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Cell Growth & Differentiation Vol. 11, 83-90, February 2000
© 2000 American Association for Cancer Research


Articles

Adherence of Human Erythroleukemia Cells Inhibits Proliferation without Inducing Differentiation1

Annie Molla2 and Marc R. Block

Laboratoire d’étude de la différenciation et de l’adhérence cellulaires, UMR Centre National de la Recherche Scientifique/UJF 5538, Institut Albert Bonniot, Faculté de Médecine Domaine de la Merci, 38706 La Tronche Cedex, France

Abstract

To investigate the effect of extracellular matrix molecules in the megakaryocytic lineage, we studied the role of integrin engagement in the proliferation and differentiation of human erythroleukemia (HEL) cells. HEL cells grew in suspension, but their adherence depended upon the presence of matrix proteins or protein kinase C signaling. Adherence by itself did not trigger commitment of these cells but accelerated phorbol 12-myristate 13-acetate-induced differentiation. HEL cells adhered to fibronectin mainly through {alpha}5ß1, and this receptor acted synergetically with {alpha}4ß1. Integrin engagement induced cell growth arrest through mitogen-activated protein kinase inactivation. Such down-regulation of the mitogen-activated protein kinase pathway by integrin engagement was suggested as a megakaryocytic-platelet lineage specificity. This signaling was not restricted to a peculiar integrin but was proposed as a general mechanism in these cells.

Introduction

Thrombopoiesis or platelet formation begins with pluripotent stem cells committed to the megakaryocytic lineage and ends with the release of circulating platelets (1) . Hematopoietic progenitors are retained in the bone marrow in intimate contact with the stroma (2) . This environmental niche regulates quiescence, proliferation, and differentiation of hematopoietic stem cells (3, 4, 5) . Long-term culture of bone marrow progenitors on stromal monolayers has highlighted the combined importance of growth factors and of the interaction between progenitor cells, stroma cells, and extracellular matrix (6, 7, 8) .

Integrins, a family of {alpha}ß heterodimeric transmembrane glycoproteins, are the major cell surface receptors for extracellular matrix molecules. They are subdivided according to their common ß chain subunit into at least eight classes (9) . Among them, the ß1 subfamily and mostly the fibronectin receptors ({alpha}4ß1 and {alpha}5ß1) have been proposed to play a crucial role in the attachment, migration, or differentiation of hematopoietic cells (10, 11, 12) . The interaction between fibronectin and its receptors was demonstrated to play a relevant role in the terminal differentiation of human B cells and megakaryocytes. Adhesion and spreading participate in the fragmentation of human activated mature megakaryocytes, leading to platelet production (13) . The effects of extracellular matrix molecules on the proliferation of hematopoietic cells are controversial. Direct interaction with bone marrow stroma via fibronectin receptors was reported to inhibit hematopoietic progenitor proliferation (14) . In myeloid cells, fibronectin-induced growth suppression via {alpha}5ß1 is coupled to the induction of apoptosis (15) , whereas the fibronectin receptor overexpression has been associated with the loss of anchorage-independent growth in human erythroleukemia cells (16) . Conversely, T-cell proliferation can be induced by the ligation of CD3 and fibronectin receptors (17) .

To investigate the effects of extracellular matrix molecules in the megakaryocytic lineage, we have studied the role of integrin receptors in the proliferation and differentiation of HEL3 cells. This erythromegakaryocytic cell line is committed to the megakaryocytic lineage after PMAtreatment (18) . Differentiated cells are adherent and polyploid. Although HEL cells grow in suspension, they express fibronectin-activated receptors, and their adhesion totally depends on the added matrix. HEL cells adhere specifically on fibrinogen and fibronectin, with {alpha}5ß1 being the major fibronectin receptor. Adherence by itself does not trigger differentiation but accelerates the PMA-induced process. Here we provide evidence that the integrin {alpha}5ß1 occupancy induces the quiescence of erythromegakaryocytic cells without promoting apoptosis or differentiation programs. These data strongly suggest that integrin signaling is lineage specific (19) .

Results

HEL Adhesion Depends on the Presence of Matrix or TPA Induction.
HEL and AP 217 phenotypes display characteristics of both erythroid and megakaryocytic lineage (20) . HEL and AP217 cells normally grow in suspension, but when they were cultured in the presence of PMA on plastic culture, HEL cells exhibit an adherent phenotype within 1 h, whereas AP217 did not, even 5 days later.

We performed adhesion assays with HEL cells on various extracellular matrix proteins. Results are reported in Fig. 1ACitation . In the absence of PMA, cells did not show any affinity for collagen I, BSA, plastic, or poly-L-lysine. However, they specifically adhered on fibronectin and fibrinogen (Fig. 1A)Citation . This interaction was dose dependent, as shown in Fig. 1BCitation . About 95% of the cells were found to be adherent within 1 h. We confirmed that PMA induced HEL adhesion as represented in Fig. 1ACitation . However, we noticed that collagen or poly-L-lysine coating prevented such an adhesion. HEL behavior toward poly-L-lysine coating is unusual because this substrate is commonly used to nonspecifically retain most adherent cells. This result opened the possibility of discriminating between the effects of PMA and adherence on cell differentiation.



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Fig. 1. Adhesion of erythromegakaryocytic cells. A, HEL and AP 217 cells were allowed to adhere for 1 h on different coated supports: poly-L-lysine (PL), BSA, collagen I (CO I), plastic culture (No), fibrinogen (FI), and fibronectin (FN) in the presence () or the absence ({square}) of PMA (100 nM). Matrix concentrations were 10 µg/ml, except for assays noted FN*, for which the fibronectin concentration was 2.5 µg/ml. In the assays No* and FN*, HEL cells were allowed to adhere for only 30 min. Adhesion was measured as described in "Materials and Methods." In this experiment, relative adhesion was calculated as follows (A 490 protein - A 490 blank), blank being MTS reagent in complete culture medium without cells. Data are the average of two independent experiments; bars, SD. B, microtitration wells were coated with increasing fibronectin concentrations (from 0.2 to 250 µg/ml), then saturated with BSA (0.5 mg/ml), and HEL cells were allowed to adhere for 1 h. The percentage of HEL adherent cells resulting from a triplicate measure was expressed as a function of the coating matrix concentration.

 
Induction of adhesion by immobilized matrix was not a general feature of megakaryocytic cells because AP217 were still nonadherent on fibronectin in the absence of PMA (Fig. 1A)Citation . In this cell line, fibronectin alone was not sufficient to trigger cell adhesion. Upon addition of PMA, 50% of AP217 were found to be adherent, suggesting that an additional PKC-dependent signaling pathway was required for cell attachment. A similar result was observed with HEL cells on fibrinogen. Indeed, PMA was required for full cell adhesion on this matrix. The additivity of the stimuli was also observed in experiments performed with HEL cells under nonsaturating conditions, i.e., 30 min of adhesion on 2.5 µg/ml fibronectin (Fig. 1A)Citation .

HEL Adhesion Depends on Integrin Receptors.
We (20) and others (21) have already reported that HEL cells express {alpha}4ß1, {alpha}5ß1, and {alpha}IIbß3 integrins at their surface. To verify that the adhesion of HEL cells on fibronectin was mediated by integrins, we performed assays in the presence of specific competitors, blocking antibodies and peptides (Fig. 2)Citation . Antibodies against ß1 integrin reduced the adhesion of HEL cells to fibronectin by 92%, whereas anti-ß3 antibodies were almost inefficient. Adhesion of HEL cells on fibronectin was completely inhibited by RGD peptide, whereas the fibronectin-related CS1 peptide was a weak inhibitor. To assess the role of specific receptors, we performed adhesion assays on fibronectin in the presence of anti-{alpha}4 and anti-{alpha}5 integrin subunit antibodies. Under our experimental conditions, antibody against {alpha}5 blocked 46% of the adhesion, whereas anti-{alpha}4 antibody blocked only 5%. The mixture of the two antibodies ({alpha}4 plus {alpha}5) gave 86.4% inhibition of the adhesion, similar to the effect obtained with the ß1 blocking antibody. These results suggest that {alpha}5ß1 is the main very late antigen receptor for fibronectin acting synergistically with {alpha}4ß1 integrin.



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Fig. 2. Involvement of ß1 integrins in HEL adherence on fibronectin. HEL cells were plated on fibronectin (10 µg/ml)-coated wells. Adhesion was measured as described in "Materials and Methods." Nonspecific adhesion on BSA was subtracted, and data are the means of two independent experiments. Competitors were as follows: antibodies anti-ß1 (A2BII, hybridoma supernatant), anti-ß3 (B2A, 50 µg/ml), anti-{alpha}5 (B2GII, hybridoma supernatant), and anti-{alpha}4 (clone HP 2/1, 20 µg/ml); peptides GRGDSP and GRGESP (50 µM) and CS1 (40 µM). Bars, SD.

 
Adhesion of Erythromegakaryocytic Cells on Fibronectin Do Not Trigger Differentiation.
PMA is known to initiate the differentiation of erythomegakaryocytic cells to the megakaryocytic lineage (18) . Differentiated cells are polyploid and express at their surface the collagen receptor {alpha}2ß1 and the platelet integrin {alpha}IIbß3, whereas {alpha}4ß1 fibronectin receptors are down-regulated (18 , 20) . By FACS analyses, the expression of three integrins was followed on HEL cells grown either in suspension or on fibronectin with and without PMA stimulation (Fig. 3)Citation . Adherence of HEL cells for 72 h on fibronectin did not modify significantly the integrin pattern (Fig. 3Citation , compare A–C and D–F; Table 1Citation ). PMA induced adherence of HEL cells on fibronectin-coated dishes within 1 h and then up-regulated the expression of {alpha}2ß1 (Fig. 3L)Citation . The percentage of positive events varied from 36% to ~83% (Table 1)Citation . Meanwhile, {alpha}IIbß3 integrin was slightly increased (Fig. 3KCitation and Table 1Citation ), and {alpha}4ß1 integrin was down-regulated and almost undetectable 72 h later (Fig. 3JCitation and Table 1Citation ). When adherence was prevented by poly-L-lysine coating (Fig. 3Citation G–I), PMA still induced {alpha}2ß1 expression (Fig. 3I)Citation and decreased {alpha}4ß1 level (Fig. 3G)Citation , but these effects were slightly delayed as compared with the adherent experiment. Our results indicated that adhesion did not trigger differentiation by itself but accelerated the PMA-induced process.



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Fig. 3. Expression of cell surface integrins on HEL cells. Three integrin subunits were analyzed by FACS: {alpha}4 in A, D, G, and J; ß3 in B, E, H, and K; {alpha}2ß1 in C, F, I, and L. Representative profiles are presented in I, and statistical data are indicated in Table 1Citation . HEL cells were cultured either in suspension in (A–C and G–I) or adherent on fibronectin (D–F and J–L). In D–F, cells were maintained for 72 h on fibronectin-coated dishes. In G–L, HEL cells were stimulated for 72 h with PMA either on poly-L-lysine-coated (G–I) or on fibronectin-coated (J–L) dishes.

 

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Table 1 Detection of cell surface receptors by flow cytometry analysis

Data indicated in the table were extracted from the corresponding histograms shown in Fig. 3Citation . Positive and negative means represent the mean of relative fluorescence intensity for the indicated antigen and the corresponding nonimmune serum.

 
Adhesion of HEL Cells on Fibronectin Prevents Cell Proliferation.
HEL serum-starved cells plated either on fibronectin or poly-L-lysine-coated wells were allowed to proliferate for 72 h in FCS-supplemented medium. As shown in Fig. 1ACitation , poly-L-lysine prevented cell attachment to plastic dishes. After 72 h of culture, cell quantification revealed that adherence on fibronectin reduced significantly cell proliferation as compared with cells grown in suspension (Fig. 4A)Citation . These data were confirmed by BrdUrd incorporation experiments. In the nonadherent population, the percentage of cells in S phase is ~70%, and this value decreased to ~5% in the adherent counterpart (Fig. 4B)Citation .



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Fig. 4. Effect of adherence on HEL cell proliferation. Microtitration wells were either coated with fibronectin () or poly-L-lysine ({square}). A, the experiment started with varying concentrations of starved cells (from 2 to 15 x 103 HEL cells). Cells grown in RPMI- FCS were quantified 72h later as described in "Materials and Methods." Bars, SD. B, the percentage of cells in S-phase was assessed by BrdUrd incorporation. Data are the mean of three independent experiments; bars, SD.

 
Although FACS analysis of HEL DNA content was somewhat complicated by the heterogeneity of the ploidy of these megakaryocytic cells, we compared the pattern of adherent versus nonadherent cells (Fig. 5)Citation . Fig. 5ACitation represents a control of nonstarved HEL cells grown in suspension, whereas starved cells are in Fig. 5BCitation . Comparison between Fig. 5, C and BCitation , confirmed that cells in suspension undergo a normal cell cycle, whereas adherent cells (Fig. 5E)Citation do not enter S phase. Adherent cells were mostly detected as nonproliferating species. PMA is known to be a potent inhibitor of cell cycling in these cells and to increase cell ploidy (22) . These features were observed independently on adherence (Fig. 5Citation , compare D and F), but the proportion of polyploid cells (>2N) was slightly higher in adherent PMA-induced cells. In the absence of PMA induction, some adherent cells were also detected as polyploid species, but chromatin was found to be compacted as compared with the PMA counterpart, thus confirming the lack of differentiation.



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Fig. 5. Cell cycle distribution of HEL cells. Cells were fixed and then analyzed for DNA content by staining with propidium iodine, followed by FACS analysis as described in "Materials and Methods." Representative profiles are shown. HEL cells grown in standard conditions (RPMI 10% FCS) are in A, whereas serum-starved cells are in B. B represents the starting material for the following experiments. After starvation cells were cultured for 72 h in complete medium either on fibronectin-coated dishes (E and F) or on poly-L-lysine support (C and D). In E and F, the analysis was performed on the adherent cell population. In D and F, cells were stimulated for 72 h with PMA in complete medium.

 
Finally, the inhibition of HEL cell growth by adhesion on matrix substratum was not specific to fibronectin-mediated signaling because similar data were obtained when the cells were plated on fibrinogen (data not shown). Cell growth arrest was found to be reversible. When HEL cells immobilized on fibronectin were detached by trypsin-EDTA treatment and then cultured in suspension in RPMI 1640 supplemented with 10% FCS, they grew normally for at least six generations (not shown). This last result indicates that fibronectin promotes quiescence of HEL cells without inducing apoptosis.

Inhibition of ERK-2 Kinase in HEL Cells by Adherence.
HEL cells were grown either on fibronectin or in suspension. They were then lysed, and MAPKs were detected in these samples by Western blotting. A specific anti-ERK-1 antibody was found to detect CHO cell kinase but gave no signal with HEL cells, suggesting that ERK-2 was the only p42 ERK protein expressed in these cells (data not shown). In HEL-starved cells, a pan-ERK antibody revealed two bands of Mr ~42,000 (Fig. 6)Citation , the upper one corresponding to the phosphorylated form of the kinase. In fibronectin-immobilized cells, only the lower band was detected after 24 and 72 h of adherence, whereas the two species were revealed in the suspension counterpart (Fig. 6)Citation . The effect did not occur immediately after engagement of integrins (Fig. 6Citation ; 1 h), suggesting that cell adhesion on fibronectin prevented the activation of p42 ERK without affecting phosphatase activity.



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Fig. 6. Detection of MAPKs in HEL cells. Starved HEL cells were suspended in FCS-supplemented medium and either cultured in suspension (S) or adherent on fibronectin (A). Cells were collected and lysed as described in "Materials and Methods." ERK proteins were revealed with a pan ERK antibody in starved cells (Lane 1), in nonadherent cells (Lanes 2, 4, and 6), and in adherent cells (Lanes 3, 5, and 7). FCS stimulation and adhesion lasted either 1, 24, or 72 h, as indicated.

 
Discussion

Integrin-mediated adhesion to extracellular matrix plays an important role in regulating cell survival and proliferation (reviewed in Ref. 23 ). Signals from these adhesion receptors are integrated with those originating from growth factor and cytokine receptors to organize the cytoskeleton, modulate MAPK cascades, and regulate immediate-early gene expression (24) . Although extracellular matrix-integrin interactions are crucial during hematopoiesis, the role of matrix proteins on cell growth and differentiation is poorly documented. Both stimulation and inhibition of proliferation have been described for hematopoietic cells. These opposite observations may suggest that there is lineage specificity (14, 15, 16, 17) . Hurley et al. (14) have reported that direct adhesion to bone marrow stroma via fibronectin receptors inhibits hematopoietic progenitor proliferation. Some hematopoietic cell lines mimicked these feature when integrins were activated by a specific antibody (clone 8A2; Refs. 16 and 25 ). Other authors who reported controversial results argued that antibodies induced artifactual conformations and that it was the combined effect of ligand occupancy and receptor clustering that was observed. To address this question, the erythroleukemia cell line HEL is an interesting model. Indeed, the integrin fibronectin receptors {alpha}5ß1 are expressed in an intermediate activation step at the HEL cell surface, which is responsive to the ligand but not fully activated (26) . HEL cells adhered to immobilized fibronectin, but actin was essentially cortical and poorly organized in these cells. Conversely, PMA promoted stress fiber organization and HEL cell spreading, suggesting that the integrin reached a fully activated state upon protein kinase C induction (21) . Interestingly, both PMA- and fibronectin-induced adhesion were found to be additive in HEL cells. Adhesion on fibronectin did not promote differentiation of HEL cells, as shown with the expression of specific cell surface markers, but it inhibited cell proliferation, suggesting that these undifferentiated cells have kept a specific characteristic of immature progenitors. Some conclusions can be drawn about the mechanism of this inhibition. The signal is partly conducted through integrins recognizing RGD motifs because {alpha}5ß1 was found to be the main fibronectin receptor acting synergetically with {alpha}4ß1. Similar data were also obtained when HEL cells adhered on fibrinogen through {alpha}IIbß3 integrin, another RGD binding receptor. Furthermore, immobilization of the cells was not a prerequisite because Sugahara et al. (15) have noticed a decrease in HEL cell proliferation by addition of soluble fibronectin. Thus, {alpha}5ß1 integrin occupancy leads to growth arrest. It was reported that ß1C, a spliced variant of ß1 integrin subunit, regulated cell proliferation (27) . This variant was detected by PCR in HEL cells as a minor component of the ß1 integrin subunits. We cannot thus exclude that this variant may be involved in the cell growth regulation.

Conversely to what was reported for other hematopoietic cells as MO7E cells (15) , fibronectin binding did not induce apoptosis in HEL cells. We found that {alpha}4ß1, although not being the main receptor, participates in fibronectin binding, and one may suggest that it is responsible for the survival advantage in HEL cells. Indeed, in early hematopoietic cells, adhesion to fibronectin via {alpha}4ß1 suppresses the apoptotic pathway, and myeloma cell lines selected for cytotoxic drug resistance overexpress {alpha}4ß1 (28) . Furthermore, in MO7E cells, where fibronectin was reported to induce cell death, {alpha}4ß1 did not participate to adherence (15) .

The next question we address is the identification of the signaling pathways that coupled growth arrest and adherence through integrins. Most mitogenic signals were integrated through the MAPK cascades (29) . MAPKs are a family of serine threonine kinases activated by many extracellular stimuli, such as growth factors or extracellular matrix. The first members of this family to be discovered in mammalian cells, the ERKs (ERK1 and ERK2) are essential for cell proliferation and differentiation (30 , 31) . We have shown that ERK2 activity was negatively regulated upon adherence of HEL cells on fibronectin, and such inhibition was sustained as long as the adherence lasted. This is in agreement with the lack of differentiation of HEL cells upon adherence. MAPK activation was indeed a prerequisite for megakaryocytic differentiation (32) . Similar negative regulation of MAPK activation was reported after {alpha}IIbß3 engagement in platelets (19) . The point raised by these authors (19) was whether it was attributable to the physical state of the ligand or to a specificity of anucleated cells or a megakaryocytic-platelet feature. Our data suggested that it was a lineage specificity, which was present in bipotential erythromegakaryocytic cells like HEL and perhaps earlier in the progenitors (14) . Such pathways must be turned off in cells committed to the erythroid lineage (16) . Mature erythroids are mostly nonadherent cells, in contrast with megakaryocytes in which functional {alpha}5ß1 and {alpha}vß3 integrins are involved in the maturation process (13 , 16 , 33) . Down-regulation of the MAPK pathway by integrin engagement was first reported for {alpha}IIbß3 (19) . Our data suggest that this signaling is not specific to the platelet integrin because {alpha}5ß1 transduces similar information. We propose that it is a general RGD-dependent transduction pathway in megakaryocytes and platelets.

It has been suggested that chronic myeloid leukemia progenitors proliferate continuously in stroma contact culture because of a defect in their adherence to fibronectin (34) . HEL cells that were also derived from malignant cells present a more sophisticated situation. The whole-cell population adhered to fibronectin and consequently did not proliferate. However, after about 1 day of adherence, cells (~20%) were released from the matrix, as was already observed with PMA-stimulated HEL cells (20) . In this peculiar cell population, integrins were engaged on fibronectin and promoted MAPK inactivation, but the adherence was only transitory. An interesting point will thus be to understand the mechanism that allows these cells to escape from the matrix because it may be relevant in chronic myelogenous leukemia.

Materials and Methods

Cell Line.
HEL is an erythromegakaryocytic cell line developed from a patient who contracted leukemia after treatment for a solid tumor (35) . This cell line was obtained from American Type Culture Collection. The AP217 cell line was established from the peripheral blood of a patient with chronic myeloid leukemia (20) . Cells were maintained in RPMI 1640 supplemented with 10% FCS at 37°C in a humidified 5% CO2 atmosphere.

Antibodies, Peptides, and Matrix.
Antibodies used in indirect immunofluorescence studies are commercially available. Mouse monoclonal integrin antibodies directed against the {alpha}4 subunit (clone HP 2/1), the {alpha}5 subunit (clone SAM1), the {alpha}2ß1 integrin (clone Gi9), and the {alpha}IIbß3 complex (clone P2) were obtained from Immunotech (Marseille, France). The monoclonal B2A recognizes the integrin ß3 subunit and inhibits platelet aggregation as well as adhesion of platelets to fibronectin (20) . Function-perturbing anti-integrin ß1 (A2BII) and anti-{alpha}5 (B2GII) antibodies were a generous gift of Dr. C. Damsky (Department of Stomatology, University of California at San Francisco, San Francisco, CA). BrdUrd and monoclonal anti-BrdUrd antibody were from Sigma Chemical Co. (St. Quentin-Fallavier, France). Pan ERK antibody was obtained from Transduction Laboratories (Lexington, KY).

Peptides were obtained by solid-phase synthesis using an Applied Biosystem synthesizer. Peptides were >95% homogeneous when analyzed by high-pressure liquid chromatography, and the amino acid composition of each peptide was consistent with its amino acid sequence. CS1 peptide was purchased from Neosystem Laboratory (Strasbourg, France).

Fibronectin was purified from bovine plasma as described by Klebe et al. (36) , and fibrinogen was purchased from Diagnostic Stago (Asnières, France). Poly-L-lysine (Mr 70,000–150,000) and collagen I were obtained from Sigma.

Adhesion Assays.
Adhesion assays were performed in plastic microtitration plates (Corning, Inc.) as described previously (20) . The wells were coated overnight at 4°C with extracellular matrix proteins and rinsed in PBS. Subsequently, free binding sites were blocked with 0.5 mg/ml BSA. Cells were suspended in synthetic culture medium (Life Technologies, Inc.; CHO-SFMII). HEL cells (1 x 105) were added per well and allowed to attach for 30 min at 37°C. Nonadherent cells were removed by three PBS washes. PBS was added in the wells with a multichannel pipette, and the liquid was gently removed by inverting the plate. The number of attached cells was quantified using a cell quantification kit (Promega Corp., Madison, WI) by adding 20 µl of MTS tetrazolium reagent to 100 µl of RPMI-FCS in each well. After color development, the plate was read on a Dynatech reader at 490 nm. Assays were run in triplicate. In each plate, wells were filled with known HEL concentrations for interplate standardization. Specific adhesion values were obtained by subtracting nonspecific attachment to BSA. Some assays were run in the presence of competitors: GRGDSP and GRGESP peptides at 50 µM; fibronectin-related peptide CS1 (40 µM); anti-ß1 (A2BII, hybridoma supernatant); anti-ß3 (B2A, 50 µg/ml); anti-{alpha}5 (B2GII, hybridoma supernatant); and anti-{alpha}4 (HP2/1, 20 µg/ml) blocking antibodies.

Detection of Cell Surface Integrins by Indirect Immunofluorescence.
Adherent cells were detached from the culture dish with trypsin-EDTA, whereas cells in suspension were recovered by centrifugation. Surface expression of individual integrins was evaluated by flow cytometry after staining of 1 x 106 cells with integrin subunit-specific antibodies, followed by a FITC-conjugated secondary antibody (antimouse IgG from Jackson Immunoresearch laboratory). Commercially available anti-integrin antibodies were used at the dilution suggested by the manufacturer. Unbound antibodies were removed by washing with PBS. By using nonimmune mouse IgG as primary antibody, nonspecific staining was assessed. Routinely, 10,000 cells were analyzed in a Becton Dickinson flow cytometer.

Mitogenic Assays and FACS Scan Analysis.
To elicit growth arrest, cells were placed for 48 h in serum-free RPMI 1640. Serum-starved cultures were stimulated to enter the cell cycle by the addition of serum (10%). The cells were either maintained in suspension or allowed to adhere on fibronectin (25 µg/ml)-coated culture plates. After 72 h of culture, cells were harvested as described previously and washed in PBS before being fixed in 1% paraformaldehyde. Cells were permeabilized in 0.25% Triton X-100, incubated in RNase (0.5 mg/ml), and then stained with propidium iodide (50 µg/ml), as described by Pierrez and Ronot (37) . The analysis was performed with a Becton Dickinson flow cytometer.

Quantitative analyses were performed in microtitration plates. Starved cells were plated on wells coated with either fibronectin (10 µg/ml) or poly-L-lysine (70 µg/ml) in RPMI-FCS medium.

Cells were quantified 72 h later using a cell quantification kit (Promega) by adding 30 µl of MTS reagent to 150 µl of RPMI-FCS in each well. After color development, the plate was read on a Dynatech reader at 490 nm. Assays were run in triplicate on several cell concentrations. Alternatively, bromodeoxyuridine incorporation was monitored on adherent and nonadherent HEL cell populations. Growth medium was replaced with fresh medium containing 30 µM BrdUrd, and cells were incubated at 37°C. After 3 h, the cells were fixed, and BrdUrd was stained with anti-BrdUrd and fluorescent secondary antibodies. The percentage of cells that had incorporated BrdUrd was evaluated microscopically.

Immunoblotting.
Cell extracts were prepared by detergent lysis in SDS sample buffer (62.5 mM Tris-HCl, 2.3% SDS, 10% glycerol, 5% ß-mercaptoethanol, and 0.005% bromphenol blue). Proteins resolved by 12% SDS-PAGE were transferred onto polyvinylidene difluoride support. The membrane was blocked for 60 min at room temperature in TBS [10 mM Tris-HCl (pH 8), 150 mM NaCl] containing 3% BSA. ERK antibody (1:5000) was incubated in the same buffer overnight at 4°C. Antibody binding was then revealed with a horseradish peroxidase antimouse secondary antibody according to Bouvard et al. (38) .

Acknowledgments

We thank Dr. C. Damsky (University of California at San Francisco, San Francisco, CA) for the gift of perturbing anti-ß1 and {alpha}5 integrin antibodies and Dr. R. Berthier (INSERM U238, Grenoble, France) for AP217 cells. We are indebted to Brigitte Peyrusse for expert editorial assistance and Sarah Linstead for the English review.

Footnotes

1 This work was supported by Centre National de la Recherche Scientifique. Back

2 To whom requests for reprints should be addressed, at LEDAC UMR 5538, Institut Albert Bonniot, Domaine de la Merci, 38 706 La Tronche Cedex, France. Phone: 33-476-54-94-74; Fax: 33-476-54-94-25; E-mail: annie.molla{at}ujf-grenoble.fr Back

3 The abbreviations used are: HEL, human erythroleukemia; PMA, phorbol 12-myristate 13-acetate; FACS, fluorescence-activated cell sorter; BrdUrd, bromodeoxyuridine; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium. Back

Received for publication 6/29/99. Revision received 10/ 7/99. Accepted for publication 12/13/99.

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Y. Saito, T. Owaki, T. Matsunaga, M. Saze, S. Miura, M. Maeda, M. Eguchi, R. Tanaka, J. Taira, H. Kodama, et al.
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