SOME CONTRIBUTIONS OF MICROBIOLOGY TO CANCER RESEARCH'
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SOME CONTRIBUTIONS OF MICROBIOLOGY TO CANCER RESEARCH'
ERICH HIRSCHBERG
Institute of Cancer Research and Department of Biochemistry, Columbia University College of Physicians
and Surgeons, New York, New York
A quarter century ago, cancer research was between bacteriology or protozoology and cancer
almost 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 experimental
examination of the cancer literature at that time material in many cancer research centers. In a
reveals that attention was focused predomi- recent analysis of the financial support of cancer
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nantly on problems, ranging from cytological and research by government and nongovernment
histological studies of spontaneous tumors to agencies (1), the total expenditure for 1953 was
investigations on homologous and heterologous placed at about ten million dollars; at least 7 per
transplantation of neoplasms, from serological cent of these funds was committed for studies
diagnosis 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 by
by its intensive rather than extensive orientation; many investigators to be primary causative
the annual meeting of the pertinent scientific agents of all cancer. The proponents of the
society was held in a single session and was de- somatic mutation theory, the other principal
voted to the presentation of about a dozen hypothesis dealing with carcinogenesis, have been
papers; 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 effect
shadows 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 examples
are few scientific disciplines which have not con- of the application of microbiology to this area of
tributed 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 other
backgrounds and interests have increased beyond microorganisms, enzyme adaptation studies in
all expectation. At the last annual meeting of the bacteria, and related subjects of current interest
American Association for Cancer Research, there are illustrations of the increasingly fruitful union
were 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 complex
searchers 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 useful
aspect 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 of
of 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
work supported innpparedy
part by ina cnearct anwth
research grant trolled growth; the first section ofirbooyt
del wihsm'otiuinso this review
(C-2354) of the National Cancer Institute, U. S. deals with some contributions of microbiology to
Public Health Service and in part by an Institu- the search for effective means of stopping the
tional Grant of the American Cancer Society to rapid proliferation of the cancer cell. Increasing
Columbia University. attention is now being given to the failure of
6566 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 most
to a consideration of certain slime molds and varied nature have been evaluated in screening
related organisms which appear to lend them- programs based on the response of one or several
selves particularly well to studies on differentia- transplanted animal tumors (8). Agents have
tion and morphogenesis. Some of these studies been selected for testing for many different
may have a more tenuous or less immediate con- reasons; these have ranged from the considered
nection 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 to
this 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 inhibit
MICROORGANISMS AND CANCER CHEMOTHERAPY
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the growth of experimental tumors or affect the
There 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 guided
effects of microorganisms and particularly of some investigators in their search for active agents has
of 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 theory
agents 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) on
these agents in bacteria and protozoa have con- the relationship between the sulfonamides and
tributed significantly to our present understand- p-aminobenzoic acid in bacterial metabolism has
ing 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 the
potential 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 of
The 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 appreciable
lytic agents will not be considered here since the number of the effective carcinostatic agents in
literature on this subject has been covered pains- current use were suggested for screening against
takingly 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, and
added here to their discussion of the mechanisms identification of pteroylglutamic acid as an
underlying the development of resistance. The essential growth factor for microorganisms is too
other two topics, however, fill be discussed in well known to bear repetition here. In 1947, the
more detail. first antimetabolites for pteroylglutamic acid
were prepared (12, 13) and shown to inhibit the
Discovery 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 treatment
principal tools for the eradication or control of of acute leukemia in children, on the basis of
neoplasms, the last decade has witnessed signifi- earlier observations of an acceleration of tumor
cant progress in chemotherapy, a form of treat- development following the administration of
ment of particular importance in disseminated pteroyldi- and triglutamic acid, and found to
cancer and in the various forms of leukemia and produce significant though temporary remis-19551 CONTRIBUTIONS OF MICROBIOLOGY TO CANCER RESEARCH 67
sions (14). The folic acid antagonists, particularly was found that 8-azaguanine inhibited tumor
aminopterin and A-methopterin, continue to be of growth profoundly. These investigators were
value in the treatment of leukemias in man and fortunate in having chosen for this demonstration
have been shown to inhibit the growth of a great mammary adenocarcinoma E0771 and two other
variety of experimental tumors. tumors which responded to 8-azaguanine, since
The road from demonstration of activity later studies have indicated that experimental
against microorganisms to introduction into neoplasms differ widely in their susceptibility to
cancer chemotherapy has been less direct for this agent.
another agent of considerable experimental in- Four other antimetabolites which have proved
terest, 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 screening
tion of the central role of nucleic acids in the on the basis of their activity in Lactobacillus casei.
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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 the
purines and pyrimidines. In a search for effective more significant therapeutic results attained by
purine antagonists, it was found in 1945 (15) that the systematic investigations of Hitchings and
8-azaguanine, which differs from guanine only by his collaborators which will be alluded to in the
the substitution of a nitrogen atom for the > CH following section.
group in position 8, was effective against The justifiable emphasis placed in these pages
Escherichia coli and Staphylococcus aureus; the on the value to cancer chemotherapy of agents
inhibition was reversed by guanine. This com- first discovered in microbiological systems should
pound was included in the first group of purines not be construed as an open invitation to the
which were screened against Sarcoma 180 but oncologist in search of new agents to rely solely or
failed 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 be
The 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 Carcinostatic
tained by a systematic study of the growth Agents in Microbiological Systems
requirements of this organism. Following the The recent review literature abounds with
demonstration 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 of
stituted purines were tested for their ability to Lactobacillus to Tetrahymena and Paramecium
replace or spare guanine or to inhibit growth in a
competitive or noncompetitive manner. Among (24-30). In many instances, the biochemical
the inhibitors, 8-azaguanine was by far the most processes in microorganisms have been compared
effective; 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 the
from normal mammalian cells by having this two types of biological systems are emerging
requirement for exogenous guanine and since the from these studies. In general, the synthesis and
possible differences in purine utilization between metabolism of the nucleic acids are carried on in a
normal and neoplastic cells were under active similar way in microbial and mammalian cells; if
investigation at that time, Kidder and his collab- the specific differences between particular systems
orators 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 for68 ERICH HIRSCHBERG [VOL. 19
the study of normal and neoplastic mammalian biochemical site of action of an inhibitor is defined
cells. 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 its
sent a definitive r6sum6 of this rapidly developing inhibitory action. A brief listing of results in
field. Rather, it is intended to illustrate the impor- microbiological systems and in a few types of
tance of this sort of investigation by citing a few experimental tumors was presented in the recent
specific reports dealing with the action of carcino- review by Skipper (4) which gave an incisive
static 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 cancer
tives 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 utilization
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related 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 its
would lead to the elucidation of the biochemical place, leading to the formation of unnatural end
pathways involved in nucleic acid synthesis and products of that pathway which may be thought
that 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 cynosure
tozoal or neoplastic diseases (31). These expecta- of cancer chemotherapy, an antipurine might
tions have been amply fulfilled; in addition to the thus be incorporated in place of the purine to
important 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 to
nificant advances in the chemotherapy of other protein to form nucleoprotein or be incapable of
diseases, 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 in
lites related to folic and folinic acids and to tumor-bearing mice (34, 35), in Tetrahymena
nucleic 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 causal
The 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 finding
of a small or an excess amount of folic acid, that the incorporation of 8-azaguanine into the
adenine, 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 the
of these media to the compounds under investiga- ribonucleic acid of an azaguanine-dependent
tion 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 armed
capacity to replace folic acid, thymine, or purine not only with his natural reluctance to generalize
or to inhibit biochemical reactions for which one from a single well substantiated illustration to a
of these entities was the substrate. On the basis general explanation of the growth inhibition, but
of these data, the 2,4-diaminopyrimidines includ- also with recent data on the incorporation of
ing the antimalarial Daraprim and its carcino- 5-bromouracil into nucleic acid of Streptococcus
static dichloro analog have been classed as power- faecaltis and Enterococcus stei (40, 41) and of
ful 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 not
antagonist in the bacterial system (21) as well as inhibited by this compound as in cells which
in 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 active
technique of inhibition analysis, in which the investigation.1955] CONTRIBUTIONS OF MICROBIOLOGY TO CANCER RESEARCH 69
This section may be concluded with a brief neoplastic cells to differentiate is as important to
mention of the interesting experiments of Stock an understanding of this disease as their capacity
and collaborators on the effect of 2,6-diamino- for unrestrained growth. The more malignant
purine 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 but
tigators (44), illustrates the wealth of material frequently also with a greater degree of anaplasia
which can be made available by judicious selec- which causes them to have little resemblance to
tion of a microbiological system for the elucida- the highly differentiated tissues from which they
tion 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 present
Recapitulation discussion, Needham's (52) definition may be the
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As ontogeny recapitulates phylogeny, so the most serviceable: "Increase in complexity and
developmental history of some of the recent agents organization. Increase in number of kinds of
of chemotherapeutic interest shares the charac- cells; invisibly, as by determination and losses of
teristic features which have been outlined in competence; visibly, as by histogenesis. Increase
these pages. This is particularly well illustrated in morphological heterogeneity by the appearance
by 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 to
perimental cancer chemotherapy. understand the mechanisms of differentiation as
This agent was isolated from laboratory broth clearly as the causes of uncontrolled growth, most
cultures of a species of Streptomyces (45) and was experimental inquiries in oncology have addressed
found 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 knowledge
activity 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 the
of 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 and
synthesis by interfering with the condensation of analyzed than the subtler defects which cause the
indole 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 biochemist
their 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 of
tion 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 for
serine-resistant line of mouse leukemia has morphogenetic studies is more restricted than the
been 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 in
many approaches are being utilized, and micro- this area, is beginning to engage the attention of
biology is only one of the numerous disciplines some experimental oncologists.
which are contributing ideas and leads. Within Practically all investigations concerned with
this 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. The
vestigation. solid progress in chemical embryology is attested
to by the monumental treatises of Needham
MICROORGANISMS AND DIFFERENTIATION (52, 53) and Brachet (54) as well as by many
The definition of "cancer" which was cited recent discussions (55, 56). Two fundamental
earlier bears witness to the fact that the failure of difficulties are inherent in this type of biological70 ERICH HIRSCHBERG [VOL. 19
: i
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 cell
lack of any quantitative measure of differentia- division.
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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 since
life. 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 mass
be investigated in the absence of cell division or over the substratum; and (e) culmination to form
at least under conditions where its role is rela- a mature structure, which consists of a basal
tively insignificant. Attention was focused on disk, a thin stalk, and a sorus containing the
microbiological 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 organism
systems for the eventual elucidation of the mech- has concerned itself with a number of problems
anisms of differentiation cannot be assessed with that will be discussed here briefly, with emphasis
assurance. 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 to
Dictyosteliurn discoideum the obvious desirability of achieving pure culture.
This organism and its close relatives among the In nature, the organism grows on decaying leaves
amoeboid slime molds, or Acrasieae, have long or in the soil, and it was quickly established
intrigued biologists with their life cycle and be- that live bacteria provide the proper physical
havior 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 grow
these organisms have appeared (57-59), and it routinely in association with a single bacterial
Dictyostelium discoideum has even been singled species (57, 64); this has been the method of
out for presentation to the general scientific choice in most subsequent studies. Numerous
fraternity (60, 61) and to the lay public (62, 63). attempts have been made to replace the living
The 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 of
organism for an examination of the mechanisms of the slime mold has not yet been achieved, solid
differentiation is the division of its life cycle into progress is being made in this direction. Raper
two 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 agar
proliferation; 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,1955] CONTRIBUTIONS OF MICROBIOLOGY TO CANCER RESEARCH 71
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 a
now been characterized as a protein of molecular single spore. If the first alternative applies, these
weight between 25,000 and 40,000 containing the studies would remain a fascinating biological
usual 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 if
organism have been postponed until pure culture the entire development could start with a single
methods become available. In the meantime, spore. In a series of ingenious experiments, it was
however, the demonstration by Bonner (58) that shown: (a) that single spores of Dictyostelium
the bacteria on which myxamoebae feed can be discoideum and two related species were indeed
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removed 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 retained
the remainder of the life cycle (aggregation, mi- this ability; (c) that at the onset of aggregation
gration, and culmination) in the absence of the only a small proportion of the cells exhibited the
bacterial 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 acrasin
pure 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 variants
Dictyostelium discoideum (58, 69, 70, 71, 72). He which failed to aggregate or to produce fruiting
has 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 abnormalities
chemotactic principle, called acrasin, which con- with changes in the enzyme pattern of these cells
tinues to be emitted by the cells even through was emphasized, but this as well as the problem
pseudoplasmodium formation, migration, and of relating them to genic mutations has not yet
culmination. In the migrating pseudoplasmodium been approached experimentally. Recent studies
the anterior region, which directs the orientation on the synergism and antagonism between
and movement of the sausage-shaped structure morphogenetically deficient variants may provide
towards light and heat, produces most of the a key to the elucidation of the metabolites and
acrasin, 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 received
structure. Isolation of acrasin for eventual chem- attention relates to the occurrence and role of cell
ical 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 a
central role in differentiation in this organism, variety of studies, an attraction noted by all
remains one of the challenging mysteries in this early observers, has been the apparent complete
field. separation of proliferation and differentiation in
Raper and Fennell (74) have carried out a the life cycle. The general conviction that all
detailed 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 discoideum
stalk. This sheath is formed by the extracellular in association with Escherichia coli, reported
deposition of cellulose in a critical circular zone, evidence of gamete fusion just prior to, and of
the dimensions of which are proportional to the meiosis during, aggregation and of a wave of
mass of cooperating myxamoebae. mitoses at the time of spore formation at the end
An 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 presence72 ERICH HIRSCHBERG [VOL. 19
of mitosis in the early migration stage but found declines to negligible levels. The same author
no cell divisions during aggregation or early culmi- (83b,c) has recently studied the carbohydrate
nation. It was suggested (82) that differentiation and nitrogen metabolism of this organism during
is not dependent on these mitoses, but further growth and morphogenesis and has demonstrated
work is clearly indicated before the role of cell by ingenious methods that protein is utilized
divisions in early migration and in spore formation to obtain energy during culmination and to
is 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 stages
formation prior to aggregation and, in general, his of the life cycle for various biochemical entities
postulate of sexuality in the development of this have been forecast by preliminary data (66) that
organism have recently come under concentrated demonstrated a decreased content of ribonucleic
attack based on a number of cogent arguments acid per mg nitrogen and a higher acid-soluble
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(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. More
least 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 in
begun along two general lines, though it must be this test system (84, 85, 86, 87). For most of these
stressed that only a small beginning has been experiments, myxamoebae were harvested before
made. Gregg (83a), studying oxygen utilization aggregation, washed free of bacteria, and allowed
during growth and differentiation, showed that to aggregate in standard salt solution containing
morphogenesis cannot proceed under anaerobiosis different concentrations of a potential inhibitor.
and that oxygen consumption increases during Figures 2 and 3 provide two views of normal
the transition from the growth phase to the aggregation patterns. In experiments on the
morphogenetic 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.1955] CONTRIBUTIONS OF MICROBIOLOGY TO CANCER RESEARCH 73
Downloaded from http://mmbr.asm.org/ on May 8, 2021 by guest
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 received
hibited by compounds of varied biochemical some attention (88), with generally analogous
action, including 2 ,4-dinitrophenol, iodoaceta- results.
mide, diethylstilbestrol, several azo dyes, and a
series of 2, 4-diaminopyrimidines, whereas com- Myxomycetes and Myxobacteria
mon 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 unusual
tion of aggregation by these agents was not a systems for investigations into differentiation and
simple irreversible process; if myxamoebae were morphogenesis. The fascinating account of
exposed to the inhibitor for several hours and morphogenesis in a wide variety of biological
then washed with salt solution, they could aggre- systems by Bonner (58) has done much to stimu
gate normally. The length of the period of per- late and direct thinking in this field. Reference
missible 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 in
energy 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 discussion
hydrolysis of organic phosphate to yield increas- has been devoted largely to the Acrasieae, because
ing amounts of inorganic phosphate. Culmination of the numerous recent contributions dealing
was more easily inhibited than aggregation under with this group. This section may be concluded by
the conditions of these tests, and the differences brief mention of two types of organisms which
in response of these two stages of morphogenesis carry on morphogenesis in a manner similar to
to certain agents provide intriguing opportuni- that which has been described.
ties-for further investigation. The myxomycetes or true slime molds differ
Although Dict yosteliurn 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 aggregate74 ERICH HIRSCHBERG [VOL. 19
of myxamoebae which in Dictyostelium and re- larly Dr. Harold P. Rusch of the University of
lated species is properly called a pseudoplasmo- Wisconsin, Dr. John T. Bonner of Princeton
dium. A number of these organisms, notably University, and Drs. Alfred Gellhorn and Stephen
Physarum polycephalum, Physarella oblonga, and Zamenhof of Columbia University.
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