Misc - 小島　隆

Different changes in expression and function of connexin 26 and connexin 32 during DNA synthesis and redifferentiation in primary rat hepatocytes using a DMSO culture system

TL Kojima, M Yamamoto, C Mochizuki, T Mitaka, N Sawada, Y Mochizuki

HEPATOLOGY ( W B SAUNDERS CO )  26 ( 3 ) 585 - 597  1997年09月

　概要を見る

In the present study, we determined in detail the changes of liver gap junctions, connexin 26 (Cx26), and connexin 32 (Cx32), during DNA synthesis and redifferentiation of hepatocytes in vitro. We used primary rat hepatocytes that expressed the liver gap junction proteins, which were cultured in the medium containing epidermal growth factor (EGF) with 2% dimethylsulfoxide (DMSO) and 10(-7) mol/L glucagon (a DMSO culture system), as we previously reported, In the present cultures, almost confluent hepatocytes cultured in the medium containing EGF with 2% DMSO and 10(-7) mol/L glucagon, underwent a nearly synchronous wave of DNA synthesis induced by the removal of 2% DMSO and 10(-7) mol/L glucagon, and the addition of 10 mmol/L nicotinamide, after which the DNA synthesis was completely re-inhibited by the re-addition of 2% DMSO and 10(-7) mol/L glucagon, During stimulation of DNA synthesis, both Cx26 and Cx32 messenger RNA (mRNAs) in hepatocytes transiently increased in the G1 phase and then markedly decreased before the onset of the S phase, while only Cx26 messenger RNA (mRNA) increased slightly in the S/M phase. Furthermore, before the onset of the S phase, a disappearance of both Cx26 and Cx32 immunoreactivities and gap junction plaques were observed, Gap junctional intercellular communication (GJIC), as measured by lucifer yellow, which indicated the function of Cx32, decreased markedly from before the onset of the S phase, GJIC measured by propidium iodide, which indicated the function of Cx26, decreased from before the onset of the S phase and then increased slightly in the S/M phase. During the re-inhibition after the stimulation of DNA synthesis, Cx32 mRNA, but not Cx26 mRNA, rapidly returned to the pretreatment control level. Cx32 immunoreactivity and gap junction plaques also recovered. However, the recovery of GJIC measured by lucifer yellow was later than that of Cx32 expression. These results indicated the different changes of expression and function of Cx26 and Cx32 in the hepatocytes during stimulation and re-inhibition of DNA synthesis. This culture system should be useful as a model in which to study liver gap junctions during hepatocyte growth and differentiation in vitro.

DOI PubMed CiNii

Effects of melatonin on proliferation, oxidative stress and Cx32 gap junction protein expression in primary cultures of adult rat hepatocytes

T Kojima, C Mochizuki, T Mitaka, Y Mochizuki

CELL STRUCTURE AND FUNCTION ( JAPAN SOC CELL BIOLOGY )  22 ( 3 ) 347 - 356  1997年06月

　概要を見る

Melatonin has antiproliferative and antioxidant effects on cells in vivo and in vitro. Gap junctions mediate the communication between adjacent cells and are closely related to cellular growth and oxidative stress. We previously reported that 2% dimethylsulfoxide (DMSO) which has antiproliferative and antioxidative effects on hepatocytes, induces connexin 32 (Cx32) gap junction protein in primary cultures of adult rat hepatocytes. In the present study, we have examined the effects of melatonin on proliferation, oxidant stress and Cx32 gap junction protein expression in the cultured rat hepatocytes as compared to 2% DMSO treatment. 10(-2) M melatonin significantly inhibited the proliferation and the oxidative stress of the cells, and markedly induced Cx32 gap junction protein expression and gap junctional intercellular communication (GJIC). These effects of 10(-2) M melatonin treatment were not due to cytotoxicity to the cultured rat hepatocytes and they were as strong as those of 2% DMSO treatment. These results suggested that melatonin might be a useful substance to maintain the functions of the hepatocytes in vitro by modulating the levels of proliferation, oxidative stress and gap junction expression.

DOI PubMed CiNii

Formation of actin filament networks in cultured rat hepatocytes treated with DMSO and glucagon

T Kojima, C Mochizuki, H Tobioka, M Saitoh, S Takahashi, T Mitaka, Y Mochizuki

CELL STRUCTURE AND FUNCTION ( JAPAN SOC CELL BIOLOGY )  22 ( 2 ) 269 - 278  1997年04月

　概要を見る

Actin filament organization may play an important role in the maintenance of differentiated functions in epithelial cells, We previously reported our success in inducing and maintaining gap junctions, which are two kinds of differentiated function, in primary rat hepatocytes cultured with 2% DMSO and 10(-7) M glucagon, In the present study, we demonstrated the formation of actin filament networks in the hepatocytes cultured with 2% DMSO and 10(-7) M glucagon, Actin filaments in hepatocytes cultured in medium with only 2% DMSO added from 96 h after plating were concentrated under the plasma membrane and were observed to be circumferential. In hepatocytes cultured in the medium with both 2% DMSO and 10(-7) M glucagon added from 96 h, not only the circumferential actin filaments but also the formation of actin filament networks were observed and the networks developed well with time in culture. The networks were observed as a dome-like structure under the cell face and terminated at the circumferential actin filaments, They were composed of electron-dense star-like vertices connected by microfilament bundles of varying length and were also very sensitive to the actin disrupter cytochalasin B, However, during the network formation, there were no significant increases in the amounts of actin protein and mRNA, The actin filament networks of the hepatocytes in this culture system might be closely related to the maintenance of differentiated functions.

DOI PubMed CiNii

Expression of p21^wafl/cipl Is Induced in the Rat Liver by Feeding of 2-Acetylaminofluorene

Watanabe Tomoyuki, Ozaki Masakazu, Kawasaki Hajime, Kojima Takashi, Sawada Norimasa

Ｊｏｕｒｎａｌ ｏｆ Ｔｏｘｉｃｏｌｏｇｉｃ Ｐａｔｈｏｌｏｇｙ ( 日本毒性病理学会 )  11 ( 3 ) 191 - 196  1997年

　概要を見る

The cyclin-dependent kinase inhibitor p21^wafl/cipl plays major role in regulation of the cell cycle arrest following DNA damage in a p53-dependent and -independent manner. To elucidate the roles of p21^wafl/cipl in selective growth of preneoplastic lesions during chemical hepatocarcinogenesis, p21^wafl/cipl expression was examined in the livers of rats fed a 0.02% 2-acetylaminofluorene (2-AAF)-containing diet for 4 weeks or subjected to the protocol of Solt and Farber consisting of a single dose of diethylnitrosamine (DEN) followed 2 weeks later by 2-AAF feeding with two-thirds hepatectomy. By feeding of 2-AAF having a strong mitoinhibitory effect, p21^wafl/cipl expression was significantly induced in rat hepatocytes. The proliferating cell nuclear antigen (PCNA) labeling index of hepatocytes was consistently and significantly decreased by 2-AAF feeding. Immunohistochemically, both p21^wafl/cipl and p53 were detected in the livers of 2-AAF-fed rats more intensely than in those of control rats. In the livers of rats subjected to the protocol of Solt and Farber, most enzyme-altered foci (EAF) were immunohistochemically negative for p21^wafl/cipl, while hepatocytes surrounding the foci were positive. These results clearly show that 2-AAF acts on hepatocytes as a mitoinhibitor by inducing p21^wafl/cipl expression, presumably in a p53-dependent manner. Furthermore, the marked difference in p21^wafl/cipl expression between non-neoplastic and preneoplastic hepatocytes led to selective growth of preneoplastic lesions during 2-AFF feeding using the protocol of Solt and Farber.

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