Proc. Natl. Acad. Sci. USA Vol. 87, pp. 123-127, January 1990
Cell Biology
Pasteurella multocida toxin: Potent mitogen for cultured fibroblasts (cell regulation/signal transduction/inositol phosphates/DNA synthesis)
ENRIQUE ROZENGURT*, THERESA HIGGINS*, NEIL CHANTERt, ALISTAIR J. LAXt,
AND
JAMES M. STADDON*
*Imperial Cancer Research Fund, P.O. Box 123, Lincoln's Inn Fields, London WC2A 3PX, United Kingdom; and tAgricultural and Food Research Council Institute for Animal Health, Compton, Newbury, Berkshire RG16 ONN, United Kingdom Communicated by Leon A. Heppel, October 16, 1989
Native Pasteurella multocida toxin (PMT) is ABSTRACT shown to be an extremely potent mitogen for Swiss 3T3 fibroblasts. Half-maximal stimulation of DNA synthesis was obtained at concentrations of 1 and 2 pM for recombinant PMT (rPMT) and PMT, respectively. The degree of rPMT-induced DNA synthesis was comparable to that elicited by 10% fetal bovine serum and, moreover, was observed in the complete absence of other factors. Cell proliferation was also enhanced by rPMT. The toxin was also a potent mitogen for BALB/c and NIH 3T3 cells, 3T6 cells, and tertiary mouse embryo or human fibroblasts. The mitogenic activity of rPMT was heat-labile. A polyclonal antiserum to PMT inhibited DNA synthesis when added early, but not late, during treatment of the Swiss 3T3 cells with rPMT. A similar time-dependent action of methylamine was also observed. Furthermore, transient exposure of the cells to rPMT at 37C, but not at 4°C, resulted in a stimulation of DNA synthesis. Thus, toxin action may require cell entry and processing via an acidic compartment. The toxin, at mitogenic concentrations, caused a large increase in the production of inositol phosphates. In contrast, rPMT did not increase the intracellular concentration of cyclic AMP in Swiss 3T3 cells. The basis of rPMT action may afford a unique insight into molecular signaling events involved in the control of cell proliferation.
The elucidation of the mechanisms by which growth factors regulate cellular mitogenesis remains one of the fundamental problems in biology and may prove crucial for understanding the unrestrained proliferation of cancer cells. In this respect cultured fibroblasts, such as murine 3T3 cells, have emerged as a model system. These cells cease to proliferate when they deplete the medium of its growth-promoting activity and enter a quiescent or nondividing state. However, such cells remain viable and can be stimulated to reinitiate DNA synthesis and cell division either by replenishing the medium with fresh serum or by the addition of polypeptide growth factors, neuropeptides, and pharmacological agents in serum-free medium (1). Stud-es performed with such growtharrested cells and defined combinations of growth factors have revealed the existence of multiple growth factoractivated signaling pathways that synergistically lead to a mitogenic response (1, 2). Hence, the discovery of additional mitogens can provide novel approaches to explore cellular signaling pathways leading to cell proliferation. Native Pasteurella multocida toxin (PMT) has been identified as a causative agent of atrophic rhinitis (3, 4), a disease of growing pigs characterized by destruction of the nasal turbinate bones (reviewed in ref. 5). The toiin has been purified in several laboratories (3, 6-8) and consists of a single polypeptide chain of ==150 kDa. Recently, the gene for the toxin has been cloned and expressed in Escherichia coli (9). Purified recombinant PMT (rPMT) is indistinguishable
from the native toxin in its antigenicity and toxicity for either experimental animals or cultured embryonic bovine lung cells (9). The molecular basis of the actions of PMT remains unknown. Bacterial toxins have provided novel approaches to elucidate cellular and molecular regulatory mechanisms (10-12). The present studies were initiated to determine whether PMT induced any cytotoxic effect on cultured fibroblasts. Surprisingly, we discovered that either PMT or rPMT is an extremely potent mitogen for 3T3 cells. The toxin, at picomolar concentrations, enters these cells and irreversibly triggers DNA synthesis and cell proliferation.
MATERIALS AND METHODS Methods. Cell culture procedures (13, 14), assays of growth promoting activity by [3H]thymidine incorporation (15) or by cell number (14), and measurements of cAMP (16) and total inositol phosphates (17) were performed as previously described. Cloning, expression, and purification of rPMT were also as described (9). rPMT was subjected to gas/liquidphase microsequencing on an Applied Biosystems 477A protein sequencer with on-line phenylthiohydantoin-conjugated amino acid analysis (Applied Biosystems model 120A phenylthiohydantoin analyzer) using the reagents and solvents supplied by the manufacturer. Materials. Bombesin, vasopressin, epidermal growth factor (EGF), 8-bromo-cAMP, cholera toxin, and insulin were obtained from Sigma. Fetal bovine serum was from GIBCO. Platelet-derived growth factor (PDGF) (recombinant homodimer of B chains), recombinant fibroblast growth factor (FGF) (basic) (rFGFb), [3H]thymidine, [2-3H]inositol, and antigens and antibodies for radioimmunoassay of cAMP were from the Radiochemical Centre. All other chemicals were of the purest grade commercially available.
RESULTS PMT and rPMT Induce DNA Synthesis in Murine Swiss 3T3 Cells. PMT was an extremely potent inducer of DNA synthesis in Swiss 3T3 cells (Fig. 1 Left). Half of the maximal effect was obtained at a concentration as low as 0.32 ng/ml (-2 pM). The maximal effect, obtained at 1.25 ng/ml (9 pM), was equivalent to the stimulation of DNA synthesis induced by medium containing 10%o (vol/vol) fetal bovine serum. Thus, in contrast to most known mitogens for Swiss 3T3 cells (1), PMT at picomolar concentrations induced maximal DNA synthesis in the absence of any other synergistic factor. Several lines of evidence indicate that the potent mitogenic effect of the PMT preparations is in fact mediated by the 150-kDa protein. The gene coding for PMT has been cloned and expressed in E. coli, and rPMT was purified as described Abbreviations: PMT, native P. multocida toxin; rPMT, recombinant PMT; PDGF, platelet-derived growth factor; EGF, epidermal growth factor; FGF, fibroblast growth factor; rFGFb, recombinant FGF (basic).
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 123
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Proc. Natl. Acad. Sci. USA 87 (1990)
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FIG. 1. Stimulation of DNA synthesis by PMT and rPMT in Swiss 3T3 cells. (Left) Dose-response curve for the stimulation of DNA synthesis by PMT. Confluent, quiescent cultures of Swiss 3T3 cells were washed and incubated at 370C in 2 ml of Dulbecco's modified Eagle's medium (DMEM)/Waymouth medium, 1:1 (vol/ vol), containing 1 /LCi (1 ;uCi = 37 kBq) of [3H]thymidine per ml and various concentrations of native toxin. After 40 hr, DNA synthesis was assessed by measuring the level of [3H]thymidine incorporated into half of the acid-precipitable material. Each point is the mean of determinations from three independent experiments expressed as a percentage of the incorporation induced by lo fetal bovine serum, which ranged from 396 x 103 to 435 x 103 cpm. (Right) Doseresponse curve for the stimulation by purified rPMT. DNA synthesis was measured as above, and each point represents the mean of determinations from five independent experiments expressed as a percentage of the incorporation given by 10%o fetal bovine serum, which ranged from 554 x 103 to 816 x 103 cpm.
for the native toxin (9). The preparation contained a major band of 150 kDa that was identical to that obtained with PMT. The N terminus of rPMT was not blocked, and hence its amino acid sequence was analyzed. The sequence obtained, Met-Lys-Ile-Lys-His-Phe-Phe-Asn-Ser-Asp-Phe-Thr-Val, is not identical or homologous to any known protein. Fig. 1 Right shows that rPMT stimulated DNA synthesis in Swiss 3T3 cells somewhat more potently than did- PMT. The half-maximal effect was achieved at 0.15 ng/ml (1 pM), with a maximum effect at 1.25 ng/ml. PMT is a heat-labile toxin (5). Fig. 2 shows that 30-min incubation at 57TC of the rPMT preparation resulted in a striking decrease in mitogenic activity. A 60-min preincubation at 57TC caused complete loss of mitogenic activity. When the toxin was incubated'at 570C for various times and then assayed at 2 ng/ml, the mitogenic activity decreased rapidly (Fig. 2 Inset). The mitogenic activity of rPMT was completely abolished by prior incubation with a polyclonal antiserum to PMT (Fig. 3 Upper). Immunoblot analysis shows that the only protein recognized by this neutralizing antiserum is rPMT (Fig. 3 Upper Inset). The antiserum did not prevent the stimulation of DNA synthesis induced by other mitogens including polypeptide'growth factors, neuropeptides, pharmacological agents, or cholera toxin (Fig. 3 Lower). The data depicted in Figs. 1-3 indicate that both PMT and rPMT are extremely potent mitogens for 3T3 cells. Induction of DNA Synthesis by Transient Exposure to rPMT. Many bacterial toxins bind to the external surface of the plasma membrane, enter the cells, and therefore cannot be removed by extensive washing or neutralized by exogenous antibodies (reviewed in ref. 18). To test whether rPMT acts in this manner to stimulate mitogenesis, quiescent 3T3 cells were incubated at 370C with rPMT at 1, 5, or 20 ng/ml for various times. When the cells were exposed for 30 min to 20 ng of rPMT per ml, washed extensively, and incubated in medium without the toxin, the level of [3H]thymidine incorporation was similar to that induced in the cultures incubated continuously with rPMT (Fig. 4 Left). Lower concentrations of rPMT required longer preincubation times to induce maximal DNA synthesis after removal of unbound toxin.
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FIG. 2. Heat lability of the mitogenic activity of rPMT. Various concentrations of toxin were incubated at 570C for 0 (M), 30 (0), and 60 (i) min. The ability of rPMT to stimulate DNA synthesis in confluent, quiescent Swiss 3T3 cells was then measured as described in the legend to Fig. 1. Each point represents the mean of two determinations, and the addition of 10%6' fetal bovine serum gave an incorporation of 674 x i03 cpm. (Inset) Time course of the heat inactivation of rPMT. Aliquots of toxin were incubated at 230C (W) or 570C (u) for various times from 0 to 30 min. DMEM/Waymouth medium (1:1) was added to each aliquot to give a final toxin concentration of 2 ng/ml, and [3H]thymidine incorporation was measured as in Fig. 1. Each point represents the mean of two determinations; 10%o fetal bovine serum gave a level-of incorporation of 744 x 103 cpm.
In other experiments, the cells were preincubated with 5 ng of rPMT per ml for various times and then transferred to media in the absence or presence of PMT antiserum (Fig. 4 Right). When the cells were treated with 5 ng of rPMT per ml for 1 hr, subsequent DNA synthesis was markedly inhibited in cultures transferred to medium containing antiserum. In contrast, after 3 hr of incubation with rPMT, the mitogenic effect of the toxin was no longer blocked by the addition of antiserum. The entry of many bacterial toxins into the cytoplasm is prevented by incubation at 40C or by addition of membranepermeant weak bases such as methylamine (18). Fig. 5 Left shows that methylamine inhibited the stimulation of DNA synthesis by rPMT in a concentration-dependent manner. At similar concentrations, methylamine had only a slight inhibitory effect on mitogenesis induced by EGF and insulin. Furthermore, methylamine became less inhibitory as the time interval between the addition of rPMT and methylamine increased; the amine did not prevent DNA synthesis when added 3 hr after rPMT (Fig. 5 Right). A transient exposure (90 min) of 3T3 cells to 5 ng of rPMT per ml at 40C, instead of at 370C, did not stimulate mitogenesis during a subsequent incubation at 370C in rPMT-free medium. rPMT Stinulates Cell Proliferation. Addition of rPMT at 10 ng/ml to the medium in which confluent and quiescent 3T3 cells were grown (depleted medium) resulted in loss of density-dependent inhibition of growth (Fig. 6A). rPMT stimulated reinitiation of cell proliferation in a concentrationdependent manner (Fig. 6B). In other experiments, addition of rPMT to subconfluent 3T3 cells resulted in a striking increase in cell proliferation; the final saturation density increased 6-fold (Fig. 6C). In view ofthe results shown in Fig. 4, we tested whether a transient exposure to rPMT would be sufficient to stimulate cell proliferation in toxin-free medium. When quiescent cultures were exposed to 10 ng of rPMT per ml for 24 hr and then washed, trypsinized, and replated in the absence of the toxin, the subsequent growth of rPMTpretreated cells was markedly enhanced (Fig. 6C Inset).
Cell
Biology: Rozengurt et al.
Proc. Natl. Acad. Sci. USA 87 (1990) C800
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FIG. 4. Transient exposure to rPMT stimulates DNA
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during a subsequent incubation in toxin-free medium. (Left) Effect of exposure time to rPMT upon its ability to induce mitogenesis in Swiss 3T3 cells. Confluent, quiescent cultures of Swiss 3T3 cells were washed twice with DMEM and incubated at 370C for various times without (o) or with 1 ng (e), 5 ng (n), or 25 ng (o) of rPMT per ml. After various incubation times, the cells were washed five times with
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FIG. 3. Inhibition of the mitogenic action of rPMT by a specific antiserum. (Upper) Dose-response curve for the effect of immune (e) and preimmune (o) serum on DNA synthesis induced by rPMT. Various concentrations of immune or preimmune serum were added to 1:1 DMEM/Waymouth medium containing 1 ,uCi of [3H]thymidine per ml and 1 ng of rPMT per ml and were incubated for 1 hr at room temperature; 2 ml of this was then added to confluent, quiescent Swiss 3T3 cells, and DNA synthesis was measured as in Fig. 1. Each point is the mean of two determinations; 10% fetal bovine serum induced an incorporation of 809 x 103 cpm. (Inset) Analysis of rPMT by SDS/8% PAGE and silver staining (lane A) or by immunoblotting (lane B). Equal aliquots of rPMT (1.5 Atg) were used. The blots were probed with the neutralizing antiserum usWl above (diluted 1:500). Bound immunoglobulins were detected by using a rabbit anti-pig IgG conjugated with alkaline phosphatase. No signal was obtained when the blots were probed with the preimmune serum instead of the immune serum. (Lower) Specificity of the antiserum tQ rPMT. Various growth promoting factors were incubated at room temperature with (hatched bars) or without (open bars) 1 Al of antiserum per ml for 1 hr in 1:1 DMEM/Waymouth medium containing insulin at 1 ,ug/ml and [3H]thymidine at 1 jiCi/ml. Then, 2 ml of each medium was added to confluent, quiescent Swiss 3T3 cells, and DNA synthesis was measured as in Fig. 1. Each point is the mean of two determinations. The concentrations used were as follows: rPMT, 1 ng/ml; PDGF, 5 ng/ml; EGF, 5 ng/mI; FGF, 2 ng/ml; bombesin (Born), 10 ng/ml; vasopressin (VP), 10 ng/ml; phorbol 12,13-dibutyrate (PDBu), 100 ng/ml; 8-bromo-cAMP (8BrcAMP), 2.5 mM; cholera toxin (CT), 10 ng/ml; and isobutylmethylxanthine (IBMX), 50 ,uM.
rPMT Induces DNA Synthesis and Cell Division in Other Cells. Several murine cell lines including BALB/c 3T3, NIH 3T3, or 3T6 cells, which were rendered quiescent by growth to confluency (BALB/c and NIH 3T3) or by incubation in 0.5% serum (3T6), responded to rPMT with a striking increase in cell proliferation. The growth-promoting effects of rPMT were not confined to established cell lines. The toxin also stimulated DNA synthesis (Fig. 7A) and proliferation (Fig. 7B) in tertiary cultures of mouse embryo cells. The mitogenic effect persisted after transient exposure to rPMT, and it was blocked by 10 mM methylamine (results not shown). Table 1 shows that rPMT stimulated [3H]thyrnidine incorporation in quiescent cultures of human fibroblasts. The toxin was more effective than PDGF, EGF, or FGF in these cells. The effect of rPMT was enhanced in the presence of these factors (Table 1). Effect of rPMT on cAMP Levels and Inositol Phosphate Production. As an initial attempt to elucidate the mode of action of PMT, we measured the effect of this toxin on two
DMEM and then incubated for 40 hr in 1:1 DMEM/Waymouth medium containing 1 ,tCi of [3H]thymidine per ml. In addition, one set of dishes was washed seven times and then continuously exposed to each concentration of toxin throughout the 40-hr incubation (values given at 40 hr). DNA synthesis was measured as in Fig. 1. In this experiment, 10%o fetal bovine serum gave a level of incorporation of 826 x 103 cpm. (Right) The effect of a transient exposure to rPMT upon its ability to induce mitogenesis in the presence or absence of specific antiserum. Confluent, quiescent cultures of Swiss 3T3 cells were washed twice with DMEM and incubated at 370C with 5 ng of rPMT for various times up to 3 hr. The cells were then washed five times with DMEM and incubated for 40 hr in DMEM/Waymouth medium containing 1 uCi of[3H]thymidine per ml with (o) or without (W) 0.5 /il of antiserum per ml. Some cells were incubated with toxin throughout the 40-hr incubation. The arrow points to the level of DNA synthesis achieved when cells were exposed to toxin preincubated with antiserum for 1 hr. Each point is the mean of two determinations; in this experiment 10% fetal bovine serum gave an incorporation of 779 x 103 cpm.
major transmembrane signaling systems, cAMP and inositol phosphate production. Fig. 8A shows that rPMT at 20 ng/ml did not increase the intracellular level of cAMP, whereas el
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FIG. 5. Early but not late addition of methylamine inhibits the mitogenic action of rPMT. (A) Various concentrations of methylamine hydrochloride (pH 7.4) were added to 1:1 DMEM/Waymouth medium containing 1 ,uCi of [3Hlthymidine per ml with either 1 ng of toxin (e) or 5 ng of EGF per ml plus 1 Aig of insulin per ml (o). Confluent, quiescent Swiss 3T3 cells were incubated in this medium for 40 hr; then DNA synthesis was measured as in Fig. 1. Each point is the mean of two determinations, and values are expressed as the percentage of the incorporation given by l10o fetal bovine serum (795 x 104 CpM). (B) Confluent, quiescent cultures of Swiss 3T3 cells were
washed twice with DMEM and incubated at 37°C in 2 ml of 1:1
DMEM/Waymouth medium containing 1 ,uCi of [H]thymidine and I ng of rPMT per ml. At various times from 0 to 6 hr, methylamine hydrochloride was added to the cultures to give a final concentration of 9 mM. DNA synthesis was measured after 40 hr. Each point is the mean of two determinations and is expressed as the percentage of the
incorporation given by 1o fetal bovine serum (684 x 103 cpm).
126
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Proc. Natl. Acad. Sci. USA 87 (1990) CF
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FIG. 6. rPMT induces cell proliferation in Swiss 3T3 cells. (A) rPMT stimulates reinitiation of growth in confluent Swiss 3T3 cells. rPMT was added directly to the medium of Swiss 3T3 cell cultures to give a final concentration of 10 ng/ml 5 days after plating. Two, 3, and 4 days after this addition, cells from both treated (hatched bars) and untreated (open bars) cultures were trypsinized and counted in a Coulter counter. The values shown are means SEM (n = 5). (B) Dose-response curve for the effect of rPMT on the growth of confluent Swiss 3T3 cells. Various concentrations of rPMT were added to the medium of Swiss 3T3 cell cultures 5 days after plating. The cells were trypsinized and counted 4 days later. Each value is the mean SEM (n = 5). (C) Effect of rPMT on the growth of subconfluent Swiss 3T3 cells. Cells (5 x 104) were seeded onto 33-mm petri dishes in 2 ml of DMEM supplemented with 10o fetal bovine serum. After 24 hr, 10 ng of rPMT per ml was added to half of these cultures, and the number of treated cells (e) or untreated cells (a) were counted over the next 11 days. Each value is the mean + SEM (n 5). (Inset) rPMT induces growth of Swiss 3T3 cells after trypsinization and subsequent replating. rPMT at 10 ng/ml (e) was added to confluent Swiss 3T3 cells 5 days after plating. An equivalent volume of toxin carrier was added to parallel dishes (o). After 24 hr the cells were typsinized and seeded at 5 x 104 cells per 33-mm dish in DMEM supplemented with 10% fetal bovine serum and in the absence oftoxin. Cells were counted over the next 8 days. Each point is the mean SEM (n = 5). =
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cholera toxin, added for comparison, caused a marked accumulation of cAMP in parallel cultures. Similar results were obtained when rPMT was added at various concentrations (Fig. 8B) or in the presence of the potent inhibitors of cAMP phosphodiesterase, 1-methyl-3-isobutylxanthine at 500 ,uM plus 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidine at 100 ttM (results not shown). In contrast to the lack of stimulatory effect of rPMT on cAMP accumulation, the toxin caused a dramatic and timedependent increase in the production of inositol phosphates (Fig. 8C). After 5 hr of incubation, rPMT at 5 ng/ml caused a 25-fold increase in the accumulation of inositol phosphates. Chromatographic analysis of the inositol phosphates revealed increases in the inositol monophosphate, bisphosphate, and trisphosphate fractions (results not shown). When the toxin was added at 1 ng/ml, the stimulation occurred after a longer lag (3 hr), but it reached the same magnitude as the rapid increase in inositol phosphate accumulation induced by a maximally effective concentration of bombesin (Fig. 8C). Methylamine (10 mM) completely blocked the striking in-
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FIG. 7. rPMT is a mitogen for tertiary mouse embryo cells. (A) Dose-response curve for the effect of rPMT on DNA synthesis in mouse embryo fibroblasts (MEF). Third passage MEFs were seeded at 105 cells per 33-mm dish in DMEM supplemented with 10%o fetal bovine serum. After 3 days the cultures were switched to DMEM containing 0.5% fetal bovine serum, and after a further 4-6 days the cultures were confluent and quiescent. The cultures were then washed twice with DMEM and incubated in 2 ml of 1:1 DMEM/ Waymouth medium containing 1 1sCi of [3H]thymidine per ml and various concentrations of rPMT. After 40 hr the level of DNA synthesis was assessed as in Fig. 1. In this experiment 10% fetal bovine serum gave a level of incorporation of 267 x 103 cpm. (B) rPMT stimulates growth of MEF cells. Third passage MEFs were seeded at 105 cells per 33-mm dish in DMEM supplemented with 10% fetal bovine serum. After 24 hr the medium was changed to DMEM supplemented with 1% fetal bovine serum with (e) or without (o) rPMT at 10 ng/ml. Cells were trypsinized and counted at intervals over the next 14 days. The values are means ± SEM (n = 5).
crease in the production of inositol phosphates caused by exposure to rPMT.
DISCUSSION The present results show that PMT is a novel and potent mitogen for cultured animal cells. Several lines of evidence, including the use of purified rPMT, indicate that this mitogenic activity is in fact mediated by the toxin, a single chain polypeptide of 150 kDa. Either PMT or rPMT induced DNA synthesis at picomolar concentrations in the absence of any other exogenously added growth-promoting factor. Maximum stimulation was equivalent to that promoted by the addition of fresh serum. These features distinguish the mitogenic effect of PMT from those of all other known growth factors for Swiss 3T3 cells (1). PDGF and bombesin, at nanomolar concentrations, induce DNA synthesis in a fraction of the cell population-i.e., they are less potent and effective than rPMT. Most other polypeptide growth factors, neuropeptides, and pharmacological agents stimulate DNA synthesis only in synergistic combinations (1, 2). So far, Table 1. Effect of various growth factors on DNA synthesis in human fibroblasts in the absence or presence of rPMT
[3H]Thymidine incorporation, Growth cpm x 10-3 With rPMT factor Without rPMT 60 9 None 75 16 Insulin 28 82 rFGFb 38 104 EGF 119 33 PDGF The concentrations used were as follows: rPMT, 10 ng/ml; insulin, 1 ,ug/ml; rFGFb, 5 ng/ml; EGF, 5 ng/ml; and PDGF, 10 ng/ml. Alone, 10% fetal bovine serum gave an incorporation of 139 X 103 cpm and 118 x 103 cpm in the presence of rPMT. The human foreskin fibroblasts were used at the 18th passage and rendered quiescent by incubation in 0.5% fetal bovine serum for 4 days. Cumulative [3H]thymidine incorporation was measured as in Fig. 1.
Cell
Proc. Natl. Acad. Sci. USA 87 (1990)
Biology: Rozengurt et al. B 100
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FIG. 8. Effect of rPMT on cAMP levels and inositol phosphate content of Swiss 3T3 fibroblasts. Cell culture and the determination
of cyclic AMP and inositol phosphates are as described. The cyclic AMP content of control cells (o) at various times and the influence ofrPMT at 20 ng/ml (e) and of cholera toxin at 100 ng/ml (A) is shown in A. Similar observations were made on several other cell preparations. (B) Cells were treated for 8 hr with the indicated concentrations of rPMT after which time the cellular content of cyclic AMP was determined. All incubations received the same concentrations of toxin carrier. The effect of a 30-min exposure to 25 AuM forskolin is illustrated for comparative purposes. The values are means SD of quadruplicate values obtained from one cell preparation. (C) Effect of 1 ng (e) and 5 ng (o) of rPMT per ml on the cellular content of inositol phosphates, with respect to control cultures (o). The cultures were labeled for 16 hr with 2 ml of 1:1 DMEM/Waymouth medium containing 10,uCi of [2-3H]inositol. rPMT was added as indicated, and 20 min prior to sampling, 1 M LiCl was added to give a final concentration of 20 mM. All cultures received the same amount of toxin carrier. Bombesin (A) was added 7 hr after the start of the experiment to give a final concentration of 10 nM. Similar time- and dose-dependent rPMT-induced increases in the cellular content of inositol phosphates were observed in several other cell preparations. ±
rPMT is the most potent and effective mitogen for Swiss 3T3 cells that has been identified. The growth-promoting effects of rPMT were not confined to established cell lines. Tertiary cultures of mouse embryo cells and human fibroblasts were also induced to resume DNA synthesis by addition of rPMT in the absence of other factors. A variety of bacterial toxins bind to the external surface of animal cells, become translocated to an intracellular compartment, acquire biological activity, and then irreversibly modify the molecular processes of the target cell (10-12). Our evidence suggests that the induction of cell proliferation by rPMT follows a similar sequence of events. Methylamine, an agent that increases endosomal and lysosomal pH (18) and thereby inhibits the processing and entry of many macromolecular ligands, viruses, and toxins into the cytoplasm (19,
127
20), completely and selectively prevented the induction of DNA synthesis by rPMT when added at early times. Similarly, after minutes of exposure to rPMT, the toxin was accessible to a neutralizing PMT antiserum. Thereafter, the mitogenic action of rPMT was no longer sensitive to either PMT antiserum or to methylamine, suggesting that the toxin is translocated to an intracellular location via an acidic compartment. A crucial prediction of this model is that a transient exposure to rPMT should be sufficient to stimulate DNA synthesis in toxin-free medium. This was indeed the case. In view of the remarkable potency, persisting action and heat lability of the toxin, it is plausible that the intracellular effects of rPMT are of an enzymatic nature, a proposition that requires further experimental work. Intracellularly acting bacterial toxins frequently alter key components in the signal transduction process (11, 12). Thus, a first step toward identifying possible intracellular targets of PMT in cultured fibroblasts requires the determination of the effects of the toxin on some of the early signals leading to mitogenesis (1). In this context, we found that rPMT does not increase the intracellular level of cAMP. In contrast, rPMT caused a striking stimulation of inositol phosphate turnover, a signal transduction mechanism leading to multiple cellular responses (21) including cell growth (1). None of the previously described bacterial toxins that act intracellularly stimulated this transmembrane signaling system. Thus, PMT may provide a novel tool to identify components of the complex cascade of molecular events involved in the initiation of cell proliferation. 1. Rozengurt, E. (1986) Science 234, 161-166. 2. Rozengurt, E., Erusalimsky, J., Mehmet, H., Morris, C., Nanberg, E. & Sinnett-Smith, J. (1988) Cold Spring Harbor Symp. Quant. Biol. 53, 945-954. 3. Chanter, N., Rutter, J. M. & Mackenzie, A. (1986) J. Gen. Microbiol. 132, 1089-1097. 4. Dominick, M. A. & Rimler, R. B. (1988) Vet. Pathol. 25, 17-27. 5. Chanter, N. & Rutter, J. M. (1989) in Pasteurella and Pasteurellosis, eds. Adlam, C. & Rutter, J. M. (Academic, New York), pp. 161-195. 6. Nakai, T., Sawata, A., Tsuji, M., Samejima, Y. & Kume, K. (1984) Infect. Immun. 46, 429-434. 7. Rimler, R. B. & Brogden, K. A. (1986) Am. J. Vet. Res. 47, 730-737. 8. Foged, N. T. (1988) Infect. Immun. 56, 1901-1906. 9. Lax, A. J. & Chanter, N. (1989) J. Gen. Microbiol., in press. 10. Simpson, L. L. (1986) Annu. Rev. Pharmacol. Toxicol. 26, 427-453. 11. Habermann, E. & Dreyer, F. (1986) Curr. Top. Microbiol. Immunol. 129, 94-179. 12. Pfeuffer, T. & Halmreich, E. J. M. (1988) Curr. Top. Cell. Regul. 29, 129-216. 13. Rozengurt, E., Mierzejewski, K. & Wigglesworth, N. (1978) J. Cell. Physiol. 97, 241-252. 14. Rozengurt, E. & Sinnett-Smith, J. (1983) Proc. Nati. Acad. Sci. USA 80, 2936-2940. 15. Dicker, P. & Rozengurt, E. (1980) Nature (London) 287, 607-612. 16. Rozengurt, E., Legg, A., Strang, G. & Courtenay-Luck, N. (1981) Proc. Natl. Acad. Sci. USA 78, 4392-4396. 17. Nanberg, E. & Rozengurt, E. (1988) EMBO J. 7, 2741-2748. 18. Middlebrook, J. L. & Dorland, R. B. (1984) Microbiol. Rev. 48, 199-221. 19. Merion, M., Schlesinger, P., Brooks, R. M., Moehring, J. M., Moehring, T. J. & Sly, W. S. (1983) Proc. Natl. Acad. Sci. USA 80, 5315-5319. 20. Goldstein, J. L., Brown, M. S., Anderson, R. G. W., Russell, D. W. & Schneider, W. J. (1985) Annu. Rev. Cell Biol. 1, 1-39. 21. Berridge, M. J. (1987) Annu. Rev. Biochem. 56, 159-193.
Cell Biology
Pasteurella multocida toxin: Potent mitogen for cultured fibroblasts (cell regulation/signal transduction/inositol phosphates/DNA synthesis)
ENRIQUE ROZENGURT*, THERESA HIGGINS*, NEIL CHANTERt, ALISTAIR J. LAXt,
AND
JAMES M. STADDON*
*Imperial Cancer Research Fund, P.O. Box 123, Lincoln's Inn Fields, London WC2A 3PX, United Kingdom; and tAgricultural and Food Research Council Institute for Animal Health, Compton, Newbury, Berkshire RG16 ONN, United Kingdom Communicated by Leon A. Heppel, October 16, 1989
Native Pasteurella multocida toxin (PMT) is ABSTRACT shown to be an extremely potent mitogen for Swiss 3T3 fibroblasts. Half-maximal stimulation of DNA synthesis was obtained at concentrations of 1 and 2 pM for recombinant PMT (rPMT) and PMT, respectively. The degree of rPMT-induced DNA synthesis was comparable to that elicited by 10% fetal bovine serum and, moreover, was observed in the complete absence of other factors. Cell proliferation was also enhanced by rPMT. The toxin was also a potent mitogen for BALB/c and NIH 3T3 cells, 3T6 cells, and tertiary mouse embryo or human fibroblasts. The mitogenic activity of rPMT was heat-labile. A polyclonal antiserum to PMT inhibited DNA synthesis when added early, but not late, during treatment of the Swiss 3T3 cells with rPMT. A similar time-dependent action of methylamine was also observed. Furthermore, transient exposure of the cells to rPMT at 37C, but not at 4°C, resulted in a stimulation of DNA synthesis. Thus, toxin action may require cell entry and processing via an acidic compartment. The toxin, at mitogenic concentrations, caused a large increase in the production of inositol phosphates. In contrast, rPMT did not increase the intracellular concentration of cyclic AMP in Swiss 3T3 cells. The basis of rPMT action may afford a unique insight into molecular signaling events involved in the control of cell proliferation.
The elucidation of the mechanisms by which growth factors regulate cellular mitogenesis remains one of the fundamental problems in biology and may prove crucial for understanding the unrestrained proliferation of cancer cells. In this respect cultured fibroblasts, such as murine 3T3 cells, have emerged as a model system. These cells cease to proliferate when they deplete the medium of its growth-promoting activity and enter a quiescent or nondividing state. However, such cells remain viable and can be stimulated to reinitiate DNA synthesis and cell division either by replenishing the medium with fresh serum or by the addition of polypeptide growth factors, neuropeptides, and pharmacological agents in serum-free medium (1). Stud-es performed with such growtharrested cells and defined combinations of growth factors have revealed the existence of multiple growth factoractivated signaling pathways that synergistically lead to a mitogenic response (1, 2). Hence, the discovery of additional mitogens can provide novel approaches to explore cellular signaling pathways leading to cell proliferation. Native Pasteurella multocida toxin (PMT) has been identified as a causative agent of atrophic rhinitis (3, 4), a disease of growing pigs characterized by destruction of the nasal turbinate bones (reviewed in ref. 5). The toiin has been purified in several laboratories (3, 6-8) and consists of a single polypeptide chain of ==150 kDa. Recently, the gene for the toxin has been cloned and expressed in Escherichia coli (9). Purified recombinant PMT (rPMT) is indistinguishable
from the native toxin in its antigenicity and toxicity for either experimental animals or cultured embryonic bovine lung cells (9). The molecular basis of the actions of PMT remains unknown. Bacterial toxins have provided novel approaches to elucidate cellular and molecular regulatory mechanisms (10-12). The present studies were initiated to determine whether PMT induced any cytotoxic effect on cultured fibroblasts. Surprisingly, we discovered that either PMT or rPMT is an extremely potent mitogen for 3T3 cells. The toxin, at picomolar concentrations, enters these cells and irreversibly triggers DNA synthesis and cell proliferation.
MATERIALS AND METHODS Methods. Cell culture procedures (13, 14), assays of growth promoting activity by [3H]thymidine incorporation (15) or by cell number (14), and measurements of cAMP (16) and total inositol phosphates (17) were performed as previously described. Cloning, expression, and purification of rPMT were also as described (9). rPMT was subjected to gas/liquidphase microsequencing on an Applied Biosystems 477A protein sequencer with on-line phenylthiohydantoin-conjugated amino acid analysis (Applied Biosystems model 120A phenylthiohydantoin analyzer) using the reagents and solvents supplied by the manufacturer. Materials. Bombesin, vasopressin, epidermal growth factor (EGF), 8-bromo-cAMP, cholera toxin, and insulin were obtained from Sigma. Fetal bovine serum was from GIBCO. Platelet-derived growth factor (PDGF) (recombinant homodimer of B chains), recombinant fibroblast growth factor (FGF) (basic) (rFGFb), [3H]thymidine, [2-3H]inositol, and antigens and antibodies for radioimmunoassay of cAMP were from the Radiochemical Centre. All other chemicals were of the purest grade commercially available.
RESULTS PMT and rPMT Induce DNA Synthesis in Murine Swiss 3T3 Cells. PMT was an extremely potent inducer of DNA synthesis in Swiss 3T3 cells (Fig. 1 Left). Half of the maximal effect was obtained at a concentration as low as 0.32 ng/ml (-2 pM). The maximal effect, obtained at 1.25 ng/ml (9 pM), was equivalent to the stimulation of DNA synthesis induced by medium containing 10%o (vol/vol) fetal bovine serum. Thus, in contrast to most known mitogens for Swiss 3T3 cells (1), PMT at picomolar concentrations induced maximal DNA synthesis in the absence of any other synergistic factor. Several lines of evidence indicate that the potent mitogenic effect of the PMT preparations is in fact mediated by the 150-kDa protein. The gene coding for PMT has been cloned and expressed in E. coli, and rPMT was purified as described Abbreviations: PMT, native P. multocida toxin; rPMT, recombinant PMT; PDGF, platelet-derived growth factor; EGF, epidermal growth factor; FGF, fibroblast growth factor; rFGFb, recombinant FGF (basic).
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 123
124
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Proc. Natl. Acad. Sci. USA 87 (1990)
.100.
-2 ~.80
.2
200U
-
200 700
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60 .~40
C2X 500
o0
x cE 400
20 20
0
0.1 1 PMT, ng/ml
10 0
0.1 1 rPMT, ng/ml
10
FIG. 1. Stimulation of DNA synthesis by PMT and rPMT in Swiss 3T3 cells. (Left) Dose-response curve for the stimulation of DNA synthesis by PMT. Confluent, quiescent cultures of Swiss 3T3 cells were washed and incubated at 370C in 2 ml of Dulbecco's modified Eagle's medium (DMEM)/Waymouth medium, 1:1 (vol/ vol), containing 1 /LCi (1 ;uCi = 37 kBq) of [3H]thymidine per ml and various concentrations of native toxin. After 40 hr, DNA synthesis was assessed by measuring the level of [3H]thymidine incorporated into half of the acid-precipitable material. Each point is the mean of determinations from three independent experiments expressed as a percentage of the incorporation induced by lo fetal bovine serum, which ranged from 396 x 103 to 435 x 103 cpm. (Right) Doseresponse curve for the stimulation by purified rPMT. DNA synthesis was measured as above, and each point represents the mean of determinations from five independent experiments expressed as a percentage of the incorporation given by 10%o fetal bovine serum, which ranged from 554 x 103 to 816 x 103 cpm.
for the native toxin (9). The preparation contained a major band of 150 kDa that was identical to that obtained with PMT. The N terminus of rPMT was not blocked, and hence its amino acid sequence was analyzed. The sequence obtained, Met-Lys-Ile-Lys-His-Phe-Phe-Asn-Ser-Asp-Phe-Thr-Val, is not identical or homologous to any known protein. Fig. 1 Right shows that rPMT stimulated DNA synthesis in Swiss 3T3 cells somewhat more potently than did- PMT. The half-maximal effect was achieved at 0.15 ng/ml (1 pM), with a maximum effect at 1.25 ng/ml. PMT is a heat-labile toxin (5). Fig. 2 shows that 30-min incubation at 57TC of the rPMT preparation resulted in a striking decrease in mitogenic activity. A 60-min preincubation at 57TC caused complete loss of mitogenic activity. When the toxin was incubated'at 570C for various times and then assayed at 2 ng/ml, the mitogenic activity decreased rapidly (Fig. 2 Inset). The mitogenic activity of rPMT was completely abolished by prior incubation with a polyclonal antiserum to PMT (Fig. 3 Upper). Immunoblot analysis shows that the only protein recognized by this neutralizing antiserum is rPMT (Fig. 3 Upper Inset). The antiserum did not prevent the stimulation of DNA synthesis induced by other mitogens including polypeptide'growth factors, neuropeptides, pharmacological agents, or cholera toxin (Fig. 3 Lower). The data depicted in Figs. 1-3 indicate that both PMT and rPMT are extremely potent mitogens for 3T3 cells. Induction of DNA Synthesis by Transient Exposure to rPMT. Many bacterial toxins bind to the external surface of the plasma membrane, enter the cells, and therefore cannot be removed by extensive washing or neutralized by exogenous antibodies (reviewed in ref. 18). To test whether rPMT acts in this manner to stimulate mitogenesis, quiescent 3T3 cells were incubated at 370C with rPMT at 1, 5, or 20 ng/ml for various times. When the cells were exposed for 30 min to 20 ng of rPMT per ml, washed extensively, and incubated in medium without the toxin, the level of [3H]thymidine incorporation was similar to that induced in the cultures incubated continuously with rPMT (Fig. 4 Left). Lower concentrations of rPMT required longer preincubation times to induce maximal DNA synthesis after removal of unbound toxin.
p
300
10 20 30 Time, min
-
200
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100 0
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1
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FIG. 2. Heat lability of the mitogenic activity of rPMT. Various concentrations of toxin were incubated at 570C for 0 (M), 30 (0), and 60 (i) min. The ability of rPMT to stimulate DNA synthesis in confluent, quiescent Swiss 3T3 cells was then measured as described in the legend to Fig. 1. Each point represents the mean of two determinations, and the addition of 10%6' fetal bovine serum gave an incorporation of 674 x i03 cpm. (Inset) Time course of the heat inactivation of rPMT. Aliquots of toxin were incubated at 230C (W) or 570C (u) for various times from 0 to 30 min. DMEM/Waymouth medium (1:1) was added to each aliquot to give a final toxin concentration of 2 ng/ml, and [3H]thymidine incorporation was measured as in Fig. 1. Each point represents the mean of two determinations; 10%o fetal bovine serum gave a level-of incorporation of 744 x 103 cpm.
In other experiments, the cells were preincubated with 5 ng of rPMT per ml for various times and then transferred to media in the absence or presence of PMT antiserum (Fig. 4 Right). When the cells were treated with 5 ng of rPMT per ml for 1 hr, subsequent DNA synthesis was markedly inhibited in cultures transferred to medium containing antiserum. In contrast, after 3 hr of incubation with rPMT, the mitogenic effect of the toxin was no longer blocked by the addition of antiserum. The entry of many bacterial toxins into the cytoplasm is prevented by incubation at 40C or by addition of membranepermeant weak bases such as methylamine (18). Fig. 5 Left shows that methylamine inhibited the stimulation of DNA synthesis by rPMT in a concentration-dependent manner. At similar concentrations, methylamine had only a slight inhibitory effect on mitogenesis induced by EGF and insulin. Furthermore, methylamine became less inhibitory as the time interval between the addition of rPMT and methylamine increased; the amine did not prevent DNA synthesis when added 3 hr after rPMT (Fig. 5 Right). A transient exposure (90 min) of 3T3 cells to 5 ng of rPMT per ml at 40C, instead of at 370C, did not stimulate mitogenesis during a subsequent incubation at 370C in rPMT-free medium. rPMT Stinulates Cell Proliferation. Addition of rPMT at 10 ng/ml to the medium in which confluent and quiescent 3T3 cells were grown (depleted medium) resulted in loss of density-dependent inhibition of growth (Fig. 6A). rPMT stimulated reinitiation of cell proliferation in a concentrationdependent manner (Fig. 6B). In other experiments, addition of rPMT to subconfluent 3T3 cells resulted in a striking increase in cell proliferation; the final saturation density increased 6-fold (Fig. 6C). In view ofthe results shown in Fig. 4, we tested whether a transient exposure to rPMT would be sufficient to stimulate cell proliferation in toxin-free medium. When quiescent cultures were exposed to 10 ng of rPMT per ml for 24 hr and then washed, trypsinized, and replated in the absence of the toxin, the subsequent growth of rPMTpretreated cells was markedly enhanced (Fig. 6C Inset).
Cell
Biology: Rozengurt et al.
Proc. Natl. Acad. Sci. USA 87 (1990) C800
125
-
0~600 E 400
.~200
Time, hr
FIG. 4. Transient exposure to rPMT stimulates DNA
synthesis
during a subsequent incubation in toxin-free medium. (Left) Effect of exposure time to rPMT upon its ability to induce mitogenesis in Swiss 3T3 cells. Confluent, quiescent cultures of Swiss 3T3 cells were washed twice with DMEM and incubated at 370C for various times without (o) or with 1 ng (e), 5 ng (n), or 25 ng (o) of rPMT per ml. After various incubation times, the cells were washed five times with
1).
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FIG. 3. Inhibition of the mitogenic action of rPMT by a specific antiserum. (Upper) Dose-response curve for the effect of immune (e) and preimmune (o) serum on DNA synthesis induced by rPMT. Various concentrations of immune or preimmune serum were added to 1:1 DMEM/Waymouth medium containing 1 ,uCi of [3H]thymidine per ml and 1 ng of rPMT per ml and were incubated for 1 hr at room temperature; 2 ml of this was then added to confluent, quiescent Swiss 3T3 cells, and DNA synthesis was measured as in Fig. 1. Each point is the mean of two determinations; 10% fetal bovine serum induced an incorporation of 809 x 103 cpm. (Inset) Analysis of rPMT by SDS/8% PAGE and silver staining (lane A) or by immunoblotting (lane B). Equal aliquots of rPMT (1.5 Atg) were used. The blots were probed with the neutralizing antiserum usWl above (diluted 1:500). Bound immunoglobulins were detected by using a rabbit anti-pig IgG conjugated with alkaline phosphatase. No signal was obtained when the blots were probed with the preimmune serum instead of the immune serum. (Lower) Specificity of the antiserum tQ rPMT. Various growth promoting factors were incubated at room temperature with (hatched bars) or without (open bars) 1 Al of antiserum per ml for 1 hr in 1:1 DMEM/Waymouth medium containing insulin at 1 ,ug/ml and [3H]thymidine at 1 jiCi/ml. Then, 2 ml of each medium was added to confluent, quiescent Swiss 3T3 cells, and DNA synthesis was measured as in Fig. 1. Each point is the mean of two determinations. The concentrations used were as follows: rPMT, 1 ng/ml; PDGF, 5 ng/ml; EGF, 5 ng/mI; FGF, 2 ng/ml; bombesin (Born), 10 ng/ml; vasopressin (VP), 10 ng/ml; phorbol 12,13-dibutyrate (PDBu), 100 ng/ml; 8-bromo-cAMP (8BrcAMP), 2.5 mM; cholera toxin (CT), 10 ng/ml; and isobutylmethylxanthine (IBMX), 50 ,uM.
rPMT Induces DNA Synthesis and Cell Division in Other Cells. Several murine cell lines including BALB/c 3T3, NIH 3T3, or 3T6 cells, which were rendered quiescent by growth to confluency (BALB/c and NIH 3T3) or by incubation in 0.5% serum (3T6), responded to rPMT with a striking increase in cell proliferation. The growth-promoting effects of rPMT were not confined to established cell lines. The toxin also stimulated DNA synthesis (Fig. 7A) and proliferation (Fig. 7B) in tertiary cultures of mouse embryo cells. The mitogenic effect persisted after transient exposure to rPMT, and it was blocked by 10 mM methylamine (results not shown). Table 1 shows that rPMT stimulated [3H]thyrnidine incorporation in quiescent cultures of human fibroblasts. The toxin was more effective than PDGF, EGF, or FGF in these cells. The effect of rPMT was enhanced in the presence of these factors (Table 1). Effect of rPMT on cAMP Levels and Inositol Phosphate Production. As an initial attempt to elucidate the mode of action of PMT, we measured the effect of this toxin on two
DMEM and then incubated for 40 hr in 1:1 DMEM/Waymouth medium containing 1 ,tCi of [3H]thymidine per ml. In addition, one set of dishes was washed seven times and then continuously exposed to each concentration of toxin throughout the 40-hr incubation (values given at 40 hr). DNA synthesis was measured as in Fig. 1. In this experiment, 10%o fetal bovine serum gave a level of incorporation of 826 x 103 cpm. (Right) The effect of a transient exposure to rPMT upon its ability to induce mitogenesis in the presence or absence of specific antiserum. Confluent, quiescent cultures of Swiss 3T3 cells were washed twice with DMEM and incubated at 370C with 5 ng of rPMT for various times up to 3 hr. The cells were then washed five times with DMEM and incubated for 40 hr in DMEM/Waymouth medium containing 1 uCi of[3H]thymidine per ml with (o) or without (W) 0.5 /il of antiserum per ml. Some cells were incubated with toxin throughout the 40-hr incubation. The arrow points to the level of DNA synthesis achieved when cells were exposed to toxin preincubated with antiserum for 1 hr. Each point is the mean of two determinations; in this experiment 10% fetal bovine serum gave an incorporation of 779 x 103 cpm.
major transmembrane signaling systems, cAMP and inositol phosphate production. Fig. 8A shows that rPMT at 20 ng/ml did not increase the intracellular level of cAMP, whereas el
A 100
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FIG. 5. Early but not late addition of methylamine inhibits the mitogenic action of rPMT. (A) Various concentrations of methylamine hydrochloride (pH 7.4) were added to 1:1 DMEM/Waymouth medium containing 1 ,uCi of [3Hlthymidine per ml with either 1 ng of toxin (e) or 5 ng of EGF per ml plus 1 Aig of insulin per ml (o). Confluent, quiescent Swiss 3T3 cells were incubated in this medium for 40 hr; then DNA synthesis was measured as in Fig. 1. Each point is the mean of two determinations, and values are expressed as the percentage of the incorporation given by l10o fetal bovine serum (795 x 104 CpM). (B) Confluent, quiescent cultures of Swiss 3T3 cells were
washed twice with DMEM and incubated at 37°C in 2 ml of 1:1
DMEM/Waymouth medium containing 1 ,uCi of [H]thymidine and I ng of rPMT per ml. At various times from 0 to 6 hr, methylamine hydrochloride was added to the cultures to give a final concentration of 9 mM. DNA synthesis was measured after 40 hr. Each point is the mean of two determinations and is expressed as the percentage of the
incorporation given by 1o fetal bovine serum (684 x 103 cpm).
126
Cell Biology: Rozengurt et al.
Proc. Natl. Acad. Sci. USA 87 (1990) CF
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FIG. 6. rPMT induces cell proliferation in Swiss 3T3 cells. (A) rPMT stimulates reinitiation of growth in confluent Swiss 3T3 cells. rPMT was added directly to the medium of Swiss 3T3 cell cultures to give a final concentration of 10 ng/ml 5 days after plating. Two, 3, and 4 days after this addition, cells from both treated (hatched bars) and untreated (open bars) cultures were trypsinized and counted in a Coulter counter. The values shown are means SEM (n = 5). (B) Dose-response curve for the effect of rPMT on the growth of confluent Swiss 3T3 cells. Various concentrations of rPMT were added to the medium of Swiss 3T3 cell cultures 5 days after plating. The cells were trypsinized and counted 4 days later. Each value is the mean SEM (n = 5). (C) Effect of rPMT on the growth of subconfluent Swiss 3T3 cells. Cells (5 x 104) were seeded onto 33-mm petri dishes in 2 ml of DMEM supplemented with 10o fetal bovine serum. After 24 hr, 10 ng of rPMT per ml was added to half of these cultures, and the number of treated cells (e) or untreated cells (a) were counted over the next 11 days. Each value is the mean + SEM (n 5). (Inset) rPMT induces growth of Swiss 3T3 cells after trypsinization and subsequent replating. rPMT at 10 ng/ml (e) was added to confluent Swiss 3T3 cells 5 days after plating. An equivalent volume of toxin carrier was added to parallel dishes (o). After 24 hr the cells were typsinized and seeded at 5 x 104 cells per 33-mm dish in DMEM supplemented with 10% fetal bovine serum and in the absence oftoxin. Cells were counted over the next 8 days. Each point is the mean SEM (n = 5). =
±
cholera toxin, added for comparison, caused a marked accumulation of cAMP in parallel cultures. Similar results were obtained when rPMT was added at various concentrations (Fig. 8B) or in the presence of the potent inhibitors of cAMP phosphodiesterase, 1-methyl-3-isobutylxanthine at 500 ,uM plus 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidine at 100 ttM (results not shown). In contrast to the lack of stimulatory effect of rPMT on cAMP accumulation, the toxin caused a dramatic and timedependent increase in the production of inositol phosphates (Fig. 8C). After 5 hr of incubation, rPMT at 5 ng/ml caused a 25-fold increase in the accumulation of inositol phosphates. Chromatographic analysis of the inositol phosphates revealed increases in the inositol monophosphate, bisphosphate, and trisphosphate fractions (results not shown). When the toxin was added at 1 ng/ml, the stimulation occurred after a longer lag (3 hr), but it reached the same magnitude as the rapid increase in inositol phosphate accumulation induced by a maximally effective concentration of bombesin (Fig. 8C). Methylamine (10 mM) completely blocked the striking in-
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FIG. 7. rPMT is a mitogen for tertiary mouse embryo cells. (A) Dose-response curve for the effect of rPMT on DNA synthesis in mouse embryo fibroblasts (MEF). Third passage MEFs were seeded at 105 cells per 33-mm dish in DMEM supplemented with 10%o fetal bovine serum. After 3 days the cultures were switched to DMEM containing 0.5% fetal bovine serum, and after a further 4-6 days the cultures were confluent and quiescent. The cultures were then washed twice with DMEM and incubated in 2 ml of 1:1 DMEM/ Waymouth medium containing 1 1sCi of [3H]thymidine per ml and various concentrations of rPMT. After 40 hr the level of DNA synthesis was assessed as in Fig. 1. In this experiment 10% fetal bovine serum gave a level of incorporation of 267 x 103 cpm. (B) rPMT stimulates growth of MEF cells. Third passage MEFs were seeded at 105 cells per 33-mm dish in DMEM supplemented with 10% fetal bovine serum. After 24 hr the medium was changed to DMEM supplemented with 1% fetal bovine serum with (e) or without (o) rPMT at 10 ng/ml. Cells were trypsinized and counted at intervals over the next 14 days. The values are means ± SEM (n = 5).
crease in the production of inositol phosphates caused by exposure to rPMT.
DISCUSSION The present results show that PMT is a novel and potent mitogen for cultured animal cells. Several lines of evidence, including the use of purified rPMT, indicate that this mitogenic activity is in fact mediated by the toxin, a single chain polypeptide of 150 kDa. Either PMT or rPMT induced DNA synthesis at picomolar concentrations in the absence of any other exogenously added growth-promoting factor. Maximum stimulation was equivalent to that promoted by the addition of fresh serum. These features distinguish the mitogenic effect of PMT from those of all other known growth factors for Swiss 3T3 cells (1). PDGF and bombesin, at nanomolar concentrations, induce DNA synthesis in a fraction of the cell population-i.e., they are less potent and effective than rPMT. Most other polypeptide growth factors, neuropeptides, and pharmacological agents stimulate DNA synthesis only in synergistic combinations (1, 2). So far, Table 1. Effect of various growth factors on DNA synthesis in human fibroblasts in the absence or presence of rPMT
[3H]Thymidine incorporation, Growth cpm x 10-3 With rPMT factor Without rPMT 60 9 None 75 16 Insulin 28 82 rFGFb 38 104 EGF 119 33 PDGF The concentrations used were as follows: rPMT, 10 ng/ml; insulin, 1 ,ug/ml; rFGFb, 5 ng/ml; EGF, 5 ng/ml; and PDGF, 10 ng/ml. Alone, 10% fetal bovine serum gave an incorporation of 139 X 103 cpm and 118 x 103 cpm in the presence of rPMT. The human foreskin fibroblasts were used at the 18th passage and rendered quiescent by incubation in 0.5% fetal bovine serum for 4 days. Cumulative [3H]thymidine incorporation was measured as in Fig. 1.
Cell
Proc. Natl. Acad. Sci. USA 87 (1990)
Biology: Rozengurt et al. B 100
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Time, hr
FIG. 8. Effect of rPMT on cAMP levels and inositol phosphate content of Swiss 3T3 fibroblasts. Cell culture and the determination
of cyclic AMP and inositol phosphates are as described. The cyclic AMP content of control cells (o) at various times and the influence ofrPMT at 20 ng/ml (e) and of cholera toxin at 100 ng/ml (A) is shown in A. Similar observations were made on several other cell preparations. (B) Cells were treated for 8 hr with the indicated concentrations of rPMT after which time the cellular content of cyclic AMP was determined. All incubations received the same concentrations of toxin carrier. The effect of a 30-min exposure to 25 AuM forskolin is illustrated for comparative purposes. The values are means SD of quadruplicate values obtained from one cell preparation. (C) Effect of 1 ng (e) and 5 ng (o) of rPMT per ml on the cellular content of inositol phosphates, with respect to control cultures (o). The cultures were labeled for 16 hr with 2 ml of 1:1 DMEM/Waymouth medium containing 10,uCi of [2-3H]inositol. rPMT was added as indicated, and 20 min prior to sampling, 1 M LiCl was added to give a final concentration of 20 mM. All cultures received the same amount of toxin carrier. Bombesin (A) was added 7 hr after the start of the experiment to give a final concentration of 10 nM. Similar time- and dose-dependent rPMT-induced increases in the cellular content of inositol phosphates were observed in several other cell preparations. ±
rPMT is the most potent and effective mitogen for Swiss 3T3 cells that has been identified. The growth-promoting effects of rPMT were not confined to established cell lines. Tertiary cultures of mouse embryo cells and human fibroblasts were also induced to resume DNA synthesis by addition of rPMT in the absence of other factors. A variety of bacterial toxins bind to the external surface of animal cells, become translocated to an intracellular compartment, acquire biological activity, and then irreversibly modify the molecular processes of the target cell (10-12). Our evidence suggests that the induction of cell proliferation by rPMT follows a similar sequence of events. Methylamine, an agent that increases endosomal and lysosomal pH (18) and thereby inhibits the processing and entry of many macromolecular ligands, viruses, and toxins into the cytoplasm (19,
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20), completely and selectively prevented the induction of DNA synthesis by rPMT when added at early times. Similarly, after minutes of exposure to rPMT, the toxin was accessible to a neutralizing PMT antiserum. Thereafter, the mitogenic action of rPMT was no longer sensitive to either PMT antiserum or to methylamine, suggesting that the toxin is translocated to an intracellular location via an acidic compartment. A crucial prediction of this model is that a transient exposure to rPMT should be sufficient to stimulate DNA synthesis in toxin-free medium. This was indeed the case. In view of the remarkable potency, persisting action and heat lability of the toxin, it is plausible that the intracellular effects of rPMT are of an enzymatic nature, a proposition that requires further experimental work. Intracellularly acting bacterial toxins frequently alter key components in the signal transduction process (11, 12). Thus, a first step toward identifying possible intracellular targets of PMT in cultured fibroblasts requires the determination of the effects of the toxin on some of the early signals leading to mitogenesis (1). In this context, we found that rPMT does not increase the intracellular level of cAMP. In contrast, rPMT caused a striking stimulation of inositol phosphate turnover, a signal transduction mechanism leading to multiple cellular responses (21) including cell growth (1). None of the previously described bacterial toxins that act intracellularly stimulated this transmembrane signaling system. Thus, PMT may provide a novel tool to identify components of the complex cascade of molecular events involved in the initiation of cell proliferation. 1. Rozengurt, E. (1986) Science 234, 161-166. 2. Rozengurt, E., Erusalimsky, J., Mehmet, H., Morris, C., Nanberg, E. & Sinnett-Smith, J. (1988) Cold Spring Harbor Symp. Quant. Biol. 53, 945-954. 3. Chanter, N., Rutter, J. M. & Mackenzie, A. (1986) J. Gen. Microbiol. 132, 1089-1097. 4. Dominick, M. A. & Rimler, R. B. (1988) Vet. Pathol. 25, 17-27. 5. Chanter, N. & Rutter, J. M. (1989) in Pasteurella and Pasteurellosis, eds. Adlam, C. & Rutter, J. M. (Academic, New York), pp. 161-195. 6. Nakai, T., Sawata, A., Tsuji, M., Samejima, Y. & Kume, K. (1984) Infect. Immun. 46, 429-434. 7. Rimler, R. B. & Brogden, K. A. (1986) Am. J. Vet. Res. 47, 730-737. 8. Foged, N. T. (1988) Infect. Immun. 56, 1901-1906. 9. Lax, A. J. & Chanter, N. (1989) J. Gen. Microbiol., in press. 10. Simpson, L. L. (1986) Annu. Rev. Pharmacol. Toxicol. 26, 427-453. 11. Habermann, E. & Dreyer, F. (1986) Curr. Top. Microbiol. Immunol. 129, 94-179. 12. Pfeuffer, T. & Halmreich, E. J. M. (1988) Curr. Top. Cell. Regul. 29, 129-216. 13. Rozengurt, E., Mierzejewski, K. & Wigglesworth, N. (1978) J. Cell. Physiol. 97, 241-252. 14. Rozengurt, E. & Sinnett-Smith, J. (1983) Proc. Nati. Acad. Sci. USA 80, 2936-2940. 15. Dicker, P. & Rozengurt, E. (1980) Nature (London) 287, 607-612. 16. Rozengurt, E., Legg, A., Strang, G. & Courtenay-Luck, N. (1981) Proc. Natl. Acad. Sci. USA 78, 4392-4396. 17. Nanberg, E. & Rozengurt, E. (1988) EMBO J. 7, 2741-2748. 18. Middlebrook, J. L. & Dorland, R. B. (1984) Microbiol. Rev. 48, 199-221. 19. Merion, M., Schlesinger, P., Brooks, R. M., Moehring, J. M., Moehring, T. J. & Sly, W. S. (1983) Proc. Natl. Acad. Sci. USA 80, 5315-5319. 20. Goldstein, J. L., Brown, M. S., Anderson, R. G. W., Russell, D. W. & Schneider, W. J. (1985) Annu. Rev. Cell Biol. 1, 1-39. 21. Berridge, M. J. (1987) Annu. Rev. Biochem. 56, 159-193.
