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

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