Influence of transfection with connexin 26 gene on malignant potential of human hepatoma cells.
We investigated the effect of transfection with connexin (Cx) 26 gene on the malignant potential of PLC/PRF/5 hepatoma cells, observing changes in their morphological features, alpha-fetoprotein (AFP) expression, cell proliferation and apoptosis in vitro, and their tumor growth in vivo. Fluorescence-activated cell sorting (FACS) analysis showed that 10.6% of PLC/PRF/5 hepatoma cells transfected with Cx26 cDNA expressed excessive Cx26, and the spread of lucifer yellow was wider in the colony of stable transfectants (PLC/Cx26) after its microinjection than in control. Nucleo-cytoplasmic (N/C) ratio was significantly lower in PLC/Cx26 (P < 0.0001). Cell proliferation assay showed significantly lower numbers in PLC/Cx26 on day 10 after seeding than in control (P = 0.0039), and AFP level /10(5) cells was significantly lower in medium of PLC/Cx26 (P = 0.0039). The number of proliferating cell nuclear antigen (PCNA)-positive cells was less in PLC/Cx26 in vitro than in control (P = 0.0039), and single-stranded DNA (ssDNA)-positive cells were more abundant in the colony of PLC/Cx26 (P = 0.029). Tumor volume in SCID mice was significantly smaller in the group of PLC/Cx26 than in the control (P < 0.01) throughout the observation period, and tumor weight of PLC/Cx26 was significantly lower (P = 0.0019) week 9 after inoculation. Transfection with Cx26 cDNA inhibited dedifferentiation, suppressed cell proliferation, and apoptosis was induced. Tumor growth of PLC/Cx26 was retarded. These findings suggest that transfection with Cx26 gene into human hepatoma cells reduces their malignant potential.
Muramatsu
Akira
A
Third Department of Internal Medicine, Kyoto Prefectural University of Medicine, Kamigyo-ku, Kyoto 602-8566, Japan. amura@sun.kpu-m.ac.jp
Iwai
Masaki
M
Morikawa
Teruhisa
T
Tanaka
Saiyu
S
Mori
Takahiro
T
Harada
Yoshinori
Y
Okanoue
Takeshi
T
D000818:Animals; D017209:Apoptosis; D006528:Carcinoma, Hepatocellular / Q000235:genetics* / Q000378:metabolism / Q000473:pathology*; D002455:Cell Division; D002467:Cell Nucleus / Q000378:metabolism; D002469:Cell Separation; D002478:Cells, Cultured; D000072259:Connexin 26; D017630:Connexins / Q000235:genetics*; D003593:Cytoplasm / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism; D005434:Flow Cytometry; D017629:Gap Junctions / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D051379:Mice; D016513:Mice, SCID; D009362:Neoplasm Metastasis; D010957:Plasmids / Q000378:metabolism; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D014407:Tumor Cells, Cultured; D000509:alpha-Fetoproteins / Q000378:metabolism
D017630:Connexins; D018076:DNA, Complementary; D004277:DNA, Single-Stranded; C000607042:GJB2 protein, human; C000607043:Gjb2 protein, mouse; D018809:Proliferating Cell Nuclear Antigen; D000509:alpha-Fetoproteins; D000072259:Connexin 26