LAMININ-5Function of the γ2 chain in epithelial cell adhesion andmigration, and expression in epithelial cells andcarcinomas

SIRPASALO

Department of Biochemistry

1999

OULUN YLIOPISTO, OULU 1999

LAMININ-5Function of the γ2 chain in epithelial celladhesion and migration, and expression inepithelial cells and carcinomas

SIRPA SALO

Academic Dissertation to be presented with the assent of the Faculty of Science, University of Oulu, for public discussion in Raahensali (Auditorium L 10), Linnamaa, on Octobet 2nd, 1999, at 12 noon.

Copyright © 1999Oulu University Library, 1999

OULU UNIVERSITY LIBRARYOULU 1999

ALSO AVAILABLE IN PRINTED FORMAT

Manuscript received 10.8.1999Accepted 31.8.1999

Communicated by Professor Nils BrünnerProfessor Jyrki Heino

ISBN 951-42-5342-6(URL: http://herkules.oulu.fi/isbn9514253426/)

ISBN 951-42-5334-5ISSN 0355-3221

Salo Sirpa, Laminin-5: Function of the γ2γ2γ2γ2 chain in epithelial cell adhesion andmigration, and expression in epithelial cells and carcinomasBiocenter Oulu and Department of Biochemistry, University of Oulu, FIN-90570 Oulu, Finland1999Oulu, Finland(Manuscript received 10.8.1999)

Abstract

Laminins are basement membrane glycoproteins consisting of three polypeptide chains α, β and γ.Until now 12 members of the protein family have been characterized and all isoforms have an αβγchain composition, but they assemble in varying combinations of chain variants. The functionalproperties of laminins include cell adhesion, proliferation, differentiation, growth and migration.Laminin-5 has a chain composition of α3β3γ2 with the distribution mainly restricted to epithelialbasement membranes, where its biological functions involve anchorage and locomotion of cells.The importance of this protein for the attachment of basal keratinocytes is clearly demonstrated bythe fact that all genes encoding its chains have been shown to be mutated in the severe skinblistering disease Epidermolysis bullosa junctionalis.

The present study focused on investigations of the role of the laminin-5 isoform and particularlyits γ2 chain in cell adhesion and migration. The role of the short arm of the laminin γ2 chain in theprocess of epithelial cell attachment is to serve as a kind of a bridging molecule to the extracellularenvironment, because it does not have any cell binding activity by itself. It was also shown that thenewly synthesized γ2 chain participates in the complex process of cell migration, probably as one ofthe first attachment components for moving cells. Thus, as a migration and differentiation-associated molecule, laminin-5 was considered a potential marker for detection of malignantprocesses where cell movement plays a role. Subsequently it was shown that the γ2 chain isexpressed not only in a restricted manner in human epithelial tissues, but also in a number of humanepithelium-derived cancers. In some carcinomas, expression of the γ2 chain appeared to be acharacteristic of cancer cells with invasive properties. Examination of over 50 dysplasias andcervical tumors revealed that γ2 chain antibodies were able to distinguish between lesions with orwithout invasive capacity. This is the first systematic study of epithelial cancers where γ2 chainantibodies have been shown to be auseful marker in the histopathological diagnostics. In addition,this study showed in a mouse tumor model that the γ2 chain of laminin-5 has a potential for being ofuse for in vivo tumor imaging.

Keywords: Basement membrane, cell anchorage, immunohistology, tumor imaging

Acknowledgements

The present work was carried out at the Biocenter Oulu, and the Department ofBiochemistry, University of Oulu. I would like to thank my supervisor, Professor KarlTryggvason, for introducing me to the scientific world, and for his support during thiswork. It has been a privilege to work in his international research group with excellentfacilities. I also wish to thank all former and present members of the basement membranegroup both in Oulu and in Stockholm. Especially, I owe my thanks to people who stillwork in Oulu, to Maarit Kangas and Tomi Ai renne for many cheerful moments, and toOlga Beltcheva, Paula Martin, Vesa Ruotsalainen, Tuire Salonurmi, Sirpa Kontusaari,Paula Reponen, Paula Väisänen, and Maire Jarva for creating a nice and relaxedatmosphere at work. I also thank Ari Tuuttila for sending me strange, but funny emailattachments from Stockholm. I wish to express my gratitude to all co-authors andcollaborators. Particularly, it has been a pleasure to work with Dr. Barbro Skyldberg andDr. Gert Auer from the Karolinska Institute. My special thanks go to Heli Haakana forcollaboration when she was still working in our research group, and for her friendshipduring that time and afterwards. I am grateful to Professor Nils Brünner and ProfessorJyrki Heino for valuable comments on the manuscript of my thesis, and to Sidney Higleyfor careful and rapid revision of the language.

Professor Kalervo Hiltunen, as well as other professors and group leaders, areacknowledged for establishing great facilities and a modern attitude towards research workat the Department of Biochemistry. I would also like to thank all colleagues and other staffat the Department for helping me in many ways.

Last, but not least, my very special thanks go to people who have taken care of meoutside the laboratory and, what is also important for a researcher, helped me to forgetwork now and then. First, I am deeply grateful to Ildikó Veress for many kinds ofentertainment, for instance, for taking me to Lapland with foreigners, and for importingUnicum to Finland several times. My sincere thanks go to my friends Jari and JuliePekkarinen, and to Kaisu Ainassaari, for many pleasant moments during these years, andto Mirka Pohjanrinne, for her nice and challenging friendship. I wish to thank AnuLeinonen for being my friend, for her numerous 5 o’clock early-morning phone calls and,especially, for being an excellent travel guide during our trips. Lahja Uitto deserves mywarmest thanks for sharing good and bad days, and for her patience in listening to mystories that can be a bit strange sometimes. Finally, I thank my dear parents Anja and

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Armas, brother Jyrki, sister-in-law Anneli, and two lovely young ladies, my favorite girls,Linda and Erika, for their support and love.

This work was granted by the Cancer Foundation of Northern Finland, the FinnishCancer Foundation, the Pohjois-Pohjanmaa Branch of Finnish Cultural Foundation, andthe University of Oulu.

Oulu, August1999

Abbreviations

AP-1 activator protein-1bp base paircDNA complementary DNAECM extracellular matrixEGF epidermal growth factorEHS Engelbrecht-Holm-SwarmGBM glomerular basement membraneGST glutathione S-transferaseHD1 hemidesmosomal protein-1IF intermediate filamentIgG immunoglobulin Gi.m. intramuscularlyi.v. intravenouslykb kilobasekDa kilodaltonmRNA messenger ribonucleic acidPBS phosphate buffered salineTGF transforming growth factoruPA urokinase plasminogen activatoruPAR urokinase plasminogen activator receptor

Origina l articles

The present work is based on the following articles:

I Pyke C, Salo S, Ralfkiær E, Rømer J, Danø K & Tryggvason K (1995) Laminin-5is a marker of invading cancer cells in some human carcinomas and iscoexpressed with the receptor for urokinase plasminogen activator in buddingcancer cells in colon adenocarcinomas. Cancer Res. 55: 4132-4139

II Salo S, Haakana H, Kontusaari S, Hujanen E, Kallunki T & Tryggvason K(1999) Laminin-5 promotes adhesion and migration of epithelial cells:identification of a migration-related element in the γ2 chain gene (LAMC2) withactivity in transgenic mice. Matrix Biol. 18: 197-210.

III Salo S, Airenne T, Heikkilä J & Tryggvason K (1999) Intravenously injectedanti-laminin γ2 chain antibody targets to epithelial tumors. Submitted forpublication.

IV Skyldberg B, Salo S, Eriksson E, Aspenblad U, Moberger B, Tryggvason K &Auer G (1999) Laminin-5 is a marker of invasiveness in cervical lesions. J NatlCancer Inst, In press.

Contents

AbstractAcknowledgementsAbbreviationsOriginal articles1. Introduction...................................................................................................................132. Review of the literature .................................................................................................15

2.1. Basement Membranes.............................................................................................152.2. Laminins.................................................................................................................16

2.2.1. Isoforms and chains .....................................................................................162.2.2. Structure, self-assembly and polymerization................................................192.2.3. Location, structure and regulation of genes.................................................242.2.4. Tissue distribution........................................................................................262.2.5. Interactions with other basement membrane ligands....................................282.2.6. Interactions with cellular receptors..............................................................292.2.7. Examples of biological functions.................................................................32

2.2.7.1. Laminin-5 as a component of the epithelial anchoring system........322.2.7.2. Laminin-2/laminin-4 and attachment of muscle cells......................342.2.7.3. β2 chain of laminin-3 in neuromuscular junctions and kidney........352.2.7.4. Importance of the α5 and γ1 chains in embryogenesis....................35

2.3. Interactions of tumor cells with ECM and basement membrane.............................362.3.1. General aspects of tumor progression..........................................................362.3.2. Laminins and cancer ....................................................................................37

3. Outlines of the present study .........................................................................................384. Materials and methods...................................................................................................39

4.1. Preparation and characterization of polyclonal antibodies.....................................394.2. Expression of recombinant laminin γ2 chain..........................................................394.3. Immunohistochemistry and in situ hybridization....................................................404.4. Cells........................................................................................................................404.5. Cell adhesion and migration assays........................................................................404.6. Generation and analyses of transgenic mice...........................................................414.7. Tumor imaging with unlabeled and radioactively labeled antibodies....................42

5. Results...........................................................................................................................445.1. Antisera against recombinant γ2 .............................................................................44

5.2. Expression of the γ2 chain in human cancers .........................................................445.3. The effect of the γ2 chain laminin-5 on cell attachment and migration of

epithelial cells........................................................................................................465.4. Identification of a migration-related element in the LAMC2 gene.........................465.5. Targeting of polyclonal antibody to tumors in KLN-205 mice...............................47

6. Discussion .....................................................................................................................496.1. Expression of the γ2 chain of laminin-5 in human epithelial tissues and

malignancies...........................................................................................................496.2. The γ2 chain of laminin-5 in epithelial cell adhesion and.......................................51

migration.................................................................................................................516.3. Potential of the γ2 chain of laminin-5 as an in vivo marker of epithelial

tumors......................................................................................................................536.4. Conclusion..............................................................................................................54

7. References.....................................................................................................................56

1. Introduction

Laminins are large heterotrimeric basement membrane glycoproteins composed ofα, β and γ chains (Timpl 1996). The number of laminins has enlarged noteworthly inrecent years, and currently at least 12 different isoforms are known (Verrando et al. 1988,Beck et al. 1990, Engvall et al. 1990, Carter et al. 1991, Rousselle et al. 1991,Marinkovich et al. 1992a, Champliaud et al. 1996, Miner et al. 1997, Koch et al. 1999).Al l isoforms have αβγ composition, but assemble in varying combinations of these chains.As an essential basement membrane component, laminins show a large variety ofbiological functions in cells and organs, including cell attachment, proliferation,differentiation, growth and migration. However, investigations of the exact functionalproperties of different laminin isoforms remain largely to be carried out.

Laminin-5, earlier also known as epiligrin, nicein, kalinin and ladsin, is one of the mostinvestigated laminin isoforms in recent years. It has a chain composition of α3β3γ2 and itstissue distribution is mainly restricted to epithelial basement membranes (Verrando et al.1988, Carter et al. 1991, Marinkovich et al. 1992b, Mizushima et al. 1996). Thebiological functions proposed for laminin-5 are as part of epithelial anchoring systems andcell locomotion. Laminin-5 is essential for the adhesion of basal keratinocytes to theunderlying basement membrane as demonstrated by the fact that the genes coding for theα3, β3 or γ2 chains have been associated with a severe skin blistering disease termedHerlitz’s junctional epidermolysis bullosa (Pulkkinen et al. 1994a, Pulkkinen et al. 1994b,Kivi rikko et al. 1995). The main symptom of this disease is disruption of the epidermisfrom its supporting basement membrane. Laminin-5, or at least some of its chains are alsoexpressed in healing skin wounds, suggesting a possible biological role in cellproliferation and migration.

The present study focused on investigations of the role of laminin-5, especially its γ2chain, in cell adhesion and migration, and the localization of a migration-related elementin the gene. In addition, it was shown that the γ2 chain protein is expressed in a number ofdifferent human carcinomas, with its expression being characteristic of some cancer cellswith a budding cell phenotype. Because γ2 was expressed in invasive tumor cells ofepithelial origin, but only marginally in mature human tissues, the question was raisedwhether or not antibodies against this chain could potentially be used as an in vivo marker

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of some epithelial tumors. This idea was also evaluated using gamma camera imaging ofradioactively labeled antibodies.

2. Review of the literature

2.1. Basement Membranes

Epithelial and endothelial cells are in close contact with highly specialized extracellularmatrices called basement membranes that are widely distributed within the body.Basement membranes also surround most muscle and fat cells, as well as peripheral nerveaxons. The basement membranes affect cell phenotypes and tissue compartmentalizationin many ways, starting from early embryonic development. During development, cellsattach and move along the basement membrane. It is needed for the polarization of cells,both in the embryo and the adult, and it serves as a substratum for cell adhesion andlocomotion during wound healing and nerve regeneration (Engvall 1995, Timpl 1996).

The basement membrane is composed of several types of collagens, laminins,proteoglycans, calcium-binding proteins, as well as some other structural or adhesiveproteins. These proteins form a thin sheet-like structure of 50-100 nm in thicknessthrough specific self-assembly mechanisms. Although electron microscopy shows a tightstructure of lamina lucida and lamina densa layers, the real structure may be morevariable. In addition to the traditional basement membrane proteins, other componentsmay also be selectively incorporated confering additional biologically important qualitiesto the membranes (Engvall 1995, Timpl 1996).

Major architectural features of basement membranes are two independent networks,one formed from type IV collagen and the other from laminin. The covalently cross-linkedcollagen network is considered to maintain the mechanical stability, while the lamininnetwork is more dynamic (Timpl & Brown 1996). The most widespread basementmembrane component is type IV collagen with the chain composition [α1(IV)] 2α2(IV).Three less frequent type IV collagen isoforms, [α3(IV)] 2α4(IV) , [α5(IV)] 2α6(IV) , andα3(IV)α4(IV)α5(IV) have also been identified and shown to be involved in someinherited or autoimmune disorders (Hudson et al. 1993). Collagen VII , which consists ofthree α1(VII ) chains, is a constituent of anchoring fibril s which connect the basementmembranes of squamous epithelia to the underlying stroma (Timpl 1996, Timpl & Brown1996). Proteoglycans, of which perlecan is the most abundant, are further importantbasement membrane constituents. Some other proteoglycans exist in basement membranes,

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but they are less well characterized. Quite recently the 250 kDa protein agrin was shown tobe a proteoglycan. It was identified in basement membranes where it is able to clusteracetylcholine receptors at neuromuscular synapses. Agrin also exists in other basementmembranes. There is also another group of basement membrane proteins, which includesnidogen, BM-40 (osteonectin/SPARC), fibulin-1 and fibulin-2 (Timpl &Brown 1996). The150 kDa protein nidogen connects collagen and laminin meshworks, thus stabilizing thenetwork structure (Foxet al. 1991, Mayeret al. 1993). Nidogen also binds to perlecan,fibulin-1 and fibulin-2 and it has been shown to exist in at least two significantly distinctforms (Timpl &Brown 1996, Kohfeldtet al.1998).

2.2. Laminins

The first laminin isoform, laminin-1, was isolated from the mouse Engelbreht-Holm-Swarm (EHS) tumor in 1979 (Timplet al. 1979), but currently at least 12 differentisoforms are known. The number of both chains and isoforms can be expected to increasefurther, and alternative splicing of some laminin gene transcripts may also raise thenumber of laminin variants. Laminins exhibit a complex variety of biological functionsand they are expressed as early as the two cell stage of embryonic development (Timpl1996). However, because of the increasing number of laminin chains and isoforms, itremains to be shown if the tissue distributions and biological functions linked earlier e.g.to laminin-1 or theα1 chain are really true or do they belong to other isoforms or chains.

2.2.1. Isoforms and chains

Five genetically distinctα, threeβ and threeγ chains have been cloned and characterizedand they can assemble into at least 12 different isoforms. The isomers and chains knownso far are summarized in Table 1.

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Table 1. Laminin isoforms.

Isoform Chain composition References

Laminin-1 α1β1γ1 Beck et al. 1990Laminin-2 α2β1γ1 Engvall et al.1990Laminin-3 α1β2γ1 Engvall et al.1990Laminin-4 α2β2γ1 Engvall et al.1990Laminin-5 α3β3γ2 Carter et al. 1991

Rousselle et al. 1991Laminin-6 α3β1γ1 Marinkovich et al. 1992aLaminin-7 α3β2γ1 Champliaud et al. 1996Laminin-8 α4β1γ1 Miner et al. 1997Laminin-9 α4β2γ1 Miner et al. 1997Laminin-10 α5β1γ1 Miner et al. 1997Laminin-11 α5β2γ1 Miner et al. 1997Laminin-12 α2β1γ3 Koch et al. 1999

The primary structure of theα1, α2, α3, α4, β1, β2, β3, γ1, γ2 andγ3 chains has beenreported from man (Pikkarainenet al. 1987, Pikkarainenet al. 1988, Carteret al. 1991,Haaparantaet al. 1991, Nissinenet al. 1991, Kallunki et al 1992, Marinkovichet al.1992a, Gereckeet al. 1994, Ryanet al. 1994, Vuolteenahoet al. 1994, Weweret al.1994, Iivanainenet al. 1995a, Iivanainenet al. 1995b, Mineret al. 1997, Kochet al.1999) and those of theα1, α3, α4, α5, β1, γ1, γ2 andγ3 chains from mouse (Sasakiet al.1987, Sasakiet al. 1988, Mineret al. 1995, Sugiyamaet al. 1995, Iivanainenet al. 1997,Iivanainenet al. 1999) andα1, β1 and γ1 chains from Drosophila as well (Montell &Goodman 1988, Chi & Hui 1989, Garrisonet al. 1991, Kusche-Gullberget al. 1992). Inaddition, the primary structure of theβ1 variant from rat has been determined (Hunteretal. 1989a, Hunteret al. 1989b). Apart from the number of possible chain associations, it isquite obvious that only certain chains can combine with each other to form trimers. Theα1 chain can be found in laminin-1 and laminin-3 isoforms, which are composed ofα1β1γ1 and α1β2γ1 chains. Laminin-1 represents the previously known EHS-typelaminin, and laminin-3 was known earlier as s-laminin (Becket al. 1990, Engvall et al.1990, Burgesonet al. 1994). Theα2 chain can associate withβ1 and γ1 chains into thelaminin-2 isoform (merosin), and also withβ2 and γ1 chains into the laminin-4 isoform,which was previously also known as s-merosin (Engvallet al. 1990). Laminin-5 (epiligrin,kalinin, nicein, ladsin) has a chain composition ofα3β3γ2 (Verrandoet al.1988, Carteretal. 1991, Marinkovichet al. 1992b, Mizushimaet al. 1996). Theα3 chain is also found inlaminin-6 (α3β1γ1) and laminin-7(α3β2γ1), which can covalently associate with laminin-5 in epidermal anchoring structures (Champliaudet al. 1996). Bothα4 andα5 chains canassemble withβ1 and γ1 chains thus forming isoforms laminin-8 (α4β1γ1), laminin-9(α4β2γ1), laminin-10α5β1γ1) and laminin-11 (α5β2γ1) (Miner et al. 1997). Kochet al.

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have suggested thatα2 and β1 could be the partner chains of the recently describedγ3chain.

Laminin-5 is initially synthesized as a 460 kD molecule which undergoes specificproteolytic processing to a smaller form after secretion into the extracellular matrix. Sofar, no other laminin isoform has been shown to be proteolytically modified aftertranslation. The reduction of size is a result of cleaving theα3 subunit from 190-200 kDato 160 kDa andγ2 subunit from 155 to 105 kD, respectively (Marinkovichet al. 1992b,Matsui et al. 1995). Recently, processing of theα3 subunit of laminin-5 was shown to bemediated by a plasmin-dependent mechanism involving tissue-type plasminogen activator(tPA)-catalyzed plasminogen activation (Goldfingeret al. 1998). The processing of theγ2chain and the components taking part in it remain to be characterized.

Some laminin chain transcripts have been shown to be alternatively spliced, which mayincrease the number of laminin variants. Nevertheless, it is not known if all transcriptvariants also exist as proteins, how they assemble in trimers, and what are their functionsin living animals. Ryanet al. reported in 1994 two distinct humanα3 transcripts thatdisplay variability within domain IIIa. Theα3EpA transcript has a smaller version ofdomain IIIa and is missing the 4th, 5th, and 6th cysteine residues that are typically found inthe beginning of domain IIIa. Theα3EpB transcript has maintained similarity to theα1chain throughout domain IIIa and into domain IV and thus has a larger amino-terminaldomain (Ryanet al. 1994). Two alternatively splicedα3 isoforms have also been reportedfor mouse (Gallianoet al. 1995) and these isoform chains are generated by usage of twopromoters (Ferrignoet al. 1997). The longer mouseα3Β transcript shows 77 % homologyto the available sequence of the humanα3B and the shorterα3A has 67.4% identity to thehomologous 5’-amino acid sequence of the humanα3Α counterpart. According toin situperformed on sections of tissue obtained from 13.5- and 17.5-day mouse embryos thesetwo transcripts also have distinct expression patterns (Gallianoet al. 1995). Miner andcoworkers have presented a third mouseα3 sequence encoded by the LAMA3 gene(Miner et al. 1997). This isoform extends the sequence reported by Gallianoet al. (1995)at the 5’end by the about 2.2 kb. Thisα3 isoform has the NH2-terminal portion of domainIVb, a complete domain V, and almost all of a domain VI (Mineret al. 1997). Theγ2chain of the laminin-5 isoform also has two alternatively spliced transcripts for which afunctional role at the protein level remains to be shown. Kallunkiet al. (Kallunki et al.1992) demonstrated two transcripts with differences at their 3’ends. The longerγ2transcript is encoded by 23 exons, which represent 5200 bp cDNA. The shorter transcriptis encoded by 22 exons together with part of the 5’end of intron 22, resulting in a cDNA of4316 bp. The longer transcript encodes a protein of 140 kDa, assuming that the protein isfully glycosylated, and the shorter codes for a protein of 130 kDa (Kallunkiet al. 1992).Later, it has been shown inin situ experiments that these transcripts have different tissuedistributions (Airenneet al. 1996). The consequence of the alternative splicing of theγ2transcript at the protein level could be that the shorter polypeptide lacks a C-terminalcysteine residue that is thought to form a disulfide bond with a cysteine residue at the samelocation in theβ3 chain (Airenneet al. 1996, Gereckeet al. 1994). It is, therefore,possible that thisγ2 chain would not be able to form stable long-arm structures withβ3chain. Until now only humanβ2 chain has been shown to have alternative splicing at the5’-untranslated region of the mRNA (Durkinet al.1996).

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2.2.2. Structure, self-assembly and polymerization

Laminins have a cruciform, T- or Y-shape structure as visualized by electron microscopyafter rotary shadowing. The structure of the laminin-1 molecule is presented in Fig 1. Theunderstanding of laminin structure is mostly based on electron microscopy and sequenceanalysis, as the flexibility and size of laminins have prevented crystallization and NMRstudies. Fragmentation by limited proteolysis, circular dichroism spectroscopy,hydrodynamic methods and atomic force microscopy have also been used for structuralcharacterization (Engel 1992, Maurer 1995, Chenet al.1998).

Fig 1. Structural model of a laminin-1 molecule (adapted and modified from Timpl & Brown1994). The domain structure is divided into regions I to VI according to the sequence, and eachdomain is further divided into smaller modular units that are presented here with LN, LE, L,LG and CC.

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The overall structure of different isoforms is quite similar among the vertebrate lamininsstudied thus far. Domains I and II, that take part in the chain assembly, are quiteconserved and differences in shape are mainly caused by the missing N-terminal regions ofα3, β3, and γ2 chains (Engel 1992, Maurer 1995). The best studied example ofinvertebrate laminins is Drosophila laminin, whose component chains have beensequenced to completion (Montell &Goodman 1988, Chi &Hui 1989, Garrisonet al.1991, Kusche-Gullberget al.1992).

The laminin polypeptide chains are folded into a large number of structurally and oftenfunctionally autonomous protein units. These units are divided according to theirsequences into domains I to VI (fig.1 and 2). The domains are further divided into smallerunits, since laminins belong to a class of multifunctional proteins designated as mosaic ormodular proteins. In mosaic proteins, domains occur as modular units in several differentextracellular matrix proteins and also in proteins of non-extracellular matrix origin. Inlaminins, anα-helical coiled-coil structure (CC) in region I and II of all three chains has aheptad repeat of nonpolar amino acid residues. Rod-like regions III and V inβ and γchains and IIIa, IIIb, and V inα chain are composed of many repeating EGF-like(epidermal growth factor) domains (LE). Regions IVa and IVb of theα chain and IVof theγ chain have been proposed to be EGF-like domains (L4). Region IV in theβ chain(LF) has a unique sequence motif and occurs only in laminin-1. Regions VI (LN) arehomologous between chains and do not show homology to the other domains in laminin.The C-terminal globular region has been defined at theα chain and it consists of five LGrepeats (Engel 1992, Maurer 1995).

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Fig. 2. Schematic representation of the domain organization and motifs in various lamininchains (adapted from Engvall 1995). The domains or their parts with homology in differentchain variants are presented with similar patterns.

The three different chains of the laminin molecule are held together by anα-helicalcoiled-coil structure. Theβ andγ chains both contain a cysteine residue at their C-terminiand are disulfide bonded, which further stabilizes the trimer structure. The coiled-coilstructure is formed via domains I and II where different chains have little sequencehomology. All polypeptide chains contain a heptad pattern of residues of the form(a,b,c,d,e,f,g)n where nonpolar hydrophobic amino acids are located preferentially inpositions a and d, charged residues in positions e and g, and polar residues in b, c and f(Beck et al. 1990). The hydrophobic residues in positions a and d are located in thecenter of the trimer structure protecting them from the aqueous environment, whereashydrophilic amino acids are exposed on the surface. The coiled-coil is further stabilizedby ionic interactions between residues e and g (Becket al. 1990, Becket al. 1993).Nomizu and coworkers (Nomizuet al. 1996) have further concluded from experimentswith laminin-2 (α2β1γ1) peptides that theβ−γ dimer forms an acidic pocket because of thenegatively charged ionic interactions. The basic residues of a short peptide representingthe C-terminal part of theα-chain then specifically interact with the acidic pocket of the

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dimer to form a stable triple-stranded coiled-coil structure. In particular theconformational instability ofγ1 chain is a driving force for this interaction (Nomizuet al.1996). Although the triple-stranded coiled-coil region of laminin consist of about 570amino acids of each chain only a short C-terminal sequence of 25-amino acids of eachchain is active for efficient initiation of trimer formation (Utaniet al. 1994, Utaniet al.1995). This sequence can also function as a nucleation site to initiate chain interactionswhich lead to the completion of assembly in a C- to N-terminal direction (Nomizuet al.1994, Nomizuet al.1996).

Laminins are glycoproteins with varying numbers of N-glycosylation sites. Thestructural studies of laminin carbohydrates suggest that laminins, or at least laminin-1,contain only N-linked oligosaccharides (Arumughamet al. 1986, Fujiwaraet al. 1988,Knibbset al. 1989, Tanzeret al. 1993). The function of laminin carbohydrates has not yetbeen definitely determined, although a functional role of glycosylation of laminin-1 hasbeen reported for tumor cell adhesion, cell spreading, neurite outgrowth and integrin-laminin interactions (Engel 1992). The glycosylation of laminins does not seem to takepart in the stability against proteases, heparin binding or chain assembly (Howe 1984, Wuet al.1988).

In most basement membranes, laminin forms a double polymer with type IV collagenand these polymers are connected via a bridging molecule nidogen (Aumailleyet al. 1989,Fox et al. 1991, Yurchencoet al. 1992). A three-arm interaction model has been proposedfor laminin polymerization (Yurchenco & Cheng 1993, Chenget al. 1997). According tothis model, laminin-1 self-assembles through interactions between N-terminal short armdomains forming a meshwork polymer. The assembly is reversible and calcium-dependentand calcium binding is believed to confer the correct conformation for favorable bindingto the other short arms. The flexible long arm of each monomer would then be free tointeract with cells or heparin or heparan sulfates out of the plane of the polymer(Yurchenco &Cheng 1993, Chenget al. 1997). Because of the large number and variety inthe structures of laminin isoforms, it has been found that there are also differences in theability to polymerize. It has also been suggested that only laminins with three “full-sized”short arms (i.e. laminins 1, 2 and 4) are able to polymerize (Chenget al. 1997) aspresented in Fig. 3. Laminin-5, which is a non-polymerizing laminin, does not bind tonidogen, but it may have other matrix binding interactions through type VII collagen,laminin-6 and 7 or fibulin-2 (Chenget al.1997, Rousselleet al.1997, Utaniet al.1997).

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Fig. 3. A model for the polymerization of different types of laminin (Cheng et al. 1997).Laminins with three full-length short-arms can polymerize via NH2-terminal parts of thechains (a), while isoforms with truncated short-arm structures cannot. Nidogen links lamininswith full-length ββββ or γγγγ chain to the type IV collagen network, but the laminin-5 isoform whichhas truncated short-arms has to be linked via other proteins, i.e. type VII collagen (b).

Neither homotrimers nor single laminin chains have been found in the form of separateproteins.β−γ dimers have been observed as biosynthetic intermediates into which theαchain is added later (Moritaet al. 1985, Peterset al. 1985). Alternatively, it has beensuggested that the laminin chains are initially assembled randomly (Wuet al. 1988).According to studies done by Yurchencoet al. (1997), expression ofβ or γ chains aloneresults in intracellular retention of a non-disulfide linked chain with dimerization of theβchain, but not theγ chain. Coexpression ofβ andγ chains results in intracellular retentionof heterodimers. Expression of theα chain results in secretion of monomeric chains withpartial proteolytic cleavage. However, coexpression of all three chains is needed forsecretion of intact trimeric laminin. Thus, the addition of theα chain also seems to driveand regulate the secretion of the trimer (Yurchencoet al.1997).

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2.2.3. Location, structure and regulation of genes

The laminin genes can be subdivided into LAMA, LAMB and LAMC families that encodeα, β and γ chains, respectively. With the exception of mouse LAMC3, all laminin geneshave been localized in the chromosomes and some gene structures have also beenreported. Table 2. summarizes the chromosomal locations of laminin genes of man, mouseandDrosophila.

Table 2. Chromosomal location of laminin genes.

Chain Gene Species Locus Reference

α1 LAMA1 man 18p11.3 Nagayoshi et al. 1989 mouse 17 Weber-Kaye et al.1990

Okazaki et al.1993Doyle et al. 1996

Drosophila 65A10-11(3L) Montell &Goodman 1998α2 LAMA2 man 6q22-23 Vuolteenaho et al .1994

mouse 10 Sunada et al. 1994α3 LAMA3 man 18q11.2 Ryan et al. 1994

mouse 18, band A Aberdam et al. 1994bα4 LAMA4 man 6q21 Richards et al. 1994

mouse 10 Miner et al. 1997α5 LAMA5 man 20q13.2-13.3 Durkin et al. 1997

mouse 2 Miner et al. 1997β1 LAMB1 man 7q22 Pikkarainen et al. 1987

mouse 12 Seldin et al. 1989Drosophila 28D(2L) Montell &Goodman 1998

β2 LAMB2 man 3p21 Iivanainen et al. 1995bWewer et al. 1994

mouse 9 Porter et al. 1993β3 LAMB3 man 1q32 Vailly et al. 1994a

mouse 1, band H2-6 Aberdam et al. 1994bγ1 LAMC1 man 1q25-q31 Fukushima et al. 1988

mouse 1 Weber-Kaye et al. 1990Drosophila 67C(3L) Montell&Goodman 1998

γ2 LAMC2 man 1q25-q31 Kallunki et al. 1992mouse 1, band H1 Aberdam et al. 1994b

γ3 LAMC3 man 9q31-34 Koch et al. 1999mouse unknown Iivanainen et al. 1999

Although five α chains have been identified, only the structures of LAMA2 andLAMA4 have been determined thus far (Zhanget al. 1996, Richardset al. 1997). Thehuman LAMA2 gene was the first structure determined as a mammalian lamininα-chaingene, and it has been shown to be affected in congenital muscular dystrophy (Helbling-

25

Leclerc et al. 1995, Zhanget al. 1996). It consists of 64 exons, which encode a 9500-nucleotide transcript and the 3110-residueα2-polypeptide chain. LAMA2 is considerablylarger and more complex than the human genes coding for theβ andγ chains (Zhanget al.1996). The LAMA4 gene consists of 39 exons and spans over 122 kb (Richardset al.1997). The LAMA gene of the 3712-residueα chain ofDrosophila is more compact thanits human counterparts, containing only 15 exons (Kusche-Gullberget al. 1992).Comparison of the human 260 kb LAMA2 with that of the 14 kbDrosophilaLAMA generevealed poor conservation of locations of intervening sequences. Thus, only introns 3 and6 in the human gene match introns 2 and 3 in theDrosophilagene (Kusche-Gullberget al.1992, Zhanget al.1996).

All known LAMB gene structures have been determined (Vuolteenahoet al. 1990,Pulkkinenet al. 1995a, Durkinet al. 1996), as well as, the structures for LAMC1 andLAMC2 (Kallunki et al. 1991, Airenneet al. 1996). The entire humanβ1chain gene(LAMB1) has a size of more than 80 kb and contains 34 exons (Vuolteenahoet al. 1990).The human and mouse LAMB2 have been found to consist of 33 exons that occupy 12 kbor less of genomic DNA (Durkinet al. 1996). LAMB3 consists of 23 exons accounting forthe full-length cDNA with an open reading frame of 3516 bp encoding 1172 amino acids(Pulkkinenet al. 1995a). LAMC1 is over 58 kb in size and has 28 exons (Kallunki et al.1991). LAMC2 has 23 exons that covers about 55 kb and 16 of them have the same size asexons in the LAMC1 gene (Kallunkiet al. 1992, Airenneet al. 1996). The two genes arelocated close to each other on chromosome 1q25-31 suggesting that they have evolvedthrough duplication of a common ancestral gene (Fukushimaet al. 1988, Kallunki et al.1991). Mutations identified in the LAMC2 gene in junctional epidermolysis bullosa werethe first description of a genetic laminin disease (Aberdamet al. 1994b, Pulkkinenet al.1994a). Later, the LAMA3 and LAMB3 genes were found to be mutated in junctionalepidermolysis bullosa (Pulkkinenet al.1994b, Kivirikkoet al. 1995). Recently, the humanLAMC3 gene has been located on chromosome 9q31-34 (Kochet al.1999).

Laminin genes are expressed at different stages during development and in a tissuespecific manner. It is apparent that this complexity demands tight control of generegulation. At present, knowledge of the regulatory patterns is still quite limited. Thesequences of the promoter regions are known and have, at least partially, beencharacterized for human LAMB1, LAMB2 and LAMC1 (Vuolteenahoet al. 1990,Kallunki et al. 1991, Durkinet al. 1996), as well as, for the mouse LAMA3 and LAMA4(Ferrignoet al. 1997, Richardset al. 1997) and LAMB2 and LAMC1 genes (Ogawaet al.1988, Durkinet al. 1996). Many of these promoter regions lack a TATA or CAAT boxneeded for the initiation of transcription (Ogawaet al. 1988, Vuolteenahoet al. 1990,Kallunki et al. 1991, Durkin et al. 1996, Ferrignoet al. 1997, Richardset al. 1997).However, the LAMA3A and LAMC2 genes have putative TATA box sites (Airenneet al.1996, Ferrignoet al. 1997) and human LAMA4 and LAMB2 have AT-rich regions whichcould fulfill this function (Durkinet al. 1996, Richardset al. 1997). The large sizes oflaminin genes have made it difficult to identify the regulatory elements of the genes and todate, they remain largely uncharacterized. Potential Sp1, AP-1, AP-2 binding sites or GC-boxes have been localized in the mouse LAMA3, human LAMC1, LAMB1 and LAMB2genes (Kallunkiet al. 1991, Vuolteenahoet al. 1990, Durkinet al. 1996, Ferrignoet al.1997). The Sp1 factor is thought to have a role in the initiation of transcription of genes

26

that contain no TATA boxes in their promoters. Recently, two reports dealing with thetranscriptional regulation of the LAMC1 and LAMA3 genes were published. Suzuki andcoworkers characterized a highly conserved enhancer element, bcn-1, in both human andmouse LAMC1 gene promoters in mesangial cells (Suzukiet al. 1996). This motifrecognizes an inducible nuclear protein(s) BCN-1, which might regulate lamininγ1 chaingene trancription. The murine laminin-5α3A and α3B isoform chains are generated byusage of two promoters, and transcription results in distinct expression patterns of thesetwo polypeptides (Gallianoet al. 1995, Ferrignoet al. 1997, Mineret al. 1997). Virolle etal. (1998) studied the transcriptional regulation of theα3A gene by transforming growthfactor-β (TGF-β), which is a prototype member in a large family of morfogens anddifferentiation factors, and which is an important regulator of connective tissue duringhealing processes (Roberts & Sporn 1996, Virolleet al. 1998). They characterized threeactivator protein-1 (AP-1) binding sites between nucleotides –297 and –54 relative to thetranscription start site of which one seems to be essential for gene expression. They alsodemonstrated a specific binding of Fra-2 and JunD to the AP-1 sites, suggesting a possibleregulatory function for this complex in a basal keratinocyte-specific gene (Virolleet al.1998).

2.2.4. Tissue distribution

Laminins are expressed in a tissue-specific manner in the embryo and adult, and theexpression changes during development (Engvallet al. 1990, Saneset al. 1990, Nissinenet al. 1991, Kallunki et al. 1992, Aberdamet al. 1994a, Vuolteenahoet al. 1994, Gallianoet al. 1995, Iivanainenet al. 1995a, Mineret al. 1995, Champliaudet al. 1996, Orian-Rousseauet al.1996, Durkinet al. 1997, Frieseret al. 1997, Iivanainenet al. 1997, Mineret al. 1997). However, in many cases the expression patterns are overlapping. Theα5 andα4 chains seem to be the most widely expressedα chains both in man and mouse, whileexpression of theα1 chain is the most restricted, only being detected in the placenta andkidney (Nissinenet al. 1991, Iivanainenet al. 1995a, Durkinet al. 1997, Frieseret al.1997, Iivanainenet al. 1997, Mineret al. 1997). Northern blot andin situ studies haveshown expression of theα5 chain in the mouse intestine, heart, lung, skeletal muscle, skinand placenta, with low levels also in kidney, and theα4 chain almost as widely expressedas theα5 chain (Mineret al. 1997). A more restricted expression pattern for theα4 chainhas also been presented (Iivanainenet al. 1997).In situ hybridization revealed expressionin mesenchymal cells of the developing (branching) lung epithelia, in the villi andsubmucosa of the intestine and in the external root sheet of vibrissae follicles. Theexpression in the developing kidney was transient. At the protein level theα4 subunit ofembryonic and postnatal mouse tissues showed it to be generally located in mesenchymaltissues (skeletal and heart muscles), lung septa, as well as, in subendothelial basementmembranes of capillaries (Iivanainenet al. 1997). Expression levels of theα2 chain ishighest in tissues with mesodermally derived components, such as skeletal and cardiacmuscle, with some expression also in kidney, whereasα3 chain is found in organs rich inepithelia (skin, lung, low levels in kidney) (Nissinenet al. 1991, Kallunki et al. 1992,

27

Aberdamet al. 1994a, Vuolteenahoet al. 1994, Gallianoet al. 1995, Mineret al. 1997).The main pattern of expression for eachα chain seems to be established before birth, butsome individual basement membranes change theα chain composition as developmentproceeds. For example, in the developing kidney, forming glomeruli express threedifferent α chains in dynamic progression. Distinct regions of a continuous basementmembrane, as found in the nephron, can contain distinct combinations ofα chains, andchange these during development (Mineret al.1997)

The humanβ1 andβ2 chains are expressed in the brain, lung, heart, muscle, adiposetissue, blood vessels, skin and kidney according to Northern andin situ analysis of humanchains. Expression of theβ2 chain can also be detected in liver (Nissinenet al. 1991,Kallunki et al. 1992, Iivanainenet al. 1995b). When the expression patterns of thesechains were compared with each other, it appeared that cell types expressing theβ1 chainalso expressed theβ2 chain, but that certain cell types expressed only theβ2 chain. Forexample, in kidney theβ2 chain is expressed in the glomeruli, while theβ1 chain is not. Inthe skin, strong expression ofβ2 has been seen in cells of both epidermis and dermis, butthe expression ofβ1 chain was prominent in stromal cells of the dermis and adnexes(Iivanainenet al. 1995b). The major difference between theγ1 and γ2 chain expression isthat theγ1 chain is expressed in both epithelial and vascular endothelial cells, while theγ2chain is restricted to epithelial cells (Kallunkiet al. 1992, Aberdamet al. 1994a,Sugiyamaet al. 1995). When theγ1 chain is expressed throughout most tissues, theγ2chain is strongly expressed in epithelial cells of skin and lung as well as in the collectingtubules in kidney, although some expression can be seen also in the thymus, choroidplexus, cerebellum and the brain intermediate zone (Kallunkiet al. 1992, Airenneet al.1996). The recently characterized murineγ3 chain has highly restricted distribution, beingmainly expressed in blood vessels and Leydig cells (Iivanainen 1999).

Laminin-5 is a unique isoform in that expression of its subunits is restricted toepithelial tissues (Kallunkiet al. 1992, Aberdamet al. 1994a). Alternatively splicedvariants have been described for theα3 chain (Gallianoet al. 1995, Mineret al. 1997), theγ2 chain (Kallunkiet al. 1992, Airenneet al. 1996) and theβ2 chain (Durkinet al. 1996)and they have some differences in their distribution. Polypeptides encoded by LAMA3 areknown but it remains to be shown ifγ2 andβ2 chain variants also exist at the protein level.The twoα3 chain transcripts have different 5’ends. The longerα3B polypeptide harbors atruncated amino-terminal end, which is missing the most amino-terminal domains V andVI. As a result of alternative splicing,α3A has an even shorter amino-terminal end, alsomissing domain IV. In mouse tissues (13.5 and 17.5 d embryos) both transcripts weredetected in the skin, teeth, respiratory and alimentary tract, kidney and central nervoussystem with a couple of differencies in their expression patterns (Aberdamet al. 1994a,Galliano et al. 1995). The longer transcript ofα3B chain was exclusively found in thebronchi and alveoli, the stomach and intestinal crypts, and the whisker bads and centralnervous system (Gallianoet al.1995).

The primary transcript of LAMC2 also undergoes an alternative splicing, resulting ashorter 3’end (Kallunkiet al. 1992, Airenneet al. 1996). The shorter chain has an evenmore restricted expression pattern than that of the longerγ2 chain. According to Northernanalysis andin situ experiments, the expression of the longerγ2 chain is seen in mostepithelial cell types except for glomeruli. It is absent in embryonic and neonatal brain,

28

endothelia and skeletal muscle. The shorter transcript ofγ2 chain can be seen, however, inthe periventricular layer of the brain, perialveolar mesenchyme of the lung, and distaltubules of the kidney (Kallunkiet al. 1992, Airenneet al. 1996). Nevertheless, it remainsto be shown if this is true also at the protein level and if so, how these chains areassembled in laminin-5 isoform or isoforms.

2.2.5. Interactions with other basement membrane ligands

Basement membrane components have been shown to interact and self-assemble to form asupramolecular network. Laminin and type IV collagen are connected to each other vianidogen, which is probably a central connecting element within basement membranestructures since it also binds perlecan (Timpl &Brown 1996). The data accumulated thusfar on the interactions of laminin with extracellular matrix proteins are mainly based onstudies with laminin-1 extracted from the EHS-tumor (see Fig. 4). The lack of purifiedproteins in large quantities has restricted studies with other isoforms, but something isknown about the binding activities of laminins 2, 4, 5 and 7 to basement membrane ligands(Timpl et al. 1995).

The laminin-1-nidogen complex has been purified in large quantities and in nativeform from the EHS-tumor. Analysis of the purified complex has demonstrated that the twoproteins occur in an equimolar ratio and that nidogen specifically interacts with the centerof the cross-shaped laminin molecule (Paulssonet al. 1987). Electron microscopy ofrecombinant nidogen shows three globular domains (G1, G2 and G3) which are connectedby a long rod and a flexible linker region. High binding to laminin-1 was assigned to theC-terminal domain, G3 (Foxet al. 1991). Later, the exact binding site in laminin-1 wasmapped by proteolytic and recombinant protein studies to a single EGF-like motif,γ1III4,in the short arm of theγ1 chain (Gerlet al. 1991, Mayeret al. 1993). Laminin-1, 2 and 4share theγ1 chain and high affinity nidogen binding (Mannet al. 1988, Foxet al. 1991,Brown et al. 1994). Recombinant nidogen, as well as the laminin-nidogen complexpurified from tissues, have also been shown to specifically bind to type IV collagen. Themajor collagen binding site in nidogen has been localized to domain G2 and in type IVcollagen two nidogen binding sites have been localized to the triple-helix of type IVcollagen about 80 and 200 nm away from its C-terminal globular domain, NC1 (Aumailleyet al. 1989, Foxet al. 1991, Reinhardtet al. 1993). Nidogen also binds to the core proteinof the BM proteoclycan, perlecan, and to fibulin-1 and fibulin-2 (Battagliaet al. 1992,Sasakiet al. 1995a, Sasakiet al. 1995b). Recently, a new laminin-1-binding humannidogen, nidogen-2, was described (Kohfeldtet al. 1998). It seems to share some of thefunctions of nidogen-1, since it binds to collagen IV, perlecan and collagen I, but it fails tobind fibulins. Furthermore, a new mouse gene for entactin/nidogen, entactin-2, has beenreported (Kimuraet al.1998).

Laminins have been shown to interact directly with heparin, perlecan, fibulin-1 andfibulin-2 (Timpl 1996). Heparin and heparan sulfate binding have been attributed to thedistal domains, G1 to G3 (Ottet al. 1982, Yurchencoet al. 1990, Yurchencoet al. 1993).The most abundant heparan sulfate proteoglycan, perlecan, has also been shown to bind

29

the proteolytic long-arm fragment E3 of laminin-1 as strongly as intact laminin (Battagliaet al. 1992). In contrast to laminin-1, which can bind perlecan either directly or vianidogen G2 domain, laminin-2 and 4 seem to depend entirely on nidogen for perlecanassociation (Brownet al. 1994). Agrin, which is also a heparan sulfate proteoglycan, isrequired for the formation and maintenance of neuromuscular junctions (Tsenet al. 1995,Gautamet al. 1996). The binding of an NH2-terminal fragment of agrin to laminin-1 isconfined to a particular region in the upper part of the triple coiled-coil domain of laminin-1 (Denzeret al.1997).

Fibulin-1 (BM-90) and fibulin-2 compose an extracellular protein family that interactswith the laminin-1-nidogen complex, type IV collagen and fibronectin and they aresuggested to function as mediators of the assembly of basement membranes (Balbonaetal. 1992, Panet al. 1993, Sasakiet al. 1995a, Sasakiet al. 1995b, Utaniet al. 1997).Binding studies with laminin-1 have demonstrated no affinity for fibulin-2, but rather adistinct affinity for fibulin-1, which is mediated through the laminin long-arm fragment E3(Sasakiet al. 1995a). Fibulin-1 has also been proposed to bind to laminin-4 but not tolaminin-2 (Brownet al. 1994). However, recently it has been demonstrated that fibulin-2binds to the short-arms of laminin-5 and laminin-1, suggesting that it could function as abridge between the laminin-1 and laminin-5 molecules, along with other extracellularmatrix proteins, providing a link between the cell surface and the basement membrane(Utani et al.1997).

Laminin-5, a component of the epithelial anchoring system, has truncated short-arms ascompared with laminin-1 (Rousselleet al. 1991, Marinkovichet al. 1992b). Theγ2 chaincannot bind nidogen (Mayeret al. 1995) and, therefore, laminin-5 cannot associate withperlecan or the type IV collagen network as do the other isoforms. Laminin-5 also lacksthe short-arm structures believed to be required to promote network assembly (Schittny &Yurchenco 1990, Yurchencoet al. 1992). Nevertheless, it can form a disulfide-bondedcomplex with laminin-6 and laminin-7 (Champliaudet al. 1996), and through thismechanism, be incorporated into the basement membrane. A second important mechanismhas been proposed by Rousselle et al. (1997) by which laminin-5 interacts with a basementmembrane or stromal component. According to this model, monomeric laminin is theprimary link between hemidesmosomal componentsα6β4 integrin and type VII collagen,with the laminin 5-6/7 complex present within the interhemidesmosomal spaces. Thebinding of laminin-5 has been localized to the NC-1 domain of type VII collagen and thebinding site of type VII collagen in laminin-5 probably occurs within the short arm of theβ3 or γ2 chains (Rousselleet al.1995, Rousselleet al.1997).

2.2.6. Interactions with cellular receptors

Laminins interact with cells via cell surface receptors, such as integrins, membrane-boundproteoglycans (e.g. dystroglycan), and other membrane-bound glycoproteins. Integrins area large family of transmembrane adhesion proteins that are composed of two subunitchainsα andβ. They can bind laminins, as well as many other extracellular ligands andmodulate intracellular signalling pathways in response to this binding. Nine different

30

integrins (α1β1, α2β1, α3β1, α6β1, α6β4, α7β1, α9β1, αvβ3, αvβ8) have beensuggested to be receptors for laminins (Hynes1992, Mercurio 1995). As an example thespecific binding sites of known integrin receptors in laminin-1 molecule are presented inFig. 4. Laminin-1 has been shown to bind all known integrin-type lamininreceptors (Sonnenberget al. 1988, Elices & Hemler 1989, Gehlsenet al. 1989, Ignatiusetal. 1990, Krameret al. 1990, Krameret al. 1991, Leeet al. 1992, Forsberget al. 1994,Venstrom & Reichardt 1995). Laminin-2 has been found to interact withα3β1, α6β1,α6β4, andα7β1 integrins (Pfaffet al. 1994, Changet al. 1995, Yaoet al. 1996), but onlyα6β1 binds to laminin-3 (Delwelet al. 1993) andα6β4 to laminin-4 (Spinardiet al.1995). Laminin-5 interacts with three laminin receptors, namely,α3β1 (Carter et al.1991), α6β1(Delwel et al. 1993, Rousselle & Aumailley 1994) andα6β4 (Niessenet al.1994). Recently, cell adhesion onto laminin-10/11 was found to be mediated by integrinα3β1(Kikkawa et al.1998).

Fig. 4. Interactions of laminin-1 with other basement membrane components and integrin-typelaminin receptors (adapted and modified from Timpl et al. 1994). The proteolytic fragmentstaking part in interactions of laminin-1 with integrins are presented as E1, P1, and E8.

Some integrins can bind more than one laminin while cells can use multiple integrins tointeract with one laminin. Within laminins, some specific domains have been identified as

31

integrin-binding regions. Theα1β1 integrin has been shown to bind to domain VI of theα-chain short arm of laminin-1 (Colognato-Pykeet al. 1995). The binding site for theα6β1 integrin in laminin-1 is located in the long-arm G domain (Sunget al. 1993). Theα2chain of laminin-2/4 has recently been reported to contain two distinct integrin bindingsites within its amino-terminal domain, recognizing both theα1β1 and α2β1 integrins.Both of these recognition sites are conserved in domain VI of the lamininα1 chainisoform (Colognatoet al.1997).

Laminin-5 has been demonstrated to be an adhesion ligand for integrinsα3β1, α6β1 and α6β4 (Carter et al. 1991, Delwel et al. 1993, Niessenet al. 1994,Rousselle &Aumailley 1994). Theα6β4 integrin is present in hemidesmosomes, whileα3β1 is recruited into focal contacts in cultured cells (Carteret al.1990, Steppet al. 1990,Jones et al. 1991, Grenzet al. 1993, DiPersioet al. 1995). These differences inlocalization betweenα3β1 and α6β4 integrins appear to reflect differences in adhesion-related functions. It has been suggested that in skin theα6β4 integrin mediates stableanchorage of keratinocytes to the substrate, while theα3β1 integrin appears to function incell spreading and migration (Carteret al. 1990, Ryanet al. 1994, Xia et al. 1996,DiPersioet al. 1997). The importance of theα6β4 integrin for attachment of epithelialcells and formation of hemidesmosomes is well demonstrated, since mice lacking theα6 or β4 integrins die at or shortly after birth with severe blistering of the skin and othersquamous epithelia reminishing the phenotype of the human disorder Epidermolysisbullosa (Georges-Labouesseet al. 1996, van der Neutet al. 1996). Studies ofα3β1deficient mice have shown that theα3β1 integrin is reguired for postattachment spreadingof keratinocytes on laminin-5 (DiPersioet al.1997).

α-dystroglycan is another ubiquitous laminin receptor in addition to integrins and, atthe moment, also the best characterized non-integrin laminin receptor. It is a 156 kDa cellsurface protein that is part of the dystrophin-receptor complex in muscle, so it provides alinkage of the basement membrane to the dystrophin-actin cytoskeleton (Campbell 1995,Timpl &Brown 1996). Dystroglycan is encoded by a single gene and cleaved intoα− andβ-dystroglycans by post-translational processing (Ibraghimov-Beskrovnayaet al. 1992).Skeletal muscleα-dystroglycan is a laminin-binding extracellular peripheral membraneglycoprotein that is anchored to the sarcolemma by a transmembrane glycoprotein,β-dystroglycan (Ibraghimov-Beskrovnayaet al. 1992, Ervasti & Campbell 1993).α-dystroglycan is also a Schwann cell receptor of laminin-2 in peripheral nerves (Matsumuraet al.1993, Yamadaet al.1994).

Some other non-integrin laminin receptors have been reported, but they are still quitepoorly characterized, and their functions in vivo are still unclear. However, carbohydrate-binding proteins that can bind specific oligosaccharide structures on the laminins and afamily of galactose-specific lectins, the galectins, that bind the lactosamine-type sugars onlaminin-1 have been discovered (Mercurio 1995). The structure and functionalsignificance of the 67 kD laminin-receptor is still an open question. The cDNA encoding acytoplasmic precursor protein of 37 kDa has been identified (Raoet al. 1989), but thestructure of this molecule has not yet been elucidated. Also the post-translationalmechanism of processing the 67 kD laminin receptor from the precursor is still unknown(Castronovoet al.1991a, Castronovoet al.1991b).

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2.2.7. Examples of biological functions

The biological functions of different laminin isoforms have been investigated in hundredsof in vitro studies. Laminins have been shown to have effect on cell attachment,proliferation, differentiation and migration (Timpl 1996). However, only recently have thefirst laminin diseases and transgenic mice been introduced that demonstrated definitiveand distinct functional roles for some laminin chains and isoforms alsoin vivo.

2.2.7.1. Laminin-5 as a component of the epithelial anchoring system

Basement membranes separate epithelial cells from the underlying stroma. In manyepithelia, including skin, specific electron-dense points of connection can be seen byelectron microscopy. These transmembrane cell-matrix junctions between the epithelialcells and the basement membrane are called hemidesmosomes and they have an importantrole in stabilizing and maintaining the epithelial structures (Joneset al. 1998). Outside thecell, laminin-5 is suggested to connect the hemidesmosomal complex to the underlyingstroma via anchoring fibrils formed from type VII collagen (Champliaudet al. 1996, Chenet al. 1997, Rousselleet al. 1997). In addition to laminin-5, the major components ofhemidesmsomes areα6β4 integrin, plectin, a 300-kD intermediate filament-associatedprotein (IFAP300), HD1, a bullous pemphigoid antigen II (BPAG2, BP180 or type XVIIcollagen) and a bullous pemphigoid antigen I (BPAG1 or BP230) (for review see (Jonesetal. 1998). As shown in the model of the hemidesmosomal complex presented in Fig. 4, theβ4 integrin subunit binds the cytoplasmic protein called plectin which, in turn, mediatesthe interaction between the hemidesmosome and the keratin cytoskeleton (Rezniczeket al.1998). IFAP300 and HD1 may also bind the same integrin chain participating in keratinanchorage, but it is not sure, however, if plectin, IFAP300 and HD1 are actually the sameor closely related proteins (Herrmann & Wiche 1987, Gacheet al. 1996). Atransmembrane component BPAG2 may also associate with theβ4 integrin (Hopkinsonetal. 1995, Borradoriet al. 1997), while the BPAG1, another bullous pemhigoid antigen,seems to also be involved in linkage of the keratin cytoskeleton to the hemidesmosome(Clarkeet al.1995, Guoet al.1995).

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Fig. 5. The structure of hemidesmosomes (Joneset al.1998).α6β4α6β4α6β4α6β4 integrin links the interactionof cytoskeleton and extracellular matrix (ECM) at the site of the hemidesmosome. In thecytoplasm, the β4β4β4β4 integrin subunit binds the cytoplasmic protein plectin, which mediates theassociation between the hemidesmosome and keratin cytoskeleton and intermediate filaments(IF). Another transmembrane protein BP180 (type XVII collagen) associates with integrinα6α6α6α6and maybe also with integrin β4β4β4β4 in the cytoplasm. The BP230 component is involved in thelinkage of the keratin cytoskeleton and intermediate filaments to the hemidesmosome via theBP180 protein. Outside the cell, integrinα6β4α6β4α6β4α6β4 associates with laminin-5, further mediating thecell attachment to the underlying ECM.

A number of skin diseases have been reported in which cell-basement membranedetachment and blister formation are characteristic symptoms, with the main cause beingperturbations in hemidesmosomal integrity. Epidermolysis bullosa is a group of skindisorders characterized by the fragility of the skin and the mucous membranes (Epstein1992, Uitto et al. 1997). In the simplex form of epidermolysis bullosa, mutated genesencode intermediate filament components of the epidermal cytoskeleton. In the dystrophicform, the type VII collagen gene is mutated, affecting the anchoring fibrils, while in thejunctional form, the split in the skin is within the epidermal-dermal basement membranezone (Epstein 1992, Uittoet al. 1997). The first demonstration of a genetic laminindisease came out when Pulkkinenet al. demonstrated mutations in theγ2 chain gene(LAMC2) in Epidermolysis bullosa junctionalis (Pulkkinenet al. 1994a). This disease wasfirst attributed to a keratinocyte protein termed nicein (Verrandoet al. 1991, Domloge-Hultschet al. 1992). Subsequently, cloning of its component chains showed this protein tocontain the epithelium-specific lamininγ2 chain (Kallunki et al. 1992, Vailly et al.1994b). Later, it has been shown that mutations in all three laminin-5 chain genes underlie

34

cause the same disease (Pulkkinenet al. 1994a, Pulkkinenet al. 1994b, Kivirikko et al.1995, Vailly et al. 1995a). As a consequense of mutations, the laminin-5 trimer assemblyor connections to other extracellular proteins are probably disturbed explaining whynormal attachment of keratinocytes to the underlying stroma cannot be formed. Based onthis, it is very obvious that laminin-5 has an important role as a structural attachmentcomponent of epithelial cells.

In addition to that seen in epidermal-dermal structures, there are also epithelial tissuesthat do not form hemidesmosomes. For example, the anchoring system of epithelial cellsand basement membrane in the gut is different from that seen in skin. Epithelial cells in thehuman intestine do not possess type VII collagen. However, laminin-5 and HD1 areexpressed, suggesting that some kind of hemidesmosome-like complexes may exist in theintestine. Laminin-5 and HD1 are located mostly at the basal pole of epithelial cellsmigrating up the villi, suggesting a role for these molecules in intestinal epithelial cellproliferation, differentiation and migration, in addition to cell anchorage (Virtanenet al.1995, Orian-Rousseauet al.1996).

2.2.7.2. Laminin-2/laminin-4 and attachment of muscle cells

The dystrophin-glycoprotein complex is composed of dystroglycan and sarcoglycancomplexes and it mediates attachment of muscle cells to the surrounding tissuecompartments. The dystroglycan complex containsα- and β-dystroglycans of whichα-dystroglycan links the sarcolemmal membrane to the extracellular matrix by binding the Gdomain of theα chain in laminin-2 (for review see Campbell 1995, Worton 1995).α-dystroglycan is linked to the sarcolemma by a complex which is composed of five integralmembrane proteins, namelyα-dystroglycan, β-dystroglycan, andα−, β− and γ-sarcoglycans (Ervastiet al. 1990, Yoshida & Ozawa 1990, Ibraghimov-Beskrovnayaet al.1992, Suzukiet al.1994).

Congenital muscular dystrophies are autosomal recessive muscle diseases andsymptoms exhibited are muscle weakness, hypotonia, delayed motor development, severeand early contractures and often joint deformities (Campbell 1995). Specific absence oftheα2 chain of laminin-2 was observed in patients affected by a classical non-Fukuyamatype of CMD (Tomeet al. 1994) and the analysis of laminin-2 deficient CMD families byhomozygosity mapping localized the CMD gene to chromosome 6q2 (Hillaireet al. 1994).Later, splice site and nonsense mutations were found that lead, presumably, to a truncatedα2 chain protein lacking domains I and II and the C-terminal G domain (Helbling-Leclercet al. 1995). A homozygous missense mutation has also been identified that affects one ofthe conserved EGF-like repeats in the short arm of theα2 chain (Nissinenet al.1996).

In addition to the dystrophin-glycoprotein complex, muscle cells and myofibers canattach to the surrounding basement membrane via integrinα7β1D-based attachmentsystem. Integrinα7β1D is a specific receptor for laminin-1 (von der Market al. 1991) aswell as laminin-2 and –4 (Yaoet al. 1996). The absence of the integrinα7 causes a novelform of muscular dystrophy demonstrating distict roles for dystroglycan-sarcoglycancomplex and integrin-mediated complex of muscle cells and basement membrane (Mayer

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et al. 1997). Analysis of integrins have shown an abnormal expression and localization ofα7β1 integrin in myofibers of laminin-2/4-deficient human patients and mice, but not indystrophin-deficient or sarcoglycan-deficient humans or mice suggesting that accurateexpression and membrane localization ofα7β1 integrins in myofibers, as well as myofibersurvival, depend on laminin-2/4 (Vachonet al.1997).

2.2.7.3.β2 chain of laminin-3 in neuromuscular junctions and kidney

Skeletal muscle fibers are surrounded by basement membrane and at neuromuscularjunctions basement membrane passes between the pre- and postsynaptic membranes. Thesynaptic and extrasynaptic regions contain different lamininβ chains. Theβ2 chain islocated at synapses, whereas theβ1 chain can be found extrasynaptically (Hunteret al.1989b). Inin vitro experiments, theβ2 chain has been shown to arrest the growth of axonspromoted by theβ1 -containing laminin trimers (Hunteret al. 1989b, Hunteret al. 1991).In mutant mice that lack theβ2 chain, neuromuscular junctions have defects in structure,function and molecular architecture (Noakeset al. 1995). Thus, the restriction of theβ2chain to the synaptic cleft and theβ1 chain to the extrasynaptic area suggests a role forthese chains in normal neuronal development. What is known of synaptic specialization, ithas been proposed that theβ2 chain could be a link in the signalling pathway in assuringthe alignment of presynaptic and postsynaptic structures (Martinet al.1995).

The glomerular basement membrane (GBM) also has variations in lamininβ1 andβ2chain localizations. Theβ1 chain is a component of the immature GBM, but is replacedwith the β2 chain as development proceeds withβ2 being the major component in theadult (Miner & Sanes 1994). In mice that lack theβ2 chain, the GBM is structurally intactand obviously theβ1 chain compensates for the missing chain. However, mutant micedevelop a massive proteinuria due to failure of the glomerular filtration barrier, whichstrongly suggest the idea that these chains are functionally different despite of theirstructural similarity (Noakeset al.1995).

2.2.7.4. Importance of theα5 andγ1 chains in embryogenesis

The lamininα5 chain associates with theγ1 chain and either theβ1 or β2 chains to formlaminins 10 and 11 (Mineret al. 1997). It is widely expressed in fetal and adult tissues,including kidney, skeletal muscle, lung and intestine (Mineret al. 1997, Pattonet al. 1997,Sorokin et al. 1997). Embryos lacking this chain die late in embryogenesis because ofseveral localized structural and developmental defects. Lamininα5 may be important inplacental endothelial cell migration, blood vessel branching, trophoblast adhesion tobasement membranes, and in formation of a proper basement membrane. At least somebasement membranes are ultrastructurally defective inα5 deficient mice despite ectopicdeposition of otherα chains, suggesting that the compensation is functionally inadequate(Miner et al.1998).

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The LAMC1-null mutation causes early embryonic lethality, as well (Smythet al.1999). Theγ1 chain is the most widely expressed laminin chain and it assembles in allother isoforms except laminin-5 (Timpl 1996). Studies of knock-out mice revealed that itis necessary for laminin assembly and that laminin is, in turn, essential for the organizationof other basement membrane componentsin vivo andin vitro (Smythet al.1999).

2.3. Interactions of tumor cells with ECM and basement membrane

The basement membranes coordinate and regulate the behaviour of cells of organizedtissues in many ways. Their composition and three-dimensional structure, as well asproteolytic remodelling are involved in the complex processes of microenvironmentalsignalling that affect cell shape, locomotion, growth, differentiation and apoptosis. Theregulatory effects exhibited by the basement membranes are, therefore, essential for thenormal function of tissues and cells associated with these matrices while disturbances inthese structures can contribute to many diseases, including cancer (Liotta & Stetler-Stevenson 1991, Werb 1997, Lukashev & Werb 1998).

2.3.1. General aspects of tumor progression

The hallmark of cancer cells is their ability to invade other tissues and locomotethroughout thebody to distant organs to form new tumors, referred to as metastases.Uncontrollable growth of metastases is the major single cause of death of cancer patients.Formation of metastasis requires the disruption of local cell-cell contacts, invasion,penetration of blood or lymphatic vessels (intravasation), escape from those vessels(extravasation), migration and growth (Liottaet al. 1991, King 1996, Ziober 1996).Although uncontrolled proliferation and growth of cells and cell colonies can result fromsome genetic changes, it does not, by itself, result in invasion and metastases. During thedevelopment of metastases, tumor cells have to pass cellular and extracellular boundariesincluding basement membranes, survive the circulatory or lymphatic system and cross intoforeign tissues (Liotta &Stetler-Stevenson 1991, Zioberet al. 1996). Thus, a complex mixof consecutive processes of cell attachment, detachment and migration are needed fortumor progression, as well as the degradation of temporary complexes formed during theseprocesses (Liotta &Stetler-Stevenson 1991, King 1996).

The interactions of tumor cells with basement membranes include cell adhesion via cellsurface receptors, matrix dissolution by degradative enzymes, and migration (Liotta&Stetler-Stevenson 1991). Integrins are the main cell surface receptors that tumor cellsuse in binding to the extracellular matrix (ECM) or the basement membrane proteins typeIV collagen, laminin and fibronectin (Hyneset al. 1992, Martinet al. 1995, Zioberet al.1996), but there are also numerous non-integrin receptors (Aznavoorianet al. 1990,Mercurio 1991, Martinet al. 1995, Mercurio 1995). The main enzymes degrading ECMand basement membrane components include matrix metalloproteinases, the adamalysin-

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related membrane proteinases, the bone morphogenetic protein 1 family ofmetalloproteinases, and tissue serine proteinases, such as thrombin, tissue plasminogenactivator (tPA), urokinase (uPA), and plasmin (Werb 1997). The third step of invasion ismigration of tumor cell across the basement membrane and stroma through the zone ofmatrix proteolysis. The induction of pseudopodial protrusion at the leading edge of themigrating cell is directional and regulated by cell surface ligand binding and it involvescoordinated mobilization of cytoskeletal elements that interact with the inner membranesurface (Liotta 1991, Liotta &Stetler-Stevenson 1991).

2.3.2. Laminins and cancer

Considering the important role of basement membranes for maintaining the architecture oforganized tissues, abnormality in the function of basement membrane components, such aslaminin, could affect tumor cell behavior. This is particularly the case for epithelialbasement membranes and the epithelium specific laminin-5, as 85 % of all cancers are ofepithelial origin (King 1996). However, the overall picture of the interactions of basementmembrane proteins, such as laminins and/or laminin-binding receptors with tumor cells isincomplete. The interaction of laminin-1 with cancer cells has been investigatedextensively (Martinet al. 1995), but not much is known concerning other isomers, exceptlaminin-5. The effects of laminin-1 on tumor cells are mediated through cell surfacereceptors (Hynes 1992, Mecham 1991, Mercurio & Shaw 1991) and, at least integrinsα1β1, α2β1, α3β1, α6β1, α6β4, and α7β1 have been shown to be differentiallyexpressed in a number of tumor cells that are believed to interact with laminins duringinvasion. However, their exact role in tumor progression is still under speculation (Martinet al 1995, Zioberet al. 1996). Among the non-integrin receptors, the 67 kDa lamininreceptor has been found to be elevated in malignant cells (Weweret al. 1986). Laminin-1has been shown to promote tumor cell adhesion and growth (Terranovaet al. 1980,Kubotaet al. 1992, Terranovaet al. 1982, Terranovaet al. 1984) and the level of laminin-1 has also been shown to correlate with malignancy in some biopsy specimens (Mafuneetal. 1990). Studies using proteolytic fragments, synthetic peptides and recombinant regionsof this molecule have revealed some active sites. A pentapeptide sequence, YIGSR of theβ1 chain of laminin-1, has been reported to bind to the 67 kDa metastasis associatedlaminin receptor (Grafet al. 1987a, Grafet al. 1987b, Mafuneet al. 1990, Massiaet al.1993). More recently, a synthetic radioiodinated peptide, YIGSR, was used in imaging ofLewis Lung carcinoma (Koliakoset al. 1997). Three more peptides in laminin-1, namely,SIKVAV in the α1 chain, PDSGR and RLVVPRL, have been connected with tumor celladhesion, growth, lung colonization, collagenase IV secretion, and angiogenesis bypromoting or reducing these activities, but they appear to be much less active that YIGSR(Skubitzet al.1990, Skubitzet al. 1991, Martinet al.1995).

3. Outlines of the present study

Laminin-5 is a highly epithelium-specific basement membrane protein that is essential foranchoring epithelial cells to the ECM. Considering the fact that about 85 % of allmalignant tumors are of epithelial origin (carcinomas), it was considered possible thatabnormal expression and turnover of laminin-5 might be altered in carcinomas. Suchalterations might involve altered epithelial adhesion or migration properties. The purposeof this study was to obtain more detailed information about the function of laminin-5,especially its γ2 chain, in adhesion and migration of epithelial cells. In addition, it wasexamined if expression of the γ2 chain becomes affected in malignant epithelial tissues,and if so whether applications of laminin-5 analysis might be useful in cancer diagnosisand therapy. The specific aims of the study were as follows:

1. To generate antibodies against the laminin γ2 chain.2. To study expression of the γ2 chain in human tissues and different cancers.3. To explore the involvement of the γ2 chain in cell attachment and migration of

epithelial and tumor cells.4. To characterize a potential migration-related element in the gene (LAMC2).5. To evaluate the potential of the γ2 chain as a marker for the detection of epithelial

tumor cells using antibodies in immunohistochemical staining and for in vivo imaging.

4. Material s and methods

The details of materials and methods used in this study are described in the originalarticles I – IV.

4.1. Preparation and characterization of polyclonal antibodies(I , II , III , IV)

Polyclonal antibodies against domain II I (anti-LNγ2-III) and the C-terminus (anti-LNγ2-I/II ) of laminin γ2 were prepared in rabbits using γ2-GST fusion proteins as antigens. Thedomain II I antigen contained 177 amino acid residues (res. #391-567) and the domain I/IIantigen 161 amino acid residues (res. #1017-1178) (Kallunki et al. 1992). Antibodiesagainst the GST-epitopes were removed from the antisera by negative immunoadsorptionwith GST-Sepharose made by coupling E.coli expressed GST protein to CNBr-activatedSepharose (Pharmacia, Uppsala, Sweden).

4.2. Expression of recombinant laminin γ2γ2γ2γ2 chain (I , II , III)

The γ2 chain of laminin-5 was expressed as a recombinant protein using the baculovirussystem. A full-length human laminin γ2 chain cDNA containing 6 bp of the 5’ UTR and822 bp of the 3’ UTR was constructed from four overlapping cDNA clones L52, HT2-7,L15 and L61 (Kallunki et al. 1992). The resulting 4,402 bp cDNA was cloned into thepVL1393 recombinant transfer plasmid prior to transfer into the AcNPV-γ2 baculovirusvector. For expression of the recombinant protein, High Five (H5) cells were infected withthe recombinant pVL1393-γ2 virus at a multiplicity of infection (MOI) of 5 – 10 pfu percell by using the standard protocol (Summers & Smith 1987). The recombinant γ2 chainwas partially purified by first resuspending the cells in 10 volumes of 50 mM Tris-HCl,pH 7.4, 100 mM NaCl, 2.5 mM EDTA, 1 % Triton X-100, 1 mM PMSF and 1 mM NEMfollowed by homogenization in a Dounce homogenizer. The solubilized proteins wereremoved by centrifugation and the pellet was extracted again with buffer containing 1 – 3M urea. The recombinant γ2 chain was extracted with a buffer containing 5 M urea, andrenatured by dialysis against 50 mM Tris-HCl, pH 7.4, 100 mM NaCl.

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4.3. Immunohistochemistry andin situ hybridization (I, II, III, IV)

Five µm thick paraffin sections were stained with polyclonal antibodies against laminin-1(a kind gift from Dr. Foidart, Liege, Belgium) or theγ2 chain of laminin-5. The paraffinsections were deparaffinized in xylene, then in some cases heated to 90oC in a microwaveoven in 10 mM citrate buffer (pH 6.0) for 10 min, and were then allowed to cool in thebuffer at room temperature for 20 min. To properly expose the lamininγ2 chain in thehighly cross-linked native basement membrane the sections were digested either with 0.03% trypsin for 10 min at 37oC or with 0.4 % pepsin in 1 mM HCl at 37oC for 20 min,blocked for nonspecific binding with 5 % newborn rabbit serum, 0,1 % BSA, and thenincubated for 1 h at 37oC with the polyclonal IgG diluted in TBS to 2 – 10µg/ml.Subsequently, a biotinylated swine-anti-rabbit antibody was applied, followed byhorseradish peroxidase-avidin-biotin complex diluted as recommended by themanufacturer (DAKO, Copenhagen, Denmark). The color was developed indiaminobentsamidine (DAB), followed by counterstaining of the slides with hematoxylin.In study IV, when visualization of newly synthesizedγ2 chain was important, the stainingwas carried out without digestion.

In situ hybridization was performed with35S-labeled RNA probes as describedpreviously (Pykeet al. 1991, Pykeet al. 1994). Plasmid constructs for theγ2 chain ofhuman laminin-5 and for human urokinase-type plasminogen activator receptor were usedas described for preparation of RNA probes (Pykeet al. 1991, Pykeet al. 1994). Theβ3chain of human laminin-5 cDNA was made using reverse transcriptase-PCR. A pool ofcDNAs from human cultured cells was made from RNA purified from cells and primedwith a poly(dT) primer with reverse transcriptase (Pykeet al. 1994). This pool was usedfor PCR amplification of aβ3 cDNA fragment with two primers designed to amplify2897-3506 of the coding sequence of the lamininβ3 chain (Gereckeet al. 1994). Senseand antisense RNA transcripts were made byin vitro transcription using T3 and T7polymerase and35S-labeled UTP (Pykeet al.1994).

4.4. Cells (II, III)

Mouse squamous cell carcinoma cells, KLN-205 (cat. No. ATCC CRL-1453), weremaintained as monolayer cultures in Eagle’s minimum essential medium (MEM)containing non-essential amino acids and Earle’s BSS, supplemented with 10 % fetal calfserum (FCS). The HaCat human keratinocyte cells (a kind gift from Dr. N. Fusenig,Heidelberg, Germany) were cultured in Dulbecco’s MEM supplemented with 10 % FCS.

4.5. Cell adhesion and migration assays (II)

Microtiter plates (96 wells; Nunc, Copenhagen, Denmark) were coated with 100µl/well oflaminin-1 (10µg/ml, Gibco BRL), or recombinant lamininγ2 chain (10µg/ml) in PBS or

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50 mM Tris-HCl, pH 7.4. Control wells were uncoated or coated with the same amounts ofBSA. In some experiments, the proteins were first denatured by dialysis overnight against5 M urea, 50 mM Tris-HCl, pH 7.4 and then renatured by dialysis against 50 mM Tris-HCl, pH 7.4. Remaining active sites on the plates were blocked with 150µl of 10 mg/mlBSA in PBS for 2 hours at room temperature. The wells were washed with PBS, and 100µl of Eagle’s MEM containing 5 mg/ml BSA was added. A total of 20 000 KLN-205 cellswere added to each well and allowed to attach for 90 min at 37oC. The extent of celladhesion was determined by measuring color yields at 600 nm, following fixation with 3% paraformaldehyde and staining with 0.1 % crystal violet (Gillieset al. 1986). Forinhibition assays with the anti-γ2 antibody, the substrate coated wells were incubated with20 µg/ml of anti-LNγ2−ΙΙΙ in PBS for 60 minutes prior to incubation with the cells.

The effect of endogenous laminin-5 on migration of KLN-205 cells was determined byusing a modified Boyden chamber assay (Hujanen & Terranova 1985), and the effect ofexogenous laminin-5, by using a modified Transwell assay (Pelletieret al. 1996). Briefly,polycarbonate filters (pore size 10µm, diameter 12 mm; Costar, Cambridge, MA) werecoated with 2.5µg of EHS type IV collagen, and used to separate the upper and lowercompartments of the 50µl chamber. A total of 1 x 105 cells in Eagle’s MEM containing0.1 % BSA were placed in the upper compartment, and the lower compartment was filledwith medium, with or without chemoattractants (50µg/ml laminin-1 or fibronectin). Tostudy the effect of the lamininγ2 chain antibodies on cell migration, anti-LNγ2−ΙΙΙ or anti-LNγ2−Ι/ΙΙ was added to the upper compartment together with the cells at a concentrationof 20 µg/ml. Normal rabbit IgG was used as a negative control. After an 8 hour incubationat 37oC in a humidified atmosphere, the filters were removed, fixed and stained (Diff-Quick, Baxter Diagnostics, Tübingen, Germany). The cells that had not migrated wereremoved from the upper surface of the filter with cotton swabs. Migration of cells wasquantified by counting the cells on the lower surface of each filter in 10 randomly selectedhigh power fields (400x). All assays were performed in triplicate.

The Transwell plate assay was used to determine the effect of exogenous laminin-5 oncell migration. The membranes of the Transwell plates had pore sizes of 12µm. The lowerside of the membrane was coated with 2.5µg of EHS type IV collagen for 3 hours at roomtemperature. Both side were blocked with 1 % bovine serum albumin for 1 hour. 1 x 105

cells were added per well to the upper compartment in Eagle’s MEM containing 10 %FCS and the lower compartment was filled with 2.5µg/ml laminin-5 as a chemoattractant.Antibodies against either theγ2 chain or normal rabbit IgG were added to the uppercompartment, together with the cells, at a concentration of 20µg/ml. Following a 16 hourincubation at 37oC, the cells were fixed and stained with Diff-Quick. Cells from the topsurface of the membrane were remowed with cotton swabs and cells that had migrated tothe lower side of the membrane were counted (12 fields +/- S.D.).

4.6. Generation and analyses of transgenic mice (II)

Two different segments of the LAMC2 5’flanking regions were subcloned into apKK2480 vector containing theβ-galactosidase gene and the SV40 polyadenylation signal(kind gift from Mikkel Rohde, Aarhus, Denmark) for expression in transgenic mice. The

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longer construct, pHH-1, contained 5946 bp (-5,900 to +46) and the shorter construct,pHH-2, contained 668 bp (-613 to +55). The 5’end of the pHH-1 insert was made byligating a 3,900 bp HindIII-PstI fragment and a 1,150 bp PstI fragment of the genomicclone, P14 (Airenneet al. 1996) The 3’end of the construct was a PstI-SalI fragment (-699 to +46) made by PCR and ligated to the 5’end 5,050 bp fragment. The full-lengthfragment was blunt ended with Klenow and subcloned into pKK2480 vector. The shorterconstruct was made by PCR from the genomic clone P14, digested with SalI and XbaI andsubcloned into pKK2480. The plasmids, pHH-1 and pHH-2, were digested with EagI andXhoI to release the inserts from the vectors. Transgenic mice were produced by pronuclearmicroinjection of (C57B1/6+DBA/2)F1 fertilized oocytes as described by Hogan et al.(1986). Expression of thelacZ gene was detected by staining with X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) as a substrate (Behringer et al. 1993).

4.7. Tumor imaging with unlabeled and radioactively labeledantibodies (III)

To estimate the specific targeting of theγ2 chain antibody into tumors, 1 x 105 KLN-205cells were injected i.m. or i.v. into four DBA/2 adult male mice and 3 or 5 weeks later,anti-LNγ2-III antibodies or normal rabbit IgG (20 – 40µg/mouse) were injected into thetail vein. Mice were killed 6, 24 or 48 hours after injection and tumor, muscle, lung, liver,kidney and spleen were prepared and examined histologically after staining with FITC-conjugated anti-rabbit IgG secondary antibody (Cappel, Durham, NC).

99mTc-labeling was carried out according to the instructions of MAP MedicalTechnologies (Tikkakoski, Finland). 1 mg of either anti-LNγ2-III or normal rabbit IgGwas dissolved in 200µl of PBS and mixed with 200µl of ascorbic acid solution, pH 5.5– 6.0. After 30 min incubation at room temperature, 150µl of freshly prepared Na-dithionite solution was added (30 mg/ml Na-dithionite in 0.15 M Na-acetate biffer, pH 6).8 mCi of 99Tc (MAP Medical Technologies OY, Tikkakoski, Finland) was added and theincubation was continued for 30 min at room temperature. The labeling efficiency wasdetermined with instant thin layer chromatography (ITLC) using 0.15 M NaCl as thesolvent.

Biodistribution studies and radioimmunoimaging were performed in KLN-205male mice having primary tumors in their left legs. 20µg of either 99mTc-labelled anti-LNγ2−ΙΙΙ IgG or normal rabbit IgG were administered into the tail vein, and groups ofthree mice were perfused 24 or 48 h after injection. Mice were imaged with an ElscintApex 409ECT gamma scintillation camera (Elscint Ltd, Israel) using a low-energy, high-resolution collimator or a low-energy all-purpose collimator. Afterwards, the tissues(tumor, muscle, lung, liver, kidney and spleen) were dissected out, weighed and theirradioactivity counted. The experiment was repeated twice.

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5. Results

5.1. Antisera against recombinant γ2γ2γ2γ2

Polyclonal antibodies against domain II I and the C-terminus of the γ2 chain were preparedusing γ2-GST fusion proteins as antigens (original articles I and II) . The recombinant γ2-GST fusion proteins were purified on a glutathione-Sepharose 4B column and the purifiedproteins were used to immunize rabbits. Antibodies against the GST-epitopes wereremoved from the antisera by negative immunoadsorption with GST-Sepharose made bycoupling E. Coli expressed GST protein to CNBr-activated Sepharose. The antisera weretested for immunoreactivity against purified human laminin γ chains, as well as, purifiedlaminin-5 and commercial mouse laminin-1 by Western blotting. Purified full-lengthrecombinant human γ1 and γ2 chains, laminin-5 and laminin-1 were electrophoresed bySDS-PAGE, transferred to nylon membranes and immunoreacted with the antisera. Bothantisera reacted with the full-length γ2 chain, while no reactivity was observed with theclosely related γ1 chain or laminin-1 chains. In addition, both antibodies reacted with thelaminin-5 chains, corresponding to the processed and unprocessed (full-length) γ2 chains.The specificity of the antibodies was also tested by ELISA. The removal of anti-GST IgGwas verified by Western analysis.

5.2. Expression of the γ2γ2γ2γ2 chain in human cancers

The presence of the laminin γ2 chain in normal human tissues was verified byimmunohistochemical analysis of a variety of adult human tissues. The γ2 chain proteinwas exclusively seen in basement membranes under the basal epihelial cells in theendocervix, endometrium, intestine, stomach, lung, skin, tongue and esophagus. Thestaining in the lung was strongest in the bronchial epithelia, although a weak signal wasalso seen in some alveolar epithelia. In the kidney, expression of the γ2 chain waslocalized to the collecting tubules. These results were confirmed in a recent study(Mizushima et al. 1998).

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Under pathological conditions, expression and immunoreactivity of laminin-5 chainshave been found to be upregulated in migrating keratinocytes of healing skin wounds(Larjavaet al. 1993, Pykeet al. 1994, Ryanet al. 1994).γ2 chain mRNA has also beenshown to be expressed in a number of cancer cells of epithelial origin, and in colonadenocarcinomas, the expression appears to be a characteristic of cancer cells showing abudding cell phenotype (Pykeet al. 1994). In the present study a variety of human tumorsamples were studied to further explore whether expression of laminin-5 is deregulated incarcinomas. A total of 72 human cancer biopsies were stained with the polyclonalantibody produced against domain III of theγ2 chain to examine the localization of thischain (original article I). These biopsies included 23 cases of colon adenocarcinomas, 16ductal breast carcinomas, 9 malignant melanomas, 14 squamous cell carcinomas of theskin and cervix, and 10 sarcomas.In situ hybridization of adjacent sections was used as acontrol for the immunostaining results. All colon adenocarcinoma specimens were positivefor theγ2 chain, and the staining was almost exclusively confined to the cancer cells. Thestaining was most prominent at the invasive front of the tumors. Eleven out of the 16 casesof mammary cancer were positive for theγ2 mRNA and protein staining, and 10 of thecases contained positive cells immediately surrounding nests of cancer cells. Six out ofnine malignant melanomas showed moderate to strong staining in apparently nonmalignantkeratinocytes, which, in all cases, were judged to be engaged in wound healing responses.All cases of squamous cell carcinomas originating from the cervix, vulva, and skin wereγ2chain immunoreactive, and the staining was seen only in the cancerous cells. In most of thecases, cancer cells located immediately adjacent to the surrounding stromal tissue showedthe most intense labeling. None of the sarcoma specimens studied showed positivestaining. In situ hybridization of both theγ2 chain and urokinase receptor (uPAR) onadjacent sections established that the cancer cell population containingγ2 chain mRNAand protein also contained uPAR mRNA.

To evaluate in more detail if the laminin-5γ2 chain can be used as a marker ofinvasiveness in carcinomas, an extensive systematic study was carried out on uterinecervical tumors. Ninety paraffin-embedded cervical lesions containing mild dysplasias toinvasive cancers were analyzed with the polyclonal anti-LNγ2-III antibody (original articleIV). The tissues were not pretreated with proteases or strong denaturing agents, in order tominimize staining of the native basement membrane. With one exception, all lesions ofmild and moderate dysplasia did not show any positivity for the anti-LNγ2-III antibody. Atotal of 21 out of 32 samples diagnosed with cancerin situ (CIS), were negative, whereasall lesions with invasive and microinvasive cancers were positive for the antibody with thestaining confined to the cancer cells at the invasive front of the tumor. Interestingly, one ofthe lesions diagnosed as moderate dysplasia was strongly positive for theγ2 chainantibody, suggesting that the synthesis ofγ2 is not always indicative of malignanttransformation of epithelial cells. Histopathologically, the squamous epithelia was judgedas of metaplastic type with considerable degenerative changes and with an atypia difficultto grade. Importantly, however, one month later a cervical cone from the same patient wasdiagnosed being microinvasive,or alternatively, a moderately differentiated invasivecancer. This specific case showed a strong cytoplasmicγ2 chain immunostaining and alsoa tendency to stain extracellularly. Thus, this study strongly supports the use of lamininγ2as a marker for microinvasive cervical cancers.

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5.3. The effect of theγ2γ2γ2γ2 chain laminin-5 on cell attachment andmigration of epithelial cells

Having observed increased expression of theγ2 chain in carcinomas, we wished toexamine the role of laminin-5 and itsγ2 chain in epithelial cell adhesion and migration.Laminin-5 isolated from human keratinocyte cell culture medium and commercial laminin-1 were used as substrata in attachment assays (original article II). Adherance of twoepithelium-derived cell lines, HaCat and KLN-205, to laminin-5 was about 2.5 timeshigher than to plastic, and was slightly higher than that to laminin-1. The attachment tolaminin-5 was not significantly decreased in the precence of two different polyclonalantibodies raised against the short and long arms of theγ2 chain or by non-immune sera.When the cells were plated on the recombinantγ2 chain alone, the number of cellsattached was not significantly higher than to plastic, and this attachment was notinfluenced by the polyclonal antibodies mentioned above. Thus, these results confirm theearlier results suggesting that trimeric laminin-5 promotes adhesion of epithelial cells, butfurther strongly suggest that this adhesion is not mediated by theγ2 chain.

The potential role of theγ2 chain of laminin-5 in the cell migration process wasexamined for the KLN-205 cellsin vitro using Boyden chamber and Transwell assays.Migration of cells in the presence of preimmune IgG was arbitrarily set as 100 %. Whenlaminin-1 and fibronectin were used as chemoattractants the migration of cells through thefilter was decreased to about 35 to 45 % of that observed with the preimmune IgG whenpolyclonal antibody against the domain III of theγ2 chain was added. In contrast, thepolyclonal IgG against the C-terminal domain I/II did not affect migration. When nativelaminin-5 was used as a chemoattractant in the lower compartment of the chamber, theresults were essentially the same as above. The addition of IgG raised against domain IIIinhibited migration to about 50 % as compared to the preimmune IgG. The polyclonal IgGagainst domain I/II did not have any effect. Thesein vitro results demonstrated thatlaminin-5 can have a role in the locomotion of epithelium-derived cells, and that thisfunction can be inhibited by antibodies directed against domain III of theγ2 chain.

5.4. Identification of a migration-related element in the LAMC2 gene

In order to search for potential epithelium-specific enhancer elements in the LAMC2 gene,transgenic mice containing the LAMC2 promoter and a reporter gene were produced.DNA constructs containing varying lengths of the 5’ flanking region of the LAMC2 genewere cloned with a downstream LacZ reporter gene (original article II). This reporter genecodes for bacterialβ-galactosidase. The first construct, HH-1, contained about 5900 bp,including 55 bp from the 5’ untranslated region and the 5’ flanking region. The secondconstruct, HH-2, contained 55 bp of the 5’ untranslated region and 613 bp of the 5’flanking region. In mouse embryos, very little expression was observed with either

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construct, demonstrating that the reporter gene constructs did not contain the cis-actingelements needed for epithelial expression. Previous results had shown strong expression ofthe LAMC2 gene in epithelial layers of skin, respiratory tract and kidney in humanembryos, as determined byin situ hybridization (Kallunkiet al. 1992). When tissues ofadult transgenic mice were studied, both constructs also yielded as highly restricted and astemporary an expression as in mouse embryos, although in adult tissues the distribution ofexpression was slightly more extensive. The limited expression was confined to epithelialcells, but, for example, in skin, discontinuous expression was observed in somekeratinocytes of the epidermis and the epithelial cells of some hair follicles. In thestomach, intense expression was seen in some villi of the gastric mucosa, but it was absentin most areas.

To examine if thelacZ gene was expressed in migrating keratinocytes during woundhealing, small incision wounds were made in the dorsal skin and tails of mice madetransgenic with these two constructs. No color reaction could be seen in keratinocytesresting on a normal epithelial basement membrane. However, intense staining was seen inseveral layers of keratinocytes migrating over the healing wound, indicating that theenhancer element(s) directs expression in proliferating and migrating keratinocytes.Identical patterns were obtained with both constructs. Thus, the element necessary fordriving the expression is probably located within the 613 bp region immediately upstreamof the transcription site of the LAMC2 gene.

5.5. Targeting of polyclonal antibody to tumors in KLN-205 mice

Having observed high association of lamininγ2 chain expression with carcinomas, wewished to explore whether anti-γ2 antibodies target to carcinomasin vivo, and thus mightserve as a potential markers forin vivo imaging. Such a study was then carried out in aKLN-205 mouse model (original article III). Following intramuscular or subcutaneousinoculations of KLN-205 cells, largeγ2 chain positive primary tumors developed in 4weeks with numerous lung metastases after 4 to 6 weeks. Cells injected into the tail veinproduce multiple lung tumors (experimental metastases) in 3 to 4 weeks. When injectedinto tail vein of KLN-205 mice the unlabeled anti-LNγ2-III targeted specifically toprimary tumors and lung metastases as examined by histochemical staining of normal andtumor tissues with FITC-conjugated anti-rabbit IgG secondary antibody. No staining wasobserved in epithelial basement membranes that do contain some amount of laminin-5.Normal rabbit IgG revealed negative results. The results clearly demonstrated thatintravenously administered anti-γ2 antibodies bind newly synthesized laminin-5in vivo butpoorly the protein in native basement membranes.

The above results indicated that radioactively labeled anti-γ2 antibodies might be usedfor in vivo imaging of carcinomas. For example, antibodies labeled withγ-rays emittingisotopes could be detected by a gamma camera. The biodistribution of99mTc-labeled anti-LNγ2-III antibodies and normal rabbit IgG were first studied in mice carrying KLN-205tumors. Samples containing 20µg of specific antibody or normal rabbit IgG were injectedintravenously and the mice were perfused 24 or 48 hours after injection. The mice were

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first imaged using a gamma camera, after which the tissues (tumor, muscle, lung, liver,kidney and spleen) were excised, separated, and their radioactivity was counted. Theaccumulation of the99mTc-labeled anti-LNγ2-III antibody in the tumor was about 4.5%ID/g tissue, or about twice as much as that of labeled normal rabbit IgG. Fine contrastbetween tumors and normal could also be obtained with the gamma camera using a99mTc-labeled anti-LNγ2-III antibody and normal rabbit IgG. However, perfusion of the animalwas necessary for imaging with the gamma camera, because the background could not beremoved by diminishing the amount of injected polyclonal antibody. The staining ofhistological sections of tumors with FITC-conjugated anti-rabbit IgG secondary antibodyconfirmed further the specificity of biodistribution of the radioactively labeled antibody inthese animals.

6. Discussion

The present work provided new information on the role of laminin-5 in epithelial celladhesion and, particularly, on the role of its γ2 chain in cell migration. The role inmigration was emphasized by identifying the presence of gene elements induced in cellmigration using transgenic mice as a model system. The present work further demonstratedincreased levels of γ2 chain protein in cells of epithelial cancers and, notably, that suchantibodies can be used for both histochemical and in vivo imaging of malignant epithelialtumors.

6.1. Expression of the γ2γ2γ2γ2 chain of laminin-5 in human epithelialtissues and malignancies

The results of this study demonstrated a strong increase amount of the laminin γ2 chain ininvading tumor cells of most epithelial cancers, supporting a previous finding of theupregulation of laminin-5 expression in such tumors (Pyke et al. 1994). It was also shownin this study that in some human carcinomas, upregulated expression of laminin-5 or itssubunit chains is characteristic of budding cancer cells located at the tip of invadingmalignant epithelia. The results of this work also demonstrated that the basementmembranes of normal human skin, lung, stomach, small intestine, kidney, and germinalepithelium were positive for γ2 chain staining and that the staining was always continuousunder the basal epithelial cells, except in the stomach and small intestine where the γ2chain localized only to the microvill i tips. Several reports have described the expressionof laminin-5 also in malignancies (Pyke et al. 1994, Hao et al. 1996, Tani et al. 1996, Taniet al. 1997, Fukushima et al. 1998, Martin 1998, Orian-Rousseau et al. 1998, Sordat et al.1998). There is, however, some differencies in the expression patterns of laminin-5 inepithelial cancers. Expression of laminin-5 chains is up-regulated quite generally inepithelial cancers, such as squamous cell carcinomas and gastric carcinoma, but not inmesenchymally derived cancers (Larjava et al.1993, Pyke et al. 1994, Tani et al. 1996,Orian-Rousseau et al. 1998, Sordat et al. 1998). Down-regulation has been reported inepithelial prostate and breast carcinomas (Hao et al. 1996, Martin et al. 1998). In thisstudy the cancer cells of ductal mammary carcinomas and malignant melanomas were

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shown to be negative forγ2 and the weak or moderate staining was judged to be engagedin wound healing responses. Similar kind of down-regulation of laminin-5 in breastcarcinoma cells has been reported recently by others (Martinet al. 1998). Urokinaseplasminogen activator (uPA) is a protease involved in cancer invasion and metastasis. Theproteolytic enzyme plasmin is generated by the action of uPA that binds to cell membranereceptor urokinase plasminogen activator (uPAR). The activation is proposed to lead toextracellular matrix degradation and to facilitate cancer cell invasion (King 1996). In thisstudy uPAR was shown to be coexpressed with theγ2 chain of laminin-5 in the buddingcancer cells at the invasion front in colon cancer. uPAR has previously been shown to beexpressed in stromal cells and in cancer cells at invasive foci being a marker ofinvasiveness (Grøndahl-Hansenet al. 1991, Pykeet al. 1991). Theγ2 chain was notexpressed in cancer cells of ductal mammary carcinomas and malignant melanomas.Interestingly, recently it has been shown that in breast carcinomas uPA is locatedpredominantly also in the stromal cells, although some expression can be detected inmalignant cells, as well. uPAR localized principally to stromal cells, especially whenthese cells surrounded invasive breast cancer (Kennedyet al. 1998). Malignant epithelialcells do not seem to express uPA, either (Grøndahl-Hansenet al. 1991). Alltogether, theseresults suggest that probably stromal cells collaborate with malignant cells to mediatemetastases. However, the reason for the differencies in the up- or down-regulation oflaminin-5 in some epithelial cancers, as well as the possible biological function of theexpression of laminin-5 in stromal cell in breast carcinoma remains to be solved.Nevertheless, because almost 85 % of all cancers are of epithelial origin (King 1996) theconnection between the early expression of laminin-5 in most carcinomas and adhesionand migration of carcinoma cells raises interesting questions about the role of this proteinin these processes and the potential of this protein as a marker of epithelial malignancy.

This study also provided the first example of a specific type of cancer in whichantibodies against laminin-5 or its subunit chains can be used for histopathologicaldiagnosis. Localization of theγ2 chain was examined in 90 specimens of bothprecancerous stages and manifest cancer from the uterine cervix. The high incidence ofcervical cancer worldwide, about half a million women each year, constitutes a majorproblem in clinical diagnostics (Pisaniet al. 1993). The precursor stages of invasivecervical carcinoma are defined as different types of atypia; mild cervical intraepithelialneoplasia, moderate and severe or cancerin situ (Richard et al. 1973). Many clinicalquestions and issues concern whether the cancer isin situ, microinvasive or frank invasiveand, depending on the diagnosis, the patient will have different treatments. Markers ofcellular proliferation, including such as Ki-67 and PCNA, and more recently otherbiomarkers such as nm23-H1, MN protein and matrix metalloproteinase-9 have been usedin diagnostics of cervical disease (Bultenet al. 1996, Maroneet al. 1996, Resnicket al.1996, Davidsonet al. 1998). However, so far no single marker has been found to haveoutstanding prognostic or diagnostic significance. In this study, all microinvasive andfrank invasive cancers showedγ2 chain immunopositivity located almost exclusively to thecytoplasm of the cancer cells at the invasion front of the cancerous area. In 11 out of 32carcinomasin situ, theγ2 chain was localized to the cells close to the basement membrane.The investigation of the material with the Ki-67 and cyclin A proliferation markers,showed an increasing proliferative activity with increasing advencement in lesion withcancer in situ and invasive cancer. The degree of proliferative activity was independent of

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laminin-5 positivity in both lesion with cancer in situ and invasive cancer. It has beensuggested that approximately 30 % of all CIS, when left untreated, develop later intoinvasive cancer (Petersen 1956), although later it has been reported that 12.2 % of cases ofnew cancer in situ progress to invasive cancer (Gustafssonet al. 1989). Interestingly, inthis study it was shown that the proportion of lamininγ2 positive CIS samples was roughly30 %. Thus, it could be speculated that these cases will progress to invasive cancer or are,possibly, already invasive, and, thus, need therapy. All mild and moderate dysplasialesions were negative for theγ2 chain in immunohistology, except one which was, onemonth later, associated with a microinvasive cancer. By means of hematoxylin staining,the invasive cells could easily be missed because of the numerous inflammatory cells, butthey were clearly indentified by positive staining for theγ2 chain. Thus, the results showedthat antibodies against theγ2 chain of laminin-5 are able to distinguish lesions with orwithout invasive capacity and could become a useful help in the histopathologicaldiagnostics of cervical cancer. Naturally, one of the future goals is to do a statisticallyrelevant study with hundreds of samples to validate whether this marker has real clinicalutility.

6.2. Theγ2γ2γ2γ2 chain of laminin-5 in epithelial cell adhesion andmigration

The results of this study showed that the short arm of the laminin-5γ2 chain does notpromote epithelial cell adhesion, but that it does have an effect on epithelial cell migration.Adhesion of human keratinocytes and KLN-205 mouse squamous cell carcinoma cells tolaminin-5 was similar to that of laminin-1, but the full-length recombinantγ2 chainproduced in insect cells, did not show any significant effect on cell adhesion. In addition,the polyclonal antibody raised against domain III of theγ2 chain did not inhibit theadhesion, which further supported the idea that the short arm of theγ2 chain does notinteract with cellular receptors.

All genes coding for the chains of laminin-5 have been shown to be mutated in aninherited skin disease called Epidermolysis bullosa junctionalis (Aberdamet al. 1994b,Pulkkinenet al. 1994a, Pulkkinenet al. 1994b, Kivirikko et al. 1995, Vailly et al. 1995b,Vailly et al. 1995a, Vidalet al. 1995) demonstrating the essential role of this protein inboth epidermal, as well as, general epithelial cell attachment. The G-domain in theα3chain has been proposed to be the major cell attachment site (Rousselle &Aumailley 1994,Rousselleet al. 1995), and the integrins,α6β4 and α3β1, mediating the adhesionproperties, both bind to the long-arm of this molecyle (Carteret al. 1991, Niessenet al.1994, Rousselle &Aumailley 1994, Pulkkinenet al. 1995b). However, there has also beenindications that the short arm domains are involved in the anchorage of epithelial cells, aswell. An in-frame mutation removing 73 amino acid residues from domains III and IV ofthe short-arm of theγ2 chain results in lethal Herlitz’s junctional epidermolysis bullosa(Pulkkinenet al. 1994a). It remains to be shown if the important binding site of the short-arm, lost in Epidermolysis bullosa junctionalis, interacts with some extracellular matrixproteins. Laminin-5 has been shown to bind type VII collagen, as well as fibulin-2

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(Rousselleet al. 1997, Utaniet al. 1997). The NC-1 domain of type VII collagen binds tolaminin-5, but not to laminin-6, and because these isoforms share theα3 chain, it issuggested that the binding activity is mediated by theβ3 or γ2 chain (Rousselleet al.1997). Recently, the NC-1 domain of type VII collagen was reported to bindpredominantly to theβ3 chain, but also to theγ2 chain, when examined by a proteinoverlay assay (Chen et al. 1999). Fibulin-2 has been demonstrated to bind to laminin-1 andlaminin-5 through theα1 andγ2 chain short arms. The 9 amino acid sequence in domainIV of the γ2 chain was defined as a critical site for fibulin-2 binding (Utaniet al. 1997).Because the processing of theγ2 chain removes most of the short-arm of this chain,including domain IV, it raises questions if there are laminin-5 or other laminin isoformwith differently processedγ2 chains that could have different functions. So far, only theα3 and γ2 chains of laminin-5 have been shown to be proteolytically processed(Marinkovichet al.1992b, Goldfingeret al.1998).

The present results suggest that theγ2 chain has a role in epithelial cell migration.Polyclonal antibodies against domain III of theγ2 chain inhibited the migration of KLN-205 squamous cell carcinoma cells by about 55 to 65 %. The antibody used was raisedagainst the residues mutated in Epidermolysis bullosa junctionalis. The results withtransgenic animals further supported the suggested role of laminin-5 and theγ2 chain incell locomotion. The reporter gene was strongly expressed in healing skin wounds and themigration-related element was located in the 613 bp upstream region flanking the LAMC2gene. However, the results also demonstrated that all the enhancer elements necessary fornormal tissue specific expression in epithelial cells are not present in a sequence reachingas far as 5900 bp upstream of the transcription site.

Already the first reports characterizing laminin-5 demonstrated that it appeared to bemade primarily by dividing and migrating cells and not by stationary keratinocytes(Marinkovich et al. 1992a, Ryanet al. 1994). In normal skin, laminin-5 is expressed as apart of the basement membrane that connects the epidermal cells to the underlying stroma(Carteret al. 1991). The wounding of skin results in a transition of keratinocytes from astationary to a migratory state and these proliferating and migrating cells start to stronglyexpress the subunit chains of laminin-5 (Carteret al. 1991, Larjavaet al. 1993, Pykeet al.1994, Ryanet al. 1994). Also in the mature cut the differentiating epithelial cellsmigrating from crypt to villus tip express actively laminin-5 (Virtanenet al. 1995, Leivoetal. 1996, Orian-Rousseauet al. 1996, Orian-Rousseauet al. 1998). The involvement oflaminin-5 or some of its chains in the process of cell migration has been demonstrated inseveralin vitro and immunohistological studies, but they have revealed, in some cases,controversial results (Kikkawaet al. 1994, Verrandoet al. 1994, Zhang & Kramer 1996,Tani et al.1997, O'Tooleet al.1997a, Giannelliet al. 1997). Some studies have suggestedthat exogenous laminin-5 inhibits cell locomotion (O'Tooleet al. 1997a, O'Tooleet al.1997b), while others propose that endogenous or newly secreted laminin-5 promotesmotility of keratinocytes or carcinoma cells (Taniet al. 1996, Zhang &Kramer 1996, Taniet al. 1997). It has also been suggested that the optimal migration occurs at intermediateadhesion strengths (Verrandoet al. 1994) which could partially explain the opposingresults. Another explanation to the controversial results could be that cells used indifferent in vitro studies express different laminin-5 receptors (i.e. eitherα6β4, α3β1 orboth), which might have different functional roles in cell attachment, spreading and

53

migration (Carteret al. 1990, Ryanet al. 1994, Xiaet al. 1996, DiPersioet al. 1997). Cellmigration has been reported to be induced by the cleavage of laminin-5 by matrixmetalloproteinase-2 which cleaves theγ2 subunit of rat laminin-5 at residue 587, removingthe short arm (Giannelliet al. 1997). Nevertheless, it is not known if the cleavage alsooccurs with the human protein because the sequence of the cleavage area is differentbetween the rat and human. This study, however, support the hypotheses that the newlydepositedγ2 chain of laminin-5 is important for the interaction of this laminin isoformwith the extracellular environment and that laminin-5 could be one of the first ECManchorage proteins of epitelial cells expressed during cellular movement. However, itremains to be confirmed if the migration promoting activity is based on the interactionsbetween theγ2 chain and some extracellular ligands or the possible different functions ofthe processed and unprocessed (full-length)γ2 chain.

6.3. Potential of the γ2γ2γ2γ2 chain of laminin-5 as anin vivo marker ofepithelial tumors

To estimate the potential of this isomer forin vivo imaging, the detection of epithelialtumors was studied using polyclonal antibodies raised against theγ2 chain of laminin-5.Both the unlabeled and radioactively labeled antibodies produced against the short armdomain III of theγ2 chain targeted specifically into primary tumors and lung tumors inKLN-205 mouse model when injected into the tail vein. Accumulation of the radioactivelylabeled antibody in the primary tumor was about 4.5 % (ID%/g tissue), being almost twiceas high as that of radioactively labeled normal rabbit IgG. A fine contrast could also bedetected by gamma camera in tumor tissue of the KLN-205 mice. Thus, the results of thisstudy demonstrated the potential of theγ2 chain molecule inin vivo imaging.

More than a decade ago antibodies were thought to provide a spesific and accurate toolfor tumor imaging and to bring help for different therapies by carrying drugs to targetsites. A lot of setbacks have come out since then mainly because of the problems withbackground, spesificity, unstability, toxicity and poor penetration into tissues (Bischor-Delaloyeet al. 1996). In recent years the delivery of antibodies to tumors have, however,become more promising because antibody fragments Fab, Fv, scFv or bispesific diabodieshave been used (Holliger and Hoogenboom 1998). New detection techniques using e.g.magnetic resonance imaging and new contrast agents for that (Sipkinset al. 1998) mayalso improve the results of tumor imaging with antibodies.

A mix of the consecutive processes of cell attachment, detachment, and migration, aswell as, the degradation of the temporary complexes formed for these processes areneeded for the locomotion of cancer cells to distant locations (Liotta 1991, Liotta &Stetler-Stevenson 1991). The degree of differentiation is a significant factor in predictingthe behavior of tumor cells. Because laminin-5 or its subunit chains seem to be a marker ofdifferentiation stage or malignant growth of epithelial cells, it could have prognostic ordiagnostic value. Early and accurate detection of malignant growth is still important forgood results in cancer therapy. Although the effects of laminin-1 on cancer cells has beeninvestigated quite efficiently (Martinet al. 1995), not much is known so far concerning

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other laminin isomers, except laminin-5. In addition, the overall picture of the linksbetween the expression of laminin-1 and/or some of its receptors and malignant growth isstill very much incomplete. Laminin-5 appears to be a very promising protein for tumorimaging because of its restricted and clear tissue specificity and aberrant expression ininvasive carcinomas, as in the cervical cancers presented in this study. Monoclonalantibodies or antibody fragments need to be produced for future studies and foroptimization of the critical parameters ofin vivo visualization. Probably another animalmodel is also needed to investigate the potentialin vivo detection of metastasis. In amouse model the detection of at least lung metastasis may be impossible, if the backgoundin kidney, liver and spleen cannot be removed. In addition, the expression patterns oflaminin-5 in different human carcinomas need to be investigated more accurately toidentify the most potential cancers that could benefit from this laminin isomer as a markerprotein in detection.

6.4. Conclusion

The present study demonstrated the role of laminin-5 for cell locomotionin vitro andconfirmed its role as an attachment component of epithelial cells. The short arm of theγ2chain is thought to serve as a bridging molecule to the extracellular environment, becauseit does not have any cell binding activity by itself. An important challenge for futurestudies is to determine molecules that interact with the short-arms of the trimer and theγ2 chain. As a migration-associated molecule, laminin-5 may also be an interesting targetin pathological processes where cell locomotion plays a role. Theγ2 chain of laminin-5 is,so far, known to assemble only in laminin-5 isoforms and is, therefore, a good marker forthis epithelium cell specific protein. In this study, theγ2 chain was shown to be expressedin a highly restricted manner in human epithelial tissues and in carcinomas and shown tobe a predictive marker for microinvasive epithelial cancer. The gene and proteinexpression in some human carcinomas, especially in colon adenocarcinomas, appeared tobe characteristic of cancer cells showing a budding cell phenotype, suggesting that thismolecule could be useful as a marker for these type of invasive cells. Importantly, inuterine cervical tumors, antibodies against theγ2 chain of laminin-5 were able todistinguish between lesions with or without invasive capacity and, thus, laminin-5 mayhave the potential to become useful help in histopathological diagnostics of cervixsquamous carcinomas. Unlabeled and radioactively labeled polyclonal antibodies raisedagainst domain III of theγ2 chain targeted specifically to primary tumors and metastasesof epithelial origin, demonstrating the potential of this molecule inin vivo imaging. Inconclusion, since the differentiation stage is an important criterium in diagnosis ofmalignancy and invasiveness of carcinoma cells, laminin-5, as a marker of differentiationof epithelial cells, might be useful as a marker, not only in immunohistology, but also as atarget forin vivo imaging.

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