©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Lineage-specific Induction of B Cell Apoptosis and Altered Signal Transduction by the Phosphotyrosine Phosphatase Inhibitor Bis(maltolato)oxovanadium(IV) (*)

(Received for publication, May 10, 1995; and in revised form, June 27, 1995)

Gary L. Schieven (§) Alan F. Wahl Sigrid Myrdal Laura Grosmaire Jeffrey A. Ledbetter

From the Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle, Washington 98121

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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(2), 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.


INTRODUCTION

Tyrosine kinase activation and the phosphorylation of key signaling molecules such as PLC (^1)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(2) 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.


MATERIALS AND METHODS

Cells and Reagents

The human B cell lymphoma line Ramos, the human T cell leukemia lines CEM and Jurkat, and the human acute promyelocytic leukemia line HL-60 were obtained from the American Type Culture Collection and were grown in RPMI 1640 media (Life Technologies, Inc.) with 10% fetal calf serum. The murine B cell lymphoma line A20 was obtained from the American Type Culture Collection and was grown in RPMI 1640 media, 10% fetal bovine serum plus 50 µM beta-mercaptoethanol. The human colon carcinoma cell line H3347 (22) was grown in Iscove's modified Dulbecco's media (Life Technologies, Inc.) with 10% fetal bovine serum. Human peripheral blood T lymphocytes and phytohemagglutinin-induced human T cell blasts were prepared from peripheral blood mononuclear cells from normal volunteers as described previously(23) . Anti-human CD3 chain monoclonal G19-4, anti-human CD45 monoclonal 9.4, and anti-human CD40 monoclonal G28-5 have been described previously(24, 25, 26, 27) . Anti-human Syk and ZAP-70 were from Upstate Biotechnology (Lake Placid, New York) and anti-CD25 was from Becton Dickinson. Anti-PLC(2) has been described previously(7) . Rabbit anti-human IgM F(ab)`(2) was from Jackson ImmunoResearch Laboratories (West Grove, PA). IL-2 and IL-4 were from R & D Systems (Minneapolis, MN). Protein tyrosine phosphatase PTP1B, and serine phosphatases PP1 and PP2A were from Upstate Biotechnology. Calf intestinal alkaline phosphatase was from Sigma. Bis(maltolato)oxovanadium(IV) was synthesized as described previously (21, 28) , and the product was characterized by infrared and mass spectroscopy.

Analysis of Tyrosine Phosphorylation and Phosphatase Activity

Anti-phosphotyrosine immunoblotting was performed using either affinity-purified polyclonal anti-phosphotyrosine antibodies as described previously (29) or with monoclonal antibody 4G10 (Upstate Biotechnology). Antibody binding was detected either by utilizing I-protein A followed by autoradiography or by enhanced chemiluminescence (ECL) (Amersham) in accordance with the manufacturer's directions. CD45 and PTP1B were assayed using the substrate-phosphorylated myelin basic protein as described(30) , except that 2-min assays were performed. The bovine brain myelin basic protein (Sigma) was P-labeled on tyrosine by phosphorylation with recombinant Lck tyrosine kinase expressed in a baculovirus-Sf9 insect cell system. Lck specifically phosphorylates bovine myelin basic protein on the tyrosine 67 residue(31) . Baculoviruses encoding the lck gene were generously provided by Dr. Joseph Bolen (Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ). CD45 was immunoprecipitated from Jurkat T cells with mAb 9.4 and assayed as the immune complex. PP1 and PP2A and calf intestinal alkaline phosphatase were assayed with p-nitrophenyl phosphate as a substrate.

Measurement of Cytoplasmic Calcium Concentration

Ca responses were measured using indo-1 (Molecular Probes) and a model 50HH/2150 flow cytometer (Ortho, Westwood, MA) as described previously(32) . The histograms were analyzed by programs (Cicero, Cytomation Inc., Fort Collins, CO) that calculate the mean indo-1 violet/blue fluorescence ratio versus time. There are 100 data points on the X (time) axis for all flow cytometric data.

DNA Fragmentation and Cell Death

Cellular DNA was extracted, and then the state of DNA fragmentation was analyzed by agarose gel electrophoresis as described previously(33) . Cell death was measured by staining cells with propidium iodide followed by flow cytometry (EPICS C, Coulter) as described previously(34, 35) . Flow cytometric data on forward scatter and red fluorescence arising from propidium iodide binding to cellular DNA were collected for 5000 cells and analyzed by the Quadstat program(35) . The membranes of dead cells are permeable to propidium iodide(34) , but cells undergoing the process of apoptosis can shrink in size while temporarily maintaining plasma membrane integrity(36) . Dead cells exhibiting both reduced size (lower forward scatter) and staining by propidium iodide (elevated red fluorescence) are found in the upper left quadrant, cells with damaged membranes but of normal size are found in the upper right quadrant, and compromised cells of reduced size but impermeable to propidium iodide are found in the lower left quadrant. Only cells displaying normal size and resistance to staining by propidium iodide are considered viable cells (lower right quadrant)(35) . The percentage of cells in each quadrant was calculated by the Quadstat program.


RESULTS

Bis(maltolato)oxovanadium(IV) (BMLOV) Inhibits Phosphotyrosine Phosphatases and Induces Cellular Tyrosine Phosphorylation

BMLOV and other vanadium compounds of similar structure have been reported to be potent orally active insulin mimetic agents in animal models(21) . However, the mechanism of action of these compounds has not been defined. Since the well-known PTP inhibitor sodium orthovanadate is also known to have insulin mimetic activity(37) , we examined the ability of BMLOV to inhibit various types of phosphatases (Fig. 1). BMLOV was a potent and selective inhibitor of PTPs such as CD45 and PTP1B, displaying IC values of approximately 25 nM for these PTPs while requiring over 100-fold greater concentrations to give similar inhibition of the serine/threonine phosphatases PP1 and PP2A. The selective inhibition of phosphotyrosine phosphatases relative to serine/threonine phosphatases by BMLOV is similar to results from previous studies on sodium orthovanadate(38) . Calf intestinal alkaline phosphatase was inhibited less than 10% by 500 µM BMLOV, whereas sodium orthovanadate has been reported to inhibit this enzyme strongly at concentrations of less than 20 µM(39) , indicating a greater degree of selectivity by BMLOV relative to orthovanadate.


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. (^2)We have observed strong induction of cellular tyrosine phosphorylation within 30 min,^2 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)`(2) (alpha-µ) for 1 min. The alphalane at right is a 6-fold shorter autoradiographic exposure of the left alphalane 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.



Lineage-specific Alteration of Antigen Receptor Signal Transduction by BMLOV

The above results suggested that since BMLOV displayed significantly greater biological activity than orthovanadate, particularly in B cells, BMLOV would be a useful probe in investigating the effects of PTP inhibition on antigen receptor signal transduction in B cells. BMLOV treatment of Ramos B cells for 16 h resulted in elevation of basal Ca levels (Fig. 3). We have not observed an acute Ca response to the addition of BMLOV to the Ramos cells,^2 in contrast to the rapid elevation of Ca observed following treatment with pervanadate or H(2)O(2)(17, 40) . However, Ca signals induced by cross-linking sIgM were strongly inhibited in a dose-dependent manner by the BMLOV treatment. Complete inhibition was observed in cells treated with 25 µM 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)`(2) (alpha-µ). The gap in the trace reflects the addition of alpha-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 alpha-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 alpha-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 alpha-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 alpha-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 alpha-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(2) that leads to Ca signaling in B cells following antigen receptor stimulation(43) . BMLOV treatment strongly induced the tyrosine phosphorylation of PLC(2) and associated proteins in a dose-dependent manner (Fig. 5A) in the absence of antigen receptor stimulation, while the amount of PLC(2) protein remained constant (Fig. 5B). The extent of tyrosine phosphorylation of PLC(2) induced by BMLOV was substantially more than that observed following a maximal sIgM stimulation, and no additional tyrosine phosphorylation of PLC(2) could be induced by sIgM stimulation of BMLOV-treated cells.


Figure 5: Tyrosine phosphorylation of PLC(2) and associated proteins. Ramos B cells were treated for 16 h with the indicated concentrations of BMLOV and then stimulated for 2 min with alpha-µ. Antibody binding in immunoblots was detected by ECL. A, anti-phosphotyrosine immunoblot of anti-PLC(2) immunoprecipitates. B, the blot was stripped and reprobed with anti-PLC(2) antibody to show recovery of the PLC(2) 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(2) and other cellular proteins. Our results suggest that inhibition of Ca signaling by BMLOV appears to be due to activation-induced desensitization, since PLC(2) 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.^2 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.



Lineage-specific Induction of Apoptosis by BMLOV

The contrasting effects of the PTP inhibitor BMLOV on T and B cell signaling suggested that BMLOV might have lineage-specific biological effects as well. Since many immature B cells and B cell lines, including Ramos(14) , respond to sIgM-induced signals by undergoing activation-induced programmed cell death, we investigated whether treatment with the PTP inhibitor BMLOV that induced activation signals could also induce apoptosis. BMLOV induced extensive DNA fragmentation in Ramos B cell lymphoma cells within 48 h at a dose of 10 µM (Fig. 8). This pattern of DNA fragmentation is a hallmark of apoptosis in many cell types, including lymphocytes, as a result of cleavage of DNA between nucleosomes(36) . Higher doses induced DNA fragmentation within 24 h of treatment. DNA fragmentation was also induced in the human acute promyelocytic leukemia cell line HL-60 in a dose-dependent manner (Fig. 8). The induction of apoptosis was specific for myeloid and B cell lineages in that DNA fragmentation was not observed in the human T cell leukemia cell lines CEM or Jurkat, nor was it observed in the human colon carcinoma cell line H3347 (Fig. 8). The extent of DNA fragmentation and the cell type selectivity was quantitated by TUNEL analysis(31) . DNA fragmentation was induced extensively in a dose-dependent manner in the majority of the Ramos B cells treated with BMLOV (data not shown), confirming the observation of DNA laddering shown in Fig. 8. However, little labeling was observed in TUNEL analysis of Jurkat cells treated with BMLOV, confirming that these cells are resistant to BMLOV-induced DNA fragmentation. Taken together, the DNA laddering observed on agarose gel electrophoresis and the TUNEL analysis indicate that BMLOV strongly induces DNA fragmentation in B cell lines such as Ramos but not in T cells such as Jurkat.


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. (^3)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.^2 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. (^4)


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-2Ralpha 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-2Ralpha 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; bullet bullet bullet bullet bullet, 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.

BMLOV Increases T Cell IL-2 Receptor Expression Induced by Antigen Receptor Stimulation

The expression of the IL-2 receptor alpha chain (IL-2Ralpha or CD25) is an essential biological response of T cells for productive activation following T cell receptor stimulation(45) . In addition, the PTP inhibitor pervanadate has been reported to induce IL-2Ralpha expression in the absence of biological stimulation(15) . We therefore examined whether BMLOV could alter IL-2Ralpha expression alone or in combination with CD3 stimulation by solid phase anti-CD3 mAb G19-4. BMLOV alone or in combination with IL-2 did not induce IL-2Ralpha expression (Fig. 10). However, when used in combination with anti-CD3 stimulation, 25 µM BMLOV enhanced IL-2Ralpha expression, giving a 1.7-fold increase over anti-CD3 stimulation alone (Fig. 10A). The combination of anti-CD3 and IL-2 treatment gave additional IL-2Ralpha expression, and BMLOV treatment further enhanced this expression 3-fold (Fig. 10B). These results indicate that the PTP inhibitor cannot only increase T cell receptor- induced signals, but that it also can enhance the cells' productive biological response.


DISCUSSION

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(2) was induced. PLC(2) 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(2). Under conditions of PTP inhibition, even moderate Syk activation could induce substantial phosphorylation. We previously found that H(2)O(2) activates Syk in B cells, but not following direct treatment of isolated Syk(40) . H(2)O(2) inhibits cellular PTP activity(49) , and this PTP inhibition may be a mechanism by which H(2)O(2) 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(2) 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,^4 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.^2 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(2) 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(2) 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.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed. Tel.: 206-727-3718; Fax: 206-727-3600.

(^1)
The abbreviations used are: PLC, phospholipase C; PTP, phosphotyrosine phosphatase; BMLOV, bis(maltolato)oxovanadium(IV); TUNEL, terminal deoxytransferase-mediated dUTP-biotin nick end labeling; sIgM, surface IgM; IL, interleukin; mAb, monoclonal antibody.

(^2)
G. Schieven, unpublished results.

(^3)
G. Schieven and S. Myrdal, unpublished results.

(^4)
L. Grosmaire, unpublished results.


ACKNOWLEDGEMENTS

We thank Jean Kirihara for technical assistance, Karen Donaldson for flow cytometry of cells for TUNEL analysis, Murthy Vrudhula for advice on chemical synthesis, Steve Klohr for chemical analysis of BMLOV, and Peter Kiener for advice on serine phosphatase assays.


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