Cyr61, a Member of CCN Family, Is a Tumor Suppressor in Non-Small Cell Lung Cancer

Abstract

Cysteine-rich protein 61 (Cyr61) is a member of a family of growth factor-inducible immediate-early genes. It regulates cell adhesion, migration, proliferation, and differentiation and is involved in tumor growth. In our experiments, the role of Cyr61 in non-small cell lung cancer (NSCLC) was examined. Expression of Cyr61 mRNA was decreased markedly in four of five human lung tumor samples compared with their normal matched lung samples. NSCLC cell lines NCI-H520 and H460, which have no endogenous Cyr61, formed 60–90% fewer colonies after being transfected with a Cyr61 cDNA expression vector than cells transfected with the same amount of empty vector. After stable transfection of a Cyr61 cDNA expression vector, proliferation of both H520-Cyr61 and H460-Cyr61 sublines decreased remarkably compared with the cells stably transfected with empty vector. The addition of antibody against Cyr61 partially rescued the growth suppression of both H520-Cyr61 and H460-Cyr61 cells. Cell cycle analysis revealed that both H520-Cyr61 and H460-Cyr61 cells developed G1 arrest, prominently up-regulated expression of p53 and p21 WAF1, and had decreased activity of cyclin-dependent kinase 2. The increase of pocket protein pRB2/p130 was also detected in these cells. Notably, both of the Cyr61-stably transfected lung cancer cell lines developed smaller tumors than those formed by the wild-type cells in nude mice. Taken together, we conclude that Cyr61 may play a role as a tumor suppressor in NSCLC. Cysteine-rich protein 61 (Cyr61) is a member of a family of growth factor-inducible immediate-early genes. It regulates cell adhesion, migration, proliferation, and differentiation and is involved in tumor growth. In our experiments, the role of Cyr61 in non-small cell lung cancer (NSCLC) was examined. Expression of Cyr61 mRNA was decreased markedly in four of five human lung tumor samples compared with their normal matched lung samples. NSCLC cell lines NCI-H520 and H460, which have no endogenous Cyr61, formed 60–90% fewer colonies after being transfected with a Cyr61 cDNA expression vector than cells transfected with the same amount of empty vector. After stable transfection of a Cyr61 cDNA expression vector, proliferation of both H520-Cyr61 and H460-Cyr61 sublines decreased remarkably compared with the cells stably transfected with empty vector. The addition of antibody against Cyr61 partially rescued the growth suppression of both H520-Cyr61 and H460-Cyr61 cells. Cell cycle analysis revealed that both H520-Cyr61 and H460-Cyr61 cells developed G1 arrest, prominently up-regulated expression of p53 and p21 WAF1, and had decreased activity of cyclin-dependent kinase 2. The increase of pocket protein pRB2/p130 was also detected in these cells. Notably, both of the Cyr61-stably transfected lung cancer cell lines developed smaller tumors than those formed by the wild-type cells in nude mice. Taken together, we conclude that Cyr61 may play a role as a tumor suppressor in NSCLC. CTGF, Cyr61 and Nov cysteine-rich protein 61 non-small cell lung cancer retinoblastoma related protein cyclin-dependent kinase neomycin-resistant cells that are transfected with pcDNA3.1/Neo 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide The immediate response of cells to exposure to polypeptide growth factors includes the rapid and transient activation of a set of immediate-early genes, which are categorized into several different families (1Kireeva M.L. Mo F.E. Yang G.P. Lau L.F. Mol. Cell. Biol. 1996; 16: 1326-1334Crossref PubMed Scopus (303) Google Scholar). One of these families is called CCN1 (CTGF (connective tissue growth factor), Cyr61 (cysteine-rich protein 61), Nov (nephroblastoma overexpressed)). CCN proteins are secreted, extracellular matrix-associated proteins, which regulate cell adhesion, migration, mitogenesis, differentiation, and survival (2Lau L.F. Lam S.C. Exp. Cell Res. 1999; 248: 44-57Crossref PubMed Scopus (575) Google Scholar). The proteins of this family are divided into two groups: one includes CTGF and Cyr61, which usually stimulates cell proliferation; the other cohort is the putative growth arrest/suppression genes, including nov, elm1, and HICP, which are often involved in the negative regulation of cell growth (3Brigstock D.R. Endocr. Rev. 1999; 20: 189-206Crossref PubMed Scopus (534) Google Scholar). The first cloned member of the CCN family is Cyr61, which was identified by differential hybridization screening of a cDNA library from serum-stimulated murine fibroblasts (4Lau L.F. Nathans D. EMBO J. 1985; 4: 3145-3151Crossref PubMed Scopus (365) Google Scholar). Cyr61 shares with most members of CCN family a uniform modular architecture and 38 completely conserved cysteines. After secretion, Cyr61 is associated with the extracellular matrix and cell surfaces (1Kireeva M.L. Mo F.E. Yang G.P. Lau L.F. Mol. Cell. Biol. 1996; 16: 1326-1334Crossref PubMed Scopus (303) Google Scholar, 5O'Brien T.P. Yang G.P. Sanders L. Lau L.F. Mol. Cell. Biol. 1990; 10: 3569-3577Crossref PubMed Scopus (268) Google Scholar, 6Latinkic B.V. O'Brien T.P. Lau L.F. Nucleic Acids Res. 1991; 19: 3261-3267Crossref PubMed Scopus (74) Google Scholar, 7Lechner A. Schutze N. Siggelkow H. Seufert J. Jakob F. Bone. 2000; 27: 53-60Crossref PubMed Scopus (29) Google Scholar). Integrins, such as αvβ3, αvβ5, α6β1, and αIIbβ3, have been identified as the receptors of Cyr61 (8Grzeszkiewicz T.M. Kirschling D.J. Chen N. Lau L.F. J. Biol. Chem. 2001; 276: 21943-21950Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 9Chen N. Chen C.C. Lau L.F. J. Biol. Chem. 2000; 275: 24953-24961Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 10Jedsadayanmata A. Chen C.C. Kireeva M.L. Lau L.F. Lam S.C. J. Biol. Chem. 1999; 274: 24321-24327Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 11Kireeva M.L. Lam S.C. Lau L.F. J. Biol. Chem. 1998; 273: 3090-3096Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). Through integrins as well as other incompletely characterized non-integrin receptors, Cyr61 promotes several cellular activities in a variety cell types (1Kireeva M.L. Mo F.E. Yang G.P. Lau L.F. Mol. Cell. Biol. 1996; 16: 1326-1334Crossref PubMed Scopus (303) Google Scholar, 12Sampath D. Winneker R.C. Zhang Z. Endocrinology. 2001; 142: 2540-2548Crossref PubMed Scopus (93) Google Scholar, 13Sampath D. Zhu Y. Winneker R.C. Zhang Z. J. Clin. Endocrinol. Metab. 2001; 86: 1707-1715Crossref PubMed Scopus (78) Google Scholar, 14Xie D. Miller C.W. O'Kelly J. Nakachi K. Sakashita A. Said J.W. Gornbein J. Koeffler H.P. J. Biol. Chem. 2001; 276: 14187-14194Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 15Tsai M.S. Hornby A.E. Lakins J. Lupu R. Cancer Res. 2000; 60: 5603-5607PubMed Google Scholar). During embryogenesis, Cyr61 exhibits tissue-specific distribution that closely associates with the development of cartilage and the circulatory system (16O'Brien T.P. Lau L.F. Cell Growth Differ. 1992; 3: 645-654PubMed Google Scholar). It also promotes the differentiation of murine bud mesenchymal cells into chondrocytes (17Wong M. Kireeva M.L. Kolesnikova T.V. Lau L.F. Dev. Biol. 1997; 192: 492-508Crossref PubMed Scopus (132) Google Scholar). Cyr61 also plays a role in tumorigenesis. In breast cancer, it stimulates tumor progression (14Xie D. Miller C.W. O'Kelly J. Nakachi K. Sakashita A. Said J.W. Gornbein J. Koeffler H.P. J. Biol. Chem. 2001; 276: 14187-14194Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 15Tsai M.S. Hornby A.E. Lakins J. Lupu R. Cancer Res. 2000; 60: 5603-5607PubMed Google Scholar). It also enhances the malignant phenotype of the gastric adenocarcinoma cell line RF-1 (18Babic A.M. Kireeva M.L. Kolesnikova T.V. Lau L.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6355-6360Crossref PubMed Scopus (426) Google Scholar). Interestingly, expression of Cyr61 is down-regulated in prostate cancer (19Pilarsky C.P. Schmidt U. Eissrich C. Stade J. Froschermaier S.E. Haase M. Faller G. Kirchner T.W. Wirth M.P. Prostate. 1998; 36: 85-91Crossref PubMed Scopus (78) Google Scholar) and leiomyoma (13Sampath D. Zhu Y. Winneker R.C. Zhang Z. J. Clin. Endocrinol. Metab. 2001; 86: 1707-1715Crossref PubMed Scopus (78) Google Scholar), which suggests that Cyr61 might under certain circumstances behave as a tumor suppressor in several tissue types. In this investigation, we examined for the first time the expression and behavior of Cyr61 in lung cancer cells, including its effect on the proliferation of two non-small cell lung cancer (NSCLC) cell lines. Levels of Cyr61 were low in some lung cancer cells, and forced expression of Cyr61 in these cells suppressed cellular proliferationin vitro and in vivo, associated with enhanced expression of p53, p21 WAF1, and pRB2/p130, and decreased expression of cyclin-dependent kinase 2 (CDK2). Total RNA was isolated from a variety of lung cancer cell lines and five matched normal and cancerous tissues by using Trizol reagent (Life Technology, Inc.) according to the standard protocol. The RNA was separated on 1.0% formaldehyde-agarose gel, transferred onto Hybond-N+membrane, and fixed by cross-linking with U.V. Human multiple tissue Northern blot was purchased from CLONTECH. Expression of Cyr61 was detected by 32P-labeled Cyr61 cDNA probe. NCI-H446, H460, H520, H125, H157, H1299, H187, and H526 lung cancer cells were purchased from ATCC and maintained in RPMI 1640 medium (Life Technologies, Inc.) supplemented with 10% fetal calf serum. To make Cyr61 stably expressing lines, H520 (squamous lung cancer) and H460 (large cell lung cancer) were plated in 100-mm dishes and transfected with Cyr61 cDNA cloned into a pcDNA3.1 expression vector by using GenePorter transfection reagent (GTS, Inc.) according to the protocol provided by GTS. After 48 h, the cells were replated in the medium containing 400 μg/ml G418. Two weeks later, well separated colonies were isolated and plated into 24-well plates. The expression of Cyr61 was detected by either Northern (H520 cells) or Western (H460 cells) blot. Neomycin-resistant cells (Neo cells) were obtained by transfecting them with empty pcDNA3.1 vector. The H520 or H460 cells were split evenly into two 100-mm dishes. After growing to ∼60% confluence, cells were transfected with 8 μg of either pcDNA3.1-Cyr61 or pcDNA3.1 empty vector. After 48 h, cells were resuspended in the medium containing 400 μg/ml G418 and replated into 12-well plates. After treatment with G418 for 2 weeks, the cells were stained with 0.1% crystal violet to assess colony formation. Colonies containing more than 40 cells were counted. The cells, which had been stably transfected with either Cyr61 (H520-Cyr61 and H460-Cyr61) or pcDNA3.1 empty vector, which contains the neomycin-resistant gene (H520-Neo and H460-Neo), were plated into 96-well plates at 3.0 × 103 cells/well. After culturing for various durations, cell numbers were measured by MTT assay according to the protocol provided by Roche Molecular Biochemicals. To examine the effect of Cyr61 antibody (14Xie D. Miller C.W. O'Kelly J. Nakachi K. Sakashita A. Said J.W. Gornbein J. Koeffler H.P. J. Biol. Chem. 2001; 276: 14187-14194Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar) on the growth of the cells, the antibody was added at a ratio of 1:50 (v/v) into the wells after the cells were plated as described above. Two days later, the growth of the cells was assessed by MTT assay, and the cell number was calculated according to the standard curve. Cells were plated in 100-mm dishes and trypsinized when they reached 60% confluence. After washing twice with phosphate-buffered saline, cells were fixed in 70% ice-cold ethanol overnight. After staining with propidium iodide, samples were analyzed by flow cytometry. Cells were harvested for total cell lysates with RIPA buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mm Tris-HCl, pH 7.5) containing protease inhibitor mixture (Roche Molecular Biochemicals) and 1 mm NaF, 1 mm NaVO4. Cell lysates were centrifuged at 13,000 rpm for 10 min at 4 °C. The supernatant was collected, and the protein concentration was measured. The same amount of protein was added to each lane, resolved on 4–15% SDS-polyacrylamide gel electrophoresis, and transferred to polyvinylidene difluoride membranes. Antibodies against p21 WAF1 (Oncogene, OP64), p53 (Santa Cruz, sc-126), pRB2/P130 (Santa Cruz, sc-317), pRB (Santa Cruz, sc-102), cyclin D1 (Santa Cruz, sc-7533), and cyclin E (Santa Cruz, sc-247) were used for detecting these proteins. Cell lysates for immunoprecipitation were made as described above. Endogenous CDK2 was precipitated from an equal amount of total protein using anti-CDK2 antibody (Santa Cruz, sc-163) and protein A/G-Sepharose (Santa Cruz, sc-2003) at 4 °C for 1 h. The Sepharose beads were washed three times with protease containing RIPA buffer. Kinase activity was assayed by using histone H1 (Roche Molecular Biochemicals) as the substrate in a 10-μl mixture containing 20 mm Tris-HCl, pH 7.5, 75 mm NaCl, 10 mm MgCl2, 1 mm dithiothreitol, 20 μm ATP, 2 μg of histone H1, and 5 μCi of [γ-32P]ATP. The mixture was incubated at 30 °C for 15 min and then stopped by adding 10 μl of 2× Laemmli loading buffer. Labeled substrate was separated from free by 4–15% SDS-polyacrylamide gel The gel was and to D. M. D. Cancer Res. 2001; Google Scholar). H520-Cyr61 and cells × were in the and of nude mice. were using H460-Cyr61 and cells. weeks after the were and the tumors were Expression of Cyr61 in various normal tissues and cell lines was examined by Northern blot Cyr61 was in normal lung and were in and and were in and 1 also examined Cyr61 in five matched samples of normal lung and NSCLC from the same with normal lung expression of Cyr61 was decreased markedly in four of the five NSCLC samples 1 In lung cancer cell lines, expression of Cyr61 the various lines 1 It was in three cell lung cancer lines H187, and and in the NSCLC cell lines H520 and H460 (large of these lines been to have a to tumors in nude from In the NSCLC cell lines and (large which had low to tumors had expression of Cyr61 H520 and H460 cells had low expression of Cyr61, these two cell lines were transfected with a Cyr61 expression vector to examine the of this assay that H520 cells transfected with formed fewer colonies compared with those transfected with the same amount of pcDNA3.1 empty vector 2 Cyr61 expression H460 cells also formed fewer colonies compared with those transfected with empty vector 2 After stably transfecting H520 and H460 NSCLC cell lines with Cyr61, we isolated two sublines for each cell expression of Cyr61 in these sublines was by Northern or Western 2 of the sublines of H520 and H460 had a markedly than the cells in as measured by MTT assay 2 against Cyr61 was added to containing H520 and H460 cells. with the cells in normal the addition of the Cyr61 antibody had no effect on the growth of the cells, the proliferation of both H520-Cyr61 and H460-Cyr61 cells by and 2 our proliferation that the growth of the cells cell cycle analysis was to which of the cell cycle were H520-Cyr61 and H460-Cyr61 cells had a of cells in the G1 and compared with the cells and a the of cells in decreased The number of cells in markedly these Cyr61-stably transfected lung cancer cells and with a amount of than the cells Cell cycle is often by of examined several proteins related to the cell cycle and p21 WAF1, a was up-regulated in both H520-Cyr61 and H460-Cyr61 cells. Levels of the tumor suppressor proteins pRB and pRB2/p130 were also examined by Western blot Expression of pRB was different the NSCLC cell lines and their pRB2/p130, member of pRB was prominently up-regulated in H520-Cyr61 cells and in the H460-Cyr61 cells. Levels of the tumor suppressor protein p53 were also examined. p53 is in NSCLC cell lines, analysis was and we that both H520 and H460 cells had the wild-type p53 gene increase of expression of p53 was in Cyr61-stably transfected H520 cells, and a increase of p53 was in H460-Cyr61 The NSCLC cell had no p53 protein as D. D. S. 1992; Google Scholar). In the activity of CDK2 was markedly in both H460-Cyr61 cells and H520-Cyr61 cells compared with the cells To this protein tumor the and as well as and were in nude as described under nude was with the cells on one and the cells on the other After 5 weeks, the tumors were and each the cells developed a smaller tumor than the cells and analysis that the of the H520-Cyr61 tumors was than those formed by the cells 4 the of the tumors formed by H460-Cyr61 was than those formed by cells 4 Cyr61 was the first member cloned in the CCN expression is several by growth factors (2Lau L.F. Lam S.C. Exp. Cell Res. 1999; 248: 44-57Crossref PubMed Scopus (575) Google Scholar) and by the receptors C. H. M. J. M. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar), receptors (14Xie D. Miller C.W. O'Kelly J. Nakachi K. Sakashita A. Said J.W. Gornbein J. Koeffler H.P. J. Biol. Chem. 2001; 276: 14187-14194Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar), as well as by and M. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). The Cyr61 protein associates with the extracellular matrix and several integrins (8Grzeszkiewicz T.M. Kirschling D.J. Chen N. Lau L.F. J. Biol. Chem. 2001; 276: 21943-21950Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 9Chen N. Chen C.C. Lau L.F. J. Biol. Chem. 2000; 275: 24953-24961Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 10Jedsadayanmata A. Chen C.C. Kireeva M.L. Lau L.F. Lam S.C. J. Biol. Chem. 1999; 274: 24321-24327Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 11Kireeva M.L. Lam S.C. Lau L.F. J. Biol. Chem. 1998; 273: 3090-3096Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar) and promotes cellular that Cyr61 tumor growth of a gastric adenocarcinoma cell line RF-1 and breast cancer cell lines (14Xie D. Miller C.W. O'Kelly J. Nakachi K. Sakashita A. Said J.W. Gornbein J. Koeffler H.P. J. Biol. Chem. 2001; 276: 14187-14194Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 15Tsai M.S. Hornby A.E. Lakins J. Lupu R. Cancer Res. 2000; 60: 5603-5607PubMed Google Scholar, A.M. Kireeva M.L. Kolesnikova T.V. Lau L.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6355-6360Crossref PubMed Scopus (426) Google Scholar). of Cyr61 are in breast that are at an at the time of (14Xie D. Miller C.W. O'Kelly J. Nakachi K. Sakashita A. Said J.W. Gornbein J. Koeffler H.P. J. Biol. Chem. 2001; 276: 14187-14194Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). Cyr61 are or are decreased in and prostate (13Sampath D. Zhu Y. Winneker R.C. Zhang Z. J. Clin. Endocrinol. Metab. 2001; 86: 1707-1715Crossref PubMed Scopus (78) Google Scholar, C.P. Schmidt U. Eissrich C. Stade J. Froschermaier S.E. Haase M. Faller G. Kirchner T.W. Wirth M.P. Prostate. 1998; 36: 85-91Crossref PubMed Scopus (78) Google Scholar, S. N. K. Res. 2001; 19: PubMed Scopus Google Scholar). The role of Cyr61 in NSCLC was examined in our Expression of this CCN protein was in normal human lung tissue than in their matched NSCLC samples. cells formed fewer colonies than cells transfected with empty vector, which that this protein growth. was also in the H520-Cyr61 and H460-Cyr61 cells stably transfected with Cyr61 expression vector. The addition of Cyr61 antibody to the medium was partially to the growth that growth by Cyr61 is at in a of Cyr61 being and then as a for an that is associated with growth in revealed that cells formed smaller tumors in nude compared with the cells. Taken together, the suggests that Cyr61 is a tumor suppressor protein for lung H520-Cyr61 and H460-Cyr61 cells had a of cells in the G1 and a in the of the cell cycle compared with matched cells. To the of the G1 arrest, we examined the expression of cell proteins. The of both p53 and p21 WAF1 was detected in the cells, which also had the most of cell growth. expression of p21 is related to G1 cell cycle by the R. Clin. Cancer Res. 1998; 4: Google Scholar, Cancer Res. Google Scholar, G. A. N. L. R. D. 1998; PubMed Scopus Google Scholar, J. K. Y. Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google Scholar, Cancer 1999; PubMed Scopus Google Scholar). CDK2 is one of the proteins of The of cyclin plays a role in the from G1 to of p21 with cyclin and the kinase in a of the In our in with the of a remarkably decreased kinase activity of CDK2 was detected in both H520-Cyr61 and H460-Cyr61 cells. Interestingly, the pocket protein pRB2/p130 was also up-regulated in cells, in H520-Cyr61 cells. a member of the pRB cell proliferation and tumor J. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar, M. S. C. R. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google Scholar, L. F. D. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar, A. M. K. A. Cancer Res. 2000; 60: Google Scholar, L. C. A. S. C. G. L. M. S. C. A. Clin. Cancer Res. 1999; Google Scholar, J. 1998; PubMed Scopus Google Scholar, J. K. A. J. Natl. Cancer 1998; PubMed Scopus Google Scholar, Y. A. K. K. M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: PubMed Scopus Google Scholar), including in lung tissue M. A. Clin. Cancer Res. 2000; Google Scholar, C. C. G. C. L. L. M. A. Cancer Res. 2000; 60: Google Scholar, A. A. G. F. M. A. Clin. Cancer Res. 1996; Google Scholar, A. A. Y. M. F. A. Clin. Cancer Res. 1997; 3: Google Scholar, K. K. A. H. N. S. M. Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google Scholar). pRB and is the pocket protein in and cells J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar, A. 1999; Google Scholar). The of in the of The is to a G1 by of and of cyclin K. J. K. L. J. EMBO J. 2001; 20: PubMed Scopus Google Scholar). pRB2/p130 and p21 WAF1 the G1 in Cyr61-stably transfected NSCLC cells. The of of Cyr61 is that Cyr61 was a of integrins (8Grzeszkiewicz T.M. Kirschling D.J. Chen N. Lau L.F. J. Biol. Chem. 2001; 276: 21943-21950Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 9Chen N. Chen C.C. Lau L.F. J. Biol. Chem. 2000; 275: 24953-24961Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 10Jedsadayanmata A. Chen C.C. Kireeva M.L. Lau L.F. Lam S.C. J. Biol. Chem. 1999; 274: 24321-24327Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 11Kireeva M.L. Lam S.C. Lau L.F. J. Biol. Chem. 1998; 273: 3090-3096Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). to Cyr61 several cellular including the enhanced cellular response to growth factors and (2Lau L.F. Lam S.C. Exp. Cell Res. 1999; 248: 44-57Crossref PubMed Scopus (575) Google Scholar, D.R. Endocr. Rev. 1999; 20: 189-206Crossref PubMed Scopus (534) Google Scholar). the after Cyr61 to integrins are Cyr61 to have different in various types of cells, and by expression of integrins in the cells or various that by the same in different types of cells. are In our expression of Cyr61 in a in the cell cycle and of both p53 and the Cyr61 and either p53 or p21 is that the was to p53 and p21 and cell cycle A. R. S. A.M. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar, C. A.M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). are to Cyr61 also to and p53 or Interestingly, as member of the CCN to Cyr61, have on the cell that tumor growth in a cell line Y. N. M. Y. K. H. J. J. Exp. 1998; PubMed Scopus Google Scholar) proliferation of fibroblasts L. J.W. D. L. Dev. 2000; PubMed Google Scholar). the of of the CCN family in cell growth and Taken together, we have that Cyr61 as a tumor suppressor gene in NSCLC. Expression of Cyr61 in the NSCLC cell lines H520 and H460 in their growth and of p53, p21 WAF1, and The cells also formed markedly smaller tumors in nude compared with the cells. have provided the first that Cyr61 a negative regulation of cell growth.