Proteome analysis of rat hepatomas: Carcinogen-dependent tumor-associated protein variants

Abstract

Proteome analysis led to the identification and characterization of tumor-associated protein variants by two-dimensional electrophoresis and mass spectrometry. We focused on comparing the influence of genotoxic nitroso compounds N-methyl-N-nitrosourea, diethylnitrosamine and N-nitrosomorpholine and the nongenotoxic peroxisome proliferator Nafenopin as tumor-inducing agents on the protein pattern of rat hepatomas. We found several tumor-associated variants that represent members of the aldo-keto reductase superfamily. Their induction and/or inhibition was specifically related to the carcinogen used for tumor induction. The most prominent tumor-associated protein, rat aldose reductase-like protein-1 (rARLP-1) (69% sequence identity to lens aldose reductase) and three additional types of rARLP-1 were detected in nitroso compound-induced rat hepatomas, while rat aldo-keto reductase protein-c (Rak-c), a novel tumor-associated variant (65% sequence identity with 3alpha-hydroxysteroid dehydrogenase) was discovered in N-methyl-N-nitrosourea-induced hepatomas only. 3Alpha-hydroxysteroid dehydrogenase and delta4-3-ketosteroid-5beta-reductase, both liver-specific enzymes, were reduced in amount in all hepatomas investigated, independent of their mode of induction. We conclude, that detoxification enzymes like 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD) and delta4-3-ketosteroid-5beta-reductase (5beta-Red) might be replaced in hepatomas by tumor-associated proteins that are often present in the embryonal state, like the rARLPs or the Rak-c protein. Their induction appears to reflect an altered constitutive pattern of detoxification enzymes, detoxifying toxic aldehydes being induced by nitroso compounds. In contrast, members of the aldo-keto reductase superfamily have not been found in Nafenopin-induced hepatomas. The pattern of tumor-associated protein variants is apparently characteristic for a given group of initiating carcinogens. The hypothesis is proposed that carcinogens leave specific fingerprints at the proteome level of manifest liver tumors.

Detection and identification of tumor-associated protein variants in human hepatocellular carcinomas

“…The extraction method of the soluble protein fraction was the same for all samples following the method of Klose 28 as previously described. 5 Two-Dimensional Electrophoresis. Soluble proteins were separated by high-resolution 2-DE according to Klose,29 using Iso-Dalt -equipment (Hoefer Scientific Instruments, San Francisco, CA).…”

“…In group I, 2-DE gels of 10 different HCC samples were compared with 3 different normal liver tissues; in group II, 5 different HCC samples were compared with their corresponding normal liver tissues. The selected area in 2-DE gels, Ϸ30 -40 kd/pI Ϸ6.2-7.8, according to the rat model, 5 was evaluated visually at first according to several criteria. All protein spots that were changed in quantity and/or quality in at least 80% of all HCCs investigated (n ϭ 15) were defined as candidates for tumor-associated protein variants.…”

“…These protein alterations might represent tumor markers that are useful in the diagnosis of human tumors and may also help the understanding of mechanisms of tumor induction and development. Proteome analysis of liver proteins and HCC were predominantly performed using either chemically induced hepatomas in animals (predominantly the rat [5][6][7][8][9][10] ) or human HCC cell lines, such as HepG2 and Huh7 cells, 11 BEL-7404 cells, 12 or HCC-M cells. 2,13,14 Numerous so-called tumor-associated or cancer-related proteins were identified; these provide valuable information for the establishment of HCC protein databases.…”

“…These are 4 aldose reductase-like proteins (ARLPs) predominantly detected in nitroso-compound-induced rat hepatomas. 5 In human HCC, an aldose reductase-like-1 (ARL-1) mRNA was expressed. 24,25 Two aflatoxin B1-inducible aldehyde reductases were discovered in aflatoxin-B1-induced rat hepatomas 26 and in human colonic carcinomas.…”

Hepatitis B virus X protein mutant upregulates CENP-A expression in hepatoma cells

“…Briefly, culture cells or tissues were lysed in buffer containing 10 mmol/l Tris (pH 7.4), 1% sodium dodecyl sulfate (SDS), 1 mmol/l sodium orthovanadate, and complete protease inhibitors (Roche). The extraction was accomplished using the Klose method as described previously (22,23). Samples of the lysates (50 µg) were then separated using SDS-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA), and blotted with the primary antibodies.…”