Cyclooxygenase (COX) is a key enzyme in the synthesis of prostanoids. Two isoforms of this enzyme have been identified: COX-1 and COX-2. Recent studies have suggested that COX-2, but not COX-1, may play a role in colorectal tumorigenesis. In the present study, we investigated the expression of COX-2 as well as COX-1 in human hepatocellular carcinoma (HCC) tissues using immunohistochemistry and immunoblotting. Forty-four surgically resected HCC tissues with adjacent nontumorous livers (NTs), involving 17 cases of chronic viral hepatitis and 27 cases of cirrhosis, and 7 surgically resected, histologically normal liver tissues were used. The well-differentiated HCC expressed COX-2 more frequently and strongly than less-differentiated HCC or hepatocytes of NTs. Lessdifferentiated HCCs expressed less COX-2 than hepatocytes of NTs, which showed scattered, strong COX-2 expression. Histologically normal liver was weakly positive for COX-2. The expression of COX-1 was weaker than that of COX-2 in hepatic neoplastic and non-neoplastic parenchymal cells. An enhanced expression of COX-1 was not observed in well-differentiated HCCs. Immunoblotting also confirmed up-regulation of COX-2, but not COX-1, in well-differentiated HCCs. The present study is the first to demonstrate a high expression of COX-2 in well-differentiated HCC and a low expression in advanced HCC, in contrast to its continuous expression during colorectal carcinogenesis. These findings suggested that COX-2 may play a role in the early stages of hepatocarcinogenesis, but not in the advanced stages, and may consequently be related to HCC dedifferentiation. (HEPATOLOGY 1999;29:688-696.) Cyclooxygenases (COXs) are key enzymes in the conversion of arachidonic acid to prostaglandins and other eicosanoids. Recently, two isoforms of the enzyme have been identified.

Members of the epidermal growth factor (EGF) family are the most important growth factors involved in epithelialization during cutaneous wound healing. Heparin-binding EGF-like growth factor (HB-EGF), a member of the EGF family, is thought to play an important role in skin wound healing. To investigate the in vivo function of HB-EGF in skin wound healing, we generated keratinocyte-specific HB-EGF-deficient mice using Cre/loxP technology in combination with the keratin 5 promoter. Studies of wound healing revealed that wound closure was markedly impaired in keratinocyte-specific HB-EGF-deficient mice. HB-EGF mRNA was upregulated at the migrating epidermal edge, although cell growth was not altered. Of the members of the EGF family, HB-EGF mRNA expression was induced the most rapidly and dramatically as a result of scraping in vitro. Combined, these findings clearly demonstrate, for the first time, that HB-EGF is the predominant growth factor involved in epithelialization in skin wound healing in vivo and that it functions by accelerating keratinocyte migration, rather than proliferation.

Heparin-binding EGF-like growth factor (HB-EGF) is first synthesized as a membrane-anchored form (proHB-EGF), and its soluble form (sHB-EGF) is released by ectodomain shedding from proHB-EGF. To examine the significance of proHB-EGF processing in vivo, we generated mutant mice by targeted gene replacement, expressing either an uncleavable form (HBuc) or a transmembrane domain–truncated form (HBΔtm) of the molecule. HBuc/uc mice developed severe heart failure and enlarged heart valves, phenotypes similar to those in proHB-EGF null mice. On the other hand, mice carrying HBΔtm exhibited severe hyperplasia in both skin and heart. These results indicate that ectodomain shedding of proHB-EGF is essential for HB-EGF function in vivo, and that this process requires strict control.

Peroxisome proliferator-activated receptor ␥ (PPAR␥) regulates cell growth and differentiation. Recent evidence has suggested that PPAR␥ ligands had anti-tumor effects through inhibiting cell growth and inducing cell differentiation in several types of malignant neoplasm. In the present study, we investigated: 1) the expression of PPAR␥ in both human hepatoma cell lines and 5 resected human hepatocellular carcinoma (HCC) tissues; 2) the growth-inhibitory effect of troglitazone, a PPAR␥ ligand, on those hepatoma cells; and 3) the molecular mechanisms of troglitazone-induced cell-cycle arrest. Five hepatoma cell lines, HLF, HuH-7, HAK-1A, HAK-1B, and HAK-5, were used. The mRNA expression levels of PPAR␥, p21 WAF1/Cip1 , and p27 Kip1 were determined by real-time quantitative reverse transcription-polymerase chain reaction. The expression of cell cycle-regulating proteins, such as p21, p27, p18 INK4c , cyclin E, and pRb, was examined using Western blotting. PPAR␥ was constitutively expressed in all the cell lines and the HCC tissues used in this study. A cytostatic effect of troglitazone was found in those cell lines, and this inhibition of cell growth was dosage-dependent. G0/G1 arrest was apparently demonstrated in flow cytometric analysis in HLF, HAK-1A, HAK-1B, and HAK-5, all of which showed an increased expression of p21 protein. However, HuH-7, lacking p21 protein expression, did not demonstrate clear arrest in the cellcycle analysis. HLF, which was deficient in the protein product of the retinoblastoma tumor-suppressor gene (pRb), responded most profoundly to troglitazone, showing an increased expression in not only p21, but also in p27 and in p18. These findings suggested that p21, p27, and p18 might be involved in troglitazone-induced cell-cycle arrest in human hepatoma cells. (HEPATOLOGY 2001;33:1087-1097.)Peroxisome proliferator-activated receptor ␥ (PPAR␥), a member of the nuclear hormone receptor superfamily, is known to regulate growth arrest and terminal differentiation of adipocytes. 1,2 Recently, PPAR␥ ligands, some of which are clinically used as a new class of antidiabetic drugs, have been shown to inhibit cell growth in several malignant cell types. [3][4][5] Indeed, PPAR␥ ligands have been found to inhibit cell growth and to induce terminal differentiation of human liposarcoma cells in vitro and in patients suffering from advanced liposarcoma. 3,6 PPAR␥ ligands also promoted terminal differentiation of breast cancer cells in vitro, induced apoptosis of injected breast cancer cells in mice, and reduced tumor incidence in rats treated with nitrosomethylurea. 7,8 However, such growth-inhibitory effect via PPAR␥ activation has not yet been assessed in human hepatoma cells, nor have the expression levels of PPAR␥ yet been examined in either human hepatocellular carcinoma (HCC) tissues or in the liver of chronic viral hepatitis or cirrhosis.In eukaryotes, the cell cycle is tightly regulated by several protein kinases composed of a cyclin-dependent kinase (CDK) subunit(s) and corresponding regulatory ...

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