Chromosome Evolution of Near-Haploid Clones in an Established
Human Acute Lymphoblastic Leukemia Cell Line (NALM-16) 1.2.3.4
Sei-ichi Kohno,
5
Jun Minowada,
5
and Avery A. Sandberg
Cells with a haploid chromosome constitution seem
to have difficulty retaining their viability and main"
taining their karyotype. Only a few reports have
appeared concerning haploid cells in vertebrates, i.e.,
haploid chick embryos (1, 2), haploid mouse, Syrian
golden hamster, and Chinese hamster cells in cleavage
(3-5), and haploid mouse cells in culture (6, 7). The
only known established and stable haploid cell line
was derived from an artificially produced haploid
embryo of Rana pipiens (8-10).
Haploid cell lines provide interesting and informative material for somatic cell and cancer genetic studies, especially in humans, because the contribution of
haploid alleles, whether dominant or recessive, is an
unnecessary concern.
In recent years, reports of near-haploid cell clones in
human hematopoietic disorders have appeared (11-16),
but reports of near-haploid established cell lines in
vitro have not. We (12) reported an ALL patient with a
near-haploid cell clone of 27 chromosomes. From the
lymphoblasts of this patient, a cell line, including
near-haploid cell clones, was established. We present
here the results of cytogenetic and immunologic studies
of this cell line.
tory, and cytogenetic findings of the patient with nearhaploid ALL from whom the cells were obtained have
been detailed in (12). Blood lymphoblasts from this 12year-old white female in relapse were cultured on
February 8, 1977; from this culture a permanent cell
line, NALM-16, was subsequently produced. The cytogenetic study (12) of clones of her bone marrow showed
near-haploid cells with 27 chromosomes. The cell line
was maintained in RPMI-1640 with 10% heat-inactivated fetal calf serum at 37° C in a S% C02 incubator.
At the time of culture, the patient's peripheral blood
findings were: white blood cells, 123,SOO/mm3 ; platelets, 37,SOO/mm3 ; hematocrit, 32.S%; and lymphoblasts.
90.0%.
Cytogenetic studies. - W e studied the chromosomes
of this cell line on five occasions: February 8, 1977
(start of culture), June 7, 1977 (4 mo later), September
28, 1977 (=8 mo later), December IS, 1977 (=10 mo
later), and May 17, 1978 (=IS mo later). The cells
examined on February 8 and September 28, 1977, were
frozen in liquid nitrogen on these dates. Active cultures
were reestablished on May 18 and May 23, 1978,
respectively. Except for these 2 lines, examined cells
were kept in continuous culture from the initiation
date (2-8-77).
Cells were harvested after exposure to 0.01 p.g Colcemid/ml for 2 hours, processed with hypotonic O.S%
KCl solution at 37° C for 20 minutes, and fixed with
acetic acid-methanol (l:3). The slides for chromosome
- studies were made by the usual air-drying method. Qbands and BrdUrd-acridine orange fluorescence patterns were determined with methods modified from
Caspers son et al. (17) and Zakharov and Egolina (18),
respectively.
ABBREVIATIONS lISED: ALL=acute lymphoblastic leukemia; BrdUrd=
5-bromodeoxyuridine; cALL=antigen associated with null-cell ALL;
CML = chronic myleocytic leukemia; E = sheep erythrocyte; EAC =
erythrocyte-anti body-complement; la = la-like human B-cell-associated antigen; Q=quinacrine fluorescence; Smlg=surface membrane
immunoglobulin; T-LCL=T-cells from Iymphoblastoid culture line.
Received May 3, 1979; accepted July 27, 1979.
Supported in part by Public Health Service grant CAI4555 from
the National Cancer Institute.
3 Presented at the Annual Meeting of the American Society of
Human Genetics held in November 1978 in Vancouver, Canada.
4 Research procedures were in accord with the ethical standards of
the Committee on Human Experimentation, Roswell Park Memorial
Institute.
5 Roswell Park Memorial Institute, Buffalo, N.Y. 14263.
6 Address reprint requests to: Dr. Sandberg, Roswell Park Memorial Institute, 666 Elm St., Buffalo, N.Y. 14263.
I
2
MATERIALS AND METHODS
Establishment of cell line.- The clinical, labora-
485
lNCI, VOL. 64, NO.3, MARCH 1980
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ABSTRACT -A cell line has been established from blood Iymphoblasts of a female patient with acute lymphoblastic leukemia
(ALL) shown to have near-haploid (27 chromosomes) cells In the
bone marrow. The findings about the cell line were: 1) The
frequency of near-haploid cells In culture decreased with time
from 98.2% when the culture was started to 5.4% 15 months later.
2) Most of the other cells except the near-haploid ones were
hyperdiploid, I.e., duplicates of the cells with near-haploid chromosome constitutions. 3) Chromosome evolution was seen In the
near-haploid clones. The possible ancestor clone (clone A) had
27 chromosomes, one of each pair except #10, 14, 18, and 21,
which were dlsomlc. A suggested evolution process Is: clone
A~clone B (26 chromosomes: clone A, -#10)~clone C (27
chromosomes: clone B, +X)~clone D (26 chromosomes: clone C,
-#21), clone E (28 chromosomes: clone C, +#20). Clones Band
D, each with 26 chromosomes, appeared to contain the lowest
number of chromosomes ever described for human somatic cell
clones In vitro. 4) Changes In the constitutions of the hyperdiploid celi clones were preceded by evolution and changes In the
near-haploid clones. 5) In near-haploid celis with 2 X-chromosomes, 1 exhibited late DNA replication; In hyperdiploid cells
with 3-5 X-chromosomes, 2 were non-late DNA-replicating. 6)
Fresh (uncultured) and cultured leukemia cells were antlgenlcaliy
typical non-T, non-B or common type ALL cells (positive for lalike and nUll-type ALL antigens and negative for surface
membrane Immunoglobulin).-JNCI 64: 485-493, 1980.
5. 6
486
Kohno, Minowada, and Sandberg
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Frequency of Near-Haploid Cells
The number of chromosomes in NALM-16 cells was
studied five times in 15 months. The data are summarized in table 1, including the data previously
obtained from fresh blood lymphoblasts. The distribution of NALM-16 cells in the near-haploid and hyperdiploid ranges is shown in text-figure 1. All the fresh
blood lymphoblasts obtained 1 month before NALM-16
was started were near-haploid except for 2 (2/135 == 1.5%)
with normal karyotypes. Cells with 27 chromosomes
constituted 83.0% (112/135) of the observed metaphases,
and no hyperdiploid cells were observed.
In the fresh blood sample obtained on February 8,
1977, from which NALM-16 was started, 86.5% (96/1 U)
of the cells had a near-haploid constitution with 27
chromosomes. Two hyperdiploid metaphases (2/111 ==
1.8%) with 54 chromosomes were encountered among
the blood cells. After 4 months (6-7-77), two chromosome modes, 27 (311190== 16.3%) and 54 (44/190==
23.1 %), were observed. If we consider the near-haploid
range as 27±3 chromosomes and the hyperdiploid as
54±6 chromosomes, 35.8% of the cells were near-haploid in this sampling, an extreme decrease from the
98.2% in the previous observation; 47.4% of the cells
were in the hyperdiploid range, a substantial increase
from the low value of 1.8%.
The frequency of cells in the near-haploid range
progressively decreased, i.e., 35.8% (67/190) at 4 months,
31.2% (62/199) at 8 months, and 19.5% (411210) at 10
months after culture, whereas the frequency of cells in
the hyperdiploid range progressively increased, i.e.,
50.5% (96/190), 59.3% (1111199), and 67.6% (142/210),
respectively. Also, on September 28, 1977, hyperdiploid
cells with more than 54 chromosomes were observed,
and their frequency increased with time. After 15
months, the cells with a near-haploid number of chromosomes constituted only 5.4% (111203) of the cells;
most of the cells (186/203 == 91.2%) were hyperdiploid.
JNCI, VOL. 64, NO.3, MARCH 1980
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Cell surface marker analysis.-Mononuclear cell populations were isolated from heparinized blood by FicollHypaque gradient centrifugation. The cells were washed
three times with phosphate-buffered saline and tes·ted
for various cell surface markers. Specificity of marker
expression and test procedures have been detailed in
(19, 20). We considered E-rosette formation as a
definitive T-cell marker, and the presence of Smlg as a
definiti ve B-cell marker. Other cell surface markers,
though each may not have been specific for T- or Bcell subpopulations, were used to define specific
immunologic subtypes of particular ALL cells.
Receptors for EAC were detected by IgM antibodysensitized bovine erythrocytes coated with mouse
complement, and for Smlg by direct membrane
immunofluorescence. Antigens specific for T-LCL, la,
and cALL were detected by indirect membrane
immunofluorescence with respective specific antisera.
Near Haploidy in ALL Cell Line
NUMBER
OF CELLS
OBSERVED
DATE
90
487
Ii I I I
50
2-8-77
10
111
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o
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190
0>
.-<
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to-
.-<
<0
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to
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199
5-17-78~03
27
46
50
54
59
NUMBER OF CHROMOSOMES
TEXT-FIGURE I.-Distribution of cells from NALM-16 in near-haploid and hyperdiploid ranges_ The top graph shows the cell distribution in the parent blood from which NALM-16 was derived_
The lower four graphs show the distributions 4, 8, 10, and 15
rna after NALM-16 was established_
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Clonal Evolution of Near-Haploid Cells
During the period of observation, the karyotypes of
the near-haploid cells underwent changes without significant (if any) alterations in the number of chromosomes_ In the near-haploid population, we found 5
clones that had different chromosome constitutions;
these are summarized in table 2, which also includes
data from the 6 published leukemia cases with nearhaploid clones in vivo_ The clonal evolution of NALM16 is also shown in text-figure 2_ The near-haploid
clone A with 27 chromosomes was found in the bone
marrow of the patient when first examined (12) and
was present also in her blood before culture_ This
clone had only 4 disomic pairs of chromosomes: #10,
14, 18, and 21; all other groups were monosomic (case
2, table 2)_ Clone B, observed in the blood and in
cultured cells (fig_ 1), was produced by the loss of one
disomic #10 chromosome from clone A_ Clone C, also
seen in the blood and cultured cells (fig_ 2a), originated
from clone B by duplication of one X-chromosome_
Clone C led to clones D (fig_ 3) and E (fig_ 2b)_ Clone
D was produced by loss of a #21 chromosome, and
clone E by duplication of a #20_ Clones D and E were
observed only in culture_ Hyperdiploid cell clones,
representing duplication of the near-haploid cell clones,
were also observed; further chromosome changes took
place within these hyperdiploid clones_
In five samples of the NALM-16 line examined, the
proportion of cells in clones A-E changed with time:
In the initial sample on February 8, 1977, more than
90% of the cells were in clone A and only a small
MM
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JNCI, VOL. 64, NO_ 3, MARCH 1980
Downloaded from http://jnci.oxfordjournals.org/ at Pennsylvania State University on May 10, 2016
~210
12-15-77
488
Kohno, Minowada, and Sandberg
OATE
/~6"
/@If
"\
27
TABLE 3.-Percent of cells having cell surface antigen markers a
Date
E
EAC
Smlg
T-LCL
Ia
5-13-76b
12-27-76'
2_8_77d
6-8-77
6-30-77
9-25-77
1-10-78
4-11-78
8-24-78
9-1-78
8
11
0
0
0
0
0
0
1
3
0
0
0
0
0
0
2
0
0
0
4
39
0
0
0
3
0
90
90
100
85
89
95
100
100
0
0
/'
o
Jt'
CLONE A
.!!:
\ \ @]
ti9 f27\--~
~\tV~~7.X.+IO,+14,+18,+2I,7~ Q
>
.!':
@
I
27,X,+IO,+14,+18,+21
12-27-76 -
~,+X'+14,+18.+21
2-8 -77 -
~
6-7-77-~·
\E.9-
@]
-21
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i
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CLONE E
12-15-77-
I
28, X.+X.+14.+18.+20,+21
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5-17-78-1
TEXT·FIGURE 2.-Schematic presentation of the clonal evolution of
NALM-16. The description of the chromosome numbers and
clones is based on the Paris Conference nomenclature (\971) modified for haploid cells; e.g., 26,X,+14,+18,+21 indicates a haploid
set plus extra chromosomes in groups #14, 18, and 21. 0 = Chromosome number, 0 = chromosome change, D = duplication,
- - = course of chromosome evolution, and - - - - - = possible
cause of chromosome evolution.
percentage was in clones Band C. On June 7, 1977,
however, clones D and E constituted more than 60% of
the cells, and by May 17, 1978, only clones D and E
were encountered.
Chromosome Constitution of Hyperdiploid Cells and
X-Chromosome Inactivation
The changes in the constitution of the hyperdiploid
clones were preceded by evolutionary changes in the
near-haploid clones. A hyperdiploid clone with 2 #7p+
chromosomes, originally described by Oshimura et al.
(12), was not seen in NALM-16. The hyperdiploid
clones with 2 X-chromosomes, probably duplications
of clones A and B, were the first hyperdiploid clones
observed in NALM-16. These clones were replaced by
clones with 4 X-chromosomes, probably duplications
of clones C, D, and E. Hyperdiploid clones with 3 and
5 X-chromosomes were also observed: The former may
be produced by the addition of 1 X-chromosome to the
cells in clones with 2 X-chromosomes or by a deletion
from the clone with 4 X-chromosomes; the latter may
be produced by addition of 1 X-chromosome to the
clone with 4 X-chromosomes. The autosomal groups
with trisomy or tetrasomy in the hyperdiploid cells
were #10, 14, 18, 19, 20, and 21.
JNCI. VOL. 64, NO.3. MARCH 1980
85
100
80
80
91
100
100
91
100
-=Not done.
Fresh blood; percent of lymphoblasts= 95.
, Fresh blood; percent of lymphoblasts = 75.
d Fresh blood samples from which NALM-16 originated; percent of lymphoblasts= 100.
a
b
Cytogenetic and Immunologic Findings
BrdUrd-acridine orange banding revealed 1 of the 2
X-chromosomes of the near-haploid cells to be late
DNA-replicating (fig. 5). In hyperdiploid cells with 3,
4, or 5 X-chromosomes, 2 such chromosomes exhibited
early DNA replication and those remaining exhibited
late DNA replication (fig. 6). In the duplicated hyperdiploid cells, the inactivation of the X-chromosomes
repeated the phenomenon encountered in the original
near-haploid cells.
The cell surface and antigen markers in NALM-16
are summarized in table 3. Values for E-rosettes (8%,
11 %), SmIg (2%, 0%), and antigen specific for T-LCL
(4%, 39%) showed some residual normal T- and B-cells
in the fresh blood sample of 1976. When NALM-16
was started, almost all the cells were antigenically
typical non-T, non-B common type ALL cells (Erosette-, EAC-, SmIg-, antigen specific for T-LCL-,
Ia+, and cALL+). Throughout the 19 months of subculture, despite the shift to a hyperdiploid karyotype,
the antigenic characteristics have remained consistent
and stable. This observation indicates that the expression of these cell surface markers is apparently independent of the ploidy of the cells.
DISCUSSION
To date, near-haploid clones have been described for
6 patients with hematopoietic disorders (4 with ALL
and 2 with CML). The chromosome constitution in the
clones of these 6 patients is summarized in table 2.
Kessous et al. (11) reported a clone with 27 chromosomes in a 4l1-year-old female ALL patient (table 2,
case 1). Oshimura et al. (12) reported 2 cell clones with
27 chromosomes in a 12-year-old female ALL patient
(case 2), the donor of the cells of NALM-16. In these 2
cases, the near-haploid cells were accompanied by cells
with 54 chromosomes. The chromosome constitution
of case 1 resembled that of case 2 (table 2). Except for
an addition to the short arm of chromosome #7 in one
of the clones of case 2, only two differences existed
Downloaded from http://jnci.oxfordjournals.org/ at Pennsylvania State University on May 10, 2016
\,,'H""r~ ~ ~5~
9- 28-77-
100
cALL
Near Haploidy in ALL Cell Line
(11-29-76 and 12-27-76). At diagnosis, no cytogenetically normal cells were encountered; instead, of the 210
cells in metaphase examined, about 20% were near-haploid and the remaining were hyperdiploid (54 chromosomes). During remission only diploid cells were observed. At relapse, 20-40% of the cells in the marrow
were diploid and the remaining were near-haploid. On
November 29, 1976, 2 of the 30 cells examined had 54
chromosomes. Clone A', containing an abnormal #7 in
all cells, was seen only at diagnosis and was never
encountered again. In contrast to these findings, the
line containing 54 chromosomes, present in more than
70% of the marrow cells at diagnosis, occurred in only
a few of the cells during relapse 6 months later,
whereas the near-haploid line (clone A) increased from
about 20% at diagnosis to 75% on relapse. In vitro,
however, the frequency of near-haploid cells decreased
significantly, and hyperdiploid cells, representing duplicates of the near-haploid chromosome constitutions,
increased proportionately.
Initial examination of the bone marrow revealed a
mixture of small and large lymphoblasts, and we
thought that the small cells gave rise to the near-haploid metaphases because the percentage of such small
lymphoblasts tended to change in proportion to the
number of near-haploid cells that we observed. A
similar situation existed under culture conditions; very
few small cells were present in the last sample examined on May 17, 1978, when the percent of nearhaploid cells was the lowest «5%).
The chromosome evolution from near haploidy to
hyperdiploidy in the NALM-16 line was reflected by
the number of some autosomes or X-chromosomes.
Oshimura and coworkers (12) pointed out that the
most likely possibility is that the cells with 27 chromosomes constituted the stem (s) line of the leukemia and
that the cells with 54 chromosomes represented 2s cells.
They listed the following reason for this hypothesis:
The hyperdiploid cells with 54 chromosomes had 2
intact #7p+ marker chromosomes, which are unlikely
to have been produced by two separate events but must
have been produced by duplication of a near-haploid
cell with one marker.
Our observations of the hyperdiploid clones support
this opinion because: 1) The appearance of a hyperdiploid clone was preceded by the existence of a near-haploid clone, the latter serving as a parent for the former;
and 2) 2 active (early-replicating) X-chromosomes existed in hyperdiploid cells, representing a duplicate
pattern of the near-haploid cells.
We observed a NALM-16 clone with 49 chromosomes (fig. 4). Three chromosome groups (#5, 6, and 9)
were monosomic, and 4 (#14, 18, 20, and 21) were
trisomic. Most likely, a clone with duplicated chromosomes of a near-haploid cell was originally produced,
with a subsequent loss of one of the #5, 6, 9, or other
chromosomes. This suggests that the hyperdiploid
clones were not stable and developed their chromosome
constitutions because of this instability.
In a recently published case of CML (21), a hypolNCI, VOL. 64, NO.3, MARCH 1980
Downloaded from http://jnci.oxfordjournals.org/ at Pennsylvania State University on May 10, 2016
between these 2 cases; i.e., the monosomy of #14 in case
I contrasted with its disomy in case 2, and the disomy
of the X-chromosome in case I contrasted with its
monosomy in case 2. A near-haploid clone in an ALL
patient (l4-year-old male, case 3) was reported by
Prieto et al. (13). Except for one chromosome in group
D (#14) and I in group G (#21), all other autosomes
were monosomic, and the X- and Y-chromosomes
persisted in all cells. Kaneko and Sakurai (16) described a young girl with near-haploid ALL in whom
most of the marrow cells had 28 chromosomes (table
2); some metaphases containing 56 chromosomes were
also encountered. Except for disomy of #6, this case
had a chromosome distribution very similar to that of
the other cases of near-haploid ALL.
Daniel et al. (14) observed a near-haploid cell line
from a 58-year-old male in the blast phase of CML
(case 4) and reported 24 chromosomes with a PhI
translocation between #9 and 22. In this clone, all the
22 autosomes were monosomic; each cell contained an
X- and a Y-chromosome. These findings suggest the
possibility that human somatic cells are capable of
proliferating and maintaining their characteristics under an autosomally haploid condition.
In another case of CML in the blast phase in a 55year-old female (case 6, table 2), most unstimulated
(without phytohemagglutinin) cells in the blood and
marrow contained a near-haploid karyotype [26,XX,
t(9;22)(q34;qll),+8,+2I] (15). The near-haploid cells
(23) in this case contained 2 X-chromosomes and were
disomic for #8 and 21; all other groups contained a
haploid set. Most of the blasts were small lymphoblast-like cells. As in the case in Daniel et al. (14), the
PhI persisted in all of the near-haploid cells. These
authors (14, 15) indicated that a haploidy event is
probably responsible for generating these clones, with
selective survival of the PhI-positive clone having taken
place rather than the generation of near-haploid cells
through chromosome loss.
An analysis of the 10 near-haploid clones (5 in vivo
and 5 in vitro) of the 4 cases of ALL and of cell line
NALM-16, as well as those of the 2 near-haploid CML
cases (table 2) revealed the following: I) The chromosome numbers of these near-haploid clones varied from
24 to 28; 2) neither was the karyotype consistent in
these clones in vitro or in vivo nor were chromosome
groups always disomic; 3) some chromosome groups
tended to be disomic in near-haploid ALL-#IO, 18,
and 21 were disomic in 3 cases in vivo and #18 and 21
were disomic in most of the clones derived from case 2;
4) #10 was disomic in 3 cases, but 4 of 5 clones of
NALM-16 were monosomic for #10; and 5) 5 of the 6
cases (table 2) and 3 in vitro clones had a full set of sex
chromosomes. Clones C, D, and E in NALM-16 had 2
X-chromosomes. These clones were derived from case
2, which is the only one of the 4 described with cells
with a single sex chromosome.
The bone marrow chromosomes of case 2 were
studied on four occasions (12): at diagnosis (5-13-76),
following full remission (10-14-76), and during relapse
489
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REFERENCES
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1974;71 :4416-4420.
(9) - - . Chromosomal evolution in a haploid frog cell line: Implications for the origin of karyotypic variants. Chromosoma 1977;62:1-15.
(10) - - . Haploid vertebrate cell cultures. In: Rothblat GH,
Christofolo VJ, eds. Growth, nutrition and metabolism of
cells in culture, vol. 3. New York: Academic Press, 1977:59-82.
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Fr Hematol Blood Cells 1975;15:73-82 (in French).
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clone: 24,XY,t(9;22)(q34;qIl) from a patient in blast crisis of
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(15) HARTLEY SE, COOK MK. Near-haploidy in a case of chronic
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(16) KANEKO Y, SAKURAI M. Acute lymphocytic leukemia (ALL) with
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(21) CoMO RM, GRAZE PRo Emergence of a cell line with extreme
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diploid line with 35 chromosomes appeared at blast
crisis. Most of the blasts were of the myeloid variety.
Chromosome groups (#10, 14, 18, and 21) that were
found to be disomic in previously published cases.of
leukemia with near haploidy (table 2) were also disomic
in this case (21). In addition, due to a t(9;22), the Phi
and the X- and V-chromosomes were still present in
the cells of this patient, as in the cases of CML in the
blast phase described by Daniel et al. (14) and by
Hartley and Cook (15). This case (21) may provide
evidence for the evolution of near-haploid cells, particularly because cells with less than 35 chromosomes
were also observed.
Oshimura and co-workers (12) postulated several
mechanisms for the formation of the original leukemia
near-haploid stem line: double reduction division of
tetraploid nuclei in "hybrid-cell types" (7), somatic
pairing (22), or multipolar mitosis (23); they suggested
multipolar mitosis as the most likely mechanism by
which the near-haploid cells were produced.
The clonal evolution of NALM-16, especially its
shift to hyperdiploid cells, could be a reaction to in
vitro conditions. Throughout the 15 months of culture,
only 2 near-haploid cells with chromosome rearrangements were observed. One was a cell with a translocation between #9 and 18, and the other had an additional part on the long arm of a #21 chromosome.
Neither of these cells proliferated sufficiently to produce a large clone. In contrast to the clonal evolution
in a haploid frog cell line (9), in which the chromosome changes primarily involved translocations, chromosome evolution in NALM-16 proceeded through
loss or gain of whole chromosomes or through duplication of the whole chromosome set.
Near Haploidy in ALL Cell Line
491
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l.-Q-banded metaphase and its karyotype of clone B with 26 chromosomes showing monosomy of #10 and only I X-chromosome.
2.-Q-banded karyotypes. a) Clone C, with 27 chromosomes, shows chromosome constitution of clone B but with duplication of Xchromosome. b) Clone E, with 28 chromosomes, shows chromosome constitution of clone C but with duplication of #20.
FIGURE
FIGURE
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Kohno, Minowada, and Sandberg
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3.-Q-banded metaphase and its karyotype of clone D showing the chromosome constitution of clone C but with a loss of I #21 chromosome.
FIGURE 4.-Q-banded hyperdiploid clone with 49 chromosomes showing monosomy of #5, 6, and 9, trisomy of #14, 18, 20, and 21, and
tetrasomy of the X-chromosome.
FIGURE
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Near Haploidy in ALL Cell Line
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FIGURE
5.-BrdUrd-acridine orange-banded metaphase and its karyotype of clone E showing the late-replicating X-chromosome as pale and
long.
FIGURE
6.-BrdUrd-acridine orange-banded metaphase with 5 X-chromosomes, 2 of which are early-replicating and 3 are late-replicating.
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