comparative antiproliferative activity in vitro of - cancer research

[CANCER RESEARCH 42, 4948-4953, December 1982]0008-5472/82/0042-OOOOS02.00

Comparative Antiproliferative Activity in Vitro of Natural Interferonsa and ÃŸfor Diploid and Transformed Human Cells1

E. C. Borden,2 T. F. Hogan, and J. G. Voelkel

Departments of Human Oncology [E. C. B., T. F. H., J. G. V.I, Medicine [E. C. B., T. F. H.Â¡,and Biostatistics [J. G. V.I, The University of Wisconsin. Madison,Wisconsin 53792

ABSTRACT

The relative antiproliferative activity of natural interferons aand ÃŸwas compared in 43 in vitro assays of 25 human celllines or strains. After 120 hr of continuous exposure to 100units/ml, Interferon ÃŸproduced >20% growth inhibition in 22cells (88%), and Interferon a produced 20% growth inhibitionin 9 cells (36%). Only Daudi (Burkitt's lymphoma) cells were

consistently more inhibited by Interferon a. In the other 24human cells, the effect of Interferon ÃŸwas greater or equal toInterferon a. Although no tissue specificity for interferon ÃŸwasevident, interferon a generally had greater antiproliferativeeffects in cells of hematopoietic origin. The effect of interferona was usually established by 72 hr with little further growthinhibition at 120 hr. Conversely, interferon ÃŸoften had a greaterantiproliferative effect at 120 than at 72 hr. These findingssupport the hypothesis that various interferons may differ intheir biological, cell-regulatory, and clinical effects.

INTRODUCTION

Interferons inhibit the multiplication in vitro of both diploidand transformed human cells, including those from lymphomas(1,11,16), myelomas (8, 11), lymphoid leukemias (12), breastcarcinomas (2, 8, 11), osteo- and soft-tissue sarcomas (22),

ovarian carcinoma (13), melanomas (9), and urinary tract cancers (5,18). Cell growth inhibition has usually been a reversiblecytostatic effect. Since antiproliferative activity has been observed with highly purified interferons (10, 20) and interferonsproduced by recombinant DNA techniques (21), growth inhibition is considered a property of interferons per se. Relativetissue affinities for the different interferons have been postulated (12). Interferon Â«(leukocyte) inhibited proliferation ofcells from Burkitt's lymphoma to a greater degree than osteo-

sarcoma cells; conversely, osteosarcoma cells were more inhibited by interferon ÃŸ(fibroblast) (12).

Studies in vitro of the relative antiproliferative activity ofnatural human interferons might detect different cell-regulatory

effects and provide useful data for clinical trial design. We haveconducted such studies with clinically achievable interferonconcentrations (0 to 1000 units/ml). Our objectives were todetermine (a) the comparative antiproliferative potency of naturally induced interferons a and ÃŸ,(b) the type of human cellsexhibiting growth inhibition (i.e., whether tissue affinities existfor different interferons), and (c) the effect of in vitro pharmacological factors such as interferon concentration and celltreatment duration.

1Assisted by grants from the Veterans Administration and the American

Cancer Society. Data presented in part at the Fifth International Congress ofVirology. Strasbourg, France. August 1981 (4).

2 To whom requests for reprints should be addressed.

Received April 2, 1982; accepted August 19. 1982.

MATERIALS AND METHODS

Cells. Adherent cells were grown in 75-sq cm plastic flasks. Cellswere fed twice weekly with Eagle's minimal essential medium withEarle's salts and 10% heat-inactivated FCS.3 Lymphoid and myeloid

cells were grown in suspension in 25-sq cm plastic flasks (Corning

Glass Works, Corning, N. Y.) using Roswell Park Memorial InstituteTissue Culture Medium 1640 with Earle's salts and 10% FCS. Media

and sera were obtained from Grand Island Biological Co. (Grand IslandN. Y.). Gentamicin (50 fig/ml) was added to all culture media.

The source of tested cells was as follows: HT 1080, RD, A549,WI26VA4, WI38VA13, L132, HEL, MRC-5, and L929 (American Type

Culture Collection, Rockville, Md.); RT112 (L. Franks, Imperial CancerResearch Fund, London, England); L1210S6 (I. Gresser, Institute deRecherches Scientifiques sur le Cancer, Villejuif, France); HS0768,HS0803, TE85, and HS0303 (Naval Biological Laboratory, Oakland,Calif.); Daudi (S. Leong, Roswell Park Memorial Institute, Buffalo, N.Y.); MOLT-3, HT1376, and 647V (J. Fogh, Sloan-Kettering Institute,

Rye, N. Y.); T98G (G. Stein, University of Colorado, Boulder, Col.);K562, LCL664, LCL719, LCL721, and LIBR (A. Coates, R. DeMars,and J. Zarling, University of Wisconsin, Madison, Wis.); and HUT-23

(A. Gazdar, Veterans Administration Hospital, Washington, D. C.).Characterization of each of these cells has been reported independently in the literature. LUBC and ACHN were secondary cell linesderived in our laboratory from patients with malignant effusions. LUBCwas a line of mixed morphology from a woman with breast carcinoma.ACHN was from a patient with renal carcinoma and has been acceptedas an established cell line with uniform morphology (CRL 1611 ) by theAmerican Type Culture Collection.

Antiproliferative Assays. Adherent cells were trypsinized from T75flasks at -24 hr; 10" to 10s cells/well were seeded in to 16-mm wells

in 96-well trays (Catalog No. 76-000-04; Flow Laboratories, Rockville,

Md.). Stock interferon aliquots were thawed and diluted to test concentrations in minimal essential medium (Eagle's):2% FCS. On Day 0,

the growth medium was aspirated from each well and 0.5 ml of freshmedium with interferon was added. Ten-fold dilutions of from 1 to 2 up

to 1000 to 2000 units of each interferon were tested in triplicate.Control wells (without interferon) were used on each tray during everyexperiment. Trays were incubated at 37Â°(95% humidiÂ»y:5% CO2).

For wells containing adherent cells, the cell number was determinedat 0-, 72-, and 120-hr growth. Medium was aspirated from each well,cells were washed once with calcium-free Hanks' balanced salt solution

(Grand Island Biological Co.), and 0.2 ml of 0.25% trypsin (GrandIsland Biological Co.) were added. After thorough trypsinization at 37Â°,

cells were transferred by pipet until single-cell suspensions, defined

by light microscopy, were obtained. Cells were then counted in a ModelB Coulter Counter (Coulter Electronics, Inc., Hialeah, Fla.).

For lymphoid and myeloid cells growing in suspension, 104 to 10s

cells/well were seeded to 96-well trays on Day 0. A 2 x concentration

of each interferon preparation in Roswell Park Memorial Institute TissueCulture Medium 1640 with 2% FCS was added to an equal volume ofcell suspension to yield the desired interferon concentration. Cell

control wells were included, and trays were incubated as above. Cellgrowth was determined after 0, 72, and 120 hr by aspirating the well

3 The abbreviations used are: FCS, fetal calf serum; NIAID. National Institute

of Allergy and Infectious Diseases.

4948 CANCER RESEARCH VOL. 42

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Antiproliferative Effects of Interferons

contents, transferring by pipet to suspend the cells (no trypsinization),and counting in the Coulter Counter.

For every cell type at the specified times and interferon concentrations, cells from each of 3 replicate wells were counted (3 repeatedcounts/well). For control monolayers, 6 replicate wells were counted.Results were expressed as the percentage of cell growth in the mono-

layers treated with interferons when compared to control monolayers.Cell viability was measured after 120 hr growth with trypan blue dyeexclusion. For each cell type, one to 5 separate antiproliferative assayswere performed.

Interferons. Stock interferons were kept frozen at -80Â°. Aliquots

were thawed, and final dilutions were made in appropriate cell culturemedia on the day each antiproliferative assay was initiated. Interferonconcentration was determined in our laboratory for each preparationby repetitive titration on BG-9 human foreskin fibroblasts by the neutral

red vital dye uptake method (6). This assay used vesicular stomatitisvirus, Indiana strain (0.1 plaque-forming unit per cell for 48 hr), as

cytolytic challenge. It defines that interferon sample dilution, whichprotects 50% of the cells, as containing one unit of interferon per ml.The titer of each preparation was directly compared to a correspondinginternational standard preparation in repetitive assays.

For interferon a (leukocyte), NIAID Preparation G-023-901-527 was

the reference standard; American Cancer Society interferon a (K.Cantell) (specific activity, 106 units/mg protein) was the interferon a

used for antiproliferative assays. Over a 2-year period, 14 assays

directly comparing the antiviral activity of these 2 preparations resultedin geometric mean titers of 0.50 IU and 0.28 units, respectively, for50% monolayer protection from viral cytopathic effect. A correctionfactor was thus derived and applied to the interferon a (lU/ml) used inantiproliferative assays.

For interferon ÃŸ(fibroblast), NIAID Preparation G-023-902-527 was

the reference standard; National Cancer Institute interferon ÃŸ(Lot 3;Rentschler, Laupheim, Federal Republic of Germany) (specific activity,

1 x 105 units/mg protein) was the reagent interferon ÃŸused in most

experiments. Over a 2-year period, 16 assays directly comparing the

antiviral activity of these preparations resulted in geometric mean titersof 0.30 IU and 1.80 units, respectively, for 50% monolayer protectionfrom viral cytopathic effect. Thus, a correction factor was applied tothe interferon ÃŸused in the antiproliferative assays. Two other interferon ÃŸpreparations were used for selected experiments: Roswell ParkMemorial Institute Lots 3 and 5 (specific activity, 107 units/mg protein).

They were standardized as described above in 4 separate assays.For mouse interferon a:ÃŸ,NIAID Preparation G-002-904-511 was

the reference standard. Mouse interferon was prepared by infection ofL929 cells with Newcastle disease virus and partial purification ofculture supernatants (7). It was assayed on L929 cells (6).

Statistics. Since in about one-half of the experiments one of the

interferons was not tested at 100 units/ml (the concentration used forfinal comparisons in Table 1), the effects of interferons on cell growthwere compared and expressed with a regression analysis. For eachexperiment (one 96-well tray), the logarithm of cell counts at 72 and

120 hr was regressed on the logarithm of interferon concentrations. In30% of the experiments, a lack of linear fit occurred. However, mostoften this reflected small well-to-well count variation rather than animportant dose-response nonlinearity. Interassay variability was cal

culated by pooling estimated interassay standard deviations for all celllines that were tested 2 or more times. This pooling was done separatelyfor 72- and 120-hr measurements. Estimated standard errors for per

centage inhibition measurements were thus

SEtime â€”¿�(100% - inhibition %) x s

where n is the number of experiments run with a given cell line, and sis the pooled average interexperiment variation. For 72 hr s was 0.19,and for 120 hr s was 0.27 (comparable intraassay standard errorswere 0.04 for both 72 and 120 hr).

Table 1

Growth of cells after treatment with human interferons a or ÃŸ( 100 units/ml)

Cell growth (as % ofcontrol)Cell

name8Mouse

L929transformedOsteosarcomaHS0768Mouse

L1210S6leukemiaRhabdomyosarcomaRDDiploid

fibroblastsMRC-5AstrocytomaT48GDiploid

fibroblastsHS0303OsteosarcomaHS0803Transitional

carcinoma bladder HT1376OsteosarcomaTE85Breast

carcinomaLUBCLymphoblastoidLCL664Bronchogenic

carcinomaA549FibrosarcomaHT1080Adenocarcinoma,

lungHUT23Transitionalcarcinoma, bladder RT112Transitionalcarcinoma, bladder647VDiploid

fibroblastsHELLymphoblastoidLCL719LymphoblastoidLCL721MelanomaLIBRLymphoid

leukemiaMOLT-3ErythroleukemiaK562SV40-transformed

fibroblastsWI38VA13CervixL132(HeLa)Renal

adenocarcinomaACHNDaudiBurkitt's lymphomaNo.

ofexperi

ments11112111121221341211115122372

hrInterferon

a107Â±20C115

Â±2299Â±1992Â±17104Â±14107Â±2080Â±15100Â±1996Â±1889Â±1280Â±1582Â±11102Â±1485Â±1694Â±1080Â±889Â±17103Â±1493Â±1879Â±1577Â±1576Â±1472Â±677Â±1590Â±1260Â±850Â± 6Interferon

ÃŸ94

Â±18113Â±2199Â±1961Â±1277Â±1086Â±1684Â±1682Â±1687Â±1656Â±884Â±1684Â±1162Â±870Â±1361Â±748Â±576Â±1481Â±1182Â±1681Â±1561Â±1272Â±1471Â±652Â±1085Â±1126Â±464Â± 76120hrInterferon

a116

Â±31115Â±31110Â±30109Â±29105Â±20104Â±28103Â±2899Â±2798Â±2797Â±1994Â±2593Â±1892Â±1792Â±2589Â±1486Â±1284Â±2382Â±1680Â±2280Â±2278Â±2171Â±1970Â±868Â±1862Â±1243Â±841Â± 6Interferon

ÃŸ100

Â±27107Â±2998Â±2769Â±1977Â±1577Â±2182Â±2270Â±1983Â±2347Â±967Â±1880Â±1549Â±962Â±1751Â±838Â±573Â±2070Â±1369Â±1966Â±1860Â±1667Â±1864Â±835Â±936Â±711Â±252Â±8a

All of human origin except L929 andL1210S.j>

_ â€ž¿� No. of cells in treated wellsCe arowth = â€”¿�v 1nnc Mean Â±S.E. No. of cells in control wells

DECEMBER 1982 4949



Â£.C. Borden et al.

RESULTS

The antiproliferative effects of human interferons a and /?were compared in 48 separate experiments involving 25 humanand 2 murine cell lines (Table 1). At 100 units of interferon aper ml, 10 (40%) human cell lines were inhibted >20% after a72-hr treatment, and 9 (36%) were inhibited 2:20% after a 120-

hr treatment. At 100 units of interferon ÃŸper ml, 14 (52%)human cell lines were inhibited >20% after a 72-hr treatment,and 22 (88%) were inhibited >20% after a 120-hr treatment.

The antiproliferative effects of interferon a were usually established by 72 hr with little additional effect at 120 hr (Table 1).However, mitotic inhibitory effects of interferon ÃŸwere oftengreater at 120 hr (Table 1). Determinations of stability ofinterferons in media at 37Â°were undertaken. Interferon a lost

no antiviral activity after 120 hr, whereas the titer of interferonÃŸdecreased 50 to 90%.

Increasing the concentration of interferons resulted in dose-

dependent inhibition of cell growth at both 72 and 120 hr(Chart 1). In almost all cells tested, the antiproliferative effectsof interferon ÃŸexceeded those of interferon a (Chart 1; Table1). Results with 3 different interferon ÃŸpreparations from 2different sources were indistinguishable. Only Daudi (Burkitt's

lymphoma) was more inhibited by interferon a than by interferon ÃŸ(Chart 1). Neither mouse L929 nor L1210S cells wereinhibited in growth by human interferons (Table 1). However,mouse L121 OS cells were inhibited by mouse interferon (Chart1).

The antiproliferative effects of Interferon ÃŸthus exceededthose of interferon a (Chart 2). After 72 hr interferon ÃŸwasmore inhibitory of cell growth for 20 of 25 cell lines tested (signtest, 2-sided, p < 0.005) and at 120 hr for 24 of 25 cell lines(sign test, 2-sided, p < 0.0001 ). Interferon ÃŸat 120 hr resultedin growth inhibition >20% in 9 cell lines in which interferon ahad little or no Â«10%) antiproliferative effect. In 6 of these celllines, interferon ÃŸresulted in >30% inhibition. In no cell linedid interferon a result in >20% growth inhibition in the absence

of a similar antiproliferative effect of interferon ÃŸ.Interferon a at 100 units/ml produced >20% growth inhibi

tion in 5 of 6 myeloid cell lines (Table 1). Only occasionalactivity was noted against other cell types. In this sense, arelative tissue specificity for the antiproliferative effect of inter-

feron a existed for myeloid cells. Interferon ÃŸexhibited greaterantimitotic activity against a broader spectrum of cells. However, interferon ÃŸalso exerted antiproliferative effects for myeloid cells. Interferon ÃŸdid not have augmented effectivenessagainst homologous histolÃ³gica! cell types, human sarcomacells, or diploid fibroblasts. Thus, no tissue specificity existedamong the cell lines for interferon ÃŸ.

Cell growth inhibition was usually due to cytostasis withoutcell lysis or death. For most cells, viability testing with 4%trypan blue showed that >70% of adherent cells remainedviable after 5 days exposure to either natural interferon a or ÃŸat 100 units/ml. When well supernatants were counted andtested with trypan blue, few nonviable floating cells were noted.Further, frequent inspection of the growing cells by light microscopy revealed that interferon exposure did not inducedetachment of adherent cells which then remained viable insuspension. However, after 72-hr exposure at 1000 units/ml,

interferon a produced a decrease in cell number in Daudi cells,while interferon ÃŸcaused a decrease in the number of ACHN

and HEL cells. Further, after 120-hr exposure, interferon ÃŸat1000 units/ml produced a decrease in the number of TE85osteosarcoma cells. Therefore, in 4 of the 25 human cell typestested (16%), continuous exposure to high concentrations ofhuman interferon (1000 units/ml) did result in cytotoxicity withdecreases from starting cell numbers.

Although culture media and growth conditions were keptuniform, heterogenous human cell types were tested. Here, 0-to 17-fold growth was observed across cell types, but no

correlation between cell growth rate with the antimitotic effectof interferons was detected (data not shown). In addition, nosuch correlation was found within the 12 cell types that weretested 2 or more times.

DISCUSSION

These data were obtained with partially purified natural interferons. However, it seems unlikely that nonspecific toxicfactors inhibited cell growth. Human Interferon ÃŸhad no antiproliferative effect against murine cells, although these cellswere inhibited by mouse interferon. Furthermore, not all humancells were growth inhibited at the interferon a and ÃŸconcentrations tested (Table 1). The partially purified human interferonÃŸpreparations used had up to 100-fold differences in specific

activity. When these preparations and a reference Â¡nterferonÃŸpurified to homogeneity (kindly provided by E. Knight, DuPontCo.) were tested at equivalent antiviral concentrations againstselected cells, the observed antiproliferative activities weresimilar. Therefore, the results suggest that the antiproliferativeeffects were of interferons per se and were not the effect ofimpurities in the preparations.

Differences in antiproliferative effects of interferons occurredby using as a base for comparison the standard biologicalassay for interferons. This assay defines an interferon unit onthe basis of inhibition of virus replication. Assuming that theentire antiproliferative effect resulted from interferons (and noimpurities), it could be concluded conversely that interferon ÃŸhas a weaker antiviral activity when compared to interferon a.To compare absolute antiproliferative potency of 2 interferons,the number of interferon molecules per antiviral unit must beknown. Until these data are available, the results should onlybe interpreted as antiproliferative activity relative to antiviralactivity.

Interferons exert antiproliferative effects throughout the cellcycle and lengthen all cycle phases. However, maximum cellsensitivity has been reported in the G0-Gi phases (9, 14, 15,

18), and greater antiproliferative effects have been noted onquiescent cells (at high-saturation densities) than on rapidly

proliferating cells (9, 14, 17). The minor growth variation weobserved within each of the 12 repetitively tested cell types didnot demonstrate that growth rate influenced the antiproliferative effects measured. However, deliberately altering cellgrowth rates by using different cell or growth factors or conditions might detect further differences in the antiproliferativeeffects of interferons a and ÃŸ.

An i.m. dose of 3 x 106 units of interferon a produces mean

peak serum titers in humans of 50 to 60 units/ml. If antitumoractivity of interferons results from direct cell antiproliferativeeffects (rather than immune modulation or other cellular effects), achieving serum and intratumor interferon levels ofgreater than 100 units/ml may be clinically important. Although




A) MRC-5 DIPLOID FIBROBLASTS

I20r

INTERFERON (I U IlOOO

B)

HTIOeO FIBROSARCOMA

IO lOO Â¡000INTERFERON (I U)

C)A549 BRONCHOGENICCARCINOMA

IO 100 lOOOINTERFERON (I U)

D)HUT23 LUNG ADENOCARCINOMA

IO 100 lOOOINTERFERON (IU )

E)ACHN, RENAL CARCINOMA

F)

G)

DAUDI BURKITT'S LYMPHOMA

I20r

10 100 1000INTERFERON (lu)

LI2IOS6 MOUSE LYMPHOCYTIC LEUKEMIA

IO 100 1000INTERFERON d.U.)

IO lOO lOOOINTERFERON iU)

Chart 1. A to G, Â¡nvitro, dose-dependent antiprcHrferativeeffect of natural human interferons (IFN) a (leukocyte) and ÃŸ(fibroblast) against selected human celltypes {A to H Each panel depicts averaged data from 2 or more experiments. At equal antiviral concentrations, Interferon /; had greater activity than did inferierona except for Daudi cells (F). Human Interferon /( had no antÂ¡proliferative effect against mouse cells (G).

4951



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120r

O 100Â£Ã¶

UJÃ¼

2 80

_i rrO UJ 60or i-(- z

20erUJ0.

' '

/ 72 hrj_

O 20 40 60 80 100 120

PERCENT CONTROL CELL GROWTHHUMAN INTERFERON^

120

O _ 100er Ã¶o

80UJ

O UJ 60er H

20crUJo.

"V/

â€¢¿�

â€¢¿�

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0 20 40 60 80 100 120

PERCENT CONTROL CELL GROWTHHUMAN INTERFERON^

Chart 2. Relative growth inhibition of 25 human cells at 72 (A) and 120 (8)hr (expressed as percentage of control cell growth). Points, a single cell line. Theantiproliferative effect of interferon ÃŸsignificantly exceeded that of interferon o(72 hr, p < 0.005; 120 hr, p < 0.0001 ; sign test, 2-sided).

antiproliferative activity in the above experiments was usuallyapparent by 3 days, selected tumor types may require longerexposure at high concentrations for maximal antiproliferativeeffects (e.g., Chart 2, C and E).

Antiproliferative studies of induced human interferons a andÃŸagainst 9 types of human cells have concurrently been inprogress in Japan (19). These studies provide important, independent confirmation of our data. At concentrations of interferons of 1 to 500 units/ml, 2 of 4 cell lines derived from

/ normal tissues and 4 of 5 cell lines of tumor origin were growthinhibited by human interferon. Interferon a was more potentthan interferon ÃŸfor only Daudi cells. No tissue specificity forthe interferons was evident, and cell growth rate was not clearlyrelated to antiproliferative effectiveness of interferons. Fragmentary data in earlier reports also corroborate these findings(3, 8).

Since an interferon a subspecies, present in only smallamounts in buffy coat preparations, may have very potentantiproliferative activity, it is not possible to predict what mayresult when interferon a or ÃŸsubspecies are compared. However, the in vitro antiproliferative advantage of natural interferonÃŸover interferon a defined by our data and those of Kataokaet al. (19) has both biological and clinical implications. Theresults support the hypothesis that various interferons mayhave differing biological and cell-regulatory effects.

ACKNOWLEDGMENTS

The authors thank Kim Sack and Jack McBain for technical laboratory assistance, Susan Anderson and Lynn Maertz for assistance with data collection andanalysis, and Kathy Edge for manuscript preparation.

REFERENCES

1. Adams, A., Strander, H., and Cantei!, K. Sensitivity of the Epstein-Barr virustransformed human lymphoid cell lines to interferon. J. Gen. Virol., 28. 207-217, 1975.

2. Balkwill, F.. Watling, D., and Taylor-Papadimitriou. J. Inhibition by lympho-

/ blastoid inferieron of growth of cells derived from the human breast. Int. J./ Cancer, 22. 258-265, 1978.

3. Blalock, J. E., Georgiades, J. A., Langford, M. P., and Johnson, H. M.Purified human immune inferieron has more potent anticellular activity thanfibroblast or leukocyte interferon. Cell. Immunol., 49: 390-394, 1980.

4. Borden, E. C. Antitumor and antiproliferative effects of human interferons aand ÃŸ.Int. Virol. Cong., (Abstract) 5. 93. 1981.

5. Borden, E. C., Hogan, T. F.. Yamamoto, N., Edwards, B. S., and Bryan. G.T. Interferons: rationale for use in human urinary tract malignancies. In: K.Munk and H. Kirchner, (eds.), Interferon, pp. 42-52. Basel: S. Karger-Verlag. 1981.

6. Borden, E. C., and Leonhardt, P. H. A quantitative semimicro, semi-automated colorimetrie assay for interferon. J. Lab. Clin. Med., 81:1036-1042,1977.

7. Borden, E. C., and Murphy, F. A. The interferon refractory state: in vivo andin vitro studies of its mechanism. J. Immunol., )06. 134-142, 1971.

8. Bradley, E. C.. and Ruscecetti, F. W. Effect of fibroblast, lymphoid, andmyeloid interferons on human tumor colony formation. Cancer Res., 41:244-249, 1981.

9. Creasey. A. A.. Bartholomew, J. C., and Merigan, T. C. Tole of Go-Gi arrestI?O hr 'n 'ne inhibition of tumor cell growth by interferon. Proc. Nati. Acad. Sci.Â¿ U. S. A., 77. 1471-1475, 1980.

10. DeMaeyer-Guignard. J., Tovey. M. G.. Gresser, I., and DeMaeyer, E. Purification of mouse interferon by sequential affinity chromatography onpoly(U)- and antibody-agarose columns. Nature (Lond.), 27Â». 622-625,1978.

11. Einhorn, S., and Strander, H. Interferon therapy for neoplastic disease inman. In: W. R. Stinebring and P. J. Chappie (eds.). Human InterferonProduction and Clinical Use. pp. 159-174. New York: Plenum PublishingCorp., 1977.

12. Einhorn, S., and Strander, H. Is interferon tissue specific? Effect of humanleukocyte and fibroblast interfÃ©rons on the growth of lymphoblastoid andosteosarcoma cell lines. J. Gen. Virol., 35: 573-577, 1977.

13. Epstein, L., Shen, J-T., Abele, J. S. and Reese, C. C. Sensitivity of humanovarian carcinoma cells to interferon and other antitumor agents as assessedby an in vitro semi-solid agar technique. Ann. N. Y. Acad. Sci.. 350. 228-

244, 1980.14. Fuse, A., and Kuwata, T. Inhibition of DNA synthesis of synchronized RSa

cells by human leukocyte Interferon. J. Nati. Cancer Inst., 58. 891-896,1977.

15. Greenberg. P. L., and Mosny. S. A. Cytotoxic effects of interferon in vitro ongranulocytic progenitor cells. Cancer Res.. 37. 1794-1799, 1977.

16. Hilfenhaus, J., Damm, H . and Johannsen, R. Sensivities of various lymphoblastoid cells to the antiviral and anticellular activity of human leukocyteinterferon. Arch. Virol.. 54: 271-277, 1977.

17. Horoszewicz, J. S., Leong, S. S.. and Carter, W. A. Noncyling tumor cells




Antiproliferative Effects of Interferons

are sensitive targets for the antiproliferative activity of human inferieron. 20. Knight. E., Jr. Interferon: purification and initial characterization from humanScience (Wash. D. C.), 206. 1091-1092, 1979. diploid cells. Proc. Nati. Acad. Sei. U. S. A. 73. 520-523, 1976.

18. Horoszewicz, J. S., Leong, S. S.. Ito, M.. Buffett, R. F., Karakousis, C., 21. Masucci, M. G., Szigeti, R., Klein, E., Klein, G., Gruest, J., Montagnier, L.,Holyoke, E.. Job, L., DÃ¶len, J. G., and Carter, W. A. Human fibroblast Taira, H., Hall, A., Magata, S., and Weissmann, C. Effect of interferon-oiInterferon in human neoplasia: clinical and laboratory study. Cancer Treat. from E. coli on some cell functions. Science (Wash. D. C.), 209. 1431-Rep., 62. 1899-1906, 1978. 1435, 1980.

19. Kataoka. T., Oh-hashi, F., Sakuqai, Y., and Ida, N. Characteristics of in vitro 22. Strander, H., and Einhorn, S. Effect of human leukocyte interferon on theantiproliferative activity of human interferon-yS. Cancer Chemother. Phar- growth of human osteosarcoma cells in tissue culture. Int. J. Cancer, 79:macol., in press, 1982. 468-473, 1977.

DECEMBER 1982 4953



1982;42:4948-4953. Cancer Res E. C. Borden, T. F. Hogan and J. G. Voelkel

for Diploid and Transformed Human Cellsβ and αInterferons of Naturalin VitroComparative Antiproliferative Activity

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