Insulin-like Growth Factor I Regulation of Swarm Rat Chondrosarcoma Chondrocytes in Culture

Insulin-like growth factor I (IGF-I) is anabolic for chondrocytes and is thought to be important in regulating such normal cartilaginous tissues as the epiphyseal growth plate. In the present studies, we have investigated the role of IGF-I in the regulation of neoplastic cartilage. Chondrocytes cultured from a transplantable rat chondrosarcoma were analyzed for responsiveness to IGF-I with respect to DNA and glycosaminoglycan synthesis as determined by labeling with radioactive thymidine and sulfate, respectively. Stimulation of [³H]thymidine and [³⁵S]sulfate incorporation by IGF-I was two to four times that in serum-free controls, with half-maximal stimulation at 1 × 10^-9M. The efficacy of IGF-I was approximately one-half of that of serum in stimulating [³H]thymidine incorporation and was comparable to that of serum for [³⁵S]sulfate incorporation. When Swarm rat chondrosarcoma chondrocytes were cultured in the presence of IGF-I and exposed to graded concentrations of anti-IGF-I antibody, [³H]thymidine incorporation and [³⁵S]sulfate incorporation were attenuated in a dose-dependent fashion to 29 and 25% of antibody-free controls, respectively. Nonspecific antibody not raised against IGF-I was not inhibitory. These observations suggest that the majority of IGF-I action on these cells is susceptible to immunoinhibition. To estimate the contribution of IGF-I to the regulation of these cells by serum, Swarm rat chondrosarcoma chondrocytes were cultured with graded concentrations of either calf serum or fetal calf serum in the presence of anti-IGF-I antibody, nonspecific antibody, or no other additives. Specific antibody attenuated the effect of calf serum on both [³H]thymidine and [³⁵S]sulfate incorporation with overall inhibition of 52% (P < 0.01) and 48% (P < 0.001), respectively. Nonspecific antibody superimposed small, variably stimulatory or inhibitory effects on those of calf serum. When chondrosarcoma chondrocytes were incubated with fetal calf serum, anti-IGF-I antibody exerted a minimal inhibitory effect, reducing both [³H]thymidine and [³⁵S]sulfate incorporation by less than 25%. The immunoinhibition of both pre- and postnatal serum could be overcome in a dose-dependent fashion by increasing serum concentrations. These results suggest that the factors influencing Swarm rat chondrosarcoma chondrocytes may be developmentally regulated and that the contribution of IGF-I to the action of serum increases between fetal and postnatal life. These data support the hypothesis that chondrosarcoma is a somatomedin-responsive neoplasm and suggest that this tumor may be susceptible to interventions directed toward mechanisms that block insulin-like growth factor action.     

Recruitment of null cells during graft versus host reactions in the chicken

Untreated SC (B2/B2) chicken spleen or thymus cells (2 × 10⁷) caused significantly increased [³H]thymidine incorporation in spleens of heavily irradiated FP (B15/B21) recipient chicks on Day 4 after iv injection. Mitomycin-treated SC spleen cells or spleen cells treated with rabbit anti-T-cell serum and complement failed to raise the [³H]thymidine incorporation over that in uninjected, bursa cell-injected or FP spleen cell-injected controls. However, the combination of mitomycin-treated spleen or thymus cells and anti-T-treated spleen cells caused an increased [³H]thymidine uptake, suggesting the recruitment of non-T cells into proliferation by alloreactive mitomycin-treated T cells. Bursa cells did not proliferate during GVH reactions even though they could be shown to undergo proliferation in vivo upon mitogen (lipopolysaccharide and dextran sulfate) stimulation. In contrast, anti-T-treated spleen cells from agammaglobulinemic chickens were recruited into proliferation, suggesting that the recruited cell was not only not a T cell, but also no pre-B or B cell and most likely represented a cell of the monocyte-macrophage series.     

Role of serum and hormones during the growth and development of rat mammary tumor epithelial cells in collagen gel culture

Summary The characteristics of hormone-dependent rat mammary tumors in response to serum and hormones were determined in collagen gel matrix culture. Epithelial cells from 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary adenocarcinomas were embedded in collagen gel and the effect of estrogen, progesterone, prolactin, insulin, and serum was tested. The total cell number and [³H]thymidine incorporation were used to determine the growth pattern of the cells in culture. It was found that in medium containing 20% porcine serum and supplemented with insulin, estrogen, progesterone and prolactin, both the cell number and [³H]thymidine labeling index increased with time, after an initial lag. Serum seemed to be essential to maintain growth of the tumor cells, because hormones alone, in the absence of serum, were unable to sustain growth of the cells. When estrogen, progesterone, prolactin, and insulin were tested individually in the presence of 20% porcine serum, only estrogen demonstrated a significant stimulatory effect.     

Serum glucocorticoids have persistent and controlling effects on insulinlike growth factor i action under serum-free assay conditions in cultured human fibroblasts

Summary The biological actions of insulinlike growth factor I (IGF-I) measured under serum-free assay conditions were found to be significantly influenced by prior subculture conditions for adult human fibroblasts. Glucocorticoids seemed to be the major medium variable affecting IGF-I action. IGF-I added to serum-free cultures had little or no effect on [¹⁴C]aminoisobutyric acid uptake or [³H]thymidine incorporation in human fibroblasts previously maintained in media containing serum with low glucocorticoid levels or in serum stripped of endogenous steroids. However, a 48-h preincubation with dexamethasone resulted in a marked synergistic increase in IGF-I stimulation of [¹⁴C]aminoisobutyric acid uptake and [³Hthymidine incorporation in these cultures. In contrast, IGF-I in serum-free medium seemed to be a potent mitogenic and metabolic stimulus for human fibroblasts which had been subcultured in media with a high glucocorticoid content, either endogenous, or supplemented. After these culture conditions, a 48-h preexposure to dexamethasone had no further enhancing effect on IGF-I action. Dexamethasone also potentiated IGF-I, insulin, and epidermal growth factor stimulation of fibroblast replication depending on the earlier subcultivation conditions. Thus, glucocorticoids are important modulators of IGF-I bioactivity in cultured human fibroblasts. Serum glucocorticoids can exert a profound influence on the biological phenomena measured in cell culture, even when the serum has been removed before the actual experiment, and must be carefully taken into account for accurate evaluation of the biological function of IGF-I and other growth factors. This work was supported in part by grants DK28229, DK36054, CA42106, RCDA01275, and DK24085 from the National Institutes of Health, Bethesda, MD.     

Differential effects of glucocorticoids on insulin-like growth factor I action in cultured human fibroblasts

Glucocorticoids act synergistically with insulin-like growth factor I (IGF-I) to stimulate DNA synthesis and replication of cultured human fibroblasts. In the present study, we further define glucocorticoid and IGF-I interactive effects on human fibroblast metabolism and growth. IGF-I stimulated dose-dependent increases in early metabolic events. Half-maximal effectiveness was seen at 5–8 ng/ml IGF-I, with mean maximal responses of 1.5-, 2-, and 6-fold for [³H]2-deoxyglucose uptake, [¹⁴C]glucose incorporation, and [¹⁴C]aminoisobutyric acid (AIB) uptake, respectively. A 48-hour preincubation with 10^?7 M dexamethasone markedly enhanced both the sensitivity and maximal effectiveness of IGF-I stimulation of AIB uptake. In contrast, dexamethasone had no effect on IGF-I-stimulated glucose uptake and utilization. Maximum specific binding of [125I]IGF-I to fibroblast monolayers was identical in ethanol control and glucocorticoid-treated cells, with 50% displacement at ～5 ng/ml IGF-I. In addition to its synergism with IGF-I, preincubation with dexamethasone augmented insulin and epidermal growth factor (EGF) stimulation of [³H]thymidine incorporation; dexamethasone had no effect on platelet-derived growth factor or fibroblast growth factor action. Two-dimensional gel electrophoresis identified two specific glucocorticoid-induced proteins in human fibroblast cell extracts with molecular weights of 45K and 53K and pls of 6.8 and 6.3, respectively. These data indicate that IGF-I receptor-mediated actions in human fibroblasts are differentially modulated by glucocorticoids. Glucocorticoids are synergistic with IGF-I in stimulating mitogenesis and amino acid uptake, without having any apparent effect on IGF-I-stimulated glucose metabolism. Glucocorticoid enhancement of growth factor bioactivity may involve modulation of a regulatory event in the mitogenic signaling pathway subsequent to cell surface receptor activation. © 1995 Wiley-Liss, Inc.     

A New Liquid Medium for Trypanosoma (Schizotrypanum) cruzi*

A new liquid medium, with fetal calf serum as the sole undefined component, was devised for the cultivation of Trypanosoma cruzi. The need for the serum is ascribed to its mitogenic proteins, which stimulate division of, and the uptake of [³H]thymidine by the parasites. In the new medium, T. cruzi has a cycle culminating in the appearance of up to 90% metacyclic forms in the stationary phase. This cycle is repeated on each serial transfer.     

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.     

Studied on ribonucleoside-diphosphate reductase in permeable animal cells. I. Reversible permeabilization of mouse L cells with dextran sulfate

The treatment of mouse L cells with sodium dextran sulfate 500 for 20 min at 4 °C rendered them selectively permeable to small molecules. The degree of permeabilization was reproducible and depended on dextran sulfate concentration: as the dextran sulfate concentration increased, the cells became more permeable to the dye erythrosin B, lost the ability to incorporate [³H]thymidine into DNA, and acquired the capacity for nucleotide-dependent incorporation of [³H]dTTP into DNA and for K⁺- and ATP-dependent incorporation of [³H]lysine into protein. The ability to engage in macromolecular synthesis indicated that the permeabilized cells had retained a considerable amount of integrity, and this was confirmed by the observation that the cells could regain viability after an appropriate repair treatment. Permeabilization was rapidly reversed by incubating the cells in suspension culture medium plus 10% calf serum or in a defined medium composed of NaCl, NaH₂PO₄, glucose, glutamine, and CaCl₂, calcium ion being the most critical component. Using this minimally disruptive permeabilization procedure, it was possible to assay the enzyme ribonucleoside-diphosphate reductase in situ. The enzymatic conversion of CDP and GDP to the corresponding deoxyribonucleotides was studied; enzyme activity was almost entirely intracellular and responded to the allosteric activators and inhibitors that are known to modulate the activity of ribonucleotide reductase in vitro.     

The relative contribution of somatomedin to the serum-stimulated growth of human fibroblasts

Culture conditions were defined allowing to demonstrate a stimulatory effect of both serum-contained and purified Somatomedin activity on incorporation of [3H]thymidine and replication of cultured normal human fibroblasts. The use of dialyzed human serum in MEM medium supplemented by 0.2 mM serine offered the necessary and sufficient culture conditions. A significant difference between normal and hypopituitary patients sera was found in their effect on the rate of [3H]thymidine incorporation (p < 0.0001) and on cell replication (p < 0.01). Purified Somatomedin-C, in MEM without serum, is a poor mitogen. Its activity was strongly enhanced by the addition of 0.1 % dialyzed serum and 0.2 mM serine without, however, exceeding the stimulatory level of 1 % whole normal serum. The requirement of concomitant presence, for optimal $\text{in}\text{vitro}$ cell growth, of different low and high MW serum components is discussed.     

Glial conditioned media inhibit the proliferation of cultured rat cerebellar astrocytes

Conditioned medium (CM) obtained from rat cerebellar astrocytes cultured in a serumcontaining medium was able to inhibit [³H]thymidine incorporation into proliferating astrocytes, when compared to fresh medium. This effect could be attributed to two fractions of the CM with different molecular weights. The low molecular weight fraction (M_r<1,000) inhibited the cellular transport of the labeled precursor, without significantly affecting cell proliferation. The high molecular weight fraction (M_r>10,000) showed a strong inhibitory effect on astrocyte proliferation, which was documented using different assay techniques: i) [³H]thymidine incorporation performed in conditions preventing the effects of CM on transport; ii) [³H]thymidine autoradiography; iii) determination of the DNA content of the cultures. The inhibitory activity was present in media conditioned by non proliferating astrocytes treated with the antimitotic cytosine arabinoside, but not in media conditioned by neuron-enriched cultures nor in a chemically defined (N2) CM. The antiproliferative activity of astrocyte CM could be due either to a rapid depletion of mitogenic factors present in serum, or, to a secretion of growth inhibitory factor(s) by cultured astrocytes.Special Issue Dedicated to Dr. Abel Lajtha.     

Site-directed mutagenesis of bovine FGF-2 cDNA allows the production of the human-form of FGF-2 in Escherichia coli

Human fibroblast growth factor-2 can be used to induce angiogenesis in ischemias and wound healing. Site-directed mutagenesis of bovine FGF-2 cDNA was performed in order to produce the human-form of FGF-2 in E. coli. The mitogenic, angiogenic and neurotrophic activities of the recovered protein were analysed by [³H]thymidine uptake to DNA of cultured fibroblasts, rabbit ear dermal ulcers wound healing and neuronal differentiation of PC12 cells.     

Electron microscope analysis of amplifying ribosomal DNA from Xenopus laevis.

The inhibition of human prostatic epithelial cell (MA-160) replication by cAMP and certain analogs was explored in tissue cultures. When untreated fetal bovine serum was used to supplement the culture medium, cyclic AMP (cAMP) markedly inhibited cell growth. The inhibition was reversed by equimolar concentrations of uridine. Inhibition by 8-methyl-thio-cAMP (MES) was somewhat less effective and was not reversed by uridine. After heat treatment of the fetal bovine serum, which inactivated the cAMP phosphodiesterases, cAMP became less effective in cell growth inhibition, whereas the activity of MES remained unaltered. Dibutyryl cAMP (db-cAMP) had no effect on cell growth, however, when combined with the phosphodiesterase inhibitor, 1-methyl-3-isobutylxanthine (MIX), significant retardation of cell replication was observed. Cells treated for 24 h with 0.5 mM MES took up and incorporated significantly less [³H]TdR and [³H]uridine than control cells. Treatment of cells with 0.5 mM cAMP for 24 h, on the other hand, resulted in both substantially increased [³H]TdR uptake and increased [³H]uridine incorporation into RNA. The effects of similar treatment with db-cAMP plus MIX closely paralleled those of MES with marked inhibition of the uptake and incorporation of both thymidine and uridine.     

Labeling of Crithidia fasciculata DNA with [3H]Thymidine

Attempts at continuous labeling of Crithidia fasciculata DNA with [³H]thymidine led to a pulse-chase situation due to a cell-mediated conversion of thymidine to thymine in the medium. The uptake of thymine was slow compared to that of thymidine. Neither the addition of deoxyadenosine nor the sequential addition of several aliquots of [³H]thymidine had an effect on the pattern of labeling.     

Alterations of the incorporation of [3H]thymidine in cells in culture regulated by nucleoplasmic extract

Porcine skin nucleoplasmic extract (PSNE) was shown to alter the incorporation of [³H]thymidine into DNA of selected porcine, bovine, and human cell populations in culture. PSNE stimulated incorporation of [³H]thymidine into DNA of porcine and bovine dermal cells an average of 300 and 200% of control value, respectively. When porcine and bovine epidermal cells were exposed to PSNE the treatment inhibited [³H]thymidine incorporation into DNA by an average of 48 and 45%, respectively. Similar inhibitions were observed for porcine and bovine kidney, porcine lung, and human KB cells. Thus, the effect of PSNE on the incorporation of [³H]thymidine into DNA of various cultured cells was either stimulatory to dermal cells or inhibitory to a variety of other cell types, including skin epidermal cells. The stimulatory and inhibitory effects of PSNE were abolished by heating PSNE for 5 min in boiling water before its addition to cell cultures. This suggests that macromolecular structure is important in the action of PSNE. This project was supported by a grant from the Research Advisory Board, University of Nevada, Reno, NV.     

NuSerum,a synthetic serum replacement,supports chondrogenesis of embryonic chick limb bud mesenchymal cells in micromass culture

Summary In an effort to establish a more chemically defined culture system to study the regulation of chondrogenic differentiation in vitro, two commercially available serum replacements, NuSerum and NuSerum IV, were tested on embryonic limb mesenchyme. Limb bud (LB) mesenchymal cells were isolated from Hamilton-Hamburger stage 23–24 chick embryos and plated at various densities (1, 5, 10, or 20 × 10⁶ cells/ml) in micromass culture for 4 days in media supplemented with 10% fetal bovine serum (FBS), NuSerum or NuSerum IV. Cell growth was assessed by the incorporation of [³H]leucine and [³H]thymidine. Chondrogenesis was determined by the incorporation of [³⁵S]sulfate and by the number of Alcian blue-staining cartilage nodules. In high density (20 × 10⁶ cells/ml) cultures, which favored chondrogenic differentiation, both serum replacements supported protein synthesis and chondrogenesis equally well as FBS. In cultures plated at 5 × 10⁶ cells/ml, a cell density in which was chondrogenesis-limiting, both NuSerum and NuSerum IV significantly enhanced incorporation of [³⁵S]sulfate (2.6-fold), [³H]leucine (1.4-fold), and [³H]thymidine (1.9-fold), compared to FBS. Enhancement of chondrogenesis was also apparent by the increases in the number of Alcian blue-staining cartilage nodules and the ratio of sulfate: leucine incorporation in cultures plated at 5 × 10⁶ cells/ml. Interestingly, the localization of cartilage nodules was extended out to the periphery of micromass cultures fed with NuSerum or NuSerum IV. The observed effects of NuSerum and NuSerum IV may be attributed to a combination of factors, including lower concentrations of serum and its associated proteins, as well as supplemented growth factors and hormones known to promote cell proliferation and differentiation. Therefore, NuSerum and NuSerum IV are excellent, low-cost replacements for FBS in maintaining cellular growth and promoting chondrogenesis in LB mesenchymal cell cultures in vitro.     

Control mechanism of lymphocyte traffic. A study of the action of two sulfated polysaccharides on the distribution of 51Cr- and [3H]adenosine-labeled mouse lymph node cells

The effects of dextrans of varying molecular weights and of pentosan sulfate on the distribution of ⁵¹Cr-labeled mouse lymph node cells were studied in vivo, i.e., in recipients treated with the sulfated polysaccharides, and in vitro, i.e., by following the fate of cells treated in vitro, in intact syngeneic recipients. Both types of experiments demonstrate that dextrans, especially dextran sulfate (DXS) and pentosan sulfate (PS), considerably reduce lymph node entry of lymphocytes, with concomitant increases in the blood and, in the case of DXS, in both the blood and lungs. A parallel quantitative autoradiographic analysis of the distribution of [³H]adenosine-labeled cells confirmed the data with the ⁵¹Cr-labeled cells and, in adidtion, indicated that DXS and PS slow down circulation of lymphocytes through the marginal zone and red pulp of the spleen and, in the case of DXS, in the pulmonary capillary bed. Unusually large numbers of unlabeled lymphocytes were found in the endothelial wall of the post-capillary venules in lymph nodes of PS-treated mice.     

DNA REPLICATION: DIRECTION AND RATE OF CHAIN GROWTH IN MAMMALIAN CELLS

Using pulse labeling techniques with [³H]thymidine or [³H]cytidine, combined with DNA fiber autoradiography, we have investigated the direction and rate of DNA chain growth in mammalian cells. In general, chain elongation proceeds bidirectionally from the common origin of pairs of adjacent replication sections. This type of replication is noted whether the DNA is labeled first with [³H]thymidine of high specific activity, followed by [³H]thymidine of low specific activity or the sequence is reversed. Approximately one-fifth of the growing points have unique origins and in these replication units, chain growth proceeds in one direction only. Fluorodeoxyuridine and hydroxyurea both inhibit DNA chain propagation. Fluorodeoxyuridine exerts its effect on chain growth within 15–23 min, while the effect of hydroxyurea is evident within 15 min under conditions where the endogenous thymidine pool has been depleted by prior treatment with fluorodeoxyuridine. Puromycin has no effect on chain growth until 60 min after addition of the compound, even though thymidine incorporation is more than 50% reduced within 15 min. After 2 h of treatment with puromycin, the rate of chain growth is reduced by 50%, whereas thymidine incorporation is reduced by 75%. Cycloheximide reduces the rates of DNA chain growth and thymidine incorporation 50% within 15 min, and, on prolonged treatment, the decrease in rate of chain growth generally parallels the reduction in thymidine incorporation.     

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.     

Adenosine stimulation of proliferation of breast carcinoma cell lines: evaluation of the [3H]thymidine assay system and modulatory effects of the cellular microenvironment in vitro

The purine nucleoside adenosine is produced at increased levels in the tissues of solid cancers as a result of local hypoxia. Adenosine inhibits the cell-mediated anti-tumor immune response, promotes tumor cell migration and angiogenesis, and stimulates the proliferation of tumor cells. We examined the stimulatory effect of adenosine on DNA synthesis, cell cycle progression, and cell proliferation in MCF7 and T-47D breast carcinoma cell lines in culture, and identified factors that modulate the growth response. The ability of adenosine to stimulate DNA synthesis, as measured by the incorporation of [(3)H]thymidine, was independent of the total radioactivity of the [(3)H]thymidine up to 10 microCi/ml, total thymidine concentrations up to 100 microM, and the labeling interval. It was also not affected by the presence of low-molecular-weight compounds (such as thymidine and adenosine) in the serum used to supplement the medium. Adenosine stimulated DNA synthesis and cell proliferation with an EC(50) of 4-6 microM and a maximum response at 30-100 microM, when given as a single addition. The stimulatory effect of adenosine involved progression through the cell cycle and a genuine increase in cell number, in the absence of significant apoptotic or necrotic cell death. The mitogenic effect of adenosine was dependent upon the culture cell density, with an optimum adenosine response at around 50% of confluent density. The response was also highly dependent upon the form of the serum addition to the growth medium, with the best response elicited in the presence of low concentrations of nonfetal bovine serum, although adenosine was mitogenic under standard culture conditions. The effects of serum supplementation and cell density were not due to differences in the rate of adenosine metabolism by either serum or cellular enzymes, but appeared to result from changes in the sensitivity to adenosine of the cell population in response to environmental cues. We, therefore, find that adenosine is consistently mitogenic for human breast carcinoma cells, and that the [(3)H]thymidine incorporation assay is a valid measure of this response. The data are consistent with the stimulatory effect of adenosine on cell proliferation being modulated by the local cellular environment.     

[3H]Thymidine Incorporation To Estimate Growth Rates of Anaerobic Bacterial Strains

The incorporation of [³H]thymidine by axenic cultures of anaerobic bacteria was investigated as a means to measure growth. The three fermentative strains and one of the methanogenic strains tested incorporated [³H]thymidine, whereas the sulfate-reducing bacterium and two of the methanogenic bacteria were unable to incorporate [³H]thymidine during growth. It is concluded that the [³H]thymidine incorporation method underestimates bacterial growth in anaerobic environments.