(Received for publication, May 10, 1995; and in revised form, June 27, 1995)
From the
Protein tyrosine phosphorylation is known to play key roles in
lymphocyte signal transduction, and phosphotyrosine phosphatases (PTP)
can act as both positive and negative regulators of these lymphocyte
signals. We sought to examine the role of PTP further in these
processes by characterizing the effects of
bis(maltolato)oxovanadium(IV) (BMLOV), previously known to be a
nontoxic insulin mimetic agent in vivo. BMLOV was found to be
a potent phosphotyrosine phosphatase inhibitor. BMLOV induced cellular
tyrosine phosphorylation in B cells in a pattern similar to that
observed following antigen receptor stimulation, whereas little
tyrosine phosphorylation was induced in T cells. In B cells, BMLOV
treatment resulted in tyrosine phosphorylation of Syk and phospholipase
C, while sIgM-induced signals were inhibited. By
contrast, T cell receptor signals were moderately increased by BMLOV,
and the cells displayed greater induction of IL-2 receptor without
toxicity. The compound selectively induced apoptosis in B cell lymphoma
and myeloid leukemia cell lines, but not in T cell leukemia or colon
carcinoma cells. Interleukin-4 plus anti-CD40 antibody treatment of
normal human peripheral B cells rescued the cells from BMLOV-induced
death. These results suggest that phosphotyrosine phosphatase
inhibitors can activate B cell signal pathways in a lineage-specific
manner, resulting in desensitization of receptor-mediated signaling and
induction of apoptosis.
Tyrosine kinase activation and the phosphorylation of key
signaling molecules such as PLC (
)are early and
essential steps in lymphocyte antigen receptor signal
transduction(1) . Src family kinases are believed to act first,
followed by the Syk family kinases Syk in B cells (2) and
ZAP-70 in T cells(3) . Phosphotyrosine phosphatases (PTP)
provide both positive and negative regulation of these signals. CD45 is
essential for signaling in both T and B cells, acting at least in part
by dephosphorylating the negative regulatory C-terminal phosphorylation
site in Src family kinases, permitting them to be
activated(1, 4) . However, CD45 can also negatively
regulate T cell signaling by dephosphorylating specific substrates such
as the
chain of the T cell receptor(5) . In addition,
PTP1C has recently been reported to negatively regulate B cell antigen
receptor signals(6) . It is likely that additional PTPs are
also involved in regulating lymphocyte signal transduction. PTP
inhibitors might thus be expected to either inhibit or augment antigen
receptor signaling in lymphocytes.
Although mature B cells usually respond to antigen receptor-induced signals by proliferation, immature B cell lines frequently respond by undergoing apoptosis or programmed cell death(7, 8, 9) . This response permits the process of negative selection that eliminates self-reactive immature B cells. This activation-induced B cell death requires specific tyrosine kinases involved in antigen receptor signaling, such as the Blk tyrosine kinase(10) . Recently, it has been reported that mice deficient in PTP1C more readily eliminate self-reactive B cells (6) , indicating a role for phosphotyrosine phosphatases in this process. Tyrosine kinase activation has also been found to be essential for the ability of therapeutically relevant doses of ionizing radiation to induce tyrosine phosphorylation and apoptosis in human leukemic B cells(11) . Tyrosine kinase inhibitors blocked the radiation-induced tyrosine phosphorylation and apoptosis. The PTP inhibitor vanadate, which when used alone had little effect, greatly augmented the radiation-induced tyrosine phosphorylation and cell death(11) . These results indicate that not only is tyrosine kinase activation essential for radiation-induced apoptosis in B cells, but also suggest that phosphatases can act to limit these responses.
Taken together, these findings suggest that PTP inhibition might
either augment or inhibit antigen receptor signal transduction in
lymphocytes and could affect lymphocyte apoptosis. Although the PTP
inhibitors phenylarsine oxide (12, 13) and pervanadate (14, 15, 16, 17) have provided
valuable insights into T cell function, these two compounds have
properties that can limit their utility for biological studies.
Phenylarsine oxide is a highly toxic molecule that has the potential to
react with vicinal sulfhydryl groups on a wide variety of proteins (18) . Pervanadate is an unstable compound that can give
extraordinary induction of tyrosine phosphorylation in the absence of
biological stimulation in all cell types
examined(19, 20) . Furthermore, the effect of PTP
inhibition on B cell antigen receptor signaling has not been addressed
extensively. There is thus a need for new PTP inhibitors to compare the
effects of PTP inhibition on T and B cell signaling. We have employed
the insulin mimetic bis(maltolato)oxovanadium(IV) (BMLOV), which we
found to be an inhibitor of PTP. BMLOV acted in a lineage-specific
manner on T and B lymphocytes. In B cells, BMLOV induced tyrosine
phosphorylation of Syk and PLC while inhibiting
antigen receptor-induced signals. By contrast, the PTP inhibitor had
little effect on basal tyrosine phosphorylation in T cells but
augmented antigen receptor-mediated signals. The biological responses
of the T and B cells to the PTP inhibitor were also divergent, with T
cells displaying augmented induction of IL-2 receptor, whereas B cells
responded by undergoing programmed cell death.
Figure 1: Selective inhibition of phosphotyrosine phosphatases by BMLOV. Phosphatases were assayed as described under ``Materials and Methods.'' Error bars show the standard error of the mean. Data for PTP1B is from 5 experiments, for CD45 from 3 experiments, and for PP1, PP2A, and alkaline phosphatase from 2 experiments each.
The potent PTP
inhibitor phenylarsine oxide has been reported to induce tyrosine
phosphorylation strongly in T cells, but the high reactivity of this
compound with protein sulfhydryl groups limits its utility for
biological studies(12, 13) . Since this provided a
precedent for induction of tyrosine phosphorylation by a PTP inhibitor,
we examined whether BMLOV might also induce cellular tyrosine
phosphorylation. BMLOV displayed a dose-dependent induction of tyrosine
phosphorylation in Ramos B cells (Fig. 2A). At 25
µM, the pattern of tyrosine phosphorylation was very
similar to that induced by cross-linking surface IgM, but the level of
phosphorylation was substantially less than that obtained following a
maximal stimulation of the antigen receptor (anti-µ). The
phosphorylation was prolonged, however, as evidenced by the results of
continuous exposure to the drug shown in Fig. 2A. We
compared the induction of cellular tyrosine phosphorylation by BMLOV to
that induced by the widely used PTP inhibitor sodium orthovanadate.
While orthovanadate was able to induce some cellular tyrosine
phosphorylation, BMLOV was much more potent than orthovanadate in the
induction of tyrosine phosphorylation in Ramos B cells. ()We
have observed strong induction of cellular tyrosine phosphorylation
within 30 min,
indicating that BMLOV can act rapidly to
induce cellular tyrosine phosphorylation. These results indicate that,
like sodium orthovanadate, BMLOV is a PTP inhibitor active both in
vitro and in cells. However, the ability of BMLOV to induce
tyrosine phosphorylation was cell type-specific. BMLOV had little
effect on tyrosine phosphorylation in the human T cell leukemia lines
CEM and Jurkat, whereas the murine B cell lymphoma line A20 was highly
responsive (Fig. 2B).
Figure 2:
Induction of cellular tyrosine
phosphorylation by BMLOV. Anti-phosphotyrosine immunoblotting of
cellular proteins was performed using I-protein A
followed by autoradiography to detect antibody binding. A,
Ramos B cells were treated with the indicated concentrations of BMLOV
for 16 h or were stimulated with goat anti-human IgM F(ab)`
(
-µ) for 1 min. The
-µ lane at right is a 6-fold shorter autoradiographic exposure of the left
-µ lane to show greater detail. B, murine B cell lymphoma A20 cells and the human T cell
leukemia lines CEM and Jurkat were treated for 16 h with the indicated
concentrations of BMLOV.
Figure 3:
BMLOV inhibits sIgM-induced Ca signals. Ramos B cells were treated overnight with the indicated
concentrations of BMLOV. The base-line level of intracellular free
Ca
([Ca
]
) was
established for 1 min, and then the cells were treated with anti-human
IgM F(ab)`
(
-µ). The gap in the trace
reflects the addition of
-µ.
Analysis of cellular tyrosine phosphorylation revealed
that the BMLOV treatment affected both the amplitude and the timing of
the cells' response to sIgM cross-linking (Fig. 4A). The basal level of tyrosine phosphorylation
increased in a dose-dependent manner. In cells treated with 10
µM BMLOV, the -µ-induced signal was stronger than
in the control cells, and the signal was maintained at a high level at
both the 5- and 30-min time points. By contrast, the control cells
showed a substantial decline at 30 min. Cells treated with 25
µM BMLOV showed only small increases following
-µ
treatment, and these occurred at late time points.
Figure 4:
BMLOV activates Syk in Ramos B cells,
disregulating sIgM-induced signals. Cells were treated for 16 h with
BMLOV and then stimulated with -µ for 5 min. Cells were then
washed and resuspended in fresh media at equal concentrations of BMLOV
at 37° C. Cells were harvested and lysed at times following the
initiation of
-µ stimulation as indicated. Antibody binding in
immunoblots was detected by ECL. A, anti-phosphotyrosine
immunoblot of whole cell lysates. B, anti-phosphotyrosine
immunoblots of anti-Syk immunoprecipitates prepared from the same
samples. C, the blot in B was stripped and reprobed
with anti-Syk antibody to show recovery of the Syk
protein.
We next examined
the activation state of the Syk tyrosine kinase, since Syk is a key B
cell signaling kinase that shows prolonged activation following
-µ stimulation(2, 41) . BMLOV treatment
induced tyrosine phosphorylation of Syk in a dose-dependent manner (Fig. 4B) in the absence of antigen receptor
stimulation, while the amount of Syk protein remained constant (Fig. 4C). These results indicate that BMLOV treatment
activates the Syk tyrosine kinase, since the tyrosine phosphorylation
of Syk has been found to reflect the activation of its enzymatic
activity(2, 41, 42) . The activation of Syk
correlated well with cellular tyrosine phosphorylation. Syk was more
strongly activated at the 5- and 10-min time points in the 10
µM BMLOV samples and was strongly activated at later time
points in the 25 µM BMLOV samples.
Syk is essential for
the tyrosine phosphorylation of PLC that leads to
Ca
signaling in B cells following antigen receptor
stimulation(43) . BMLOV treatment strongly induced the tyrosine
phosphorylation of PLC
and associated proteins in a
dose-dependent manner (Fig. 5A) in the absence of
antigen receptor stimulation, while the amount of PLC
protein remained constant (Fig. 5B). The extent
of tyrosine phosphorylation of PLC
induced by BMLOV
was substantially more than that observed following a maximal sIgM
stimulation, and no additional tyrosine phosphorylation of
PLC
could be induced by sIgM stimulation of
BMLOV-treated cells.
Figure 5:
Tyrosine phosphorylation of PLC and associated proteins. Ramos B cells were treated for 16 h with
the indicated concentrations of BMLOV and then stimulated for 2 min
with
-µ. Antibody binding in immunoblots was detected by ECL. A, anti-phosphotyrosine immunoblot of anti-PLC
immunoprecipitates. B, the blot was stripped and
reprobed with anti-PLC
antibody to show recovery of
the PLC
protein.
Taken together, these results indicate that
treatment of the B cells with the PTP inhibitor BMLOV leads to
activation of signal pathways normally under antigen receptor
regulation, including the activation of Syk and the tyrosine
phosphorylation of PLC and other cellular proteins.
Our results suggest that inhibition of Ca
signaling
by BMLOV appears to be due to activation-induced desensitization, since
PLC
is strongly phosphorylated and basal
Ca
levels are elevated in the absence of receptor
stimulation.
In contrast to the results observed in the B cells,
Ca signaling in T cells was increased and prolonged
by treatment with BMLOV (Fig. 6A). Similar increases
were observed for Ca
signaling resulting from the
cross-linking of CD3 with the accessory molecules CD2 or CD4.
An increase also occurred in the presence of EGTA (Fig. 6B), indicating that BMLOV increased the release
of Ca
from intracellular stores. BMLOV had little
effect on basal or T cell receptor-induced cellular tyrosine
phosphorylation, even when cells were treated with 50 µM BMLOV (Fig. 7A). Similarly, little effect was
observed on the tyrosine phosphorylation of the key T cell signaling
kinase ZAP-70 or the associated
chain, except for a small
enhancement of tyrosine phosphorylation of ZAP-70 at 20 min (Fig. 7B). Taken together, these results indicate that
BMLOV affects lymphocyte signaling in a lineage-specific manner,
displaying disregulation of B cell antigen receptor signal pathways
while T cell antigen receptor signals can be enhanced modestly.
Figure 6:
Ca signaling in human
phytohemagglutinin-induced T cell blasts. Cells were treated overnight
with the indicated concentrations of BMLOV. A, the base-line
level of intracellular free Ca
([Ca
]
) was
established for 1 min, and then the cells were treated with
biotinylated anti-CD3 mAb (G19-4). After 5 min, avidin was added (gap in the trace) to cross-link the antibody. B,
assays were performed as in A, but in the presence of 5 mM EGTA.
Figure 7: BMLOV effect on T cell tyrosine phosphorylation. Jurkat cells were treated 16 h with BMLOV and then stimulated with anti-CD3 mAb G19-4 for 5 min. Cells were then washed and resuspended in fresh media at equal concentrations of BMLOV at 37° C. Cells were harvested and lysed at times following the initiation of CD3 stimulation as indicated. Antibody binding in immunoblots was detected by ECL. A, anti-phosphotyrosine immunoblot of whole cell lysates. B, anti-phosphotyrosine immunoblots of anti-ZAP-70 immunoprecipitates prepared from the same samples. C, anti-ZAP-70 immunoblots of anti-ZAP-70 immunoprecipitates prepared from the same samples.
Figure 8: Selective induction of DNA fragmentation by BMLOV. Cells were treated with BMLOV for the times and concentrations indicated. DNA was extracted, and the state of DNA fragmentation was examined by agarose gel electrophoresis.
In
addition to DNA fragmentation, apoptotic lymphocytes display
condensation of chromatin, loss of DNA, and fragmentation of nuclei
that can be detected by confocal laser scanning microscopy of propidium
iodide-stained cells(44) . We therefore used confocal laser
scanning microscopy to examine Ramos cells stained for DNA with
propidium iodide and for phosphotyrosine with anti-phosphotyrosine
antibodies(29) . We observed that control cells displayed
strong staining of DNA but only low levels of phosphotyrosine, whereas
the BMLOV-treated cells showed extensive loss of DNA, condensation of
chromatin, and altered nuclear morphology indicating initiation of
nuclear fragmentation. ()The cells displaying these changes
simultaneously showed strong staining with anti-phosphotyrosine
antibody, confirming that the PTP inhibitor BMLOV induces tyrosine
phosphorylation in the cells undergoing programmed cell death.
To
provide a final measure of cell death, we used propidium iodide
staining of cells. Normal healthy unpermeabilized cells exclude the
dye, whereas cells that have completed the process of programmed cell
death are stained(34, 35) . We employed this method to
compare the killing of Ramos B cells to normal human peripheral B cells
and Jurkat T cells. As shown in Fig. 9, the Ramos B cells were
completely killed by treatment with 25 µM BMLOV for 5
days, whereas the Jurkat cells were highly resistant even to this
prolonged treatment. Although data for the 5-day treatment are shown
here for comparison to the other cell types, Ramos cells were killed
extensively by shorter times of BMLOV treatment as well. Untreated control normal human peripheral B cells showed 67%
viability during 5 days of culture, but only 27% of the cells remained
viable when exposed to 25 µM BMLOV. These results indicate
that BMLOV can kill normal mature B cells, but these cells are somewhat
less sensitive than transformed immature B cell lines. By contrast, T
cells such as Jurkat, as well as normal human peripheral blood T
lymphocytes (Fig. 10), are highly resistant to killing by the
PTP inhibitor. (
)
Figure 9: IL-4 + anti-CD40 rescues B cells from BMLOV-induced death. Cells were grown for 5 days either in the absence (control) or continuous presence of 25 µM BMLOV. Viability was measured by staining with propidium iodide followed by flow cytometric analysis as described under ``Materials and Methods.'' The percentage of cells in each quadrant is indicated. The lower right quadrant contains the viable cells. A, comparison of Ramos and Jurkat cells. B, normal human peripheral B cells were either untreated or treated with 1 µg/ml anti-CD40 (G28-5) plus 10 ng/ml IL-4 as indicated.
Figure 10:
BMLOV increases anti-CD3-induced
IL-2R expression. Normal peripheral blood T cells were incubated
for 24 h in the presence or absence of 25 µM BMLOV either
in plates precoated with 10 µg/ml anti-CD3 mAb G19-4 and then
washed twice prior to addition of cells or in uncoated plates.
IL-2R
expression was analyzed by flow cytometry after staining the
cells with fluoresceinated anti-CD25. Cells not receiving any treatment
are shown by the shaded profile. - - -, 25 µM BMLOV only; --, G19-4 only;
, G19-4 plus BMLOV. A, no IL-2 added. B,
incubations were performed with 1 ng/ml
IL-2.
Anti-CD40 antibodies are known to protect B cells in germinal centers from undergoing apoptosis(2) . Furthermore, the combination of IL-4 plus anti-CD40 has been reported to be more effective than either treatment alone in protecting B cells from apoptosis induced by hyper-cross-linking of surface IgM or IgD receptors(5) . We therefore examined whether BMLOV-induced cell death in the normal B cells could be reduced by these treatments. Treatment with anti-CD40 mAb G28-5 plus IL-4 protected the cells from the BMLOV-induced death, with 58% of the cells remaining viable as compared to only 27% viability for cells treated with BMLOV alone. Interestingly, the viability was slightly higher than the 50% viability observed in cells treated with IL-4 plus anti-CD40 alone. These results suggest that BMLOV-induced death can be blocked by appropriate biological stimulation that is known to protect against activation-induced cell death in vivo.
We have identified BMLOV as a PTP inhibitor with selectivity for PTP such as CD45 and PTP1B relative to serine/threonine phosphatases such as PP1 and PP2A or to alkaline phosphatase. In addition, BMLOV also appears to be active as a PTP inhibitor in cells, since it induced cellular tyrosine phosphorylation and altered antigen receptor signal transduction in a manner consistent with PTP inhibition. Most interestingly, BMLOV displayed lineage-specific effects on B versus T lymphocyte signal transduction.
Our
data provide three related lines of evidence that BMLOV acted to induce
signal pathways normally under control of the antigen receptor in B
cells. First, BMLOV induced cellular tyrosine phosphorylation in an
overall pattern similar to that observed following sIgM cross-linking.
Second, BMLOV treatment induced the activation of the Syk tyrosine
kinase, which is known to be normally activated in B cells by antigen
receptor stimulation(46) . Third, the tyrosine phosphorylation
of PLC was induced. PLC
is regulated
by Syk (43) and is normally activated by antigen receptor
stimulation in B cells(47, 48) . Consistent with this
pathway, elevated basal Ca
levels were observed.
The exact mechanism by which PTP inhibition would have these effects
is not clear, particularly since normal antigen receptor signal
transduction is a complex and as yet incompletely understood process (1) . Previous studies with phenylarsine oxide have indicated
that tyrosine phosphorylation in T cells is regulated by phosphatase
activity, with the PTP counteracting constitutive levels of protein
tyrosine kinase activity by dephosphorylating their
substrates(12, 13) . We believe that inhibition of PTP
activity by BMLOV leads to the accumulation of cellular tyrosine
phosphorylation as a result of basal levels of tyrosine kinase
activity. The increase in the amount and duration of Syk tyrosine
phosphorylation observed in response to sIgM cross-linking in the
presence of 10 µM BMLOV is consistent with BMLOV
inhibition of the PTP that normally dephosphorylate Syk. When the
accumulated tyrosine phosphorylation would include direct Syk tyrosine
phosphorylation, Syk would be activated, increasing the phosphorylation
of its substrates such as PLC. Under conditions of PTP
inhibition, even moderate Syk activation could induce substantial
phosphorylation. We previously found that H
O
activates Syk in B cells, but not following direct treatment of
isolated Syk(40) . H
O
inhibits cellular
PTP activity(49) , and this PTP inhibition may be a mechanism
by which H
O
activates Syk in B cells.
Similarly, the PTP inhibitor pervanadate activates Src family tyrosine
kinases in cells(17, 50) , but does not directly
activate the isolated kinases (17) . However, we cannot exclude
the possibility that BMLOV might act directly on Syk or other tyrosine
kinases. These possibilities are currently under investigation.
While BMLOV treatment appeared to activate the signal pathways of
the B cells, the cells' response to antigen receptor signals was
inhibited. Specifically, anti-IgM-mediated Ca signals
were inhibited, and increases in cellular tyrosine phosphorylation were
impaired. We propose that these effects are due to an
activation-induced desensitization of the cells to biological signals.
This unresponsiveness may be similar to the desensitization of B cells
that occurs after cross-linking a small fraction of either sIgM or sIgD
receptors. Desensitized cells do not display Ca
mobilization or protein kinase C translocation in response to
subsequent cross-linking of the reciprocal isotype(51) . In
support of this hypothesis, we observed that BMLOV treatment of cells
resulted in Syk activation and strong PLC
tyrosine
phosphorylation with little or no increase occurring following
subsequent sIgM stimulation. In contrast to the B cells, T cells did
not show basal activation but did show moderate increases in some
signals, consistent with an inhibition of phosphatases that would
terminate these signals. The increased IL-2 receptor expression
observed following combined anti-CD3 and BMLOV treatment is consistent
with the enhancement of signaling. We have observed normal
proliferation,
so no long term toxicity is apparent.
BMLOV selectively induced apoptosis in the B cells, as demonstrated by the combined results of four different experimental approaches. First, DNA ladders, a hallmark of lymphocyte apoptosis, were observed following BMLOV treatment of B cells but not T cells. Second, TUNEL analysis demonstrated that BMLOV induced of apoptosis in the majority of Ramos B cells, whereas identical treatment of Jurkat T cells had little effect. Third, microscopy revealed that BMLOV induced in B cells the condensation of chromatin and distortion of nuclear morphology known to occur in programmed cell death. A final measure of cell death, propidium iodide staining, demonstrated the selective killing of B versus T cells and also showed that BMLOV treatment could induce the death of normal human peripheral B cells.
Previous studies have indicated that tyrosine phosphorylation plays a key role in lymphocyte apoptosis. Treatment of immature B cell lymphomas with soluble anti-Ig antibodies often induces apoptosis(52, 53) , and hyper-cross-linking of surface IgM or IgD receptors on mature B cells also results in apoptosis(5) . Signaling by anti-Ig antibodies requires tyrosine phosphorylation as an early and essential step(54) . Specifically, expression of the Blk tyrosine kinase has been found to be necessary for antigen receptor-induced apoptosis in CH31 lymphoma cells(10) . The correlation of the ability of monoclonal anti-idiotypic antibodies to induce tyrosine phosphorylation signaling and their ability to produce lymphoma regression in human patients also supports the role of tyrosine phosphorylation in these processes(55) . In the case of T cells, the tyrosine kinase inhibitor herbimycin A prevented superantigen-induced cell death that otherwise resulted from cross-linking multiple antigen receptors(35) . Cross-linking of Fas antigen on Jurkat T cell leukemia cells with anti-Fas antibodies induces both apoptosis and activation of tyrosine kinases leading to cellular tyrosine phosphorylation(41) . The tyrosine kinase inhibitor herbimycin A blocked both the Fas-induced tyrosine phosphorylation and death in the cells. Finally, tyrosine kinase inhibitors have been found to block tyrosine phosphorylation and apoptosis induced in B cells by ionizing radiation, while the PTP inhibitor vanadate increased these effects(11) .
We propose that BMLOV induces B cell apoptosis
by a mechanism similar to activation-induced cell death rather than by
a general toxic effect. Our data support this hypothesis in several
ways. First, the induction of apoptosis was lineage-specific and
matched the specificity of tyrosine phosphorylation, whereas such
specificity would not be expected for a general toxic effect. In
addition to B cells, we have also observed strong tyrosine
phosphorylation in the myeloid HL-60 cells that are killed by
BMLOV. Second, the combination of IL-4 and anti-CD40 that
is known to specifically rescue B cells from activation-induced
programmed cell death (5) also rescued the cells from
BMLOV-induced death. Third, BMLOV activated specific pathways and
molecules known to be important in B cell activation-induced apoptosis.
It has recently been reported that both Syk and PLC
are essential for activation-induced apoptosis in B
cells(56) , and BMLOV acted on both of these signaling
molecules. Finally, elevated intracellular Ca
is
known to induce apoptosis in Ramos as well as other B
cells(57) , and BMLOV was found to elevate basal Ca
levels. Taken together, these results suggest that an important
role of PTP in B cells would be to prevent apoptosis by preventing the
activation of signaling molecules such as Syk and PLC
in the absence of receptor activation.
The reason for the selective effects of BMLOV on B cells versus T cells or other cell types remains to be identified. The phosphatases regulating B cell tyrosine phosphorylation may be more sensitive to BMLOV. Alternatively, B cells might have more closely balanced levels of basal tyrosine kinase and PTP activity, making the cells more susceptible to PTP inhibition. It is likely that multiple PTPs are involved in these effects. CD45 may play an important role since immature B cells negative for CD45 are much more sensitive to apoptosis induced by anti-IgM stimulation(58) . The expression of oncogenes and tumor suppressor genes might also be expected to influence the sensitivity of cells to PTP inhibitor-induced apoptosis. HL-60 cells lack an intact p53 gene, so wild type p53 does not appear to be essential for BMLOV-induced apoptosis. The lineage-specific effects of BMLOV on antigen receptor signaling suggest that this compound may be of value in the analysis of other receptor signal pathways. Furthermore, the lineage-specific induction of apoptosis we have observed raises the possibility that PTP inhibitors such as BMLOV might be of value in selectively inducing programmed cell death in B cell or other malignancies.