Download Mitotic Ability of Leukemic Leukocytes in Chronic

Mitotic Ability of Leukemic Leukocytes
Myelocytic Leukemia*
AVERY
A.
SANDBERG,
YASUMOTO
KIKTJCHI,
(Ro8well Park Memorial Institute,
AND Lois
in Chronic
H.
CROSSWHITE
Buffalo, New York)
SUMMARY
In vitro culture of leukemic blood leukocytes from patients with chronic myelocytic
leukemia (CML) showed them to be capable of division in the absence of phyto
hemagglutinin.
In the presence of the latter, lymphocytes
in the blood of these sub
jects were preferentially
stimulated
into mitosis.
In the absence of the mitogenic
stimulant only the immature leukemic granulocytic
cells underwent division.
The
capacity of the leukemic leukocytes in CML to divide in vitro without an exogenous
mitogenic agent was a characteristic
of both Ph'-positive and Ph'-negative CML cells.
The results indicate that immature granulocytic cells in the blood of patients with
CML,
and possibly
in the blood
out exogenous mitogenic
of nonleukemic
It has been amply demonstrated that a factor in phyto
In previous studies we have shown that acute leukemic
blood cells did not undergo mitosis, either with or without
PHA, to a sufficient degree to gain a representative picture
of the chromosome patterns of these cells from blood
cultures (17). In the presence of PHA, the majority of
metaphases originated from nonleukemic cells and had a
normal karyotype.
On the other hand, the leukemic cells,
which were characterized by aneuploid or pseudodiploid
modes in the bone marrow, constituted, at best, a very
of the metaphases,
are capable
of division
with
possible influence of this abnormal
hemagglutinin
(PHA) or other mitogenic substances are
necessary for the growth of certain blood cells (i.e., lympho
cytes) in short-term culture (48—72hr.), rendering them
suitable for karyotypic examination (1—3,5—10,12, 13, 18).
small percentage
subjects,
substances.
or failed to divide
autosome on cell
division was investigated.
MATERIALS
AND METHODS
Blood cells were cultured for 48—72hr. according to
technics previously described (11, 15—17). Blood was
obtained from seven patients with Ph'-positive CML and
from three subjects with Ph'-negative CML. In each case
the blood contained substantial numbers of immature
leukemic cells (myeloblasts, promyelocytes, myelocytes)
and lymphocytes.
The presence of the latter was neces
sary, since the division of these cells had to serve as an
indicator
of the number of metaphases
with
karyotypes
observed under various conditions.
normal
All the
patients
chemo
with CML had been treated
with various
altogether. Our observations have recently been sub
stantiated by Fitzgerald et al. (4) in a group of adult
therapeutic agents, and their white blood cell counts,
when elevated, were not excessively so (8—30,000/cumm).
patients
Whether
with
acute
leukemia.
These
authors
found
a
the patients
dichotomy between the very aneuploid cells observed in
leukemic bone marrows and the preponderantly diploid
tion
metaphases
blood cells (16).
seen following culture of blood from the same
patients.
leukemic
In the absence of PHA, both normal and acute
blood cells failed, for all practical
purposes,
from patients
with chronic myelocytic
quire exogenous mitogenic
factors.
leukemia
(CML)
re
Since not all patients
with CML have the Philadelphia (Ph') chromosome, the
*
This
study
has
been
supported
in
part
by
grant
T-182
of metaphases
to
undergo mitosis, at least in our hands and under the condi
tions employed (24- to 72-hr. cultures). The present
study was undertaken to ascertain whether blood cells
from
the American Cancer Society.
Parts of this study were presented at the Annual Meeting of
the American Society of Hematology, December 9, 1963, Washing
ton, D. C., and were abstracted in Blood, 22:816—17,1963.
were Ph'-positive
or Ph'-negative
had been established in the past on the basis of examina
from
bone
marrow
and in cultured
RESULTS
A study
of the morphology
revealed immature
of the
lymphocytic
cells
following
culture
and lymphoblastic-like
cells in the smears derived from normal blood (3, 9) and
immature granulocytic cells (myeloblasts, promyelocytes,
and myelocytes) in the blood of patients with CML grown
without phytohemagglutinin.
The CML blood cultured
with phytohemagglutinin revealed a mixture of immature
lymphocytic
and
immature
granulocytic
cells.
In all
smears dividing cells at various stages of mitosis were
observed.
Received for publication April 8, 1964.
Crucial to the interpretation of the initial results was the
1468
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SANDBERG et al.—Mitotic
Ability
of Leukemic
Leukocytes
1469
identification
of metaphases
with the Ph' chromosome.
Cytogenetic
evidence indicates that the abnormal Ph'autosome is confined to those hemopoietic elements which
Nevertheless,
the nearly total absence of metaphases
in
cultured normal blood incubated
without PHA would
militate against the metaphases observed in CML bloods
are of bone marrow origin (granulocytic, megakaryocytic,
and possibly the erythroblastic series) and is not present
in lymphocytes and in cultured cells of skin and other tis
as being nonleukemic.
sues (16, 19).
metaphases observed in the three blood samples did not dif
fer materially whether or not PHA was added to the cul
ture medium.
The experiments cited above indicate that cells present
Metaphases
were labeled
as Ph'-positive
only if the abnormal autosome could be identified with
certainty
and when it met the morphologic
criteria
of the
Ph' chromosome. Metaphases and karyotypes with and
without the Ph' chromosome are shown in Figures 1, 2.
As can be seen, the abnormal Ph' can be easily differ
entiated from the other G-group acrocentrics and from
the Y.
Preliminary
studies showed that the number of meta
phases in CML blood grown in vitro without phytohemag
glutinin was higher (twenty per 1000 cells) after 48 hr. of
culture than after 72 hr. (ten per 1000 cells) ; and that in
the presence of PHA there was no significant difference.
Hence, the results to be presented are based on cells
cultured for 48 hr.
The incidence of the Ph' chromosome in blood grown for
48 hr. with and without phytohemagglutinin and the per
centage of cells in the blood of patients with CML capable
of division
the absence
are shown
in Table
of PHA,
1.
It is apparent
a much higher
that,
in
(92 per cent) per
centage of metaphases was Ph'-positive as compared with
the incidence of metaphases with the Ph'-autosome (39 per
cent)
in PITA-treated
granulocytic
(range : fifteen
In addition, in smears only im
cells were observed.
to twenty
metaphases
per
The number
1000
cells)
to undergo mitosis in the absence of added mito
genic factors, blood cells from three subjects with CML and
devoid of the Ph' autosomes were cultured with and with
out PHA. Since the incubated cells did not contain an ab
normal “marker―
chromosome and were not aneuploid, it
cannot be assumed with certainty that the metaphases ob
served in the absence of PHA are of leukemic origin.
in the blood of patients with CML are capable of division
in the absence of PITA regardless of whether the Ph' is pres
ent, in contrast to cells present in normal blood, which fail
to undergo mitosis unless a mitogenic substance is added
to the medium.
A series of experiments was performed with the aim of
determining whether a mitogenic substance was present in
the plasma of subjects with CML or was confined to the
cells. Normal and CML leukocytes were incubated with
normal or CML serum, in the presence or absence of PITA,
and the number of metaphases was determined.
leukocytes
underwent
division
in both normal
CML
and CML
serum, with the number of metaphases being higher, in this
series of experiments, in the absence of PITA. Interest
ingly, in several instances a significant number (five to ten
per 1000 cells) of metaphases was observed when normal
leukocytes were incubated with CML serum and no
PHA.
Under the same conditions,
and with PHA, the
absence of PHA.
Not all (four out of seven) of the CML
sera had this mitogenic effect. Whether this is related to
previous therapy, stage or complications of the disease,
quantity
or quality
of cells in the blood or marrow,
or other
factors is at present unknown.
An attempt was made next to determine whether the
capacity of CML leukocytes to divide in vitro in the
absence of PHA was peculiar to the leukemic state.
To
this end blood was obtained from several subjects who had
TABLE 1
THE INCIDENCE OF METAPHASES WITH THE PH' CHROMOSOMEIN CML BLOOD CULTURES WITH
AND WITHOUT
Also shown in the table are the percentages
PHA
of cells capable of division present
in the bloods as
determined from differential counts.
DIVISIONWith CELLS
CASENO.SEXNuuzaa
monocYtes1
@
@
@
@
2
3
4
5
6
7
Total13
phytohe
magglutinin
Ph1-pos.
c?
d'
a,'
of
number of metaphases was somewhat smaller than in the
cultures.
To test the possibility that the presence of the Ph'
chromosome increased the mitotic capacity of the leukemic
leukocytes
mature
Ph'-neg.Per
15
8
17
16
25
9
35
22
13
34
5
41
10337
18726
ca@iT
czu.s CAPABLE
@NUMBER
CELLSPaz
01
phytohe
magglutmin
cent
cent
Ph-'pos.Per
Ph1-pos.Without
Ph'-pos.
Ph'-neg.Per
30
27
57
32
83
1820
18
29
27
42
30
44
3
1
3
8
0
1
2103
1987
cent
ST8flUin@YbC
86
97
90
84
5.0
20.5
23.5
3.2
100
29.0
984.0
cent
lymphocytes
cellsPer
27.019.5
and
11.0
18.0
10.5
20.5
2.0
5.0
Downloaded from cancerres.aacrjournals.org on June 11, 2017. © 1964 American Association for Cancer Research.
Cancer Research
1470
myelocytes and other immature granulocytic
blood (two subjects with myeloid metaplasia,
cells in the
one subject
may
Vol. 24, September
be of considerable
importance
1964
in relation
to the
development of disorders involving these cells or their
with a high leukocyte count due to infection complicating
cancer of the lung). Even though the number of meta
precursors.
The fact that serum from CML was capable
of initiating
mitosis
phases was not as high as that observed with CML cells,
there was a significant number of metaphases (four to seven
that such serum contains mitogenic substances, which
may have originated from the granulocytic cells, capable
of inducing division in normal cells.
per 1000 cells) observed in the three blood samples in
cubated
without
PHA.
Even
DISCUSSION
Recently published results and the data of the present
experiments elucidate several parameters of leukocytic
division in vitro.
It would appear that the leukocytes
of
normal blood which are capable of mitosis—i.e., the
though
of normal
lymphocytes
immunological
may indicate
mechanisms
may
be
responsible for initiating and sustaining mitoses in blood
lymphocytes, it appears that each antigen may provoke
the cells into division by diverse processes (2). For
example, it has been shown that phytohemagglutinin
and
tuberculin induce mitosis in normal blood lymphocytes at
different times and rates, suggesting different loci of action
of these two substances.
These observations
indicate that
lymphocytes—are
triggered into that process by sub
stances acting through immune mechanisms (1—3,5—10, the factors controlling and initiating mitosis of leukocytic
12, 13, 18). In the absence of mitogenic substances, for
all practical purposes no mitotic cells can be seen in nor
mal blood cells cultured for short periods of time (48—72
hr.).
In the present study, in six out of seven cases the non
leukemic leukocyte metaphases outnumber the leukemic
ones (Ph'-positive cells) in the presence of PITA. It is
unclear whether
this is due to stimulating
on lymphocytes
and/or
inhibition
cells may be highly diversified.
ACKNOWLEDGMENTS
We wish to thank Mr. Robert N. Holdsworth, Mrs. Regina
Joyce, and Mrs. Catherine Egri for valuable technical assistance;
also Mrs. Anne Caimano and Miss Noreen Sominer for their help
in the clerical and photographic aspects of the work. We wish to
thank Dr. Theodore S. Hauschka
effects of PHA
of division
for his advice.
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of the leu
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chromosome did not seem to endow the leukemic leuko
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with leukemic leukocytes devoid of the Ph' autosome.
In
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1471
Downloaded from cancerres.aacrjournals.org on June 11, 2017. © 1964 American Association for Cancer Research.
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Mitotic Ability of Leukemic Leukocytes in Chronic Myelocytic
Leukemia
Avery A. Sandberg, Yasumoto Kikuchi and Lois H. Crosswhite
Cancer Res 1964;24:1468-1473.
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