Characterization and use of monoclonal antibodies for isolation of phosphotyrosyl proteins from retrovirus-transformed cells and growth factor-stimulated cells.

Protein kinases that phosphorylate the hydroxyl group of tyrosine residues of proteins have been implicated in cell transformation by some retroviruses and in regulation of normal cell growth by some polypeptide growth factors. To facilitate the identification of tyrosine kinase substrates, we developed monoclonal antibodies to the hapten azobenzylphosphonate. One of these antibodies, MA-2G8, proved to be especially attractive in that it bound a derivative of aminophenylphosphate, a close phosphotyrosine analog, with higher affinity than it bound the corresponding derivative of aminobenzylphosphonate; however, its affinity for phosphoserine was negligible. In this paper we describe the optimal conditions for using this antibody to isolate phosphotyrosine proteins, emphasizing particularly that its interaction with phosphotyrosyl proteins is sensitive to ionic detergents and to antibody density on the immunosorbent matrix. The antibody also bound ATP citrate lyase; this enzyme lacks phosphotyrosine but contains phosphohistidine, which is similar structurally to phosphotyrosine. By attaching the antibody at high density to Sepharose beads and omitting ionic detergents from the buffers, it was possible by microbatch immunoadsorption (followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) to isolate the 120,000-dalton transforming protein and several other phosphotyrosyl proteins from cells transformed by Abelson murine leukemia virus. Under the same conditions, phosphotyrosyl proteins were also isolated from human epidermal carcinoma cells (A431) that had been stimulated with epidermal growth factor; most prominent among these proteins was the 170,000-dalton receptor for epidermal growth factor.

Application of a galactosidase immunosorbent test to carcinoembryonic antigen in plasma.

The galactosidase immunosorbent test for carcinoembryonic antigen is simple to perform, uses stable reagents, does not require radioactive reagents, and is adaptable to large numbers of samples. Concentration of carcinoembryonic antigen in sera or plasma was determined by the galactosidase immunosorbent test and by the Egan-Todd double antibody assay (93% agreement), indirect Z-gel (83% agreement), and direct Z-gel assay (ps = 0.97). The galactosidase immunosorbent test has potential as a clinically useful nonisotopic assay for carcinoembryonic antigen.

A galactosidase immunosorbent test for carcinoembryonic antigen.

A galactosidase immunosorbent test for carcinoembryonic antigen (CEA) is described in which the amount of galactosidase adsorbed to a cellulose disc is a hyperbolic function of CEA concentration. Thus, molecules with CEA-like activity can be characterized by mathematical analysis of data obtained from the galactosidase immunosorbent test. By such analysis, CEA-reactive molecules in normal human plasma were distinguished from normal cross-reacting antigen and from authentic CEA. Variation of the amount of antibody-enzyme conjugate used in the galactosidase immunosorbent test permitted CEA-reactive material in plasma of a patient with rectal carcinoma to be antigenically distinguished from the CEA-reactive material in urine of a patient with bladder carcinoma. The galactosidase immunosorbent test is a useful tool for analysis of CEA-reactive molecules.

Modulation of p53 expression by human recombinant interferon-alpha2a correlates with abrogation of cisplatin resistance in a human melanoma cell line.

G3361/CP cells, a cisplatin (CDDP)-resistant subclone of the human melanoma cell line G3361, overexpress wild-type p53 protein and demonstrate an increase in the percentage of cells in G0--G1 arrest compared to parental cells. Exposing G3361/CP cells to human recombinant IFN-alpha2a reduces the high basal levels of p53, releases G3361/CP cells from G0-G1 into S phase, and abrogates CDDP resistance. These findings suggest that recombinant IFN-alpha2a disrupts p53-mediated cell cycle regulation to restore CDDP sensitivity in G3361/CP cells.

Protein-tyrosine kinase activity and pp60v-src expression in whole cells measured by flow cytometry.

The expression and phosphotyrosine activity of pp60v-src were measured in the B31 avian sarcoma virus-transformed rat cell line by flow cytometry using monoclonal antibodies against pp60v-src (EB7) and phosphotyrosine (1G2). Although the immunocytochemical staining was markedly heterogeneous, binding of both antibodies was significantly greater to B31 cells than to untransformed Rat 1 cells. Binding of 1G2 to phosphotyrosine residues was specific; it was entirely inhibited by adding excess phenylphosphate but was not affected by phosphoserine or phosphothreonine. The relationship between the amount of phosphorylated tyrosine measured by our FCM technique and total cellular phosphotyrosine measured by phosphoamino acid analysis was linear in vanadate-treated BALB/c 3T3 cells. Treatment of B31 cells for 48 h with herbimycin A, a benzenoid ansamycin antibiotic, to decrease the expression and tyrosine kinase activity of pp60v-src caused reductions of 42% in anti-pp60v-src and 58% in anti-phosphotyrosine antibody immunofluorescence. DNA staining with the fluorescent dye propidium iodide showed no cell cycle specificity in the binding of either antibody. Herbimycin A also caused the transformed cell line to revert to the morphology, actin configuration, and growth behavior of untransformed cells; these changes were reversed within 12 h after removal of the drug. Flow cytometric evaluation of tyrosine kinase expression and activity was fast and easy, and the results correlated well with other measures of cell phenotype. This technique can be used to quantitate the effects of drugs on oncogenic proteins such as pp60v-src and their associated tyrosine kinase activity.

Human monoclonal antibodies reactive with human myelomonocytic leukemia cells.

Peripheral blood mononuclear cells from a patient with chronic myelogenous leukemia (CML), in remission, were depleted of CD8-positive T-cells and cultured with Epstein-Barr virus. Four of 20 cultures (20%) secreted human IgG antibodies selectively reactive with the cell surfaces of certain human leukemia cell lines. Three polyclonal, Epstein-Barr virus-transformed, B-cell lines were expanded and fused with the human-mouse myeloma analogue HMMA2.11TG/O. Antibody from secreting clones HL 1.2 (IgG1), HL 2.1 (IgG3), and HL 3.1 (IgG1) have been characterized. All three react with HL-60 (promyelocytic), RWLeu4 (CML promyelocytic), and U937 (monocytic), but not with KG-1 (myeloblastic) or K562 (CML erythroid). There is no reactivity with T-cell lines, Burkitt's cell lines, pre-B-leukemia cell lines, or an undifferentiated CML cell line, BV173. Leukemic cells from two of seven patients with acute myelogenous leukemia and one of five with acute lymphocytic leukemia react with all three antibodies. Normal lymphocytes, monocytes, polymorphonuclear cells, red blood cells, bone marrow cells, and platelets do not react. Samples from patients with other diverse hematopoietic malignancies showed no reactivity. Immunoprecipitations suggest that the reactive antigen(s) is a lactoperoxidase iodinatable series of cell surface proteins with molecular weights of 42,000-54,000 and a noniodinatable protein with a molecular weight of 82,000. Based on these data these human monoclonal antibodies appear to react with myelomonocytic leukemic cells and may detect a leukemia-specific antigen or a highly restricted differentiation antigen.

Differentiation-induced changes in protein-tyrosine phosphatase activity and commensurate expression of CD45 in human leukemia cell lines.

Pharmacologic differentiation of the promyelocytic leukemia HL60 is associated with an increase in cellular tyrosine phosphatase activity. We asked (a) if this increase might, at least in part, be due to changes in a transmembranous protein-tyrosine phosphatase, CD45; and (b) if CD45 changes similarly in other differentiating leukemias. Differentiation of HL60, several chronic myelogenous leukemias, a monocytic leukemia (THP-1), and a monoblastoid leukemia (U-937) could be induced by phorbol ester, 1,25-dihydroxy vitamin D3, dimethyl sulfoxide, or cyclic AMP analogues. This differentiation was associated with a marked increase in (a) total cellular tyrosine phosphatase activity (2-4-fold as measured by the ability to dephosphorylate a tyrosine-phosphorylated peptide); (b) CD45-specific tyrosine phosphatase activity (2-4-fold); (c) CD45 cell surface expression by flow cytometry (2-5-fold); (d) synthesis of both exon B-dependent M(r) 205,000 and exon ABC- M(r) 185,000 CD45 proteins, as revealed by immunoprecipitation with antisera specific for CD45 isoforms. Both isoforms have enhanced electrophoretic mobility when isolated from the differentiated cells. This enhanced mobility did not appear to be due to decreased stoichiometry of CD45 phosphorylation on serine/threonine residues. Interestingly, 12-O-tetradecanoylphorbol-13-acetate transiently reduced CD45 protein-tyrosine phosphatase activity in the chronic myelogenous leukemia cell RWLeu4 without altering the CD45 amount (as measured by cell surface immunofluorescence). Modulation of CD45 tyrosine phosphatase activity (and protein levels) may play a role in differentiation or in maintaining cells in a nonproliferative state or may represent a phenotypic marker of differentiation.

Antitumor activity of basic fibroblast growth factor-saporin mitotoxin in vitro and in vivo.

Many cancer cell lines express basic fibroblast growth factor (FGF) receptors, making them potential targets for the delivery of FGF-based cytotoxic compounds. To this end, we have investigated the antitumor activity of a novel mitotoxin, Fibroblast Growth Factor-saporin (FGF-SAP), a conjugate of FGF and the ribosome-inactivating protein, saporin. In vitro, FGF-SAP is cytotoxic for human melanoma, teratocarcinoma, and neuroblastoma cells expressing FGF-receptors. Mice treated with FGF-SAP i.v., on a variety of schedules, showed dramatic tumor growth inhibition with minimal toxicity. Thus, FGF-SAP appears to be a well-tolerated and potent antitumor agent. The potential of FGF-targeted cytotoxicity is discussed.

Estrogen-induced activation of Erk-1 and Erk-2 requires the G protein-coupled receptor homolog, GPR30, and occurs via trans-activation of the epidermal growth factor receptor through release of HB-EGF.

Estrogen rapidly activates the mitogen-activated protein kinases, Erk-1 and Erk-2, via an as yet unknown mechanism. Here, evidence is provided that estrogen-induced Erk-1/-2 activation occurs independently of known estrogen receptors, but requires the expression of the G protein-coupled receptor homolog, GPR30. We show that 17beta-estradiol activates Erk-1/-2 not only in MCF-7 cells, which express both estrogen receptor alpha (ER alpha) and ER beta, but also in SKBR3 breast cancer cells, which fail to express either receptor. Immunoblot analysis using GPR30 peptide antibodies showed that this estrogen response was associated with the presence of GPR30 protein in these cells. MDA-MB-231 breast cancer cells (ER alpha-, ER beta+) are GPR30 deficient and insensitive to Erk-1/-2 activation by 17beta-estradiol. Transfection of MDA-MB-231 cells with a GPR30 complementary DNA resulted in overexpression of GPR30 protein and conversion to an estrogen-responsive phenotype. In addition, GPR30-dependent Erk-1/-2 activation was triggered by ER antagonists, including ICI 182,780, yet not by 17alpha-estradiol or progesterone. Consistent with acting through a G protein-coupled receptor, estradiol signaling to Erk-1/-2 occurred via a Gbetagamma-dependent, pertussis toxin-sensitive pathway that required Src-related tyrosine kinase activity and tyrosine phosphorylation of tyrosine 317 of the Shc adapter protein. Reinforcing this idea, estradiol signaling to Erk-1/-2 was dependent upon trans-activation of the epidermal growth factor (EGF) receptor via release of heparan-bound EGF (HB-EGF). Estradiol signaling to Erk-1/-2 could be blocked by: 1) inhibiting EGF-receptor tyrosine kinase activity, 2) neutralizing HB-EGF with antibodies, or 3) down-modulating HB-EGF from the cell surface with the diphtheria toxin mutant, CRM-197. Our data imply that ER-negative breast tumors that continue to express GPR30 may use estrogen to drive growth factor-dependent cellular responses.

The critical role of Shc in insulin-like growth factor-I-mediated mitogenesis and differentiation in 3T3-L1 preadipocytes.

Insulin-like growth factor-I (IGF-I) stimulates mitogenesis in proliferating preadipocytes, but when cells reach confluence and become growth arrested, IGF-I stimulates differentiation into adipocytes. IGF-I induces signaling pathways that involve IGF-I receptor-mediated tyrosine phosphorylation of Shc and insulin receptor substrate 1 (IRS-1). Either of these adaptor proteins can lead to activation of the three-kinase cascade ending in activation of the extracellular signal-regulated kinase 1 and -2 (ERK-1 and -2) mitogen-activated protein kinases (MAPKs). Several lines of evidence suggest that activation of MAPK inhibits 3T3-L1 preadipocyte differentiation. We have shown that IGF-I stimulation of MAPK activity is lost as 3T3-L1 preadipocytes begin to differentiate. This change in MAPK signaling coincides with loss of IGF-I-mediated Shc, but not IRS-1, tyrosine phosphorylation. We hypothesized that down-regulation of MAPK via loss of proximal signaling through Shc is an early component in the IGF-I switch from mitogenesis to differentiation in 3T3-L1 preadipocytes. Treatment of subconfluent cells with the MEK inhibitor PD098059 inhibited both IGF-I-activation of MAPK as well as 3H-thymidine incorporation. PD098059, in the presence of differentiation-inducing media, accelerated differentiation in subconfluent cells as measured by expression of adipocyte protein-2 (aP-2), peroxisome proliferator-activated receptor gamma (PPARgamma) and lipoprotein lipase (LPL). Transient transfection of subconfluent cells with Shc-Y317F, a dominant-negative mutant, attenuated IGF-I-mediated MAPK activation, inhibited DNA synthesis, and accelerated expression of differentiation markers aP-2, PPARgamma, and LPL. We conclude that signaling through Shc to MAPK plays a critical role in mediating IGF-I-stimulated 3T3-L1 mitogenesis. Our results suggest that loss of the ability of IGF-I to activate Shc signaling to MAPK may be an early component of adipogenesis in 3T3-L1 cells.

Autocrine down-regulation of basic fibroblast growth factor receptors causes mitotoxin resistance in a human melanoma cell line.

The ability of melanoma to develop resistance to mitotoxins, growth-factor-directed anti-neoplastic agents that offer potential for the treatment of this highly refractory disease, may limit therapeutic efficacy. To address this problem, we developed a subcloned human melanoma cell line that is resistant to the mitotoxin composed of basic fibroblast growth factor conjugated to the ribosome-inactivating protein saporin. Resistance was caused by autocrine FGF ligands, which down-regulate bFGF receptors and reduce bFGF-saporin binding. Inhibiting the autocrine loop with suramin or with neutralizing antibodies to FGF up-regulated receptors and decreased resistance in vitro. Furthermore, suramin restored sensitivity in resistant tumor xenografts. These results suggest the potential of therapeutic modalities combining agents that neutralize growth factors with receptor-directed mitotoxins for targeting malignant melanoma either to prevent emergence of resistance or to circumvent resistance once it occurs.

Actions of TNF and IFN-gamma on angiogenesis in vitro.

We have developed a model system for studying angiogenesis in which microvascular fragments and myofibroblasts (Mf) isolated from lipid tissues are grown in co-culture. We have found that Mf induce capillary formation by producing an endothelial cell growth factor and by secreting an extracellular matrix that causes endothelial cells to form a cordlike structure. This system appeared to be well suited for examining the effects of vasoactive substances such as the TNF and INF-gamma on capillary growth. TNF-alpha,beta, and IFN-gamma not only significantly inhibited capillary growth induced by Mf, but also blocked capillary development induced by fibroblast growth factors (FGF), well-known potent angiogenic factors. Recently, we have found that platelet-derived growth factor (PDGF) enhances in vitro capillary formation, probably at least in part by acting on Mf. Just as with FGF, capillary growth in the presence of PDGF was almost completely blocked by IFN-gamma. We examined the mode by which IFN-gamma inhibits angiogenesis and found that IFN-gamma inhibits both the proliferation of endothelial cells and collagen(s) synthesis by Mf. These actions of TNF or IFN-gamma could limit vascular formation in solid tumors.

Receptors for platelet-derived growth factor on microvascular endothelial cells.

Endothelial cells are widely thought to be unresponsive to platelet-derived growth factor (PDGF) and devoid of PDGF receptors. However, in examining the growth factor responses of microvascular endothelial cells isolated from human omental adipose tissue, we detected PDGF-induced tyrosine phosphorylation of an 180-kD glycoprotein, subsequently identified as the cellular receptor for PDGF by specific immunoprecipitation. Scatchard analysis of 125I-PDGF binding to human microvascular endothelial cells revealed 30,000 PDGF receptors/cell with a kD of 0.14 nM. PDGF stimulated tyrosine phosphorylation of PDGF and other cellular proteins in a dose- and time-dependent manner, with half-maximal receptor phosphorylation occurring at 0.3 nM recombinant human PDGF-BB within 1 min of PDGF exposure. Normal cellular consequences of receptor activation were also observed, including tyrosine phosphorylation of a 42-kD protein and serine phosphorylation of ribosomal protein S6. Furthermore, PDGF was mitogenic for these cells. The finding of functional PDGF receptors on human microvascular endothelial cells suggests an important direct role for PDGF in neovascularization.

Tyrosine phosphorylated proteins in chronic myelogenous leukemia.

An aberrantly expressed and highly active abl tyrosine kinase (p210bcr-abl) appears critical for the development and pathogenesis of chronic myelogenous-leukemia (CML). CML cells and cell lines each displayed a similar spectrum of phosphotyrosyl proteins. Analysis of these proteins by glycerol-gradient ultracentrifugation showed that many apparently existed as multimeric complexes. Confirming this, several of these proteins co-immunoprecipitated, along with the p210bcr-abl, with antibody to abl. Included were co-precipitating proteins identified as the p120 ras GTPase-activating protein (GAP) and the p62 protein that binds both to GAP and to a number of other tyrosine-phosphorylated proteins having peptide regions homologous to the second domain of src. Because p62, ras GAP and ras are involved in growth-factor and oncogene activation of cells, this pathway may also play an important role in CML.

Wortmannin, a phosphoinositide 3-kinase inhibitor, selectively enhances cytotoxicity of receptor-directed-toxin chimeras in vitro and in vivo.

BACKGROUND: Generalized resistance of some neoplastic cell lines to treatment with ligand-toxin chimeras has been attributed to an increased rate of lysosomal uptake and degradation following endocytosis of the chimera-receptor complex. Because phosphoinositide 3-kinase (Pl 3-kinase) activity is known to play a role in intracellular trafficking, particularly from endosomes to lysosomes, we hypothesized that co-exposing cells to the Pl 3-kinase inhibitor, wortmannin, might enhance cytotoxicity of ligand-toxin chimeras. METHODS: In vitro, cytotoxicity of five receptor directed-toxin chimeras (bFGF-SAP, bFGF-PE, aFGF-PE, HBEGF-SAP, bFGF-gelonin) and an immunotoxin (11A8-SAP) was examined in the presence or absence of this Pl 3-kinase inhibitor against a panel of human neoplastic cell lines: SK-MEL-5 (melanoma), PA-1 (ovarian teratocarcinoma), DU145 (prostatic carcinoma) and MCF-7 (breast carcinoma). In vivo, antitumor activity of a treatment regimen combining wortmannin (1 or 2 mg/kg i.p.) and bFGF-SAP (10 micrograms/kg i.v.) once a week for 4 weeks was evaluated compared to administration of each agent alone in C3H/HeN mice implanted with the FSallC murine fibrosarcoma. RESULTS: At concentrations greater than the reported Ki for Pl 3-kinase inhibition (1-10 microM), wortmannin enhanced cytotoxicity when combined with saporin or gelonin chimeras, but produced subadditive cytotoxicity when combined with Pseudomonas exotoxin chimeras. When low nanomolar concentrations selective for Pl 3-kinase inhibition (5-100 nM) were examined for effects on one receptor directed-toxin chimera, wortmannin dramatically enhanced bFGF-SAP cytotoxicity in three of the four cell lines. A different Pl 3-kinase inhibitor, LY294002 (Ki approximately 1 microM), however, failed to potentiate bFGF-SAP. When administered to mice, wortmannin combined with bFGF-SAP resulted in a significant decrease in tumor volumes compared to vehicle-treated controls that was not observed in mice treated with either agent alone. CONCLUSIONS: Taken together, these results suggest that although wortmannin increases the cytotoxic efficacy of some receptor-directed chimeras, potentiation may occur through an alternative pathway not involving Pl 3-kinase inhibition.

Targeting human prostatic carcinoma through basic fibroblast growth factor receptors in an animal model: characterizing and circumventing mechanisms of tumor resistance.

BACKGROUND: Basic fibroblast growth factor receptors on DU145 human prostatic carcinoma xenografts serve as targets for the delivery of a growth factor-toxin chimera, basic fibroblast growth factor-saporin (bFGF-SAP), which produces significant antitumor activity in a nude mouse model. However, DU145 tumors often become resistant to prolonged treatment. METHODS: Nude mice bearing DU145 xenografts were intravenously administered bFGF-SAP (0.05 microg/kg weekly for 4 weeks), and a panel of eight tumors was isolated from the treated animals and established in monolayer culture. RESULTS: In cell-survival assays, sensitivity of the treated tumor-derived cell lines to bFGF-SAP (IC50 = 12-100 nM) varied widely from cells derived from a vehicle-treated control tumor (IC50 = 10 nM). A significant inverse correlation was observed between increased IC50 values in vitro and increased tumor growth delay in vivo. Pretreatment of tumor cells with suramin or neutralizing antibodies to bFGF or keratinocyte growth factor (KGF) circumvented resistance in one of the tumor lines, confirming autocrine-mediated resistance. In another tumor subline, a 3-fold decrease in bFGF high-affinity receptor sites, which concurred with a 4-fold decrease in ability to internalize the bFGF ligand, was consistent with a decrease in total cellular expression of the FGF2 receptor (Bek). Resistance was circumvented by alternatively targeting FGF1 receptor (Flg) on these cells with a saporin immunotoxin. CONCLUSIONS: These studies identify alterations in the ligand-targeted receptor as a frequent contributor to resistance arising in DU145 tumors to in vivo treatment with a bFGF receptor-directed-toxin chimera, and provide the basis for designing methods to circumvent resistance for the purpose of enhancing efficacy of receptor-directed therapies in the treatment of prostate cancer.

Diffusion control of the binding of carcinoembryonic antigen (CEA) with insoluble anti-CEA antibody.

Analysis of the kinetics of CEA binding to rabbit anti-CEA antibody insolubilized on filter paper discs provided six lines of evidence indicating diffusion control of this reaction: 1) The observed forward rate constant, k1obs, decreased when solvent viscosity was increased with sucrose or Ficoll. 2) k1obs per receptor site increased from 1.2 x 10(4) M-1 sec-1 to 2.6 x 10(4) M-1 sec-1 as receptor site density on immunosorbent discs decreased from 5.2 x 10(-13) to 2.1 x 10(-13) moles/disc. 3) Stirring the reacting mixture of CEA and insoluble anti-CEA increased k1obs from 1.2 x 10(4) M-1 sec-1 to 3.4 x 10(4) M-1 sec-1. 4) The activation energy for CEA-insoluble anti-CEA binding was indistinguishable (p greater than 0.1 by t-test) from 4.1 Kcal/mole expected for diffusion controlled reactions. 5) Bimolecular reaction theory predicted a diffusion limited forward rate constant, k1max, for CEA binding to insoluble anti-CEA, which was consistent with our observed k1 of 1.2 x 10(4) M-1 sec-1. 6) k1obs for CEA binding to soluble rabbit anti-CEA antibody was 6.4 x 10(4) M-1 sec-1, 5 times faster than the k1obs of the heterogeneous phase reaction for receptor site density of 5.2 x 10(-13) moles/disc. Diffusion control of ligand binding to insoluble receptors, as exemplified by the CEA-insoluble anti-CEA model system, may be a very general biologic phenomenon.

Constitutively tyrosine phosphorylated p52 Shc in breast cancer cells: correlation with ErbB2 and p66 Shc expression.

Breast cancer cell lines display a wide variety of growth factor receptors, and considerable evidence implicates signaling from these receptors, especially ErbB2, in the important early stages of this disease, contributing to malignant progression. If this is true, then we would hypothesize that a useful prognostic indicator would be the level of activity of a second messenger protein used in common by these receptors. One such second messenger is the Shc adapter protein, which is activated when tyrosine phosphorylated by receptors. Therefore, one prediction from the hypothesis is that the level of tyrosine-phosphorylated Shc (PY-Shc) in breast cancer cell lines would correlate with total receptor tyrosine kinase activity. To begin to test this prediction, we examined Shc tyrosine phosphorylation in a diverse group of breast cancer cell lines that display varied levels of ErbB2. Using Shc immunoprecipitation and anti-phosphotyrosine immunoblotting analysis, we found a strong correlation between the level of ErbB2 overexpression (r = 0.91, p < 0.0002) and PY-ErbB2 levels (r = 0.89, p = 0.0005) compared with the level of tyrosine phosphorylation of the p52 and p46 Shc isoforms. Consistent with Shc tyrosine phosphorylation being driven by ErbB2, an ErbB2-specific tyrosine kinase inhibitor markedly reduced Shc tyrosine phosphorylation. Unexpectedly, although all cell lines had comparable total amounts of p52 and p46 Shc, the amount of an inhibitory Shc isoform, p66, was inversely related to the level of ErbB2 expression (r = -0.86, p = 0.0013). This suggests that reduced p66 Shc expression may play a role in ErbB2-positive breast cancer. In summary, these data are consistent with our prediction that the cellular level of PY-Shc would correlate with the levels of activated ErbB2 displayed by cell lines derived from breast cancers.