Differences in the Urea-extracted Proteins of Mouse Epidermis and Squamous cell Carcinomata Determined by Fluorescence Microscopy

Abstract

Summary.-Fluorescence microscopy was used to demonstrate differences in the urea-extractable antigens of mouse epidermis and squamous cell carcinoma. When serum-and normal tissue sediment-absorbed antisera prepared against mouse epidermal urea-extracted proteins were further absorbed with carcinoma urea antigens, antisera specific for epidermis resulted. When antisera raised against the urea-extractable proteins of mouse squamous cell carcinomata were serumand normal tissue sediment-absorbed and then further absorbed with epidermal urea antigens, antisera were prepared which stained papilloma and carcinoma, but not epidermis, and thus these antisera were not specific for carcinoma. Antisera prepared against the urea-extractable proteins of human epidermis reacted in immunodiffusion in agar with the epidermal urea proteins, but not with human squamous cell carcinoma urea proteins. Also antisera prepared against the carcinoma urea-extractable proteins reacted with these proteins in agar, but no reaction occurred with the epidermal urea-extractable proteins.

Differences in the Urea-Extracted or Fibrous Proteins of Mouse Epidermis at Various Stages of Malignant Transformation

“…On the other hand, when a mixture of IgG preparations or antisera raised against the papilloma and carcinoma urea proteins reacted with the gel or supernatant fractions of these tissues, precipitin bands of identity resulted. Thus these immunodiffusion results on the gel and supernatant fractions of the urea proteins of epidermis and related tissues are in agreement with previous studies on antigenic differences between the urea proteins of epidermis and papilloma orcarcinoma[l, 3,7]. The urea proteins of papilloma and carcinoma appear to be similar as judged from the immunodiffusion ex periments given above and as is suggested by the amino acid composition of the urea proteins (table III).…”

“…Recent studies employing immunodiffusion in agar suggested that the urea proteins of normal human epidermis and squamous-cell carcinoma were nonidentical [3]. These differences suggest the possibility of developing fluorescent labelled antisera specific for squamous-cell carcinoma and their detection in specific staining of tissue sections by fluorescence microscopy.…”

“…Procedures have been given previously for the following: (a) preparation of normal and hyperplastic epidermis, papilloma, and carcinoma: (b) isolation and purification of the urea proteins from these tissues; (c) and preparation of antisera in rabbits against the urea proteins [2, 3,7], The IgG fractions were obtained from antisera through precipitation with 50% (N H^SO ,, followed by DEAE-cellulose chromatography [12], Immunodiffusion. Immunodiffusion experiments were carried out on microscopic slides in 1.5% agar buffered with pH 7.4 barbital (tx = 0.15).…”

“…The various antisera and the IgG prep arations obtained from these antisera were adsorbed with pooled normal mouse serum, liver, kidney, and dermis sediments in the same manner as detailed previously [3], Cel filtration. Gel filtration was carried out with Sepharose 4B in conventional columns, essentially in the same way as indicated previously [8], Prior to gel filtration the various urea proteins were thoroughly dialyzed against 0.05 m barbital buffer, pH 8.5, containing 0.1 m NaCl.…”

“…The nature of these antigenic differences was demonstrated by using thoroughly adsorbed antisera or IgG preparations raised in rabbits against the urea proteins of epidermis, papilloma, and carcinoma by (a) fluorescence microscopy [3,5,6]; (b) immunodiffusion in agar [I, 2,7]; and (c) the paired label technique using 13lI and 125I [I]. The 1 The author acknowledges the excellent technical assistance of Mrs. A. Heining and McKenna and Miss A. Baumler.…”