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A protein detected in N-methyl-N-nitrosourea-initiated rat hepatomas by two-dimensional electrophoresis at 35 kDdpI 7.4 was identified in a previous study by internal amino acid micro sequencing as an aldose-reductase-like protein [Zeindl-Eberhart, E., Jungblut, P. R., Otto, A. & Rabes, H. M. (1994) Identification of tumor-associated protein variants during rat hepatocarcinogenesis, J. Biol. Chem. 269, 14 589-14 5941. Two-dimensional electrophoresis of rat lens proteins revealed a spot at 37 kDa/pI 6.8 that showed a high degree of identity (98.5%) with rat lens aldose reductase after amino acid sequencing and 80% sequence identity to the rat-hepatoma-derived aldose-reductase-like protein. This suggests that hepatomaderived aldose-reductase-like protein and rat lens aldose reductase are related proteins encoded by different genes. A different expression profile of these proteins was found in various rat organs. Rat lens aldose reductase is present, in addition to in lens, in heart, brain, muscle, lung, duodenum, kidney, spleen and bone marrow, while the hepatoma-derived aldose-reductase-like protein is found preferentially in hepatomas and in embryonic liver. Though different in organ expression, an identical response was found for both proteins after stimulation with fibroblast growth factor-1 and after exposure to increased glucose concentrations. Since rat hepatoma-derived aldose-reductase-like protein is expressed in embryonic, but not in adult liver, it is assumed that it is expressed in hepatomas as a functionally active embryonal type of aldose reductase during hepatocarcinogenesis. Immunohistochemistry revealed that the hepatomaderived aldose-reductase-like protein is expressed already in the preneoplastic stage of hepatocarcinogenesis and might potentially serve as a marker enzyme in early hepatic neoplasia.Keywords: two-dimensional electrophoresis ; rat hepatoma; aldose-reductase-like protein.Misprogramming of genetic information in cancer may lead to quantitative andor qualitative protein alterations. Such changes can be studied by high resolution two-dimensional electrophoresis [ 1-31, in combination with ultrasensitive silver staining [4, 51. Two-dimensional electrophoresis has successfully been used to detect tumor associated proteins in chemically induced rat hepatomas [6-121.We detected previously several protein variants in N-methyl-N-nitrosourea-induced rat hepatomas by two-dimensional electrophoresis [ I l l and identified one of these variants (spot 17) by amino acid sequencing as an enzyme apparently related to rat lens aldose reductase (AR) [13]. In a different system, NIH 3T3 mouse fibroblasts, it was shown that fibroblast growth factor-I (FGF-I) induced temporarily an increased level of FGF-1-regulated (FR-1) mRNA [14]. The amino acid sequence deduced from FR-1 mRNA revealed 97% sequence identity [I41 to the rat hepatoma-derived AR-like protein of chemically induced hepatomas [ 131. These findings of putative relations between rat hepatoma-derived AR-like protein, rat lens AR and FR-1 prot...

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To compare the formation and persistence of alkylated DNA bases in the G1- and S-phase compartments in liver in vivo, regenerating rat liver was exposed to [14C]dimethylnitrosamine (0.57 mg/kg, IP injection) or N-[methyl14C]-N-nitrosourea (3.3 mg/kg, intraportal injection) during the G1 phase of the cell cycle (12 h after partial hepatectomy), or at 24 h after partial hepatectomy with 30% hepatocytes in DNA synthesis, or at 43 h after partial hepatectomy, 4 h after an hydroxyurea block from 14 to 39 h after operation with 80% hepatocytes in DNA synthesis. At 120 min after dimethylnitrosamine and 90 s, 5, 10, or 60 min after the intraportal pulse of N-methyl-N-nitrosourea the molar fractions of 7-methylguanine (7megua), O6-methylguanine (O6megua), and 3-methyladenine (3mead) and of metabolically labeled guanine were determined from DNA hydrolysates by Sephadex-G10 radiochromatography. After dimethylnitrosamine only minor differences were observed for 7megua formation in the three groups; the 3mead/7megua ratio remained constant irrespective of the number of cells in S phase. In contrast, the O6megua/7megua ratio revealed a loss of O6megua, the extent of which appeared proportional to the fraction of DNA-synthesizing cells in the liver. The rapid loss of O6megua in S-phase cells was confirmed after intraportal administration of N-methyl-N-nitrosourea. During the first 10 min after the methylnitrosourea pulse the O6megua/7megua ratio was constant in G1 cells and dropped from 90 s to 10 min by about 15% in liver containing 30% S-phase cells and by about 40% with 80% cells in DNA synthesis. DNA-synthesizing hepatocytes are apparently endowed with a higher O6megua DNA transferase activity than nonproliferating liver cells. The rapid, though exhaustible elimination of O6megua during S-phase might result in partial protection of DNA-synthesizing cells from base-mispairing and/or from hypomethylation at G-C sites.

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