645 Comparison of Uroplakin Expression During Urothelial Carcinogenesis Induced by N -Butyl-N-(4-Hydroxybutyl)Nitrosamine in Rats and Mice* KUMIKO OGAWA, 1,2 MARGARET ST. JOHN, 1 MARIA LUIZA DE OLIVEIRA, 1,4 LORA ARNOLD, 1 TOMOYUKI SHIRAI, 2 TUNG-TIEN SUN, 3 AND SAMUEL M. COHEN 1 1 Department of Pathology and Microbiology and the Eppley Institute for Research in Cancer, University of Nebraska Medical Center, Omaha, Nebraska 68198-3135, USA, 2 First Department of Pathology, Nagoya City University Medical School, Nagoya 454, Japan, and 3 Epithelial Biology Unit, The Ronald O. Perelman Department of Dermatology, Department of Pharmacology and Kaplan Comprehensive Cancer Center, New York University School of Medicine, New York, New York 10016, USA * Address correspondence to: Dr. Samuel M. Cohen, Department of Pathology and Microbiology, University of Nebraska Medical Center, 983135 Nebraska Medical Center, Omaha, Nebraska 68198-3135; e-mail: scohen@unmc.edu. 4 Present address: Departamento de Patologia, Faculdade de Medici- na, Unesp, Botucatu-SP, Brazil, CEP, 18618-000. ABSTRACT The expression of uroplakins, the tissue-specific and differentiation-dependent membrane proteins of the urothelium, was analyzed immunohistochemically in N- butyl -N- (4-hydroxybutyl)nitrosamine (BBN)-treated rats and mice during bladder carcinogenesis. Male Fischer 344 rats were treated with 0.05% BBN in the drinking water for 10 wk and were euthanatized at week 20 of the experiment. BBN was administered to male B6D2F 1 mice; it was either provided at a rate of 0.05% in the drinking water (for 26 wk) or 5 mg BBN was administered by intragastric gavage twice weekly for 10 wk, followed by 20 wk without treatment. In rats, BBN-induced, noninvasive, low-grade, papillary, transitional cell carcinoma (TCC) showed decreased uroplakin-staining of cells lining the lumen but showed increased expression in some nonluminal cells. In mice, nonpapillary, high-grade dysplasia, carcinoma in situ, and invasive carcinoma were induced. There was a marked decrease in the number of uroplakin-positive cells lining the lumen and in nonluminal cells. This occurred in normal-appearing urothelium in BBN-treated mice and in dysplasic urothelium, in carcinoma in situ, and in invasive TCC. The percentage of uroplakin-positive nonluminal cells was higher in control mice than in rats, but it was lower in the mouse than in the rat after BBN treatment. Uroplakin expression was disorderly and focal in BBN-treated urothelium in both species. These results indicate that BBN treatment changed the expression of uroplakins during bladder carcinogenesis, with differences in rats and mice being related to degree of tumor differentiation. Keywords. Uroplakins; differentiation; bladder cancer; cell membranes INTRODUCTION Uroplakins are the major protein components of the asymmetric unit membrane (AUM). The AUM is a spe- cialized plasma membrane representing urothelial termi- nal differentiation, which is unique to the luminal surface of superficial cells and of the intracytoplasmic vesicles (17, 20). Possible functions of the AUM include the fol- lowing : it may act as a permeability barrier; it may act as a means of stabilizing the apical bladder surface; and it may act as a supramolecular device to change the sur- face area of the plasma membrane during expansion or contraction of the bladder. Uroplakins are synthesized in the endoplasmic reticulum, concentrated and modified in the Golgi complex, and then incorporated into the mem- brane of intracytoplasmic fusiform vesicles and into the plasmalemma. Uroplakins are mainly produced in cells of the superficial layer of the urothelium. Uroplakins con- sist of 4 components: the 27-kDa uroplakin Ia; the 28- kDa uroplakin Ib; the 15-kDa uroplakin II; and the 47- kDa uroplakin III (10, 17-20). Together they form 16- nm twisted ribbon-shaped particles that are arranged in a well-ordered, hexagonal lattice with p6 symmetry (15). These 4 uroplakins are conserved through a broad range of mammalian species, including human, monkey, cattle, sheep, pig, dog, rabbit, rat, and mouse (16). Previous experiments investigating uroplakins in vari- ous carcinogen-treated rat bladders showed that the pat- terns of expression of these proteins were modified dur- ing carcinogenesis (13). In the control bladder, the lu- minal surface membrane of the superficial cells stains strongly positive for uroplakins, but there is no cytoplas- mic staining and no staining of basal intermediate cells. The genotoxic carcinogen N-[4-(5-nitro-2-furyl)-2-thia- zolyl]formamide (FANFT) induces simple hyperplasia, in which uroplakin expression is discontinuous and disor- derly, not only in the superficial cells but also in the intermediate cells. The nongenotoxic carcinogens uracil, sodium saccharin, and sodium ascorbate also induce sim- ple hyperplasia, but the staining of the superficial cells remains continuous and orderly. Occasional intermediate cells of the bladder also stained positive for uroplakins in uracil-fed rats (13). In carcinoma induced by FANFT in rats, uroplakin expression is scarce, disorderly, and fo- cal and is usually not present in the cells lining the blad- der lumen (13). N-Butyl-N-(4-hydroxybutyl)nitrosamine (BBN), like