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Human
Chronic
With
M yelogenous
Positive
Leukemia
Philadelphia
By Carmen
B. Lozzio
A
TTEMPTS
TO
ESTABLISH
Chromosome
and
Bismarck
A cell-line
derived
from a patient
with
chronic
myelogenous
leukemia
(CML)
is
described.
The new cell-line,
which
has
over 175 serial passages
in a 3k-yr period,
has the following
characteristics:
(1) CML
cells started
to proliferate
actively
since
they were first incubated
in culture media.
A threefold
increase in the total number
of
cells was observed
during
the first seven
passages;
the cell population
increased
by
a factor of 10 to 20 every 7 days from passage 8 through
85; from 20 to 40 times
from passage
86 through
150, and more
than 40 times after 150 passages.
(2) The
majority
of the nononucleated
cells are
undifferentiated
blasts. (3) The karyotype
of all the cells examined
show the Philadelphia
(Ph’)
chromosome
and
a long
acrocentric
marker
plus aneuploidy.
The
B. Lozzio
studies
identified
the Ph’
chromosome
as a terminal
deletion
of the
long arm of chromosome
22:del(22)(q12)
and the long acrocentric
marker
as an
unbalanced
reciprocal
translocation
of one
chromosome
17 and the long arm of one
chromosome
15. (4) The CML cells do not
produce
immunoglobulins,
are
free
of
mycoplasma,
Epstein-Barr
virus,
and
herpes-like
virus particles.
(5) CML cells
have no alkaline
phosphatase
and myeloperoxidase
activities
and did not engulf
inert
particles.
(6)
Cultured
CML
cells
provide a constant
source of a specific antigen.
This CML
cell-line
represents
a
unique
source of CML cells with meaningful indicators
of malignancy
for clinical
and experimental
studies.
Giemsa-banding
permanent
of patients
with chronic
myelogenous
adelphia
(Ph’)
chromosome
marker,
have
cultures showing
the Ph’ chromosome
chromosome
after serial subcultivation
cultures,’5
(lymphoid)
Cell-Line
cell-lines derived
from
the blood
leukemia
(CML),
having
been consistently
unsuccessful,’
the
Philand
for several months5
have lost the Ph’
for longer periods of time.4 In all these
the leukemic
cells were replaced
by immunoglobulin-producing
cells. Recently, the growth
and maturation
of CML
cells was stud-
ied in liquid
media.
tients
with
Ph’-positive
Bone
marrow
CML
were
days.
The
Ph’
chromosome
some
was
also
found
was
and peripheral
blood
cells from
seven
pacultured
in diffusion
chambers
for up to 45
found
in short-term
in only
soft-gel
two
media
cultures.6
The
Ph’
chromo-
cultures.7
Preliminary reports from our laboratory showed
persistence of the Ph’ chromosome
20 mo after establishment
of a CML
cell-line from a pleural effusion
of a terminal case in blastic crisis. These cells have
very rapidly and maintain their positive Ph’ chromosome
rearrangement
The purpose
after continuous
of this report
characteristics of this unique
From
the Laboratories
of Tennessee
Memorial
Submitted
June
Supported
in part
Address
Research
reprint
Center
© 1975
Blood,
for
14. 1974;
by Grune
and
accepted
& Stratton,
Vol. 45, No. 3 (March),
and
Carmen
Knoxville.
22.
Society
B.
and
Spleen
Hospital,
September
Cancer
requests:
Hospital.
(CBL)
Center
by American
culture for 3 yr and over 175 serial passages.
to
describe
the origin,
culture
method,
and
cell-line.
of Cytogenetics
Research
is
continued
proliferating
and a long acrocentric
Tenn.
(BBL).
The
University
37920.
1974.
Grant
Lozzio,
Tenn.
Pathophysiology
Knoxville,
CI 87B
M.D.,
and Grant
The
University
FR 5540.
of
Tennessee
Memorial
37920.
Inc.
1975
321
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322LOZZIO
AND
MATERIAL
Case
AND
LOZZIO
METHODS
History
The cell line described
herein
a pleural effusion
of a 53-yr-old
with
busulfan
for
3 yr and
was derived
from leukemic
cells obtained
in December
female suffering
from CML for about 4 yr. She had
pipobroman
for
I yr.
In
October
of
1970,
all
1970 from
been treated
peripheral
blood
cells
showed
of45
Soon
tumor
pleural
tamed
tures.
served
a positive
Ph’ chromosome
and a clonal evolution
of the leukemia
with a new cell-line
chromosomes
and a translocation
D/E.
In November
of 1970, a splenectomy
was made.
after surgery
the patient
underwent
blastic crisis, and the malignant
cells accumulated
as
masses
of highly
undifferentiated
cells through
most tissues
of the body
and produced
effusion.
Nine days prior to the patient’s
death
a sample
of the pleural
fluid was oband used for chromosome
analysis
in short-term
cultures,
and to start the long-term
culA positive
Ph’ chromosome
and a long acrocentric
chromosomal
rearrangement
were obin all cells. The aggressiveness
and extensiveness
of the malignant
infiltrate
suggested
marked
alteration of the original leukemic
cells, and the presence
of a positive
Ph’ chromosome
in addition
origin
to the
of these
lack
of structural
neoplastic
Establishment
patterns
of the
tumor
masses
provided
evidence
of the
myeloid
cells.
of a CML
Cell-line
in Suspension
Cultures
The pleural
fluid was collected
with heparin
and aliquots
containing
15-20 x 106 cells each
were diluted with Eagle’s minimal
essential
medium
(MEM)
plus additional
amino acids and 15%
fetal bovine serum.
All cultures
were incubated
at 37’ in a humidified
atmosphere
with constant
flow of 5% CO2 in air. The cells grew as a free-floating
cell suspension
since they were first incubated
in liquid cultures.
Serial
subcultivations
of aliquot
samples
were carried
out weekly
after determination
of the total number
of cells per culture.
The cell-line
was named
CM L- I,
K-562.
Freezing
and
Thawing
of Cultured
Samples
of cells were
glycerol. The cells were
ampules
were thawed
of their viability with
Colony
The
of only
were
Growth
method
one
frozen
at various
frozen
at l’C
incubated
trypan
and Isolation
and
of CMU
cells
under
the plating
suspended
medium
supplemented
stored
in culture
in liquid
medium
with
nitrogen.
5%
The
after determination
blue.
in Agar-gel
at 3TC
per plate and
passages using Eagle’s
per mm
and the ampules
rapidly, and the cells were
0.5%
of Robinson
layer
Cells
Pike10
mixed
was
with
controlled
of Cell
followed
with
Clones
slight
3 ml of semisolid
pH
and
humidity
efficiency of the CML
cell-line
modifications.
agar
for
diluted
12-14
were
The
with
days.
The
determined
plates,
McCoy’s
number
at
various
made
5A
up
medium
of colonies
passages.
plating efficiency
was also investigated
in plates containing
a feeder layer of peripheral
leukocytes (2 x 106) from normal humans.
The agar-gel
method
was used also to isolate
clones
of cells derived
from single cells. The
clones were isolated
by picking
individual
colonies
which
were transferred
to Leighton
tubes
with
I ml of medium.
Each colony grew as a suspension
culture
and was transferred
to culture
The
bottles
line
with
K-562.
clones,
increased
Each
samples
Cytogenetic
Chromosome
amounts
clone
from
was
of
media
maintained
each clone
until
by
were stored
the
passage.
frozen
in liquid
et
density
After
was
similar
chromosome
to
the
analysis
original
cell
of
all
the
of
Moor-
nitrogen.
studies
preparations
were
made
at
various
These smears were stained
with Giemsa
mosome
analyses
were also performed
in smears
banding
thfliqu,2
and by the fluorescence
method
The length
of the cell cycle was determined
at
mitosis of Quastler
and Sherman.14
A suspension
head
cell
weekly
passages
according
to the
procedure
dye after hydrolysis
with I N HCI.
Chrostained
by the Sumner
and Evans’
Giemsaof Caspersson
et al.13
various
passages
by the technique
of labeled
containing
10 cells/mI
was pulse labeled
for
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MYELOGENOUS
mm
15
burg,
with
CELL-LINE
1 zCi/ml
of
323
3H-thymidine
(specific
activity,
1.9
Ci/mM:
Schwarz/Mann,
Orange-
N.Y.).
These
cells
culture
30
LEUKEMIA
were
medium
mm,
centrifuged,
without
the
1,2,3,4,5,7,9,1
rinsed
isotope.
with
balanced
salt
of
were
Samples
1,13,15,17,19,21,23,
and
The radioautographs
were exposed
for
The per cent of labeled versus unlabeled
10 ml
25
hr
solution,
and
incubated
after
pulse
resuspended
at
37’C
labeling
with
10 days, developed
with D I I, stained
metaphases
was determined.
in fresh
until
fixation
at
3H-thymidine.
with
Giemsa
dye.
was
studied
peri-
Cytology
The
morphology
odically
of CML
on smears
Enzyme
stained
cells
with
growing
Giemsa
either
normal
peripheral
Viral
Testing
leukocytes
cells were
Cultured
Henle
liquid
or
semisolid
media
Studies
The Graham-Knoll
peroxidase
reaction
tion of alkaline
phosphatase
according
antigen
in
dye.
referred
of the
(control)
tested
to as
for
and
Gomori
cultured
Epstein-Barr
ENBA.’6
Division
for general
to
These
of Virology,
differentiation
technique’5
CML
cells.
virus
tests
were
Childrens
(EBV)
kindly
Hospital,
and cytochemical
were performed
genome
made
by
and
Dr.
University
of
on
demonstrasmears
of
EBV-associated
Gertrude
nuclear
and
Pennsylvania,
Dr.
Werner
Philadelphia,
Pa. Mycoplasm
(PPLO)
contamination
was tested using media and supplements
from Microbiological Associates,
Bethesda,
Md., following
the method
of Hayflick
and Stanbridge,’7
and Hayflick.18 The media and cells were monitored
for PPLO continuously.
Tissue culture
reagents
were
also tested for mycoplasm
before use following
the procedures
of Barile and Kern.19
Immunoglobulin
The
plate
(Ig)
technique
of
contained
agar
Determination
Mancini
al.20 was
et
impregnated
with
followed
goat
throughout
monospecific
with
antiserum.
slight
modifications.
Cells,
cultured
Each
for
7 days,
were centrifuged.
The concentration
of Ig was determined
in nonconcentrated
and 10 x concentrated
media.
The media
were concentrated
by ultrafiltration.
A cell pellet
containing
20 x 106 CML
cells was washed
once with cold saline disrupted
by sonic vibrations,
and an
aliquot assayed for 1g.
Phagocytosis
The
ability
of CML
cells
to
ingest
polystyrene
of Cline and Lehrer.21
The culture
medium
Polystyrene
particles
(1.3-1.7
.t in
diameter)
of 1 mg/mI.
Five milliliters
of the suspension
Ieukocytes
bated
(control)
1 hr
for
contained
at 37’C
in 5 ml
with
particles
of medium.
shaking.
was
was the same
were added
were mixed
The
Intracellular
determined
following
the
technique
used to grow the cells in suspension.
to tissue culture
medium
at the rate
with either 2 x 106 CML or normal
cells
and
particles
polystyrene
were
studied
particles
were
by
phase-contrast
incu-
microscopy.
RESULTS
Growth
Characteristics
in Free-floating
Suspension
Cultures
The CML
cells started
to proliferate
actively
as soon
as they were incubated
in culture medium.
No latent period was observed, but the rate of growth varied
at various
passages
as follows: one to three times
the firstseven passages,
10-20 times from
increase
per passage
the 8th to the 85th, 20-40
during
times from
the 86th to the 150th, and 40-70 times from the 150th to the 175th passage. A
corresponding
decrease in the mean
generation
time was observed. This was
approximately
50 hr during
the first seven
passages,
and
10-12
hr after passage
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324
LOZZIO
85. The
decrease
in the
total
as seen in cells studied
hr, including
a period
of2
hr,
and
Cytologic
a 0,
The
mitotic
imately
ofthese
cells
had
20-35
with
basophilic,
promyelocytes
interphase.
their
cell
was
two
These
cells
Growth
The
plated,
10%
,
of granules,
eccentric
had
and
nucleus
7-8
nucleoli
Large-sized
was
were
very
characterized
by
blue
developed
and
easily
nucleoli
larger
and
a pale
and
skeinlike
particularly
A bility
cells. Small
incubation,
of culture.
of 582 ± 67
than
those
In five experiments,
to the hard
agar layer.
not
differentiate
growing
vacuoles
nucleus.
In
to
Two
so
was
well
main
into
general,
colonies
three
cells
were
on agar were
in the narrow
Giemsa.
distinct
nucleus,
threads
of
on agar
(Fig.
con-
chromatin.
I B).
to
the number
made
with
of
250,
arose
present
from
and
cells
500,
cells
cells
and
with
were
were
60%,
1000
single
cells,
originated
but
even
above.
layer
and
cells
highly
more
a large
that
irregularly
in Agar
described
a feeder
on CML
cell differentiation
The morphology
of all
lB-D).
nucleolus,
round
colonies
containing
less than
100
and colonies
containing
more
than
2000
The plating
efficiency
was between
50%
(1 SD) colonies
in the plates
inoculated
agar underlayer,
and
The plating
efficiency
(Fig.
vacuoles.
granules
The nuclei
shape
dur-
in each
chromatin
the
in-
Several cells,
buds which
uncovered
often
cytoplasm
long
the
s. Poly-
few small
azurophilic
discernible
The
blue
within
proliferated
a feeder
layer.
groups
of two
colonies
5% 40-55
were
number
of colonies
was directly
proportional
as demonstrated
in triplicate
plates
simultaneously
cells without
occasionally
of
(Fig.
IA).
approx-
of the cell diameter.
of semi-moon-like
prominent.
nucleoli
and Colony-forming
1000 CML
by day 5 of
on day 14
an average
passages
fine laced
threads
which
stained
reddish
violet
with
figures
and bin ucleated
cells were also seen.
Another
six
12
G2
delimitation
between
The
cytoplasm
was
contained
and scanty
large: one-third
and, occasionally,
size.
color
to
at different
There
was not a sharp
majority
of the cells.
to four
cell
or formed
mitotic
type
and
seen
seen
with
One
blue-violet
taining
devoid
extremely
indented,
of the
granular
Numerous
cells
account for less than 2% of the cell population.
of 20 or over, had deeply basophilic cytoplasmic
in some cells were
were round,
slightly
stage
,
lasted
stage
hr.
of CML
tensely
Atypical
regardless
to a shortened
the total
cycle
a postsynthetic
of highly
undifferentiated
cells of variable
size
round
or oval cells measured
14-17
z in diameter,
seen.
in the
ing
due
LOZZIO
Cells
ploid cells were frequently
nucleus
and cytoplasm
in the cytoplasm
with a diameter
probably
of2
characteristics
those
ofthe
25%
period
ofCML
morphologic
culture
were
The majority
was
at passage
96. At this passage,
of DNA
synthesis
(5) of 8 hr,
plus
Features
cell cycle
AND
of normal
peripheral
CML
cells
was reduced
leukocytes
were
added
by a factor
on top
of 5-6,
maturation
was
in 156 colonies
observed.
examined
was
undifferentiated
cell
proliferated
mature
forms
very large (40-60
band
of deeply
of
the
in diameter)
basophilic
types
granulocytic
was
with
and
nearly
series.
added
the soft
no effect
identical
but
All
did
cells
and exhibited
numerous
cytoplasm
surrounding
the
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MYELOGENOUS
LEUKEMIA
CELL-LINE
325
Fig. 1. (A) Compositive
photograph
showing chronic myelogenous
Ieukemic (CML) cells growing in 7-day-old suspension cultures. (8-0)
CML cells developing
in semisolid
agar in a 15-day
period. A small part of a large colony is seen in (D). Both liquid and agar cultures were made
on passage 168 of the CML cell line K-562. Giemsa stain. x 540.
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326
LOZZIO
Cytogenetic
Studies
A description
a control
of the Patient
Cells
studies
of the cytogenetic
for the
cultured
abnormal
karyotypes.
phases
studied
from
chromosomes
were
chromosomes.
The
CML
cells
observed:
cells
28 cells
with
46
E (17-18)
with
apparent
gain
was interpreted
of the
to show
A positive
Ph’
peripheral
blood
patient’s
of
cells
origin
chromosome
in October
had
is given
in clones
herein
of cells
as
with
was seen in all the metha1970. Two modal
numbers
of
45 chromosomes
had
and the cells
of the group
one
LOZZIO
Cultures
their
chromosomes
otherwise normal
female karyotype,
positive
Ph’ and had lost one member
(Fig. 2), which
in Short-term
AND
and
22 cells had 46
a Ph’ chromosome
in an
with 45 chromosomes
D (13-15)
and one
had a
of group
submetacentric
of
group
C
(6-l2-X)
D/E.
as a reciprocal translocation
a’s’
-s
I
‘a”
a
II
U
U
t D;E
a
01
1
**
$1
Fig. 2. Karyotype
45,XX,-D,
-E,+t(D;E)
observed
in 24-hr
cultures
of peripheral
blood ofrom the patient 2 mo before the long-term cultures were started. Note the absence of one
chromosome from group
D and one from
group
E (probably
a number 17) and the presence of
an extra C-like chromosome. The arrow indicates this chromosome,
which was interpreted
as a
translocation
between
one chromosome of group D and one of group E. The Philadelphia
chromosome (Ph’)
is also identified
by an arrow. Fifty per cent of the cells cultured from peripheral
blood had this karyotype
and the other 50% had a karyotype 46,XX,Ph1
+.
tamed
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MYELOGENOUS
LEUKEMIA
CELL-LINE
327
,tj
4
I
V
8
11
H
t
‘
II
61
II
U
U
E;D
4.
1
-
#{149}8
Fig. 3. Karyotype
of an hypodiploid
cell showing a long acrocentric marker and a positive
Ph’ chromosome. This marker and the Ph’ chromosome were observed
in all the cells analyzed
in short-term
cultures of 24 hr from the same
pleural effusion used
to start the long-term
cultures.
The
cells
from
the
pleural
marked
aneuploidy
one hundred
cells
with a large
in metaphase
45 chromosomes.
All
chromosome
(Fig. 3) and
significantly
a consistent
Cytogenetic
Studies
Various
Passages
the
cells
effusion,
obtained
had
a Ph’
December
chromosome
larger
than
the other
loss of one chromosome
in Long-term
in
proportion
of hypodiploid
had a chromosome
number
Cultures
Chromosome
analyses
of the cultured
5, 10, 20, 40, 90, 110, 144, and 171. One
and
members
of group
of the CML
1970,
showed
cells. Ninety-two
varying
from
a long
of group
E (16-18).
Cell-line
of
40 to
acrocentric
D (13-15)
at
CML
cells were performed
at passages
hundred
cells were studied, and five to
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328
LOZZIO
ten karyotypes
were analyzed
from each passage.
the cells contained
at least
one
Ph’ chromosome,
marker
described
in the short-term
cultures
of
percentage
not directly
of cells
related
Changes
clones
with
in the modal
additional
These
the
with three or four Ph’ chromosomes
to either
the age of culture
or the
number
of chromosomes,
chromosome
markers,
were
AND
LOZZIO
studies
revealed
that all
plus
the long
acrocentric
pleural
effusion.
A small
age
was seen, but
of the cell-line
they were
K-562.
as well as appearance
of new
observed
at different
pas-
1
‘I’.
Ur
ii
U
XI
U
ii
a
ii
t(15;17)
Li
Fig. 4. Karyotype of a pseudodiploid
cell with 46 chromosomes
technique. This cell was observed
in long-term cultures of the cell-line
The banding
pattern
shows that the long acrocentric chromosome
between one chromosome 17 and one chromosome number 15, and
is a deletion of half of the long arm of one chromosome
number
extra chromosome number 1 and one number 22 and the absence
11 andonenumberl2.
..
U
stained with the G-banding
CML K-562 at passage 110.
is probably
a translocation
the Philadelphia
chromosome
22. Note the presence of an
of one chromosome
number
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MYEIOGENOUS
sages.
and
LEUKEMIA
the
By
the
the
marker
cells
maintained
have
lost
majority
sublines
in one
5, the
their
one
group
of
the
At
passage
at different
days
were
studied
subline
and
chromosome
chromosome
in addition
cells
84,
had
an
two
sublines
of the
and
At
modal
number
started
passage
1 10,
karyotypes
near
triploid
(90-96)
tetraploid
by
if cells
growing
on
agar
had
while
of the
10 and
of
50-52
performing
serial
of
the
mode
two
(69-73)
in the other
subline.
A new
subline.
These
cells had a
number
2 with
a significant
elongation
of the long
to the long acrocentric and one to four Ph’ chromosomes.
To demonstrate
(40-45),
1, or one
On passages
were
a predominant
a near tetraploid
mode
appeared
in the near
mode
(6- 12-X),
number
F (19-20).
hyperdiploid
week.
revealed
hypodiploid
C chromosome
of the cells have
lost a chromosome
ofgroup
D (13-15),
E (16-18),
or
chromosomes.
passages
329
of the cells
passage
majority
the minority
chromosomes
20,
CELL-LINE
the
same
arm
chromosome
(2q+)
markers
as those cultured in liquid medium,
several individual colonies were removed
from the agar and cultured in suspension.
The cells isolated from semisolid
agar showed
the same Ph’ chromosome
and the marker
present in the blasts
proliferating in the pleural effusion before the suspension cultures were started.
Ten
triploid
diploid
clones
were
isolated:
seven
mode
(67-73),
two
cells with
a modal
were
had two
number
aneuploid
analyses
of the hyperdiploid
clone
showed
that
grown
the hyperdiploid
cells 4 mo after the clone
types analyzed
from these clones
had one to four
acrocentric
marker.
Chromosome
analyses
at passages
the original
cloning.
studies
with
Q
the segment
22. Therefore,
that
distal
the
translocation
15 (Fig.
the
Ph’
near
one clone
had hyperRepeat
chromosome
near
triploid
cells
had
overwas established.
All the karyoPh’ chromosomes
and a long
technique
chromosome
to band
12 of the long
Ph’ chromosome
may
cording
to the nomenclature
ing studies
also
showed
number
G-banding
a predominant
and
(Fig.
4) were
performed
110 (January
1973),
144 (September
1973),
and 171 (April
1974) of
line. The hyperdiploid
clone
was studied
at passages
8 and 21 after
bands
were
investigated
at passage
144. The
G- and
Q-banding
demonstrated
reciprocal
the
with
modal
numbers,
of 52 chromosomes.
that
established
the long
between
5). This
one
translocation
at the Paris
acrocentric
24 and
17 and
the
the
one
breakage
long
of
The bandunbalanced
chromosome
of
arm
(del)
number
(q12),
ac-
Conference.22*
marker
is an
chromosome
involved
number
deletion
the
long
arm
(q) of chromosome
17 at the
15 at band
number
(q21;q24)
according
in vivo as the result
21. Therefore,
the translocation
is reported
as t(15;17)
to the Paris
Conference.22
This
translocation
originated
of clonal
evolution
and persisted
in vitro
for over
175 serial
passages.
G-bands
rangements
band
is a terminal
arm (q) of one chromosome
be designated
as a del(22)
Other
chromosomes,
including
chromosome
number
in contrast
to reports
from
other
cases
with
CML,23-24
appeared
as the result
of in vitro clonal
evolution.
In summary,
the
banding
*Explanation
of terminology
t, translocation;
for additional
studies
established
explanation
have
identified
several
at the Paris Conference:
of the chromosome
of chromosome
9, had normal
but new rear-
chromosomal
del, deletion;
aberrations
reported
struc-
q, long
see Fig. 5.
arm;
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
330
LOZZIO
AND
LOZZIO
t(15;17)(q21;q24)
17
hIIftb*bI
15
J
21
#{149}I6’O*
22
#{149}
0 A
Ph1:deI(22)(q12)fi
Fig. 5.
karyotype
Partial
of six
mitotic
cells
from
passage
171
of the
CML
fr
cell-line
stained
with the Giemsa-banding
technique
and diagrammatic
representation
of the Giemsa
bands.
The
first row shows
the translocation
of the distal
half of the long arm of one chromosome
15 to
the end of the long arm of chromosome
17. This translocation
was seen as a marker
in all the
cells studied
in the short-term
cultures
and in all the cells analyzed
at various
passages
of this
cell line. The normal
chromosomes
numbers
17, 15, 21, and 22 are shown
on the second,
third,
fourth,
and fIfth
rows. The law
row demonstrates
the terminal
deletion
seen
in all the Ph
chromosomes.
tural
aberrations,
others
which
The
two
were
modal
of
them
developed
number
present
before
during
in vitro
of chromosomes
has
the
changed
evolution
of new sublines
and clones
with
faster
numbers
of chromosomes.
Thus,
clones
containing
grown
by the cell type with the largest
chromosome
repeated
cloning
clones
with
pseudodiploid
for experimental
of the
original
pseudodiploid
CML
cells
use.
cell-line
karyotypes
with characteristic
in agar
are
cultures
were
started,
with
spontaneous
and
subcultures.
in vitro
growth
rates
and
increased
two cell types
are overnumber.
For this reason,
plates
required
chromosome
and
to
selective
maintain
aberrations
isolation
of
a source
of
available
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
MYELOGENOUS
Enzyme
in ten
Viral
CELL-LINE
331
Studies
Normal
tase
LEUKEMIA
granulocytes
exhibited
a strongly
reaction,
whereas
cultured
CML
slides
containing
several
hundred
cells
cells
positive
had
peroxidase
consistently
and
phospha-
negative
reactions
each.
Antigens
The CML
cell-line
was found
to be free of the EBV-associated
gen which
is present
in most
lymphoblastoid
cell-lines
carrying
genome.
Routine
tests for mycoplasma
were always
negative.
nuclear
the EB
antiviral
Immunoglobulins
A search
detectable
Phagocytic
Monocytes
ticles.
After
for Ig in the CML
cells
and
culture
amount
of IgA, IgG, and 1gM (Fig. 6).
media
failed
to
reveal
any
Activity
were
60 mm
more
efficient
of incubation,
than
a greater
neutrophils
number
to
(six
engulf
or more)
polystyrene
par-
of polystyrene
Fig.
6.
Quantitative
determination
of immunoglobulins
(lg)
by single
radial
diffusion.
(A-C)
Reference
sera: lgG, 4, 10, and 20 iig/ml;
lgM, 0.32,
0.80,
and 1.60 pg/mI;
and IgA,
0.8, 2.0, and 4.0 pg/mI.
(0-F)
Test samples:
(D) Undiluted
culture
medium,
(E) tenfold
concentrated
culture
medium,
and (F) disrupted
CML cell suspension
containing
20 x 106 cells/mI.
Media
and cells were from 5-day-old
cultures of the CML cell-line
K-562.
No detectable
amounts
of
IgG, 1gM, and IgA could be demonstrated
in the media and the CML cell pellet at several passages
of the culture.
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
332
LOZZIO
were
seen
in monocytes,
phils
and
eosinophils.
solid
media
did
mononuclear
whereas
two
In marked
not
cells
ingest
to five
contrast,
polystyrene
developing
particles
CML
particles.
in semisolid
were
cells
medium
observed
cultured
Also,
AND
in neutro-
in liquid
none
of
the
phagocytize
LOZZIO
or semi-
large
CML
effusion
of
agar.
DISCUSSION
We
described
herein
patient
with
CML.
permanent
cell-line
prolonged
in vitro
this cell-line
is due
cells obtained
These
cells
a unique
from
the patient
were
derived
from
chromosome
Rearrangements
rearrangement
ofthe
long
patients25’26
in blastic
rate.
The
leukemic
blood4
may
crisis
from
a pleural
at the terminal
a clone
with
and
might
stage
a Ph’
long
be another
origin of the cultures
derived
of an acute
chromosome
blastic
and
proliferative
pleural
fluid
capacity
and were
with
a
most
were
chromosome
17.
reported
in CML
cause
increased
of the
by others
from
growth
CML
leukemic
of normal
the critical factor could be the absence of normal
used
to start
our
cultures.
The
malignant
CML
well in suspension
derived
from
which
were
not overgrown
permanent
myeloblastic
crisis.
a new
arm
of one
17 have been
be the result of differential in vitro proliferative capacity
blasts
(K-562)
proliferated
karyotype
because
they
The
derived
involving
the
arm ofchromosome
and leukemic cells. Indeed,
cells in the pleural
effusion
occurred
blood.4
cell-line
As far as we are aware,
the CML-l,
K-562
is the first
available
with
a persistent
positive
Ph’ chromosome
after
cultivation.
We believe
that the successful
establishment
of
to the active
proliferative
capacity
of highly
undifferentiated
cultures
and retained
the original
a clone
of malignant
cells with high
already
growing
as
by the proliferation
cell-lines
cell-line (K-562)
a cell suspension
of normal
cells,
established
described
from
human
herein differs from
blastoid
cell lines described
by others
(see reference
The CML
cells have a positive
Ph’ chromosome,
in the
as has
leukemic
the lympho-
4) in many
characteristics.
they
lack immunoglobulins,
they are negative
for EBV or herpes-like
virus,
and began
to proliferate
actively
as soon
as the suspension
cultures
were
started.
In contrast,
lymphoblastoid
cell-lines
established
at other
laboratories
began
to proliferate
rapidly
after
a
latent
period
of 4-15
wk,4’27#{176}during
which
the number
of cells decreased
significantly.
particles
After
were
establishment
detected
of the
lymphoblastoid
cell-line,
by electromicroscopy,4’27-30’33
and
the EBV
genome.4”6’3”
It is believed
that
an interaction
human
diploid
cells is essential
for the establishment
of
blastoid
cell-line.
Also,
the majority
of these
cell-lines
globulins.4’30’3536
Preliminary
(K-562)
fail to show
herpes-like
electron
virus
and
failure
herpeslike
virus
globulins
are three
line (K-562)
is not
presence
of
the
Ph’
is of CML
origin.
As expected,
the
and
the
important
another
microscopic
particles.
of this
differential
culture
of
chromosome
undifferentiated
CML
CML
cell-line
cells
were
devoid
virus
carried
EBV
and
lymphoimmuno-
of CML
cells
absence
of EBV
to produce
that
cells.
demonstrates
herpeslike
cell-lines
between
a permanent
synthesize
examinations
Therefore,
the
characteristics
lymphoblastoid
conclusively
most
immuno-
indicate
Furthermore,
that
that
cell-line
of alkaline
this
phospha-
the
the
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
MYELOGENOUS
tase
LEUKEMIA
and
333
CELL-LINE
peroxidase
activity,
and
lacked
a property
of more differentiated
The CML
cell-line
has been
antigen.37
Work
in progress
sorbed
on normal
granulocytes
CML
cells
obtained
from
may
be of value
for
indicates
that
is cytotoxic
untreated
ity of the rabbit
anti-CML
from patients
with CML
the
cell-line
nancy:
gen(s)
chromosome
after
prolonged
human
cells
inert
patients
with
this
the detection,
prognosis,
disease.
and/or
which
has
aberrations,
cultivation.
derived
clonal
of
The
retained
meaningful
and
clinical
adfor
cross-reactiv-
of CML.
undifferentiated
is the first
indicators
cloning
efficiency
on agar,
Therefore,
it represents
for experimental
a specific
those
obtained
antigen,
which
immunotherapy
from
evolution
particles,
K-562
serum
CML
cells and
serum
with cultured
CML
cells and
before
treatment
suggests
a common
available
CML
of ingesting
cells and macrophages.
a continuous
source
a rabbit
anti-CML
for cultivated
In summary,
this new CML
cell-line
logenous
leukemic
cells with
karyotypic
CML
capacity
granulocytic
found
to be
myehuman
of
malig-
and a specific
antia unique
source
of
studies.
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1975 45: 321-334
Human chronic myelogenous leukemia cell-line with positive Philadelphia
chromosome
CB Lozzio and BB Lozzio
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