Cytokeratin 7/19 expression in N-diethylnitrosamine-induced mouse hepatocellular lesions: implications for histogenesis

Abstract

Hepatocellular carcinoma (HCC) is a common malignancy with poor clinical outcome, whose histogenesis is the subject of intense debate. Specifically, expression of cytokeratins (CKs) 7 and 19, associated with aggressive biological behaviour, is proposed to reflect a possible progenitor cell origin or tumour dedifferentiation towards a primitive phenotype. This work addresses that problem by studying CKs 7 and 19 expression in N-diethylnitrosamine (DEN)-induced mouse HCCs. ICR mice were divided into six DEN-exposed and six matched control groups. Samples were taken from each group at consecutive time points. Hyperplastic foci (13 lesions) occurred at 29-40 weeks (groups 8, 10 and 12) with diffuse dysplastic areas (19 lesions) and with one hepatocellular adenoma (HCA) (at 29 weeks). HCCs (4 lesions) were observed 40 weeks after the first DEN administration (group 12). CKs 7 and 19 showed identical expression patterns and located to large, mature hepatocytes, isolated or in small clusters. Hyperplastic foci and the single HCA were consistently negative for both markers, while dysplastic areas and HCCs were positive. These results support the hypothesis that CKs 7 and 19 expression in hepatocellular malignancies results from a dedifferentiation process rather than from a possible progenitor cell origin.

Keywords: N-diethylnitrosamine; cytokeratin; hepatic progenitor cell; hepatocellular carcinoma; histogenesis; liver.

Figure 1

Experimental design and animal groups. Experimental design; Time in weeks (postexposure): T1 – 8 weeks; T2 – 15 weeks; T3 – 22 weeks; T4 – 29 weeks; T5 – 36 weeks; T6 – 40 weeks; black arrows indicate euthanasia. Control animals received saline solution injections and were euthanized at the same time as the N-diethylnitrosamine-exposed group.

Figure 2

Histological and immunohistochemical features of the livers of control mice, 40W, 100×, bar = 100 μm. (a) H&E. (b) immunohistochemistry for CK7. (c) immunohistochemistry for CK19.

Figure 3

N-diethylnitrosamine-exposed mouse liver. H&E, 100×, bars = 100 μm. Inserts 400×, bars = 20 μm. (a) 8W animal. Hepatocellular hydropic degeneration. Note intense cytoplasmic vacuolization. (b) 15W animal. Note coagulative necrosis focus. (c) 22W animal. Note nuclear changes with condensed, fuzzy chromatin. (d) 29W animal. Note small, mixed type hyperplastic nodule. (e) 36W animal. Note pleomorphic nuclei with pseudonucleoli over a dysplastic area. (f) 40W animal. Note a pseudo-acinar structure amidst a hepatocellular carcinoma.

Figure 4

N-diethylnitrosamine-exposed mouse liver. Immunostaining for CK7-Mayer's haematoxylin, 100×, bars = 100 μm. Inserts 400×, bars = 20 μm. (a) 8W animal. (b) 15W animal. Note mitotic figure. (c) 22W animal. (d) 29W animal. Note hyperplastic nodule on the upper half of the picture. (e) 36W animal. Note scattered, large, mature, immunopositive hepatocytes over a dysplastic area. (f) 40W animal. Note variably stained, mature, immunopositive hepatocytes scattered amidst a hepatocellular carcinoma.

Figure 5

N-diethylnitrosamine-exposed mouse liver. Immunostaining for CK19-Mayer's haematoxylin, 100×, bars = 100 μm. Inserts 400×, bars = 20 μm. (a) 8W animal. (b) 15W animal. Note immunopositive biliary epithelium. (c) 22W animal. (d) 29W animal. Note hyperplastic nodule on the lower half of the picture. (e) 36W animal. Note scattered, large, mature, immunopositive hepatocytes over a dysplastic area. (f) 40W animal. Note variably stained, mature, immunopositive hepatocytes scattered amidst a hepatocellular carcinoma.

Figure 6

Macroscopic features of livers from control and N-diethylnitrosamine-exposed ICR mice. (a) 40W control animal. (b) 36W exposed animal. Note hepatomegally with grey discoloration and irregular hepatic surface. (c) 40W exposed animal. Note distortion of hepatic lobes, haemorrhagic foci and multiple, grey nodular lesions.