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Contact Inhibitory Factor Also Restores Anchorage and Serum Dependence to Hamster Melanoma Cells
George Lipkin, M.D., Martin Rosenberg, Ph.D., and Vera Klaus-Kovtun, B.S. Department of Dermatology, N ew York University Medical Center, New York, New York, U.S.A.
Conditioned medium (CM) from confluent cultures of the contact-inhibited hamster melanocytic cell line, FF, contains a biologic activity, contact inhibitory factor (CIF) , which reversibly restores density-dependent growth to melanoma cells.
When a hydrophobic affll1ity-concentrated extract of CIFcontaining CM was incorporated in agarose at a concentration of 1000 }-Lg protein/ml, it restored anchorage-dependent growth to RPMI 1846 hamster melanoma cells. Colony-forming efficiency in CIF-treated wells decreased to 5% from levels of 51.5% in controls prepared with regular growth medium. In addition, CIF-containing CM
Various in vitro characteristics of the transformed phenotype have been examined to determine whether one or more can be correlated reliably with the capacity for neoplastic growth in vivo . Properties analyzed have included morphology [1], saturation den
sity [2-5]. fibrinolytic activity [6-10], nutrient uptake [11]. fibronectin production [12-13], lectin agglutinability [11], surface structure [14-15]. cytoskeleton [16]. cell shape [17]. serum requirement for growth [18-21], and anchorage dependence [22-25].
Evidence thus far suggests a noncoordinate control of these various phenotypic properties [10,26] . Nevertheless loss of one of these, the requirement for attachment to a substrate in order to grow (anchorage dependence), has, despite exceptions [27,28], been shown repeatedly to be significantly correlated with malignant behavior in vivo [9,23,29,30] . In a quantitative study of hamster cell lines, anchorage independence was the in vitro prope rty most highly correlated (p < 0.01) with tumorigenicity [10] . Another property of most transformed cells, the ability to grow progressively in 1 % serum, was also significantly correlated (p < O.OS) with in vivo growth of 7 transformed hamster cell lines [10].
A diffusible factor capable of restoring density-dependent growth to hamster malignant melanoma cells was originally detected in culture medium of an established line of contact-inhibited hamster melanoctyes [31] . The origin of this cell line has been described
Manuscript received November 4, 1985; accepted for publication February 24, 1986.
Supported in part by grants from The Skin Cancer Foundation; The Berger Foundation for Cancer Research; The RudolfL. Baer Foundation for Skin Diseases, Inc. ; The Orentreich Medical Group; the Department of Dermatology Cancer Prize Fund; and Bion Corporation.
Reprint requests to: George Lipkin, M.D'., Department of Dermatology, New York University Medical Center, 562 First Avenue, New York. New York 10016.
AbbreVIatIOns: CIF: contact inhibitory factor CM: conditioned medium PBS: phosphate-buffered saline
restored serum-dependent growth to RPMI 1846 cells, markedly restricting proliferation in 1 % calf serum-containing medium. Control cultures containing 1 % calf serum and either complete growth medium or CM from the noncontact-inhibited hamster melanoma line itself, supported proliferation of RPM I 1846 cells to levels 3.9 x and 3.7 x that of CIF-treated cultures, respectively.
CIF is the first factor derived from contact-inhibited mammalian cell cultures that has been shown to restore density-, anchorage-, and serum-dependent growth to malignant melanoma cells.] Invest Dermato/87:305-308, 1986
in detail [32-34] . Effects of the melanocyte contact inhibitory factor (CIF) subsequently were shown to transcend both species [35] and tissue [36] barriers, to arrest melanoma cells in G 1 phase of the cell cycle [35]. and to be a potent inhibitor of growth of a broad spectrum of tumor cell types of ectodermal , mesodermal, and endodermal origins [36]. Similar biologic activity was also detected in culture medium from a human cell line [36] . Recently CIF was shown to induce the expression, following phenotypic reversion, of vitiligo-related pigment cell differentiation antigens on both hamster and mouse melanoma cells [37]. It also JTIarkedly reduced susceptibility of human melanoma, colon carcinoma, and erythroleukemia cells to lysis by natural killer (NK) cells [38].
We now report that CIF also restores both anchorage dependence and serum dependence to hamster melanoma cells.
MATERIALS AND METHODS
Cell Cultures The hamster amelanotic malignant melanoma cell line RPMI 1846 and its contact-inhibited (density-dependent) derivative line FF were maintained as stock cultures in 7S-cm2
polystyrene culture flasks (Corning) at 37°C in a humidified atmosphere containing 5% CO2 in balanced air. Cultures were fed biweekly with RPMI Medium 1640 supplemented with 10% calf serum (Irvine Scientific) and antibiotics (penicillin, 100 U/ml; streptomycin , 100 ,ug/ml; gentamicin, 10 ,ug/ml; Fungizone, 2.5 ,ug/ml) , and subcultured at weekly intervals following removal of attached cells with trypsin-EDTA solution (Gibco). All experiments were performed with cells subcultured into wells of Falcon MicroTest n plates .
Contact Inhibitory Factor Confluent FF cultures, which served as a source of CIF, were prepared by seeding freshly trypsinized FF cells in multiple 7S-cm2 flasks at a density of 500,000 cells per flask in 15 ml of complete medium. At confluence, cells were washed twice with phosphate-buffered saline (PBS; Gibco), and refed with serum-free medium. After 48 h, the serum-free conditioned medium (CM), containing CIF, was collected and centrifuged gently to remove cells and debris. Some of this CM was stored at 4°C for up to 14 days prior to use in serum-dependence studies, while some was immediately lyophiJized and stored as a
0022-202X/86/S03.50 Copyright © 1986 by The Society for Investigative Dermatology, Inc.
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306 LIPKIN, ROSENBERG, AND KLAUS-KOVTUN
dry powder until further processed by hydrophobic affinity chromatography for use in anchorage-dependence studies.
A phenyl sepharose (Pharmacia, Piscataway, New Jersey) column was equilibrated with 4 M NaCI, 10 mM Tris, 10 mM EDTA at pH 8.0. The lyophilized CM was dissolved in the same buffer and run through the column using the same buffer until a peak (monitored at 280 nm) appeared. After return of the peak to baseline, a new elution buffer (Tris and EDT A) was started and a second peak eluted. [n a similar manner, a third peak was obtained using unbuffered distilled water as the eluant.
Each fraction was desalted by Amicon filtration with a 10,000 D membrane, then lyophilized and stored at 4°C.
For bioassay, each sample was dissolved in complete culture medium and applied directly to hamster RPMI 1846 melanoma cells in Falcon 96-well micro titer plates . The contact inhibitory biologic activity was found to be concentrated in the third, i.e. , relatively most hydrophobic, peak.
Anchorage Dependence Agarose cultures were prepared using a micro-well modification of the method of MacPherson and Montagnier [22] . The underlayer contained 0.5% agarose, the overlay 0.33% . For experimental wells, the phenyl sepharoseconcentrated material containing C[F, was incorporated into the overlay mixture at concentrations ranging from 0-1000 p.g/ml protein (Bio-Rad Laboratories, Rockville Center, New York). The warmed overlay mixture was added to a pellet ofRPM[ 1846 cells to give a final concentration of 50,000 cells/ml. Two-tenths (0.2 ml) of this warmed mixture was then layered over 0.2 ml of semisolidified underlayer in each well to give a final concentration of 10,000 cells per well, and allowed to harden. All cells were then observed daily and scored at 6 days for clones of 8 or more cells. At the same time that wells were being prepared for anchorage-dependence studies, similar aliquots of the phenyl sepharose-concentrated material were also tested over the entire concentration range, on freshly plated RPMl 1846 cells in Falcon MicroTest II wells for effects on cell viability. The latter was judged by dye exclusion using trypan blue both immediately after exposure of cells to C[F and 24 h later.
Serum Dependence RPM11846 cells were plated out in Falcon MicroTest II wells at initial concentrations of 3500 cells/0.2 ml complete medium per well . Following attachment of cells after 2 h, cells were washed with PBS, and medium was replaced to define 6 experimental groups: cultures containing RPMI 1640, antibiotics, and 1 % or 10% calf serum; those containing 48-h conditioned FF medium, antibiotics, and 1 % or 10% calf serum; and cultures containing 48-h conditioned RPMl 1846 medium, antibiotics, and 1 % or 10% calf serum. All groups were refed after 3 days, and growth and morphology observed for up to 6 days.
RESULTS
Anchorage Dependence In wells containing CIF, clonal growth was inhibited (Fig la, Table 1) in a concentration-dependent manner. At the highest concentration (1000 p.g/ml) some 95% of cells failed to proliferate, only 4.9% forming aggregates of 8 or more cells . Control wells lacking CIF exhibited excellent clonal growth of RPM 1 1846 cells (Fig Ib, Table 1), with 51.5% of cells forming aggregates of 8 cells or more. Simultaneous testing in vitro of the FF-conditioned medium used in preparing the agarose showed all concentrations to be nontoxic to RPMI 1846 cells as judged by dye exclusion (90% viability at the highest concentration immediately after plating, 88% after 24 h).
Serum Dependence Results are shown in Figs 2 and 3. RPM! 1846 cells grew well in 1 % serum in the presence of either unconditioned complete growth medil!m or conditioned medium from cultures of the non-contact-inhibited melanoma line itself. The almost identical population densities reached represent about 3 cell doublings. By contrast, RPMI 1846 cells grown in 1 % serum-containing conditioned medium from contact-inhibited FF
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Figure 1. Effect ofCIF upon anchorage dependence of RPM I 1846 cells. a, RPMI 1846 cells are unable to form clones in agarose at 6 days in the presence of CIF (1000 p.g protein/ml). b, Anchorage independence of control RPMI 1846 cells. Clonal growth occurs in agarose at 6 days in the absence of CIF. Bars = 35 p.m.
cultures failed to proliferate at all . As expected, RPM! 1846 cells grew to much higher densities in 10% calf serum with both the complete growth medium and conditioned medium from the melanoma line itself, but did exhibit the usual marked reduction in saturation density when grown to confluence in 10% calf serum and conditioned medium fr0111 FF cultures .
DISCUSSION
Melanocyte contact inhibitory factor, previously shown to mediate density-dependent growth of melanoma cells of human, murine, and hamster origins, has now been found, in hamster melanoma, to restore anchorage and serum dependence as well. Loss of one or more of these properties is a prominent accompaniment of the transformed phenotype; the ability of ClF to restore all 3 suggests that it is acting at a central or critical point to modulate growth control. That such a mechanism for regu-
VOL. 87, NO.3 SEPTEMBER 1986
Table I. Effect of Affinity-Concentrated FF Conditioned Medium upon Cloning of RPMI 1846 Cells
Protein (I-'g/ml)
0.0 67.5
125.0 250.0 500.0
1000.0
Cloning Efficiency (%)
51.5 38.0 25.0 13.5 9.7 4.9
Figure 2. Effect of CIF-containing FF conditioned medium upon serum dependence of hamster melanoma cells. a, RPMI 1846 cells growing in control RPMI 1640 medium containing 1 % feta l calf serum. b, RPMI 1846 cells unable to divide in FF conditioned RPM I 1640 medium (500 I-'g protein/ml) containing added 1 % fetal calf serum. c, RPM I 1846 cells growing in FF conditioned RPMI 1640 medium (500 I-'g protein/ml) containing added 10% fetal calf serum. Cells exhibit characteristic flattening and bipolar morphologic change. Bars = 70l-'m.
RESTORING GROWTH CONTROL TO HAMSTER MELANOMA 307
~
'0
X
(f) ....J ....J W U
1.0
o 2 3 4 DAY
5 6 7
Figure 3. Effect ofFF-conditioned medium upon serum-dependent growth of RPMI 1846 cells in culture. Triallgles, RPMI 1846 cells in complete medium. Circles, RPM I 1846 cells in complete medium conditioned by non-contact-inhibited RPMI 1846 hamster elanoma cells. Sqllares, RPM I 1846 cells in complete medium conditioned by contact-inhibited hamster FF cel ls. Opell symbols, Growth in 10% calfserum. Closed symbols, Growth in I % calf serum.
lation of growth may be a genera l one is suggested by the lack of species or tissue restrictions upon the biologic effects of hamster CIF, as well as by our earlier demonstration of apparently identical biologic activity, mediating contact inhibition of growth, in culture medium of an entirely different cell type, viz., a human epidermal cell line [36] . Such modulation probably affects the cell surface since both density and anchorage dependence reflect surface-mediated events. Furthermore, in view of the very close correlations between loss of anchorage, density, and serum dependence and tumorigenicity , it is possible that CIF migh t predictably alter melanoma cells so as to m ake them more subject in vivo to normal growth regulatory mechanisms . Studies of this question are now in progress . Preliminary data indicate a marked retardation of hamster melanoma growth in an in situ model (manuscript in prepara tion).
It is pertinent to note that retinoic acid , a vitamin A analogue which promotes differentiation of human HL-60 leukemic cells [39] and teratocarcinoma cells [40], was shown to restore both density [41] and anchorage [42] dependence to transformed L-929 mouse fibroblasts , as well as anchorage dependence to a mouse melanoma line [42]. Retinol (vitamin A) induced density dependent growth in viral-transformed hamster fibroblasts in correlation with striking alterations of surface glycolipids [43]. Both density and anchorage dependence were also restored coordinately to SV 403T3 transformants by succinylated concanavalin A [44] . However, to the best to our knowledge, CIF is the first material derived from contact-inhibited mammalian cell cultures which has been shown to restore these 3 significant aspects of in vitro growth control to a malignant cell type, viz, melanoma. Purification studies are in progress, and the m anner in which CIF interacts with tumor cells will be the subject of future studies. Earlier observations suggest that C IF is not a protease inhibitor itself, but may alter the ability of melanoma cells to take up serum protease inhibitors [45]. It also elevates cAMP levels while decreasing intracellular plasminogen activator, but exogenous cAMP alone, or its dibutyryl derivative, fails to miI?1ic the morphologic or contact inhibitory growth effects of CIF (unpublished data).
TI,e expert teelmical assistallce of MOllica GidJ,md alld Georgette Dllralld is gratejilily ackllolll/edged .
308 LIPKIN, ROSENBERG, AND KLAUS-KOVTUN
REFERENCES
1. Tucker RW, Sanford KK, Handelman SL, Jones GM: Colony morphology and growth in agarose as tests for spontaneous neoplastIc transformation i/1 vivo . Cancer Res 37:1571-1579, 1977
2. Todaro GJ, Lazar GK, Green H : The initiation of cell division in a contact-inhibited mammalian cell line. J Cell Comp PhyslOl 6:325-333, 1965
3. Aaronson SA, Todaro G: Basis for the acquisition of malignant potential by mouse cells cultivated in vivo. Science 162:1024-1026, 1968
4. Pollack RE, Green H, Todaro GJ : Growth control in cultured cells: selection of sublines with increased sensitivity to contact inhibition and decreased tumor-producing activity. Proc Natl Acad Sci USA 60:126-133, 1968
5. Pollack RE, Teebor GW: Relationship of contact inhibition to tumor transplantability, morphology, and growth rate. Cancer Res 29:1770-1772,1969
6. Ossowski L, Quigley JP, Kellerman GM, Reich E: Fibrinolysis ~ssocia ted with oncogenic transformation-requirement of plasmmogen for correlated changes in cellular morphology, colony formation in agar, and cell migration. J Exp Med 138:1056-1064, 1973
7. Laug WE, Jones PA, Benedict WF: Relationship between fibrinolysis of cultured cells and malignacy . J Natl Cancer Inst 54:173-:-179, 1975
8. Pearlstein E, Hynes RO , Franks LM, Hemmings V]: Surface proteins and fibrinolytic activity of cultured mammalian cells. Cancer Res 36:1475-1480, 1976
9. Montesano R, Drevon C, Kuroki T , Saint Vincent L, Handelman S, Sanford KK, Defoe 0, Weinstein IB : Test for malignant transformation of rat liver cells in culture: cytology, growth m soft agar and production of plasminogen activator. J Nat! Cancer Inst 59:1651-1658, 1977
10. BarretJC, Crawford BD, Mixter LO, Schechtman LM, Ts'o PO~, Pollack R: Correlation of in vivo growth propertIes and tumorigenicity of Syrian hamster cell lines. Cancer Res 39:1504-1510, 1979
11. San RHC, Laspia MF, Soiefer AI, Maslansky Cj, RiceJM, Williams GM: A survey of growth in soft agar and cell surface properties as markers of transformation in adult rat liver epithelial-like cell cultures . Cancer Res 39:1026-1034, 1979
12. Canary PK, Jackson MJ, Shin S: Association between cell surface fibronectin (LETS protein), anchorage independence and tumorigenicity in animal cells. J Cell Bioi 79(2, part 2) :397a, 1978
13. Kahn P: Cellular tumorigenicity in nude mice: test of associations among loss of cell-surface fibronectin, anchorage independence, and tumor-forming ability. J Cell Bioi 82:1-16, 1979
14. Hynes RO: Cell surface proteins and malignant transformation. Biochim Biophys Acta 485:73-107, 1976
15. Nicolson GL: Trans-membrane control of the receptors on normal and tumor cells . II . Surface changes associated with transformation and malignancy. Biochim Biophys Acta 458:1-72, 1976
16. Pollack R, Osborn M, Weber K: Patterns of organization of actin and myosin in normal and transformed cultured cells. Proc Nat! Acad Sci USA 72:994-998, 1975
17. FolkmanJ, Moscona A: Role of cell shape in growth control. Nature 273:345-349, 1978
18. PaulO, Lipton A, Klinger I: Serum factor requirements of normal and simian virus 40-transformed 3T3 mouse fibroblasts. Proc Nat! Acad Sci USA 68:645-648, 1971
19. Smith CH, Scher CD, Todaro G]: Induction of cell division in medium lacking serum growth factor by SV 40. Virology 44:359-370, 1971
20. Holley RW, KiernanJW: Control of the initiation of DNA synthesis in 3T3 cells: serum factors. Proc Natl Acad Sci USA 71 :2908-2911, 1974
21. Dulbecco R: Topoinhibition and serum requirement of transformed and un transformed cells. Nature 227:802-806, 1970
22. MacPherson I, Montagnier L: Agar suspension culture for the selective assay of cells transformed by polyoma virus. Virology 23:291-294, 1964
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23. Freedman VH, Shin S: Cellular tumorigenicity in nude mice: Correlation with cell growth in semi-solid medium. Cell 3:355-359, 1974
24.
25.
Shin S, Freedman VH, Risser R, Pollack R: Tumorigenicity of virus_ transformed cells in nude mice is correlated specifically with anchorage independent growth itl vivo . Proc Nat! Acad Sci USA 11 :4435-4439, 1975
Jones PA, Laug WE, Gardner A, Ny~ CA, Fink LM, Benedict WF: Itl vivo correlates of transformatIon m C3H/I0T1/2 clone 8 mouse cells. Cancer Res 36:2863-2867, 1976
26. Ruben RL, Rafferty KA: Colony formation by simian virus 40-transformed human parapharyngeal cells cultured in semisolid agar. J Nat! Cancer Inst 61 :993-1000, 1978
27. Marshall Cj, Franks LM, Carbonell A W: Markers of neoplastic transformation of epithelial cell lines derived from human carcinomas. J Natl Cancer Inst 58:1743-1751, 1977
28. Stanbridge Ej, Wilkinson T : Analysis of malignancy in human cells: malignant and transformed phel)otypes are under separate genetic control. Proc Natl Acad Sci USA 75:1466-1469, 1978
29. Tucker RW, Stanford KK, Handelman SL, Jones GM: Colony morphology and growth in agarose as tests for spontaneous neoplastic transformation in vitro. Cancer Res 37:1571-1579, 1977
30. Colburn NH, Bruegge WFV, BatesJR, Gray RH, RossenJD, Kelsey WH, Shimada T : Correlation of anchorage-independent growth with tumorigenicity of chemically transformed mouse epidermal cells. Cancer Res 38:624-634, 1978
31. Lipkin G, Knecht M: A diffusible factor restoring contact inhibition of growth to malignant melanocytes. Proc Nat! Acad SCI USA 71:849-853, 1974
32. Lipkin G: Altered malignancy and incre.ased contact inhibition of amelanotic melanoma cell line after pigment transformation by nucleic acids from benign blue nevi. Proc Am Assoc Cancer Res 12:26, 1971
33. Lipkin G: Pigment transformation and induction in hamster malignant amelanotic melanocytes: an effect of nucleiC aCids from hamster benign blue nevi. J Invest Dermatol 57:49-65 , 1971
34. Lipkin G: Use of mammalian nucleic acids in studies on transformation of tumor cells, Methods in Cancer Research, vol 8. Edited by H Busch. New York, Academic Press, 1973, pp 339-384
35. Lipkin G, Knecht ME: Contact inhibition of growth is restored .to malignant melanocytes of man and mouse by a hamster protem. Exp Cell Res 102:341-348, 1976
36. Lipkin G, Knecht ME, Rosenberg M : A potent inhibito~ of normal and transformed cell growth derived from contact mhlblted cells. Cancer Res 38:635-643, 1978
37. Lipkin G, Naughton GK, Rosenberg M, Bystryn J-C: Vitiligo-~elated pigment cell differentiation antigens are expressed on malignant melanoma cells following phenotypic reversion induced by contact inhibitory factor. Differentiation 30:35-39, 1985
38. Nabi ZF, Zucker-Franklin D, Lipkin G, Rosenberg M: Susceptibility to NK cell lysis is abolished in tumor cells on restoratIon of contact inhibited growth. Cancer 1986
39. Breitman TR Selonick SE, Collins S]: Induction of differentiation of the hum~n promyelocytic leukemia cell line (HL-60) by retinoic acid. Proc Nat! Acad Sci USA 77:2936-2940, 1980
40. Strickland S, Mahdavi V : The induction of differentiation in teratocarcinoma stem cells by retinoic acid. Cell 15:393-403, 1978
41. Dion LD, Blalock JE, Gifford GE: Vitamin A-induced density-dependent inhibition of L-cell proliferation. J Natl Cancer Inst 58:795-801, 1977
42. Dion LD, BlalockJE, Gifford GE: Retinoic acid and the restoration of anchorage dependent growth to transformed mammalian cells. Exp Cell Res 117:15-22, 1978
43. Patt LM, Itaya K, Hakomori S: Retinol induces density-dependent growth inhibition and changes in glycolipids and LETS. Nature 273(5661) :379-381, 1978
44. Mannino Rj, Ballmer K, Burger MM: Growth in~ibition of t~ansformed cells with succinylated concanavalin A. SCience 201 (4358):824-826, 1978
45. Lipkin G, Knecht ME, Rosenberg M : Glycoprotein-containing fac~ tor that mediates contact inhibition of growth. Ann NY Acad Sa 312:382-391, 1978