Effects of 5-azacytidine on the progressive nature of cell transformation.

C3H 10T1/2 mouse embryo fibroblasts were exposed to 3 microM 5-azacytidine for 24 h and then serially passaged in the absence of 5-azacytidine and examined for subsequent changes in growth properties. The treated cells showed changes in morphology, saturation density, growth rate, and serum dependence. By the 5th passage they acquired the ability to grow in 0.3% agarose, and by the 30th passage they gave rise to fully transformed foci that grew in agarose, in agar, and in liquid suspension. This progression was rapidly accelerated if the cultures derived from 5-azacytidine-treated cells were exposed for 48 h to the carcinogen benzo[a]pyrene. Results of these studies provide evidence that aberrations in DNA methylation may be one of a series of critical events during the course of multistage carcinogenesis and thus enhance the evolution of tumor cells.     

Primary culture and maintenance of steroid-secreting human placenatal monolayers.

A simple method is described for primary culture and for maintenance of hormone-producing cells from normal human placenta. A consistent yield of cells was obtained and an average survival of 3 to 4 months in culture using 1 mm3 explants from the most vascular area of the placentas. These explants were placed in a variety of culture media in 30 ml flasks and incubated at 37 degrees C in an atmosphere of 5% CO2 and 95% air. The best yields in terms of cell growth were observed with Eagle's MEM (minimum essential medium) with supplements of horse serum and fetal calf serum or human cord serum. (Ham's F-10 with supplement of horse serum and fetal calf serum supports growth for the longest period and media containing human cord serum had the best yield of steroids.     

Establishment of a continuous embryonic cell line from Japanese flounder Paralichthys olivaceus for virus isolation

A continuous cell line, the flounder embryonic cell line (FEC), was established from gastrula-stage embryos of a marine cultured fish, the Japanese flounder Paralichthys olivaceus and cultured for more than 200 d with more than 60 passages. FEC cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with antibiotics, fetal bovine serum (FBS), sea perch serum (SPS), and basic fibroblast growth factor (bFGF). The cells were small and round, and grew actively and stably in culture. The effect of temperature, FBS concentration and bFGF on FEC cell growth was examined. Cells grew well between 24 and 30 degrees C, but had a reduced growth rate below 18 degrees C. The growth rate of FEC cells in medium containing 15% FBS was higher than that in medium containing 7.5% FBS. Addition of bFGF to the medium also significantly increased the growth rate. Chromosome analysis revealed that FEC cells have a normal diploid karyotype with 2n = 48. High survival rate was obtained after cryopreservation of cell cultures. The susceptibility of the cell line to piscine viruses was examined. Two viruses tested were shown to induce CPE (cytopathic effect) on FEC cells. FEC cell culture infected with fish iridovirus was further elucidated by electron microscopy. Many virus particles were found in the cytoplasm of the virus-infected FEC cells. These results indicated that the FEC cell line could be potentially used to isolate and study fish viruses.     

Recombinant platelet-derived growth factor-BB stimulates growth and inhibits differentiation of rat L6 myoblasts.

We previously found that L6 myoblasts and skeletal muscle isolated from developing rats express the platelet-derived growth factor (PDGF) beta-receptor gene (Jin, P., Rahm, M., Claesson-Welsh, L., Heldin, C.-H., and Sejersen, T. (1990) J. Cell Biol. 110, 1665-1672). We now report that recombinant human PDGF-BB is a mitogen for L6 myoblasts and also a potent inhibitor of myogenic differentiation. Treatment of L6J1 myoblasts with PDGF-BB increased the rate of DNA synthesis and stimulated cell proliferation. In differentiation medium (Dulbecco's modified Eagle's medium/0.5% fetal calf serum or Dulbecco's modified Eagle's medium/insulin), PDGF-BB prevented fusion of confluent myoblasts and suppressed biochemical differentiation in L6J1 cells. Inhibition of myoblast differentiation was, however, reversible. Withdrawal of PDGF-BB from the medium allowed myoblast fusion to occur. Northern blot hybridization showed that the PDGF beta-receptor mRNA was down-regulated to an undetectable level when confluent cultures of L6J1 myoblasts in growth medium (Dulbecco's modified Eagle's medium/5% fetal calf serum) were shifted to differentiation medium. Receptor binding assays further indicated that binding of PDGF-BB to its receptors on L6J1 myoblasts declined rapidly before creatine kinase activity rose. Our results provide the first demonstration that PDGF-BB is a potent regulator of myogenesis of L6 rat myoblasts and suggest that it may regulate muscle differentiation in vivo.     

Some properties of an established fish cell line fromXiphophorus helleri (red swordtail)

Summary A cell line (SWT) was established from embryonic tissue of the red swordtailXiphophorus helleri. The SWT cells grew optimally, at 26°C to 30°C in Eagle's basal medium plus 10% fetal calf serum but failed to grow at 16°C and 37°C. After 50 subcultivations, the cells remained contact-inhibited and were pseudodiploid with a chromosomal modal number of 46. Virological studies demonstrated that SWT cells supported replication of the following viruses at the indicated temperatures: IPN virus (22°C), FV-3 (30°C), and VSV (33°C). The following mammalian, viruses failed to replicate at 33°C: vaccinia, poliovirus 2, herpes simplex, and reovirus 2. Although not replicating, reovirus induced interferon in there cells. This work was supported in part by a grant from the University Research Council. A part of these results was presented at the 22nd Annual Meeting of the Tissue Culture Association, Lake Placid, N. Y. 1971.     

Class Specificity of Avian and Mammalian Sera in Regards to Myogenic Cell Growth in vitro

Chick myogenic cells grew in the presence of a small amount of avian serum in a culture medium composed of Eagle's minimum essential medium (MEM) and horse serum. Mammalian sera, except for fetal bovine serum at high concentrations, could not substitute for the avian serum.
Rat myogenic cells grew in the presence of a small amount of mammalian serum in a culture medium composed of MEM and chick serum: avian sera, except for dove serum at high concentrations, could not substitute for the mammalian serum.
Serum from animals of the class from which the myoblasts were obtained was needed for cell growth. It is thus concluded that there is a class specificity among sera in regards to myogenic cell growth. The only exceptions to this hypothesis found so far were fetal bovine and dove sera.     

Effect of insulin, thyroxine and oestradiol on growth of normal human diploid cells and human heteroploid HeLa cells

Insulin (5 micrograms/ml), thyroxine (1 microgram/ml) and beta-oestradiol (0.1 microgram/ml), if added separately to the medium, did not significantly influence the growth of normal human diploid LEP 19 cells cultivated in Eagle's MEM medium with 1% foetal calf serum. Combined, they did not influence the growth of LEP 19 cells in serum-free MEM medium either. Under the same cultivation conditions, insulin caused 37%, thyroxine 24% and a mixture of insulin, thyroxine and beta-oestradiol 325% (without serum) or 15% (with 1% foetal calf serum) stimulation of the growth of human heteroploid HeLa cells. Isolated beta-oestradiol significantly inhibited the growth of HeLa cells.     

Cytochalasin B releases a major surface-associated glycoprotein, fibronectin, from cultured fibroblasts

BHK21 cells cultured in minimal essential medium (Eagle) supplemented with 10% dialyzed fetal calf serum did not grow as they did in whole serum containing medium. Logarithmic growth was, however, initiated after a lag period, the length of which was dependent upon the cell density: medium volume ratio. The quiescent cells conditioned the medium during this lag period, and growth stimulation was apparently due to the release of serine into the medium. Cells cultured in 10% dialyzed serum plus the low molecular weight fraction of serum (serum dialysate), grew with kinetics similar to cells cultured in serum containing medium. When serum dialysate was chromatographed on Bio-gel P-2 the growth promoting activity eluted with the amino acids. Each of the non-essential amino acids was tested for its ability to stimulate the growth of cells in 10% dialyzed serum. Serine was capable of stimulating cell growth to the same extent as 10% serum dialysate and its concentration optimum was similar to its concentration in 10% serum dialysate. The remaining non-essential amino acids were either slightly stimulatory or had no effect on cell growth. Shifting a logarithmically growing population of cells to serine-free medium resulted in the accumulation of 95% of the cells in the G1 phase of the cell cycle within 24 h. Escape from the G1 block could occur if serine was added to the medium or if the cells were allowed to condition the medium. Entry of cells into S phase after the addition of 0.05 μmoles/ml of serine followed a 4–6 h lag and 80% of the cells were synthesizing DNA 12 h after shift-up.     

Establishment and characterization of a new human germ cell tumor strain (NOC-Ts) in culture

We have derived and characterized a new cell line from a teratoma with a embryonal carcinoma and seminoma. The medium used for the cell culture was Eagle's MEM synthetic culture medium (Gibco Inc.) supplemented with 10% new calf serum (Tissue plus, Mitsubishi Kasei Co.). Subcultures were performed on 3 to 4 days basis at 1:2 split by the use of 0.25% trypsin solution. The morphology of obtained cells was a epithelial-like shape and the cell grew in a monolayered sheet with about 30-32 hrs of population doubling time. The model chromosome number of this cell line was 73 including one large submetacentric marker chromosome. The temperature sensitivity and the tumorigenicity of this cell were tested in this experiment.     

Serum and substratum-dependent coupled loss of differentiated and tumorigenic phenotypes in B16-conv melanoma cells

Clonal B16 mouse melanoma conv cells are tumorigenic spindle-shaped cells (S-type cells) exhibiting tyrosinase activity and melanosomes under usual culture conditions. When the cells passaged on glass substratum were plated for colony formation on plastic substratum in Eagle's minimum essential medium (MEM) with 10% calf bovine serum, most of them converted to fibroblastlike cells (F-type cells) with the coupled loss of differentiated and tumorigenic phenotypes. However, they continued to be S-type cells provided that they were plated on glass substratum. The conversion from S- to F-type cell was not induced with high frequency even on plastic substratum when the concentration of calf serum in the medium was low (1-2%). These results indicate that both plastic substratum and serum factor are requisites for converting the phenotypic expression of the conv cells. Partial characterization of the serum factor indicates that it is adsorbable to plastic substratum, inactivated at 70 degrees C for 10 min, salted out at 40% of saturated ammonium sulfate; in addition the factor seems to act on cells within 1 day after plating.     

Demonstration of growth in porcine thyroid cell culture

Eagle's minimum essential medium supplemented with 20 per cent newborn calf serum (N.C.S.) allows porcine thyroid cell survival but not cell growth in vitro. In NCTC 109 medium supplemented with 20 per cent N.C.S. these cells actively grow and may be serially propagated. Cell population doubling time expressed as DNA doubling value is 3.5 days at 37 degrees C in 95 per cent air-5 per cent CO2. Thyrotropin does not affect porcine thyroid cell multiplication in vitro but stimulates the plating efficiency in primary cultures to about 130 per cent of controls. Cell selection was obtained by replacing media with Earle's balanced salt solution. This operation provoked death of nearly all cells by day 18 but subsequent addition of growth medium resulted in proliferation of epithelial cell clones. From generation 2 to generation 8, cells produce thyroglobulin but they do not actively trap iodide nor form follicles when thyrotropin is added to the media. Cell selection, demonstration of growth, as well as freeze-storage techniques described in this paper permit selection and storage of porcine thyroid cells and the potential constitution of cell collections.     

Normal mouse serum contains peptides which induce fibroblasts to grow in soft agar

The untransformed mouse fibroblast cells NIH/3T3, C3H/10T1/2, and rat NRK cells do not grow in soft agar in medium supplemented with 10% fetal calf serum. When fetal calf serum in the growth medium was supplemented with less than 1% of sera from mice or other vertebrates, however, these cells responded, forming large colonies. The morphology of soft agar colonies was a function of the treated cell type. In the presence of 10% serum from C57BL/6 mice, NRK cells grew to smooth-surfaced spherical colonies, while NIH/3T3 colonies showed individual round cells on their surface and C3H/10T1/2 cells grew as extended cells forming columns of end to end connected fibroblasts. Mus Musculus Castaneus-Epithelial (MMC- E) cells were not stimulated to grow in soft agar under these conditions. The major fibroblast colony-inducing factor (F-CIF) was partially purified from mouse serum by acid/ethanol-extraction, gel permeation chromatography, and reverse-phase high-pressure liquid chromatography. F-CIF is a polypeptide, which does not compete for binding to epidermal growth factor (EGF) receptors, but stimulates normal fibroblasts to form small colonies in semisolid medium and very large colonies in the presence of added EGF (2 ng/ml). In contrast to unfractionated mouse serum, purified F-CIF did not induce C3H/10T1/2 cells to grow in soft agar, suggesting that serum contains additional cell type-specific agar growth-stimulating activities.     

Evaluation of storage conditions for refrigerated rabbit skin

An evaluation of various refrigerated (4 °C) storage solutions and conditions was conducted using rabbit skin. Two in vitro methods to assay skin viability are presented: one which directly measures basal cell viability and one which assesses the skin's ability to grow in culture following storage. The superiority of storage in nutrient medium supplemented with fetal bovine serum over conventional storage in saline is clearly demonstrated. Storage in nutrient medium with 10% fetal calf serum resulted in basal cell viabilities which were over 30% higher than viabilities of skin stored by conventional methods in saline. Skin stored in saline failed to grow in culture, while 100% of the cultures of skin stored in medium plus fetal calf serum grew. Although addition of fetal calf serum to the saline improved the basal cell viability, growth in culture occurred only when the skin was stored in a capped tube. Skin stored in medium without serum gave viability results which were not significantly different from the unstored control, but growth rates in culture did differ significantly from the control values. Our study shows that the viability of rabbit skin and its ability to grow in vitro are depressed when the tissue is maintained at 4 °C in saline or in petri dishes, and optimal when refrigerated in nutrient medium supplemented with FBS in a sealed tube.     

Melanocyte-stimulating hormone and prolactin secretion in cell culture of estrogen-induced pituitary tumors in the hamster

Pituitary cells from hamsters bearing diethylstilbestrol induced renal adenocarcinomas were cultured in vitro. Dispersed cells in plastic dishes were viable for up to two weeks in Dulbecco's modified Eagle's medium supplemented with 17.5% of 6:1 horse serum to fetal calf serum. The secretion of alpha-melanocyte stimulating hormone and prolactin into the medium were measured by radioimmunoassay. The concentrations of both were elevated by day 3 in the medium from the hyperplastic pituitaries obtained from the estrogen treated, tumor bearing hamsters. Neither DES (10(-8)M) nor tamoxifen (10(-7)M) influenced the secretion of either hormone and neither altered either cell number or DNA synthetic activity as measured by thymidine incorporation. The secretion of hormones and the growth of the pituitary cells were, however, decreased by charcoal treatment of the serum. The results suggest that the elevation of serum alpha-MSH and prolactin observed in DES implanted hamsters is due to pituitary secretion of the hormones but that DES probably does not act directly on the pituitary to control the secretion.     

Actions of exogenous histones and other proteins on [3H]-thymidine incorporation into DNA of Novikoff hepatoma cells.

The effects of exogenous proteins on the incorporation of [3H]-thymidine into DNA was studied in Novikoff hepatoma ascites cells incubated in Eagle's minimal essential medium. A liver cytosol fraction (8 mg protein/ml) caused approximately 80% inhibition of isotope incorporation. The inhibitory activity of cytosol fractions from Morris hepatomas 9618A2, 5123C, and 20 were inversely related to their growth rate. Under conditions in which there appeared to be a density dependent inhibition of growth, a mean 10-20% stimulation of isotope incorporation was observed after addition of total calf thymus histones and individual fractions in the concentration range of 100-400 microgram/ml. In experiments with lower cell concentrations, a 60% or greater increase in [3H]-thymidine incorporation could be obtained with total calf thymus histone and with F1 and arginine-rich histones from rat liver. At concentrations of 1-2 mg/ml, histones inhibited DNA synthesis. Bovine serum albumin had little effect on DNA synthesis. Polylysine caused an 80-90% inhibition at a concentration of 1 mg/ml, but stimulatory effects were detected under certain conditions at 10 microgram/ml. The results suggest critical dependence on the ratio of cell and exogenous protein concentration in the action of proteins on DNA synthesis.     

Lipid Composition of Chlamydia psittaci Growth in Monkey Kidney Cells in Defined Medium

The lipid compositions of (i) monkey kidney (MK-2) cells cultivated in Eagle's minimal essential medium (MEM) with 5% calf serum, (ii) MK-2 cells cultivated in Waymouth medium supplemented with 20 mug of sodium oleate and 2 mg of bovine albumin per ml, (iii) Chlamydia psittaci strain 6BC grown in the latter host system, and (iv) calf serum were compared. Strain 6BC contains 31% phosphatidyl ethanolamine (PE) and 15% phosphatidyl glycerol (PG), whereas the host cell contains almost the same amount of PE (27%) and no PG. A high concentration of total lipid was observed in strain 6BC (29 to 34%), whereas MK-2 cells contain only 9 to 15% and calf serum contains 4.5% total lipid. The fatty acids of the total lipid from strain 6BC contain branched-chain acids. These fatty acids were found mostly in PE (33.0%) and PG (37.0%). No branched-chain fatty acid was found in the MK-2 cells. There was an increase in triglyceride content when MK-2 cells cultivated in MEM (19.2%) were compared with cells cultivated in Waymouth medium (28.0%). A high concentration (62.0%) of octadecenoic acid (C18:1) was found in the triglyceride of MK-2 cells cultivated in Waymouth medium. The level of polyunsaturated fatty acids observed in MK-2 cells cultivated in Waymouth medium (10.8%) and in the chlamydiae grown in these cells (13.3%) was low compared with the level in MK-2 cells (28.8%) cultivated in MEM with 5% calf serum and the level in calf serum itself (50.8%). A higher ratio of sterol ester to free sterol was found in calf serum than in MK-2 cells or in chlamydiae. Host contribution to lipid composition of strain 6BC is discussed.     

Optimal conditions for cryopreservation of primary chicken embryo kidney cells with dimethyl sulfoxide

Conditions were evaluated for optimum cryopreservation of primary chicken embryo kidney (CEK) cells. The recovery of viable CEK cells was best (50.8% viability) when the concentration of dimethyl sulfoxide (DMSO) in the freezing medium was 20% (v/v). The viability of primary CEK cells was not influenced by the concentration of calf serum in the freezing medium, the duration of storage at −70°C before storage in liquid nitrogen, cell concentration, or the method of addition or dilution of DMSO. Thawed cells recovered and grew in complete growth medium similarly to cells freshly isolated from kidney, and influenza viruses produced plaques in the monolayer. The cryopreservation procedures described here may facilitate maintenance of a standard stock of primary CEK cells for laboratories where preparation of primary CEK cells is not an option.     

The clonogenic response of bovine aortic endothelial cells in culture to radiation

The effect of ionizing radiation on the survival of bovine aortic endothelial (BAE) cells was determined by the in vitro colony formation method. The BAE cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% calf serum, antibiotics, and growth factors obtained from the culture of mouse S-180 cells. The cultured BAE cells were positive to the staining of antibodies against human factor VIII and formed clones in plastic culture flasks with a plating efficiency of about 11%. The survival curve of the BAE cells following an exposure to a single dose of X rays was characterized by D0 = 101 rad, Dq = 65 rad, and an extrapolation number (n) of 1.9. These parameters were not modified by the absence of growth factors at the time of irradiation. The response of BAE cells to radiation was dose-rate dependent. The split-dose studies demonstrated that the BAE cells were able to repair sublethal radiation damage within 1 h after irradiation.     

High density lipoproteins and the growth of vascular endothelial cells in serum-free medium

Summary Low density bovine vacular endothelial cell cultures maintained on dishes coated with an extracellular matrix can be grown in serum-free Dulbecco's modified Eagle's medium supplemented with high density lipoprotein (HDL) and transferrin. Such cultures do not require insulin. Early passage cultures exposed to HDL and transferrin grew as well as cultures exposed to optimal serum concentrations and could be passaged repeatedly in total absence of serum. A requirement for fibroblast growth factor to ensure an optimal growth could be observed only with late-passage cultures. The present results suggest strongly that HDL is involved in supporting the proliferation of vascular endothelial cells in vitro. This may be important for our understanding of the biological role of HDL “in vivo”. This work was supported by Grants HL 23678 and 20192 from the National Institutes of Health, Bethesda, MD.     

KLN205—A murine lung carcinoma cell line

Summary KLN205 cells, a cloned cell line established from the Nettesheim lung carcinoma, grow in various synthetic media such as MEM, Fisher's or Roswell Park Memorial Institute Medium (RPMI) with the addition of 5 to 20% fetal bovine serum (FBS), calf serum (CS) or horse serum (HS). They grow optimally in minimum Eagle's medium plus nonessential amino acids (NEAA) plus 5 to 10% FBS or HS. The cells are transplantable to DBA/2, BDF₁, AKD₂F₁, and BALB/c, but not to C3H/He or ICR mice. The growth curves, plating efficiency, ultrastructural characteristics, modal number of chromosomes and transplantability to mice of various strains are almost the same for early and late passages of cells passaged in vitro. These parameters for 16th and 36th passages were: doubling time, 31 and 33 hr; plating efficiency, 12.4±1.2 and 14.6±2.6%; modal number of chromosomes, 73 and 76; lung colony formation in DBA/2, 50 and 45.9/mouse; and subcutaneous tumor diameter 24.5 and 27.4 mm, respectively. Only the numbers of lung colonies formed in BDF₁ mice were different: 24.4/mouse with 16th passage cells, and 10.2/mouse with 36th passage cells. The results suggest that KLN205 is a relatively stable cultured cell line through 36 passages. As was expected, immunosuppression by higher concentrations of triaminolone acetonide (TA) enhanced lung colony formation in BDF₁ mice. On the other hand, a low concentration of TA inhibited lung colony formation in DBA/2 mice, which was unexpected. These results suggest that KLN205 offers a model for investigations on metastases to lungs as well as chemotherapy for lung carcinoma. This work was supported by the National Cancer Institute of Canada.