Use of growth-attachment correlation plots to analyse human melanoma cell dependency on 2-oxocarboxylates and serum

Modulation of the population-dependent growth of the human melanoma line MM96 by serum and a series of 2-oxocarboxylates was analysed by a method offering several useful features. Initial cell attachment was measured by the use of cells prelabelled with [14C] thymidine (C) while the size of the replicating cell population on any particular day was monitored by [3H] thymidine (H) incorporation for that particular 24 hr. The 3H incorporation was normalized for the initial cell attachment as the 3H/14C ratio (H/C), which was found to be little affected by artifacts of precursor equilibration. The method allows large numbers of replicate samples so that quantitation of dose-response relationships is simple and statistically robust, and simultaneous monitoring of cell attachment and growth within the same sample is routine. Not only cell growth, but also attachment to the culture vessel surface was found to be increased by higher seeded population densities, by increasing serum concentration and by pyruvate supplementation. Further investigation of the pyruvate effect showed that all nine of the soluble 2-oxocarboxylates tested were effective for both parameters, with little difference between them which was attributable to molecular structure. Quantitative studies of the potentiation of growth and attachment by increasing cell density or a range of concentrations of 2-oxocarboxylates led to the finding that increments in the growth parameter (H/C) were directly proportional to increments in attachment (C). Such a relationship would be unexpected in ordinary logarithmically-growing cultures. The observation is consistent with the hypothesis that increases in initial cell attachment, whether induced by 2 oxocarboxylates or higher inoculation densities, lead to conditioning of the medium by cell factors which stimulate growth and are produced in amounts proportional to the attached cell numbers. Pyruvate, when present with higher serum concentrations, stimulated growth above the levels accounted for by concomitant increases in cell attachment. Serum also appears to have other potentiating factors in addition to those attributable to its content of 2-oxocarboxylates. Differences in the response to 2-oxocarboxylates of human melanoma cells and human diploid fibroblasts were noted, and suggest that the present observations may be due to unique properties of the melanoma cell.     

Nonhaem complexes of FeIII stimulate cell attachment and growth by a mechanism different from that of serum, 2-oxocarboxylates, and haemproteins

Most cell lines, even those producing their own growth factors, need a serum supplement when growing in several commonly used media. The requirement for serum to sustain attachment and growth in RPMI 1640 and MEM has been found to be met by a range of 2-oxocarboxylates, by diverse coordination complexes of FeIII, and by a variety of haem-containing proteins including catalase. The latter directly implicates H2O2 in the serum shift-down effects. H2O2 was found to accumulate in low serum media under normal laboratory lighting conditions to levels that were shown to be sufficient, when added to freshly prepared media, to explain the depressed cell performance. With the exception of some of the nonhaem FeIII coordination complexes, substances found to stimulate cell attachment and growth were capable of scavenging H2O2. This suggests that an important function of serum and the 2-oxocarboxylates (alpha-keto acids) frequently used as "nonessential" medium additives is to remove H2O2 produced photodynamically during the storage and manipulation of media containing a high content of riboflavin. However, the nonhaem FeIII complexes with saturated coordination shells, although capable of reducing photodynamic generation of H2O2 to a greater or lesser extent, have their prime effect by an unknown, intriguing mechanism, probably based on a common redox function.     

LIMITATIONS IN THE USE OF [3H]THYMIDINE INCORPORATION INTO DNA AS AN INDICATOR OF EPIDERMAL KERATINOCYTE PROLIFERATION IN VITRO

The validity of using the incorporation of [³H]thymidine into DNA as an indicator of epidermal keratinocyte proliferation in vitro has been investigated. Other parameters of cell proliferation, direct count of cell number and measurement of DNA content, consistently fail to correlate with changes in [³H]thymidine incorporation into DNA in primary and first passage cultures of rabbit and human epidermal keratinocytes. Maximum incorporation of [³H]thymidine precedes the active growth period by three days. Incorporation declines markedly during the proliferative period. Thymidine kinase activity decreases during the proliferative growth phase. Incorporation of another pyrimidine nucleotide precursor, [¹⁴C]aspartic acid, suggests that in epidermal keratinocytes in vitro the extent of utilization of the salvage and the de novo pathways may be inversely related. In such cases [³H]thymidine incorporation into TCA precipitable material fails to reflect accurately cell proliferation.     

In vitro stimulation of human endothelial cells by derivatized dextrans

Summary Derivatized dextrans exert a stimulatory effect on the in vitro growth of human umbilical vein endothelial cells (HUVEC). Measurements of growth were monitored by [³H]thymidine uptake and cell numbers. Our results show that some derivatized dextrans at 4 μg/ml (88 nM) increase the [³H]thymidine incorporation, whereas starting dextran (40 000 Da), dextran sulfate, and carboxymethyl dextran have no effect. In addition, heparin under similar experimental conditions shows a slight inhibitory effect on the HUVEC growth. The stimulatory effect of derivatized dextrans was also found when HUVEC grew during 7 days in medium containing 2% fetal bovine serum. We also observed that derivatized dextrans had no effect on the mitogenic activity of acidic fibroblast growth factor, a mitogenic factor for several cell types including HUVEC. By assessment of [³H]thymidine uptake at 48 h without serum, we concluded that the exogenous growth factors were not involved in the proliferative activity of these components. The stimulatory effects are related to the chemical nature and the proportion of substituents on the synthetic polysaccharides. The data indicate that benzylamide sulfonated groups play a key role in the stimulation of HUVEC growth. Neither carboxyl nor sulfate groups alone exhibit this effect. Thus, the stimulatory capacity of dextran derivatives depends strongly on the respective ratios of the functional groups.     

Gene transfection of the HuLy-m2 (Leu-9) antigen into mouse L cells

Human DNA was transfected into mouse L cells and tk⁺ HuLy-m2⁺ (= CD7⁺) transfectants isolated after growth in hypoxanthine, aminopterin, thymidine medium and repeated cloning. After several cycles of transfection, > 90% of HuLy-m2⁺ L cells could be detected, by rosetting and by cytofluorography, which showed the transfectants to have a density of CD7 two to five times that found on peripheral blood lymphocytes. Despite this, the 37 kd CD7⁺ dimer could only be identified with difficulty using cell-surface radioiodination and sodium dodecyl sulfate-polyacrylamide gel electrophoresis techniques. An antiserum was produced (C3H anti-HuLy-m2⁺ L cells) which, after absorption, was shown to react with HuLy-m2 antigens present on human thymocytes and lymphocytes and on CD7⁺ transfected L cells.Abbreviations BSA bovine serum albumin - DME Dulbecco's modified Eagle's medium - EDTA ethylenediamine-tetraacetate - HAT hypoxanthine, aminopterin, thymidine - HSV herpes simplex virus - PBL peripheral blood lymphocyte - PBS phosphate-buffered saline - RFC rosette-forming cell - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - tk thymidine kinase     

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.     

Identification and characterization of a soluble suppressor factor(s) in the serum of AKR mice bearing lymphocytic leukemia

Leukemia in AKR mice was found to be associated with the presence of a serum factor(s) termed AKR leukemic suppressor factor (AKR-LSF). Suppression was quantitated by measuring the inhibition of PHA-stimulated [³H]thymidine incorporation by normal AKR spleen cells at various dilutions of leukemic mouse serum (LMS). AKR-LSF activity was expressed as units per milliliter, which is the reciprocal of the LMS dilution that inhibited [³H]thymidine uptake by 50% with respect to fetal calf serum control cultures. The amount of activity in the serum directly correlated to the rate of tumor cell growth. Mice receiving 10⁷ BW5147 transplanted leukemia cells had 130 ± 12 units of AKR-LSF activity/ml of serum compared to 40 ± 8 units/ ml for mice with spontaneous leukemia. Normal mouse serum contained 33 ± 11 units/ml. The leukemic serum exhibited no strain specificity in either phytohemagglutinin or lipopolysaccharide assays, but was found to be twofold more inhibitory against mouse spleen cells than that against rat spleen cells. Human lymphocyte blastogenesis was not inhibited by the leukemic serum. LMS did not inhibit the growth of L929 fibroblasts or murine tumor cells in vitro. Further work is necessary to determine what role the suppressor factor may play in the regulation of antitumor cell immunity.     

4-Borono-2-[18F]fluoro-d,l-phenylalanine: a possible tracer for melanoma diagnosis with PET

The potential of 4-borono-2-[¹⁸F]fluoro-d,l-phenylalanine ([¹⁸F]FBPA), a fluorinated derivative of a target compound for boron neutron capture therapy, for melanoma imaging by positron emission tomography (PET) was studied using animal models. A high uptake of [¹⁸F]FBPA was found in murine B16 melanoma or in Greene's melanoma No. 179, a melanotic cell line in hamsters, for the first 6 h after injection. Whole body autoradiography using [¹⁸F]FBPA gave a clear image of the B16 tumor. The acid-insoluble ¹⁸F in the B16 increased to 27% by 6h, and most of the free ¹⁸F was detected as [¹⁸F]FBPA in both B16 and plasma. In the hamster models, No. 179 showed a 1.7 times higher uptake than amelanotic Greene's melanoma No. 178 at 6 h post-injection, although both melanomas indicated similar metabolic activities when examined by a tracer uptake study using l-[¹⁴C]methionine, 2-deoxy-d-[¹⁴C]glucose and [³H]thymidine. [¹⁸F]FBPA may be a very promising PET tracer for melanoma imaging.     

Differential patterns of mitochondrial,chloroplastic and nuclear DNA synthesis in the synchronous cell cycle of Chlamydomonas reinhardtii

Nuclear DNA (ncDNA) synthesis in Chlamydomonas reinhardtii was measured by both ³²P[or-thophosphoric acid] (³²P) and [¹⁴C]adenine incorporation and found to be highly synchronous. Ca. 85% of incorporation was confined to the first 6 h of the dark period of a synchronized regime consisting of an alternating light-dark period of 12 h each. In contrast, no such synchronous incorporation pattern was found for chloroplast (cp) and mitochondrial (mt) DNAs in the same cell population. These two organellar DNAs also exhibited different ³²P-incorporation patterns in the cell cycle. Considerable amounts of ³²P were incorporated into cpDNA throughout the light-dark synchronous cycle under both mixo- and phototrophic growth conditions, although the second 6-h light period under phototrophy showed an increase not apparent under mixotrophy. This change in growth conditions did not affect ³²P incorporation into mtDNA, which was found throughout the cell cycle, with a modest peak in the first 6-h of the dark period. The pattern of [³H]thymidine incorporation into cpDNA was also determined. Under synchronous phototrophic conditions, this pattern was quite different from that obtained with ³²P. Most [³H]thymidine incorporation occurred during the light period of the synchronous cycle; this period had been shown previously by density transfer experiments to be the time of cpDNA duplication. Such preferential [³H]thymidine incorporation into cpDNA in the light period was not observed under mixotrophic synchronous growth conditions; in these, [³H]thymidine incorporation was detected throughout the cell cycle. This lack of coincidence between the patterns of ³²P- and of [³H]thymidine incorporation into cpDNA during the synchronous cell cycle indicates that in addition to replication, the considerably reiterated organelle-DNA molecules may also regularly undergo an extensive repair process during each cell cycle.     

Growth and differentiation in cultured human thyroid cells: Effects of epidermal growth factor and thyrotropin

Summary Human thyroid cells were grown and subcultured in vitro to examine their responses to known hormones and growth factors, and to serum. The cells were obtained from surgical specimens and were either neoplastic or nonneoplastic. The effects of culture conditions on cell growth were measured by changes in cell numbers and by stimulation of [³H]thymidine incorporation. The results showed that serum (0.5%) was essential for cell proliferation, and that a mixture of insulin (10 μg/ml), transferrin (5 μg/ml), hydrocortisone (10 μg/ml), somatostatin (10 ng/ml), and glycyl-histidyl-lysine (10 ng/ml) enhanced the effect of serum. Maximum growth of the cells was obtained when epidermal growth factor was present at 10⁻⁹ M. Differentiation was measured by production of thyroglobulin, which was found to be stimulated by thyrotropin. This system provides a means to study the hormonal control of growth and differentiation in human thyroid cells. This work was supported by grants from the Medical Research Council of Canada; the Department of Medicine, University of Toronto; and the National Cancer Institute of Canada. J. E. E. is a C.H. Best Foundation and Department of Medicine postdoctoral fellow.     

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.     

Steroid hormone receptor studies in melanoma model systems

The Transplantable B-16 melanotic melanoma carried in syngeneic C57B1/6J female mice and the Syrian hamster melanoma cell line, RPMI 3460, were utilized to determine whether steroid-hormone receptors are present in animal melanomas. In the B-16 melanoma, a cytoplasmic-estrogen receptor is detectable, but there is no evidence for androgen or progestin receptors. Some tumors contain a glucocorticoid-binding macromolecule. Sucrosedensity gradient centrifugation of cytosol after incubation with [³H]-estradiol revealed an 8S peak that was suppressed by excess radioinert diethylstilbesterol. Binding varied from 5–35 fmoles per mg cytosol protein. Scatchard analysis of [³H]-estradiol binding in cytosol yielded a single class of high-affinity binding sites; the dissociation constant is 6 × 10^?10 M. The receptor molecule is shown to be estrogen-specific by ligand competition assays. In contrast to B-16 melanoma, no estrogen, androgen, or progestin receptor can be found in the Syrian hamster melanoma cell line. However, a substantial level of specific binding is observed using [³H]-dexamethasone. Sucrose-gradient centrifugation of cytosol from this cell line after incubation with [³H]-dexamethasone revealed a 7S peak that was suppressed by excess radioinert dexamethasone. Scatchard analysis indicated a single class of high affinity sites with a dissociation constant of 2 × 10^?9 M. Binding levels from 70–610 fmoles per mg cytosol protein were observed. The Syrian hamster melanoma cells also exhibit a biological response to glucocorticoids: Dexamethasone causes both an inhibition of growth and a decrease in final-cell density in these cells.     

Polypeptide inhibitors of DNA synthesis and cellular proliferation produced by cultured BRL-3A rat liver cells

Variants of the NIL8 hamster cell line have been isolated by a procedure that selects against cells that incorporate [³H]thymidine into DNA in response to stimulation with multiplication-stimulating activity (MSA, an insulin-like growth factor produced by cultured BRL-3A rat liver cells). Some of the variant clones proved to be hypersensitive to two inhibitors of [³H]thymidine incorporation that were present in partially purified MSA preparations. The inhibitors have been found to be heat-labile polypeptides. The inhibitors copurify with MSA through ion exchange chromatography on Dowex 50, but are separable from MSA by gel filtration on Sephadex G-75. One of these factors (IN I) has been shown to inhibit DNA synthesis (as determined by autoradiography) and proliferation of the variant cells. Among several other rodent tumor cell lines tested for sensitivity to inhibition by INI, Reuber H-35 rat hepatoma cells and HSV-transformed hamster fibroblasts were not inhibited by the factor, whereas PG19 mouse melanoma cells were slightly inhibited.     

Incorporation and Degradation of C and H-labeled Thymidine by Sugarcane Cells in Suspension Culture

Sugarcane cells growing in suspension culture degrade exogenous thymidine, releasing thymine. Thymine is not utilized for DNA synthesis. Thymine is rapidly catabolized to β-aminoisobutyric acid which is found within the cell. Thymidine in the medium is used for DNA synthesis. The label of [2-¹⁴C]thymidine is lost as ¹⁴CO₂, but the label of [³H]methylthymidine is found in the cell as [³H]β-aminoisobutyric acid, some of which is used for the synthesis of other cell components. The degradation of thymidine can be partially inhibited by addition of certain substituted pyrimidines.     

Lack of effect of butyrate on S phase DNA synthesis despite presence of histone hyperacetylation

The effects of sodium butyrate on [³H]thymidine incorporation and cell growth characteristics in randomly growing and synchronized HeLa S₃ cells have been examined in an attempt to determine what effects, if any, butyrate has on S phase cells. Whereas 5 mM sodium butyrate rapidly inhibits [⁵H]thymidine incorporation in a randomly growing cell populations, it has no effect on incorporation during the S phase in cells synchronized by double thymidine block techniques. This lack of effect does not result from an impaired ability of the S phase cells to take up butyrate, since butyrate administration during this period leads to histone hyperacetylation that is identical with that seen with butyrate treatment of randomly growing cells. Furthermore, the ability to induce such hyperacetylation with butyrate during an apparently normal progression through S phase indicates that histone hyperacetylation probably has no effect on the overall process of DNA replication. Temporal patterns of [³H]thymidine incorporation and cell growth following release from a 24-h exposure to butyrate confirm blockage of cell growth in the G1 phase of the cell cycle. Thus, the inhibition by butyrate of [³H]thymidine incorporation in randomly growing HeLa S₃ cell populations can be accounted for solely on the basis of a G1 phase block, with no inhibitory effects on cells already engaged in DNA synthesis or cells beyond the G1 phase block at the time of butyrate administration.     

Experimental Evaluation of Conversion Factors for the [3H]Thymidine Incorporation Assay of Bacterial Secondary Productivity

The relationship between bacterial growth and incorporation of [methyl-³H]thymidine in oligotrophic lake water cultures was investigated. Prescreening, dilution, and addition of organic and inorganic nutrients were treatments used to prevent bacterivory and stimulate bacterial growth. Growth in unmanipulated samples was estimated through separate measurements of grazing losses. Both bacterial number and biovolume growth responses were measured, and incorporation of [³H]thymidine in both total macromolecules and nucleic acids was assayed. The treatments had significant effects on conversion factors used to relate thymidine incorporation to bacterial growth. Cell number-based factors ranged from 1.1 × 10¹⁸ to 38 × 10¹⁸ cells mol of total thymidine incorporation⁻¹ and varied with treatment up to 10-fold for the same initial bacterial assemblage. In contrast, cell biovolume-based conversion factors were similar for two treatment groups across a 16-fold range of [³H]thymidine incorporation rates: 5.54 × 10¹⁷ μm³ mol of total thymidine incorporation⁻¹ and 15.2 × 10¹⁷ μm³ mol of nucleic acid incorporation⁻¹. Much of the variation in cell number-based conversion factors was related to changes in apparent mean cell volume of produced bacteria. Phosphorus addition stimulated [³H]thymidine incorporation more than it increased bacterial growth, which resulted in low conversion factors.     

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.     

Inhibition of isoproterenol-induced DNA and RNA synthesis in rat parotid and pancreas following removal of submandibular-sublingual glands

Summary [³H] thymidine incorporation into DNA of the parotid (PA) gland of adult and 20-day-old rats and into DNA of the pancreas (PANC) of 20-day-old rats was increased markedly following a 2-day regimen of isoproterenol (ISO) administration. However, when the submandibular-sublingual (SM-SL) glands had been removed just prior to initiation of the ISO injections, the [³H] thymidine incorporation into PA and PANC was inhibited, and cpm/mg protein of these organs was even lower than that of organs of untreated rats with SM-SL glands present. Removal of the PA glands just prior to initiation of the ISO regimen had no effect on the ISO-induced [³H] thymidine incorporation into DNA of PANC but partially inhibited that of the submandibular (SM) gland. It is suggested that the inhibitory effects on DNA and RNA synthesis that follow removal of SM-SL glands are attributable to the growth factors (epidermal growth factor and nerve growth factor) found in the rat SM gland. These factors appear to regulate normal DNA synthetic activity of exocrine glands as well as ₁-adrenoceptor mediated DNA synthesis. Cellular hypertrophy induced by the ISO was less markedly affected by absence of the SM glands, but a partial inhibition of [³H] uridine incorporation into RNA of PA of adult rats also occurred when SM-SL glands were removed prior to initiation of the ISO-regimen.     

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.     

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.