A model of cell cycle control: effects of thymidine on synchronous cell cultures.

Further evidence is presented in support of a model for growth control in which commitment for cell division is determined by an event in the preceding cell cycle. A study was made of conditions affecting synchronous growth following treatment of murine mastocytoma cells with excess thymidine at different phases of the cell cycle. Cells were synchronized by a physical procedure involving velocity sedimentation in a zonal rotor. Pulse treatment of such cultures with thymidine at times corresponding to the S, G2, and M periods had no effect on further growth. However, addition at G1, although having no immediate effect, arrested cell growth in the next cell cycle. This temporal effect may account for the decay of synchrony observed during double thymidine blockade or thymidine-FUdR blockade. When the time interval between two such blocks was 7 hr or less, P815Y cells were arrested after one synchronous division. At this critical time a majority of cells were at, or near, G1. It is suggested that thymidine exerts a hitherto unrecognized effect at the G1 interval.     

Effect of interferon on growth and division cycle of Friend erythroleukemic murine cells in vitro

The administration of appropriate doses of interferon to cultures of Friend leukemia cells causes a pronounced inhibition of cell growth. Several lines of evidence indicate that this effect is due to interferon itself, rather than to unknown contaminants of interferon preparations. Autoradiograph analysis of growth parameters of Friend leukemia cells during treatment with interferon demonstrates that the rate of entry into the S phase, the percent decline of unlabeled mitoses, and the mitotic indexes are significantly lower in interferon- treated cell cultures than in control untreated cultures when tritiated thymidine was added 12 h after the administration of interferon. These data indicate that fractions of interferon-treated cell population are delayed in both G1 and in G2 phases of the cell cycle. This was confirmed by exact measurements of the length of the various phases of the cycle. The interferon-induced inhibition of growth of Friend leukemia cells is reversible after removal of the compound. Autoradiograph data obtained from control cultures and from cultures previously treated with interferon that had been washed free of interferon and reseeded in interferon-free medium, demonstrate that during the first 12 h after removal of interferon, a large majority of the cells previously treated with interferon had a deranged flow into the S phase, a high number of unlabeled mitoses, and a low mitotic index. These data provide further evidence for the above-mentioned prolongations of G1 and G2 phases of the cell cycle. All growth parameters tested reverted to normal values within 12 h after washing out interferon.     

Control of cell division in hepatoma cells by exogenous heparan sulfate proteoglycan

The effects of cell surface heparan sulfate proteoglycan (HSPG) prepared from log and confluent monolayers of a rat hepatoma cell line on hepatoma cell growth were studied. When HSPG isolated from confluent cells was added exogenously to log phase cells, it was internalized and free heparan sulfate (HS) chains appeared transiently in the nucleus. Concurrently, the growth of the treated cells was inhibited, but the cells resumed logarithmic growth as the level of nuclear HS fell, and the cells grew to confluence and became contact inhibited. When HSPG prepared from log-phase hepatoma cells was added exogenously to log phase cells, it was internalized but very little of the internalized HS appeared in the nucleus, and there was no change in the rate of cell growth. However, when the rate of cell growth was reduced by culture of the cells in serum- and insulin-deficient medium, HSPG prepared from log-phase cells stimulated the growth rate of these slow-growing cells. The cell cycle dependency of HSPG uptake and growth inhibition was studied in cultures synchronized by a thymidine/aphidicolin double block. When [35SO4]HSPG from confluent cells was added to synchronized cells just as they were released from the second block, a portion of the [35SO4]HSPG was internalized and [35SO4]HS appeared in the nucleus. However, at mitosis the [35SO4]HS disappeared almost completely from all of the cellular pools, and after mitosis, more of the [35SO4]HSPG was taken up and [35SO4]HS reappeared in the nucleus and remained in the nucleus until the cells divided again. When cultures were released from the aphidicolin block, both control and HSPG-treated cells progressed through the S, the G2, and the M phases of the cell cycle. However, the length of the G1 phase of the cycle was increased in the HSPG-treated cells. The treated cultures then progressed through the second S, G2, and M phases. Thus, the inhibition of cell division occurred in the G1 phase of the cell cycle, prior to the G1/S boundary. Addition of the HSPG to the synchronized cultures just after the first mitosis resulted in an immediate arrest of the cell cycle in G1.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cell cycle analysis of Taxus suspension cultures at the single cell level as an indicator of culture heterogeneity

Single cell growth and division was measured via flow cytometry in order to characterize the metabolic variability of Taxus cuspidata suspension cultures, which produce the valuable secondary metabolite Taxol. Good agreement was observed between the cell cycle distribution and biomass accumulation over the batch culture period. Specific growth rates of 0.13 days(-1) by fresh weight and 0.15 days(-1) by dry weight were measured. Elicitation with methyl jasmonate (MJ) significantly decreased both cell cycle progression and biomass accumulation, as the specific growth rate decreased to 0.027 days(-1) by fresh and dry weight. Despite the decrease in biomass accumulation for MJ elicited cultures, sucrose utilization was not significantly different from control cultures. MJ elicitation also increased the accumulation of paclitaxel and other taxanes. The accumulation of upstream taxanes (baccatin III and 10-deactylbaccatin III) increased during exponential growth, reached a maximum around day 12, and then declined throughout the stationary phase. The paclitaxel concentration increased during both exponential growth and stationary phase, reaching a maximum around days 20-25. Throughout the culture period, greater than 70% of the cells were in G(0)/G(1) phase of the cell cycle. Studies using bromodeoxyuridine (BrdU) incorporation showed that approximately 65% of the Taxus cells are noncycling, even during exponential growth. Although the role of these cells is currently unknown, the presence of a large, noncycling subpopulation can have a significant impact on the utilization of plant cell culture technology for the large-scale production of paclitaxel. These results demonstrate that there is a high degree of metabolic heterogeneity in Taxus cuspidata suspension cultures. Understanding this heterogeneity is important for the optimization of plant cell cultures, particularly the reduction of production variability.     

The radiation response of cells recovering after chronic hypoxia

Experiments were performed to study the influence of hypoxic pretreatment on the radiation response of A431 human squamous carcinoma cells. Reaeration for 10 min after chronic hypoxia (greater than 2 h) was found to enhance the radiosensitivity of A431 cells, and the maximal effect was seen for those cells reaerated after 12 h of hypoxia. The radiosensitivity enhancement for reaerated cells after 12 h of hypoxia was maximized by 5 min after the return to aerobic conditions and reached the control level by 12 h of reaeration. This enhanced radiosensitive state was characterized by a reduced shoulder region and increased slope of the radiation dose-response curve for cells in both the exponential and plateau phases of growth. There was a slight increase in the number of G1 and decrease in the number of S and G2 + M cells for both exponential- and plateau-phase cultures following 12 h hypoxic treatment. Although growth inhibition induced by 12 h of hypoxia was seen for cells in the exponential phase, there was no cell number change in the plateau-phase culture after hypoxia. Plating efficiency (PE) of cells in both growth phases was reduced by 30% after hypoxia. Furthermore, in the exponential-phase culture, the extent of reduction in PE after hypoxia was similar among cells in different phases of the cell cycle. Although S-phase cells in exponentially growing cultures were relatively more resistant to radiation than G1 and G2 + M cells, the cell age-response pattern was the same whether the cells had been aerobic or hypoxic before reaeration and irradiation. Furthermore, the enhancement ratio associated with reaeration after 12 h of hypoxia for these three subpopulations of cells was 1.3. Our results indicate that the increase in radiosensitivity due to reaeration after chronic hypoxia is unlikely to be related to the changes of cell cycle stage and growth phase during hypoxic treatment.     

Polysomes in various phases of the cell cycle in synchronized plasmacytoma cells

The fraction of membrane-bound and free polysomes during different phases of the cell cycle was determined in suspension cultures of mouse plasmacytoma cells, synchronized by growth in isoleucine-deficient medium. The membrane-bound polysomes reached a maximum value (about 28 % of total polysomes) during the G 1 phase. In the S phase and G 2 phase only 18 to 20 % of the total polysomes were found to be membrane-bound. A high percentage of membrane-bound polysomes in the G 1 phase of the cell cycle agrees with the earlier finding that maximum synthesis of immunoglobulin light chain takes place on polysomes bound to the membrane in the G 1 phase of the cell cycle. The presence of a significant fraction of membrane-bound polysomes in the S and G 2 phases of the cell cycle would suggest that membrane-bound polysomes are also involved in the synthesis of proteins other than immunoglobulins.The ultrastructure of the cells during the various phases of the cell cycle was also studied. During the G 1 phase the surface of the majority of cells was distinguished by the presence of ruffles and slender villus-like cytoplasmic projections. In the S phase the surface contour tended to become smooth and even. These differences in the surface morphology may reflect the change in function which occurs during the transition from the G 1 to the S phase.     

Combined in silico modeling and metabolomics analysis to characterize fed-batch CHO cell culture

The increasing demand for recombinant therapeutic proteins highlights the need to constantly improve the efficiency and yield of these biopharmaceutical products from mammalian cells, which is fully achievable only through proper understanding of cellular functioning. Towards this end, the current study exploited a combined metabolomics and in silico modeling approach to gain a deeper insight into the cellular mechanisms of Chinese hamster ovary (CHO) fed-batch cultures. Initially, extracellular and intracellular metabolite profiling analysis shortlisted key metabolites associated with cell growth limitation within the energy, glutathione, and glycerophospholipid pathways that have distinct changes at the exponential-stationary transition phase of the cultures. In addition, biomass compositional analysis newly revealed different amino acid content in the CHO cells from other mammalian cells, indicating the significance of accurate protein composition data in metabolite balancing across required nutrient assimilation, metabolic utilization, and cell growth. Subsequent in silico modeling of CHO cells characterized internal metabolic behaviors attaining physiological changes during growth and non-growth phases, thereby allowing us to explore relevant pathways to growth limitation and identify major growth-limiting factors including the oxidative stress and depletion of lipid metabolites. Such key information on growth-related mechanisms derived from the current approach can potentially guide the development of new strategies to enhance CHO culture performance.     

Adaptive control at low glucose concentration of HEK-293 cell serum-free cultures.

Fed-batch cultures were implemented to study the metabolism of HEK-293 cells. Glucose, measured every 30 min by a FIA biosensor system, was maintained at 1 mM throughout the culture using an adaptive nonlinear controller based on minimal process modeling. The controller performed satisfactorily at both low and high cell concentrations without the need for retuning between different culture phases. Overall, lactate production was significantly reduced by maintaining a low glucose concentration, thus decreasing the rate of glycolysis. The rates of glucose and glutamine uptake as well as the lactate and ammonia production were compared to those obtained in batch mode with an initial glucose concentration of 21 mM. Basically, three phases were observed in both culture modes. The metabolic shift from the first to the second phase was characterized by a significant reduction in glucose consumption and lactate production while maximum growth rate was maintained. The specific respiration rate appeared unchanged during the first two phases, suggesting that no change occurred in the oxidative pathway capacity. In the third phase, cell growth became slower very likely due to glutamine limitation.     

Coordination of growth rate, cell cycle, stress response, and metabolic activity in yeast

We studied the relationship between growth rate and genome-wide gene expression, cell cycle progression, and glucose metabolism in 36 steady-state continuous cultures limited by one of six different nutrients (glucose, ammonium, sulfate, phosphate, uracil, or leucine). The expression of more than one quarter of all yeast genes is linearly correlated with growth rate, independent of the limiting nutrient. The subset of negatively growth-correlated genes is most enriched for peroxisomal functions, whereas positively correlated genes mainly encode ribosomal functions. Many (not all) genes associated with stress response are strongly correlated with growth rate, as are genes that are periodically expressed under conditions of metabolic cycling. We confirmed a linear relationship between growth rate and the fraction of the cell population in the G0/G1 cell cycle phase, independent of limiting nutrient. Cultures limited by auxotrophic requirements wasted excess glucose, whereas those limited on phosphate, sulfate, or ammonia did not; this phenomenon (reminiscent of the "Warburg effect" in cancer cells) was confirmed in batch cultures. Using an aggregate of gene expression values, we predict (in both continuous and batch cultures) an "instantaneous growth rate." This concept is useful in interpreting the system-level connections among growth rate, metabolism, stress, and the cell cycle.     

Cell cycle-dependent vimentin expression in elutriator-synchronized, TPA-treated MPC-11 mouse plasmacytoma cells.

We correlated cell cycle progression and vimentin expression at the single cell level by multiparameter flow cytometry in populations of MPC-11 cells enriched in different cell cycle phases by centrifugal elutriation and subsequently treated with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Synchronized, untreated cultures showed a uniform, synchronous progression through the cell cycle during further cultivation. A 6-h TPA treatment of G1-phase-enriched cultures induced both a partial G1-phase arrest in the same cycle and a moderate fraction of cells to become vimentin positive. However, nearly all cells of the cultures enriched in S- or in G2/M-phase cells could be arrested by TPA treatment at the earliest in the G1 phase of the second cell cycle and displayed higher fractions of positive cells as well as higher average levels of vimentin. After 20 h of treatment, the G1-phase arrest was almost complete. In terms of fractions of vimentin-positive cells as well as of average cellular vimentin content, the differences between the cultures resembled, albeit on a higher level, those between the respective cultures treated with TPA for 6 h. These observations might explain the striking bimodal distribution of individual cellular vimentin content detectable in G1-phase fractions of asynchronous, TPA-treated cultures. The pattern of vimentin mRNA accumulation in synchronized cultures after short-term TPA treatment strongly suggests that the cell cycle-dependent pattern of vimentin expression is caused, at least in part, by different levels of vimentin mRNA accumulated in the cells. Since proteinaceous mediator(s) are obviously involved in TPA-induced vimentin expression in MPC-11 cells, cell cycle-dependent vimentin expression in these cells may be dependent on cell cycle-dependent regulation of the activity and/or concentration of such mediator(s).     

Metabolic Profiling of Geobacter sulfurreducens during Industrial Bioprocess Scale-Up

During the industrial scale-up of bioprocesses it is important to establish that the biological system has not changed significantly when moving from small laboratory-scale shake flasks or culturing bottles to an industrially relevant production level. Therefore, during upscaling of biomass production for a range of metal transformations, including the production of biogenic magnetite nanoparticles by Geobacter sulfurreducens, from 100-ml bench-scale to 5-liter fermentors, we applied Fourier transform infrared (FTIR) spectroscopy as a metabolic fingerprinting approach followed by the analysis of bacterial cell extracts by gas chromatography-mass spectrometry (GC-MS) for metabolic profiling. FTIR results clearly differentiated between the phenotypic changes associated with different growth phases as well as the two culturing conditions. Furthermore, the clustering patterns displayed by multivariate analysis were in agreement with the turbidimetric measurements, which displayed an extended lag phase for cells grown in a 5-liter bioreactor (24 h) compared to those grown in 100-ml serum bottles (6 h). GC-MS analysis of the cell extracts demonstrated an overall accumulation of fumarate during the lag phase under both culturing conditions, coinciding with the detected concentrations of oxaloacetate, pyruvate, nicotinamide, and glycerol-3-phosphate being at their lowest levels compared to other growth phases. These metabolites were overlaid onto a metabolic network of G. sulfurreducens, and taking into account the levels of these metabolites throughout the fermentation process, the limited availability of oxaloacetate and nicotinamide would seem to be the main metabolic bottleneck resulting from this scale-up process. Additional metabolite-feeding experiments were carried out to validate the above hypothesis. Nicotinamide supplementation (1 mM) did not display any significant effects on the lag phase of G. sulfurreducens cells grown in the 100-ml serum bottles. However, it significantly improved the growth behavior of cells grown in the 5-liter bioreactor by reducing the lag phase from 24 h to 6 h, while providing higher yield than in the 100-ml serum bottles.     

Synchronous Arabidopsis suspension cultures for analysis of cell-cycle gene activity

Synchronized suspension cultures are powerful tools in plant cell-cycle studies. However, few Arabidopsis cell cultures are available, and synchrony extending over several sequential phases of the cell cycle has not been reported. Here we describe the first useful synchrony in Arabidopsis, achieved by selecting the rapidly dividing Arabidopsis cell suspensions MM1 and MM2d. Synchrony may be achieved either by removing and re-supplying sucrose to the growth media or by applying an aphidicolin block/release. Synchronization with aphidicolin produced up to 80% S-phase cells and up to 92% G2 cells, together with clear separation of different cell-cycle phases. These synchronization procedures can be used for analysis of gene expression and protein activity. We show that representatives of three CDK gene classes of Arabidopsis (CDKA, CDKB1 and CDKB2) show differential expression timing, and that three CDK inhibitor genes show strikingly different expression patterns during cell-cycle re-entry. We propose that ICK2 (KRP2) may have a specific role in this process.     

Characterization of functionally active interleukin‐18/eGFP fusion protein expression during cell cycle phases in recombinant chicken DF1 Cells

The dependence of foreign gene expression on cell cycle phases in mammalian cells has been described. In this study, a DF1/chIL‐18a cell line that stably expresses the fusion protein chIL‐18 was constructed and the enhanced green fluorescence protein connected through a (G₄S)₃ linker sequence investigated the relationship between cell cycle phases and fusion protein production. DF1/chIL‐18a cells (1 × 10⁵) were inoculated in 60‐mm culture dishes containing 5 mL of media to achieve 50%–60% confluence and were cultured in the presence of the cycle‐specific inhibitors 10058‐F4, aphidicolin, and colchicine for 24 and 48 h. The percentage of cell density and mean fluorescence intensity in each cell cycle phase were assessed using flow cytometry. The inhibitors effectively arrested cell growth. The fusion protein production rate was higher in the S phase than in the G0/G1 and G2/M phases. When cell cycle progression was blocked in the G0/G1, S, and G2/M phases by the addition of 10058‐F4, aphidicolin, and colchicine, respectively, the aphidicolin‐induced single cells showed higher fusion protein levels than did the 10058‐F4‐ or colchicine‐induced phase cells and the uninduced control cells. Although the cells did not proliferate after the drug additions, the amount of total fusion protein accumulated in aphidicolin‐treated cells was similar to that in the untreated cultures. Fusion protein is biologically active because it induces IFN‐γ production in splenocyte cultures of chicken. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:581–591, 2016     

Cell cycle parameters of Chinese hamster ovary cells during exponential, polyamine-limited growth.

We have previously shown that Chinese hamster ovary cells made polyamine deficient by treatment with alpha-methylornithine, an inhibitor of ornithine decarboxylase, grow exponentially in culture at low densities at one-half the rate observed in untreated (control) cultures. In this study, the cell cycle of polyamine-limited cells was examined by using thymidine autoradiography, mitotic index analysis, and fraction labeled mitoses analysis. We found that the longer doubling time of inhibitor-treated cultures was a consequence of increases in the lengths of the G1 and S phases. The expansion of the S phase was proportional to the increase in doubling time (twofold), whereas the G1 phase was lengthened by slightly more than a factor of 2. The lengths of the G2 and M phases were essentially unchanged. Putrescine stimulated the growth of inhibitor-treated cultures and restored the cell cycle parameters to those of untreated cells.     

Segmented linear modeling of CHO fed‐batch culture and its application to large scale production

We describe a systematic approach to model CHO metabolism during biopharmaceutical production across a wide range of cell culture conditions. To this end, we applied the metabolic steady state concept. We analyzed and modeled the production rates of metabolites as a function of the specific growth rate. First, the total number of metabolic steady state phases and the location of the breakpoints were determined by recursive partitioning. For this, the smoothed derivative of the metabolic rates with respect to the growth rate were used followed by hierarchical clustering of the obtained partition. We then applied a piecewise regression to the metabolic rates with the previously determined number of phases. This allowed identifying the growth rates at which the cells underwent a metabolic shift. The resulting model with piecewise linear relationships between metabolic rates and the growth rate did well describe cellular metabolism in the fed‐batch cultures. Using the model structure and parameter values from a small‐scale cell culture (2 L) training dataset, it was possible to predict metabolic rates of new fed‐batch cultures just using the experimental specific growth rates. Such prediction was successful both at the laboratory scale with 2 L bioreactors but also at the production scale of 2000 L. This type of modeling provides a flexible framework to set a solid foundation for metabolic flux analysis and mechanistic type of modeling. Biotechnol. Bioeng. 2017;114: 785–797. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.     

UNCOUPLING OF SILICON COMPARED WITH CARBON AND NITROGEN METABOLISMS AND THE ROLE OF THE CELL CYCLE IN CONTINUOUS CULTURES OF THALASSIOSIRA PSEUDONANA (BACILLARIOPHYCEAE) UNDER LIGHT,NITROGEN, AND PHOSPHORUS CONTROL1

The elemental composition and the cell cycle stages of the marine diatom Thalassiosira pseudonana Hasle and Heimdal were studied in continuous cultures over a range of different light‐ (E), nitrogen‐ (N), and phosphorus‐ (P) limited growth rates. In all growth conditions investigated, the decrease in the growth rate was linked with a higher relative contribution of the G2+M phase. The other phases of the cell cycle, G1 and S, showed different patterns, depending on the type of limitation. All experiments showed a highly significant increase in the amount of biogenic silica per cell and per cell surface with decreasing growth rates. At low growth rates, the G2+M elongation allowed an increase of the silicification of the cells. This pattern could be explained by the major uptake of silicon during the G2+M phase and by the independence of this process on the requirements of the other elements. This was illustrated by the elemental ratios Si/C and Si/N that increased from 2‐ to 6‐fold, depending of the type of limitation, whereas the C/N ratio decreased by 10% (E limitation) or increased by 50% (P limitation). The variations of the ratios clearly demonstrate the uncoupling of the Si metabolism compared with the C and N metabolisms. This uncoupling enabled us to explain that in any of the growth condition investigated, the silicification of the cells increased at low growth rates, whereas carbon and nitrogen cellular content are differently regulated, depending of the growth conditions.     

Flow cytometric study of cultured mammalian cells.

Flow cytometry provides a rapid, sensitive and accurate analytical means to monitor hybridoma cell cultures. The use of flow cytometry has enabled us to study the changes in DNA, RNA, protein, IgG, mitochondrial activity and cell size that take place during the growth cycle of batch culture. The temporal changes in the levels of these analytes and their heterogeneity have been related to the growth/death kinetics. The maximum proportion of S-cells was reached early in the growth phase while a population of low fluorescence cells with lower polidy than G1, dead cells and fragmented nuclei emerged during the death phase. Supplementation with amino acids during the exponential phase prolonged the growth cycle by enhancing cell proliferation. The fraction of S/G2 cells was much reduced by a reduction in serum concentration but was maintained during the prolonged non-proliferating "stationary" phase. The magnitude of Rhodamine 123 staining showed a consistent and general decrease during late exponential and decline phases. This trend was accompanied by an increase in the fraction of the Propidium Iodide-stained population which reflected the deteriorating metabolic and membrane integrity. Decrease in mean fluorescence intensity for DNA, RNA, protein and intracellular IgG was noted at the decline phase. Intracellular immunofluorescence was a more reliable indicator of antibody productivity than surface immunofluorescence.     

Retroviral-mediated gene transfer of the peripheral myelin protein PMP22 in Schwann cells: modulation of cell growth.

The peripheral myelin gene PMP22 is the rat and human homologue of the murine growth arrest-specific gene gas3. Besides a putative role of PMP22 in myelination, a regulatory function in cell growth has been suspected. Here we have investigated both the expression of PMP22 during cell cycle progression of cultured rat Schwann cells and the influence of altered levels of PMP22 on Schwann cell growth. When resting cells were stimulated to begin the cell cycle, the regulation of PMP22 mRNA resembled the growth arrest-specific pattern of gas3 expression observed previously in NIH3T3 fibroblasts. To prove a growth regulatory function of PMP22, we generated Schwann cell cultures by infection with retroviral PMP22 expression vectors that constitutively expressed PMP22 cDNA sequences, in either the sense or antisense orientation. Transduced cells carrying the sense construct overexpressed PMP22 mRNA and protein, whereas in cells infected with an antisense PMP22 expression vector PMP22 mRNA levels were reduced markedly. Altered levels of PMP22 significantly modulated Schwann cell proliferation, as judged by 5-bromo-2'-deoxy-uridine incorporation into replicated DNA. In asynchronously dividing cultures enhanced expression of PMP22 decreased DNA synthesis to 60% of the control level. Conversely, reduced levels of PMP22 mRNA led to enhanced DNA synthesis of approximately 150%. Further cell cycle analyses by flow cytometry revealed that overexpression of PMP22 delayed serum- and forskolin-stimulated entry of resting Schwann cells from G0/G1 into the S + G2/M phases by approximately 8 h, whereas underexpression of PMP22 mRNA slightly increased the proportion of cells that entered the S + G2/M phases.(ABSTRACT TRUNCATED AT 250 WORDS)     

Receptors for B-melanocyte-stimulating hormone exhibit positive cooperativity in synchronized melanoma cells

Cloudman S91 mouse melanoma cells respond in culture to B-melanocyte-stimulating hormone (B-MSH) with changes in morphology, growth rates, and melanin production. The effects of MSH appear to be mediated through a stimulation of the cyclic AMP system. It was reported earlier that at least some of the responses to MSH (increased cyclic AMP production and tyrosinase activity) occur in the G2 phase of the cell cycle [Wong, G., Pawelek, J., Sansone, M., & Morowitz, J. (1974) Nature (London) 248, 351-354] and that the apparent reason for this cell cycle restriction is that receptors for MSH are most active in the G2 phase [Varga, J. M., DiPasquale, A., Pawelek, J., McGuire, J., & Lerner, A. (1974) Proc. Natl. Acad. Sci. U.S.A. 71, 1590-1593]. In this report, we found that by two separate methods of obtaining populations of cells in the G2 phase of their cycle--centrifugal elutriation or synchronization with thymidine--we observed increased binding of MSH by cells in the G2 and possibly late S phases of their cycle. However, cultures of cells passing through their cycle in synchrony were quite different from nonsynchronized (random) cultures. Both synchronized and random cultures expressed receptors for MSH in the G2 and possibly late S phases of their cycle, but synchronized cultures bound severalfold more MSH per cell than random cultures. This increased binding of MSH by synchronized cells was accompanied by an increase in tyrosinase activity and pigment production.(ABSTRACT TRUNCATED AT 250 WORDS)