Characterization of insulin-like-growth factor II (IGF II) mRNA positive hepatic altered foci and IGF II expression in hepatocellular carcinoma during diethylnitrosamine-induced hepatocarcinogenesis in rats

Abstract

Background: Insulin-like-growth factor II (IGF II) has been implicated in the pathogenesis of neoplasm of different tissues, including liver of rats and men. This growth factor is believed to exert its effect during cellular proliferation. During the process of development of hepatocellular carcinoma (HCC), different hepatic altered foci appear. They are believed to be the putative precursors of HCC in rats and in men. Thus, to study the role of the gene in a defined model of hepatocarcinogenesis was the target to elucidate its role in various cancer phenotypes during the entire development stage of cancer, right from earlier preneoplastic lesions to HCC.

Methods: Antisense in situ hybridization technique was used here to characterize the type(s) of foci in which IGF II mRNA had expressed during the development of hepatocarcinogenesis-induced by diethylnitrosamine and promoted by phenobarbital in rats. Various focal lesions have been categorized depending on the stages and sizes along with IGF II expression patterns in them. Immunohistochemical detection for proliferating cell nuclear antigen (PCNA) was made to detect the role of the gene in preneoplastic and neoplastic cellular proliferation.

Results: IGF II expression was located in the glycogen-storage acidophilic cell foci maximally followed by mixed cell lesions and the least in basophilic lesions. The expression of IGF II was found to be predominant in the HCC. The expression of gene was also located at the peripheral cells of spongiosis hepatis which are believed to be the precursor of ito cell carcinoma. It was noted that there is a direct correlation between IGF II expression and immunohistochemical detection for PCNA.

Conclusion: It may be concluded that IGF II gene expression plays an important role during the development of neoplasia and the gene expresses in the sequence of events leading from glycogen-rich-acidophilic lesions to glycogen poor basophilic lesions to HCC with an expression pattern of "high-low-high" in terms of degree of expression. Moreover, the essential role of the gene at the immediate initiation stage of carcinogenesis (first few weeks) and during HCC development cannot be ignored. Thus this expression can be used as a suitable marker for very early detection of the cancerous process and can save numbers of future cancer victims by very early detection of this disease.

Figure 1

Quantification of DIG-labeled sense and antisense IGFII mRNA by spot test against standard labeled mRNA (Boehringer-Mannheim RNA detection kit).

Figure 2

Detection of DIG-labeled IGFII sense and antisense mRNA (860 bp) using gel electrophoresis. Lane 1; DIG-labeled sense IGFII mRNA, Lane 2; DIG-labeled antisense IGFII mRNA.

Figure 3

Glycogen storage foci (shown by yellow arrows) in rat hepatic tissue with Periodic Acid Schiff reaction, ×100.

Figure 4

Mixed cell focal lesion (shown by green arrows) and basophilic lesion (shown by blue arrows) in DENA treated rat liver, ×100.

Figure 5

Hepatic section of DENA treated experimental animals showing glycogen storage foci with PAS staining, ×100.

Figure 6

IGFII mRNA expressed glycogen storage lesion detected with Digoxigenin-labeled antisense IGFII, mRNA ×100.

Figure 7

Glycogen storage lesion shown after Digoxigenin-labeled sense IGFII mRNA treatment during in situ hybridization method ×100. Pv- portal vein. Bd – bile duct.

Figure 8

Section of hepatic tumor (shown by arrows) in HCC; using toluedine blue ×40.

Figure 9

Section of hepatic tumor (shown by arrows) in HCC; using PAS reaction ×40.

Figure 10

Section of hepatic tumor (shown by arrows) in HCC; IGFII mRNA expression detected with DIG-labeled IGFII antisense mRNA in tumor as well as peripheral tissue. ×40.

Figure 11

IGFII mRNA expression in hepatocytes in tumor area during HCC in experimental animals (see protocols) ×97.5.

Figure 12

Serial sections of experimental rat hepatic tissue showing spongiosis hepatis with haematoxylin and eosin (shown by white arrows) ×40.

Figure 13

Section of experimental rat hepatic tissue showing spongiosis hepatis with toluedine blue ×120.

Figure 14

Section of experimental rat hepatic tissue showing spongiosis hepatis with PAS reaction) ×40.

Figure 15

IGFII mRNA expression detected by DIG- labeled antisense IGFII mRNA in the peripheral cells of the spongiocyst hepatis ×120.