Diphtheria Toxin Effects on Human Cells in Tissue Culture1

[CANCER RESEARCH 36, 4590-4594, December 1976] Diphtheria Toxin Effects on Human Cells in Tissue Culture1

Barbara R. Venter2 and Nathan 0. Kaplan3

Departments of Biology (B. A. V.) and Chemistry (N. 0. K.), University of California, San Diego, La Jolla, California 92093

SUMMARY component that renders the hybrids retaining chromosome 5 much more sensitive to diphtheria toxin. HeLa calls exposed to a single sublethal concentration of Several reports have appeared in the recent literature diphtheria toxin were found to have diminished sensitivity exploring the possible use of diphtheria toxin as an anhineo when subsequently reaxposad to the toxin. Three calls plastic agent (2, 8). In 1974, lglewski and Rithenbeng (5) strains exhibiting toxin resistance were developed. In the reported that diphtheria toxin produced a greater inhibitory calls that had previously been exposed to toxin at 0.015 j.@g/ effect on protein synthesisof humanand mousetumor cells ml, 50% inhibition of protein synthesis required a toxin than on normal tissues from the same species. Their paper concentration of 0.3 pg/mI, which is more than 10 times expanded upon an earlier study by Buzzi and Maistrallo (1) that required in normal HeLa cells. in which it was reported that striking results could be ob There appears to be a threshold level of diphtheria toxin tamed with diphtheria toxin treatment of experimental hu action.Concentrationsof toxingreaterthan that required momsin Swiss mica. Papenheiman and Randall (9), in 1975, for 50% inhibition of protein synthesis (0.01 @g/ml)are while confirming that diphtheria toxin causes some tempo associated with cytotoxicity, whereas those below this con nary regression of Ehrlich ascihas tumors in mice, were less cantration may not be lethal. optimistic as to the potential usefulness of diphtheria toxin Several established human cell lines of both normal and as an antineoplashic agent for human use. In their study, neoplastic origin ware tested for their sensitivity to the these workers also questioned the validity of the assay used effects of the toxin. No special sensitivity was observed with by lglewski and Rithanbeng (5). the calls of tumor origin. Fifty % inhibition of protein syn In this paper, we report that HaLa cells exposed to a thesis in HeLa cells was achieved with diphtheria toxin (0.01 single sublethal dose of diphtheria toxin exhibit resistance pg/mI) as compared to the normal human cell lines tested when subsequently reaxposed to the toxin. A positive comma (0.03 and 0.5 p.g/ml) and a cell line derived from a human lation is demonstrated between the degree of the acquired pancreatic adanocarcinoma (0.2 @g/ml).A human breast resistanceto diphtheria toxin and the concentration of toxin carcinoma call line showed a maximum of 45% inhibition of to which the calls were initially exposed. In addition, the protein synthesis. This required a diphtheria toxin concen effects of diphtheria toxin wanestudied on cultured human tration of 5 j.@g/ml. These results suggest that different tumor cells as opposed to cultured normal tissue of human human call lines show wide variation in their sensitivity to origin. In comparing a number of human cell lines, no thetoxin. consistent level of in vitro sensitivity, which may be related to whether the calls wane normal or neoplastic, was noted. INTRODUCTION MATERIALS AND METHODS Diphtheriatoxinisaprotein(M.W.62,000)releasedextra callularly from Corynabactarium diphtheriaa. The toxin is Cell Cultures. HeLa calls (CCL-2), normal human fibro composed of 2 fragments, A and B, into which it may be blasts (CAL-i 121), and mouse connective tissue cells (CCL split by trypsin in the presence of a reducing agent. The 1 ) were obtained from the American Type Culture Collec cytoloxic activity is thought to reside in the A fragment, lion, Rockvilla, Md. Human breast carcinoma cells (BT-20) whereas the ability of the toxin to bind to sensitive cells is and human pancreatic adenocarcinoma calls (A-i 165) ware thought to reside in the B fragment (8). In the intact toxin, provided by Contract E-73-2001-NO1 within the special Vi the B fragment is believed to interact with a specific site on rus-Cancar Program, NIH, USPHS, through the courtesy of the plasma membrane. Creagen at a!. (2), using mouse Dr. W. Nelson-Aees at the Naval Biomedical Research Labo human somatic call hybrids, have demonstrated that human ratory, Oakland, Calif. Normal (VF)and transformed (SV-VF) chrdmosoma 5 codas for a presumed membrane membrane human fibroblast calls were obtained from the Cone C tissue culture facility at the University of California, San Diego,

1 This work was supported in part by USPHS Grant CA 1 1683 from the NIH Calif. , courtesy of Dr. W. L. Nyhan. All the cell lines were and American Cancer Society Grant BC-60-R. grown in DME4 (Flow Laboratories, Inglewood, Calif.) sup 2 Portions of the work reported here were taken from a dissertation sub mitted to the University of California, San Diego, Calif., in partial fulfillment plamanled with 10% FBS (Irvine Scientific, Irvine, Calif.). of the requirements for the Ph.D. degree (12). Present address: Department of Breast Surgery, Roswell Park Memorial Institute, 666 Elm St. , Buffalo, N. 4 The abbreviations used are: DME, Dulbecco's modified Eagle's minimum Y. 14263. essential medium; FBS, fetal bovine serum; TCA, trichloroacetic acid; ED@, 3 To whom requests for reprints should be addressed. concentration of diphtheria toxin that produced 50% inhibition of protein Received June 7, 1976; accepted September 9, 1976. synthesis.

4590 CANCER RESEARCH VOL. 36

Toxins. Diphtheria toxin (Connaught Medical Research subcultuned (1:5 dilution) as required . Three cell lines, HaLa Laboratories, Ontario, Canada) Lots D-2905 and F-370 con nC, mE,and rF, previously exposed to toxin concentrations tamed 200 Lf/mI (7 mg protein per ml). For the growth of 0.015, 0.003, and 0.0015 @g/ml,respectively, ware devel studies and development of toxin-resistant cell lines, the oped. Six weeks after the initial exposure to diphtheria toxin was restanilized by filtration through a 0.22-j.@mMilli toxin, the calls ware hashedfor resistance to the toxin using pore filter. Ricin was a gift from Dr. Garth Nicholson of the the protein synthesis assay and have subsequently bean Salk Institute, La Jolla, Calif. rehashed3 times oven a period of 9 months. Protein Synthesis Assay. The protein synthesis assay is a modification of that described in Ref. 5, and was done as follows. The calls to be tested were seeded at 2 x 10@cells/ RESULTS plate in 35-mm tissue culture dishes (Falcon; obtained from Scientific Products, Los Angeles, Calif.) in regular DME Effect of DME Containing 1% of the Normal Amino Acid supplemented with 10% FBS and incubated overnight. The Complement, Supplemented with 10% Undialyzed Serum, cells were then washed twice with warm phosphate on Protein Synthesis. The protein synthesis assay we have buffered saline containing, in g/litan: NaCI, 8; KCI, 0.2; Na2 used is similar hothe assay reported by lglewski and Rithen HPO4, 1.15; and KH2PO4,0.2. DME containing 1% of the berg (5); however, we did find it necessary (see â€œDiscus normal concentration of amino acids was added, 2 mI/dish. sionâ€•)toreplace the dialyzed serum with undialyzad serum. The low-amino acid medium was supplemented with 10% Incorporation of labeled amino acids into cell protein in undialyzed FBS. The calls ware incubated at 37Â°for 2 hr, uninhoxicahed HaLa cells is shown in Chart 1A to be assen then 10 @Iof the appropriate toxin concentration wane tially linear over the time course of the assay (5 hr). Protein added. Each concentration was lasted in duplicate; 4 dishes synthesis in the HaLa cells was completely inhibited by were left untreated with toxin. After 3 hm,10 @l(1MCi)of a diphtheria toxin (3 @g/ml)within 3 to 4 hr. No effects wane â€œC-aminoacidmix (New England Nuclear, Boston, Mass.) noted with this toxin concentration on â€˜4C-labeledamino were added. After an additional 2 hr, the cells wanewashed acid incorporation into the mouse fibmoblast (L-929) cell twice with cold DME(minusserum)and fixed with 4 ml cold protein over a 6-hr period (Chart iB). The results with the 10% TCA. The TCA was aspirated after 15 mm, the calls HeLa cells are similar ho those reported by Pappenhaimer were dissolved overnight in 4 ml 0.5 N NaOH, and the and Randall (9) using complete medium supplemented with solution was transferred ho large hashtubas. One ml cold 5% FBS. 50% TCA and 4 ml cold 10% TCA ware added ho precipitate Cytotoxic Effects of Diphtheria Toxin. The affect of diph the protein, and the solution was filtered (Millipore filtration thenia toxin on the growth of HeLa cells over a period of 6 apparatus) on 0.45-zm, HAWP fillers (Millipora; obtained days was examined according ho the procedure outlined in from Scientific Products). The tubes wanerinsed with 4 ml â€œMaterials and Methods â€œunder â€œGrowth Studies.â€• At con cold 5% TCA, and the rinse was added to the filters. The centrations of diphtheria toxin greater than 0.05 pg/mI, the filters ware washed with cold 0.5% TCA, than they ware cells wane lysed, and after 4 days essentially no viable calls dried and counted using loluana-based Omnifluom (New remained on the plates. At concentrations of diphtheria England Nuclear GalPacks) in a Beckman scintillation toxin between 0.015 and 0.005 @g/ml,the growth pattern counter. approached that of untreated HeLa calls. However, at con Cytotoxic ActIvity of DIphtheria Toxin. HaLa cells ware cantrahions of toxin between 0.05 and 0.015 j.@g/mI,the total seeded at 0.5 x i0@ cells/60-mm tissue culture dish, in number of cells remained relatively constant over the time normal DME supplemented with 10% FBS. The cells ware course of the study. These results are summarized in Chart allowed to plate out for 3 hr before diphtheria toxin was 2. added. Duplicate dishes for each toxin concentration were Effect of Diphtheria Toxin on Protein Synthesis in Cells trypsinized and counted by hamacytometar at 24-hr inter Previously Exposed to Toxin. Three cell strains ware davel vals. z @ Toxin-resistant Cell Lines. HaLa cells were seeded at 0.5 2 32r 6 B x i0@ cells/60-mm tissue culture dish (Falcon) in normal 0 DME supplemented with 10% FBS. As in the growth studies, 0*a-..241 the cells were allowed to plate out for 3 hr prior to toxin

addition. Ten @lofrestenilized diphtheria toxin ware added 0 161 to duplicate dishes ho give a final toxin concentration of 0 between 0.0015 and 0.05 @g/ml.Atotal of 6 concentrations z were tested. Four plates of cells untreated with toxin were C.) used as control cultures. After 96 hn, some viable cells were Y 0 I 2 3456 0 I 234S6 still observed in all plates. These surviving calls ware TIME (hours) washed gently with warm phosphate-buffered saline, and Chart 1. Incorporation of â€˜4C-Iabeledaminoacids into protein using me fresh medium without toxin was added ho the plates. The dium containing 1% of the normal amino acid complement supplemented with 10% FBS. A, HeLa cells with (â€¢)andwithout (0) addition of diphtheria mediumwas changed at weekly intervals,and the cells wane toxin (3 pg/mI); B, L-929 cells with (â€¢)and without ( 0) the addition of diphtheria toxin (3 @g/ml).Theassay was performed as described in â€œMateri S Batch 0-290 was less active than Batch F-370 as tested on HeLa cells; als and Methodsâ€•(protein synthesis assay) except that the radioactivity was Batch D-290 was used exclusively for the experiments on the toxin-resistant added concurrently with the diphtheria toxin. Duplicate cultures were as cell strains. sessed for incorporation at hourly intervals.

DECEMBER1976 4591

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0003 001 003 01 03 0 30 0 DIPHTHERIAT0X@N(@gImI) Chart 3. Comparison of the effects of diphtheria toxin on protein synthe sis in normal HeLa cells and those cells previously exposed to diphtheria toxin. The cell strains (toxin resistant) were obtained and the protein assay was performed as described in â€œMaterialsandMethods.â€•The cell strains 110.2 used were normal HeLa cells (A) and HeLa cells previously exposed to toxin at: 0.015 @g/ml(â€¢),0.003 @g/ml(0), and 0.015 pg/mI (tx).

t@'@ TIME (DAYS) Chart 2. The effects of various concentrations of diphtheria toxin on the growth of HeLa cells over a period of 6 days. The experiment was performed as described in â€œMaterialsandMethodsâ€•(cytotoxic activity). Toxin concen @ trations used were: 5 pg/mI (); 2.5 pg/mI ( ); 1.5 @g/ml(C>);0.5 @g/ml (V); 0.15 @g/ml(V); 0.05 ag/mI (U); 0.015 pg/mI (0); 0.005 .tg/ml (A); 0.0015 pg/mI (0); 0.0005 pg/mI (â€¢);and0.00015 @g/ml(s). x , untreated control cultures.

oped according to the procedure outlined in â€œMaterialsand Methodsâ€•under â€œToxin-resistantCallLinesâ€•fromsurviving 0.I 1.0 0.0 00.0 cells in cultures exposed for 4 days to diphtheria toxin ng/mt RICIN concentrationsof0.015,0.003,and0.0015 @g/ml.These3 Chart 4. The effect of ricin on protein synthesis in normal HeLa cells (0) lines ware hashedfor resistance to diphtheria toxin using the as compared to HeLa cells previously exposed to diphtheria toxin (0.015 protein synthesis assay. In 4 separate experiments over a @.tg/mI)(C). The protein synthesis assay was performed as described in â€œMaterialsandMethods.â€• period of 9 months, those cells previously exposed to diph thenia toxin at 0.0015 and 0.003 @g/mlshowed 3- and 5-fold Two cell lines derived from normal human skin also dam decreases in sensitivity to the toxin with ED@concentra onstrated differing degrees of sensitivity to diphtheria toxin. lions of 0.06 and 0.1 @g/ml,respectively. Those cells pravi In one (CRL-i 12), protein synthesis was inhibited 50% by a ously exposed to toxin at 0.015 @g/mlexhibiheda decrease toxin concentration of0.5 pg/mI; however,complete inhibi in sensitivity of more than 1 order of magnitude, the ED@ lion of protein synthesis could not be achieved even with being 0.3 pg/mI with these cells as compared to 0.025 @g/ toxin concentrations as high as 105 @g/ml.The2nd normal m15in normal HeLa cells. Even at high doses of toxin (up to cell line (VF) showed 50% inhibition at 0.03 @g/ml.This 10 @g/ml),protein synthesis was never inhibited mona than same cell line transformed with simian virus 40 (SV-VF) 65%. These results are summarized in Chart 3. showed a similar sensitivity to hhe toxin; there was a 50% Those calls previously exposed to diphtheria toxin HeLa inhibition of protein synthesis with a toxin dose of 0.02 @g/ mC(0.015 @g/ml)were hasted for sensitivity to nicin, a toxin ml. HeLa cells amathe most sensitive of all the cell lines present in the seeds of Ricinus communis L. (Euphorbi hasted, with 50% inhibition of protein synthesis achieved aceaa)which also inhibits protein synthesis. The sensitivity wihh diphtheria toxin at a dose of only 0.01 p.g/ml. Protein of the HaLa nC calls ho micin was identical ho that of the synthesis in an established line of mouse fibmoblasts (L-929) parent HaLa cells, as shown in Chart 4. was unaffected by diphtheria toxin at all concentrations Effect of Diphtheria Toxin on Protein Synthesis of Estab tested (0.OOi to 100 @g/ml).These results are summarized lished Human Tumor Lines and Normal Cell Lines. Two in Chart 5. established human tumor call lines ware tested using the protein synthesis assay for their sensitivity to diphtheria toxin, as compared to established cell lines derived from DISCUSSION biopsy of normal skin and HeLa cells. The 2 tumor cell lines Pappenhaimer and Randall (9) reported that incorpona displayed different degrees of sensitivity hodiphtheria toxin. lionof radioactiveamino acids intoproteinisdecreased in A cell line derived from a human pancreatic adanocarci isolated Enlich ascitas cells independent of toxin addition noma (A-i 165) showed 50% inhibition with a diphtheria when dialyzed fetal calf serum rather than undialyzed serum toxin dose of 0.2 @g/ml;a call line derived from a primary is used in the assay medium. We had experienced pmob breast carcinoma (BT-20) showed a maximum of 45% inhi bition of protein synthesis with a dose of 5 @g/ml.The S Recent work by J. Beglau and F. Stolzenbach of our laboratory have shown strong cytotoxic action of diphtherIa toxin on isolated Erlich ascites addition of higher toxin concentrations did not further in cells as well as on the tumor when grown In both Swiss and athymic mice. creasethe inhibition in these cells. Details of this study will be presented elsewhere.

4592 CANCERRESEARCHVOL. 36

I00 saturation of the receptor, or a transport system occurring at between 1 and 3 @g/ml.Itis also possible that themeis an z 0 80 increased synthesis of elongation factor II in these cells, or that the ADP-nibose-elongation factor II complex retains 60 z some biological activity. The development of resistance to the effects of diphtheria z 40 Li C.) toxin has bean reported by those working with experimental

Li tumors in animals (1, 5, 9). Moahring and Moehning (6) have a. 20 also demonstrated the development of resistance in KB calls in tissue culture. In single-call clones of cells exposed 000l 0003 4 times to toxin, they were able to obtain calls that ware

DIPHTHERIATOXIN(pg/mI) 1000 times less sensitive than the parent cell line. Chart 5. The inhibition of protein synthesis by diphtheria toxin is com Ricin (M.W. 65,000) sensitivity was unchanged in the pared in the following established cell lines: HeLa (U), normal human fibro HeLa rC-cells. Differences in the transport mechanism for blast (VF) (A), SV4O-transformed human fibroblast (SV-VF) (C), human pan creatic adenocarcinoma (â€¢),normalhuman fibroblast (CCRL-1) (0), human the 2 toxins could account for the observed responses. breast carcinoma (BT-20) (ti), and normal mouse fibroblast (0). The protein The pH differential in the environment of a rapidly divid synthesis assay was performed as described in â€œMaterialsand Methods.â€• ing tumor (10, ii) and the requirement of leukemia calls for L-asparagine (4, 7) have prompted the development of treat lems with the use of dialyzed serum in the protein synthesis mant regimens and drugs which might exploit these small assay medium. Our HaLa cells did not plate out (settle and differences between normal and neoplastic calls. The report attach to the culture dish) in medium supplemented with by Buzzi and Maistrello in 1973 (i) ofa differential sensitivity dialyzed fatal calf serum. The cells were plated out over of mouse tumor cells and normal cells to the effects of night in medium containing undialyzed serum, and the fol diphtheria toxin therefore stirred interest in the possible use lowing day the medium was changed to the low (1%)-amino of diphtheria toxin as an anhineoplastic agent for treating acid medium again supplemented with undialyzed serum. human tumors. In 1973, lglewski and Rittanbang reported With this combination, 14C-labeled amino acid incorpora that themewas a differential sensitivity of human tumors and lion is essentially linear over the period of the assay (Chart normal calls to diphtheria toxin (5). Our results both with 1), indicating the effectiveness of this medium for support HeLa calls and with human breast carcinoma calls (BT-20) ing protein synthesis under our assay conditions. Our me are essentially identical with those obtained by these work suits with the protein synthesisassaywith the HeLacells in ens, yet we feel that our data suggest there is no in vitro monolayer are very similar to those reported by lglewski and classifiable difference in sensitivity that correlates with Rittanbeng, who used dialyzed serum with HeLa cells in whether the tissue is normal or cancerous. However, mdi suspension culture (5). vidual differences may exist. Normal and neoplastic tissue Inhibition of protein synthesis is 1 of the earliest measura taken from a single patient may show differing sensitivities ble biochemical affects of diphtheria toxin on the cell. We to the toxin (5). II has been reported that primary cells may felt it was important to relate this inhibition of protein syn be more sensitive to diphtheria toxin than are established thesis ho the long-term affects of intoxication. Using in call strains derived from the same species (3). creasing doses of diphtheria toxin , as indicated in Chart 2, it It is important that the potential role of diphtheria toxin in appears that there may be a threshold level of diphtheria cancer chemotherapy by carefully evaluated. The develop toxinactionrequiredforcytotoxicily.Atconcentrationsof ment of resistance in HeLa calls following a single exposure diphtheria toxin of 0.05 @g/mlandhigher, the calls lysed . In to diphtheria toxin is a somewhat disquieting finding, par those cultures exposed to 0.015 on 0.005 j@g/ml, the total ticularly since, of the cells we tasted, HaLa cells were onigi number of cells remaining at the end of 6 days was essen nally the most sensitive.7 The resultant shift of the dose tially unchanged from the starting number. At 0.0015 @g/ml response curve obtained with the HeLa cells more than 1 and lass, the growth pattern approached that of untreated log unit means that previously exposed cells now show lass cultures. It would appear from these results that concentra sensitivity to the toxin than either of the normal human skin lions of diphtheria toxin greaten than the ED50for protein cell lines we tested. We were unable to demonstrate a synthesis (0.01 @g/ml)(Chart 5, closed square) are associ consistent difference in the sensitivity of normal and malig ated with cytoloxicily, whereas those below the ED50may nant calls in culture to diphtheria toxin and are currently not be lethal. exploring the possibility that diphtheria toxin analogs may It was of interest to us to determine whether the surviving exhibit a selective action on cancer cells (12). Furthermore, cells would exhibit an altered response when subsequently it is evident that a clearer understanding of why different reexposed to diphtheria toxin. Three cell lines ware deval cells (whether normal or malignant) react differently is as oped from calls that survived 4 days of exposure to the senhial before the toxin can be applied as a utilizable antitu effects of toxin .The change hasbeanstable ovena period of mom agent. 9 months, during which time the cells have bean frozen and thawed, and are now at the 27th passage postdiphthamia toxin exposure. The changed responseto diphtheria toxin 7 P. Viancour of our group has shown that HeLa tumors grown in athymic mice can be completely eradicated without reoccurrence of the tumor after a exhibited by these calls possibly reflects decrease in the period of a year with no observable toxic symptoms. Details of this investiga number of toxin receptor sites on the plasma membrane, tion will be published shortly.

DECEMBER 1976 4593

ACKNOWLEDGMENTS Toxin for Malignant Cells. Proc. NatI. Aced. Sci. U. S., 71: 2707-2710, 1974. 6. Moehnng, T. J., and Mcehring, J. M. Response of Cultured Mammalian We thank Judith Gawarecki and MonÃ©eMcGill for excellent technical Cells to Diphtheria Toxin. IV. Isolation of KB Cells Resistant to Diphtheria assistance. Toxin. Infect. Immunity, 6: 487-492, 1972. 7. Oettgen, H. F., Old, L. J., Boyse, E. A., Campbell, H. A., Philips, F. S., Clarkson, B. D., Tallal, L., Leeper, R. D., Schwarts, M. D., and Kim, J. H. Inhibition of Leukemias in Man by L-Asparaginase. Cancer Res., 27: REFERENCES 2619-2631, 1967. 8. Pappenhelmer, A. M., Jr., and Brown, R. Studies on the Mode of Action 1. Buzzi, S., and Maistrello, I. Inhibition of Growth of Ehrlich Tumors in of Diphtheria Toxin. VI. Site of the Action of Toxin in Living Cells. J. Swiss Mice by Diphtheria Toxin. Cancer Res., 33: 2349-2353, 1973. Exptl. Med., 127: 1073-1086, 1968. 2. Creagen, R. P., Chen, S., and Ruddle, F. H. Genetic Analysis of the Cell 9. Pappenhelmer, A. M., Jr., and Randall, V. On the Alleged High Sensitivity Surface: Association of Human Chromosome 5 with Sensitivity to Diph of Mouse Ehrlich-Lettre Ascites Tumor Cells to Diphtheria Toxin. Proc. theria Toxin in Mouse-Human Somatic Cell Hybrids. Proc. NatI. Acad. NatI. Aced. Sd. U. S., 72: 3149-3152, 1975. Sci. U. S., 72: 2237-2241, 1975. 10. Papanastassiou, z. B., Bruni, R. J., White, E@,Levine,V., and Levine, P. 3. Gabliks, J., and Solotrovsky, M. Cell Culture Reactivity to Diphtheria L. Potential Carcinolytic Agents. IV. Diazoamino Mustards. J. Med. Staphylococcus, Tetanus and Escherichia coil Toxins. J. Immunol., 88: Chem., 9: 725-729, 1966. 505-512, 1962. I 1. Stevens, C. D., and Mosteller, R. C. Enhancement by Glucose of the 4. Hill, J. M., Roberts, J., Loeb, E., Khan, A., MacLellan, A., and Hill, R. W. Inhibition of An Ehrlich Ascites Tumor. Cancer Res., 29: 1132-1136, L-Asparaginase Therapy for Leukemia and Other Malignant Neoplasms. 1969. Remission in Human Leukemia. J. Am. Med. Assoc., 202: 882-888, 1967. 12. Venter, B. R. Ph.D. Dissertation, University of California, San Diego, 5. lglewski, B. H., and Rittenberg, M. B. Selective Toxicity of Diphtheria Calif., 1976.

4594 CANCERRESEARCHVOL. 36

Barbara R. Venter and Nathan O. Kaplan

Cancer Res 1976;36:4590-4594.

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