Increasing evidence has indicated that perturbation of cyclins is one of the major factors leading to cancer. The aim of this study was not only to investigate various cell cycle-related kinase activities in hepatocellular carcinoma (HCC), but also to analyze the difference of cell cycle-related kinase activity levels between hepatitis C virus (HCV)-induced HCC and HCVinduced cirrhosis. The protein levels of cyclins D1, E, A, and H, and of cyclin dependent kinase 1 (Cdk1), Cdk2, Cdk4, Cdk6, and Cdk7 in HCC and in surrounding nontumorous cirrhosis were determined by Western blot. The enzymatic activities of cyclins D1, E, A, Cdk1, Cdk4, Cdk6, Cdk7, and Wee1 were measured using in vitro kinase assays. Protein levels and kinase activities of cyclin D1, Cdk4, cyclin E, cyclin A, and Wee1 were significantly elevated in HCC compared with surrounding cirrhotic tissues. The enhanced cyclin D1-related kinase activity in HCC was accompanied by the up-regulation of Cdk4 activity, but not Cdk6 activity. The kinase activities of Cdk6, Cdk7, and Cdk1 did not differ between HCC and surrounding cirrhotic tissues. In addition, the protein levels and kinase activities of cyclin D1, Cdk4, and cyclin E were higher in poorly differentiated HCC and advanced HCC. In conclusion, the increases of cyclin D1, Cdk4, cyclin E, cyclin A, and Wee1 play an important role in the development of HCC from cirrhosis. Cyclin D1, Cdk4, and cyclin E activation may be closely related to the histopathologic grade and progression of HCC. R ecent studies, including our research, have revealed that the deranged expression of cell cyclerelated proteins is one of the major factors contributing to hepatocellular carcinoma (HCC) development. 1-6 Here we investigated biochemical differences of various cell cycle-related kinase activities and protein levels in cirrhosis and HCC.Specific cyclin/cyclin-dependent kinase (Cdk) complexes are activated at different intervals during the cell cycle. 7-15 Cyclin D1/Cdk4 and cyclin D1/Cdk6 are activated in mid-G1 (Fig. 1, phase 1), whereas cyclin E/Cdk2 complexes are required for the G1/S transition (Fig. 1, phase 2), cyclin A/Cdk2 for the progression of DNA synthesis (Fig. 1, phase 3), and cyclin A-B/Cdk1 for the G2/M transition (Fig. 1, phase 4) of mitosis. The activation of cyclin D1/Cdk4 and cyclin D1/Cdk6 complexes at mid-G1 is responsible for the phosphorylation of retinoblastoma protein (pRb), and 2 Rb related proteins, p107, and p130. Members of the pRb family form complexes with transcription factors of the E2F family. 16 This interaction with pRb family members blocks the transcriptional activity of E2Fs; the complexes formed also function as active transcriptional repressor complexes at the promoters of some cell cycle genes. Phosphorylation of the pRb family inhibits the interaction with E2F family transcription factor and permits the expression of genes necessary for S-phase entry (cyclin A, proliferating cell nuclear antigen, and so on). 17 The expression of cyclins E and A at mid-to late-G1 permits the form...

For the related Src kinases, a close correlation exists between elevated tyrosine kinase activity and cell transformation. However, the involvement of pp60 c-src in hepatocellular carcinoma (HCC) remains obscure. The aim of this study was to evaluate whether pp60 c-src tyrosine kinase activity is elevated in HCC. We analyzed the kinase activity of pp60 c-src in normal liver tissue, chronic hepatitis liver tissue, and tumorous and adjacent nontumorous portions of HCC tissue from patients and Long-Evans cinnamon (LEC) rats that are known to develop liver cancer spontaneously. The kinase activity of pp60 c-src was rarely detected in the normal human liver tissue and chronic hepatitis liver tissue, but it was elevated in tumorous and nontumorous portions of HCC tissue. Furthermore, the kinase activity of pp60 c-src was significantly elevated in tumorous tissues compared with nontumorous tissues. The kinase activity of pp60 c-src was also higher in poorly differentiated HCC. In addition, the kinase activity of pp60 c-src increased proportionately with the development of HCC of LEC rats. Our results suggest that activation of the protooncogene product pp60 c-src may play an important role in the malignant transformation of hepatocytes in human and LEC rats, and that it may be closely related to the histopathological grading of human HCC. (HEPATOLOGY 1998;27:1257-1264.) Hepatocellular carcinoma (HCC) is thought to develop through a multistep process. 1 A long history of viral hepatitis or prolonged exposure to environmental toxins predisposes liver cells to mutations of genes critical in the control of hepatocyte growth. In fact, both activation of cellular oncogenes and inactivation of tumor-suppressor genes are involved in HCC. Activation of oncogenes by hepatitis virus integration has been shown in the woodchuck animal model, 2,3 although its significance in hepatocarcinogenesis in humans and in Long-Evans cinnamon (LEC) rats is still under investigation. A large number of cellular protein tyrosine kinase (PTK) genes have been cloned and sequenced. These kinases are classified into two major groups: the first comprises growth factor receptor tyrosine kinases; the second includes retroviral protein PTKs and their cellular homologues. 4 The main representatives of the latter group are non-receptor-linked and -membrane-associated src-related tyrosine kinases. 5 At least nine src-related tyrosine kinases have been identified. These include c-src, c-yes, c-lck, c-fyn, c-hck, c-lyn, c-blk, c-fgr, and c-yrk protooncogene products. 6 All members of the PTK src family have a molecular weight ranging between 55 and 62 kd and myristoylated glycine residues at the amino termini. 7,8 Among these PTKs, the protooncogene pp60 c-src is the cellular homologue of the Rous sarcoma transforming gene, v-src. 9 Both c-src and v-src encode 60-kd, membrane-associated PTKs. For pp60 c-src , a close correlation exists between elevated activity of tyrosine kinase and cell transformation. [10][11][12][13][14][15][16][17] Bolen et al. [10][11][...

We investigated the frequency and mechanism of |bT‐catenin/T cell factor (Tcf) signaling activation in a panel of 36 human gastrointestinal and liver cancer cell lines. Reporter assay and electrophoretic mobility shift assay revealed that the β‐catenin/Tcf signaling was upregulated in 12 of 12 (100%) colorectal, 5 of 8 (68%) gastric, 2 of 7 (29%) hepatic, and none of 9 pancreatic cancer cell lines. The activation of the pathway was mainly due to the mutation of adenomatous polyposis coli (APC) or β‐catenin, and Tcf‐4 was highly expressed in these cell lines with upregulated signaling. Nuclear β‐catenin was observed not only in the signaling‐activated cell lines, but also in 14 of 25 (56%) primary gastric cancers, 15 of 20 (75%) colon cancers, 5 of 19 (26%) hepatocellular carcinomas, and none of 13 pancreatic cancers. The presence of signaling‐upregulated gastric cancer cell lines with intact APC and β‐catenin suggests the involvement of other mechanisms than mutations of APC or β‐catenin.

Amplification found in a number of cyclin genes, especially in cyclin D and E, is an important event process that takes place in cancers, including hepatocellular carcinoma (HCC). The activities of a wide range of cell cycle-related kinases remain obscure in HCC. The purpose of the present study is to determine the cyclins and kinase activities of HCC in Long-Evans Cinnamon (LEC) rats. Cyclin D1, E, A, H, Cdk1(cyclin-dependent kinase; Cdc2), Cdk4, and Cdk6 protein levels were determined by Western blot analysis at different pathologic stages of liver tissues exhibiting HCC. Enzymatic activities of cyclin D1, E, A, Cdk4, Cdk6, Cdc2, Cdk7, and Wee1 kinase were measured by in-gel kinase assay. Protein levels and kinase activities of cyclin D1, E, Cdk4, cyclin A, and Wee1 increased proportionally with the development of HCC, especially in the transition process from chronic hepatitis to HCC. Although Cdc2 kinase activity was found to increase slightly from normal liver to chronic hepatitis, its activity remained unchanged in the process from chronic hepatitis to HCC. Cdk6 and Cdk7 activities remained unchanged in the process from normal liver to HCC. These data suggest that the increase in Cdc2 kinase may play a role in the process from normal liver to chronic hepatitis, whereas the predominant increase in cyclin D1, Cdk4, cyclin E, cyclin A, and Wee1 suggests involvement not only in the process from normal liver to chronic hepatitis, but also during transition into HCC. (HEPATOLOGY 2000;32:711-720.)The inbred strain of Long-Evans Cinnamon (LEC) rats was established from the closed colony of Long Evans rats. 1 These rats exhibit severe acute liver damage with jaundice spontaneously at the age of 4 to 5 months, leading to fulminant hepatic failure in more than 50%. The rats usually survive chronic hepatitis and within a year develop cholangiofibrosis and hepatocellular carcinoma (HCC). [2][3][4][5] Because the natural history of liver disease in LEC rats resembles that of human liver disease in which HCC follows persistent chronic liver disease, LEC rats are regarded as one of the most useful animal models of HCC. 3 Hitherto, little is known about the mechanism that leads to the progression of HCC. Recently, it has been known that changes do occur that alter the cell cycle of related proteins during the malignant transformation process. [6][7][8][9] Because cyclins are prime cell regulating events leading to proliferation in animal cells, the deranged expression of different cyclins may also be the key to carcinogenesis in LEC rats.We have previously reported on the importance of protein phosphorylation in signal transduction in relation to proliferation, differentiation, and carcinogenesis of hepatocytes. [10][11][12][13] Most cell cycle-related proteins show phosphorylation enzymatic activity contributing to cell cycle transition. 6,14-16 Cyclins and their catalytic subunit, the cell-dependent kinases, play key roles in the regulation of animal cell cycle events. 14 Progress of cells through the cycle is governed b...

Summary Cyclin D1 gene amplification is an important event in many cancers, but it is rarely found in non-small-cell lung cancer (NSCLC). This study was conducted in an attempt to clarify any other mechanisms related to cyclin D1 involvement in the malignant transformation of NSCLC, and we clearly showed for the first time that cyclin D1-related kinases are activated in NSCLC, especially in adenocarcinoma but not in squamous cell carcinoma. The results of this study strongly suggest that enhanced cyclin D1-related kinase activity could contribute to a progression of adenocarcinoma in NSCLC.