Prevention of mammary tumorigenesis in acatalasemic mice by vitamin E supplementation.

Adult male and female acatalasemic (C3H/AnLCsbCsb),hypocatalasemic (C3H/AnLCscCsc) and normal mice of C3H strain fed on regular laboratory chow for 15 months showed an increased incidence of spontaneous mammary tumor in the decreasing order of female acatalasemic, male acatalasemic, female hypocatalasemic and male hypocatalasemic mice. Normal mice did not develop mammary tumor. We conducted a prospective study with female acatalasemic mice, which showed the highest incidence of mammary tumor, to examine the preventive effect of vitamin E on mammary tumor. Female acatalasemic mice were fed on vitamin E-deficient (28 animals) and vitamin E-supplemented diet (25 animals) for 29 months. The incidence of mammary tumor in mice given the vitamin E-supplemented diet was 47%, while that in mice given vitamin E-deficient diet was 82% (P < 0.002). Mammary tumors were apparent after 9 months of vitamin E deprivation and after 14 months of vitamin E supplementation. Female normal mice did not develop mammary tumor during a comparable period of time. The mean catalase activity of mammary gland in acatalasemic mice was 18.8% of that in normal mice. The results indicate that vitamin E protects acatalasemic mice against the development of mammary tumor.

Phenotypical stability of a human hepatoma cell line, HuH-7, in long-term culture with chemically defined medium.

A chemically defined medium containing selenium was further improved, and the improved medium showed excellent ability to support growth and colony formation of a human hepatoma cell line, HuH-7. The improved defined medium (designated as IS-RPMI) contained small amounts of unsaturated fatty acids, inorganic trace elements including selenium, and HEPES buffer. Many of these additions exert only a small individual effect on the growth of cells, but their cumulative effects were substantial. HuH-7 cells replicated continuously with a doubling time of 47-51 hr in IS-RPMI. A conditioned medium from a semiconfluent population increased the colony-forming ability of HuH-7 cells in IS-RPMI. The addition of insulin to the medium gave good colony growth, thus duplicating the effect of the conditioned medium. Although some alterations of morphological and growth characteristics of HuH-7 cells were observed, the differentiated liver functions, as revealed by production of plasma proteins including high levels of alpha 1-fetoprotein, were maintained for a period of more than 3 years and through 70 passages under the above culture conditions.

Growth of human hepatoma cells lines with differentiated functions in chemically defined medium.

A human hepatoma cell line, HuH-7, which was established from a hepatocellular carcinoma, was found to replicate continuously in a chemically defined medium when the medium was supplemented with Na2SeO3. The cells grew better in this medium than in serum-containing medium without any adaptation period. Other established human hepatoma and hepatoblastoma cell lines, HuH-6 cl-5, PLC/PRF/5, huH-1, and huH-4, also grew in the defined medium. Although HLEC-1 cells failed to proliferate continuously with Na2SeO3 alone, they grew if a cell-free conditioned medium from HuH-7 cells was added to the medium. These cell lines, except the HLEC-1 cell line, produced the following human plasma proteins among those examined: albumin, prealbumin, alpha 1-antitrypsin, ceruloplasmin, fibrinogen, fibronectin, haptoglobin, hemopexin, beta-lipoprotein, alpha 2-macroglobulin, beta 2-microglobulin, transferrin, lipoprotein, alpha 2-macroglobulin, beta 2-microglobulin, transferrin, Complement Components 3 and 4, and alpha 1-fetoprotein. Beside plasma proteins, the media from HuH-7, HuH-6 cl-5, PLC/PRF/5, and huH-1 contained anti-carcinoembryonic antigen-reactive proteins, and those from PLC/PRF/5, huH-1, and huH-4 medium contained hepatitis B surface antigen. These proteins were detected during periods of serial cultivation over 9 months under the above culture conditions. The hepatoma cell lines grown in the fully defined synthetic medium may provide a new approach for investigating the growth and metabolism of human hepatoma cells in vitro.