Histochem Cell Biol (2011) 135:581–591 Given these background ﬁndings, we performed a study on the growth pattern of normal and nodular hepatocytes transplanted into RS-treated host rats, taking advantage of the fact that we could compare two rapidly expanding cell populations under similar experimental conditions. As expected, both cell types exhibited selective clonal prolif- eration in the recipient liver. The distribution of DPPIV enzyme activity was remarkably altered in nodular hepa- tocyte clusters, as shown in Fig. 1. Aberrant expression of this enzyme has been reported in different types of cancers, including hepatocellular carcinoma (Stecca et al. 1997). It is reasonable to hypothesize that the altered distribution of DPPIV enzyme activity in the expanding nodular hepato- cytes may reﬂect, at least in part, an altered polarity of these cells. On the other hand, DPPIV has also been proposed as a possible marker for early diagnosis of HCC in hepatitis C virus-positive cirrhotic patients (Archer et al. 2009). Low levels of Cx32 expression have long been reported in early focal lesions developing during liver carcinogen- esis in the rat (Krutovskikh et al. 1991). However, this alteration was found to be reversibly associated with the proliferative rate of individual foci (Neveu et al. 1994). Furthermore, normal proliferating hepatocytes also express Cx32 to a low extent (Kren et al. 1993; Yamamoto et al. 2005), questioning whether altered expression of this pro- tein is a primary phenomenon in some nodular lesions or it is rather a consequence of their increased growth rate (Neveu et al. 1994). Our results provide unequivocal evi- dence indicating that decreased Cx32 expression is in fact an inherent property of nodular hepatocyte lesions, at least under the experimental conditions of the present studies. Clusters of clonally expanding normal hepatocytes, with a Ki67 labelling index comparable to that of transplanted nodular clusters, displayed normal levels of Cx32 protein, while Cx32 expression was low in nodular lesions origi- nating from transplanted cells, regardless of high or low proliferative rate (Fig. 3). A similar pattern of results was visible on analysis of the expression of E-cadherin. While expanding clusters of normal transplanted hepatocytes displayed a regular dis- tribution of this membrane protein, focal lesions resulting from transplantation of nodular hepatocytes demonstrated low levels and altered localization of E-cadherin. Studies on the expression of E-cadherin in human hepatocellular carcinoma (HCC) have generated rather conﬂicting results. Earlier reports described elevated levels of E-cadherin in the majority of thin-trabecular HCC (Ihara et al. 1996). More recently, decreased expression of E-cadherin was included in a panel of 12 molecular alterations that sig- niﬁcantly discriminate between dysplastic nodules and early human HCC (Llovet et al. 2006). Consistent with the latter ﬁnding, E-cadherin has also been proposed as a marker in the differential diagnosis between adenoma and 589 HCC (Tretiakova et al. 2009). Importantly, our results provide direct evidence that these alterations appear early in the process and are independent of cell proliferation per se. Thus, it is possible that the failure of nodular hepato- cytes to integrate into the surrounding parenchyma is due, at least in part, to the altered expression of proteins involved in cell-to-cell communication and adhesion. Such alterations could involve different pathways. E-cadherin is part of a multifunctional scaffolding complex playing a key role in establishing cell polarity in several tissues (Cai and Mostov 2009). This complex includes proteins such as IQGAP2 and Cdc42. Interestingly, both IQGAP2-deﬁcient and Cdc42-deﬁcient mice are highly prone to developing HCC (Schmidt et al. 2008; van Hengel et al. 2008), thereby suggesting that alterations in tissue pattern formation are mechanistically linked to the pathogenesis of neoplastic disease. We also analysed the expression of MMP-2 following transplantation of either normal or nodular hepatocyte in RS-treated animals. It was found that this enzyme was mostly expressed at the leading edge of normal hepatocyte clones, as already reported (Koenig et al. 2005), although scattered activity could also be detected within DPPIV- positive clusters. However, the pattern of MMP-2 expres- sion was different in relation to nodular hepatocyte clusters. A sharp demarcation could be observed between the expression of MMP-2 protein and expanding nodular lesion. While the enzyme was expressed in liver tissue surrounding nodules, virtually no MMP-2 protein could be detected within such lesions. In addition, a markedly decreased expression of MMP-2 RNA was present in nodular hepa- tocytes as compared to normal counterparts. As already mentioned, MMPs are a family of enzymes that collectively play fundamental roles in several processes involved in neoplastic progression, including cell migration and metastasis (Ellerbroek and Stack 1999). MMPs exist in the matrix as pro-enzymes and are activated upon proteolytic cleavage. Unlike other MMPs, the activation of MMP-2 occurs at the cell surface (Montironi et al. 2002), a feature which confers to this enzyme a central role in cell migra- tion. It requires the binding of membrane type 1 MMPs (MT1-MMPs) with the tissue inhibitor of metalloproteinase type 2 (TIMP2) (Lehti et al. 1998; Nagase et al. 2006). Native MMP-2 then binds to this complex and this favours its proteolytic activation by a second MT1-MMP molecule on the cell membrane. The activity of MMPs is increased during tumour progression and is associated with invasion and metastases (Ellerbroek and Stack 1999). Our ﬁndings indicating low MMP-2 expression in early nodular lesions are therefore difﬁcult to reconcile with the proposed role of MMPs in the evolution of neoplastic disease. However, it is important to point out that early hepatic nodules do not display any signs of invasive capacity and they are sharply 123