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Diphtheria Toxin Effects on Human Cells in Tissue Culture1

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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 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1976 American Association for Cancer Research. Diphtheria Toxin Effects on Human Calls 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 “Discus normal concentration of amino acids was added, 2 mI/dish. sion―)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 37°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 “C-aminoacidmix (New England Nuclear, Boston, Mass.) noted with this toxin concentration on ‘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 “Materials and Methods “under “Growth Studies.― 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.
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