[CANCER
RESEARCH
28, 608-614, March 1968]
The Cytologie and Growth Characteristics
Normal Clones of Picea glauca1
of Tumor and
Denes de Torok
Department
of Biology,
The Pennsylvania
State University,
McKeesport,
SUMMARY
Comparative chromosomal, mitotic, and growth studies were
made utilizing several series of tumor and normal clones of
Picea glauca (white spruce) tissue grown in vitro. Both pri
mary and secondary cloned lines, descending from one primary
tumor expiant, exhibited a much greater heterogeneity with
respect to chromosome number, rate of cell division, and rate
of growth, among themselves, than those yielded from one nor
mal expiant.
The establishment of clonal derivatives through single-cell
isolations was found to be the most effective method of securing
cell populations with homogenous genetic make-up. Clones with
both normal and irregular chromosome numbers obtained
from one primary expiant of normal and one primary explant of tumor cells kept their chromosome complements for
over thirty passages. Throughout this study highly signifi
cant correlations were found among growth rate, rate of cell
division, and chromosome number in both the tumor and the
normal clones. Clones with higher chromosome numbers ex
hibited higher rates of growth and higher rates of cell division.
All types of tumor clones, including those with varying degrees
of aneuploidy, appeared to be able to grow indefinitely.
It is postulated that, in the fully developed tumor cell, a
series of biosynthetic systems are permanently accelerated and
that the degree of acceleration determines the growth poten
tials, which can be predicted with a fair degree of accuracy
from the cells' mitotic frequency or chromosomal alterations.
INTRODUCTION
Tumor formation occurs at the cellular level, where the simi
larities among all living forms are much more striking than
are the differences. Thus the phenomenon of neoplastic change
is a basic biologic problem, the understanding of which can
be endeavored through studies of malignant cells whether they
belong to plants, animals, or humans. Tumors of all kinds can
be brought about by agents of the most diverse types, yet
many of the physiologic and biochemical properties for the
uncontrolled growth of the malignant cell appear to be similar.
1 Supported by Grants E-319 and E-319A from the American
Cancer Society, by the NSF Institutional Fund for Research, and
by a grant from The Society of the Sigma Xi and the Scientific
Research Society of America.
Received July 27, 1967; accepted November 26, 1967.
608
Pennsylvania
15132
A number of plant neoplasms have been studied; this field has
been reviewed. (4). Having peculiarities of their own, massive
types of tumors occurring on roots, trunks, branches, and twigs
on trees of Picea glauca, white spruce, have been the experi
mental subject of plant tumor studies, especially in vitro, by
a number of plant tumor specialists. Sterile tissue culture
studies, carried out on spruce tumors and on their normal
equivalents, revealed the existence of certain marked differ
ences at the nutritional level between the two types of cells.
Of special interest is their divergent hormonal relation in vitro,
which is exactly the opposite of the results obtained with other
pairs of tumor and normal tissues (11). Accordingly, the amino
acid and vitamin requirements of the normal and tumor spruce
tissues differ in vitro (11, 36). Furthermore, the author has
isolated much higher amounts of DNA from the cells of tu
mor tissues than from normal ones. These findings are exactly
consonant with results obtained from chromosomal and growth
studies, which revealed the random peculiarities of the chro
mosomes of tumor cells; whereas the overwhelming majority
of normal cells were found to have unchanged diploid numbers
(9, 12).
The demonstration that subcultures, derived from one pri
mary tumor culture of white spruce tissue, showed greater vari
ation than the subcultures originating from one primary normal
culture with respect to chromosome number, rate of cell di
vision, and rate of growth (10) has led to studies on the same
phenomena, ultilizing both tumor and normal cloned strains
of P. glauca. The establishment of cloned strains through singlecell isolation was found to be the most effective method of
securing cell populations with a high degree of homogenous
genetic make-up (9), meaning that at least 75 percent of the
cells have the same karyotype.
MATERIALS AND METHODS
Both tumor and normal P. glauca primary cultures were
obtained by the method previously described by de Torok and
Roderick (10) and were cultured and maintained on the
medium described by de Torok and Thimann (11). After the
establishment of these tumor and normal cell lines, noninjured
single cells were obtained by the method described by the
author elsewhere (8). Using the single cell plating technic (8),
10 normal and 14 tumor clones, isolated from subcultures of
one heterogeneous tumor and one fairly homogenous normal
expiant, were established and studied. In the initial normal
expiant, 78 percent of the normal cells counted clearly had
CANCER RESEARCH VOL. 28
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Cytologie and Growth Characteristics of Clones of Picca glauca
the diploid number, 22 chromosomes. Thus, chromosomal ir
regularity of these normal cells is an atypical feature; never
theless, 9 normal cells with aberrated chromosome complements
were isolated to establish their clones, so as to be able to study
the features associated with such irregularities and to compare
the same phenomena with a single-diploid isolate and with the
tumor clones where a wide range of random peculiarities in
chromosome numbers characterized the individual cells of the
initial expiant in over 80 percent of the cases. These tissues
grew as populations of single cells, each with a different chro
mosome number. The chromosomal studies of each primary
clone were systematically continued from the four-cell stage
by the method previously reported (12). Although no note
worthy chromosomal aberration within a given primary clone
was observed, after 8 months, 3 sister clones from each primary
clone were established giving rise to a total of 30 normal and
42 secondary tumor clones, representing 10 and 14 different
chromosome complements respectively. During the 8 months
of the primary clone stage the chromosome numbers of ran
domly selected cells within each clone were counted weekly.
In order to determine the validity of the typical number thus
assigned to each clone, after new single cells were successfully
isolated from them, the entire remaining tissue of each primary
clone was searched for dividing cells, whose chromosome num
bers were counted. The results clearly demonstrated a remark
able degree of chromosomal stability. The newly isolated single
cells gave rise to the secondary clones whose chromosome com
plements were known from the outset. The present study re
fers to these secondary clones, which were transferred regu
larly for over 2.5 years by the same method as that used for
the primary clones. These tissues, originating from single cells,
which in turn were isolated from tissues derived from single
cells, followed a similar course of growth increase and exhibited
a remarkable degree of chromosomal stability.
RESULTS
Chart 1 shows that clones of tumor origin varied significantly
in the rate of growth, numbers of chromosomes, and dividing
cells. Considering that all the clones are derivatives of the cells
of a single expiant of a block of tumor tissue about 2x2 cm,
the variation of the phenomena studied among the clones is
remarkably large, yet remarkably uniform within the indi
vidual cloned lines. In examining (Charts 2, 4, 6, 8) the results
obtained from the normal clones, it must be remembered that
in the original normal expiant the number of cells carrying
irregular chromosome numbers was very low. While the dip
loid number, 22, is the characteristic feature of these cells,
single-cell isolates with irregular chromosome numbers were
plated out and were grown only to be able to make compara
tive studies between these irregularities and those associated
with neoplasms. Thus, Chart 2 shows that the variations and
their magnitude for the same components were much lower in
the normal clones, but still noteworthy considering that these
clones, too, descended from the cells of a single normal explant. The compared values of the normal clones, though not
typical, also exhibited a spread, but the magnitude of variance
among these clones, representing extreme aberrations from the
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Chart 1. Histogram of secondary tumor clones over thirty trans
fers. Each number on the horizontal axis refers to the chromosome
numbers of 3 sister clones. The numbers in parentheses refer to the
correlative number of dividing cells per 1,000 and the heights are
equal to the corresponding average monthly growth increment
(mg) in 30 months.
typical diploid number and being very few in number, was
strikingly lower than in the tumor lines.
Chart 3 shows the extremely high positive correlation of
growth increment and chromosome numbers of the tumor
clones and the computer-analyzed regression lines of this cor
relation. It can be seen that even the quadratic line becomes
almost linear at higher chromosomal values, and that the
mathematical correlation between chromosome numbers and
growth rates is strikingly high. Chart 4 reveals that, although
the component of variance among the normal clones was only
a fraction of those for the tumor clones, a linear regression
line fits the analyzed normal data.
The association found among the tumor clones between the
increment in weight and the number of dividing cells per 1,000
is shown on Chart 5. Although the quadratic line is the correct
one, still this high correlation between growth rate and rate
of cell division indicates that growth rate here is largely a func
tion of rate of division of cells. Of course the complex phe
nomenon of growth consists not merely of cell division, yet it
seems that the number of daughter cells formed does play an
essential role in determining the actual amount of growth.
Chart 6 reveals that the correlation of the same components
is even higher for the normal clones, where the linear line is
more applicable.
It was felt desirable to compare the number of dividing cells
with the number of chromosomes. The results for the tumor
MARCH 1968
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609
Denes de Torok
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Chart 2. Histogram of secondary normal clones over thirty trans
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DISCUSSION
The experimental results in the present study support our
previous findings (10), in that there was an overwhelming
heterogeneity among the cells of the tumor expiant, which, when
plated out as single isolates, gave rise to cloned lines which
maintained their initial characteristics indefinitely; thus, one
sees the obvious advantages of single-cell cultures resulting in
greatly reduced variations within cloned lines. Hildebrandt et
al. (21), Caplin and Steward (6), and Tulecke (44) are only
a few who also experienced the great variability of subcultures,
but, on the other hand, in the studies of Cooper et al. (7),
the majority of the cells in single-cell clones of tobacco retained
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their diploid character for a period of over 8 years. The re
sults obtained by Sievert and Hildebrandt (37), as well as those
obtained by Arya et al. (2), clearly demonstrated existing dif
ferences among single-cell clones, as did single-cell clones of
CANCER
RESEARCH
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VOL. 28
Cytologie and Growth Characteristics of Clones of Picea glauca
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ing clones has not been revealed. As far as the author is aware,
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normal origin plated out from single cell derivatives of single
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1968
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1968 American Association for Cancer Research.
611
Denes de Torok
ent karyotypes not as a result of cultivation. It is also demon
strated that normal cells, too, are subject to chromosomal aber
rations and that this type of abnormality is entirely different
in consequences, magnitude, and frequency from those caused
by neoplasm formation, leaving little doubt that chromosomal
irregularities in neoplasm formation are consequential in the
case studied and suggesting a difference in origin. Quite strik
ing is the remarkable degree of stability of the studied cloned
lines, in each of which a remarkable degree of mathematical
correlation was found among chromosome number, mitotic fre
quency, and growth rate. Apparently, permanently increased
metabolic efficiency accompanies increased chromosome com
plements, resulting in increased mitotic activity and growth.
On the other hand, chromosomal losses bring about opposite
results, but still can be an important factor in the transition
of a normal cell to an eventual tumor cell (5, 9, 10).
The work of Kehr and Smith (23) and Smith and Stevenson
(38) indicate that tumor formation in hybrids of certain spe
cies of tobacco is under genetic control and that specific com
binations of chromosomes are necessary for tumor formation.
Although there is a voluminous literature on chromosomal
studies of animal and human neoplasm, and the direct evidence
for visible changes in chromosomal population and morphology
is quite extensive (35), it is also apparent that a number of
neoplasms do not exhibit any visible chromosomal abnormali
ties (35) ; thus the possible relationship between chromosomal
abnormalities and malignancy remains unresolved. The results
presented here reveal a close relationship between growth
"aggressivity" and aneuploidy. Concordantly, Levan and Biesele (26) suggested that aneuploid cells may grow faster than
diploid cells; similar results were reported by Hauschka and
Levan (20), Kaziwara (22), Levan (25), Hauschka (19), and
Wakoning (45). Winge (47) postulated that the tetraploidy
which he observed in tumors of sugar beets might satisfactorily
explain the "growth energy" exhibited by tumor tissues. The
results of this study seem to be in accordance with this
suggestion.
The variation between chromosome numbers in different
clones from the same original tumor tissue (Chart 1) is strik
ing; it is perhaps paralleled by the variation reported by
Yoshida and Ishihara (48) with very different materials,
though their cultures were not all derived from the same trans
plant. Foulds (15) emphasized the random and unpredictable
nature of tumor cell progression; and the fact that there seems
to be no real "end-point" for progression and all established
tumors may undergo sequential changes. These findings with
animal tumors show some similarity to the results of the pres
ent study, but in the plant material described, each isola
tion of tumors does not seem to undergo a different develop
ment; rather, although the details differ from case to case, the
developmental tendency remains similar. In the normal tissues
studied, different isolates are also found to grow at different
rates in vitro, but the extent of the differences was greatly
restricted compared to the wide variation in the tumor tissue.
While there are reports of frequent aneuploidy (43, 46) and
other chromosomal irregularities (3, 10, 29, 30, 42) in normal
tissues, Ford (14), working with material other than plants,
found a remarkable degree of constancy of the karyotype in
612
normal somatic cells. The fact that normal cells can show cy
tologie characteristics of true tumor cells but are themselves
self-limiting indicates that the observed chromosomal abnor
malities are the result rather than the cause of the tumor state.
Cytologie studies on fern tumors revealed an initial haploidy
which changed to tetraploidy only with subsequent cultures.
Superimposed upon this polyploidy, variable aneuploidy was
commonly observed (39). Partanen (32), through studies of
the DNA in individual nuclei of normal and tumor fern, con
cluded that polyploidy is neither causal to nor even present
in the initiation of that tumor. The secondary, diagnostic char
acteristic of polyploidy became apparent by increased DNA
values obtained only after subsequent passages in culture.
There are, however, observations indicating increase in nuclear
DNA content (27, 41) proportional to increase in cell volume;
this may or may not be indicative of polyploidy. Wakoning
(45) presented over 60 cases from the literature where neo
plasms have been found predominantly diploid; this also argues
convincingly against aneuploidy as the universal cause of neo
plasm formation.
A high frequency of unbalanced chromosomal changes, dupli
cations, deficiencies, and aneuploidy characterized these tumor
cells. While the relationship of neoplasm formation to quanti
tative chromosomal changes is quite apparent from the results,
in order to establish the origin of this relationship, it would
be necessary to study the tumor cell cytologically from its be
ginning. Thus, one would have to go back to the intact tree
where tumor formation occurs. The success of such an in
vestigation must be presumed highly improbable until even,
in complex organisms, a tumor cell can be recognized at its
earliest stage, or even before tumor formation occurs. Further
difficulty in pinpointing the cytologie differences between tu
mor and normal cells lies in the fact that the chromosomal
aberration of the normal cell, though infrequent, can be very
similar numerically to a tumor cell, at least up to 5 n. Yet nor
mal tissues with aberrated chromosomal complements did not
cause tumor formation when grafted on healthy seedlings of P.
glauca; on the other hand, tumor tissues with altered chromo
some numbers developed into tumors comparable in every re
spect to those from which they were cultured upon similar
transplantations (8), suggesting the existence of a basic physio
logic difference between the two types of irregularities. Cor
respondingly, the auxin relations of the two types of tissues
revealed the existence of qualitative differences and that appar
ently normal tissues, even with irregular chromosomal comple
ments, did not follow the hormonal pattern characteristic of tu
mor tissues only (8). Much time and effort has been devoted to
grow, study, and analyze these tissues, but there is a long way
to go to find the origin of the apparent qualitative distinctions,
aside from the quantitative changes, characterizing the cy
tology of tumor cells. While the cytologie difference between
the slow-growing normal and the rapid-growing tumor cell is
quantitative in consequence and the qualitative nature of this
difference becomes apparent only upon further experimenta
tion (8), at the nutritional level, the qualitative difference is
immediately apparent, since cultured P. glauca tumors de
pend upon the availability of added auxin whereas cultures
from normal tissues do not require auxin; this has been demonCANCER RESEARCH VOL. 28
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Cytologie and Growth Characteristics of Clones of Picea glauca
strated by several workers independently in different labora
tories (11). Also, at the nutritional level, it has been found
that for tumor tissues all amino acids can be omitted without
any decrease in the growth rate (11) ; and were it not unique,
the failure of Risser and White (36) of successfully growing
normal cells in the medium that supported rapid growth of
tumor cells would also be promising. The possibility that a
specific chromosome complement is related to the biosynthetic
abilities of certain tobacco pith tissues has been pointed out
by Fox (17); similarly, Nicotiana tumors which were cytologically different from the nontumor derivatives of the same
population were found to have different nutritional require
ments (1).
No explanation is attempted for the puzzling fact that diploid cells isolated from tumor tissues increased in growth rate
almost four and a half times as much as did diploid cells iso
lated from normal tissues; at the same time, neither diploid
tissues revealed tumor characteristics. They both grew on
auxin-free medium and the transplantation of either failed to
produce tumors on healthy host (8).
Another significant result of the present study is the corre
lation between cells with high chromosome numbers and rapid
cell division. That neoplastic cells posses a high mitotic rate
was noted by the early investigators of tumor pathology (18,
34) ; Ludford (28) suggested that a high frequency of abnor
mal division may indeed be an almost universal phenomenon
in tumors. In 1943 Kostoff (24) proposed a theory of tumor
induction based on abnormal mitosis. However, the conclusion
of Kehr and Smith (23) that tumor development is at least
associated with abnormal cell division and that the condition
which ultimately brings about the development of tumor
growth is also the condition which results in abnormal mitosis
seems to be more reasonable. Steward and Shantz (40) con
cluded that tumor growth occurs when the set of factors which
promote cell division are unregulated or in excess. Yet when
these cells are kept in vitro, even normal cells lose their spe
cialization for function and the cells revert to mitotic activity.
As a result of this activity, a large enough cell mass is built up
in which it is the innermost cells that first cease mitotic activity
while the peripheral cells continue to undergo mitosis. A possi
bility exists that the process by which these cells reacquire a
mitotic ability is the same process by which similar cells in vivo
reacquire a mitotic ability upon wounding or during regenera
tion. It is important to note, however, that the overall mitotic
frequency of the tumor tissues in vitro is about 10 times as high
as observed for the normal cells (Charts 5, 6). Thus the evi
dence suggests that in tumor cells either or both the mecha
nisms controlling the mitotic rate and the degree of synthesis of
metabolites necessary for this abnormally high degree of mi
totic efficiency depend upon factors which are different either
in origin or in quantity from those that promote these activities
in normal cells. But no real understanding of the changes oc
curring during the transformation of a normal cell to a tumor
cell is likely to be achieved until a thorough understanding of
the manner in which normal tissues maintain their organization
is first accomplished. A number of known cytologie mechanisms
can account for the formation of abnormal chromosome number
of a cell and, as Partanen (32) points out, once polyploidy is
MARCH
established it can be the forerunner of yet other differences;
but, quite clearly, the factors causing karyotype alterations are
many and not completely known.
From all the foregoing one is led to the concept that in carcinogenesis there are not only a large number of agents capable
of causing cancer but also a large number of diverse accessory
factors, and that a number of combinations of agents with ap
propriate accessory factors is possible, many of which can en
hance cancer whose characteristics thus can be various and
unrelated. The results of this work offer much support for
Fould's (16) conclusion that "tumors derived from the same
tissue by the same carcinogenic procedure may be extremely
varied and integrated" so that "it is probable indeed that no
two tumors are exactly alike in every respect." In conclusion,
it seems that in the present case a variety of genetic and epigenetic mechanisms are being altered by either one or more as
yet unknown tumor-inducing factor(s), which could account for
the wide degree of cytologie changes. As a result of this trans
formation of the normal P. glauca cell to a tumor cell, a
series of biosynthetic systems become permanently accelerated
and the degree of acceleration of those systems within the cell
determines the rate at which a cell is altered to a tumor type ;
at the point of full alteration its growth potentials are deter
mined and can be predicted with a fair degree of accuracy from
its chromosomal alterations.
The terms "expiant," "primary culture," "clone," "cloned
strain or line," and "karyotype" have been used here in agree
ment with the terminology outlined by S. Fedoroff (13).
ACKNOWLEDGMENTS
The technical assistance of Mrs. Eva Suchant is gratefully
acknowledged. The author is also pleased to thank Dr. P. R. White
for his kindness in sending the material used to initiate this study
and Dr. I. S. Tuba of the Carnegie-Mellon University for his help
in programming the material for the computer.
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CANCER RESEARCH VOL. 28
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The Cytologic and Growth Characteristics of Tumor and Normal
Clones of Picea glauca
Denes de Torok
Cancer Res 1968;28:608-614.
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