JOURNAL OF INTERFERON AND CYTOKINE RESEARCH 15:285-289 (1995)Mary Ann fiebert. Inc., Publishers

Antiproliferative and Recovery Effects During Treatment ofBreast and Ovarian Carcinoma Cell Cultures with Interferon-7

MARIAM KLOUCHE,1,2 HOLGER KIRCHNER,2 and FRITZ HÖLZEL1

ABSTRACT

We studied the antiproliferative effects of human interferon-7 (IFN-7) on cell lines derived from humancarcinomas (three breast, two ovarian, and one renal) and recovery from these effects when IFN-7 wasremoved after 6 or 72 h. IFN-7 led to a dose-dependent and time-dependent cytostatic inhibition of all sixtumor cell lines; the renal carcinoma cells were by far the most sensitive, and with these, cytotoxic effects werealso seen. The 50% inhibitory dose (ID50) for each cell line was different and remarkably constant over manymonths. When cells were exposed to IFN-7 for only 9 or 72 h, those from three lines recovered completely fromthe growth inhibitory effects, but from three only partially. When cultured for several weeks in the presence of1600 U/ml of IFN-7, two lines developed increased resistance to IFN-7, one became much less sensitive, andtwo showed no changes in sensitivity. We saw no correlations between these changes during continuousexposure to IFN-7 and the antiproliferative IDS0 for each cell or whether the cells recovered completely fromthe inhibitory effects of IFN-7 after short-term exposure. Nevertheless, cells with a population doubling time ofless than 48 h had low to moderate sensitivity to IFN-7 and seemed to recover more completely than thosedoubling in more than 61 h. Our results indicate great individual variation in the in vitro sensitivity ofcarcinoma cells to the antiproliferative effects of IFN-7.

INTRODUCTION

THE DIRECT ANTIPROLIFERATIVE EFFECT of interferon-7(IFN-7) as a component of its in vivo activity against can-

cer has been subject of several in vitro studies."'2' IFN-7 led to

cytostatic inhibition of the growth of most human and murinecells,'3-5' which in many cultures was completely reversible.<6)Cytotoxic effects have been less common/7' but were noted incultures originating from human renal'8' and ovarian'" carcino-mas: their occurrence seems to depend on such factors as thetumor cell type, exposure to IFN-7 of only a small number ofcells in vitro, or a low tumor burden in vivo.(9) Few studies havelooked at the recovery of cells from the antiproliferative effectsof IFN-7 after short-term exposure, followed by cultivation inthe absence of the lymphokine; MCF-7 breast carcinoma cellsrecovered completely, but there was no recovery of ME-180cervix carcinoma cells.'8' The effect of long-term treatment hasbeen studied in some human and murine tumor cell cultures,and in the continued presence of IFN-7, resistance to its an-

tiproliferative effects was induced in human breast'10' and incolon carcinoma cells." "

In this study, we have looked at the direct antiproliferativeeffect of IFN-7 after treatment of human carcinoma cell cul-tures in a standardized in vitro proliferation assay both forlimited and extended periods. We hoped that assessment ofrecovery effects and changes in cell sensitivity dependent on theduration of exposure to IFN-7 would help to evaluate the rela-tive importance of the various antiproliferative effects of IFN-7in vitro and to decide whether, in the clinic, pulsed or continu-ous treatment schedules would give the best results.

MATERIALS AND METHODS

Cells and their originsFive of the six cell lines investigated were derived from

various tumors: MCF-7 cells"2' and EFM-19 cells,"3' from thepleural effusion of two patients with a recurrent breast carci-noma; EFO-21 cells,"4' from the ascitic fluid of a poorly differ-entiated adenocarcinoma of the ovary; EFO-27 cells,"4' from

This work was part of the doctoral thesis of Mariam Klouche.'University of Hamburg, Departments of Physiological Chemistry and Obstetrics and Gynecology, Hamburg 20246, Germany.institute of Immunology and Transfusion Medicine, University of Lübeck Medical School, Lübeck 23538, Germany.

285

286 KLOUCHE ET AL.

the solid omental metastasis of an adenocarcinoma of the ovary;and EGI-4 cells from the ascitic fluid of a metastasized renalcell carcinoma (unpublished data from this laboratory). Thevariant line MCF-7 Ml"5' appeared after prolonged cultivationof MCF-7 cells at high density.

Cell cultures

Cells were grown as monolayer cultures in an atmosphere of5% C02 at 37°C and maintained and subcultured by standardtechniques. The growth medium was based on minimum essen-

tial medium (MEM) with Earle's salts supplemented with 10%fetal calf serum, double-strength essential and nonessentialMEM amino acids and vitamins, and 4 mM glutamine (all fromSeromed, Munich); 5 p,g/ml of fetuin and 2.5 p-g/ml of transfer-rin (from Sigma, Munich); 0.1 mM glycyl-L-histidyl-L-lysine,and 2.5 u,g/mi of amphotericin B. NaHC03 was used to adjustthe pH to 7.4.

Interferon-yRecombinant human IFN-7 (Bioferon GmbH, Laupheim)

had a specific activity of 2.6 x 107 antiviral units (U)/mg pro-tein.

Proliferation assay and determination of the 1D50Cell proliferation was directly measured as described re-

cently."6' Cells were seeded at an initial density of 2.5 x 103cells/cm2 in 12-well cluster petri dishes (Costar; Becton-Dick-inson, Heidelberg) and propagated for 2-3 days in growth me-

dium to ensure exponential growth. The supernatants were thenreplaced by fresh growth medium (control cultures) or mediumcontaining IFN-7 at the desired concentrations. At the desiredtimes, the numbers of viable cells in triplicate cultures were

determined in a hemocytometer. Incubation was continued forup to a maximum of 9 days, because previous studies hadshown that cells from all six lines remained in the logarithmicgrowth phase for this time. The 50% cell growth inhibitory dose(ID50) of IFN-7 was determined on day 9 from curves plottingthe IFN-7 dose against the observed cell number.

Recovery from short-term exposure to IFN-yTriplicate cultures were grown for 6 or 72 h in the presence of

1600 U/ml of IFN-7 and then rinsed twice with medium andcultured in the absence of IFN-7 until day 9. Cell culturestreated continuously with IFN-7, and control cultures not ex-

posed to IFN-7 were tested in parallel. The medium was

changed on days 3 and 6.To compare the growth inhibitory effects of IFN-7 on the

different cells, the cell doubling times (DT) were calculatedfrom the equation

ln2 (duration of incubation in days)ln2 (cell number at day 9/cell number at day 3)

Long-term exposure to IFN-yCells were cultured in the presence of 1600 U/ml of IFN-7

for 35 or 42 days. Parallel cultures grown in medium without

IFN-7 served as controls. The growth medium with or withoutIFN-7 was changed every 3 days, and cultures were subdividedwhen confluent.

Statistical considerations

The mean value was calculated for the cell count in triplicatecultures. The calculated standard deviation, expressed as a per-centage of the mean value, ranged from 0 to 7%. Experimentswere performed at least twice.

RESULTS

Sensitivity to IFN-yIFN-7 inhibited the growth of cells of all the six lines studied

and to an extent dependent on its concentration and the durationof exposure. The dose causing 50% inhibition of cell growthdiffered greatly between the various cell lines (Fig. 1), rangingfrom 60 to 650 U/ml for the breast and ovarian carcinoma cells.The ID50 for the renal carcinoma line EGI-4 was 2 U/ml, andIFN-7 at 5 U/ml or more was cytotoxic. Interestingly, cells ofthe variant line MCF-7 Ml were more resistant to IFN-7 thanthose of the parental line, MCF-7.

When the ID50 were determined for cells of each line at threedifferent passage levels, they were found to be very reproduc-ible (Fig. 1); exceptionally, MCF-7 cells were less sensitive atpassage 35 than at passage 18 or 50.

Recovery effects after short-term exposure to IFN-yUnder the standard conditions described, the breast and ova-

rian carcinoma cells were exposed for periods of 6 h or 3 or 9days to 1600 U/ml of IFN-7 and the highly sensitive renalcarcinoma EGI-4 cells to 1 U/ml of IFN-7. Results withEFM-19 breast carcinoma cells are shown in Fig. 2a. Duringexposure to IFN-7 for 6 or 72 h, the cells continued to prolifer-ate, but more slowly than untreated control cells; when culturedin the continuous presence of 1600 U/ml of IFN-7 for the entire

ID 50 (U/ml IFN gamma)

passage level 18 35 50 26 30 35 36 42 60 28 28 37 34 39 52 17 18 19MCF-7 MCF-7 M1 EFM-19 EFO-21 EFO-27 EGI-4l_l I-1 -l-

breast ovarian renal

FIG. 1. Antiproliferative effects of human IFN-7 on cells ofsix human tumor cell lines. ID50 were calculated after incuba-tion for 9 days in the presence of different concentrations ofIFN-7. Cells from each line were tested at three different pas-sage levels.

IFN-7 AND CARCINOMA CELL GROWTH

,6 Cell number/cm10c

101

10"

10c

-©- Control-*- 6h-A- 72h

-8- 9 day»

0 3 6 9

Days of incubation

106 Cell number/cm2

3 6

Days of incubation

FIG. 2. (A) EFM-19 breast cancer cells and (B) EFO-27ovarian cancer cells: growth during exposure to 1600 units/mlof IFN-7 and recovery after its removal. Cells exposed to IFN-7for 6 h, for 72 h, and for 9 days; exposed to medium only(controls). Mean results from triplicate experiments plotted.

9 day experimental period, their growth remained depressed. Incontrast, IFN-7 reduced the proliferation of EFO-27 cells onlyduring the first 3 days of exposure, and thereafter their rate ofgrowth recovered completely even during continuous exposure(Fig. 2b). Because of the initial inhibition, the total number ofcells on day 9 was less than in untreated control cultures.

287

To compare recovery from the effects of IFN-7 in the differ-ent lines, the cell doubling times between days 3 and 9 werecalculated. Three carcinoma lines recovered completely, withdoubling times comparable in IFN-7-treated and untreated con-trol cultures, but this did not appear to depend on the initialIFN-7 sensitivity. It was seen with the breast carcinoma line,MCF-7 Ml, and the ovarian carcinoma line EFO-27, whichwas four times more sensitive to IFN-7, and even with the cellsof the highly sensitive renal carcinoma line EGI-4 (see Fig. 1).In contrast, EFM-19 and MCF-7 breast carcinoma and EFO-21ovarian carcinoma cells recovered only partially, and their dou-bling times were greatly increased.

Alteration of cellular sensitivity after long-termtreatment with ¡FN-y

To determine the effect of more prolonged exposure, cultureswere maintained in the continued presence of 1600 U/ml ofIFN-7 for 35-42 days. All the cultures continued to proliferatein the presence of IFN-7. MCF-7 and EFO-27 cells grew more

slowly than in the presence of IFN-7 before, but the growth ofMCF-7 Ml, EFM-19, and EFO-21 cells was not significantlyless than that of the corresponding untreated controls.

To compare the sensitivity of the cultures before and afterexposure to IFN-7, ID50 concentrations were determined. Theresults (Table 1) showed essentially no change in the sensitivityof MCF-7 and EFM-19 breast carcinoma cells after exposure to1600 U/ml of IFN-7 for 42 days in comparison with untreatedcontrol cultures. Unexpectedly, EFO-27 and in particular,MCF-7 Ml cells, were found to have become more sensitive tothe antiproliferative effects of IFN-7: the ID50 values deter-mined after the prolonged exposure to IFN-7 were, 5 and 18times less, respectively, than in untreated cultures. Conversely,cells of the EFO-21 line became 12.5 times less sensitive thanbefore exposure to IFN-7.

It should be noted that the ID50 values determined for thecontrol cultures correlated well with those determined in earlierexperiments (Fig. 1).

DISCUSSION

Direct in vitro antiproliferative effects of IFN-7 have beendemonstrated in cells from several solid tumors, includingbreast,'2' ovarian,'1' and renal'5' carcinomas. Some studieshave reported complete reversal of growth inhibition after with-drawal of IFN-7<6,17) or induction of resistance to its effectsafter long-term treatment"01"; in other tumor cell cultures, a

brief exposure to IFN-7 was cytotoxic.'8' These widely differ-ent results prompted us to look at the direct antiproliferativeeffects of IFN-7 after a limited (9 days) or more extended(35-42 days) exposure in a standardized in vitro assay system.We chose for study six lines of human cells, derived frombreast, ovarian, and renal carcinomas, four of which were re-

cently established in our laboratory.IFN-7 induced a dose- and time-dependent inhibition of the

growth of all six cell lines, but these differed widely in sensitiv-ity. Antiproliferative effects of IFN-7 have similarly been ob-served in most of the other carcinoma cell lines tested previ-

288 KLOUCHE ET AL.

Table 1. Antiproliferative ID50Values for IFN-7 Determined on Cells of SixHuman Tumor Lines After Incubation for 35-42 Days in the Presence of 1600 U/ml

of IFN-7 (Treated) or Medium Only (Untreated Control)

50% Cell growth inhibitorydose (units/ml)

Cell line OriginUntreated

controlTreated with1600 U/ml Control!treated

MCF-7MCF-7 MlEFM-19EPO-21EPO-27

BreastBreastBreastOvaryOvary

550550

7060

180

90030

100750

35

1:1.618.3

1:1.41:12.5

5.1

ously,'3,617' again with a range of sensitivities."5' Our renalcarcinoma cells were particularly sensitive to IFN-7, and cyto-toxic effects were readily induced, as with other renal carci-noma cells.'8'

ID50 values were determined for the different tumor cellcultures at intervals over a period of up to 2 years and provedremarkably constant, even though there were cytogeneticchanges during long-term cultivation."8' Measurement of celldoubling times provided a useful way of assessing the durationof the antiproliferative effects of IFN-7 and detecting recoveryof cell growth after exposure to it for various defined times.Other studies have followed recovery effects in terms of thegrowth kinetics of the cells or colony formation.

Unexpectedly, three of our cell lines recovered completelyfrom the growth inhibitory effects of IFN-7 even when culturedin its continued presence. Doubling times were identical for thetreated and untreated control cultures. However, the observedcomplete recovery of EGI-4 renal carcinoma cells should beinterpreted with caution: these cells were exposed only to 1U/ml of IFN-7 because of their great sensitivity. Similar revers-

ibility of the cytostatic effect has also been shown in terms ofrestoration of the original growth rates'6' and by the differencesin the degree of inhibition of colony formation depending on theexposure time.'8'

In the other three cultures, there was a persistent reduction ingrowth in cells briefly exposed to IFN-7 and subsequent cul-tured in fresh medium without the cytokine.

Our results suggest a relation between sensitivity to IFN-7and the rate at which cells grow. Those with population dou-bling times of less than 48 h had low to moderate sensitivity toIFN-7 and were likely to recover completely from IFN-7-in-duced growth inhibition. In contrast, cells with a low prolifera-tive potential, reflected by doubling times of more than 61 h,were much more sensitive to the antiproliferative effect ofIFN-7 and continued to grow more slowly even when culturedin the absence of IFN-7. This relation may reflect the relativelylengthy time required for IFN-7 to activate cells, as opposed to

type I interferons'19'; cells proliferating slowly will remain in an

IFN-7 sensitive phase for a relatively long time.Several previous studies have shown that IFN-7 can inhibit

the proliferation of tumor cells'5' or even increase it.<6-20-2l) Wefound that EFO-21 ovarian carcinoma cells exposed to IFN-7for 40 days developed resistance. Other resistant cultures have

similarly resulted from the continuous presence of IFN-7, butonly after 60 days"1' or as long as 18 months.'10' The differ-ences in the responsiveness of parental MCF-7 cells and itsvariant MCF-7 Ml to the antiproliferative effects of IFN-7 havealso been seen in studies with other related tumor cell lines.<22)

Unexpectedly, MCF-7 Ml and EFO-27 cells cultured in thecontinuous presence of IFN-7 became more sensitive to itsantiproliferative effects than before. There have been no previ-ous reports of tumor cells thus becoming more sensitive toIFN-7 after long-term exposure. Downregulation of functionalIFN-7 receptors was observed in resistant human breast'10' andcolon"" carcinoma cells during continuous IFN-7 treatment,and an autoregulatory increase in the number of specific recep-tors after long-term treatment with IFN-7'2324' might explainincreased sensitivity. However, IFN-7 sensitive and resistantvariants of the same cell lines have been found with identicalreceptor binding,(25) which argues against an important role forthe receptor number in this context, and other mechanisms,perhaps deficiencies in enzymes induced by IFN-7,'26' may beinvolved in changes in sensitivity. Interestingly, cells of theMCF-7 and EFM-19 breast carcinoma cell cultures did not

change their sensitivity to IFN-7 during the continuous treat-ment regimen. Perhaps these, like other breast carcinomacells,'27' secrete autocrine growth factors that overcome theantiproliferative effects of IFN-7.'28'

Our results show that our tumor cell cultures have a charac-teristic and individual sensitivity to the direct antiproliferativeaction of IFN-7 that can change after exposure for varioustimes. There was no correlation between their sensitivity,whether or not there was recovery after limited exposure, andthe development of resistance or increased sensitivity duringprolonged exposure. However, greater sensitivity to inhibitionby IFN-7 and a reduced capacity to recover from its effectswere associated with relatively long cell doubling times. Disap-pointingly, our results do not help to show how best to use

IFN-7 in cancer patients.

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IFN-7 AND CARCINOMA CELL GROWTH 289

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Address reprint requests to:Dr. Mariam Klouche

Institute of Immunology and Transfusion MedicineUniversity of Lübeck Medical School

Ratzeburger Allee 16023538 Lübeck, Germany

Received 10 June 1994/Accepted 26 October 1994