Effect of formaldehyde at a low concentration on proliferation and organization of cytoskeleton of cultured cells

Formaldehyde at a concentration of up to 3–4% (1.07–1.42 M) is one of the most widespread and well-known fixatives of organs, tissues, and cells. In the present work, it was shown that formaldehyde at a concentration of up to 60 μM (0.0002%) did not produce negative effect on the viability of cells of lines of A431, HEK293, and primary fibroblasts, but increased the proliferative activity of the A431 cells. This action on the A431 cells can be explained by the activation of a receptor of the epidermal growth factor as a result of its interaction with formaldehyde.     

A431 cell variants lacking the blood group A antigen display increased high affinity epidermal growth factor-receptor number, protein-tyrosine kinase activity, and receptor turnover

The epidermal growth factor receptor (EGF-R) of human A431 cells bears an antigenic determinant that is closely related to the human blood group A carbohydrate structure. Labeling studies with blood group A reactive anti-EGF-R monoclonal antibodies and various lectins revealed that A431 cultures are heterogeneous with respect to blood group A expression. We have isolated clonal variants of these cells that either express (A431A+ cells) or completely lack (A431A- cells) the blood group A specific N-acetyl-D-galactosamine (GalNAc) residue. We show that this difference is due to the absence of a UDP-GalNAc:Gal transferase activity in A431A- cells. Subsequently, we have compared EGF-R functioning in these cell lines. Scatchard analysis of EGF-binding shows that in A431A- cells 6.3% of the EGF-R belongs to a high affinity subclass (Kd = 0.4 nM) while in A431A+ this subclass represents only 3.2% of the total receptor pool. The elevated level of high affinity receptors in A431A- cells is accompanied by a parallel increase in receptor protein- tyrosine kinase activity. In membrane preparations of A431A- cells, receptor autophosphorylation as well as phosphorylation of a tyrosine-containing peptide substrate is 2-3-fold higher as compared with A431A+ cells. In intact A431A-cells, the difference in receptor activity is measured as a 2-3-fold elevated level of receptor phosphorylation and a 2-3-fold higher abundance of phosphotyrosine in total cellular protein in A431A- cells. In addition, [35S]methionine pulse-chase experiments showed a ligand-independent increase in turnover of EGF-R in A431A- cells: the receptor's half life in these cells is 10 h as compared with 17 h in A431A+ cells. Our results suggest a possible involvement of GalNAc residue(s) in determining EGF-R affinity, protein-tyrosine kinase activity and turnover in A431 cells. Furthermore, our results indicate that high affinity EGF-R are the biologically active species with respect to protein-tyrosine kinase activity.     

Monoclonal antibodies against the human epidermal growth factor receptor from A431 cells. Isolation, characterization, and use in the purification of active epidermal growth factor receptor

A431 cells have been used as an immunogen for generating monoclonal antibodies against the epidermal growth factor (EGF) receptor. Two immunoglobulin M and eight immunoglobulin G3 anti-EGF receptor antibodies were cloned. All ten antibodies immunoprecipitated biosynthetically labeled mature A431 cell EGF receptor and were able to recognize the receptor in Western blotting. However, none of the antibodies immunoprecipitated precursor polypeptides of the A431 cell EGF receptor, neither did they recognize EGF receptors from human foreskin fibroblasts, human placenta, nor a human-mouse hybrid cell expressing EGF receptor. The antibodies were found to bind to glycolipids from A431 cells and it was shown that the determinant involved was the blood group A antigen. It appears that this determinant is present on both the EGF receptor and glycolipids of A431 cells but is not expressed on EGF receptors from other human cells tested. One of the monoclonal antibodies raised was used for immunoaffinity purification of the EGF receptor. The procedure took advantage of the carbohydrate nature of the antigenic determinant by employing sugar-specific elution. The mild conditions permitted the purification of A431 cell EGF receptor (70-80% pure) that possessed an intrinsic EGF-stimulated tyrosine kinase activity with a specific activity of about 20 nmol/min/mg.     

The titration of tetanus antitoxin V. Effect of formalization method for the fixation of sheep erythrocytes on the indirect haemagglutination test

Two methods of fixation of sheep erythrocytes with formaldehyde for the titration of tetanus antitoxin by the indirect haemagglutination (IHA) test have been compared. The cells fixed with 3% formaldehyde at 4-8 degrees C for 24 h (formaldehyde (I) fixed cells) were less sensitive than the cells fixed with 3% formaldehyde at 4-8 degrees C for 24 h and subsequently treated with 40% formaldehyde at 4-8 degrees C for a further 24 h (formaldehyde (II) fixed cells). The correlation between the toxin neutralization (TN) and IHA titres using formaldehyde (I) fixed cells was better than that obtained with formaldehyde (II) fixed cells. There was no statistically significant difference between TN and IHA titres after treatment of the sera with 2-Mercaptoethanol using formaldehyde (I) fixed cells. Formaldehyde (I) fixed cells can be used for two months with adequate sensitivity to detect the minimum protective level of tetanus antitoxin in the sera.     

Ligand-induced association of epidermal growth factor receptor to the cytoskeleton of A431 cells

Recently, we have obtained evidence in favor of a structural interaction between the epidermal growth factor (EGF) receptor and the Triton X-100-insoluble cytoskeleton of epidermoid carcinoma A431 cells. Here we present a further analysis of the properties of EGF receptors attached to the cytoskeleton. Steady-state EGF binding studies, analyzed according to the Scatchard method, showed that A431 cells contain two classes of EGF-binding sites: a high-affinity site with an apparent dissociation constant (KD) of 0.7 nM (7.5 x 10(4) sites per cell) and a low-affinity site with a KD of 8.5 nM (1.9 x 10(6) sites per cell). Non-equilibrium binding studies revealed the existence of two kinetically distinguishable sites: a fast-dissociating site, with a dissociation rate constant (k-1) of 1.1 x 10(-3) s-1 (1.0-1.3 x 10(6) sites per cell) and a slow-dissociating site, with a k-1 of 3.5 x 10(-5) s-1 (0.6-0.7 x 10(6) sites per cell). The cytoskeleton of A431 cells was isolated by Triton X-100 extraction. Scatchard analysis revealed that approximately 5% of the original number of receptors were associated with the cytoskeleton predominantly via high-affinity sites (KD = 1.5 nM). This class of receptors is further characterized by the presence of a fast-dissociating component (k-1 = 2.0 x 10(-3) s-1) and a slow-dissociating component (k-1 = 9.1 x 10(-5) s-1). The distribution between fast and slow sites of the cytoskeleton was similar to that of intact cells (65% fast and 35% slow sites). Incubation of A431 cells for 2 h at 4 degrees C in the presence of EGF resulted in a dramatic increase in the number of EGF receptors associated to the cytoskeleton. These newly cytoskeleton-associated receptors appeared to represent low-affinity binding sites (KD = 7 nM). Dissociation kinetics also revealed an increase of fast-dissociating sites. These results indicate that at 4 degrees C EGF induces the binding of low-affinity, fast-dissociating sites to the cytoskeleton of A431 cells.     

Localisation of EGF-Receptor mRNA in the nucleus of A431 cells by light microscopy.

We have localized the mRNA of the epidermal growth factor receptor (EGF-receptor) in nuclei of A431 cells by non-radioactive in situ hybridization at the light microscopical level using digoxigenin-labelled DNA probes. Both formaldehyde and glutaraldehyde fixations were tested before the hybridization was performed. Glutaraldehyde, compared with formaldehyde fixation, gives a less diffuse hybridization signal, which is easier to localize. Therefore, glutaraldehyde was used as a fixative in the hybridization experiments. It is demonstrated that the mRNA of the EGF-receptor is present in restricted domains mainly located around the nucleolus. This location of the EGF-receptor mRNA was unaltered after extraction of chromatin. Therefore it is concluded that the messenger RNA of the EGF-receptor is attached to the nuclear matrix. A possible biological role for the location of mRNA of the EGF-receptor around the nucleolus is discussed.     

A proteomic approach to cisplatin resistance in the cervix squamous cell carcinoma cell line A431

Since drug resistance is a complex and multifactorial event involving activation/repression of multiple biochemical pathways, we used a proteomic approach to study cisplatin resistance and drug response in human tumor cell lines. The cervix squamous cell carcinoma cell line A431 and its cisplatin-resistant subline, A431/Pt, were used as a model system. The experimental set-up involved not just a two-way comparison of the control vs. the drug-resistant cell line, but also an acute cisplatin treatment of both cell lines, leading to a four-way comparison, as follows: 1) A431 vs. A431/Pt cells; 2) A431 vs. A431 cisplatin exposed cells; 3) A431/Pt vs. A431/Pt cisplatin exposed cells; 4) A431 cisplatin exposed cells vs. A431/Pt cisplatin exposed cells. We found modulation of proteins, which could be classified under various categories, such as molecular chaperones (e.g. heat-shock proteins HSP60, HSP90, HSC71, heat-shock cognate 71 kDa protein), Ca2+-binding proteins (e.g. calmodulin, calumenin), proteins involved in drug detoxification (such as peroxiredoxins PRX 2 and PRX 6, and glutathione-S-transferase, GST), anti-apoptotic proteins (such as 14-3-3 switched on in cisplatin-exposed cells) and ion channels (such as VDAC-1, voltage-dependent anion-selective channel). In particular, the basal levels of HSC71 and HSP60 were increased in A431/Pt cells as compared to A431 cells, and cisplatin exposure resulted in up-regulation of HSP60 and HSP90 only in A431 cells. Moreover, cisplatin exposure up-regulated the anti-apoptotic 14-3-3 protein in both cell lines, GST in sensitive cells and PRX6 in A431/Pt cells. These findings are consistent with a constitutive expression of defence factors by resistant cells and with activation by cisplatin of mechanisms acting to protect cells from drug-induced damage. This pattern of response, also observed in parental cells, could reflect an intrinsic resistance of this tumor type.     

Epidermal growth factor induces terminal differentiation in human epidermoid carcinoma cells

Previous studies have reported that the proliferation of A431 cells, a human squamous cell carcinoma cell line, was stimulated by picomolar epidermal growth factor (EGF) but inhibited by nanomolar EGF. This biphasic dose-response phenomenon is not observed in normal human epithelial cells where nanomolar EGF is usually mitogenic. We have examined the effects of inhibitory and stimulatory concentrations of EGF on the growth and differentiation of A431 cells. In the presence of 100 pM EGF, A431 cells showed a mild increase in growth rate (129% of control) compared to cells grown in the absence of EGF. At 10 nM EGF, growth inhibition to 63% of control was observed. EGF at 10 nM stimulates a twofold increase both in cornified envelope formation and in epidermal transglutaminase activity, suggesting that high concentrations of EGF induce terminal differentiation in A431 cells. Mitogenic concentrations of EGF (100 pM) had no significant effect on these differentiation markers. Chronic exposure of A431 cells to 20 or 50 nM EGF resulted in EGF-resistant A431 variants that are neither growth arrested nor induced to terminally differentiate by 10 nM EGF. Removal of EGF from the growth medium of the EGF-resistant cells resulted in the reversion of these cells back to the wild-type A431 biphasic response pattern within 2 weeks. Our results suggest that A431 cells have the capacity to non-mutatively alter their response pattern to EGF in vitro to maintain themselves in a state of optimum proliferation and away from terminal differentiation. © 1993 Wiley-Liss, Inc.     

Regulation by ceramide of epidermal growth factor signal transduction and mitogenesis in cell lines overexpressing the growth factor receptor.

Ceramide has emerged as a pleiotropic signal mediator of cellular responses including differentiation, proliferation, cell cycle arrest and apoptosis. In the present study we evaluated the effect of cell permeant ceramide analogues on ligand-induced tyrosine phosphorylation of the EGF receptor (EGFR), phospholipase Cy (PLCgamma) activity and cell proliferation. Treatment with N-acetylsphingosine (C2-cer) and N-hexanoylceramide (C6-cer) prevented EGF-induced tyrosine trans-phosphorylation of the receptor in two different cell lines overexpressing the human EGFR (A431 and EGF-T17 cells). In contrast, treatment of A431 and EGFR-T17 cells with C2-cer or C6-cer did not affect the ligand binding capacity of the receptor, an effect that was however observed after TPA-induced activation of PKC. In addition EGF-stimulated PLCgamma activity was transiently decreased in A431 cells treated with C6-cer and only a modest, albeit significant reduction on ligand-induced 3H-InsP3 generation was observed in EGFR-T17 cells pretreated with ceramide. We also examined the effect of C2-cer on serum (A431)- or EGF (EGFR-T 17)-induced cell proliferation. Treatment of EGFR-TI7 cells with C2-cer (0.1-10 microM) did not affect cell viability, but prevented EGF-induced 3H-thymidine incorporation in a dose-dependent manner. In contrast, 3H-thymidine incorporation in serum-stimulated A431 cells decreased only at the higher doses of C2-cer used (1-10 microM), being this effect accompanied by a slight, albeit significant (20-25%), reduction in cell viability.     

Similarities and differences between the effects of epidermal growth factor and Rous sarcoma virus

We have derived a line of A431 human tumor cells infected with Rous sarcoma virus (RSV). The infected cells contain the RSV-transforming protein, pp60src, which has characteristic tyrosine specific protein kinase activity. As in other RSV-transformed cells, a 36,000-dalton protein is phosphorylated in RSV-infected A431 cells. Addition of epidermal growth factor (EGF) to the cells induces further phosphorylation of this protein. In contrast, this phosphoprotein is not detected in uninfected A431 cells, except when treated with EGF. Increased phosphorylation of the EGF receptor protein and of an 81,000- dalton cellular protein is dependent upon addition of EGF to the culture fluids, in both control and RSV-infected A431 cells. The results are discussed with reference to the similarities and differences between the tyrosine-specific protein kinases induced by RSV and activated by EGF.     

Transforming growth factor beta modulates phosphorylation of the epidermal growth factor receptor and proliferation of A431 cells.

Transforming growth factor beta (TGF-beta) increased the phosphorylation of the epidermal growth factor (EGF) receptor and inhibited the growth of A431 cells. Incubation with TGF-beta induced maximal EGF receptor phosphorylation to levels 1.5-fold higher than controls. Phosphorylation increased more prominently (4-5-fold) on tyrosine residues as determined by phosphoamino acid analysis and antiphosphotyrosine antibody immunoblotting. The kinase activity of EGF receptor was also elevated 2.5-fold when cells were cultured in the presence of TGF-beta. The antiproliferative effect of TGF-beta on A431 cells was accompanied by prolongation of G0-G1 phase and by morphological changes. TGF-beta augmented the growth inhibition of A431 cells which could be induced by EGF. In parallel, the specific EGF-induced increase in total phosphorylation of the EGF receptor was also augmented in the presence of TGF-beta. In cells cultured with TGF-beta, the phosphorylation of EGF receptor tyrosines induced by 20-min exposure to EGF was further increased 2-3-fold, suggesting additive effects upon receptor phosphorylation. EGF receptor activation by TGF-beta is characterized by kinetics quite distinct from that induced by EGF and therefore appears to take place through an independent mechanism. The TGF-beta-induced elevation in the phosphorylation of the EGF receptor may have a role in the augmented growth inhibition of A431 cells observed in the presence of EGF and TGF-beta.     

A role for plakophilin-1 in the initiation of desmosome assembly

Plakophilins (pkp-1, -2, and -3) comprise a family of armadillo-repeat containing proteins that are found in the desmosomal plaque and in the nucleus. Plakophilin-1 is most highly expressed in the suprabasal layers of the epidermis and loss of plakophilin-1 expression results in skin fragility-ectodermal dysplasia syndrome, which is characterized by a reduction in the number and size of desmosomes in the epithelia of affected individuals. To investigate the role of plakophilin-1 during desmosome formation, we fused plakophilin-1 to the hormone-binding domain of the estrogen receptor to create a fusion protein (plakophilin-1/ER) that can be activated in cell culture by the addition of 4-hydroxytamoxifen. When plakophilin-1/ER was expressed in A431 cells it was incorporated into endogenous desmosomes and did not disrupt desmosome formation. A derivative of A431 cells (A431D) do not form desmosomes, even though they express all the components believed to be necessary for desmosome assembly. Expression and activation of plakophilin-1/ER in A431D cells resulted in punctate desmoplakin staining on the cell surface. Co-expression of a classical cadherin (N-cadherin) and plakophilin-1/ER in A431D cells resulted in punctate desmoplakin staining at cell-cell borders. These data suggest that plakophilin-1 can induce assembly of desmosomal components in A431D cells in the absence of a classical cadherin; however a classical cadherin (N-cadherin) is required to direct assembly of desmosomes between adjacent cells. The activatable plakophilin-1/ER system provides a unique culture system to study the assembly of the desmosomal plaque in culture.     

Relation of epidermal growth factor receptor concentration to growth of human epidermoid carcinoma A431 cells

The relation between the concentration of epidermal growth factor (EGF) receptor/kinase and effects of EGF on cell proliferation has been studied using variant A431 cells and antagonist anti-EGF receptor monoclonal antibodies. Clonal A431 cell variants selected for escape from the EGF-mediated growth inhibition of parental A431 cells all have reduced concentrations of EGF receptor/kinase; Harvey sarcoma virus-transformed A431 cells, which have escaped from EGF-mediated growth inhibition, also have reduced EGF receptors. Three clonal variants which have reacquired EGF-mediated growth inhibition have 2- to 4-fold more EGF receptor than their respective parent variant. A biphasic response with stimulation at low and inhibition at high concentrations of EGF was especially evident in revertants of clone 29. Three separate antagonist monoclonal anti-EGF receptor antibodies block the growth inhibitory effects of EGF and uncover EGF-mediated growth stimulation. These studies indicate that in A431 cell variants a continuum of ligand-activated EGF receptors determines proliferative responses from low concentrations of active receptors under basal conditions to intermediate concentrations causing growth stimulation to high concentrations, causing inhibition of cell proliferation.     

Demonstration of epidermal growth factor-induced receptor dimerization in living cells using a chemical covalent cross-linking agent

We have used the soluble covalent cross-linking agent 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDAC) to examine the capacity of epidermal growth factor (EGF) to stimulate the dimerization of purified EGF receptor, of EGF receptor in membrane preparations and in intact A431 cells. The addition of EGF either to membranes from A431 cells or to EGF receptor which was purified from A431 cells by immunoaffinity chromatography caused the appearance of a cross-linked product of Mr 340,000 which was identified using EGF receptor-specific antibodies as an EGF receptor dimer. Three independent approaches including biosynthetic labeling, surface iodination, and immunoblotting experiments were utilized to follow EGF receptor dimerization in living A431 cells. These approaches provided consistent results indicating that EGF induced rapid dimerization of EGF receptor in living cells, suggesting that this process may play a role in transmembrane signalling mediated by EGF.     

Modulation of type alpha transforming growth factor receptors by a phorbol ester tumor promoter

Epidermal growth factor (EGF) and an EGF-like transforming growth factor (eTGF) from retrovirally transformed cells bind to a common receptor type in A431 cells. We have investigated the effects of the tumor promoter phorbol myristate acetate [PMA] on EGF/eTGF receptors in intact A431 cells. Treatment with PMA at 37 degrees C induces a complete loss of high-affinity (Kd = 35-50 pM) binding sites for eTGF and EGF on the cell surface of A431 cells. This effect is half-maximal at 0.1 nM PMA, exhibits rapid kinetics, and persists for at least 4 hr in the presence of PMA. eTGF and PMA added to intact A431 cells induce the phosphorylation of immunoprecipitable 170kd EGF/eTGF receptors. The EGF/eTGF receptor isolated from control cells was found to contain phosphoserine and phosphothreonine. PMA and eTGF caused a marked increase in the level of these two phosphoamino acids. In addition, eTGF but not PMA caused the appearance of phosphotyrosine in the EGF/eTGF receptor in vivo. We conclude that the tumor-promoting phorbol diester regulates both the affinity and phosphorylation state of the A431 cell receptor for the type alpha transforming growth factors, eTGF and EGF.     

Effects of vasoactive intestinal peptide (VIP) and somatostatin (SST) on lipoprotein receptor expression by A431 tumor cells

A variety of tumor cells have been shown to express lipoprotein receptors. Recent data suggest that lipoprotein receptors may play a regulatory role in the growth of certain tumor cells. We investigated the effects of vasoactive intestinal peptide (VIP) and somatostatin-14 (SST-14) on the binding of 111Indium-labeled lipoproteins [(111)In-low density lipoprotein ((111)In-LDL), (111)In-high density lipoprotein ((111)In-HDL) and (111)In-very low density lipoprotein ((111)In-VLDL)] onto the epidermoid mammary carcinoma cell line A431. Scatchard analyses of the binding data indicated one class of specific high affinity binding sites for LDL, HDL and VLDL expressed by A431 cells, respectively. VIP increased significantly the binding capacity for (111)In-LDL on A431 cells. The VIP-induced increase of (111)In-LDL binding sites was inhibited by SST-14. Furthermore, SST-14 inhibited VIP-induced 3H-thymidine incorporation and adenosine 3'-5' cyclic monophosphate (cAMP) formation in A431 cells with IC50 values in the range of 5-7 nM. However, SST-14 showed no effect on dibutyryl-cAMP-induced increase of (111)In-LDL binding sites expressed on A431 cells. In contrast to (111)In-LDL binding, no effects of VIP or SST-14 on HDL or VLDL binding to A431 tumor cells were found. Our results suggest a direct effect of VIP and SST-14 on LDL-binding onto tumor cells. The complex interactions between VIP and SST-14 on LDL receptor expression of tumor cells may play a role in tumor cell lipid metabolism.     

Effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on the growth inhibitory and increased phosphatidylinositol (PI) responses induced by epidermal growth factor (EGF) in A431 cells

Epidermal growth factor (EGF) inhibited the growth of A431 human epidermoid carcinoma cells. The tumor promoting, phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA) also retarded A431 cell growth. Addition of both TPA and EGF inhibited cell growth in an additive or synergistic manner depending upon the initial plating density of the cultures. EGF increased the production of diacylglycerol (60-70%) and stimulated the synthesis of phosphatidylinositol (PI) from 3H-inositol (three- to fourfold increase). Both of these responses were attenuated in the presence of TPA. TPA alone stimulated the production of diacylglycerol (DG) but had little effect on PI synthesis. The biological effect of TPA appeared to be mediated by the presence of a high-affinity receptor for phorbol esters on A431 cells. Moreover, the binding of 125I-EGF to A431 cells was unaffected by TPA, suggesting that the antagonistic effects of TPA were occurring distal to the EGF receptor. These findings also indicated that although TPA and EGF both inhibited A431 cell growth, this effect could be dissociated from changes in PI synthesis but may be dependent upon transient changes in DG production.     

Comparison of epidermal growth factor (EGF) receptor-receptor interactions in intact A431 cells and isolated plasma membranes. Large scale receptor micro-aggregation is not detected during EGF-stimulated early events

We have used resonance energy transfer to monitor epidermal growth factor (EGF) receptor micro-aggregation at the surface of intact human epidermoid carcinoma (A431) cells. EGF molecules labeled with fluorescein isothiocyanate and eosin isothiocyanate were demonstrated to bind tightly to cellsurface receptors, to elicit immediate changes in cytosolic free [Ca2+], and to undergo endocytosis. Under conditions which maintain the integrity of the cell, we observed no energy transfer between the donor fluorescein isothiocyanate-labeled EGF molecules and the acceptor eosin isothiocyanate-labeled growth factors bound to receptors. However, after disruption of cells by Dounce homogenization, a significant degree of energy transfer was observed (approximately 10-20%) with membranes, indicative of receptor aggregation. These results suggest that EGF does not cause micro-aggregation of the majority of its receptors on the surface of intact A431 cells within the time period of the early events associated with growth factor action. Moreover, it appears that the A431 cells contain some component which imparts a constraint on the ability of EGF receptors to aggregate, and that some of this component is lost upon the disruption of cells.     

Reversible G(1) arrest induced by inhibition of the epidermal growth factor receptor tyrosine kinase requires up-regulation of p27(KIP1) independent of MAPK activity

We have used quinazoline inhibitors of the epidermal growth factor receptor (EGFR) tyrosine kinase to study the link between EGFR signaling and G(1) to S traverse. Treatment of A431 and MDA-468 human tumor cells with 0.1-10 microM AG-1478 inhibited basal and ligand-stimulated EGFR phosphorylation without a decrease in receptor content, EGF-binding sites, or binding affinity. Incubation of A431 cells with 0.1-1 microM AG-1517 abrogated (125)I-EGF internalization. Both AG-1478 and AG-1517 markedly inhibited A431 and MDA-468 colony formation in soft agarose at concentrations between 0.01 and 1 microM. Daily injections of AG-1478 at 50 mg/kg delayed A431 tumor formation in athymic nude mice. A transient exposure of A431 cells to AG-1478 resulted in a dose-dependent up-regulation of the cyclin-dependent kinase inhibitor p27, down-regulation of cyclin D1 and of active MAPK, and hypophosphorylation of the retinoblastoma protein (Rb). These changes were temporally associated with recruitment of tumor cells in G(1) phase and a marked reduction of the proportion of cells in S phase. Upon removal of the kinase inhibitor, EGFR and Rb phosphorylation and the levels of cyclin D1 protein were quickly restored, but the cells did not reenter S phase until p27 protein levels were decreased. Phosphorothioate p27 oligonucleotides decreased p27 protein in A431 cells and abrogated the quinazoline-mediated G(1) arrest. Treatment of A431 cells with PD 098509, a synthetic inhibitor of MEK1, inhibited MAPK activity without inducing G(1) arrest or increasing the levels of p27. However, treatment with LY 294002, an inhibitor of phosphatidylinositol 3-kinase (PI3K), inhibited basal Akt activity, up-regulated p27, and recruited cells in G(1). These data suggest that p27 is required for the growth arrest that follows interruption of the EGFR kinase in receptor-overexpressing cells. In addition, the G(1) arrest and up-regulation of p27 resulting from EGFR blockade are not due to the interruption of MAPK, but to the interruption of constitutively active PI3K function.