frog pnkt-4b cells express specific extracellular matrix-degrading enzymes and cytokines correlated...

THE JOURNAL OF EXPERIMENTAL ZOOLOGY 278:201–214 (1997)

© 1997 WILEY-LISS, INC.

JEZ 786

Frog PNKT-4B Cells Express Specific ExtracellularMatrix-Degrading Enzymes and CytokinesCorrelated With an Invasive Phenotype

KATHERINE J. STRISSEL,1* MARILYN J. TESSIER,2NAVEED B. SHAMS,3 STEFAN GRABBE,2 JEROME GROSS,2AND M. ELIZABETH FINI1

1Vision Research Laboratories of New England Medical Center and theDepartment of Ophthalmology, Tufts University School of Medicine,Boston Massachusetts 02111

2Cutaneous Biology Research Center, Massachusetts General Hospital andthe Department of Dermatology, Harvard Medical School, Charlestown,Massachusetts 02129

3The Schepens Eye Research Institute, Boston, Massachusetts 02114

ABSTRACT A temperature-dependent metastatic phenotype reported for a frog cell line, PNKT-4B, provided a means for studying potential mediators of cell-matrix interaction involved in meta-static invasion. Zymography revealed that these cells secreted enzyme species with properties andcharacteristics of mammalian metalloproteinases: collagenase, stromelysin, gelatinase A, andgelatinase B. These enzymes were produced by PNKT-4B cultures maintained at both invasive-permissive (28°C), and invasion-restrictive (20°C) temperatures. However, under the invasive-per-missive culture condition cells produced more of the putative gelatinase B and A enzymes. Inaddition, an activated form of gelatinase A was produced only in invasion-permissive cultures.DNA synthesis bioassays (Mv1Lu cell line and mouse thymocytes) to detect growth promotingand/or inhibitory cytokines, revealed that PNKT-4B cultures kept at 28°C released significantlyhigher levels of stimulatory (interleukin-1-like) and latent inhibitory (transforming growth factor-beta-like) substances into the medium compared to 20°C cultures. Pre-absorption of media sampleswith heparin-sepharose indicated a second stimulatory cytokine as well. A corneal fibroblast bio-assay that tests for mediators of collagenase synthesis, detected a stimulatory substance whoseactivity was greatly reduced in the presence of interleukin-1 receptor antagonist protein. Collage-nase stimulatory activity present in 28°C culture medium was significantly higher than equalsamples from 20°C cultures. These studies provide a molecular correlation between release ofcytokines with properties of the metastatic phenotype seen in vivo. They further provide some ofthe first characterizations of frog MMPs and cytokines, which are likely to be involved in othertissue remodeling events. J. Exp. Zool. 278:201–214, 1997. © 1997 Wiley-Liss, Inc.

The capacity to invade the surrounding extra-cellular matrix and to metastasize to distant tis-sues distinguishes the malignant neoplasm fromthe benign growth. The metastatic cascade is acomplex, multi-step process and a tumor cell isthought to acquire metastatic capacity by progres-sive mutation (Fidler and Balch, ’87). To metas-tasize, tumor cells must be able to locally invadethe surrounding tissue, penetrate blood or lym-phatic vessels, and exit vessels at distant sites toform secondary tumors. Thus, it is widely acceptedthat pathways leading to metastatic invasion nec-essarily require the production of enzymes capableof degrading connective tissue barriers (Liotta,’90). Several classes of matrix-degrading enzymes

have been implicated in this role including MMPs,serine proteinases, and cathepsins (Liotta et al,’80; Liotta, ’90).

MMPs are a family of zinc-containing endopep-tidases with reactivity against a broad spectrum

Abbreviations used: APMA, 4-aminophenylmercuric acetate; bFGF,basic fibroblast growth factor; IL-1, interleukin-1; IL-1ra, interleukin-1 receptor antagonist; MMP, matrix metalloproteinase; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-dipheynyl tetrazolium bromide; PDGF,platelet-derived growth factor; TGF-beta, transforming growth fac-tor beta; TNF-alpha, tumor necrosis factor alpha.

*Correpsondence to: Katherine J. Strissel, Vision Research Labo-ratories, New England Medical Center, 750 Washington St., Box 450,Boston, MA 02111.

Received 20 June 1996; Revision accepted 9 January 1997

202 K.J. STRISSEL ET AL.

of extracellular matrix components. As a rule, theyare not produced in adult tissues except when re-quired for remodeling. However, these enzymeshave been found in naturally occurring tumorsfrom a variety of tissue sources and species, aswell as tumorigenic cell lines (Mignatti and Rifkin,’93). They are also found in the stroma surround-ing tumors, where they are produced by residentcells of the tissue being invaded (Liotta, ’90;Goldberg et al, ’90). Cell culture studies have dem-onstrated that numerous agents can affect MMPexpression; these include growth regulatory andinflammatory cytokines such as TGF-beta, PDGF,bFGF, IL-1, and TNF-alpha (Brinckerhoff, ’92;Matrisian, ’92). Once MMPs are synthesized andreleased from the cell, their activity is subject toregulation at several control points (Birkedal-Hansen et al, ’93). Thus, all MMPs with the ex-ception of membrane-bound MMPs are secretedas latent proenzymes, which must be convertedto their active form in the extracellular space.Active MMPs are inhibited by members of thetissue inhibitors of metalloproteinases family(TIMPs), or by less selective inhibitors such asalpha-2-macroglobulin. Changes at many ofthese control points have been associated withneoplastic transformation and with competence formetastasis (Mullins and Rohrlich, ’83). Mecha-nisms which lead to these changes during progres-sion to metastasis need to be identified.

A naturally occurring metastatic renal adeno-carcinoma discovered in a population of NorthAmerican leopard frogs (Rana pipiens) offers auseful model for studying changes in the regula-tion of MMP expression during progression to me-tastasis. This herpes virus–inducted neoplasm(Luké, ’52; McKinnell and Cunningham, ’82) growsas a localized tumor mass in frogs during the win-ter months when these animals live at tempera-tures close to freezing. However, in the spring, asambient temperatures begin to climb, tumors be-gin to metastasize (McKinnell and McKinnell, ’68).Temperature-dependent metastatic behavior wasfound to be reproducible in the laboratory wherefrogs could be maintained in metastatic-permissive(28°C) or metastatic-inhibitory (20°C) tempera-tures. It is this unique temperature-dependent prop-erty of the frog tumors that could be furtherexploited to identify mediators of the metastatic pro-cess (McKinnell and Tarin, ’84). Properties of thefrog tumors associated with the shift to invasive-permissive temperatures include elaboration ofcollagenolytic activity, tumor cell dissociation, andin vitro invasive potential (Ogilvie et al, ’84;

Seppanen et al, ’84; McKinnell et al, ’86). ThePNKT-4B cell line, derived from a Lucke virus-induced pronephric carcinoma of a Rana pipienstadpole (Tweedell, ’78), maintains a temperature-dependent metastatic phenotype similar to wholetumors (McKinnell et al, ’88), providing a reliablemodel for further study.

The reports of an increase in collagenolytic ac-tivity associated with the invasive phenotype ofamphibian tumors described by McKinnell andTarin (’84) provided a starting point for this study.Using the PNKT-4B cell line, we attempted to de-fine some of the molecular changes associatedwith the shift to higher temperature that couldexplain changes in the metastatic properties ofthese cells. We hypothesized that PNKT-4B cellsproduce different types or quantities of matrixdegrading enzymes (MMPs) at the invasive-per-missive temperature, and that this might bedue to alteration in the production of specificautocrine cytokines. We report our findings on al-tered metalloproteinase activity and specific cyto-kine release by PNKT-4B cells associated with atemperature-dependent invasive-phenotype.

MATERIALS AND METHODSCell culture

PNKT-4B cell line was cultured in Leibovitz’sL-15 medium with 10 mM HEPES supplementedwith 10% calf serum, L-glutamine (0.05%), andantibiotic/antimycotic (Gibco, Gaithersburg, MD).The cultures were propagated in sealed 25 cm cul-ture flasks at 20°C in a non-humidified ambientair incubator. Mv1Lu cells were maintained as rec-ommended by American Type Culture Collection(Rockville, MD). Primary rabbit corneal fibroblastswere isolated and cultured as described previouslyby our laboratory (Fini and Girard, ’90).

Preparation of conditioned mediaPNKT-4B cells were plated to 100 mm culture

dishes at 90% confluence in the presence of serumto promote attachment and spreading. The next day,the plating medium was removed and cells werewashed three times with serum-free L-15 medium.An equal volume of fresh, serum-free medium (8ml) was added and cultures were placed at either20° or 28°C. The cells were allowed to conditionthe medium for 48 hours. The conditioned mediumwas collected, centrifuged briefly to remove celldebris, and the supernatants were aliquotted andstored at –70°C until needed. In some experimentsbFGF-like substances were removed from samples

MEDIATORS CORRELATED WITH INVASIVE PHENOTYPE 203

by mixing them with 200 µl of Heparin-Sepharose(Pharmacia, Piscataway, NJ) beads for a 30-minute incubation. The heparin-sepharose slurrywas prepared in non-supplemented MEM or L-15medium, by first swelling the beads in excess me-dium (1 g yields about 4 ml of gel), followed by 2washes of fresh medium.

ZymographyZymography was used to visualize specific pro-

teinase species in samples of conditioned media.With this technique, proteinases in crude samplesare resolved by their electrophoretic mobility un-der non-reducing conditions in SDS-polyacryla-mide gels. Specific enzymes are then visualizedby their ability to degrade and “clear” a discretearea of the gel matrix within which a substrate,0.1% bovine skin gelatin or 0.1% beta casein(Sigma Chemical Co., St. Louis, MO), is incorpo-rated following removal of SDS. Both proenzymeand active forms can be visualized using this tech-nique since many proenzyme forms (includingmembers of the MMP family) renature into ac-tive configuration after removal of SDS.

Zymography was performed using our standardlab protocol (Fini and Girard, ’90). Briefly, 11%SDS-polyacrylamide slab gels were prepared inthe V16 vertical gel apparatus purchased fromLife Technologies (Bethesda, MD; 17 × 15 cm) us-ing 1.5 mm spacers. An acrylamide stock of 30%w/v Acrylamide: 0.8% w/v Bisacrylamide was used.Gelatin (2% aqueous stock from bovine skin, LifeTechnologies) or β-casein (2% aqueous stock,Sigma) were co-polymerized in the gel at a finalconcentration of 0.1%. Conditioned media sampleswere diluted in SDS-sample buffer prepared with-out mercaptan and equal volume samples wereloaded onto each gel lane. In some experiments,samples were first incubated for 1 hour at 37°Cwith APMA (4-aminophenylmercuric acetate) toconvert them to their auto-proteolytically acti-vated forms (Matsubara et al., ’91). Molecularweight standards (Gibco) were run one lane apartfrom media samples in reducing sample buffer(0.5% mercaptoethanol) as an electrophoretic mo-bility standard. Gels were then shaken gently ina 2.5% Triton X-100 solution for 1 hour at roomtemperature to remove SDS. This was followedby overnight incubation at 37°C in reaction buffer(50 mM Tris, pH 7.5, 10 mM CaCl2). The nextday the gel was stained with Coomassie brilliantblue (Sigma) and destained in 10% isopropanoluntil clear bands were visible in the blue gel back-ground.

Pulse labeling with 3H thymidineA single cell suspension of PNKT-4B cells

(brought to a concentration of 1 × 106) was seri-ally diluted 1:1 and plated into triplicate wells ofa 96-well culture dish. The cells were allowed toattach to the culture surface overnight in serum-containing L-15 medium (described earlier in Cellculture) at which time the medium was changedto serum-free medium and [3H]thymidine (1 µCi/well) was added to each well for a further incuba-tion of 48 hours (New England Nuclear, Boston,MA). The plate was then frozen at –70°C. To col-lect cellular material, plates were thawed and 10µl trypsin (0.25%) was added to each of the wellsto incubate at room temperature for 60 minutes.A 96-well plate harvester was used to separatesoluble material from cellular material in eachwell by filtration. The amount of [3H]thymidineincorporated into DNA and collected on the fil-ters was measured in a 1205 beta plate liquid scin-tillation counter (Pharmacia). The means oftriplicates and corresponding standard errors werecalculated for comparison.

DNA synthesis bioassay forTGF-beta-like activity

The Mv1Lu cell line was utilized, essentially aswe described previously (Strissel et al, ’95), to de-tect TGF-beta-like cell growth inhibitors in PNKT-4B conditioned medium. Mv1Lu cells are highlysensitive to inhibitory activity of all three of themammalian TGF-beta forms (Cheifetz et al., ’90).Cells were suspended in MEM containing 3% fe-tal bovine serum (Gibco) at 106 cell/ml and 100 µlof this suspension was added to each well of a 96-well culture plate. Media samples were then addedto triplicate wells. Serving as positive controls,TGF-beta standards (human recombinant TGF-beta1) (R & D Systems, Minneapolis, MN) seri-ally diluted at a concentration range of 1 to 0.03µg/ml, were included in separate sets of triplicatewells. The plates were incubated under cultureconditions for 18 hours. After this time, [3H]-thymidine (1 µCi/well) was added to each well fora further incubation of 6 hours (New EnglandNuclear, Boston, MA). The plate was then frozenat –70°C. To collect cellular material, the plateswere thawed and 10 µl trypsin (0.25%) was addedto each of the wells to incubate at room tempera-ture for 60 minutes. A 96-well plate harvesterwas used to separate soluble material from cel-lular material in each well by filtration. Theamount of [3H]thymidine incorporated into DNA


and collected on the filters was measured in a1205 beta plate liquid scintillation counter (Phar-macia). The mean of triplicates was calculatedand compared using the Student’s T-test. A valueof P<0.05 was considered to indicate significantdifference.

When activation of potential latent TGF-betaswas desired, 10 µl of 1N HCl was added to 0.5–1.5 ml of each conditioned media and control me-dium sample. The samples were incubated for 1hour at room temperature, then neutralized with10 µl of 1N NaOH and tested in the DNA synthe-sis assay. When required, neutralizing antibodyagainst human TGF-beta1 (R & D Systems) wasadded to the appropriate wells at concentrationsup to 1,000 pg/ml.

Mouse thymocyte proliferation/MTTreduction bioassay for IL-1-like activity

Conditioned culture media samples were as-sayed in quadruplicate for the presence of IL-1-like stimulators of thymocyte growth according toMosmann (’83). The thymus from a 6–8 week-oldmouse was isolated and a single cell suspensionmade by passing the thymus through a 60 gaugewire sieve into (RPMI) medium (Gibco); red bloodcells were removed, then thymocytes were washedtwice with RPMI medium and resuspended at 107

cells/ml in complete medium. Thymocytes wereplated at 1.0 × 106 cells per well of a 96-well flat-bottom culture plate. Conditioned media samplesto be treated or unsupplemented RPMI as a con-trol were then added. As a routine assessment ofcell responsiveness, some control wells receiveda serial dilution of concavalin A (from 25 to 1.2µg/ml). Another set of wells received human re-combinant IL-1beta (3 pg/ml, R & D Systems)serving as a positive control. Thymocytes werecultured for 72 hours after which time 150 µl ofmedium was removed from the wells and 10 µl ofMTT solution (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) (Sigma) at 5 mg/ml in PBS was added. After the 6-hour reduction/incubation period, 150 µl of acidified-isopropanol(0.04N HCl in isopropanol) was added to solubi-lize the formazan reaction product. Samples wereincubated overnight at room temperature andread at 570 nm test, and 600 nm reference wave-lengths using a Dynatech enzyme-linked immuno-sorbent assay (ELISA) plate reader (Dynatech,Chantilly, VA). Blank reference wells contained 10µl MTT, 50 µl assay medium, and 150 µl acidifiedisopropanol. The mean of quadruplicate sampleswas calculated and compared using the Student’s

T-test. A value of P<0.05 was considered to indi-cate significant difference.

Bioassay for regulators ofcollagenase expression

We followed the assay procedure essentially asdescribed previously by our laboratory (West-Mayset al., ’95). Freshly isolated cultures of cornealstromal fibroblasts were utilized for a bioassay todetect stimulators of collagenase synthesis in cell-conditioned media. Stromal fibroblasts wereplated in equal numbers (2.0 × 105) into 24-wellplastic culture dishes (Costar, Cambridge, MA) inMEM containing 10% supplemented calf serumand incubated under culture conditions overnight.The cells were washed three times with serum-free MEM and wells were brought to 300 µl vol-ume by adding either serum-free MEM, or one ofthe conditioned media test samples. In some cases,cultures were further treated with IL-1alpha orhuman recombinant IL-1ra, which were added toappropriate wells at 1 and 100 ng/ml, respectively(R & D Systems). 35[S]methionine (New EnglandNuclear) was added to the wells at 80 µCi/ml tolabel newly synthesized proteins. After 24 hours,the medium containing radio-labeled, secreted pro-teins was removed and centrifuged briefly. Thetotal labeled proteins in an equal volume (15 µl)from each culture well were then displayed bySDS polyacrylamide gel electrophoresis using BRLvertical gel apparatus (8% polyacrylamide gels 7.5× 6’, 0.75 mm) and autoradiography. After elec-trophoresis, the gels were stained with Coomassiebrilliant blue for 1 hour followed by destaining in10% isopropanol to visualize molecular weightstandard proteins. The gels were dried down toWhatman filter paper using a gel dryer and thenplaced with Kodak radiography film for a 12- to14-hour exposure. The specific 53 and 57 kD col-lagenase bands were quantitated by computinglaser densitometry (Molecular Dynamics, Sunny-vale, CA) from the autoradiographs exposed in thelinear range of film sensitivity. Identity of the col-lagenase band was verified by immunoprecipita-tion analysis using polyclonal antisera specific forrabbit collagenase and stromelysin proteins (Finiand Girard, ’90).

Protein labeling with 35S-methioninePNKT-4B cells were plated to 24-well plastic cul-

ture dishes (Costar) in equal numbers (2.0 × 105

cells/well) in L-15 medium containing 10% supple-mented calf serum and incubated at the two testtemperatures under culture conditions overnight.


The cells were washed three times with serum-free MEM and wells were brought to 300 µl vol-ume by adding serum-free L-15 medium and35[S]methionine (New England Nuclear) at 80 µCi/ml, then returned to the appropriate temperatureto label newly synthesized proteins. After 24hours, the medium containing radio-labeled, se-creted proteins was removed and centrifugedbriefly, and the adherent cells were lysed by threerounds of freeze/thaw in serum-free L-15, the wellcontents collected, vortexed, centrifuged at 2,000rpm in an Eppendorf bench top microfuge, andthe supernatants collected for comparison. The to-tal labeled proteins in an equal volume (contain-ing equal TCA-precipitable CPCM) from eachculture well conditioned medium and cell extractsamples were then displayed by SDS polyacryla-mide gel electrophoresis (8% polyacrylamide gels7.5 × 6’, 0.75 mm) and autoradiography. After elec-trophoresis, the gels were stained with Coomassiebrilliant blue for 1 hour followed by destaining in10% isopropanol to visualize molecular weightstandard proteins. The gels were dried down toWhatman filter paper using a gel dryer and thenplaced with Kodak radiography film for a 12- to14-hour exposure.

RESULTS

Specific proteinases detected in PNKT-4Bconditioned medium

We used zymography to define any differencesin proteinases secreted by PNKT-4B cells at in-vasion-restrictive (20°C) vs. invasion-permissive(28°C) temperatures. In zymography, proteinasespecies are separated by polyacrylamide gel elec-trophoresis and then specifically identified by theirability to degrade a selected protein substrate co-polymerized in the gel under chosen conditions.Figure 1 depicts a representative analysis usinggelatin as the substrate. In all such experimentsthat we performed, including this one, samples ofmedium conditioned by rabbit cell cultures wereused as standards; these are shown in Figure 1,R. The enzyme species in the rabbit standardswere previously identified by antibody binding ex-periments (Fini and Girard, ’90; Matsubara et al.,’91). The predominant proteinase found in the rab-bit epithelial cell standard (Ep) is the matrixmetalloproteinase gelatinase B (MMP-9); this en-zyme is present in the 92 kD proenzyme form.Gelatinase B proenzyme is also a component ofthe rabbit fibroblast standard (RF), however, thepredominant proteinase in the fibroblast samples

is the MMP, Gelatinase A. This proteinase is foundas three molecular species: the major 65 kDproenzyme form, and two minor species of 62 and58 kD, which represent proteolytically cleaved,activated enzyme.

The left panels of Figure 1 depict zymographicanalysis of culture medium samples conditionedby six different PNKT-4B cultures plated at equalcell density. Three of these cultures were main-tained at 20°C and three cultures were main-tained at 28°C for 48 hours before the mediumwas collected for analysis. All samples were foundto produce sharply defined cleared bands in thegelatin substrate co-migrating with the rabbitgelatinase B (92 kD) and gelatinase A (65 kDproenzyme and lower molecular weight forms). Inaddition, a broader band of partial substrate clear-ing was observed at 50 kD. A corresponding bandwas not present in the rabbit samples used for

Fig. 1. Zymographic analysis of gelatinases present inPNKT-4B culture medium from cells maintained at 20° and28°C and, for comparison, medium from rabbit corneal cell(R), epithelial (Ep), and stromal fibroblast (RF) cultures. Equalloading of unconcentrated samples are shown in each lane.Arrows at left indicate bands of enzymatic activity of 92, 65,and 50 kD, and small arrows at right indicate activated pro-teolytic products of the 65 kD enzyme species against a gela-tin substrate. Migration positions of protein molecular weightstandards (kD) are indicated at the right.


this experiment. However, the molecular sizeand diffuse character of the 50 kD band fromPNKT-4B cultures suggested its correspondenceto rabbit collagenase-1 (MMP-1) and/or stromel-ysin-1 (MMP-3), two additional MMPs that canbe produced by rabbit fibroblasts and comigrateon zymograms. Further experiments with rab-bit samples containing these enzymes confirmedtheir comigration with the 50 kD gelatinolyticactivity present in the PNKT-4B samples (datanot shown). The 50 kD activity from PNKT-4Bmedia could also be detected when casein wasused as the zymography substrate, while theother gelatin-degrading enzymes were not vis-ible, as expected on this substrate. The 50 kDband in both rabbit and PNKT-4B samples had adifferent character on casein zymograms than ongelatin zymograms, appearing sharp rather thanfuzzy (data not shown). All of these bands disap-pear when gels are developed in EDTA-contain-ing buffer, a characteristic of MMPs. Theseanalyses indicate that frog PNKT-4B cells produceproteinases with properties suggesting their iden-tity as specific members of the MMP family.

There appeared to be no difference in theamount of the 50 kD activity produced by cellsgrown at 20° vs. 28°C in the analysis shown inFigure 1, and this was also the case in repeti-tions of this experiment. However, differences inthe amounts of the other gelatin-degrading pro-teinases are apparent. The first difference is inthe amount of the putative gelatinase B proen-zyme; considerably more 92 kD enzyme is presentin the samples from cells grown at 28°C in com-parison to those grown at 20°C. The second differ-ence is in the putative gelatinase A species. Mediafrom cells grown at both 20° and 28°C containedequivalent amounts of the 65 and 58 kD forms.However, cells grown at 28°C also produced a 62kD enzyme species, not found in the medium fromcells grown at 20°C. That this represents a pro-teolytic fragment of the 65 kD enzyme species wasconfirmed by experiments with the organic mercu-rial, APMA, which converts gelatinase A to the pro-teolytically activated form (data not shown). In arepetition of the experiment shown in Figure 1, theincrease in the amounts of putative gelatinase B at28°C was also observed. However, slightly differentobservations were made with regard to the puta-tive gelatinase A forms; there was very little of theactivated forms in media from cells grown at either20° or 28°C, but there was more of the putativeproenzyme form at 28°C (data not shown).

From these results, it can be concluded that

PNKT-4B cultures maintained at the invasive-permissive temperature secrete more of certaingelatinase species, preliminarily identified by sizeand substrate specificity as the MMPs, gelatinaseB, and gelatinase A.

Bioassay for growth-regulatory andinflammatory cytokines

We performed bioassays with the Mv1Lu mam-malian cell line to test the hypothesis that PNKT-4B cells release greater amounts of TGF-beta-likegrowth inhibitory factors into the culture mediumwhen grown at 28° vs. 20°C. The results of a rep-resentative experiment are graphed in Figure 2.Crude culture medium conditioned by cells grownat 20°C stimulated DNA synthesis of the Mv1Lucultures, causing an increase in 3H-thymidine in-corporation to 139% of the level stimulated by con-trol medium, which had not been incubated withcells (Fig. 2, Con). Medium from 28°C cultures waseven more stimulatory, causing 3H-thymidine in-corporation at 166% of the control values. The dif-ference in stimulatory activity between culturesgrown at 20° and 28°C was statistically significant(P = 0.018). Because of these high levels of mask-ing stimulatory activity present in the conditionedmedia samples, we were unable to determinewhether there existed any active TGF-beta-like in-hibitory activity in the samples. However, when con-ditioned media were treated with acid to activatelatent TGF-beta-like molecules (Massague, ’90),a potent growth-inhibiting activity was revealed.Acid activation of control medium had no effectupon DNA synthesis. However, medium from 20°Ccultures decreased 3H-thymidine incorporation to

Fig. 2. Mv1Lu DNA synthesis bioassay for detection ofTGF-beta-like activity in PNKT-4B conditioned media fromcultures maintained at 20° and 28°C. Con = amphibian cul-ture medium; c.m. = conditioned medium; acid act. c.m. =conditioned medium treated with HCl for activation of latentTGF-beta molecules. Error bars represent 1 standard devia-tion of the mean.


6% of control values. Medium from 28°C cultureswas even more inhibitory for DNA synthesis, de-creasing 3H-thymidine incorporation to 1% of con-trol values. The difference in inhibitory activitybetween cultures grown at 20° and 28°C was sta-tistically significant (P = 0.001). This result indi-cates that higher levels of total TGF-beta ispresent in 28° than at 20°C cultures.

We next tested the hypothesis that the growth-stimulatory activity in the PNKT-4B mediummight be due to molecules that bind to heparin,which would include bFGF-like cytokines alsoknown to affect MMP expression. This was accom-plished by pre-treating conditioned media sampleswith heparin-sepharose, which binds bFGF-likegrowth factors (Saksela et al., ’88). Samples werethen assayed in the Mv1Lu system. Heparin treat-ment of PNKT-4B samples removed stimulatoryactivity, but only partially (data not shown). A sec-ond cytokine produced by PNKT-4B cells must,therefore, also contribute to the net growth-stimu-latory effect.

To test for temperature-dependent differencesin inflammatory cytokine release, we used a DNAsynthesis bioassay which detects IL-1-like sub-stances by their ability to stimulate DNA syn-thesis in mouse thymocytes. Figure 3 shows agraph of a representative experiment. Mediafrom PNKT-4B cultures at 20°C did not signifi-cantly stimulate cell growth when compared to

control medium (P = 0.757). However, media from28°C cultures did significantly stimulate DNA syn-thesis over the background level of controls (P =0.038). The level of stimulation was close to thevalue obtained by the addition of 3 pg of recombi-nant IL-1 to parallel test wells.

PNKT-4B media stimulates fibroblastcollagenase synthesis

A corneal fibroblast bioassay (West-Mays et al.,’95) was used to test the hypothesis that PNKT-4B cells produce substances at invasion-permis-sive temperatures that can act as paracrines tostimulate collagenase and stromelysin synthesisby neighboring cells. A representative experimentis shown in Figure 4. The control panel (Fig. 4A,left) demonstrates the basis of the assay. As wehave previously shown (West-Mays et al., ’95), fi-broblasts freshly isolated from the corneal stromado not constitutively synthesize collagenase orstromelysin (Fig. 4A, —). However, treatment withrecombinant interleukin-1 (Fig. 4A, IL-1) selec-tively stimulates synthesis of a protein doublet of53/51 kD, which immunoprecipitation analysis(Fig. 4B) identifies as the proenzyme forms of col-lagenase-1 (CL; Fig. 4B, lane 1) and stromelysin-1 (SL; Fig. 4B, lane 2), respectively. Treatmentwith a competitive antagonist of IL-1, IL-1 recep-tor antagonist (Fig. 4A, IL-1ra), has no effect onprotein synthesis. However, when cells are treatedwith IL-1ra at the same time as IL-1alpha, theIL-1 stimulated synthesis of the collagenase/stromelysin doublet is blocked.

Synthesis of the collagenase/stromelysin proteindoublet was selectively stimulated when primarycorneal fibroblasts were treated with mediasamples of PNKT-4B cultures grown at 20° or28°C (Fig. 4A, right panel). Interestingly, the me-dium from cells grown at 28°C stimulated at alevel 3.2-fold greater than the medium from cellsgrown at 20°C. Addition of IL-1 receptor antago-nist protein (IL-1ra) along with either of thePNKT-4B media samples markedly reduced thestimulatory effect of either 20° or 28°C media.These results suggest that the stimulatory sub-stance produced by PNKT-4B cells competes withIL-1ra for binding to the IL-1 receptor and areconsistent with the identity of the substance asan IL-1-like cytokine.

Metabolic changes in PNKT-4B culturesdue to temperature shift

The differences in gelatinase species seen at 20°and 28°C were selective, i.e., only some of the spe-

Fig. 3. Mouse thymocyte proliferation assay to detect IL-1-like activity in conditioned medium from PNKT-4B culturesmaintained at the two test temperatures, 20° and 28°C. Pro-liferation is analyzed by MTT reduction of triplicate samplesresulting in a color change detected at optical density 570nm. Control = cell culture medium alone; IL-1beta = humanrecombinant IL-1beta added to test wells at 3 pg/ml; PNKT20°C and PNKT 28°C = conditioned medium of cultures fromthe two test temperatures. Error bars represent 1 standarddeviation of the mean of triplicate test samples.


cies were increased. This suggested that thechanges in release of MMPs and cytokines couldnot be accounted for simply by general metabolicchanges due to the switch to a higher tempera-ture. To provide further support for this hypoth-esis, we compared overall DNA and proteinsynthetic rates of PNKT-4B cells cultured at thetwo test temperatures.

Results of a representative experiment measur-ing the incorporation of 3H thymidine into DNAby PNKT-4B cultures at the two test temperaturesare shown in Table 1. We found that cells grownat 28°C incorporated 3H thymidine more slowlythan cells grown at 20°C (by roughly 2-fold), indi-cating that the cell replication rate was slower at

Fig. 4. A: Effects of PNKT-4B conditioned mediumupon corneal fibroblast collagenase synthesis. Total 35Smethionine-labeled proteins, secreted by corneal fibro-blasts into culture medium from control wells (left panel,first eight lanes) with cells left untreated (-), treated withinterleukin-1 (IL-1), interleukin-1receptor antagonist (IL-1ra), or from cells treated with conditioned medium (rightpanel, last eight lanes) from cultures of PNKT-4B cellsgrown at 20° and 28°C, and of IL-1ra combined with con-ditioned medium (20° + IL-1ra and 28° +IL-1ra), were ana-lyzed by gel electrophoresis and autoradiography. Samples

were equally loaded to each lane and represent 5% of thetotal secreted proteins from each treatment well. Arrowspoint to a protein doublet of 53 and 41 kD of the correctmolecular size to be collagenase (CL) and stromelysin (SL),respectively. Optical densitometric readings of the CL andSL doublet appear below each of the duplicate samplesfor relative quantitative comparisons. B: Immunoprecipi-tation of pooled samples of conditioned medium using CLand SL antiserum confirm the identify of CL and SL pro-teins. Molecular weight protein standards are indicatedto the left in kilodaltons.

TABLE 1. Growth of PNKT-4B cultures at different temperatures1

Incorporation ofCell number

3H at different temperaturesat plating (× 104) 20°C 28°C

4.00 109,200 (± 20,070) 75,180 (± 11,990)2.00 59,300 (± 9,290) 29,900 (± 8,800)1.00 23,360 (± 2,870) 9,490 (± 2,100)0.25 3,400 (± 900) 1,340 (± 200)0.06 900 (± 180) 360 (± 56)1Incorporation of 3H thymidine by PNKT 4B cultures plated atvarious densities at two test temperatures during a 48-hour timeperiod. Values represent CPM of the mean of triplicate samples.Values corresponding to the standard error of the mean are includedto the right of each. Differences in DNA synthesis between the twotemperatures averages 2-fold greater for the 20°C cultures.


the higher temperature. These results are simi-lar to those previously reported by McKinnell andcolleagues (’88) and eliminate the possibility thatthe observed increase in released MMPs andcytokines at the higher test temperature are dueto an increase in the cell number over the 48-hourperiod in which conditioned media were collectedfor an experiment.

Results of a representative experiment exam-ining the incorporation of 35S-methionine intoprotein by PNKT-4B cultures at the two testtemperatures are shown in Figure 5. SDS-PAGEand autoradiography (Fig. 5, Lysates) displayeda 35S-methionine-labeled intracellular proteinprofile for cells grown at 28°C that was essen-tially identical to that of cells grown at 20°C.Quantitation using optical densitometry of pro-teins in each gel lane revealed no significantdifference in the amount of total 35S-methion-ine-labeled intracellular proteins between thetwo test temperatures. Maintenance of the pro-tein synthetic rate with the shift to the higher

temperature is not consistent with the hypoth-esis that increases in proteins released into thecell culture medium might be due to increasedcell death. The profile of 35S-methionine-labeledreleased proteins (Fig. 5, Medium) showed dif-ferences in the amounts of certain bands, sug-gesting an increased production of some proteinsat the higher temperature, which would be con-sistent with the zymogram and bioassay results.The increased radioactivity observed for some pro-teins at the higher temperature when comparedto the intracellular protein profile at both tem-peratures, further indicated that cell lysis did notoccur to any greater degree at the higher test tem-perature. Densitometry of individual gel lanes re-vealed an overall increase of 2.5-fold betweenradiolabel in proteins in PNKT-4B cultures at 28°Ccompared to cultures at 20°C. However, this in-crease did not occur across the board; the level ofsome proteins was increased but the level of othersremained unchanged. Together, these results sup-port the hypothesis that PNKT-4B cells are caused

Fig. 5. Comparison of total newly synthesized 35S methion-ine-labeled proteins from PNKT-4B cells cultured at 20° and28°C displayed by SDS-PAGE and autoradiography. Left panelshows quadruplicate cell lysate samples of PNKT-4B culturesincubated at the test temperatures for 24 hours. The right

panel shows corresponding conditioned medium samples con-taining total proteins released by PNKT-4B cells cultured at20° and 28°C. Positions of molecular weight standards areindicated in kilodaltons (kD).


to release increased amounts of certain proteins,including specific MMPs and cytokines, with anincrease in temperature.

DISCUSSIONThe PNKT-4B cell line from Rana pipiens has

an unusual, temperature-dependent metastaticphenotype (McKinnell and Tarin, ’84), which pro-vides a unique opportunity to study the mecha-nisms of tumor cell invasion and disseminationto distant sites. In this study, we have attemptedto understand some of the molecular changes thatoccur with the shift to the metastasis-permissivetemperature, which might contribute to metastaticbehavior. We found that PNKT-4B cells releaseincreased amounts of specific extracellular matrix-degrading enzyme species at the invasion-permis-sive temperature. We also measured an increasein the release of growth-promoting and inflam-matory cytokines known to affect the expressionand activity of matrix-degrading enzymes in mam-malian cells. Finally, we directly demonstrated anincrease in stimulatory activity for collagenasesynthesis at the invasion permissive temperature.

These changes we observed were specific; therewas no general increase in the synthesis or secre-tion of proteins when cells were grown at themetastatic-permissive temperature vs. the meta-static-restrictive temperature.

Previously, metalloproteinase activity againsttype IV and I collagens was detected in culturemedium conditioned by Lucke tumor explants(Shield et al., ’84). We used the technique of gela-tin zymography in order to characterize the en-zymes secreted by the cells that were responsiblefor this activity. The gelatin-degrading enzymespecies produced by Rana pipiens PNKT-4B cellsshowed similarity to several distinct rabbit en-zymes of the MMP family. Based on molecular sizeand substrate specificity, the 92 kD gelatinase ap-pears to be the Rana pipiens version of gelatinaseB, and the enzyme species between 58 and 65 kDprobably represents gelatinase A. In addition, a50 kD gelatinolytic band with a broad, diffusecharacter was quite similar to the band producedon zymograms by the combination of mammalianstromelysin-1 and collagenase-1, which co-electro-phorese under the non-reducing conditions ofzymography. Our results differed from a previousreport which showed an increased collagenolyticactivity against type I collagen released by renaltumor explants at higher temperatures (Ogilvieet al., ’84). Zymography did not show any appar-ent increase in MMP activity by molecular spe-

cies migrating at approximately 50 kD (expectedsize for type I collagenase, MMP1) at the highertest temperature. This difference in results couldbe due to the amount of specific inhibitors present,or the relative amounts of activated vs. pro-enzymespecies present in the media samples tested in theearlier study; both could affect measurements ofenzyme activity. Differences in our results fromthose of the earlier work could simply be due topotential alterations of phenotype that might oc-cur in an immortalized cell line compared to tu-mor cells of explant cultures, and although thisis a consideration in the use of an in vitro model,it does not alter the interpretations of tempera-ture differences we observed.

The enzyme characterization described here rep-resents one of the few reports on the molecularproperties of amphibian MMPs. Surprisingly, al-though the first characterized member of theMMP family, a type I collagenase, was purifiedfrom the resorbing tail of the Rana catesbiana tad-pole in the 1960s (Gross and Lapiere, ’62; Nagalet al., ’66), molecular characterization of MMPssince that time have been almost exclusivelyperformed in mammalian species. Since theearly tadpole experiments, later biochemicalstudies identified metalloproteinase activitiesagainst type I and type IV collagens to be re-leased into culture medium from amphibian tu-mor explants (Ogilvie et al., ’84; Shields et al., ’84).It is only very recently that reports on frog MMPsidentified on the molecular level have begun to ap-pear in the literature and a striking conservationof molecular character between mammals and frogshas been reported. The first of these reports de-scribed cDNAs isolated as part of a screen forgenes whose expression is induced during meta-morphosis in Xenopus laevis (Wang and Brown,’93). Two cDNAs were isolated with sequence fea-tures indicating their likely membership in theMMP family; homologies were strongest to mam-malian collagenase-3 and stromelysin-3, respec-tively. In a second report, a cDNA isolated byscreening a Rana castebeiana library with a mam-malian cDNA for collagenase-1 revealed about70% homology to human and porcine sequencesat the amino acid level (Oofusa et al., ’94). Theprotein product of this cDNA was further shownto have collagenolytic activity. A third report sug-gests that there is strong conservation of sequencebetween mammals and Xenopus for gelatinase Aas evidenced by cross-hybridization of a mamma-lian cDNA to a Xenopus mRNA (Patterton et al.,’95). In addition, a 65 kD enzyme likely to be


gelatinase A was detected by zymography of ex-tracts from metamorphosing Xenopus tadpoles.This is the first report on an amphibian MMP re-sembling gelatinase B of which we are aware. To-gether, these studies indicate a strong degree ofconservation of MMPs across the large evolution-ary distance separating mammals and frogs.

All of the enzymes identified in medium condi-tioned by PNKT-4B cells at the metastasis-restric-tive temperature were also present in mediumfrom cells grown at the metastasis-permissivetemperature. However, we saw specific increasesin the levels of MMP species that we have pre-liminarily identified as gelatinase B and gela-tinase A. In addition, in some culture mediumsamples from metastasis-permissive cultures, theputative gelatinase A proenzyme was shifted to asmaller size, suggesting its conversion to an acti-vated form. These changes provide a potentialmolecular explanation for the previous determi-nation that temperature shift causes an increasein matrix-degrading activity (Ogilvie et al., ’84).The results of this study have identified sev-eral different MMPs which could participate inmatrix degradation and are consistent with thereport of higher collagenase (MMP1) activityproduced by cultured tumor explant tissuesmaintained at higher temperatures taken fromafflicted frogs in the wild (Ogilvie et al., ’84).Our findings of activated enzyme species presentin the PNKT-4B culture media samples, weresimilar to the observation by Shields and cowork-ers (’84) that amphibian tumor explant culturesamples did not require trypsin activation in or-der for enzymatic activity to be detectable. Inter-estingly, an increase in the amounts of gelatinaseB has been correlated with tumorigenicity andmetastatic behaviors of oncogene-transformedmammalian cells (Bernhard et al., ’90; Sato et al.,’92). Also, in a study of MMP regulation and in-vasive potential of human tumorigenic cell lines,it was found that the production of processedforms of gelatinase A correlated more closely withinvasive potential than expression level of the en-zymes as a whole (Brown et al., ’90).

What might mediate an increase in MMP ex-pression and activation in response to a shiftin temperature? MMP expression in cell cul-tures has been shown to be stimulated by anumber of different growth-promoting and in-flammatory cytokines of physiological relevance.However, expression is also stimulated by di-verse agents and conditions, which seem to havelittle in common, including disruption of the ac-

tin cytoskeleton, phagocytosis, and an elevationin temperature (Alexander and Werb, ’92; Vanceet al., ’89). Recent evidence has been accumulat-ing to suggest that regulation of MMP expressionin response to this second class of stimulators iscontrolled through the release of cytokine inter-mediates, which then act in an autocrine mannerto promote MMP synthesis (Fini et al., ’94; West-Mays et al., ’95). For this reason, we assayed fortemperature-dependent release of growth-promot-ing and inflammatory cytokines known to stimu-late MMP expression in mammalian cells (Overallet al., ’89; Salo et al., ’91; Brinckerhoff, ’92). Thesestudies revealed that specific cytokine activitiesare released by PNKT-4B cells in significantlygreater quantities at the invasion-permissive tem-perature as compared to the invasion-restrictivetemperature. The cytokines have properties simi-lar to members of the mammalian TGF-beta,bFGF, and IL-1 cytokine families.

The frog PNKT-4B cytokines were characterizedby their ability to act cross-species on mamma-lian cells indicating a high level of functional con-servation across a large evolutionary distance.This is consistent with current reports. Thus,mammalian bFGF can promote mesoderm induc-tion during early embryological development inXenopus (Slack, ’90). Similarly, exogenous treat-ment of Xenopus embryonic tissue with humanrecombinant TGF-beta1 and 3 was found to actas weak mesoderm inducers in a Xenopus animalcap assay, a standard assay used to study howcells become specified in embryogenesis (Slack,’90). Several Xenopus bFGF family members havebeen identified (Slack, ’90) each of which shareswith the bFGF activity produced by the PNKT-4B cells, the capacity to bind to heparin-sepharose.In addition, several Xenopus members of the TGF-beta family have been identified (Slack, ’90).

We are aware of only one report describing anIL-1 family member in frogs. In this study, IL-1activity was detected in macrophage-enriched peri-toneal cells from Xenopus laevis, but no functionalcross-reactivity was observed when tested onmammalian cells (Watkins et al., ’87). This dif-fers from our finding that the IL-1-like cytokineproduced by PNKT-4B cells could act on bothmouse thymocytes and rabbit corneal fibroblasts.In addition, the mammalian form of the naturalinhibitor, IL-1ra, was able to interfere with thisactivity. IL-1ra competes with IL-1 binding to theIL-1 receptor, but it cannot produce a transduc-ing signal (Dripps et al., ’91).

There are two mammalian forms of IL-1, IL-1


alpha and IL-1 beta, which are only 23% homolo-gous at the amino acid level (Eisenberg et al., ’91).These two cytokines are often regulated differentlyand have distinct tissue distribution (Turner etal., ’89; Dinarello and Thompson, ’91). Neverthe-less, the two IL-1 forms are often thought of asfunctionally interchangeable since they both seemto activate signal transduction pathways by bind-ing the same cellular receptor (Dower et al., ’86).There is evidence that the two mammalian IL-1forms may not be equally able to bind the IL-1receptor from a second mammalian species, how-ever (Dinarello and Thompson, ’91). Thus, the IL-1family member released by PNKT-4B cells mighthave greater similarity or affinity for the rabbitreceptor than the form released by the peritonealcells. An alternative explanation could simply bethat the IL-1 of Rana pipiens has greater simi-larity to mammalian IL-1 than does the Xenopuscytokine.

In addition to their known capacity to act asautocrine regulators of MMP expression, thecytokines that we identified might also act as me-diators of the metastatic phenotype through theirability to control cell growth. In fact, their capac-ity for growth regulation was directly demon-strated here in several of the bioassays that wechose for cytokine detection. They also have otherproperties which could contribute to the meta-static phenotype. For example, bFGFs can stimu-late cell motility (Gospodarowicz et al., ’87) andangiogenesis (Folkman and Klagsbrun, ’87). Rolesfor TGF-beta in tumor growth and metastasishave also been demonstrated in mammals (Spornand Roberts, ’85; Knabbe et al., ’87). A third pos-sible role for the cytokines might be as paracrinestimulators of MMP expression in the tissue tobe invaded. The capacity of the IL-1-like cytokineto act as a paracrine stimulator of collagenase syn-thesis was directly demonstrated here, in the cor-neal fibroblast bioassay.

The identification of an increase in release ofspecific cytokines triggered by the shift to meta-static temperature raises the question of mecha-nism. A number of important paracrine andautocrine signaling molecules such as interleukin-1 (Auron et al., ’84; March et al., ’85) and bFGF(Mignatti and Rifkin, ’93) lack a leader sequencerequired for secretion via the endoplasmic reticu-lum and Golgi pathway. Studies have suggestedpossible cytokine release mechanisms in such in-stances that include cell death and lysis, releasethrough plasma membrane disruption of viablecells (McNeil et al., ’89), cell migration-associated

release, and exocytosis (Mignatti and Rifkin, ’93).In monocytes, a number of physiologic agents cantrigger cytokine release including mediators ofcell-cell contacts (Webb et al., ’90) and plasmin-like proteases, which may be involved in both ac-tivating latent cytokine and facilitating cytokinerelease from the cell (Matsushima et al., ’86). Afew of these cytokines, fibroblast growth factor(Mignatti et al., ’91; Jackson et al., ’92) andinterleukin-1 beta (Rubartelli et al., ’90) have alsobeen found to be released in greater amounts bycells undergoing heat shock or temperature-relatedstress response. The mechanisms controlling the re-lease of cytokines by these diverse stimuli is notunderstood (Mignatti and Rifkin, ’93).

The focus of the experiments in this study hascentered on mediators of matrix metalloproteinaseexpression and potential temperature-related re-lease mechanisms. However, there are a numberof consequences of temperature regulation andmetabolism in poikilothermic animals, which bearmentioning. Classic studies of metabolic rates inpoikilotherms had led to the view that the rela-tionship of temperature to metabolic rate couldbe stated by approximately doubling basal meta-bolic rate with every 10° rise in temperature (Bul-lock, ’55). Although a variety of studies continueto support this basic view (for example, Nathan,’89), more recently, results suggest that the ef-fects of temperature on metabolism should be in-vestigated in greater detail. For instance, theinfluence of temperature on synthesis or activi-ties of particular neuromolecules (Gabriel andBudai, ’92), or on the respiratory exchange ratioof some species can vary significantly dependingon which specific organs or tissues are being mea-sured (Stinner et al., ’94). These studies point tothe possibility that complex mechanisms, whichare directly influenced or regulated by tempera-ture, may be controlling normal metabolic pro-cesses, one or several of which could be involvedin metastatic and invasive tumor progression inpoikilotherms. Such organ- or tissue-specific ef-fects could contribute to why certain organs maybe preferred sites of metastasis. The PNKT-4Bcells might provide a useful model for further stud-ies along these lines.

ACKNOWLEDGMENTSThe authors thank Dr. Constance E. Brincker-

hoff (Dartmouth Medical School, Hanover, NH) forthe collagenase and stromelysin antisera and Dr.Robert McKinnell (U. Minnesota) for the PNKT-4B cells. This work was supported by grants from


the National Institutes of Health R01-EY08408(M.E.F.), R01-EY09828 (M.E.F.), T32-AR07908(K.J.S.), and F32-EY06719 (K.J.S.). Further sup-port was provided by the Cutaneous Biology Re-search Center through the MGH/Shisiedo Co. Ltd.Agreement. M.E.F. is a Research to Prevent Blind-ness Jules and Doris Stein Professor.

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frog pnkt-4b cells express specific extracellular matrix-degrading enzymes and cytokines correlated...

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