SOME CONTRIBUTIONS MICROBIOLOGY TO CANCER RESEARCH' · SOME CONTRIBUTIONS OF MICROBIOLOGY TO CANCERRESEARCH' ERICH HIRSCHBERG Institute of Cancer Research andDepartment ofBiochemistry,

SOME CONTRIBUTIONS OF MICROBIOLOGY TO CANCER RESEARCH'

ERICH HIRSCHBERG

Institute of Cancer Research and Department of Biochemistry, Columbia University College of Physiciansand Surgeons, New York, New York

A quarter century ago, cancer research was between bacteriology or protozoology and canceralmost exclusively in the domain of the patholo- research. Today, microbiological systems consti-gist, the biologist, and the clinician. A cursory tute a considerable portion of the experimentalexamination of the cancer literature at that time material in many cancer research centers. In areveals that attention was focused predomi- recent analysis of the financial support of cancernantly on problems, ranging from cytological and research by government and nongovernmenthistological studies of spontaneous tumors to agencies (1), the total expenditure for 1953 wasinvestigations on homologous and heterologous placed at about ten million dollars; at least 7 pertransplantation of neoplasms, from serological cent of these funds was committed for studiesdiagnosis of human cancer to immunological which were devoted predominantly to some micro-studies in fowl sarcoma, from blood chemistry of biological system.tumor-bearing hosts to the role of heredity in Viruses have long been known to cause a vari-carcinogenesis. Cancer research was characterized ety of neoplastic conditions and are considered byby its intensive rather than extensive orientation; many investigators to be primary causativethe annual meeting of the pertinent scientific agents of all cancer. The proponents of thesociety was held in a single session and was de- somatic mutation theory, the other principalvoted to the presentation of about a dozen hypothesis dealing with carcinogenesis, have beenpapers; many of the problems which occupy the interested in the contributions of bacterial genet-center of the stage today were little more than ics and bacterial mutations. Studies on the effectshadows waiting in the wings. of chemical carcinogens on yeasts, ciliates, and

In the intervening years,the elusive problems other organisms and on the action of carcino-of neoplastic growth have been attacked from so genic and mutagenic agents on Hemophilus influ-many different points of view that today there enzae transforming principle are other examplesare few scientific disciplines which have not con- of the application of microbiology to this area oftributed in some way to its understanding. The research. Oncolytic viruses, crown-gall tumors,number of active investigators devoting their bacteriophage metabolism, elucidation of meta-full time to oncology and the diversity of their bolic pathways with the neurospora and otherbackgrounds and interests have increased beyond microorganisms, enzyme adaptation studies inall expectation. At the last annual meeting of the bacteria, and related subjects of current interestAmerican Association for Cancer Research, there are illustrations of the increasingly fruitful unionwere as many as four concurrent sessions on each of the two disciplines.of three days, and the scientific program consisted No single short review can begin to deal ade-of close to 200 reports. The efforts of cancer re- quately with these diverse and often complexsearchers have remained intensive, but they have problems. In selecting a few topics for discussion,now extended to practically every conceivable the reviewer was guided by one of the more usefulaspect of biology and biochemistry. definitions of "cancer" which have been coined

Microbiology has also been drawn into this over the years. In this definition, cancer is con-development. A few decades, ago, even a collector sidered to involve the uncontrolled growth ofof biological curiosities would have demurred at cells that have failed to differentiate completely.the suggestion that there was common ground Most of the investigations in oncology have con-

' Thisreviewwas preparedinconnectionwith cerned themselves with the problem of uncon-

worksupporteview npparedy in cnearct anwth trolled growth; the first section of this reviewwork supported in part by a research grant del wihsm'otiuinso irbooyt(C-2354) of the National Cancer Institute, U. S. deals with some contributions of microbiology toPublic Health Service and in part by an Institu- the search for effective means of stopping thetional Grant of the American Cancer Society to rapid proliferation of the cancer cell. IncreasingColumbia University. attention is now being given to the failure of

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66 ERICH HIRSCHBERG [voL. 19

differentiation which also characterizes neoplastic lymphoma. During this time, thousands of chem-cells; the second section of this review is devoted ical compounds and preparations of the mostto a consideration of certain slime molds and varied nature have been evaluated in screeningrelated organisms which appear to lend them- programs based on the response of one or severalselves particularly well to studies on differentia- transplanted animal tumors (8). Agents havetion and morphogenesis. Some of these studies been selected for testing for many differentmay have a more tenuous or less immediate con- reasons; these have ranged from the considerednection with the cancer problem than others, but conclusions of elaborate studies on the bio-no attempt has been made to apportion space in chemical properties and functions of tumors tothis review in relation to considerations of this the results of unashamed and enthusiastic specu-sort. lation. From these countless trials there have

emerged a handful of compounds which inhibitMICROORGANISMS AND CANCER CHEMOTHERAPY the growth of experimental tumors or affect theThere are at least four areas of research where clinical course of human neoplasms without

microbiology has made significant contributions forbidding toxicity to the host.to cancer chemotherapy. The tumor-inhibitory One of the main concepts which has guidedeffects of microorganisms and particularly of some investigators in their search for active agents hasof their products have been widely studied. A been visualized as the "lock-and-key" hypothe-number of the more useful chemotherapeutic sis. The development of the antimetabolite theoryagents were first discovered in microbiological from the observations of Fildes (9) on the anti-systems. Studies on the mechanism of action of bacterial action of mercury and of Woods (10) onthese agents in bacteria and protozoa have con- the relationship between the sulfonamides andtributed significantly to our present understand- p-aminobenzoic acid in bacterial metabolism hasing of their anticancer activity. The development been amply documented (11). The idea, decep-of resistance to initially effective chemicals by tively simple in retrospect, that an essential bio-animal and human leukemic cells, which has been chemical reaction can be blocked by the introduc-a major obstacle to the realization of the full tion of an agent which closely resembles thepotential usefulness of the agents we now have substrate of the reaction and competes success-is closely similar to the equally vexing develop: fully with it for the enzyme controlling the reac-ment of resistance to antibiotics by bacteria. tion, has led to the design and preparation ofThe use of bacteria, fungi, and protozoa and of numerous series of potential inhibitors of bac-

some of their products as carcinostatic or carcino- terial growth. It is of interest that an appreciablelytic agents will not be considered here since the number of the effective carcinostatic agents inliterature on this subject has been covered pains- current use were suggested for screening againsttakingly and critically in a recent review by tumors on the basis of their demonstrated anti-Reilly (2; cf also 3). The striking parallels between bacterial activity and that many of the com-bacterial and neoplastic cell resistance to chemo- pounds in this group may be classed as antimetab-therapeutic agents have been pointed out by olites.several reviewers (4, 5, 6, 7), and little could be The history of the recognition, isolation, andadded here to their discussion of the mechanisms identification of pteroylglutamic acid as anunderlying the development of resistance. The essential growth factor for microorganisms is tooother two topics, however, fill be discussed in well known to bear repetition here. In 1947, themore detail. first antimetabolites for pteroylglutamic acid

were prepared (12, 13) and shown to inhibit theDiscovery of Carcinostatic Agents in growth of Lactobacillus casei and Streptococcus

Microbiological Studies faecalis in a competitive manner. These com-

While surgery and radiation continue to be the pounds were promptly employed in the treatmentprincipal tools for the eradication or control of of acute leukemia in children, on the basis ofneoplasms, the last decade has witnessed signifi- earlier observations of an acceleration of tumorcant progress in chemotherapy, a form of treat- development following the administration ofment of particular importance in disseminated pteroyldi- and triglutamic acid, and found tocancer and in the various forms of leukemia and produce significant though temporary remis-


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19551 CONTRIBUTIONS OF MICROBIOLOGY TO CANCER RESEARCH 67

sions (14). The folic acid antagonists, particularly was found that 8-azaguanine inhibited tumoraminopterin and A-methopterin, continue to be of growth profoundly. These investigators werevalue in the treatment of leukemias in man and fortunate in having chosen for this demonstrationhave been shown to inhibit the growth of a great mammary adenocarcinoma E0771 and two othervariety of experimental tumors. tumors which responded to 8-azaguanine, sinceThe road from demonstration of activity later studies have indicated that experimental

against microorganisms to introduction into neoplasms differ widely in their susceptibility tocancer chemotherapy has been less direct for this agent.another agent of considerable experimental in- Four other antimetabolites which have provedterest, 5-amino-7-hydroxy-lH-v-triazolo(d)-pyr- to be effective in inhibiting the growth of experi-imidine or 8-azaguanine. The gradual elucida- mental tumors were first suggested for screeningtion of the central role of nucleic acids in the on the basis of their activity in Lactobacillus casei.economy of the cell and of the pathways of their These compounds, 2,6-diaminopurine (20), thio-synthesis from simpler precursors had focused guanine (21), 2,4-diamino-5-(3',4'-dichlorophen-increasing attention on the vast array of chem- yl)-6-ethylpyrimidine (22), and particularly 6-icals which might act as antimetabolites for mercaptopurine (21), represent some of thepurines and pyrimidines. In a search for effective more significant therapeutic results attained bypurine antagonists, it was found in 1945 (15) that the systematic investigations of Hitchings and8-azaguanine, which differs from guanine only by his collaborators which will be alluded to in thethe substitution of a nitrogen atom for the >CH following section.group in position 8, was effective against The justifiable emphasis placed in these pagesEscherichia coli and Staphylococcus aureus; the on the value to cancer chemotherapy of agentsinhibition was reversed by guanine. This com- first discovered in microbiological systems shouldpound was included in the first group of purines not be construed as an open invitation to thewhich were screened against Sarcoma 180 but oncologist in search of new agents to rely solely orfailed to inhibit the growth of this tumor (16) and even predominantly on bacteria, fungi or proto-might well have been relegated to the large group zoa for an effective screening method. Mlany com-of agents which have not been studied further had pounds which inhibit a wide variety of micro-it not been for the renewed interest occasioned organisms, including the most potent antibiotics,later by its unusually high activity in an entirely have failed to exhibit any antitumor activity;different microbiological system. effective carcinostatic agents which cannot beThe animal microorganism, Tetrahymena geleii, considered as antimetabolites have frequently

has long been the subject of extensive investiga- shown no striking antimicrobial activity(23).tion from many different points of view (17).Some of the most interesting data have been ob- Studies on Mechanism of Action of Carcinostatictained by a systematic study of the growth Agents in Microbiological Systemsrequirements of this organism. Following the The recent review literature abounds withdemonstration that an exogenous source of purine summaries and discussions of nucleic acid metab-was required for growth, about two dozen sub- olism in microorganisms ranging from species ofstituted purines were tested for their ability to Lactobacillus to Tetrahymena and Parameciumreplace or spare guanine or to inhibit growth in acompetitive or noncompetitive manner. Among (24-30). In many instances, the biochemicalthe inhibitors, 8-azaguanine was by far the most processes in microorganisms have been comparedeffective; the inhibition was completely reversed and contrasted with those in mammalian tissues,by guanine (18). Since Tetrahymena geleii differed and the differences and similarities between thefrom normal mammalian cells by having this two types of biological systems are emergingrequirement for exogenous guanine and since the from these studies. In general, the synthesis andpossible differences in purine utilization between metabolism of the nucleic acids are carried on in anormal and neoplastic cells were under active similar way in microbial and mammalian cells; ifinvestigation at that time, Kidder and his collab- the specific differences between particular systemsorators extended their studies from the animal are clearly understood (29), much of the informa-microorganism to experimental tumors (19). It tion gained in microorganisms can be a guide for


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68 ERICH HIRSCHBERG [VOL. 19

the study of normal and neoplastic mammalian biochemical site of action of an inhibitor is definedcells. in a more or less general way by the identity of a

In this section, no attempt will be made to pre- normal metabolite which prevents or reverses itssent a definitive r6sum6 of this rapidly developing inhibitory action. A brief listing of results infield. Rather, it is intended to illustrate the impor- microbiological systems and in a few types oftance of this sort of investigation by citing a few experimental tumors was presented in the recentspecific reports dealing with the action of carcino- review by Skipper (4) which gave an incisivestatic antimetabolites in microbiological systems. summary of the evidence for the attractive hy-In the past decade or so, a comprehensive pro- pothesis that the mechanism of carcinostasis

gram of the biosynthesis of nucleic acid deriva- primarily involves the nucleic acids of the cancertives in Lactobacillus casesi has been developed by cell.Hitchings and his collaborators. An impressive It has recently been realized that an antimetab-number of purines, pyrimidines, pteridines and olite may not only interfere with the utilizationrelated compounds have been prepared and tested of the normal metabolite for a particular syn-in the expectation that the discovery of inhibitors thetic pathway but may itself be utilized in itswould lead to the elucidation of the biochemical place, leading to the formation of unnatural endpathways involved in nucleic acid synthesis and products of that pathway which may be thoughtthat this program would yield new chemothera- of as antimetabolites at that more complex level.peutic agents for bacterial, rickettsial, viral, pro- In nucleic acid biosynthesis, the present cynosuretozoal or neoplastic diseases (31). These expecta- of cancer chemotherapy, an antipurine mighttions have been amply fulfilled; in addition to the thus be incorporated in place of the purine toimportant contributions to cancer chemotherapy form a nucleic acid molecule which would inter-mentioned earlier, this program has caused sig- fere with mitosis or fail to be attached properly tonificant advances in the chemotherapy of other protein to form nucleoprotein or be incapable ofdiseases, particularly malaria. The Lactobacillus reaching the correct degree of polymerization.casei model has served, moreover, to shed some An example of this type of reaction is the incor-light on the mechanism of action of antimetabo- poration of 8-azaguanine into nucleic acid inlites related to folic and folinic acids and to tumor-bearing mice (34, 35), in Tetrahymenanucleic acid derivatives. A brief consideration of geleii (36, 37), and in tobacco mosaic virus (38).this model system may therefore be in order. It is, of course, tempting to postulate a causalThe organism was grown on a basal medium de- relationship between incorporation of the anti-

ficient in purines, uracil, and folic acid and on six metabolite and inhibition of growth of the neo-variants of this medium achieved by the addition plastic cell, particularly in view of the findingof a small or an excess amount of folic acid, that the incorporation of 8-azaguanine into theadenine, thymine, adenine + thymine, or adenine ribonucleic acid of an azaguanine-susceptible+ folic acid. The response of the bacteria on each strain of leukemia was much greater than into theof these media to the compounds under investiga- ribonucleic acid of an azaguanine-dependenttion permitted a rapid definition of their mecha- variant (39). However, in resisting this tempta-nism of action in this system in terms of their tion at the present time the investigator is armedcapacity to replace folic acid, thymine, or purine not only with his natural reluctance to generalizeor to inhibit biochemical reactions for which one from a single well substantiated illustration to aof these entities was the substrate. On the basis general explanation of the growth inhibition, butof these data, the 2,4-diaminopyrimidines includ- also with recent data on the incorporation ofing the antimalarial Daraprim and its carcino- 5-bromouracil into nucleic acid of Streptococcusstatic dichloro analog have been classed as power- faecaltis and Enterococcus stei (40, 41) and offul antagonists of folic or folinic acid, a conclusion Escherichia coli (42, 43), data that clearly indi-which has been confirmed in mammals (32); cate that the incorporation of this thymine antag-6-mercaptopurine has been shown to be a purine onist may be as great in cells whose growth is notantagonist in the bacterial system (21) as well as inhibited by this compound as in cells whichin experimental neoplasms (33). respond to it. The full significance of information

Several other studies have employed the same from this type of experiment is still under activetechnique of inhibition analysis, in which the investigation.


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1955] CONTRIBUTIONS OF MICROBIOLOGY TO CANCER RESEARCH 69

This section may be concluded with a brief neoplastic cells to differentiate is as important tomention of the interesting experiments of Stock an understanding of this disease as their capacityand collaborators on the effect of 2,6-diamino- for unrestrained growth. The more malignantpurine on kappa in Paramecium aurelia. This tumors are endowed not only with greater in-work, which was recently reviewed by these inves- vasiveness and a higher rate of proliferation buttigators (44), illustrates the wealth of material frequently also with a greater degree of anaplasiawhich can be made available by judicious selec- which causes them to have little resemblance totion of a microbiological system for the elucida- the highly differentiated tissues from which theytion of the mechanism of action of carcinostatic are derived.agents. The term "differentiation" is widely used, but

its meaning is rarely spelled out. For the presentRecapitulation discussion, Needham's (52) definition may be the

As ontogeny recapitulates phylogeny, so the most serviceable: "Increase in complexity anddevelopmental history of some of the recent agents organization. Increase in number of kinds ofof chemotherapeutic interest shares the charac- cells; invisibly, as by determination and losses ofteristic features which have been outlined in competence; visibly, as by histogenesis. Increasethese pages. This is particularly well illustrated in morphological heterogeneity by the appearanceby O-diazoacetyl-L-serine, or azaserine, one of the of form and pattern."newest additions to the armamentarium of ex- Despite the abstract acceptance of the need toperimental cancer chemotherapy. understand the mechanisms of differentiation as

This agent was isolated from laboratory broth clearly as the causes of uncontrolled growth, mostcultures of a species of Streptomyces (45) and was experimental inquiries in oncology have addressedfound to inhibit the growth of Sarcoma 180 (46) themselves to the latter aspect of the problem.and other neoplasms (47). It has shown antibiotic There are many reasons for this. Our knowledgeactivity against a variety of microorganisms of synthetic processes in biochemistry is much(45, 48), one of which has been used for the assay more extensive than our understanding of theof activity during isolation. Studies on the mech- more nebulous events which take place in organi-anism of action of azaserine in Escherichia coli zation and morphogenesis. The rapid multiplica-(49, 50) suggest that it may inhibit amino acid tion of tumor cells is more easily observed andsynthesis by interfering with the condensation of analyzed than the subtler defects which cause theindole and serine to form tryptophan and with the cells to lose their highly differentiated character.formation of tyrosine and phenylalanine from The analytical approach, which to a biochemisttheir precursors. Analagous studies in tumors usually holds the greatest promise for the elucida-have not been reported; the only data available tion of a problem, has already been used exten-in neoplastic tissues implicate formate incorpora- sively in studying proliferation, whereas most oftion into nucleic acids as a site of inhibition by the work on differentiation has been descriptive.azaserine (51). The development of an aza- The range of biological systems available forserine-resistant line of mouse leukemia has morphogenetic studies is more restricted than thebeen reported (47). biological material which lends itself readily to

It should not be inferred from this discussion experiments on proliferation. This last difficulty,that all research in cancer chemotherapy is which may be an underlying or at least contribu-moving along these paths. In this active field, tory cause of the other obstacles to progress inmany approaches are being utilized, and micro- this area, is beginning to engage the attention ofbiology is only one of the numerous disciplines some experimental oncologists.which are contributing ideas and leads. Within Practically all investigations concerned withthis framework, however, it can be concluded differentiation have been carried out in embryo-that it is one of the most rewarding areas of in- logical systems, from the sea urchin to man. Thevestigation. solid progress in chemical embryology is attested

to by the monumental treatises of NeedhamMICROORGANISMS AND DIFFERENTIATION (52, 53) and Brachet (54) as well as by many

The definition of "cancer" which was cited recent discussions (55, 56). Two fundamentalearlier bears witness to the fact that the failure of difficulties are inherent in this type of biological


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Figure 1. The life cycle of Dictyosteliuni discoideum, from a drawing by Mr. James Lewicki repro-duced in Life Magazine of April 10, 1950. Reproduced by permission of copyright owner.

material, as repeatedly stressed by Needham: the etic movements in the virtual absence of celllack of any quantitative measure of differentia- division.tion and the simultaneous occurrence of growth A detailed description of the life cycle of Dic-and differentiation at all stages of embryonic tyostelium discoideum may be dispensed with sincelife. The analytical approach to the problem of it has been presented repeatedly (57, 58). Briefly,mechanisms of differentiation is seriously ham- the following stages are observed (figure 1): (a)pered by these difficulties; the desirability of germination of spores to liberate myxamoebae;studying biochemical changes caused by morpho- (b) growth and proliferation of myxamoebae,genesis in the absence of cell proliferation is self- which feed on their bacterial associates; (c) aggre-evident. gation of the previously independent myxamoe-These considerations prompted a search for bae towards a center, by chemotaxis, to form a

biological materials in which differentiation might pseudoplasmodium; (d) migration of this massbe investigated in the absence of cell division or over the substratum; and (e) culmination to format least under conditions where its role is rela- a mature structure, which consists of a basaltively insignificant. Attention was focused on disk, a thin stalk, and a sorus containing themicrobiological systems, particularly certain pro- spores that are ready to release new mvxamoebae.tozoa. At the present time, the value of these The rapidly expanding literature on this organismsystems for the eventual elucidation of the mech- has concerned itself with a number of problemsanisms of differentiation cannot be assessed with that will be discussed here briefly, with emphasisassurance. They are discussed here in the hope on the more recent contributions in this field.that more widespread interest in this approach In connection with the elegant series of investi-may speed the day when such an assessment can gations by Raper and Bonner on the life cycle,be made. growth, and organization of Dictyostelium dis-

coideum (57), attention was drawn repeatedly toDictyosteliurn discoideum the obvious desirability of achieving pure culture.

This organism and its close relatives among the In nature, the organism grows on decaying leavesamoeboid slime molds, or Acrasieae, have long or in the soil, and it was quickly establishedintrigued biologists with their life cycle and be- that live bacteria provide the proper physicalhavior patterns. In the past half dozen years, and nutritional conditions for its development.several reviews of experimental studies with In the laboratory, it has been possible to growthese organisms have appeared (57-59), and it routinely in association with a single bacterialDictyostelium discoideum has even been singled species (57, 64); this has been the method ofout for presentation to the general scientific choice in most subsequent studies. Numerousfraternity (60, 61) and to the lay public (62, 63). attempts have been made to replace the livingThe aspect which has fascinated a number of bacteria with a variety of supplements (65, 66),observers and which led to the selection of this and although routine large-scale pure culture oforganism for an examination of the mechanisms of the slime mold has not yet been achieved, soliddifferentiation is the division of its life cycle into progress is being made in this direction. Rapertwo distinct parts. In the first part, the organism (57) has reported successful growth and develop-is unicellular and carries on active feeding and ment of the slime mold on lactose-peptone agarproliferation; in the second, it becomes multicel- streaked with Escherichia coli killed by steriliza-lular and exhibits a complex series of morphogen- tion for 20 minutes at 121°C. More recently,


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Bradley and Sussman (67) obtained some growth the complex pattern of differentiation in this(10-20 per cent of normal) on a sterile medium organism could be brought about by the fortui-enriched with a fraction of Aerobacter aerogenes tous association of two or more genetically hetero-which after isolation in homogeneous form has geneous parent spores or could proceed from anow been characterized as a protein of molecular single spore. If the first alternative applies, theseweight between 25,000 and 40,000 containing the studies would remain a fascinating biologicalusual spectrum of amino acids (68). occupation, but the results would have general

Experiments on the proliferative stage of this significance for problems in differentiation only iforganism have been postponed until pure culture the entire development could start with a singlemethods become available. In the meantime, spore. In a series of ingenious experiments, it washowever, the demonstration by Bonner (58) that shown: (a) that single spores of Dictyosteliumthe bacteria on which myxamoebae feed can be discoideum and two related species were indeedremoved by washing and differential centrifuga- capable of initiating the normal course of develop-tion and that the myxamoebae will go through ment; (b) that vegetative myxamoebae retainedthe remainder of the life cycle (aggregation, mi- this ability; (c) that at the onset of aggregationgration, and culmination) in the absence of the only a small proportion of the cells exhibited thebacterial associates has made it possible to study capacity of initiating a center; (d) that, however,the various stages of differentiation in essentially the ability of the myxamoebae to produce acrasinpure culture. was not restricted to the "initiator" cells. Irradia-Bonner has been particularly interested in the tion of spores or myxamoebae of the wild type

control of the morphogenetic movements in Dictyosteliaceae produced a number of variantsDictyostelium discoideum (58, 69, 70, 71, 72). He which failed to aggregate or to produce fruitinghas demonstrated that aggregation of the inde- structures or which had an abnormal appearance.pendent myxamoebae is brought about by a The importance of correlating these abnormalitieschemotactic principle, called acrasin, which con- with changes in the enzyme pattern of these cellstinues to be emitted by the cells even through was emphasized, but this as well as the problempseudoplasmodium formation, migration, and of relating them to genic mutations has not yetculmination. In the migrating pseudoplasmodium been approached experimentally. Recent studiesthe anterior region, which directs the orientation on the synergism and antagonism betweenand movement of the sausage-shaped structure morphogenetically deficient variants may providetowards light and heat, produces most of the a key to the elucidation of the metabolites andacrasin, and during culmination the capacity to biosynthetic pathways involved in the morpho-produce this chemotactic principleis retained only genetic cycle.by the cells located at the tip of the fruiting Another problem which has recently receivedstructure. Isolation of acrasin for eventual chem- attention relates to the occurrence and role of cellical identification has been attempted repeatedly division during differentiation. One of the prin-(73), but the identity of this agent, which plays a cipal attractions of Dictyostelium discoideum for acentral role in differentiation in this organism, variety of studies, an attraction noted by allremains one of the challenging mysteries in this early observers, has been the apparent completefield. separation of proliferation and differentiation in

Raper and Fennell (74) have carried out a the life cycle. The general conviction that alldetailed investigation of stalk formation during mitotic activity ceased before aggregation, how-culmination of the slime mold. Particular atten- ever, was recently shaken by the findings of Wil-tion was given to the nature of the outer sheath son (80, 81) and Bonner and Frascella (82).surrounding a column of vacuolated cells in the Wilson, using smears of Dictyostelium discoideumstalk. This sheath is formed by the extracellular in association with Escherichia coli, reporteddeposition of cellulose in a critical circular zone, evidence of gamete fusion just prior to, and ofthe dimensions of which are proportional to the meiosis during, aggregation and of a wave ofmass of cooperating myxamoebae. mitoses at the time of spore formation at the endAn important contribution has been made by of culmination. Bonner, in experiments on myx-

Sussman in a series of reports over the last three amoebae freed of bacteria by washing and differ-years (59, 75, 76, 77, 78, 79). It was realized that ential centrifugation, demonstrated the presence


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of mitosis in the early migration stage but found declines to negligible levels. The same authorno cell divisions during aggregation or early culmi- (83b,c) has recently studied the carbohydratenation. It was suggested (82) that differentiation and nitrogen metabolism of this organism duringis not dependent on these mitoses, but further growth and morphogenesis and has demonstratedwork is clearly indicated before the role of cell by ingenious methods that protein is utilizeddivisions in early migration and in spore formation to obtain energy during culmination and tois clarified. Some of this new information appears satisfy other morphogenetic requests. Further,to be well founded, but Wilson's claim of zygote comparative analyses of cells at different stagesformation prior to aggregation and, in general, his of the life cycle for various biochemical entitiespostulate of sexuality in the development of this have been forecast by preliminary data (66) thatorganism have recently come under concentrated demonstrated a decreased content of ribonucleicattack based on a number of cogent arguments acid per mg nitrogen and a higher acid-soluble(59). Until more convincing evidence for sexuality fraction of the total phosphorus in pseudoplas-is presented, these claims must be considered at modia than in preaggregation myxamoebae. Moreleast somewhat speculative. extensive work on these subjects is in progress.The application of biochemical techniques to A different, more indirect biochemical approach

the problems of differentiation in Dictyostelium consists in the inhibition of aggregation or cul-discoideum promises to contribute valuable infor- mination by compounds whose general mecha-mation on the underlying mechanisms. Work has nism of action is known or can be further studied inbegun along two general lines, though it must be this test system (84, 85, 86, 87). For most of thesestressed that only a small beginning has been experiments, myxamoebae were harvested beforemade. Gregg (83a), studying oxygen utilization aggregation, washed free of bacteria, and allowedduring growth and differentiation, showed that to aggregate in standard salt solution containingmorphogenesis cannot proceed under anaerobiosis different concentrations of a potential inhibitor.and that oxygen consumption increases during Figures 2 and 3 provide two views of normalthe transition from the growth phase to the aggregation patterns. In experiments on themorphogenetic phases, remains constant through inhibition of culmination, aggregation was al-the middle of the culmination phase, and then lowed to proceed, and an agar disk bearing the

Figure 2. Early aggregation pattern of Dictyostelium discoideum.


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Figure S. Late aggregation pattern of Dictyostelium discoideum.

aggregates was placed in the salt solution with since its isolation by Raper in 1935, other mem-

and without the inhibitor. Aggregation was in- hers of the Dictyosteliaceae have also receivedhibited by compounds of varied biochemical some attention (88), with generally analogousaction, including 2 ,4-dinitrophenol, iodoaceta- results.mide, diethylstilbestrol, several azo dyes, and a

series of 2, 4-diaminopyrimidines, whereas com- Myxomycetes and Myxobacteriamon inhibitors of glycolysis and of mitosis gener- The biological literature of the past half cen-ally had no effect. In most instances, the inhibi- tury is a treasure house filled with other unusualtion of aggregation by these agents was not a systems for investigations into differentiation andsimple irreversible process; if myxamoebae were morphogenesis. The fascinating account ofexposed to the inhibitor for several hours and morphogenesis in a wide variety of biologicalthen washed with salt solution, they could aggre- systems by Bonner (58) has done much to stimugate normally. The length of the period of per- late and direct thinking in this field. Referencemissible exposure to the inhibitor varied from should also be made to the work of Weisz (89, 90),~agent to agent. A more detailed study of the who developed a general statement on the mech-effect of 2, 4-dinitrophenol suggested that high- anism of differentiation based on studies inenergy phosphate is necessary for aggregation ciliates but of tentative applicability to multicel-and that inhibition of this process relates to the lular organisms as well. The present discussionhydrolysis of organic phosphate to yield increas- has been devoted largely to the Acrasieae, becauseing amounts of inorganic phosphate. Culmination of the numerous recent contributions dealingwas more easily inhibited than aggregation under with this group. This section may be concluded bythe conditions of these tests, and the differences brief mention of two types of organisms whichin response of these two stages of morphogenesis carry on morphogenesis in a manner similar toto certain agents provide intriguing opportuni- that which has been described.ties-for further investigation. The myxomycetes or true slime molds differAlthough Dictyosteliurn discoideum has been the from the Acrasieae in that they have a true plas-

organism used most extensively in these studies modium rather than the multicellular aggregate


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of myxamoebae which in Dictyostelium and re- larly Dr. Harold P. Rusch of the University oflated species is properly called a pseudoplasmo- Wisconsin, Dr. John T. Bonner of Princetondium. A number of these organisms, notably University, and Drs. Alfred Gellhorn and StephenPhysarum polycephalum, Physarella oblonga, and Zamenhof of Columbia University.Fuligo septica, have been examined from the point REFERENCESof view of nutrition, physiology, taxonomy, andmorphogenesis (58, 91, 92, 93, 94, 95). Physarumt 1. DEIGNAN, STELLA L., AND MILLER, ESTHERpolycephalum has long been a favorite for studies 1954. The support of research in the bio-on protoplasmic streaming and chemotaxis (96- sciences for the fiscal years 1952 and 1953.101). Many of the problems that have been en- Science, 119, 661-671.2. REILLY, H. CHRISTINE 1953 Microbiologycountered and the sort of data which have been and c e therapy: A riew.bCancerobtained are similar to the information available Research, 13, 821-834.in the Acrasieae, but less thought has perhaps 3. GREENSTEIN, J. P. 1954 Biochemistry ofbeen given to the possible applications of these cancer, pp 298-302. 2nd ed. Academicresults to general mechanisms of differentiation. Press, Inc., N. Y.The recent demonstration of the suitability of 4. SKIPPER, H. E. 1953 A review: On themyxomycetes for biochemical studies (102, 103) mechanism of action of certain temporaryand the current advances towards pure culture of anticancer agents. Cancer Research, 13,the plasmodium (H. P. Rusch, unpublished re- 545-551.suits) greatly increase the attractiveness of these 5. LETTR", H. 1954 Neuere Ergebnisse der

organisms for such investigations. Krebsforschung. Naturw. Rundschau, 3,Finally, brief mentionmneslatsbe made of theIn101-110.Finally,briefmentionmaybemadeofthemyxo- 6. GELLHORN, A. 1953 Recent developments

bacteria, which exhibit striking similarities to in laboratory and clinical cancer chemo-the Acrasieae though they are totally unrelated therapy. J. Urol., 70, 813-826.to them from the point of view of taxonomy. 7. GELLHORN, A. 1954 Clinical cancer chemo-These similarities have been emphasized by most therapy. Acta Unio Intern. contra Can-observers (58, 104, 105, 106), particularly with crum, in press.regard to the aggregation patterns and, in the 8. STOCK, C. C. 1954 Experimental cancerhigher myxobacteria, the formation of elaborate chemotherapy. Advances in Cancer Re-fruiting bodies. The application to these organ- search, 2, 425-492. Academic Press, Inc.,isms of the investigative techniques Which have 9 N. Y.beeanarbeng iretedat he craiea

9.FILDES, P. 1940 The mechanism of thebeen and are being directed at the Acrasieae anti-bacterial action of mercury. Brit. J.appears to be one of the most promising tasks for Exptl. Pathol., 21, 67-73.the immediate future. 10. WOODS, D. D. 1940 The relation of

It was stated at the beginning of this section p-aminobenzoic acid to the mechanism ofthat the value of these studies to experimental the action of sulphanilamide. Brit. J.oncology cannot yet be assessed. Results in these Exptl. Pathol., 21, 74-90.microbiological systems have correlated reason- 11. WOOLLEY, D. W. 1952 A study of anti-ably well with findings in multicellular organisms metabolites. John Wiley and Sons, Newwhere such a comparison has been possible; the York, N. Y.

12. SEEGER, D. R., SMITH, J. M., JR., AND HULT-basic biochemical equipment of these organisms QUIST, M. E. 1947 Antagonist foris generally the same as that of mammalian cells. pteroylglutamic acid. J. Am. Chem. Soc.,Nevertheless, even the more enthusiastic advo- 69, 2567.cates of these fascinating microorganisms will 13. HUTCHINGS, B. L., MOWAT, J. H., OLESON,defer final judgment on the applicability of these J. J., STOKSTAD, E. L. R., BOOTHE, J. H.,systems to an explanation of the failure of differ- WALLER, C. W., ANGIER, R. B., SEMB, J.,entiation in neoplasia until a good deal of further AND SUBBAROW, Y. 1947 Pteroylasparticevidence can be assembled. acid, an antagonist for pteroylglutamic

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ACKNOWLEDGEMENT 14. FARBER, S., DIAMOND, L. K., MERCER, R. D.,SYLVESTER, R. F., JR., AND WOLFF, J. A.

The author is indebted for many helpful dis- 1948 Temporary remissions in acute leu-cussions to a number of his colleagues, particu- kemia in children produced by folic acid


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1955J CONTRIBUTIONS OF MICROBIOLOGY TO CANCER RESEARCH 75

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SOME CONTRIBUTIONS MICROBIOLOGY TO CANCER RESEARCH' · SOME CONTRIBUTIONS OF MICROBIOLOGY TO CANCERRESEARCH' ERICH HIRSCHBERG Institute of Cancer Research andDepartment ofBiochemistry,