Characteristic upregulation of glucose-regulated protein 78 in an early lesion negative for hitherto established cytochemical markers in rat hepatocarcinogenesis

Abstract

Previously, we reported α(2)-macroglobulin (α(2)M) to be a novel marker characteristic of rat hepatocellular preneoplastic and neoplastic lesions negative for hitherto well-established markers. In the present study, we further examined other candidate markers with specificity for the same type of lesions. Glutathione S-transferase-placental form (GST-P)-negative hepatocellular altered foci (HAF) were generated using a two-stage (initiation and promotion) carcinogenesis protocol with N,N-diethylnitrosamine (DEN) and either Wy-14,643 or clofibrate, two peroxisome proliferators. Microarray analysis using total RNAs isolated from laser-microdissected GST-P-negative HAF (amphophilic cell foci) and adjacent normal tissues was conducted along with immunohistochemistry and real-time RT-PCR. Staining for glucose-regulated protein 78 (GRP78) was detected in GST-P-negative HAF and hepatocellular adenomas, and slightly increased GRP78 mRNA expression was observed in the lesions by real-time RT-PCR analysis. Thus, an early increase of GRP78 expression in hepatocarcinogenesis is likely a feature of the amphophilic subset of HAF.

Keywords: glucose-regulated protein 78; molecular marker; preneoplastic lesion; rat hepatocarcinogenesis; α2-macroglobulin.

Fig. 1

The relative expression of GRP78 mRNA in GST-P-negative HAF induced by DEN→clofibrate was slightly increased compared with the adjacent normal tissue. The vertical axis represents the ratio of GRP78 mRNA expression versus GAPDH mRNA expression in each region. The white bars represent GRP78 mRNA expression in the normal tissue, and the left hatched and black bars represent GST-P-positive and GST-P-negative HAF, respectively.

Fig. 2

Immunohistochemistry for GRP78 and GST-P in HAF induced by DEN→clofibrate. (a) GST-P-negative lesion: 1, HE staining; 2, GRP78; 3, GST-P. (b) GST-P positive lesion: 1, HE staining; 2, GRP78; 3, GST-P. Lens × 4.

Fig. 3

Immunohistochemistry for GRP78 in GST-P-negative HCAs induced by DEN→clofibrate. (a) HE staining. (b) GST-P. (c), (d) GRP78. (a)-(c) lens × 4; (d) lens × 10.