Proc. Nati. Acad. Sci. USA Vol. 89, pp. 4240-4244, May 1992 Cell Biology
Pasteurella multocida toxin is a potent inducer of anchorageindependent cell growth (cell proliferation/transformed phenotype/signal transduction/inositol phosphates/protein kinase C)
THERESA E. HIGGINS*, ANNE C. MURPHY*, JAMES M. STADDON*, ALISTAIR J. LAXt, AND ENRIQUE ROZENGURT*t *Imperial Cancer Research Fund, P.O. Box 123, 44 Lincoln's Inn Fields, London WC2A 3PX, United Kingdom; and tAgriculture and Food Research Council, Institute for Animal Health, Compton, Newbury, Berkshire RG16 ONN, United Kingdom
Communicated by Leon A. Heppel, February 10, 1992 (received for review December 15, 1991)
protein kinase C, and stimulated the phosphorylation of an 80-kDa protein (16) that is a major substrate of protein kinase C (refs. 17-19, and see ref. 20). Furthermore, the binding of epidermal growth factor (EGF) to its receptor was decreased by rPMT, an action attributable in part to protein kinase C activation (16). The stimulation of these early events by rPMT, like its mitogenic action (4), required cellular entry and activation of the toxin (15, 16). The toxin did not increase the cellular content of cAMP (4). PMT is the first identified intracellularly acting toxin that leads to an activation of phosphatidylinositol-specific phospholipase C, a major transducer of transmembrane signaling (21). Hence, PMT may provide a tool to study the cellular effects of this signaling pathway under conditions free from constraints such as ligand-induced cellular desensitization. Most normal cells require contact with an adhesive substratum to proliferate, and oncogenic transformation removes this requirement for adherence (22). The signaltransduction pathways that induce anchorage-independent growth have remained unclear. In the present study we determined the effects of rPMT on the growth of Rat-i cells, a cell line that can be induced to form colonies in semisolid medium (e.g., see ref. 23). We report that rPMT induces a potent and dramatic stimulation of anchorage-independent growth.
The growth of many normal cells requires ABSTRACT contact with an adhesive substratum, a requirement that is frequently abrogated in the transformed phenotype. We have explored pathways that can lead to the anchorage-independent growth of cultured Rat-i fibroblasts. PasteureUa mulocida toxin (PMT), a 146-kDa mitogenic protein, caused a striking increase in the formation of colonies (>200 jam) from single cells in soft agar. The magnitude of the effect of PMT was greater than that achieved by epidermal growth factor or platelet-derived growth factor. The toxin was extremely potent, with half-maximal and maximal effects observed at 1 and 10 pM PMT, respectively. This concentration dependence of the action of the toxin is similar to that for the stimulation of DNA synthesis in adherent cultures of the cells. Stimulation of colony formation could be achieved by a transient exposure of the cells to PMT and it was blocked by methylamine, indicating that the toxin enters the cells to act. Colony formation was stimulated equally by native and recombinant PMT, but a truncated version (33.5 kDa) of the recombinant toxin was ineffective. PMT antiserum blocked colony formation in response to PMT. In the Rat-1 cells, PMT stimulated the phospholipase C-mediated hydrolysis of inositolphospholipids, as indicated by the stimulation of inositol phosphate release, Ca2+ mobilization, and phosphorylation of a protein kinase C substrate. The results indicate that the deregulation of signal-transduction pathways as elicited by an intracellularly acting bacterial toxin can induce a malignant phenotype.
MATERIALS AND METHODS Materials. Fetal bovine serum (FBS) was obtained from GIBCO. EGF, phorbol 12,13-dibutyrate, and nitroblue tetrazolium were from Sigma. Platelet-derived growth factor (PDGF) (recombinant homodimer of B chains), [3H]thymidine, [2-3H]inositol, 1II-labeled EGF (='100 ,uCi/mg; 1 Ci = 37 GBq), carrier-free [32p]p;, and 45CaC12 (10-40 Ci/mg of calcium) were supplied by Amersham. Electrophoresis reagents and molecular mass standards were from Bio-Rad. Ampholines were from LKB and SeaKem agarose was from FMC. All other reagents were of the highest grade commercially available. Cell Culture. Stock cultures of Rat-1 cells were maintained in 90-mm Nunc dishes in Dulbecco's modified Eagle's medium (DMEM) containing penicillin (100 units/ml) and streptomycin sulfate (100 pug/ml) and supplemented with 10o FBS. The cultures were incubated in a humidified atmosphere of lo0 C02/90% air at 370C. For experiments requiring confluent and quiescent cultures, the cells were seeded at 5 x 104 cells per 33-mm Nunc dish and incubated in 10% FBS in DMEM. Two days later the medium was changed to 2 ml
The mechanisms of action of bacterial toxins have provided insights into the control of cellular regulatory processes, including signal transduction and cell proliferation (1-3). Pasteurella multocida toxin (PMT) has been shown to be an extremely potent and effective mitogen for fibroblast cell lines and early-passage cultures (4). The toxin is a monomeric 146-kDa protein that has been purified (5-8), cloned (9-11), and sequenced (12-14). The deduced amino acid sequence of PMT did not reveal any significant homologies with other toxins or proteins (13, 14). Both native and recombinant PMT (rPMT) are mitogenic at picomolar concentrations (4). Several lines of evidence indicate that PMT enters the cells and acts intracellularly to initiate and sustain DNA synthesis. Thus, a transient exposure of the cells to the toxin was sufficient to commit them to S phase and cell division. Furthermore, early but not late addition of the lysosomotrophic agent methylamine selectively blocked the mitogenic action of rPMT (4). Prior to the stimulation of DNA synthesis, rPMT enhanced the formation of inositol phosphates (15), increased the cellular content of diacylglycerol, caused the translocation of
Abbreviations: PMT, Pasteurella multocida toxin; rPMT, recombinant PMT; FBS, fetal bovine serum; EGF, epidermal growth factor; PDGF, platelet-derived growth factor. tTo whom reprint requests should be addressed.
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. 4240
Cell
~n
Proc. NatL. Acad. Sci. USA 89 (1992)
Biology: Higgins et al.
profoundly inhibited the stimulation of DNA synthesis by rPMT when added together with the toxin. This lysosomotrophic agent became progressively less inhibitory as the time interval between the addition of rPMT and methylamine increased; it did not prevent DNA synthesis when added 6 hr after rPMT (Fig. 1 Inset). Addition of rPMT to cultures of Rat-1 cells maintained in the presence of 1% FBS induced a striking increase in cell proliferation (Fig. 1 Right). Next we determined the effect of rPMT on inositol phosphate formation, Ca2+ mobilization, and protein kinase C activation in quiescent cultures of Rat-i cells. rPMT caused a dramatic and dose-dependent increase in total inositol phosphate production by [2-3H]inositol-labeled Rat-i cells (Fig. 2 Left). Methylamine completely blocked this response to rPMT (Fig. 2 Center). In cultured fibroblasts, intracellular Ca2l mobilized in response to factors acting via inositol 1,4,5-trisphosphate is released into the extracellular medium, leading to a decrease in the cellular Ca2+ content (15, 24-26). In Rat-i cells, the 45Ca2+ content of 45Ca2+-equilibrated cells was decreased by rPMT to 43 ± 1% of the control value (Fig. 2 Right), consistent with mobilization of an intracellular store of Ca2'. This action of rPMT was blocked by methylamine. To investigate the effects of rPMT on protein phosphorylation, Rat-1 cells were labeled with [32p]pi and treated with rPMT, and cellular proteins were extracted and separated by two-dimensional gel electrophoresis. Phorbol 12,13dibutyrate, a direct activator of protein kinase C, induced the phosphorylation of an 80-kDa protein that is a major substrate for protein kinase C (Fig. 3). rPMT markedly stimulated a similar response (Fig. 3). rPMT also caused a dosedependent decrease in the binding of 125I-EGF to Rat-1 cells, an effect blocked by methylamine (Fig. 3). This action of methylamine was selective since it did not block EGF receptor transmodulation induced by the phorbol ester. Thus, rPMT is a potent inducer of DNA synthesis, inositol phosphate production, Ca2+ mobilization, and activation of protein kinase C in quiescent cultures of Rat-1 cells. PMT Induces Anchorage-Independent Growth. When Rat-1 cells are suspended in semisolid medium, they are able to proceed through only a few rounds of cell division and consequently fail to form colonies (Fig. 4). As with many other nontumorigenic cell lines, Rat-1 cells exhibit anchorage-dependent growth. To test whether rPMT stimulates
of DMEM containing 0.5% FBS and the cells became quiescent after a further 4-6 days of incubation. Assays in Adherent Cultures of Rat-i Cells. Assays of growth-promoting activity either by [3Hlthymidine incorporation into DNA (4) or by cell number (4) and measurements of total inositol phosphate production, residual 45Ca2' content, and 125I-EGF binding (15, 16) were performed as described. To investigate the effects of rPMT on protein phosphorylation, quiescent cultures of Rat-1 cells were labeled with [32p]Pi and cellular proteins were extracted and separated by two-dimensional gel electrophoresis (16). PMT and rPMT were purified as described (5, 10). Clonogenic Assay. Stock cultures of Rat-1 cells, 3-4 days after passage, were trypsinized and gently dispersed to ensure a single-cell suspension. Cells were added to DMEM supplemented with FBS and other factors, as described for each experiment, to give 5 x 103 cells per ml. Agarose was added at 1:10 dilution to give a final concentration of 0.3%. An aliquot (1 ml) of this mixture was plated onto 33-mm plastic dishes containing a 2-ml base layer of hardened 0.3% agarose. In all cases the composition of the base layer was identical to that of the top layer, as indicated. The cultures were incubated in an atmosphere of 10%o C02/90o air at 370C for 14 days and then stained for 18 hr with the vital stain nitroblue tetrazolium. Colonies with diameters of >200 ,um were counted using a BioTranII automated colony counter (New Brunswick Scientific).
RESULTS PMT Stimulates DNA Synthesis and Early Events in Adherent Cultures of Rat-i Cells. To determine the effects of rPMT on DNA synthesis by Rat-1 cells, quiescent cultures of these cells were transferred to serum-free medium containing various concentrations ofrPMT. [3H]Thymidine was added 20 hr after the addition of the toxin and its incorporation into DNA was measured 5 hr later. rPMT was an extremely potent inducer of DNA synthesis in cultures of Rat-1 cells (Fig. 1 Left). The half-maximal response was obtained with rPMT at 0.3 ng/ml (2 pM). The maximal effect, obtained at 1 ng/ml, was equivalent to the DNA synthesis stimulated by medium containing 10% (vol/vol) FBS (Fig. 1 legend). Methylamine, a weak base that increases endosomal and lysosomal pH (1), C
_0 too E3
*0.
A
o2
°
0
02 46
SO2
.100
20
.
II
'X E
24
/9 0~~~~~~5 S~~~~~~~~~~~~~~~~~~.
HOURS
.Uu I
4241
10
III
rPMT (ng/ml)
0
2
4
6
TIME (Days)
FIG. 1. Stimulation of DNA synthesis and cell growth by rPMT in Rat-i cells. (Left) Dose dependence of the stimulation of DNA synthesis by rPMT. Confluent, quiescent cultures of Rat-1 cells were washed and incubated at 37°C in 2 ml of DMEM/Waymouth's medium, 1:1 (vol/vol), containing various concentrations of rPMT. The cultures were labeled with [3H]thymidine (1 uCi/ml) from 20 to 25 hr. DNA synthesis was assessed by measuring the [3H]thymidine incorporated into half of the acid-precipitable material. (Inset) Early but not late addition of methylamine inhibits the mitogenic action of rPMT. Confluent, quiescent cultures of Rat-1 cells were washed twice with DMEM and incubated at 37°C in 2 ml of 1:1 DMEM/Waymouth's medium containing rPMT (1 ng/ml). At various times from 0 to 25 hr, methylamine hydrochloride (pH 7.4) was added to the cultures to give a final concentration of 9 mM. The cultures were incubated with [3H]thymidine (1 ,uCi/ml) from 20 to 25 hr and DNA synthesis was measured after this time. Ten percent FBS gave an incorporation of 8 x 105 cpm and 7.9 x 105 cpm in the presence of methylamine hydrochloride. (Right) Effect of rPMT on the growth of subconfluent Rat-1 cells. Cells (5 x 104) were seeded onto 33-mm Petri dishes in 2 ml of DMEM supplemented with 10%o FBS. After 24 hr the cultures were switched to 1% FBS in the absence (o) or presence (o) of rPMT (10 ng/ml). Cells were counted (mean ± SEM, n = 5) over the next 7 days.
4242
Cell Biology:
Higgins et al.
Proc. Natl. Acad. Sci. USA 89 (1992) Total Inositol Phosphates
Residual
81
0
Ca
15
61 '70
10
4
b-
5-
21
0. U-
O0
1 10 rPMT (ng/ml)
_
MA
_
MA
FIG. 2. Effect of rPMT on inositol phosphate production and mobilization of Ca2+ in Rat-1 cells. (Left) Cells were prelabeled with [2-3H]inositol (10 kCi/ml) for 16 hr. Various amounts of rPMT were then added and the cultures were incubated at 370C for 4.5 hr. LiCl (20 mM) was then added and after a further 30 min the cellular inositol phosphate content was determined. (Center) Effect of methylamine on the production of inositol phosphates by rPMT. After prelabeling for 16 hr with [3H]inositol (10 ,uCi/ml), cells were incubated for 5 hr at 370C in the absence (open bars) or presence (filled bars) of rPMT (10 ng/ml) either without or with 10 mM methylamine (MA). LiCl (20 mM) was added 30 min prior to extraction. Values shown for the inositol phosphate are means + SEM (n = 3). (Right) Effect of rPMT on Ca2+ mobilization. Rat-1 cells were equilibrated with 45Ca2+ for 16 hr at 370C. The cultures were then incubated for a further 4 hr in the absence (open bars) or SEM (n = 5). presence (filled bars) of rPMT (20 ng/ml) either without or with 10 mM methylamine (MA). Each value is the mean
colony growth of Rat-1 cells, cultures of these cells were treated with rPMT for 24 hr, trypsinized, and suspended (5 x 103 cells) in semisolid medium containing rPMT. rPMT markedly stimulated Rat-1 cells to form large colonies (i.e., >200 Am in diameter) in semisolid medium (Fig. 4). The stimulation of colony formation by rPMT was abolished by a polyclonal antiserum to PMT (Table 1). Immunoblot analysis showed that the only protein recognized by this neutralizing antiserum was rPMT (4). The antiserum did not prevent colony formation induced by PDGF and EGF (results not shown). Extracts of Escherichia coli transformed either by a plasmid without any insert (pAT153) or by the recombinant [pAJL14 (13)] that encodes only part of the PMT gene (amino acids 1-287, to produce a 33.5-kDa protein) were completely ineffective in inducing colony growth (Table 1). In contrast, _120 ~~~-~
o100- q 0
U
PDBu
_
80 60 40 20
rPMT
.1
1
10
PDBu
rPMT (ng/ml)
FIG. 3. Stimulation of protein phosphorylation and inhibition of EGF binding by rPMT and phorbol 12,13-dibutyrate (PDBu) in Rat-1 cells. (Left) Confluent, quiescent cultures of Rat-1 cells were labeled with [32p]p, (400 ,Ci/ml) for 20 hr. Prior to extraction, rPMT (20 ng/ml) was added for 4 hr or PDBu (100 ng/ml) for 10 min. Phosphoproteins were extracted and separated by two-dimensional gel electrophoresis. The autoradiograms show phosphoproteins derived from control (-), PDBu-treated, or rPMT-treated cultures. The large arrow identifies the acidic 80-kDa phosphoprotein substrate of protein kinase C; arrowhead indicates a phosphoprotein with similar intensity under all conditions. (Right) Confluent, quiescent cultures of Rat-1 cells were treated for 4 hr with various concentrations of rPMT in the absence (o) or presence (e) of 9 mM methylamine hydrochloride. Specific binding of 1251-EGF at 4°C was then determined. The effect of a 30-min exposure to PDBu (100 ng/ml) in the absence (open bar) or presence (filled bar) of methylamine hydrochloride is also shown.
crude or purified preparations of rPMT were as active as the native toxin purified from P. multocida (Table 1). The effect of rPMT on colony formation was tested in the presence of various concentrations of serum in the semisolid medium. The toxin induced a 50-fold increase in anchorageindependent growth in the presence of 5% serum (Fig. 5 Left). We also tested whether a transient exposure to rPMT would be sufficient to stimulate anchorage-independent growth in toxin-free medium. When Rat-1 cells were treated with 10 ng of rPMT per ml for 24 hr and then washed, trypsinized, and suspended in semisolid medium in the absence of toxin, the subsequent colony formation of rPMT-pretreated cells was markedly enhanced (Fig. 5 Right). Methylamine added during the 24-hr exposure to rPMT profoundly inhibited the ability of PMT to promote colony growth. Thus, induction of anchorage-independent growth, like proliferation of adherent cells (4), can be induced by transient exposure to rPMT and requires processing of the toxin. PMT was an extremely potent inducer of anchorageindependent growth; half-maximal effect was achieved at a concentration as low as 1 pM (Fig. 6). Maximal effect was induced at a concentration of 10 pM. The ability of PMT to induce colony formation was compared with that of EGF and PDGF alone or together. PMT was more effective than either of these factors (Fig. 6). Interestingly, the number of colonies formed by cells exposed to rPMT was virtually identical to that formed by cells treated with both PDGF and EGF (Table 1).
DISCUSSION Cells acquire the ability to grow in an anchorage-independent fashion through various mechanisms. These include production of growth factors that act in an autocrine or paracrine manner, alterations in the number or structure of cellular receptors, and changes in the activity of postreceptor signaling pathways (27). For example, the receptors for EGF or PDGF, which signal through an intrinsic tyrosine kinase, can induce cellular transformation when constitutively activated (28, 29). In addition, transfected G-protein-coupled receptors, including muscarinic and serotonin receptors, can also confer transformed properties (30, 31). In the present study we exploited the unique properties of the intracellularly acting toxin from P. multocida to investigate whether persistent activation of signal-transduction pathways induces anchorage-independent growth. PMT is an extremely potent mitogen for adherent cultures of mesenchymal cells (4). Recent studies on the mechanism
Cell
Biology: Higgins et al.
Proc. Natl. Acad. Sci. USA 89 (1992)
9. /*
4243
q
0
'@
rPM (10 ng/m)
0 0
N
No addition FIG. 4. rPMT induces anchorage-independent growth of Rat-1 cells. (Upper) Direct photograph of colonies of Rat-i cells grown for 14 days in 0.3% agarose in DMEM containing 5% FBS in the absence (Left) or presence (Right) of rPMT (10 ng/ml). (Lower) Colonies grown as above, but photographed prior to staining, using a Leitz Labovert microscope. (x50.)
of action of rPMT reveal that this toxin enters the cell to activate inositolphospholipid breakdown causing the release of inositol 1,4,5-trisphosphate, Ca2+ mobilization from intracellular stores, an increase in the level of diacylglycerol, translocation of protein kinase C, and phosphorylation of an 80-kDa substrate of protein kinase C substrate (15, 16). PMT is the first bacterial toxin that has been shown to activate this transmembrane signaling pathway. In the present study, we verified that rPMT initiates a similar sequence of signaling events in Rat-1 cells and that this toxin is a potent mitogen for adherent cultures of these cells. We have shown here that rPMT induces a dramatic increase in the anchorage-independent growth of Rat-1 cells. Table 1. Effects of PMT antiserum, native PMT, rPMT, and truncated PMT on colony formation by Rat-i cells No. of colonies Addition(s) None 9± 1 Anti-PMT 18 ± 2 rPMT 184 ± 6 rPMT + anti-PMT 24 ± 4 13 ± 2 pAT153 10 ± 1 pAJL14 193 ± 11 pAJL13 PMT 174 7 PDGF+ EGF 181 4 The antiserum was added at 1 ,.l/ml. Extracts of Escherichia coli HB101 transformed with pAT153 (plasmid without insert), pAJL14 (plasmid encoding a truncated PMT), or pAL13 (plasmid encoding rPMT) were used at 100 ng of protein per ml. Purified native toxin (PMT) or rPMT was added at 10 ng/ml. The concentrations of PDGF and EGF were 25 and 10 ng/ml, respectively. The PMT antiserum did not inhibit (results not shown) colony formation by PDGF plus EGF. Colony formation (mean + SEM, n = 5) of Rat-1 cells was determined in 0.3% agarose containing DMEM and 5% FBS in the absence or presence of the extracts, pure toxins, or growth factors. as indicated.
Colony formation was stimulated by rPMT at picomolar concentrations and even after transient exposure to the toxin. Furthermore, PMT was more effective than either EGF or PDGF added at nanomolar concentrations. Thus, rPMT is an extremely potent and effective inducer of anchorageindependent growth, a hallmark of the malignant phenotype. Methylamine, a membrane-permeant weak base that increases endosomal and lysosomal pH (1) and thereby inhibits the processing and entry of many macromolecular ligands 0
0 0
0 E
z
250
-8 0
200
6100
1 50
-4110
100. 50.
210O -
FBS (%)
rPMT MA rPMT + MA
0
FIG. 5. (Left) Effect of serum concentration (%, vol/vol) on colony growth of Rat-1 cells in semisolid medium. Cultures of Rat-i cells 3-4 days after passage were treated with rPMT (10 ng/ml) (0) or untreated (o). After 24 hr they were trypsinized and carefully mixed to ensure a single-cell suspension. Colony formation was determined in 0.3% agarose containing DMEM and various amounts of FBS with or without rPMT (10 ng/ml). Colonies >200 Am were counted with the automatic colony counter. (Right) Effect of rPMT pretreatment on subsequent colony formation in Rat-1 cells. Adherent cultures of Rat-1 cells 3-4 days after passage were pretreated in the absence (-) or presence of rPMT (10 ng/ml) without or with 9 mM methylamine hydrochloride, pH 7.4 (MA). After 24 hr the cells were trypsinized and colony formation was determined in 0.3% agarose containing DMEM and 5% FBS in the absence of added rPMT. Bars show means SEM of values from five replicate dishes in a representative experiment. ±
4244
Cell Biology:
Proc. NatL. Acad. Sci. USA 89 (1992)
Higgins et al. CDin
1 60
._
o 1 20
1200
0 L) 0
.0 E
80 100
40
z
0
-it .
1
1n10 M.1 rPMT (ng/mI)
-
PDGF
EGF
rPMT
FIG. 6. (Left) Effect of rPMT concentration on colony growth of Rat-1 cells in semisolid medium. Cultures of Rat-1 cells 3-4 days after trypsinized and colony formation was determined in 0.3% agarose containing DMEM plus 5% FBS and various concentrations of rPMT. Values shown are means SEM of 10 replicates. (Right) Effect of various factors on colony growth of Rat-1 cells. Cultures of Rat-i cells 3-4 days after passage were left untreated (-) or were pretreated with EGF (25 ng/ml), PDGF (25 ng/ml), or rPMT (10 ng/ml). After 24 hr, the cultures were trypsinized and colony formation was determined in 0.3% agarose containing DMEM, 5% FBS, and growth factors or rPMT exactly as during the 24-hr pretreatment. Each bar represents the mean SEM of values from 5 replicate dishes. passage were
into the cytoplasm, prevented the stimulation ofearly signals, monolayer DNA synthesis, and anchorage-independent growth in semisolid medium. Thus, induction of colony formation, as for all other known biological responses elicited by rPMT, requires cellular entry and processing of the toxin through acidic vesicular compartments. The pathological basis of the action of certain bacteria lies in their ability to produce toxins that enter eukaryotic cells and subvert cellular regulatory processes. The results presented here emphasize that toxin-induced deregulation of signal-transduction pathways can elicit a malignant phenotype. To our knowledge, this is the first time that a bacterial toxin has been shown to potently induce anchorageindependent cell proliferation. 1. Middlebrook, J. C. & Dorland, R. B. (1984) Microbiol. Rev. 48, 199-221. 2. Moss, J. & Vaughan, M. (1988) Adv. Enzymol. 61, 303-379. 3. Bourne, H. R., Sanders, D. A. & McCormick, F. (1990) Nature (London) 348, 125-131. 4. Rozengurt, E., Higgins, T., Chanter, N., Lax, A. J. & Staddon, J. M. (1990) Proc. Nat!. Acad. Sci. USA 87, 123-127. 5. Chanter, N., Rutter, J. M. & Mackenzie, A. (1986) J. Gen. Microbiol. 132, 1089-1097. 6. Nakai, T., Sawata, A., Tsuji, M., Samejima, Y. & Kume, K. (1984) Infect. Immun. 46, 429-434. 7. Foged, N. T. (1988) Infect. Immun. 56, 1901-1906. 8. Foged, N. T., Pedersen, K. B. & Elling, F. (1987) FEMS Microbiol. Lett. 43, 45-51. 9. Peterson, S. K. & Foged, N. T. (1989) Infect. Immun. 57, 3907-3913. 10. Lax, A. J. & Chanter, N. (1990) J. Gen. Microbiol. 136, 81-87. 11. Kamps, A. M. I. E., Kamp, E. M. & Smits, M. A. (1990) FEMS Microbiol. Lett. 67, 187-190.
12. Buys, W. E. C. M., Smith, H. E., Kamps, A. M. I. E., Kamp, E. M. & Smits, M. A. (1990) Nucleic Acids Res. 18, 2815-2816. 13. Lax, A. J., Chanter, N., Pullinger, G. D., Higgins, T., Staddon, J. M. & Rozengurt, E. (1990) FEBS Lett. 227, 59-64. 14. Petersen, S. K. (1990) Mol. Microbiol. 4, 821-830. 15. Staddon, J. M., Barker, C. J., Murphy, A. C., Chanter, N., Lax, A. J., Michell, R. H. & Rozengurt, E. (1991) J. Biol. Chem. 266, 4840-4847. 16. Staddon, J. M., Chanter, N., Lax, A. J., Higgins, T. E. & Rozengurt, E. (1990) J. Biol. Chem. 265, 11841-11848. 17. Rozengurt, E., Rodriguez-Pena, A. & Smith, K. A. (1983) Proc. Nat!. Acad. Sci. USA 80, 7224-7248. 18. Rodriguez-Pena, A. & Rozengurt, E. (1986) EMBO J. 5, 77-83. 19. Blackshear, P. J., Wen, L., Glynn, B. P. & Witters, L. A. (1986) J. Biol. Chem. 261, 1459-1469. 20. Brooks, S. F., Herget, T., Erusalimsky, J. D. & Rozengurt, E. (1991) EMBO J. 10, 2497-2505. 21. Berridge, M. J. (1987) Annu. Rev. Biochem. 56, 159-193. 22. Varmus, H. E. (1984) Annu. Rev. Genet. 18, 553-612. 23. Pace, A. M., Wong, Y. H. & Bourne, H. R. (1991) Proc. Nat!. Acad. Sci. USA 88, 7031-7035. 24. Lopez-Rivas, A., Mendoza, S. A., NAnberg, E., SinnettSmith, J. W. & Rozengurt, E. (1987) Proc. Natl. Acad. Sci. USA 84, 5768-5772. 25. Nanberg, E. & Rozengurt, E. (1988) EMBO J. 7, 2741-2747. 26. Mendoza, S. A., Schneider, J. A., Lopez-Rivas, A., SinnettSmith, J. W. & Rozengurt, E. (1986) J. Cell Biol. 102, 22232233. 27. Bishop, J. M. (1991) Cell 64, 235-248. 28.
Westermark, B. & Heldin, C.-H. (1991) Cancer Res. 51,
5087-5092. 29. Yarden, Y. & Ullrich, A. (1988) Annu. Rev. Biochem. 57,
443-478. 30. Julius, D., Livelli, T. J., Jessell, T. M. & Axel, R. (1989) Science 244, 1057-1062. 31. Gutkind, J. S., Novotny, E. A., Brann, M. R. & Robbins, K. C. (1991) Proc. Nat!. Acad. Sci. USA 88, 4703-4707.
Pasteurella multocida toxin is a potent inducer of anchorageindependent cell growth (cell proliferation/transformed phenotype/signal transduction/inositol phosphates/protein kinase C)
THERESA E. HIGGINS*, ANNE C. MURPHY*, JAMES M. STADDON*, ALISTAIR J. LAXt, AND ENRIQUE ROZENGURT*t *Imperial Cancer Research Fund, P.O. Box 123, 44 Lincoln's Inn Fields, London WC2A 3PX, United Kingdom; and tAgriculture and Food Research Council, Institute for Animal Health, Compton, Newbury, Berkshire RG16 ONN, United Kingdom
Communicated by Leon A. Heppel, February 10, 1992 (received for review December 15, 1991)
protein kinase C, and stimulated the phosphorylation of an 80-kDa protein (16) that is a major substrate of protein kinase C (refs. 17-19, and see ref. 20). Furthermore, the binding of epidermal growth factor (EGF) to its receptor was decreased by rPMT, an action attributable in part to protein kinase C activation (16). The stimulation of these early events by rPMT, like its mitogenic action (4), required cellular entry and activation of the toxin (15, 16). The toxin did not increase the cellular content of cAMP (4). PMT is the first identified intracellularly acting toxin that leads to an activation of phosphatidylinositol-specific phospholipase C, a major transducer of transmembrane signaling (21). Hence, PMT may provide a tool to study the cellular effects of this signaling pathway under conditions free from constraints such as ligand-induced cellular desensitization. Most normal cells require contact with an adhesive substratum to proliferate, and oncogenic transformation removes this requirement for adherence (22). The signaltransduction pathways that induce anchorage-independent growth have remained unclear. In the present study we determined the effects of rPMT on the growth of Rat-i cells, a cell line that can be induced to form colonies in semisolid medium (e.g., see ref. 23). We report that rPMT induces a potent and dramatic stimulation of anchorage-independent growth.
The growth of many normal cells requires ABSTRACT contact with an adhesive substratum, a requirement that is frequently abrogated in the transformed phenotype. We have explored pathways that can lead to the anchorage-independent growth of cultured Rat-i fibroblasts. PasteureUa mulocida toxin (PMT), a 146-kDa mitogenic protein, caused a striking increase in the formation of colonies (>200 jam) from single cells in soft agar. The magnitude of the effect of PMT was greater than that achieved by epidermal growth factor or platelet-derived growth factor. The toxin was extremely potent, with half-maximal and maximal effects observed at 1 and 10 pM PMT, respectively. This concentration dependence of the action of the toxin is similar to that for the stimulation of DNA synthesis in adherent cultures of the cells. Stimulation of colony formation could be achieved by a transient exposure of the cells to PMT and it was blocked by methylamine, indicating that the toxin enters the cells to act. Colony formation was stimulated equally by native and recombinant PMT, but a truncated version (33.5 kDa) of the recombinant toxin was ineffective. PMT antiserum blocked colony formation in response to PMT. In the Rat-1 cells, PMT stimulated the phospholipase C-mediated hydrolysis of inositolphospholipids, as indicated by the stimulation of inositol phosphate release, Ca2+ mobilization, and phosphorylation of a protein kinase C substrate. The results indicate that the deregulation of signal-transduction pathways as elicited by an intracellularly acting bacterial toxin can induce a malignant phenotype.
MATERIALS AND METHODS Materials. Fetal bovine serum (FBS) was obtained from GIBCO. EGF, phorbol 12,13-dibutyrate, and nitroblue tetrazolium were from Sigma. Platelet-derived growth factor (PDGF) (recombinant homodimer of B chains), [3H]thymidine, [2-3H]inositol, 1II-labeled EGF (='100 ,uCi/mg; 1 Ci = 37 GBq), carrier-free [32p]p;, and 45CaC12 (10-40 Ci/mg of calcium) were supplied by Amersham. Electrophoresis reagents and molecular mass standards were from Bio-Rad. Ampholines were from LKB and SeaKem agarose was from FMC. All other reagents were of the highest grade commercially available. Cell Culture. Stock cultures of Rat-1 cells were maintained in 90-mm Nunc dishes in Dulbecco's modified Eagle's medium (DMEM) containing penicillin (100 units/ml) and streptomycin sulfate (100 pug/ml) and supplemented with 10o FBS. The cultures were incubated in a humidified atmosphere of lo0 C02/90% air at 370C. For experiments requiring confluent and quiescent cultures, the cells were seeded at 5 x 104 cells per 33-mm Nunc dish and incubated in 10% FBS in DMEM. Two days later the medium was changed to 2 ml
The mechanisms of action of bacterial toxins have provided insights into the control of cellular regulatory processes, including signal transduction and cell proliferation (1-3). Pasteurella multocida toxin (PMT) has been shown to be an extremely potent and effective mitogen for fibroblast cell lines and early-passage cultures (4). The toxin is a monomeric 146-kDa protein that has been purified (5-8), cloned (9-11), and sequenced (12-14). The deduced amino acid sequence of PMT did not reveal any significant homologies with other toxins or proteins (13, 14). Both native and recombinant PMT (rPMT) are mitogenic at picomolar concentrations (4). Several lines of evidence indicate that PMT enters the cells and acts intracellularly to initiate and sustain DNA synthesis. Thus, a transient exposure of the cells to the toxin was sufficient to commit them to S phase and cell division. Furthermore, early but not late addition of the lysosomotrophic agent methylamine selectively blocked the mitogenic action of rPMT (4). Prior to the stimulation of DNA synthesis, rPMT enhanced the formation of inositol phosphates (15), increased the cellular content of diacylglycerol, caused the translocation of
Abbreviations: PMT, Pasteurella multocida toxin; rPMT, recombinant PMT; FBS, fetal bovine serum; EGF, epidermal growth factor; PDGF, platelet-derived growth factor. tTo whom reprint requests should be addressed.
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. 4240
Cell
~n
Proc. NatL. Acad. Sci. USA 89 (1992)
Biology: Higgins et al.
profoundly inhibited the stimulation of DNA synthesis by rPMT when added together with the toxin. This lysosomotrophic agent became progressively less inhibitory as the time interval between the addition of rPMT and methylamine increased; it did not prevent DNA synthesis when added 6 hr after rPMT (Fig. 1 Inset). Addition of rPMT to cultures of Rat-1 cells maintained in the presence of 1% FBS induced a striking increase in cell proliferation (Fig. 1 Right). Next we determined the effect of rPMT on inositol phosphate formation, Ca2+ mobilization, and protein kinase C activation in quiescent cultures of Rat-i cells. rPMT caused a dramatic and dose-dependent increase in total inositol phosphate production by [2-3H]inositol-labeled Rat-i cells (Fig. 2 Left). Methylamine completely blocked this response to rPMT (Fig. 2 Center). In cultured fibroblasts, intracellular Ca2l mobilized in response to factors acting via inositol 1,4,5-trisphosphate is released into the extracellular medium, leading to a decrease in the cellular Ca2+ content (15, 24-26). In Rat-i cells, the 45Ca2+ content of 45Ca2+-equilibrated cells was decreased by rPMT to 43 ± 1% of the control value (Fig. 2 Right), consistent with mobilization of an intracellular store of Ca2'. This action of rPMT was blocked by methylamine. To investigate the effects of rPMT on protein phosphorylation, Rat-1 cells were labeled with [32p]pi and treated with rPMT, and cellular proteins were extracted and separated by two-dimensional gel electrophoresis. Phorbol 12,13dibutyrate, a direct activator of protein kinase C, induced the phosphorylation of an 80-kDa protein that is a major substrate for protein kinase C (Fig. 3). rPMT markedly stimulated a similar response (Fig. 3). rPMT also caused a dosedependent decrease in the binding of 125I-EGF to Rat-1 cells, an effect blocked by methylamine (Fig. 3). This action of methylamine was selective since it did not block EGF receptor transmodulation induced by the phorbol ester. Thus, rPMT is a potent inducer of DNA synthesis, inositol phosphate production, Ca2+ mobilization, and activation of protein kinase C in quiescent cultures of Rat-1 cells. PMT Induces Anchorage-Independent Growth. When Rat-1 cells are suspended in semisolid medium, they are able to proceed through only a few rounds of cell division and consequently fail to form colonies (Fig. 4). As with many other nontumorigenic cell lines, Rat-1 cells exhibit anchorage-dependent growth. To test whether rPMT stimulates
of DMEM containing 0.5% FBS and the cells became quiescent after a further 4-6 days of incubation. Assays in Adherent Cultures of Rat-i Cells. Assays of growth-promoting activity either by [3Hlthymidine incorporation into DNA (4) or by cell number (4) and measurements of total inositol phosphate production, residual 45Ca2' content, and 125I-EGF binding (15, 16) were performed as described. To investigate the effects of rPMT on protein phosphorylation, quiescent cultures of Rat-1 cells were labeled with [32p]Pi and cellular proteins were extracted and separated by two-dimensional gel electrophoresis (16). PMT and rPMT were purified as described (5, 10). Clonogenic Assay. Stock cultures of Rat-1 cells, 3-4 days after passage, were trypsinized and gently dispersed to ensure a single-cell suspension. Cells were added to DMEM supplemented with FBS and other factors, as described for each experiment, to give 5 x 103 cells per ml. Agarose was added at 1:10 dilution to give a final concentration of 0.3%. An aliquot (1 ml) of this mixture was plated onto 33-mm plastic dishes containing a 2-ml base layer of hardened 0.3% agarose. In all cases the composition of the base layer was identical to that of the top layer, as indicated. The cultures were incubated in an atmosphere of 10%o C02/90o air at 370C for 14 days and then stained for 18 hr with the vital stain nitroblue tetrazolium. Colonies with diameters of >200 ,um were counted using a BioTranII automated colony counter (New Brunswick Scientific).
RESULTS PMT Stimulates DNA Synthesis and Early Events in Adherent Cultures of Rat-i Cells. To determine the effects of rPMT on DNA synthesis by Rat-1 cells, quiescent cultures of these cells were transferred to serum-free medium containing various concentrations ofrPMT. [3H]Thymidine was added 20 hr after the addition of the toxin and its incorporation into DNA was measured 5 hr later. rPMT was an extremely potent inducer of DNA synthesis in cultures of Rat-1 cells (Fig. 1 Left). The half-maximal response was obtained with rPMT at 0.3 ng/ml (2 pM). The maximal effect, obtained at 1 ng/ml, was equivalent to the DNA synthesis stimulated by medium containing 10% (vol/vol) FBS (Fig. 1 legend). Methylamine, a weak base that increases endosomal and lysosomal pH (1), C
_0 too E3
*0.
A
o2
°
0
02 46
SO2
.100
20
.
II
'X E
24
/9 0~~~~~~5 S~~~~~~~~~~~~~~~~~~.
HOURS
.Uu I
4241
10
III
rPMT (ng/ml)
0
2
4
6
TIME (Days)
FIG. 1. Stimulation of DNA synthesis and cell growth by rPMT in Rat-i cells. (Left) Dose dependence of the stimulation of DNA synthesis by rPMT. Confluent, quiescent cultures of Rat-1 cells were washed and incubated at 37°C in 2 ml of DMEM/Waymouth's medium, 1:1 (vol/vol), containing various concentrations of rPMT. The cultures were labeled with [3H]thymidine (1 uCi/ml) from 20 to 25 hr. DNA synthesis was assessed by measuring the [3H]thymidine incorporated into half of the acid-precipitable material. (Inset) Early but not late addition of methylamine inhibits the mitogenic action of rPMT. Confluent, quiescent cultures of Rat-1 cells were washed twice with DMEM and incubated at 37°C in 2 ml of 1:1 DMEM/Waymouth's medium containing rPMT (1 ng/ml). At various times from 0 to 25 hr, methylamine hydrochloride (pH 7.4) was added to the cultures to give a final concentration of 9 mM. The cultures were incubated with [3H]thymidine (1 ,uCi/ml) from 20 to 25 hr and DNA synthesis was measured after this time. Ten percent FBS gave an incorporation of 8 x 105 cpm and 7.9 x 105 cpm in the presence of methylamine hydrochloride. (Right) Effect of rPMT on the growth of subconfluent Rat-1 cells. Cells (5 x 104) were seeded onto 33-mm Petri dishes in 2 ml of DMEM supplemented with 10%o FBS. After 24 hr the cultures were switched to 1% FBS in the absence (o) or presence (o) of rPMT (10 ng/ml). Cells were counted (mean ± SEM, n = 5) over the next 7 days.
4242
Cell Biology:
Higgins et al.
Proc. Natl. Acad. Sci. USA 89 (1992) Total Inositol Phosphates
Residual
81
0
Ca
15
61 '70
10
4
b-
5-
21
0. U-
O0
1 10 rPMT (ng/ml)
_
MA
_
MA
FIG. 2. Effect of rPMT on inositol phosphate production and mobilization of Ca2+ in Rat-1 cells. (Left) Cells were prelabeled with [2-3H]inositol (10 kCi/ml) for 16 hr. Various amounts of rPMT were then added and the cultures were incubated at 370C for 4.5 hr. LiCl (20 mM) was then added and after a further 30 min the cellular inositol phosphate content was determined. (Center) Effect of methylamine on the production of inositol phosphates by rPMT. After prelabeling for 16 hr with [3H]inositol (10 ,uCi/ml), cells were incubated for 5 hr at 370C in the absence (open bars) or presence (filled bars) of rPMT (10 ng/ml) either without or with 10 mM methylamine (MA). LiCl (20 mM) was added 30 min prior to extraction. Values shown for the inositol phosphate are means + SEM (n = 3). (Right) Effect of rPMT on Ca2+ mobilization. Rat-1 cells were equilibrated with 45Ca2+ for 16 hr at 370C. The cultures were then incubated for a further 4 hr in the absence (open bars) or SEM (n = 5). presence (filled bars) of rPMT (20 ng/ml) either without or with 10 mM methylamine (MA). Each value is the mean
colony growth of Rat-1 cells, cultures of these cells were treated with rPMT for 24 hr, trypsinized, and suspended (5 x 103 cells) in semisolid medium containing rPMT. rPMT markedly stimulated Rat-1 cells to form large colonies (i.e., >200 Am in diameter) in semisolid medium (Fig. 4). The stimulation of colony formation by rPMT was abolished by a polyclonal antiserum to PMT (Table 1). Immunoblot analysis showed that the only protein recognized by this neutralizing antiserum was rPMT (4). The antiserum did not prevent colony formation induced by PDGF and EGF (results not shown). Extracts of Escherichia coli transformed either by a plasmid without any insert (pAT153) or by the recombinant [pAJL14 (13)] that encodes only part of the PMT gene (amino acids 1-287, to produce a 33.5-kDa protein) were completely ineffective in inducing colony growth (Table 1). In contrast, _120 ~~~-~
o100- q 0
U
PDBu
_
80 60 40 20
rPMT
.1
1
10
PDBu
rPMT (ng/ml)
FIG. 3. Stimulation of protein phosphorylation and inhibition of EGF binding by rPMT and phorbol 12,13-dibutyrate (PDBu) in Rat-1 cells. (Left) Confluent, quiescent cultures of Rat-1 cells were labeled with [32p]p, (400 ,Ci/ml) for 20 hr. Prior to extraction, rPMT (20 ng/ml) was added for 4 hr or PDBu (100 ng/ml) for 10 min. Phosphoproteins were extracted and separated by two-dimensional gel electrophoresis. The autoradiograms show phosphoproteins derived from control (-), PDBu-treated, or rPMT-treated cultures. The large arrow identifies the acidic 80-kDa phosphoprotein substrate of protein kinase C; arrowhead indicates a phosphoprotein with similar intensity under all conditions. (Right) Confluent, quiescent cultures of Rat-1 cells were treated for 4 hr with various concentrations of rPMT in the absence (o) or presence (e) of 9 mM methylamine hydrochloride. Specific binding of 1251-EGF at 4°C was then determined. The effect of a 30-min exposure to PDBu (100 ng/ml) in the absence (open bar) or presence (filled bar) of methylamine hydrochloride is also shown.
crude or purified preparations of rPMT were as active as the native toxin purified from P. multocida (Table 1). The effect of rPMT on colony formation was tested in the presence of various concentrations of serum in the semisolid medium. The toxin induced a 50-fold increase in anchorageindependent growth in the presence of 5% serum (Fig. 5 Left). We also tested whether a transient exposure to rPMT would be sufficient to stimulate anchorage-independent growth in toxin-free medium. When Rat-1 cells were treated with 10 ng of rPMT per ml for 24 hr and then washed, trypsinized, and suspended in semisolid medium in the absence of toxin, the subsequent colony formation of rPMT-pretreated cells was markedly enhanced (Fig. 5 Right). Methylamine added during the 24-hr exposure to rPMT profoundly inhibited the ability of PMT to promote colony growth. Thus, induction of anchorage-independent growth, like proliferation of adherent cells (4), can be induced by transient exposure to rPMT and requires processing of the toxin. PMT was an extremely potent inducer of anchorageindependent growth; half-maximal effect was achieved at a concentration as low as 1 pM (Fig. 6). Maximal effect was induced at a concentration of 10 pM. The ability of PMT to induce colony formation was compared with that of EGF and PDGF alone or together. PMT was more effective than either of these factors (Fig. 6). Interestingly, the number of colonies formed by cells exposed to rPMT was virtually identical to that formed by cells treated with both PDGF and EGF (Table 1).
DISCUSSION Cells acquire the ability to grow in an anchorage-independent fashion through various mechanisms. These include production of growth factors that act in an autocrine or paracrine manner, alterations in the number or structure of cellular receptors, and changes in the activity of postreceptor signaling pathways (27). For example, the receptors for EGF or PDGF, which signal through an intrinsic tyrosine kinase, can induce cellular transformation when constitutively activated (28, 29). In addition, transfected G-protein-coupled receptors, including muscarinic and serotonin receptors, can also confer transformed properties (30, 31). In the present study we exploited the unique properties of the intracellularly acting toxin from P. multocida to investigate whether persistent activation of signal-transduction pathways induces anchorage-independent growth. PMT is an extremely potent mitogen for adherent cultures of mesenchymal cells (4). Recent studies on the mechanism
Cell
Biology: Higgins et al.
Proc. Natl. Acad. Sci. USA 89 (1992)
9. /*
4243
q
0
'@
rPM (10 ng/m)
0 0
N
No addition FIG. 4. rPMT induces anchorage-independent growth of Rat-1 cells. (Upper) Direct photograph of colonies of Rat-i cells grown for 14 days in 0.3% agarose in DMEM containing 5% FBS in the absence (Left) or presence (Right) of rPMT (10 ng/ml). (Lower) Colonies grown as above, but photographed prior to staining, using a Leitz Labovert microscope. (x50.)
of action of rPMT reveal that this toxin enters the cell to activate inositolphospholipid breakdown causing the release of inositol 1,4,5-trisphosphate, Ca2+ mobilization from intracellular stores, an increase in the level of diacylglycerol, translocation of protein kinase C, and phosphorylation of an 80-kDa substrate of protein kinase C substrate (15, 16). PMT is the first bacterial toxin that has been shown to activate this transmembrane signaling pathway. In the present study, we verified that rPMT initiates a similar sequence of signaling events in Rat-1 cells and that this toxin is a potent mitogen for adherent cultures of these cells. We have shown here that rPMT induces a dramatic increase in the anchorage-independent growth of Rat-1 cells. Table 1. Effects of PMT antiserum, native PMT, rPMT, and truncated PMT on colony formation by Rat-i cells No. of colonies Addition(s) None 9± 1 Anti-PMT 18 ± 2 rPMT 184 ± 6 rPMT + anti-PMT 24 ± 4 13 ± 2 pAT153 10 ± 1 pAJL14 193 ± 11 pAJL13 PMT 174 7 PDGF+ EGF 181 4 The antiserum was added at 1 ,.l/ml. Extracts of Escherichia coli HB101 transformed with pAT153 (plasmid without insert), pAJL14 (plasmid encoding a truncated PMT), or pAL13 (plasmid encoding rPMT) were used at 100 ng of protein per ml. Purified native toxin (PMT) or rPMT was added at 10 ng/ml. The concentrations of PDGF and EGF were 25 and 10 ng/ml, respectively. The PMT antiserum did not inhibit (results not shown) colony formation by PDGF plus EGF. Colony formation (mean + SEM, n = 5) of Rat-1 cells was determined in 0.3% agarose containing DMEM and 5% FBS in the absence or presence of the extracts, pure toxins, or growth factors. as indicated.
Colony formation was stimulated by rPMT at picomolar concentrations and even after transient exposure to the toxin. Furthermore, PMT was more effective than either EGF or PDGF added at nanomolar concentrations. Thus, rPMT is an extremely potent and effective inducer of anchorageindependent growth, a hallmark of the malignant phenotype. Methylamine, a membrane-permeant weak base that increases endosomal and lysosomal pH (1) and thereby inhibits the processing and entry of many macromolecular ligands 0
0 0
0 E
z
250
-8 0
200
6100
1 50
-4110
100. 50.
210O -
FBS (%)
rPMT MA rPMT + MA
0
FIG. 5. (Left) Effect of serum concentration (%, vol/vol) on colony growth of Rat-1 cells in semisolid medium. Cultures of Rat-i cells 3-4 days after passage were treated with rPMT (10 ng/ml) (0) or untreated (o). After 24 hr they were trypsinized and carefully mixed to ensure a single-cell suspension. Colony formation was determined in 0.3% agarose containing DMEM and various amounts of FBS with or without rPMT (10 ng/ml). Colonies >200 Am were counted with the automatic colony counter. (Right) Effect of rPMT pretreatment on subsequent colony formation in Rat-1 cells. Adherent cultures of Rat-1 cells 3-4 days after passage were pretreated in the absence (-) or presence of rPMT (10 ng/ml) without or with 9 mM methylamine hydrochloride, pH 7.4 (MA). After 24 hr the cells were trypsinized and colony formation was determined in 0.3% agarose containing DMEM and 5% FBS in the absence of added rPMT. Bars show means SEM of values from five replicate dishes in a representative experiment. ±
4244
Cell Biology:
Proc. NatL. Acad. Sci. USA 89 (1992)
Higgins et al. CDin
1 60
._
o 1 20
1200
0 L) 0
.0 E
80 100
40
z
0
-it .
1
1n10 M.1 rPMT (ng/mI)
-
PDGF
EGF
rPMT
FIG. 6. (Left) Effect of rPMT concentration on colony growth of Rat-1 cells in semisolid medium. Cultures of Rat-1 cells 3-4 days after trypsinized and colony formation was determined in 0.3% agarose containing DMEM plus 5% FBS and various concentrations of rPMT. Values shown are means SEM of 10 replicates. (Right) Effect of various factors on colony growth of Rat-1 cells. Cultures of Rat-i cells 3-4 days after passage were left untreated (-) or were pretreated with EGF (25 ng/ml), PDGF (25 ng/ml), or rPMT (10 ng/ml). After 24 hr, the cultures were trypsinized and colony formation was determined in 0.3% agarose containing DMEM, 5% FBS, and growth factors or rPMT exactly as during the 24-hr pretreatment. Each bar represents the mean SEM of values from 5 replicate dishes. passage were
into the cytoplasm, prevented the stimulation ofearly signals, monolayer DNA synthesis, and anchorage-independent growth in semisolid medium. Thus, induction of colony formation, as for all other known biological responses elicited by rPMT, requires cellular entry and processing of the toxin through acidic vesicular compartments. The pathological basis of the action of certain bacteria lies in their ability to produce toxins that enter eukaryotic cells and subvert cellular regulatory processes. The results presented here emphasize that toxin-induced deregulation of signal-transduction pathways can elicit a malignant phenotype. To our knowledge, this is the first time that a bacterial toxin has been shown to potently induce anchorageindependent cell proliferation. 1. Middlebrook, J. C. & Dorland, R. B. (1984) Microbiol. Rev. 48, 199-221. 2. Moss, J. & Vaughan, M. (1988) Adv. Enzymol. 61, 303-379. 3. Bourne, H. R., Sanders, D. A. & McCormick, F. (1990) Nature (London) 348, 125-131. 4. Rozengurt, E., Higgins, T., Chanter, N., Lax, A. J. & Staddon, J. M. (1990) Proc. Nat!. Acad. Sci. USA 87, 123-127. 5. Chanter, N., Rutter, J. M. & Mackenzie, A. (1986) J. Gen. Microbiol. 132, 1089-1097. 6. Nakai, T., Sawata, A., Tsuji, M., Samejima, Y. & Kume, K. (1984) Infect. Immun. 46, 429-434. 7. Foged, N. T. (1988) Infect. Immun. 56, 1901-1906. 8. Foged, N. T., Pedersen, K. B. & Elling, F. (1987) FEMS Microbiol. Lett. 43, 45-51. 9. Peterson, S. K. & Foged, N. T. (1989) Infect. Immun. 57, 3907-3913. 10. Lax, A. J. & Chanter, N. (1990) J. Gen. Microbiol. 136, 81-87. 11. Kamps, A. M. I. E., Kamp, E. M. & Smits, M. A. (1990) FEMS Microbiol. Lett. 67, 187-190.
12. Buys, W. E. C. M., Smith, H. E., Kamps, A. M. I. E., Kamp, E. M. & Smits, M. A. (1990) Nucleic Acids Res. 18, 2815-2816. 13. Lax, A. J., Chanter, N., Pullinger, G. D., Higgins, T., Staddon, J. M. & Rozengurt, E. (1990) FEBS Lett. 227, 59-64. 14. Petersen, S. K. (1990) Mol. Microbiol. 4, 821-830. 15. Staddon, J. M., Barker, C. J., Murphy, A. C., Chanter, N., Lax, A. J., Michell, R. H. & Rozengurt, E. (1991) J. Biol. Chem. 266, 4840-4847. 16. Staddon, J. M., Chanter, N., Lax, A. J., Higgins, T. E. & Rozengurt, E. (1990) J. Biol. Chem. 265, 11841-11848. 17. Rozengurt, E., Rodriguez-Pena, A. & Smith, K. A. (1983) Proc. Nat!. Acad. Sci. USA 80, 7224-7248. 18. Rodriguez-Pena, A. & Rozengurt, E. (1986) EMBO J. 5, 77-83. 19. Blackshear, P. J., Wen, L., Glynn, B. P. & Witters, L. A. (1986) J. Biol. Chem. 261, 1459-1469. 20. Brooks, S. F., Herget, T., Erusalimsky, J. D. & Rozengurt, E. (1991) EMBO J. 10, 2497-2505. 21. Berridge, M. J. (1987) Annu. Rev. Biochem. 56, 159-193. 22. Varmus, H. E. (1984) Annu. Rev. Genet. 18, 553-612. 23. Pace, A. M., Wong, Y. H. & Bourne, H. R. (1991) Proc. Nat!. Acad. Sci. USA 88, 7031-7035. 24. Lopez-Rivas, A., Mendoza, S. A., NAnberg, E., SinnettSmith, J. W. & Rozengurt, E. (1987) Proc. Natl. Acad. Sci. USA 84, 5768-5772. 25. Nanberg, E. & Rozengurt, E. (1988) EMBO J. 7, 2741-2747. 26. Mendoza, S. A., Schneider, J. A., Lopez-Rivas, A., SinnettSmith, J. W. & Rozengurt, E. (1986) J. Cell Biol. 102, 22232233. 27. Bishop, J. M. (1991) Cell 64, 235-248. 28.
Westermark, B. & Heldin, C.-H. (1991) Cancer Res. 51,
5087-5092. 29. Yarden, Y. & Ullrich, A. (1988) Annu. Rev. Biochem. 57,
443-478. 30. Julius, D., Livelli, T. J., Jessell, T. M. & Axel, R. (1989) Science 244, 1057-1062. 31. Gutkind, J. S., Novotny, E. A., Brann, M. R. & Robbins, K. C. (1991) Proc. Nat!. Acad. Sci. USA 88, 4703-4707.
