(CANCER RESEARCH 37, 1432-1437, May 1977]

pig tumor cells in media deficient in essential nutrients orwith certain inhibitors of cellular energy metabolism failedto increase the sensitivity of the cells to killing by antibodyand complement (2). These studies raised the question ofwhether the appearance of drug-induced sensitivity to humoral immune attack could be correlated with the inhibitionof macromolecular synthesis. This prompted us to investigate whether the mechanism of action of the effective drugsin increasing sensitivity to antibody-complement-mediatedkilling could be attributed to their primary action of inhibitingmacromolecularsynthesis.

In this paper we have compared the ability of line 1 tumorcells to synthesize DNA, RNA, and protein to the susceptibility of these cells to killing by antibody and GPC.2 We nowreport the existence of conditions for inhibition of the metabolism of line 1 cells by chemical agents (i.e., actinomycinD, puromycin, on adniamycmn)or by physical treatment (i.e.,heat or X-inradiation) that do not affect the sensitivity of thecells to antibody-GPC-mediated killing.

MATERIALS AND METHODS

Animals. Inbred Sewall-Wnight strain 2 male guinea pigswere obtained from the Frederick Cancer Research Center,Frederick, Md. The animals weighed approximately 250g.

Cells and Reagents. Line 1, a diethylnitrosamine-induced hepatic tumor passed in strain 2 guinea pigs,' wascollected as described in Refs. 13, 24, and 25 and suspendedin RPMI 1640-20% FCS (Grand Island Biological Co., GrandIsland, N. V.). All cytotoxicity assays were performed inisotonic Venonal-buffened saline containing 0.1% gelatin,0.001 M Mg2@and 0.00015 M Ca2t

Treatment of Line 1 Tumor Cells with Drugs. The metabolic inhibitors used in this study, actinomycin D (NSC3053) and adniamycin (NSC 123127), were obtained from theNational Cancer Institute Cancer Therapy EvaluationBranch, Bethesda, Md. Puromycin dihydmochlonide (Lot43C-0430) was obtained from Sigma Chemical Co. , St.Louis, Mo.

The treatment of tumor cells with drugs has been descnibed previously (18—20).Briefly, line 1 tumor cells (5 x10@cells/mi) in RPMI 1640-20% FCS with added drugs (see

2 The abbreviations used are: GPC, guinea pig complement; RPMI 1640-20% FCS, Roswell Park Memorial Institute Tissue Culture Medium 1640containing 20% fetal calf serum and 1 % antibiotic-antimycotic mixture; RPMI1640, Roswell Park Memorial Institute Tissue Culture Medium 1640 alone.

1432 CANCERRESEARCHVOL. 37

Effect of Inhibiting DNA, RNA, and Protein Synthesis of TumorCells on Their Susceptibility to Killing by Antibody andComplement

Seymour I. Schlager, Michael D. P. Boyle, Sarkis H. Ohanian, and Tibor Borsos

Laboratory of Immunobiology, National Cancer Institute, NIH, Bethesda, Maryland 20014

SUMMARY

A number of metabolic inhibitors and chemothenapeuticagents have been found to increase the sensitivity of achemically induced guinea pig hepatoma (line 1) to killin@by antibody and complement. We have investigatedwhether the mechanism whereby these drugs increase sensitivity to killing is attributable to their primary action ofinhibiting DNA, RNA, or protein synthesis. Line 1 cells incubated for 1, 4, or 17 hr with actinomycin D (25 pg/mI),adniamycin (40 @g/ml),on punomycin (5 pg/mI) on with 5-fold lower concentrations of these drugs were maximallyinhibited (>90%) in their ability to synthesize DNA, RNA,and protein within 1 hr. However, only cells incubated for 17hr with the high concentrations of drugs showed increasedsensitivity to killing by antibody and complement. Line 1cells incubated with high concentrations of these drugs for17 hr, washed, and resuspended in drug-free medium me

covered their resistance to killing by antibody and complement within 4 hr. These cells even after culture for 24 hr indrug-free medium did not regain their ability to synthesizeDNA, RNA, on protein. A similar lack of correlation betweensynthesis of these macromolecules and sensitivity to antibody-complement-mediated killing was observed after thecells were treated with physical agents that inhibit macnomolecular synthesis. Both heat-treated and X-innadiatedcells were inhibited in their ability to synthesize DNA, RNA,and protein immediately after treatment; however, only Xirradiated cells (6 and 16 hr postinnadiation) were increasedin their sensitivity to antibody-complement-mediated killing. Our data show that the ability of line 1 tumor cells toresist humoral immune attack does not depend solely ontheir ability to synthesize DNA, RNA, on protein.

INTRODUCTION

A number of metabolic inhibitors or chemotherapeuticagents used in the treatment of neoplasia have been foundto increase the sensitivity of guinea pig hepatoma cells andhuman lymphoid cells to killing by antibody and complement (4, 18-20). These effects required relatively high drugconcentrations and a long incubation time of the cells withdrugs. In addition, it has been shown that culture of guinea

I This is Paper 10 of the series, “Lysis of Tumor Cells by Antibody and

Complement.―Received December 2, 1976; accepted February 7, 1977.

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Macromolecular Synthesis and Sensitivity to Killing

legend to Chart 1 for concentrations) were incubated at 37°in 5% C02-air. At the appropriate time intervals, aliquots ofthe suspensions were drawn off, and the cells were washedand tested for their ability to synthesize DNA, RNA, and protein and for their susceptibility to killing by antibody andcomplement. Controls consisted of tumor cells cultured inRPMI 1640-20% FCS without added drugs.

X-irradiation of Line I Tumor Cells. Line 1 cells (10@cells/ml)suspended in 1 mlRPMll640weneimradiated inainin 12-x 75-mm polypropylene-capped tubes with a Westinghouse250 KVCP Dual Quadrocondex X-ray unit (200 kV, 15 ma, 0.9mm Cu). Cells were irradiated at a distance of 25 cm fromthe X-ray portals. After irradiation with 3000, 6000, and 9000R, the cells were suspended at 5 x 105/mI in RPMI 1640-20%FCS and incubated at 37°in 5% CO2-air. Control unirradiated cells were prepared concurrently.

Heat Treatment of Line I Tumor Cells. Line 1 cells (10@cells/mi) suspended in RPMI 1640 were heated for 30 mm at43°,44°,on 45°.Controls consisted of cells maintained atambient temperature (23°)in RPMI 1640-20% FCS. Aftertreatment an equal volume of RPMI 1640-20% FCS wasadded, and the cell suspensions were incubated at 37°in 5%CO2-air.

Measurement of Macromolecular Synthesis. Line 1 cellswere tested for their ability to incorporate precursors ofDNA, RNA, and protein. Two hundred fifty thousand cellswere suspended in 0.5 ml RPMI 1640-20% FCS containing20 @Ci[3H]thymidmne per ml (Amersham/Searle Corp. , Arlington Heights, III., 24.6 Ci/mmole) or 20 pCi [3H]unidineper ml (Amersham/Searle; 46 Ci/mmole) or in RPMI 1640deficient in essential amino acids but containing per ml 20

@Ciof a ‘4C-IabeledL-amino acid labeling mixture(Schwarz/Mann, Onangebung, N. Y.; L-anginine, 460 mCi/mmole; L-Ieucine, 312 mCi/mmole; L-Iysine, 300 mCi/mmole; L-valine, 260 mCi/mmole). After 1 hr incubation at37°,the cells were washed 2 times with 4 ml ice-cold Hanks'balanced salt solution and transferred onto 0.45-gm Millipore filter discs on a Millipone vacuum sampling manifold.The discs were rinsed 2 times with 4 ml ice-cold Hanks'balanced salt solution and 3 times with 4 ml ice-cold 20%tmichlomoacetic acid . The tnichiomoacetic acid-precipitableradioactivity was quantitated in 10 ml Aquasol (New England Nuclear, Boston, Mass.) in a Beckman scintillationcounter.

Blanks consisted of radioisotope added to tissue culturemedium alone. All experiments were performed in duplicate,and values reported have been corrected for the nadioisotope blanks (these values were <50 cpm in all experiments).

Sera and Antisera. The 1gM-richfraction of rabbit antiserum to sheep Forssman antigen was prepared as describedpreviously (13, 17). Tumor-specific rabbit anti-line 1 antiserum, also prepared as previously described (1), was heatedfor 30 mm at 56°,absorbed with sheep erythnocytes and line10 tumor cells, filtered, and stored at —20°.Normal GPC(JEM Research , Inc. , Kensington , Md .) was stored at —30°.

Antibody-Complement-mediated Cytotoxicity of Line 1Tumor Cells. This method for determining antibody-complement-mediated killing has been described (13). Briefly,0.1 ml of diluted antiserum was added to 0.1 ml of tumorcells (106/mI). After incubation for 30 mm at 30°,the cells

were washed with Veronal-buffened saline containing 0.1%gelatin, 0.001 M Mg2@,and 0.00015 M Ca2@and incubatedwith 0.1 ml GPC (diluted 1:8) for 60 mm at 37°.One-tenth ml0.4% trypan blue was added, and the percentage of cellstaking up the dye was determined. Controls includedcells plus complement alone and cells plus buffer alone. Thecell control and complement control values remained <10%.

RESULTS

Effect of Metabolic Inhibitors on Macromolecular Synthesis and Susceptibility to Antibody-Complement-mediated Killing of Line I Tumor Cells. Five million line 1 tumorcells were cultured at 37°in 10 ml RPMI 1640-20% FCScontaining actinomycin D (25 @g/ml),adniamycin (40 @g/ml), or puromycin (5 p@g/ml).One, 4, and 17 hr later, thecells were tested for their ability to incorporate[3H]thymidmne,[3H]unidine, and ‘4C-labeledamino acids andfor their susceptibility to killing by anti-Forssman 1gM orspecific antitumor antibody plus GPC.

Chart 1 illustrates the results of a representative expeniment. Line 1 cells cultured with actinomycin D, adniamycin,on punomycin were inhibited 85 to 99% in their inconponation of labeled precursors after incubation for 1, 4, or 17 hr.These cells were resistant to killing by anti-Fonssman orspecific antitumon antibody plus GPC after incubation withthe drugs for 1 and 4 hr. A marked increase in susceptibilityto killing by either antibody plus GPC, compared to controlcells, was observed only after 17 hr of exposure to the drugs(Chart1).

We next chose conditions (as suggested from the data inChart 1) that would result in maximal inhibition of macromolecular synthesis of the cells to determine whether thesecells would at any time become susceptible to antibodycomplement-mediated killing. Line 1 cells (5 x 105/ml) wereculturedfor1 hrat37°inRPMI 1640-20%FCS containingthe appropriate concentrations of drugs (Chart 2). The cellswere then washed free of d rug , resuspended at 5 x 10@cells/mi in RPMI 1640-20% FCS without added drugs, andneincubated at37°.lmmediatelyaftertneatmentand 4and 17hr later, aliquots of the cells were tested for their ability toincorporate the nadiolabels and for their susceptibility to killing by antibody plus GPC. After 1 hr in culture with actinomycin D or puromycin, the tumor cells were inhibited 85 to95% in their ability to incorporate [3H]thymidmne,[3H]unidine,or ‘2C-labeledamino acids (Chart 2); this inhibition persistedafter incubation for 4 and 17 hr in drug-free medium. At notime during the course of the experiments were the cellsrendered susceptible to killing by either anti-Fonssman onspecific antitumon antibody plus GPC (Chart 2). Similarresults were obtained with adniamycin (data not shown).When line 1 cells were incubated for 17 hr with 5-fold-lowerconcentrations of puromycin (1 @g/ml)on actinomycin D (5pg/mI), they were inhibited 90 to 95% in their incorporationof the radiolabeled precursors but showed no increase insusceptibility to killing by antibody plus GPC (data notshown).

It has been previously reported (19, 20) that tumor cellscultured for 17 hr with metabolic inhibitors, washed free of

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drugs, and neincubated in drug-free medium will revert tothe resistant state within 4 hr. Experiments were next performed to determine whether the reversion of drug-treatedcells to a state of resistance to antibody-complement-mediated killing was related temporally with the recovery of theability of the cells to synthesize DNA, RNA, or protein. Line 1tumor cells (5 x 10@cells/mi) were cultured for 17 hr at 37°in RPMI 1640-20% FCS containing the appropriate concentrations of drugs (Chart 3). At this time the cells werewashed free of drug, resuspended at 5 x 10@cells/mi inRPMI 1640-20% FCS, and reincubated at 37°.Immediatelyand 4 and 17 hr later, aliquots of the cells were tested fortheir ability to incorporate [3H]thymidmne, [3H]unidine, and‘4C-labeledamino acids and for their susceptibility to killingby antibody plus GPC. As expected (19, 20), cells exposedto drug for 17 hn, washed, and tested immediately (0 hm)had

TIME OF INCUBATION WITH DRUG CHRI

Chart 1. Effect of metabolic inhibitorson the ability of line 1 tumor cells to incorporate 13H)thymidine (0), (3H]unidine (91),or “C-labeledamino acids (U) and on thesensitivity of the cells to killing by anti

@ body plus GPC. Anti-Forssman antibodywas diluted 1:80; anti-line 1 antibody wasdiluted 1:15; GPCwasdlluted 1:8.A, actinomycin .D (25 gtg/mI); B, adriamycin (40pg/mI); C, puromycin (5 pg/mI); •,drugtreated cells tested with anti-Forssmanantibody plus GPC; A, drug-treated cellstested with anti-line 1 antibody plus GPC;0, untreated cells tested with anti-Forssman antibody plus GPC;@ untreatedcells tested with anti-line 1 antibody plus.GPC.

Chart 2. Ability of line 1 tumor cells to incorporate [‘H]thymidine(0), [3Hlunidlne (91),or “C-labeledamino acids (U) and sensitivity ofthe àellsto killing by antibody plus GPC. Cells were pretreated withmetabolic inhibitors for 60 mm and recultured at 37°in the absence ofdrug. See Chant 1 fondilutionsofantibodyand GPC.A, actinomycin D(25 @.tg/mI);B, purornycin (5 @.tg/mI);•,drug-treated cells tested withanti-Forssman antibody plus GPC; A, drug-treated cells tested withanti-line 1 antibody plus GPC; 0, untreated cells tested with antiForssman antibody plus GPC; t@,untreated cells tested with anti-line1 antibody plus GPC.

TIMEOFINCUBATIONWITHOUTDRUG(HA)

been rendered susceptible to killing by anti-Fomssman andspecific antitumom antibody plus GPC. Reversion to theresistant state occurred within 4 to 17 hr of culture inmedium free of inhibitors (Chart 3). No recovery in theability of the cells to incorporate the labeled precursors wasobserved at any of the time intervals tested (Chart 3). Theseresults suggested that the effect of metabolic inhibitors onthe susceptibility of the line 1 cells to killing by antibody andcomplement was not due to the primary action of inhibitingthe synthesis of DNA, RNA, and protein. We next examined2 purely physical agents reported to inhibit macromoleculamsynthesis of nucleated cells, X-imradiation (21) and heat (3,5, 9, 11, 12, 15), to determine whether a nonchemical treatment could increase the susceptibility of line 1 cells tokilling by antibody and GPC. These treatments were used toobviate the need for the continued incubation of the cells

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Macromolecular Synthesis and Sensitivity to Killing

with chemical agents and to determine the exact timeof insult on the cells.

Effect of Physical Agents on Macromolecular Synthesisand Susceptibility to Antibody-Complement-mediated Killing of Line I Tumor Cells. Line I cells were exposed to3000, 6000, and 9000 A of X-immadiationas described in“Materialsand Methods.―Immediately postimmadiationand1, 6, and 16 hr later, the cells were tested for their ability toincorporate [3H]thymidine, [3Hjumidine, and ‘4C-labeledamino acids and for their susceptibility to killing by antiForssman on specific antitumor antibody plus GPC. Withcells exposed to 9000 A, maximal inhibition (60 to 80%)of incorporation of radiolabels was observed immediately

postimradiation and persisted after 16 hr in culture at 37°(Chart 4). Cells exposed to 3000 or 6000 A were not maximally inhibited in their incorporation until after 16 hr inculture at 37°.None of these cells demonstrated increasedsusceptibility to killing by either antibody plus GPC untilafter 6 hr in culture postirradiation; the increase in susceptibility of the cells to killing was most marked 16 hr afterirradiation (Chart 4).

Similar experiments were performed in which line 1 cellswere heated for 30 mm at 43°,44°,or 45°.Immediately and 1,3, 6, and 24 hr after heating, the cells were tested for theirability to incorporate [3H]thymidine, [3H]uridine, and ‘4C-labeled amino acids and for their susceptibility to killing by

IIChart 3. Ability of line 1 tumor cells to incorporate [3H]thyrnidine

(0), [‘H]unidine(91),or ‘4C-labeledamino acids (U) and sensitivity ofthe cells to killing by antibody plus GPC. Cells were pretreated withmetabolic Inhibitors for 17 hr and recultured at 37°in the absenceof drug. See Chart I for dilutions of antibody and GPC.A, actinomycm D (25 @.tg/ml);B, puromycin (5 @sg/rnI).S. drug-treated cellstested with anti-Forssman antibody plus GPC; A, drug-treated cellstested with anti-line 1 antibody plus GPC; 0, untreated cells testedwith anti-Forssman antibody plus GPC; & untreated cells testedwith anti-line 1 antibody plus GPC.

Chart 4. Effect of X-irradiation on the ability of line 1tumor cells to incorporate [3H]thymidine (0). [3H]unidine (91), or ‘IC-labeledamino acids (U) and on thesensitivity of the cells to killing by antibody plus GPC.See Chart 1 for dilutions of antibody and GPC. A, immediately after X-irradiation; B, 3 hr at 37°postirnadiation; C, 6 hr at 37°postlrradlation; D, 16 hr at375 postirradlatlon; •, cells tested with anti-Forssman

antibody plus GPC; A, cells tested with anti-line 1antibody plus GPC.

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S. I. Schlager et al.

anti-Fonssman on specific antitumon antibody plus GPC.Line 1 cells heated for 30 mm at any of these temperatureswere consistently inhibited 80 to 90% in their ability toincorporate the nadiolabels immediately after heating; thiseffectpersistedfor6 hrafterheating.After24 hrincubationat 37°,heated cells were inhibited only 30% in their ability tosynthesize DNA, RNA, and protein. At no time up to 24 hrafter heating-were these cells rendered susceptible to killingby either antibody plus GPC. Once out of a total of 9 expeniments, an increase in susceptibility to killing was observed;this occurred with anti-Forssman antibody plus GPC immediately and 1 hr after heating at 45°.After 3 hr incubation at37°,the cells were as resistant to killing as were controlcells.

DISCUSSION

Segerling et al. (18-20) have reported that pretreatment ofguinea pig hepatoma cells with certain metabolic inhibitorson chemotherapeutic agents increased the sensitivity of thecells to killing by antibody and complement. These observations suggested that some intrinsic property(s) of the cellunder metabolic control may contribute to the ability of thetumor cells to resist humoral immune attack. However, notall inhibitors of cellular metabolism were effective in increasing the sensitivity of the cells to killing (2, 19, 20). Wetherefore examined the relationship of the ability of the cellto synthesize DNA, RNA, and protein and its susceptibility tokilling by antibody and complement. For this purpose weused 3 drugs, adniamycin, actinomycin D, and punomycin,which are known to have their major inhibitory effect onDNA (8, 22, 23), RNA (10), and protein synthesis (16), respectively.

In this paper we have shown that the susceptibility of line1 tumor cells to killing by antibody and GPC does notcorrelate with the ability of the cells to synthesize DNA,RNA, on protein. We have demonstrated conditions underwhich the cells are inhibited in their capacity to synthesizethese macromolecules without being rendered sensitive tohumoral immune attack. For example, line 1 cells treatedwith high concentrations of punomycin (5 pg/mI), actinomycm D (25 @g/ml),or adniamycin (40 jtg/ml) were inhibited intheir capacity to synthesize DNA, RNA, or protein by greaterthan 90% within 1 hr, and they remained inhibited for a

‘ further 16 hr even in the absence ofthe drugs. However, the

increased sensitivity of these cells to killing by antibody andcomplement was observed only after 17 hr in the continuedpresence of the drugs. Line 1 cells incubated with 5-foldlower concentrations of any of these drugs showed similarlevels of inhibition of DNA, RNA, and protein synthesis, butan increase in sensitivity to antibody-complement killingwas never observed.

Furthermore, as previously reported (19, 20), cells treatedwith these drugs for 17 hr, washed , and resuspended indrug-free medium reverted to a state of resistance to killingby antibody and complement within 4 hr of culture at 37°.

However, these cells never regained their capacity to synthesize DNA, RNA, on protein. All these results suggest thatthe increased sensitivity of line 1 tumor cells to humonal

immune attackfollowingtreatmentwithmetabolicinhibitons is not due to the basic property of the cells ininhibiting DNA (adniamycin), RNA (actinomycin D), or protein (puromycin) synthesis.

Two physical means of inhibiting cellular metabolism(heat and X-irradiation) were also tested to determinewhether a nonchemical treatment could result in increasedsensitivity of line 1 cells to killing by antibody and GPC. Theadvantage of such physical agents is in knowing the exacttime of insult and in having no need for the continuedpresence of the agent. Our results indicated that inhibitionof macnomolecular synthesis of the tumor cells following Xirradiation preceded by at least 6 hr the appearance ofincreased sensitivity of the cells to antibody-complementmediated killing. In addition, line 1 cells exposed to hyperthermic conditions (43—45°)were uniformly inhibited immediately after heating in their ability to synthesize DNA, RNA,or protein. However, at no time interval up to 17 hr posttreatment were the cells rendered susceptible to antibodyGPC-mediated killing.

The observations presented here do not exclude the p05-sibility that increased sensitivity to antibody-complementmediated killing by tumor cells following chemical on physical treatment is associated with an effect by these agents onpathways of cellular metabolism other than DNA, RNA, onprotein synthesis. Since the action of antibody and complement occurs on on in the cell membrane (6, 7, 14), we arenow studying whether treatments that increase the sensitivity of tumor cells to immune attack can be correlated with achange in the properties of the cell membrane on withchanges in other metabolic pathways associated with membrane structure and function.

REFERENCES

1. Borsos, T., Richardson, A. K., Ohanian, S. H., and Leonard, E. J.Immunochemical Detection of Tumor-Specific and Embryonic Antigensof Diethylnitrosamine-Induced Guinea Pig Tumors. J. NatI. Cancer Inst.51: 1955-1960, 1973.

2. Boyle, M. D. P., Ohanian, S. H., and Borsos, T. Lysis of Tumor Cells byAntibody and Complement. III. Lack of Correlation between AntigenMovement and Cell Lysis. J. Immunol., 115: 473—475,1975.

3. Dickson, J. A., and Shah, D. M. The Effects of Hyperthermia (42°C)on theBiochemistry and Growth of a Malignant Cell Line. European J. Cancer,8: 561—571, 1972.

4. Ferrone, S., Pellegnino, M. A., Dienich, M. P., and Reisfeld, R. A. Effect ofInhibitors of Macromolecular Synthesis on HL-A Antibody MediatedLysis of Cultured Lymphoblasts. Tissue Antigens, 4: 275—282,1974.

5. Glovanella, B. C., Lohman, W. A., and Heidelberger, C. Effects of Elevated Temperatures and Drugs on the Viability of L1210 Leukemia Cells.CancerRes.,30:1623-1631,1970.

6. Green, H., and Goldberg, B. The Action of Antibody and Complement onMammalian Cells. Ann. N. V. Acad. Sci., 87: 352-362, 1960.

7. Hammer, C. H., Nicholson, A., and Mayer, M. M. On the Mechanism ofCytolysis by Complement: Evidence on Insertion of C5b and C7 Subunitsof the C5b,6,7 Complex into Phospholipid Bilayers of Erythocyte Membranes. Proc. NatI. Acad. Sci. U. S., 72: 5076-5080, 1975.

8. Hyman, R. W., and Davidson, N. The Binding of Actinomycin D to CrabPoly d (A-T)-poly d (T-A). II. On the Nature of the DNA Binding Site.Biochim. Biophys. Acta, 28: 38-48, 1971.

9. McCormick, W., and Penman, 5. Regulation of Protein Synthesis inHeLa Cells: Translation at Elevated Temperatures. J. Mol. Biol., 39: 315-.333, 1969.

10. Mompanler, R. L., Karon, M., Siegel, S. E., and Avila, F. Effect ofAdniarnycin on DNA, RNA. and Protein Synthesis in Cell-free Sytstemsand Intact Cells. Cancer Res., 36: 2891-2895, 1976.

11. Mondovi, B., Agno, A. F., Rotilio, G., Strom, R., Monicca, G., and Fanelli,A. R. The Biochemical Mechanism of Selective Heat Sensitivity of Cancer

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Cells. II. Studies on Nucleic Acids and Protein Synthesis. European J.Cancer, 5: 137-146, 1969.

12. Muckle, D. S., and Dickson, J. A. The Selective Inhibitory Effect ofHyperthermia on the Metabolism and Growth of Malignant Cells. Bnit. J.Cancer, 25: 771-776, 1972.

13. Ohanian, S. H., Borsos, T., and Rapp, H. J. Lysis of Tumor Cells byAntibody and Complement. I. Lack of Correlation between Antigen Content and Lytic Susceptibility. J. NatI. Cancer Inst., 50: 1313-1320, 1973.

14. Okada, H., and Okada, N. Complement Receptors on Cell Membrane. II.Separation from Mouse Plasma of Two Essential Factors for Complement Receptors on Cell Membranes. Japan. J. Exptl. Med., 44: 301-310,1974.

15. Palzer, A. J., and Heidelbenger, C. Studies on the Quantitative Biology ofHyperthermic Killing of HeLa Cells. Cancer Res., 33: 415-421 , 1973.

16. Pestka, S. Inhibitors of Ribosome Functions. Ann. Rev. Microbiol., 25:487-562, 1971.

17. Rapp, H. J., and Borsos, T. Molecular Basis of Complement Action, pp.75-109. New York: Appleton-Century-Cnofts, Inc., 1970.

18. Segerling, M., Ohanian, S. H., and Bonsos, T. Effect of Metabolic Inhibitons on Killing of Tumor Cells by Antibody and Complement. J. NatI.Cancer Inst., 53: 1411-1413, 1974.

19. Segenling. M., Ohanian, S. H., and Borsos, T. Chemotherapeutic DrugsIncrease Killing of Tumor Cells by Antibody and Complement. Science,188:55-57,1975.

20. Segenling,M., Ohanian,S. H., and Borsos,T. EnhancingEffectbyMetabolic Inhibitors on the Killing of Tumor Cells by Antibody andComplement.CancerRes.,35:3199-3203,1975.

21. Shipley, W. U. Immune Cytolysis and X-lrradiation: Independent LethalAction in Chinese Hamster Cells In Vitro. J. NatI. Cancer Inst., 48: 651-655, 1972.

22. Wells, R. D. Actinomycin Binding to DNA: Inability of a DNA ContainingGuanine to Bind Actinomycin D. Science, 165: 75-76, 1969.

23. Wells, R. D., and Larson, J. E. Studies on the Binding of Actinomycin Dto DNA and DNA Model Polymers. J. Mol. Biol., 49: 319-342, 1970.

24. Zbar, B., @e!nstein,I. D., and Rape, H. J. Suppression ofTumor Growthat the 5ite of Infection with Living Bacillus Calmette-Gudrin. J. NatI.Cancer Inst., 46: 831-839, 1971.

25. Zban, B., Wepsic, H. T., Rapp, H. J., Whang-Peng, J., and Borsos, T.Transplantable Hepatomas Induced in Strain-2 Guinea Pigs by Diethylnitrosamine: Characterization by Histology, Growth, and Chromosomes. J.NatI. Cancer Inst., 43: 821-831 , 1969.

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1977;37:1432-1437. Cancer Res   Seymour I. Schlager, Michael D. P. Boyle, Sarkis H. Ohanian, et al.   ComplementCells on Their Susceptibility to Killing by Antibody and Effect of Inhibiting DNA, RNA, and Protein Synthesis of Tumor

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