neuraminidase digestion studies which demonstrated differences in the saccharide moieties on both virion and cell surface-associated viral glycoproteins. Further evidence of structural differences was provided by limited digestion with trypsin and V8 protease of the Mr 64 000 (Nov gp64) and Mr 68 000 (WRC gp68) viral glycoproteins immunoprecipitated from Novikoff and WRC cells, respectively, with either monospecific anti-Rauscher murine leukaemia virus anti-gp70 serum or monospecific antiserum against Nov gp64 (anti-gp64). Results from digestion studies showed that all the major cleavage fragments from WRC gp68 were of higher molecular weight than their Nov gp64-derived counterparts. Evidence that Nov gp64 and WRC gp68 both share structural homology with other murine viral gp70s was suggested by results from immunoprecipitation analysis with anti-gp70 and anti-gp64 sera under reducing and non-reducing conditions which demonstrated the presence of an interchain disulphide bond in both glycoproteins and showed that at least some of these molecules exist on the cell surface as disulphide-linked heterodimers of Mr 78 000 and 82 000.