Regulation of the in vitro anamnestic antibody response by cyclic AMP: I. Antigen-induced uptake of nucleotides and nucleosides by lymphocytes

Addition of N⁶, O²′-dibutyryl cAMP (DbcAMP) to keyhole limpet hemocyanin (KLH)-primed rabbit lymph node cells for 1 hr, followed by its removal and the addition of KLH, had no effect on the subsequent antibody response, whereas addition of KLH for 1 hr followed by DbcAMP resulted in a 100% enhancement of antibody synthesis. Addition of cholera enterotoxin (CT), which rapidly and irreversibly binds to lymphocytes and activates adenylate cyclase, either before or after the addition of antigen, elevated the antibody response by 100%. These results suggested that some antigen-induced event(s) may be required for DbcAMP to exert its enhancing effects on the antibody response. The effect of KLH on the uptake of DbcAMP by KLH-primed lymph node cells was investigated. One and one hundred micrograms of KLH, which induce optimal and supraoptimal antibody synthesis, respectively, promoted maximal uptake of DbcAMP. This induced uptake was first detectable about 12 hr after addition of KLH, and it peaked during 24–48 hr of culture. DbcAMP uptake induced by a brief exposure of KLH (0–1 hr) was equivalent to that observed with long-term KLH addition (0–24 hr). KLH-induced DbcAMP uptake required KLH-reactive lymphocytes and represented active transport. Antibody to rabbit T lymphocytes inhibited this antigen-induced uptake. The mitogens concanavalin A (Con A) (T cells) and goat anti-rabbit Fab' (anti-Fab') (B cells) also stimulated DbcAMP uptake, as did human serum albumin (HSA) and myoglobulin (Mb) when added to homologously primed cells, indicating the generality of the phenomenon. [³H]DbcAMP entered the cells as di- or monobutyryl cAMP with about 40% metabolized to 5′AMP. This uptake could be competitively inhibited by other adenine or guanine nucleotides and nucleosides.     

Evidence for cellular but not serologic cross-reactivity between keyhole limpet hemocyanin and sperm-whale myoglobin.

The addition of keyhole limpet hemocyanin (KLH) to cultures of rabbit lymph node cells (LNC) primed with KLH and sperm-whale myoglobin (Mb) induced the synthesis of antibody to Mb as well as to KLH. Several mechanisms for this heterologous induction were considered. It was established that KLH does not nonspecifically activate rabbit T or B lymphocytes. It was also shown that KLH and Mb do not cross-react serologically by several sensitive and specific criteria. Therefore, it was surmised that heterologous induction of Mb antibody synthesis by KLH was due to cellular cross-reactivity between these proteins. Rabbits were primed by the injection of Mb-complete Freund's adjuvant (CFA), alum-Mb, or alum-KLH, and their LNC challenged with KLH, Mb, and synthetic antigenic sites of Mb. These experiments yielded much and diverse evidence for cellular cross-reactivity between KLH and Mb, and especially between KLH and the Mb peptides: KLH plus Mb-primed LNC evoked enhanced anti-KLH and anti-Mb syntheses. KLH plus KLH-sensitized LNC resulted in a lowered anti-Mb antibody response. Mb added to Mb-educated LNC either enhanced or inhibited the anti-KLH antibody response, depending on whether the priming adjuvant was CFA or alum. The addition of Mb to KLH-primed cells enhanced or inhibited the ensuing anti-Mb antibody synthesis; KLH did not affect or inhibit anti-KLH antibody synthesis. Addition of synthetic Mb antigenic sites to Mb-sensitized LNC elevated or suppressed anti-KLH antibody production, depending on the length of time between priming and in vitro challenge. A mixture of KLH and Mb peptide lowered the anti-Mb antibody response of Mb-educated LNC compared to KLH alone. A combination of KLH and Mb peptide also reduced the anti-KLH antibody synthesis of KLH-primed cells compared to KLH per se. The addition of KLH to Mb-sensitized LNC enhanced their uptake of tritiated thymidine, and their transport of tritiated cyclic AMP and protein synthesis. Added Mb induced the synthesis of protein and nonspecific IgG by KLH-primed LNC; Mb peptides evoked protein synthesis by these cells. It is postulated that cross-reactivity at the T-cell level is responsible for the induction of Mb antibody synthesis by adding KLH to either Mb-primed or KLH/Mb-primed LNC. The implications of these findings with respect to cellular and humoral immunity are discussed.     

Double labelling of tissue combining tritiated thymidine autoradiography with immunodetection of bromodeoxyuridine: the autoradiographic significance of inhibition of thymidine incorporation into DNA by bromodeoxyuridine given simultaneously

We describe a reproducible method for combining tritiated thymidine ([H]TdR) autoradiography with immunoperoxidase detection of bromodeoxyuridine (BrdU) in paraffin-embedded tissues. The technique has been used to examine, in mouse tongue epithelium, the inhibition of incorporation into DNA of [3H]TdR by a simultaneous injection of BrdU in the doses that both compounds are likely to be used in cell proliferation studies. The significance that this inhibition has on prolongation of autoradiograph exposure times, to ensure that all cells that incorporate [3H]TdR are scored as positive, in particular the most lightly labelled cells, has been quantified. The inhibition of uptake into DNA of [3H]TdR from 0.23 to 1.85 MBq (6.25 to 50 mu Ci) per animal, produced by a simultaneous injection of 2.5 mg BrdU shows a linear, dose-dependent relationship. Provided the injected dose (in mu Ci per animal) multiplied by the autoradiographic exposure time (in days) is greater than a value of 700, then all cells that are labelled after incorporation of [3H]TdR alone are also labelled after simultaneous double labelling, despite the latter producing a lower average grain count.     

In vitro anamnestic immune responses and modulating factors

Summary The term anamnestic refers to the specific and enhanced immune responses of antigen-immunized (primed) lymphoid memory cells to secondary challenge with a foreign substance (antigen). These responses include the accelerated and quantitatively greater syntheses of antibody and other macromolecules than upon primary challenge of such cells. Rabbits were primarily immunized with keyhole limpet hemocyanin (KLH). Six days later their memory lymph node cells (LNC) were removed, and upon culture with KLH, responded with the synthesis of antibody, immunoglobulin (Ig), protein, DNA and RNA, as well as with active transport of dibutryl cyclic AMP (DbcAMP). Purified thymus-derived (T) LNC were prepared on anti-rabbit Ig affinity columns. Bursal-equivalent (B) cells were prepared by binding to a complex of sheep erythrocytes (SRBC)-antibody to SRBC-complement and centrifugation of these complexes on suitable gradients. When these T and B KLH-primed LNC were mixed and challenged with KLH the aforementioned macromolecular syntheses and active transport occurred. Indeed, by a variety of criteria, the reconstituted anamnestic immune responses were indistinguishable from these responses of unfractionated LNC. Antigenic stimulation of KLH-primed T cells induced the synthesis of proteins and DNA, but not antibody, but antigenic challenge of KLH-primed B cells did not evoke these syntheses. However, added KLH induced a mixture of T and B antigen-primed LNC to synthesize more protein, Ig, DNA than either population alone and more antibody than T cells per se; B cells required help for all of these responses. The thymus (T) cell-dependent phase of in vitro anamnestic antibody response lasted the first 24–36 hr.The antibody response was regulated by antigen-concentration. One g KLH evoked maximal antibody synthesis, 10 and 100 g KLH much less. Challenge of the separated T and B cell populations with different KLH concentrations, followed by recombination and eventual assay of antibody synthesis revealed different optima. The optimal concentration for T cell help was 0.01–0.1 g KLH; higher amounts induced much less antibody production. The optimum for B cells was 1–10 g KLH; 100 g inhibited antibody formation.The antibody response to KLH and human serum albumin (HSA) was regulated nonspecifically utilizing LNC from rabbits immunized simultaneously with these two antigens. Thus stimulation of LNC from these rabbits with either antigen induced the synthesis of antibodies to both antigens. HSA and KLH did not cross-react either serologically or cellularly. Cross-stimulation of antibody synthesis also was observed when rabbit LNC were primed with KLH and Mb. However, in this instance, cross-reaction between KLH and sperm-whale myoglobulin (Mb) was observed at the cellular, presumably the T cell, level, although not at the antibody (B cell) level. The antibody response could also be modulated by exogenous cholera enterotoxin (CT), dibutyryl cyclic AMP (DbcAMP) and prostaglandins of the E series. The addition of each substance together with 1–100 g KLH to KLH-primed LNC enhanced the antibody response many-fold. CT-induced non-immunized LNC to produce soluble factor(s) (SF) which, when added to KLH-primed LNC together with KLH, enhanced antibody synthesis significantly. The addition of Indomethacin, an inhibitor of PGE synthesis to KLH-immunized cells together with KLH inhibited antibody production, suggesting that PGE was involved in this response. Evidence was adduced that neither cyclic AMP nor PGE was required for the antibody response: Ca²⁺ was not required for induction of this response by KLH, but only its regulation by cAMP.Moreover, when KLH-primed LNC were fractionated on Nylon columns, the effluent cells were induced by KLH to synthesize antibody, but this synthesis was not enhanced by added DbcAMP or PGE; presumably, regulatory cells were removed on the column. Added KLH induced PGE synthsis in these cultures; this synthesis required macrophages. In all of the LNC cultures — including cultures from rabbits immunized with KLH, HSA, and MB months or a year earlier — much antibody synthesis occurred even when antigen was not added to the cultures. This spontaneous antibody was anamnestic, thymus (T cell)-dependent and involved the interaction of residual immunogen on dendritic cells with T and B memory cells. This spontaneous antibody response provides a model for the study of the factors involved in the longterm maintenance of humoral immunity.Mb was employed as a source of more refined antigenic determinants. Rabbits were immunized with Mb in complete Freunds adjuvant. The addition of small synthetic peptides corresponding to the five antigenic sites of Mb to the Mb-primed LNC induced the synthesis of antibody, Ig, protein, DNA, RNA, and macrophage migration inhibitory factor (MIF). The N terminal 1–6 peptide, which is not antigenic, i.e. does not combine with antibody to Mb, also induced all of these syntheses, except MIF. These peptide-induced responses appeared to be thymus-dependent.Abbreviations AP alum-precipitated - AFab goat IgG antibody to rabbit Fab - ATG goat IgG antibody to rabbit thymocytes - BGG bovine gamma globulin - Bsa bovine serum albumin - BAC bromo acetyl cellulose - B bursalequivalent lymphocytes - CT cholera enterotoxin - CRL complement receptor lymphocytes - DFA complete Freund's adjuvant-, - cAMP adenosine 3:5-cyclic monophosphate - cGMP guanosine 3:5-cyclic monophosphate - DbcAMP N⁶,O²-dibutryl cyclic AMP - EAC sheep erythrocytes sensitized with antibody and complement - FITC fluorescein isothiocyanate - HSA human serum albumin - KLH keyhole limpet hemocyanin - LNC lymph node cells - MEM minimum essectial Eagle's medium - medium; MIF m crophage migration inhibitory factor - Mb sperm-whale myoglobin - PHA phytohemagglutinin - PGE prostaglandins of the E series - PGF prostaglandins of the F series - PGSI inhibitors of prostaglandin systhesis - Slg surface immunoglobulin - T thymus-derived lymphocytes     

Measuring bacterial production via rate of incorporation of [3H]thymidine into DNA

Thymidine (TdR) incorporation into DNA as a measure of bacterial production in environmental samples relies on assumptions about what organisms incorporate exogenous thymidine, extent of dilution of labelled thymidine by internal and external pools, and analytical methods for recovery and purification of bacterial DNA. We have examined these assumptions with regard to the feasibility of using [³H]TdR incorporation in the water column and sediments of a blackwater river. The extent of dilution of added [³H]TdR may be determined with isotope dilution plots (Moriarty and Pollard, 1981 and 1982) and these indicate a wide range of degree of participation of added [³H]TdR. Previously described methods for extracting DNA from sediment bacteria may lead to underestimates and we described a more efficient recovery scheme.     

Effect of protease inhibitors on mitogen stimulation of hamster lymphoid cells

Hamster lymph node and spleen cells can be stimulated to incorporate tritiated thymidine ([³H]TdR) in vitro under serum-free conditions by the proteases trypsin and chymotrypsin. Under similar conditions, thymocytes could be stimulated by concanavalin A (ConA) but not lipopolysaccharide (LPS) or the proteases. The subpopulation of cells responding to the proteases correlated with the cells responding to LPS on fractionation of spleen and lymph node cells on discontinuous bovine serum albumin (BSA) gradients or on nylon-wool columns. The stimulation induced by trypsin was completely blocked by soybean trypsin inhibitor (SBTI) while that induced by chymotrypsin was only partially blocked. The inhibition by SBTI of protease activation was not effective when added 24 h after initiation of stimulation. On the other hand, addition of clarified isologous serum to protease activated cultures after 24 h still lead to greater than 50% inhibition of [³H]TdR incorporation.     

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)     

Regulation of the in vitro anamnestic antibody response by cyclic AMP. III. Cholera enterotoxin induces lymph node cells to release soluble factor(s) which enhance(s) antibody synthesis by antigen-treated lymph node cells.

Unprimed or KLH-primed rabbit lymph node cells were pulsed with cholera enterotoxin or KLH for 2 hr and washed. KLH-treated LNC were mixed with equal numbers of CT-treated LNC or boiled CT-treated LNC. Cocultivation of CT-treated LNC with KLH-treated cells resulted in at least a 100% increase in antibody synthesis compared to control cultures. Delaying cocultivation for 24 hr reduced enhancement to 25%. Thus it appears that an early event—before 24 hr—is involved in CT enhancement. Using ¹²⁵I-CT, it was shown that these effects were not due to CT carry-over. When KLH- and CT-pulsed LNC were cultured in chambers separated by polycarbonate membranes (0.2- to 0.4-μm pore size) antibody production was enhanced 50–80%. Supernates of CT-treated LNC also enhanced antibody production by KLH-treated LNC. These results suggest that CT triggers the release of soluble factor(s) which enhance(s) antibody synthesis by antigen-primed and antigen-challenged LNC.     

Different Sensitivities of Granulocyte-Macrophage and Erythroid Progenitor Cells of Normal Human Bone Marrow to S-Phase-Specific Agents

The extraordinary sensitivity of early erythroid progenitor cells (BFU-e) of normal human bone marrow to tritiated thymidine ([³H]TdR) was studied. While exposure of bone-marrow cells to [³H]TdR for 1 hr resulted in the death of only 40% of the granulocyte-macrophage progenitor cells (CFU-c), 90% of BFU-e were killed. Experiments in which normal bone-marrow cells were mixed with bone-marrow cells which had been exposed to [³H]TdR demonstrated that the excessive killing of BFU-e by [³H]TdR reflected carry-over of the [³H]TdR by the exposed cells. A carry-over effect was not observed for CFU-c, suggesting the presence of a fundamental difference in the metabolism of TdR between CFU-c and BFU-e. There was a suggestion of a carry-over effect regarding two other S-phase-specific agents, hydroxyurea and 1-β-D-arabinofuranosylcytosine.     

Double labelling of tissue combining tritiated thymidine autoradiography with immunodetection of bromodeoxyuridine: the autoradiographic significance of inhibition of thymidine incorporation into DNA by bromodeoxyuridine given simultaneously

Abstract We describe a reproducible method for combining tritiated thymidine ([³H]TdR) autoradiography with immunoperoxidase detection of bromodeoxyuridine (BrdU) in paraffin-embedded tissues. The technique has been used to examine, in mouse tongue epithelium, the inhibition of incorporation into DNA of [³H]TdR by a simultaneous injection of BrdU in the doses that both compounds are likely to be used in cell proliferation studies. The significance that this inhibition has on prolongation of autoradiograph exposure times, to ensure that all cells that incorporate [³H]TdR are scored as positive, in particular the most lightly labelled cells, has been quantified.
The inhibition of uptake into DNA of [³H]TdR from 0.23 to 1.85 MBq (6.25 to 50 μCi) per animal, produced by a simultaneous injection of 2.5 mg BrdU shows a linear, dose-dependent relationship. Provided the injected dose (in μCi per animal) multiplied by the autoradiographic exposure time (in days) is greater than a value of 700, then all cells that are labelled after incorporation of [³H]TdR alone are also labelled after simultaneous double labelling, despite the latter producing a lower average grain count.     

Inorganic Phosphorus Stimulation of Bacterioplankton Production in a Meso-Eutrophic Lake

Experiments were conducted to determine whether production of heterotrophic bacterioplankton in a small meso-eutrophic lake was influenced by the dissolved inorganic phosphorus (DIP) supply. DIP may indirectly limit bacterial production by limiting phytoplankton, which in turn may limit the carbon available to bacteria. Direct DIP limitation of bacteria occurs where the availability of DIP for bacteria is insufficient to maintain growth. This work examined direct DIP limitation of bacteria by removing phytoplankton and incubating flasks with or without added P in the dark. Bacterial production was measured via the rate of incorporation of [³H]thymidine ([³H]TdR) into DNA. Bacterial abundance was followed with epifluorescent direct counts. Rates of [³H]TdR incorporation were significantly greater in flasks with added DIP, and changes in cell abundances generally paralleled increases in [³H]TdR incorporation. Even very small additions of P (0.05 μM) were sufficient to stimulate production. DIP addition to whole lakewater also stimulated [³H]TdR incorporation relative to that in zero-addition controls, but there was not a concurrent increase in bacterial cell numbers. The stimulation of [³H]TdR incorporation after DIP addition to whole lakewater was significantly less than the stimulation due to DIP addition to 1-μm-pore-size-filtered lakewater. In this study, addition of DIP caused as much as an eightfold stimulation of [³H]TdR incorporation.     

DIFFERENT LABELLING PATTERNS IN MOUSE LYMPHOID TISSUES WITH [3H]DEOXYCYTIDINE AND [3H]THYMIDINE

The percentages of labelled lymphocytes in smear preparations of mouse thymus were higher than those in similar preparations of mesenteric lymph nodes with either generally labelled tritiated deoxycytidine, [³H]CdR, or tritiated thymidine, [³H]TdR. Lymphocytes in the thymus cortex and in germinal centres of mesenteric lymph nodes were intensely labelled with [³H]CdR, whereas with [³H]TdR lymphocytes in the peripheral region of thymus and medullary cords of mesenteric lymph nodes were heavily labelled. The majority of lymphocytes in thymic cortex and germinal centres of mesenteric lymph nodes were labelled weakly with [³H]TdR. Thus, labelling patterns with [³H]CdR differed from those with [³H]TdR in lymphoid tissues of the mouse. Mouse lymphocytes can utilize [³H]CdR as a precursor molecule for cytosine and thymine in DNA. The ratio of radioactivity of thymine to that of cytosine was measured biochemically in DNA extracted from lymphocytes labelled with [³H]CdR. This radioactivity ratio in thymus was higher than that in mesenteric lymph nodes. These results suggest that the metabolic activities of utilizing CdR for DNA synthesis differ within lymphocyte populations in various lymphoid tissues in the mouse.     

Synchronization of mouse 3T3 and SV40 3T3 cells by way of centrifugal elutriation

Populations of G1 phase 3T3 and SV40 3T3 mouse fibroblasts have been isolated from exponentially growing cultures by the technique of centrifugal elutriation. Return of the G1 phase cells to growth conditions results in their synchronous passage through the cell cycle, as determined from monitoring of cell number, [³H]thymidine ([³H]TdR) incorporation and fraction of [³H]TdR labeled nuclei. The durations of G1, S and G2 phases are consistent with values obtained by previous investigators using conventional induction techniques for synchronization. The method for isolation of the G1 phase cells is rapid, the yield is high and the process does not appear to alter the temporal aspects of the cell cycle in either cell type.     

Regulation of the in vitro anamnestic antibody response by cyclic AMP. IV. Evidence for participation of prostaglandins of the E series in the early events.

The addition of KLH to KLH-primed rabbit lymph node cell cultures induced an anamnestic antibody response. The further addition of prostaglandins of the E series, but not PGF1^α, enhanced this antibody response manifold. The addition to these cultures of prostaglandin synthetase inhibitors together with KLH inhibited antibody production. At the concentration (10^?4) required to inhibit antibody synthesis, by a variety of criteria one of these inhibitors, indomethacin, was shown not to exert its effects through cytotoxicity. By contrast, two other inhibitors of prostaglandin synthesis, Ro-20-5720 and Ro-3-1314, inhibited antibody synthesis because of their cytotoxicity. The inhibition of the antibody response by indomethacin did not occur when PGE1 or PGE2 was added concurrently to these cultures, clearly showing that inhibition was due to a deficiency of prostaglandins. These findings strongly suggest that induction and/or regulation of the in vitro anamnestic antibody response of KLH-primed lymph node cells to 1 and 100 μg KLH requires continued prostaglandin synthesis. Potential mechanisms for the regulation of the antibody response by prostaglandins are discussed.     

SYNCHRONIZATION OF MITOCHONDRIAL DNA SYNTHESIS IN CHINESE HAMSTER CELLS (LINE CHO) DEPRIVED OF ISOLEUCINE

Mitochondrial DNA (mit-DNA) synthesis was compared in suspension cultures of Chinese hamster cells (line CHO) whose cell cycle events had been synchronized by isoleucine deprivation or mitotic selection. At hourly intervals during cell cycle progression, synchronized cells were exposed to tritiated thymidine ([³H]TdR), homogenized, and nuclei and mitochondria isolated by differential centrifugation. Mit-DNA and nuclear DNA were isolated and incorporation of radioisotope measured as counts per minute ([³H]TdR) per microgram DNA. Mit-DNA synthesis in cells synchronized by mitotic selection began after 4 h and continued for approximately 9 h. This time-course pattern resembled that of nuclear DNA synthesis. In contrast, mit-DNA synthesis in cells synchronized by isoleucine deprivation did not begin until 9–12 h after addition of isoleucine and virtually all [³H]TdR was incorporated during a 3-h interval. We have concluded from these results that mit-DNA synthesis is inhibited in CHO cells which are arrested in G₁ because of isoleucine deprivation and that addition of isoleucine stimulates synchronous synthesis of mit-DNA. We believe this method of synchronizing mit-DNA synthesis may be of value in studies of factors which regulate synthesis of mit-DNA.     

LOW LEVEL INCORPORATION OF TRITIATED THYMIDINE INTO THE NUCLEAR DNA OF PURKINJE NEURONS OF ADULT MICE

Adult mice were pulse labeled with tritiated thymidine [³H]TdR and killed 9 hr later. A low level incorporation of [³H]TdR into the nuclear DNA of Purkinje neurons was found in autoradiographs. Enzymatic digestions with DNase and with RNase in combination with autoradiographic grain counts indicate that a portion of nuclear DNA is not stable in the Purkinje nucleus. These results are discussed in light of reports of the stable nature of DNA in Purkinje neurons of adult mice.     

Endogenous Regulation of Endothelial Cell Proliferation

Bovine aortic endothelial cells (BAEC) in culture have the ability to regulate their own proliferation. We have found that a fraction below 100,000 daltons obtained from the media of confluent cultures of BAEC inhibits tritiated thymidine [³H]TdR incorporation as well as their proliferation. the inhibition is dose- and time-dependent; maximum inhibition of [³H]TdR incorporation occurs 8 hr after cells are released from synchronization and the inhibitory fraction is added. Inhibition is evident at concentrations as low as 50 μg/ml and reaches a maximum at 600 μg/ml. the blockage of [³H]TdR incorporation is reflected in the inhibition of cell proliferation. In the presence of 400 μg of endogenous inhibitor per ml of media, added at the time of plating, the average population doubling time increases from 19 to 41 hr. These findings indicate that, in culture, BAEC can regulate their own proliferation by synthesizing an endogenous inhibitor(s) of proliferation.     

An anti-HLA class I monoclonal antibody alters the progression in the cell cycle of phytohemagglutinin-activated human T lymphocytes

The monomorphic anti-HLA Class I monoclonal antibody 01.65 inhibits the incorporation of tritiated thymidine ([3H]TdR) in Phytohemagglutinin (PHA)-activated human T lymphocytes. Our data indicate that 01.65 affects the average duration of the cell cycle by increasing the length of the early S subphase. As a consequence of the increase in the doubling time of the cell population, the absolute number of cells at harvesting time was reduced in 01.65-treated cultures compared to that of untreated cultures. The lengthening of the S-phase and the decrease in the cell number can together quantitatively account for the reduction of [3H]TdR incorporation observed in 01.65-treated cultures.     

Augmentation by thymidine of the incorporation and distribution of 5-fluorouracil in ribosomal RNA.

The effect of high dose thymidine (TdR) pretreatment on the action of 5-fluorouracil (5-FU) on rRNA was measured in regenerating liver after partial hepatectomy. TdR did not enhance the inhibitory or non-inhibitory effects of 5-FU on the synthesis of rRNA and poly(A)RNA, respectively. However, TdR pretreatment intensified the effect of 5-FU on interference with the processing of rRNA from precursor rRNA. The distribution of [³H]5FU, as well as [³H]-orotic acid was enhanced in the 2′-0-methylated sequences of rRNA following pretreatment with TdR. Thus, the combination regimen of TdR plus 5-FU resulted in enhanced interference with the processing of rRNA.     

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.