DNA In Chick Embryo Fibroblasts Research Articles

Studies on the cytosolic dna of chick embryo fibroblasts and its uptake by recipient cultured cells

1. 1. Cytosol and extruded DNA complexes from cultured chick embryo fibroblast cells have been separated by agarose gel chromatography at intervals after pulse labelling with [ 3H]thymidine. 2. 2. The proportion of the various cytosol components changed markedly with time: there was a lag period of 3 hr before the major labelled (5 x 10 5 dalton) DNA complex appeared in the cytosol, and a further lag period of 5 hr before it was extruded from the cell. 3. 3. Cultured chick embryo fibroblast, and rat spleen, cells rapidly and very efficiently import their own or each others cytosolic DNA complexes into their respective cytosol fractions: the material recovered from the cytosol of recipient cells is characteristic of the presented material. 4. 4. Homologous cytosolic DNA complex presented to chick embryo fibroblast cells also becomes associated with the nucleus. The rat at which this occurs is comparable with the rate of incorporation of [ 3H]thymidine into nuclear DNA.

HORMONAL REGULATION OF SOMATOMEDIN SECRETION BY FETAL RAT HEPATOCYTES IN CULTURE

To determine which hormones might regulate fetal somatomedin (SM) secretion, we measured SM levels in conditioned medium from primary cultures of fetal rat hepatocytes. We employed a bioassay (3H-thymidine incorporation into DNA of chick embryo fibroblasts), a displacement assay (competition for binding of a radiolabeled rat IGF-II to the SM binding protein) for total somatomedin, and the radioimmunoassay for SM-C. Epidermal growth factor (EGF) and dexamethasone were the most active hormones tested, as measured by the displacement assay. Rat growth hormone (rGH) was much less stimulatory. Human placental lactogen (hPL), glucagon and insulin had little or no effect. Stimulation of SM secretion by both EGF and dexamethasone was time- and dose-dependent. The maximal response occurred at 48 hours, at a concentration of about 1 × 10−7 M of either hormone. In the bioassay, the stimulation by EGF, but not dexamethasone, could be detected. The steroid had enhanced the secretion of a heat-labile inhibitor that completely masked the mitogenic activity of the increased SM levels. The fetal SM secreted exhibited immunological cross-reactivity with human SM-C, but the levels were 500-fold less than those measured by our displacement assay. This suggests that the predominant fetal rat SM is not SM-C. We conclude that EGF and dexamethasone, but not rGH or hPL, stimulated the secretion by fetal hepatocytes of a fetal SM which resembled IGF-II.

Synthesis of multiplication-stimulating activity (rat insulin-like growth factor II) by rat embryo fibroblasts.

Multiplication-stimulating activity, a family of insulin-like growth factors previously identified in medium conditioned by the BRL-3A rat liver cell line, is also synthesized by third passage cultures of rat embryo fibroblasts (REFs) maintained in serum-free medium. Conditioned serum-free medium from REFs was chromatographed on Sephadex G-75 in 1 m acetic acid, and fractions in the size range of BRL-MSA contained MSA immunoreactivity. A dose-response curve of pooled G-75 fractions was parallel to BRL-MSA standard, and levels in the REF-conditioned medium were 0.5-1 microgram/ml. REF-MSA from the Sephadex G-75 pool was equipotent to BRL-MSA in stimulating [3H]thymidine incorporation into DNA in chick embryo fibroblasts and in stimulating DNA synthesis in REFs, as measured by autoradiography. In receptor binding assays using REFs, chick embryo fibroblasts, Swarm rat chondrosarcoma cells, and rat liver membranes, REF MSA was equal to BRL MSA in competition for binding of [125I]iodo-MSA. REF MSA also behaved identically to BRL MSA in a competitive binding protein assay using binding proteins in rat serum. Further characterization of REF MSA using Bio-Gel P-10 chromatography, followed by high pressure liquid chromatography or polyacrylamide gel electrophoresis, indicated that immunoreactive polypeptides in the fibroblast medium corresponded to BRL MSA II (mol wt, 8700) and BRL MSA III (mol wt, 7100). The amount of REF MSA released into the medium increased linearly over time. Cycloheximide decreased the amount of MSA in the medium, and during a recovery period, the amount of MSA returned nearly to control levels. In summary, rat embryo fibroblasts synthesize MSA which is biologically, immunologically, and chemically identical to MSA produced by the BRL-3A rat liver cell line.

Thermal inhibition of repair of methylmethane sulfonate-damaged DNA in chick embryo fibroblasts

Chick embryo fibroblasts were treated with the monofunctional alkylating agent methylmethane sulfonate at various concentrations for 1 h at 42°C, rinsed and then incubated post-treatment at various temperatures at which the kinetics of alkali-labile bond disappearance was followed. Growth experiments showed that these cells grew similarly at temperatures of either 37°C or 42°C. Repair as assessed by removal of alkali-labile bond was also similar for postincubation in the temperature range 37–42°C for damage due to methylmethane sulfonate treatment at concentrations less than 1.5 mM. When the postincubation temperature was raised higher than 42.5–43°C, this type of repair was stopped. The normal internal body temperature of adult chickens is about 41.6°C. Hence the present finding indicates that chick cells are much more severely restricted in DNA repair at temperatures above normal than are mammalian cells, which can function in this respect for several deg. C above 37°C.

Increased efficiency of photoreversal of UV-induced dimers in the DNA of chick embryo fibroblasts with post-UV dark time

The kinetics of photoreversal of UV-induced dimers in the DNA of early passage chick embryo fibroblasts was studied by monitoring disappearance of UV-endonuleae-sensitive sites. Photorepair was found to increase in efficiency when cells were incubated in the dark for several hours at 37°C following the dimer-inducing short-wavelength (254 nm) UV treatment, but prior to the photoreactivating black light (365 nm). Folllowing a UV dose of 10 J/m 2 it took at least 4 h in the dark to saturate this effect. This UV dose inserts roughly 2.4 dimer/10 7 daltons of DNA. Dark repair removes about 0.08 dimers /h/10 7 daltons. After 6 h in the dark, exposure to black light removes an additional 1.4 dimers /10 7 daltons leaving about 0.5 dimers unaffected by this treatment. After saturation of the dark effect, the amount of photoreactivation depends only on total black light fluence and not on fluence rate for the range of rates studied. This indicates that during 30 min, the maximum time of black light exposure, no appreciable reattachment of the photorepair molecule to additional unrepaired dimer sites occurs. We estimate that the number of effective photorepair molecules per chick chick cell is at least of the order of 2 × 10 5.

Comparison of pancreatic human and biosynthetic human insulin with respect to their action on adipocytes and chick embryo fibroblasts.

Biosynthetic human insulin (BHI) was compared to pancreatic human insulin and sperm whale insulin in terms of ability to stimulate incorporation of glucose into isolated rat adipocytes and thymidine into DNA in chick embryo fibroblasts. The human insulins were identical in their effects in both assays. Sperm whale insulin was more potent than the human insulins in stimulating glucose incorporation into rat adipocytes. All three insulins showed identical stimulation of DNA synthesis in the fibroblast assay. That action, however, is mediated via the receptor for insulin-like growth factor (IGF-I). Therefore, both human insulins were evaluated in terms of binding to the IGF-1 receptor in chick embryo fibroblasts. The two human insulins behaved identically (and agreed with previous findings for sperm whale insulin). All insulins, however, were approximately 200-fold less potent than IGF-I itself in this binding assay.

Characterization of a somatomedin (insulin-like growth factor) synthesized by fetal rat liver organ cultures.

Explants of 19- to 20-day fetal rat liver synthesize polypeptides biochemically and immunologically related to the well characterized somatomedin (insulin-like growth factor) BRL-MSA, multiplication-stimulating activity. Fetal MSA was purified from media conditioned by fetal liver explants by chromatography on Sephadex G-75 under acid conditions. Partially purified fetal MSA: 1) inhibited the binding of BRL-MSA to the MSA receptor of rat liver plasma membranes, to somatomedin-binding proteins from rat serum, and to rabbit anti-BRL-MSA serum; 2) had a molecular weight of 4,500 to 12,500 determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate; 3) stimulated the incorporation of [3H]thymidine into the DNA of chick embryo fibroblasts and induced cell multiplication; 4) stimulated glucose oxidation in rat adipocytes and weakly inhibited the binding of insulin to the insulin receptors of IM-9 lymphocytes; and 5) stimulated sulfate uptake in costal cartilage from hypophysectomized rats. These activities were associated with the same molecular species in fetal MSA preparations following disc acrylamide electrophoresis and co-migrated with active BRL-MSA peptides.

Proinsulin Binds to a Growth Peptide Receptor and Stimulates DNA Synthesis in Chick Embryo Fibroblasts1

Proinsulin, the biosynthetic precursor of insulin, has 3–5% the potency of insulin in various in vitro assays for insulin-like metabolicactivity. We now report that proinsulin was approximately 60% as potent as insulin and 20% as potent as the growth polypeptide, multiplication stimulating activity (MSA), in stimulating [3H]thymidine incorporation into DNA in chick embryo fibroblasts (CEFs). The stimulation by maximally effective concentrations of proinsulin plus MSA or insulin was not additive. The [3H]thymidine incorporation data reflected DNA synthesis since proinsulin also stimulated DNA synthesis as measured by flow microfluorometry and promoted the multiplication of CEFs. Proinsulin also competed with [125I]- iodoMSA for binding to CEFs, and with 60% the potency of insulin. Moreover, MSA binding was inhibited by the same concentrations of proinsulin that were required to stimulate [3H]thymidine incorporation into DNA. The activity of proinsulin in the [3H]thymidine incorporation and MSA radiorecepto...

Proliferation of buffalo rat liver cells in serum-free medium does not depend upon multiplication-stimulating activity (MSA)

A line of Buffalo rat liver cells (BRL 3A) that multiplies in the absence of serum produces a family of polypeptides termed MSA that can partially satisfy the serum requirement for growth of chick embryo fibroblasts. Temin, Pierson and Dulak (1972) proposed that BRL cells multiply in serum-free medium because they produce MSA. This does not appear to be the case. We have studied three BRL cell lines: 3A2 and 3A have diverged from the same original isolate from normal liver; 61t is a spontaneous transformant of a different isolate. All three cell lines showed a 10 fold increase in cell number during 5 days in serum-free medium. However, 3A-conditioned medium stimulated 3H-thymidine incorporation into DNA in chick embryo fibroblasts and human skin fibroblasts; 3A2- and 61t-conditioned media did not. After ion-exchange chromatography or gel filtration of the conditioned media and measurement of MSA by 3H-thymidine incorporation or radioreceptor assay, MSA again was found in the 3A medium but not in the 3A2 or 61t media. The absence of MSA in the 3A2 and 61t media was not due to inactivation of MSA by these two cell lines. Addition of partially purified MSA to 3A2 cells did not increase their multiplication rate in serum-free medium. We conclude that the ability of the BRL cells to multiply in serum-free medium is independent of the level of MSA in the medium.

Specific changes in the synthesis of mitochondrial DNA in chick embryo fibroblasts transformed by Rous sarcoma viruses

In chick-embryo fibroblasts infected with the Schmidt-Ruppin strain of Rous sarcoma virus, subgroup A (wild type), or with a thermosensitive mutant of this virus, T5, the rates of mitochondrial DNA synthesis differ in cells that exhibit normal and malignant phenotypes. In wild type virus-infected cells grown at 36 or 41 degrees C, morphological transformation is expressed, the rate of 2-deoxy-D-[3H]glucose uptake is stimulated, and mitochondrial DNA synthesis in vivo is stimulated three- to fivefold over that in uninfected cells. In T5-infected cells these changes occur only at the permissive temperature (36 degrees C); a shift to the nonpermissive temperature (41 degrees C) causes the reversal of these effects, and the specific activity of purified mitochondrial DNA is characteristic of that from uninfected cells. In contrast, the specific activities of nuclear DNA purified from cells maximally transformed under the permissive conditions do not differ between wild type-infected and uninfected with the T5 virus. In parallel experiments with isolated mitochondria, the rate of mtDNA synthesis in vitro is again greater in mitochondria isolated from transformed cells. In addition, mitochondrial DNA synthesis in vitro in mitochondria from nontransformed and virus-transformed cells exhibits differential sensitivity to inhibition by mercaptoethanol. Furthermore, the ntDNAP polymerase activity in mitochondrial extracts prepared from cells with transformed phenotypes is about sevenfold higher than in extracts from cells with nontransformed phenotypes.

Polypeptides with nonsuppressible insulin-like and cell-growth promoting activities in human serum: isolation, chemical characterization, and some biological properties of forms I and II.

Serum contains a polypeptide with insulin-like activity not suppressible by insulin antibodies (NSILA). A large-scale isolation procedure for NSILA is described, starting from an acid ethanol extract of a Cohn fraction (precipitate B) obtained from human plasma. Two homogenous polypeptides with insulin-like and cell-growth promoting activities could be isolated by gel filtration, ion exchange chromatography, and preparative polyacrylamide gel electrophoresis. Both components are slightly basic polypeptides with a minimal molecular weight of 5800 +/- 400. Both are single-chain molecules with two intrachain disulfide bridges each and no free sulfhydryl groups. NSILA I and II differ, however, in their amino acid compositions. The N-terminal amino acid sequences are Gly-Pro-Glu- in NSILA I, and Ala-Tyr-Arg- and Tyr-Arg- in NSILA II. Both NSILA I and II enhance net gas exchange in adipose tissue with a specific activity 60 times lower than that of insulin. In the range of 1-50 ng/ml, both substances stimulate [3H]thymidine incorporation into DNA of chick embryo fibroblasts. The same effect can be obtained with insulin but only at concentrations 50-100 times higher than those of NSILA. These results suggest that NSILA I and II are two forms of an insulin-like hormone with predominating effects on cell and tissue growth parameters.