Association of inhibition of cell growth by O-phospho-l -tyrosine with decreased tyrosine phosphorylation

ELSEVIER

CANCER LETTERS Cancer Letters 102 ( 1996) 65-7 1

Association of inhibition of cell growth by O-phospho-L-tyrosine with decreasedtyrosine phosphorylation Shrikant Mishra, Anne W. Hamburger* University of Maryland Cancer Center; Department of Pathology, 9-051 Bressler Research Building, 655 WestBaltimore Street, Baltinwre, MD 21201, USA

Received26 December1995;revisionreceived19January1996;accepted19January1996

Abstract

We have previously demonstratedthat 0-phospho-L-tyrosine (P-‘Qr), a substratefor a wide range of PTPases,inhibits the growth of human renal cell carcinoma and human breastcancercell lines and suppressesEGF-mediatedEGFR tyrosine phosphorylation. We now show that P-Qr inhibited the growth of the human hepatomacell line HEPG2, and src transformedNIH 3T3 cells, but did not inhibit the growth of human ovarian carcinoma SKOV-3 cells. Addition of exogenous P-Tyr inhibited the insulin triggered insulin receptor (IR) tyrosine phosphorylation in the HEPGZ cell line and the tyrosine phosphorylation of a variety of cellular proteins in src-transformedNIH3T3 cells. P-‘Qr did not inhibit the tyrosine phosphorylation of gpta5 erbB-2 in P-‘Qr resistant SKOV-3 cells. Thus, inhibition of cell growth by P-tyr was associatedwith decreasedtyrosine phosphorylation of cellular proteins. Keyworcis:

0-Phospho-L-tyrosine; Tyrosine phosphorylation; Cell growth

1. Introduction

Phosphorylation at protein tyrosyl residues,a major mechanismof cellular signal transduction, is involved in mitogenesis and neoplastic transformation [ 11. Phosphotyrosine levels are regulated by the balance between protein tyrosine kinases (PTK) and protein tyrosine phosphatase (FTPase) functions. Increased

PTK activity, accompaniedby the appearanceof tyrosine phosphorylated cellular proteins, is associated

* Correspondingauthor.Tel.: +l 410 3283911;fax: +l 410 3286559.

with mitogenesisand/or transformation.In contrast,an increase in cellular PTPaselevels is associatedwith arrestof cell growth [2-6]. We demonstrated that 0-phospho-L-tyrosine (PTyr), a substratefor a wide rangeof PTPases,inhibited growth of breastand renal carcinomacells in vitro [7]. The inhibition of cell growth was shown, in part, to be due to activation of cellular PTPases.P-tyr decreased EGF mediatedtyrosine phosphorylation of EGFK, and enhanced the dephosphorylation of previously tyrphosphorylatedEGFR [8]. P-Tyr was also shown to inhibit cell growth in a synergistic manner with doxorubicin and etoposide. The aim of the current investigation was to deter-

0304-3835/96/$12.000 1996ElsevierScienceIrelandLtd. All rightsreserved PII: SO304-3835(96)04164-X

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S. Mishra, A. W. Hamburger I Cancer Letters 102 (1996) 65-71

mine if P-Tyr could modulate the tyrosine phosphorylation of different classes of cellular tyrosine kinases, including other growth factor receptor tyrosine kinases, and non-receptor tyrosine kinases. The tyrosine phosphorylation of the insulin receptor (IR) in response to insulin treatment was studied in a human hepatoma cell line, HEPG2. The tyrosine phosphorylation of erbB-2 was studied in a human ovarian carcinoma cell line, SKOV-3, known to overexpress the erbB-2 protein. Lastly, the tyrosine phosphorylation of srcm was measured in src transformed NIH 3T3 cells. Our investigation showed that the growth inhibitory effects of P-Tyr were associated with its ability to inhibit the tyrosine phosphorylation of cellular proteins.

2. Materials and methods 2.1. Materials The human hepatoma cell line HEPG2, the human ovarian carcinoma cell line SKOV-3 and src transformed NM 3T3 cells were purchased from the American Type Culture Collection (Rockville, MD). Culture media were purchased from Gibco Laboratories (Gaithersburg, MD). Calf serum (CS) and fetal bovine serum (FBS) vere from Intergen Co. (Purchase, NY). Anti-phosphotyrosine antibody was from Boehringer Mannheim (Indianapolis, IN). Grthovanadate and P-Tyr were from Sigma Chemical Co. (St. Louis, MO). The ECL chemiluminescent detection kit was from Amersham Corp. (Arlington Heights, IL). Anti-mouse goat IgG (Fc specific) peroxidase conjugated antibody was from Sigma Chemical Co. Antiinsulin receptor, Anti-V-src and Anti-c-neu antibodies were purchased from Oncogene Science Inc. (Uniondale, NY). 2.2. Cell culture The NIH 3’IXsrc cells were grown in DMEM (50%) + F12 (50%), 10% CS, 10 units/ml penicillin, and lO,@ml streptomycin in 5% CO2 at 37°C in a humidified atmosphere. HEPG2 and SKOV-3 cells were grown in DMEM (50%) F-12 (50%) and 10% FBS. For the cell growth inhibition assays, the cells were plated and grown in 6 well plates, washed in Dulbecco’s phosphate buffered saline, trypsinized, and counted in a Coulter counter.

2.3. Measurement of insulin receptor (IR) tyrosine phosphorylation To determine whether incubation with P-Tyr could modulate insulin induced IR tyrosine phosphorylation, HEPG2 cells were grown to semiconfluence in 25 cm2 flasks. The semiconfluent cells were washed and incubated overnight with culture medium with 0.5% FCS. Cells were then incubated in 0, 0.5, 1.0, 5.0 and 10x P-Tyr containing medium (1 X = 1.67 mM) for 24 h. At the end of the P-Tyr incubation, cells were further incubated with 1 U/ml of insulin for 5 min. At the end of the insulin incubation, the culture medium was removed and the cells were washed with sterile Hank’s balanced salt solution and subsequently lysed with 1 ml of Laemmli’s buffer. The samples were briefly sonicated and stored at -20°C prior to determination of insulin induced IR tyrosine phosphorylation by Western blotting using anti-phosphotyrosine antibody. The IR levels were determined using an anti-IR monoclonal mouse antibody. 2.4. Effect of P-Tyr incubation on tyrosine phosphorylation of NIH 3T3 and NIH 3T3-scr cells Semiconfluent NIH 3T3-src cells in 25 cm2 flasks were incubated with 5.0X P-Tyr for 24 h. The samples were briefly sonicated and stored at -20°C prior to determination of the tyrosine phosphorylation profile by Western blotting. The p60src protein levels were determined using an anti-v-src antibody. 2.5. Effect of P-Tyr incubation on erbB-2 tyrosine phosphorylation in SKOV-3 Cells Semiconfluent SKOV-3 cells in 25 cm2 flasks were incubated with 5.0~ P-Tyr for 24 h. At the end of the P-Tyr incubation, the SKOV-3 cells were washed with Hank’s balanced salt solution and lysed in R/PA buffer [7]. ErbB-2 was immuno-precipitated using anti-new antibodies and Protein A-Protein G. 2.6. Western blotting Ten microliters of cell lysates were resolved in 7.5% SDS-polyacrylamide gels. The gels were washed extensively to remove residual P-Tyr. The gels were blotted at 40 V for 8 h onto nitrocellulose filters. The

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S. Mishra, A. W. Hamburger I Cancer Letters 102 (1996) 65-71

filters were washed extensively with phosphatebuffered saline with 0.5% Tween 20 (PBST), blocked in 5.0% milk in PBST and incubated with appropriate dilutions of either anti-phosphotyrosine,anti-IR, antisrc, or anti-C-neu mouse monoclonal first antibodies. The blots were washed in four changes of PBST (10 ml/wash) and were incubated with horseradish peroxidase coupled anti-mouse IgG. The blots were developedusing an ECL Westernblotting kit asper the manufacturer’sdirections.

fected by P-Tyr treatment (Fig. 2b). A band of -170 kDa was tyr-phosphorylated in a similar pattern.

50000 =: N 40000 A al x

3. Results

9

3.1. Inhibition of cell growth by P-Tyr

= 3

A

SRC-c SRC-01x SRC-05x WC-1.0x SAC-5 ox

1 30000 4

?t 20000 i

We determined the effect of P-Tyr incubation on the growth of NIH 3T3, NIH 3T3-src, HEPGZ and SKOV-3 cells (Fig. 1). P-Tyr significantly inhibited the growth of the NIH3T3-src and the HEPG2 cell lines. The P-Tyr mediated growth inhibition was dose dependentover the concentration (0-5X) range of PTyr tested. In contrast, there was little growth inhibition of the ovarian carcinoma SK-OV3 cells over the samerange of P-Tyr concentration.

10000 ‘/i-: rG&

0 1

0

1

2

3

4

5

6

Days 10000 -

HEP-C HEP-01X HEP-1.0X HEP-3.0X

3.2. Effect of P-Tyr on modulation of tyrosine phosphorylation of IR in responseto insulin We next investigated if the growth inhibition of HEPG2 cells by P-Tyr was accompanied by the modulation of tyrosine phosphorylation of the IR @Isubunit = 95 000 Da) in responseto insulin. HEPG2 cells were incubated without and with P-Tyr (&10x) for 1 day, and the ability of insulin (1 unit/ml for 5 min) to induce tyrosine phosphorylation of the /3 subunit of the IR was determined. As shown (Fig. 2a), insulin induced tyrosine phosphorylation of the fi subunit of IR and other proteins in HEPG2 cells. The insulin-induced IR tyrosine phosphorylation was inhibited in P-Tyr pretreated cells. P-Tyr inhibited insulin-induced IR phosphotylation maximally at the lowest P-Tyr concentration (= 0.1 X) tested.Treatment with o-vanadate (lOOpM, 4 h) prior to insulin incubation increased the tyrosine phosphorylation of IR and other protein substratesin control cells. The P-Tyr mediated decrease of insulin-induced tyrosine phosphorylation, was inhibited in o-vanadate pretreatedcells. The amount of IR protein was unaf-

~10000 Lo o) 6000 X

5

6000

* =

4000

3 2000

Fig. 1. Effect of P-Tyr on growth of cells in culture. Src transformed NIH 3T3 (A), human hepatoma HEPG2 (B), and human ovarian carcinoma SKOV-3 (C) cells were plated as described. Triplicate wells were treated with the indicated concentrations of P-Tyr. Cells were counted as described in Section 2. Each point representsmean + SD (6 replicates).

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S. Mishra, A. W. Hamburger I Cancer Letters 102 (1996) 65-71

b 123456

78

Fig. 2. (a) Inhibition of tyrosine phosphorylation of IR with P-Tyr in HEPG2 cells, and restoration of tyrosine phosphorylation by orthovanadate. Lane I, control cells; 2, P-Tyr (1 X, 24 h) treated cells; 3, vanadate (lOOpM, 4 h) treated cells; 4, P-Tyr and vanadate pretreated cells; 5, cells treated with insulin (1 unit/ml, 5 min); 6-10, P-Tyr (0.1X, 24 h) 0.5, 1, 5, and 10~ treated cells incubated with insulin; 11, HEPG2 cells treated with o-vanadate (100,~M. 4 h) followed by insulin incubation (1 unit/ml, 5 min); 12-14, P-Tyr (0.1,l, and 5 x 24 h) treated cells pretreated with o-vanadate (IOOpM, 4 h) followed by insulin incubation (1 unit/ml, 5 mm), arrow indicates 95 ma IR /I subunit. (b) IR levels in control and P-Tyr treated cells were detected by Western blotting using an anti-IR antibody. Lane 1, control HEPG2 lysate; 2, lysate of P-Tyr (1 X, 24 h) treated cells; 3, lysate of control cells treated with o-vanadate (IOOyM, 4 h); 4, P-Tyr (1 x, 24 h) treated cells treated with o-vanadate (IOOpM, 4 h); 5, control cells treated with insulin; 6, P-Tyr (1 X, 24 h) treated cells incubated with insulin; 7, cells treated with o-vanadate (100,~M, 4 h) followed by insulin incubation (1 unit/ml, 5 min); 8. P-Tyr (I x, 24 h) and 0. vanadatepretreated cells (IOOpM, 4 h) followed by insulin incubation,

3.3. P-Tyr modulation of tyrosine phosphorylation in NIH 3T3-src cells We also examined the ability of P-Tyr to modulate tyrosine phosphorylation in NIH 3T3 src cells (Fig.

a

160,

12

3a). Basal tyr-phosphorylation of 160, 116, 60 and 40 kDa proteins was observed in NIH 3T3-src cells. The p60 band was shown to be src by blotting with an anti-src antibody (Fig. 4b). As seen in Fig. 3a, basal ~60s”: tyr phosphorylation was decreased in P-Tyr

34

b

1234

116, 60kDm

Fig. 3. (a) Modulation of tyrosine phosphotylation in src-transformedNIH-3T3 cells with P-Tyr. Lane 1, lysate of control cells; 2, lysate of P-Tyr (5.0x, 24 h) treated cells; 3, lysate of control cells treated with o-vanadate(lOOpM, 4 h); 4, lysate of P-Tyr (5.0x. 24 h) treated cells preincubated with o-vanadate (4 h prior to lysis). (b) The p60srcprotein levels, as determined by immunoblotting with an anti-v-xc antibody. The lane designations for the sampleswere identical to (a).

S. Mishra, A. W. Hamburger / Cancer Letters 102 (1996) 65-71

a

1234

Kd 185)

-

b 1234

Fig. 4. (a) Modulation of tyrosine phosphorylation in SK-OV3 ovarian carcinoma cells with P-Tyr. Lane 1, lysate of cells pretreated wtth o-vanadate; 2, lysate of untreated cells; 3, lysate of cells preincubated with o-vanadate and P-Tyr (5X, 24 h) prior to lysis; 4, lysate of P-Tyr (5.0X. 24 h) treated cells not incubated with o-vanadate. (b) ErbB-2 protein levels were determined by immunoblotting using an anti-erbB-2 antibody. The lane designations are identical to those in (a).

treated cells. The P-Tyr-mediated dephosphorylation of ~60”‘~ was only partially inhibitable by incubating the P-Tyr treated cells with o-vanadate. The levels of p60srcprotein were not changed with P-Tyr incubation (Fig. 3b). 3.4. P-Tyr does not modulate the tyrosine phosphorylation of erbB-2 in SKOV-3 cells The effect of P-Tyr incubation on erbB-2 tyrosine phosphorylation was studied in SKOV-3 cells. SKOV3 cells were treated with P-Tyr (5X) for 24 h in the presence or absence of o-vanadate (lOOpM, 4 h). As observed by Jallal et al. [9], erbB-2 is tyr-phosphorylated in SK-OV3 cells in the absence of vanadate (Fig. 4a). There was no difference in the tyrosine phosphorylation of erbB-2 protein in control or P-Tyr treated cells. P-Tyr incubation did not change the level of erbB-2 protein in these cells (Fig. 4b).

4. Discussion The significance of tyrosine phosphorylation of growth factor receptors and signal transduction proteins in the control of mitogenic growth and cellular

69

transformation has been a major area of investigation in recent years. The activity of cellular PTKs and PTPases are delicately balanced to control the overall phosphotyrosyl profile of cellular proteins. The role of PTKs in neoplastic transformation has been known for some time [lo], but it is only recently that the significance of PTPases as potential antioncogenic proteins has been studied [ 111. Many recent investigations have determined that increased activity of PTPases leads to inhibition of cell growth [2-6]. Microinjection of an intracellular PTPase, PTPase-I#?, into Xenopus oocytes delays insulininduced maturation and inhibits insulin-stimulated S6 peptide phosphorylation [ 121. Cellular PTPases regulate insulin action by dephosphorylating the active autophosphorylated form of the insulin receptor, and thus attenuating its tyrosine kinase activity [ 131. PTPases also modulate post-receptor signalling by catalyzing the dephosphorylation of the cellular substrates of both EGFR receptor kinase [8] and the insulin receptor kinase [12]. It is known that insulin rapidly induces tyr-phosphorylation of the 95 kDa t!I subunit of IR. This phosphorylation renders IR kinase active, and results in phosphorylation of insulin-receptor substrate-l (IRS1). In this study, we demonstrate that insulin, added to P-Tyr treated cells, is not able to induce phosphorylation of the IR /3 subunit or a 170 kDa protein that maybe IRS-l. P-Tyr may have directly enhanced PTPase mediated dephosphotylation of IR, resulting in the subsequent inhibition of IR kinase activity. We have previously shown that P-Tyr increases overall PTPase activity in lysates of human breast carcinoma MDA-MB 468 cells. Alternatively P-Tyr may have increased PTPase activity by competing for the P-Tyr binding sites in SH2 domain containing proteins that protect phosphotyrosyl groups from PI’Pase activity. Thus, Rotin et al. [14] demonstrated that SH2--SHs constructs of PLC-K inhibit PTPase activity found in A-431 cell lysates for tyr-phosphorylated EGFR. A stable interaction between P-Tyr and SH2 domain containing proteins would expose cellular phosphotyrosy1 proteins to dephosphorylation by PTPases. Thus, exogenous P-Tyr could have resulted in increased susceptibility of tyr-phosphorylated proteins to PTPase. When o-vanadate was added to P-Tyr treated cells, tyrosyl phosphorylation of IR and other cellular substrates was restored, further substantiating the hy-

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S. Mishra, A. W. Hamburger!

pothesis that P-Tyr inhibits tyrosyl phosphorylation via PTPases. The transforming gene (v-src) of Rous Sarcoma Virus (RSV) encodes a highly active 60 kDa protein tyrosine kinase (pp60v-sr9, capable of phosphorylating a variety of cellular proteins [ 151. As p~60”-~“: has been implicated in the establishment and maintenance of the neoplastic phenotype [ 15,161, depression of its activity via activation of PTPase might be expected to reverse the transformed phenotype. For example, overexpression of a 5 l&Da protein tyrosine phosphatase (PTPl), in src-transformed NIH 3T3 cells, resulted in cells that are more flattened, less refractile and have decreased phosphotyrosyl content of a 70 kDa protein. However, little apparent change in the rate of cell proliferation or tyrosine phosphorylation of pp60v-srcis observed [ 171. In our study, addition of P-Tyr resulted in considerable dephosphorylation of pp60v-s’cprotein, which was only partially inhibitable by o-vanadate. We have previously demonstrated that P-Tyr may also inhibit kinase function [8]. Perhaps the activity of tyrosine kinases of the src-transformed cells were sensitive to P-Tyr inhibition. This would result in inhibition of tyrosine phosphorylation in src-transformed cells even in the presence of vanadate. P-Tyr had little effect on erbB-2 tyr-phosphorylation in SKOV-3 cells, and failed to inhibit growth of the cell line. Thus, P-Tyr mediated dephosphorylation of cellular phosphoproteins at tyrosine residues, may be selective and associated with growth inhibition. However, inhibition of tyrosyl phosphorylation by overexpression of a cytosolic PTPase in erbB-2 transformed fibroblasts decreased cell proliferation indicating erbB-2 transformed cells can be inhibited by activation of PTPases other methods [ 181. In conclusion, we have demonstrated that exogenous P-Tyr inhibits insulin-induced tyrosyl phosphorylation of IR in HEPG2 cells, and basal levels of tyrphosphorylation in v-src transformed NIH 3T3 cells. Cell growth is inhibited in association with decreased levels of protein tyrosyl phosphorylation. Tyr-phosphorylation in SK-OV3 cells is unaffected by P-Tyr, as is cell growth. The mechanism by which P-Tyr inhibits EGFR, IR, and p~60”-~” tyrosine phosphorylation in PTyr sensitive tumor cells is not known. However, these studies suggest that decreasing cellular tyrosyl phosphorylation by exogenous compounds may result in cell growth inhibition.

Cancer Letters 102 (1996) 65-71

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ity associatedwith the avian sarcomavirus src gene product. Proc. Natl. Acad. Sci. USA, 75, 2021-2024. [16] Levinson, A.D., Oppermann, H., Levintow, L., Varmus, H.E. and Bishop, J.M. (1978) Evidence that the transforming gene of avian sarcomavirus encodesa protein kinase associated with a phosphoprotein. Cell, 15, 561-572. [17] Woodford-Thomas, T.A., Rhodes, J.D. and Dixon, J.E.

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(1992) Expression of a protein tyrosine phosphatasein norma1 and v-src-transformed mouse 3T3 tibroblasts. J. Cell Biol., 117,401414. [18] Ramoni, G., Ruggiero, M., Raugei, G., Berti, et al. (1992) Overexpression of a synthetic phosphotyrosine protein phosphatase gene inhibits normal and transformed cell growth. Inst. J. Cancer, 51.652656.