, . 183: 325–329 (1997)

Abl EXPRESSION IN HUMAN FETAL AND ADULTTISSUES, TUMOURS, AND TUMOUR MICROVESSELS

. . ’1, . . 2, . . 1 . . 1*1Department of Histopathology, St James’s Hospital and Trinity College, Dublin, Ireland

2Department of Biochemistry, University College, Cork, Ireland

SUMMARY

Abl kinases encoded by the abl oncogenes inhibit apoptosis without affecting cell proliferation. The aim of this study was to examinea wide range of normal fetal and adult human tissues and a variety of tumour types for Abl immunoreactivity. Sections from 193 paraffinblocks of normal fetal and adult tissues and 72 blocks from representative tumours were stained immunohistochemically using apolyclonal antibody to c-Abl/Bcr–Abl oncoprotein. Weak Abl immunoreactivity was observed in many adult tissues. Moderately intenseor strong staining (cytoplasmic, nuclear or membranous) was consistently seen in hyaline cartilage, adipocytes, and ciliated epithelium.In fetal tissues, there was a broadly similar staining pattern, but Abl expression was also seen in muscle (all types) and occasionally inendothelial cells. The most intense staining was seen in sites of endochondral ossification and in the umbilical cord stroma. Negativelystaining tissues included epidermis and squamous mucosa, lymph nodes, tonsil, spleen, hepatocytes, and adrenals. Most tumours showedfocal or weak Abl immunoreactivity. The most intense staining was seen in chondrosarcoma, liposarcoma, and diffuse gastric (signetring) adenocarcinoma. In the latter two tumour types, Abl expression was also observed in tumour microvessels. These results suggestthat Abl not only functions as an apoptosis inhibitor, but also may have a role in connective tissue maturation and differentiation andin tumour growth and angiogenesis. ? 1997 John Wiley & Sons, Ltd.

J. Pathol. 183: 325–329, 1997.No. of Figures: 7. No. of Tables: 1. No. of References: 22.

KEY WORDS—c-Abl; apoptosis; connective tissue differentiation; chondrocytes; fetal tissues; normal adult tissues; tumours; angiogenesis

INTRODUCTION

The Abl oncogenes—cellular homologues of the v-abloncogene of the Abelson murine leukaemia retrovirus—encode intracellular protein tyrosine kinases that arethought to have roles in signal transduction, cell cycleregulation, and inhibition of apoptosis.1–4 In 90 per centof patients with chronic myeloid leukaemia (CML), areciprocal translocation t(9;22)(q34;q11) of the c-ablgene from chromosome 9 to the breakpoint clusterregion (bcr) on chromosome 22 results in fusion of thebcr and abl genes.3 The resulting bcr–abl fusion gene onthe Philadelphia chromosome5 codes for chimeric Bcr–Abl fusion proteins, which have increased tyrosinekinase activity. 6 The activated Abl moiety is thought tobe responsible for the inhibition of apoptosis character-istic of the indolent phase of CML.7,8 Clonal expansionof mature myeloid cells is thought to be due to Abl-induced apoptosis inhibition rather than increased cellproliferation.3,4 Blast crisis is probably associated withadditional genetic events.4The precise functions of c-abl are poorly understood3

and there have been no detailed observations on Ablexpression in normal human tissues or non-haematopoietic tumours. The purpose of this study was

to examine a wide range of normal fetal and adulthuman tissues and a variety of different tumour types forAbl immunoreactivity.

MATERIALS AND METHODS

Tissue samplesNinety paraffin blocks of formalin-fixed normal adult

tissues were obtained from the files of the Department ofHistopathology, St James’s Hospital, for the purpose ofthis study. Most blocks were from surgical resectionspecimens, but minimally autolysed autopsy tissue wasused where necessary. In addition, 103 paraffin blocksfrom autopsies on six 20–23 week fetuses were madeavailable by Dr J. Gillan, Rotunda Hospital, Dublin. Inall, there were two to six blocks for each of a wide rangeof fetal and adult human tissues. Seventy-two paraffinblocks from various tumour types were sectioned for thesecond part of the study.

Immunohistochemistry

Sections (4 ìm) were cut and mounted on3-aminopropyltriethoxysilane-coated slides, oven-dried,deparaffinized in xylene, and rehydrated in alcohol.Sections were incubated with sheep anti-c-Abl/Bcr–Abloncoprotein antibody (polyclonal; Serotec, U.K.; 1/100dilution) for 1 h at room temperature and thenincubated with biotinylated rabbit anti-sheep antibody(Serotec, U.K.; 1/400), followed by peroxidase-conjugated streptavidin (Dakopatts, Denmark; 1/400).

Presented in part at the 173rd Meeting of the Pathological Societyof Great Britain and Ireland, Southampton, 11 July 1996.

*Correspondence to: Dr Eoin F. Gaffney, Department of Histo-pathology, St James’s Hospital, Dublin 8, Ireland.

Contract grant sponsors: Irish Association for Cancer Research;C.R.A.B. (Irish Cancer Society); Health Research Board.

CCC 0022–3417/97/110325–05 $17.50 Received 25 June 1996? 1997 John Wiley & Sons, Ltd. Accepted 3 June 1997

1 2

3

5 6

7

4

Figs 1–7.

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The reaction product was visualized with 3,3*-diaminobenzidene and a haematoxylin counterstain.Optimal immunoreactivity was determined by using

dilutions of the primary antibody between 1/10 (unac-ceptable background staining) and 1/400 (specific butweak staining). Sections of normal bone marrow con-taining immunoreactive mature myeloid cells were usedas positive controls. For negative controls, the primaryantibody was either omitted or preabsorbed with thepeptide (Serotec) against which it was directed. In thelatter case, immunoreactivity was completely ablated.Abl immunoreactivity in normal tissues and tumourswas assessed using a simple three-tiered scale: +, focal ordiffuse weak staining; ++, moderately intense staining;and +++, intense staining.

RESULTS

Expression of Abl in normal adult tissuesWeak or focal Abl expression was observed, as antici-

pated, in many normal cell types and tissues. Theseincluded small mucous glands and ductules, gastriccrypts, endocervix, ovarian follicles, breast myoepithe-lial cells, prostatic acini, renal tubules, transitional epi-thelium, skin adnexae, myeloid cells, and osteoblasts.Certain cell types, e.g., adipocytes and chondrocytes(Fig. 1), showed consistent strong staining, which wasablated by preabsorption with the immunizing peptide(Fig. 2). The staining pattern was consistent in each ofthe tissues examined. In most cell types, Abl immuno-reactivity was cytoplasmic. However, mucous cells andchondrocytes often had nuclear staining. There was verydistinctive staining of the apical aspect of ciliated epithe-lium in the Fallopian tube (Fig. 3), epididymis, and, to alesser extent, bronchi and vas deferens. Membranousstaining was characteristic of mature adipocytes andchondrocytes. There was no staining for Abl in eryth-roid precursors or megakaryocytes, neurones (Fig. 4),epidermis, squamous mucosa, smooth, cardiac, or skel-etal muscle, hepatocytes, thyroid, adrenals, renalglomeruli, blood vessels, lymph nodes, spleen, or tonsil.

Expression of Abl in fetal tissues

Fetal tissues showed a broadly similar pattern of Ablimmunoreactivity. There was, in addition, weak or focalstaining of squamous mucosa, sex cords, adrenal cortexand medulla, small intestinal and colonic epithelium,skeletal, cardiac, and smooth muscle, capillary endothe-

lial cells, and endocardial intima. Developing whiteadipose tissue was moderately stained. As in adult tissue,chondrocytes were strongly stained. The most strikingAbl immunoreactivity was seen in osteoblasts andtheir associated neovasculature in sites of endochondralossification (Fig. 5). This was associated with greatlydiminished Abl staining in adjacent hypertrophicchondrocytes, and (very infrequent) chondrocyte apop-tosis. Umbilical cord blood vessels and Wharton’s jellyfibroblasts were also moderately to intensely stained(Fig. 6), as were the amnion and chorion. There was nostaining for Abl in fetal skin, hepatocytes, thyroid,neurones, or metanephric blastema.

Expression of Abl in tumours

Most tumours showed weak Abl immunoreactivity(Table I). The most intensely staining tumours wereliposarcoma, chondrosarcoma, and diffuse (signet ring)carcinoma of the stomach. In signet ring carcinoma,colloid carcinoma, liposarcoma, and pleomorphicadenoma, Abl expression was also observed to a vari-able extent in tumour microvessels (Fig. 7). Tumourmicrovessel staining was generally more prominent inless cellular, mucinous or myxoid areas. In myxoidliposarcoma, the fibroblast-like pre-adipocytes wereunstained, whereas lipoblasts, adipocytes, and theirintimately associated vasculature showed Abl expres-sion. Tissues and structures that normally showed nostaining for Abl (colonic crypts, breast lobular epithe-lium, visceral smooth muscle, and small autonomicnerves) occasionally showed Abl immunoreactivity inthe vicinity of Abl-positive tumour cells.

DISCUSSION

In this immunohistochemical study we found that Ablis weakly or focally expressed in many different humantissues, as it is in murine tissues.9,10 However, intenseAbl immunoreactivity was virtually restricted to certainconnective tissue cells, such as adipocytes, chondrocytes,and umbilical cord fibroblasts. In the fetus, endothelialcells and osteoblasts in the growth plates of immaturelong bones were intensely stained. Small depots ofdeveloping white adipose tissue11 also showed moderateAbl expression. Cytoplasmic staining was more commonthan nuclear staining, but nuclear staining was seen inaddition to cytoplasmic staining in mucous cells and

Fig. 1—Individual chondrocytes show intense nuclear, membranous or cytoplasmic Abl immunoreactivity in adult tracheal cartilage

Fig. 2—Negative control (the primary antibody is preabsorbed with the immunizing peptide)

Fig. 3—Abl immunoreactivity is confined to the apical surface of ciliated epithelium in adult Fallopian tube

Fig. 4—Absence of Abl immunoreactivity in adult cerebral cortex

Fig. 5—Intense Abl immunoreactivity in osteoblasts and proliferating blood vessels in endochondral ossification of fetal rib. Adjacent hypertrophicchondrocytes, above, show minimal or no staining, and one (left of centre, adjacent to osteoblasts) has undergone apoptosis

Fig. 6—Umbilical cord fibroblasts of Wharton’s jelly show variable Abl expression

Fig. 7—Intense Abl expression in both tumour cells and microvessels in gastric adenocarcinoma, diffuse type

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chondrocytes. In ciliated epithelial cells of the Fallopiantube, bronchus, epididymis, and vas deferens, there wasdistinctive apical cell membrane staining, possibly theresult of N-terminal myristoylation of c-Abl protein.12Nuclear immunoreactivity represents localization ofc-Abl,1,12,13 whereas cytoplasmic staining might theo-retically represent either c-Abl or Bcr–Abl.12 However,there is no evidence that the bcr–abl translocation occursin the normal tissues that we examined.Intense Abl expression in differentiating chondro-

cytes, growth plates, adipose cells, and the umbilicalcord strongly suggests hitherto undescribed functionsfor Abl in the differentiation and survival of specificconnective tissues. During endochondral ossification,chondrocytes hypertrophy, proliferate, and secreteabundant matrix and undergo apoptosis. This coincideswith osteoblast proliferation and vascular invasion.14 Itis likely that Abl, normally inhibits apoptosis inchondrocytes because we found that Abl expression,although conspicuous in both fetal and adult chondro-cytes, is greatly diminished or absent in hypertrophicchondrocytes about to undergo apoptosis at growthplates. Although positive immunostaining does notimply specific functional activities, strong Abl stainingin proliferating osteoblasts and associated vasculaturesuggests a complementary survival role for Abl in thegrowth plate itself.

Abl was strongly expressed in liposarcoma andchondrosarcoma, tumours in which there is negligibleapoptosis15 and a very low percentage of proliferatingcells.16,17 However, Abl was also strongly expressed indiffuse (signet ring) adenocarcinoma of the stomachand, less intensely, in infiltrating breast carcinoma andangiosarcoma, each of which has an intermediate levelof apoptosis.15 We found no significant differencebetween apoptotic indices in diffuse adenocarcinomaand intestinal-type gastric adenocarcinoma, or betweensignet ring cell and undifferentiated areas in diffusegastric adenocarcinoma (data not shown). Expression ofa single apoptosis-modulating oncogene/suppressor geneproduct does not always correlate with net apoptosisin tumours,18–20 and a contributory direct or indirectrole for Abl in the regulation of apoptosis innon-haematopoietic tumours cannot be excluded.Abl expression in tumour microvessels of diffuse

gastric adenocarcinoma and liposarcoma was an unex-pected finding. Abl immunoreactivity was not seen innormal blood vessels in adult tissues, but Abl stainingwas seen occasionally in certain fetal capillary endo-thelial cells, most notably in proliferating vascular chan-nels in sites of endochondral ossification, and also inumbilical cord blood vessels. This raises the possibilitythat Abl, like certain other oncogene products,21 may bedirectly or indirectly angiogenic. The other putativefunctions of Abl in connective tissues—cell differentia-tion and inhibition of apoptosis—have been reported tobe linked with the two predominant c-abl isoforms.22 Wetherefore plan to extend the present immunohistochemi-cal observations and investigate more fully the potentialfunctions of Abl in connective tissues and tumourangiogenesis using in situ and PCR techniques.

ACKNOWLEDGEMENTS

This work was supported by an Irish Association forCancer Research Studentship to Amanda O’Neill and byC.R.A.B. (Irish Cancer Society). Jennifer Russell issupported by the Health Research Board (Ireland). Weare very grateful to Professor Andrew Wyllie for hiscomments and for invaluable assistance with relatedwork. We wish to thank Dr John O’Leary for practicaladvice and assistance in applying PCR techniques. MrsK. Kiely typed the manuscript.

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Table I—Abl expression in tumours

Tumour typeAbl

expressionNo. ofcases

Breast, infiltrating lobular carcinoma +/++ 3/3Breast, infiltrating ductal carcinoma +/++ 3/3Breast, in situ ductal carcinoma + 1/1Ovarian serous carcinoma + 4/4Ovarian small cell carcinoma " 0/1Gastric adenocarcinoma, diffuse type ++ 6/6Gastric adenocarcinoma, intestinal type + 5/5Colonic adenocarcinoma + 6/6Lung, squamous cell carcinoma + 3/3Lung, adenocarcinoma + 1/1Renal clear cell carcinoma + 3/3Renal cell carcinoma, papillary " 0/2Bladder transitional cell carcinoma + 4/4Seminoma + 1/6Chronic myeloid leukaemia + 2/2Chronic lymphocytic leukaemia " 0/2Acute myeloid leukaemia + 1/1Burkitt’s lymphoma " 0/1Immunoblastic lymphoma + 1/1Liposarcoma ++/+++ 2/2Chondrosarcoma ++/+++ 2/2Angiosarcoma +/++ 2/2

MiscellaneousPleomorphic adenoma ++ 3/3Haemangioma + 4/4Granulation tissue "/+ 4/4

Total 72

"=no staining; +=weak focal or diffuse staining; ++=moderatestaining; +++=intense staining.

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