alpha(2)-Macroglobulin: a novel cytochemical marker characterizing preneoplastic and neoplastic rat liver lesions negative for hitherto established cytochemical markers

Abstract

We tried to identify a novel marker characteristic for rat hepatocellular preneoplastic and neoplastic lesions, undetectable by well established cytochemical markers. Glutathione S-transferase placental (GST-P)-negative hepatocellular altered foci (HAF), hepatocellular adenoma (HCA), and hepatocellular carcinoma (HCC) were generated by two initiation-promotion models with N-nitrosodiethylamine (NDEN) and peroxisome proliferators, Wy-14,643 and clofibrate. Total RNAs isolated from laser-microdissected GST-P-negative HAF (amphophilic cell foci) and adjacent normal tissues were applied to microarray analysis. As a result, five up-regulated genes were identified, and further detailed examinations of the gene demonstrating most fluctuation, ie, that for alpha(2)-macroglobulin (alpha(2)M) were performed. In reverse transcriptase-polymerase chain reaction, alpha(2)M mRNA was overexpressed not only in amphophilic GST-P-negative HAF but also in amphophilic GST-P-negative HCA and HCC. In situ hybridization showed accumulation of alpha(2)M mRNA to be evenly distributed within GST-P-negative HAF (predominantly amphophilic cell foci). Distinctive immunohistochemical staining for alpha(2)M could be consistently demonstrated in GST-P-negative HAF, HCA, and HCC induced not only by peroxisome proliferators but also N-nitrosodiethylamine alone. Thus our findings suggest that alpha(2)M is an important novel cytochemical marker to identify hepatocellular preneoplastic and neoplastic lesions, particularly amphophilic cell foci, undetectable by established cytochemical markers and is tightly linked to rat hepatocarcinogenesis.

Figure 1

Semiquantitative RT-PCR for α₂M mRNA in HAF, HCA, HCC, and adjacent normal liver tissues. Po, GST-P-positive; Ne, GST-P-negative (amphophilic phenotype); N, normal tissue; F, hepatocellular altered foci; A, hepatocellular adenoma; C, hepatocellular carcinoma; WY, Wy-14,643; CF, clofibrate.

Figure 2

In situ hybridization for α₂M mRNA in GST-P-negative HAF (amphophilic cell foci) induced by NDEN + clofibrate. a: H&E staining. b: Accumulation of α₂-M mRNA. Original magnifications, ×125.

Figure 3

Immunohistochemistry for α₂M and GST-P. A: HAF induced by NDEN + clofibrate. a: Amphophilic GST-P-negative HAF: 1, H&E staining; 2, α₂-M; 3, GST-P. b: GST-P-positive HAF: 1, H&E staining; 2, α₂-M; 3, GST-P. B: GST-P-negative HAF induced by NDEN alone. 1, H&E staining; 2, α₂-M; 3, GST-P. Original magnifications: ×40 (A); ×30 (B).

Figure 4

Immunohistochemistry for α₂M and GST-P in liver tumors induced by NDEN + clofibrate. a and c: GST-P-negative HCA and HCC (amphophilic phenotype), respectively. b and d: GST-P-positive HCA and HCC, respectively. 1, H&E staining; 2, α₂-M; 3, GST-P. Arrow illustrates invasive tumor cells into blood vessel in c2. Original magnifications, ×85.