Normal human fibroblasts (the OUMS-24 strain), derived from a 6-week-old human embryo, were transformed (into the OUMS-24F line) and immortalized by repeated treatments (59 times) with 4-nitroquinoline 1-oxide (4NQO). Treatment began during primary culture and ended at the 51st population doubling level (PDL). At the 57th PDL (146 days after the last treatment), morphologically altered, epithelial-type cells appeared, began to grow and became immortal (now past the 100th PDL). However, the control fibroblasts, which were not treated with 4NQO, senesced at the 62nd PDL. The finding that extensive, repeated treatments with 4NQO are required for the immortalization of normal human cells, indicates that multiple mutational events are involved in the immortalization of human cells in general. In other words, immortalization itself seems to be a multi-step process. Karyotypic analysis showed that many cells were hypodiploid before immortalization, but that afterwards chromosomes were distributed broadly in the diploid to tetraploid regions. The immortalized cells showed amplification and enhanced expression of c-myc. Two-dimensional electrophoretic analysis showed that the number of disappearing cellular proteins was greater than the number of the newly appearing ones after the cells became immortalized. Since the immortalized cells showed neither anchorage-independent growth nor tumorigenicity, they are useful for studying factors that can contribute to multi-step carcinogenesis in human cells. In addition, genetically matched normal (OUMS-24) and immortalized (OUMS-24F) cells will be useful for analyzing the genes related to cellular mortality and immortalization.

4-Nitroquinoline-1-oxide↗

PubMed1993

Immortalization of epithelial-like cells from human liver tissue with SV40 T-antigen gene.

The cells derived from the human embryo liver tissue were transfected with a plasmid pSV3neo containing both the large and small T-antigen gene of the early region of simian virus 40 (SV40), and two cell strains, OUMS-21 and -22, were obtained. OUMS-22 cells, to date, have reached over 100 population doublings through a culture crisis and are considered to have become an immortal cell line. However, OUMS-21 cells failed to become an immortal cell line. Both OUMS-21 and -22 cells were SV40 T-antigen-positive, epithelial-like, and immunoreactive against an anti-keratin 18 monoclonal antibody but against neither an anti-vimentin nor an anti-von Willebrandt factor VIII monoclonal antibody. The staining pattern of cytokeratin in these cells was similar to that in the differentiated human hepatoblastoma and hepatocellular carcinoma cell lines but not to that in the human cholangiocellular carcinoma cell lines. OUMS-21 and -22 cells expressed neither alpha-fetoprotein nor albumin mRNAs. These cells showed no tyrosine aminotransferase activity. However, both OUMS-21 and -22 cells were sensitive to cytotoxicity of aflatoxin B1, 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole, and benzo[a]pyrene, whereas human embryo lung fibroblasts were insensitive to the cytotoxicity of these carcinogens. These findings suggest that OUMS-21 and -22 cells may arise from undifferentiated liver stem cells or from hepatocytes that lost their ability to express the liver-specific functions prior to immortalization. Both OUMS-21 and -22 cells expressed glutathione S-transferase pi (GST-pi) mRNA. The expression of GST-pi mRNA highly increased in OUMS-22 cells with their immortalization. Karyotypic analysis showed that numerical and structural aberrations of the chromosomes were profound, but neither specific events nor marker chromosomes were found in OUMS-21 and -22 cells. Both OUMS-21 and -22 cells could grow in soft agar, but they were not tumorigenic when transplanted into nude mice.

Aflatoxin B1↗

PubMed1993

Alteration in the retinoblastoma gene associated with immortalization of human fibroblasts treated with 60Co gamma rays.

Genetic analysis was carried out in human fibroblasts (KMST-6) immortalized by treatment with 60Co gamma rays in order to determine if any genetic change was involved in the immortal transformation of human cells. Analysis by restriction fragment length polymorphism revealed an alteration in chromosome 13q12-14, in which the retinoblastoma (RB) gene locus (13q14) is located. Then the RB gene itself was examined. Structural abnormalities in the RB gene were detected by Southern blot analysis. Furthermore, abnormal RB protein (pRB) was expressed in immortalized KMST-6 cells, as shown by in vitro phosphorylation, whereas normal KMS-6 cells expressed the intact pRB. These findings indicated that inactivation of the RB gene is one of the key events of the immortalization of human cells.

Cell Transformation, Neoplastic↗

PubMed1993

Neoplastic transformation and characterization of human fibroblasts by treatment with 60Co gamma rays and the human c-Ha-ras oncogene.

Human fibroblasts (KMST-6) immortalized by treatment with 60Co gamma rays were further neoplastically transformed by transfection of the c-Ha-ras oncogene from human lung cancer. The ras-transfected cells formed undifferentiated fibrosarcoma in nude mice. One of the tumors was recultured and a neoplastic human fibroblast line, KMST-6/RAS, was established. To analyze multistep carcinogenesis of human cells, the cellular characteristics of these genetically matched immortalized (KMST-6) and neoplastic (KMST-6/RAS) cell lines were studied in detail. KMST-6/RAS cells showed an increased saturation density, colony formation on confluent monolayers of normal human fibroblasts, proliferation in neomycin-containing medium, anchorage-independent growth, and enhanced expression of the transfected c-Ha-ras oncogene, whereas the immortalized cells did not demonstrate these characteristics. Unexpectedly, growth of KMST-6/RAS cells was serum-dependent, although they were neoplastic. Interestingly, the neoplastic cells did not show the criss-crossing or piling up growth pattern characteristic of transformed rodent fibroblasts.

Animals↗

PubMed1993

Clinical evaluation of biosynthetic glucagon treatment for recovery from hypoglycemia developed in diabetic patients. The GL-G Hypoglycemia Study Group.

Biosynthetic glucagon (GL-G) produced by recombinant DNA technology with transformed yeast strains is already available for clinical use. We studied the effects of 1 mg GL-G injection on plasma glucose level and hypoglycemic symptoms in 38 diabetic patients treated with insulin or oral hypoglycemic agents during spontaneous hypoglycemic episodes. In both intramuscularly and intravenously administered GL-G groups, plasma glucose significantly increased from 58.1 +/- 11.4 to 113.2 +/- 6.9 mg/dl (i.m., n = 17, P < 0.01) and from 76.4 +/- 4.4 to 125.7 +/- 5.9 mg/dl (i.v., n = 15, P < 0.01), respectively 20 min after the administration and the symptoms due to hypoglycemia subsided promptly after the injection of GL-G in 27 cases. The hyperglycemic effect of intramuscularly injected GL-G was more potent and long-standing than when intravenously injected, particularly in insulin-dependent diabetic (IDDM) patients. Neither significant changes of antibody levels against yeast proteins nor serious adverse effects were observed after GL-G administration. Biosynthetic glucagon is safe and useful for the treatment of hypoglycemia developing in diabetic patients.

Diabetes Mellitus↗

PubMed1993

Chromosome 7 suppresses indefinite division of nontumorigenic immortalized human fibroblast cell lines KMST-6 and SUSM-1.

Using nontumorigenic immortalized human cell lines KMST-6 (KMST) and SUSM-1 (SUSM), we attempted to identify the chromosome that carries a putative senescence-related gene(s). These cell lines are the only ones that have been established independently from normal human diploid fibroblasts following in vitro mutagenesis. We first examined restriction fragment length polymorphisms on each chromosome of these immortalized cell lines and their parental cell lines and found specific chromosomal alterations common to these cell lines (a loss of heterozygosity in KMST and a deletion in SUSM) on the long arm of chromosome 7. In addition to these, we also found that introduction of chromosome 7 into these cell lines by means of microcell fusion resulted in the cessation of cell division, giving rise to cells resembling cells in senescence. Introduction of other chromosomes, such as chromosomes 1 and 11, on which losses of heterozygosity were also detected in one of the cell lines (KMST), to either KMST or SUSM cells or of chromosome 7 to several tumor-derived cell lines had no effect on their division potential. These results strongly suggest that a gene(s) affecting limited-division potential or senescence of normal human fibroblasts is located on chromosome 7, probably at the long arm of the chromosome, representing the first case in which a specific chromosome reverses the immortal phenotype of otherwise normal human cell lines.

Animals↗

PubMed1993

Establishment and characterization of a SV40 T-antigen immortalized epithelial-like cell line derived from the newborn rat colorectum and its malignant transformation by the ras oncogene.

Epithelial-like cells from the colorectum of one-day-old newborn rats were immortalized by transfection with the simian virus 40 (SV40) T-antigen gene, and a cell line OUMS-25 was established. The cells were positive for the SV40 T-antigen, and immunoreactive to a colonic epithelial cell monoclonal antibody and a keratin-18 monoclonal antibody. Ultrastructural studies revealed the presence of microvilli on the cell surface and desmosomes between the adjacent cells. Karyotypic analysis showed that OUMS-25 cells were aneuploid. Cloning efficiency of the cells was 0.01% in soft agar. However, the cells were not tumorigenic in the syngeneic newborn rats. The cells were further transformed by transfection with the cloned activated c-Ha-ras oncogene containing a point mutation within codon 61. Characteristics of the activated-c-Ha-ras transfected cells (OUMS-25/RAS) were different in some respects from those of the parent cells (OUMS-25). OUMS-25/RAS cells demonstrated more malignant morphology, elevated cloning efficiency in soft agar, and tumorigenicity. This is the first report on the immortalization and malignant transformation of colorectal epithelial-like cells by transfection with a combination of SV40 T-antigen gene and cloned activated c-Ha-ras oncogene.

Animals↗

PubMed1993

Methanol stimulates the colony-formation rate in a human hepatoma cell line (HLE).

Effects of methanol on colony-formation of human hepatoma cells were investigated. Among five human hepatoma cell lines (Hep G2, HLE, HuH-6, HuH-7, and PLC/PRF/5), only HLE cells showed enhanced colony formation due to methanol. The effective concentrations of methanol were around 1%. The enhancement occurred in a greater degree when the cells were seeded in the culture medium containing methanol than when methanol was added 24h after the cells were seeded. Methanol itself, however, did not enhance the cell proliferation.

Carcinoma, Hepatocellular↗

PubMed1993

Immortalization of normal human cells is a multistep process and a rate limiting step of neoplastic transformation of the cells.

Three normal human fibroblast strains were immortalized by treatment with either 4-nitroquinoline 1-oxide (SUSM-1 and OUMS-24F lines) or 60Co gamma rays (KMST-6 line). Immortalization of normal human cells with these agents required repeated treatments. Before immortalization, most of the cells treated with these mutagenic agents showed more or less chromosome aberrations, but immortalized cells appeared very rarely. This suggests that several mutational events are involved in the immortalization process itself. The immortalized cells showed polygonal or epithelial-like morphology, prominent numerical and structural abnormalities of chromosomes, requirement of serum growth factors for their growth, and no tumorigenicity. The immortalized KMST-6 and OUMS-24F were further transformed into neoplastic ones by the ras oncogene, but not by transfection with the mutant p53 gene. Normal human fibroblasts which are mortal were not immortalized nor neoplastically transformed with the ras oncogene or the mutant p53. These findings strongly indicate that the immortalization of human cells is prerequisite for neoplastic transformation of the cells.

4-Nitroquinoline-1-oxide↗

PubMed1993

[Effects of cisplatin, carboplatin, etoposide and human hepatocyte growth factor on the colony formation of four human liver cancer cell lines].

Antitumor activity of cisplatin, carboplatin, etoposide, or human hepatocyte growth factor (hHGF), was compared by examining the colony formation ability of four liver cancer cell lines (PLC/PRF/5 and HuH-7, hepatocellular carcinoma; HuH-6 and HepG2, hepatoblastoma). Antitumor activity was evaluated from the drug concentration causing 50% cell death by a colony assay. PLC/PRF/5 cells were most effectively killed by cisplatin and etoposide, HuH-7 cells by cisplatin and carboplatin, HuH-6 cells by etoposide, and Hep G2 cells by cisplatin. These results indicate that among four liver cancer cell lines, the three were the most sensitive to cisplatin, the two were to etoposide and the only one cell line was to carboplatin. There was no significant relationship between each drug and types of liver cancer. Combined treatment with cisplatin (0.01-1.0 microgram/ml) and etoposide (0.1 microgram/ml) showed synergistic cytotoxic effects on the colony formation of PLC/PRF/5 cells, while combination of carboplatin (0.01-0.1 microgram/ml) and etoposide (0.1 microgram/ml) caused subadditive cytotoxic effects. hHGF stimulated the colony formation of HuH-6 cells, while it inhibited that of Hep G2 cells. The treatment of HuH-6 with cisplatin and hHGF showed a higher cell survival percentage compared with the treatment with cisplatin alone. On the other hand, cell survival of Hep G2 cells was remarkably decreased by the combined treatment with cisplatin and hHGF.

Carboplatin↗

PubMed1992

Glucagonlike peptide-1(7-36)amide suppresses glucagon secretion and decreases cyclic AMP concentration in cultured In-R1-G9 cells.

We previously reported that GLP-1(7-36)amide had glucagonostatic action as well as insulinotropic action in the perfused rat pancreas. In this study, we examined the effect of GLP-1(7-36)amide on glucagon secretion and cAMP concentration in glucagon-secreting cell line, In-R1-G9. GLP-1(7-36)amide (1nM) significantly suppressed glucagon secretion and decreased cAMP concentration in the cells. GLP-1(1-37) did not affect glucagon secretion. It is suggested that inhibitory effect of GLP-1(7-36)amide on glucagon secretion is at least partly mediated by adenylate cyclase system.

1-Methyl-3-isobutylxanthine↗

PubMed1992

Malignant transformation of human fibroblasts previously immortalized with 60Co gamma rays.

Normal human fibroblasts obtained from a whole embryo were malignantly transformed with only 60Co gamma rays, but after extensive passage. The fibroblasts were exposed intermittently to 60Co gamma rays, 13 times, 2,800 rads in total, from the primary culture to the 40th passage level. At the 50th passage level the cells became morphologically changed, showing chromosomal abnormalities, and serial passages showed them to be immortalized. They were not tumorigenic on transplantation into nude mice, but became malignant after extensive passage; i.e., at the 547th passage level and 2,800 days after initiation of the culture. Our results indicate that several different genotypic changes are necessary for malignant transformation of human cells over long periods. No mutation at codons 12 and 61 of H-, K- and N-ras was detected in the malignant cells. Thus, this system may be useful to detect other cellular genes that may contribute to the malignant phenotypes of human fibroblasts.

Base Sequence↗

PubMed1992

[Study on uric acid metabolism in patients with primary aldosteronism].

This study was conducted to elucidate renal uric acid metabolism in patients with primary aldosteronism (PA;16 cases) as compared with normotensive subjects (NT;25 cases) and essential hypertensives (EHT;51 cases). All subjects were hospitalized and received a regular diet(Na;120 mEq,K;75 mEq,daily) for more than two weeks, after which renal clearance tests were performed, and serum uric acid(SUA), fractional excretions of uric acid(FEUA), sodium(FENa), and inorganic phosphorus(FEP) were evaluated. Plasma aldosterone concentration(PAC) was measured in 16 patients with PA before treatment and in 8 patients after adrenalectomy. SUA was lower in PA than in either NT or EHT, and this lowering was more obvious in male subjects. In NT, PA and EHT, FEUA, an index of renal excretion of uric acid, correlated negatively with SUA and positively with FENa and FEP, which reflected sodium reabsorption at the renal total tubules and proximal tubules, respectively. Although FENa was nearly the same in all the three groups, FEUA and FEP were significantly higher in PA than in EHT or NT. However, no significant correlation was found between PAC and SUA or FEUA in PA. In PA a significant increase of SUA, and decreases of FEUA and FEP were observed after the removal of adenoma compared to before the surgery. These results suggest that uric acid transport might be closely related to sodium transport in the renal tubules, particularly at the proximal site, and also lead to the conclusion that the lower SUA in PA resulted from the suppression of reabsorption and/or an enhancement of secretion of uric acid in the proximal tubules, being related to the so-called escape phenomenon.

Adenoma↗