ALBERT

Description: Bone morphogenetic proteins (BMPs), members of the transforming growth factor (TGF)–β superfamily, are a group of related proteins that are capable of inducing the formation of cartilage and bone but are now regarded as multifunctional cytokines. We show in this report a novel function of BMPs in hematopoietic cells: BMP-2 induces apoptosis not only in human myeloma cell lines (U266, RPMI 8226, HS-Sultan, IM-9, OPM-2, and KMS-12 cells), but also in primary samples from patients with multiple myeloma. The mechanism of BMP-2–induced apoptosis was investigated with the use of U266 cells, which are dependent on the interleukin-6 autocrine loop. We showed that BMP-2 caused cell-cycle arrest in the G1 phase and the subsequent apoptosis of myeloma cells. BMP-2 up-regulated the expression of cyclin-dependent kinase inhibitors (p21CIP1/WAF1 and p27KIP1) and caused hypophosphorylation of retinoblastoma (Rb) protein. In studies of apoptosis-associated proteins, BMP-2 was seen to down-regulate the expression of Bcl-xL; however, BMP-2 had no effects on the expression of Bcl-2, Bax, or Bad. Therefore, BMP-2 induces apoptosis in various human myeloma cells by means of the down-regulation of Bcl-xL and by cell-cycle arrest through the up-regulation of p21CIP1/WAF1 and p27KIP1 and by the hypophosphorylation of Rb. Further analysis showed that the signal transducer and activator of transcription 3 (STAT3) was inactivated immediately after BMP-2 treatment. We conclude that BMP-2 would be useful as a novel therapeutic agent in the treatment of multiple myeloma both by means of its antitumor effect of inducing apoptotis and through its original bone-inducing activity, because bone lesions are frequently seen in myeloma patients.

Description: The therapeutic approach to acute myelogenous leukemia (AML) is usually chemotherapy, but severe side effects and complications, including infection and bleeding induced by the anticancer drugs, are major problems in the clinical setting. Recently, more specifically targeted agents have been developed for the treatment of AML; however, most candidate agents for targeted therapy have yet to be translated into clinical application. In the future, it will be important to identify other highly specific therapeutic agents based on our evolving understanding of the biology of AML. We have reported that reactive oxygen species (ROS) are key mediators of apoptosis induced by a green tea polyphenol, (-)-epigallocatechin-3-gallate (EGCG), and other ROS-producing agents, in myeloid leukemic cells (Haematologica2005;90:317–325). In this study, we further examined the mechanism of oxidative stress-induced apoptosis and its relationship to the heme enzyme myeloperoxidase (MPO). EGCG rapidly induced apoptosis in MPO-positive (HL-60, UF-1, NB4, Kasumi-1), but not MPO-negative (U937, THP-1, KG-1, K562) leukemia cell lines as well as fresh samples from AML. Pre-incubation of MPO-positive leukemic cells with the MPO-specific inhibitor, 4-aminobenzoic acid hydrazide, and the heme biosynthesis inhibitor, succinylacetone, resulted in inhibition of the intracellular MPO activity, ROS production and induction of apoptosis following addition of EGCG. To investigate the role of MPO in EGCG-induced apoptosis, we transfected the full length MPO cDNA and empty vector into MPO-negative K562 cells (designated K562/MPO and K562/Cont cells, respectively). In contrast to K562/Cont cells, K562/MPO cells enhanced MPO activity and ROS production, and sensitized EGCG-resistant K562 cells to apoptosis induced by ROS-producing agents. In addition, an enzymatically inactive MPO mutant expressing K562 (K562/H502A) cells could not respond to EGCG, suggesting that MPO is important for determining the sensitivity to EGCG-induced oxidative stress. MPO catalyzes the formation of HOCl, a powerful oxidant formed from chloride ions and H2O2. We next examined the relationship between EGCG-induced apoptosis and the H2O2/MPO/halide system in MPO-positive myeloid leukemic cells. Further examination revealed that both HOCl scavengers (methionine, taurine) and the hydroxyl radical (·OH) scavenger thiourea inhibited EGCG-induced apoptosis in myeloid leukemic cells. To determine which ROS play a key role in oxidative stress-induced apoptosis mediated through MPO, we used the novel fluorescence probes AFP and HPF to detect highly toxic ROS (·OH). It is noteworthy that the fluorescence intensity of both APF- and HPF-loaded myeloid leukemic cells significantly increased upon stimulation with EGCG, suggesting that EGCG generated highly toxic ROS (·OH) in MPO-positive leukemic cells. Interestingly, the combination of EGCG and other ROS-producing agents (As2O3, doxorubicin, and daunorubicin) enhanced apoptosis through an increase in the production of highly toxic ROS in myeloid leukemic cells. Taken together, these observations indicate that highly toxic ROS (·OH) generated via the H2O2/MPO/halide system induce apoptosis, and that such ROS may be the direct mediators of oxidative stress-induced apoptosis in MPO-positive leukemic cells. “MPO-targeted therapy” will lead to new insights that should help in overcoming drug resistance in refractory AML.

Description: Retinoic acid (RA) resistance is a serious problem for patients with acute promyelocytic leukemia (APL) who are receiving all-transRA. However, the mechanisms and strategies to overcome RA resistance by APL cells are still unclear. The biologic effects of RA are mediated by two distinct families of transcriptional factors: RA receptors (RARs) and retinoid X receptors (RXRs). RXRs heterodimerize with 1,25-dihydroxyvitamin D3[1,25(OH)2D3] receptor (VDR), enabling their efficient transcriptional activation. The cyclin-dependent kinase (cdk) inhibitor p21WAF1/CIP1 has a vitamin D3–responsive element (VDRE) in its promoter, and 1,25(OH)2D3 enhances the expression of p21WAF1/CIP1 and induces differentiation of selected myeloid leukemic cell lines. We have recently established a novel APL cell line (UF-1) with features of RA resistance. 1,25(OH)2D3 can induce growth inhibition and G1 arrest of UF-1 cells, resulting in differentiation of these cells toward granulocytes. This 1,25(OH)2D3-induced G1 arrest is enhanced by all-trans RA. Also, 1,25(OH)2D3 (10−10 to 10−7 mol/L) in combination with RA markedly inhibits cellular proliferation in a dose- and time-dependent manner. Associated with these findings, the levels of p21WAF1/CIP1 and p27KIP1 mRNA and protein increased in these cells. Northern blot analysis showed that p21WAF1/CIP1 and p27KIP1 mRNA and protein increased in these cells. Northern blot analysis showed that p21WAF1/CIP1 and p27KIP1 transcripts were induced after 6 hours’ exposure to 1,25(OH)2D3 and then decreased to basal levels over 48 hours. Western blot experiments showed that p21WAF1/CIP1 protein levels increased and became detectable after 12 hours of 1,25(OH)2D3treatment and induction of p27KIP1 protein was much more gradual and sustained in UF-1 cells. Interestingly, the combination of 1,25(OH)2D3 and RA markedly enhanced the levels of p27KIP1 transcript and protein as compared with levels induced by 1,25(OH)2D3 alone. In addition, exogenous p27KIP1 expression can enhance the level of CD11b antigen in myeloid leukemic cells. In contrast, RA alone can induce G1 arrest of UF-1 cells; however, it did not result in an increase of p21WAF1/CIP1 and p27KIP1transcript and protein expression in RA-resistant cells. Taken together, we conclude that 1,25(OH)2D3 induces increased expression of cdk inhibitors, which mediates a G1 arrest, and this may be associated with differentiation of RA-resistant UF-1 cells toward mature granulocytes.

Description: Bone morphogenetic proteins (BMPs), members of the transforming growth factor (TGF)–β superfamily, are a group of related proteins that are capable of inducing the formation of cartilage and bone but are now regarded as multifunctional cytokines. We show in this report a novel function of BMPs in hematopoietic cells: BMP-2 induces apoptosis not only in human myeloma cell lines (U266, RPMI 8226, HS-Sultan, IM-9, OPM-2, and KMS-12 cells), but also in primary samples from patients with multiple myeloma. The mechanism of BMP-2–induced apoptosis was investigated with the use of U266 cells, which are dependent on the interleukin-6 autocrine loop. We showed that BMP-2 caused cell-cycle arrest in the G1 phase and the subsequent apoptosis of myeloma cells. BMP-2 up-regulated the expression of cyclin-dependent kinase inhibitors (p21CIP1/WAF1 and p27KIP1) and caused hypophosphorylation of retinoblastoma (Rb) protein. In studies of apoptosis-associated proteins, BMP-2 was seen to down-regulate the expression of Bcl-xL; however, BMP-2 had no effects on the expression of Bcl-2, Bax, or Bad. Therefore, BMP-2 induces apoptosis in various human myeloma cells by means of the down-regulation of Bcl-xL and by cell-cycle arrest through the up-regulation of p21CIP1/WAF1 and p27KIP1 and by the hypophosphorylation of Rb. Further analysis showed that the signal transducer and activator of transcription 3 (STAT3) was inactivated immediately after BMP-2 treatment. We conclude that BMP-2 would be useful as a novel therapeutic agent in the treatment of multiple myeloma both by means of its antitumor effect of inducing apoptotis and through its original bone-inducing activity, because bone lesions are frequently seen in myeloma patients.

Description: Green tea polyphenol, (−)-epigallocatechin-3-gallate (EGCG), has a potent chemopreventive effects against various tumors, and epidemiologic studies have suggested that green tea consumption might be effective for reducing the incidence of certain cancers. We have reported that EGCG rapidly induces apoptosis of myeloid leukemic cells via modulation of reactive oxygen species (ROS) production in vitro and in vivo (Haematologica2005;90:317). In this study, we further examined the precise mechanism of EGCG-induced apoptosis and its relationship to the heme enzyme myeloperoxidase (MPO). EGCG inhibited cellular growth of various myeloid leukemic cells via induction of apoptosis in dose- and time-dependent manners. Interestingly, EGCG rapidly induced apoptosis in MPO-positive myeloid leukemic cell lines (HL-60, UF-1, NB4, Kasumi-1) and fresh myeloid leukemic cells from patients, whereas EGCG failed to induce apoptosis in MPO-negative leukemic cells (U937, THP-1, KG-1, K562). Pre-incubation of MPO-positive myeloid leukemic cells with the MPO-specific inhibitor, 4-aminobenzoic acid hydrazide (50 μM), and the heme biosynthesis inhibitor, succinylacetone (0.5 mM), resulted in significant inhibition of intracellular MPO activity, ROS production, and induction of apoptosis after treatment with 50 μM EGCG. Pre-incubation of MPO-positive myeloid leukemic cells with anti-oxidant, catalase, completely suppressed EGCG-induced ROS production and apoptosis. These results indicate that EGCG-induced apoptosis is mediated through the generation of hydrogen peroxide (H2O2). To investigate the role of MPO in EGCG-induced apoptosis of leukemic cells, MPO-negative K562 cells were stably transfected with full length of MPO cDNA (K562/MPO cells). Marked interest, K562/MPO cells induced MPO activity, intracellular ROS production, and enhanced susceptibility of cells to EGCG-induced apoptosis compared to wild-type K562 cells. These results suggested that MPO positivity may be important to determine the sensitivity to EGCG-induced apoptosis, and MPO-derived ROS are involved in apoptosis in myeloid leukemic cells. MPO catalyzes the formation of hypochlorous acid (HOCl), a powerful oxidant formed from Cl− and H2O2. Therefore, we next examined the relationship between EGCG-induced apoptosis and H2O2/MPO/halide system in MPO-positive HL-60 cells. Addition of HOCl scavengers, methionine (10 mM) and taurine (25 mM), inhibited EGCG-induced apoptosis in HL-60 and K562/MPO cells, but not wild-type K562 cells, suggesting that HOCl is the mediator of EGCG-induced apoptosis. Interestingly, hydroxyl radical (•OH) scavenger, thiourea, also inhibited EGCG-induced apoptosis in HL-60 cells. To determine which reactive oxygen species play a key role in EGCG-induced apoptosis mediated through MPO, we used novel fluorescence probes APF and HPF, which can detect selectively highly ROS (hROS). It is noteworthy that the fluorescence intensity of both APF- and HPF-loaded HL-60 cells significantly increased upon stimulation with EGCG, suggesting that EGCG generated hROS (•OH, ONOO−) and OCl−, but not other ROS (H2O2, NO, O2−, 1O2) in HL-60 cells. Taken together, these results indicated that highly toxic ROS such as hydroxyl radical generated via H2O2/MPO/halide system induces apoptosis, and that it may be the direct mediator of EGCG-induced apoptosis in MPO-positive myeloid leukemic cells.

Description: Retinoic acid (RA) resistance is a serious problem for patients with acute promyelocytic leukemia (APL) who are receiving all-transRA. However, the mechanisms and strategies to overcome RA resistance by APL cells are still unclear. The biologic effects of RA are mediated by two distinct families of transcriptional factors: RA receptors (RARs) and retinoid X receptors (RXRs). RXRs heterodimerize with 1,25-dihydroxyvitamin D3[1,25(OH)2D3] receptor (VDR), enabling their efficient transcriptional activation. The cyclin-dependent kinase (cdk) inhibitor p21WAF1/CIP1 has a vitamin D3–responsive element (VDRE) in its promoter, and 1,25(OH)2D3 enhances the expression of p21WAF1/CIP1 and induces differentiation of selected myeloid leukemic cell lines. We have recently established a novel APL cell line (UF-1) with features of RA resistance. 1,25(OH)2D3 can induce growth inhibition and G1 arrest of UF-1 cells, resulting in differentiation of these cells toward granulocytes. This 1,25(OH)2D3-induced G1 arrest is enhanced by all-trans RA. Also, 1,25(OH)2D3 (10−10 to 10−7 mol/L) in combination with RA markedly inhibits cellular proliferation in a dose- and time-dependent manner. Associated with these findings, the levels of p21WAF1/CIP1 and p27KIP1 mRNA and protein increased in these cells. Northern blot analysis showed that p21WAF1/CIP1 and p27KIP1 mRNA and protein increased in these cells. Northern blot analysis showed that p21WAF1/CIP1 and p27KIP1 transcripts were induced after 6 hours’ exposure to 1,25(OH)2D3 and then decreased to basal levels over 48 hours. Western blot experiments showed that p21WAF1/CIP1 protein levels increased and became detectable after 12 hours of 1,25(OH)2D3treatment and induction of p27KIP1 protein was much more gradual and sustained in UF-1 cells. Interestingly, the combination of 1,25(OH)2D3 and RA markedly enhanced the levels of p27KIP1 transcript and protein as compared with levels induced by 1,25(OH)2D3 alone. In addition, exogenous p27KIP1 expression can enhance the level of CD11b antigen in myeloid leukemic cells. In contrast, RA alone can induce G1 arrest of UF-1 cells; however, it did not result in an increase of p21WAF1/CIP1 and p27KIP1transcript and protein expression in RA-resistant cells. Taken together, we conclude that 1,25(OH)2D3 induces increased expression of cdk inhibitors, which mediates a G1 arrest, and this may be associated with differentiation of RA-resistant UF-1 cells toward mature granulocytes.