- Email: [email protected]
The role of fibronectin in adhesion of metastatic melanoma cells to endothelial cells and their basal lamina
Preliminary 23. Beebe, 24.
D C, Feagans, D E, Blanchette-Mackie. E J & Nau. M E, Science 206 (1979) 836. Mousa. G Y & Trevithick, J R. Dev biol 60 (1977)
14. 25. Ramaekers.
F C S, Hukkelhoven, M W A C. A & Bloemendal. H. Ophthal res 11
Groeneveld,
(1980) 283.
26. Wessels, 27. 28. 29.
N K, Spooner. B S. Ash, J F, Bradley, M 0, Luduena, M A, Taylor. E L, Wrenn. J T & Yamada, K M, Science 171 (1971) 135. Blikstad, 1, Markey, F, Carlsson. L, Persson. T & Lindberg, U, Cell 15 (1978) 135. Lowry, 0 H, Rosebrough, N J. Farr, A L & Randall, R J, J biol them 193 (1951) 265. Dulley, J R & Grieve. P A. Anal biochem 64 (1975)
136. 30. Laemmli, U K. Nature 227 (1970) 680. 31. Weber, K & Osborn. M, J biol them 244 (1969) 4406. 32. O’Farrell, P H. J biol them 250 (1975) 4007. Received November 11, 1980 Revised version received May Accepted May 27, 1981
15, 1981
CopyrIght 0 1981 h) Academic Pre\\. Inc. All right\ of reproductwn in any form rewrved ~XlI4-4X?7.‘XI:loll4hI-n~‘;02.0oio
The role of fihronectin in adhesion of metastatic melanoma cells to endothelial cells and their basal lamina GARTH ROBERT LAHTI,2
L.
NICOLSON,’ GONZALEZ’,
*
TATSURO IRIMURA,’ and ERKKI RUOS-
‘Deoartmeni of‘ Tumor Biolopy, The University of Texk System dower Center. k. D. Anderson HosDituI and Tumor Institute. Houston, TX 77030. und ‘Ln’Jolla Crrncrr Resrnrch Foundation. La Jolla. CA 92038, USA Summury. Vascular
endothelial cells synthesize an extracellular matrix or basal lamina composed of collagens, proteoglycans and glycoproteins such as fibronectin (FN). Using affinity-purified anti-FN, we have examined the role of FN in adherence of metastatic B16 melanoma cells to endothelial cell monolayers which lack FN on apical cell surfaces and to their basal lamina which contains FN. B16 melanoma ceils, which do not contain significant amounts of FN. attached at much higher rates to endothelial basal lamina and polyvinyl-immobilized FN compared with intact endothelial cell monolayers. Anti-FN failed to inhibit attachment of melanoma sublines of low (B16-Fl) or high (B16-FlO) metastatic potential to intact endothelial cell monolayers, inhibited slightly B16 cell attachment to basal- lamina and completely abolished attachment of Bl6 cells to polyvinyl-immobilized FN. The antibiotic tunicamycin which inhibits glycosylation of B16 cell surface glycoproteins and blocks experimental metastasis [18] inhibited B16 attachment
notes
461
to endothelial cells, basal lamina and immobilized FN. The results suggest that FN mediates, only in part, the adhesion of B16 melanoma cells to basal lamina through glycoprotein receptors on Bl6 cells.
Crucial steps in blood-borne tumor metastasis are the arrest of malignant cells in the microcirculation, often in specific organs [1], and subsequent extravasation or invasion of surrounding endothelium and escape to extravascular sites [2-41. Metastatic tumor cells and invasive normal cells such as leukocytes attach to endothelial monolayers, cause rupture of endothelial cell-cell interactions and retraction of the endothelial cells from their underlying basal lamina, whereupon the invasive cells move from the endothelial cells and bind tightly to basal lamina [S-7]. Eventually the metastatic cells solubilize components such as fibronectin (FN) and sulfated proteoglycans in the endothelial cell basal lamina [8]. We have developed an in vitro model for extravasation [5, 61 that utilizes vascular endothelial cell monolayers which synthesize a basal lamina containing collagens [9, lo], sulfated proteoglycans and glycosaminoglycans [S, 111 and glycoproteins such as FN [9, 121 and laminin [13]. We report here that attachment of B16 melanoma cells to endothelial cell basal lamina (but not to endothelial cells) appears to be mediated, in part, by FN but also by other adhesive molecules in the extracellular matrix. Materials
and Methods
Cell cultures. Murine melanoma sublines selected once (B16-Fl) or ten times (Bl6-FlO) for blood-borne lung implantation. survival and growth [14] were obtained from Dr I. J. Fidler (NCI-Frederick Cancer Research Center, Frederick, Md) and were grown in Dulbeccomodified Eagle’s minimum essential medium (Gibco, Grand Island, N.Y.) supplemented with I % nonessential amino acids (Gibco) (complete medium), and 5 % heat-inactivated fetal bovine serum (Flow Laboratories, Inc., Inglewood. Calif.). Low passage cell cul* Present address: Cancer Research Institute, University of California, San Francisco. CA 94143, USA.
462
Preliminary
60
notes
ta
I
0’
I 10
0
1 20
I / 30 40 Time (mid
I 50
I
Fig. 1. Kinetics of adhesion of B16 melanoma cells to (a) vascular endothelial cell monolayer; or(b) endothelial cell basal lamina in the absence or presence of afftnitv mnified anti-tibronectin. O-O, B16-FlO; O-O, Bi&Fl; A-A, B16-FlO, monolayer or matrix pretreated with anti-tibronectin (400 Fg/ml); A-A, B16-FI, monolayer or matrix pretreated with antitibronectin (400 pg/ml).
tures (<8 passages) were grown to subcontluency and harvested by 10-15 min treatment with 2 mM EDTA in Ca’+-, Mg2+-free Dulbecco’s phosphate-buffered saline (DPBS). Cell viabilities were determined by Trypan blue ‘exclusion, and cell lines were tested routinely for mycoplasma by use of Hoechst stain 33258 [15]. Bovine aortic endothelial cells were obtained from Dr D. Gosnodarowicz (Universitv of California, San Francisco)tl6] and were cultured-in alphamodified minimum essential medium (Gibco) supplemented with 10% calf serum (Gibco). Fibroblast growth factor was ouritied as described f171 and added lo endothelial cells every other day ai a-concentration of 100-500 rig/ml until confluency and at 5 rig/ml thereafter. For the adhesion assays endothelial cells were grown to confluency in 24-well Costar tissue culture dishes [7]. Tunicamycin treatment. Tunicamycin (lot 177382) was obtained from the US National Cancer Institute, solubilized in 10 mM sodium hydroxide at a concentration of 2 mg/ml and kept at -20°C for up to 3 weeks. Immediately before each experiment the tunicamycin solution was diluted into culture medium which was then sterilized by passing through Acrodisc (0.2 pm; Gelman, Ann Arbor, Mich.). From the results of dose-response experiments described elsewhere for B16 melanoma cells f181 a concentration of 0.5 pg/ml was used during a-26 h incubation. At the end of the tunicamycin treatment cell viability remained 90-95 %. Exp Cd
Res
135 (1981)
Adhesion crsscrq’s.B16 melanoma cells were radiolabeled with 0.25 &i Na[J’Cr]O, (carrier-free in sterile saline solution; New England Nuclear) per T-75 tissue culture flask for 3 h in complete medium. 816 cells (2-4xlO”lflask) were harvested with 2 mM EDTA in Ca’+-, Mg”-free DPBS, washed three times with complete medium containing I % bovine serum albumin (BSA). pH 7.4, and suspended in the same solution at a concentration of 4~ 10” cells/ml. Endothelial cells were washed carefully with complete medium containing I % BSA, pH 7.4, 30 min prior to the assays. Endothelial basal lamina was .prepared as described previously [7]. Bovine plasma fibronectin puritied from calf serum (Irvine Scientific, Irvine, Calif.) according to Engvall & Ruoslahti [I93 was used for coating polyvinyl dishes (Linbro, Hamden. Conn.), as described elsewhere [7]. Tumor cell suspensions were allowed to settle for 1 min on endothelial monolayers, endothelial basal lamina or polyvinyl-immobilized tibronectin and were then incubated with or without agitation at 37°C. At given time intervals the samoles were washed eentlv 3 times with 0.5 ml complete medium containmg I-% BSA, pH 7.4, at 37°C. Adhering “‘Cr-labeled B16 cells were counted in a Beckman Model 8000 automatic gamma counter. Afifnitv mrrified trnti-bovine fibuonrctin. Bovine FN was purified by affinity chromatography on Sepharoseimmobilized gelatin as described previously [l9]. Antibodies were purified from antisera prepared against purified FN by affinity chromatography on Sepharose-iminobilized bovine FN with or without prior adsorption on Sepharose-immobilized murine FN. The affinity-purified anti-bovine FN was eluted from Sepharose-immobilized bovine FN in glycine-HCI buffer, pH 2.6 as described [20, 211. Affinity-purified anti-FN in DPBS was radiolabeled with lrJI using the chloroamine-T method [22]. AntiFN binding assays were performed with ‘Y-labeled anti-FN as described elsewhere [23]. These studies indicated that basal lamina bind a maximum amount of anti-FN (initial cont. 400 pg/ml) during a 45 min incubation at 37°C; therefore, all subsequent experiments utilized this concentration of anti-FN. .,”
,,
I
Results Murine melanoma sublines B 16-F 1 and B 16FlO attached without shear at similar rates to intact endothelial cell monolayers such that after 60 min approx. 50% of the cells were adherent and not removed during the subsequent washing steps (fig. 1a). Preincubation of endothelial cell monolayers with affinity-purified anti-FN (300 pg/ml) for 45 min at 37°C had little effect on B16Fl or Bl6-FlO cell adhesion (fig. la). The B16 melanoma cells adhered at much faster rates to isolated endothelial basal lamina such that by 20-24 min >90% of the cells
Preliminag
notes
463
0
20 30 40 Time (min) Fig. 2. Kinetics of adhesion of Bl6-Fl melanoma cells to various substratum. O-O, Polyvinyl-immobilized tibronectin; O-0, polyvinyl-immobilized tibronectin in the presence of 400 pg/ml anti-tibronectin; A-A, polyvinyl-immobilized bovine serum albumin; O-0. polyvinyl.
10 20 Time (min) Fig. .3. Kinetics of adhesion of untreated and tunicamycin-treated melanoma cells to endothelial cell basal lamina. O-O, Bl6-FlO; O-O, B16-Fl without tunicamycin treatment; A-A, B16-FlO; A-A, Bl6-Fl with tunicamycin (0.5 Fg/ml) treatment for 24 h.
were tightly adherent (fig. 16) similar to previous data [7]. When endothelial cell basal lamina was pre-incubated with antiFN as before, the kinetics of B16 cell adhesion were reduced slightly (approx. 2030% initial inhibition in four separate experiments) (fig. 1 b) suggesting that matrix FN may mediate, in part, the adhesion of B16 melanoma cells to endothelial cell basal lamina. Dose-response experiments indicated that higher concentrations of anti-FN were no more effective in inhibiting B16 cell attachment to endothelial basal lamina. Essentially no difference in cell attachment rates were found between sublines B16-Fl and B16-FlO under non-shear conditions (fig. la, h). The kinetics of B16-Fl melanoma attachment to polyvinyl-immobilized FN were the same as for B16-Fl adhesion to endothelial basal lamina (fig. 2); however, anti-FN abolished completely B16-Fl cell attachment to the polyvinyl-immobilized FN. Polyvinyl surfaces or polyvinylimmobilized BSA failed to bind B16 cells (fig. 2). Tunicamycin pretreatment of B16 melanoma cells modified their adhesive behavior
without modifying cell viability [18]. When B16 cells were incubated with 0.5 pglml tunicamycin for 24 h, their attachment rates to both endothelial basal lamina and polyvinyl-immobilized FN were dramatically reduced (fig. 3) suggesting that tunicamycinsensitive glycoproteins on B16 cells bind to FN and other endothelial basal lamina components.
“0
IO
0
Discussion The apical surfaces of confluent endothelial cell monolayers were found to be much less adhesive to malignant melanoma cells than the basal lamina synthesized by endothelial cells ([7] and fig. 1). Since FN is a major constituent of the endothelial basal lamina [7, 8, 121 and is involved in cell-substratum interactions in several systems [24-261, we hypothesized that the enhanced rates of adhesion of metastatic murine and human melanoma cells to basal lamina might be due to the high concentration of FN in the extracellular matrix. We noted that the rates of malignant cell attachment to basal lamina were identical with those found with polyvinyl-immobilized FN [7]. However,
464
Preliminary
notes
here we found that saturating concentrations of affinity-purified anti-FN inhibited only slightly (approx. 20-30%) the kinetics of B16 melanoma attachment to endothelial basal lamina, while this same concentration of anti-FN blocked completely B16 cell attachment to polyvinyl-immobilized FN. Anti-FN had no effect on B16 cell attachment to apical endothelial cell surfaces. Thus FN may have mediated, only in part, the enhanced attachment of metastatic B16 cells to endothelial basal lamina, and it was apparently not involved in tumor cell adhesion to the apical surfaces of endothelial cells. That the anti-FN bound to endothelial basal lamina but not to the apical surfaces of confluent endothelial cell monolayers was shown by indirect immunofluorescence experiments [12] and binding of ‘2”I-labeled anti-FN. Since B16 melanoma cells do not display significant amounts of cell surface FN (unpublished observations) similar to most transformed and tumor cells [27, 281, the possibility that some anti-FN eluted during the assays and blocked adhesion by binding to B16 cells was considered unlikely. In addition, in some experiments the anti-FN was absorbed to remove any crossreacting activity against murine FN. The antibiotic tunicamycin inhibited adhesion of B16 melanoma cells to endothelial basal lamina as well as to polyvinylimmobilized FN. Tunicamycin-treated B16 cells show alterations in cellular morphologies such as cell rounding and formation of surface blebs [28], reduced binding of Is51labeled Ricinus communis agglutinin to cell surface asialogalactoproteins [ 18, 291 and depression of experimental metastasis of B16-Fl and B16-FlO cells [18]. These effects were completely reversible within 24 h after drug removal [ 181. Our prior results and the experiments presented here suggest that the B16 cell surface receptors for FN Exp
Cdl
Res
I35 (1981)
are tunicamycin-sensitive glycoproteins, perhaps one of the sialogalactoproteins known to be modified by tunicamycin [ 181. The endothelial basal lamina contains, in addition to FN, other molecules which could be important in metastatic cell adhesion. Murray et al. [30] determined that the binding of B16 melanoma cells to basal lamina type IV collagen correlated with metastatic potential, and Liotta et al. [31] found that the abilities of B16 cells to degrade type IV collagen was related to their ability to invade and colonize distant bloodborne sites. Although endothelial basal lamina contains some laminin [13], a molecule known to be involved in cell adhesion [32, 331, in preliminary experiments we were unable to inhibit significantly the binding of B16 cells to endothelial basal lamina with anti-laminin (unpublished results). Endothelial cells also synthesize and secrete large amounts of sulfated glycosaminoglycans [8, 1 l] which are present in the form of basal lamina proteoglycans. FN [34, 351 and laminin [36] are known to interact with sulfated proteoglycans and glycosaminoglycans, and it was not certain from our experiments whether FN might bind to B 16 cells via this type of interaction; however, glycosaminoglycan binding does not appear to be directly involved in FNmediated cell adhesion [37, 381. Alternatively, metastatic cells could possess receptors for basal lamina glycosaminoglycans not expressed on the apical surfaces of endothelial cells. These possibilities have led us to speculate that metastatic cells probably use a variety of adhesive mechanisms to attach to endothelial basal lamina, and each class of adhesive interaction may independently play only a partial role (but collectively a major role) in successful blood-borne arrest and extravasation of metastatic tumor cells.
Preliminary These studies were supported by US National Cancer Institute grants ROl-CA28867 and ROl-CA28844 (to G.L.N.) and ROl-CA27455 and ROl-CA25392 (to E. R.).
References 1. Nicolson, G L, Bioscience 28 (1978) 441. 2. Fidler, 1 J, Gersten, D M & Hart, I R. Adv cancer res 28 (1978) 149. 3. Poste. G & Fidler, I J, Nature 283 (1980) 139. 4. Fidler, I J & Nicolson. G L. Cancer biol rev 2 (1981) 1. 5. Kramer, R H & Nicolson, G L, Proc natl acad sci US 76 (1979) 5704. 6. - International cell biology (ed S Schweiger) p. 794. Springer-Verlag. Heidelberg (1981). 7. Kramer, R H, Gonzalez, R & Nicolson, G L, lnt j cancer 26 (1980) 639. 8. Kramer, R H, Vogel, K & Nicolson, G L, J biol them. In press (1981). 9. Sage, H & Borstein, P, J cell biol 83 (1979) 97a. 10. Foidart. J M, Bere, E W, Yaar, M, Rennard, S 1, Gullino, M, Martin, G R & Katz, S I, Lab invest 42 (1980) 336. 11. Gamse, G, Fromme, H G & Kresse, H, Biochim biophys acta 554 (1978) 514. 12. Birdwell, C R, Gospodarowicz, D & Nicolson, G L, Proc natl acad sci US 75 (1978) 3273. 13. Gospodarowicz, D, Greenburg, G, Foidart. J M & Savion, N. J cell physiol. In press. 14. Fidler, 1 J, Nature 242 (1973) 148. 15. Chen, T R, Exp cell res 104 (1977) 225. 16. Gospodarowicz, D, Moran, J, Braun, D & Birdwell, C, Proc natl acad sci US 73 (1976) 4120. 17. Gospodarowicz, D, J biol them 250 (1975) 2515. 18. Irimura, T, Gonzalez, R & Nicolson, G L, Cancer res 41 (1981) 3411. 19. Engvall, E & Ruoslahti, E, lnt j cancer 20 (1977) 1. 20. Ruoslahti, E, Vuento, M & Engvall, E, Biochim biophys acta 534 (1978) 210. 21. Hayman, E G & Ruoslahti, E, J cell biol 83 (1979) 7cc LJJ). 22. Cuatrecasas, P, J biol them 12 (1973) 1312. 23. Reading, C R, Belloni, P N & Nicolson, G L, J natl cancer inst 64 (1980) 1241. 24. Yamada, K M&Olden, K, Nature 275 (1978) 179. 25. Pearlstein, E, Gold, L I & Garcia-Pardo, A, Mol cell biochem 29 (1980) 103. 26. Hook, M, Rubin, K, Oldberg, A & Vaheri, A, Biochem biophys res commun 79 (1977) 726. 27. Hynes, R 0, Biochim biophys acta 458 (1976) 73. 28. Nicolson, G L. Biochim biophys acta 458 (1976) 1. 29. Irimura, T & Nicolson, G L, J supramol struct cell biochem (1981). In press. 30. Murray, J C, Liotta, L, Rennard, S I & Martin, G R. Cancer res 40 (1980) 347. 31. Liotta, L A, Tryggvason, S, Garbisa, S, Hart, I, Foltz. C M & Shaftie, S, Nature 284 (1980) 67. 32. Terranova, V P, Rohrbach, D H & Martin, G R, Cell 22 (1980) 719. 33. Carlsson, R, Engvall, E, Freeman, A & Ruoslahti, E, Proc natl acad sci US 78 (1981) 2403. 34. Perkins, M E. Ji, T H & Hynes, R 0, Cell 16 (1979) 941. Prmted
in Sweden
notes
465
35. Ruoslahti, E & Engvall, E, Biochim biophys acta 631 (1980) 350. 36. Saskashita, S, Engvall, E & Ruoslahti, E, FEBS lett 116 (1980) 243. 37. Kleinman, H K, Martin, G & Fishman, P. Proc natl acad sci US 76 (1979) 3367. 38. Ruoslahti, E, Hayman, E G, Engvall, E, Cothran, W C & Butler, W T, J biol them 256 (1981) 7277. Received May 27, 1981 Accepted August 5, 1981
Copyright 0 1981 by Academic PET\. Inc. All rights of reproductwn m any form reserved 0014.48?7/81/100465-04$0?.0010
Differential chromatid damage induced by 64hioguanine in CHO cells JONATHAN MAYBAUM and H. GEORGE DEL, Depurtment of Pharmacology, George ington
University
Mrdicul
Center,
Washington,
MANWushDC
20037, USA Treatment of Chinese hamster ovary ceils with 6-thioguanine (TG) produces severely disrupted chromatin in G2 cells, as visualized with premature chromosome condensation. In many regions of the chromosome this damage, appearing as a gapped or diffusely staining region, occurs in only one of sister chromatids. This morphology strongly supports previous reports that TG-induced cytotoxicity is related to its incorporation into DNA and that this lesion is expressed in the cell cycle following that in which drug exposure occurs.
Summary.
Thiopurines such as 6-thioguanine (TG) and 6-mercaptopurine are clinically useful antineoplastic agents which are known to be incorporated into RNA and DNA [l] in the form of thioguanine nucleotides [2]. Much evidence shows that incorporation of TG into DNA is closely related to the delayEd cytotoxicity of this drug [2-4]. Recent repOrts indicate that cells treated with TG are arrested in the G2 phase of the cell cycle [.5, 61, and that this arrest is delayed by one full cell cycle [5]. That is, cells exposed in S phase of a given cycle complete that cycle and progress through mitosis and Gl and S of the next cycle before being arrested.
D C, Feagans, D E, Blanchette-Mackie. E J & Nau. M E, Science 206 (1979) 836. Mousa. G Y & Trevithick, J R. Dev biol 60 (1977)
14. 25. Ramaekers.
F C S, Hukkelhoven, M W A C. A & Bloemendal. H. Ophthal res 11
Groeneveld,
(1980) 283.
26. Wessels, 27. 28. 29.
N K, Spooner. B S. Ash, J F, Bradley, M 0, Luduena, M A, Taylor. E L, Wrenn. J T & Yamada, K M, Science 171 (1971) 135. Blikstad, 1, Markey, F, Carlsson. L, Persson. T & Lindberg, U, Cell 15 (1978) 135. Lowry, 0 H, Rosebrough, N J. Farr, A L & Randall, R J, J biol them 193 (1951) 265. Dulley, J R & Grieve. P A. Anal biochem 64 (1975)
136. 30. Laemmli, U K. Nature 227 (1970) 680. 31. Weber, K & Osborn. M, J biol them 244 (1969) 4406. 32. O’Farrell, P H. J biol them 250 (1975) 4007. Received November 11, 1980 Revised version received May Accepted May 27, 1981
15, 1981
CopyrIght 0 1981 h) Academic Pre\\. Inc. All right\ of reproductwn in any form rewrved ~XlI4-4X?7.‘XI:loll4hI-n~‘;02.0oio
The role of fihronectin in adhesion of metastatic melanoma cells to endothelial cells and their basal lamina GARTH ROBERT LAHTI,2
L.
NICOLSON,’ GONZALEZ’,
*
TATSURO IRIMURA,’ and ERKKI RUOS-
‘Deoartmeni of‘ Tumor Biolopy, The University of Texk System dower Center. k. D. Anderson HosDituI and Tumor Institute. Houston, TX 77030. und ‘Ln’Jolla Crrncrr Resrnrch Foundation. La Jolla. CA 92038, USA Summury. Vascular
endothelial cells synthesize an extracellular matrix or basal lamina composed of collagens, proteoglycans and glycoproteins such as fibronectin (FN). Using affinity-purified anti-FN, we have examined the role of FN in adherence of metastatic B16 melanoma cells to endothelial cell monolayers which lack FN on apical cell surfaces and to their basal lamina which contains FN. B16 melanoma ceils, which do not contain significant amounts of FN. attached at much higher rates to endothelial basal lamina and polyvinyl-immobilized FN compared with intact endothelial cell monolayers. Anti-FN failed to inhibit attachment of melanoma sublines of low (B16-Fl) or high (B16-FlO) metastatic potential to intact endothelial cell monolayers, inhibited slightly B16 cell attachment to basal- lamina and completely abolished attachment of Bl6 cells to polyvinyl-immobilized FN. The antibiotic tunicamycin which inhibits glycosylation of B16 cell surface glycoproteins and blocks experimental metastasis [18] inhibited B16 attachment
notes
461
to endothelial cells, basal lamina and immobilized FN. The results suggest that FN mediates, only in part, the adhesion of B16 melanoma cells to basal lamina through glycoprotein receptors on Bl6 cells.
Crucial steps in blood-borne tumor metastasis are the arrest of malignant cells in the microcirculation, often in specific organs [1], and subsequent extravasation or invasion of surrounding endothelium and escape to extravascular sites [2-41. Metastatic tumor cells and invasive normal cells such as leukocytes attach to endothelial monolayers, cause rupture of endothelial cell-cell interactions and retraction of the endothelial cells from their underlying basal lamina, whereupon the invasive cells move from the endothelial cells and bind tightly to basal lamina [S-7]. Eventually the metastatic cells solubilize components such as fibronectin (FN) and sulfated proteoglycans in the endothelial cell basal lamina [8]. We have developed an in vitro model for extravasation [5, 61 that utilizes vascular endothelial cell monolayers which synthesize a basal lamina containing collagens [9, lo], sulfated proteoglycans and glycosaminoglycans [S, 111 and glycoproteins such as FN [9, 121 and laminin [13]. We report here that attachment of B16 melanoma cells to endothelial cell basal lamina (but not to endothelial cells) appears to be mediated, in part, by FN but also by other adhesive molecules in the extracellular matrix. Materials
and Methods
Cell cultures. Murine melanoma sublines selected once (B16-Fl) or ten times (Bl6-FlO) for blood-borne lung implantation. survival and growth [14] were obtained from Dr I. J. Fidler (NCI-Frederick Cancer Research Center, Frederick, Md) and were grown in Dulbeccomodified Eagle’s minimum essential medium (Gibco, Grand Island, N.Y.) supplemented with I % nonessential amino acids (Gibco) (complete medium), and 5 % heat-inactivated fetal bovine serum (Flow Laboratories, Inc., Inglewood. Calif.). Low passage cell cul* Present address: Cancer Research Institute, University of California, San Francisco. CA 94143, USA.
462
Preliminary
60
notes
ta
I
0’
I 10
0
1 20
I / 30 40 Time (mid
I 50
I
Fig. 1. Kinetics of adhesion of B16 melanoma cells to (a) vascular endothelial cell monolayer; or(b) endothelial cell basal lamina in the absence or presence of afftnitv mnified anti-tibronectin. O-O, B16-FlO; O-O, Bi&Fl; A-A, B16-FlO, monolayer or matrix pretreated with anti-tibronectin (400 Fg/ml); A-A, B16-FI, monolayer or matrix pretreated with antitibronectin (400 pg/ml).
tures (<8 passages) were grown to subcontluency and harvested by 10-15 min treatment with 2 mM EDTA in Ca’+-, Mg2+-free Dulbecco’s phosphate-buffered saline (DPBS). Cell viabilities were determined by Trypan blue ‘exclusion, and cell lines were tested routinely for mycoplasma by use of Hoechst stain 33258 [15]. Bovine aortic endothelial cells were obtained from Dr D. Gosnodarowicz (Universitv of California, San Francisco)tl6] and were cultured-in alphamodified minimum essential medium (Gibco) supplemented with 10% calf serum (Gibco). Fibroblast growth factor was ouritied as described f171 and added lo endothelial cells every other day ai a-concentration of 100-500 rig/ml until confluency and at 5 rig/ml thereafter. For the adhesion assays endothelial cells were grown to confluency in 24-well Costar tissue culture dishes [7]. Tunicamycin treatment. Tunicamycin (lot 177382) was obtained from the US National Cancer Institute, solubilized in 10 mM sodium hydroxide at a concentration of 2 mg/ml and kept at -20°C for up to 3 weeks. Immediately before each experiment the tunicamycin solution was diluted into culture medium which was then sterilized by passing through Acrodisc (0.2 pm; Gelman, Ann Arbor, Mich.). From the results of dose-response experiments described elsewhere for B16 melanoma cells f181 a concentration of 0.5 pg/ml was used during a-26 h incubation. At the end of the tunicamycin treatment cell viability remained 90-95 %. Exp Cd
Res
135 (1981)
Adhesion crsscrq’s.B16 melanoma cells were radiolabeled with 0.25 &i Na[J’Cr]O, (carrier-free in sterile saline solution; New England Nuclear) per T-75 tissue culture flask for 3 h in complete medium. 816 cells (2-4xlO”lflask) were harvested with 2 mM EDTA in Ca’+-, Mg”-free DPBS, washed three times with complete medium containing I % bovine serum albumin (BSA). pH 7.4, and suspended in the same solution at a concentration of 4~ 10” cells/ml. Endothelial cells were washed carefully with complete medium containing I % BSA, pH 7.4, 30 min prior to the assays. Endothelial basal lamina was .prepared as described previously [7]. Bovine plasma fibronectin puritied from calf serum (Irvine Scientific, Irvine, Calif.) according to Engvall & Ruoslahti [I93 was used for coating polyvinyl dishes (Linbro, Hamden. Conn.), as described elsewhere [7]. Tumor cell suspensions were allowed to settle for 1 min on endothelial monolayers, endothelial basal lamina or polyvinyl-immobilized tibronectin and were then incubated with or without agitation at 37°C. At given time intervals the samoles were washed eentlv 3 times with 0.5 ml complete medium containmg I-% BSA, pH 7.4, at 37°C. Adhering “‘Cr-labeled B16 cells were counted in a Beckman Model 8000 automatic gamma counter. Afifnitv mrrified trnti-bovine fibuonrctin. Bovine FN was purified by affinity chromatography on Sepharoseimmobilized gelatin as described previously [l9]. Antibodies were purified from antisera prepared against purified FN by affinity chromatography on Sepharose-iminobilized bovine FN with or without prior adsorption on Sepharose-immobilized murine FN. The affinity-purified anti-bovine FN was eluted from Sepharose-immobilized bovine FN in glycine-HCI buffer, pH 2.6 as described [20, 211. Affinity-purified anti-FN in DPBS was radiolabeled with lrJI using the chloroamine-T method [22]. AntiFN binding assays were performed with ‘Y-labeled anti-FN as described elsewhere [23]. These studies indicated that basal lamina bind a maximum amount of anti-FN (initial cont. 400 pg/ml) during a 45 min incubation at 37°C; therefore, all subsequent experiments utilized this concentration of anti-FN. .,”
,,
I
Results Murine melanoma sublines B 16-F 1 and B 16FlO attached without shear at similar rates to intact endothelial cell monolayers such that after 60 min approx. 50% of the cells were adherent and not removed during the subsequent washing steps (fig. 1a). Preincubation of endothelial cell monolayers with affinity-purified anti-FN (300 pg/ml) for 45 min at 37°C had little effect on B16Fl or Bl6-FlO cell adhesion (fig. la). The B16 melanoma cells adhered at much faster rates to isolated endothelial basal lamina such that by 20-24 min >90% of the cells
Preliminag
notes
463
0
20 30 40 Time (min) Fig. 2. Kinetics of adhesion of Bl6-Fl melanoma cells to various substratum. O-O, Polyvinyl-immobilized tibronectin; O-0, polyvinyl-immobilized tibronectin in the presence of 400 pg/ml anti-tibronectin; A-A, polyvinyl-immobilized bovine serum albumin; O-0. polyvinyl.
10 20 Time (min) Fig. .3. Kinetics of adhesion of untreated and tunicamycin-treated melanoma cells to endothelial cell basal lamina. O-O, Bl6-FlO; O-O, B16-Fl without tunicamycin treatment; A-A, B16-FlO; A-A, Bl6-Fl with tunicamycin (0.5 Fg/ml) treatment for 24 h.
were tightly adherent (fig. 16) similar to previous data [7]. When endothelial cell basal lamina was pre-incubated with antiFN as before, the kinetics of B16 cell adhesion were reduced slightly (approx. 2030% initial inhibition in four separate experiments) (fig. 1 b) suggesting that matrix FN may mediate, in part, the adhesion of B16 melanoma cells to endothelial cell basal lamina. Dose-response experiments indicated that higher concentrations of anti-FN were no more effective in inhibiting B16 cell attachment to endothelial basal lamina. Essentially no difference in cell attachment rates were found between sublines B16-Fl and B16-FlO under non-shear conditions (fig. la, h). The kinetics of B16-Fl melanoma attachment to polyvinyl-immobilized FN were the same as for B16-Fl adhesion to endothelial basal lamina (fig. 2); however, anti-FN abolished completely B16-Fl cell attachment to the polyvinyl-immobilized FN. Polyvinyl surfaces or polyvinylimmobilized BSA failed to bind B16 cells (fig. 2). Tunicamycin pretreatment of B16 melanoma cells modified their adhesive behavior
without modifying cell viability [18]. When B16 cells were incubated with 0.5 pglml tunicamycin for 24 h, their attachment rates to both endothelial basal lamina and polyvinyl-immobilized FN were dramatically reduced (fig. 3) suggesting that tunicamycinsensitive glycoproteins on B16 cells bind to FN and other endothelial basal lamina components.
“0
IO
0
Discussion The apical surfaces of confluent endothelial cell monolayers were found to be much less adhesive to malignant melanoma cells than the basal lamina synthesized by endothelial cells ([7] and fig. 1). Since FN is a major constituent of the endothelial basal lamina [7, 8, 121 and is involved in cell-substratum interactions in several systems [24-261, we hypothesized that the enhanced rates of adhesion of metastatic murine and human melanoma cells to basal lamina might be due to the high concentration of FN in the extracellular matrix. We noted that the rates of malignant cell attachment to basal lamina were identical with those found with polyvinyl-immobilized FN [7]. However,
464
Preliminary
notes
here we found that saturating concentrations of affinity-purified anti-FN inhibited only slightly (approx. 20-30%) the kinetics of B16 melanoma attachment to endothelial basal lamina, while this same concentration of anti-FN blocked completely B16 cell attachment to polyvinyl-immobilized FN. Anti-FN had no effect on B16 cell attachment to apical endothelial cell surfaces. Thus FN may have mediated, only in part, the enhanced attachment of metastatic B16 cells to endothelial basal lamina, and it was apparently not involved in tumor cell adhesion to the apical surfaces of endothelial cells. That the anti-FN bound to endothelial basal lamina but not to the apical surfaces of confluent endothelial cell monolayers was shown by indirect immunofluorescence experiments [12] and binding of ‘2”I-labeled anti-FN. Since B16 melanoma cells do not display significant amounts of cell surface FN (unpublished observations) similar to most transformed and tumor cells [27, 281, the possibility that some anti-FN eluted during the assays and blocked adhesion by binding to B16 cells was considered unlikely. In addition, in some experiments the anti-FN was absorbed to remove any crossreacting activity against murine FN. The antibiotic tunicamycin inhibited adhesion of B16 melanoma cells to endothelial basal lamina as well as to polyvinylimmobilized FN. Tunicamycin-treated B16 cells show alterations in cellular morphologies such as cell rounding and formation of surface blebs [28], reduced binding of Is51labeled Ricinus communis agglutinin to cell surface asialogalactoproteins [ 18, 291 and depression of experimental metastasis of B16-Fl and B16-FlO cells [18]. These effects were completely reversible within 24 h after drug removal [ 181. Our prior results and the experiments presented here suggest that the B16 cell surface receptors for FN Exp
Cdl
Res
I35 (1981)
are tunicamycin-sensitive glycoproteins, perhaps one of the sialogalactoproteins known to be modified by tunicamycin [ 181. The endothelial basal lamina contains, in addition to FN, other molecules which could be important in metastatic cell adhesion. Murray et al. [30] determined that the binding of B16 melanoma cells to basal lamina type IV collagen correlated with metastatic potential, and Liotta et al. [31] found that the abilities of B16 cells to degrade type IV collagen was related to their ability to invade and colonize distant bloodborne sites. Although endothelial basal lamina contains some laminin [13], a molecule known to be involved in cell adhesion [32, 331, in preliminary experiments we were unable to inhibit significantly the binding of B16 cells to endothelial basal lamina with anti-laminin (unpublished results). Endothelial cells also synthesize and secrete large amounts of sulfated glycosaminoglycans [8, 1 l] which are present in the form of basal lamina proteoglycans. FN [34, 351 and laminin [36] are known to interact with sulfated proteoglycans and glycosaminoglycans, and it was not certain from our experiments whether FN might bind to B 16 cells via this type of interaction; however, glycosaminoglycan binding does not appear to be directly involved in FNmediated cell adhesion [37, 381. Alternatively, metastatic cells could possess receptors for basal lamina glycosaminoglycans not expressed on the apical surfaces of endothelial cells. These possibilities have led us to speculate that metastatic cells probably use a variety of adhesive mechanisms to attach to endothelial basal lamina, and each class of adhesive interaction may independently play only a partial role (but collectively a major role) in successful blood-borne arrest and extravasation of metastatic tumor cells.
Preliminary These studies were supported by US National Cancer Institute grants ROl-CA28867 and ROl-CA28844 (to G.L.N.) and ROl-CA27455 and ROl-CA25392 (to E. R.).
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35. Ruoslahti, E & Engvall, E, Biochim biophys acta 631 (1980) 350. 36. Saskashita, S, Engvall, E & Ruoslahti, E, FEBS lett 116 (1980) 243. 37. Kleinman, H K, Martin, G & Fishman, P. Proc natl acad sci US 76 (1979) 3367. 38. Ruoslahti, E, Hayman, E G, Engvall, E, Cothran, W C & Butler, W T, J biol them 256 (1981) 7277. Received May 27, 1981 Accepted August 5, 1981
Copyright 0 1981 by Academic PET\. Inc. All rights of reproductwn m any form reserved 0014.48?7/81/100465-04$0?.0010
Differential chromatid damage induced by 64hioguanine in CHO cells JONATHAN MAYBAUM and H. GEORGE DEL, Depurtment of Pharmacology, George ington
University
Mrdicul
Center,
Washington,
MANWushDC
20037, USA Treatment of Chinese hamster ovary ceils with 6-thioguanine (TG) produces severely disrupted chromatin in G2 cells, as visualized with premature chromosome condensation. In many regions of the chromosome this damage, appearing as a gapped or diffusely staining region, occurs in only one of sister chromatids. This morphology strongly supports previous reports that TG-induced cytotoxicity is related to its incorporation into DNA and that this lesion is expressed in the cell cycle following that in which drug exposure occurs.
Summary.
Thiopurines such as 6-thioguanine (TG) and 6-mercaptopurine are clinically useful antineoplastic agents which are known to be incorporated into RNA and DNA [l] in the form of thioguanine nucleotides [2]. Much evidence shows that incorporation of TG into DNA is closely related to the delayEd cytotoxicity of this drug [2-4]. Recent repOrts indicate that cells treated with TG are arrested in the G2 phase of the cell cycle [.5, 61, and that this arrest is delayed by one full cell cycle [5]. That is, cells exposed in S phase of a given cycle complete that cycle and progress through mitosis and Gl and S of the next cycle before being arrested.