sion in human breast cancer revealed the a231 inte- ..... differentiated component of breast cancer. ..... Pignatelli M, Hanby AM,Stamp GWH: Low expression.
American Journal of Pathology, Vol. 142, No. 5, May 1993 Copright ©) American Society for Itnvestigative Pathology
Altered Integrin Expression in Adenocarcinoma of the Breast Analysis by in Situ Hybridization
Mary M. Zutter, Hannah R. Krigman, and Samuel A. Santoro From the Departments of Pathology anid Medicine, Washington University School of Medicine, St. Louis, Missouri
The integrin superfamily of adhesion receptors mediates interactions between cells and the extracelular matrix. Our earlier immunohistochemical analysis showed that normal mammary epithelium expressed high levels ofthe q2fi1 coUlagen/laminin receptor and intermediate levels of the a5481fibronectin receptor. In contrast, malignant cells of adenocarcinoma of the breast exhibited marked diminution or loss ofthe a2f31 and a5.81 integrins. We have now evaluated the level of a2, aS, and 81 integrin subunit messenger (m)RNA by in situ hybridization in adenocarcinoma of the breast. Normal breast ducts and ductules expressed high levels of all three integrin subunit mRNAs. Poorly differentiated lesions expressed low to undetectable levels of a2, a5, and I31 mRNA. Well- and moderately differentiated lesions expressed al three subunits at intermediate levels. Thus, decreased expression of the a2f81 and a5131 integrins in mammary carcinoma is the result of decreased steady-state integrin subunit mRNA levels due to altered expression of the integrin genes. (Am JPathol 1993, 142:1439-1448)
The integrin superfamily of heterodimeric cell-surface receptors composed of distinct a and 3 subunits mediates the adhesion of cells to the extracellular matrix and, in some instances, the adhesion of cells to one another. 1-3 The integrins are thought to play important roles in differentiation and development,4-7 cell migration, and the complex process of tumor cell invasion and metastasis,9 12 as well as adhesion per se. Alterations in integrin receptor expression upon
malignant transformation in vitro as well as in naturally occurring human malignancies are now well established.9' 12-1 8 However, the molecular mechanisms that give rise to altered integrin protein expression following malignant transformation are poorly understood. Our earlier immunohistochemical studies revealed that the a2P1 integrin that serves as a collagen receptor on platelets and fibroblastic cells but as a receptor for both collagen and laminin on endothelial and some epithelial cell types was expressed at high levels in the proliferative layer of most epithelial cell types evaluated, including the ductular epithelium of the breast.19-25 A detailed study of integrin expression in human breast cancer revealed the a231 integrin was highly expressed on the epithelium of ducts and ductules of normal breast tissue and was expressed at normal or close-to-normal levels in benign lesions such as fibroadenoma or papilloma. 13 In contrast, markedly reduced or undetectable a2f31 expression was seen in poorly differentiated adenocarcinomas. Well-differentiated adenocarcinomas exhibited intermediate levels of expression. Similar but less extensive changes were observed for the a5f31 integrin (fibronectin receptor). Pignatelli et al15 and Koukoulis et al16 subsequently described similar differentiationdependent alterations of integrin expression in adenocarcinoma of the breast. Altered integrin expression, the degree of which was dependent upon the extent of differentiation, has also recently been described in adenocarcinomas of the colon and pancreas. 14,17,18 Both in vitro and in vivo evidence has accumulated to suggest that altered integrin expression contributes to the invasive and metastatic potential of tumor cells.9-12,26,27 Supported by grant CN-44 from the American Cancer Society and by NIH grant T32-HL07038. Accepted for publication November 18, 1992. Address reprint requests to Dr. Mary M. Zutter, Department of Pathology, Box 8118, Washington University School of Medicine, 499 South Euclid, St. Louis, MO 63110.
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To begin to examine the molecular basis underlying altered integrin expression during the malignant progression of human mammary adenocarcinoma, we have determined the levels of messenger (m)RNA encoding the a2, a5, and 13i integrin subunits by in situ hybridization and compared the results to protein expression evaluated by immunohistochemistry in a series of breast cancers.
Materials and Methods Tissue Fixation and Embedding Biopsy and mastectomy samples were processed routinely by fixation in 10% neutral buffered formalin and paraffin embedding. The specimens included representative samples from the cases for which integrin expression had been previously investigated by immunohistochemistry on frozen tissue.13 Sections (3 to 4 p) on Superfrost Plus slides (Fisher Scientific, Pittsburgh, PA) were baked at 55 C for 2 hours, deparaffinized in xylene, rehydrated in graded alcohols, and equilibrated at room temperature in phosphate-buffered saline (10 mmol/L sodium phosphate, 150 mmol/L NaCI, pH 7.4). Sections were sequentially treated as previously described28 with phosphate-buffered saline for 5 minutes at room temperature, proteinase K (1 pg/ml) in phosphate-buffered saline for 15 minutes at 37 C, triethanolamine buffer 0.1 mol/L, pH 8.0, for 5 minutes at room temperature, and then with triethanolamine buffer containing 0.25% acetic anhydride for 10 minutes at room temperature. The slides were then washed twice in 2x standard saline citrate (SSC) (1 x SSC: 150 mmol/L NaCI, 15 mmol/L Na citrate, pH 7.0), dehydrated in graded alcohols, and air-dried.28
Preparation of RNA Probes A 1296-bp segment of the a2 complementary (c) DNA corresponding to nucleotides 1-1296 of the published cDNA sequence was generated by reverse transcription of total RNA from the mammary carcinoma cell line T47D, followed by polymerase chain reaction as recently described.29'30 The cDNA segment was cloned into pBluescript SKplasmid (Stratagene Cloning Systems) and completely sequenced by the dideoxynucleotide chain termination method of Sanger31 to confirm its au-
thenticity. Partial-length cDNA clones representing 2.5 kb of the published a5 cDNA and 2.5 kb of the f,3 cDNA in M13mp18 were generously provided by Dr. Lau-
rence Fitzgerald, University of Utah.3233 The cDNA segments were initially cloned into pBluescript SKplasmid. For optimal in situ hybridization, small subclones, including a 1796-bp (bp 1502 to 3281) EcoRV/EcoRl segment of the a5 cDNA and a 1043-bp (bp 1594 to 2639) AccI/EcoRI segment of the I31 cDNA were subcloned into pBluescript SK-. Orientation was confirmed by sequence analysis.31 In vitro transcribed RNA corresponding to singlestranded either sense or anti-sense mRNA was synthesized by in vitro transcription for 3 hours (Stratagene Cloning Systems, La Jolla, CA), using bacteriophage T3 and T7 RNA polymerases (Promega Corp., Madison, WI) and labeled with a [35S]UTP (New England Nuclear, Boston, MA). Specific activity for each probe ranged from 6 x 107 to 1 x 108 cpm/pg. The DNA template was degraded with RNAse-free DNAse (Promega) for 15 minutes. The transcribed RNA was extracted with phenol/ chloroform, chloroform, ethanol precipitation, and redissolved in 20 mmol/L dithiothreitol.
Hybridization Tissue sections were preincubated in hybridization buffer (50% formamide, 20 mmol/L Tris pH 8.0, 2x SSC, 10% dextran sulfate, 1 x Denhardt's solution, 1 mmol/L ethylenediaminetetraacetic acid, 100 mmol/L dithiothreitol, and 500 pg/mI yeast transfer RNA) for 2 hours before addition of 1 x 104 cpm/pl denatured RNA probe. Hybridization proceeded overnight at 42 C in a humidified chamber. The following morning, sections were washed once with 4x SSC, 25 mmol/L 2-mercaptoethanol at room temperature, then twice with 0.1 x SSC, 25 mmol/L 2-mercaptoethanol for 30 minutes at 47 C, with vigorous shaking. The slides were then rinsed in 2x SSC for 10 minutes at room temperature, dehydrated through graded ethanols, and air-dried. The air-dried slides were coated in melted KODAK NT2B photoemulsion (diluted 1:1 with water), slowly dried, exposed in the dark, and desiccated for 4 days to 4 weeks, depending on the experiment. Slides were developed, using KODAK D-19 developer (diluted 1:1 with water) for 3 minutes at 14 C. The reaction was stopped with 1% acetic acid, and slides were fixed with KODAK Rapid-Fix (diluted 1:3 with water). The air-dried slides were then stained with hematoxylin and eosin.
Grading Each series of seven cases was hybridized in duplicate with paired sense and anti-sense probes plus
Integrin mRNA in Carcinoma of the Breast AJP May 1993,
a section of normal breast epithelium and nipple. The skin of the breast and glandular epithelium served as a 4+ positive control for each experiment. The malignant epithelium was compared to the control and graded semiquantitatively from 0 (no signal above background) to 4+ (the signal of a normal breast lobule) by two independent observers (MMZ, HK). Many of the tissue sections also contained normal mammary epithelium that served as an internal control. Fibroblasts and endothelial cells provided internal controls for tissue fixation and RNA preservation.
Results The a231 integrin protein is expressed at high levels on the cell surface of most epithelial cell types, including the ducts and ductules of normal breast tissue. As shown in earlier studies, the basal cell layer of the bilayered cuboidal epithelium of the breast expressed higher levels of a2f1 protein than the more superficial layer not in contact with the basement membrane.19 By in situ hybridization, normal breast ducts and ductules from six separate normal breast biopsies or mastectomy specimens expressed high levels of a2 mRNA similar to the high level of a2 protein demonstrated by immunohistochemistry (Figure 1, A and B). Although both layers of the bilayered epithelium expressed high levels of a2 mRNA, the intensity of a2 mRNA expression was greatest in the basal cell layer (4+) and slightly weaker in the more superficial layer (3-4+). Low-level a2 mRNA expression by fibroblasts and endothelial cells served as an internal control. The absence of background hybridization with the corresponding sense probe indicates the specificity of the hybridization observed with the anti-sense probe (Figure 1A, insert). In contrast to the high levels (4+) of a2 mRNA in normal breast, a2 mRNA expression was markedly decreased in poorly differentiated breast carcinomas ( to 2+) (Figure 1, E and F, Table 1). The low level of a2 mRNA expression in poorly differentiated carcinomas corresponded closely to the low or absent levels of a2 protein determined by immunohistochemistry. The striking contrast between a2 mRNA expression in normal breast and poorly differentiated carcinoma is best illustrated in sections where the tumor abuts normal breast lobules (Figure 1, E and F). Most well-differentiated carcinomas expressed relatively high to intermediate levels of a2 mRNA, ranging from 2 to 3+ intensity. Moderately differentiated carcinomas showed similar but
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slightly lower levels of a2 mRNA (1 to 2+ positive) (Figure 1, C and D). The diminished levels of a2 mRNA expression in more poorly differentiated carcinomas corresponded closely, but not exactly, to the low levels of a2 protein determined by immunohistochemistry. Decreased steady-state levels of a2 mRNA determined by in situ hybridization paralleled the changes in protein expression previously observed in adenocarcinoma of the breast. Because we had also observed decreased expression of a5f1 protein in our earlier study, we examined the levels of a5 and P1 mRNA. The a5 integrin subunit mRNA was expressed at very high levels similar to the a2 mRNA by the bilayered epithelium of the ducts and ductules of normal breast (Figure 2, A and B). Much greater levels of a5 mRNA than a2 mRNA were expressed by fibroblasts and endothelial cells. Smooth muscle bundles demonstrated low to intermediate levels of a!5 mRNA, whereas a2 mRNA was not detectable in these cells. In contrast to normal breast, poorly differentiated carcinomas of the breast expressed very low, but still detectable, levels of a5 mRNA (Table 1) (Figure 2, E and F). Well- or intermediately differentiated carcinomas expressed a5 at intermediate levels, from 2 to 4+ and from 1 to 2+, respectively (Figure 2, C and D). The changes in a5 mRNA determined by in situ hybridization paralleled the changes in a2 mRNA as well as the decreased level of a5 protein determined by immunohistochemistry. The absence of signal with the sense probe again illustrated the specificity of observed hybridization
(Figure 2A, insert). In situ hybridization analysis of the f3 subunit revealed very high levels of 13 integrin mRNA in normal breast and in fibroadenomas (Figure 3, A and B). The comparable P, sense probe yields an undetectable signal, as illustrated in Figure 3A, insert. Changes in the 13i subunit mRNA in malignancy were similar, although not identical, to those of the a2 and a5 subunits. Well-differentiated carcinomas showed a slight decrease in j31 mRNA expression (Figure 3, C and D); moderately differentiated carcinomas showed a more marked decrease; and poorly differentiated lesions showed a significant reduction to 1 + positive (Figure 3, E and F). The decrement in f,3 mRNA levels paralleled the changes in a2 and a5 mRNA. However, the decrease in 13 mRNA expression in moderately and poorly differentiated lesions was more pronounced than the changes in o1 integrin expression determined by immunohistochemistry. For example, in the moderately and poorly differentiated tumors, a2 and a5 protein was minimally expressed or undetectable,
1442 Zutter et al AJP MaY 1993, Vol. 142, No. 5
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I I.
42, ,.
t
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Figure 1. a2 mRNA expression determined by in situ hybridizationi ini ntormal breast ducts and dtuctuiles (A, B), moderatelv' differenitiated carcinioma (C, D), and poorly differenitiated carcitnoma (E, F). Normal breast ducts and dtctuiles express higb levels of a2 mRNA tisintg ani anti-sense a, transcript (A, bright field, B, dark field; x 100 magnification). Te intensity, of a2 mRNA uwas greatest in the basal cell layer (4+) atnd uwas slightly weaker (3 to 4+ ) in the more stuperficial laver The absence of backgrountid hYbridization in niormal breast uith a senise a, probe indicatets specificitv of hybridization (insert; X 100 magntificationi). In conitrast, poorly diffentiated carcinomas shou markedly decreased Iciels of a, mRNA (E, brightfield; F, darkfield; x 100 magntificationi). The conitrast betueen high anid lou levels of a2 mnRAA is best demonstrated in areas uhere niornial breast lobiules (arrou) abuit iniasiue carciniomna (arrouhead) (X 100 magnification). Moderately, d{Jferentiated lesions shou, ani intermediate level of a2 mKYA expression (C, brightfield; D, darkfield,; x 100 magnification). All sections are counterstained with heniatoxyilini antd eosin.
but the level of a2 and a5 mRNA remained detectable at 1 to 2+. In contrast, f1 mRNA levels were decreased to almost undetectable levels when protein was still expressed at 2+ positivity. Because the high-level expression of the 1 integrin subunit
in normal tissue necessitated short exposure times in these comparative studies of breast cancer relative to those employed for the a2 and a5 studies, the absolute level of ,13 expression may be underestimated by our analysis. It is clear, however, that rel-
Integrin mRNA in Carcinoma of the Breast 1443 AJP May 1993, Vol. 142, No. 5
Table 1. Integnin Protein versus mRNA Expression in Adenocarcinoma of the Breast: Comparative Analysis by in Situ Hybridization and Immunohistochemistry*
a2
Specimen
a5
Pi
In Situ
Immuno
In Situ
Immuno
In Situ
Immuno
A B G
++++ +++
++++ ++++
++++ ++
++++ ++++
++++
++++
++++ ++++
+++
+++
+++
+++
+++
++
H
++
+++
++++
+++
++-+++
+++
J
++ ++
+++ ++
++ ++
+++
++
+++
++
++
+
K L
+++
+
++
+
+++ ++
+ +
+++ ++
+++ ++
M
++ ++ + ++ +
++ +-++ 0-+ +-++ ++
++ ++ + ++
+++ ++
++ +++
0-+ ++
++
0-+
++ +-++ 0-+ +-++ ++
+ +-++ ++
0-+ +
N
O P
o
R S T U V W X Y
+ + + ++-+++
Z
+
+-++
++
0
0-+
++
+
0-+ +-++
++
++ ++ ++ + + + ++-+++
++
+-++
+
0
+-++ +
++
++ + + + +-++
++ +++ ++ + ++ ++ +++ ++
++
+
+++
+
0
Tumor Histology Normal breast (n = 6) Fibroadenoma Intraductal and invasive ductal, well-differentiated Intraductal and invasive ductal,
well-differentiated Invasive colloid, well-differentiated Focal invasive and intraductal, moderately differentiated Intraductal, well- to moderately differentiated Invasive ductal with intraductal, moderately differentiated Invasive ductal, moderately differentiated
Invasive ductal, moderately differentiated Intraductal, moderately differentiated Invasive ductal, moderately differentiated Invasive ductal and intraductal, moderately differentiated Invasive ductal, poorly differentiated Invasive ductal, poorly differentiated Invasive ductal, poorly differentiated Invasive lobular Invasive ductal, poorly differentiated Invasive ductal, poorly differentiated Invasive ductal, poorly differentiated Metastatic carcinoma, lymph node, poorly differentiated Metastatic carcinoma, brain, poorly differentiated
* Specimen designations are those used in Zutter et al (Am J Patho 1 1990, 137:863-870) where the immunohistochemical results were originally described.
ative to normal breast epithelial elements, I3 expression is markedly reduced in the poorly differentiated carcinomas. Intraductal carcinomas represent the mostdifferentiated component of breast cancer. In some of our cases (cases G and Q), intraductal carcinoma co-existed with invasive carcinoma. In these examples, the intraductal component of welldifferentiated and moderately differentiated, invasive lesions expressed levels of a2, a5, and 13 mRNA indistinguishable from those of the invasive component. The similarities in level of mRNA expression of all three integrin subunits between intraductal and well or poorly differentiated regions of the same tumor suggest that the changes reflect tumor phenotype and not only the differentiation status. High levels of a2, a5, and /31 mRNA expression in adjacent normal breast lobules exclude the possibility of artifact or problems in fixation and processing.
Discussion Malignant transformation alters normal gene expression, thereby impairing control of cellular prolif-
eration and differentiation. Current thought holds that several such genetic alterations in succession culminate in progression to an invasive and metastatic phenotype.34 36 Hynes initially suggested that defects in cell adhesive receptors for components of the extracellular matrix could contribute to the invasive and metastatic potential of tumor cells.37 Since his original proposal was made, a large body of evidence has accumulated to support the role of altered expression of the integrin superfamily of adhesion receptors, as well as other adhesion receptors in invasion and metastasis.9-18 Despite a growing literature documenting decreased integrin expression at the protein level in a variety of adenocarcinomas, the mechanisms leading to altered integrin expression upon malignant transformation have not been defined. As a first step toward that understanding, in this report we describe studies employing in situ hybridization to assess the integrin subunit mRNA in adenocarcinomas of the breast. The results indicate that the levels of a2 and a5 mRNA closely parallel the expression of the a2 and a5 integrin subunits at the protein level. Both a2 and a5 mRNA levels were slightly decreased in well-differentiated lesions and were more
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Figure 2. a5 mRNA expression determined by in situ hybridization in normal breast ducts and ductules (A, B), moderately differentiated carcinoma (C, D), and poorly differentiated carcinoma (E, F). Normal mammary epithelium fibroblasts and endothelial cells express high levels ofa5 mRNA uwith the anzti-sense a2 probe (A, bight field; B, dark field; x 100 magnification). 7Te specificity of hybridization is demonstrated with the sense a5 probe (insert; X 400 magnzificationi). Moderate rediuction in aS mRNA ini moderately differentiated lesions (C, bright field; D, dark field; X 100 magnification) and marked reduction in a5 mRNA in poorly, differentiated tuimors (E, bight field; F, dark field; x 100 magniification) parallel the chanige in a2 mRlNA expression. Expression of a 5 in normal breast lobthles (arrou) contrasts uwith invasive carcinoma (arrowhead). All sectiotns are cotunterstainied with hematoxvlin and eosin.
significantly decreased in moderately differentiated lesions. In poorly differentiated tumors, both a2 and ao5 mRNA were markedly decreased, but still detectable. In all the cases examined, including both welland poorly differentiated lesions, changes in the level of a2 and a5 mRNA by in situ hybridization appeared less marked than the changes in a2 and a5
protein observed by immunohistochemistry. For example, in cases T, U, and V, no detectable protein was identified, yet the corresponding mRNA was present at a level of 1 to 2+. This apparent discordance may be attributable to the differing sensitivities of the two techniques. It may also suggest that additional mechanisms, either posttranscriptional or translational in nature, influence the level of a
Integrin mRNA in Carcinoma of the Breast
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AJP May 1993, Vol. 142, No. 5
1b. . .'
*..*.4
*
-,
6'.
A
--
-.
14j. -.
41
4.
,...,Y
.4.
C
-
14
I
-.
4.t
/
,*.
2 4
4
4-
.4'
-
Figure 3. ,B, integrin mRNA expression in normal (A, B) and malignant breast carcinoma (C to F). Very high levels of PI mRNA were detected in normal breast (A, bright field; B, dark field; x 100 magnification). Hybridization with the sense f31 probe demonstrated a very low level of nonspecific hybridization (insert; X400 magnification). (3, mRNA expression decreased in well-differentiated (C, bright field; D, dark field; x 100 magnification) and poorly differentiated (E, bright field; F, dark field; x 100 magnification) carcinoma in association with decreasing differentiation. All sections are counterstained with hematoxTylin and eosin.
subunit protein expression. In contrast to changes observed in the a subunit, the level of ,1 mRNA was decreased more substantially than the level of 13 protein. Several other recent studies are germane to these observations. Because biosynthetic studies have revealed that a large intracellular pool of 13 subunit exists,38 even quite profound decreases in
f3 mRNA may be insufficient to reduce the level of this intracellular pool to a size where f,3 subunit protein limits cell-surface expression of 1 integrin heterodimers. Our recent study of regulated integrin expression during the megakaryocytic differentiation of K562 cells established that the increased expression of the a2131 integrin accompanying megakaryocytic differentiation was a consequence of
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increased steady-state level of a2 mRNA due to transcriptional activation of the a2 integrin gene.30 The steady-state level of the long-lived 13 mRNA did not change upon differentiation.30 These data are consistent with the idea that the expression of Pf integrins is regulated by controlling expression of the a subunit gene. Other recent observations suggest that regulation of integrin expression may be both differentiationand cell type-specific. The diminished integrin expression that accompanies viral transformation of 3T3 cells reflected changes in both a and f subunit mRNAs.9 The alteration in a6j1, a231, and alp1 integrin receptors following chemical transformation of osteosarcoma cells by N-methyl-N'-nitro-Nnitrosoguanidine did not alter the steady-state level of 13 mRNA; levels of steady-state mRNA for the a subunits were not determined.12 During terminal differentiation of cultured human epidermal keratinocytes, loss of adhesion to the basement membrane was accompanied by loss of the a531 protein expression and diminution of the steady-state level of ,13 mRNA.14 The complexity of cell type and differentiation-specific expression of the multiple integrins required for normal development that may be altered by malignant transformation is apparently mediated at multiple levels of regulation of the separate a and 3 subunits. The present in situ hybridization studies suggest that the changes in cell-surface expression of the a2f1 and a5J1 integrins in human breast cancer are a consequence of decreased steady-state levels of OL2 and a-5 subunit mRNAs that decrease with decreasing degree of tumor differentiation. Because of the factors discussed above, the contribution(s) of the decreased level of f,3 mRNA is less certain. Integrins are the primary mediators of cellular interactions with the extracellular matrix. Such cellmatrix interactions are required for control of normal differentiation of a variety of cell types including epithelial, mesenchymal, and neuronal cells.3943 Overexpression of the a,5f3 integrin in Chinese hamster ovary cells restored control of cell proliferation, reduced growth in soft agar, and resulted in loss of tumorigenicity in nude mice.10 The role of the integrins in cell motility and in the ability of malignant cells to invade and metastasize has been demonstrated in a variety of experimental models. Alterations in the alf,3, a2f31, and a6/1 integrins following chemical transformation of osteosarcoma (HOS) cells resulted in increased invasiveness. 12 More recently, transfection of an a2 cDNA into the rhabdomyosarcoma cell line, RD, which normally
expresses no a291, resulted in a marked increase in the metastatic and invasive potential of the cells in vivo. i 1
Whether the changes in integrin expression documented in epithelial malignancies are secondary to the loss of the differentiated phenotype or whether malignant transformation directly alters integrin gene expression with resultant changes in differentiated phenotype and in the invasive or metastatic potential of the cells remains to be established. Our observation that similar levels of mRNA are present in both the intraductal and invasive components of a tumor suggests that changes in integrin regulation may be one step in progression of malignancy. If so, the role of the integrins in breast cancer may be that of a tumor suppressor gene like the DCC (deleted in colon carcinoma) gene, encoding a putative cell adhesion molecule, in colonic carcinoma.44
Acknowledgments We thank Dr. William C. Parks for helpful discussions.
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24. Kirchhofer D, Languino LR, Ruoslahti E, Pierschbacher MD: a231 integrins from different cell types show different binding specificities. J Biol Chem 1990, 265:615-618 25. Languino LR, Gehisen KR, Wayner E, Carter WG, Engvall E, Ruoslahti E: Endothelial cells use a231 integrin as a laminin receptor. J Cell Biol 1989, 109:2455-2462 26. Chammas R, Brentani R: Integrins and metastases: an overview. Tumor Biol 1991, 12:309-320 27. Dedhar S: Integrins and tumor invasion: Bioessays 1990, 12:583-590 28. Prosser IW, Stenmark KR, Suthar M, Crouch EC, Mecham RP, Parks WC: Regional heterogeneity of elastin and collagen gene expression in intralobar arteries in response to hypoxic pulmonary hypertension as demonstrated by in situ hybridization. Am J Pathol 1989, 135:1073-1088 29. Takada Y, Hemler ME: The primary structure of the VLA-2/collagen receptor a2 subunit (platelet GPla): homology to other integrins and the presence of a possible collagen-binding domain. J Cell Biol 1989, 109:397-407 30. Zutter MM, Fong AM, Krigman HR, Santoro SA: Differential regulation of the a2g3 and allb13 integrin genes
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