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Ann Thorac Surg 2004;78:450-457
© 2004 The Society of Thoracic Surgeons
a Division of Thoracic Surgery, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA
b Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA
c Division of Urology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA
d Department of Adult Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, USA
Accepted for publication January 28, 2004.
* Address reprint requests to Dr Jaklitsch, Division of Thoracic Surgery, Brigham and Women's Hospital, 75 Francis St, Boston, MA 02115, USA
e-mail: mjaklitsch{at}partners.org
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Abstract |
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METHODS: Four nonsmall-cell lung cancer cells were tested for adhesion, proliferation, migration and morphologic alterations on collagen type IV matrix by immunoprecipitation, Western blotting, phase contrast and time lapse video microscopy.
RESULTS: Collagen type IV promoted Calu-1 cell adhesion (< 15 minutes) and motility (< 6 hours) without any significant effect on proliferation. ß1-integrin is essential for collagen type IV adhesion and 8 to 10 fold higher expression of ß1-integrin on the surface of Calu-1 cells was identified. A protein tyrosine phosphatase inhibitor, peroxyvanadate, caused 50% inhibition in the adhesion process within 5 minutes but exposure to 60 µmol/L genistein for 72 hours, a protein tyrosine kinase inhibitor, drastically inhibits Calu-1 cell proliferation (> 70%). We examined the influence of ß1-integrin, peroxyvanadate and genistein on the spreading morphogenesis of Calu-1 cells on Collagen type IV. Use of either 1 µg of anti ß1-integrin inhibitory antibody (P5D2), 100 µmol/L peroxyvanadate or 60 µmol/L genistein had dramatic influence on the spreading of Calu-1 cells. Finally, Focal adhesion kinase was identified as a phosphoprotein target.
CONCLUSIONS: We have characterized an in vitro model of matrix-specific binding of a lung cancer cell line, Calu-1 to Coll IV. Calu-1 cells use primarily a ß1-integrin mediated intracellular tyrosine phosphorylation phenomenon as the key regulatory mechanism for its binding advantage to Coll IV matrix.
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Introduction |
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The process of metastasis involves a coordinated series of interactions between the tumor cells and the surrounding ECM. Proteins embedded in both the matrix and the plasma membranes create the phenotype of the normal cell [3]. Alterations in the binding of the cell to the ECM enables tumor cells to proliferate, migrate, invade the matrix, and colonize a distant site. Furthermore, organ-specific ECM separates different tissue compartments and may relate to clinical metastatic patterns observed in lung cancer [4].
The ECM and the surrounding microenvironment are capable of influencing the cells by protein receptors, integrins, and adhesion molecules. Activation of these proteins lead to one or more possible intracellular signals resulting in dramatic changes in the cytoskeleton, gene expression, and association of cell membrane-bound proteins.
Type IV collagen (Coll IV) is the structural backbone of the basement membrane of several solid organs including lung [5, 6]. This protein has the ability to interact with the cell surface adhesion molecules such as integrin and proteoglycans, and serves as scaffolding for the binding of other basement membrane components [7].
Many complex intracellular signaling pathways activated by ECM components have been studied. However, the intracellular signaling pathway following lung cancer cell binding to Coll IV has not been elucidated. Integrin-mediated binding to Coll IV in melanoma, ovarian, and breast carcinoma cells results in the tyrosine phosphorylation of numerous intracellular proteins including both cytoskeleton [8, 9] and signaling molecules [10, 11].
In this study we describe that a lung squamous carcinoma cell line (Calu-1) exhibits an inherent adhesion affinity for Coll IV. In addition, we report the role of tyrosine phosphorylation-dephosphorylation mechanisms in Calu-1 cell adhesion, proliferation, motility, and morphology. Our results indicate that ß1-integrin plays an essential role in this process. Understanding the molecular basis of the lung cancer cell-extracellular matrix interactions could lead to the identification of novel therapeutic strategies.
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Material and methods |
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2 and V blocking antibodies (Ab) are from Cal Biochem (San Diego, CA). Bovine serum albumin (BSA), genistein, Soyabin trypsin inhibitor, mouse IgG, glycerol, Nonidate P-40 (NP40), dithiothretol, phenylmethylsulphonyl fluoride (PMSF), leupeptin, aprotinin, and ß-mercaptoethanol were purchased from Sigma Chemical Co (St. Louis, MO).
Cell culture
The human nonsmall-cell lung cancer lines used in these studies (Calu-1, NCI-520, NCI-H441, and NCI-H23) were kindly provided by Dr. Ian Anderson (Dana-Farber Cancer Institute) but are also available through American Type Culture collection (www.atcc.org). All the above cell lines were grown in either DMEM or RPMI supplemented with 10% heat inactivated fetal bovine serum, 1% penicillin-streptomycin, and 2% glutamine at 37°C and 5% CO2.
Cell adhesion and proliferation assay
Six-well polystyrene plates (9.6 cm2/well) were coated either with Coll IV (5 µg per well) or fibronectin (5 µg per well) suspended in 1-mL coating buffer containing 100 mmol/L tris (pH 8.0) and incubated at 4°C for 16 hours. The plates were washed with PBS and incubated with 3% BSA in PBS for 2 hours at 37°C in order to block nonspecific binding sites. The plates were then washed twice more with PBS and kept at 4°C until needed.
Adhesion assays were performed with serum-starved cells (14 to 16 hours), detached with 0.25% trypsin/EDTA (ethylene diamine tetra acetic acid) followed by suspension in medium containing DMEM, 20 mmol/L hepes, and 2% BSA. A 1-mL suspension containing 1 to 2x105 cells was added to each well and incubated at 37°C and 5% CO2. At the end of each incubation time point, the plates were washed twice with PBS to remove the nonadherent cells, trypsinized, and counted. The data were means of triplicate wells and were expressed as a percentage of deposited cells.
Cell proliferation was determined by adding 1x105 serum-starved cells on either BSA, fibronectin or on Coll IV-coated plates and incubated for several days. The cells were counted every 24 hours as discussed earlier. The data were means of triplicate wells.
Immunoprecipitation and Western blotting
Cells from BSA-coated or Coll IV-coated plates were lysed at 4°C in the lysis buffer containing 20 mmol/L Tris-HCl (pH 8.0), 137 mmol/L NaCl, 2 mmol/L EDTA, 10% glycerol, 1% NP-40, 200 µmol/L sodium orthovanadate, 1 mmol/L dithiothretol (DTT), 1 mmol/L phenylmethylsulfonyl fluoride (PMSF), 2 mg/mL leupeptin, and 5 mg/mL aprotinin. The lysates were centrifuged at 14,000 rpm for 15 minutes at 4°C, and the supernatants (500 µg –1 mg of protein) were used for immunoprecipitations with either antiphosphotyrosine (4G10) or polyclonal anti-FAK antibody at 4°C either overnight or for 3 to 5 hours. After incubation for 1 hour with antibody, either protein A-sepharose or protein G-sepharose was added to the lysates and was incubated for required time. The bead bound complexes were pelleted, washed several times with lysis buffer and PBS, and boiled with SDS (sodium dodecyl sulfate) sample buffer for 3 to 5 minutes before loading on SDS-PAGE (polyacrylamide gel electrophoresis, 7.5% to 10%).
For Western blot analysis, the proteins were transferred to nitrocellulose membranes after SDS-PAGE and blocked with 5% dry milk in TBST (10 mmol/L Tris-HCl, pH8.0, 150 mmol/L NaCl, 0.05% Tween 20). The blots were incubated with specific primary antibody from 1 hour at room temperature to overnight at 4°C. Membranes were washed briefly with TBST and incubated with the horseradish peroxidase conjugated secondary antibody for 1 hour at room temperature (1:5000 dilution). Following extensive washing, immunoreactive bands were visualized by chemiluminescence (ECL reagent; New England Nuclear, Wellesley, MA). When required, the membranes were stripped in 62.5 mmol/L Tris-HCL (pH 6.8), 2% SDS and 1.0 mmol/L ß-mercaptoethanol for 30 minutes at 50°C and reblotted again.
Time lapse video microscopy
The time lapse video microscopy (TLVM) procedure was performed as previously described [10]. Cells were cultured on uncoated, fibronectin-coated or Coll IV-coated tissue culture plates in a temperature and CO2 controlled chamber in their standard growth media. The cells were observed in a time-lapse fashion using an Olympus IX70 inverted device (Therm-Omega-Tech, Warmington, PA), Optronic Engineering DEI-750 3CCD digital video camera (Optronicsw, Galeta, CA), and Sony SVT-S3100 time lapse S-VHS video recorder (Sony, Tokyo, Japan). For image presentation, video images were captured and printed in a Sony Color Video Printer P-56,000 MD.
Densitometry and statistical analysis
Western blots were quantified by using a densitometric-based analysis performed on scanned fluorograms using Scion Image Beta 4.02 software from Scion Corporation (Frederick, MD; info@scioncorp.com). GraphPad Prism v.3.02 (GraphPad Software, San Diego, CA) was used to assess quantified differences in the number of adherent cells to Coll IV using a two-tailed Students (parametric) t test and the differences were determined to be statistically significant if p less than 0.05.
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Results |
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TopAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsReferences |
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Adhesion of Calu-1 cells to collagen type IV is integrin dependent
Cell lysates from all four cell lines were analyzed for the presence of ß1-integrin. Two proteins of apparent molecular mass 120 and 135 kDa were immunoreactive in the Western blot by anti ß1-integrin subunit monoclonal antibodies (mAb; Fig 2A). Although the 135-kDa ß1-integrin subunit was present in H441, H23 and H520, it was expressed approximately tenfold higher in Calu-1 cells. The 120-kDa band was present in equivalent amounts in H520, H23 and H441 cells, but is less concentrated in Calu-1 cells and the control cell extract.
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2 or V integrin blocking antibodies were used.
Peroxyvanadate-Induced phosphorylation negatively regulates the adhesion of Calu-1 cells to human collagen type IV
Serum-starved Calu-1 cells were treated with 100 µmol/L peroxyvanadate for various time intervals, and an adhesion assay was performed for 1 hour at 37°C with Coll IV-coated plates. Gradual induction of tyrosine phosphorylation induced by peroxyvanadate negatively regulated Calu-1 cell attachment to Coll IV (Fig 3A). More than 50% reduction in adhesion occurred after 3 minutes of treatment and less than 25% of the cells were attached to the Coll IV after 5 minutes of induction.
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Protein-tyrosine kinase inhibitior prevents proliferation but not attachment of Calu-1 cells
Treatment of Calu-1 cells with 60 µmol/L of genistein for 3 days did not alter the attachment rate to Coll IV-coated wells as compared to the cells exposed to DMSO for 3 days (Fig 4A). However, genistein retarded the proliferation of Calu-1 cells severely with 70% to 80% reduction in 48 hours (Fig 4B). Western blot analysis of the untreated and genistein-treated cells revealed that protein tyrosine phosphorylation was globally suppressed by genistein treatment (Fig 4C)
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Comment |
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TopAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsReferences |
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Coll IV improved adhesion of Calu-1 cells 50% to 70% over control, but did not provide a proliferation advantage nor did it cause inhibition of proliferation. Furthermore, Coll IV has been noted to have disparate effects on adhesion and proliferation in other cell lines. For instance, adhesion of melanoma cells to Coll IV was enhanced by 50%, but proliferation was inhibited by 40% when compared to control [13]. Proliferation of rat cortical progenitor cells was also inhibited on Coll IV matrix [14]. In the case of hepatoma cells, both adhesion and proliferation increased on Coll IV matrix [15], and Coll IV played a critical role in conditioning glomerular endothelial cells to respond to proliferative stimuli [16].
Bredin and colleagues [17], and other investigators [18–20], demonstrated that most lung cancer cell lines express a variety of ß1-integrins. In our present study we not only reported that all four NSCLC cell lines examined expressed ß1-integrin, but also demonstrated that high levels of expression in Calu-1 cells differed clearly with respect to the other lung cancer cell lines tested and contributed to its adhesion to Coll IV.
The role of ß1-integrin in lung cancer cell adhesion is not clear. We have established that it was the ß1-integrin expression on the surface of the Calu-1 cells that was responsible for interaction with Coll IV. However, other ß1-integrin positive NSCLC lines (including H441, H23, and H520) marginally adhere to Coll IV matrix, indicating that either the amount of ß1-integrin on the surface of these cells was not sufficient or other molecules on the surface of these cells besides ß1-integrin also play a role in adhesion.
By altering the level of intracellular phosphorylation, we further elucidate the role of tyrosine phosphorylation in the adhesion process. Several reports have indicated that tyrosine phosphatase inhibitors, such as vanadate and peroxyvanadate, activate tyrosine phosphorylation of FAK and paxillin [21–23]. Our data also confirms that the inhibition of tyrosine phosphatase influenced the adhesion process with a delicate balance.
Surprisingly, genistein had no effect on Calu-1 cell adhesion at the same concentration that dramatically attenuated its proliferation. It has been reported that the effective inhibitory concentration of genistein in vivo is several fold higher than in vitro, due to differences in intracellular ATP concentration and drug accumulation within cells [24]. The concentration of genistein used in these studies did not produce a large change in protein phosphorylation intensity even after 72 hours, but did affect the proliferative rate of Calu-1 cells, indicating that a fine balance of tyrosine phosphorylation events were important for proliferation in this cell line.
Change in morphology is an indication of alteration of intracellular events. Spreading of Calu-1 cells on Coll IV, dramatically altered by treatment with either ß1-integrin blocking antibody, or peroxyvanadate and genistein, indicates that a ß1-integrin dependent phosphorylation event is the control mechanism for the morphologic alteration of Calu-1 cells on Coll IV matrix.
In addition to providing a molecular "glue"' essential for tissue organization, integrins are dynamic signaling molecules. Binding and clustering of integrin at the cell surface induces protein tyrosine phosphorylation of intracellular proteins. One of the central phosphorylated molecules in these events is FAK. Accumulating evidence supports a critical role for FAK in promoting cell migration stimulated by different types of cell surface ligand receptors. Activation of FAK following adhesion to Coll IV and integrin engagement has been reported for many different cell types [8, 9]. Our observation of phosphorylation of FAK after Calu-1 cell adhesion to Coll IV might be important for integrin signaling in this lung cancer cell line. It will be important to identify the associated events following phosphorylation and activation of FAK in lung cancer cells. For example, FAK has been reported to phosphorylate and associate with many cytoskeleton and cytoplasmic signaling molecules including paxillin, p130 Cas, C-Src [25], and ERKs [11].
It remains to be established whether signal transduction pathways downstream of the Coll IV receptors participate directly in the tyrosine phosphorylation of FAK in this system. However, we have established that the phosphorylation of FAK in Calu-1 cells is dependent on ß1-integrin function although the involvement of other integrin subtypes can not be ignored. It is highly likely that additional signaling molecules downstream of FAK are involved in the regulation of proliferation, migration and morphology of Calu-1 cells.
In conclusion, we have characterized an in vitro model of matrix-specific binding of a lung cancer cell line to Coll IV. We have demonstrated that Calu-1 cells use primarily a ß1-integrin mediated intracellular tyrosine phosphorylation phenomenon as the key regulatory mechanism for its binding advantage to Coll IV matrix. FAK may be a promising target for future therapeutic intervention strategies in lung cancer. Further understanding of the molecular basis of lung cancer cell adhesion, proliferation and motility with respect to the extracellular matrix could lead to new insights into the molecular mechanism of lung cancer metastsais leading to better therapy.
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Acknowledgments |
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TopAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsReferences |
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References |
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TopAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsReferences |
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), and bovine serum albumin (
) coated matrix. The data were expressed as either percentage of control (A, % of total number of attached cells compared to total) or the actual cell number (B). (C) Represents the time-lapsed video microscopy of Calu-1 cells on fibronectin (left) or on Collagen IV (right). Pictures were taken every 3 hours as described in Materials and Methods. The arrow indicates a long actin extension.
). Triplicate wells of each time point were counted for cell growth by hemocytometer. (C) Phosphotyrosine blot of whole cell extracts of control and genistein treated cells shows suppressed phosphorylation of several proteins ranging from 30 to 200 kDa (as indicated by arrows). ß-actin was used as a loading control.
