Limited engraftment capacity of bone marrow-derived mesenchymal cells following T-cell-depleted hematopoietic stem cell transplantation.

The engraftment capacity of bone marrow-derived mesenchymal cells was investigated in 41 patients who had received a sex-mismatched, T-cell-depleted allograft from human leukocyte antigen (HLA)-matched or -mismatched family donors. Polymerase chain reaction (PCR) analysis of the human androgen receptor (HUMARA) or the amelogenin genes was used to detect donor-derived mesenchymal cells. Only 14 marrow samples (34%) from 41 consenting patients generated a marrow stromal layer adequate for PCR analysis. Monocyte-macrophage contamination of marrow stromal layers was reduced below the levels of sensitivity of HUMARA and amelogenin assays (5% and 3%, respectively) by repeated trypsinizations and treatment with the leucyl-leucine (leu-leu) methyl ester. Patients who received allografts from 12 female donors were analyzed by means of the HUMARA assay, and in 5 of 12 cases a partial female origin of stromal cells was demonstrated. Two patients who received allografts from male donors were analyzed by amplifying the amelogenin gene, and in both cases a partial male origin of stromal cells was shown. Fluorescent in situ hybridization analysis using a Y probe confirmed the results of PCR analysis and demonstrated in 2 cases the existence of a mixed chimerism at the stromal cell level. There was no statistical difference detected between the dose of fibroblast progenitors (colony-forming unit-F [CFU-F]) infused to patients with donor- or host-derived stromal cells (1.18 +/- 0.13 x 10(4)/kg vs 1. 19 +/- 0.19 x 10(4)/kg; P >/=.97). In conclusion, marrow stromal progenitors reinfused in patients receiving a T-cell-depleted allograft have a limited capacity of reconstituting marrow mesenchymal cells.

Amifostine (WR-2721) selective protection against melphalan genotoxicity.

Amifostine (WR-2721) is an aminothiol compound dephosphorylated at the tissue site by alkaline phosphatase to the active metabolite, which is able to inactivate electrophilic substances and scavenge free radicals. Amifostine effects against melphalan-induced DNA strand breaks were studied in normal human white blood cells (WBC) and K562 leukemic cells using the single cell gel electrophoresis (SCGE) or Comet assay, a reported method for measuring DNA damage in individual cells. Prior to treatment (1 h, 37 degrees C) with increasing doses of melphalan, with or without S9, the cells were treated (15 min, 37 degrees C) with a control medium or amifostine (3 mg/ml). Treatment of normal and leukemic cells with melphalan induced a dose-dependent 'comet formation'. Melphalan-induced DNA damage follows a normal distribution in WBC. On the other hand, in K562, a significant proportion of undamaged cells remains even with doses at which mean DNA damage is serious. Pretreatment with WR-2721 protects WBC, but not K562, against the genotoxic effect of melphalan. Amifostine might even strengthen the action of the antiblastic drug against K562 cells. S9 addition appears to enhance melphalan effectiveness. SCGE appears as a suitable primary screening method for in vitro and in vivo studies on drug-DNA interactions and their modulations by endogenous/exogenous factors.

Peripheral blood progenitor cell mobilization in healthy donors receiving recombinant human granulocyte colony-stimulating factor.

OBJECTIVE: We analyzed the incidence of primitive (LTC-IC) and committed (CFU-mix, BFU-E, CFU-GM) hematopoietic progenitors detected under steady-state conditions and upon progenitor cell mobilization in a cohort of healthy donors receiving recombinant human granulocyte colony-stimulating factor (rhG-CSF). MATERIALS AND METHODS: Healthy donors (n = 30) of HLA-mismatched or -matched stem cell transplants were mobilized with rhG-CSF (8 microg/Kg body weight subcutaneously twice daily until completion of leukapheresis). PBPC collections were started after 4 days of rhG-CSF therapy. RESULTS: Steady-state incidence of bone marrow LTC-IC, but not committed progenitors, significantly correlated with the numbers of mobilized CD34+ cells (r = 0.6, p = 0.004), CFU-GM (r = 0.79, p = 0.0005) and CFC (r = 0.76, p = 0.001) detected after 4 days of rhG-CSF therapy. Statistically significant correlations were also found between steady-state blood CFU-GM and peak numbers of CD341 cells (r = 0.68, p = 0.001), numbers of day 4 CD341 cells (r = 0.52, p = 0.005), CFU-GM (r = 0.63, p = 0.002), and CFC (r = 0.61, p = 0.003). CONCLUSION: Our data show that in normal volunteers baseline marrow LTC-IC and blood CFU-GM correlate with rhG-CSF-mobilized PBPC. The potential clinical relevance of these findings in the identification of poor mobilizers will be tested in a prospective study.

Induction of apoptosis by arachidonic acid in chronic myeloid leukemia cells.

The hallmark of chronic myeloid leukemia (CML) is the presence of the bcr-abl oncogene, which is associated with transforming ability and an intrinsic resistance to induction of apoptosis by genotoxic agents. Arachidonic acid (AA), a biologically active fatty acid, plays a crucial role as a mediator of signaling pathways involved in cell proliferation and survival. In this study, we investigated the potential role of AA as a proapoptotic agent in CML. Pretreatment of human CML isolated progenitor cells with AA (100 microM for 18 h) induced 71-75% inhibition of in vitro colony formation of granulocyte-macrophage colony-forming units, multilineage colony-forming units, and erythroid burst-forming units. This inhibition was significantly greater than the effect on normal progenitor cells (19-39% growth inhibition of erythroid burst-forming units, multilineage colony-forming units, and granulocyte-macrophage colony-forming units). AA also inhibited growth of the bcr-abl-transformed cell line H7.bcr-abl A54. In contrast, a minimal effect of AA on inhibition of cell growth was observed in the parental nontransformed NSF/N1.H7 cell line. The antiproliferative effect of AA was associated with apoptosis. Gamma-linolenic acid, a precursor of AA, also inhibited cell growth, whereas other unsaturated and saturated fatty acids had no effect. Pharmacological inhibition of cyclooxygenase, lipooxygenase, and cytochrome P450 monooxygenase enzymes prior to exposure to AA did not rescue cells from the inhibitory effect of AA. Moreover, 5,8,11,14-eicosatetraynoic acid, a nonmetabolizable arachidonate analogue, also inhibited cell growth, suggesting that the effect of AA did not require further metabolism. Treatment with antioxidants prior to stimulation with AA was also ineffective in preventing its antiproliferative effect. Thus, AA inhibited proliferation of CML cells by inducing apoptotic cell death. The signaling mechanisms of AA-induced inhibition of cell growth appeared to be independent of its conversion into eicosanoids or free radical generation.

Clonogenic potential and phenotypic analysis of CD34+ cells mobilized by different chemotherapy regimens.

BACKGROUND AND OBJECTIVE: Since limited data concerning quantitative and qualitative differences of CD34+ cells collected after different mobilization schedules are available, we investigated phenotype, proliferative capacity and primitive progenitor cell content of CD34+ cells mobilized with four different regimens. DESIGN AND METHODS: The number, phenotype, and progenitor cell content of CD34+ cells were investigated in 46 patients mobilized with cyclophosphamide (CY) 7 g/m(2) plus granulocyte colony-stimulating factor (G-CSF, 5 microg/kg) (CY7+G-CSF) (n=16), CY 4 g/m(2) plus G-CSF (CY4+G-CSF) (n=8), IVE [ifosphamide (2.5 g/m(2) for 3 d), etoposide (150 mg/m(2) for 3 d), epirubicin (100 mg/m(2) on day 1)] plus G-CSF (IVE+G-CSF) (n=9), or G-CSF (10 microg/kg) alone (n=13). RESULTS: The number of CD34+ cells collected per liter of processed blood was significantly higher in the CY7+G-CSF group than in the CY4+G-CSF and G-CSF groups (p

Effects of the tyrosine kinase inhibitor AG957 and an Anti-Fas receptor antibody on CD34(+) chronic myelogenous leukemia progenitor cells.

The hallmark of chronic myelogenous leukemia (CML) is the Philadelphia (Ph) chromosome that fuses genetic sequences of the BCR gene on chromosome 22 with c-ABL sequences translocated from chromosome 9. BCR/ABL fusion proteins have a dysregulated protein tyrosine kinase (PTK) activity exerting a key role in malignant transformation. Targeting the tyrosine kinase activity of BCR/ABL or using agents capable of triggering apoptosis might represent attractive therapeutic approaches for ex vivo purging. AG957, a member of the tyrphostin compounds, exerts a selective inhibition of p210(BCR/ABL) tyrosine phosphorylation. We report here that preincubation of CML or normal CD34(+) cells with graded concentration of AG957 (1 to 100 micromol/L) resulted in a statistically significant, dose-dependent suppression of colony growth from multipotent, erythroid, and granulocyte-macrophage progenitors as well as the more primitive long-term culture-initiating cells (LTC-IC). However, AG957 doses causing 50% inhibition (ID50) of CML and normal progenitors were significantly different for multilineage colony-forming units (CFU-Mix; 12 v 64 micromol/L; P =.008), burst-forming unit-erythroid (BFU-E; 29 v 89 micromol/L; P =.004), colony-forming unit-granulocyte-macrophage (CFU-GM; 34 v 85 micromol/L; P =.004), and LTC-IC (43 v 181 micromol/L; P =.004). In 5 of 10 patients, analysis of BCR/ABL mRNA on single progenitors by reverse transcription-polymerase chain reaction showed that AG957 at 50 micromol/L significantly reduced the mean (+/-SD) percentage of BCR/ABL-positive progenitors (92% +/- 10% v 33 +/- 5%; P =.001). Because AG957 treatment resulted in significantly higher percentages of apoptotic cells (30% v 9%) in the BCR/ABL-transfected 32DLG7 cells as compared with 32D-T2/93 cells (BCR/ABL-negative), we investigated the combined effects of AG957 with the anti-Fas receptor (Fas-R) monoclonal antibody CH11 that triggers apoptosis. As compared with AG957 alone, the sequential treatment of CML CD34(+) cells with AG957 (1 micromol/L) and CH11 (1 microgram/mL) increased CFU-Mix, BFU-E, and CFU-GM growth inhibition by 1.6-fold, 3-fold, and 4-fold, respectively. In contrast, the treatment of normal CD34(+) cells with AG957 and CH11 failed to enhance AG957-induced colony growth inhibition. We conclude that (1) AG957 inhibits in a dose-dependent manner CML CD34-derived colony formation by both primitive LTC-IC as well as committed CFU-Mix, BFU-E, and CFU-GM; (2) this growth inhibition is associated with the selection of a substantial amount of BCR/ABL-negative progenitors; and (3) the antiproliferative effect of AG957 is dramatically increased by combining this compound with the anti-Fas-R antibody CH11. These data may have significant therapeutic applications.

Primitive hematopoietic progenitors within mobilized blood are spared by uncontrolled rate freezing.

Uncontrolled-rate freezing techniques represent an attractive alternative to controlled-rate cryopreservation procedures which are time-consuming and require high-level technical expertise. In this study, we report our experience using uncontrolled-rate cryopreservation and mechanical freezer storage at -140 degrees C. Twenty-eight PBPC samples (10 cryovials, 18 freezing bags) from 23 patients were cryopreserved in a cryoprotectant solution composed of phosphate-buffered saline (80%, v/v) supplemented with human serum albumin (10%, v/v) and dimethylsulfoxide (10%, v/v). The cryopreservation procedure required on average 1.5 h. The mean (+/- s.e.m.) storage time of cryovials and bags was 344+/-40 and 299+57 days, respectively. Although cell thawing was associated with a statistically significant reduction of the absolute number of nucleated cells (vials: 0.3x10(9) vs. 0.2x10(9), P< or =0.02; bags: 14x10(9) vs. 11x10(9), P< or =0.0003), the growth of committed progenitors was substantially unaffected by the freezing-thawing procedure, with mean recoveries of CFU-Mix, BFU-E, and CFU-GM ranging from 60+/- 29% to 134+/-15%. Mean recoveries of LTC-IC from cryovials and bags were 262+/-101% and 155+/-27% (P< or =0.2), respectively. In 14 out of 23 patients who underwent high-dose chemotherapy and PBPC reinfusion, the pre-and post-freezing absolute numbers of hematopoietic progenitors cryopreserved in bags were compared. A significant reduction was detected for CFU-Mix (11 vs. 7.4x10(5)), but no significant loss of BFU-E (180 vs. 150x10(5)), CFU-GM (400 vs. 290x10(5)) and LTC-IC (15 vs. 16x10(5)) could be demonstrated. When these patients were reinfused with uncontrolled-rate cryopreserved PBPC, the mean number of days to reach 1x10(9)/l white blood cells and 50x10(9)/l platelets were 9 and 13, respectively. In conclusion, the procedure described here is characterized by short execution time, allows a substantial recovery of primitive and committed progenitors and is associated with prompt hematopoietic recovery following myeloablative therapy even after long-term storage.

CD34+ cells mobilized by cyclophosphamide and granulocyte colony-stimulating factor (G-CSF) are functionally different from CD34+ cells mobilized by G-CSF.

Mobilized peripheral blood progenitor cells (PBPC) are increasingly used as an alternative to bone marrow for autografting procedures. Currently, cyclophosphamide (CY) followed by granulocyte colony-stimulating factor (G-CSF) or G-CSF alone are the most commonly used PBPC mobilization schedules. In an attempt to investigate whether the use of these two mobilization regimens could result in the collection of functionally different CD34+ cells, we analyzed nucleated cells (NC), CD34+ cells, committed progenitor cells and long-term culture initiating-cells (LTC-IC) in 52 leukaphereses from 26 patients with lymphoid malignancies, mobilized either by CY+G-CSF (n=16) or G-CSF alone (n=10). Thirty-four aphereses from the CY+G-CSF group and 18 aphereses from the G-CSF group were investigated. According to the study design, leukaphereses were carried out until an average number of 7 x 10(6) CD34+ cells/kg body weight were collected. The mean (+/-s.e.m.) numbers of CD34+ cells mobilized per apheresis by CY+G-CSF and G-CSF were not significantly different (2.76+/-0.6 x 10(8) vs 2.53+/-0.4 x 10(8), P < or = 0.7). This resulted from a mean number of NC that was significantly lower in the CY+G-CSF products than in the G-CSF products (12.4+/-1.7 x 10(9) vs 32+/-5.4 x 10(9), P < or = 0.0001) and a mean incidence of CD34+ cells that was significantly higher in the CY+G-CSF products than in the G-CSF products (2.9+/-0.6% vs 0.9+/-0.2%, P < or = 0.0018). The mean (+/-s.e.m.) number of CFU-GM collected per apheresis was significantly higher in the CY+G-CSF group than in the G-CSF group (37+/-7 x 10(6) vs 14+/-2 x 10(6), P < or = 0.03). Interestingly, CY+G-CSF-mobilized CD34+ cells had a significantly higher plating efficiency than G-CSF-mobilized CD34+ cells (25.5+/-2.9% vs 10.8+/-1.9%, P < or = 0.0006). In addition, the mean number of LTC-IC was significantly higher in the CY+G-CSF products than in the G-CSF products (6.3+/-1 x 10[6] vs 3.3+/-0.3 x 10[6], P < or = 0.05). In conclusion, our data provide evidence that CY+G-CSF and G-CSF induce the mobilization of CD34+ cells with different clonogenic potential. As mobilized PBPC containing large numbers of progenitors lead to safer transplantation, this issue may have implications for planning mobilization strategies.

Reverse transcription polymerase chain reaction is a reliable assay for detecting leukemic colonies generated by chronic myelogenous leukemia cells.

Single-colony karyotyping (SCK) and reverse-transcription polymerase chain reaction (RT-PCR) are two increasingly used techniques for the quantification of leukemic colonies generated by chronic myelogenous leukemia (CML) cell fractions purged or selected in vitro. Recently, the existence of Philadelphia (Ph) chromosome positive progenitors with a silent BCR-ABL gene has been reported, thus raising concerns on the use of RT-PCR for detecting BCR-ABL positive progenitors. In order to investigate this issue further, colonies (n = 204) generated by mononuclear (MNC) or CD34+ CML cells were individually harvested, divided into two aliquots and analyzed both at the cytogenetic level to detect the Ph chromosome, and the molecular level to detect BCR-ABL transcripts. The mean (+/- s.d.) percentages of colonies analyzable by either SCK or RT-PCR were 74 +/- 16% and 86 +/- 16%, respectively. A significant percentage of colonies (67 +/- 19%) could be successfully analyzed by both SCK and RT-PCR. Although the majority of these colonies (97 +/- 5%) were Ph-positive and BCR-ABL-positive, a negligible percentage (4%) of progenitors were Ph-positive but BCR-ABL-negative. In order to test the influence of colony size on the outcome of molecular analysis, the efficiency of our RT-PCR assay in detecting BCR-ABL transcripts was investigated by means of experiments in which the number of cells used to start RNA extraction was serially reduced. These experiments showed that at least 150 cells were necessary to achieve a reproducible amplification of BCR-ABL transcripts. By correlating the size of harvested colonies with the outcome of molecular analysis, it was evident that BCR-ABL-negative but Ph-positive colonies represented false negative results occurring when a number of leukemic cells below the detection limit of our RT-PCR assay was analyzed. In conclusion, our data demonstrate that individual CML colonies grown in semisolid culture assays can be indifferently analyzed by SCK or RT-PCR, and support an extensive use of a carefully standardized RT-PCR assay to estimate the leukemic burden within samples which have been purged and selected in vitro.

Effect of chemotherapy for acute myelogenous leukemia on hematopoietic and fibroblast marrow progenitors.

Since reduced marrow cellularity and prolonged pancytopenia following autologous bone marrow transplantation (ABMT) have been frequently observed in patients with acute myelogenous leukemia (AML) included in the AML10 GIMEMA/EORTC trial, the question was raised to what extent hematopoietic and microenvironmental progenitor cells were involved in these patients. Marrow hematopoietic progenitors were investigated by a short-term methylcellulose assay quantitating multipotent CFU-Mix, erythroid BFU-E and granulocyte-macrophage CFU-GM, as well as a long-term assay quantitating long-term culture-initiating cells (LTC-IC). The marrow microenvironment was studied by evaluating the incidence of fibroblastoid progenitors (CFU-F) and the capacity of stromal layers to support allogeneic hematopoietic progenitors. As compared to normal controls (n = 57), AML patients (n = 26) showed a statistically significant reduction of the mean (+/-s.e.m.) number of CFU-Mix (5.3 +/- 0.6 vs 0.8 +/- 0.2, P < or = 0.0001), BFU-E (68 +/- 5 vs 20 +/- 4, P < or = 0.0001), CFU-GM (198 +/- 11 vs 144 +/- 15, P < or = 0.008), and LTC-IC (302 +/- 46 vs 50 +/- 8, P < or = 0.001). The mean (+/-s.e.m.) incidence of marrow CFU-F was not significantly reduced as compared to normal controls (48 +/- 6 vs 52 +/- 7, P < or = 0.73). Seventeen AML stromal layers were tested for their capacity to support the growth of allogeneic hematopoietic progenitors. Seven samples failed to support any progenitor cell growth, seven had a significantly lower supportive activity as compared to normal stromal layers (13 +/- 5 vs 249 +/- 56, P < or = 0.002), whereas three cultures could not be analyzed due to contamination. In conclusion, induction and consolidation regimens used in AML patients of the AML10 protocol induce a markedly defective in vitro growth of primitive hematopoietic progenitors and a severe functional defect of marrow stroma. The association of hematopoietic with microenvironmental damage might play a key role in the delayed hematopoietic regeneration observed following ABMT in patients of the AML10 trial.

Use of granulocyte-macrophage colony-stimulating factor (GM-CSF) in combination with hydroxyurea as post-transplant therapy in chronic myelogenous leukemia patients autografted with unmanipulated hematopoietic cells.

BACKGROUND AND OBJECTIVE: Allogeneic bone marrow transplantation remains the only potentially curative treatment for CML, but more than 70% of patients will be ineligible for allogeneic marrow transplant either because they do not have a suitable HLA-matched related or unrelated donor or because they are more than 50 years old. Several experimental and clinical findings support a role for autologous stem cell transplantation (ASCT) in CML. It has been suggested that in the early phase following autografting the Ph-negative clone has a proliferative advantage over the Ph-positive clone. We hypothesized that post-transplant GM-CSF administration could reactivate the functional activity of quiescent normal progenitors and prolong the duration of the post-transplant proliferative advantage of Ph-negative over Ph-positive progenitors. In order to evaluate the effect of post-transplant GM-CSF administration, a pilot clinical study was performed in which CML patients resistant to IFN-alpha therapy were autografted with unmanipulated marrow or blood cells and given prolonged GM-CSF therapy post-transplant. METHODS: Five adult CML patients conditioned with the BAVC regimen were reinfused with either marrow (n = 2) or blood (n = 3) cells and given granulocyte-macrophage colony-stimulating factor (GM-CSF). Recombinant GM-CSF was initially administered at standard dosage (5 micrograms/kg/day) until a white blood cell count > or = 2 x 10(9)/L was achieved on two consecutive examinations, and thereafter at a low dose (1 microgram/kg/day) for 5 to 9 months. On a weekly basis, GM-CSF was discontinued and hydroxyurea (1,000 mg/d) was given for two days. RESULTS: Evidence of trilineage engraftment was observed in all cases. At autografting, 3 out of the 5 patients revealed 8-9% Ph-negative metaphases. During the initial phase of hematopoietic regeneration, direct cytogenetic analysis revealed 81% and 100% Ph-negative metaphases in two cases; nonleukemic hematopoiesis progressively decreased and was no longer detectable at +9 months. One patient showed cyclic Ph-negative hematopoiesis that appeared 3 months following autografting and peaked at +4 and +8 months. The fourth patient showed a low percentage (20%) of Ph-negative metaphases 1 month after ASCT, followed by a significant expansion of nonleukemic hematopoiesis, which could be detected up to month +13. No evidence of Ph-negative hematopoiesis could be detected in one patient. Three patients are in chronic phase 28, 30 and 31 months after autografting, respectively, and two patients evolved into blast crisis. INTERPRETATION AND CONCLUSIONS: This pilot study demonstrates that combined GM-CSF and hydroxyurea therapy seems to be effective in inducing and/or prolonging a transient period of Ph-negative hematopoiesis. The late appearance of Ph-negative hematopoiesis detected in two patients suggests an antileukemic activity of the combined GM-CSF/hydroxyurea therapy rather than an antileukemic effect of the conditioning regimen.

In vitro growth of mobilized peripheral blood progenitor cells is significantly enhanced by stem cell factor.

The existence of primitive hematopoietic progenitors in mobilized peripheral blood is suggested by clinical, phenotypic and in vitro cell culture evidences. In order to quantify primitive progenitors, 32 leukaphereses from 15 patients with lymphoid malignancies were investigated for the growth of multilineage colony-forming units (CFU-Mix), erythroid burst-forming units (BFU-E) and granulocyte-macrophage colony-forming units (CFU-GM) in the absence or presence of recombinant stem cell factor (SCF), a cytokine which selectively controls stem cell self-renewal, proliferation and differentiation. Primitive progenitors were also quantitated by means of a long-term assay which allows the growth of cells capable of initiating and sustaining hematopoiesis in long-term culture (LTC-IC). Addition of SCF (50 ng/ml) to methyl-cellulose cultures stimulated with maximal concentrations of G-CSF, GM-CSF, interleukin 3 and erythropoietin significantly increased the growth (mean +/- SE) of CFU-Mix (7.7 +/- 1.7 versus 2.4 +/- 0.6, p < or = 0.0001), BFU-E (47 +/- 10 versus 32 +/- 6, p < or = 0.002) and CFU-GM (173 +/- 31 versus 112 +/- 20, p < or = 0.0001). Mean (+/- SE) percentages of SCF-dependent CFU-Mix, BFU-E and CFU-GM were 60 +/- 5%, 19 +/- 5%, and 33 +/- 4%, respectively. Mean (+/- SE) LTC-IC growth per 2 x 10(6) nucleated cells was 221 +/- 53 (range, 2 to 704). Linear regression analysis demonstrated a statistically significant correlation (r = .87; p < or = 0.0001) between LTC-IC and SCF-dependent progenitors. In conclusion, our data suggest that: A) the optimal quantification of mobilized progenitors requires supplementation of methylcellulose cultures with SCF, and B) in vitro detection of SCF-dependent progenitors might represent a reliable and technically simple method to assess the primitive progenitor cell content of blood cell autografts. Such in vitro evaluation of immature hematopoietic progenitors might be clinically relevant for predicting the reconstituting potential of autografts.

Selection of myeloid progenitors lacking BCR/ABL mRNA in chronic myelogenous leukemia patients after in vitro treatment with the tyrosine kinase inhibitor genistein.

Chronic myelogenous leukemia (CML) is a clonal disorder of the hematopoietic stem cell characterized by a chimeric BCR/ABL gene giving rise to a 210-kD fusion protein with dysregulated tyrosine kinase activity. We investigated the effect of genistein, a protein tyrosine kinase inhibitor, on the in vitro growth of CML and normal marrow-derived multi-potent (colony-forming unit-mix [CFU-Mix]), erythroid (burst-forming unit-erythroid [BFU-E]), and granulocyte-macrophage (colony-forming unit-granulocyte-macrophage [CFU-GM]) hematopoietic progenitors. Continuous exposure of CML and normal marrow to genistein induced a statistically significant and dose-dependent suppression of colony formation. Genistein doses causing 50% inhibition of CML and normal progenitors were not significantly different for CFU-Mix (27 mumol/L v 23 mumol/L), BFU-E (31 mumol/L v 29 mumol/L), and CFU-GM (40 mumol/L v 32 mumol/L v 32 mumol/L). Preincubation of CML and normal marrow with genistein (200 mumol/ L for 1 to 18 hours) induced a time-dependent suppression of progenitor cell growth, while sparing a substantial proportion of long-term culture-initiating cells (LTC-IC) from CML (range, 91% +/- 9% to 32% +/- 3%) and normal marrow (range, 85% +/- 8% to 38% +/- 9%). Analysis of individual CML colonies for the presence of the hybrid BCR/ABL mRNA by reverse transcription-polymerase chain reaction (RT-PCR) showed that genistein treatment significantly reduced the mean +/- SD percentage of marrow BCR/ABL+ progenitors both by continuous exposure (76% +/- 18% v 24% +/- 12%, P < or = .004) or preincubation (75% +/- 16% v 21% +/- 10%, P < or = .002) experiments. Preincubation with genistein reduced the percentage of leukemic LTC-IC from 87% +/- 12% to 37% +/- 12% (P < or = .003). Analysis of individual colonies by cytogenetics and RT-PCR confirmed that genistein-induced increase in the percentage of nonleukemic progenitors was not due to suppression of BCR/ABL transcription. Analysis of nuclear DNA fragmentation by DNA gel electrophoresis and terminal deoxynucleotidyl transferase assay showed that preincubation of CML mononuclear and CD34+ cells with genistein induced significant evidence of apoptosis. These observations show that genistein is capable of (1) exerting a strong antiproliferative effect on CFU-Mix, BFU-E, and CFU-GM while sparing the more primitive LTC-IC and (2) selecting benign hematopoietic progenitors from CML marrow, probably through an apoptotic mechanism.

Effect of the protein tyrosine kinase inhibitor genistein on normal and leukaemic haemopoietic progenitor cells.

Receptor and nonreceptor protein tyrosine kinases (PTKs) play a key role in the control of normal and neoplastic cell growth. The availability of PTK inhibitors prompted us to evaluate the effects of genistein, a natural inhibitor of PTKs, on in vitro colony formation by normal multilineage colony-forming units (CFU-Mix), erythroid bursts (BFU-E), granulocyte-macrophage colony-forming units (CFU-GM), long-term culture-initiating cells (LTC-IC) and acute myelogenous leukaemia colony-forming units (CFU-AML). Continuous exposure of normal marrow and blood mononuclear non-adherent cells, blood CD34+CD45RA- cells, and leukaemic blasts to increasing doses of genistein (1-100 microM) resulted in a statistically significant (P < or = 0.05) dose-dependent suppression of CFU-Mix, BFU-E, CFU-GM and CFU-AML growth. Regression analysis showed that growth inhibition was linearly related to genistein concentration. Genistein dose causing 50% inhibition (ID50) of CFU-AML was significantly lower compared to CFU-GM ID50 for marrow (19 v 32 microM, P < or = 0.017), unseparated blood (19 v 44 microM, P < or = 0.028) or CD34+CD45RA- blood (19 v 36, P < or = 0.04). Preincubation of leukaemic blasts with genistein (200 microM) for 1-2h confirmed that CFU-AML were significantly more sensitive than normal marrow and blood CFU-GM to genistein. Preincubation conditions which maximally suppressed leukaemic and normal colony growth spared a substantial percentage of marrow (29 +/- 4%) and blood (40 +/- 3%) LTC-IC. In conclusion, our data demonstrate that: (a) genistein strongly inhibits the growth of normal and leukaemic haemopoietic progenitors; (b) growth inhibition is dose- and time-dependent; (c) leukaemic progenitors are more sensitive than normal progenitors to genistein-induced growth inhibition; (d) genistein exerts a direct toxic effect on haemopoietic cells while sparing a substantial proportion of LTC-IC. The potent CFU-AML growth inhibition associated with the relative resistance of normal LTC-IC strongly supports the use of genistein for marrow purging.