134integrintransfectionofum-uc-2… · um-uc-2 grown in mlo was transfected with 2â€”[email protected]...

ICANCERRESEARCH57.38-42. January1, 19971

Advances in Brief

134IntegrinTransfectionofUM-UC-2(HumanBladderCarcinoma)Cells:StableExpression of a Spontaneous Cytoplasmic Truncation Mutant with RapidLoss of Clones Expressing Intact I@4@

Sang Yoon Kim,2'@Nancy J. Bachman,2 Thankam S. Nair, Scott Goldsmith, Monica Liebert, H. Barton Grossman,Margaret I. Lomax, and Thomas E. Carey4

Laboratories of Head and Neck Cancer Biology [S. Y. K., T. S. N., S. G., T. E. C.J and Molecular Biology (N. I. B., M. 1. LI, Department of Otolaryngology/Head and NeckSurgery, The University ofMichigan, Ann Arbor, Michigan 48/09-0506. and Department of Urology, M. D. Anderson Cancer Center, Houston, Texas 77030 (M. L, H. B. G.j

Abstract

The a6@34integrin is a component of the hemidesmosome, the anchoring structure in the basal membrane of epithelial ceUs.a6@l4expression isfrequenfly altered In neoplastic cells. It Is sometimes lost and sometimesoverexpressed, which suggests that disruption of normal function is involved in neoplastic transformation. To examine the effect of this Integrinon the growth and behavior of malignant cells that have lost @l4,wetransfected a full-length fi4 cDNA into the UM-UC-2 cell line that expresses a6 but not f34. Although large numbers of clones were obtainedwhen a control vector was used Inthe transfection, only 12 clones could beIsolated that expressed g34.Of these, only two @i4.posltiveclones, clones 8and 11, persisted long enough for further study. aone 8 cells initiallyexpressed 04, but within 2 weeks, all positive cells were lost from theculture. Clone 11 persisted in culture and retained strong surface expression [email protected] analysis and Western blotting revealed that thisclone contained a truncated form of@34that had lost the distal cytoplasmicdomain. We conclude that expression ofwild-type @J4in UM-UC-2 inhibitscell growth, presumably by an integrin-medlated signaling pathway.Clone 11 escaped from normal signaling because the cytoplasntic domain,a region essential for basal polar localization, was lost. The a6@i4integrinappears to have tumor suppressor activity in epithellal tumors.

Introduction

The a6f34 integrin is a laminin./kalinin receptor (1, 2) and a component of the hemidesmosome (3, 4). The @34subunit is a Mr 205,000protein characterized by an unusually large cytoplasmic domain of Mr118,000 that contains four type III fibronectin-like repeats in the

COOH terminus (5, 6). This domain, unique among integrins, isessential for interactions with other cytoplasmic proteins and neces

sary for localization in hemidesmosomes (6â€”8)but not for associationwith a6 or adhesion to laminin (7). Furthermore, Clarke et a!. (9)observed that the intact @4integrin is part of a signaling pathway thatup-regulates p21/Cipi (cyclin-dependent kinase inhibitor) expressionand induces apoptosis.

The a6f34 integrin was initially identified as a tumor-associatedantigen by monoclonal antibodies raised to murine lung and humanpancreatic, squamous cell, and bladder carcinomas (10â€”13). High

Received 10/10/96; accepted 11/7/96.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

I Supported by grants from the Office of the Vice President for Research of the

University of Michigan, the NIH-NCI CA56973 and NCI CA56973, the General ClinicalResearch Center Grant MOl-RR00042, and from the Asan Medical Center and AsanInstitute for Life Science.

2@ contributions as first author.

3 Present address: Department of Otolaryngology, Asan Medical Center, University of

Ulsan College of Medicine, 388â€”I,Poongnap-Dong, Songpa-Ku, Seoul 138â€”040Korea.4 To whom requests for reprints should be addressed, at Laboratory of Head/Neck

Cancer Biology, The University of Michigan, 6020 KHRI Campus Zip 0506, 1301 EastAnn Street, Ann Arbor, Ml 48109-0506.

expression is associated with tumor progression (14), poor prognosis(15), or metastasis (16). However, in some cases, expression of a6@34is lost, suggesting that either overexpression or loss may contribute tomalignant progression. The aim of this study is to analyze the effectof expression of the full-length j34 cDNA in a human bladder carcinoma cell line, UM-UC-2 ( 17), that expresses a6 but not @34(13).Clones expressing the full-length f34 subunit did not persist in culture,but a clone expressing a spontaneously mutated @34subunit, in whichthe distal cytoplasmic domain was lost, continued to proliferate andexpress the mutant form of f34 in association with the endogenous a6.These results suggest that the (34 integrin may function as a tumor

suppressor gene.

Materials and Methods

Monoclonal Antibodies. Hybridoma supematants containing the anti-@34monoclonal antibody UM-A9 (12) and the anti-a6 antibody BQ-l6 (13) were

used undiluted. The rat anti-@34monoclonal antibodies 439-9B, against theextracellular domain, and 450-llA, against the cytoplasmic domain, were

generous gifts from Drs. S. J. Kennel and T. Langford of Oak Ridge NationalLaboratory (10, 18). Lyophilized ascites fluids were rehydrated and diluted to

1:1000 for use in Western blot analysis.Cell Culture. Squamouscarcinomacell lines UM-SCC-22Band-38 were

established in our laboratory (12, 14) and were used as a6(34-positive controls.

The bladder carcinoma cell line, UM-UC-2 (13, 17),was used for transfection.Cells are maintained in Dulbecco's modification of Eagle's minimal essentialmedium supplemented with 2 mM L-glutamine, I % nonessential amino acids(Sigma Chemical Co.), penicillin (100 units/mI), streptomycin (100 ,.tg/ml),and 10% FCS (Hyclone, Logan, UT; MlO medium). Confluent cultures are

passaged using porcine trypsin (0.1% w/v; Sigma) and 0.02% EDTA in Puck'ssaline A.

Expression Constructs and Transfection. The pRc-CMV' eukaryoticexpression vector containing the full-length f34subunit cDNA, pCMV-f34,wasobtained from Dr. Filippo Giancotti (6, 7). This vector (InVitrogen, San Diego,CA) contains the CMV promoter,which drives high level expression ofmammalian genes, and the neomycin resistance gene as a selectable marker.

UM-UC-2 grown in MlO was transfected with 2â€”[email protected] pCMV-f34 orpRc-CMV by the calcium phosphate precipitation method (6â€”9).Neomycinresistant clones were selected in 600 mg/ml geneticin (0418; Life Technolo

gies, Inc., Grand Island, NY), a concentration previously determined to kill theparental UM-UC-2 cells within 7â€”10days. Resistant colonies were transferredto 24-well plates and then to coverslips in 6-well plates and tested for f34expression using immunofluorescence. The clones were subcultured and testedby immunoprecipitation and Western blot assays. Clones transfected withvector alone were selected and analyzed in parallel. Flow cytometry was usedto assess intensity of surface ct6f34 expression. While in culture, the cells werefed periodically with 0418-containing medium to prevent outgrowth of revertant clones. All of the clones were frozen at early passage for subsequent study.

5 The abbreviations used are: CMV, cytomegalovirus; IF, immunofluorescence; RIPA,

radioimmunoprecipitation assay.

38

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GROWTh SUPPRESSION BY THE B4 INTEGRIN

IF StainingandFlowCytometry.Therelativelevelof expressionof @34was compared by IF staining and flow cytometry. For IF, cells were grown oncoverslips or in eight-well Lab-Tek chamber slides (Nunc, Naperville, IL). Thecells were washed in cold PBS containing 0.9 mM CaC12 and 0.5 mM MgC12,

incubated with 3% (v/v) normal goat serum (Vector Laboratories, Burlingame,CA) for45 mm,washedthreetimes,andincubatedwithprimaryantibodiesfor2 h. After washing, specimens were incubated for 45 mm with trimethylrhodamine isothiocyanate-conjugated affinity purified F(Ab')2 fragment goat an

timouse IgG (Accurate Chemical & Scientific Co., Westbury, NY) diluted1:200.All incubationswereat4Â°Conarotatingplatform.Afterfixingthecellsin cold acetone for 3 mm, coverslips were mounted using Vectashield medium(Vector).

For flow cytometry, cells were detached in trypsin/EDTA, collected in MlO,counted, aliquoted at 106cells/tube, and washed once in PBS containing 0.5%(w/v) BSA (PBS-B). The cells were incubated with 100 @lofprimary antibodyfor 45 mm on ice. After washing in PBS-B, 100 pi of fluorescein isothiocyanate-conjugated affinity purified F(Ab')2 fragment goat antimouse IgG (Accurate; 1:20) were added for 30 mm on ice. Then cells were washed, fixed in200 @lof 1%paraformaldehyde,andanalyzedby flow cytometry(CoulterEpics Co., Hialeah, FL) calibrated with standard beads at an excitation wavelength of 488 nm.

Immunopreclpltation. Cells were metabolically labeled for 4 h with 100mCi/mI of [35Slmethionine (DuPont NEN, Wilmington, DE) in methionine

free medium as described previously (12). Labeled cells were rinsed with PBS

and harvested by scraping in 200 pJ of lysis buffer (1% NP4O in PBScontaining protease inhibitors (13). After 30 mm on ice, the lysates wereclarified by centrifugation for 5 mm at 2000 rpm at 4Â°C,mixed with 100 piPBS-B (1% w/v) and 300 @lof RIPA [1% NP4O,50 mMTris-CI (pH 8.0), 150mM NaCl, 0.1% sodium deoxycholate, 0.1% SDS, and 1 mM phenylmethyl

sulfonyl fluoride], precleared twice with protein A-Sepharose beads (Sigma),and incubated overnight at 4Â°Cwith primary antibodies UM-A9 (anti-@4)andBQ-16 (anti-a6). Antigen-antibody complexes were incubated with 30 @lofprotein A-Sepharose beads for 2 h at 4Â°C,and washed three times in high saltRIPA (500 mM NaCl) and once in 150 mr@isaline RIPA. The precipitatedsamples were boiled for 3 mm in 60 p.1 reducing buffer (5% v/v 2-mercaptoethanol, 2% SDS, 10% glycerol, 0.0625 MTris-Cl, and 0.005% bromphenolblue) and the proteins were separated by electrophoresis in 7% polyacrylamide.

Immunoblotting and ECL Detection. UM-A9 immunoprecipitates wereseparated by SDS-PAGE and were transferred to nitrocellulose filters inWestern blot buffer (20 mt@iTris, 150 mM glycine, and 15% methanol; Ref.12). The filter strips were blocked for 1 h at room temperature in 3% nonfatdried milk dissolved in PBS with 0.1% Tween (PBS-T) and 0.1% antirat IgG(Sigma), washed, and then incubated for 1 h with the rat anti-@4antibodies439-9B or 450-1 1A diluted 1:1000 in PBS-T. After PBS-T washes, the stripswere incubated for 1h with biotinylated goat antirat antibody (1:1000) (VectorLabs, Burlingame, CA), washed, incubated with avidin-biotin horseradishperoxidase complex (Vectastain ABC-HRP reagent; Vector Labs) for 30 mm,washed in PBS-T, and immersed in enhanced chemiluminescence (ECL)detection reagent (Amersham Corp., Arlington Heights, IL) for 1 mm; chemiluminescence was detected on Kodak X-OMAT AR film.

Results

Selection of Clones Expressing Integrin @34Subunit. Cloneswere selected for G4l8 resistance and tested for fri surface expressionusing IF on unfixed cells. Forty-two G4l8-resistant clones wereobtained from the pCMV-@34 transfection. Of these, 12 were positive

for @34by IF. Ten clones were randomly selected from the numerous(>150) pRc-CMV-transfected cells; all were negative for f34 IF. ThepCMV-f34-transfected clones demonstrated a range of @34intensity, asillustrated in Fig. 1. Examples of the IF results are shown for onecontrol pRc-CMV-transfected clone, pCMVcl (clone 1P; Fig. la),and three of the (34-positive clones, pCMV(34c1 (clone 1; Fig. lb),pCMVI34C8(clone 8; Fig. 1, c and d), pCMVf34C11 (clone 11; Fig. 1,e and f). Most of the clones were weakly positive like that shown inFig. lb. Two pCMV-f34 clones, clones 8 and 11, exhibited strong IFstaining with UM-A9 antibody. However, within 2 weeks, all positivecells were lost from the clone 8 culture. Clone 11 retained strong

a PCMVC1 b @MVr34-c1@

@1@

J.

st

J

@CrAV@4c8 d pCMVI34c8C

@:..

e pCMVI34-cll f pCMV(34-cllFig. 1. IF photomicrographs showing expression of j34 integrin in transfected clones

derived from the p34-negativeUM-UC-2 bladder carcinoma cell line. a, pCMV-cl plasmidcontrol clone I; b-f (34-transfectedclones (pCMV-@34-cI,[email protected] pCMV-(34-cli).

expression of a6j34. A vial of clone 8 cells that had been previouslyfrozen was thawed and retested. It was positive for (34 expression byIF, but in spite of selection of the 5% most positive cells by flowcytometry, again, within 2 weeks positive cells could no longer bedetected.

Biochemical Analysis and Flow Cytometry. Immunoprecipitation with the UM-A9 anti-j34 antibody followed by Western blotanalysis with the 439-9B anti-(34 antibody directed against the extracellular domain of (34 (18) confirmed loss of (34 expression in clone8 and revealed a change in the structure of the @34protein in clone 11.In the first experiment (Fig. 2, left panel), both clones 8 and 11expressed the @34complex, with the expected @34bands at Mr 205,000(intact f34), Mr 185,000, and Mr 155,000 (proteolytic breakdownproducts; Refs. 12, 13, and 19). In clone I 1, there was an additionalprominent band of Mr 175,000. When this experiment was repeated 2weeks later on the progeny of the same clones, the intact fri proteincould no longer be detected in either clone 8 or I 1. In clone I 1, theprominent band of Mr 175,000 and the Mr 155,000 bands were stilldetectable. In clone 8, only a very faint Mr 155,000 band could beobserved. This suggested that the @34gene had been truncated in clone11, resulting in a smaller gene product. To further assess this possibility, cells were labeled with [35S]methionine, immunoprecipitatedwith UM-A9, and analyzed by autoradiography (Fig. 3). UM-SCC22B cells were used as a positive control. In clone 8 (Fig. 3, left panel,Lane C8), the a6@34complex was undetectable, indicating that thecells in this clone were no longer expressing the transfected @34protein. Immunoprecipitation with the BQ-l6 anti-a6 antibody confirmed this. An a6 doublet OfMr 125,000 (processed heavy chain) andMr 150,000 (a6 precursor; Refs. 12 and 13) was observed in clone 8

immunoprecipitates (Fig. 3, right panel, Lane C8). These a6 bandsare also present in the parent UM-UC-2 (Fig. 3, Lanes UC2) andcontrol plasmid transfected cells (Fig. 3, Lanes CJP). UM-A9 immunoprecipitates of clone 11 cells (Fig. 3, leftpanel, Lane ci 1) exhibited

39

@:@



GROWrH SUPPRESSION BY THE B4 1NTEGRIN

of the clone I 1 lane. This was not present in a repeat experiment. Flowcytometry confirmed that clone I 1 cells expressing the truncated (34subunit do express (34 on the cell surface (Fig. 4B).

In normal resting epithelia, the a6f34 integrin is found within thehemidesmosome, an organelle involved in anchoring the epithehum to the basement membrane (4). Many epithelial tumor cellsfail to form hemidesmosome-like structures, suggesting that theremay be loss of function associated with aberrant (34 expression.The function of the hemidesmosome can probably be disrupted bya variety of mechanisms, including loss of expression of the j34subunit as in the UM-UC-2 cell line. When UM-UC-2 cells weretransfected, abundant plasmid control clones were obtained, butvery few /34-expressing clones were recovered. Of the clones that

expressed (34, most ceased to express the integrin subunit within2â€”3weeks in culture. These clones retained the plasmid becausethey remained resistant to G4l8. These observations suggest thatj34 gene expression suppresses growth and that those cells that losethe ability to express the intact (34 subunit have a growth advantage. This is consistent with the observations by Clarke et al. (9),who found that transfection of (34 into the (34-negative RKO coloncarcinoma cells induced the expression of p2 1 and concomitantapoptosis. In contrast, clone 11 continued to express (34 on thesurface, which seems to contradict the growth-inhibitory hypothesis. However, immunoprecipitation analysis of the (34 proteindemonstrated that the clone 11 cells were producing a truncatedprotein of Mr 175,000. Western blot analysis of (34 immunoprecipitates with antibodies specific for the extracellular and distal

cytoplasmic domains demonstrated that the distal cytoplasmic domain of 13@was lost. This spontaneous truncation is reminiscent of

40

l.p. __________-@ UM-A9(anti @l4)

W.B. 439-9B 43996 anti-rat IgGonly@ @11 @1I

MW C8 C11C1PUC2 C8 C11C1PUC2 C8 CliCIPUC22OOâ€”--@'@ -

97â€”@@

69â€”S

@ ._@@

Discussion

46â€”

:=m@@!@â€˜@V...

the truncated Mr 175,000 and the Mr 155,000 134 protein bandsobserved previously in the Western blots. In addition, the Mr 125,000

processed a6 band was coprecipitated, indicating that a6f34 heterodimers were being formed in clone 1 1. Immunoprecipitation of the

clone 11 extract with anti-ca6 antibody confirmed this finding (Fig. 3,rightpanel, Lane CII). The truncated Mr 175,000 and Mr 155,000 (34bands were coprecipitated with a6. A Mr 200,000 band was alsopresent in this immunoprecipitate and in that from the parental UMUC-2 cells (Fig. 3, righipanel, Lane UC2); however, the nature of thisband is not known since it was not precipitated or Western blotted bythe anti-f34 antibodies.

To further examine the nature of the truncated protein in clone 11,134immunoprecipitateswereWesternblottedwitheitherthe439-9Banti-(34 antibody to the NH2-terminal extracellular domain of (34 orthe 450-1 1A anti-(34 antibody to the COOH-terminal cytoplasmicdomain of the (34 protein (18). UM-SCC-22B and UM-SCC-38 cellswere used as positive controls. As shown in Fig. 4A, 439-9B stainedthe Mr 175,000 and the Mr 155,000 (34 bands in clone I 1 extracts (Fig.4A, left panel, Lane Cli), as well as the Mr 205,000, Mr 185,000, Mr175,000, and Mr 155,000 bands in the UM-SCC-22B extracts (Fig.4A, left panel, Lane 22B). In contrast, the 450-I lA antibody to theCOOH-terminal domain failed to stain the Mr 175,000 band in (34immunoprecipitates of clone I I in either of two experiments (Fig. 4A,center panel, Lane Cli, and right panel, Lane Cli). Note that thisantibody does stain the prominent Mr 205,000 band corresponding tothe intact (34 protein in both UM-SCC-22B and UM-SCC-38 extracts.There was a slight overflow from the UM-SCC-22B lane during theloading of the gel, which resulted in a faint Mr 205,000 band at the top

Exp. 1 Exp.2Fig. 2. Western blot of 134 integrin complexes immunoprecipitated from cell extracts

oftransfected clones of UM-UC-2. Cell extracts (CIP, pCMV-cl), a plasmid only controlclone; C8 (pCMV-f34-c8) and Cl 1 (pCMV-@4-c 11). @4-transfectedclones; UC2 (UMUC-2) parental nontransfected cells) were immunoprecipitated with the UM-A9 mousemonoclonal antibody to the extracellular domain of @4and protein A-Sepharose beads asdescribed in â€œMaterialsand Methods.â€•The precipitated proteins were separated bySDS-PAGE under reducing conditions, transferred to nitrocellulose membranes, andstained with the 439-9B rat antibody to the extracellular domain of the j34 protein. Twoexperiments are shown (Ezp. i and Erp. 2). In Exp. 1, the @34-transfectedclones C8 andCl 1 each expressed the intact Mr 205,000 frI protein and the proteolytic breakdownproducts of Mr 180,000and Mr 150,000,but 2 weeks later (Exp. 2), both had lost the intactform. Cl I also expressed a Mr 175,000 (solid arrowhead) and a Mr 155,000 (openarrowhead) doublet of @34-positivebands that were also present in Exp. 2. The laneslabeled antirat IgG are from Exp. I and are identical to the control lanes from Exp. 2. Thedark bands of Mr 50,000 and Mr 28,000 @flthe control lanes and the experimental lanes arefrom cross-reactivity of the anti-rat lgG reagent with the heavy and light chains of theUM-A9 antibody used to precipitate (34.

anti @4 anti a6

MW C8 Cli C1PUC222B

200â€”

97â€”

69-@@ i

46â€”

30â€” 30â€”

Fig. 3. Immunoprecipitation and SDS-PAGE analysis of a6(34 proteins from clones 8and I I. Cells from clone 8 (C8), clone 11 (Cl 1), plasmid control clone I (C1P),UM-UC-2, and UM-SCC-22B ((34-positive control) were labeled metabolically with[35Slmethionine,lysed, immunoprecipitated with the UM-A9 anti-(34antibody (leftpanel)or the BQ16 anti-a6 antibody (right panel), resolved by gel electrophoresis underreducing conditions, and developed by autoradiography. The intact a6j34 integrin complex is indicated by braces next to the UM-SCC-22B lane. The M, 175,000 truncated (34protein expressed by clone 11 is indicated by an arrowhead in the second lane of the leftpanel. The M, 155,000 protein of the doublet observed in Fig. 2, as well as a bandcorresponding to the cr6 heavy chain at Mr 125,000, are also present. Similarly, in the Cl 1lane precipitated by the a6 antibody (right panel), the M@175,000 and Mr I55,000 134complex are present. These experiments indicate that the truncated (34protein assemblesinto the a6J34 complex. The dark band at Mr 200,000 lfl the Cl I lane precipitated by thea6 antibody is most likely a fibronectin band that is sometimes coprecipitated by anti-a6antibodies and is also present in the UM-UC-2 precipitates.

C8 Cli C1PUC2

1@i@4200â€”@]@u6 â€”

97â€”

69â€”

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10 â€” â€” â€”â€”

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GROWTH SUPPRESSION BY ThE B4 INTEGRIN

A BI.P. UM-A9(anti @4) C1P

W.B. 439-9B 450-hA 450.11AI I I I I

MW Cli 22B Cli 22B Cli 38

46â€” 1

@;j@â€¢Fr@Tâ€¢@1@@ @==

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0.1 I tO 100 1000

Fluorescence Intensity

Fig. 4. Western blot and flow cytometric analysis of 134integrin expression in clone I 1cells. A, Western blot of the 134integrin immunoprecipitated from clone I I and from positivecontrol cell lines UM-SCC-22B and UM-SCC-38. Immunoprecipitated proteins were identified with the 439-9B antibody to the extracellular domain of 134(left panel) or the 450-lIAantibody to the distal cytoplasmic domain of (34(center and right lanes). Both antibodies identify the 134proteins identified in the control cell lines, but in clone I 1, only the antibodyto the extracellular domain can bind to the 134protein ofclone I I, indicating that the truncated (34protein in these cells has lost the distal cytoplasmic domain. B, flow cytometric analysisof surface 134expression in the vector-only transfected clone C1P, in the 134transfected Cl 1 clone, and in the positive control cell line UM-SCC-22B cell line. The results show thatthe truncatedformof 134expressedin cloneII is presenton thecell surface.

mutant constructs of (34 that have the ability to disrupt a6(34polarization in rat bladder carcinoma cells (6â€”8).Similarly, Shawet a!. (21) showed that a cytoplasmic deletion mutant of /34 couldact as an inhibitor of cs6j3l function in breast carcinoma cells bycompeting for the a6 subunit and blocking a6(3l-mediated attachment and migration. Taken together, these results indicate that the1.34cytoplasmic domain is required for integrin function.

We postulate that disrupted function of the hemidesmosome is acommon characteristic of tumors. It appears that hemidesmosomefunction may be inhibited by loss of the (34 subunit as in the RKOcolon carcinoma cells and UM-UC-2 bladder carcinoma cells. In thesecells, when the (34 protein is restored by transfection, programmedcell death and/or growth inhibition ensues. However, in the hemidesmosome-forming 804G rat bladder carcinoma cell line, the a6 and (34subunits are intact, and addition of (34 to these cells has no growthinhibitory effects (6â€”8),suggesting that some other component of theanchoring signaling pathway has been compromised. The nature ofthese aberrations remains to be characterized. However, we recentlyfound a poorly differentiated carcinoma of the parotid gland thatexpresses a6j34 but fails to express the BPAG2 or bullous pemphig

oid-l80 protein.6 Just as with the E-cadherin/catenin interaction, avariety of disruptions in the a6(34 pathway may result in subversionof normal regulatory function.

References

1. Lee, E. C., Lots, M. M., Steele, G. D. Jr., and Mercurio, A. M. The integrin a6134isa laminin receptor. J. Cell Biol., ii7: 671â€”678,1992.

2. Niessen, C. M., Hogervorst, F., Jaspars, L. H., de Melker, A. A., Delwel, G. 0.,Hulsman, E. H. M., Kuikman, I., and Sonnenberg, A. The a6134 integrin is a receptorfor both laminm and kalinin. Exp. Cell Res., 211: 360â€”367,1994.

3. Hogervorst, F., Kuikman, I., von dem Borne, A. E. G., and Sonnenberg, A. Cloningand sequence analysis of beta-4 cDNA: an integrin subunit that contains a unique I 18kd cytoplasmicdomain.EMBOJ., 9: 765â€”770,1990.

4. Stepp, M. A., Spurr-Michaud, S., Tisadale, A., Elwell, J., and Gipson, K. a6134integrin heterodimer is a component of hemidesmosomes. Proc. Nat. Aced. Sci. USA,87: 8970â€”8974, 1990.

5. Carter, W. G., Kaur, P., Gil, S. G., and Wayner, E. A. Distinct function for integrinsa3f3l in focal adhesions and a6(34/bullous pemphigoid antigen in a new stableanchoring contact (SAC) of keratinocytes: relation to hemidesmosomes. J. Cell Biol.,lii:3141â€”3154,1990.

6. Spinardi, L., Ren, Y-L., Sanders, R., and Giancotti, F. G. The 134subunit cytoplasmic

6 5 Y. Kim, Chu, K-C., Lee, H. R., Lee, K-S. and Carey, T. E. Establishment and

characterization of nine new head and neck cancer cell lines, submitted for publication.

41

200â€”

â€˜S

@1o

UM.SCC.22B



GROWTH SUPPRESSION BY THE B4 INTEGR1N

domain mediates the interaction of the a6(34 integrin with the cytoskeleton ofhemidesmosomes. Mol. Biol. Cell, 4: 871â€”884,1993.

7. Spinardi, L., Einheber, S., Cullen, T., Milner, 1., and Giancotti, F. G. A recombinanttail-less integrin 134subunit disrupts hemidesmosome but does not suppress a6134-mediated cell adhesion to laminins. J. Cell Biol., 129: 473â€”487,1995.

8. Mainiero, F., Pepe, A., Wary, K. K., Spinardi, L., Mohammadi, M., Schlessinger, J.,and Gianconi, F. G. Signal transduction by the a6134 integrin: distinct 134subunit sitesmediate recruitment of Shc/Grb2 and association with the cytoskeleton of hemidesmosomes. EMBO J., 14: 4470â€”4481,1995.

9. Clarke, A. S., Lotz, M. M., Chao, C., and Mercurio, A. M. Activation of the p21pathway of growth arrest and apoptosis by the 134integrin cytoplasmic domain.J. Biol. Chem., 270: 22673â€”22676,1995.

10. Kennel, S. J., Foote, L. J.. Falcioni, R., Sonnenberg, A., Stringer, C. D., Crouse, C.,and Hemler, M. E. Analysis of the tumor-associated antigen TSP-180: identity withcr6/34 in the integrin superfamily. J. Biol. Chem., 264: 155l5â€”1552l, 1989.

I 1. Kajiji, S., Tamura, R. N., and Quaranta, V. A novel integrin (a@fl4)from humanepithelial cells suggests a fourth family of integrin adhesion receptors. EMBO J., 8:673â€”680,1989.

12. Van Waes, C., Kozarsky, K. F., Warren, A. B., Kidd, L, Paugh, D., Liebert, M., andCarey, 1. E. The A9 antigen associated with aggressive human squamous carcinomahas structural and functional similarity to the newly defined integrin cr6134.CancerRes., 51: 2395â€”2404,1991.

13. Liebert, M., Wedemeyer, G., Stein, J. A., Washington, R. W., Jr., Van Waes, C.,Carey. 1. E., and Grossman, H. B. The monoclonal antibody BQ 16 identifies thea6134 integrin on bladder cancer. Hybridoma. 12: 67â€”80,1993.

14. Kimmel, K. A., and Carey, 1. E. Altered expression in squamous cells of anorientation restricted epithelial antigen detected by monoclonal antibody A9. CancerRes., 46: 3614â€”3623,1986.

15. Wolf, G. T., Carey, T. E., Schmaltz, S. P., McClatchey, K. D., Poore, J., Glaser, L.,Hayashida, D. J. S., and Hsu, S. Altered antigen expression predicts outcome insquamous cell carcinoma of the head and neck. J. Nail. Cancer Inst., 82: 1566â€”1572,1990.

16. Falcioni, R., Kennel, S. J., Giacornini, P., Zupi, G., and Sacchi, A. Expression oftumor antigen correlated with metastatic potential of Lewis lung carcinoma and B16melanoma clones in mice. Cancer Res., 46: 5772â€”5778,1986.

17. Grossman, H. B., Wedemeyer, G., and Ren. L. UM-UC-l and UM-UC-2: characterization of two new human transitional cell carcinoma cell lines. J. Urol., 132:834â€”837,1984.

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1997;57:38-42. Cancer Res Sang Yoon Kim, Nancy J. Bachman, Thankam S. Nair, et al.

4βExpressing Intact Cytoplasmic Truncation Mutant with Rapid Loss of ClonesCarcinoma) Cells: Stable Expression of a Spontaneous 4 Integrin Transfection of UM-UC-2 (Human Bladderβ

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134integrintransfectionofum-uc-2… · um-uc-2 grown in mlo was transfected with 2â€”[email protected]...

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