Studies of Liver Cells: W hat are "Small Hepatocytes"?

In this review the research we have performed,will be mainly described through my studies. In particular, the study of hepatic progenitor cells, "small hepatocytes (SHs)," which were first found by me, is detailed. Until now, our oratory has focused its research on the issue of the "liver", i.e., development, regeneration, stem/progenitor cells, and carcinogenesis.I will summarize our research by 8 themes. In each theme I only quote the references that we have reported

Effects of Cholesteryl Hemisuccinate on the Phalloidin Sensitivity of Normal and Preneoplastic Rat Hepatocytes

Normal and preneoplastic hepatocytes were isolated from the untreated liver and carcinogen-induced hyperplastic liver nodules of rats, respectively. Phalloidin sensitivity of the 1-hour cultured cells decreased in the presence of cholesteryl hemisuccinate (CH). This was seen more markedly in the order of peneoplastic hepatocytes positive in gamma-glutamyltransferase (GGT), the preneoplastic cells negative in GGT, GGT-positive normal hepatocytes, and GGT-negative normal hepatocytes. Coexistence of phosphatidylcholine and CH inhibited the CH-inducible decrease completely in GGT-negative normal hepatocytes and partially in the normal cells positive in GGT, but failed to recover the decrease in both GGT-negative and positive preneoplastic hepatocytes. These phenomena are suggested to be ascribed to the differences of the cell membrane fluidity among these cells

Susceptibility of Rat Preneoplastic Hepatocytes to Liver-tumor Promoters : Effects of Phenobarbital and Ethyl-α-p-chlorophenoxyisobutyrate on Phalloidin Sensitivity

The effects of the liver-tumor promoters phenobarbital (PB) and ethyl-α-p-chlorophenoxyisobutyrate (CPIB) on phalloidin sensitivity of normal and preneoplastic hepatocytes were examined. Hepatocytes were isolated by a collagenase perfusion method from rats fed either 0.05% PB or 0.25% CPIB for 14 weeks, and from those treated for 12 weeks with these agents followed by 2-week withdrawal of the agents. Preneoplastic hepatocytes were isolated from enucleated nodules induced by the Solt-Farber method in the livers of rats, either treated or not treated with PB or CPIB. The phalloidin sensitivity of hepatocytes of the rats treated with PB or CPIB deceased, showing 73 to 90% of the control value in the former and 88 to 97% in the latter, depending on the concentration of phalloidin. After the 2-week withdrawal of the agents, the sensitivity recovered completely. The sensitivity of the preneoplastic hepatocytes of rats treated with PB or CPIB were far less sensitive to phalloidin than the untreated preneoplastic cells. The sensitivity was 28 to 54% in the preneoplastic cells of PB-treated and 54 to 59% in the cells of CPIB-treated rats compared to the values in the untreated preneoplastic cells. After the 2-week withdrawal of PB or CPIB, the sensitivity increased but was not completely recovered. Thus, preneoplastic hepatocytes were more susceptible to PB and CPIB than normal hepatocytes. Histological examinations showed that PB and CPIB promoted preneoplastic lesions in the livers. The enhanced susceptibility is suggested to be responsible for the promotion of hepatocarcinogenesis

Inhibition of Tumor-induced Angiogenesis by Ribonuclease Inhibitor

The effect of recombinant ribonuclease inhibitor on angiogenesis induced by B16-F10 cells was examined in syngeneic mice. Ribonuclease inhibitor coated with a slow release substance caused a marked decrease of the number of vessels toward tumor mass (angiogenic response), however, injection of ribonuclease in-hibitor did not. Ribonuclease inhibitor did not directly affect the growth of tumor cell or endothelial cell in vitro. These results suggest that ribonuclease inhibitor could be an inhibitor of tumor-induced angiogenesis by using an appro-priate delivery system and stabilizing its activity

Prolonged oxidative stress and delayed tissue repair exacerbate acetaminophen-induced liver injury in aged mice

マウスにアセトアミノフェンを投与して急性劇症肝炎を誘導すると，中心静脈域にネクローシスによる大量の肝細胞死が誘導される．障害後は，速やかに障害が解消され，障害後48 時間後には活発な肝細胞増殖が誘導され，組織再生が進行する．老齢マウスでは，アセトアミノフェン代謝産物の処理能力低下による障害の持続，障害部位へのマクロファージ集積低下による死細胞の残存，肝細胞増殖能の低下，などによって障害の持続と再生の遅延が認められた．本研究の結果は，加齢が薬剤の肝毒性を高めるリスクファクターとなる場合があることを示している

A three-dimensional microfluidic tumor cell migration assay to screen the effect of anti-migratory drugs and interstitial flow

Most anti-cancer drug screening assays are currently performed in two dimensions, on flat, rigid surfaces. However, there are increasing indications that three-dimensional (3D) platforms provide a more realistic setting to investigate accurate morphology, growth, and sensitivity of tumor cells to chemical factors. Moreover, interstitial flow plays a pivotal role in tumor growth. Here, we present a microfluidic 3D platform to investigate behaviors of tumor cells in flow conditions with anti-migratory compounds. Our results show that interstitial flow and its direction have significant impact on migration and growth of hepatocellular carcinoma cell lines such as HepG2 and HLE. In particular, HepG2/HLE cells tend to migrate against interstitial flow, and their growth increases in interstitial flow conditions regardless of the flow direction. Furthermore, this migratory activity of HepG2 cells is enhanced when they are co-cultured with human umbilical vein endothelial cells. We also found that migration activity of HepG2 cells attenuates under hypoxic conditions. In addition, the effect of Artemisinin, an anti-migratory compound, on HepG2 cells was quantitatively analyzed. The microfluidic 3D platform described here is useful to investigate more accurately the effect of anti-migratory drugs on tumor cells and the critical influence of interstitial flow than 2D culture models.Japan Society for the Promotion of Science (22680037)Japan Society for the Promotion of Science (G2212)National Cancer Institute (U.S.) (R21CA140096)Japan. Ministry of Education, Culture, Sports, Science and Technology (2009-00631)Japan. Ministry of Education, Culture, Sports, Science and Technology (2012-0009565)Korea (South). Ministry of Education & Human Resources Development (MOEHRD)Korea (South). Ministry of Education & Human Resources Development (MOEHRD) (20124010203250

A Case of Retroperitoneal Malignant Mesenchymoma

A rare case of malignant mesenchymoma of a retroperitoneal lesion, com-posed of liposarcoma and osteosarcoma, is reported. For complete resection of the tumor, two surgical operations were performed. The first operative material showed a mass measuring 20 x 20 x 10 cm, weighing 1607g, arising from the soft tissue of the left retroperitoneum and the tumor had a smooth surface and elastic-hard consistency. The secondary operative materials exhi-bited a mass measuring 10 x 5 x 3 cm, weighing 268g. The tumor was com-posed of soft gelatinous tissues and adhered to the tail of the pancreas but was separate from the spleen. More than 3 years after the secondary operation, no recurrence has been observed. Immunohistochemical examinations showed that S-100 protein and non-specific enolase were present in the liposarcoma-tous area and that vimentin was positive in the osteosarcomatous area

Effects of the Protein Phosphatase Inhibitors, Okadaic Acid and Vanadate, on Localization of Occludin in Primary Cultures of Rat Hepatocytes

To elucidate whether protein phosphorylation is associated with the loca-lization of the tight junction protein occludin, we determined the changes of occludin protein expression in primary cultures of rat hepatocytes after treat-ment with the protein phosphatase inhibitors okadaic acid and vanadate. After 2 h of treatment with 1myu M okadaic acid or 5 mM vanadate, occludin immunoreactivity showing continuous lines in non-treated cells changed to a few spots on the plasma membrane. In western blots, broad bands above the occludin protein (65 kD) became conspicuous after treatment with okadaic acid and vanadate. We treated the same samples with alkaline phosphatase to examine whether the broad bands depended on the changes in the phos-phorylation states of occludin protein. The broad bands disappeared and the occludin was observed as a narrow band corresponding to 65 kD. Neither a significant change in the mRNA of occludin nor a change in the immunoreac-tivity of the tight junction associated protein, ZO-1, was observed after treatment with okadaic acid or vanadate. These results suggested that the phosphorylation of occludin is closely associated with localization of the protein in cultured hepatocytes and that protein phosphatase inhibitors affect the loca-lization of occludin but not ZO-1 on the plasma membrane