A KINETIC ANALYSIS OF MYOGENESIS IN VITRO

Conditions which yielded reproducible growth kinetics with extensive, relatively synchronous differentiation are described for chick muscle cultures. The effects of cell density and medium changes on the timing of cell fusion were examined. Low-density cultures which received a change of medium at 24 hr after plating show the highest rate of cell fusion, increasing from 15 to 80% fused cells in a 10 hr period. These optimal culture conditions were employed to reexamine two questions from the earlier literature on muscle culture: (a) can cells which normally would fuse at the end of one cell cycle be forced to go through another cell cycle before fusion; and (b) how soon after its final S period can a cell complete fusion? In answer to the first question, it was found that if the medium is changed, many cells which would otherwise fuse can be made to undergo another cell cycle before fusion. In the second case, radioautographs were made from cultures incubated with tritiated thymidine for various times at the beginning of the fusion period. These show labeled nuclei in myotubes as early as 3 hr after the beginning of the incubation period. This indicates that cells can fuse as early as the beginning of the G₁ period, and suggests that there is not an obligatory exit from the cell cycle or a prolonged G₁ period before cell fusion and differentiation during myogenesis.     

Cell Cycle Kinetics of Aerated, Hypoxic and Re-Aerated Cells In Vitro Using Flow Cytometric Determination of Cellular Dna and Incorporated Bromodeoxyuridine

Abstract. The effects of extreme hypoxia on cell cycle progression were studied by simultaneous determination of DNA and bromodeoxyuridine (BrdU) contents of individual cells. V79-379A cells were pulse-labelled with BrdU (1 μM, 20 min, 37°C) and then incubated for up to 12 hr in BrdU-free medium under either aerated or extremely hypoxic conditions. After the incubation interval (0-12 hr), the cells were trypsinized and fixed in 50% EtOH. Propidium iodide and a fluorescein-labelled monoclonal antibody to BrdU were then used to quantify DNA content and incorporated BrdU, respectively. Measurements in individual cells were made by simultaneous detection of green and red fluorescence upon excitation at 488 nm using flow cytometry. Bivariate analysis revealed progression of BrdU-labelled cells in aerated cultures out of S phase, into G₂ and cell division, with halving of mean fluorescence, and back into S phase by approximately 9 hr after the BrdU pulse. Hypoxia immediately arrested cells in all phases of the cell cycle. Both the DNA distribution and the bivariate profile of cells that were fixed from 2 to 12 hr after induction of hypoxia were identical to the 0 hr controls. the percent of cells with green fluorescence in a mid-S phase window remained 100% and the mean fluorescence of these cells remained at control (0 hr) levels. This indicates that, under hypoxic conditions, cells were moving neither into nor out of S phase. Cultures that had been hypoxic for 12 hr exhibited an increasing rate of BrdU uptake with time after re-aeration. Re-aerated cells were able to complete or initiate DNA synthesis, but their rates of progression through the cell cycle were markedly reduced. A large fraction of cells appeared unable to divide up to 12 hr following release from hypoxia.     

Induction of erythroid differentiation in Friend leukemia cells by bromodeoxyuridine

Treatment of Friend leukemia cells with BrdU, the thymidine analog which interferes with DMSO induced differentiation in these cells as well as the expression of differentiated character in many other cell systems, is capable of inducing erythroid differentiation. Globin mRNA, as assayed by hybridization to globin cDNA, increases 2.5- to 30-fold after appropriate treatment with BrdU. This effect was observed with several different subclones of three independent Friend tumor cell lines. After BrdU treatment, globin mRNA content may reach up to 10-20% of the levels in DMSO induced cultures. The induction of erythroid differentiation is also apparent when accumulated heme content or the appearance of benzidine positive cells is monitored. One Friend cell line (745) we examined was not induced by BrdU although it incorporated an amount of BrdU into its DNA comparable to that incorporated by the other cell lines. In addition, BrdU did interfere with DMSO induction in this cell line. These results suggest that two different mechanisms may be operative in regulating erythroid differentiation in Friend leukemia cells. While BrdU interferes with the mechanism activated by DMSO treatment, this analog could independently activate an alternative mechanism.     

Cell cycle kinetics of aerated, hypoxic and re-aerated cells in vitro using flow cytometric determination of cellular DNA and incorporated bromodeoxyuridine

The effects of extreme hypoxia on cell cycle progression were studied by simultaneous determination of DNA and bromodeoxyuridine (BrdU) contents of individual cells. V79-379A cells were pulse-labelled with BrdU (1 microM, 20 min, 37 degrees C) and then incubated for up to 12 hr in BrdU-free medium under either aerated or extremely hypoxic conditions. After the incubation interval (0-12 hr), the cells were trypsinized and fixed in 50% EtOH. Propidium iodide and a fluorescein-labelled monoclonal antibody to BrdU were then used to quantify DNA content and incorporated BrdU, respectively. Measurements in individual cells were made by simultaneous detection of green and red fluorescence upon excitation at 488 nm using flow cytometry. Bivariate analysis revealed progression of BrdU-labelled cells in aerated cultures out of S phase, into G2 and cell division, with halving of mean fluorescence, and back into S phase by approximately 9 hr after the BrdU pulse. Hypoxia immediately arrested cells in all phases of the cell cycle. Both the DNA distribution and the bivariate profile of cells that were fixed from 2 to 12 hr after induction of hypoxia were identical to the 0 hr controls. The percent of cells with green fluorescence in a mid-S phase window remained 100% and the mean fluorescence of these cells remained at control (0 hr) levels. This indicates that, under hypoxic conditions, cells were moving neither into nor out of S phase. Cultures that had been hypoxic for 12 hr exhibited an increasing rate of BrdU uptake with time after re-aeration. Re-aerated cells were able to complete or initiate DNA synthesis, but their rates of progression through the cell cycle were markedly reduced. A large fraction of cells appeared unable to divide up to 12 hr following release from hypoxia.     

Effect of Penicillin on the Multiplication of Meningopneumonitis Organisms (Chlamydia psittaci)

Although formation of infectious particles of meningopneumonitis organism in L cells was completely inhibited by 1 or more units of penicillin per ml, multiplication of reticulate bodies was observed, by light microscopy, in the presence of 200 units of penicillin per ml in stained smears of infected cells. When reticulate bodies were purified from cultures containing penicillin after 18, 30, and 45 hr of incubation, continuously increasing yields were obtained. When penicillin was added to infected cultures 0 to 15 hr after infection, no increase in infectivity was observed at 40 hr, but when antibiotic was added between 20 and 35 hr, partial synthesis of infectious particles was observed at 40 hr. On the other hand, removal of penicillin from an infected culture before 15 hr after infection did not affect the final yields of infectivity when assayed at 40 hr, but elimination of penicillin after 20 hr resulted in a decrease in infectivity. In suspensions of (32)P-labeled purified reticulate bodies grown in cultures containing penicillin and harvested 18 and 40 hr after infection, the (32)P distributions obtained by acid fractionation were similar to those of reticulate bodies from penicillin-free cultures. Cell membranes of reticulate bodies were also prepared from 40-hr cultures with penicillin. The size and shape of purified membranes, as seen by electron microscopy, and their amino acid compositions were similar to membranes prepared from reticulate bodies grown without penicillin, except that very small structures were observed in membranes from cultures containing penicillin. These results indicated that penicillin does not inhibit reproduction of reticulate bodies and formation of their cell membranes, but does inhibit the formation of elementary body cell envelopes.     

Perturbation of growth and differentiation of Friend murine erythroleukemia cells by 5-bromodeoxyuridine incorporation in early S phase.

Cultured Friend murine erythroleukemia cells (Friend cells) are induced to undergo erythroid differentiation when grown in the presence of dimethylsulfoxide (DMSO) and other compounds. The effects of unifilar substitution of bromouracil (BU) for thymidine in the DNA (BU-DNA) of Friend cells were examined. Cells were grown in the presence of 5-bromodeoxy-uridine (BrdU) for one generation, then centrifuged and resuspended in medium containing DMSO without BrdU. These cells exhibited a delay in the appearance of heme-producing, benzidine-reative (B+) cells and a decreased rate of cell proliferation in comparison to the control not containing BU-DNA. A transient inhibition of entry into S phase was observed when control cells or cells containing BU-DNA were grown in the presence of DMSO) for 10 to 20 hours. This transient inhibition was increased in the BrdU culture. Thus BU-substitution in Friend cells alters other cellular functions in addition to erythroid differentiation. The rate of increase in the percent of cells committed to differentiate (those forming B+ colonies in plasma clots) was similar in the BrdU and control cultures until 40 to 50 hours. After this time, a delay in the appearance of committed cells was observed in the BrdU culture. The effect of BrdU on the appearance of B+ cells was more pronounced and occurred earlier than its effect on the rate of commitment. Therefore, the delay in the appearance of B+ cells in the BrdU culture was due primarily to perturbation of post-commitment events such as the accumulation of hemoglobin. We also examined the effect on growth and differentiation after BrdU was incorporated during different intervals of S phase in cells synchronized by centrifugal elutriation or by double thymidine block and hydroxyurea treatment. The delay in the appearance of B+ cells and inhibition of cell proliferation were only observed when BrdU was incorporated in the first half of S phase. BrdU (10 muM) had no effect on growth or differentiation when present during late S or G1 and G2. These results, using two very different methods to achieve cell synchrony, indicate that the effects of BrdU on growth and differentiation described above are due to its incorporation into DNA sequences replicating during early S.     

High resolution analysis of cell cycle-correlated vimentin expression in asynchronously grown,TPA-treated MPC-11 cells by the novel flow cytometric multiparameter BrdU-hoechst/PI and immunolabeling technique

High resolution, multiparameter analysis using the flow cytometric BrdU/Hoechst quenching technique has been applied to study cell cycle kinetics and vimentin expression in individual cells of asynchronously grown MPC-11 mouse plasmacytoma cell cultures treated with 12-O-tetradecanoylphorbol-13-acetate (TPA) to induce in vitro differentiation. BrdU treatment up to 16 h in the absence or presence of TPA did not affect either cell cycle progression or the kinetics or quantity of vimentin expression. TPA-treated cells became arrested in G1 phase of the second cell cycle; however, this G1 phase arrest was transient only. In addition, G1 phase cells located prior to a putative transition point at the beginning of TPA treatment were completely blocked in cell cycle progression. There is also evidence that cells located in G1 or G2/M phase at the beginning of TPA treatment finally expressed low levels of vimentin. On the contrary, cells located in S phase at TPA exposure showed high vimentin levels after treatment. The results presented here show that, with the flow cytometric BrdU/Hoechst quenching technique, one can correlate time-dependent protein expression at the single cell level in asynchronously grown cultures not only with the actual cell cycle state, but also with the history of cell replication. © 1994 Wiley-Liss, Inc.     

Molecular signals in B cell activation. II. IL-2-mediated signals are required in late G1 for transition to S phase after ionomycin and PMA treatment

We report that sustained increase of intracellular calcium ion concentration and protein kinase C (PKC) activation maintained throughout the G1 phase of cell cycle do not provide sufficient signals to cause S-phase entry in rabbit B cells, and that additional signals transduced by IL-2 and IL-2 receptor interaction are essential for G1 to S transition. We have shown earlier that rabbit B cells can be activated to produce IL-2 and express functional IL-2 receptors after treatment with ionomycin and PMA. Herein we have compared the response of rabbit PBLs, which contain about 50% T cells, with those of purified B cells. After activation with ionomycin or PMA, comparable numbers of PBLs and B cells entered the cell cycle; but DNA synthesis by the PBL cultures was three to four times higher than that of cultures of purified B cells. Interestingly, IL-2 production by the PBL cultures was also three to four times higher than in B cell cultures, suggesting an involvement of IL-2 in inducing DNA synthesis in these cells. The hypothesis that IL-2, which is produced in early G1, acts in late G1 and is required for G1 to S transition in B cells was supported by the following observations: (i) IL-2 production by B cells was detected as early as 6 hr after activation and preceded DNA synthesis by at least 24 hr. (ii) B cell blasts in G1 (produced by treatment of resting B cells with ionomycin and PMA) showed DNA synthesis in response to IL-2, but showed very little DNA synthesis in response to restimulation with ionomycin and PMA. (iii) A polyclonal rabbit anti-human IL-2 antibody caused nearly complete inhibition of DNA synthesis by B cells activated by ionomycin and PMA. (iv) A PKC inhibitor, K252b, inhibited DNA synthesis in ionomycin and PMA-stimulated cells if added at the beginning of culture but was not inhibitory if added 16 hr later. We conclude that increased [Ca2+]i and PKC activation are not sufficient signals for G1 to S transition in B cells; entry into S is signaled by IL-2, and IL-2-mediated signal transduction probably does not involve increased [Ca2+]i or PKC activation.     

Cytochrome synthesis in synchronous cultures of the yeast, Saccharomyces cerevisiae.

The synthesis of cytochromes aa3, b, and c has been investigated during synchronous growth in the yeast, Saccharomyces cerevisiae. These cytochromes increase in concentration continuously throughout each cell cycle, with an approximate doubling in rate during successive cycles. The rates of cytochrome formation are considerably higher in galactose-grown cultures than in cells grown in glucose. Although cytochrome aa3 increases at a continuous rate, its functional counterpart, cytochrome c oxidase, increases in stepwise fashion, with the increments occurring at the beginning of each new cell cycle. Chloramphenicol, a specific inhibitor of intramitochondrial protein synthesis, inhibits the formation of cytochrome aa3 at all stages of the cell cycle, but does not inhibit cytochrome c. Chloramphenicol exhibits a somewhat intermediate effect on cytochrome b synthesis, with transient inhibition occurring only when the drug is added prior to or during the initial part of the first cell cycle. After this time, chloramphenicol had no effect on the rate of cytochrome b synthesis. The data indicate that under our conditions of cell synchrony mitochondrial membrane formation as reflected by increments in mitochondrial cytochromes occurs by continuous accretion of new material throughout the cell cycle. Intramitochondrially synthesized polypeptide products, responsible for the formation of new cytochrome aa3, appear to be synthesized throughout the cell cycle.     

Inhibitor effects during the cell cycle in Chlamydomonas reinhardtii. Determination of transition points in asynchronous cultures

A wide variety of inhibitors (drugs, antibiotics, and antimetabolites) will block cell division within an ongoing cell cycle in autotrophic cultures of Chlamydomonas reinhardtii. To determine when during the cell cycle a given inhibitor is effective in preventing cell division, a technique is described which does not rely on the use of synchronous cultures. The technique permits the measurement of transition points, the cell cycle stage at which the subsequent cell division becomes insensitive to the effects of an inhibitor. A map of transition points in the cell cycle reveals that they are grouped into two broad periods, the second and fourth quarters. In general, inhibitors which block organellar DNA, RNA, and protein synthesis have second-quarter transition points, while those which inhibit nuclear cytoplasmic macromolecular synthesis have fourth-quarter transition points. The specific grouping of these transition points into two periods suggests that the synthesis of organellar components is completed midway through the cell cycle and that the synthesis of nonorganellar components required for cell division is not completed until late in the cell cycle.     

Reovirus-directed Ribonucleic Acid Synthesis in Infected L Cells

Reovirus replication in L-929 mouse fibroblasts was unaffected by 0.5 mug of actinomycin per ml, a concentration which inhibited cell ribonucleic acid (RNA) synthesis by more than 90%. Under these conditions of selective inhibition, the formation of both single-stranded and double-stranded virus-specific RNA was detected beginning at 6 hr after infection. The purified double-stranded RNA was similar in size and base composition to virus RNA and presumably was incorporated into mature virus. The single-stranded RNA formed ribonuclease-resistant duplexes when annealed with denatured virus RNA but did not self-anneal, thus indicating that it includes copies of only one strand of the duplex. The single-stranded RNA was polyribosome-associated and may function as the virus messenger RNA. Production of both types of virus-induced RNA required protein synthesis 6 to 9 hr after infection. At later times in the infectious cycle, only double-stranded RNA synthesis was dependent on continued protein formation.     

Sister-chromatid exchanges and gene mutations are induced by the replication of 5-bromo- and 5-chloro-deoxyuridine substituted DNA

The thymidine (dT) analogue 5-chlorodeoxyuridine (CldU) induces 7–8-fold more sister-chromatid exchanges (SCE) than does 5-bromodeoxyuridine (BrdU) at equal substitution for dT in Chinese hamster ovary cells in culture. This difference facilitates study of the mechanism of induction of SCE by these analoques. Cultures were incubated with either BrdU or CldU for one cell cycle, followed by incubation in the presence of dT alone or BrdU or CldU for the second cell cycle and the SCE frequency determined in M2 cells. The results suggest that the induction of SCE is dependent only on the replication of the analogue-substituted DNA during the second cell cycle. Additional studies employed cultures grown in the presence of BrdU or CldU for 7 days to obtain mainly bifilarly substituted DNA, followed by 2 rounds of replication in the presence of dT alone. The SCE frequencies were approximately twice those found in cultures which had undergone the usual 2 rounds in the presence of the analogue; this is consistent with the replication of twice the amount of analogue-substituted DNA. Furthermore, such long-term growth in the presence of BrdU or CldU also results in concentration-dependent increases in the frequency of 6-thioguanine-resistant mutants, suggesting that gene mutations also result from the replication of analogue-substituted DNA.     

THE SEXUAL AND ASEXUAL LIFE CYCLE OF GLENODINIOPSIS STEINII (DINOPHYCEAE)

The sexual and asexual portions of the life cycle of Glenodiniopsis steinii were examined at both the light and scanning electron microscopic levels. Asexual reproduction by cell division occurs every 2 to 3 d under optimal conditions. Gamete formation and sexual reproduction can be induced by either nitrogen deprivation or aging of cultures. Fusion of gametes peaks about 10 hr into the light cycle and requires about 10 hr for completion. Cells remain motile during fusion but the zygote loses motility. The cell then undergoes changes that give it a thick-walled, warty appearance.     

Thymidine as a synchronizing agent. I. Nucleic acid and protein formation in synchronous HeLa cultures treated with excess thymidine

Synchronous cultures of HeLa cells obtained by selective detachment of mitoses were treated with high concentrations of thymidine. The inhibitor was added soon after completion of cell division and rates of cell enlargement and accumulation of DNA, RNA and protein were compared for untreated and thymidine-treated cultures at various points of the cell cycle. It was found that concentrations of thymidine which in randomly growing cultures inhibit the rate of cell division by more than 90% allowed a considerable degree of DNA synthesis and did not affect the rate of accumulation of RNA and protein, when applied to cells in the G1 phase of synchronous culture. Treated and untreated cells enlarged at the same rate throughout their life cycle. The results show that concentrations of thymidine commonly employed to produce cell synchrony do not arrest the cells at the G1-S boundary, but allow slow progress through S in respect to DNA synthesis, and near-normal progress towards G2 as regards RNA and protein accumulation and cell enlargement.     

Exposure to continuous bromodeoxyuridine (BrdU) differentially affects cell cycle progression of human breast and bladder cancer cell lines

Incorporation of bromodeoxyuridine (BrdU) during DNA replication is frequently used for cell cycle analysis. The flow cytometric BrdU/Hoechst quenching technique is conducive to high-resolution assessment of cell cycle kinetics, but requires continuous BrdU treatment, which may have cytostatic or cytotoxic effects. Here, we have examined the impact of BrdU on the proliferation of BT474 and SK-BR-3 breast cancer cell lines and compared the observed effects with cell proliferation of RT4 and J82 bladder carcinoma cells, previously described to be sensitive and insensitive to BrdU, respectively. Both uni- and bi-parametric DNA measurements were performed to identify BrdU-induced alterations in the S-phase fraction and in cell cycle progression. An annexinV/propidium iodide (PI) assay was used to identify potential induction of apoptosis by BrdU. Proliferative activity in BT474, SK-BR-3, and RT4 cultures was reduced in different cell cycle phases due to continuous treatment with 60, 5.0, and 3.5 micro m BrdU. This effect, which was not found in J82 cultures, was dependent on exposure time (96 versus 48 h) and was also dose-dependent for RT4 and SK-BR-3. BrdU application does not induce apoptosis or necrosis as revealed with the annexin V/PI assay. We concluded that continuous BrdU treatment did not affect cell viability, but essentially alters cell cycle progression in three out of four cell lines tested. Cell-type specific validation of the feasibility of the powerful BrdU/Hoechst quenching technique is required and recommended.     

The effect of nalidixic acid on growth and reproductive events in nucleocytosolic and chloroplast compartments in the alga<Emphasis Type="Italic">Scenedesmus quadricauda</Emphasis>

The courses of rRNA accumulation, DNA replication, and nuclear division were followed both in the chloroplast and the nucleocytosolic compartments during the cell cycle in synchronized populations of the chlorococcal alga Scenedesmus quadricauda. Control and nalidixic acid-treated cultures were compared. Nalidixic acid (150 mg/L) was added either at the beginning of the cell cycle or consecutively during the cell cycle to subcultures transferred into the dark. If the inhibitor was applied at the beginning of the cell cycle, chloroplast DNA did not replicate and nucleoids did not divide. Chloroplast division, however, was coordinated in a timely fashion with cytokinesis even under conditions of blocked chloroplast DNA replication. While the growth rate was slowed down, the courses of reproductive processes in the nucleocytosolic compartment were not affected and their timing and the number of rounds were coordinated with growth rate as in the control culture. The rate of cytosolic rRNA synthesis was lower but no apparent effect was seen on the amount of rRNA that accumulated during the cell cycle. In contrast, lower levels of chloroplast rRNA were found at the end of the cell cycle compared with the control culture. Experiments in which cells were transferred to the dark during the cell cycle showed that the inhibitor affected none of the reproductive events in the nucleocytosolic compartment. In the chloroplast compartment, DNA replication was inhibited in inhibitor-treated cultures, but was unaffected in controls. The chloroplast nucleoids themselves divided even in the presence of the inhibitor, reducing their DNA content to a level which corresponded to that in freshly formed control daughter cells.     

Mechanism of lymphocyte activation. II. Requirements for macromolecular synthesis in the production of lymphokines

Reversible inhibitors of protein synthesis, cycloheximide and puromycin, and an irreversible inhibitor of RNA synthesis, actinomycin D, were employed to study the kinetics and types of macromolecular synthetic events required for the production of migration inhibitory factor (MIF) and macrophage activating factor (MAF) by Con A-stimulated lymphocytes. Reversible inhibition of protein synthesis during the first 2 hr of stimulation completely inhibited MIF and MAF production. The same treatment, performed 4 hr after the beginning of the stimulation, had no effect. When the inhibitors of protein synthesis were left in the cultures, a block of lymphokine production was observed when the drugs were added at 6 hr as well as at time 0. In contrast, irreversible inhibition of RNA synthesis at 6 hr was ineffective and only treatment at the beginning of culture blocked lymphokine production. These data suggest that a critical protein is synthesized during the first few hours of stimulation, which is required for subsequent production of lymphokines. After this special early requirement, however, continued protein synthesis is needed for lymphokine production. In contrast, the RNA required for MIF and MAF production seemed to be completely synthesized within 4 to 6 hr of stimulation. The possibility that suppressor macrophages inhibit lymphokine production via modulation of macromolecular synthesis is discussed.     

Increase in globin chains and globin mRNA in erythroleukemia cells in response to hemin.

Addition of 50 μm hemin to mouse erythroleukemia cells cultured in 0.5% dimethyl-sulfoxide (DMSO) resulted in >10-fold stimulation of globin chain synthesis as a percentage of acid precipitable protein. In cultures fully induced with 1.5% DMSO, addition of 15 mm 3-amino-1,2,4-triazole (AT), an inhibitor of heme synthesis, reduced globin chain synthesis to uninduced levels and reduced globin mRNA levels to less than 20% of induced values. The inhibition of AT was prevented by simultaneous addition of 25 μm hemin to the cultures. Using RNA-DNA hybridization analysis, the amount of globin mRNA sequences as a fraction of total cytoplasmic RNA was also increased by addition of 50 μm hemin to cultures with 0.5% DMSO. The results suggest that exogenous hemin can promote globin chain synthesis, that endogenously synthesized heme can be required for globin chain synthesis, and that hemin directly or indirectly also alters the appearance or degradation of globin mRNA sequences in the cytoplasm.     

Studies of mechanisms of antibody formation: III. Effects of 5-bromodeoxyuridine upon normal and neoplastic B-lymphocyte function

Although 5-bromodeoxyuridine (BrdU) does not interfere with the rate of proliferation, or with total protein, DNA, or RNA synthesis in normal mouse spleen B cells exposed to specific antigenic excitation or to polyclonal activators, this agent has the capacity to block the appearance of IgM antibody-forming cells (AFC) in response to antigenic challenge in vitro with sheep red blood cells (sRBC), as well as the generation of anti-sRBC or 2,4,6-trinitrobenzene sulfonate-AFC in the course of polyclonal expansion. The effect of BrdU can be blocked competitively by a 10-fold excess of thymidine (TdR). CsCl density equilibrium analysis indicates that B cells with 55% BrdU substitution of TdR in a single DNA strand could participate in at least one additional division cycle, resulting in a double DNA strand substitution. BrdU substitution in a single DNA strand was sufficient, however, to block antibody secretion in B cells (unifilar dominance). When cells with single-strand BrdU substitution were allowed to proliferate in the presence of a 10-fold excess of TdR, the cells regained the capacity to synthesize and secrete specific antibody, demonstrating the reversibility of the inhibitory effect of BrdU upon this specialized B-cell function. Cells stimulated with LPS in the presence of BrdU exhibited higher rates of synthesis of total RNA as well as of the RNA species of the translation machinery (28, 18, and 5S and tRNA), measured by the rate of incorporation of radioactive precursors at the time of peak proliferation. Exposure to BrdU blocked the secretion of μ and light chains, although such cells had high levels of membrane IgM (mIgM) and were able to regenerate mIgM after enzymatic stripping. The regeneration of mIgM could be suppressed with α-amanitine and by actinomycin D, suggesting that the incorporation of BrdU into DNA does not affect the transcripition of nucleotide sequences for mIgM. MOPC 315 and 70 plasmocytoma cells cultured in the presence of BrdU for 60 hr regularly incorporated BrdU into one and two DNA strands. There was a 55–60% substitution of BrdU in single DNA strands. This situation produces complete inhibition of the production of IgM, μ, or light chains in normal cells. In contrast, plasmocytoma cells treated with BrdU secreted as many α, γ, and light chains as the corresponding control cells grown in the presence of TdR. This result points to possibly profound differences in the regulation of transcripition of Ig nucleotide sequences in lymphocytes which have undergone malignant transformation.     

The cell cycle of glial cells grown in vitro: an immunocytochemical method of analysis.

Studies of cell cycles have traditionally employed [3H]- and [14C]-thymidine to label the DNA of proliferating cells and autoradiography to reveal the thymidine label. The development of antibodies to the thymidine analogue 5-bromodeoxyuridine (BrdU) has allowed the development of an immunocytochemical method analogous to the thymidine autoradiographic technique. In direct comparisons, we found that the immunocytochemical method consistently detected a larger number of proliferating cells. This suggests that it may be a more sensitive index of proliferation than thymidine autoradiography in some systems. We used the BrdU method to analyze the cycle of astroglia cultured from neonatal mouse cerebral cortex. Cells were exposed to BrdU for 1 hr to label a discrete subpopulation of proliferating cells. At 2-36 hr after the pulse, a combination of anti-BrdU immunocytochemistry and counterstaining with propidium iodide was used to identify proliferating cells. The length of the cell cycle was determined by charting the percent of BrdU-labeled mitotic cells vs time after the pulse. We found the average length of the cell cycle of astrocytes grown in vitro to be 20.5 hr. The combined G2 + M phases were 2-3 hr. These values are virtually identical with those found for glial cells in vivo, suggesting that the culture environment does not interfere with the normal control of cell cycle length.