Distribution of the VLA Family of Integrins in Normal Salivary Gland and in Pleomorphic Adenoma

Path. Res. Pract. 190, 600-608 (1994)

Distribution of the VLA Family of Integrins in Normal Salivary Gland and in Pleomorphic Adenoma S. Sunardhi-Widyaputra and B. Van Damme Laboratory of Histo- & Cytochemistry, Department of Pathology, Sint Raphael University Hospital, Catholic University of Leuven, Leuven, Belgium

Summary The extracellular matrix (ECM) and the alterations in tumour stroma affect the biologic behaviour of tumours. These interactions are, at least in part, mediated by the integrin superfamily. In this study we examined the immunohistochemical expression of the "very late activation" (VLA)-integrins in pleomorphic adenomas and in the corresponding normal tissues. Normal salivary gland expresses integrins in the myoepithelial cells (MEC) and in the basal and dark cells of ducts. This pattern is retained in pleomorphic adenoma. Regardless of the staining intensity, these tumour cells have a VLA expression similar to normal basal and dark cells, with only a few cells bearing MEC characteristics. We suggest that the tumour cells of pleomorphic adenoma have a common origin but show different stages of differentiation. Only cells bearing basement membrane (BM)-associated material form typical tubulo-ductal structures.

Introduction The extracellular matrix (ECM) affects the biologic behaviour of tumours in several ways and therefore plays an important role in the attachment, growth, and spreading of epithelial cells in embryogenesis, tumour development and metastasis 13 ,17,28,29. Interactions of ECM with cells are, at least in part, mediated by a group of cell surface receptor molecules belonging to the integrin superfamil y2,22,24,35,44. The term "integrin" was originally adopted to indicate that these receptors "integrate" signals between extra- and intracellular environments across the cell membrane. Integrins consist of one li- and one ~-chain that form characteristic heterodimer combinations. Current reports have identified 14 different li-subunits and eight different ~-subunits23. The li chains can be associated with ~1 chain to form "very late activation" (VLA)-integrin antigens 19 ,41. Different integrin subunits have different roles in cell adhesion. These differences can be distinguished functionally because they bind to different ligands. Collagens are ligands for VLA1, 2, and 3; fibronectin for VLA3, 4, and 5; and laminin for VLA 0344-0338/94/0190-0600$3.50/0

1,2 and 6 20 ,22,27,34,35,37,41. Furthermore, modulation of integrin expression has been associated in some instances with tumour progression or with the degree of differentiation 4o . Although a large number of ECM components have been described in salivary pleomorphic adenoma, information about their receptors and their correlation to morphogenesis is only available for a few members of this important class of molecules. It is thus of interest to compare the integrin expression in salivary pleomorphic adenomas and the corresponding normal tissues, in order to gain information on the possible role of. specific integrin complexes in tumour morphogeneSIS.

Materials and Methods Frozen material from 10 normal salivary glands and from 27 pleomorphic adenomas were obtained from the surgical pathology files from the Department of Pathology, Sint Raphael University Hospital - Catholic University of Leuven, Leuven, Belgium. Diagnoses were based on haematoxylin and eosin-stained sections. Serial frozen sections (5 Ilm) were © 1994 by Gustav Fischer Verlag, Stuttgart

VLA Integrins in Pleomorphic Adenoma· 601 dried overnight and fixed in acetone for 10 minutes. A threestep indirect immunoperoxidase procedure was used in this study. The following primary monoclonal antibodies (mcab) with subunits of VLA (~1) integrins were used: TS27 (VLAl), Gi9 (VLA2), PIB5 (VLA3), CDW49d (VLA4), )311G2 (VLA5), GoH3 (VLA6) (Table 1). A representative sample of material was also studied for the common ~1 subunit (DH12) and for ~4 subunit (3El). Incubation with the mcab was followed by incubation with peroxidase-labelled rabbit anti-mouse IgG (diluted 1:50; Dakopatts, Denmark), or, following VLA6, which is a rat mcab, goat anti-rat IgG antiserum (diluted 1:50, Dakopatts, Denmark). The third step consisted of peroxidase-conjugated swine-anti rabbit Ig (diluted 1:100; Dakopatts, Denmark) or swine-anti goat (diluted 1:100; Dakopatts, Denmark), respectively. To reduce unwanted background staining, both secondary and tertiary antibodies were diluted in PBS, pH 7.2, containing 10% normal human AB-serum. Each incubation with antibody was performed for 30 minutes at room temperature and was followed by a wash in three changes of PBS, pH 7.2. Sections were then incubated for 15 minutes in 0.05 M acetate buffer (pH 4.9) containing 0.05% 3-amino-9-ethylcarbazole and 0.01 % H 2 0 2 resulting

Table 1. Monoclonal antibodies used in this study Mabs

Working Source dilution

TS27 Gi9 PIB5

1:500 1:10 1:10

CDW49d 1:10 B11G2 1:10 GoH3 1:10 4b4 3El

1:20 1 :10

Specificity

Dr. ME Hemler Immunotech Telios Pharmaceutical Inc. Immunotech Dr. C. Damsky Dr. AEGK von dem Borne

VLA-cxl chain VLA -cx2 chain VLA-cx3 chain

Coulter Immunology Telios Pharmaceutical Inc.

VLA-~l VLA-~4

VLA-cx4 chain VLA-cx5 chain VLA-cx6 chain chain chain

in a red precipitate, and lightly counterstained with Harris' haematoxylin. For each case, control sections in which the primary antibody was omitted, were included.

Table 2. Distribution of the various single VLA chains in normal salivary gland VLA-integrins subunit

Histology

MEC Light cell Basal cell Dark cell

VLAI cs cy

VLA2 cs cy

+++

+++

+++ + + +

VLA3 cs cy

+++ + ++ ++

+++ +++

+++ +++ +++

VLA4 cs cy

+++ + +++ +++

VLA5 cs cy

VLA6 cs cy +++ +++ +++

+ + +

~1

+++ + + +

~-

~4

cs

cy

cs

cy

+++

+++ + ++ ++

+++ +++ +++

+++ + + +

+++ +++

~-

Table 3. Distribution of the various single VLA chains in pleomorphic adenoma Histology

VLA-integrins subunit VLAI

VLA2 ~!-

cs

cy

VLA3

:!-

cs

VLA4

cy

cs

cy

1) without spindle cells in the outer zone - inner layer +++ - outer layer +++

+++ +++

+++ +++

+++ +++

2) with spindle cells in the outer zone - inner layer - outer layer +++ - squamous metaplasia :;. :1- dense cellular clusters

+ ++ + +

Cyst-like structures - inner layer - outer layer

+++ +++

VLA5

cs

cy

*

+ +

;{.

+ + + +

cs

VLA6

* :}

cy

~1

~4 *::-*

~:-

cs

cy

cs

cy

cs

cy

+++

+ +

+++ +++

+++ +++

+++ +++

+++ +

+++

+

+++

+++

+ + +

+

+++

+ ++ + ++

+ +

+++ +++

+++ +++

+++ +++

+++ +++

Tubulo-ductal structures

+++ +++

Squamous metaplasia Tumor cells in myxoid and in chondroid" Matrices':- ':-"-"-

+++

+ ++ + ++

+++ +++

+++ +++

+++

+

+

:}

:}

:;.

"::.

" ;,

:;-

+

+ + +

-/

-/

-/

-/

+++

+++

+++

+++

+ ::.

-/

-/

+++

+++

++++

++++

+

-/ +++

cs =cell surface; cy =cytoplasm; - =no staining; + =slight; ++ =moderate; +++ =strong; * only few tumor cells stained strongly; ,';. stained only at the cell-to-cell contact; ':-,' ,:- stained only at the cell-to-stromal junction; ,',',',' in these areas the positive cells were mixed with negative cells

602 . S. Sunardhi-Widyaputra and B. Van Damme

Results The distribution of the various VLA chains in normal human salivary gland and in pleomorphic adenoma is shown in Table 2 and Table 3 respectively. Although the VLA-integrins are cell-surface receptors, cytoplasms also show positivity.

Normal Salivary Glands (Fig. 1, A-D) Normal salivary glands consist of acinar cells, myoepithelial cel1s (MEC), and ductal cel1s. The ducts comprise three types of epithelial cells: light cells, dark cells, and basal cells. In the tissues used, the immunohistochemical demonstration ofVLA-integrins reveals negative staining in the cytoplasm of acinar cells and only slight positivity in the light cells. Generally, the cytoplasm of light cells is weaker and less consistently positive than other cells.

MEC differ in VLA expression from ductal cells (light, basal and dark cells), in that they stain strongly with VLAI but are completely negative with VLA4. Basal cells have VLA expressions similar to dark cells, except for VLA6 and B4 that cannot be demonstrated in the dark cells. The positivity for VLA6 and B4 is exclusively present at cell-to-basement membrane (BM) contact of light and basal cells, whereas VLA2 is present at the cell-to-cell contacts. Moreover, a slight diffuse staining for VLA3, 5, and Bl is seen in the stroma. VLA5 stained both stromal fibroblasts and matrix, while VLA4 only stained the fibroblasts. Fibroblasts are also positive for VLAl (data not shown).

Salivary pleomorphic adenoma Tubulo-ductal structures (Fig. 2, A-E; Fig. 3, A-E; Fig. 4, A-C). Two different patterns of VLA expression were recognised.

Fig. 1. Normal salivary gland. The presence of basal ductal and dark cells in the striated and excretory ducts (arrows) stained for VLA2 (A), VLA3 (B), PI (C), and P4(D). Three-step immunoperoxidase method, lightly counterstained with Harris' haematoxylin.

VLA Integrins in Pleomorphic Adenoma . 603

Fig. 2. Pleomorphic adenoma. Cyst-like and tubulo-ductal structures without spindle cells in the outer zone, stained for VLA2 (A), VLA (B), VLA6 (C), ~1 (D), and ~4 (E). Three-step immunoperoxidase method, lightly counterstained with Harris' haematoxylin.

First, tubulo-ductal structures without spindle cells in the outer zone (type 1) (Fig. 2, A-E). Tumour cells in both layers expressed similar positivity, except for VLA6 which is absent in the cell surface of the inner layer, and ~4 which stained slightly in the cytoplasm of the outer layer but is strongly positive in the inner layer cells. Second, tubulo-ductal structures with spindle cells in the outer zone (type 2). These structures occasionally show squamous metaplasia (Fig. 3, A-E), or dense cellular clusters. The squamous metaplastic cells are surrounded by cuboidal tumour cells with characteristics of tumour cells of the outer layer. The inner layer of the tubulo-ductal structures have VLA expressions similar to that of squamous metaplasia (see below), and often show a structural connection with the tubulo-ductal structures of type 2. The tubulo-ductal structures (type 2) are formed by cells that are strongly positive for VLA6 and ~4 in their BM-associated poles, suggesting the presence of centres

of ductal development (Fig. 4, A-C). In peripheral areas the tumour cells show a spreading into the myxoid or chondroid matrices, and are therefore called "spreading" tumour cells. Here VLA6 and ~4 stain the tumour cells in a limited area of the cell surface. Occasionally the dense cellular clusters are also related to the outer layers of tubulo-ductal structures and share similarities in VLA-integrins expression. A few tumour cells stain for VLA 1 and VLA2 on cell surface but not VLA6 and ~4, present only in scattered tumour cells. The positivity for VLA5 on cell surfaces is seen in a few spindle cells, scattered in the dense cellular clusters, and in the stromal tumour tissue.

Cyst-like structures (Fig. 2, A-E). Both layers in this structures present an expression similar to the tubuloductal structures without spindle cells (type 1) in the outer zone as shown in Table 3.

604 . S. Sunardhi-Widyaputra and B. Van Damme

Fig. 3. Pleomorphic adenoma. Tubulo-ductal structure with squamous metaplasia stained for VLA2 (A), VLA3 (B), VLA6 (C), ~1 (D), and ~4 (E). The cytoplasm of the tumor cells in the inner layer cells stained slightly, in connection with the squamous metaplasia, and demarcated by the strongly stained outer cells. Note the expression of VLA2 lacking at the cell-to-stromal junction (arrows), and the presence of VLA6 and ~4 at the BM-associated poles. Three-step immunoperoxidase method, lightly counterstained with Harris' haematoxylin.

Squamous metaplasia (Fig. 5, A-E). Areas with squamous metaplasia have VLA-integrin expression similar to that of the inner layer of the tubulo-ductal structures with spindle cells in the outer zone. These structures are surrounded by rows of cuboidal tumour cells with characteristics of the outer layer that strain strongly for VLA2, 3, and ~l, and stain at the BM-associated poles for VLA6 and ~4. The VLA2 expression lacks the enhancement at the cell-to-stromal junction. VLAI and VLA6 are undetectable on the tumour cell surface, and VLA4 is seen in some scattered tumour cells. Myxoid and chondroid matrices (Fig. 6, A-D). Here the tumour cells with characteristics of the outer layer are mixed with the tumour cells that are devoid of staining. A few tumour cells, strongly positive for

VLAI and VLA4 on their cell surfaces, are also present. This feature resembles a mixture of tumour cells from each layer of the tubulo-ductal structure. Some positive cells are present in sm<'!.ll clusters, the others as individual cells. VLA2, 6, and ~4 are only present in a limited area of the cell surface of single cells. Similar features are seen in the chondroid matrix, but the VLA6-positive cells are found in small clusters rather than individual cells. In all structures described above, cells positive for VLAl, 4, and 5 are sparsely seen. These cells are spindled or dendritic in shape, and are located mainly in the stromal areas, in the dense cellular clusters, and in the myxoid and chondroid matrices, and are present as small clusters or as single cells.

VLA Integrins in Pleomorphic Adenoma . 605

Fig. 4. Pleomorphic adenoma. Tubulo-ductal structure (the lumen marked by asterix) with centers of developmentformed by the cells bearing VLA2 (A), VLA6 (B) and P4 (C) positivity, and a spreading appearance where the stained and the negative tumor cells are mixed. Three-step immunoperoxidase method, lightly counterstained with Harris' haematoxylin.

Discussion It is generally accepted that myoepithelial cells (MEC) and light cells of normal salivary glands show clear differences in ultrastructure, cytoskeletal protein and enzymatic organisation. The VLA-in~egrin expression described in this study apparently reflects some phenotypic distinctions. The normal immunoprofiles, as seen in Table 3, are generally maintained in pleomorphic adenoma. In this study, we found in virtually all tumour cells in pleomorphic adenoma characteristics of basal ductal and dark cells of normal salivary glands, and only few cells bearing the integrin expression characteristic of MEC. The exclusive staining for VLA 1 in MEC of normal salivary glands is in line with other studies in normal salivary and breast tissue 8,26, 30,31. Although in some instances basal cells have phenotypic similarities to MEC7,9,39, it is evident that basal cells are distinct from MEC, as shown by the expression of VLAintegrins in this study and by an ultrastructural study33. The presence ofVLA6 and ~4 in (apical) dark cells cannot be recognised by light microscopy, but since the slender infranuclear processes of this cell extend to the basement membrane 4 3, we suppose that these receptors could be present, but not be visible at the light microscopic level. Many previous studies concluded that pleomorphic adenoma consists mainly of tumour cells with MEC characteristics rather than ductal features. Orily in a few studies is pleomorpnic adenoma

suggested as being composed of small numbers of tumour cells with MEC characteristics 12,32. The fact that in pleomorphic adenoma almost all tumour cells bear basal cell characteristics supports the concept of the basal "reserve" ductal cells 3, 14. The exclusive staining for VLA5 along with the coexpression ofVLAl and VLA4 in stromal fibroblasts of normal salivary gland distinguishes them from MEC and from basal and dark cells. Even after using the demasking agent hyaluronidase we failed to expose the presence of VLA5 in ductal cells (data now shown). The expression of VLA4 and VLA5 in stromal fibroblasts of normal salivary gland and in breast tissue has been described I5 ,18,41. In pleomorphic adenoma, in which the formation of ductal structures initiates the development of a tumourlO, the localisation of VLA2 and VLA6 in these structures could be related to the various degrees of disruption in the basement membrane 6 and the spreading of tumour cells in the stroma. Since the VLA2 enhances the spreading of tumour cells in vitro 5 , and VLA6 receptors mediate the attachment of tumour cells to neoplastic BM material 4 , 11, the presence of these integrins in "spreading" tumour cells may endow , these cells with an increased ability to spread through the ECM. The expression of VLA-integrins in almost all tumour cells in pleomorphic adenoma is similar to that of ductal basal cells, suggesting a more important histogenetic role for basal cells than previously accepted.

606 . S., Sunardhi-Widyaputra and B. Van Damme

Fig. 5. Pleomorphic adenoma. Squamous metaplasia area stained for VLA2 (A), VLA3 (B) VLA6 (C), P4 (D), and P4 (E), demarcated by the strongly stained outer cells. Note the expression of VLA2 lacking at the cell-to-stromal junction, and the presence of VLA6 and P4 at the BM-associated poles. Three-step immunoperoxidase method, lightly counterstained with Harris' haematoxylin.

Acknowledgements We thank C. Van den Broeck, E. Van Dessel and K. Van Meerbeek for technical assistance, and M. Rooseleers for preparation of the micrographs. The author (SS-W) appreciates the financial support from the Belgian Administration for Developmental Cooperation.

References J Akiyama SK, Yamada KM (1987) Fibronectin. Adv Enzymol 59: 1-57 2 Albelda SM (1993) Biology of disease. Role of integrins and other cell adhesion molecules in tumor progression and metastasis. Lab Invest 68: 4-17

3 Batsakis JG (1980) Salivary gland neoplasia: an outcome of modified morphogenesis and cytodifferentiation. Oral Surg 49:229-232 4 Bonkhoff H, Stein U, Remberger K (1993) Differential expression of :x6 and ':x2 very late antigen integrins in the normal, hyperplastic, and neoplastic prostate: simultaneous demonstration of cell surface receptors and their extracellular ligands. Hum Pathol 24: 243-248 5 Bosco M, Chan C, Matsuura N, Takada Y, Zetter BR, Hemler ME (1991) In vitro and in vivo consequences of VLA2 expression on rhabdomyosarcoma cells. Science 251: 1600-1602 • 6 Caselitz], Schmitt P, Seifert G, Wuwtrow J, Schuppan D (1988) Basal membrane associated substances in human salivary gland and salivary gland tumours. Pathol Res Pract 183: 386-394 7 Cutler LS, Chaudhry AP, Innes DJ (1977) Ultrastructure of the parotid duct. Arch Pathol Lab Med 101: 420-424

VLA Integrins in Pleomorphic Adenoma . 607

Fig. 6. Pleomorphic adenoma. A representative area shows the myxoid and chondroid matrices, stained for VLA2 (A), VLA6 (B), ~1 (C), and ~4 (D). Three-step immunoperoxidase method, lightly counterstained with Harris' haematoxylin. 8 d'Ardenne AJ, Richman PI, Horton MA, McAulay AE, Jordan S (1991) Co-ordinate expression of the alpha-6 integrin laminin receptor subunit and laminin in breast cancer. J Pathol165: 213-220 9 Dardick I, Rippenstein P, Skimming L, Dairkee SH (1987) Immunohistochemistry and ultrastructure of myoepithelium and modified myoepithelium of the ducts of human major salivary glands. Histogenetic emplications for salivary gland tumors. Oral Surg 64: 703 - 715 10 Dardick I, Van Nostrand AWP, Jeans MTD, Rippstein P, Edwards V (1983) Pleomorphic adenoma, I: Ultrastructural organization of "epithelial" regions. Hum Pathol 14: 780797 11 Dedhar S, Saulnier R (1990) Alterations in integrin receptor expression on chemically transformed human cells: specific enhancement of laminin and collagen receptor complexes. J Cell Bioi 110: 481-489 12 Draeger A, Nathrath WBJ, Lane EB, Sundstrom BE, Stigbrand TI (1991) Cytokeratins, smooth muscle actin and vimentin in human normal salivary gland and pleomorphic adenomas. Immunohistochemical studies with particular reference to myoepithelial and basal cells. APMIS 99: 405-415 13 Duband JL, Rooner S, Chen WT, Yamada KM, Thiery JP (1986) Cell adhesion and migration in the early vertebrate embryo: location and possible role of the putative fibronectin receptor complex. J Cell Bioi 102: 160-178 14 Eversole LR (1971) Histogenetic classification of salivary tumors. Arch Path 92: 433-443

15 Gailit J, Pierschbacher M, Clark RAF (1993) Expression of functional cz4~1 integrin by human dermal fibroblast. J Invest dermatol100: 323-328 16 Guan ]-L, Hynes RO (1990) Lymphoid cells recognize an alternatively spliced segment of fibronectin via the integrin receptor cz4~1. Cell 60: 53-61 17 Hay ED (ed.) (1981) Cell Biology of extracellular matrix. Plenum Press, New York and London, pp. 379-405 18 Hemler ME (1990) VLA proteins in the integrin family: structures, functions, and their role on leucocytes: Annu Rev Immunol 8: 365 -400 19 Hemler ME, Elices MJ, Parker C, Takada Y (1990) Structure of the integrin VLA-4 and its cell-cell and cell-matrix adhesion functions. Immunol Rev 114: 45-65 20 Hemler ME, HuangC, Takada Y, Schwarz L, Strom inger JL, Clabby ML (1987) Characterization of the cell surface heterodimer VLA-4 and related peptides. J BioI. Chern 262: 11478-11485 21 Humphries MJ (1990) The molecular basis and specificity of integrin-ligand interactions. J Cell Sci 97: 585 -592 22 Hynes RO (1987) Integrins: a family of cell surface receptors. Cell 48: 549-554 23 Hynes RO (1992) Integrins: versatility, modulation, and signaling in cell adhesion. Cell 69: 11-25 24 Juliano RL (1987) Membrane receptors for extracellular matrix macromolecules: relationships to cell adhesion and tumor metastasis. Biochim Biophys Acta 907: 261178

608 . S. Sunardhi-Widyaputra and B. Van Damme 25 Kawakatsu H, Shiurba R, Obara M, Hiraiwa H, Kusakabe M, Sakakura T (1992) Human carcinoma cells synthesize and secrete tenascin in vitro. Jpn J Cancer Res 83: 10731080 26 Koukoulis GK, Virtanen I, Korhonen M, Laitinen L, Quar,a nta V, Gould VE (1991) Immunohistochemicallocalization of integrins in normal, hyperplastic, and neoplastic breast. Am J Pathol 139: 787-799 27 Languino LR, Gehlsen KR, Wayner E, Carter WG, Engvall E, Ruoslahti E (1989) Endothelial cells use Cl2~1 integrins as a laminin receptor. J Cell Bioi 109: 2455-2462 28 Lee K, Hynes RO, Kirschner M (1984) Temporal and spatial regulation of fibronectin in early Xenopus development. Cell 36: 729-740 29 Martinez-Hernandez A (1988) Editorial: The extracellular matrix and neoplasia. Lab Invest 58: 609-611 30 Mechtersheimer G, Munk M, Barth T, Koretz K, Moller P (1993) Expression of ~1 integrins in non-neoplastic mammary epithelium, fibroadenoma and carcinoma of the breast. Virchows Archiv A Pathol Anat 422: 203-210 31 Miettinen M, Castello R, Wayner E, Schwarting R (1993) Distribution of VLA integrins in solid tumors. Emergence of tumor-type-related expression patterns in carcinomas and sarcomas. Am J Pathol142: 1009-1018 32 Palmer RM, Lucas RB, Knight J, Gusterson B (1985) Immunocytochemical identification of cell types in pleomorphic adenoma, with particular reference tomyoepithelial cells. J Path 146: 213 - 220 33 Riva A, Serra GP, Proto E, Faa G, Puxeddu R, Riva FT (1992) The myoepithelial and basal cells of ducts of human major salivary glands: A SEM study. Arch Histol Cytol 55 (Supp!.): 115 -124, 34 Ruoslahti E (1991) Integrins. J Clin Invest 87: 1-5

35 Ruoslahti E, Pierschbacher MD (1987) New perspectives in cell adhesion: RGD and integrins. Science 238: 491-497 36 Soini Y, Piiiikko P, Virtanen I, Lehto VeP (1992) Tenascin in salivary gland tumors. Virchows Arch A Pathol Anat 421: 217-222 37 Springer TA (1990) Adhesion receptors of the immune system. Nature 346: 425 -434 38 Sunardhi-Widyaputra S, Van Damme B. (1993) Immunohistochemical expression of tenascin in normal human salivary glands and in pleomorphic adenomas. Path Res Pract 189: 138-143 39 Tandler B (1965) Ultrastructure of the human submaxillary gland. III. Myoepithelium. Z Zellforsch 68: 852-883 40 Virtanen I, Korhonen M, Kariniemi AL, Gould VA, Laitinen L, Ylanne J (1990) Integrins in human cells and tumors. Cell Diff Develop 32: 215 - 228 41 Wayner EA, Carter WG (1987) Identification of multiple cell adhesion receptors for collagen and fibronectin in human fibrosarcoma cells possessing unique Cl and ~ subunits. J Cell Bioi 105: 1873-1884 42 Wayner EA, Garcia-Pardo A, Humphries MJ, McDonald JA, Carter WG (1989) Identification and characterization of the T lymphocytes adhesion receptor for an alternative cell attachment domain (CS-1) in plasma fibronectin. J Cell Bioi 109: 1321-1330 43 Wilborn WH, Shackleford JM (1980) Microanatomy of human salivary glands. In: L. Menaker (ed.) The biologic basis of dental caries. Hagerstown, Harper & Row, pp. 3-63 44 Yamada KM (1988) Fibronectin domains and receptors. In: Fibronectin. DF Mosher (ed.), Academic Press, Inc. New York, pp. 47-121

Received December 8, 1993 . Accepted in revised form March 7, 1994

Key words: VLA - Integrin - Pleomorphic adenoma - Salivary gland - Receptor - Morphogenesis S. Sunardhi-Widyaputra, Laboratory of Histo- & Cytochemistry, Department of Pathology, Sint Raphael University Hospital, Catholic University of Leuven, Minderbroedersstraat 12, B-3000 Leuven, Belgium, Phone 016/33.65.88, Fax. 016/33.65.48