Expression of human hepatic genes in somatic cell hybrids

Four diploid human cell types (lymphocytes, fibroblasts, amniotic fluid cells, and hepatocytes) were fused to mouse hepatoma cells, HH. HH synthesized and secreted several liver-specific gene products including albumin, transferrin, and alpha-fetoprotein. The resulting interspecific hybrids were compared to determine whether or not the pattern of human hepatic gene expression was similar when these various cells were fused with the mouse hepatoma line. The expression of six human hepatic genes was examined, including albumin, alpha-fetoprotein, ceruloplasmin, transferrin, alpha-1-antitrypsin, and haptoglobin. Albumin was most frequently expressed while alpha-fetoprotein was not detected in any of the hybrids studied. The patterns of expression of human serum proteins differed between the hybrid series. Hybrids derived from human fibroblasts produced primarily albumin, while those derived from lymphoblastoid cells and amniocytes had a higher frequency of clones secreting alpha-1-antitrypsin. The findings reported here suggest that the frequency of hybrid clones expressing human hepatic gene products and the array of proteins produced are influenced by the histogenetic state of the human parental cell type.     

Control of angiotensinogen production by H4 rat hepatoma cells in serum-free culture

Summary A serum-free, hormone and attachment factor supplemented culture for rat H4 hepatoma cells was established. In the defined medium (Dulbecco's Modified Eagle's +Ham's F12+insulin, transferrin, fibronectin liver cell growth factor, and sodium selenite), H4 cells grew equally well as in 10% fetal bovine serum supplemented medium. H4 cells in either defined or serum-containing culture conditions produce transferrin but not albumin or alpha-fetoprotein. In this paper we have studied the effect of various hormones and pressor peptides on the production of angiotensinogen by H4 cells cultured in defined conditions. Only glucocorticoid hormone had a significant effect on the production of angiotensinogen, whereas other hormones previously reported to exert their effect on angiotensinogen production had little or no effect. This work was supported by grant P01 CA37589 from the National Institutes of Health, Bethesda, MD.     

Ability of the cells of intra- and interspecific hybrids of murine hepatoma XXIIa to synthesize the serum proteins albumin and transferrin

Independent hybrid clones resulted from the whole cell and microcell-mediated transfer of hamster or mouse fibroblast chromosomes into mouse hepatoma XXIIa cells. The fusion was promoted with PEG, ethidium bromide alone, or in combination with HAT and ouabain, was used for selecting the hybrids. Using indirect immunoautoradiography, three clones (one intra- and one interspecies microcellular; one interspecies, whole cell fusion) have been found to express their hepatic function to synthesize transferrin. The liver specific protein--albumin--was extinguished in all the hybrid combinations. Possible mechanisms of gene expression are discussed. The hybrids selected could be used for mapping chromosomes, coding proteins, as well as for studying regulation in the tandem of albumin and alpha-fetoprotein genes in the mouse genome. The microcell mediated chromosome transfer into differentiated cells has been used to construct original genetical combinations of regulatory and structural elements of the mouse genome.     

Expression of alpha-fetoprotein and albumin genes in a rat hepatoma cell line SY/1/80: Evidence for the need of species specific serum factors

The expression of alpha-fetoprotein and albumin genes has been studied in a rat hepatoma cell line (SY/1/80) developed from a liver cell tumor induced with di-ethylnitrosamine. This original tumor produces both proteins. However, in $\text{in vitro}$ propagated hepatoma cells, after passages in growth medium containing new born calf serum, the mRNAs of both proteins were undetectable. Supplementation with rat serum, but not serum from calf, horse or human, in growth medium for this cell line led to resynthesis of albumin and AFPmRNAs. These findings suggest that species specific serum factor(s) play an important role in the regulation of gene expression. Although the nature of the factor(s) and of the mechanism of action remains to be elucidated, this phenomenon may explain the general feature of diminshing abilities of cells to produce specific proteins in continuous subculture using standard calf serum.     

The control of serum protein synthesis in hepatoma-fibroblast hybrids.

Hybrids between mouse hepatoma cells (which secrete several serum proteins) and mouse or rat fibroblasts (which do not secrete these proteins) produce transferrin and the third component of complement (C3) like the parental hepatoma cells, while they do not secrete either albumin or alpha-fetoprotein (AFP). This lack of albumin and AFP secretion is probably due to a lack of synthesis, rather than to a simple defect in secretion. The cessation of albumin and AFP production is not dependent upon the parental fibroblast nor upon the selection conditions; it is best explained by a shut-off synthesis and could thus reflect the existence of a regulatory mechanism. This would imply a difference between the control of albumin and AFP synthesis and that of transferrin and C3 synthesis. On the other hand, in agreement with Peterson and Weiss (1972), hybrids between rat hepatoma cells and mouse fibroblasts continue to product rat albumin. This suggests that the mouse hepatoma cells differ from the rat hepatoma cells in the way they control albumin production.     

Synthesis and Secretion of Transferrin by Cultured Mouse Hepatoma Cells

The mouse hepatoma cell (Hepa-1) in tissue culture has been shown to synthesize and secrete three electrophoretically distinct transferrins. Each of these forms of transferrin has a molecular weight of 77,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The concentration of each form is indicated by its staining intensity, which is highest in the form with the fastest mobility and lowest in the form with the slowest mobility. The relative rate of transferrin synthesis has been determined in log-phase and stationary-phase cells; the data indicate that the relative rate of synthesis increases twofold in stationary-phase cells. When the incorporation of [³H]leucine into transferrin reaches steady state, the rate of secretion is equal to the rate of synthesis; the rate of secretion also increases twofold in stationary-phase cells. Our studies also show that transferrin synthesis accounts for 0.98% of the total protein synthesis in log-phase cells and for 1.8% in stationary-phase cells. This is the level of synthesis that has been determined by in vivo studies. We conclude that after continuous culture for several years these hepatoma cells have maintained one of the characteristics of the differentiated liver cell, namely, the ability to synthesize and secrete transferrin.     

Synthesis and secretion of transferrin by cultured mouse hepatoma cells.

The mouse hepatoma cell (Hepa-1) in tissue culture has been shown to synthesize and secrete three electrophoretically distinct transferrins. Each of these forms of transferrin has a molecular weight of 77,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The concentration of each form is indicated by its staining intensity, which is highest in the form with the fastest mobility and lowest in the form with the slowest mobility. The relative rate of transferrin synthesis has been determined in log-phase and stationary-phase cells; the data indicate that the relative rate of synthesis increases twofold in stationary-phase cells. When the incorporation of [3H]leucine into transferrin reaches steady state, the rate of secretion is equal to the rate of synthesis; the rate of secretion also increases twofold in stationary-phase cells. Our studies also show that transferrin synthesis accounts for 0.98% of the total protein synthesis in log-phase cells and for 1.8% in stationary-phase cells. This is the level of synthesis that has been determined by in vivo studies. We conclude that after continuous culture for several years these hepatoma cells have maintained one of the characteristics of the differentiated liver cell, namely, the ability to synthesize and secrete transferrin.     

Production and secretion of retinol-binding protein by a human hepatoma cell line, HepG2

Retinol-binding protein (RBP) that is synthesized and secreted by the human hepatoma cell HepG2 has been measured using a sensitive radioimmunoassay in which RBP in media and hepatoma cell sonicates reacts identically to human serum RBP. RBP was synthesized and secreted when cells were grown in retinol-depleted as well as retinol-containing media. However, immunoreactive transthyretin (prealbumin) could not be detected in concentrated HepG2 medium. RBP secretion and accumulation per mg of cell protein could be modulated by the concentration of fetal calf serum in the growth medium: secreted RBP equaled 782 +/- 123 ng/mg of cell protein per 8 hr after preincubation with 10% fetal calf serum versus 555 +/- 86 ng/mg per 8 hr in the absence of serum, whereas RBP in cell sonicates decreased only slightly. When HepG2 cells were cultured for two or more passages in medium containing fetal calf serum depleted of retinol by ultraviolet irradiation, the amounts of RBP in the cells and released to the medium were both significantly increased. When vitamin A (90% as retinyl esters) in the form of chylomicron remnants was presented to cells, there was a significant, dose-dependent redistribution of RBP from cells to medium, both in cells grown in normal fetal calf serum and in retinol-depleted serum. These data indicate that the secretion of RBP by HepG2 can occur constitutively in the absence of retinol, but that secretion can be enhanced and regulated by retinol delivered by the chylomicron remnant.     

The growth requirements of SV40 virus transformed Balb/c-3T3 cells in serum-free monolayer culture

The growth requirements of SV40 transformed Balb/c-3T3 cells have been studied in the absence of serum. For growth in serum-free medium, the cells require (i) insulin, (ii) transferrin, and (iii) cis-unsaturated fatty acids added in combination with fatty acid free bovine serum albumin. The growth rate, saturation density, and morphology of cells grown in this serum-free medium are the same as those of cells grown in serum supplemented medium. This mixture also supports the growth of SV40 transformed Swiss-3T3 cells and SV40 transformed primary mouse embryo cells, but does not support the growth of untransformed Balb/c-3T3 cells. The addition of fibronectin to this mixture allows routine subculture, repeated passage, and indefinite propagation of SV40 transformed Balb/c-3T3 cells. Cells grown in this medium for a period of two months retain their ability to induce tumors when injected into athymic nude mice.     

Growth of B95-8 cells and expression of Epstein-Barr virus lytic phase in serum-free medium.

Epstein-Barr virus (EBV)-transformed tamarin (Saguinus oedipus) cells (B95-8) were selected for growth in medium with reduced serum and then transferred to serum-free medium which consisted of RPMI 1640 supplemented with insulin, transferrin, and selenium. Serum-free cells in continuous passage for 1 year had a morphology, growth rate, and culture density which approached those of B95-8 cells grown with serum. The cells expressed virus-induced antigens, including the EBV-associated DNA polymerase. Cells exposed to EBV-inducing agents, n-butyric acid and phorbol 12-myristate-13-acetate, produced transforming virus with titers comparable to those of cultures grown with serum. These findings demonstrate that serum is neither required for the growth of B95-8 cells nor necessary for induction or full expression of the EBV lytic phase in these cells.     

Serum inhibition of proliferation of serum-free mouse embryo cells

Serum-free mouse embryo (SFME) cells, derived in medium supplemented with insulin, transferrin, high density lipoprotein, epidermal growth factor, and fibronectin, do not undergo crisis, maintain a predominantly diploid karyotype with no detectable chromosomal abnormalities for well over 100 population doublings in vitro, and are growth inhibited by concentrations of serum that are growth-stimulatory for most cell lines in culture. Serum inhibition of SFME cell proliferation was reversible and was not prevented by addition of the supplements of the serum-free medium, even when added repeatedly during the culture period. The serum effect on SFME cell proliferation could be detected after incubation in serum-containing medium for as little as 8 h. SFME cells in serum-containing medium were arrested in the G1 phase of the cell cycle with a greatly reduced rate of incorporation of precursors into DNA and thymidine kinase activity, while a reduction in rate of incorporation of amino acids into protein was not observed. SFME cultures maintained for extended periods in serum-containing medium underwent a crisis-like period followed by the appearance of variant cells capable of growing in serum-supplemented medium. These cells exhibited abnormal karyotype and were resistant to several inhibitors of proliferation active on the parent SFME cell type.     

人OC-3-VGH卵巢癌细胞裸小鼠肿瘤模型的建立

目的建立人OC-3-VGH细胞株卵巢癌裸小鼠模型并观察该肿瘤生物学生长特性。方法OC-3-VGH细胞株复苏后加入10 mL RPMI-1640培养液,放入培养箱,传2～3代后,取细胞悬液,均以4×106个细胞,每只0.2 mL分别接种至BALB/c雌性裸小鼠皮下,2月后处死取材,观察肿瘤生长特性和转移情况。结果皮下接种一周后,裸鼠长出肿瘤,并随时间而增大,体积呈指数增长,第42天始,明显增大（P〈0.05）。组织学检查发现裸鼠皮下肿瘤细胞均细胞体积较大,细胞核大而染色深,核分裂相较多、异型性明显,接种2个月时,未发生其他组织转移。结论建立了新的卵巢癌动物模型,并初步研究了其生物学特性,为卵巢癌治疗方法的研究拓宽了道路。     

Colony formation in agar by murine plasmacytoma cells: potentiation by hemopoietic cells and serum

Clonal growth in semisolid agar medium was obtained using cells from 19 of 25 transplanted murine plasmacytomas when the medium was supplemented by whole mouse blood or washed red cells. With different tumors cloning efficiency ranged from 0.01% to 21.6%. With two exceptions, mouse blood did not potentiate colony formation in agar by cells from transplantable myelomonocytic, myeloid, and lymphoid leukemias, reticulum cell sarcomas and fibrosarcomas. The clonal growth of some plasmacytomas was also potentiated by syngeneic thymic, spleen or bone marrow cells. Plasmacytoma colony growth was not stimulated by normal mouse serum but serum from mice injected with endotoxin or polymerised flagellin stimulated colony growth by some plasmacytomas. The active serum factor was not the colony stimulating factor (CSF) and its appearance after antigenic stimulation was not T cell-dependent. Preimmunised mice failed tq respond to antigenic stimulation. Whole body irradiation did not induce a rise in the capacity of serum to stimulate colony formation by plasmacytoma cells.     

Growth of Myeloma MPC-11 Cells in Serum-free Growth Factor Supplemented Medium

A myeloma MPC-11 cell line, which originated in a mouse plasma cell tumor, was able to proliferate in the absence of serum in a synthetic medium supplemented with transferrin, ethanolamine and selenium. The cells showed almost the same growth rate in this medium as in medium with 10% fetal calf serum added. Immunoglobulins secreted into the serum-free medium by the cells were easily separated from medium components by polyacrylamide gel electrophoresis.     

Long-term multiplication of the Chinese hamster ovary (CHO) cell line in a serum-free medium

Summary A new synthetic medium (referred to as GC₃) that supports the growth of the Chinese hamster ovary cell line has been developed. It is composed of a 1∶1 mixture of Ham's F12 and modified Eagle's minimum essential (MEM.S) mediums supplemented with transferrin (10 μg/ml), insulin (80 mU/ml), and selenium (1×10⁻⁷ M). Other more simple supplementations of our basal medium MEM.S/F12 (transferrin+insulin, transferrin+selenium, ferrous iron+selenium) also give good cell growth responses. Fibronectin or serum pretreatment is not needed for cellular attachment and spreading. Our culture system is characterized by a continuous serum-free cultivation (more than 200 doublings), a clonal growth, a high density proliferation, and a rapid growth rate near that of cells in serum-supplemented medium.     

Altered growth factor requirements and cell cycle control in rat hepatoma cells versus adult rat hepatocytes in culture

Adult rat hepatocytes multiply in primary cultures when incubated in arginine-free MX-83 medium supplemented with dialyzed fetal calf serum, insulin, glucagon, hydrocortisone, epidermal growth factor, and transferrin. In the absence of mitogens, the fraction of the cells engaged in DNA synthesis dropped sharply. However, cells initiated DNA synthesis in response to the mitogenic mixture indicating that hepatocyte proliferation is controlled by G1----S transition rates. In contrast, rat hepatoma line DTH-3, derived from Morris 7777 "minimal deviation" hepatoma, required only insulin for proliferation in chemically defined MX-83 medium. The lengths of their cell cycle phases varied with the growth rate. The phases of the growth cycle were proportionately shortened (expanded) when the growth rate was increased (decreased). It is concluded that DTH-3 hepatoma cells, which display a decreased growth factor requirement as compared with adult rat hepatocytes differ from normal hepatocytes by fundamental alterations in the mechanisms controlling the progression of the cell cycle.     

Growth of H-35 rat hepatoma cells in unsupplemented serum-free media: Effect of transferrin,insulin and cell density

Summary Serum-free tissue culture medium consisting of a 1∶1 mixture of Dulbecco's modified Eagle's medium (DMEM) and Ham's F12 medium is herein shown to support growth of Reuber H-35 cells over several days in culture. Cells were initially plated in serum containing DMEM medium for 3 h. After cell attachment, serum is removed and replaced with a serum-free 1∶1 mixture of these two commercially available tissue culture media. The doubling time of cell growth in this unsupplemented serum-free medium was 46 h in lightly plated cultures over the first 5 d. The presence of transferrin (5 μg/ml) and insulin (3.3 nM) results in a cell doubling time of 17 h, which equaled the growth rate in medium containing 10% fetal bovine serum. In the absence of transferrin, growth rates in serum-free medium were correlated with the cell density of cultures. Conditioned medium from dense, serum-free cultures has growth-stimulating activity in recipient lightly plated cultures. This simple, serum-free culture medium will facilitate studies on the growth regulation of H-35 rat hepatoma cells. This work was funded by a feasibility grant from the American Diabetes Association, as well as by the National Institutes of Health grants CA 24604-09 and CA 16463-14.     

Production of an auto-stimulatory growth factor by human hepatoma cells abrogates requirement for a brain-derived factor

Summary Human hepatoma cells grow at high cell density in the absence of exogenous growth factors. At low cell density, two different hepatoma cell lines required a novel growth factor from brain tissue. A factor with similar physico-chemical properties in the concentrated medium from high density cultures completely substituted for the brain extract. The autogenous secretion of a novel liver cell growth factor that is concentrated in brain tissue may underlie in part the unregulated growth of hepatomas. EDITOR'S STATEMENT Collective properties of growth factor activities for hepatoma cells in bovine brain extract and the medium of hepatoma cells suggest a neural tissue-derived liver cell growth factor that may be autogenously produced by liver tumor cells. Purification and characterization of such a factor may lead to selective inhibition of hepatoma, cell proliferation. David W. Barnes     

A mouse hepatoma cell line which secretes several serum proteins including albumin and alpha-foetoprotein.

A permanent cell line (BW) was established from a transplantable mouse hepatoma, BW7756, which produces alpha-foetoprotein (AFP). Three clones were isolated from the uncloned culture: BW1, BW2 and BWTG3. The cells of the latter clone, which was isolated after selection in the presence of thioguanine, are deficient in the enzyme hypoxanthine-guanine-phosphoribosyl transferase. Both BW1 and BWTG3 cells have mean chromosome number of 64 (60 telocentric and 4 metacentric chromosomes). All three clones secrete at least five serum proteins into the culture medium: albumin, AFP, and alpha 2 globulin, transferrin and C3, the third component of complement. The approximate rate of albumin secretion by BW1 and BWTG3 cells is 10 mug/24 h/10(6) cells. Both albumin and AFP can easily be detected in cell extracts. The simultaneous production of AFP and a hepatocyte specific marker (albumin) by cloned hepatoma cells show that the production of AFP by the tumour is due to the tumoural hepatocytes themselves.     

Growth of mouse vaginal epithelial cells in culture: Effect of sera and supplemented serum-free media

Summary Normal mouse vaginal epithelial cells isolated from ovariectomized ca. 35-d-old BALB/cCrgl mice were grown in primary culture using collagen gel metrix and a serum-free medium composed of a 1∶1 mixture of Dulbecco’s modified Eagle’s medium and Ham’s F12 (D:H) medium supplemented with insulin (IN), epidermal growth factor (EGF), cholera toxin, transferrin, and bovine serum albumin V (BSA). Three-dimensional cellular outgrowths occurred inside the collagen gel matrix. The contribution of each factor to cell growth was examined by individual addition to the basic D:H medium and by individual deletion from the complete serum-free medium. When added individually, only IN promoted growth. Deletion of IN from the complete serum-free medium markedly, diminished growth; deletion of EGF or BSA slightly diminished growth. When horse, fetal bovine, or chicken serum was added to the basal D:H medium, only with increasing doses of horse serum was there enhanced cell growth. The effect of 17?-estradiol and diethylstilbestrol on the growth of cells was also tested, using a suboptimal medium of D:H supplemented with BSA and IN, or a minimal medium supplemented with IN alone. During the 8-d time period, addition of estrogen did not enhance cell growth in either medium. To date, we have been unable to demonstrate a mitogenic effect of estrogen; rather a dose-dependent inhibition of proliferation is seen. This investigation was supported by grants CA-05388 and CA-09041, awarded by the National Institutes of Health, DHHS, Bethesda, MD.