[CANCER RESEARCH 50. 686-690, February 1, 1990]

Induction by Transforming Growth Factor ßof Pemphigus Vulgaris AntigenActivity in Mouse Papilloma Cells'

Molly Kulesz-Martin,2 Ingrid Glurich, Barbara Lisafeld, and Paul Kozlowski

Grace Cancer Drug Center, Roswell Park Memorial institute, Buffalo, New York 14263

ABSTRACT

Induction of a marker of epidermal spinous cells, pemphigus antigenactivity, was detected by indirect immunofluorescence in murine papil-loma cells exposed to human transforming growth factor ./ ( H.I -//}.

Detection of pemphigus antigen activity required exposure of cells to 1.4mivi Ca2* for 3 h just prior to immunoassay. The brief exposure to Ca2+

may be necessary for translocation of intracellular pemphigus antigen tothe cell surface, where it is accessible to antibody. Cells grown in mediumcontaining 0.02-0.04 HIM ( ;r* were shown previously to be primarily

basal cells characterized by pemphigoid antigen activity. Following treatment with 0.25-25 pg/ml TGF-0 for 44 h under 0.02-0.04 mivi Ca2*

conditions, 63 ±9% (SD) of cells were pemphigus positive. This percentage was comparable to that of positive control cultures exposed to1.4 mivi Ca2* for 44 h (70 ±10%) and was up to 2-fold that of solvent

control cultures. Pemphigus antigen activity was significantly induced by0.1-25 pg/ml TGF-A out of a tested range of 10"5-10' pg/ml. The total

number of papilloma cell colonies was unaffected by treatment with 0.1-25 pg/ml TGF-0 but was reduced greater than 90% by treatment with103-5 x IO3 pg/ml TGF-/9. The described immunofluorescence assay for

pemphigus antigen activity may be useful for preliminary evaluation ofdifferentiation-inducing agents in anticarcinoma therapy.

INTRODUCTION

Most human cancers are solid tumors of differentiating epi-thelia. In normal epidermis, differentiation involves programmed transition via RNA and protein synthesis (1) fromthe proliferative basal stage to the spinous, granular, and cor-nified cell stages. Noncytotoxic agents which induce differentiation in tumor cells could be important in anticarcinomatherapy. Prerequisites for identifying such agents were: (a)determining the kinetics of spontaneous transition from thebasal to spinous cell type in epidermal tumor cell cultures; and(¿>)developing an assay for drug-induced expression of a spinouscell marker which required extracellular Ca2* for immunode-

tection.Two differentiation stage-specific antigens of mouse and

human epidermis are recognized by human sera from patientswith the skin diseases huilons pemphigoid and pemphigus vul-garis. Pemphigoid antiserum recognizes a hemidesmosomal-associated antigen in basal cells of normal epidermis (2). Pemphigus antiserum specifically stains the plasma membrane junctions of spinous cells (3, 4). In cultured normal cells, pemphigoid antigen activity is expressed under conditions favoringbasal cell proliferation (0.02-0.04 miviCa2+ in culture medium),

whereas pemphigus antigen activity is expressed under conditions favoring differentiation [1.4 mM Ca2+ (5, 6)]. Recently,we have reported that, under low Ca2* conditions, 291.09RA-

T papilloma cells are primarily basal cells expressing pemphigoid antigen activity and that elevation of Ca2+ to 1.4 m\i

results in decreasing pemphigoid antigen activity and increasing

Received 2/14/89; revised 7/17/89, 10/10/89; accepted 10/31/89.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

'This work was supported by USPHS Grants CA-42852. CA-31101, CA-13038. and CA-24S38 awarded by the National Cancer Institute.

2To whom requests for reprints should be addressed, at Grace Cancer DrugCenter, Roswell Park Memorial Institute. 666 Elm Street. Buffalo. NY 14263.

pemphigus antigen activity (7). Since pemphigus antigen activity was not detectable under low Ca2+ conditions (7), it wasconjectured that pemphigus antigen, like certain desmosomal-associated antigens, may require extracellular Ca2* for trans-

location to the cell surface, where it is reactive with antibody(8).

The present experiments were designed to determine whethera short exposure of 291.09RA-T cells to 1.4 mM Ca2* would

permit the detection of increased pemphigus antigen activity inresponse to a potential therapeutic agent. TGF-ß,-1a homodi-

meric polypeptide produced by normal and neoplastic cells, wasexamined because it inhibits the growth of certain epithelialcells, including human epidermal cells (9). It was of interest todetermine whether this effect was associated with spinous celldifferentiation. Using the modified immunofluorescence assay,induction of pemphigus antigen activity following exposure ofmouse papilloma cells to TGF-/Õwas detected.

MATERIALS AND METHODS

Culture Medium. The tumor cell line was derived and maintained inEagle's medium with 5% fetal bovine serum and standard Ca2* concen

tration (1.4 mM) as described previously (10, 11). All experiments wereplated under low Ca2* conditions, i.e.. Eagle's medium containing 5%fetal bovine serum, with a final concentration of 0.02-0.04 mM Ca2*

(6), supplemented with 10 ng/ml epidermal growth factor (Collaborative Research, Bedford, MA).

Cell Line. Cell line 291.09RA-T was derived from expiants of apapilloma [a highly keratinizing, benign epidermal tumor (10)]. Thistumor arose in athymic nu/nu mice (Harlan-Sprague-Dawley, Indianapolis, IN) after i.p. injection of initiated cells which had been selectedunder standard Ca2* conditions after treatment of the normal cell line291 with 7,12-dimethylbenz(a)anthracene as previously described (11).Cell line 291.09RA-T was used between passages 50 and 64 in vitroand was stable in its tumorigenic phenotype during this period (10)."

Sera. Normal human sera from 15 donors (Roswell Park MemorialInstitute Blood Bank) and pemphigus vulgaris patient sera (gift ofIMMCO, Buffalo. NY) were pooled prior to use in these experiments.The specificity of each patient serum was defined by pemphigus antigenactivity at the plasma membrane in one to two layers of suprabasalcells of monkey esophagus by IIP as reported previously (12). Serumtiters were 1:640 (three sera) and 1:80 (one serum). Patient sera werefree of discernible autoantibody reactivity to nuclear, smooth muscle,or cytoplasmic antigens. Secondary' antibody was FITC-labeled goatanti-human IgG (1:64 dilution; IMMCO).

Experimental Design. Papilloma cells were plated under low Ca2*conditions as described above. Plating of 2.7 x IO4291.09RA-T cells/18-mm glass coverslip resulted in approximately 10-15% confluence24 h after plating, 20-30% 48 h after plating, and 50% 72 h afterplating. Four h after plating, low Ca2* medium without EGF wasexchanged, and TGF-fi or solvent (5 mM HC1) was added in 15 ^1/3 mlof medium to give the final concentration indicated. Replicate coverslipswere exposed to 1.4 mM Ca2* beginning at 4 h as a positive control.

'The abbreviations used are: TGF-ji, human transforming growth factor fi;EGF, epidermal growth factor: FITC. fluorescein isothiocyanate: IIP. indirectimmunofluorescence assay; PBS/0.5TÕ sodium azide, 0.116 M NaCl, 0.01 MNa2HPO4, and 0.003 M KH¡PO4(pH 7.4) with 0.5'í sodium azidc and 1.4 mMCa2*.

' M. Kulcsz Martin. I. Glurich. B. Lisafeld, and P. Kozlowski. unpublished

results.

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EPIDERMAL DIFFERENTIATION MARKER IN TUMOR CELLS TREATED WITH TGF-fi

Unless otherwise indicated, IIP was performed 48 h after plating,following 3-h exposure of cells to 1.4 mM Ca2* by addition of 14 ¿ilof

CaCI2 (0.3 M stock). TGF-/J derived from human platelets was obtainedfrom Collaborative Research.

Indirect Immunofluorescence Assay. None of the permeabilization orfixation techniques tested previously permitted retention of specificpemphigus antibody reactivity above nonspecific background fluorescence of normal human serum (7). Therefore, IIP for pemphigus wasperformed without fixation according to Beutner et al. (13). Sera werediluted 1:10 in PBS-0.5% sodium azide and applied to coverslips in ahumidified chamber at room temperature for 30 min. After washing inPBS-0.5% sodium azide for 15 min, FITC-labeled goat anti-humanIgG was applied for 30 min. Coverslips were mounted in Gelvatol(IMMCO) and viewed using a Nikon Labophot microscope equippedwith a mercury vapor lamp and a B2E filter block. Fluorescenceintensity was rated from 1+ to 4+ with normal human serum as acontrol. Percentages of pemphigus-positive cells were calculated basedon numbers of cells exhibiting 3+ or 4+ fluorescence intensity per totalcells viewed by phase contrast microscopy. Counts of fluorescent-labeled cells were made only on cells in contact in areas of comparabledensity among treatment groups. Duplicate coverslips were evaluatedper treatment group representing a minimum of 100 cells/slip.

RESULTS

Ca2+ Dependence of Pemphigus Antigen Activity. Watt et al.(8) reported that extracellular Ca2* concentration above 0.1 HIM

was required for translocation of certain intracellular antigensto desmosomal locations at the cell surface. As a basis for drugtreatment studies, it was necessary to determine the time ofexposure to 1.4 mivi Ca2* required for maximal immunodetec-tion of pemphigus antigen activity in 291.09RA-T papillomacells. As shown in Table 1, pemphigus antigen activity wasdetectable 2 or 3 h after exposure of cells to 1.4 m\i Ca2* and

did not increase significantly by further time of exposure up to6 h. In addition, spontaneous increase in pemphigus antigenactivity detectable by 3 h of exposure to elevated Ca2* was

minimal up to 96 h after plating (data not shown). Therefore,the effects of TGF-/J were examined 48 h after plating using a3-h exposure to 1.4 mivi Ca2*.

TGF-/S Effect on Pemphigus Antigen Activity. A significantincrease in pemphigus antigen activity occurred within therange of 0.1-25 pg/ml TGF-/ÃŒout of a tested range of 10~5-103

pg/ml (Fig. 1). The percentage of pemphigus-positive cellsincreased from a basal value of 39 ±6% (n = 7 experiments)to a value of 63 ±9% after exposure of cells to TGF-/ÌThisincrease was comparable to that observed in cells exposed to1.4 mivi Ca2* during the same time period (44 h) as a positive

Table 1 Effect of duration of exposure to 1.4 m\i Co2* on pemphigus antigenactivity in 291.09R.4-T papilloma cells

IIP assay was performed at the indicated times after plating. Coverslips werewashed with PBS, reacted with primary antisera for 30 min at room temperature,washed, and reacted with FITC-labeled goat anti-human IgG. Values given arethe mean ±SD/approximately 100 cells from multiple fields in each of 2coverslips. The requirement for 2-3-h exposure to 1.4 mM Ca2* and the presenceof pemphigus antigen activity in only a minority of cells exposed to Ca2* for 6 hor less were reproduced in two other experiments.

Time (h) ofexposure

to 1.4 mMCa2*0

123456%

of cells exhibitingpemphigus antigen

activity/time (h) afterplating24

487200

517 ±2.1 2015 ±0.7 2221 ±7.0 2415 ±1.4 2619 ±2.1 300

00.7 05.7 2 ±2.83.5 34 ±3.50.7 32 ±3.52.8 37 ±7.07.0 45 ±4.9

„¿�80J»

3601MJ.

40E•E,-

/<TT*¿T"

AK i•

20 -

-5 -4-3210123TGF-/3(logpg/mU44h)

Fig. 1. Percentage of 291.09RA-T papilloma cells expressing pemphigusantigen activity following exposure to TGF-ji. Cells were plated under 0.02-0.04mM Ca2* conditions. Beginning at 4 h after plating, low Ca2* medium withoutEGF was exchanged, and TGF-tf or 5 mM HCI was added in 15 yl/3 ml to givethe final concentration indicated. For 3 h immediately prior to III cells in allgroups were exposed to 1.4 mM extracellular Ca2* to permit immunodetection of

pemphigus vulgaris antigen activity at the cell surface. IIP assay was performedat 48 h as described in Table 1. Dashed line labeled (+). mean value (n = 7experiments) ¡ngroups of cells exposed to 1.4 m\i Ca2* beginning at 4 h afterplating, as a positive control. Dashed line labeled (-). mean value in groupsexposed to solvent as a negative control. Values are mean ±SD for a minimumof 100 cells from multiple fields in each of 2 coverslips. Results of 3 of 7comparable experiments (A. •¿�.and •¿�)are shown. Significant increase of percentage of pemphigus-positive cells was observed in the range of 0.1-25 pg/mlTGF-f) as determined using the Fisher exact test (P < 0.001 ).

control (70 ±10%). As shown in Fig. 2, pemphigus antigenactivity in TGF-/i-treated cells was localized at the cell surface(Fig. IB) as in cells exposed to 1.4 mM Ca2* for 48 h (Fig. 1C).

Specific pemphigus antigen activity was not detectable in replicate cultures maintained under low Ca2* conditions, withoutexposure to elevated extracellular Ca2* (Fig. 2, G and H).

In order to verify that the induction of pemphigus antigenactivity occurred during the 4-45 h under low Ca2* conditions,

TGF-/3 was added according to each of the three schedulesshown in Fig. 3. The results indicated that TGF-/3 was ineffective at inducing pemphigus antigen activity when applied onlyduring the 3-h exposure of cells to 1.4 mM extracellular Ca2*(treatment schedule C). Treatment of cells with TGF-0 between4 and 45 h under low Ca2* conditions only, followed by twowashes with PBS and 3-h exposure of cells to 1.4 mM Ca2* infresh medium without added TGF-/J (treatment B) was aseffective as treatment between 4 and 48 h, including the 3-hexposure of cells to 1.4 mM Ca2* (standard treatment A). Thus,

pemphigus antigen activity was not induced by TGF-/ÃŒduringthe 3-h exposure of cells to 1.4 mM Ca2*. In addition, exposureof cells to cycloheximide (50 Mg/ml) during the 3-h incubationwith 1.4 mM Ca2* did not prevent the detection of pemphigus

antigen activity in untreated cells, nor did it inhibit the 2-foldinduction of activity by 1.4 mM Ca2* or TGF-/3 (data not

shown). These results are consistent with induction of pemphigus antigen synthesis under low Ca2* conditions, followed by

translocation of antigen activity to the cell membrane duringthe 3-h exposure to 1.4 mM Ca2*.

Effects of TGF-/9 in Colony Assays. Concentrations of TGF-ßwhich resulted in peak induction of pemphigus antigen activity had little effect on the total number of papilloma colonies,which were counted using an electronic image analyzer system(14) (Fig. 4). Thus, pemphigus antigen activity was induced atconcentrations of TGF-/Õwhich were not cytotoxic. A decreasein the number of colonies occurred above 100 pg/ml TGF-0,approaching 0 colonies/dish in cultures treated with 103-5 x10' pg/ml TGF-/3. However, since TGF-ßadheres strongly to

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EPIDERMAL DIFFERENTIATION MARKER IN TUMOR CELLS TREATED WITH TGF-ti

Fig. 2. Localization of pemphigus antigenactivity at the cell membrane. Ill assay wasperformed as described in Table 1 and Fig. I.2.5 pg/ml TGF-0 x 44 h (B, E, //) or solvent x44 h (A, D, G) were added 4 h after plating under0.02-0.04 mM Ca2* conditions; 1.4 mM Ca2* x

44 h (C, F) was used as a positive control.Primary antisera were pemphigus vulgaris patient serum (A-C, G, H) or normal human serum(D-F). Exposure to 1.4 mM Ca2* for 3 h wasdone immediately prior to IIP (A-F). Withoutexposure to 1.4 mM Ca2*, no specific reactivity

with pemphigus antiserum was detectable (G,H), x 320.

•¿�o 60

»40

E •¿�il

-(+)

•¿�(-

100

« 80Q

8 60

o 40oo-= 20

-20

O 4Time After Plating (h)

48

Fig. 3. Effect of treatment schedule on induction of pemphigus antigen activityby TGF-ti. Plating and I1F were as described in Fig. 1. Schedules depicted at thebottom were: treatment with TGF-rf for 41 h under 0.02-0.04 mM Ca2*conditionsand during 3-h exposure to 1.4 mM Ca2* (treatment A, standard treatment as inFigs. 1 and 2). treatment for 41 h under 0.02-0.04 mM Ca2* conditions only(treatment B), and treatment for 3 h during exposure to 1.4 mM Ca2* only(treatment C). m. 1.4 mM Ca2* x 3 h; D, 5 pg/ml TGF-fl; •¿�5 pg/ml TGF-d plus1.4mMCa2*x 3 h.

0 10' IO2 IO3

TGF-/9 pg/mL

Fig. 4. TGF-iJ effect on survival of 291.09RA-T papilloma cell colonies. Cellswere plated at a density of 700/60-mm Petri dish. On the fourth day after plating,when colonies contained no more than 16 cells. TGF-t) or 5 mM HC1 was added(15 nl/3 ml) in low Ca2* medium without EGF. On the seventh day. standardgrowth medium containing 1.4 mM Ca2* was exchanged. Cultures were terminated

on the 14th day. fixed in methanol. and stained with Gicmsa. Total colonies perdish were counted using a computer-assisted electronic imaging system as recentlyreported (14) (DISHMS Petri dish measurement system. John Peach, developer;Spectra Services. Rochester. NY). These results were reproduced in two additionalexperiments.

glass and plastic, it was possible that TGF-/Õmight not be duplicated the IIP assays, except that cells were trypsinized attotally removed by media exchange. Therefore, cells were plated the 48-h point and replated at clonal density ( l O1cells/60-mm

on coverslips and treated with TGF-/3 under conditions which Petri dish) in culture medium containing 10 ng/ml EGF. Col-

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EPIDERMAL DIFFERENTIATION MARKER IN TUMOR CELLS TREATED WITH TGF-0

ony-forming efficiency (number of colonies per total cellsplated) was unaffected by exposure of cells to less than 100 pg/ml TGF-/3 and was decreased to 40 ±12% of solvent controlby exposure of cells to 5 x IO3 pg/ml TGF-/3.

DISCUSSION

Reports of an intracellular and extracellular Ca2* gradient in

epidermis suggest a physiological role for this ion in normalepidermal differentiation in vivo as well as in vitro (15). Lowextracellular Ca2+ concentration (less than 0.1 mM Ca2+) has

been used to select for basal cell populations in culture (5-7).Elevation of Ca2+ concentration has been shown to induceepidermal cells to synthesize spinous cell-specific proteins such

as pemphigus antigen (5, 16) and keratins Kl and K10 (17). Inaddition, Watt et al. (8) reported that 0.1 mM Ca2+ or greateris required for translocation of certain desmosomal-associatedantigens in human keratinocytes from the cytoplasm to theplasma membrane, where they are reactive with antibody.

Our results indicated that exposure to Ça2*for 2 or 3 h prior

to the IIP assay was required for detection of pemphigus antigenactivity at the cell membrane of 291.09RA-T papilloma cells.The spinous cell subpopulation detectable in untreated or solvent-exposed cultures from 24 to 96 h after plating may haveoriginated in the high density cultures used to plate the cover-slips. The data presented in Table 1 indicated that 4-6 h ofexposure to 1.4 mM Ca2+ did not increase pemphigus antigenactivity significantly, whereas exposure of cells to 1.4 mM Ca2+

for 24 h did induce activity (7). Protein synthesis during the 3h prior to IIP was not required for detection of pemphigusantigen activity in untreated cultures or in cultures induced toexpress activity by TGF-0 or 1.4 mM Ca2+. This is consistent

with translocation of antigen during the short exposure to 1.4mM Ca2+as the mechanism by which pemphigus antigen activity

becomes detectable. It implies, too, that any synthesis of pemphigus protein responsible for its immunodetection in the cellsexposed to TGF-/3 must have occurred under low Ca2+ conditions. Coffey et al. (18) reported that TGF-/3 did not induce theexpression of pemphigus antigen activity in cultured epidermalcells when low Ca2+ conditions were used for the culture, drug

incubation, and immunofluorescence steps in the experiment.The current results suggest that this lack of detectable inductionof pemphigus antigen activity in response to TGF-/3 was due tothe Ca2+ requirement for immunoreactivity.

While induction of pemphigus antigen activity in normalepidermal cell populations is indicative of commitment to terminal differentiation, this is not necessarily also true of tumorcells. An alternative explanation is that tumor cells expressingpemphigus antigen activity are still capable of proliferation.The lack of effect on total colony number at concentrations ofTGF-/} with maximal effect on pemphigus antigen activity couldbe explained either by irreversible loss of growth potential inthe pemphigus-positive subpopulation (63%) within a colony atthe time of treatment, with subsequent growth of the remainingunresponsive cells, or by reversible growth arrest of treatedcells. Irreversible growth arrest and induction of terminal differentiation is consistent with the data of Masui et al. (19),which indicated that TGF-/3 treatment of human bronchialepithelial cultures increased the proportion of cells with corni-fied envelopes, a marker of late-stage squamous cell differentiation. In the current study, a decrease in colony number byconcentrations of TGF-0 greater than IO2pg/ml was not asso

ciated with pemphigus antigen activity in cells. This couldreflect progress of cells through the spinous cell stage to a later

differentiation stage (7). On the other hand, reversible growtharrest has been suggested by Wilke et al. (20) to occur in humankeratinocyte cultures treated with 5 x IO3pg/ml TGF-0. In thecurrent study, the reduction in colony-forming efficiency in291.09RA-T papilloma cells trypsinized and replated aftertreatment with 5 x IO3pg/ml TGF-/3 suggested irreversible loss

of proliferative potential in the majority of clonagenic cellstreated. These seemingly contradictory results can be explainedif cell cycle arrest by TGF-/3 in basal cells is reversible undergrowth conditions, e.g., in the presence of EGF and low extracellular Ca2+and in the absence of serum differentiation factors,

but irreversible in the majority of cells in the presence of serumor endogenous differentiation factors. The influence of cultureconditions on the cellular response to TGF-/3 should be resolvable by analysis of proliferation and differentiation parametersin the same cell by double-label IIF. In addition, future studiesshould address whether an increase in intracellular Ca2+ con

centration, for example due to mobilization from intracellularCa2+ stores, is a mediator of the effects of TGF-0 on induction

of pemphigus antigen activity.The modification of an indirect immunofluorescence assay

for detection of pemphigus antigen activity has permitted themeasurement of a drug effect on induction of a differentiationmarker in epidermal tumor cells. This assay may be useful inthe initial evaluation of other drugs or biologicals for potentialto induce differentiation in squamous cell tumors. The resultsare significant to differentiation therapy even in light of the lowinduction level (up to 2-fold) for two reasons: (a) increasedduration of treatment may increase the proportion of responding cells, and (b) induction of differentiation in quiescent tumorcells which otherwise would remain capable of proliferation ispotentially therapeutic.

ACKNOWLEDGMENTS

We thank Dr. Vijay Kumar of IMMCO, Buffalo, New York, forproviding the antisera and for his advice and assistance throughout thecourse of this work. We are grateful to Dr. John Stanley and Dr. DennisRoop for helpful discussion, and to Mae Brown for assistance in thepreparation of this manuscript.

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1990;50:686-690. Cancer Res   Molly Kulesz-Martin, Ingrid Glurich, Barbara Lisafeld, et al.   Vulgaris Antigen Activity in Mouse Papilloma Cells

of PemphigusβInduction by Transforming Growth Factor

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