|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 2 (January 15), 1998:
pp. 641-648
Suppression of Cell Proliferation and the Expression of a
bcr-abl Fusion Gene and Apoptotic Cell Death in a New Human
Chronic Myelogenous Leukemia Cell Line, KT-1, by Interferon-
By
Kohsuke Yanagisawa,
Hayato Yamauchi,
Masahiko Kaneko,
Hidehisa Kohno,
Hitoshi Hasegawa, and
Shigeru Fujita
From the First Department of Internal Medicine, School of Medicine,
Ehime University, Ehime; and the Department of Internal Medicine,
Uwajima City Hospital, Ehime, Japan
 |
ABSTRACT |
A new human leukemia cell line, KT-1, was established from a patient
in the blastic crisis phase of chronic myelogenous leukemia (CML). This
cell line had a positive reaction for intracytoplasmic myeloperoxidase
and two Philadelphia chromosomes (Ph1) [t(9;22)(q34;q11)] and lacked normal copies of chromosomes 9 and 22. Molecular
characterization of the breakpoint in the t(9;22)(q34;q11) showed that
KT-1 had a bcr-2/abl-2 splice junction. When the KT-1 cells
were cultured with interferon (IFN)- or IFN- , the growth of the
cells were dose-dependently suppressed. IFN- and IFN- exerted
synergistic suppressive effects on the growth of KT-1 cells.
Furthermore, IFN- suppressed the expression of the bcr-abl
fusion gene in KT-1 cells, and induced G1 cell-cycle arrest and
apoptotic cell death. The KT-1 cell line should be a valuable tool for
studying the molecular mechanism of the suppression of Ph1
clone cells from CML by IFN.
| |
INTRODUCTION |
CHRONIC MYELOGENOUS leukemia (CML) is a
clonal myeloproliferative disorder resulting from oncogenic
transformation of a hematopoietic stem cells. The hallmarks of CML
include Philadelphia chromosome (Ph1) translocation
[t(9;22)(q34;q11)]1 and consistent molecular genetic
aberrations. The c-abl proto-oncogene, normally located on
chromosome 9q34, is moved to chromosome 22q11 and juxtaposed with a
breakpoint cluster region (bcr).2 The abnormal
configuration results in the creation of a hybrid bcr-abl fusion gene, which transcribes a novel 8.5-kb mRNA.3 The
bcr-abl fusion transcript forms an abnormal 210-kD protein with
augmented tyrosine kinase enzymic activity.4,5 These
molecular changes are thought to play a significant role in the
pathogenetics of this malignancy and confer a growth advantage on
Ph1-positive CML cells over normal hematopoietic
precursors. On the other hand, bcr-abl fusion protein induces
the suppression of apoptotic cell death that results in the
accumulation of myeloid cells to a greater extent.6-8
Indeed, the bcr-abl fusion protein has shown its oncogenic
potential by transforming hematopoietic progenitor cells in vitro and
in vivo.9,10 Interferon (IFN) is the most efficient
treatment for CML patients who cannot undergo allogeneic bone marrow
transplantation.11-14 In approximately 10% to 20% of
patients under IFN- therapy, the bone marrow is rendered negative
for the Ph1 chromosome, indicating cytogenetical
remission.12,14,15 Moreover, reverse-transcriptase
polymerase chain reaction (RT-PCR) amplification does not detect
bcr-abl mRNA transcripts in bone marrow cells from some CML
patients who maintain cytogenetic remission,16,17 although,
even in these cases, bcr-abl transcripts persist in single
progenitor colonies. However, the exact mechanism of the inhibitory
action of IFN has been unknown. Here, we describe the establishment and
characterization of a new human leukemia cell line derived from a CML
patient in the blastic crisis phase, which we designated KT-1. KT-1
carried two copies of the Ph1 chromosome and lacked normal
copies of chromosomes 9 and 22. Either IFN- or IFN- suppressed
the growth of KT-1 cells. Furthermore, we have demonstrated that
IFN- suppressed the expression level of bcr-abl mRNA in KT-1
cells, and induced G1 cell-cycle arrest and apoptotic cell death. We
suggest that the KT-1 cell line will be valuable for investigating the
mechanism of the suppressive effects of IFN on Ph1 clones
from CML.
| |
MATERIALS AND METHODS |
Case report.
The cell line described here was derived from peripheral blood from a
32-year-old man with CML in blastic crisis. In December 1991, he was
diagnosed with CML. Karyotypes of his bone marrow cells showed that
they were 46, XY, t(9;22)(q34;q11). The patient was treated with
busulfan and 6-mercaptopurine. On February 24, 1994, he was admitted
because of fever and back pain. His peripheral blood count results were
as follows: red blood cells (RBCs), 312 × 104/µL;
hemoglobin, 7.9 g/dL; hematocrit, 23.7%; platelets, 2.8 × 104/µL; and white blood cells (WBCs), 1.8 × 104/µL, with 49% blasts. The bone marrow had an 8.8 × 104/µL nucleated cell count (NCC), with 67% blasts.
Cytogenetic analysis of bone marrow cells showed 50, XY, +8, +8,
t(9;22)(q34;q11), t(9;22)(q34;q11), +19, +19. The patient's blasts
were negative for myeloperoxidase (MPO), terminal deoxynucleotidyl
transferase (TdT), and lymphoid antigens, except for CD4 and CD7, and
were positive for CD33 antigen. He was diagnosed as having CML in
blastic crisis. Chemotherapy, initially with vincristine and
prednisolone and subsequently with cytarabine and aclarubicin, showed
no effects. The patient died of cerebral bleeding on March 18, 1994.
Cell culture.
Mononuclear cells (MNCs) were isolated from the patient's peripheral
blood in the blastic crisis phase, using a Ficoll-conray gradient. The
MNCs were suspended in RPMI 1640 medium supplemented with 10% fetal
calf serum (FCS; GIBCO, Grand Island, NY) in 24-well tissue culture
plates (Corning Glass Works, Corning, NY) and incubated at 37°C in a
humidified atmosphere of 5% CO2. Half of the medium was
replaced twice weekly. When the cell line had been established, cells
were maintained in tissue culture flasks (no. 3013; Falcon, Oxnard,
CA).
Cell morphology and cytochemical stainings.
A cytocentrifuge preparation of cells was made by using a Shanden
Cytospin (Shandon Southern Product, Cheshire, UK) and stained with
May-Grünwald-Giemsa solution. The cells were also cytochemically stained for MPO, naphtol ASD chloroacetate esterase (CAE),
alpha-naphthyl butyrate esterase (NBE), periodic acid-Schiff (PAS),
and TdT.
Intracytoplasmic staining.
Immunocytochemical staining for intracytoplasmic MPO was determined by
means of the alkaline phosphatase/anti-alkaline phosphatase (APAAP)
technique using the monoclonal antibody, MPO7 (Dakopatts A/S, Santa
Barbara, CA).18
Surface-marker analysis.
Surface markers of cells were analyzed by fluorescence-activated cell
sorting (FACS). The monoclonal antibodies used recognized the following
antigens: CD2, CD3, CD4, CD8, and CD11b, which were purchased from
Ortho (Ortho Diagnostic Systems, Raritan, NJ); CD5, CD10, CD19, CD20,
HLA-DR, CD34, and CD41a, which were purchased from Becton Dickinson
(Mountain View, CA); and CD13, CD14, and CD33, which were purchased
from Coulter Immunology (Hialeah, FL). These antibodies were used in a
direct staining technique.
Cytogenetic analysis.
Cytogenetic analysis was performed on KT-1 cells in the logarithmic
growth phase. Cells were treated with 0.075 mol/L potassium chloride
hypotonic solution for 30 minutes at 37°C and fixed in methanol
acetate. Chromosomes were banded by the trypsin-Giemsa method.19
Analysis for bcr rearrangement.
Fifteen micrograms of DNA of KT-1 cells was digested with the
restriction enzymes, BglII and BamHI, electrophoresed
on 0.8% agarose gel, blotted, and hybridized. After hybridization, the filters were washed, dried, and autoradiographed.
Analysis of the bcr-abl splice junction.
RNA extraction and RT-PCR for bcr-abl were performed by the
techniques, and using the primers, described
elsewhere.20,21
Northern blot analysis of bcr-abl mRNA expression.
KT-1 cells or K562 cells (5 × 107 cells) were incubated
with IFN- (1,000 U/mL) or IFN- (1,000 U/mL) for various times.
IFN- and IFN- were kindly provided by Sumitomo Pharmaceutical
(Tokyo, Japan) and Shionogi Pharmaceutical (Tokyo, Japan). Total
cellular RNA was isolated from the cells by the hot-phenol method. The cellular RNA was run on a 1.1% agarose gel, transferred to
nitrocellulose filters, and hybridized to 32P-labeled DNA
probes. The 1.2-kb bcr probe (Oncogene Science, Uniondale,
NY) was isolated from the 5 portion of bcr. Human glyceraldehyde-3-phosphate dehydrogenase (G3PDH) cDNA was used as a
control to assess differences in the amount of RNA loaded on the gel.
The filters were then washed, air-dried, and autoradiographed.
Cell-cycle analysis.
KT-1 cells were incubated with IFN- (1,000 U/mL) or IFN- (1,000 U/mL) for various times. The percentage of KT-1 cells in each phase of
the cell cycle was determined by flow cytometry using a FACScan (Becton
Dickinson). The cellular DNA content was measured after propidium
iodide staining, and the percentage of cells in each phase was
calculated by the sum of broadened rectangles method.
Detection of apoptotic cells.
To identify apoptotic cell death, the method of TdT-mediated
dUTP-biotin nick end labeling (TUNEL) was tested.22 KT-1
cells were incubated with IFN- (1,000 U/mL) or IFN- (1,000 U/mL)
for various times. At the end of the incubation, cells were fixed in
4% buffered formaldehyde and spread on polylysine-coated slides. Subsequently, TUNEL staining was tested on cells. TUNEL-positive cells
were determined by stained nuclei.
Effects of IFN- and/or IFN- in liquid culture.
To examine the effects of IFN- and/or IFN-, the
patient's leukemic cells, KT-1, K562, KU812, KCL-22, or YOS-M, were
seeded at 2 × 105 cells/mL in 2 mL RPMI 1640 containing
10% FCS with or without IFN- and/or IFN-. After culture
for 7 days, viable cells were counted by the trypan blue dye exclusion
test. In some experiments, viable cells were counted every day after
IFN- or IFN- treatment. For morphologic analysis, cytospin slides
were prepared in a Shanden Cytospin and stained with
May-Grünwald-Giemsa solution. In some experiments, granulocyte
colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), or interleukin-3 (IL-3) was added
to the cultures. G-CSF, GM-CSF, and IL-3 were kindly provided by Kirin
Brewery (Gunma, Japan).
Effects of IFN- and/or IFN-
in semisolid culture.
KT-1 cells and K562 cells were cultured in semisolid medium with or
without IFN- and/or IFN-. Cells (1 × 103)
were mixed with 1 mL Iscove's modified Dulbecco medium (IMDM) containing 0.8% methylcellulose and 20% FCS with or without IFN- and/or IFN-. Cells were then plated onto a plastic culture
dish (no. 1008; Falcon). After incubation for 14 days, colonies that had more than 40 cells were counted under an inverted microscope. Smears of cells from each colony were prepared by cytospin and stained
with May-Grünwald-Giemsa solution.
| |
RESULTS |
Establishment of the KT-1 cell line.
Two months after culture of the patient's peripheral blood was
initiated, expansive proliferation of the cells was observed. Cells
were subsequently maintained in continuous culture for 18 months. They
grew in suspension with a little aggregation. The established cell line
was designated KT-1. The KT-1 cells have since been maintained in RPMI
1640 supplemented 10% FCS with a doubling time of 18 to 24 hours.
Morphological and cytochemical characteristics of KT-1 cells.
In May-Grünwald-Giemsa-stained preparations, KT-1 cells were of
blastic appearance. The cells had fine chromatin and round nuclei, and
lacked granules in the slightly basophilic cytoplasm. Cytoplasmic
protrusions were observed. KT-1 cells were negative for MPO, CAE, NBE,
and TdT, and weakly positive for PAS (14.4%). Intracytoplasmic MPO was
positive in both KT-1 cells (98.6%) and original leukemic cells
(99.2%).
Cell-surface markers.
The original leukemic cells were positive for CD4 (52.7%), CD7
(65.4%), and CD33 (89.4%), and negative for the other lymphoid or
myeloid markers. KT-1 cells were also positive for CD4 (58.9%) and
CD33 (69.2%), but were negative for CD7 (0.8%). No Epstein-Barr virus
nuclear antigen (EBNA) was detected in KT-1 cells (data not shown).
Cytogenetic analysis.
Cytogenetic analysis was performed on more than 100 metaphases of KT-1
cells. All of them showed the same chromosomal abnormality: 51, XXYY,
+X, +Y,  5, 6, +8, +8, t(9;22)(q34;q11), t(9;22)(q34;q11), +19,
+19, + unidentified chromosome. The chromosomal abnormality was similar
to that of the patient's bone marrow cells: 50, XY, +8, +8,
t(9;22)(q34;q11), t(9;22)(q34;q11), +19, +19.
Bcr rearrangement.
The 3 bcr probe detected a single rearranged band without a
germ line band in KT-1 cells, digested by BglII or
BamHI. This result was consistent with the cytogenetic
analysis, which showed no normal chromosome 22. The T-cell receptor
(TCR) and immunoglobulin H (IgH) genes were in germ-line configuration
in KT-1 cells (data not shown).
Detection of the bcr/abl splice junction.
RT-PCR showed that the bcr-2/abl-2 junction probe detected a
182-bp product in RNA extracted from KT-1 cells and that KT-1 cells
carried a bcr-2/abl-2 splice junction.
Suppression of cell growth by IFN- and/or
IFN-.
Dose-dependent growth suppression of the patient's original leukemic
cells and KT-1 cells by IFN- or IFN- was shown in liquid 7-day
culture (Fig 1A and B). The time course of
growth suppression demonstrated that the suppressive effects were noted
during the first 2 days of IFN- or IFN- induction and continued
for 6 days (Fig 2). Furthermore, IFN-
and IFN- exerted synergistic growth-suppressive effects on the
original leukemic cells and on KT-1 cells (Fig 3). IFN- and/or IFN- did not
induce cell differentiation of KT-1 cells (data not shown). In
semisolid culture, KT-1 cells formed colonies that consisted of
undifferentiated blasts without exogenous cytokines. The plating
efficiency was 10% to 20%. In semisolid cultures, IFN-
and/or IFN- had similar inhibitory and synergistic effects
on colony formation by KT-1 cells (Table 1). In contrast, IFN- and/or
IFN- did not inhibit cell growth of K562 cells in either liquid (Fig
3) or semisolid culture (Table 1). IFN- and/or IFN- also
did not affect cell growth of KU812, KCL-22, and YOS-M cells (data not
shown). The cell growth and differentiation of KT-1 cells in liquid or
semisolid culture was not affected by G-CSF, GM-CSF, or IL-3. In
addition, these cytokines could not reverse the growth suppression of
KT-1 cells by IFN- (data not shown).
View larger version (16K):
[in this window]
[in a new window]
| Fig 2.
Time course of growth-suppressive effects of IFN- or
IFN- on KT-1 cells. Cultures were initiated at 0.1 × 106 cells/mL. Cultures were exposed to either IFN- or
IFN- for time indicated. Results are comparable with those in 3 experiments.
|
|
View larger version (28K):
[in this window]
[in a new window]
| Fig 3.
Growth-suppressive effects of IFN- and/or
IFN- on KT-1 cells or K562 cells. Results are percentages relative
to each control culture. Values are means ± SD of 3 experiments.
|
|
Effects on bcr-abl gene expression by IFN- or
IFN-.
KT-1 cells and K562 cells expressed 8.5-kb aberrant bcr-abl
fusion transcripts. K562 cells also expressed a normal 4.5-kb bcr transcript, which KT-1 cells did not
express (Fig 4A and B). IFN- (1,000 U/mL) significantly suppressed bcr-abl gene
expression in KT-1 cells from 6 hours on, and its suppressive effects
had increased 24 hours after exposure (Fig 4A). IFN- (1,000 U/mL) also suppressed bcr-abl gene expression in KT-1 cells. However, its suppressive effect was weak compared with that of IFN- (Fig 4C).
IFN- (1,000 U/mL) did not affect bcr-abl gene expression in
K562 cells (Fig 4B).
View larger version (57K):
[in this window]
[in a new window]
View larger version (57K):
[in this window]
[in a new window]
View larger version (61K):
[in this window]
[in a new window]
| Fig 4.
(A) Northern blot analysis of IFN--treated KT-1
cells. The blot was transferred to nitrocellulose and successfully
hybridized to the indicated probes. KT-1 cells were exposed to IFN-
(1,000 U/mL) for indicated times. Time points were 6 and 24 hours'
exposure to IFN-. (B) Northern blot analysis of IFN--treated
K562 cells. K562 cells were exposed to IFN- (1,000 U/mL) for 24 hours. (C) Northern blot analysis of IFN--treated KT-1 cells. KT-1
cells were exposed to IFN- (1,000 U/mL) for indicated times. Time
points were 6 and 24 hours' exposure to IFN-.
|
|
Effects of IFN- or IFN- on the cell cycle of KT-1 cells.
When KT-1 cells were treated with IFN- (1,000 U/mL), a gradual
accumulation of cells in the G1 phase of the cell cycle was observed
(Fig 5A). Greater than 35% of the treated
cells were in G1 at 96 hours after treatment, whereas 18.8% of
untreated cells were in G1. In addition, a decrease in the number of
cells in the S and G2/M phases was observed in treated cells. IFN- also induced G1 cell-cycle accumulation in KT-1 cells (Fig 5B).
View larger version (27K):
[in this window]
[in a new window]
View larger version (20K):
[in this window]
[in a new window]
| Fig 5.
Flow-cytometric analysis of KT-1 cells for analysis of
cell-cycle distribution. KT-1 cells were cultured with (A) IFN-
(1,000 U/mL) or (B) IFN- (1,000 U/mL) for indicated times. Results
are comparable in 3 experiments.
|
|
Effects of IFN- or IFN- on apoptotic cell death of KT-1 cells.
The percentage of TUNEL-positive cells was less than 3% in untreated
KT-1 cells. Incubation with IFN- for 48 hours increased the fraction
of TUNEL-stained nuclei to 20%. A longer incubation (96 hours) with
IFN- increased the percentage of TUNEL-positive KT-1 cells to
greater than 60% (Fig 6). IFN- also
induced similar apoptotic cell death of KT-1 cells (data not shown).
View larger version (52K):
[in this window]
[in a new window]
| Fig 6.
TUNEL staining of IFN--induced apoptosis in KT-1
cells. KT-1 were cultured with IFN- (1,000 U/mL) for (A) 0 hours,
(B) 48 hours, and (C) 96 hours.
|
|
| |
DISCUSSION |
IFNs exert antiviral and antiproliferative activities against a variety
of malignant cells.23,24 In particular, IFN- suppresses the proliferation of Ph1-positive CML cells, induces both
hematologic remission and cytogenetic remission with disappearance of
Ph1 clones, and provides an improved
prognosis.12,14,15 IFN- is also clinically
useful25,26 and is occasionally administered to CML
patients in combination with IFN-.27 Despite intensive
efforts, the mechanism responsible for the specific antiproliferative
activity of IFNs in CML remains unknown. Immortalized, continuously
growing cell lines facilitate studies of the biology of malignant
cells. However, cell lines from CML patients, K562 and KCL-22, are not
appropriate for studying the mechanism of growth suppression by IFN,
because IFN- dose not significantly affect the growth of these cell
lines.28,29 Furthermore, IFN- and/or IFN- did
not inhibit the growth of K562, KCL-22, KU812, and YOS-M cells in our
laboratory. These cell lines were derived from leukemic cells in the
blastic crisis phase of CML, on which IFNs generally exert no effects
in vivo or in vitro, and seem to have lost their growth-suppressive
sensitivity to IFNs. Fortunately, leukemic cells from our patient with
CML were sensitive to IFN- or IFN- in vitro, even though they
were in the blastic crisis phase. Therefore, we tried to obtain an immortalized cell line from the patient and succeeded in establishing a
novel cell line, designated KT-1. KT-1 had undifferentiated morphology
that exhibited a high nuclear/cytoplasm ratio without cytoplasmic
granules. Positive reactions for intracellular MPO and the myeloid
marker CD33 indicated that KT-1 cells possessed the features of
myeloid-lineage cells. Cytogenetic analysis of KT-1 cells showed that
the chromosome abnormalities of KT-1 cells were similar to those of the
original leukemic cells. KT-1 cells showed two Ph1
chromosomes and an absence of normal copies of chromosomes 9 and 22. These results demonstrate that KT-1 cells acquired two Ph1
chromosomes from the patient's leukemic cells. The 3 bcr
probe detected a single rearrangement band without a germ-line band in
KT-1 cells. This finding is consistent with the cytogenetic analysis
that showed no normal chromosome 22.
IFN- suppressed the growth of KT-1 cells dose-dependently both in
suspension and in semisolid culture. The bcr-abl fusion transcript, p210bcr/abl protein,4,5 confers a
growth advantage to CML cells over normal precursors and also
suppresses the apoptotic machinery of CML cells.6-8
Therefore, we have studied the effects of IFN- on the expression of
the bcr-abl fusion gene in KT-1 cells. IFN- suppressed the
expression of the bcr-abl fusion gene significantly in KT-1
cells within 6 hours and its suppressive effects had increased after 24 hours, whereas IFN- did not affect bcr-abl fusion gene in
K562 cells in our laboratory, as previously reported.28
IFN- did not affect the proliferation of KT-1 cells within 24 hours, so the suppressive effects of IFN- on the expression of the
bcr-abl gene preceded the proliferation-inhibition of KT-1
cells. Decreased bcr-abl expression of fresh cells from CML
patients accompanied by cell differentiation has been
reported.30 However, IFN- did not induce differentiation
of KT-1 cells. Therefore, it seems that the decrease in bcr-abl
fusion gene expression induced by IFN- resulted in the proliferation
suppression, but not differentiation, of KT-1 cells. IFN- induced G1
cell-cycle arrest in KT-1 cells. Furthermore, the rate of apoptotic
cells by the TUNEL method was less than 3% without IFN- treatment,
but greater than 60% with IFN- treatment for 96 hours. The effect
of bcr-abl on apoptosis in cells that contain this fusion gene
has studied by inhibiting its expression using antisence
oligonucleotides. Using murine and human cell lines, bcr-abl
antisense oligonucleotides induced a decline in cell number in culture
associated with apoptosis.6,8 Therefore, our results
suggested that part of the proliferation-suppression mechanism of KT-1
cells by IFN- depended on the apoptosis associated with the
suppression of bcr-abl expression. It has been recently demonstrated that IFN- increased Fas receptor expression on
CML progenitors and Fas-mediated apoptosis was involved in the
inhibitory effects of IFN- in CML.31 Further studies
containing Fas-mediated apoptosis are recommended to elucidate
the proliferation-suppression mechanism of KT-1 cells by IFN-.
Synergistic growth-suppressive effects were observed when IFN- and
IFN- were combined in vitro.32 In vivo, IFN- or
IFN- induced hematologic remission of patients with CML who had
failed to improve after therapy with either IFN.25
Therefore, treatment with IFN- in combination with IFN- has been
tried in CML patients.27 In culture, IFN- suppressed
KT-1 cell proliferation dose-dependently, as did IFN-. Moreover, a
synergistic effect on proliferation suppression of KT-1 cells was
observed between IFN- and IFN-. IFN- and IFN- use different
signal-transduction pathways. Binding of IFN- or IFN- to their
specific receptors activates different sets of kinases within the JAK
family (Jak 1 and Tyk 2 for IFN-,33,34 or Jak 1 and Jak
2 for IFN-34,35), which then leads to the activation of
different combinations of Stat proteins.36-38 The
synergistic effects between IFN- and IFN- on growth suppression
of KT-1 cells might depend on these different signal-transduction
pathways. Indeed, IFN-, which suppressed the growth of KT-1 cells
and induced G1 arrest and apoptosis, as did IFN-, did not affect the
bcr-abl fusion gene mRNA expression in KT-1 cells
significantly, whereas IFN- suppressed it markedly. These results
suggest that the growth suppression of KT-1 cells does not always
result in the suppression of bcr-abl gene expression and that
the antipoliferative effect of IFN- in KT-1 cells relies on a
genetic mechanism other than modulation of bcr-abl expression, differing from that of IFN-. More detailed studies of the
signal-transduction mechanisms of IFN- and IFN- in KT-1 cells are
recommended.
We have reported the establishment and characterization of a new human
leukemia cell line, KT-1, derived from a patient with CML in blastic
crisis. IFN- suppressed the proliferation of KT-1 cells and their
expression of the bcr-abl fusion gene, and induced their
apoptotic cell death. We anticipate that this cell line will be
valuable to investigations of the molecular mechanism of suppression of
CML cells by IFN-.
| |
FOOTNOTES |
Submitted May 27, 1997;
accepted September 18, 1997.
Address reprint requests to Kohsuke Yanagisawa, MD, First
Department of Internal Medicine, Ehime University, Shigenobu, Ehime, Japan, 791-02.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely
to indicate this fact.
| |
REFERENCES |
1.
Rowley J:
A new consistent chromosomal abnormality in chronic myelogenous leukemia identified by quinacrine fluorescence and giemsa staining.
Nature
243:290,
1973[Medline]
[Order article via Infotrieve]
2.
Kurzrock R,
Gutterman JU,
Talpaz M:
The molecular genetics of Philadelphia chromosome positive leukemia.
N Engl J Med
319:990,
1988[Medline]
[Order article via Infotrieve]
3.
Shtivelman E,
Lifshitz B,
Gale RP,
Canaani E:
Fused transcripts of abl and bcr genes in chronic myelogenous leukemia.
Nature
315:550,
1985[Medline]
[Order article via Infotrieve]
4.
Ben-Neriah Y,
Daley GQ,
Mes-Masson AM,
Witte ON,
Baltimore D:
The chronic myelogenous leukemia-specific P210 protein is the product of the bcr/abl hybrid gene.
Science
233:212,
1986[Abstract/Free Full Text]
5.
Konopa JB,
Watanabe SM,
Whitte ON:
An alternation of the human c-abl protein in K562 leukemia cells unmasks associated tyrosine kinase activity.
Cell
37:1035,
1984[Medline]
[Order article via Infotrieve]
6.
Bedi A,
Zehnbauer BA,
Barber JP,
Sharkis SJ,
Jones RJ:
Inhibition of apoptosis by bcr-abl in chronic myeloid leukemia.
Blood
83:2038,
1994[Abstract/Free Full Text]
7.
Cortez D,
Kadlec L,
Pendergast AM:
Structural and signaling requirements for BCR-ABL-mediated transformation and inhibition of apoptosis.
Mol Cell Biol
15:5531,
1995[Abstract]
8.
McGahon A,
Bissonette R,
Schmitt M,
Cotter KM,
Green DR,
Cotter TG:
BCR-ABL maintains resistance of chronic myelogenous leukemia cells to apoptotic cell death.
Blood
83:1179,
1994[Abstract/Free Full Text]
9.
Wetzler M,
Kurzrock R,
Lowe DG,
Kantarjian H,
Gutterman JU,
Talpaz M:
Alternation in bone marrow adherent layer growth factor expression: A novel mechanism of chronic myelogenous leukemia progression.
Blood
78:2400,
1991[Abstract/Free Full Text]
10.
Daley GQ,
Van Etten RA,
Baltimore D:
Induction of chronic myelogenous leukemia in mice by the P210 bcr-abl gene of the Philadelphia chromosome.
Science
247:824,
1990[Abstract/Free Full Text]
11.
Talpaz M,
Kantarjian H,
McCredie K,
Trujillo JM,
Keating MJ,
Gutterman JU:
Hematological remission and cytogenetic improvement induced by recombinant interferon alpha in chronic myelogenous leukemia.
N Engl J Med
314:1065,
1986[Abstract]
12.
Kantarjian H,
Deisseroth A,
Kurzrock R,
Estrov Z,
Talpaz M:
Chronic myelogenous leukemia: A concise update.
Blood
82:691,
1993[Free Full Text]
13.
Ozer H,
George SL,
Schiffer CA,
Rao K,
Rao PN,
Wurster-Hill DH,
Arther DD,
Powell B,
Gottlieb A,
Peterson BA,
Rai K,
Testa JR,
LeBeau M,
Tantravahi R,
Bloomfield CD:
Prolonged subcutaneous administration of recombinant ab2 interferon in patients with previously untreated Philadelphia chromosome-positive chronic-phase myelogenous leukemia: Effect on remission duration and survival: Cancer and Leukemia Group B study 8583.
Blood
82:2975,
1993[Abstract/Free Full Text]
14. Tura S, Baccarani M, Zuffa E, Russo D, Fanin R, Zaccaria A,
Fiacchini M, Italian Cooperative Study Group on Chronic Myeloid
Leukemia: Interferon alfa-2a as compared with conventional chemotherapy
for treatment of chronic myeloid leukemia. N Engl J Med 330:820, 1994
15.
Talpaz M,
Kantarjian H,
Kurzrock R,
Trujillo JM,
Gutterman JU:
Interferon alpha produces sustained cytogenetic responses in chronic myelogenous leukemia.
Ann Intern Med
114:532,
1991
16.
Talpaz M,
Estrov Z,
Kantarjian H,
Ku S,
Foteh A,
Kurzrock R:
Persistence of dormant leukemic progenitors during interferon-induced remission in chronic myelogenous leukemia: Analysis by polymerase chain reaction of individual colonies.
J Clin Invest
94:1383,
1994
17.
Schulze E,
Krahl R,
Thalmeier K,
Helbig W:
Detection of bcr-abl mRNA in single progenitor colonies from patients with chronic myeloid leukemia by PCR: Comparison with cytogenetics and PCR from uncultured cells.
Exp Hematol
23:1649,
1995[Medline]
[Order article via Infotrieve]
18.
Davey FR,
Erber WN,
Gatter KC,
Mason DY:
Immunophenotyping of acute myeloid leukemia by immuno alkaline phosphatase (APAAP) labeling with a panel of antibodies.
Am J Hematol
26:157,
1987[Medline]
[Order article via Infotrieve]
19.
Seabright M:
A rapid banding technique for human chromosomes.
Lancet
2:971,
1971[Medline]
[Order article via Infotrieve]
20.
Sagloi G,
Guerrasio A,
Rosso C,
Zaccaria A,
Tassinari A,
Serra A,
Rege-Cambrin G,
Mazza U,
Gavosto F:
New type of BCR/ABL junction in Philadelphia chromosome-positive chronic myelogenous leukemia.
Blood
76:1819,
1990[Abstract/Free Full Text]
21.
Zaccaria A,
Martinelli G,
Buzzi M,
Testoni N,
Farabegoli P,
Zuffa E,
Zamagni MD,
Russo D,
Baccarani M,
Ambrosetti A,
Guerrasio A,
Saglio G,
Tura S:
The type of bcr/abl junction dose not predict the survival of patients with Ph1-positive chronic myeloid leukemia.
Br J Haematol
84:265,
1993[Medline]
[Order article via Infotrieve]
22.
Gavrieli Y,
Sherman Y,
Ben-Sasson SA:
Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation.
J Cell Biol
119:493,
1992[Abstract/Free Full Text]
23.
Borden EC,
Fall LA:
Interferons: Biochemical, cell growth inhibitory and immunological effects.
Prog Hematol
350:16,
1981
24.
Gresser I:
On the varied biological effects of interferon.
Cell Immunol
34:406,
1977[Medline]
[Order article via Infotrieve]
25.
Kurzrock R,
Talpaz M,
Kantarjian H,
Walters R,
Saks S,
Trujillo JM,
Gutterman JU:
Therapy of chronic myelogenous leukemia with interferon-.
Blood
70:943,
1987[Abstract/Free Full Text]
26.
Russo D,
Zuffa E,
Fanin R:
Treatment of chronic myeloid leukemia by gamma interferon.
Blut
59:15,
1989[Medline]
[Order article via Infotrieve]
27.
Opalka B,
Wandl UB,
Becher R,
Kloke O,
Nagel-Hiemke M,
Moritz T,
Beer U,
Seeber S,
Niederle N:
Minimal residual disease in patients with chronic myelogenous leukemia undergoing long-term treatment with recombinant interferon -2b alone or in combination with interferon .
Blood
78:2188,
1991[Abstract/Free Full Text]
28.
Andrews DF,
Singer JW,
Collins SJ:
Effect of recombinant -interferon on the expression of the bcr-abl fusion gene in human chronic myelogenous leukemia cell lines.
Cancer Res
47:6629,
1987[Medline]
[Order article via Infotrieve]
29.
Shibata K,
Nishimura J,
Takahira H,
Nawata H:
-Interferon reduces in vivo phosphorylation of P210bcr/abl protein during hemin-induced erythroid differentiation of K-562 cells.
Exp Hematol
19:161,
1991[Medline]
[Order article via Infotrieve]
30.
Wetzler M,
Talpaz M,
Van Etten RA,
Hirsh-Ginsberg C,
Beran M,
Kurzrock R:
Subcellular localization of bcr, abl, and bcr-abl proteins in normal and leukemic cells and correlation of expression with myeloid differentiation.
J Clin Invest
92:1925,
1993
31.
Selleri C,
Sato T,
Vecchio LD,
Luciano L,
Barrett AJ,
Rotoli B,
Young NS,
Maciejewski JP:
Involvement of Fas-mediated apoptosis in the inhibitory effects of interferon- in chronic myelogenous leukemia.
Blood
89:957,
1997[Abstract/Free Full Text]
32.
Klimpl GR,
Fleischmann WR Jr,
Klimpl KD:
Gamma interferon (IFN-gamma) and IFN-alpha/beta suppress murine myeloid colony formation (CFU-C): Magnitude of suppression is dependent upon level of colony-stimulating factor (CSF).
J Immunol
129:76,
1982[Abstract]
33.
Velazquez L,
Fellows M,
Stark GR,
Pellegrini S:
A protein tyrosine kinase in the interferon / signaling pathway.
Cell
70:313,
1992[Medline]
[Order article via Infotrieve]
34.
Muller M,
Briscoe J,
Laxton C,
Gushuin D,
Ziemiecki A,
Silvennoinen O,
Harpur AG,
Barbieri G,
Witthuhn BA,
Schindler C:
The protein tyrosine kinase JAK1 complements defects in interferon / and - signal transduction.
Nature
366:129,
1933
35.
Watling D,
Gushuin D,
Muller M,
Slivennoinen O,
Witthuhn BA,
Quellw FW,
Rogers NC,
Schindler C,
Stark GR,
Ihle JN:
Complementation of a mutant cell line defective in the interferon- signal transduction pathway by the protein tyrosine kinase Jak2.
Nature
366:166,
1993[Medline]
[Order article via Infotrieve]
36.
Schindler C,
Fu X-Y,
Improta T,
Aebersold R,
Darnell JE Jr:
Proteins of transcription factor ISGF-3: One gene encodes the 91 and 84 kDA ISGF-3 proteins that are activated by Interferon-.
Proc Natl Acad Sci USA
89:7836,
1992[Abstract/Free Full Text]
37.
Fu X-Y,
Schindler C,
Improta T,
Aebersold R,
Darnell JE Jr:
The proteins of ISGF-3: The interferon -induced transcriptional activator, define a gene family involved in signal transduction.
Proc Natl Acad Sci USA
89:7840,
1992[Abstract/Free Full Text]
38.
Shuai K,
Schindler C,
Prezioso VR,
Darnell JE Jr:
Activation of transcription by IFN-. Tyrosine phosphorylation of a 91kD DNA binding protein.
Science
259:1808,
1992
 CiteULike  Connotea  Del.icio.us  Digg |
Abstract
Introduction
Abstract
