CANCER RESEARCH
VOLUME22
Experimental
AUGUST
and Therapeutic
1962
Modification
NUMBER7
of Mitosis'
JOHNJ. BIESELE
(Dejmrtment of Zoology, The Unieernly
INTRODUCTION
This review is concerned with two questions:
(a) how can mitosis be modified experimentally
and (6) of what value in cancer therapy can such
modification be? It pretends to be neither exhaus
tive nor very systematic.
From one point of view, a great deal of experi
mental and practical cancer therapy can be said
to be antimitotic in effect if not primarily in in
tent. Any measure that halts growth of cancer cells
would necessarily impede their division. Although
one might be led to restrict the term "mitotic
poisons" to include only those chemical agents
that affect cells during the visible prophase-metaphase-anaphase-telophase portion of the mitotic
cycle, the newer knowledge that important events
of the total mitotic or cell cycle occur during
interphase would lead us to do otherwise. Chèvremont (16) has pointed out that a toxic substance
that suppresses growth or inhibits metabolism is
not to be considered an antimitotic agent, proper
ly speaking. I find this distinction sometimes
difficult to draw; but our interest in the cancer
field is to a considerable extent in end-results.
The literature of experimental cancer chemo
therapy lies open to the inquiring student of
mitosis. Mazia (48) has written that review of the
vast literature on mitotic stimulation and inhibi
tion by such agents as carcinogens, antimitotic
drugs, hormones, and radiation might provide im
portant clues to the biochemistry of cell division.
This literature is vast, indeed, as anyone who
* Presented in the symposium on "Perspectives on Cell Di
vision in Neoplastic Growth" held during the annual meeting
of the American Association for Cancer Research
City on April 14, 1962.
Received for publication April 30, 196-2.
in Atlantic
of Texas, Aiuttin, Texas)
glances through the products of the abstracting
services can see.
One is sometimes tempted to think of cancer
cells, apart from any question of the normality or
abnormality of their other attributes, as growing
and dividing beyond any possible need of the body,
and of cancer as a disease of excessive mitosis. Re
cent work of Malmgren and Mills (45) tends to
support this view: a liver mitotic stimulant, rich
in nucleic acids, which they found in tumors, was
detectable in normal tissues only after treatment
that might have destroyed an inhibitor or released
the stimulant from a suppressing complex. The
concept of cancer cells expressed in Swann's re
view (71) was also of this nature: Swann looked
on cancer cells as becoming "streamlined" for
proliferation by natural selection in a varying
population.
If cancer is regarded as a disease of excessive
mitosis, then restraint of mitosis would be the di
rect therapeutic measure. This restraint of mitosis,
however, must be selective. Otherwise, for the
organism as a whole, the therapeutic gain from
inhibition of mitosis in cancer cells may be offset
by a loss from the restraint of cell division in those
tissues in which the continued production of new
cells is a necessity for bodily well being, such as
certain epithelia and the blood-forming regions.
It may be questioned whether absolute selectiv
ity against cancer cell mitosis has ever been
achieved. There are reports, of course, concerned
with individual tumors, such as that from France
(14), which reported selective inhibitory effects of
estradici and testosterone on HeLa cells in culture,
including reduction of mitotic index, with no re
straint on normal uterine epithelium in culture.
Welch (76) has pointed out in a recent review
779
This
One
FR8G-5BG-UYOF
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Cancer Research
that there is at least one chemotherapeutic agent
capable of selective action on certain neoplastia
cells and without harmful effect on proliferating
normal cells of man: namely, 6-azauridine. It must
be remarked, however, that pregnancy in mice can
be interrupted by this agent (63). Although Taylor
et cd. (72) found that 6-azauridine was without
effect on mitosis in bean seedling roots, perhaps
because the cotyledons had an adequate reserve of
pyrimidines, I would expect that, under other cir
cumstances, 6-azauridine might be found to in
hibit certain interphasic syntheses that are neces
sary preliminaries to mitosis. Its active derivative,
the 5'-phosphate, interferes in the de novo pathway
of nucleic acid pyrimidine biosynthesis (76). Its
free base, 6-azauracil, upsets proper chromosome
movement in the mitotic apparatus in onion root
cells (44).
For cancer therapy, however, it may be only
relative selectivity that is needed, and this may be
obtained to some degree by such means as localized
irradiation (to mention another sort of therapy)
or the infusion of tumorous regions of the body
with chemical agents.
What about the possibilities of experimental
modification of mitosis? Nature has already shown
us, with modifications that occur in the normal
course of events, that the possibilities are numer
ous. To cite an extreme example, one could men
tion meiosis in the formation of germ cells. The
reduction division of meiosis, with its prolonged
prophase, synapsis of homologous chromosomes,
and their separation to opposite poles, may be
regarded as a derivative or variant of mitosis; and
in oogénesisthe unequal cell division by which
small polar bodies are cut off from their much
larger sister oocyte merely emphasizes the differ
ence from the usual mitotic division in somatic
cells. Endomitosis, or other endoreduplicative
phenomena, by which regular polyploid series of
nuclei are built up in some organs, including
mammalian liver and the nurse cells of the
dipteran ovary (57), furnishes an example of the
normal suppression of the achromatic apparatus.
The building up of polytene chromosomes in some
insect tissues is a related phenomenon (38). What
could be more bizarre than the mitoses in which
certain chromosomes are eliminated from the
somatic cell lines of certain organisms (56)? The
answer is, perhaps only the spermatogenic divi
sions in the fungus fly, Sciara, in which the chro
mosomes of paternal origin appear to back away
from the single center of the division figure, only
to be discarded (49). Here we see that a chromo
some is not merely a chromosome—it is also its
recent history. Where it has been, and its en
Vol. 22, August 1962
vironment in a previous generation, may have a
determining influence on its behavior now. Some
what similarly, with respect to environment here
and now, one X-chromosome may undergo heteropyknosis to form the sex chromatin body, if the
resting nucleus contains another X-chromosome, as
Ohno and Hauschka (54) have determined.
This is not to say that the mitosis-wreckers in
laboratory smocks have as yet achieved such
startling results. It does tell us that much is pos
sible in the modification of mitosis and chromo
somal behavior, if we but knew how.
What sorts of mitotic defects do we usually
recognize in our treated cells, in our tissue cul
tures, in our sectioned tissues (9) ? There may be,
first of all, a change in frequency of mitotic figures.
There may be a wave-like increase of mitoses re
sulting from stimulation (or release from inhibi
tion), or there may be an only apparent increase
in mitotic rate resulting from the accumulation of
mitotic figures blocked in some recognizable stage.
The best-known block is the metaphase arrest.
There may be accumulations in other stages, as in
prophase, or of the pseudoprophases that pile up
from treatment with ribonuclease (17). There may
be a decreased frequency of mitotic figures be
cause of arrest at some point in interphase, per
haps from inhibition of some essential synthesis
by some preprophasic (i.e., interphasic) inhibitor.
Damage to chromosomes may be observed in
metaphase or anaphase; chromosome breaks,
chromatid breaks, various sorts of rearrangements,
fragmentations, chromosome bridges, and chro
mosomal "stickiness" may be evident. Chromo
somes may be lost, or fail to divide, or move
precociously to the poles, or fail to move to the
poles. Mitoses may be multipolar, and daughter
cells formed in irregular number may have irregu
lar numbers of chromosomes. Cells may become
pyknotic and die at various stages of the cell
cycle. In short, it appears that we can merely make
accidents happen, increasing in fact the frequency,
often not inconsiderable, with which these same
mitotic abnormalities spontaneously occur in the
cells of many cancers (10).
AGENTS THAT MODIFY MITOSIS
Most investigators, when confronted with the
welter of data on effects of agents on mitosis, find
themselves impelled to classify. First let us con
sider the agents themselves.
A primary step in classification would be to set
stimulators apart from inhibitors, disregarding the
pharmacological dictum about stimulation from
minute quantities of inhibitors. I have little to say
about agents that stimulate mitosis, because with
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BiESELE—Modification of Mitosis
reference to cancer therapy they have the opposite
of what appears to be the desired effect. This is the
case, unless one wishes to follow the novel pro
posal of Berglas (7) and induce cancer cells to
divide themselves to oblivion. That there may be
merit in this general idea is seen from the work of
Braun (15) : hypothetical epigenetic particles re
sponsible for the tumorous nature of crown-gall
cells did not divide as rapidly as the cells contain
ing them, and thus "cured" cells could be ob
tained. What of the possibility, moreover, that
cancer chromosomes have some, not high, degree
of multiple-strandedness, and that the chromo
somes might be induced to divide down through
this strandedness more rapidly than the strands
could be replicated? This possibility is suggested
by Lindner's work (42) with 5-fluorouracil treat
ment of Ehrlich ascites cells, which suffered a de
crease by one-half in DNA per cell with no drop in
number of chromosomes. I am reminded of my
own dissertation research (13), which suggested a
moderate polyteny in cancer chromosomes. The
5-fluorouracil is an inhibitor, of course, but rela
tive to it the normal set of circumstances in pro
liferating Ehrlich cells constituted stimulation;
shifting the base-line allows us to think in terms
of relative stimulation in this case.
Stimulators of mitosis have been considered by
Swann (71). He wrote of hormones, of special sub
stances for each cell type, and of the stimulus to
cell division as partaking of the nature of an in
ductive phenomenon, with the cell being steered
away from synthesis supporting its previous dif
ferentiated function and toward synthesis of the
materials needed for cell division, such as rep
licated chromosomes and a mitotic apparatus.
A few specific agents capable of stimulating
mitosis may be noted. Agmatine, or decarboxylated arginine, was shown by St. Amand, Ander
son, and Gaulden (69) to speed up mitotic activity
in grasshopper neuroblasts; it had been chosen for
test in the light of a theory of mitosis initiation
based on polycation-polyanion balance (3). Phytohemagglutinin, a mucoprotein from beans, in
duces mitotic activity in peripheral leukocytes in
vitro, or at least puts them into a state conducive
to mitotic activity (52), and the glucocorticoid,
prednisolone 21-phosphate, tends to prevent this
change (53). Administration of thymidine to adult
mice speeds up mitotic activity in their intestinal
crypts, where the methylation reaction for the
production of thymidine for DNA is thought to be
rate-limiting (32). For plant cells, there is the
well known influence of kinetin, or 6-furfurylaminopurine (50). Finally, this audience is hardly
the one to forget the mitotic stimulation that
781
occurs in mouse epidermis shortly after it is
painted with carcinogenic hydrocarbons (61).
A second step in classification would be to con
sider the nature of the agents, and initially whether
they are physical or chemical. Among the physical
agents are various forms of radiant energy, sonic
vibrations, and the like. The mechanisms of action
include gross denaturation, as by microbeams of
ultraviolet (25) ; scission of the DNA double helix
(46); thymine-dimerization in DNA, by ultra
violet (8, 75), which could involve cross-linking of
strands; hydration of the cytosine ring in DNA
under the influence of ultraviolet (77) ; formation
of free radicals (39, 59), of peroxyl radicals (18), of
peroxides (66, 79), and of derivatives of nitroge
nous base precursors of nucleic acids (70).
An analysis of the mechanisms of cell death
from radiations, and of the part played in this by
mitosis or attempts at mitosis, has been presented
by Howard (34). She reviewed the evidence that
the site most sensitive to radiation in the cell is the
nucleus. Mitotic delay is produced more effective
ly by ultraviolet irradiation of the nucleolus than
of other portions of the nucleus in the grasshopper
neuroblast, according to Gaulden and Perry (30) ;
when the microbeam was applied during the period
from late telophase to mid-prophasc, mitosis could
be permanently halted. Howard (34) suggested,
therefore, that nuclear RNA and possibly also
DNA might be involved in mitotic delay from
irradiation. Mitotic inhibition was not accounted
for by delay in synthesis of new DNA (and this is
also the conclusion of Alfert and Das [2, 19]).
When DNA synthesis was delayed by irradiation,
however, this might result from an untoward
effect on nuclear phosphorylation. Loss of viabil
ity of a cell line was probably produced by chro
mosomal structural changes. Errors in replication
ultimately caused by traversal of the template by
an ionizing particle could lead to chromosome
breakage. Studies of the loss of reproductive abil
ity with respect to ploidy suggest that it is the re
sult of upsets in genomic balance. Daughter cells
resulting from mitosis of irradiated cells would re
ceive effectively unbalanced genetic complements
and might shortly die out. Death might not liter
ally ensue, however, until several cell generations
have passed (24). Reversible mitotic delay in S3
HeLa cells given low doses of x-ray has been at
tributed to chromosomal damage expressed in
interference in the condensation of chromosomes
in the G2 period (80).
The state of nutrition can influence the re
sponse of cells to irradiation. Alexander and
Mikulski (1) found that, with lymphoma cells
growing in a completely adequate medium, all
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Vol. 22, August 1962
cells divided at least one time, after receiving
x-ray doses up to 500 r, before autolyzing; but with
lymphoma cells growing about half as rapidly in a
restricted medium, death usually occurred in the
absence of any mitosis or attempt at division.
They concluded, therefore, that for the latter cells
chromosomal injury was not involved in death to
an important extent. Howard (34) has also re
marked on the puzzling great radiation-sensitivity
of small lymphocytes, despite their mitotic in
activity.
It is intriguing that some of the effects of
physical agents are mediated by altered chemicals
from the cellular environment or from the cells'
patulin, a lactone, which was also a strong chromo
some poison.
The agents whose primary effect was on chro
mosomal integrity included triethylene melamine,
nitrogen mustard, and various ethyleneiminoquinones, which might also influence the achro
matic apparatus.
The substances of mixed and complex effects on
spindle, nucleus, and chromatin included phenol
and various of its derivatives, some khellin de
rivatives, para-aminosalicylates, phenylmercuric
borate, and caffeine.
It is noteworthy in this classification by Sentein
that nearly all agents had multiple effects, not
own stock of metabolic intermediates. In short, merely those in the third category. This fact
we are provided with a ready-made transition to should help to disabuse us of over-simplified no
a consideration of chemical agents that influence tions about the mitotic poisoning actions of
mitosis.
chemicals.
The chemical agents that affect mitosis include
It is not only with respect to site of action that
various alkylating agents; some quiñones;certain there is heterogeneity of effect; this is also true for
alkaloids; carbamates; respiratory poisons; certain stage of action. Sentein (65) objected to calling
antibiotics; a variety of analogs of amino acids, colchicine purely a metaphase poison, because his
vitamins, and precursors of nucleic acids; and material included arrested prophases, anaphases,
and telophases as well as C-metaphases. Lehinann
others (9).
(41) raised a similar objection to terming col
These heterogeneous chemicals can be further
classified from the physiological or mitotic- chicine purely a spindle poison, because in Tubifex
eggs it appeared to affect the centriole in stages
pharmacological point of view. Our current inter
pretations of the actions of mitotic inhibitors are prior to development of the spindle.
influenced by newer knowledge of the cell cycle—
The centriole, we must note, has been suggested
i.e., of the total mitotic cycle including the triply- by Mazia (47) as perhaps a most logical target of
partitioned interphase between telophase and the antimitotic action, as its duplication appears to
following prophase. Mitotic poisons can be classi
lead the way in mitosis. Rustad (62) has mar
fied by the stages they affect—by the stages in shaled evidence in support of the concept of
which their actions cause the mitosis to be delayed division delay resulting from an effect of x-rays or
or arrested. Mitotic poisons can also be roughly ultraviolet on replication or separation of the
classified by the site of their major effects in the centrioles. Because acridine orange produces a
cell, as on the chromosomes or on some part of the similar effect, it was suggested that the damage to
achromatic apparatus, for example.
the centriole might be mediated by interference in
Thus Sentein (65), using the segmenting egg of nucleic acid metabolism. This is an attractive
the urodele as his biological test system, has classi
hypothesis, but it should be recalled that Fogg and
fied antimitotic agents into three categories: Warren (26) in 1941 found an x-ray dose of 2,400 r
spindle poisons, chromosome poisons, and agents to Sarcoma 180 or Walker 256 tumors to increase
of mixed effect.
frequency of cells with more than two centrioles
Among the agents whose primary effect was considerably, as though centriolar duplication had
depolarization of the spindle, Sentein (65) in
not been inhibited by the treatment.
cluded (a) the slowly reversible agents, colchicine,
Other examples may be given of multiple stages
and sites of sensitivity to antimitotic agents. Actiits derivatives, podophyllin, and podophyllotoxin
(the latter two could also cause secondary break
dione appears to act at several stages in the
mitotic cycle (33), and its effects include a proage of chromosomes); (6) agents of moderate
reversibility, such as chloral hydrate, which pro
phase arrest. Maleuric acid was reported (55) to
duced monocentric mitoses and some chromo
inhibit not only DNA synthesis in Ehrlich ascites
some breakage; (c) rapidly reversible ethyl-, tumor cells, but also progression through the Go
phenyl-, and monomethylurethans,
which also period of interphase into prophase, as well as
damaged chromosomes; (d) feebly acting sodium passage through metaphase. Among the effects of
cacodylate, sarcomycin, aloin, and phenylaminoantifolic acids that have been described in various
propane sulfate—these affected cleavage; and (e) mitotic systems (9) are interphasic inhibition, ex-
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BiESELE—Modification of Mitosis
pressed as a general decrease of mitotic incidence;
a metaphasic arrest in certain cells (6, 36); de
layed, three-group metaphases in cultured H.Ep.
#2 cells (11), and chromosomal damage in certain
tissues (23, 73). A dissent should be noted: Gelfant
(31) has failed to confirm the effects of maleuric
acid and the metaphase arrest by antifolics in ex
cised mouse ear epidermis.
A comprehensive study by Deysson and Truhaut (20) of the mitotic effects on plant root tips
of a variety of agents used in experimental cancer
chemotherapy is noteworthy. The agents tested
included mustards, ethyleneimine compounds, urethans, certain antibiotics, and various purine and
pyrimidine bases. Compounds of these several
classes tended to have similar effects. At the
lowest active concentrations (about 10~5Mfor the
urethans and about 10~7to 10~9M for the others)
there was a depression of mitotic incidence. With
progression to higher concentrations, chromo
somal breakage and bridge formation set in. At
higher concentrations, all entry into mitosis was
prevented as the result of preprophasic inhibition.
Still higher concentrations or prolonged exposure
caused cell death. Near the lethal concentrations,
a few compounds among those tested produced
spindle inhibition or prevented cytokinesis. It was
of interest that the so-called "radiomimetic"
agents and the presumed antimetabolites had no
essential differences in effect. All of them were
preprophasic poisons, and most of them damaged
chromosomes. Very few of them damaged both
chromosomes and spindle, and in this respect the
findings of Deysson and Truhaut are not in strict
accord with those of Sentein (65). Poussel et al.
(58) have also found alkylating agents and antipurine agents to have much the same final effects.
This should not be taken to mean that nucleic
acid base analogs and alkylating agents necessari
ly have the same mechanisms of action. Perhaps it
means that the cell has limited morphological
avenues down which to move in response to dif
ferent agents. Nevertheless, it is well to bear in
mind the findings of Lorkiewicz and Szybalski (43)
that triethylene melamine reacts primarily with
phosphorylated pyrimidine precursors of DNA,
converting thymidylate into a potent mutagen,
rather than with DNA itself.
We are still much in the dark with respect to
the mechanisms of action of many antimitotic
chemical agents. For some that are known to be
mutagenic or to interfere in nucleic acid metabo
lism, we suspect that an attack on DNA or in some
cases on RNA is the basis for the antimitotic ef
fect. For others an interference in the synthesis of
special proteins is suspected. There may be inter
783
ference at a higher level, too, in putting together
the structural components of the chromosomes or
of the achromatic apparatus. Perhaps colchicine
and other C-mitotic agents operate at this level of
interference in intermolecular bonding (64), but
exactly how is not known.
Discussions of the mechanisms of action of
nitrogen mustard and other alkylating agents in
their inhibition of cell division have tended, de
spite the high reactivity of these agents with many
molecular constituents of cells, to center on their
reactions with nucleic acids. There are conflicting
claims, such as that DNA synthesis may be in
hibited or stimulated by alkylating agents. Gelfant
(31) has attempted to resolve these discordant re
ports by suggesting that nitrogen mustard, for ex
ample, inhibits cell division during the 62 period
of interphase and not in the preceding S period of
DNA replication; it is only because passage into
the first mitosis and subsequent cycles is prevented
that there is an illusion of the inhibition of DNA
synthesis. In some respects, then, there is a
parallel to the effects of x-rays. It is difficult to
anticipate, however, that DNA cross-linked by bifunctional alkylating agents (68) could be rep
licated to completion. In this connection we
should consider Auerbach's statement (5) that the
induction of mutation by alkylating agents need
not occur by cross-linking, because certain monofunctional agents are highly mutagenic.
Another interesting parallel between the effects
of x-rays and radiomimetic chemicals is seen with
respect to the state of nutrition of treated micro
organisms: after treatment, starved animals ex
hibit immediate delay of cell division, whereas
well nourished animals go through a division or
two before inhibition (60).
Another parallelism may exist in the repair of
DNA damaged by irradiation or by chemical
agents. Chèvremont (16) has reported an interest
ing phenomenon in certain cells that remain
mitotically active in cultures of chick fibroblasts
despite inhibitory treatments with the alkylating
agent Myleran. In the presence of Myleran, these
cells quadruple their DNA content instead of
merely doubling it in preparation for mitosis.
Chèvremont suggested that the Myleran damages
the old DNA, which the cell replaces, not by
replication with the existing material as a tem
plate, but by another process, in which the mito
chondria may participate. This hypothesis reminds
one of that advanced by Doudney (22), according
to which there are two mechanisms for synthesis
of DNA, a primitive "mutagenic" mechanism in
volving coding transfer of new DNA through an
intermediate RNA-protein structure, and the
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Cancer Research
"Kornberg mechanism," which can be blocked by
cross-linking of DXA strands by ultraviolet and
can then be photoreactivated with white light.
Taylor et al. (72) have an illuminating interpre
tation for this problem, based on their work with
5-fluoro-2'-deoxyuridine (FUDR) and x-radiation.
It is that DNA polymerase repairs and rejoins
DXA with broken chains. Single strands grow in
one direction, copying the antiparallel strand, to
repair the break, and the broken pieces are dis
carded, perhaps as single-stranded fragments. It
will be recalled that Anderson (3) proposed that
the discarding of defective DNA strands might
occur during chromosomal condensation; the
presence of these defective strands would explain
chromosomal "stickiness" after damage. It is in
the sense of a repair of broken and frayed strands,
which make more primer DNA available, that the
recent finding of Alfert and Das (2, 19) that xradiation accelerates DNA synthesis in the early
part of the S period can be interpreted.
An interesting aspect of Taylor's hypothesis
(72) is that chromosomal repair, although involving
DNA polymerization, need not be restricted to the
S period of DNA synthesis during the cell cycle.
Sheldon Wolff (78) has remarked on chromosome
repair as involving protein synthesis rather than
DNA synthesis, because it goes on at all stages of
the cell cycle and is prevented by inhibitors of
protein synthesis. It is sensitive to various meta
bolic inhibitors and is stimulated by exogenous
adenosine triphosphate. Perhaps some of the pro
tein involved is part of the machinery for repair,
however.
It is difficult to assess our current understanding
of the actions of certain groups of antimetabolites
with respect to cell division. Furine analogs, for
instance, may interfere in the biosynthesis and
utilization of normal purines and thus depress the
synthesis of nucleic acids; if they are incorpo
rated into nucleotides and polynucleotides, fraudu
lent coenzymes and nucleic acids may result (9,
74). It has been suggested (4) that 6-mercaptopurine riboside triphosphate, formed in vivo from
6-mercaptopurine, may inhibit the formation of
the nicotinamide nucleotide coenzymes I and II.
This possibility is not easily reconciled with the
theory of Morton (51) that mitosis is initiated
when diphosphopyridine nucleotide (DPN) con
centration falls to a critically low level, or with
the theory of Fujii (29) that nicotinamide inhibits
mitosis by inhibiting DPNase activity and thus
allowing the intracellular concentration of DPN
to increase.
Consideration of 2-aminopurine may put us on
Vol. 22, August 1962
more solid ground. This agent is very active in
mouse tissue cultures (12), producing chromo
somal bridges and breaks and many laggard
chromosomes that lie near the spindle poles in
stead of on the equatorial plate. This abnormality
is related to the "three-group metaphase," which
is often delayed in passage to anaphase (11).
It would be interesting to ascertain whether
the considerable activity displayed by 2-amino
purine in the production of mitotic abnormalities
is related to the observations by Freese (27, 28) on
mutagenesis by this agent. 2-Aminopurine, incor
porated in place of adenine in DNA, can hydro
gen-bond with and lead to the incorporation of
hydroxymethylcytosine in the new complemen
tary strand of DNA in bacteriophage T4. Thus, an
adenine-thymine base pair can be changed, over
several cycles of DNA replication, to a guaninehydroxymethylcytosine pair in this phage and
presumably to a guanine-cytosine pair in organ
isms with more conventional nucleic acid bases.
This change constitutes a genetic mutation.
The cytological effects of base pair-shifting
mutagens are under study by Hsu and Somers
(35). An outstanding effect of the thymidine
analog, 5-bromo-2'-deoxyuridine (BUDR), was
the stretching of kinetochore regions and sec
ondary constrictions. Chromosome breakage and
translocation occurred at these sites, as well as at
telemeres and a few other regions, which Hsu and
Somers suggested might contain high concen
trations of adenine-thymine pairs, with much of
the thymine replaced by 5-bromouracil by this
treatment. The instability basic to the grossly
visible chromosome damage appeared to result im
mediately from the incorporation of BUDIl into
DNA, because chromosome breakage was manifest
in cells of a hamster cell line in the first metaphase
succeeding exposure to BUDR during the S
period 6 or 8 hours before (67).
Fluorodeoxyuridine, whose mechanism of action
involves production of an endogenous thymidylate
deficiency, has been applied to Vida faba seedling
roots by Taylor et al. (72). Chromosome breaks
and gaps (interpreted as relatively uncoiled single
strands of DNA) were noted in the mitotic figures
after 3 or 4 hours. The extreme effect of shattered
chromosomes in anaphase had much the appear
ance of the superfragmented mitoses Koller (40)
noted in Walker carcinoma 256 cells after treat
ment with certain alkylating agents, and which
he attributed to metabolic disturbances beyond
the truly radiomimetic effect. Chromosomal frag
mentation and anaphasic bridging caused by
treatment with FUDR and its 5'-monophosphate
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BiESELE—Modification, of Mitosis
were noted by Biesele (11) in H.Ep. #2 cultures, as
well as some three-group metaphases and delayed
metaphases with scattered chromosomes.
In H.Ep. #2 cultures, BUDR was less inhibitory
than FUDR (11). BUDR relieved some of the
metaphase arrest produced by aminopterin, and
cultures treated with both agents together con
tained anaphases and telophases with chromo
somal bridges and fragments. By itself, BUDR had
little effect; but this exposure was for 24 hours
only, and Hsu and Somers (35) found that strain
L cells show low frequency of chromosomal
breakage during the first 10 days of exposure to
BUDR. Djordjevic and Szybalski (21) also noted
few chromosome breaks in the treated human cell
line D98S, but they did observe a great increase in
sensitivity to ultraviolet radiation.
DISCUSSION
What is to be made of all this? We have con
sidered only a small percentage of even the rela
tively recent data, concepts, and hypotheses that
bear on our subject. Even so, what conclusions can
be drawn about the therapeutic application of
experimental modifications of mitosis in cancer
cells?
Before drawing conclusions, I would state my
conviction that cancer cannot be looked on simply
as a disease of excessive mitosis. This oversimpli
fied view can hardly gain adherents. In treating
cancers with antimitotic agents per se one is
probably treating a major symptom rather than
the disease. It would be our hope that alleviation
of this symptom would indicate that one is on the
right track toward a definitive therapy. On the
other hand, the distinct possibility must be
acknowledged that the mechanisms of therapy of
some agents may differ from the mechanisms by
which they exert their effects on mitosis. For ex
ample, Johnson and co-workers (37) considered
the C-mitotic effect of vincaleukoblastine to be
only coincidental with the tumor-inhibiting prop
erties of this agent. Koller (40) concluded that the
inhibition of growth in experimental tumors by
means of alkylating agents was not brought about
solely by the radiomimetic injuries to the chromo
somes. In comparing a number of related alkylat
ing agents, Koller saw that growth was probably
also suppressed by mitotic inhibition, interphasic
pyknosis, and stremai reaction. Two isomers pro
ducing equal chromosomal damage were not
necessarily equal in suppressing tumor growth.
Be that as it may, it appears that chromosomal
damage probably plays some role in cancer
therapy, whether for mechanical reasons, such as
785
those having to do with cross-linking or with dicentric chromosomes and bridge formation in anaphase, or for genetic reasons, with nonviable gene
combinations resulting from abnormal mitosis or
from gene mutations.
In the matter of gene mutations, there are
several aspects to be considered. Some mutations
may be produced directly, whereas others may re
quire mitosis to become established. With mutagens that shift base pairs, for example, several
cycles of DNA synthesis after the base analog has
been incorporated may be required to establish the
altered base pair, and a cell division may be
needed to have the mutation come to expression.
As for chromosome breakage after incorpora
tion of an analog, subsequent DNA syntheses with
certain illicit replications may not be needed, ac
cording to the work of Hsu and Somers. Chromo
somal breakage in sites of considerable analog
incorporation may occur in the course of mitosis,
however, by virtue of the locally changed physical
properties of the chromosomes and their response
to the tensions and stresses of chromosomal con
densation incident to mitosis.
Once chromosomal or genie imbalance is ex
perimentally established in tumor cells, does that
contribute to therapy? Certainly, nonviable chro
mosomal or genie combinations may be set up,
just as they often arise spontaneously in tumor
cells; but it is also possible that the genetic in
stability will contribute to tumor progression,
greater malignancy, and the origin of new stemlines resistant to one means of therapy or an
other.
In the research we have considered above in
volving radiation, it is evident that cell death or
reproductive incapacitation induced by irradiation
can occur either in the absence of mitosis or after
mitotic activity, the abnormality of which we
may consider as contributing to extinction of the
cell line.
In summary, mitosis may be a point of weak
ness and a good point of attack against cancer
cells, just because proliferation is one of their
common and prime concerns; mitosis may be a
necessary means to cell death through incorrect or
inadequate replication of DNA; mitotic disturb
ances may help to establish unbalanced and nonviable chromosome complements in daughter cells
by one means or another; but at the same time it
is evident that cell death can be produced by some
of the agents concerned without recourse to
mitosis. The mechanisms of action involved in
successful cancer therapy with these agents may
not be the same as those by which they derange
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1962 American Association for Cancer Research.
786
Cancer Research
mitosis, or therapy and mitotic derangement may
be coincidental. In any event, our level of under
standing needs to be raised with respect to funda
mentals of cell division, mitotic poisoning action of
drugs, radiation effects, and cancer therapy—all
four.
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Experimental and Therapeutic Modification of Mitosis
John J. Biesele
Cancer Res 1962;22:779-787.
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