CANCER
VOLUME24
The
RESEARCH
MAY 1964
Significance
of Invertebrate
NUMBER4
Hormones
to Differentiation*
in Relation
WALTERJ. BÃœROBOTE
(Department of Surgery and Laboratory of Clinical Biology, University of Utah College of Medicine, Salt Lake City, Utah)
The manner in which specialized form and function are
exchanged in a predictable pattern for the pluripotentiality
of the zygote poses one of the most refractory riddles in
biology. A few generalizations concerning the steps in
converting the polymorphic to the differentiated organism
have emerged, but the total concept remains inadequate.
On the assumption that common mechanisms, with
differences only hi the details, may operate in all bio
logical systems of differentiation, it may be profitable to
examine information contributed by invertebrate systems
of metamorphosis to the general knowledge.
The endocrine systems that exist hi invertebrates
constitute a fascinating chapter hi the history of science.
Some of them are exotic but not germane to the topic
under discussion. For example, pheromones (3, 27, 28,
32, 66, 68), such as the sex attractants (26, 30, 31), behave
in many respects like hormones but exert their effect
at a distance to produce a lepidopteran romantic telemetry
that is the envy of purveyors of perfume from Paris to
Cairo. Others are intimately related to the process of
differentiation.
INVERTEBRATE
HORMONES
In invertebrates, the process of growth necessitates
moulting and the periodic renewal of the exoskeleton.
Attention will be directed toward three groups of hor
mones and the tissues that produce them, since they are
directly concerned with the regulation of events taking
place during moulting and metamorphosis. The orderly
initiation and succession of the usual pupal, larval, and
imaginai stages in the process depend on neurosecretion
from the brain, juvenile hormone (neotenin) from the
paired corpora aliata, and ecdysones from the prothoracic
glands.
Brain hormone.—Early in the twentieth century the
conviction prevailed that insects do not secrete hormones.
However, Kopeöwas able to show by 1917 that pupation
is initiated by a hormone (86) secreted in the brain.
Wigglesworth (117) confirmed the presence of a hormone
in the pars intercerebralis of the protocerebrum that
induced each cycle of growth and formation of successive
cuticles in Rhodnius prolixus, and Fraenkel (43) reported
similar findings for Calliphora. Weyer (115) demon* Aided by a grant from the Department of Health, Education,
and Welfare, U. S. Public Health Service.
strated secretory activity in the brain of Apis nielli/era
at about the same tune. Others (70, 103, 114) have
confirmed and extended these observations. Fukuda
(44) demonstrated that the moulting hormone hi the
silkworm was produced by the prothoracic glands. The
relationship between this discovery and the known effects
of neurosecretion on development was worked out by
Williams (120) in Platysamia. He found that the prothoracic glands secreted hormone hi response to stimula
tion by secretion from the brain.
Neurosecretory droplets have been noted hi the corpora
cardiaca at the termination of axons from the brain by
both Hanström (57) and Scharrer (101). It is usually
assumed that the pathway for release is through the
corpora cardiaca. However, noting that two pathways
for neurosecretion (70) are visible from the onset of fourth
and fifth larval stage of the silkworm, one through the
axon of the A-cell and the other to the open circulation
via the cortex, Kobayashi suggested that the prothoracotropic hormone concerned with imaginai differentiation is
probably released through the latter pathway (71). Ichikawa and Nishiitsutsuji-Uwo found that corpora aliata
also store neurosecretory material transported via the
nerves connecting them with the brain (60), and Kobaya
shi et al. (72, 77, 84) have reported imaginai differentiation
of Dauer-pupae in the presence of corpora aliata. With
his collaborators at the Sericultural Institute in Tokyo
(69, 78, 79), Kobayashi was able to isolate (from thousands
of brains dissected from silkworms) cholesterol, hi addi
tion to a protein fraction, as an active material producing
this effect. This action of cholesterol has been confirmed
by Schneiderman and co-workers. However, hormone
from the brain may also have a direct effect on tissues
(61, 89) hi influencing the course of metamorphosis. For
example, Kobayashi and Burdette (73) found that pupa
tion of Calliphora was produced when brain hormone
was injected with hormone from prothoracic glands in
concentrations of the latter that produced no effect. Con
tamination of the former with the latter in the process of
extraction does not seem probable, since high concentra
tions of brain hormone did not produce a similar positive
bioassay.
Both Bergman (12) and Karlson (65) report isolation
of cholesterol in relatively large amounts from the silk
moth, and the latter questions whether cholesterol repre
sents the active principle secreted bv the brain. The
521
This
One
20BJ-ZJY-1NWN
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for Cancer Research.
522
Cancer Research
likelihood that cholesterol is a precursor of ecdysone has
been enhanced by the observation of Church (35) and
Wigglesworth (118) that continued neurosecretion is
necessary for the orderly progression of metamorphosis.
By using labeled cholesterol, Karlson and Hoffmeister
(67) have demonstrated recently that this is the case. It
should be noted, however, that Kobayashi suggests that
cholesterol from the brain may be different from that
found elsewhere in the insect, since the latter is present
during diapause. A number of active extracts of brain
hormone have been obtained by investigators other than
the group at the Sericultural Institute.
The extracts of
Gilbert and Schneiderman (53) as well as Kobayashi et al.
(69, 78-80) are lipide-soluble, whereas Gersch (46, 47)
and Ichikawa (59) and co-workers report activity in
fractions soluble in water. Thus the hormonal chemistry
of neurosecretion in insects remains partially unresolved.
Activity resembling that of the brain hormone on Dauerpupae of Bombyx has been found by Kobayashi and coinvestigators (83) for progesterone, hexestrol, diethylstilbestrol, cholestanol, 7-dehydrocholesterol, campesterol,
and noradrenalin.
Periodicity in insects is explained by external stimuli
such as a change in temperature (82, 121), feedings (118),
etc. (81) activating the brain to secrete. Apparently
low temperature does not induce imaginai differentiation
of Dauer-pupae (76). In Platysamia, vanderKloot (112)
showed that the brain, which is dormant and electrically
silent during the winter, produces cholinesterase and shows
electrical activity at the time neurosecretion appears.
This is then followed by changes in morphology of the
prothoracic gland. Transplantation
experiments indi
cate that larval brain is as effective as pupal brain in
affecting the imaginai differentiation of Dauer-pupae (71).
Ecdysones.—The prothoracic gland in lepidoptera is a
more favorable object for ablation and transplantation
than corresponding cells in diptera where they lie in
proximity to cells of the corpora aliata to form the ring
gland. From experiments of this type it is apparent that
successive larval molts are brought about by balances
between levels of hormone from corpora aliata and prothoracic glands, and transition between larval and pupal
and pupal and imaginai (adult) stages are induced by
hormone from the prothoracic glands. Extraction of the
hormone of the prothoracic glands is based on pioneer
work of Becker and Plagge (6) on isolating active material.
Active hormone was first crystallized from chrysalides of
the silkworm by Butenandt and Karlson (31), who named
it ecdysone. This water-soluble material has the same
action as the secretion from transplanted prothoracic
glands. Recently, Karlson (65) has published a tentative
and partial formula for the ecdysone he has isolated. We
(23) have obtained five separate, active fractions of ecdy
sone from Bombyx, two (a and /3) previously reported by
Butenandt and Karlson and three (7, 5, and e) previously
not isolated. The relationship of the latter to the chemi
cal structure reported by Karlson and to the activity of
the gland is not currently known. The likelihood that
these are steroids with cholesterol a precursor is great.
We (20) have carefully followed the levels of ecdysones
in Bombyx during various stages in the life cycle and have
Vol. 24, May 1964
°=C.U./6.Wet Weight
•=
C U/Individuo!
F-G-LPP
I
Larval—•+*
2
3
4
5
Pupal
6
7
8
9
IO II
J
12
H* Imaginai—
LEVELS OF ECDYSONEDURINGMETAMORPHOSIS
CHART 1.—Levels of ecdysones
Bombyx mori.
during
metamorphosis
STAGES
of
found increased titer before pupation, with precipitous
fall and gradual rise thereafter (Chart 1). Samples from
full-grown larvae, silkworms in the prepupal stage, and
silkworms at 1, 2, 3-4, 5-6, and 6-7 days of age were used
in the tests and extracts bioassayed by means of the
Calliphora test. The prothoracic glands from which the
fractions we have isolated presumably originate undergo
dissolution soon after the adult stage is reached.
Apparently ecdysones are the substances responsible
(39) for eliciting puffs (89) on the chromosomes1 which
Becker (5, 6) discovered to appear in response to hormone
from the ring gland. Recently Kroeger2 has found that
several simple substances, including ZnCl2, chloroform,
butanol, and urethan, mimic action of ecdysones on
chromosomes. He has also proposed that successive
Na+:K+ ratios activate respective genetic loci and that
ecdysones act by increasing the concentration of K+ ions
in the nuclear "sap" whereas juvenile hormone is effective
by maintaining a high level of Na+ ions.
Juvenile hormone.—Wigglesworth established some years
ago (116) that the corpora aliata are the source of juvenile
hormone. This hormone is a mandatory component of
the mechanism to induce larval molting. It acts in op
position to ecdysone in the direction of retaining less
mature characteristics of the organism. Although this
is probably not a simple inhibitory effect (118), its exact
mechanism remains speculative (56). Kobayashi and
Burdette (74) found that heterologous transplantation
of corpora aliata between lepidoptera diapausing in egg
and pupal stages resulted in different effectiveness in a
foreign environment. Also similar transplantations (60,
75) suggest that the corpora aliata may act in pupal and
in imaginai differentiation under certain circumstances to
retard differentiation3.
Large quantities of the hormone (49, 54) are present in
the abdomen of adult male Hyalophora (Platysamia)
1W. J. Burdette and R. Anderson, Sequence of Puffing in
Salivary Glands Following Administration
of Ecdysones, un
published.
! H. Kroeger, personal communication.
»
M. Kobayashi and W. J. Burdette, Histophysiologic Studies
on the Corpora Aliata in Dauer Pupae of the Silkworm, Bombyx
mori, to be published.
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BuRDETTE—Invertebrate Hormones and Differentiation
523
cecropia (122-124). Also, Gilbert and Schneiderman
transplanted or a combination of the two used. Ligation
(51, 52, 105) and Williams and co-workers (124) isolated has also been used to impede the distribution of hormone.
material from a number of diverse biological sources To recapitulate events in metamorphosis (Chart 2), the
including bacteria and vertebrate adrenal cortex and brain becomes electrically active as a result of some ex
ternal stimulus, such as a change in temperature; neuro
thymus that gave a positive bioassay for juvenile hor
mone activity. Subsequently, Schmialek (104), noting secretion occurs; and the prothoracic glands then become
that similar active material could be isolated from the active to secrete the ecdysones. This is reminiscent of the
feces of Tenebrio, was successful in extracting farnesol analogous tropic action of the hypophysis and the glands
and its oxidation product, farnesal, from this source. responsive to it in vertebrates (102, 103). The balance
Wigglesworth confirmed that these were active on bio- between the amount of ecdysones and juvenile hormones
assay. However, the activity of farnesol is not great (possibly in the form of congener|s] of farnesol from cor
enough for one to assume that this is identical with the pora aliata) acts on sensitive tissue in such a way as to
natural hormone. Farnesyl phosphate is also not very regulate metamorphosis from one larval instar to the next.
active, but the methyl ether of farnesol and N, N-diethylContinued action of ecdysones brings about differentiation
farnesyl amine are very active (65). Schneiderman et al. from the last larval instar onward. Hormone from the
also find a number of compounds including phytol, corpora aliata may also play a role in imaginai differentia
linaloöl,solanesol, and nerolidol to have activity similar tion.3 In addition to the hormonal mechanics discussed
to juvenile hormone on bioassay. The intermediate
previously, the subesophageal ganglion of the silk moth
position of farnesol in the metabolism of cholesterol is of produces a hormone that regulates the egg diapause, and
interest in view of the report of Kobayashi that cholesterol females given injections of extracts are induced to lay
is the product of neurosecretion of the brain and is winter eggs (59).4
The significance of positive bioassay for brain hormone
probably a precursor of the ecdysones.
In the adult, the corpora aliata secrete a substance
and juvenile hormone in the case of a large number of
necessary for the formation of the yolk (92, 110, 116). steroids, alcohols, and other substances known in verte
Also, Schneiderman has suggested that secretion from brates (13) is difficult to assess. Whether the bioassay is
corpora aliata may stimulate the prothoracic gland since simply not very specific or there is some similarity in
Krishnakumaran and he found that farnesol can activate
structure and/or action between them and natural hor
the prothoracic glands of A. polyphemus.
mones is open to question. We have assayed a number of
Bioassay.—Several bioassays have been useful in the water-soluble steroids by the Calliphora test and so far
course of investigations on hormonal control of meta
have obtained negative or equivocal results. The chemi
morphosis. Pupae with brains ablated (Dauer-pupae)
cal nature of those hormones related to metamorphosis
have been used to test the effect of hormone extracted
that have been studied most thoroughly are cholesterol
from the brain (50). The cuticle of various species of (79), a precursor of cholesterol (104), or a possible deriva
moths (50, 55, 106) as well as Tenebrio have been used in tive (66). Sterols are essential components of the diet of
the bioassay of juvenile hormone; and Calliphora, the insects, since biosynthesis otherwise proceeds no farther
blow fly, has been used as a means for detecting ecdysone. than squalene. Although cholesterol may be an active
The Tenebrio test (65) is carried out by injecting 0.5 component of the brain and may act by stimulating secre
pi. of the substance to be assayed into the abdominal wall tion of the prothoracic gland in addition to providing the
of pupae of meal beetles when they are 24-48 hours old. molecule from which ecdysones are elaborated; although
Activity for juvenile hormone is present if a portion of the ecdysone with formula similar to that published by Karlcuticle has the pale color characteristic of the pupal stage son may be secreted by the prothoracic glands; and al
instead of the dark brown appearance characteristic of though a substance structurally similar to farnesol may
the adult when the beetles emerge in 8-10 days.
be produced by the corpora aliata, in every case there is
The bioassay for ecdysone is carried out by ligating reasonable likelihood that other components of the secre
larvae of this carnivorous fly posterior to the ring gland. tory product are active as hormones. In addition, the
If this has been done at the appropriate stage, the anterior specter that the biological action described for each hor
portion pupates, and the posterior portion remains in the mone is rather nonspecific has been raised by the extensive
distribution of material having brain- and juvenile-hor
larval stage. The latter is then injected with the extract
to be tested. A positive test consists of pupation within mone activity in nature and the report that rather simple
24-48 hours. A weighted average is used, and 50 per compounds have an action on chromosomes similar to
cent pupation of 20 posterior segments or more consti
that of ecdysones. Nevertheless, the chemical story
tutes a positive test. The test is essentially a refinement emerging is a rather coherent one, and for the first time
by Karlson of that developed by Becker and Plagge (4), an experimental approach for probing differentiation in
based in turn on earlier work by Fraenkel.
chemical terms is becoming available.
Metamorphosis.—The holometabolous insects have
TUMORS IN DROSOPHILA
larval instars followed by transition from larva to pupa
and then emergence from pupa to adult. The adult stage
It is of interest to determine how the regulatory systems
is entered from the last larval instar in the hemimetabolous
The
group. Information about the control (14, 15) of these described affect atypical growth in invertebrates.
types of differentiation has come principally from experi
4 Other neurohormones extracted by Cameron (33), Carlisle
ments in which various glands have either been ablated or (34), and Gersch et al. (48) affect muscular contraction.
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BRAIN
-CH2
-C = CH-CH2
-CH2
-C--CH-CH2
OH
NEOTENIN(S)
LARVAL
INSTARS
IMAGO
METAMORPHOSIS
CHART2.—Diagram of the interaction of hormones from brain, corpora aliata, and prothoracic
glands during metamorphosis.
524
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BuRDETTE—Invertebrate Hormones and Differentiation
tumors that appear in Drosophila (19) usually consist of
aggregations of cells that do not divide or divide very
seldom. They appear early in larval life and undergo
dissolution later. Pigmentation of the inert residuum
remains throughout the remainder of the life cycle and
provides a convenient marker of those animals that have
had tumors in the larval stage. As a rule, the process
does not interfere with the viability of the individuals
affected. The phenomenon is inherited, and more than 50
strains have been described. The genes responsible for
susceptibility are present on from one to four chromosomes
and consist of main genes, suppressors, and enhancers in
various combinations. For some reason, more are located
on the second chromosome, but few are allelomorphic.
The mutants that have been examined show no evidence
of position effect and are point mutations, most of which
have occurred spontaneously.
The effect of the hormonal system regulating meta
morphosis on these tumors has been tried in two ways:
first, by ligating larvae posterior to brain and ring gland
(17); and, second, by introducing a gene on the second
chromosome (18) that arrests development, resulting in
giant larvae due to a congenital defect of the ring gland
which contains cells analogous to those both in the corpora
cardiaca and prothoracic glands of lepidoptera and other
invertebrates.
When hormone from the ring gland is
excluded by either means, the number of individuals with
tumors is greater than in those with normal glands, in
dicating that there is a release of hormonal inhibition.
This has been confirmed by Oster (89).
525
TABLE 1
EFFECT OF FARNESOL ON SARCOMA180
REGRESSIONSNone509107679Partial2144337Complete30135016PERCENTA
Or
DEATHS
DOSE
PERINJECTIONControl.05.10.10.10.20.20NO.
(ml.)
IN
WITHIN
48007111313NO.
JECTIONS353131No.
REGRESSION9103350573072
A-strain mice were given inoculations subcutaneoualy
at 60
days of age of a small (1 mm.) fragment of Sarcoma 180. When
the tumor was established and growing, farnesol was injected
subcutaneously
in the opposite flank in the respective schedule
of dosages given in the table. Observations were made daily
and weight of the animals and measurements
of the tumors
recorded at least twice each week thereafter.
to cholesterol (79). Various dosages of ecdysone were
injected intraperitoneally into mice bearing transplanted
tumors; and, although there was suggestive regression of
21 per cent of the tumors, this was not proportional to the
dosage (21).
Considerable toxicity was attendant on the injection of
farnesol. As high as 72 per cent of the tumors regressed
either completely or partially when up to 0.2 ml. was
injected (21)5 (Table 1). Divided doses did not seem to
reduce the toxicity in all cases, and there was some loss
in the cancerocidal effect by giving the material in this
manner.
Since juvenile hormone tends to perpetuate larval
characteristics and possibly inhibit differentiation, it was
thought that it might be teratogenic in mammals. So
far results of investigations on this point are negative.6
Farnesol injected into pregnant mice failed to induce more
lethals in utero than were found in mice without treatment,
and no malformations were found in the offspring of those
females allowed to reach parturition.
Experiments in vitro.—From these experiments it
seemed likely that pursuit of the problem with biological
systems in vitro would be more rewarding and that the
effect suggested by ecdysone would be most likely to show
specific effects on mammalian tissue. Therefore, both
embryonic fibroblasts and Sarcoma 180 were cultured in
hanging-drop cultures, and the effect of ecdysones was
tested. The degree of growth was scored uniformly,
and it was found that the growth of embryonic fibroblasts
and neoplastic tissue was inhibited (25). The sequence of
inhibition was somewhat different, however, when the
growth of the two tissues was compared. In the case of
embryonic fibroblasts, increasing concentrations of ecdy
sone progressively delayed the onset of growth. On the
other hand, Sarcoma 180 began to grow and persisted for
a time inversely related to the strength of the dose.
HORMONAL HETEROPHYLY
Experiments in vivo.—Since the processes that have
been described in insects constitute a powerful regulatory
system for differentiation and the control of growth which
is distributed widely throughout the animal kingdom
including not only diptera and lepidoptera but also crus
taceans and other invertebrates, the possibility that these
materials may have an effect on mammalian tissue seemed
worthy of scrutiny. The place of at least one of the active
components produced by the brain (cholesterol) and of the
corpora aliata (farnesol) in mammalian metabolism is
already well known. On the other hand, we extracted a
number of human tissues and failed to obtain a positive
bioassay of the product for ecdysone (22). The portion
of the formula reported by Karlson also fails to suggest that
it occurs naturally in mammals, although structurally it is
related to bile acids and some of the other steroids known
to be important in mammalian metabolism. It should
be noted, however, that other components secreted by
both brain and corpora aliata may be important in the
process of differentiation and yet remain undiscovered.
The additional ecdysones we have recently described are
examples.
The first experiment was performed by injecting respec
tive active extracts of brain hormone, juvenile hormone,
and ecdysones into animals bearing transplanted tumors.
6 W. J. Burdette, Effect of Farnesol on the Growth of Sarcoma
Sarcoma 180 was transplanted into C3H mice, and be
180, unpublished.
cause of the small amount of brain hormone available
•
W. J. Burdette, J. Simmons, and R. Anderson, The Lethal
only a few mice were given injections. The negative Mutation Rate in Mice Following the Administration of Farnesol,
results are understandable if the principal activity is due unpublished.
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Vol. 24, May 1964
Cancer Research
526
TABLE 2
EFFECT OF ECDYSONES*ON PROLIFERATION OF HELA CELLS!
IN VITRO
TABLE 3
EFFECT OF ECDYSONESON HELA CELLS
DOSEcu./ml.t100500100015002000GROUPControlTestControlTestControlTestControlTestControlTestINOCULUMJ3.13.10.40.41.01.00.70.70.90.9DAY§410.910.51.41.53.93.41.00.91.51.4s
ControlEcdysones*DXAtWg/cell1629pg/mg20.087.8RNAtWig/cell6582Mg/mg70.1
* 600 Calliphora
units/ml,
t Diphenylamine method.
ÃŽ
Orcinol method.
•
Hormone added on day 3.
t Cells grown in Eagle's minimal essential
medium with calf
serum.
ÕCalliphora
units/ml.
§Number of cells X IO6 (counted with Coulter counter after
trypsinization).
In order to obtain less subjective evaluation of changes
in rates of proliferation, a Coulter counter was used to
determine the number of HeLa cells in culture, and the
experiment was repeated.7 Cultures with a known num
ber of cells in suspension were treated with ecdysone on
the 3d day of growth. Aliquots of the cells in suspension
were then counted, and the mean size of the cells was
determined on successive days after the cells had been
treated with trypsin. It was found (Table 2) that in
creasing dosage progressively shortened the time of pro
liferation and the number of cells in the culture, and a
threshold of dosage was found below which the hormone
was not effective in inhibiting growth. The same type of
experiment was performed utilizing E. coli and Staphylococcus aureus.* In the concentrations of hormone used,
there was no significant alteration in the growth of the
cultures when hormone was added during the log phase of
growth. The amount of DNA and RNA in HeLa cells
cultured with and without ecdysones was determined, and
an increase was found in both when means were compared9
(Table 3). These and the experiments described sub
sequently were all carried out with ecdysone extracts
carefully bioassayed.
Metabolic studies.—Since the addition of ecdysone to
Rhodnius results in the proliferation of mitochondria and
a change in their gross appearance under the light microscrope according to Wiggelsworth (119), tissue was selected
that is adapted to aerobic metabolism and known to have
7 W. J. Burdette, Effect of Ecdysones on the Growth of HeLa
Cells in Tissue Culture, unpublished.
•
W. J. Burdette, The Growth of Bacteria in Cultures Con
taining Ecdysones, unpublished.
•
W. J. Burdette, DNA and RNA of HeLa Cells Cultured in
Medium Containing Ecdvsones. unpublished.
a huge number of active mitochondria in order to test the
effect of ecdysone on aerobic respiration. Slices of
mammalian cardiac muscle were placed in Warburg vessels,
and Qo2 was determined under various conditions with
and without the addition of ecdysones (21). There was
a small but suggestive increase in Qo2 when the exogenous
substrate used was lactic acid. Otherwise the ecdysones
have not given any changes of note so far, and mitochon
dria of mammalian liver are unchanged in appearance
under the electron microscope during hepatic perfusion
with ecdysones.8
Protein synthesis (118, 126).—It also seemed desirable
to determine the effect of ecdysones on protein synthesis
in the cytoplasm. A fraction of mammalian liver ob
tained by centrifugation of homogenates at 14,000 X
g was incubated with hormone, and leucine labeled in
position one with carbon 14 was added. The fine structure
of this fraction,9 sedimented at 20,000 X g and recogniz
able with the electron microscope, consisted of smooth
and rough endoplasmic reticulum, glycogen, and a few
intact mitochondria (Fig. 1). The incorporation into
protein from zero time was determined by plating the
material precipitated by trichloroacetic acid and counting
in the proportional range. These results were compared
with those in preparations without hormone. There was
a significant increase in the incorporation of leucine into
protein when 250 Calliphora units of ecdysone per ml. or
more were used (24). Although there was a disturbing
variation in the uptake from one sample to the next,
incorporation increased in every instance in those dosages
when sufficient hormone was present in the preparation.
However, it is noted that there is apparently a maximum
mean beyond which the uptake did not progress and that
the control varied more than the experimental group. The
significance of this is not known. The limitations of such
a system are well known, and the fact that messenger
RNA is unstable (113) in such a situation has been docu
mented hi studies on microorganisms. However, the
fact that there was an increase is suggestive.
Ultrastructure of mammalian liver perfused with
ecdysone.™—Since
results in vitro with the ribosomal frac
tion of mammalian liver suggest enhancement of the rate
of protein synthesis when ecdysones are added and the
morphology and rate of growth of mammalian cells are
altered by extracts containing the hormone, the ultrastructure of mammalian tissue perfused with ecdysone
10W. J. Burdette and T. P. Ashford, Fine Structure of Mam
malian Liver Following Perfusion with Ecdysones, unpublished.
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BuRDETTE—Invertebrate Hormones and Differentiation
527
has also been studied. Livers from fasted rats were intraccllular permeabilities in insects so as to allow access
perfused with blood containing ecdysone, and samples of enzyme to substrate for the assimilation of amino acids.
were taken at intervals of 10 minutes. The fine structure
He does not regard the action of neotenin as inhibitory
but as a substance reacting with gene-controlled enzymatic
was then compared with that in sections from liver per
fused without hormone. Preliminary observations reveal systems to provide the realization of a choice of alternative
the following picture after perfusion (Fig. 2): coarse morphologic forms.
granules are prominent in nucleoli; large cytoplasmic
vacuoles are present; the rough endoplasmic reticulum is
CHROMOSOMAL RESPONSE TO ECDYSONES
prominent and is usually arranged centrally in the cyto
Very little direct information is available on the state
plasm; the mitochondria are aligned with the rough
of the mammalian chromosome during interphase or early
endoplasmic reticulum in the usual fashion; the smooth
prophase. Three special cases have attracted attention as
endoplasmic reticulum is distributed diffusely throughout
the cytoplasm; and pinocytosis at the cellular surface is visible structures from which inferences applicable to less
favorable material may be drawn concerning chromosomal
easily seen.
Organization of the rough endoplasmic reticulum (Fig. structure and function. They are the spiral structures
3) in chemical terms may take the form illustrated in the observed in amoebae (94), the lampbrush chromosomes
in oöcytesof certain amphibia, and the dipteran salivary
diagram (Chart 3), since polypeptide synthesis from acti
(1, 65, 93, 107). The significance of the
vated amino acids takes place through the interaction of chromosomes
first of these remains controversial. In the case of lampmessenger RNA, aminoacyl sRNA, and heavy ribosomes
(109, 113). That ecdysones may have a direct effect on brush chromosomes, peculiar loops extend outward from
this organization rather than a secondary effect as a result the chromosomes of these leptotene structures. Signifi
of its activity on the chromosome is suggested by the cant amounts of RNA are present in the loops, and one
side is thicker than the other. A prodigious amount of
evidence that ribosomal fractions incubated in vitro with
out intact chromosomes incorporate leucine into protein study has been devoted to the prophase chromosomes of
the salivary glands of insects, since the significance of
at an increased rate as well as the morphologic appearance
mentioned. In some of the preparations from fasted these structures was first recognized by Painter (90).
animals perfused with ecdysones the rough endoplasmic Similar structures are seen in the midgut, rectum, testes,
reticulum was organized and prominent, reminiscent of and malpighian tubules but have not been examined as
the picture in the liver of animals that were fed.
The pinocytosis observed at the cellular surface suggests
ample transport at the periphery of the cell. The idea p^ RNAC=0(de)'-^~
p^
'
that this is facilitated in primary or secondaiy fashion by
C=CKde)s/^\
action of ecdysones is attractive but has not been proved.
"^0SRNA~
,
'
"NRNA-A
i
^V
NAA'-'SRNA20^ ^Y*^0SRNA•~AAA.
Whether the large vacuoles observed represent a toxic
effect such as that observed with hypoxia or whether this
A,
AAv
represents transport of product from the cell or ingress
and accumulation of substrate from without awaits
J_^H1-C=00e)RNA,
J^^^—
¿<.AA~SRNAyC=0(de)RNARNA00<de)^
further study. These vacuoles are the most consistent
N-AA,„H^-PxH—
difference between liver from fasted rats perfused with
PRNA<
ecdysones and those without addition of extract to perfusate.
These observations of mammalian hepatic fine structure
AA,-,
NRNAC=0(de)^
after perfusion with ecdysones provides an interesting
comparison to the effect of the same substances on the
architecture of the cell in insects. Wigglesworth (118)
Pf
P^
•
noted enlarged nuclei and nucleoli, increased RNA be
C=0(de)0SRNA~
tween the nuclei, increased size and number of mito
^*AA~S.RNA0^
chondria, large amount of protein, and "highly developed
i
N0SRNA—
i
^AA-SRNAQV S0SRNA—AAV
and often lamellar ergastoplasm" in the cells of Rhodnius
following the administration of ecdysone. Cells of the
AAX
AA/.
fat body appeared to have a lower threshold for the action
^C=0(de)RNARNAC=0(de)f~
'
iC=OWe)RNARNA/<
^A^
^<^~\r^
i/of ecdysone than did other tissues studied in decapitation
experiments. The similarity between the changes induced
— p/-—
p\f~~~
P
by ecdysone and those appearing as a result of injury to
insects continues to be intriguing. (We11 have found
that the tensile strength of mammalian wounds is not
CHART3.—Theoretical chemical arrangement of polyribosomes
enhanced by extracts containing ecdysones, however.) during protein synthesis. (This represents a chemical scheme
Wigglesworth has suggested that ecdysone may influence for an area in the cell corresponding to that enclosed in the rec
" W. J. Burdette and R. Price, The Effect of Ecdysones
Healing of Mammalian Wounds, unpublished.
on
tangle in Figure 3.) The letters, de, are added to the carboxyl
group as a convention to indicate that messenger RNA (gray)
carries the coded message that is being replicated.
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1964 American Association for Cancer Research.
528
Cancer Research
extensively. The salivary chromosomes in Drosophila,
Chironomus, and Rhynchosciara have been favorite
objects for cytologie study for many years.
The salivary chromosomes consist of parallel duplica
tions (99) of the paired somatic chromosomes so that 64,
128, or more lie side by side with the matching chromomères giving the large, elongated structures a banded
appearance. These bands are constant in their inter
relationships and morphology and may be used to identify
specific regions. Chromosomal aberrations have been
useful in identifying the location of mutants, most of which
cause no visible structural alteration. The bands are
Feulgen-positive and contain DNA.
Painter was the first to note knoblike enlargements or
puffs on the salivary chromosomes. These puffs appear
quickly in the salivary chromosomes and regress rapidly
after a characteristic period of persistence. Changes in
the pattern of puffing are correlated with the progression
of events during the life cycle and presumably are some
how a reflection of genie action responsible for the changes
known as metamorphosis. Becker (5) has carefully
recorded the temporal-morphologic relationships of puffing
in Drosophila melanogaster and is responsible for establish
ing that the hormone of the cells in the ring gland corres
ponding to prothoracic gland is responsible for eliciting
the puffs. He was able to identify approximately 70
regions showing distinct puffing on the X, second, and
third chromosomes. Glands transferred to Ringer solu
tion for a short period of time regressed to an earlier pat
tern of puffing when explantation was carried out before
a certain critical period in the life cycle. Later, develop
ment toward a more advanced pattern of puffing occurred
in vitro. Also puffing was observed in vitro that did not
occur at that site in the intact animal. When the l(2)gl
gene is homozygous, the normal puffing pattern is deranged
so that no puffs characteristic of the corresponding phase
of larval development are found. Different types of
cells in the same or different organs may have a different
pattern of chromosomal puffing. Even though the pattern
may be similar in the same tissue, we have often observed
salivary cells side by side presenting different stages of the
same pattern so that the synchrony is obviously imperfect.
Also Becker (5) noted a puff in the anterior portion of the
salivary glands of Drosophila not appearing farther pos
teriorly. Kroeger (86) reported the induction of a differ
ent pattern of puffing in the chromosomes of Drosophila
when the salivary-gland nucleus was transplanted into
the cytoplasm of the egg.
Many investigators have been interested in the chemical
components and their concentration in the puffs (95,
97, 105). Recently Edstrom and Beermann (11, 42)
concluded from studies on the RNA of single giant puffs
that this RNA is different from that in the nucleolus and
from most of that in the cytoplasm. During the first
half hour after injection of tritiated uridine and other
precursors of RNA, the puffs are labeled more heavily
than any other part of the cell according to Felling (91).
Rudkin (95, 98, 99) found a consistent but not sig
nificant increase of RNA formation concomitant with
formation of a specific puff in Drosophila melanogaster, and
Rudkin and Corlette (98) found larger amounts of DNA
Vol. 24, May 1964
(108) in puffs than elsewhere during the prepupal stage.
Earlier, Breuer and Pavan (16) reported an increase in
Feulgen staining in puffs of Rhynchosciara. Labeled
cytidine but not thymidine was incorporated to a greater
extent into puff 60B of Drosophila than elsewhere, ac
cording to Rudkin and Woods (100). Breuer and Pavan
(16) found indications that disproportionate DNA syn
thesis occurred in puffs in Rhynchosciara, and the capac
ity to synthesize RNA and the amount of DNA contained
was found by Stich and Naylor (110) to be variable in
puffed regions of the salivary chromosomes of one of the
Chironomids. Therefore the chemical composition of
puffs varies from one to another, may not be the same
when identical regions are compared in different tissues,
and may vary from one stage to another in the life cycle.
The puffs showed no consistent response to treatment
with an analog, amethopterin, expected to alter metabo
lism of nucleic acid in experiments by Corlette (40).
There was also no influence of colchicine on these struc
tures. These and other observations have tended to
focus attention on the RNA content of the puffs, al
though their exact chemical composition is difficult to
determine because it is not constant and because of defi
ciencies in methods of analysis.
A type of secretion granule unique to a small region of
the salivary gland in Chironomus was found by Beermann
(9) to be due to a genetic factor located at the locus of a
puff on the fourth chromosome, appearing only when the
secretion granules appeared. He looks upon this as an
example of the cellular specificity of the puff and the
function (7, 8) it controls. It is not clear why puffs and
granules do not occur in the remainder of the salivary
gland bearing the gene in question.
As an extension of the observation that the defective
ring gland associated with the l(2)gl gene results in
chromosomes without puffs, the results of injections with
ecdysones are of interest. Clever and Karlson (36, 37,
39) observed that puffing in the salivary chromosomes of
Chironomus is enhanced in certain regions, and the in
tensity of the effect depends on the dosage of hormone
(38). This effect appears within 30-60 minutes. A
secondary sequence of puffing then appears 6-48 hours
later, also related to dosage. Therefore, as one would
expect from projecting the results of Becker, ecdysones
are the substances responsible for the effect of secretion of
prothoracic glands on the puffing pattern of the salivary
chromosomes.
In our laboratory the appearance of salivary chromo
somes in Drosophila (Fig. 4) after injection of ecdysones
or culturing the glands in saline containing ecdysones has
been changed.1 The size of the puffs has been increased,
sometimes to gigantic size with margins fading into the
cytoplasm, suggesting a release of material from the region
of the puff. So far only regions forming these enlarge
ments in the ordinary course of development have been
observed to do so following treatment.
Larger doses of
ecdysones are probably required to produce the effect in
Drosophila than in Chironomus, and the effect of hormone
appears suddenly in approximately 20 minutes. When a
puff has a different appearance in pupal and larval stages,
ecdysones injected during larval instars may produce a
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BuRDETTE—Invertebrate Hormones and Differentiation
puff with pupal characteristics.
Large doses of hormone
cause the chromosomes to swell and apparently to assume
more adhesive qualities. The clear areas between bands
become somewhat distended, but the width of the chromo
somes is not changed appreciably.
An examination of mutants in or about the region of the
puffs in Drosophila melanogasler fails to give any uniform
picture of a type of genie action or adaptation to the
specific function of the tissue in which the puffs appear.
This is not particularly surprising, since genes situated in
close proximity may have very different action, and the
genie action signified by appearance of a puff may occur
at a time remote from the action responsible for the mu
tants listed and detected in the adult. In Drosophila a
threshold of dosage exists, but otherwise the appearance
of the chromosomes is not changed by alterations in dosage
of hormone unless exceedingly large amounts are used.
Theoretically the locus where the puffs appear represents
the site of increased genie activity, and the genes con
cerned in events taking place at the time during the life
cycle when they appear are located in the same regions
of the chromosomes.
INVERTEBRATE
HORMONES AND
DIFFERENTIATION
The current status of knowledge concerning events in
metamorphosis of insects (56, 63, 64, 88) may be com
bined with evidence from mammalian and microbial
systems (2) to construct a hypothesis applying to the
general process of differentiation, if license is permitted
to overlook certain deficiencies in current information.
A balance between the effect of neotenin and the ecdysones
is apparently responsible for a number of the events in
metamorphosis of insects. The concentration of each
hormone in relation to the other affects larval and adult
cells in such a fashion that an orderly process of growth,
replacement, and differentiation ensues. How this is
accomplished at the cellular and molecular level is more
apparent for the ecdysones. Also this group of hormones
probably has effects that are more than nonspecific on
mammalian cells, so that the influence of invertebrate
hormones on molecular events may have some general
significance.
The ecdysones activate sensitive loci on the chromo
somes and possibly increase the rate of protein synthesis
in the cytoplasm. The question whether the material
selectively produced as a result of the action of ecdysones
is exclusively messenger RNA (10) or other substances as
well requires much additional work. Also whether they
increase transport at the surface of the cell is the subject
of current investigation. These mechanisms obviously
enhance synthesis of proteins of specific type, some of
which in turn may be regulatory. The effect on the
chromosome as well as elsewhere may constitute a mecha
nism of release such as increased permeability or removal
of inhibition. An inductive activation of enzymes (62)
could also be involved.
These observations yield one example of some of the
steps in differentiation in molecular and morphologic
terms and suggest how the cellular product, a hormone
in this case, may constitute a feedback mechanism selec
529
tive for individual opéronson the chromosome. It
emphasizes the importance of the sequence of events, but
it does not divulge general secrets of irreversibility and
fails to give the ultimate answer about why the template
is activated or sensitive at one chromosomal locus and not
at others. Evidently the explanation for different steps
in differentiation must be solved in terms of individual
molecules, chemical reactions, genes, cistrons, opérons,
etc. Invertebrate material offers advantages in bridging
the méthodologiegap between microbiologie and mam
malian systems. Retrogressions so obvious in malignant
cells should be understood the better as a result of such
an approach.
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FIG. 1.—Ultrastructure of homogenate of liver from the rat
used to test effect of ecdysones on the rate of incorporation of
leucine into protein.
Smooth endoplasmic reticulum and glycogen are easily seen. The latter obscures the rough endoplasmic
reticulum.
Occasional mitochondria are also present.
FIG. 2.—Hepatic cell of the rat following perfusion with ecdy
sones. (N: nucleus; n: nucleolus; V: vacuole; RE R: rough
endoplasmic reticulum; M : mitochondrium)
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©
v
"
- PER
531
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Fio. 3.—Rough endoplasrnic reticuluni (RER) of liver (rat)
after perfusion with ecdysones.
Fio. 4.—Puff (65) on chromosome III L of Drosophila melanogaster. (Puff is in center of plate.)
532
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R ER
533
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BuRDETTE—Invertebrate Hormones and Differentiation
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M. A Nomenclature Proposal
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The Significance of Invertebrate Hormones in Relation to
Differentiation
Walter J. Burdette
Cancer Res 1964;24:521-536.
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