LYSOGENIC BACTERIA and TUMOR CELLS to CHEMICAL AGENTS* Which Are Effective in Altering the Growth of Lysogenic Bacteria and Then

VOL. 55, 1966 PATHOLOGY: GELDERMAN ET AL. 289

4Margolin, P., and F. H. Mukai, Z. Vererbungslehre, 92, 330 (1961). 5 Margolin, P., Genetics, 48, 441 (1963). 6 Grigg, G. W., Australian J. Biol. Sci., 11, 69 (1958). 7Demerec, M., and E. Cahn, J. Bacteriol., 65, 27 (1953). 8 Freese, E., in Molecular Genetics, ed. J. H. Taylor (New York: Academic Press, 1963), part 1, p. 207. 9Krieg, D. R., Progr. Nucleic Acid Res., 2, 125 (1963). 10 Rudner, Rt., Z. Vererbungslehre, 92, 336 (1961). 11 Ronen, A., J. Gen. Microbiol., 37, 49 (1964). 12 Cavalieri, L. F., and B. H. Rosenberg, Progr. Nucleic Acid Res., 2, 1 (1963). 13 Hamilton, L. D., W. Fuller, and E. Reich, Nature, 198, 538 (1963). 14 Margolin, P., Science, 147, 1456 (1965). 16 Freese, E. B., and E. Freese, Virology, 13, 19 (1961). 16 Freese, E., E. Bautz, and E. B. Freese, these PROCEEDINGS, 47, 845 (1961). 17 Goodgal, S. H., and E. H. Postel, Science, 148, 1095 (1965). 18 Strelzoff, E., and F. J. Ryan, Biochem. Biophys. Res. Commun., 7, 471 (1962). 19 Loftfield, R. B., Biochem. J., 89, 82 (1963). 20 Berg, P., and M. Chamberlin, Bull. Soc. Chim. Biol., 46, 1427 (1964). 21 Chamberlin, M., and P. Berg, J. Mol. Biol., 8, 708 (1964). 22 Speyer, J. F., Biochem. Biophys. Res. Commun., 21, 6 (1965). 23 Stent, G. S., Advan. Virus Res., 5, 95 (1958). 24 Zubay, G., these PROCEEDINGS, 48, 456 (1962).

A FURTHER CORRELATION BETWEEN THE RESPONSE OF LYSOGENIC BACTERIA AND TUMOR CELLS TO CHEMICAL AGENTS* BY ALBERT H. GELDERMAN, THOMAS L. LINCOLN, DEAN B. CoWIE, AND RICHARD B. ROBERTS PATHOLOGIC ANATOMY BRANCH, NATIONAL CANCER INSTITUTE, NATIONAL INSTITUTES OF HEALTH, DEPARTMENT OF PATHOLOGY, JOHNS HOPKINS UNIVERSITY SCHOOL OF MEDICINE, AND DEPARTMENT OF TERRESTRIAL MAGNETISM, CARNEGIE INSTITUTION OF WASHINGTON Communicated December 23, 1965 Price et al. have shown a strong correlation between drugs capable of induction of virus in lysogenic bacteria and drugs effective as antitumor agents.1 If this correlation is not fortuitous but is due to an underlying mechanism common to lysogenic bacteria and certain tumor cells,2 then studies of the lysogenic bacteria might indicate which conditions are favorable for inhibiting the growth of these tumor cells. We have tested this hypothesis by finding combinations of drugs which are effective in altering the growth of lysogenic bacteria and then observing the effect of the same combination on ascites tumors growing in mice. Methods.-Cultures of Escherichia coli were grown overnight in a glucose-salts medium with a limited supply of glucose. Growth was initiated by the addition of glucose, and the cells were allowed to double in mass to ensure exponential growth. They were then centrifuged and resuspended in 10 ml of the growth medium at a density of roughly 10 mg wet weight per ml. Mito- mycin C was added and the suspension was incubated without aeration for 15 min at 370C. The treatment with mitomycin C was terminated by adding 20 ml of the growth medium, centrifug- ing, and washing once. The cells were then resuspended and distributed to aerated flasks con- taining the growth medium and various additives. The initial density was 0.1-0.2 mg per ml. Downloaded by guest on September 28, 2021 290 PAPHOLO6Ol: 6ELDEJRMAN E? AL. PROC. N. A. S.

Alternatively, the mitomycin C could be added directly to the aerated cultures. The con- tinuous aerobic exposure results in approximately the same degree of lysis as a 15-min anaerobic treatment with the same concentration of mitomycin C. The growth of the culture and the induction of lysis were observed by measurements of the optical density of 650 mg. Under conditions giving complete lysis the optical density at 650 mji is reduced tenfold or more. In addition, DNA synthesis and loss were observed by usual tracer techniques. E. coli strain BB was used as an example of a nonlysogenic bacterium. E. coli K-12X and E. coli strain 15TAU- (which requires thymine, arginine, and uracil) were used as examples of lysogenic bacteria. Initially, we were not aware that E. coli strain 15TAU- was lysogenic. This strain could, however, be lysed by mitomycin C, by UV radiation, by azaserine, or by transient deprivation of thymine.3 Pellets obtained by centrifugation of lysates of E. coli 15TAU- indicated the presence of viruslike material. These pellets contained UV-absorbing material showing an ab- sorption spectrum similar to nucleoprotein but which was not rendered acid-soluble by DNase. Electron micrographs prepared by Dr. Morowitz of Yale University showed viruslike particles similar to T2 bacteriophage. Finally, use of the DNA agar technique indicated that the viral DNA was related to the DNA of its host, E. coli. Virus-host genetic relatedness appears to be a feature common to lysogenic systems.4 6 Similar results have recently been reported by other authors,6 and our experiments confirm their findings. Conditions Influencing the Induction of Virus Synthesis in Lysogenic Bacteria. Figure 1 shows the growth and lysis of E. coli 15TAU_ after exposure to various concentrations of mitomycin C. Except at the high concentrations, the initial growth

T 1 r 1 0.0 Y/ml 0. I FIG. 1.-Growth and lysis of E. coli strain 15TAU after exposure to mitomycin C. A culture of E. coli strain 15TAU -, growing exponentially in a glucose- .= 0. --0.25 - salts medium supplemented with thymine, arginine, C v / and uridine, was centrifuged and resuspended in 10 ml of the same medium. Mitomycin C was added to give 0.2 , the indicated concentrations, and the cells were treated 08.. for 15 min at 370 without aeration. At the end of >0f 0.5 this period they were centrifuged and washed. They 0.1 were then suspended in 100 ml of the growth medium ~

more, cyclohexane 1,2 diamino sodium tetra- acetate (CDT) a stronger chelator, is effective Control at 10-4 M, which is only one fifth the mo- larity of the Mg++. CDT even at concentra- 0.4 tions as low as 10-5 M causes a shift from ex- *EDT 10-3M ponential to linear growth, but exponential G u growth can be restored by the addition of Mn++ X CDT 10-4 M or Fe+++. Also, in the absence of chelators the 'o. I addition of Mn++ (10-3 M) reduces the lysis caused by mitomycin C. Evidently, a heavy CDT 10-3 M 0.041 ion is required both for exponential growth and ) 0 1 2 .3 4 5 6 for recovery from treatment with mitomycin C. Hours The effect of the chelators is not limited to FIG. 2.-Effect of chelators on lysis augmenting the lysis induced by mitomycin C. inducedcells wereby treatedmitomycinwithC.E.mitomycincoli 15TAU-C Growth recovery remains slower in the pres- at 0.25 iAg/ml for 15 min as described in Fig. 1. They were then suspended ence of EDTA after lysis induced. by UY radia- growth medium with chelators tion,tionazaserie, orordeiciecy.inthymie defciency. added at the indicated concentrations. DNA *, Control (no chelator); o, M Effect of inhibitors of synthesis: Figure 10O4 M 10-310-1 M 3 shows that the extent of lysis and the rate EDTA;CDT. c, CDT; A, of recovery can be markedly influenced by agents that affect the rate of nucleic acid synthesis. Lysis is considerably reduced by the addition of nucleic acid precursors. In contrast, the lysis is enhanced and recovery is delayed by agents which inhibit DNA synthesis. Deprivation of thymine for 90 min has a drastic effect, but this prolonged deficiency of thymine

1.0 1,-Adenouine Control 0.8 0 MC 0.252MC + 0.4 ~--Thymine 0.6 rinn -'0.05 YMC 0.04 --~-- t0.4 -~~~~--C-o -~~~~~~~~-0.1~~~IYMC .2 ~-yxyurea a

O 1 2 3 4 5 6 Hu Hours FIG.inhibitio-4.-Effect of of protein FIG. 3.-Effect of inhibitors on lysis synthesis. E. coli 15TAU - cells were induced by mitomycin C. E. coli 15TAU - treated for 1,5 min with various concen- cells were treated with mitomycin C at trations of mitomycin C and then sub- 0.25 jug/ml for 15 min as described in jected to a period of inhibition of protein Fig. 1. They were then suspended in synthesis resulting from arginine priva- media with the indicated additives. tion. For this experiment the cells were For inhibition by thymine privation, washed and treated in medium lacking the cells were washed and treated in arginine. 0, 0.25 ,ug/ml mitomycin C; media lacking thymine, and the thy- arginine restored at 0 min. n, 0.25 mine was restored after 40 min incu- Ag/ml mitomycin C; arginine restored at bation. *, Control (complete growth 45 min. *, 0.1 Asg/ml mitomycin C; medium); c, adenosine 0.15 mg/ml; arginine restored at 45 min. A, 0.05 o, hydroxyurea 3.8 mg/ml; *, pheti- jug/ml mitomycin C; arginine restored at ethyl alcohol 1 mg/ml; A, 40 min 45 min. o, 0.0 /ug/ml mitomycin C; without thymine. arginine restored at 45 min. Downloaded by guest on September 28, 2021 292 PATHOLOGY: GELDERMAN ET AL. PROC. N. A. S.

is sufficient in itself to induce lysis (after restoration of thymine) in cells not exposed to mitomycin C. Deprivation for 40 min does not induce lysis but does enhance the effect of mitomycin C. Hydroxyurea and phenethyl alcohol, when )resent in concentration sufficientt to cause a partial inhibition of DNA synthesis,8' 9 I)otentiate the mitomycin C, even though they cause only a slight inhibition of the growth rate of untreated cells. Other agents which have been found effective in- clude azaserine (0.04 Ag/ml), 8 azaadenine (40 Ag/ml), mercaptoethanol (20,/g/ nl), aminopterin (330 ,g/ml). These results can be interpreted in terms of the repair mechanism which has been shown to come into play after DNA is damaged by radiation or radiomimetic drugs. According to present evidence, the defects caused by these agents are excised, leaving a gap ill one of the strands of the DNA molecule. The gap is then enlarged (presumably by an exonuclease), and a considerable degradation of the DNA strand may ensue. Finally, the processes of degradation are reversed by a DNA polymerase which fills in the missing material by inserting bases complementary to the remaining intact strand. 10-12 These mechanisms are sufficient to explain the effects observed in lysogenic cells if one additional assumption is made, namely, that the initial defect is not sufficient to release a lysogenic virus but further degradation of the DNA is also required. IIi this situation, when conditions are favorable for DNA synthesis, the defects are filled in rapidly and the virus remains associated with the genome of the host. When DNA synthesis is inhibited, the degradation proceeds further, and the virus is released to multiply independently. Effect of inhibitors of protein synthesis: With E. coli 15TAU- the synthesis of protein can be stopped by removing arginine from the medium. As shown in Figure 4, growth resumes promptly when the arginine is restored. If, however, the cells have previously been treated with mitomycin C, a prolonged lag ensues. A period of inhibition as short as 30 min is sufficient to induce the prolonged lag. Moreover, the inhibition need not be during the initial period after mitomycin C treatment. Cells allowed to grow in the complete medium for 45 min and then subjected to 45 min inhibition of protein synthesis show the same effect. The effect is quite general; the protein inhibition may be induced by 5-methyltryptophan or chloramphenicol, as well as by lack of arginine. Lack of uracil causes cessation of RNA synthesis and a subsequent protein inhibition; p-fluorophenylalanine and 5- fluorouracil cause the synthesis of imperfect proteins; all these agents give long lags to mitomycin C-treated cells (Fig. 5). In addition, similar lags are noted in cells exposed to UV radiation or to azaserine, if they are subjected to a period of l)roteiri inhibitiomi. IIi comitrast, no such lag is observed when the nonlysogenic bacteria, E. coli strain BB, is treated with mitomycin C followed by 5-methyltryptophan. Growth resumes as soon as the inhibition is reversed by the addition of tryptophan. These results can again be interpreted, partially at least, in terms of inhibiting the repair mechanism. It was established several years ago that a period of protein synthesis was required before bacteria could resume DNA synthesis after exposure to UVI3' 14 or nitrogen mustard."5 This effect is undoubtedly present. There seem to be differences; however, the prolonged lag after the removal of inhibition is seen only in lysogenic cells. An initial period of protein synthesis is not sufficient to ensure subsequent growth. We, therefore, believe that viral Downloaded by guest on September 28, 2021 VOL. 55, 1966 PATHOLOGY:. GELDERMAN ET AL. 293

Control +D 0.4 504 1 3 2 4 5 6n n - Arginine +

Hours 5 lurnI-ine FIG. 5.-Effect of protein synthesis in- 0 1 2 3 4 5 6 hibitors on mitomycin-treated cells. E. coli 15TAU- cells washed and treated for Hours 15 miin with mitomycin C (0.25 Aug/ml) FIG. 6.-Combined effect of EDTA and in medium lacking arginine and uracil. inhibition of protein synthesis on mito- *, Uracil restored at 0 min, arginine at mycin-treated cells. E. coli 1STAU- cells 910 min. co, Uracil restored at 90 min, washed and treated in medium lacking arginine at 0 min. A, 5-Fluorouracil arginine. Mitomycin C concentratjon present throughout, reversed by addition 0).15 Aug/ml to give limited lysis. a, of uracil at 90 mmn arginine restored at 0 Arginine restored at 0 mmn (Control); min. A, Arginine and uracil restored at 0, arginine restored at 0 mm EDTA 0 mi. p-fluorophenylalanine (1 mg/nd) (10-3 M); A, argininerestoredat60 nun; present throughout, reversed by addition A, arginine restored at 60 min EDTA of tyrosine and phenylalanine at mhi.90 (10-0 M).

DNA may be released during the period of protein inhibition and then block the cell's metabolism so that neither cell nor virus is synthesized, a situation similar to that caused by the entry of defective virus. 6 Figure 6 shows a result of potential significance for chemotherapy. The addition of EDTA makes the lag after protein inhibition more prolonged. Ehrlich Ascites Tumor in Mice.-The Ehrlich ascites tumor was chosen because these tumor cells will readily transplant in almost 100 per cent of the recipient animals and grow exponentially for the first 8-10 days, causing death of the animal in an average of 14 days. Young adult mice were used and carefully observed for complications that would alter the weight gain associated with tumor growth, such as drug toxicity, diarrhea, or dehydration. The daily weight of mice with ascites tumor increased linearly and correlated with the growth of tumor cells. Further, the ascites tumor offered the advantage that intraperitoneal injections of drugs were known to reach the tumor cells. Eight-to ten-week-old C3H/HEN mice, born on the same day and maintained at the National Institutes of Health in a disease-free environment, were used in each experiment. The tumor was grown for 6 days in a separate group of Swiss carrier mice, and then the cells were removed and counted in a counting chamber. Tumor cells (2 X10s ) were injected intraperitoneally into the recipients. In 3 days a 2-sgm mouse would contain an average of 2-3 gm of ascites cells and fluid. In the initial experiments 57 complete autopsies were pefformed to evaluate the extent of tumor invasion and toxicity. The object of these experiments was not to cure the mice, but to establish whether or not the combinations of drugs found effective iln lysogenic bacteria were equally effective against tumor cells. Thus, when exponentially growing tumor cells were treated with single marginal doses of antitumor agents, such as mitomycin C, Downloaded by guest on September 28, 2021 294 PATHOLOGY: GELDERMAN ET AL. Pioc. N. A. S.

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0123 4 5 6 7 0 9 ~~~10 II 12 13 14 15 0 2 3 4 5 6 7 8 9 10 .l 12 13 14 FIG. 7.-Growth of ascites tumor in mice after various single treatments C3H mice were injected with 2 X 107 Ehrlich ascites cells on day 0. When Niv2CaEDTA was Used, 50 mg/Kg of Na2CaEDTA was injected intraperitoneally and was added to the drinking water (10-2 M) thereafter. Puromycin (when used) was injected (IP) on day 3 at 10 mg/Kg mouse Mitomycin C (when used) was injected (IP) on day 3 an hr after the puromycin at2u5 mg/Kg. Curves a, b, and c show the weights of individual mice given mitomycin alone, mitomycin C plus Na2Ca- EDTA, and mitomycin C plus Na2CaEDTA plus puromycin, respectively. Curve d shows the averages of the weights of these mice and of other groups of mice given other combinations. and the daily weight changes in the mice were followed, the data from the animal experiments closely approximated that of the bacterial experiments. To date 14 experiments have been carried out. In every case, the combinations suggested by the bacterial work have been more effective than using the drugs sep- arately, and seem to act synergetically. The results of an experiment which tested the combination of mitomycin C, puromycin, and Na2CaEDTA are shown in Fig- ure 7. The use of puromycin to inhibit protein synthesis was suggested by the effects of protein inhibition on the lysogenic bacteria shown in Figures 5 and 6. The results of Figure 7 bear out the predictions from the bacterial system. The combination of mitomycin C, puromycin, and Na2CaEDTA produced prolonged inhibitions of tumor growth, even though the individual agents had little antitumor effect when used singly. Figure 8 shows the results of a similar experiment testing the combination mitomycin C, chloramphenicol, and EDT. Again, the triple therapy using a relatively Downloaded by guest on September 28, 2021 VOL. 55, 1966 PAHlIOLOC: 0ELDERMAN ET AL. 295 8- Begin No2CaEDTA MC Chioro 06 1/ I- I /MC ci-X , ~ ~~// ~ ~~~~~MCIA42CGEDTAI = 4 No Tre/rnennf// Ch/oro'5doysl

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- 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 DAYS AFTER TRANSPLANTATION FIG. 8.-Growth of ascites tumor in mice after various treatments. The curves are of the averages of weights of 10 mice in each group. The tumor was injected as in Fig. 7 and, when used, the Na2CaEDTA was similarly administered. Mitomycin C (when used) was-injected (IP), 5 mg/Kg, on day 3. Chloramphenicol (when used, 100 mg/Kg) was injected (subcutaneously) daily for 3 and 5 days as indicated.

less toxic inhibitor of protein synthesis gives a considerably greater antitumor effect than that of mitomycin C used alone. Na2CaEDTA is the strongest example of synergism. It has no effect alone, is nontoxic, and markedly enhances the antitumor effect of mitomycin C. In the doses used, the same may be said of chloramphenicol and puromycin. While the weight of the mice provides a simple and easy measure of the effect of the treatment, it does not give an absolute proof that the growth of the tumor is inhibited; the failure to gain weight could be attributed to an enhanced loss of body weight. The weight curves do, however, correlate with survival times. Table 1 shows the survival of mice in five different experiments testing the combinations of mitomycin C, Na2CaEDTA, and puromycin or chloramphenicol. In Table 2 the data from the five experiments are grouped in different categories and expressed as per cent survival. In addition, all the pairs of groups which dif- fered only in the presence or absence of Na2CaEDTA have been selected and com- pared. In round numbers mitomycin C, at the level used, added less than 1 day to the survival time. Mitomycin C + Na2CaEDTA added 2 days, and mitomycin C + Na2CaEDTA + puromycin or chloramphenicol added 4 days. In addition to the experiments above actinomycin D, azaserine, 5-fluorouracil, and 5-fluorodeoxyuridine have been tested in combination with mitomycin C and EDTA. Also X radiation and nitrogen mustard have been tested as the primary agents with EDTA and chloramphenicol as potentiating agents. The weight curves have indicated improved effectiveness with the combinations, but the groups tested were too small to yield significant survival data. However, in no instance were the combinations less effective than any individual drug. Downloaded by guest on September 28, 2021 296 PATHOLOGY: GELDERMAN ET AL. PROC. N. A. S.

TABLE 1 SURVIVAL DATE FROM MITOMYCIN, EDTA, PUROMYCIN, AND CHLORAMPHENICOL EXPERIMENTS Survivors on Day Treatment 0 10 12 14 17 20+ Expt. 4/12 P 4 3 2 1 0 0 M 4 4 4 3 2 1 P+M 4 4 4 4 4 4 Expt. 4/27 P 5 4 3 0 0 -0 P + E 5 3 3 1 0 0 M 10 10 7 2 0 0 M + E 10 10 10 9 2 0 M + P 10 10 8 4 2 1 M + F, + P 10 10 10 10 7 3 Expt. 5/3 P 5 4 3 0 0 0 P + E 5 4 4 0 0 0 C 5 4 2 1 0 0 C+E 4 4 4 2 0 0 M 5 5 4 2 0 0 M+E 5 5 5 3 0 0 M+P 5 5 5 1 0 0 M + C 10 10 10 4 2 1 M+P+E 5 5 5 3 3 2 M + C + E 10 10 8 6 3 1 Expt. 5/12 None 5 4 2 1 0 0 M 10 8 4 3 1 0 M + E 10 10 7 4 4 1 I + C + E 10 8 8 5 4 4 Expt. 5/31 None 4 4 4 0 0 0 WI 5 5 5 1 0 0 + E 5 5 5 3 2 1 M + C + E 20 20 20 20 19 7 M, mitomycin (5 mg/kg, day 3). P, puromycin (10 mg/kg, day 3). C, chloramphenicol (100 mg/kg). E, Na2CaEDTA (50 mg/kg, days 2 and 3, plus drinking water 10 -3 M). We believe that these experiments extend the correlation between lysis-inducing agents and antitumor agents' to a correlation between combinations of drugs which affect lysogenic bacteria and combinations which inhibit the ascites tumor. They TABLE 2 also demonstrate the value of testing first in bacteria where the ex- PBEPer Cent SurvivalSurvivalonDayon Day agents, Treattment No. 10 12 14 17 20+ periments can be completed in a day, Nothing, C, CE 42 1 64 14 3 and second in ascites tumors where iP, 34 94 71 32 9 3 the drug reaches the tumor cells di- ME 30 100 90 63 27 7 rectly and where the results are in- MCE, MPE 65 97 91 72 57 28 PMCrs EM9 2 dicated by weight changes. The com- +E 70 94 87 58 30 11 binations which prove hopeful in these -E 69 93 71 26 10 3 preliminary screening tests can then M, mitomycin C. be used to treat a variety of tumors P,C, puromycin.chlo3raipphenicol. ini experiments which need months for E, Na2CaEDTA (doses as indicated in Table 1). completion. We also hope that the * Treatments indicated ineffective by weight curves. rationale of using one drug to in- troduce defects in the DNA and others to inhibit the repair of the defects may be a useful guide in cancer chemotherapy. Downloaded by guest on September 28, 2021 VOL. 55, 1966 PATHOLOGY: DALLDORF ET AL. 297

* The work with bacteria was carried out by Cowie and Roberts at the Carnegie Institution of Washington, and the experiments with mice were done at the National Institutes of Health by Gelderman and Lincoln. 1 Price, K. E., R. E. Buck, and J. Lein, Antimicrobial Agents and Chemotherapy (1964), pp. 505-517. 2 Axelrod, D., K. Habel, and E. T. Bolton, Science, 146, 1466-1469 (1964). 3Cowie, D. B., and R. B. Roberts, Carnegie Inst. Wash. Year Book, 64, On press (1965). 4Cowie, D. B., and B. J. McCarthy, the3e PROCESDINGS, 50, 537-543 (1963). 5 Cowie, D. B., Carnegie Inst. Wash. Year B9ok, 63, 380-385 (1964). 6Endo, H., K. Ayabe, K. Amako, and K. Takeya, Virolo7y, 25, 469-471 (1965). I Frampton, E. W., and R. R. Brinkley, J. Bacteriol., 90, 446-452 (1965). 8 Rosenkranz, H. S., A. J. Gavro, J. A. Levy, and H. S. Carr, Bioehim. Biophys. Acta, in Tress. 9 Rosenkranz, H. S., H. S. Carr, and H. M. Ro3e, J. Bacteriol., 89, 1354-1369 and 1370-1373 (1965). 10 Setlow, R. B., and W. L. Carrier, these PROCEEDINGS, 51, 226-231 (1964). 11 Boyce, R. P., and P. Howard-Flanders, these PROCEEDINGS, 51, 293-300 (1964). 12 Pettijohn, D., and P. Hanawalt, J. Mol. Biol., 9, 395-410 (1964). 13 Harold, F. M., and Z. Z. Ziporin, Biochim. Biophys. Acta, 29, 439-440 (1958). 14 l)rakulic, M., and M. Errera, Biochim. Biophys. Acta, 31, 459-463 (1959). 15 Harold, F. M., and Z. Z. Ziporin, Biochim. Biophys. Acta, 28, 144-155 (t61). 16 Cowie, D. B., Carnegie Inst. Wash. Publ., 624, 515-518 (1964).

FURTHER OBSERVATIONS OF THE LYMPHOMAS OF AFRICAN CHILDREN BY GILBERT DALLDORF, FERNANDA BERGAMINI, AND PATRICIA FROST

SLOAN-KETTERING INSTITUTE FOR CANCER RESEARCH, NEW YORK, NEW YORK Communicated December 6, 1965 During 1963, cytopathogenic, filtrable agents were frequently encountered in cultures of embryonic human kidney cells following their inoculation with. super- natant fluids from primary human amnion cultures which had previously been exposed to extracts of tumors and other specimens from East African children with malignant lymphomas of the kind described by Burkitt and others.1 The results seemed noteworthy because they suggested an intimate association between the agents and the disease, and also because of the nature of the isolations which involved two phenomena, an initial induction of a peculiar spindling and twisting of the amnion cells (Fig. 1) and subsequently destructive changes in kidney cells inoculated with fluid from such altered amnion cultures. The direct inoculation of embryonic kidney cells with extracts of tumors or bone marrow never caused cytopathogenic effects nor did the isolated, transmissible agents have the capacity to induce the amnion lesions. Nevertheless, the two effects were closely associated and clearly related to the specimens.2 The cytopathogenic agents were later cultivated on protein-rich media and found to have the characteristics of mycoplasma.3 They failed to induce tumors in a variety of animals, and serologic tests in which they served as antigen gave suggestive but inconclusive evidence of a relationship to the disease. The mycoplasma also failed to induce the amnion lesions caused by the specimens. The following year five additional East African patients were investigated. A Downloaded by guest on September 28, 2021