[CANCER RESEARCH 44, 2942-2946, July 1984]
Presence of a-2-Macroglobulin in Normal but not in Malignant Cervical Epithelium1
Olii Saksela,2 Torsten Wahlström, Bengt Meyer, and Antti Vaheri
Departments of Virology [0. S., A. V.], Pathology [T. W.], and Obstetrics and Gynecology [B. M.], University of Helsinki, 00290 Helsinki, Finland
ABSTRACT obtained from surgical preparations after hysterectomy performed be cause of benign tumors of the uterus. Informed consent for further study o-2-Macroglobulin («2M)was demonstrated in normal and of the removed tissue was obtained from the patients. Cell cultures were mildly dysplastic cervical epithelium using immunohistochemical prepared as described in detail elsewhere (22). Briefly, the ectocervical part of the epithelium was detached and cut in 1- to 2-mm pieces. methods. In contrast, staining for «2Mdiminished gradually with Epithelial cells grew out from the expiants in Eagle's minimal essential advancing epithelial dysplasia and was totally negative in truly medium supplemented with 10% fetal bovine serum and formed a neoplastic lesions. No in vitro synthesis could be detected in confluent monolayer on plastic Retri dishes within 1 to 2 weeks. After cultured ectocervical cells or in a corresponding malignant cell that, the tissue pieces were removed, and the cell cultures were used line (HeLa). However, «2Mwas taken up from added human for experiments. The cervical carcinoma cell line (HeLa, ATCC CCL2; serum by cultured normal ectocervical cells, whereas HeLa cells American Type Culture Collection, Rockville, MD) and a fetal lung fibre- remained negative also under these conditions. These and pre blast cell line (WI-38, CCL 75) were also studied. For the uptake experi vious results suggest that normal cells may have the capacity to ments, the cells were maintained for 4 h in minimal essential medium synthesize or endocytose «2M,thus possibly regulating their supplemented with 10% human serum. All cell cultures were kept in a humidified 5% CO2 atmosphere at 37°.For staining, the cells were grown pericellular proteolysis, while these functions may be defective in malignantly transformed cells. on glass coverslips. Antiserum. Rabbit antiserum to human «2Mwas from a commercial source (Behringwerke, Marburg/Lahn, Federal Republic of Germany). Its INTRODUCTION specificity was verified by crossed immunoelectrophoresis against nor mal human plasma and by removing its reactivity with agarose-coupled «2M3is a high-molecular-weight wide-spectrum endoprotei- a2M purified as described previously (16). The antiserum used for staining nase inhibitor normally found in plasma in relatively high concen of cultured cells was first absorbed with fetal bovine serum to remove trations (20). It is also present in other body fluids (lymph, pleural, its reactivity with bovine a2M. synovial, and amniotic fluids), indicating a role in control of Immunohistochemical Techniques. The tissue sections were stained proteolysis also in the extracellular space. The liver is considered using a modification of the immunoperoxidase-staining procedure (19, to be the main source of the circulating a2M. However, in culture 23). In short, the sections were deparaffinized in xylene, their endoge conditions, human embryonic lung fibroblasts (16), blood mono- nous peroxidase activity was extinguished by incubation in methanol containing 0.3% hydrogen peroxide, and they were washed in PBS cyte-macrophages (6), and alveolar macrophages (24) also have containing 2% normal rabbit serum. The sections were incubated in a been shown to synthesize it, suggesting that a2M may have a moist chamber at 37°for 30 min with the antiserum to «2M(diluted role at the cellular level. 1:100), washed as above, and incubated at 37°for 30 min with swine Several cell types possess membrane receptors for «2Mor anti-rabbit IgG serum (diluted 1:50, Lot 069A; Dako, Copenhagen, Den a2M:proteinase complexes (10, 25). Fibroblasts have been mark). After washing, the sections were incubated at 37°for 30 min with shown to endocytose «2Mtogether with epidermal growth factor horseradish peroxidase:rabbit anti-peroxidase IgG complex (diluted and insulin (14), and macrophages are obviously responsible for 1:500, Lot 019; Dako) containing 2% normal swine serum and, subse the rapid removal of «2M:proteinase complexes from the circu quently, washed in PBS. The chromogenic substrate was 3,3-diaminob- lation. a2M has been detected inside certain cells in vivo, namely, enzidine tetrahydrochloride. Adjacent sections were stained similarly with in the endothelium of the vessel walls (3) and the syncytiotro- the fully absorbed antiserum, and all other appropriate controls as defined by De Lellis ef al. (5) were included. phoblasts of the placenta (19). <*2Mwas, however, not found in For immunofluorescence staining, the cultured cells on glass cover- invasive moles or choriocarcinomas. These findings led us to slips were washed with cold PBS and fixed with 3% paraformaldehyde study «2Min uterine cervical lesions of different stages of dys for 20 min. After a second wash, the cells were treated with 0.05% plasia together with the corresponding cell culture model. Triton X-100 to expose intracellular structures. The indirect immunoflu orescence staining technique was utilized as described (2). Control preparations were stained with antiserum absorbed with purified human MATERIALS AND METHODS «2Mand nonimmune rabbit serum. Tissue Sections. Sections 5 ng thick were prepared from formalin- Immunoprecipitation and Ammonium Sulfate Precipitation of Pro teins Labeled with [35S]Methionine. For metabolic labeling, epithelial fixed paraffin-embedded routine tissue samples taken at colposcopy cell cultures were used as confluent 1- to 2-week-old monolayers. WI- from the uterine cervix for histological examination. Cell Cultures. Tissue for cultivation of normal ectocervical cells was 38 cells were labeled as subconfluent 3 days after subculture. Growth medium was removed, and the cells were washed with PBS and incu bated in methionine-free Eagle's minimal essential medium containing 1This work was supported by the Medical Research Council of the Academy of Finland, the Finnish Cancer Foundation, and the Emil Aaltonen Foundation. 0.05% bovine serum albumin and 10 ¿¿CiL-^SJmethionine/ml (800 Ci/ 2To whom requests for reprints should be addressed, at Department of Virology, mmol; Amersham, United Kingdom) for 12 hr. After centrifugation of the University of Helsinki, Haartmaninkatu 3, SF-00290 Helsinki, Finland. 3The abbreviations used are: ajM, a-2-macroglobulin; PBS, phosphate-buffered medium at 2000 x g for 20 min, ammonium sulfate precipitation and saline (0.01 M sodium phosphate, 0.14 M sodium chloride, pH 7.4). immunoprecipitation were carried out as described (19). The samples Received October 19, 1983; accepted March 20,1984. were analyzed by electrophoresis in a discontinuous sodium dodecyl
2942 CANCER RESEARCH VOL. 44
Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1984 American Association for Cancer Research. Presence of azM Cervical Epithelium sulfate: polyacrylamide (6 to 12%) slab gel followed by autoradiography Table 1 (12). ciiM in normal, dysplastic, and malignant lesions of the uterine cervical epithelium Radioimmunoassay of «2M.Human a2M was purified as described of a2MPositive43for earlier (16) and ¡odinatedaccording to the method of Krohn eÃal. (11). cases The reaction mixture contained 200 ¡¡\ofprotein solution (3 mg/ml in TissueNormal studied43 PBS), 10 n\ of carrier-free Na125l(1 mCi), and 25 M!of 0.7 mw chloramine- epithelium T freshly dissolved in 0.4 M sodium phosphate, pH 7.8. Immediately Mild dysplasia 10 6 4 Moderate dysplasia after addition of chloramine-T, the reaction mixture was chromato- 9 3 6 Severe dysplasia 11 1 10 graphed on Sephadex G-25. Void volume fractions were pooled and Carcinoma in situ 10 0 10 diluted with PBS containing bovine serum albumin (1 mg/ml). The anti- Invasive epidermoid carcinoma 13 0 13 serum was absorbed with fetal bovine serum to remove reactivity with CondylomasNo. 11Staining 11Negative0 0 bovine «2Mand tested for monospecificity as described above. Eighty % of the ¡odinatedprotein was precipitated by the absorbed antiserum. The dose-dependent curve for a2M in this assay was linear between 10 diffuse readily to the extravascular space. The relatively heavy and 300 ng/ml. «2Mis sometimes precipitated also with nonspecific staining of the protein in the normal cervical epithelium suggests immune complexes (13). This could be avoided by using a solid-phase local synthesis inside the epithelium. In culture, the proliferating second antibody (Dasp Anti-rabbit; Organon, Oss, The Netherlands) for ectocervical cells presumably represent the basal cell layer of precipitation of the primary immune complexes. the stratified epithelium. Under culture conditions, extensive multilayer structures do not seem to be formed. Thus, the in RESULTS vitro model may not adequately represent the in vivo situation, and direct conclusions regarding the functions and activities of In the immunoperoxidase staining for a2M a distinct intracy- the normal epithelium cannot be drawn on the basis of the in toplasmic stain was seen in all samples containing normal cer vitro findings, a conclusion also made in studies on the produc vical epithelium, in 6 of 10 cases of mild dysplasia, in 3 of 9 of tion of extracellular matrix components by these cells (1). The moderate dysplasia, and in 1 of 11 of severe dysplasia. In the lack of measurable a2M synthesis in cultures of normal cervical normal epithelium, most cells, except for the basal 2 to 3 layers, epithelium could thus be due to a lower degree of differentiation were positive. The stain was seen to be totally absent from all of the cells in vitro. cases of invasive epidermoid carcinoma (Table 1). Preparations Uptake of o2M has been demonstrated in cultures of various stained with «2M-absorbedantiserum or nonimmune serum were normal types of cells (15,17,19) but not in cultures of malignant negative. The demarcation at the border between the dysplastic cells such as fibrosarcoma, rhabdomyosarcoma (26), osteosar- and normal epithelia (Fig. 1, A and C) was clear-cut. Benign coma, lung carcinoma cells (21), or cells transformed by DMA or condylomas stained positively for a2M. As expected, «2Mwas RNA viruses (17, 18). Similarly, HeLa cells, unlike their normal also seen in the subepithelial blood and lymph vessels. counterparts human cervical epithelial cells, did not take up «2M No synthesis of a2M was detected in the ectocervical epithelial from culture medium. Thus, the possibility that the differential cells under culture conditions. Immunoprecipitation of «2Mfrom uptake of «2Mbetween normal and malignant cells could explain the growth medium did not reveal any radioactivity labeled our in vivo results cannot be excluded. If so, it can be speculated protein (data not shown), nor did the radioimmunoassay for a2M that the proliferating cells, such as the HeLa cells, lack the show any accumulation of the protein in the growth medium. functional receptors for «2Muptake. Immunofluorescent staining for a2M was also negative in the The amount of a2M decreased with advancing architectural epithelial cells (Fig. 2). On the contrary, all 3 methods, in agree disturbances in the epithelium and with neoplastic changes of its ment with previous findings (16), demonstrated synthesis of a2M cells. Cervical epithelial cells are reported to be proteolytically in the WI-38 lung fibroblast cell line (about 200 ng of «2M/mgof active (4, 9), and the lack of <>-.Minpotentially neoplastic lesions cellular protein during 24 hr). may reflect the commonly observed association of proteolytic Uptake to the cells of added human «2Mfrom the culture activity with malignant transformation. Whether this results from medium could be demonstrated in the cultured normal cervical altered synthesis or defective uptake by the malignant cells cells but not in the corresponding malignant cell line (HeLa), remains to be solved. although in a lower degree than in fibroblasts (Fig. 2). The possible functions of «2Maccumulating in the normal cervical epithelium are only a matter of speculation. a2M is known DISCUSSION to inhibit a wide variety of microbial proteinases of invasive pathogens of Pseudomonas aeruginosa, Staphylococcus aureus, In this paper, we demonstrate a2M in normal human uterine and Proteus vulgaris. It is also the only plasma inhibitor of cervical epithelium in vivo. In invasive epidermoid cervical carci collagenase of Clostridium histolyticum and the keratinase of noma or carcinoma in situ, this proteinase inhibitor was com Trichophyton mentagrophytes (20). The a2M acute-phase protein pletely absent. No synthesis of a2M was detected in cultures of found in some species (19) has no apparent counterpart in the ectocervical cells or HeLa cells. humans. The human «2Mpresent, e.g., in cervical epithelium, Earlier studies on the distribution of «2Min human tissues could represent a locally active defense protein, which does not describe the presence of this proteinase inhibitor in several body affect the overall plasma level and thus passes unnoticed. Re fluids (20), endothelium of the vessel walls (3), and the syncytio- cently, a role for plasminogen and plasminogen activator in trophoblast layer of the placenta (19). Certain cell types synthe differentiation of squamous epithelium has been suggested (7, size the protein in vitro, e.g., blood monocyte macrophages, 8), which indicates that control of these proteolytical events is alveolar macrophages, and lung fibroblasts (6,16, 24). needed also. In the cervical epithelium, <*2Mis a proper candidate Being a high-molecular-weight protein, «2Mis not likely to for such a function.
JULY 1984 2943
Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1984 American Association for Cancer Research. O. Saksela et al.
14. Maxfield, F. R., Schlessinger, J., Schecter, Y., Pastan, I., and Willingham, M. ACKNOWLEDGMENTS C. Collection of insulin, EGF, and a2-macroglobulin in the same patches on the surface of cultured fibroblasts and common intemalization. Cell, 74: 805-810, We thank Anja Virtanen, Tuula Halmesvaara, and Anne Brisk for expert technical 1978. and secretarial assistance. 15. Maxfield, F. R., Willingham, M. C., Haigler, H. T., Dragsten, P., and Pastan, I. H. Binding, surface mobility, intemalization, and degradation of rhodamine- REFERENCES labeled a2-macroglobulin. Biochemistry, 20: 5353-5358,1981. 16. Mosher, D. F., Saksela, O., and Vaheri, A. Synthesis and secretion of alpha- 1. Alitalo, K., Halila, H., Vesterinen, E., and Vahen, A. Ende- and ectocervical 2-macroglobulin by cultured adherent lung cells. J. Clin. Invest., 60: 1036- human uterine epithelial cells distinguished by fibronectin production and 1045,1977. keratinization in culture. Cancer Res., 42: 1142-1145,1982. 17. Mosher, D. F., and Vaheri, A. Binding degradation of alpha-2-macroglobulin by 2. Alitalo, K., Kurkinen, M. Vaheri, A., Krieg, T., and Timpl, R. Extracellular matrix cultured fibroblasts. Biochim. Biophys. Acta, 647: 113-120, 1980. components produced by human amniotic epithelial cells in cultures. Cell, 79: 18. Pastan, I., Willingham, M., Anderson, W., and Gallo, M. Localization of serum- 1053-1062, 1980. derived aa-macroglobulin in cultured cells and decrease after Moloney sarcoma 3. Becker, C. G., and Harpel, P. C. «j-Macroglobulin on human vascular endo- virus transformation. Cell, 72: 609-617,1977. thelium. J. Exp. Med., 744: 1-9, 1976. 19. Saksela, O., Wahlström, T., Lehtovirta, P., Seppälä,M.,and Vaheri, A. Pres 4. Bigbee, W. L, and Jensen, R. H. Characterization of plasminogen activate in ence of a2-macroglobulin in normal but not in malignant human syncytiotro- human cervical cells. Biochim. Biophys. Acta, 540: 285-294, 1978. phoblasts. Cancer Res., 47: 2507-2513,1981. 5. De Lellis, R. A., Stemberger, L. A., Mann, R. B., Banks, P. M., and Nakane, 20. Starkey, P. M., and Barrett, A. J. az-Macroglobulin, a physiological regulator P. K. Immunoperoxidase techniques in diagnostic pathology. Am. J. Clin. of proteinase activity. In: A. J. Barrett (ed.), Proteinases in Mammalian Cells Pathol., 77:483-488, 1979. and Tissues, pp. 663-696. Amsterdam: Elsevier/North-Holland Biomédical 6. Hovi, T., Mosher, D., and Vaheri, A. Cultured human monocytes synthesize Press, 1977. and secrete
Fig. 1. Immunoperoxidase staining of the ectocervical squamous epithelium of «2M.The arrow indicates demarcation between the severely dysplastic lesion (left) and the metaplastic squamous epithelium. Staining of «zMis seen in the normal epithelium (c) and in the underlying vessels (c and d), but not in the severely dysplastic (c and d) epithelium, a and b, hematoxylin:eosin staining; c and d, peroxidase:antiperoxidase staining; e and /, control staining with nonimmune serum, a, c, and e are the same field as are b, d, and 1. Original magnifications: x 250 (a, c, e); x 400 (b, d, r).
2944 CANCER RESEARCH VOL. 44
Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1984 American Association for Cancer Research. 2945 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1984 American Association for Cancer Research. O. Saksela et al.
Fig. 2. Immunofluorescent staining of cultured cells for a,M. WI-38 fibroblasts show a diffuse cytoplasmic fluoresœnce (a), whereas the cervical epithelial cells are negative (6). After incubation of the cells with human serum, vacudar fluorescence indicating endocytosis in seen in both WI-38 cells (c) and cervical epithelial cells (d) HeLa cells remained negative, also, under these conditions (r). e, control staining of WI-38 cells with nonimmune serum x 1000
2946 CANCER RESEARCH VOL. 44
Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1984 American Association for Cancer Research. Presence of α-2-Macroglobulin in Normal but not in Malignant Cervical Epithelium
Olli Saksela, Torsten Wahlström, Bengt Meyer, et al.
Cancer Res 1984;44:2942-2946.
Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/44/7/2942
E-mail alerts Sign up to receive free email-alerts related to this article or journal.
Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].
Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/44/7/2942. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.
Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1984 American Association for Cancer Research.