[BAFF level in bone marrow and expression of BAFF receptor on B cells in multiple myeloma patients].

This study was purposed to investigate the B cell-activating factor belonging to the TNF family (BAFF) and a proliferation-inducing ligand (APRIL) levels in bone marrow, and the BAFF receptor expression level on B cells in multiple myeloma (MM) patients, in order to explore the characteristics of B cells in bone marrow of MM patients. MM patients were studied before treatment (newly diagnosed group, 19 patients) and after treatment with improvement (stable group, 17 patients), 10 non-hematologic patients were selected as control (control group). The BAFF receptors (BAFF-R) and transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI) on B cell (CD19(+)), naive B cell (CD19(+)IgD(+)) and memory B cell (CD19(+)CD27(+)) of bone marrow in all groups were detected by flow cytometry. The BAFF, APRIL level in bone marrow supernatant were tested with ELISA. The results showed that the BAFF-R expression level on CD19(+) cells in newly diagnosed group were higher than that in stable group and control group; there was no significant difference between the BAFF-R expression level on CD19(+)IgD(+) cells in newly diagnosed group and stable group, but BAFF-R expression level on CD19(+)IgD(+) cells in newly diagnosed group was higher than that in control group; the BAFF-R expression level on CD19(+)CD27(+) cells in newly group was higher than that in stable group and control group; there was no significant difference between the BAFF-R expression level on CD19(+) cells, CD19(+)IgD(+) cells or CD19(+)CD27(+) cells in stable group and control group. There was no significant difference among the TACI expression level on CD19(+) cells, CD19(+)IgD(+) cells or CD19(+)CD27(+) cells in newly diagnosed group, stable group and control group. The bone marrow supernatant BAFF level in newly diagnosed group was higher than that in stable group and control group, but there was no significant difference between stable group and control group. There was no significant difference among the bone marrow TACI levels in newly diagnosed group, stable group and control group. It is concluded that both the bone marrow BAFF level and the BAFF-R expression level on CD19(+) cell, CD19(+)IgD(+) cells and CD19(+)CD27(+) cells in MM patients increase, which may help to stimulate B cells, thereby may relate with to MM pathogenesis.

Molecular mechanisms of the biphasic effects of interferon-γ on osteoclastogenesis.

Although interferon-γ (IFN-γ) potently inhibits osteoclastogenesis, the suppressive effect is significantly reduced when osteoclast precursors are pre-exposed to the receptor activator of NF-κB (RANK) ligand (RANKL). However, the molecular mechanism underlying the biphasic effects of IFN-γ on osteoclastogenesis remains elusive. Here, we recapitulate the biphasic functions of IFN-γ in osteoclastogenesis in both tissue culture dishes and on bone slices. We further demonstrate that IFN-γ markedly suppresses the RANKL-induced expression of nuclear factor of activated T-cells c1 (NFATc1) in normal, but not RANKL-pretreated bone marrow macrophages (BMMs). Similarly, IFN-γ impairs the activation of the nuclear factor-κB (NF-κB) and c-Jun N-terminal kinase (JNK) pathways in normal, but not RANKL-pretreated, BMMs. These findings indicate that IFN-γ inhibits osteoclastogenesis partially by suppressing the expression of NFATc1 and the activation of the NF-κB and JNK pathways. Moreover, IFN-γ inhibits the RANKL-induced expression of osteoclast genes, but RANKL pretreatment reprograms osteoclast genes into a state in which they can no longer be suppressed by IFN-γ, indicating that IFN-γ inhibits osteoclastogenesis by blocking the expression of osteoclast genes. Finally, the IVVY(535-538) motif in the cytoplasmic domain of RANK is responsible for rendering BMMs refractory to the inhibitory effect of IFN-γ. Taken together, these findings provide important mechanistic insights into the biphasic effects of IFN-γ on osteoclastogenesis.

Interleukin-4 inhibits RANKL-induced NFATc1 expression via STAT6: a novel mechanism mediating its blockade of osteoclastogenesis.

Interleukin-4 (IL-4) is an important immune regulatory protein that possesses potent anti-osteoclastogenic properties, and does so via the transcription factor STAT6. Previous studies have shown that IL-4 selectively blocks RANKL-induced activation of NF-κB and mitogen-activated protein kinase (MAPK) pathway molecules, suggesting that the cytokine arrests osteoclastogenesis by blockade of these signaling cascades. However, the fact that the inhibitory effect on these pathways requires prolonged IL-4 pretreatment, and that the cytokine fails to exert an anti-osteoclastogenic effect after short-term pre-exposure of RANKL to osteoclast precursors, suggests that an additional, more immediate mechanism may also be involved. In this study, we found that simultaneous exposure of IL-4 did not alter RANKL-dependent activation of NF-κB or MAPKs, whereas the cytokine did block RANKL-induced nuclear factor activated T cells c1 (NFATc1), a master osteoclastogenic transcription factor. This inhibitory effect of IL-4 required STAT6, consistent with its functional role in osteoclastogenesis. In addition, the cytokine also partially impaired RANKL-stimulated bone resorption. Furthermore, IL-4 suppressed expression of RANKL-induced osteoclast specific genes in a STAT6-dependent manner, but failed to do so when osteoclast precursors were pre-exposed to RANKL. Thus, we provide the first evidence that IL-4 inhibits osteoclast formation by inhibiting RANKL induction of NFATc1 via STAT6 as an early event, in addition to its suppression of other signaling pathways. The inhibitory effect is ultimately regulated at the gene expression transcriptional level.

[Correlation of CD19 positive cell counts in bone marrow with therapeutic efficacy in patients with multiple myeloma].

This study was aimed to analyze the correlation of CD19 positive cell counts in bone marrow of multiple myeloma(MM) patients with therapeutic efficacy and investigate the characteristics of CD19 cell change in MM bone marrow. The CD19(+) and CD38(++)CD45(-), CD38(++)CD45(-)CD56(+) cells in bone marrow of 63 MM patients were detected by flow cytometry. The difference of CD19(+), CD38(++)CD45(-), CD38(++)CD45(-)CD56(+) cell counts at different stages and types, as well as their relation with results of 4 course of VADM or VD chemotherapy were analyzed. The results showed that in 63 MM patients, CD19(+) cell ratio at stage II were higher than those at stage III; CD38(++)CD45(-)CD56(+) cell ratio at stage II were lower than those at stage III; CD19(+) cell ratio in type IgA were higher than those in type IgD; the CD38(++)CD45(-), CD38(++)CD45(-)CD56(+) cell counts in type IgA were obviously lower than those in type IgG, IgD and light chain which showed a negative correlation between cell counts of CD19(+) against CD38(++)CD45(-), CD38(++)CD45(-)CD56(+). CD19(+) cell counts in effective treatment group of all 43 patients and the effective treatment group with VD were both higher than those in the ineffective treatment group; CD38(++)CD45(-) cell counts in effective treatment group with VD was obviously lower than those in ineffective treatment group, and CD38(++)CD45(-), CD38(++)CD45(-)CD56(+) in effective treatment group of all 43 patients were lower than those in ineffective treatment group. It is concluded that CD19(+) cell counts in bone marrow may be related to disease status and development stage of MM, which may be useful to predict treatment efficacy and prognosis.

[The role of B cell-activating factor secreted by peripheral blood monocyte-derived dendritic cell in chronic idiopathic thrombocytopenic purpura].

OBJECTIVE: To explore the characteristics of B cell-activating factor (BAFF) secreted by peripheral blood monocyte-derived dendritic cell (MoDC) in chronic idiopathic thrombocytopenic purpura (cITP) and the function of MoDC on B cell proliferation. METHODS: Ten cITP patients were studied dynamically before and after treatment. The BAFF levels in serum and the supernatant of LPS stimulated MoDC were tested with ELISA. The BAFF gene expression in LPS stimulated MoDC was tested with RQ-PCR, the B cell proliferation co-cultured with the supernatant of LPS stimulated MoDC for 5 days was tested with flow cytometry for CFSE and (3)H thymidine incorporation. RESULTS: The BAFF level in serum (serum BAFF) \[(2461 ± 483) ng/L\], and supernatant of LPS stimulated MoDC (supernatant BAFF) \[(1113 ± 113) ng/L\] and BAFF mRNA in LPS stimulated MoDC (BAFF mRNA) (1.70 ± 0.23) before treatment were higher than that after treatment \[(621 ± 53) ng/L, (490 ± 49) ng/L and 0.37 ± 0.12\] and normal group \[(742 ± 77) ng/L, (582 ± 63) ng/L and 0.52 ± 0.08\]. There was a positive correlation among serum BAFF, supernatant BAFF and BAFF mRNA, and a negative correlation among serum BAFF, supernatant BAFF and BAFF mRNA and blood platelet count (BPC) in all ITP patients. The supernatant of LPS-stimulated MoDC from untreated patients enhanced B cell proliferation as compared with the supernatant of LPS-stimulated MoDC from treated patients and normal group. CONCLUSION: BAFF might contribute to disease development in cITP. MoDC may directly increase B cell proliferation by secreting BAFF without T cell help, playing an important role in the antibody production in cITP.

[Dynamic observations of beta-catenin in chronic myeloid leukemia and its relationship with cytogenetic response].

OBJECTIVE: To investigate the changes in the expression of beta-catenin in patients with chronic myeloid leukemia (CML) in different phases, and explore the relationship between beta-catenin and the cytogenetic response to imatinib mesylate. METHODS: Beta-catenin mRNA and protein expressions were detected by RT-PCR and Western blotting in the bone marrow mononuclear cells (BMMNCs) from 99 CML patients. The expressions of BCR-ABL fusion gene at both the mRNA and protein levels were detected by fluorescence in situ hybridization (FISH) in 94 patients before and during the one-year treatment with imatinib mesylate at the interval of 3 months, and the relationship between beta-catenin and cytogenetic response to imatinib mesylate was analyzed. RESULTS: The expression of beta-catenin increased significantly in patients with blast crisis and accelerated phase (P<0.001), but showed no significant difference between normal subjects and CML patients in the chronic phase (P>0.05). The main cytogenetic remission rate was significantly higher in patients who were consistently negative for beta-catenin than in those consistently positive for beta-catenin or those with a positive transformation (P<0.001). CONCLUSION: Beta-catenin overexpression in the progression of CML, consistent high level of beta-catenin or a positive transformation may indicate a poor response to imatinib, and early measures should be taken to increase the remission rate.

Direct B-cell stimulation by peripheral blood monocyte-derived dendritic cells in idiopathic thrombocytopenic purpura patients.

OBJECTIVE: Dendritic cells (DCs) play a major role in regulating lymphocytes. The emergence of antiplatelet autoantibodies remains the central pathogenetic mechanism in idiopathic thrombocytopenic purpura (ITP); defective DC functions have been implicated in ITP. The purpose of this study was to assess the contribution of DCs to B-cell hyperactivity in ITP patients. METHODS: Ten ITP patients were studied before treatment (group untreated) and after treatment (group treated ) with improvement. We compared the effects of monocyte-derived DC from ITP and healthy control (group control) on naive B cells in the presence of lipopolysaccharide, or anti-CD40. We measured the proliferation, antibody production, and expression of activation markers for B cells, as well as DC-secreted B-cell activating factor (BAFF) production. We also measured the serum BAFF level in ITP patients and control. The role of DC-produced BAFF was evaluated with anti-BAFF. RESULTS: Lipopolysaccharide, or anti-CD40, stimulated DCs from group untreated increased B-cell proliferation and antibody production as compared with DCs from group treated and group control. Cell-to-cell contact was not necessary for the augmented effect of the ITP DCs. Anti-CD40 treatment induced a higher production of BAFF in group untreated DCs. Serum BAFF levels, supernatant BAFF from anti-CD40-activated DCs, and BAFF mRNA expression of anti-CD40-activated DCs in group untreated were significantly higher than that in group treated and group control. In ITP patients, there was positive correlation among serum BAFF, supernatant BAFF, and BAFF mRNA levels, and there was negative correlation between serum BAFF, supernatant BAFF, BAFF mRNA levels, and blood platelet count. Blocking BAFF abrogated the effects of ITP DCs on naive B partially. CONCLUSION: Activated monocyte-derived DCs from ITP patients directly increase B-cell effector functions; this effect depends on soluble factors released by activated DCs and cell-to-cell contact. This might play an important role in the antibody production in ITP. DC-secreted BAFF is involved and might contribute to disease development.

[Significance of sRANKL/OPG ratio in diagnosis of multiple myeloma bone disease].

This study was purposed to investigate the relationship between the levels of soluble receptor activator of nuclear factor kappa B ligand (sRANKL) and osteoprotegerin (OPG) in serum of the patients with multiple myeloma (MM) and multiple myeloma bone disease (MBD). The serum levels of sRANKL, OPG, tartrate-resistant acid phosphatase-5b (TRAP-5b) and C-terminal telopeptide of collagen I (CTP-I) which both are indexes for metabolism of osteoclast (OC) in newly diagnosed MM patients (n=42, experimental group) and healthy persons (n=25, control group) were detected by enzyme-linked immunosorbent assay. The roentgenography was used to determine bone damage in MM patients at the same time. According to these results acquired, the correlation of sRANKL/OPG ratio with levels of TRAP-5b/CTP-I, the incidence and degree of bone destruction were analyzed. The results indicated that the level of sRANKL (median value 9.33 microg/L) increased and level of OPG (median value 4.93 microg/L) decreased and the sRANKL/OPG ratio (2.65) increased significantly in experimental group. Compared with control group, the differences in all the corresponding indicators were statistically significant (p<0.05). The sRANKL/OPG ratio was closely related to levels of TRAP-5b (r=0.512, p<0.05) and CTP-I (r=0.481, p<0.05) in MM patients. After all patients in experimental groups were divided into group with bone destruction (n=29) and without bone destruction (n=13), the sRANKL/OPG ratio in the group with bone destruction was 5.13 and much higher than that in group without bone destruction (1.12) (p<0.05). A close correlation between the sRANKL/OPG ratio and degree of bone destruction (r=0.445, p<0.05) was acquired when all MM patients were divided into three groups according to degree of bone destruction, but no difference between the ratio and clinical classification and International Staging System (ISS) in MM patients was found. It is concluded that the sRANKL/OPG ratio in serum of MM patients is significantly elevated, which may be closely related to increase metabolism of OC along with the incidence and degree of bone destruction. In short, the sRANKL/OPG ratio can be used as a reference index for the diagnosis of MBD.

New targets of PS-341: BAFF and APRIL.

Multiple myeloma (MM) is an incurable B-cell malignancy, characterized by the proliferation of malignant plasma cells. B-cell-activating factor (BAFF, also known as BlyS or B-lymphocyte stimulator) and a proliferation-inducing ligand (APRIL), the two members of the tumor necrosis factor ligand superfamily, enhance the production of antibodies and regulate and promote proliferation and survival. Both BAFF and APRIL have an important role in B cell lymphoma and chronic lymphocytic leukemia cell survival. This study evaluates the expression of BAFF, APRIL, and their three receptors in two human MM cell lines (KM3 and PRMI 8226) and ten primary MM cell lines, and their effects on the growth of MM cells in vivo. MM cells were found to express BAFF, APRIL, and their three receptor genes both at the gene and protein levels. ELISA found high concentrations of BAFF and APRIL in the supernatants of these cultured cells. Treatment with PS-341 decreased the concentration of BAFF and APRIL. The WST-1 analysis of growth found that BAFF and APRIL stimulated the proliferation of MM cells. PS-341 inhibited this enhanced proliferation and induced apoptosis. Making use of the rhTACI-Fc to block the BAFF- and APRIL-induced activation of NF-kappaB2, we demonstrated that the NF-kappaB2-p52/RelB signal contributes to MM cell survival. In summary, PS-341 was shown to block this pathway. PS-341 inhibits the autocrine secretion of BAFF and APRIL and thereby MM cell proliferation by the alterative NF-kappaB2 pathway. BAFF and APRIL appear to be the targets of PS-341.

[Osteoclast differentiation regulated by receptor activator of nuclear factor kappaB probably through a novel signaling pathway].

This study was aimed to investigate the signaling pathways regulating osteoclast (OC) differentiation by receptor activator of nuclear factor kappa (RANK) under physiological condition so as to provide some theoretical basis for clarifying mechanism of bone destruction in multiple myeloma. A mutant TNFR(1)/RANK(2) (named RANK-Mu) chimera consisting of tumor necrosis factor receptor 1 (TNFR(1)) and RANK intramembrane domain was constructed by using deletion mutation for deleting IVVY amino acids in RANK intramembrane domain in accordance with (535-)IVVY(-538) as specific domain regulating OC differentiation by RANK. The RANK-Mu and TNFR(1)/RANK chimera without mutation (RANK-WT) were packaged by using plat E cell line to produce the retrovirus, which were transfected into bone marrow macrophages (BMMs) of TNFR(1)/TNFR(2) double knockout mice. After stimulation of these transfected BMMs with TNF-alpha, the differentiation of BMMs into OCs were observed, meanwhile the phosphorylation of NF-kappab, JNK, p38 and ERK was detected by Western blot after stimulation of these BMMs with TNF-alpha. The results showed that BMMs transfected with RANK-WT could be differentiated into OCs and phosphorylation of NF-kappaB, JNK, P38 and ERK were activated at 5 - 10 minutes after being stimulated by TNFalpha. BMMs transfected with RANK-Mu could not be differentiated into OCs, but phosphorylation of NF-kappaB, JNK, P38 and ERK were activated also. It is concluded that RANK regulates osteoclast differentiation probably not through 4 typical signaling pathways, named as NF-kappaB, JNK, P38 and ERK, in this process other new signaling pathways maybe participate.

[The clinical features, chemotherapy responses and survival of 223 patients with newly diagnosed multiple myeloma].

OBJECTIVE: To explore the proportion, clinical and laboratory features, chemotherapy responses and long term survival of different kinds of newly diagnosed multiple myeloma (MM) in China. METHODS: The clinical data of 223 cases of newly diagnosed MM patients were gathered in the First Affiliated Hospital of Sun Yat-sen University from Jan, 2000 to Seb, 2007 were retrospectively analyzed. RESULTS: The proportions of each kind of MM including IgG, IgA, light chain, IgD, IgM and biclonal MM were 48.0%, 20.6%, 25.6%, 4.0%, 0.9% and 0.9% respectively. No IgE and nonsecretory myeloma was found. The median age of onset was 58 years, of which that of the IgA type was the oldest one and the light chain type was the youngest (P = 0.004). Bone pain, renal insufficiency, and anemia were the most common symptoms which accounted for 67.7%, 61.0% and 45.3% respectively. The incidences of renal inadequacy, hypercalcemia and pathological fracture in light chain type were higher than those in IgG and IgA types. Besides, no M protein were found in serum protein electrophoresis and no elevation of total globulin. The clinical features of IgD type were similar to that of the light-chain type. The total chemotherapy efficacy rate of 89 patients who were treated with more than 3 cycles in our hospital is 61.8%, which has no difference in all types. Median overall survival of the 89 patients was 33.0 months. CONCLUSION: IgG is the most common type in MM. Bone pain, anemia, hypercalcemia and renal insufficiency are common symptoms. Immunofixation electrophoresis should be performed routinely to avoid missed diagnosis of light-chain and IgD types of MM.

[Imatinib induces c-kit positive myeloma cells apoptosis].

OBJECTIVE: To explore the influence of Imatinib on multiple myeloma cells expressing c-kit in vitro and its mechanism. METHODS: KM3 cells were treated with Imatinib at different concentrations, and cell growth index were evaluated by XTT assay, cell cycle by flow cytometry, apoptosis by Annexin V/ PI and DNA ladder, and change in protein level by Western blot. RESULTS: Imatinib inhibited proliferation of KM3 cells at concentrations more than 0.25 micromol/L in a dose-dependent manner, and the 48 h IC50 was 0.33 micromol/L (P < 0.01). Imatinib arrested cell in C0/G1 phase. Annexin V/PI staining and DNA ladder indicated that Imatinib had a substantial effect on inducing apoptosis of KM3 cells in a dose-dependent manner and induced pro-caspase-3 and poly ADP-ribose polymerase (PARP) cleaved. Imatinib inhibited expression of c-kit and provoked a decrease of IL-6 induced c-kit phosphorylation in vitro. CONCLUSION: Imatinib inhibits KM3 cells proliferation and induces the cells apoptosis by inhibiting c-kit signalling transduction.

[Therapeutic effects of imatinib on chronic myeloid leukemia in different phases and the factors affecting the effects].

OBJECTIVE: To evaluate the therapeutic effect of imatinib on chronic myeloid leukemia (CML) in different phases and analyze the factors that may affect the effects. METHODS: Eighty-five patients with CML in chronic phase, 24 in accelerated phase and 19 in blastic phase patients were treated with imitinib. The hematologic response, cytogenetic response, molecular response, overall survival (OS), progression-free survival (PFS) and adverse events were analyzed in these groups. RESULTS: The rates of complete hematologic response (CHR), complete cytogenetic response (CCyR) and complete molecular response (CMoR) of the patients in chronic phase were 100%, 82.4% and 21.2%, respectively, and the 5-year OS and PFS of these patients were 92.1% and 84.7%. All these rates were significantly higher than those in patients in accelerated and blastic phases (P<0.0001). The CCyR, CMoR, 5-year OS and PFS in the 42 newly diagnosed patients in chronic phase were 92.9%, 26.3%, 100% and 95.2%, respectively, all significantly higher than those in patients with interferon therapy failure (P<0.001). Severe leukocytopenia and thrombocytopenia occurred at greater frequencey in AP and BP patients than in chronic phase patients (P<0.0001). Non-hematologic toxicity was rarer and milder in patients in chronic phase. Multivariate analysis showed that interferon therapy prior to imitinib treatment and prolonged drug cessation were the independent factors that affected the achievement of cytogenetic response and PFS. CONCLUSION: Early imitinib therapy can be effective and safe, and should be used as the first line drug for CML.

[Efficacy of bortezomib combined dexamethasone in 24 patients with multiple myeloma].

BACKGROUND & OBJECTIVE: Bortezomib, a potent and reversible proteasome inhibitor, induces apoptosis of myeloma cells, resulting in durable responses in patients with multiple myeloma (MM). This study was to explore the medical effects and side effects of bortezomib combined dexamethasone in treating newly diagnosed and relapsed or refractory MM, and evaluate the safety of this regimen in the patients with renal impairment. METHODS: Twenty-four MM patients were treated with bortezomib and dexamethasone in a 21-day cycle. The patients received a median of 3 cycles (range, 1-8 cycles) of the treatment. Response to bortezomib was evaluated according to the criteria of the European Group for Blood and Marrow Transplantation (EBMT); adverse events were graded according to the National Cancer Institute Common Toxicity Criteria. RESULTS: During the follow-up with a median of 4 months, 19 (79.2%) patients responded to the treatment. The complete remission (CR) rate was significantly higher in the patients of light-chain type than in those of non-light-chain type (57.1% vs. 5.9%, P=0.014). The response rates of the patients with and without renal impairment were similar (100% vs. 70.6%, P=0.272), and the renal functions were ameliorated in the patients with renal impairment during chemotherapy. Grade III-IV adverse events, including leucocytopenia (8.3%), thrombocytopenia (33.3%), diarrhea (8.3%) and debility (4.2%), could be relieved by symptomatic treatment or delayed chemotherapy. CONCLUSIONS: The combination of bortezomib and dexamethasone shows obvious effects on MM, especially in the patients with light-chain type. The adverse events can be tolerant in most patients, and this regimen is also safe in the patients with renal impairment.

Aurora kinase inhibitory VX-680 increases Bax/Bcl-2 ratio and induces apoptosis in Aurora-A-high acute myeloid leukemia.

Previously, we and others showed that mitotic Aurora-A kinase (Aur-A) was required for accurate mitotic entry and proper spindle assembly. In this study, we found that expression of Aur-A was markedly elevated in bone marrow mononuclear cells (BMMCs) obtained from a significant portion of de novo acute myeloid leukemia (AML) patients. Targeting human primary AML cells with Aur-A kinase inhibitory VX-680 led to apoptotic cell death in a dose-dependent manner. Importantly, VX-680-induced cell death was preferentially higher in Aur-A-high primary leukemic blasts compared with Aur-A-low AML (P < .001) or normal BMMCs (P < .001), suggesting the possible pharmacologic window in targeting Aurora kinase among Aur-A-high VX-680-sensitive leukemia patients. VX-680-induced cell death in AML cell lines was accompanied by formation of monopolar mitotic spindles, G(2)/M phase arrest, decreased phosphorylated(p)-Akt-1, and increased proteolytic cleavage of procaspase-3 and poly(ADP)ribose polymerase. Notably, VX-680 increased Bax/Bcl-2 expression ratio, a favorable proapoptotic predictor for drug response and survival in AML. Lastly, VX-680 enhanced the cytotoxic effect of the chemotherapeutic agent etoposide (VP16) on AML cells. Together, we concluded that Aurora kinases were potentially therapeutic targets for AML and that Aur-A-high expression may serve as a differential marker for selective treatment.

Expression of soluble Flt-1 gene in Pichia pastoris and the effect of the product on multiple-myeloma cells in vitro.

VEGF (vascular endothelial growth factor), a potent angiogenic molecule specific for vascular endothelial cells, is overexpressed in most tumours including MM (multiple myeloma) and closely associated with tumour growth and prognosis. It has been shown that a soluble fragment of the VEGF receptor Flt-1 (Fms-like tyrosine kinase-1) [sFlt-1 (soluble Flt-1)] has antiangiogenic properties by way of its antagonist activity against VEGF. VEGF and its receptors have been shown to be targets for treating tumours. In the present study, sFlt-1 gene was expressed in Pichia pastoris and the product was applied for studying the effect on KM3 MM cells. sFlt-1 gene was inserted into the pPICZalphaA vector and the expressed product was analysed by SDS/PAGE, immunoblot and ELISA. The sFlt-1 protein was expressed by 0.5% (v/v) methanol induction and it accumulated up to 23% of total proteins in the supernatant. The product was further purified with metal-chelating resin [Ni-NTA (Ni(2+)-nitrilotriacetate)]. The functional analysis of the sFlt-1 protein was performed with HUVEC (human umbilical-vein endothelial cells) proliferation assay. We next showed that the sFlt-1 protein acted directly on MM cells and inhibited the VEGF-induced proliferation of MM cells with MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] and (3)H uptake assay. The sFlt-1 protein blocked VEGF-induced ERK (extracellular-signal-regulated kinase) phosphorylation and inhibited the MAPK (mitogen-activated protein kinase) signalling cascades. The present study demonstrated that anti-MM activity of the sFlt-1 protein, coupled with its antiangiogenic effects, provides the basis for clinical trials of this agent to improve the outcome in MM.

[Expression and clinical significance of beta-catenin in multiple myeloma].

BACKGROUND & OBJECTIVE: beta-catenin is the pivotal regulator in Wnt pathway and in charge of cellular adhesion and signal conduction. Overexpression of beta-catenin has been observed in various human tumors. This study was to investigate the expression and clinical significance of beta-catenin in multiple myeloma (MM). METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot were used to detect mRNA and protein expression of beta-catenin in bone marrow samples from 12 newly diagnosed MM patients, 14 relapsed/refractory MM patients, and 11 healthy donors. The clinical data and treatment outcomes of the MM patient were also analyzed. RESULTS: The positive rate and the expression level of beta-catenin mRNA were significantly lower in healthy donors than in newly diagnosed patients and relapsed/refractory patients (27.3% vs. 75.0% and 100%, P=5.01e(-4); 0.35+/-0.17 vs. 0.72+/-0.11 and 0.85+/-0.16, P=5.88e(-5)); the mRNA level of beta-catenin was significantly lower in newly diagnosed patients than in relapsed/refractory patients (P=0.045). beta-catenin protein was not detected in healthy donors; while the positive rate and the expression level of beta-catenin protein were significantly lower in newly diagnosed patients than in relapsed/refractory patients (50.0% vs. 85.7%, 0.32+/-0.11 vs. 0.21+/-0.08, P=0.039). In the 10 newly diagnosed MM patients with evaluable treatment outcomes, the positive rate of beta-catenin protein was significant lower in the 7 patients without response than in the 3 patients showed response (14.3% vs. 100%, P=0.033). The positive rate of beta-catenin protein was significant higher in the 16 Durie/Salmon stage III patients than in the 10 stage II patients (87.5% vs. 40.0%, P=0.026), and significant higher in ISS stage III patients than in stage I and II patients (100% vs. 45.5% and 33.3%, P=0.006, P=0.032). The protein level of beta-catenin was positively correlated to serum levels of beta2-MG (r=0.688, P=0.002) and lactate dehydrogenase (LDH) (r=0.502, P=0.034). CONCLUSION: The expression of beta-catenin is related with treatment outcome, Durin-Salmon stage and ISS stage of MM, and is positively correlated to serum levels of LDH and beta2-MG.

[Efficacy and toxicity of intravenous busulfan-based conditioning before allogeneic peripheral blood stem cell transplantation for leukemia].

BACKGROUND & OBJECTIVE: Busulfan (Bu) is commonly used as a component of conditioning regimens for hematopoietic stem cell transplantation. Erratic gastrointestinal absorption as a result of oral administration of Bu not only affects the efficacy, but also increases the risk of toxicity. This study was to analyze the efficacy and toxicity of intravenous Bu and cyclophosphamide (Cy) conditioning before allogeneic peripheral blood stem cell transplantation (allo-PBSCT) for leukemia. METHODS: Fifteen leukemia patients received intravenous Bu/Cy conditioning before allo-PBSCT, while 20 patients received oral Bu/Cy conditioning. The responses and adverse events of the 2 groups were assessed. RESULTS: All 15 patients in intravenous Bu/Cy group had hematopoietic engraftment. The median time of engraftment was 12 (9-15) days for neutrophils and 15 (11-24) days for platelets. Of the 15 patients, 6 (40.0%) developed acute graft-versus-host disease (aGVHD), including 4 cases of grade I-II aGVHD and 2 cases of grade III-IV aGVHD; during conditioning, 7 (46.6%) had vomiting, 1 (6.7%) had oral mucositis, 1 (6.7%) had hemorrhagic cystitis, 2 (13.3%) had hepatic damage, none developed seizure. With a median follow-up of 180 days (range, 35-420 days), 14 (93.3%) patients were alive, 1 died of severe aGVHD accompanied fungal infection of the lung and central nerve system. The occurrence rates of hepatic damage and oral mucositis were significantly lower in intravenous Bu/Cy group than in oral Bu/Cy group (13.3% vs. 60.0%, 6.7% vs. 80.0%, P<0.01). There were no significant differences in hematopoietic reconstruction, aGVHD, stomatitis, gastrointestinal reaction, and hemorrhagic cystitis between the 2 groups (P>0.05). CONCLUSION: The intravenous Bu/Cy conditioning before allo-PBSCT for leukemia has clear efficacy with low extramedullary toxicity.

Soluble Fms-like tyrosine kinase-1 expression inhibits the growth of multiple myeloma in nude mice.

Angiogenesis is an essential factor in the growth and progression of hematological malignancies including multiple myeloma (MM). Vascular endothelial growth factor and its receptors have been shown to be targets for treating tumors. This study explores the effect of adenovirus-mediated delivery of soluble vascular endothelial growth factor receptor Fms-like tyrosine kinase-1 (sFLT-1) on the growth of MM cell line KM3 in nude mice. sFLT-1 cDNA was amplified by reverse transcription-polymerase chain reaction from human umbilical vein endothelial cells and was used as a transgene to construct an adenoviral vector carrying sFLT-1 (ADV-sFLT). Cell proliferation and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assays were carried out to evaluate the effect of ADV-sFLT on human umbilical vein endothelial cells and KM3 cells in vitro. Eighteen female BALB/c nude mice were inoculated subcutaneously with KM3 cells, and they were randomly divided into three groups and injected intravenously with ADV-sFLT, ADV-LacZ, or phosphate-buffered saline (PBS). The volume of KM3 xenografts was measured twice a week. Three weeks after the initial treatment, the volume of MM xenografts in the mice treated with ADV-sFLT, ADV-LacZ, or PBS was 770.32+/-28.73 mm3, 1983.36+/-43.72 mm3, and 2042.05+/-82.31 mm3, respectively (P<0.01, ADV-sFLT versus ADV-LacZ or PBS). The value of microvessel density was 29.17+/-6.85, 79.17+/-7.35, and 78.83+/-8.54 in the tumors treated with ADV-sFLT, ADV-LacZ, and PBS, respectively (P<0.01, ADV-sFLT versus ADV-LacZ or PBS). This study suggested that the adenovirus-mediated sFLT-1 gene greatly inhibits MM-derived tumor growth and angiogenesis in mouse xenograft, and might serve as a new therapy for MM.

[Expression and clinical significance of vascular endothelial growth factor and its receptors in multiple myeloma].

BACKGROUND & OBJECTIVE: Bone marrow angiogenesis plays an important role in the development and prognosis of multiple myeloma (MM). Vascular endothelial growth factor (VEGF) is the most potent stimulatory factor in angiogenesis. This study was to examine the expression of VEGF and its receptors in MM, and analyze their correlations to the tumorigenesis and development of MM. METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect mRNA expression of VEGF, Flt-1, and KDR in bone marrow samples from 12 newly diagnosed MM patients, 23 relapsed/refractory MM patients, 16 control people, and in KM3 cells. RESULTS: The positive rates of VEGF and Flt-1 were significantly higher in MM patients than in control people (62.9% vs. 18.8%, P<0.01; 80% vs. 31.3%, P<0.01). The mRNA levels of VEGF and Flt-1 were significantly higher in MM patients than in control people (0.41+/-0.19 vs. 0.06+/-0.01, P<0.05; 0.60+/-0.33 vs. 0.08+/-0.03, P<0.01). The positive rates of VEGF and Flt-1 mRNA were not significantly different between newly diagnosed and relapsed/refractory MM patients (66.7% vs. 60.9%, P>0.05; 83.3% vs. 78.3%, P>0.05), and between stage II MM and stage III MM (50% vs. 73.7%, P>0.05; 81.3% vs. 78.9%, P>0.05). The mRNA levels of VEGF and Flt-1 were significantly higher in relapsed/refractory MM patients than in newly diagnosed MM patients (0.49+/-0.20 vs. 0.28+/-0.04, P<0.05; 0.70+/-0.38 vs. 0.41+/-0.06, P<0.05), and were significantly higher in stage III MM than that in stage II MM (0.48+/-0.19 vs. 0.28+/-0.09, P<0.05; 0.75+/-0.35 vs. 0.41+/-0.21, P<0.05). KDR was detected only in 3 MM patients and was not detected in control group. CONCLUSION: High expression of VEGF and Flt-1 mRNA is associated with the development of MM.