Phosphoinositide metabolism and oncogenes

309

Clinica Chimica Acta, 185 (1989) 309-316 Elsevier

CCA 04602

Enzymes in Cancer

Phosphoinositide

metabolism and oncogenes

Tadaomi Takenawa and Kiyoko Fukami Department of Molecular Pharmacology, Tokyo Metropolitan Institute of Gerontology, Tokyo (Japan) (Received

28 March

1989; revision received

Key war&:

Oncogene;

17 July 1989; accepted

Transformation;

18 August

1989)

Inositolphospholipid

sumulary

The hydrolysis of phosphatidylinositol4,Sbisphosphate plays important roles in growth factor- or oncogene-induced cell proliferation. However, it is still unknown whether the hydrolysis of phosphatidylinositol 4,Sbisphosphate is an essential step for cell growth. To solve this problem, we developed a monoclonal antibody against the lipid. Injection of the antibody into ras-transformed cells caused reversible and dose-dependent decrease in DNA synthesis and reverted the cell morphology to that of the untransformed cells. The antibody also inhibited the proliferation of erbBand src-transformed cells but not the proliferation of untransformed or myc-transformed cells.

Introduction

Recent studies on transmembrane signaling pathways have demonstrated that mitogens such as platelete derived growth factor (PDGF), bombesin, PGF,,, thrombin and bradykinin [l-4] rapidly induce the hydrolysis of phosphatidylinosito1 4,Sbisphosphate by phospholipase C, resulting in the formation of inositol 1,4,5-trisphosphate (IP,) and diacylglycerol (DG). 1s mobilizes Ca2+ from intracellular Ca2+ stores and DG stimulates protein kinase C activity [5,6]. These signaling pathways have been considered to play important role in cellular responses induced by various hormones, neurotransmitters and autacoids. But, some types of growth factors, FGF and insulin do not elicit PIP, breakdown [7,8]. EGF is also considered not to evoke PIP, breakdown except in some cells such as A431 cells [9]. These findings raise the question_whether PIP, breakdown is an indispensable process for

Correspondence to: Dr. T. Takenawa, Department of Molecular Pharmacology, Institute of Gerontology, Sakae-cho, Itabashi-ku, Tokyo 173, Japan.

0009-8981/89/%03.50

0 1989 Elsevier Science Publishers

B.V. (Biomedical

Division)

Tokyo

Metropolitan

310

the promotion of cell proliferation. As well, there is much evidence that some types of oncogenes and tumor viruses enhance inositolphospholipid metabolism during the transformation process and modulate the activity of enzymes that catalyze the metabolism of inositolphospholipids [lo-151. But, oncogenes such as myc and fos, do not evoke PIP, breakdown. Generally, oncogene products have been classified into 5 groups: (1) proteins related to growth factor; (2) proteins which have tyrosine kinase activity and are located on plasma membranes; (3) proteins which are located in cytoplasma and have serine-threonine kinase activity; (4) proteins homologous to G-proteins; (5) proteins which interact with DNA and are located in nucleus. Thus, most of oncogenes seem to encode the proteins involved in signal transfuctions of growth factors. Therefore, it is very important to know the mechanisms of signal transduction system of growth factors to understand oncogene-induced transformation. Considering the fact that inositolphospholipid metabolism is enhanced in tumor cells, signaling pathways mediated by inositolphospholipid must be involved in some kind of tumor cells. However, it is uncertain whether inositolphospholipid metabolism is actually important in oncogene-induced cell proliferation. Therefore, to solve these questions, we developed the antibody against PIP, to inhibit PIP, hydrolysis which is a trigger reaction of the signal transduction. Materials and methods Materials Phosphatidyl[Z 3Hlinositol Cphosphate, phosphatidyl[Z 3Hlinositol 4,5_bisphosphate and [6-3H]thymidine were from Amersham. PDGF was from Collaborative Research. Peroxidase-conjugated IgG(goat) anti-mouse IgG was from Cappel Laboratories. PIP, and PIP were prepared from bovine spinal cord by the method of Schacht [16]. Cells and cell culture NIH 3T3 cells were transformed by transfection with cloned human activated c-Ki-ras (Calu-PFl cells) and v-Ha-ras. Chicken embryo fibroblastic cells were transformed by infection with gag-erbB (GEV cells) gag-myc (MC29), avian erythroblastosis virus (AEV), or the Schmidt-Ruppin strain of Rous sarcoma virus of subgroup A (SRA). Cells were cultured in Dulbecco-modified Eagle’s medium supplemented with 10% (v/v) bovine calf serum. Monoclonal antibody The antibody against PIP, was developed and characterized as described before [17]. The antibody bound to PIP, very specifically and had little affinity for PIP, PI, IP, or other lipids. Inhibition of PIP, hydrolysis by anti-PIP, antibody Phospholipase C activity was measured by incubation for 20 min at 37’ C of a reaction mixture containing 50 pmol/l Mes buffer (pH 6.8), 100 pmol/l CaCl,, 1 mg/ml bovine serum albumin, rat liver total lipid extract (75 pg including 0.2 pmol of PIP, and 0.36 pmol of PIP) and 50 pmol/l phosphatidyl ethanolamine in a total volume of 100 ~1 in the presence of purified rat liver phospholipase C. Inhibitory effect of the antibody on PIP, hydrolysis was examined by preincubation with the reaction mixture at room temperature for 1 h.

311

Microinjection of antibodies and [‘HJthymidine

labeling

Cells were inoculated onto plastic overslips compartmentalized by scratches of a needle and were logarithmically growing in the 10% bovine calf serum. Antibody was prepared as 0.2-6 mg/ml solutions in phosphate buffer (140 mmol/l [+K] pH 7.25) and microinjected into the cytoplasm of the cells with an injectoscope (IMT-ZSYF, Olympus). After injection, the cells were washed and cultured in the presence of 10% serum. At the indicated time, cells were pulsed with [3H]thymidine (1 @Zi/ml) and processed for autoradiography. The labelling indices (percentages of the cells that incorporated [3H]thymidine) were determined by examining about 100 injected cells or > 200 uninjected cells. The labeling efficiency was calculated as the percentage of the labeling index of the injected cells to that of uninjected cells on the same cover slip.

Specificity of the antibody

ELISA showed that the antibody only reacted with PIP, but not with PIP, PI, IP, or other lipids (Fig. 1). Moreover, on TLC immunostaining, the antibody reacted only with PIP, even when rat brain total lipids (12.6 pg phosphorus/spot) were used as antigens. In addition, on Western blotting and dot blotting, the antibody did not react with any proteins or nucleic acid extracted from NIH 3T3 cells. To establish the specificity of the action of antibody on PIP,, we examined its effect on the breakdown of PIP, and PIP. Preincubation of PIP, with the antibody strongly inhibited the hydrolysis of PIP, by phospholipase C but scarcely affected the hydrolysis of PIP. Moreover, IgG from unimmunized mice had no effect on the enzymatic breakdown of PIP,. Effect of anti-PIP, antibody on cell proliferation

Injection of antibody into c-Ki-ras transformed cells cultured in the presence of serum caused reversible and dose-dependent decrease in the rate of cell proliferation. The decrease in the labeling efficiency was obvious within 3 h after injection of antibody at a concentration of 2 mg/ml. The labeling efficiency decreased for upto 17 h and thereafter reverted to the level of uninjected cells by 64 h after injection (Fig. 2). Since the cell cycle of Calu-PFl has a duration of 15 h, including 6.5 in G, phase, the decrease in the labelling efficiency within 3 h after injection suggests that the action of the antibody is on G, phase, where cells are sensitive to signals for the initiation of mitogenesis. However, S phase entry of Calu-PFl cells was not completely inhibited by the antibody even after all cells underwent G, phase in the presence of the antibody. Since injection of unimmunized mouse IgG did not effect the labelling efficiency, the above change is ascribable to a specific action of the antibody on a PIP,-related cellular events rather than to an artifact caused by the manipulation for microinjection or to a non-specific effect of the injected IgG molecules.

312

1.5

1.0 :: 4

0.5

10Ong

long

lOpg/well

bg

Fig. 1. Specificity of anti-PIP, antibody. The specificity of anti-PIP, antibody was examined by ELISA. Microtiter wells were coated with various concentrations of PIP,, PIP, PI, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidic acid, cardiolipin or IP, and then the reactivities of these materials with the antibody were examined. 0, PIP,; 0, PIP; A, PI; A, PA; 0, phosphatidylcholine; n, phosphatidylethanolamine; v, phosphatidylserine; V, cardiolipin; 0, IP,.

0

10

20

30

Time after

40

injection

50

60

70

(h)

Fig. 2. Inhibitory effect of anti-PIP, antibody in the labeling efficiency of ras-transformed cells. After injection of the antibody (2 mg/ml), Calu-PFI cells were pulsed at the indicated times with [3H]thymidine for 1 h (A) or 3 h (0). 0, control without injection; A, 0, injection.

313

TABLE

I

Changes

in entry into the S phase of various

Cell strain

(Oncogene)

NIH 3T3 variants Untransformed clone 5611 (c-Ki-ras) Calu-PF (v-Ha-ras) ras-BF Chick embryo fibroblastic cells Normal (v-erbB) GEV-transformed (v-erbB) AEV-transformed (v-src) SBA-transformed MC29-transformed (v-myc) MC29-transformed ’ (v-myc)

cell types after microinjection Labelling

of anti-PIP,

index (%) b

Labelling efficiency

No injection

Injection

69.2kl.O 68.8rt2.1 43.5 f 1.0

71.3 * 2.9 44.7 f 2.6 25.7 f 4.0

103.0 65.0 59.1

40.7k1.4 46.7kO.4 52.7 + 1.2 41.3 f 0.9 37.3&-2.7 30.2 + 3.2

38.3 f 1.6 22.2k1.4 34.3 f 2.6 28.8 f 2.6 36.1 f 3.6 30.4 f 3.9

94.1 47.5 65.1 69.7 96.8 100.7

a The antibody (2 mg/ml) was injected into cells growing in the Then 15 h later, the cells were pulsed with [3H]thymidine for 3 The methods, cell strains, and definitions of the terms ‘labelling described in ‘Materials and Methods’. Injection of unimmunized types had no effect (labelling efficiency, 96.9% to 98.5%). b MeanfSE(n=3). ’ Cultured in the presence of 5% calf serum.

antibody

a

(W)

presence of 10% fetal bovine serum. h and analyzed by autoradiography. index’ and ‘labelling efficiency’ are mouse IgG (2 mg/ml) into these cell

Morphological change by anti-PIP, antibody In addition to the change in proliferation, injection of the antibody into Cam-PFl cells also induced a reversible conversion in the cellular morphological appearance to that of the normal phenotype. The injection cells began to flatten within 3 h and their nuclei were clearly visible 12 h after injection. The similarity in appearance to untransformed cells lasted until 17 h after injection, but thereafter the morphological appearance reverted by 38 h. This change in morphology is parallel to that in cell proliferation, implying that anti-PIP, antibody elicits, not a mere inhibition of cell proliferation, but abolishment of transformed phenotypes. To confirm the effect of anti-PIP, antibody, the antibody was injected into cells transformed by various types of oncogenes (Table I). Consistent with the above results, injection of the antibody caused the decrease in the labeling efficiency of cells transformed by c-Ki-ras, v-Ha-ras, v-erbB, and v-src by 30-52%. However, the antibody had virtually no effect on untransformed cells and v-myc-transformed cells.

Discussion The results of the present study strongly suggest that cells in which inositolphospholipid metabolism is enhanced are sensitive to anti-PIP, antibody, since transformation by the src, ras, and erbB oncogenes has been demonstrated to induce enhancement of inositolphospholipid metabolism in the presence of serum. This presumption is consistent with our recent finding [18] that injection of the antibody

314

Fig. 3. Signal transduction systems of growth factors and oncogne products. R, receptor; G, GTP binding protein; PLC, phospholipase C; Ty-K, tyrosine kinase; PI-K, PI kinase; CK, C Kinase.

completely abolishes the mitogenesis induced by PDGF and bombesin (both eliciting PIP, breakdown), but not that induced by FGF, EGF or insulin (all lacking in the ability to elicit PIP, breakdown). It is noteworthy that in a great contrast to the complete inhibition of PDGF-induced mitogenesis, anti-PIP, antibody exerted only a partial decrease in S phase entry of serum exposed ras-, erbB and src-transformed cells. This finding supports the idea that signal transduction systems dependent on and independent of PIP, both function in these types of transformed cells in the presence of serum. Fig. 3 shows the summary of signal transduction system of growth factors and oncogene products. Growth factors such as PDGF, bombesin, vasopressin, thrombin, bradykinin and CSF-1 cause PIP, breakdown, whose signal seems to be essential for cell proliferation. But, FGF, EGF and insulin generate other signals independent of PIP, hydrolysis. It has been widely accepted that these growth factors mediate the signals through tyrosine kinases of their receptor. However, PDGF and CSF-1 can cause PIP, hydrolysis though their receptors have a tyrosine kinase activity. The fact may implicate the close association between tyrosine kinase and inositolphospholipid metabolism in the signal transduction system of these growth factors. For instance, some enzyme which catalyzes inositolphospholipid metabolism may be phosphorylated by these receptor-tyrosine kinases. In fact, Kaplan et al. [13] have reported that PDGF stimulates

315 slgnalmg

pathway up stream oncogene plasma

in

membrane ras

CytOSOL

by anti-

Located

PIP2 antibody

rot mos

wb

nudeus

inhibited

products membrane

w

fos

not

Inhibited

by anti-PIP2

antlbody

down stream

Fig. 4. Stream of the signals generated by oncogene products.

the phosphorylation of phosphatidylinositol kinase in tyrosine residues, resulting in the activation of the enzyme. On the other hand, oncogenes of src family such as src, erbB, ros and fms also code tyrosine kinases for their oncogene products. Moreover, it has been clear that tyrosine kinase activity is necessary for cell transformation. Interestingly, in all cells transformed by these oncogenes, inositolphospholipid metabolism is enhanced compared to untransformed cells. These results also suggest that signals generated by tyrosine kinases are switched over to inositolphospholipid-mediated signals. Therefore, anti-PIP, antibody may inhibit the cell proliferation caused by src family oncogenes. In Fig. 4, the signals are arranged according to stream of signal transduction. In up stream, a lot of oncogene products are located in plasma membranes. Signal transductions mediated by these oncogene products seem to be very similar with those of PDGF and CSF-1. These signals can be shut off by anti-PIP, antibody. On the contrary, in down stream, there are several oncogene products which are located in nucleus. These oncogene products appears to interact with DNA directly. The functions of these oncogene products could not be inhibited by anti-PIP, antibody. Therefore, inositolphospholipid metabolism may play a crucial role in tumorigenesis induced by oncogenes of which products are located on plasma membranes. Among oncogenes located on plasma membranes, activated ras-family oncogene has been most frequently detected in human tumors. Since inositolphospholipid metabolism appears to be involved in the signal transduction of ras-induced transformation, to clarify the mechanism of inositolphospholipid-mediated signal transduction system contributes further understanding for human tumor. Especially development of new agents which inhibit inositolphospholipid metabolism may be useful for treatment of human cancer. References 1

Benidge MJ, Heslop JP, Irvine RF, Brown KD. Inositol triphosphate formation and calcium mobilization in Swiss 3T3 cells in response to platelet derived growth factor. Biochem J 1984;222:195-201.

316

2 Takuwa N, Takuwa Y, Bollag WE, Rasmussen H. The effects of bomb&n on polyphosphoinositide and calcium metabolism in Swiss 3T3 cells. J Biol Chem 1987;262:182-183. 3 Macphee CH, Drummond AH, Otto AM, Asua L. Prostaglandin Fsn stimulates phosphatidylinositol turnover and increases the cellular content of 1,2-diacylglycerol in confluent resting Swiss 3T3 cells. J Cell Physiol 1984;119:35-40. 4 Rasben DM, Yasuda KM, Cunningham DD. Relationship of thrombin-stimulated arachidonic acid release and metabolism to mitogenesis and phosphatidylinositol synthesis. J Cell Physiol 1987;130:466-473, 5 Nishizuka Y. The role of protein kinase C in cell surface signal transduction and tumor promotion. Nature 1984;308:693-698. 6 Beridge MF, Irvine RF. Inositol t&phosphate, a novel second messenger in cellular signal transduction. Nature 1984;312:315-321. 7 Magnaldo I, L’Allemain G, Chambard JC, Moemrer M, Barritault D, Pouyssegur J. The mitogenicsignaling pathway of FGF is not mediated through polyphosphoinositide hydrolysis and protein kinase C activation in hamster fibroblast. J Biol Chem 1986;261:16916-16922. 8 L’Allemain G, Pouyssegur J. EGF and insulin action in fibroblast: evidence that phosphoinositide hydrolysis is not an essential mitogenic signaling pathway. FEBS Lett 198;197:344-348. 9 Pike LJ, Eakes AT. Epidermal growth factor stimulates the production of phosphatidylinositol monophosphate and the breakdown of polyphosphoinositides in A431 cells. J Biol Chem 1986;262:16441651. 10 Diringer H, Friis RR. Changes in phosphatidylinositol metabolism correlated to growth state of normal and rous sarcoma virus-transformed cells. Cancer Res 1977;37:2979-2984. 11 Fleishman LF, Chashwala SB, Cantley L. Ras-transformed cells altered levels of phosphatidylinositol 4,5_bisphosphate and catabolites. Science 1986;231:407-410, 12 Kato M, Kawai S, Takenawa T. Altered signal transduction in erbB-transformed cells. J Biol Chem 1987;262:5696-5704. 13 Kaplan DR, Whitman M, Schatthausen B, et al. Common elements in growth factor stimulation and oncogenic transformation: 85 kd phosphoprotein and phosphatidylinositol kinase activity. Cell 1987;50:1021-1029. 14 Whitman M, Kaplan DR, Schaffhausen BS, Cantley L, Roberts TM. Association of phosphatidylinositol kinase activity with polyoma middle-T component for transformation. Nature 1985;315:239-242. 15 Jackowski S, Rettenmier CW, Sherr CW, Rock C. A guanine nucleotide-dependent phosphatidylinositol4,5-diphosphate phospholipase C in cells transformed by the v-fms and v-fes oncogenes. J Biol Chem 1986;261:4978-4985. 16 Schacht J. Extraction and purification of polyphosphoinositides. Methods Enzymol 1981;27:623-631. 17 Fukami K, Matsuoka K, Nakanishi 0, Yamakawa A, Kawai S, Takenawa T. Antibody to phosphatidylinositol 4,5-bisphosphate inhibits oncogene-induced mitogenesis. Proc Nat1 Acad Sci USA 1988;85:9057-9061. 18 Matsuoka K, Fukami K, Nakanishi 0, Kawai S, Takenawa T. Mitogenesis in response to PDGF and bombesin abolished by microinjection of antibody to PIP,. Science 1988;239:640-643.