Homing and mobilization of hematopoietic stem cells and hematopoietic cancer cells are mirror image processes, utilizing similar signaling pathways and occurring concurrently: circulating cancer cells constitute an ideal target for concurrent treatment with chemotherapy and antilineage-specific antibodies.

Adhesion molecules and stromal cell-derived factor-1 (SDF-1)/CXCR4 signaling play key role in homing and mobilization of hematopoietic progenitor (HPC) and hematopoietic cancer clonogenic cells (HCC). High expression of VLA-4 is required for homing of HPC and HCC, whereas downregulation of these molecules is required for successful mobilization of HPC and HCC. Upregulation and activation of the SDF-1/CXCR4 signaling is required for homing of HPC and HCC, whereas disruption of the SDF-1 signaling is required for mobilization of HPC and HCC. Hence, mobilizations of HPC and HCC occur concurrently. It is proposed that drug resistance evolves as a result of repeated cycles of chemotherapy. Following each cycle of chemotherapy, HCC lose adhesion molecules and SDF-1 signaling. Surviving cells, released from tumor sites, circulate until re-expression of adhesion molecules and CXCR4 occurs, then homing to stroma of distal tissues occurs. Cytokines secreted by cells in the new microenvironment induce proliferation and drug resistance of HCC. This process is amplified in each cycle of chemotherapy resulting in disease progression. A novel model for treatment is proposed in which circulating HCC are the target for clinical intervention, and concurrent treatment with chemotherapy and antilineage-specific antibodies will result in abrogation of the 'vicious cycle' of conventional anticancer therapy.

Antisense inhibition of macrophage inflammatory protein 1-alpha blocks bone destruction in a model of myeloma bone disease.

We recently identified macrophage inflammatory protein 1-alpha (MIP-1alpha) as a factor produced by multiple myeloma (MM) cells that may be responsible for the bone destruction in MM (1). To investigate the role of MIP-1alpha in MM bone disease in vivo, the human MM-derived cell line ARH was stably transfected with an antisense construct to MIP-1alpha (AS-ARH) and tested for its capacity to induce MM bone disease in SCID mice. Human MIP-1alpha levels in marrow plasma from AS-ARH mice were markedly decreased compared with controls treated with ARH cells transfected with empty vector (EV-ARH). Mice treated with AS-ARH cells lived longer than controls and, unlike the controls, they showed no radiologically identifiable lytic lesions. Histomorphometric analysis demonstrated that osteoclasts (OCLs) per square millimeter of bone and OCLs per millimeter of bone surface of AS-ARH mice were significantly less than in EV-ARH mice, and the percentage of tumors per total bone area was also significantly decreased. AS-ARH cells demonstrated decreased adherence to marrow stromal cells, due to reduced expression of the alpha(5)beta(1) integrin and diminished homing capacity and survival. These data support an important role for MIP-1alpha in cell homing, survival, and bone destruction in MM.

Additive effect of Apo2L/TRAIL and Adeno-p53 in the induction of apoptosis in myeloma cell lines.

OBJECTIVE: We have previously shown that Adenovirus-p53 (Ad-p53) is a potent inducer of apoptosis in myeloma cells expressing nonfunctional p53 and low levels of bcl-2 and that Apo2L/TRAIL is a potent inducer of apoptosis, independent of bcl-2. A study was designed to test the synergy between Ad-p53 and Apo2L/TRAIL in the induction of apoptosis in relation to the expression of DR4/DR5 and DcR1, in cells undergoing Ad-p53-induced apoptosis. METHODS: Replication deficient Ad-p53 and human recombinant Apo2L/TRAIL were used. Myeloma cells with mutated/w.t. p53 and varying expression of bcl-2 were used to test the effect of Ad-p53, Apo2L/TRAIL, or both, on apoptosis, measured by annexin V. RESULTS: Treatment with Ad-p53 resulted in a dose-dependent apoptosis concomitant with a dose-dependent increase in the expression of DR4/DR5 and a decrease in the expression of DcR1, in Ad-p53-sensitive cell lines. In these cells, addition of Apo2L/TRAIL to cells treated with Ad-p53 resulted in a dose-dependent increase in apoptosis. Myeloma cells resistant to Ad-p53 had high levels of DR4/DR5 and high levels of DcR1 and treatment with Ad-p53 did not reduce the expression of DcR1. Also, addition of Apo2L/TRAIL to Ad-p53 did not affect the level of apoptosis beyond the level of apoptosis observed with Apo2L/TRAIL alone. CONCLUSIONS: 1) Cotreatment with Ad-p53 and Apo2L/TRAIL resulted in additive apoptosis in myeloma cells expressing nonfunctional p53 and low levels of bcl-2. 2) Resistance to Ad-p53 or to the combination of Ad-p53 and Apo2L/TRAIL was not due to the lack of adenovirus receptor (CAR) or low expression of DR4/DR5 but rather due to the relatively high expression of DcR1 receptor.

Myeloablation and autologous peripheral blood stem cell rescue results in hematologic and clinical responses in patients with myeloid metaplasia with myelofibrosis.

Current therapeutic options for myeloid metaplasia with myelofibrosis (MMM) are limited. A pilot study was conducted of autologous peripheral blood stem cell (PBSC) collection in 27, followed by transplantation in 21 patients with MMM. The median age was 59 (range 45-75) years. PBSCs were mobilized at steady state (n = 2), after granulocyte colony-stimulating factor (G-CSF) alone (n = 17), or after anthracycline-cytarabine induction plus G-CSF (n = 8). A median of 11.6 x 10(6) (range 0 to 410 x 10(6)) CD34(+) cells per kilogram were collected. Twenty-one patients then underwent myeloablation with oral busulfan (16 mg/kg) and PBSC transplantation. The median times to neutrophil and platelet recovery after transplantation were 21 (range 10-96) and 21 (range, 13 to > or = 246) days, respectively. Five patients received back-up PBSC infusion because of delayed neutrophil or platelet recovery. The median follow-up is 390 (range 70-1623) days after transplantation, and the 2-year actuarial survival is 61%. After transplantion, 6 patients died: 3 of nonrelapse causes (1 within 100 days of PBSC infusion) and 3 of disease progression. Erythroid response (hemoglobin > or = 100 g/L [10 gm/dL] without transfusion for > or = 8 weeks) occurred in 10 of 17 anemic patients. Four of 8 patients with a platelet count less than 100 x 10(9)/L (100 000/microL) responded with a durable platelet count more than 100 x 10(9)/L (100 000/microL). Symptomatic splenomegaly improved in 7 of 10 patients. It is concluded that (1) PBSC collection was feasible and stable engraftment occurred after transplantation in most patients with MMM, (2) myeloablation with busulfan was associated with acceptable toxicity, (3) a significant proportion of patients derived clinical benefit after treatment, and (4) further investigation of this novel approach is warranted. (Blood. 2001;98:586-593)

Recent developments in the regulation of peripheral blood stem cell mobilization and engraftment by cytokines, chemokines, and adhesion molecules.

Peripheral blood stem cells (PBSC) have become the preferred source of stem cells for autologous transplantation because of the technical advantage and the shorter time to engraftment. Administration of hematopoietic growth factors such as granulocyte colony-stimulating factor (G-CSF) or granulocyte-macrophage colony-stimulating factor (GM-CSF) results in mobilization of PBSCs into the peripheral blood. G-CSF and GM-CSF differ somewhat in the number and composition of CD34(+) cells and effector cells mobilized to the peripheral blood; however, the molecular mechanism underlying the release and engraftment of CD34(+) cells by these growth factors is poorly understood. This review provides a recent update on the involvement of hematopoietic growth factors, chemokines, adhesion molecules, and chemokine receptors in the regulation of stem cell release and engraftment. The involvement of very late antigen-4 (VLA-4), VLA-5, leukocyte function associated-1 molecule (LFA-1), and L-selectin and their receptors CXCR4 and its ligand SDF-1 will be discussed, and cross talk between these factors will also be reviewed in the context of stem cell release and engraftment. Finally, PBSC mobilization by chemokines will be reviewed in relation to hematopoietic growth factors.

Expression of adhesion molecules on CD34(+) cells in peripheral blood of non-hodgkin's lymphoma patients mobilized with different growth factors.

Adhesion molecules on CD34(+) cells were implicated in the process of peripheral blood stem cell (PBSC) mobilization and homing. We studied the mobilization of CD34(+)Thy1(+) cells, CD34(+) very late-acting antigen (VLA)4(+) cells, and CD34(+)L-selectin(+) cells in non-Hodgkin's lymphoma patients mobilized with cyclophosphamide plus G-CSF, GM-CSF, or GM-CSF followed by G-CSF. The mean percentage of CD34(+) cells in the bone marrow (BM) expressing Thy1 was 23.6% +/- 11% and 17.8% +/- 8% in the PB before mobilization, and was markedly decreased to 4.5% +/- 3.3% in the apheresis collections. Similarly, the mean percentage of CD34(+) cells expressing L-selectin was 35.8% +/- 4.3% in the BM, 21.6% +/- 4.1% in the PB before mobilization and was markedly decreased to 9.1% +/- 2.5% in the apheresis collections. Patients in the three arms of the study had a similar pattern of CD34(+)Thy1(+) and CD34(+)L-selectin(+) cell mobilization. Also, a similar pattern of coexpression of CD34(+)Thy1(+) and CD34(+)L-selectin(+) cells was observed when the patients were regrouped as "good mobilizers" (> or =2 x 10(6) CD34(+)CD45(dim) cells/kg, in four collections) and "poor mobilizers" (<0.4 x 10(6) CD34(+)CD45(dim) cells/kg, in two collections). The mean percentage of CD34(+) cells expressing VLA-4 in the BM and PB was relatively high (73.4% +/- 12% and 65.4% +/- 6.6%, respectively) and dropped considerably in the PBSC collections to 43.5% +/- 7.1% with a similar pattern observed for patients in arms A, B, and C. However, when the patients were regrouped as "good mobilizers" and "poor mobilizers," a higher percentage of CD34(+) cells expressing VLA-4 was observed in the PBSC of the pooled "good mobilizers" (50.5% +/- 9% versus 36.3% +/- 6.4%; p = 0.01). We conclude that release of CD34(+) cells to the PB involves a general downregulation of Thy1, L-selectin and VLA-4 on CD34(+) cells, irrespective of the growth factor used for mobilization. However, good mobilizers had a relatively higher percentage of CD34(+) cells expressing the VLA-4 antigen.

A new molecular role for iron in regulation of cell cycling and differentiation of HL-60 human leukemia cells: iron is required for transcription of p21(WAF1/CIP1) in cells induced by phorbol myristate acetate.

To investigate the role of iron in hematopoiesis, we studied effects of iron deprivation on PMA-induced monocyte/macrophage differentiation in HL-60 cells. Iron deprivation induced by desferrioxamine (DF) blocked PMA-induced differentiation and induced S-phase arrest and apoptosis in up to 60% of cells. Apoptosis was not related to a decrease of bcl-2 or to c-myc overexpression. In the presence of DF, PMA-induced upregulation of the cyclin dependent kinase inhibitor (CDKI), p21(WAF1/CIP1), was blocked and its expression could be restored in the presence of DF by supplementation with ferric citrate. Furthermore, ferrioxamine (iron saturated DF) did not block induction of p21(WAF1/CIP1) indicating that the changes were not due to a nonspecific toxic effect of DF. Similarly, hydroxyurea, an inhibitor of ribonucleotide reductase, did not block p21 expression. p21(WAF1/CIP1) antisense oligonucleotides caused cell cycle alterations similar to DF and p21 overexpression overcame effects of iron deprivation on both cell cycling and differentiation. Therefore, p21 is a key target for the effects of iron deprivation on HL-60 cell cycling and differentiation. Nuclear run-on transcription assays and p21 mRNA half-life studies indicated that iron was required to support transcriptional activation of p21(WAF1/CIP1) after a PMA stimulus. By contrast, iron deprivation did not inhibit expression of a second CDKI, p27(KIP1). These data demonstrate a new role for iron during monocyte/macrophage differentiation. A key role of iron is to allow induction of p21(WAF1/CIP1) in response to a differentiation stimulus subsequently blocking cells at the G(1)/S cell cycle interface and preventing premature apoptosis. This effect of iron is independent of its requirement in supporting the activity of the enzyme, ribonucleotide reductase. Because of the central role of p21(WAF1/CIP1) as regulator of the G(1)/S cell cycle checkpoint this requirement for iron to support p21 expression represents an important mechanism by which iron may modulate hematopoietic cell growth and differentiation. Published 2001 Wiley-Liss, Inc.

Differential mobilization of CD34+ cells and lymphoma cells in non-Hodgkin's lymphoma patients mobilized with different growth factors.

Co-mobilization of CD34(+) cells and tumor has been documented in patients with different types of cancer undergoing peripheral blood stem cell transplantation (PBSCT). Conflicting reports were published regarding the role of various growth factors in tumor cells mobilization, hence we studied the extent of CD34(+) cells and lymphoma cell mobilization in 35 non-Hodgkin's (NHL) patients primed by cyclophosphamide (Cy) in combination with granulocyte colony-stimulating factor (GCSF) (A, 13 patients), granulocyte-macrophage (GM)-CSF (B, 10 patients), or GM-CSF followed by G-CSF (C, 12 patients). CD34(+) cells were quantitated by flow cytometry and lymphoma cells by the TaqMan Real Time PCR for bcl-2 gene rearrangement. Successful collection in 4 days of > or = 2 x 10(6) CD34(+) cells/kg needed for prompt engraftment was obtained in 76%, 60%, and 58% of patients in arms A, B, and C, respectively. Lymphoma cell mobilization was detected in 35% patients tested, 78% of which had follicular lymphoma. Lymphoma cell mobilization was similar in the three arms of the study, however, presence of lymphoma cells was prevalent in patients who failed to mobilize the amount of 0.4 x 10(6) CD34(+) cells/kg in 2 days ("poor mobilizers") and reached 42%, compared to 17% in the "successful mobilizers" group of patients. Lymphoma cell contamination in PBSCs was detected proportionately in the peripheral blood and in the bone marrow. We conclude that bcl-2 gene rearrangement is prevalent in patients with follicular histology, and, in these patients, an inverse relationship was observed between mobilization of CD34(+) cells and lymphoma cells. Our results explain the high relative risk (1.98) for mobilization in patients with follicular histology.

Mobilization of dendritic cells and NK cells in non-Hodgkin's lymphoma patients mobilized with different growth factors.

Conflicting results have been reported regarding the effect of various growth factors on the mobilization of natural killer (NK) cells and dendritic cells in patients undergoing stem cell mobilization for autotransplantation. We compared the extent of mobilization of NK cells and dendritic cells in non-Hodgkin's (NHL) patients undergoing mobilization with granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage (GM)-CSF, or GM-CSF followed by G-CSF. Overall, 35 patients were studied. NK cells and dendritic were quantitated by flow cytometry. NK cells were defined as the sum of CD56(+) cells and CD56/CD16(+) cells. Dendritic cells were defined as the sum of CD80(+) and CD80(+)/CD14(+) cells. NK activity was determined by by microcytotoxicity assay. NK activity correlated well with the total amount of CD56(+) cells mobilized to the peripheral blood. Patients in the three arms of the study mobilized similar amounts of NK cells and NK activity, and patients who lacked NK activity in the peripheral blood, before mobilization, lacked NK activity in their apheresis collections. In contrast to NK cell mobilization, mobilization of dendritic cells/kg was three- to five-fold higher in patients mobilized with GM-CSF-containing regimens compared to patients mobilized with G-CSF alone. We conclude that GM-CSF-containing mobilization regimens are superior for dendritic cell mobilization but similar in the mobilization of NK cells. Therefore, we recommend using GM-CSF-containing regimens for patients undergoing ex vivo or in vivo manipulation of dendritic cells.

Enhanced TRAIL sensitivity by p53 overexpression in human cancer but not normal cell lines.

The cytotoxic ligand TRAIL is a promising anti-cancer agent that is entering into clinical trials. We previously identified a major subgroup of TRAIL resistant cancer cell lines with absent, or reduced DR4 expression containing a K441R polymorphism or harboring elevated levels of the caspase activation inhibitor FLIP. In the present study, we explored the use of a gene therapeutic approach utilizing p53, delivered by an adenovirus-p53 (Ad-p53) vector, which directly controls expression of the TRAIL receptor KILLER/DR5 in a panel of 8 cell lines including normal and TRAIL sensitive or resistant cancers. The functional status of the delivered p53 was monitored by detection of induced p21WAF1 expression by immunocytochemistry. In normal cells, which are TRAIL resistant, TRAIL did not reduce cell viability over and above the effect of Ad-p53 alone. All cancer cell lines were sensitive to Ad-p53 and up-regulated expression of the TRAIL receptor KILLER/DR5. TRAIL-resistant cancer cells became more sensitive to TRAIL at low Ad-p53 multiplicities of infection but TRAIL resistance was not completely overcome in one TRAIL-resistant cell line probably because of a high level of expression of FLIP. The results reveal that Ad-p53 induces the TRAIL receptor KILLER/DR5 and, like radiation or chemotherapy may effectively reverse TRAIL resistance.

Plasma levels of SDF-1 and expression of SDF-1 receptor on CD34+ cells in mobilized peripheral blood of non-Hodgkin's lymphoma patients.

CXCR4 is the receptor for the chemokine stromal derived factor-1 (SDF-1), is expressed on CD34+ cells, and has been implicated in the process of CD34+ cell migration and homing. We studied the mobilization of CD34/CXCR4 cells and the plasma levels of SDF-1 and flt3-ligand (flt3-L) in 36 non-Hodgkin's lymphoma patients receiving cyclophosphamide (Cy) plus G-CSF (arm A), Cy plus GM-CSF (arm B), or Cy plus GM-CSF followed by G-CSF (arm C) for peripheral blood stem cell (PBSC) mobilization and autotransplantation. We observed lower plasma levels of SDF-1 in PBSCs compared to premobilization bone marrow samples. The mean plasma SDF-1 levels were similar in PBSC collections in the three arms of the study. In contrast, SDF-1 levels in the apheresis collections of the "good mobilizers" (patients who collected a minimum of 2 x 10(6) CD34+ cells/kg in one to four PBSC collections) were significantly lower than the apheresis collections of the "poor mobilizers" (> or = 0.4 x 10(6) CD34+ cells/kg in the first two PBSC collections; 288 +/- 82 pg/ml versus 583 +/- 217 pg/ml; p = 0.0009). The mean percentage of CD34+ cells expressing CXCR4 in the apheresis collections was decreased in the PBSC collections compared with premobilization values from 28% to 19.4%. Furthermore, the percentage of CD34+ cells expressing CXCR4 in the good mobilizers was significantly lower compared with the poor mobilizers (14.7 +/- 2.1% versus 33.6 +/- 2.1%; p = 0.002). The good mobilizers had also significantly lower levels of flt3-L compared with the poor mobilizers (34 +/- 4 pg/ml versus 106 +/- 11 pg/ml; p = 0.006), Finally, the levels of flt3-L strongly correlated with SDF-1 levels (r = 0.8; p < 0.0001). We conclude: A) low plasma levels of SDF-1 and low expression of CXCR4 characterize patients with good mobilization outcome, and B) the levels of SDF-1 correlate with flt3-L, suggesting an association of these cytokines in mobilization of CD34+ cells.

Peripheral blood stem cell mobilization with cyclophosphamide in combination with G-CSF, GM-CSF, or sequential GM-CSF/G-CSF in non-Hodgkin's lymphoma patients: a randomized prospective study.

We designed a randomized, prospective three-arm mobilization study to determine the kinetics of peripheral blood stem cell (PBSC) mobilization in 60 non-Hodgkin's lymphoma (NHL) patients primed with cyclophosphamide (CTX) in combination with granulocyte colony-stimulating factor (G-CSF) (arm A), granulocyte-macrophage (GM)-CSF (arm B) or GM-CSF/G-CSF (arm C). We also compared mobilization and transplant-related toxicities, pre- and post-transplant support and the probability of survival among the three arms. To date, 35 patients have been enrolled in the study; 13 patients have been enrolled in arm A, 10 patients in arm B, and 13 patients in arm C. Successful collection of the target of > or = 2 X 10(6) CD34+ cells/kg in one to four apheresis collections was 10/13, 6/10, and 7/12 in arms A, B, and C, respectively. The differences between arms were not statistically significant. The median time to achieve the target CD34+ cells in patients who successfully mobilized the target CD34+ cells was 3 days, 2 days, and 1 day, in patients in arms A, B, and C, respectively. The time for neutrophil engraftment was 11, 10, and 10 days in arms A, B, and C, respectively. The time for platelet engraftment was 11 days for patients in all arms of the study. Most importantly, no significant differences were observed among the three arms in the duration of neutropenic fever, the extent of mucositis, diarrhea, and nausea/vomiting, or in the number of units of platelets or red cells transfused after transplantation. Risk factors associated with poor mobilization were > or = 3 regimens of chemotherapy prior to mobilization, older age, and disease histology (follicular versus diffuse). Therefore, we conclude that the type of growth factor used for mobilization did not play a major role in the outcome of mobilization and recommend mobilizing NHL patients before they receive multiple regimens of chemotherapy.

Immunologic profiles of effector cells and peripheral blood stem cells mobilized with different hematopoietic growth factors.

BACKGROUND: Peripheral blood stem cells (PBSC) have become the preferred source of stem cells for autologous transplantation because of the technical advantage and the shorter time to engraftment. Mobilization of CD34(+) cells into the peripheral blood can be achieved by the administration of G-CSF or GM-CSF, or both, alone or in combination with chemotherapy. G-CSF and GM-CSF differ somewhat in the number and composition of CD34(+) cells and effector cells mobilized to the peripheral blood. However, the molecular mechanism underlying the release and engraftment of CD34(+) cells is poorly understood. PURPOSE: The purpose of this review is to give a recent update on the type and immunological properties of effector cells and CD34(+) cells mobilized by the different growth factors with emphasis on A) mobilization of T cells, natural killer cells, and dendritic cells; B) coexpression of adhesion molecules such as VLA-4 and L-selectin in mobilized PBSC collection, and C) coexpression of CXCR4-the receptor for the stromal-derived differentiation factor 1-with latest information shedding light on the molecular mechanism underlying the release and subsequent engraftment of CD34(+) cells. CONCLUSIONS: A) The reported suppression of T cell and NK cell functions in PBSC apheresis collections in patients primed with G-CSF or GM-CSF is controversial and may merely reflect low effector cell activity before mobilization. B) A decrease in the expression of adhesion molecules such as VLA-4 and L-selectin is a necessary requirement for the release of CD34(+) cells to the peripheral blood. C) A decrease in the expression of CXCR4 is a necessary requirement for the release of CD34(+) cells to the peripheral blood and correlates with mobilization success.

High steady-state plasma levels of flt3-ligand in the peripheral blood is a good predictor for poor mobilization of CD34+ PBSC in patients undergoing high-dose chemotherapy and stem cell rescue.

flt3-ligand (flt3-L) is a very effective mobilizer of hematopoietic stem cells (HSC) and is capable of inducing multilineage hematopoietic cell differentiation both in vivo and in vitro. We measured, by ELISA, the plasma peripheral blood flt3-L concentrations in 28 non-Hodgkin's lymphoma (NHL) patients before and after mobilization with three different mobilization regimens, including priming with cyclophosphamide (CY) plus G-CSF, CY plus GM-CSF, and CY plus GM-CSF (8 days) followed by G-CSF. We also determined the levels of flt3-L in the peripheral blood during four apheresis collections and 6 months after transplantation. The steady-state level of flt3-L in NHL patients (n = 18) who mobilized > or =2 x 10(6) CD34+ cells/kg in four apheresis collections was 34 +/- 4 pg/ml and was similar to the levels observed in 10 normal controls (27 +/- 7, p = 0.1) regardless of the mobilization protocol used. In contrast, patients who failed to mobilize a total of >0.4 x 10(6) CD34+ cells/kg in two consecutive apheresis collections (n = 10) had flt3-L levels of 106 +/- 11 pg/ml, significantly higher (p = 0.006) than that of the good mobilizers group, regardless of the mobilization protocol used. Similar results were observed in 29 multiple myeloma (MM) patients. A mean of 23.8 +/- 7.9 pg/ml and 450 +/- 85 pg/ml flt3-L was obtained in the good mobilizers (n = 24) and the nonmobilizers (n = 5) groups of patients, respectively. Statistical analysis revealed a significant difference (p = 0.0006) between the two groups of MM patients, but no correlation was observed between the levels of flt3-L and CD34+ cell/microl, in mobilized peripheral blood. Our results also suggest that measurement of plasma levels of flt3-L before mobilization can be clinically useful to predict for patients with poor mobilization outcome.

Adenovirus-mediated delivery of p53 results in substantial apoptosis to myeloma cells and is not cytotoxic to flow-sorted CD34(+) hematopoietic progenitor cells and normal lymphocytes.

Multiple myeloma (MM) is an incurable disease; therefore, there is a need for new modalities of treatment for this disease. We designed a study to test the sensitivity of MM cell lines, freshly isolated myeloma cells, and CD34(+) hematopoietic progenitor cells to adenovirus-mediated delivery of wild-type p53 (Ad-p53). Replication-deficient Ad-p53, previously used in phase I-II clinical trial for treatment of patients with solid tumors, was used in this study. Myeloma cells from seven MM cell lines with mutated or w.t. p53 and varying expression of bcl-2 were used. Fresh myeloma cells (CD38(bright)CD45(-)) and fresh CD34(+) hematopoietic stem cells and CD34(-) cells were purified by flow sorting of apheresis collections of MM patients undergoing high-dose chemotherapy and stem cell rescue. The effect of Ad-p53 on colony-forming unit granulocyte-macrophage (CFU-GM) and burst-forming unit erythroid (BFU-E) colony formation in methylcellulose was tested on purified CD34(+) and CD34(-) cells to evaluate bone marrow toxicity. Myeloma cells from cell lines, or freshly isolated myeloma cells, were sensitive to Ad-p53 only if they had mutated p53 and had low expression of bcl-2. CD34(+) cells were resistant to Ad-p53-mediated apoptosis, and CFU-GM and BFU-E colony formation was not affected by treatment with Ad-p53.Ad-p53 is a potent inducer of apoptosis in MM cell lines and in freshly isolated myeloma cells expressing low levels of bcl-2. Ad-p53 is not overtly cytotoxic to normal hematopoietic stem cells or normal lymphocytes; therefore, it could be considered for a phase I clinical trial of MM patients with mutated p53.

TRAIL is a potent inducer of apoptosis in myeloma cells derived from multiple myeloma patients and is not cytotoxic to hematopoietic stem cells.

TRAIL, the ligand for the newly discovered DR-4 and DR-5 receptor, is a member of the TNF family of death signal transduction proteins with a mechanism of cell death similar to that of Fas and Fas ligand (Fas-L) system. We provide first time evidence that TRAIL is a potent inducer of apoptosis in multiple myeloma (MM) cell lines. TRAIL effectively induced extensive apoptosis in 8226 and ARP-1 MM cells in a time- and dose-dependent manner. Apoptosis with TRAIL reached about 80% within 48 h of treatment with a dose of 160 ng/ml. Furthermore, we provide first time evidence that similar to Fas, TRAIL-induced apoptosis is not blocked by bcl-2 in MM cell lines. Most importantly, TRAIL induced substantial apoptosis in freshly isolated, flow-sorted myeloma cells obtained from different MM patients expressing variable levels of bcl-2. Finally, we demonstrate for the first time that TRAIL is not cytotoxic to purified CD34+/CD45dim hematopoietic stem cells and does not inhibit CFU-GM or BFU-E colony formation in methylcellulose.

Successful PBSC mobilization with high-dose G-CSF for patients failing a first round of mobilization.

PBSC are the preferred source of stem cells for autologous transplantation. However, regardless of the mobilization procedure used, 10%-20% of patients fail to collect an adequate number to ensure prompt engraftment. There is as yet no standard mobilization procedure for patients who fail a first mobilization attempt. Here, we describe a highly efficient strategy to obtain an adequate number of stem cells for patients who failed a first mobilization attempt. Seventy-four patients with various hematologic malignancies underwent initial mobilization with various regimens including hematopoietic growth factors with or without chemotherapy. In 72% of patients, > or =2 x 10(6) CD34+ stem cells/kg were collected in the initial mobilization attempt, and patients engrafted in a median of 10 days for neutrophils and 12 days for platelets. Eighteen patients failed to mobilize adequate numbers of stem cells, defined as the inability to collect 0.2 x 10(6) CD34+ stem cells/kg/day in the first 2-3 days. These patients had their apheresis halted. Patients were immediately given G-CSF (32 microg/kg/day) for 4 days as a second attempt at mobilization. Eighty-eight percent of these patients achieved the target of > or =2 x 10(6) CD34+ cells/kg, with a median duration of apheresis of 5 days (including the first and second mobilizations). The mean CD34+ cells/kg/day increased after administration of high-dose G-CSF from 0.16 after the first mobilization attempt to 0.61 (p = 0.0002) after the second mobilization. All patients engrafted in a median of 11 and 13 days for neutrophils and platelets, respectively. We conclude that patients whose apheresis yield is <0.4 x 10(6) CD34+ cells/kg after the first two apheresis collections can be successfully mobilized if high-dose G-CSF is administered immediately and continued until achieving > or =2 x 10(6) CD34+ stem cells/kg.

Annexin II increases osteoclast formation by stimulating the proliferation of osteoclast precursors in human marrow cultures.

Annexin II (AXII), a calcium-dependent phospholipid-binding protein, has been recently found to be an osteoclast (OCL) stimulatory factor that is also secreted by OCLs. In vitro studies showed that AXII induced OCL formation and bone resorption. However, the mechanism of action by which AXII acts as a soluble extracellular protein to induce OCL formation is unknown. In this paper, we demonstrate that AXII gene expression is upregulated by 1,25-dihydroxyvitamin D3 [1, 25-(OH)2D3] and that addition of AXII significantly increased OCL-like multinucleated cell formation. Time-course studies suggested that AXII acted on the proliferative stage of OCL precursors and that AXII increased thymidine incorporation in OCL precursors. Moreover, AXII enhanced the growth of CFU-GM, the earliest identifiable OCL precursor, when bone marrow cultures were treated with low concentrations of GM-CSF. This capacity of AXII to induce OCL precursor proliferation was due to induction of GM-CSF expression, because the addition of neutralizing antibodies to GM-CSF blocked the stimulatory effect of AXII on OCL formation. RT-PCR analysis using RNA from highly purified subpopulations of marrow cells demonstrated that T cells, especially CD4(+) T cells, produced GM-CSF in response to AXII. Furthermore, FACS(R) analysis of T-cell subpopulations treated with fluorescein-labeled AXII suggested that the CD4(+), but not CD8(+), subpopulation of T cells express an AXII receptor. Taken together, these data suggest that AXII stimulates OCL formation by activating T cells through a putative receptor to secrete GM-CSF. GM-CSF then expands the OCL precursor pool to enhance OCL formation.

Apoptosis-induced by TRAIL AND TNF-alpha in human multiple myeloma cells is not blocked by BCL-2.

TRAIL, the ligand for the newly discovered DR-4 and DR-5 receptor is a member of the tumour necrosis factor (TNF) family of death signal tranduction proteins with a mechanism of cell death, similar to the Fas and Fas ligand (Fas-L) system. Here, we provide first time evidence that TRAIL and TNF-alpha are potent inducers of apoptosis in multiple myeloma (MM) cell lines and freshly isolated myeloma cells. TRAIL effectively induced extensive apoptosis in 8226 and ARP-1 MM cells in a time- and dose-dependent manner reaching 80% within 48 h of treatment with a dose of 160 ng/ml. Bcl-2 transfected 8226 and ARP-1 cells were equally sensitive to apoptosis by TRAIL. Apoptosis with TNFalpha, reached >60% within 48 h of treatment with a dose of 160 ng/ml. In addition to MM cell lines, freshly isolated, flow-sorted myeloma cells from 8 different MM patients expressing variable levels of bcl-2 were equally sensitive to both TRAIL and TNF-alpha. We have previously shown that anti-Fas-induced apoptosis is not blocked by endogenous or ectopic bcl-2 in MM cell lines. Here we extend our observation with Fas to include TNF-alpha and TRAIL to the apoptotic signals that are not be blocked by bcl-2, in MM cells.

Bcl-2 overexpression is associated with resistance to paclitaxel, but not gemcitabine, in multiple myeloma cells.

Multiple myeloma (MM) is an incurable disease despite an initial response-rate of >60% with conventional or high-dose chemotherapy using glucocorticosteroids (i.e. dexamethasone), or alkylating agents (i.e. melphalan). Although these agents are capable of inducing complete remission (CR) in >50% of MM patients, resistance develops rapidly, in >90% of patients, within 2 years of treatment. Therefore, there is a need for new drugs for the treatment of relapsing and refractory MM patients. Gemcitabine (GEM) is a pyrimidine analog that blocks DNA synthesis, whereas, paclitaxel (TAX) is a mitotic spindle poison that promotes microtubular aggregation. Since it appears that these two drugs have different cellular targets, we examined the effect of each drug individually for several parameters and for possible synergy. We studied the cytotoxic effect of TAX and GEM on MM cells expressing varying levels of the antiapoptotic protein bcl-2, which is overexpressed in the majority of myeloma cell from MM patients. We found that both drugs are cytotoxic by inducing apoptosis, however, the extent of apoptosis with TAX, but not with GEM was dependent on the levels of bcl-2 expression. We further investigated the effect of TAX and GEM on the cell cycle distribution and on the levels of bcl-2. The results indicate that the two drugs have different modes of action with respect to each parameter tested. TAX induced arrest of the cells in the G2/M phase of the cell cycle, regardless of bcl-2 levels, however, apoptosis was induced in mitotic cells expressing relatively low levels of bcl-2. In contrast, GEM caused apoptosis of cells in the S-phase, regardless of level of bcl-2 expression. A major difference between TAX and GEM was in their effects on the levels of bcl-2. Whereas, TAX induced an early downregulation of bcl-2 (only in the cells with relatively low levels of bcl-2), treatment with GEM did not affect bcl-2 levels. The effects of TAX on both the cell cycle and bcl-2 were detected very early (4-8 h) and preceded the onset of apoptosis. GEM and TAX act synergistically, at low doses (IC50 of 0.5 microM for GEM and 0.025 microM for TAX), to effectively kill bcl-2 overexpressing cells that are resistant to higher doses (0.25 microM) of TAX alone. Therefore, we have initiated a phase II clinical trial of TAX and GEM for MM patients refractory to current therapy.