(Received for publication, August 2, 1995; and in revised form, January 4, 1996)
From the
Exposure of human B-cell precursors (BCP) to ionizing radiation
results in cell cycle arrest at the G-M checkpoint as a
result of inhibitory tyrosine phosphorylation of
p34
. Here, we show that ionizing radiation
promotes physical interactions between p34
and
the Src family protein-tyrosine kinase Lyn in the cytoplasm of human
BCP leading to tyrosine phosphorylation of p34
.
Lyn kinase immunoprecipitated from lysates of irradiated BCP as well as
a full-length glutathione S-transferase (GST)-Lyn fusion
protein-phosphorylated recombinant human p34
on
tyrosine 15. Furthermore, Lyn kinase physically associated with and
tyrosine-phosphorylated p34
kinase in vivo when co-expressed in COS-7 cells. Binding experiments with
truncated GST-Lyn fusion proteins suggested a functional role for the
SH3 rather than the SH2 domain of Lyn in Lyn-p34
interactions in BCP. The first 27 residues of the unique
amino-terminal domain of Lyn were also essential for the ability of
GST-Lyn fusion proteins to bind to p34
from BCP
lysates. Ionizing radiation failed to cause tyrosine phosphorylation of
p34
or G
arrest in Lyn
kinase-deficient BCP, supporting an important role of Lyn kinase in
radiation-induced G
phase-specific cell cycle arrest. Our
findings implicate Lyn as an important cytoplasmic suppressor of
p34
function.
B-cell precursor (BCP) ()leukemia is the most common
childhood malignancy and represents one of the most radiation-resistant
forms of human
cancer(1, 2, 3, 4, 5, 6, 7, 8) .
Recent studies demonstrated that >75% of clonogenic BCP leukemia
cells from more than one-third of the newly diagnosed patients and
virtually all of the relapsed patients are able to repair potentially
lethal or sublethal DNA damage induced by radiation doses that
correspond to the clinical total body irradiation dose fractions (i.e. 2-3 Gy)(6) . Consequently, the vast
majority of BCP leukemia patients undergoing total body irradiation in
the context of bone marrow transplantation relapse within the first 12
months and only 15-20% survive disease-free beyond the first 2
years(9, 10) .
Ionizing radiation and various DNA
damaging agents cause an accumulation of cells in G phase
of the cell cycle (11, 12, 13, 14) .
Several lines of evidence indicate that this transient G
arrest allows the cells to repair potentially lethal or sublethal
DNA lesions induced by radiation or other DNA damaging agents. Cells
that are unable to show this response are more sensitive to DNA
damaging agents, and drugs that abolish this response sensitize cells
to DNA damaging
agents(11, 15, 16, 17, 18, 19, 20, 21, 22) .
A human lymphoma cell line that displayed markedly enhanced sensitivity
to DNA damage by nitrogen mustard was found to be defective in the
G
phase checkpoint control(14) . The elucidation of
the mechanism by which ionizing radiation induces G
arrest
in BCP leukemia cells could lead to a rational design of radiation
sensitizers that impair the repair of radiation-induced DNA damage by
leukemia cells and improve the outcome after total body irradiation and
bone marrow transplantation.
The molecular mechanism by which
ionizing radiation induces G arrest in the human cell cycle
and prevents entry into mitosis has not yet been deciphered, but
preliminary evidence suggested that it may involve the inactivation of
p34
kinase by inhibitory tyrosine
phosphorylation on tyrosine 15(23, 24, 25) .
p34
kinase is the catalytic subunit of mitosis
promoting factor (MPF), and its activation is a prerequisite for
induction of M phase(26, 27, 28) . Recent
studies demonstrated that exposure of BCP leukemia cells to
-rays
results in enhanced tyrosine phosphorylation of multiple substrates
including p34
kinase(25, 29) .
Furthermore, the protein-tyrosine kinase (PTK) inhibitor herbimycin A
was able to prevent radiation-induced tyrosine phosphorylation and
inactivation of p34
-linked histone H1 kinase
activity as well as mitotic arrest(25) , supporting the notion
that radiation-induced cell cycle arrest of BCP leukemia cells at
G
-M transition is likely triggered by inhibitory tyrosine
phosphorylation of p34
kinase.
Several
mitotic control genes encoding for protein-tyrosine kinases or
protein-tyrosine phosphatases have been shown to coordinately regulate
MPF function by altering tyrosine phosphorylation of
p34kinase(30, 31, 32, 33) .
Genetic experiments in fission yeast have shown that the WEE1 kinase
negatively regulates mitosis by phosphorylating p34
on Tyr
, thereby inactivating
p34
-cyclin B complex(32, 33) .
Preliminary genetic studies in fission yeast initially suggested an
important role for WEE1 kinase in radiation-induced mitotic arrest at
G
-M transition (34) . However, a more recent study
using Schizosaccharomyces pombe cells lacking functional wee1 gene product provided convincing evidence that fission
yeast WEE1 kinase is not required for radiation-induced mitotic
arrest(35) . Furthermore, we detected no increase of human WEE1
kinase activity after radiation of BCP leukemia cells, as measured by
autophosphorylation, tyrosine phosphorylation of (a)
recombinant human p34
-cyclin B complex isolated
from lysates of insect cells coinfected with recombinant viruses
encoding GST-cyclin B and
[Arg
]p34
, an inactive
mutant of p34
, (b)
p34
-cyclin B complex biochemically purified
from starfish oocytes, or (c) a synthetic peptide derived from
the p34
amino-terminal region,
[Lys
]Cdc2(6-20)NH
(25) .
Human WEE1 kinase isolated from unirradiated or irradiated BCP leukemia
cells had minimal PTK activity toward the aforementioned
substrates(25) . Thus, the identity of radiation-responsive
kinases which inactivate MPF in human BCP leukemia cells remains
unknown.
Lyn kinase is the predominant PTK in human BCP leukemia
cells(36, 37) . The enzymatic activity of Lyn in human
BCP leukemia cells is rapidly stimulated by ionizing
radiation(38) . Similarly, exposure of myeloid leukemia cells
to ionizing radiation has been reported to cause Lyn kinase
activation(39) . Lyn kinase was shown to physically associate
with p34 kinase in lysates of irradiated
myeloid leukemia cells, however the significance of Lyn kinase
activation or its association with p34
kinase
in myeloid cells has not been examined(39) . These recent
observations prompted the hypothesis that p34
kinase may associate with and serve as a substrate for Lyn
in BCP leukemia cells.
Here, we show that the Lyn kinase associates
physically and functionally with p34 in the
cytoplasm of BCP. Immunoblotting of Lyn immune complexes with an
anti-p34
-Cter antibody (where Cter indicates
COOH terminus) and immunoblotting of p34
immune
complexes with an anti-Lyn antibody provided evidence for an
association between Lyn and p34
kinases in
lysates of BCP even before radiation exposure. Irradiation of BCP
stimulated the Lyn kinase, and concomitant with Lyn kinase activation
following radiation exposure, p34
became
detectable in the Lyn immune complexes as a tyrosine-phosphorylated
protein substrate. The abundance of the Lyn protein, as estimated by
anti-Lyn Western blot analysis, did not change during the course of the
experiment, suggesting increased enzymatic activity of Lyn. However,
the abundance of the p34
protein in the same
Lyn immune complexes, as determined by anti-Cdc2-Cter Western blot
analysis, was significantly increased after radiation exposure,
suggesting that enhanced tyrosine phosphorylation of p34
which parallels the Lyn activation is at least in part due
to radiation-induced promotion of the physical association between Lyn
and p34
in NALM-6 cells. Binding experiments
with truncated GST-Lyn fusion proteins suggested a functional role for
the SH3 rather than the SH2 domain of Lyn in Lyn-p34
interactions in BCP. The first 27 residues of the unique
amino-terminal domain of Lyn were also essential for the ability of
GST-Lyn fusion proteins to bind to p34
from BCP
lysates. Lyn kinase immunoprecipitated from lysates of irradiated BCP
as well as a full-length GST-Lyn fusion protein-phosphorylated
recombinant human p34
on tyrosine 15. The
ability of the Lyn kinase to phosphorylate recombinant human
p34
on Tyr
was amplified following
radiation exposure. Lyn kinase interacts with and
tyrosine-phosphorylates p34
in vivo when these kinases are coexpressed in COS-7 cells. Ionizing
radiation failed to induce p34
tyrosine
phosphorylation or G
arrest in Lyn kinase-deficient BCP
leukemia cells expressing Fyn, Blk, and Lck kinases. These convergent
observations constitute a strong argument for an important role of a
cytoplasmic signal transduction pathway intimately linked to the Lyn
kinase in radiation-induced G
phase-specific cell cycle
arrest of human BCP leukemia cells. Since the duration of the G
arrest is a major determinant of radiation resistance in BCP
leukemias, this knowledge may lead to the design of a leukemia-specific
radiosensitization method.
Our findings implicate Lyn as an
important cytoplasmic suppressor of p34 function. Lyn kinase may serve as an integral component of a
physiologically important surveillance and repair mechanism for DNA
damage by delaying the G
-M transition in cells exposed to
mutagenic oxygen free radicals, thereby allowing them to repair their
DNA damage prior to mitosis. Lyn kinase may also protect the cell from
the potentially catastrophic consequences of premature cytoplasmic
p34
activation by maintaining the
p34
-cyclin B complex in its inactive, tyrosine
phoshorylated state.
Figure 1:
Lyn kinase associates with
p34 in unirradiated BCP leukemia cells. A, unirradiated NALM-6 cells were lysed in Nonidet P-40 lysis
buffer and 200-µg samples of the cell lysate were
immunoprecipitated with a polyclonal rabbit anti-Lyn antibody or
anti-Cdc2-Cter antibody. Immune complexes were assayed for kinase
activity during a 10-min incubation in the presence of 0.1 mM [
-
P]ATP to allow autophosphorylation
of the 53- and 56-kDa Lyn isoforms. Samples were boiled in 2
SDS sample buffer and fractionated on 12.5% polyacrylamide gels. B, unirradiated NALM-6 cells were lysed as in A and
100 µg of the lysate was immunoprecipitated with anti-Cdc2-Cter
antibody, whereas 500 µg of the lysate was immunoprecipitated with
anti-Lyn antibody, as described in A. The immune complexes
were collected, boiled in 2
SDS sample buffer, fractionated on
15% polyacrylamide gels, transferred to an Immobilon-PVDF membrane, and
immunoblotted for 90 min with anti-Cdc2-Cter antibody.
I-Labeled protein A was used to detect
p34
. C, unirradiated NALM-6 cells were
lysed in Nonidet P-40 lysis buffer and 200-µg samples of the cell
lysate were immunoprecipitated with anti-Cdc2-Cter antibody; immune
complexes were collected, washed, boiled in 2
SDS sample
buffer, fractionated on 15% polyacrylamide gels, transferred to an
Immobilon-PVDF membrane, and immunoblotted with anti-Cdc2-Cter antibody (lanes 1 and 2) or with an anti-Lyn antibody raised
against a GST-Lyn fusion protein corresponding to the 56-kDa isoform of
Lyn (lanes 3 and 4).
I-Labeled protein
A was used to detect p34
and the 56-kDa isoform
of Lyn. The upper line above lanes 1-4 indicates the antibody used for immunoprecipitation, whereas the lower line indicates the antibody used for immunoblotting. D, Lyn immune complexes from whole cell (WC,
cytoplasm + membranes), membrane (M), cytoplasmic (C), and nuclear (N) fractions of Nonidet P-40
lysates (200 µg of protein was used for each immunoprecipitation)
of unirradiated NALM-6 cells were examined in kinase assays (KA) (as in A) for autophosphorylation of Lyn (upper panel), in Western blots (as in C) for
presence of Lyn protein (middle panel) as well as for presence
of p34
protein (lower
panel).
Figure 2:
Partial mapping of the sites of
interaction between Lyn and Cdc2 kinases in BCP leukemia cells. A, schematic diagrams of truncated GST-Lyn fusion proteins
corresponding to various domains of Lyn. The inclusive amino acid (A.A.) sequence is indicated for each truncation mutant. Hatched boxes, SH3 domain; solid boxes, SH2 domain. B, functional role for the SH3 domain of Lyn in Lyn-cdc2
interactions. GST-Lyn fusion proteins non-covalently bound to
glutathione-agarose beads were used in binding assays to examine their
ability to interact with p34 in NALM-6 cells,
as described under ``Experimental Procedures.'' Samples (250
µg) of the Nonidet P-40 lysates from unirradiated NALM-6 cells were
incubated with the GST-Lyn fusion protein-coupled beads. The fusion
protein adsorbates were washed, resuspended in SDS sample buffer,
boiled, fractionated on 12.5% SDS-PAGE gels, transferred to Immobilon-P
membranes, and membranes were immunoblotted with anti-Cdc2-Cter,
followed by visualization using
I-labeled protein A and
autoradiography.
We next examined the effects of ionizing
radiation on the ability of Lyn kinase to phosphorylate a recombinant
human p34-cyclin B complex preparation in the presence
of [
-
P]ATP.This complex was isolated from
lysates of insect cells coinfected with recombinant viruses encoding
GST-cyclin B and [Arg
]p34
, an
inactive mutant form of p34
mutated at lysine
33(25, 43) . Lyn kinase was immunoprecipitated from
unirradiated as well as irradiated NALM-6 cells and examined in kinase
assays for autophosphorylation as well as its ability to phosphorylate
recombinant human p34
on tyrosine. As shown in Fig. 3A, ionizing radiation resulted in a >4-fold
increase in Lyn kinase activity, as measured by autophosphorylation.
The increased autophosphorylation was accompanied by >1.8-fold
increased phosphorylation of
[Arg
]p34
. Two-dimensional
phosphoamino acid analysis of the excised Cdc2 bands confirmed that the
increased label on p34
reacted with Lyn from irradiated
cells was caused by enhanced tyrosine phosphorylation (data not shown).
Thus, exposure of NALM-6 cells to
-rays prior to the
immunoprecipitation augmented the ability of Lyn kinase to utilize
recombinant human p34
as an exogenous substrate during
the in vitro kinase reactions.
Figure 3:
Ionizing radiation promotes the
interaction between Lyn and Cdc2 kinases in BCP leukemia cells. A, -rays stimulate the PTK activity of Lyn toward
recombinant human
[Arg
]p34
. Lyn kinase was
immunoprecipitated from Nonidet P-40 lysates of unirradiated (lane
1) and irradiated (lane 2, 5 min after 1 Gy
-rays; lane 3, 5 min after 2 Gy
-rays) NALM-6 cells. In
vitro kinase assays were performed to examine the
immunoprecipitated Lyn kinase for autophosphorylation as well as its
ability to phosphorylate recombinant human
p34
-cyclin B complex, which was used as an
exogenous substrate, on tyrosine. B,
-rays promote the
physical and functional interactions between Lyn and p34
in BCP leukemia cells. b1, Lyn kinase was
immunoprecipitated from the Nonidet P-40 lysates (600 µg/sample) of
unirradiated (lane 2) as well as irradiated (lane 3,
10 min after 2 Gy
-rays; lane 4, 20 min after 2 Gy
-rays) NALM-6 cells and in vitro kinase assays were
performed using one-third of the samples, as described in the legend of Fig. 1A, to examine Lyn autophosphorylation as well as
phosphorylation of co-immunoprecipitated p34
kinase. Arrows indicate the positions of the Lyn
and p34
kinases. b2, another third of
the samples from the Lyn immunoprecipitations shown in b1 were
boiled in 2
SDS sample buffer, fractionated on 12.5%
polyacrylamide gels, transferred to an Immobilon-PVDF membrane, and
immunoblotted with an anti-Lyn antibody raised against a GST-Lyn fusion
protein corresponding to the 56-kDa isoform of Lyn.
I-Labeled protein A was used to detect the 56 kDa isoform
of Lyn. b3, the remaining one-third of the samples from the
Lyn immunoprecipitations shown in b1 were boiled in 2
SDS sample buffer, fractionated on 12.5% polyacrylamide gels,
transferred to an Immobilon-PVDF membrane, and immunoblotted with
anti-Cdc2-Cter antibody.
I-Labeled protein A was used to
detect the p34
kinase in the Lyn immune
complexes. The purpose of the b2 portion of the experiment was
to confirm that lanes 2, 3, and 4 contained
equal amounts of Lyn and the lane-lane differences in Lyn
autophosphorylation or amount of Cdc2 kinase detected by immunoblotting
were not caused by loading unequal amounts of Lyn immune complexes in
each lane. In b1-b3, no primary immunoprecipitating
antibody was added to the control samples shown in lanes
1.
Subsequently, we evaluated
the effects of ionizing radiation on the intracellular physical and
functional interactions between Lyn and p34 in NALM-6
cells. To this end, NALM-6 cells were irradiated, lysed with Nonidet
P-40 lysis buffer, and Lyn kinase was immunoprecipitated from the
lysates of unirradiated as well as irradiated cells. In vitro kinase assays were performed to examine Lyn autophosphorylation as
well as phosphorylation of any co-immunoprecipitated p34
kinase. As shown in Fig. 3B (b1),
irradiation of NALM-6 cells stimulated the the Lyn kinase, as measured
by autophosphorylation. Concomitant with Lyn kinase activation at 10 or
20 min following radiation exposure, p34
became
detectable in the Lyn immune complexes as a tyrosine-phosphorylated
protein substrate (Fig. 3B, b1). The abundance
of the Lyn protein, as estimated by anti-Lyn Western blot analysis, did
not change during the course of the experiment, suggesting increased
enzymatic activity of Lyn (Fig. 3B, b2).
However, the abundance of the p34
protein in the same
Lyn immune complexes, as determined by anti-Cdc2-Cter Western blot
analysis, was significantly increased after radiation exposure (Fig. 3, B, b3), suggesting that enhanced
tyrosine phosphorylation of p34
, which parallels the Lyn
activation, is at least in part due to radiation-induced promotion of
the physical association between Lyn and p34
in NALM-6
cells.
Figure 4:
Tyrosine phosphorylation of recombinant
human p34 by GST-Lyn fusion protein. A, PTK activity of GST-Lyn was examined at 1:500 and 1:100
final dilutions during a 10-min in vitro kinase reaction by
measuring its autophosphorylation as well as phosphorylation of
acid-denatured rabbit enolase, which was used as an exogenous PTK
substrate, as previously reported(29, 36) . B, [Arg
]p34
was
used as a substrate for GST-Lyn, as described under ``Experimental
Procedures.'' C, GST-Lyn-phosphorylated
[Arg
]p34
was subjected
to a two-dimensional phosphoamino acid analysis, as described under
``Experimental Procedures.'' Y, tyrosine; T, threonine; S, serine. D, the ability of
GST-Lyn and GST-p49
to phosphorylate
[Arg
]p34
was measured in
a 20-min kinase reaction. Following the kinase reactions, samples were
boiled in 2
SDS reducing sample buffer, and proteins were
fractionated on 15% polyacrylamide gels and visualized by
autoradiography. The left lane (labeled as lane 1)
was loaded with the control sample, which contained
[Arg
]p34
-GST-cyclin B
complexes only. The unlabeled middle lane was loaded with the
positive control sample, which contained
[Arg
]p34
-GST-cyclin B
plus GST-p49
. The right lane (labeled as lane 2) was loaded with the test sample, which contained
[Arg
]p34
-GST-cyclin B
plus GST-Lyn. Whereas GST-p49
and GST-cyclin B (CYCB) are discernible as electrophoretically distinct bands,
the size differences between GST-cyclin B and GST-Lyn do not permit
separation on these 15% polyacrylamide gels. Thus, the phosphorylated
upper band in lane 2 contains both GST-Lyn and GST-cyclin B. E, [Arg
]p34
bands from lanes 1 (untreated) and 2 (GST-Lyn-treated) in D were excised and subjected to
two-dimensional phosphoamino acid analysis, as described under
``Experimental Procedures.''
To further evaluate
the effects of GST-Lyn as well as Lyn kinase immunoprecipitated from
unirradiated and irradiated NALM-6 pre-B leukemia cells on the
phosphorylation state of
[Arg]p34
, we subjected
p34
excised from the gels of kinase reactions to
two-dimensional tryptic phosphopeptide mapping. As shown in Fig. 5, a single threonine-containing phosphopeptide was
detected upon phosphotryptic mapping of untreated p34
.
Consistent with a previous report, which identified Tyr
as
the site of GST-WEE1-induced phosphorylation of
[Arg
]p34
(43) , one major
tyrosine-containing phosphopeptide was detected after treatment of
[Arg
]p34
with GST-WEE1. The
position of this Tyr
-containing peptide in each
phosphotryptic map shown in Fig. 5is indicated with an arrowhead. Notably, treatment of
[Arg
]p34
with GST-Lyn or Lyn
immunoprecipitated from irradiated NALM-6 cells resulted in increased
phosphorylation of the same Tyr
-containing peptide.
Figure 5:
GST-Lyn and Lyn kinase from irradiated BCP
leukemia cells phosphorylate recombinant
[Arg]p34
on
Tyr
. Top panel, p34
bands
excised from the gels shown in Fig. 4D were subjected
to two-dimensional tryptic phosphopeptide mapping, as described under
``Experimental Procedures.'' The position of
Tyr
-containing peptide was identified as the site of
GST-WEE1-induced phosphorylation of
[Arg
]p34
(30, 43) . Bottom panel, [Arg
]p34
was also used as a substrate for Lyn kinase, which was
immunoprecipitated from Nonidet P-40 lysates of unirradiated (N6, 0 Gy) and irradiated (N6, 1 Gy =
5 min after 1 Gy
-rays; N6, 2 Gy = 5 min after 2
Gy
-rays) NALM-6 cells. Two-dimensional tryptic phosphopeptide
mapping was performed as for the samples shown in the top
panel. In both the top and bottom panels, the
position of this Tyr
-containing peptide in each
phosphotryptic map shown is indicated with an arrowhead.
Taken together, these experiments provided direct evidence that Lyn
kinase can directly phosphorylate p34 on
Tyr
. The radiation-enhanced ability of Lyn kinase from
NALM-6 cells to phosphorylate recombinant p34
on
Tyr
strongly supports the hypothesis that Lyn may be one
of the PTK responsible for radiation-induced inhibitory tyrosine
phosphorylation and inactivation of p34
kinase in human
BCP leukemia cells.
Figure 6:
Reconstitution of
Lyn-p34complexes in COS-7 cells. cDNAs
encoding Lyn and p34
were transiently
co-expressed in COS-7 cells using the Lipofectamine reagent. Upper
panel, Western blot analysis of Lyn and p34
expression in whole cell lysates of COS-7 cells transfected
with cdc2 cDNA or cdc2 cDNA plus lyn cDNA. Lower panel, immune complex kinase assays and anti-Cdc2-Cter
Western blot analysis of Lyn immunoprecipitates from Nonidet P-40
lysates of COS-7 cells transfected with cdc2 cDNA or cdc2 cDNA plus lyn cDNA, or mock-transfected with the empty
expression vectors used for cdc2 (Vector1) and lyn (Vector2) cDNA. WB, Western blot; KA, kinase assay.
Figure 7:
Ionizing radiation does not trigger
tyrosine phosphorylation of p34 in Lyn
kinase-deficient BCP leukemia cells. A, Lyn immunoprecipitates
from Nonidet P-40 lysates of BCP leukemia cells from patient
UPN1
and NALM-6 pre-B leukemia cell line were
examined for the presence of autophosphorylated
p53
/p56
by immune complex
kinase assays and for the presence of Lyn protein by Western blot
analysis. B, UPN1
cells were lysed and
equal amounts of the detergent-soluble cell lysate (200 µg of
protein/reaction mixture) were used for immunoprecipitation and immune
complex kinase assays of the indicated Src family PTK. C,
p34
was immunoprecipitated from Nonidet P-40
lysates of unirradiated as well as irradiated (2 Gy delivered 5 min
prior to lysis) BCP leukemia cells of UPN1
and
UPN2
using a rabbit anti-Cdc2-Cter antibody.
Samples were run on 10.5% SDS-PAGE gels and subsequently immunoblotted
with either anti-phosphotyrosine or anti-Cdc2-Cter.
I-Labeled protein A was used to detect
tyrosine-phosphorylated proteins or p34
kinase.
The position of p34
is indicated with arrowheads. D, for comparison, using the procedures
outlined in C, radiation-induced tyrosine phosphorylation of
p34
was also examined in Lyn kinase-positive
NALM-6 pre-B leukemia cells.
We next compared the ability of ionizing
radiation to trigger tyrosine phosphorylation of p34 in
Lyn kinase expressing NALM-6 cells versus Lyn kinase-deficient
UPN1
or UPN2
cells by
anti-phosphotyrosine Western blot analysis of p34
immunoprecipitates from the Nonidet P-40 cell lysates prepared 5
min after radiation exposure. Ionizing radiation induced tyrosine
phosphorylation of p34
in NALM-6 cells, but not in
UPN1
or in UPN2
cells (Fig. 7, C and D).
We next compared the
ability of -rays to cause a G
arrest in Lyn kinase
expressing NALM-6 cells versus Lyn kinase-deficient
UPN1
cells. Asynchronously dividing NALM-6 cells
and uncultured UPN1
cells were irradiated with 2
Gy
-rays and then cultured at 5
10
cells/ml in
a clonogenic medium, as described under ``Experimental
Procedures.'' At the indicated time points, cells were stained
with Hoechst 33342 to quantify their DNA content on a FACStar Plus flow
cytometer. Prior to radiation, 25% of NALM-6 cells and 29% of
UPN1
cells were in the G
phase of the
cell cycle, which corresponds to the second peak of the DNA histogram (Fig. 8). In NALM-6 cells, a radiation-induced accumulation in
G
phase was first detectable at 8 h after radiation, when
the DNA flow cytometric analysis showed 38% of the cells to be in the
G
phase. The cell cycle arrest at the G
-M
transition checkpoint was further evident from the decreased percentage
of G
cells. The percentage of cells accumulated in
G
phase was further increased to 54% at 22 h. This cell
cycle arrest at G
-M transition checkpoint was transient, as
evidenced by the decreased percentage of G
cells and
increased percentage of G
cells at 28 h after radiation.
In contrast to NALM-6 cells, UPN1
cells did not
show any evidence of a cell cycle arrest at G
-M transition
after exposure to 2 Gy
-rays (Fig. 8). These findings
support the hypothesis that Lyn is the PTK responsible for
radiationinduced inhibitory tyrosine phosphorylation and inactivation
of p34
kinase in human BCP leukemia cells.
Figure 8:
Ionizing radiation does not cause G arrest in Lyn kinase-deficient BCP leukemia cells. Lyn
kinase-positive NALM-6 pre-B leukemia and Lyn kinase-deficient
UPN1
cells were irradiated with 2 Gy
-rays
and then cultured in clonogenic medium for 8, 22, and 28 h at 37
°C/5% CO
. Cells were washed two times in fresh
clonogenic medium and stained with the UV-excited dye, Hoechst 33342,
as described previously(5, 25) . Quantitative DNA
analysis was performed on a FACStar Plus flow cytometer equipped with a
Consort 40 computer using the COTFIT program, which includes CELLCY, a
cell cycle distribution function that fits DNA content histograms and
calculates the percentages of cells in G
, S, and
G
M phases of the cell cycle.
Radiation-induced G arrest allows the cells to repair
potentially lethal or sublethal DNA lesions induced by radiation or
other DNA damaging agents. Cells that are unable to show this response
are more sensitive to DNA damaging agents, and drugs that abolish this
response sensitize cells to DNA damaging
agents(11, 15, 16, 17, 18, 19, 20, 21, 22) .
Abrogation of radiation-induced G
arrest by caffeine
exposure induces premature mitosis before DNA repair is complete and
results in enhanced cell death(8) . Similarly, pentoxyfylline,
a caffeine analog that shortens the duration of G
arrest,
also displays radiosensitizing properties(40) . A human
lymphoma cell line that displayed markedly enhanced sensitivity to DNA
damage by nitrogen mustard was found to be defective in the G
phase checkpoint control(9) .
We provide experimental
evidence for an important role of a cytoplasmic signal transduction
pathway intimately linked to the Lyn kinase in radiation-induced
G phase-specific cell cycle arrest of human BCP leukemia
cells. Because Lyn kinase maintains p34
in an inactive
state in irradiated BCP leukemia cells, thereby allowing them to repair
sublethal radiation damage, we postulate that inhibition of Lyn kinase
in BCP leukemia cells may result in radiosensitization. To accomplish
this goal, a PTK inhibitor could be targeted to Lyn kinase in BCP
leukemia cells with a monoclonal antibody, which binds to and remains
complexed with CD19 receptor. CD19 receptor is physically associated
with the Lyn kinase(36) . Our recent results show that
treatment of CD19
BCP leukemia cells with nanomolar
concentrations of B43-Gen immunoconjugate causes sustained inhibition
of CD19-associated Lyn kinase(37) .
Several studies have
documented the ability of B-lineage lymphoid cells to produce reactive
oxygen intermediates in response to various activation
signals(53, 54, 55, 56, 57, 58) .
Recent evidence suggests that production of reactive oxygen
intermediates in response to various mitogenic stimuli may regulate the
proliferative responses of peripheral blood mononuclear
cells(59) . It has been proposed that generation of reactive
oxygen intermediates upon activation of B-lineage lymphoid cells may
contribute to somatic
mutations(53, 55, 56, 57, 60) .
Lyn kinase may serve as an integral component of a physiologically
important surveillance and repair mechanism for DNA damage by delaying
the G-M transition in cells exposed to mutagenic oxygen
free radicals, thereby allowing them to repair their DNA damage prior
to mitosis. Without this surveillance, the likelihood of malignant
transformations leading to BCP leukemias as well as impaired survival
and self-renewal capacity of BCP populations leading to
immunodeficiency disorders may be increased. Therefore, it will be
important to conduct appropriate epidemiologic studies designed to test
the hypothesis that low activity levels of Lyn in BCP populations may
be associated with increased risk of development of BCP leukemia or
B-cell immunodeficiency during childhood.