Initiation of DNA synthesis in murine B cells is independent of early changes in the cytosolic free calcium

The relationship between changes in the intracellular free Ca2+ concentration, [Ca2+]i, and the initiation of proliferation of murine B cells after the addition of mitogens and activators was studied. The effects of lipopolysaccharide (LPS), 12-O-tetradecanoyl phorbol-13-acetate (TPA), rabbit IgG antimouse Fab (IgG RAM Fab), and its F(ab')2 fragment (F(ab')2 anti-Fab) on the [Ca2+]i were measured using the fluorescent calcium indicator Fura-2. In parallel experiments, DNA and/or RNA synthesis were measured by assaying [3H]thymidine and/or [3H]uridine uptake. LPS stimulated a 20-120 X increase in the [3H]thymidine uptake, and a 3-7 X increase in [3H]uridine uptake without inducing any change in the [Ca2+]i. TPA induced a marginal increase in [3H]thymidine and [3H]uridine uptake, without effecting any change in the [Ca2+]i. In contrast, low doses of IgG RAM Fab produced a triphasic change in the [Ca2+]i, but had no effect on the [3H]thymidine or [3H]uridine uptake, even at much higher concentrations. Similarly, low doses of the F(ab')2 fragment induced sizable increases in the [Ca2+]i without affecting the [3H]nucleoside uptake. However, higher concentrations of F(ab')2 anti Fab increased the [3H]thymidine uptake and [3H]uridine uptake, while also increasing the [Ca2+]i. Significantly, pretreating the cells with TPA for 3 min virtually abolished the [Ca2+]i increase induced by IgG RAM Fab while simultaneously potentiating an increase in the IgG RAM Fab-induced [3H]thymidine uptake 85-fold. In the presence of TPA, IgG RAM Fab also induced a 2- to 30-fold increase in [3H]uridine uptake. Similarly, TPA virtually abolished the [Ca2+]i increase induced by the F(ab')2 anti-Fab fragment, yet it stimulated a F(ab')2 anti-Fab-induced uptake of [3H]thymidine and [3H]uridine by 120 and 10 times, respectively.     

Biochemical aspects of cardiac muscle differentiation. Possible control of deoxyribonucleic acid synthesis and cell differentiation by adrenergic innervation and cyclic adenosine 3':5'-monophosphate.

A single injection of either isoproternol or N6, O2'-dibutyryl adenosine 3':5'-monophosphate (dibutyryl cyclic AMP) results in an inhibition in the rate of [3H]thymidine incorporation into DNA of differentiating cardiac muscle of the neonatal rat. This inhibition is not due to substantially altered cellular uptake or catabolism of [3H]thymidine. Inhibition of [3H]thymidine incorporation by isoproterenol or dibutyryl cyclic AMP is potentiated by theophylline. Maximal inhibition (95%) is observed 24 h after administration of isoproterenol, and the rate of incorporation returns to a value 80% of control by 72 h. Norepinephrine also inhibits [3H]thymidine incorporation whereas cyclic GMP, N2, 02-Dibutyryl guanosine 3':5'-monophosphate (dibutyryl cyclic GMP), and phenylephrine have little effect. Equilibrium sedimentation analysis of cardiac muscle DNA in neutral and alkaline cesium chloride gradients using bromodeoxyuridine as a density label indicate that isoproterenol and dibutyryl cyclic AMP inhibit [3H]thymidine incorporation into DNA that is replicating semiconservatively. Administration of isoproterenol or dibutyryl cyclic AMP to neonatal rats inhibits by approximately 60% the incorporation of [3H]thymidine into DNA of tissue slices of cardiac muscle prepared 16 h later. [3H]Thymidine incorporation into DNA of tissue slices is into chains that were growing in vivo. This incorporation is linear for at least 4 h of incubation and is inhibited by isoproterenol and dibutyryl cyclic AMP. Inhibition is not due to altered cellular uptake of [3H]thymidine nor is it due to a cytotoxic action. Several other compounds which elevate intracellular levels of cyclic AMP (epinephrine, norepinephrine, glucagon, and prostaglandin E1) also inhibit [3H]thymidine incorporation into DNA or cardiac muscle tissue slices. Cyclic GMP, dibutyryl cyclic GMP, sodium butyrate, and phenylephrine have little effect. Isoproterenol administered together with theophylline to neonatal rats signficantly stimulates the in corporation of [3H]phenylalanine into total cardiac muscle protein and into myosin. This enhanced incorporation may be due in part to an increase in the cellular uptake of [3H]phenylalanine. DNA synthesis decreases progressively in differentiating cardiac muscle of the rat during postnatal development and essentially ceases by the middle of the third week (Claycomb, W. C. (1975) J. Biol. Chem. 250, 3229-3235). In reviewing the literature it was found that this decline in synthetic activity correlates temporally with a progressive increase in tissue concentrations of norepinephrine and cyclic AMP and with the anatomical and physiological development of the adrenergic nerves in this tissue. Because of these facts and data presented in this report it is proposed that cell proliferation and cell differentiation in cardiac muscle may be controlled by adrenergic innervation with norepinephrine and cyclic AMP serving as chemical mediators.     

3H]thymidine incorporation into whole liver as an alternative to [3H]thymidine incorporation into DNA as a parameter of cell proliferation in regenerating liver tissue in rats

OBJECTIVE: To monitor liver regeneration following partial hepatectomy, liver cell proliferation can be measured by assaying in vivo [3H]thymidine incorporation into liver cell DNA. We hypothesized that [3H]thymidine incorporation into whole liver tissue parallels [3H]thymidine incorporation into liver cell DNA, both in high proliferating and low proliferating liver. STUDY DESIGN: Liver cell proliferation in rats after partial hepatectomy or a sham operation was studied by measuring incorporation of [3H]thymidine into various fractions of liver tissue on days 1, 2, 3, 4 and 10 after surgery. RESULTS: [3H]thymidine incorporation into whole liver tissue and in the protein fraction correlated well with DNA-specific [3H]thymidine incorporation into regenerating (r > .80, P < .0001) and nonregenerating liver (r > .69, P < .005). [3H]thymidine incorporation into DNA was < 5% of the total amount of administered [3H]thymidine in both sham-operated and hepatectomized rats. Significant differences in [3H]thymidine incorporation into partially hepatectomized livers as compared to sham-operated rat livers were found on days 1 and 2 (whole liver tissue and protein fraction) or day 1 (DNA) after surgery. CONCLUSION: [3H]thymidine incorporation into whole liver tissue is a simple technique that can be used for the study of liver cell proliferation after partial hepatectomy in rats.     

Effect of N-trifluoroacetyladriamycin-14-valerate on [3H]thymidine uptake and DNA synthesis of human lymphoma cells

The effects of N-trifluoroacetyladriamycin-14-valerate on the uptake of [3H]thymidine and its incorporation into DNA of human P3HR-1 lymphoma cells were studied. In the absence of the drug, at 0 degrees C, [3H]thymidine was transported into the cells but not incorporated into DNA, as determined by both the trichloroacetic acid-soluble and -precipitable counts obtained with the cells. At 37 degrees C, [3H]thymidine was readily transported into the cells and incorporated into DNA. In the presence of the drug, both [3H]thymidine uptake (as shown by acid-soluble counts) and the amount of its incorporation into acid-precipitable materials were markedly reduced. However, the uptake of [3H]thymidine at 0 degrees C was found to be equally sensitive to drug inhibition as at 37 degrees C. The incorporation at 37 degrees C of [3H]thymidine into acid-precipitable materials of the cells, which had been prelabeled at 0 degrees C with [3H]thymidine, was found to be insensitive to inhibition by the drug. The in vitro activities of DNA polymerases alpha and beta purified from human P3HR-1 cells were also found not to be susceptible to inhibition. Nuclei purified from cells pretreated with the drug continued to synthesize DNA. The cytofluorograms of the cells treated with the drug indicated that the treated cells accumulated at the G2/M phase, whereas the S phase of the cells was not arrested. These results suggest that N-trifluoroacetyladriamycin-14-valerate inhibits [3H]thymidine uptake but not cellular DNA synthesis in human P3HR-1 lymphoma cells.     

Tissue-specific inhibition of [3H]thymidine incorporation into DNA by carcinogenic N-nitrosamines

The N-nitrosamines N-nitrosodimethylamine (DMN), N'-nitrosonornicotine (NNN) and 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) were injected intraperitoneally 24 h before sacrifice in F344 rats and C57BL mice in doses of 297 mumoles/kg b.w. and 148 mumoles/kg b.w., respectively. 2 h before sacrifice, the animals were given an intraperitoneal injection of [3H]thymidine. The results showed that the examined N-nitrosamines inhibited the incorporation of [3H]thymidine into DNA in a few tissues of the rats and the mice. The results indicated that the N-nitrosamines exerted a tissue-specific inhibition of the [3H]thymidine incorporation in the tissues reported to be involved in the biotransformation of these substances. The observed inhibitory effects on the incorporation of [3H]thymidine by DMN, NNN and NNK were also correlated to a considerable extent to the reported sites of carcinogenicity. The present study indicates that measurements of [3H]thymidine incorporation into DNA in various tissues of experimental animals is a useful short-term bioassay to evaluate the potential tissue-specific carcinogenicity of the N-nitrosamines. The method may also be useful as a complement to other short-term in vivo tests in the screening of potential genotoxicity of several other chemicals.     

Tumor necrosis factor stimulates DNA synthesis in the liver of intact rats

TNF is cytotoxic to tumor cell lines but enhances growth of some nontransformed cells. Because animals administered TNF have an increase in liver size, we studied the [3H]thymidine incorporation into DNA in the liver of intact rats. A significant increase in [3H]thymidine incorporation is seen 20 hours following TNF administration and peaks at 24 hours. The lowest dose of TNF that increases DNA synthesis is 10 micrograms/200 g rat with a maximal increase occurring with 25 micrograms/200 g, considerably less than the dose required for maximally increasing plasma triglycerides. The increase in [3H]thymidine incorporation was shown to be due to an increase in DNA polymerase alpha activity (associated with the replication of DNA) rather than DNA polymerases beta (associated with DNA repair) plus gamma activity. These results indicate that TNF administration stimulates DNA replication in the liver of intact animals.     

Bovine thyrotropin inhibits DNA synthesis inversely with stimulation of cyclic AMP production in cultured porcine thyroid follicles

The effects of bovine thyrotropin (TSH) on DNA synthesis and cyclic AMP production were studied in porcine thyroid follicles using suspension culture. During the early 72 hours incubation, the time-dependent uptake of [3H]thymidine by the follicles was observed. In the presence of 10 mU/ml TSH, the uptake of [3H] thymidine was significantly depressed at 72 hours incubation. TSH inhibition of [3H] thymidine incorporation was related to its concentration and the 50% inhibition was observed by using 1.0 mU/ml TSH. Under the same conditions, cyclic AMP production was stimulated by TSH and the stimulation was observed to be related to TSH concentration. In these experiments, the incubation time was 30 min. Dibutyryl cyclic AMP, an analogue of cyclic AMP, inhibited the [3H] thymidine uptake at 72 hours incubation. From these results, it is suggested that TSH inhibits DNA synthesis, and that the inhibition may be mediated by cyclic AMP that is produced by TSH stimulation.     

Stimulatory effects of epidermal growth factor on deoxyribonucleic acid synthesis in the gastrointestinal tract of the suckling mouse

The effects of epidermal growth factor (EGF), cortisone and thyroxine on deoxyribonucleic acid (DNA) synthesis in the esophagus, stomach, small intestine and colon have been studied in suckling mouse. Daily administration of EGF [4 micrograms/g body weight (bw)/day] during 3 days to 8-day-old mice induced a significant increase of the incorporation of [3H]thymidine into DNA in the stomach, the small intestine, and the two halves of the colon. The DNA synthesis in the esophagus remained unaffected by the EGF treatment. The maximal increase of [3H]thymidine incorporation into DNA was observed in the colon, and represented 112%. Daily administration of cortisone acetate (25 micrograms/g bw/day) or thyroxine (1 microgram/g bw/day) during 3 days to 8-day-old mice had no significant influence of the DNA synthesis of any part of the gastrointestinal tract. These results show that EGF is able to affect the DNA synthesis in the stomach, small intestine and colon of suckling mice.     

Cell position dependence of labelling thymidine nucleotides using the de novo and salvage pathways in the crypt of small intestine

About twice as much tritiated thymidine ([3H]TdR) is taken up by cells at the bottom of the crypt of the small intestine as by the rapidly cycling mid-crypt cells. However, the uptake of tritiated deoxyuridine ([3H]UdR) is even throughout the crypt. Exogenous thymidine is incorporated about four times and eight times more efficiently than deoxyuridine by the cells in the mid-crypt and cells at the bottom of the crypt, respectively. However all S phase cells in the crypt appear to be capable of using either precursors, i.e. either the de novo or salvage pathway. Since methotrexate (1 or 5 mg/kg) inhibits (at 5 mg/kg completely) the uptake of [3H]UdR, but has no effect on [3H]TdR uptake, the de novo and salvage pathways appear to be independent. Within the precision of the methods used in the experiments the 3 hr inhibition of the de novo pathway of deoxythymidylic acid (dTMP) synthesis by methotrexate does not produce any increase in utilization of the salvage pathway measured by incorporation of [3H]TdR into DNA. The increased efficiency of thymidine utilization by crypt base cells is not attributable to differences in accessibility of thymidine; differences in the rate of DNA synthesis or the size of the nuclei. It appears that crypt base cells (which include the putative stem cells) are efficient scavengers of [3H]TdR, and this might be related to the level of thymidine kinase activity within the cells, and/or to changes in the availability of endogenous thymidine (break-down products) which compete with exogenous [3H]TdR.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of DNA synthesis in adrenocortical cells by cytochalasin B

ACTH inhibits DNA synthesis in normal rat and mouse tumor Y-1 adrenocortical cells within the same concentration range that it stimulates steroidogenesis. These processes can be independently regulated as demonstrated by the divergent actions of cytochalasin B on these cells. In the normal cells, cytochalasin B does not increase steroidogenesis in serum-free or serum-containing media, and it decreases the stimulation produced by ACTH. In the absence of serum, the Y-1 cells respond in a similar way. However, in serum-containing media, cytochalasin B increases steroidogenesis in these cells and does not inhibit the response to ACTH. In both cell types, cytochalasin B inhibits [3H]thymidine incorporation into DNA by a mechanism different than that of ACTH. In the Y-1 cells, this inhibition is caused by a decreased uptake of [3H]thymidine into the cell, which probably reflects a decreased transport across the cell membrane. In the normal cells, cytochalasin B, like ACTH, does not affect [3H]thymidine transport, but it decreases DNA synthesis much more rapidly than does ACTH. This inhibition may be the result of the disruption of microfilaments by cytochalasinB, because our evidence indicates that it is not caused by a decrease in glucose uptake by the cells.     

Stimulation of DNA synthesis,cell multiplication,and ornithine decarboxylase in 3T3 cells by multiplication stimulating activity (MSA)

Multiplication stimulating activity (MSA) has been purified from the conditioned media of rat liver cells in culture by a modification of the procedure of Dulak and Temin. Purified MSA stimulates [³H] thymidine incorporation into DNA in subconfluent, serum starved 3T3 cells. Cell cycle analysis by the flow microfluorometer shows that the [³H] thymidine incorporation data reflects DNA synthesis. MSA also stimulates the multiplication of serum starved subconfluent 3T3 cells. MSA is approximately 10-fold less active in 3T3 cells than in chick embryo fibroblasts in stimulating [³H] thymidine incorporation into DNA. MSA causes a 2–10-fold increase in ornithine decarboxylase (ODC) activity in 3T3 cells and the dose response curve parallels the dose response curve for [³H] thymidine incorporation into DNA. The Km of ODC for ornithine is 0.12 mM. There is a 30% decrease in the activity of ornithine transaminase (OTA) during the time period in which MSA causes an increase in ODC activity. Insulin also stimulates [³H] thymidine incorporation into DNA, cell multiplication and ODC activity over the same concentration range as shown for MSA, however, the extent of stimulation by insulin is less than that observed following MSA addition.     

Effect of transforming growth factor beta on cell proliferation and glycosaminoglycan synthesis by rabbit growth-plate chondrocytes in culture

The effects of the transforming growth factor beta (TGF-beta) on the growth and glycosaminoglycan synthesis of rabbit growth plate-chondrocytes in culture were studied. In serum-free medium, TGF-beta caused dose-dependent inhibition of DNA synthesis by chondrocytes, measured as [3H]thymidine incorporation (ED50 = 0.1-0.3 ng/ml). The inhibitory effect was maximal at a dose of 1 ng/ml, and extended for a duration of 16-42 h. In contrast, TGF-beta potentiated the synthesis of DNA stimulated by fetal calf serum (FCS). Addition of TGF-beta (1 ng/ml) to cultures containing 10% FCS increased [3H]thymidine incorporation to 1.6-times that in cultures with 10% FCS alone. Consistent with this finding, TGF-beta potentiated DNA synthesis stimulated by the purified growth factors such as platelet-derived growth factor (PDGF), epidermal growth factor (EGF) and fibroblast growth factor (FGF). The maximal stimulation of DNA synthesis by FGF (0.4 ng/ml) was further potentiated dose dependently by TGF-beta (ED50 = 0.1 ng/ml, maximum at 1 ng/ml). When the cultures were treated with the optimal concentrations of TGF-beta (1 ng/ml) and FGF (0.4 ng/ml), [3H]thymidine incorporation was 3-times higher than that of cultures treated with FGF alone. This TGF-beta-induced potentiation of DNA synthesis was associated with replication of chondrocytes, as shown by a marked increase in the amount of DNA during treatment of sparse cultures of the cells with the growth factors for 5 days. In contrast, TGF-beta caused dose-dependent stimulation of glycosaminoglycan synthesis in confluent cultures of growth-plate chondrocytes (ED50 = 0.3 ng/ml, maximum at 1 ng/ml). This stimulatory effect of TGF-beta was greater than that of insulin-like growth factor I (IGF-I) or PDGF. Furthermore, TGF-beta stimulated glycosaminoglycan synthesis additively with IGF-I or PDGF. Recently, it has been suggested that bone and articular cartilage are rich sources of TGF-beta, whereas epiphyseal growth cartilage is not. Thus, the present data indicate that TGF-beta may be important in bone formation by modulating growth and phenotypic expression of chondrocytes in the growth plate, possibly via a paracrine mechanism.     

Reversible permeabilization of lymphocytes destroys the incorporation of deoxythymidine but not of deoxycytidine

The total uptake, phosphorylation and incorporation of thymidine (dThd) and deoxycytidine (dCyd) were compared in intact and reversibly permeabilized human tonsillar lymphocytes. The total uptake of [3H]dThd was lower than that of [5-3H]dCyd, but almost all of [3H]dThd was incorporated into DNA. However, the main part of [5-3H]dCyd taken up by the lymphocytes was found in the pool as phosphorylated nucleoside (55%), and only a smaller part (13%) was incorporated into DNA. Phosphorylated nucleosides were determined by DEAE-cellulose sheets in the ethanol-soluble fraction of the cells. The reversible permeabilization of lymphocytes by Dextran T-150 destroys totally the [3H]dThd incorporation, while [5-3H]dCyd incorporation decreased only to 60% of intact cells. During permeabilization the phosphorylation of both nucleosides increased severalfold. After permeabilization all [3H]dThd was in dTMP form, while [5-3H]dCyd was also found in dCDP (3%) and dCTP (38%) form. In the meanwhile, 22% of thymidine kinase, 63% of deoxycytidine kinase and 98% of DNA polymerase activity were measured in permeabilized cells as compared to intact cells. The results suggest different relationships between the lymphocyte plasma membrane and the salvage pathways of the two pyrimidine nucleosides.     

Herpes Simplex Virus Type 2 Functions Expressed During Stimulation of Human Cell DNA Synthesis

Experiments were designed to identify herpes simplex virus type 2 (HSV-2)-specific functions expressed during stimulation of human embryo fibroblast DNA synthesis. Cultures were partially arrested in DNA synthesis by pretreatment with 5-fluorouracil and maintenance in low-serum (0.2%) medium during virus infection. Results showed that continuous [methyl-(3)H]thymidine uptake into cellular DNA was ninefold greater in HSV-2-infected than in mock-infected cultures measured after 24 h of incubation at 42 degrees C. Shifting mock-infected cultures from low- to high-serum (10%) medium also caused some stimulation, but [methyl-(3)H]thymidine uptake was only twofold greater than in cells maintained with low serum. Plating efficiencies of both HSV-2-infected and mock-infected cells at 42 degrees C were essentially the same and ranged from 37 to 76% between zero time and 72 h of incubation. De novo RNA and protein syntheses were continuously required for HSV-2 stimulation of cellular DNA synthesis. HSV-2 infection markedly enhanced transport, phosphorylation, and rate of incorporation of [methyl-(3)H]thymidine into cellular DNA, starting at 3 h and reaching a maximum by 12 h; after 12 h, these processes gradually declined to low levels. In mock-infected cells these processes remained at low levels throughout the observation period. Pretreatment of cells with interferon or addition of arabinofuranosylthymine at the time of virus infection inhibited stimulation caused by HSV-2. 5-Bromodeoxyuridine density-labeled experiments revealed that HSV-2 stimulates predominantly semiconservative DNA replication and some DNA repair. Stimulation of [methyl-(3)H]thymidine into cellular DNA correlated with detection of virus-specific thymidine kinase activity. In conclusion, HSV-2 stimulation of cellular DNA synthesis appeared to involve at least four virus-specific functions: induction of thymidine transport, HSV-2 thymidine kinase activity, semiconservative replication, and repair of cellular DNA.     

Effects of carcinogenic halogenated aliphatic hydrocarbons on [3H]thymidine incorporation into various organs of the mouse. A comparison between 1,2-dibromoethane and 1,2-dichloroethane

The effects of 1,2-dibromoethane (DBE) and 1,2-dichloroethane (DCE) on the incorporation of [3H]thymidine into DNA were evaluated in various tissues of mice. The compounds were given intraperitoneally 24 h before sacrifice in an equimolar dose (293 mumoles/kg body weight). 2 h before the animals were killed, 0.5 mu Ci [3H]thymidine/g body weight was injected intraperitoneally. Both agents inhibited the [3H]thymidine incorporation in the forestomach, a site for their carcinogenic action. Whereas DBE also suppressed the [3H]thymidine incorporation in the nasal mucosa, the thymus, and the "glandular stomach", DCE was inhibitory only in the kidney. The observed difference in the effect of DBE and DCE on the thymus had its counterpart in a DBE-induced decrease of acid-insoluble radioactivity, demonstrated with whole-body autoradiography. The results indicate that in vivo screening of [3H]thymidine incorporation into various organs of an intact experimental animal is a sensitive technique for comparing cyto- and/or genotoxic effects of chemicals with a similar chemical structure.     

Steroidal and growth factor regulation of [3H]thymidine incorporation by cultured endosalpingeal cells of the bovine oviduct

Summary Cultured cells from the bovine endosalpinx were used to evaluate effects of estradiol-17β, progesterone, epidermal growth factor, and insulinlike growth factors I and II on [³H]thymidine incorporation. Cells were treated with hormones and growth factors when approximately 50% confluent. After 24 h, DNA synthesis was quantified by pulsing cells with [³H]thymidine for 12 h and determining uptake into DNA. Cells prepared by mechanical dispersal incorporated more [³H]thymidine than cells dispersed with collagenase. However, hormonal responses were the same for both types of cells. As compared to plastic, cells on a Matrigel substratum exhibited lower incorporation of [³H]thymidine and were unresponsive to hormones. Estradiol-17β increased [³H]thymidine incorporation slightly at 10⁻¹⁰ mol/liter and higher. Epidermal growth factor, insulinlike growth factor-I, and insulinlike growth factor-II also stimulated [³H]thymidine incorporation. Effects of insulinlike growth factor-I were greater for cells treated with estradiol-17β. In the absence of estradiol, progesterone inhibited [³H]thymidine incorporation at 1, 10, and 100 ng/ml. When estradiol-17β was present, progesterone stimulated [³H]thymidine incorporation at 1 ng/ml and reduced incorporation at 100 ng/ml. In conclusion, [³H]thymidine incorporation by cultured oviductal endosalpingeal cells can be regulated by ovarian steroids and growth factors. These molecules may represent signals through which the ovary, embryo, and oviduct regulate oviductal growth. Work conducted while on a sabbatical leave supported by the Deutsche Forschungsgemeinschaft.     

Bi-functional action of transforming growth factor-beta on DNA synthesis in early passage human fetal fibroblasts

We investigated the influence of transforming growth factor-beta (TGF-beta) on DNA synthesis in human fetal fibroblasts, as measured by the incorporation of [3H]thymidine and cell replication. In serum-free medium, without additional peptide growth factors, TGF-beta had no action on thymidine incorporation. However, in the presence of 0.1% v/v fetal calf serum, TGF-beta exhibited a bi-functional action on the cells. A dose-dependent stimulation of [3H]thymidine incorporation, and an increase in cell number, occurred with fibroblasts established from fetuses under 50 g body weight, with a maximum stimulation seen at 1.25 ng/ml. For fibroblasts from fetuses of 100 g or greater body weight, TGF-beta caused a dose-related decrease in thymidine uptake with a maximal inhibition at 2.5 ng/ml, and a small decrease in cell number. When DNA synthesis was stimulated by the addition of somatomedin-C/insulin-like growth factor I, epidermal growth factor, or platelet-derived growth factor, their actions were potentiated by the presence of TGF-beta on cells derived from fetuses under 50 g body weight, but inhibited on cells obtained from the larger fetuses weighing more than 100 g. Similar results were found for changes in cell number in response to TGF-beta when stimulated by SM-C/IGF I. The ability of TGF-beta to modulate [3H] thymidine incorporation did not involve a change in the time required for growth-restricted cells to enter the S phase of the replication cycle. These data suggest that TGF-beta may exert either a growth-promoting or growth-inhibiting action on human fetal connective tissues in the presence of other peptide growth factors, which is dependent on fetal age and development.     

Bovine placental lactogen stimulates DNA synthesis of bovine mammary tissue maintained in athymic nude mice

Mammary tissue from five midpregnant heifers was transplanted subcutaneously into ovariectomized athymic mice (eight pieces/mouse). After a recovery period of 19 days, mice were injected daily for 5 days with buffer (50 mM NH4HCO3, pH 7.8) as control, 17 beta-estradiol (1 micrograms) plus progesterone (1 mg). Concurrently with the buffer or steroid hormone injections, mice were injected with bovine placental lactogen (0, 5, or 25 micrograms), bovine prolactin (0, 3.4, or 17.2 micrograms), or bovine growth hormone (0, 3.4, or 17.2 micrograms). All mice were injected with 2-bromo-alpha-ergocryptine (0.1 mg/day). Transplanted bovine mammary tissue was incubated for 4 hr in minimum essential medium containing 1 mu Ci/ml [3H]TdR. Two pieces were processed for autoradiography and the others were used for DNA assay and total [3H]TdR uptake. Bovine placental lactogen, prolactin, and growth hormone each increased [3H]TdR incorporation into DNA in a linear, dose-response manner. Addition of ovarian steroids to bPL resulted in a significant increase over protein hormones alone. Autoradiographic analysis indicated that the observed differences in DNA synthesis were due to hormonal effects on epithelial, rather than stromal, DNA synthesis. These results provide the first evidence of a mammogenic role of bovine placental lactogen.     

Interferon inhibition of thymidine incorporation into DNA through effects on thymidine transport and uptake

Replenishment of medium after 72 hr of growth of HeLa-S3 cells in dense suspension cultures increased [³H]-thymidine uptake into cells and incorporation into DNA, with the levels reaching a peak ～ 12 hr following medium change; β interferon inhibits the enhanced uptake of [³H]-thymidine and labeling of DNA in a dose-dependent manner. Some reduction in these processes is observed at a concentration as low as 1 u/ml, and ～ 75% inhibition at 640 u/ml. Kinetic analysis has revealed that the rate of labeling of the acid-soluble pool with [³H]-thymidine, measured either at 22°C, or 37°C, is reduced in interferon-treated (640 u/ml, 24 hr) HeLa-S3 cells. At 22°C, the initial rate of thymidine transport at a high (500 μM) thymidine concentration, determined within the first 30 sec of [³H]-thymidine addition was depressed by 44% in interferon-treated HeLa cells. At 37°C, labeled precursors accumulate in acid-soluble material for ～ 8 min after the addition of [³H]-thymidine, after which an apparent equilibrium level is attained. At this temperature, the rate of thymidine uptake and the apparent equilibrium level attained were depressed by 70% in interferon-treated HeLa cells. The reduced incorporation of [³H]-thymidine into DNA in interferon-treated HeLa-S3 cells can be largely explained by interferon inhibition of thymidine transport and phosphorylation.     

Growth factors, mitogens, cytokines, and bone morphogenetic protein in induced chondrogenesis in tissue culture

Connective tissue outgrowths of neonatal muscle onto a substratum of bone matrix differentiate into cartilage in response to a bone morphogenetic protein (BMP). The BMP can be separated from bone matrix by extraction with 4 M guanidine hydrochloride (GuHCl) or degraded in situ by endogenous proteolytic enzymes to deactivate the matrix. Rat triceps muscle was minced in a suspension of noncollagenous bone matrix proteins including BMP (BMP/NCP) in culture medium. To investigate the possible synergistic interactions in induced chondrogenesis, six biosynthesized, highly purified growth factors were similarly added to the culture alone or in combination with BMP. Human interleukin-1 (IL-1) and Forskolin were also introduced to test the effects on BMP/NCP-induced chondrogenesis. On Day 14 of cultivation, [3H]thymidine incorporation into DNA and [35S]sulfate incorporation into glycosaminoglycans (GAG) were measured, and the values were expressed as percentages of the control. The quantity of induced cartilage formation was estimated by a histomorphometric scoring system. Under the influence of BMP/NCP, cultures grew on deactivated matrix, incorporated 55% more [3H]thymidine into DNA, incorporated 115% more [35S]sulfate into GAG than control cultures, and differentiated into cartilage. Without BMP/NCP, growth factors, IL-1, and Forskolin did not produce a comparable incorporation of either [3H]thymidine or [35S]sulfate, and they induced differentiation of fibrous tissue only. In the presence of BMP/NCP, cartilage developed in nearly all cultures. When the media were supplemented with growth factors, measurable increases in uptake of [3H]thymidine occurred with human epidermal growth factor (h-EGF), insulin-like growth factor-1 (IGF-1), nerve growth factor (NGF), transforming growth factor-beta (TGF-beta), bovine acidic fibroblast growth factor (baFGF), IL-1, bovine basic fibroblast growth factor (bbFGF), and Forskolin. Measurable increases in uptake of [35S]sulfate into GAG occurred with IL-1, baFGF, TGF-beta, h-EGF, IGF-1, bbFGF, NGF, and Forskolin. Synergistic interaction with BMP was considered when the quantity of cartilage developed (on a scale of 0-12 scores) in excess of the quantity of Score 4 induced by BMP/NCP alone. A cytokine, IL-1, had the greatest effect (Score 9). TGF-beta (Score 7), baFGF (Score 6), and NGF (Score 6) had relatively little effect. h-EGF, IGF-1, bbFGF, and Forskolin had no effect on cartilage development.(ABSTRACT TRUNCATED AT 400 WORDS)