Analysis of the growth kinetics of murine erythroleukaemia cells following commitment to terminal differentiation

Abstract Differentiation of murine erythroleukaemia cells by various inducers involves a step of irreversible commitment, after which the presence of the inducer is not required for completion of the process. Some cells become partially committed and give rise to differentiated as well as undifferentiated progeny. Commitment occurs asynchronously; under suboptimal inducing conditions, such as low concentration of inducer or short duration of exposure, both committed and uncommitted cells co-exist. In the present study the growth of these subpopulations was compared. Murine erythroleukaemia cells were exposed to the inducer hexamethylene-bisacetamide for 24 hr, then the inducer was removed by washing and the rate of proliferation of committed and uncommitted cells was measured. Commitment was scored by cloning the cells in inducer-free semi-solid medium and determining the cellular composition of the colonies with respect to haemoglobin content. The results indicated that following removal of the inducer the rate of proliferation was retarded similarly for both committed and uncommitted cells. Partially committed cells disappeared rapidly due to assymetrical cell division into fully committed and uncommitted cells. Both committed and uncommitted cells resumed logarithmic growth at 53 hr, but while uncommitted cells continued this pace until saturation was achieved, committed cells stopped multiplying earlier as a result of terminal differentiation.     

Analysis of the growth kinetics of murine erythroleukaemia cells following commitment to terminal differentiation

Differentiation of murine erythroleukaemia cells by various inducers involves a step of irreversible commitment, after which the presence of the inducer is not required for completion of the process. Some cells become partially committed and give rise to differentiated as well as undifferentiated progeny. Commitment occurs asynchronously; under suboptimal inducing conditions, such as low concentration of inducer or short duration of exposure, both committed and uncommitted cells co-exist. In the present study the growth of these subpopulations was compared. Murine erythroleukaemia cells were exposed to the inducer hexamethylene-bisacetamide for 24 hr, then the inducer was removed by washing and the rate of proliferation of committed and uncommitted cells was measured. Commitment was scored by cloning the cells in inducer-free semi-solid medium and determining the cellular composition of the colonies with respect to haemoglobin content. The results indicated that following removal of the inducer the rate of proliferation was retarded similarly for both committed and uncommitted cells. Partially committed cells disappeared rapidly due to assymetrical cell division into fully committed and uncommitted cells. Both committed and uncommitted cells resumed logarithmic growth at 53 hr, but while uncommitted cells continued this pace until saturation was achieved, committed cells stopped multiplying earlier as a result of terminal differentiation.     

Commitment of hydra interstitial cells to nerve cell differentiation occurs by late S-phase

Nerve cells in hydra differentiate from the interstitial cell, a multipotent stem cell. Decapitation elicits a sharp increase in the fraction of the interstitial cells committed to nerve cell differentiation in the tissue which forms the new head. To investigate when during the cell cycle nerve cell commitment can be stimulated, hydra were pulse-labeled with [³H]thymidine at times from 18 hr before to 15 hr following decapitation; the resulting cohorts of labeled interstitial cells were in the various phases of the cell cycle at the time of decapitation. Increased commitment to nerve cell differentiation within a single cell cycle (≈24 hr) was observed in those cohorts which were at least 6 hr before the end of S-phase (12 hr) at the time of decapitation. The lag time required for decapitation to produce an effective stimulus for nerve cell differentiation was measured by transplanting the stem cells from the regenerating tissue to a neutral environment. Following decapitation, 3 to 6 hr were required for increased nerve cell commitment to be stable to such transplantation. These results suggest that interstitial cells must be stimulated by late S-phase to become committed to undergo nerve cell differentiation following the subsequent mitosis. However, when head regeneration was reversed by grafting a new head onto the regenerating surface, nerve cell differentiation by such committed stem cells was greatly reduced. This indicates that an appropriate tissue environment is required for committed interstitial cells to complete the nerve cell differentiation pathway.     

Commitment to differentiation induced by retinoic acid in P19 embryonal carcinoma cells is cell cycle dependent

The rate at which P19 embryonal carcinoma cells in monolayer culture become anchorage dependent during differentiation induced by retinoic acid (RA) was investigated. In both nonsynchronized cultures and cultures synchronized by mitotic selection, the ability to grow in semisolid medium, characteristic of the malignant stem cell, decreased after a lag period of about 12 hr in the continuous presence of RA, prior to an increase in cell generation time. However, striking differences between synchronized and nonsynchronized cultures were observed in their commitment to differentiation following RA removal. After only 2 hr of exposure to RA, synchronized cells continued a program of differentiation in which they became anchorage dependent, while at least 24 hr of exposure was required for exponentially growing cells to become similarly committed. Induction of anchorage dependence by RA was also strikingly cell cycle dependent; 2 or 4 hr of exposure of synchronized cells to RA in G1 phase, when the intrinsic capacity for soft agar growth is low, was sufficient to commit cells to anchorage dependence, but a similar exposure in S phase was not. Together, these results suggested that interactions between cells in different cell cycle phases in asynchronous cultures influenced commitment since exposure to RA for more than one cycle (13 hr) was required for all cells to become anchorage dependent. Increased plasminogen activator secretion and epidermal growth factor binding, markers of certain differentiated cell types, increased only 3 and 5 days after RA addition, respectively, and were not induced by pulsed exposure to RA of less than 24 hr, even in synchronized cells.     

Erasure of the memory response in MEL cells

When MEL cells are reexposed to DMSO after an interruption in inducer treatment, they can initiate commitment to differentiation without the lag period observed after the primary exposure to inducer. This property is known as memory. Here we have employed metabolic inhibitors to analyze the basis of the memory response. Treatment of cells with cycloheximide or cordycepin during the inducer withdrawal period causes memory erasure. Cells must recapitulate an entire lag period upon reexposure to DMSO. The memory response is maintained, however, if cells are treated with metabolic inhibitors in the presence of DMSO. Our results suggest that the capacity of MEL cells for memory requires the synthesis of cell components which are normally stable in the absence of DMSO. Experiments involving reciprocal shifts between two different inhibitors have been performed. Evidence is presented that the process leading to the initiation of commitment is composed of at least three components acting in sequence.     

A system for monocytic differentiation of leukemic cells HL 60 by a short exposure to 1,25-dihydroxycholecalciferol

The human promyelocytic cell line HL 60 can be induced to differentiate toward more mature myeloid or monocytic forms by a variety of agents. This process is thought to require several days of exposure to the inducer, thus making it difficult to identify the early cellular changes which are fundamental to the differentiation program, and to relate the induction to phases of the cell cycle. In order to study the kinetics of leukemic cell differentiation we have developed a system for the induction of rapid monocytic maturation in a subpopulation of HL 60 cells. The cells are exposed to 10(-7) M 1,25-dihydroxycholecalciferol for 4 hr in serum-free medium. Subsequent incubation in a complete medium results in cellular differentiation recognizable by several criteria (phagocytosis, nonspecific esterase reaction, adherence to substratum, cell morphology) beginning at 10 hr from the exposure to the inducer. Approximately 20 hr later 30-40% of the cells in culture show the differentiated phenotype and are capable of phagocytosis. The proportion of differentiated cells in culture decreases thereafter. This system has been utilized to study the expression of c-myc oncogene in relation to the kinetics of maturation, and it was found that the inhibition of the expression of this gene precedes the onset of phenotypic differentiation by approximately 8 hr, is transient, and is accompanied by a brief retardation of cell proliferation, which resumes the normal rate within 24 hr of the exposure to the inducer.     

Commitment to erythroid differentiation by friend erythroleukemia cells: a stochastic analysis

A method for the clonal analysis of murine erythroleukemia cells has been developed which allows the precise characterization of the number of progeny produced by each cell and the degree of differentiation of each progeny cell. The potential of almost every cell in the culture can be monitored because a plating efficiency close to 100% has been achieved. The effects of treatment with an inducer of differentiation (DMSO) on the proliferative capacity of the treated cells have been studied with this technique. Cells from a mass culture treated with inducer give rise to colonies of differentiated progeny when subsequently cloned in the absence of inducer. Colonies exhibiting this phenotype represent the progeny of cells committed to the differentiation pathway by treatment with inducer. We observe that the commitment decision limits the subsequent proliferative capacity of the cell to four additional cell divisions. A quantitative analysis suggests that the commitment decision for each cell is made in a stochastic manner. Irreversible commitment to the expression of differentiated functions occurs with discrete probability per cell generation for many cell generations. The value for this probability is a function of the concentration of inducer (DMSO). A correlative biochemical study suggests that an irreversible commitment decision by a significant proportion of the population precedes or accompanies increases in cytoplasmic globin mRNA levels, one of the earliest detectable biochemical markers for erythroid differentiation in this system.A specific kinetic model based on these considerations has been developed to predict clonal phenotypes as a function of time and probability of commitment. Quantitative predictions based on this model are in excellent agreement with experimental observations. The effectiveness of a stochastic model in predicting the behavior of this system is discussed in relation to the stochastic behavior of normal hematopoiesis and the biochemical mechanisms which control these differentiation programs.     

Significance of the cell cycle in commitment of murine erythroleukemia cells to erythroid differentiation.

The relationship between differentiation of murine erythroleukemia cells (MEL) induced by DMSO and the cell division cycle has been analyzed. We demonstrate that incubation in the presence of DMSO increases the length of the G1 phase of the cell cycle. A method of synchronization of MEL cells by unit gravity sedimentation has been developed and characterized. Using this method, a series of synchronized cell populations covering the entire cell division cycle can be generated simultaneously. Cells synchronized by this technique were challenged with DMSO and analyzed for kinetics of commitment to the differentiation program. Our results indicate that populations of cells in G1 or G2 at the time of addition of inducer give rise to a greater proportion of committed cells than an unfractionated population, while cells in S phase result in a lower percentage of committed cells than the unfractionated population when cultured in DMSO.     

Alteration of folate analogue transport following induced maturation of HL-60 leukemia cells. Early decline in mediated influx, relationship to commitment, and functional dissociation of entry and exit routes

During treatment of HL-60 myeloid leukemia cells in culture with polar solvents or retinoic acid at a concentration inducing terminal maturation in 90-95% of the cells, there is a rapid decline (within 2 h) in the Vmax for influx of the folate analogue, [3H]methotrexate. Following 24 h of exposure to these agents, there is no effect on growth, but influx Vmax is reduced by 70%. After 7 days of exposure, influx Vmax is reduced 90-95%. A similar time course was seen for the reduction in intracellular levels of dihydrofolate reductase, a marker of cellular proliferation. Both the extent of terminal maturation (as determined by the extent of nitro blue tetrazolium reduction) and decrease in influx showed the same dependence on the concentration of inducer. In contrast to the effect seen on influx Vmax, both influx Km and mediated efflux of [3H]methotrexate remained unchanged in HL-60 cells exposed to inducers of maturation. Finally, evidence is presented for the coupling of this alteration on [3H]methotrexate influx with commitment of HL-60 cells to terminal maturation. This evidence shows that the effect on folate analogue influx precedes commitment and documents the irreversible nature of the reduction in influx once the majority of the cells exposed to inducer were committed to the process of maturation. The possible relevance of these results to the process of neoplastic transformation is discussed.     

Dependence of HL-60 myeloid cell differentiation on continuous and split retinoic acid exposures: Precommitment memory associated with altered nuclear structure

The cell differentiation of HL-60 human leukemic promyelocytes along the myeloid pathway due to various continuous and distributed exposures to retinoic acid was studied. HL-60 myeloid differentiation was a continuously driven process; significant terminal cell differentiation occurred only after a minimum exposure to inducer of two division cycles. Cells so committed to differentiation retained a heritable, finite memory of differentiation commitment over a further division cycle. Prior to becoming committed, cells acquired precommitment memory of exposure to inducer. Precommitment memory abbreviated the subsequent exposure to inducer needed for commitment to differentiation. Precommitment memory was semistable. It was heritable, but was lost after four division cycles. The acquisition and loss of precommitment memory correlated with alterations in nuclear architecture detected by narrow angle light scatter using flow cytometry. The altered nuclear architecture first occurred before any overt cell differentiation or growth arrest. It was thus an early event in the induced program of terminal cell differentiation. Alterations in relative abundances of cytoplasmic proteins also occurred prior to overt cell differentiation or growth arrest. One of these was a 17 kdalton, anionic, probably Ca²⁺ binding, protein. Retinoic acid thus induced early cellular changes, including cytoplasmic and nuclear alterations, within one cell cycle when cell differentiation was not yet apparent.     

Hormonal requirements for the larval-pupal transformation of the epidermis of Manduca sexta in vitro

In the tobacco hornworm, Manduca sexta, metamorphosis occurs in response to two releases of ecdysone that occur 2 days apart. Epidermis was explanted from feeding final-instar larvae before the first release of ecdysone and was cultured in Grace's medium. When exposed to 1 μg/ml of β-ecdysone for 24 hr and then to hormone-free medium for 24 hr, followed by 5 μg/ml of β-ecdysone for 4 days, the epidermis produced tanned pupal cuticle in vitro. During the first 24 hr of exposure to β-ecdysone, the epidermis first changed its cellular commitment to that for pupal cuticle formation (ET₅₀ = 14 hr), then later (by 22 hr) it became committed to tan that cuticle. Then, for most of the pupal cuticle to be tanned, at least a 12-hr period of culture in hormone-free medium was required before the cuticle synthesis was initiated. Consequently, some events prerequisite to sclerotization of pupal cuticle not only occur during the ecdysone-induced change in commitment but also during the ecdysone-free period. When the tissue was preincubated in 3 μg/ml of juvenile hormone (JH I or a mimic epoxygeranylsesamole) for 3 hr and then exposed to both ecdysone and juvenile hormone for 24 hr, it subsequently formed larval cuticle. The optimal conditions for this larval cuticle formation were exposure to 5 μg/ml of β-ecdysone in the presence of 3 μg/ml of epoxygeranylsesamole for 48 hr. When the epidermis was cultured in Grace's medium for 3 days and then exposed to 5 μg/ml of β-ecdysone for 4 days, 70% of the pieces formed pupal cuticle. By contrast, if both ecdysone and JH were added, 77% formed larval cuticle. Therefore, the change from larval to pupal commitment of the epidermal cells requires not only the absence of JH, but also exposure to ecdysone.     

Consequences of parental exposure to epidermal growth factor for progeny cell division

The consequences of parental exposure to epidermal growth factor (EGF), for progeny cell cycle times was investigated. Slowly dividing mouse 3T3 fibroblasts were exposed to EGF for 8 hr, the EGF was withdrawn, and the cell cycle times of parental and progeny cells were measured by time-lapse video microscopy. It was observed that exposure to EGF induced a round of cell division following a lag phase of approximately 8 hr. The progeny of these cells exhibited accelerated cell cycle times compared to cells that had not been exposed to EGF. Parental cell division time was significantly correlated with progeny cell cycle time. Sibling progeny cell cycle times were also significantly correlated. EGF can therefore apparently exert an effect on the cell cycle times of more than one generation of cells.     

Commitment and proliferation kinetics of human lymphocytes stimulated in vitro: Effects of α-MM addition and suboptimal dose on concanavalin a response

We have examined the effect of alpha-methylmannoside (α-MM) addition to concanavalin A (con A)-stimulated peripheral human lymphocytes. With a previously established kinetic model, we have, from the time course of proliferation, extracted the responding clone size, rate of entry of this clone into S phase, and the length of the lag period. We have studied the effect of con A dose and time of addition of α-MM to optimally stimulated cells on these kinetic parameters. We show that neither a low dose of con A nor an early addition of --MM to optimally stimulated cells results in a change in the responding clone size. That is, all of the potentially responsive cells appear to become “committed” to enter the cell cycle regardless of the presence of α-MM early in the culture or in the presence of suboptimal stimulation. However, the rate at which these committed cells enter the first S phase is a function of the dose of con A and time of addition of α-MM and varies over a wide range. It is the variation in this parameter that accounts for virtually all of the diminished response previously interpreted as a time-dependent irreversible commitment of mitogen-stimulated cells. The previous work using only fixed time points for measuring thymidine uptake and the concept of commitment must be reevaluated in light of the kinetic evidence presented.     

A clonal analysis of the differentiation of 3T3-L1 preadipose cells: Role of insulin

Cells of the established preadipose line, 3T3-L1, appear to be undifferentiated fibroblasts during exponential growth. When cells become quiescent, a small percentage of them accumulate triglyceride and become morphologically indistinguishable from mature adipocytes. When insulin is added to quiescent cultures, up to 50% of the cells differentiate into adipocytes. The distribution of lipid-containing cells which appear in clusters of varying sizes was analyzed to determine whether commitment to differentiation occurred after quiescence or during exponential growth and whether insulin was required as an inducer of commitment. The spatial arrangement of 3T3-L1 cells at quiescence on some culture dishes was destroyed by replating. This resulted in random distribution of these cells. The distribution of adipocytes among replated and nonreplated cells in these experiments was compared to a computer generated random distribution of differentiated among undifferentiated cells. Dispersal of cells at confluence resulted in a distribution of fat among nonfat cells not significantly different from the computer generated random distribution. In undisturbed cultures, the distribution of fat cells is not random and is consistent with a commitment event in single cells at any cell division during exponential growth followed by divisions of both committed and uncommitted cells. Since insulin affected the number of mature adipocytes only when added after cessation of exponential growth, insulin is not the inducer of commitment but merely enhances lipid production in previously committed cells.     

Cell density,negative proliferation control,and phosphorylation of retinoblastoma protein

Cell density negative control (CDNC) of normal human fibroblast proliferation occurs after stimulation by mitogens with different signal transduction mechanism. Delayed exposure to agents that interfere with CDNC, such as doublestranded RNA and vanadate, reveals the existence of a biochemical event, involved in CDNC, that occurs 5–8 hr after the beginning of mitogenic stimulation. This is earlier than the point of “mitogenic commitment,” defined by the duration of mitogen exposure required for cell cycle entry (8–18 hr). Phosphorylation of the retinoblastoma gene product (pRB) begins 8–10 hr after mitogen stimulation and is nearly complete at 18 hr, just as the first cells enter S-phase. CDNC prevents pRB phosphorylation. Interferon β delays pRB phosphorylation by up to 20 hr but has little effect on the timing of mitogenic commitment. Thus mitogenic commitment is located in time between CDNC and pRB phosphorylation. When agents that cause a release from CDNC are applied to dense, negatively controlled cultures after 18 hr of EGF stimulation, pRB phosporylation occurs 6–8 hr after release. This suggests that the negatively controlled cells process the mitogenic signal but accumulate at a restriction point. The relatively early timing of CDNC-related events in the prereplicative phase raises the possibility that pRB phosphorylation is a consequence rather than a prerequisite for release from cell density negative control. © 1993 Wiley-Liss, Inc.     

Advance toward S phase and retreat toward deeper "G0" states in resting 3Y1 cells with environmental changes

To elucidate conditions which affect the lag time for resting cells to enter S phase after serum stimulation, we used a wild-type 3Y1 rat fibroblast line and four temperature-sensitive mutants of 3Y1 (3Y1tsD123, 3Y1tsF121, 3Y1tsG125, and 3Y1tsH203). Among these five lines, in only tsG125 cells was there an obviously prolonged lag time with increase in time in resting state at 33.8 degrees C. The resting wild-type 3Y1 cells, preexposed to 39.8 degrees C, also showed a prolongation of lag time. The prolongation in tsG125 had a certain limit. Preexposure to 39.8 degrees C before serum stimulation accelerated such prolongation in tsG125 to its limit, but did not change the limit, per se. Resting tsG125 cells stimulated by serum at 39.8 degrees C, did not enter S phase, yet they did advance toward S phase. When they were kept at 39.8 degrees C, they retreated toward a deeper resting state ("G0") with time. These retreats correlated with the decrease in stimulating activity in the culture media. About 20% of the resting tsG125 cells stimulated by serum at 39.8 degrees C were committed to enter S phase, when the extent of commitment was examined at 33.8 degrees C. Most of the tsG125 cells committed at 33.8 degrees C did not enter S phase, when the extent of commitment was examined at 39.8 degrees C. More cells were committed after stimulation at 33.8 degrees C than at 39.8 degrees C, when the test was done at 33.8 degrees C. We suggest that resting cells may be reversibly changed within range of resting states, in either direction, that is, advance toward S phase or retreat toward deeper "G0." These changes may be determined by alterations in the balance between synthesis and decay of the preparedness for the initiation of DNA synthesis caused by cellular response to environmental changes (e.g., medium activity, temperature, etc.). The ts defect in tsG125 may affect the cell cycle progression, both before and after commitment by serum.     

Changes in cell kinetics associated with differentiation of a human promyelocytic cell line (HL60)

Abstract. Terminal cell differentiation results in an irreversible arrest in the G₁ phase of the cell cycle and loss of the capacity for cell renewal. In the murine erythroleukaemia cell line (MELC), commitment to erythroid differentiation was found also to be preceded by an early, transient, phase of inhibition of growth due to prolongation of the G₁ phase. We determined the effect of differentiation-inducing agents on the growth kinetics of a human promyelocytic cell line (HL60) which undergoes differentiation into mature granulocyte. At concentrations of inducers optimal for cell differentiation, an early, transient stimulation of cell multiplication was found. DNA synthesis was enhanced in HL60 cells as early as 5 hr after exposure to inducer. Nevertheless, HL60 cell maturation eventually also resulted in a loss of the multiplication ability. The duration of exposure to inducer required for irreversible loss of the potential for self-renewal was determined by the fall in the cloning efficiency of induced cells; the results indicate that it preceded the switch-off of the replication mechanism; the majority of the cells lost their ability to form large colonies at the time of peak DNA synthesis and were able to complete an additional two to three cell cycles at a rate similar to uninduced cells. These changes occurred before HL60 cells became committed and might play a pivotal role in the process of cell differentiation.     

A satellite cell mitogen from crushed adult muscle

Single fiber-satellite cell units from skeletal muscle of adult rats were used to study the regulation of satellite cell proliferation. The satellite cells remained quiescent during culture in serum-containing medium but could be induced to enter the cell cycle by exposure to a saline extract of crushed adult muscle. The activity in the extract has a molecular weight greater than 30K and is heat and trypsin sensitive. The mitogenic activity does not result from transferrin. Little or no activity was obtained from crushed extracts of heterologous tissues. Proliferation of myogenic cells from rat embryos was also stimulated by the muscle mitogen but growth of muscle fibroblasts was not enhanced. The time response of satellite cell proliferation after exposure to the muscle mitogen showed that the cells enter DNA synthesis after a lag period of 18 hr and proliferate with a generation time of 12 hr. This confirms that satellite cells in adult muscle are in G0, or an extended G1. The mitogen is also effective in stimulating muscle growth and myoblast fusion in vivo when injected into 1-week-old rat pups. These experiments suggest that muscle regeneration is initiated by the release of an endogenous mitogen from traumatized muscle.     

Synchronization of MEL cell commitment with cordycepin.

The response of differentiating MEL cells to the nucleotide analogue cordycepin reveals a previously unrecognized aspect of the molecular events which cause commitment of these cells to terminal erythroid differentiation. Cordycepin rapidly inhibits commitment of DMSO-treated MEL cells in a dose range which does not cause cytotoxicity. Reversal of cordycepin treatment in the presence of inducer leads to a rapid and synchronous commitment of a significant proportion of cells in the culture. These results suggest that MEL cells can be blocked just prior to the point of commitment by cordycepin treatment.     

Mechanism of action of cholera toxin: Studies on the lag period

Summary The lag period for activation of adenylate cyclase by choleragen was shorter in mouse neuroblastoma N18 cells than in rat glial C6 cells. N18 cells have 500-fold more toxin receptors than C6 cells. Treatment of C6 cells with ganglioside G_M1 increased the number of toxin receptors and decreased the lag phase. Choleragen concentration also effected the lag phase, which increased as the toxin concentration and the amount of toxin bound decreased. The concentration, however, required for half-maximal activation of adenylate cyclase depended on the exposure time; at 1.5, 24, and 48 hr, the values were 200, 1.1., and 0.35pm, respectively. Under the latter conditions, each cell was exposed to 84 molecules of toxin.The length of the lag period was temperature-dependent. When exposed to choleragen at 37, 24, and 20 °C, C6 cells began to accumulate cyclic AMP after 50, 90, and 180 min, respectively. In G_M1-treated cells, the corresponding times were 35, 60, and 120 min. Cells treated with toxin at 15 °C for up to 22 hr did not accumulate cAMP, whereas above this temperature they did. Antiserum to choleragen, when added prior to choleragen, completely blocked the activation of adenylate cyclase. When added after the toxin, the antitoxin lost its inhibitory capability in a time and temperature-dependent manner. Cells, however, could be preincubated with toxin at 15 °C, and the antitoxin was completely effective when added before the cells were warmed up. Finally, cells exposed to choleragen for >10 min at 37 °C accumulated cyclic AMP when shifted to 15 °C. Under optimum conditions at 37°C, the minimum lag period for adenylate cyclase activation in these cells was 10 min. These findings suggest that the lag period for cholerage action represents a temperature-dependent transmembrane event, during which the toxin (or its active component) gains access to adenylate cyclase.Abbreviations used: ganglioside nomenclature according to Svennerholm [32] (see Table 1 for structures) cAMP adenosine 35-monophosphate - MIX 3-isobutyl-1-methylxanthine - HEPES N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid - PBS phosphate-buffered saline (pH 7.4)