Hairy cell leukemia (HCL) is a B-cell tumor affecting the preplasma stage of B-cell differentiati... more Hairy cell leukemia (HCL) is a B-cell tumor affecting the preplasma stage of B-cell differentiation. One important feature of the disease is its exquisite sensitivity to interferon-alpha (IFN-alpha) therapy. Because we showed earlier that the CD20 molecule is consistently hyperphosphorylated in hairy cells and because previous studies showed that CD20 is involved in regulating intracytoplasmic free calcium concentrations ([Ca2+]i) in normal B lymphocytes, we measured [Ca2+]i in tumor cell samples from patients with HCL and studied the effect of IFN-alpha on this parameter. Using the Ca(2+)-sensitive fluorophore fura-2, we observed that hairy cells display a slightly but consistently higher [Ca2+]i than normal 48-hour-activated B cells or other leukemic cells. Furthermore, both in vitro preincubation of cell samples with IFN-alpha and in vivo administration of this cytokine reduced the [Ca2+]i in hairy cells. This effect was observed together with a decrease in transmembrane Ca2+ inf...
Hairy cells are stimulated to DNA synthesis by low molecular weight B cell growth factor (LMW-BCG... more Hairy cells are stimulated to DNA synthesis by low molecular weight B cell growth factor (LMW-BCGF) and this proliferative response is suppressed by interferon (IFN)-alpha, both in vitro and in vivo. The suggestion that the CD23 molecule (Fc epsilon II receptor) might be involved in the signalling pathway of LMW-BCGF prompted us to study the expression of this molecule on hairy cells and its modulation by IFN- alpha. By flow cytometry and direct binding experiments with anti CD23 monoclonal antibodies, the presence of the CD23 antigen was detected in 7 of 12 cases tested, on variable percentages of cells, ranging from low to medium expression. In vitro incubation of hairy cells with IFN- alpha, which elicits a suppression of the proliferative response of these cells to LMW-BCGF, induced a parallel significant reduction of CD23 expression in only three cases. Similarly, a transient in vivo decrease of CD23 expression, concommitant with an inhibition of the LMW- BCGF response, could b...
One of the most striking characteristics of hairy cell leukemia (HCL) is its remarkable responsiv... more One of the most striking characteristics of hairy cell leukemia (HCL) is its remarkable responsiveness to alpha-interferon (IFN-alpha) therapy. IFN-alpha is a multifunctional cytokine able to regulate the growth, differentiation and recirculation of normal and malignant B lymphocytes. These activities have been extensively studied in hairy cells, but none of these properties has been shown to fully account for IFN-alpha responsiveness. Numerous data now support the idea that IFN-alpha exerts its therapeutic effect both indirectly, through stimulation of immunocompetent cells, and directly on the hairy cells. Our approach to investigate the mode action of IFN-alpha in HCL has been to identify abnormalities which occur in these tumor cells and then to ascertain whether these abnormalities can be rectified by IFN-alpha treatment. A high level of free Ca2+ in the cytoplasm of hairy cells was identified. Increases in cytosolic Ca2+ are believed to be a pivotal signal in regulating cell p...
Hairy cell leukemia (HCL) is a pre-plasma B cell tumor which responds to interferon (IFN)-alpha t... more Hairy cell leukemia (HCL) is a pre-plasma B cell tumor which responds to interferon (IFN)-alpha therapy. In vitro, B cell growth factor (BCGF) can induce proliferation of hairy cells. We have investigated the effect of in vitro and in vivo treatments with different recombinant IFN on the capacity of hairy cells to proliferate in response to human BCGF. In vitro treatment of leukemic cells from HCL patients with recombinant IFN-alpha-2 (5/5 cases) or IFN-beta (4/5 cases) resulted in a marked inhibition of the BCGF-dependent response. This suppressive effect was obtained with IFN concentrations of 1000, 100 IU/ml, and even occasionally 10 IU/ml. In contrast, no such inhibition was observed with IFN-gamma, despite the presence of specific IFN-gamma receptors on hairy cells at densities similar to receptors for IFN-alpha/beta. The IFN-alpha-induced suppression of the proliferative response of hairy cells to BCGF was also observed in vivo in two patients within 6-12 hr after administrati...
Journal of immunology (Baltimore, Md. : 1950), Jan 15, 1990
Transduction through the CD23 molecule (Fc epsilon RII) was analyzed in human activated B lymphoc... more Transduction through the CD23 molecule (Fc epsilon RII) was analyzed in human activated B lymphocytes using anti-CD23 mAb. B cell blasts expressing an increased amount of surface CD23 molecule were obtained by stimulation of normal peripheral blood B lymphocytes with Staphylococcus aureus strain Cowan I and IL-4. Anti-CD23 mAb were found to trigger polyphosphoinositide hydrolysis in these cells (and also in tumoral B cells expressing spontaneously CD23) and a rise in [Ca2+]i which could be attributed to mobilization from cytoplasmic pools. This increase in [Ca2+]i could be mimicked, with a comparable time-course, by the addition of InsP3 to permeabilized B cell blasts indicating that the increase in inositol phosphate accumulation induced by the antibodies was due to a preferential attack of phosphatidylinositol-bisphosphate by a specific phosphoinositidase C (PIC). In permeabilized cells, raising the free calcium concentration above 3 microM was found to induce polyphosphoinositide...
Journal of immunology (Baltimore, Md. : 1950), 1991
Hairy cell leukemia (HCL) is a B cell tumor affecting the pre-plasma stage of B cell differentiat... more Hairy cell leukemia (HCL) is a B cell tumor affecting the pre-plasma stage of B cell differentiation. Hairy cells produce B cell growth factor (BCGF)-related growth factor(s) and we have previously shown that low mol wt (LMW)-BCGF-induced proliferation of hairy cells is inhibited in vitro and in vivo by IFN-alpha. We therefore suggested that this effect might contribute to the exquisite sensitivity of HCL to IFN-alpha therapy. To elucidate the mechanism involved in the therapeutic effect of IFN-alpha, we have analyzed the pattern of phosphorylated proteins in hairy cells. We detected the presence of a hyperphosphorylated protein with a molecular mass of about 35 kDa. This protein was identified as the CD20 molecule (B1), which is a structurally unique phosphoprotein exclusively detected on B cells and expressed during most stages of B cell development. Incubation of hairy cells with mitogenic concentrations of LMW-BCGF induces an additional increase in CD20 protein phosphorylation. ...
We have previously shown that addition of type 1 transforming growth factor-beta (TGFbeta1) to an... more We have previously shown that addition of type 1 transforming growth factor-beta (TGFbeta1) to an exponentially growing population of mink lung CCl64 cells increases their average intermitotic time from 14.4 to 20.3 hours, predominantly by extending G1 from 7.5 to 13.5 hours. Here we have used the DRIMAPS system (digitally recorded interference microscopy with automatic phase-shifting) for obtaining data on cellular mass distribution, cell motility and morphology. We found no significant change in the cells' rate of mass increase following TGFbeta1 treatment, which implies that the treated cells attained a higher mass during their extended cell cycle and this was confirmed by direct measurement of cell size. However, the cells showed a dramatic motile response to treatment: TGFbeta1-treated cells had a significantly higher time-averaged speed of 36.2 microm hour-1 compared to 14.5 microm hour-1 for the control cells. The time course of the response was gradual, reaching a maximum mean speed of 52.6 microm hour-1 after 15 hours exposure. We found that the gradual onset of the response was probably not due to a slow accumulation of a secondary factor but because cells were dividing throughout the experiment and most of the response to TGFbeta1 occurred only after the first cell division in its presence. Thus, taking only those cells that had not yet divided, the time-averaged speed of treated cells (26.1 micrometer hour-1) was only moderately higher than that of untreated cells (14.9 micrometer hour-1) whereas, for those cells that had divided, the difference in speed between treated cells (45.1 micrometer hour-1) and untreated cells (14.1 microm hour-1) was much greater. Increased speed was a consequence of enhanced protrusion and retraction of the cell margin coupled with an increase in cell polarity. TGFbeta1 also increased the mean spreading of the cells, measured as area-to-mass ratio, from 3.2 to 4.4 micrometer2 pg-1, and the intracellular mass distribution became more asymmetric. The observations indicate that a G2 signal may be necessary to reach maximal motility in the presence of TGFbeta1.
Transforming growth factor β (TGF-β) and related cytokines play a central role in the vascular sy... more Transforming growth factor β (TGF-β) and related cytokines play a central role in the vascular system. In vitro, TGF-β induces aortic endothelial cells to assemble subcellular actin-rich structures specialized for matrix degradation called podosomes. To explore further this TGF-β-specific response and determine in which context podosomes form, ALK5 and ALK1 TGF-β receptor signaling pathways were investigated in bovine aortic endothelial cells. We report that TGF-β drives podosome formation through ALK5 and the downstream effectors Smad2 and Smad3. Concurrent TGF-β-induced ALK1 signaling mitigates ALK5 responses through Smad1. ALK1 signaling induced by BMP9 also antagonizes TGF-β-induced podosome formation, but this occurs through both Smad1 and Smad5. Whereas ALK1 neutralization brings ALK5 signals to full potency for TGF-β-induced podosome formation, ALK1 depletion leads to cell disturbances not compatible with podosome assembly. Thus, ALK1 possesses passive and active modalities. ...
Hairy cell leukemia (HCL) is a B-cell tumor affecting the preplasma stage of B-cell differentiati... more Hairy cell leukemia (HCL) is a B-cell tumor affecting the preplasma stage of B-cell differentiation. One important feature of the disease is its exquisite sensitivity to interferon-alpha (IFN-alpha) therapy. Because we showed earlier that the CD20 molecule is consistently hyperphosphorylated in hairy cells and because previous studies showed that CD20 is involved in regulating intracytoplasmic free calcium concentrations ([Ca2+]i) in normal B lymphocytes, we measured [Ca2+]i in tumor cell samples from patients with HCL and studied the effect of IFN-alpha on this parameter. Using the Ca(2+)-sensitive fluorophore fura-2, we observed that hairy cells display a slightly but consistently higher [Ca2+]i than normal 48-hour-activated B cells or other leukemic cells. Furthermore, both in vitro preincubation of cell samples with IFN-alpha and in vivo administration of this cytokine reduced the [Ca2+]i in hairy cells. This effect was observed together with a decrease in transmembrane Ca2+ inf...
Hairy cells are stimulated to DNA synthesis by low molecular weight B cell growth factor (LMW-BCG... more Hairy cells are stimulated to DNA synthesis by low molecular weight B cell growth factor (LMW-BCGF) and this proliferative response is suppressed by interferon (IFN)-alpha, both in vitro and in vivo. The suggestion that the CD23 molecule (Fc epsilon II receptor) might be involved in the signalling pathway of LMW-BCGF prompted us to study the expression of this molecule on hairy cells and its modulation by IFN- alpha. By flow cytometry and direct binding experiments with anti CD23 monoclonal antibodies, the presence of the CD23 antigen was detected in 7 of 12 cases tested, on variable percentages of cells, ranging from low to medium expression. In vitro incubation of hairy cells with IFN- alpha, which elicits a suppression of the proliferative response of these cells to LMW-BCGF, induced a parallel significant reduction of CD23 expression in only three cases. Similarly, a transient in vivo decrease of CD23 expression, concommitant with an inhibition of the LMW- BCGF response, could b...
One of the most striking characteristics of hairy cell leukemia (HCL) is its remarkable responsiv... more One of the most striking characteristics of hairy cell leukemia (HCL) is its remarkable responsiveness to alpha-interferon (IFN-alpha) therapy. IFN-alpha is a multifunctional cytokine able to regulate the growth, differentiation and recirculation of normal and malignant B lymphocytes. These activities have been extensively studied in hairy cells, but none of these properties has been shown to fully account for IFN-alpha responsiveness. Numerous data now support the idea that IFN-alpha exerts its therapeutic effect both indirectly, through stimulation of immunocompetent cells, and directly on the hairy cells. Our approach to investigate the mode action of IFN-alpha in HCL has been to identify abnormalities which occur in these tumor cells and then to ascertain whether these abnormalities can be rectified by IFN-alpha treatment. A high level of free Ca2+ in the cytoplasm of hairy cells was identified. Increases in cytosolic Ca2+ are believed to be a pivotal signal in regulating cell p...
Hairy cell leukemia (HCL) is a pre-plasma B cell tumor which responds to interferon (IFN)-alpha t... more Hairy cell leukemia (HCL) is a pre-plasma B cell tumor which responds to interferon (IFN)-alpha therapy. In vitro, B cell growth factor (BCGF) can induce proliferation of hairy cells. We have investigated the effect of in vitro and in vivo treatments with different recombinant IFN on the capacity of hairy cells to proliferate in response to human BCGF. In vitro treatment of leukemic cells from HCL patients with recombinant IFN-alpha-2 (5/5 cases) or IFN-beta (4/5 cases) resulted in a marked inhibition of the BCGF-dependent response. This suppressive effect was obtained with IFN concentrations of 1000, 100 IU/ml, and even occasionally 10 IU/ml. In contrast, no such inhibition was observed with IFN-gamma, despite the presence of specific IFN-gamma receptors on hairy cells at densities similar to receptors for IFN-alpha/beta. The IFN-alpha-induced suppression of the proliferative response of hairy cells to BCGF was also observed in vivo in two patients within 6-12 hr after administrati...
Journal of immunology (Baltimore, Md. : 1950), Jan 15, 1990
Transduction through the CD23 molecule (Fc epsilon RII) was analyzed in human activated B lymphoc... more Transduction through the CD23 molecule (Fc epsilon RII) was analyzed in human activated B lymphocytes using anti-CD23 mAb. B cell blasts expressing an increased amount of surface CD23 molecule were obtained by stimulation of normal peripheral blood B lymphocytes with Staphylococcus aureus strain Cowan I and IL-4. Anti-CD23 mAb were found to trigger polyphosphoinositide hydrolysis in these cells (and also in tumoral B cells expressing spontaneously CD23) and a rise in [Ca2+]i which could be attributed to mobilization from cytoplasmic pools. This increase in [Ca2+]i could be mimicked, with a comparable time-course, by the addition of InsP3 to permeabilized B cell blasts indicating that the increase in inositol phosphate accumulation induced by the antibodies was due to a preferential attack of phosphatidylinositol-bisphosphate by a specific phosphoinositidase C (PIC). In permeabilized cells, raising the free calcium concentration above 3 microM was found to induce polyphosphoinositide...
Journal of immunology (Baltimore, Md. : 1950), 1991
Hairy cell leukemia (HCL) is a B cell tumor affecting the pre-plasma stage of B cell differentiat... more Hairy cell leukemia (HCL) is a B cell tumor affecting the pre-plasma stage of B cell differentiation. Hairy cells produce B cell growth factor (BCGF)-related growth factor(s) and we have previously shown that low mol wt (LMW)-BCGF-induced proliferation of hairy cells is inhibited in vitro and in vivo by IFN-alpha. We therefore suggested that this effect might contribute to the exquisite sensitivity of HCL to IFN-alpha therapy. To elucidate the mechanism involved in the therapeutic effect of IFN-alpha, we have analyzed the pattern of phosphorylated proteins in hairy cells. We detected the presence of a hyperphosphorylated protein with a molecular mass of about 35 kDa. This protein was identified as the CD20 molecule (B1), which is a structurally unique phosphoprotein exclusively detected on B cells and expressed during most stages of B cell development. Incubation of hairy cells with mitogenic concentrations of LMW-BCGF induces an additional increase in CD20 protein phosphorylation. ...
We have previously shown that addition of type 1 transforming growth factor-beta (TGFbeta1) to an... more We have previously shown that addition of type 1 transforming growth factor-beta (TGFbeta1) to an exponentially growing population of mink lung CCl64 cells increases their average intermitotic time from 14.4 to 20.3 hours, predominantly by extending G1 from 7.5 to 13.5 hours. Here we have used the DRIMAPS system (digitally recorded interference microscopy with automatic phase-shifting) for obtaining data on cellular mass distribution, cell motility and morphology. We found no significant change in the cells' rate of mass increase following TGFbeta1 treatment, which implies that the treated cells attained a higher mass during their extended cell cycle and this was confirmed by direct measurement of cell size. However, the cells showed a dramatic motile response to treatment: TGFbeta1-treated cells had a significantly higher time-averaged speed of 36.2 microm hour-1 compared to 14.5 microm hour-1 for the control cells. The time course of the response was gradual, reaching a maximum mean speed of 52.6 microm hour-1 after 15 hours exposure. We found that the gradual onset of the response was probably not due to a slow accumulation of a secondary factor but because cells were dividing throughout the experiment and most of the response to TGFbeta1 occurred only after the first cell division in its presence. Thus, taking only those cells that had not yet divided, the time-averaged speed of treated cells (26.1 micrometer hour-1) was only moderately higher than that of untreated cells (14.9 micrometer hour-1) whereas, for those cells that had divided, the difference in speed between treated cells (45.1 micrometer hour-1) and untreated cells (14.1 microm hour-1) was much greater. Increased speed was a consequence of enhanced protrusion and retraction of the cell margin coupled with an increase in cell polarity. TGFbeta1 also increased the mean spreading of the cells, measured as area-to-mass ratio, from 3.2 to 4.4 micrometer2 pg-1, and the intracellular mass distribution became more asymmetric. The observations indicate that a G2 signal may be necessary to reach maximal motility in the presence of TGFbeta1.
Transforming growth factor β (TGF-β) and related cytokines play a central role in the vascular sy... more Transforming growth factor β (TGF-β) and related cytokines play a central role in the vascular system. In vitro, TGF-β induces aortic endothelial cells to assemble subcellular actin-rich structures specialized for matrix degradation called podosomes. To explore further this TGF-β-specific response and determine in which context podosomes form, ALK5 and ALK1 TGF-β receptor signaling pathways were investigated in bovine aortic endothelial cells. We report that TGF-β drives podosome formation through ALK5 and the downstream effectors Smad2 and Smad3. Concurrent TGF-β-induced ALK1 signaling mitigates ALK5 responses through Smad1. ALK1 signaling induced by BMP9 also antagonizes TGF-β-induced podosome formation, but this occurs through both Smad1 and Smad5. Whereas ALK1 neutralization brings ALK5 signals to full potency for TGF-β-induced podosome formation, ALK1 depletion leads to cell disturbances not compatible with podosome assembly. Thus, ALK1 possesses passive and active modalities. ...
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