BCR-ABL accelerates C2-ceramide-induced apoptosis.

In patients with chronic myeloid leukemia (CML), the neoplastic (BCR-ABL+) progenitor cells are characterized by an increased proliferative activity. Whether these cells are also resistant to apoptosis and if so, under what conditions remains controversial. We now show that highly purified populations of very primitive neoplastic progenitor cells obtained directly from CML patients survive and proliferate in vitro for several weeks in the absence of any added growth factors (except insulin). In contrast, purified primary normal progenitors maintained under the same conditions die rapidly. Nevertheless, both primary CML cells and BCR-ABL+ BAF3 cells show the same dose-dependent sensitivity to TNF-alpha or ceramide-induced apoptosis as their respective normal counterparts. In fact, time course studies demonstrated an even faster onset of apoptosis in ceramide-treated BCR-ABL+ BAF3 cells as compared to normal controls. BCR-ABL+ cells treated with ceramide also showed a rapid and sequential increase in the tyrosine phosphorylation of p210(BCR-ABL), p46-56SHC and p120Cbl. These findings suggest growth factor deprivation and treatment with TNF-alpha or ceramide trigger different initial events both of which can lead to apoptosis in factor-dependent hematopoietic cells. However, in the first case, activation of apoptosis is blocked by the basal activity of p210(BCR-ABL), whereas in the second, the presence of p210(BCR-ABL) appears to accelerate the onset of apoptosis by a mechanism that may involve an activation of its kinase function.

Retroviral vector-mediated transfer of the interferon-alpha gene in chronic myeloid leukemia cells.

The transfer and expression of cytokine genes into tumor cells is reportedly a valuable approach to improve the antitumor activity of cytokines in various models. Interferon (IFN)-alpha may induce hematological remission in chronic myeloid leukemia (CML) patients, but only a small proportion of patients achieve a sustained, complete cytogenetic remission. We have investigated the possibility of transducing CML cells with the retroviral vector LIalpha2SN, which encodes the IFN-alpha2 gene. We first optimized the transduction efficiency using the CML-derived K562 cell line. A transduction efficiency of 50% and 85% after three and six infections, respectively, was obtained in K562 cells. We then expressed IFN-alpha2 in CML cells by transducing the latter with LIalpha2SN viral particles. The IFN-alpha secretion after three and six infections was 5,400 and 18,000 U/24 hours/10(6) cells for unselected K562 cells and 7,000 and 290 U/24 hours/10(6) cells for CML CD34+ cells at days 4 and 5. Moreover, the major histocompatibility complex class I antigens were overexpressed after infection with LIalpha2SN in both K562 and CML CD34+ cells. The proliferation (in liquid culture) and the cloning efficiency of these CML cells were significantly decreased after LIalpha2SN treatment. By contrast, the proliferation of cord blood CD34+ cells was not affected by transduction with LIalpha2SN. These results demonstrate the transduction efficiency of CML cells and suggest the possibility of CML cell immunotherapy with retroviral gene transfer of different cytokines such as IFN-alpha.

Ex vivo cytokine expansion of peripheral blood Ph-negative cells in chronic myeloid leukaemia.

Hematopoietic progenitors can be expanded ex vivo in the presence of various cytokine combinations. Since normal early progenitor or stem cells persist in the blood and bone marrow of patients with Philadelphia chromosome [Ph]-positive chronic myeloid leukaemia (CML), the selection of normal (Ph-negative) progenitor cells from CML patients would be of considerable clinical value for ex vivo purging and autologous transplantation. To obtain these cells, CD34-positive (progenitor) cells from the peripheral blood (PB) of CML patients were either pretreated or not with 5-fluorouracil (5FU) and then grown in suspension culture for 7 days with a combination of cytokines. We compared different combinations of cytokines containing interleukin-1 alpha (IL1), interleukin-3 (IL3), stem cell factor (SCF), leukemia inhibitor factor (LIF), Flt3-ligand (FLT3L), and thrombopoietin (TPO). 5FU decreased cell proliferation in the liquid culture but concurrently increased the expansion of CFU-GM. While the addition of cytokines such as FLT3L and TPO improved CFU-GM expansion. FISH and RT-PCR analysis showed that this method significantly favored a higher frequency of Ph-negative cells after expansion in liquid culture. Therefore ex vivo expansion of putatively normal hematopoietic progenitor cells from cytapheresis is feasible in CML.

Ex vivo cytokine expansion of peripheral blood 5-fluorouracil-treated CD34-positive chronic myeloid leukaemia cells increases the selection of Ph-negative cells.

It has been shown that normal early progenitor or stem cells persist in the blood and bone marrow of patients with Philadelphia chromosome (Ph)-positive chronic myeloid leukaemia (CML); it is also known that normal haemopoietic progenitors can be expanded ex vivo in the presence of various cytokine combinations. The selection of normal (Ph-negative) progenitor cells from CML patients would potentially be of considerable clinical value for ex vivo purging and autologous transplantation. To obtain these cells. CD34-positive (progenitor) cells from the peripheral blood (PB) of CML patients were pretreated with 5-fluorouracil (5FU) (5 microg/ml) to suppress Ph-positive cells and then grown in suspension culture for 7 d with a combination of cytokines. We compared two combinations of cytokines: interleukin-1alpha (IL1alpha) and interleukin-3 (IL3) with either Flt3-ligand (FLT3L) or stem cell factor (SCF). Using these two combinations, we obtained the same degree of day 14 CFU-GM expansion (3.1 +/- 0.5 and 3.4 +/- 0.7 fold expansion). FISH analysis showed that 5FU pretreatment significantly favoured a higher frequency of Ph-negative cells after expansion in liquid culture. Moreover, after 5FU pretreatment, the mean (+/-SEM) percentage of Ph negativity was significantly greater for IL1alpha-IL3-FLT3L compared to IL1alpha-IL3-SCF (19.1 +/- 2.5% v 14.8 +/- 2.3%, P=0.009, n = 7). The output long-term culture initiating cells (LTC-IC) which could only be detected after 5FU pretreatment and the combination, IL1alpha-IL3-FLT3L were all Ph negative by FISH analysis. Thus, a subset of Ph-negative cells was selected from CML PB by 5FU and expanded using the combination of cytokines IL1alpha-IL3-FLT3L and IL1alpha-IL3-SCF. Ex vivo expansion of putatively normal haemopoietic progenitor cells is feasible in CML.

All-trans retinoic acid potentiates the inhibitory effects of interferon alpha on chronic myeloid leukemia progenitors in vitro.

All-trans retinoic acid (ATRA) has recently been shown to synergize with the inhibitory effect of interferon alpha (IFN alpha) on the growth of malignant cells isolated from solid tumors. We investigated whether ATRA could potentiate the inhibitory effects of IFN alpha on the proliferation of leukemic progenitors in chronic myeloid leukemia (CML). CD34+ cells from chronic phase, newly diagnosed patients, were incubated in short-term liquid culture with ATRA, IFN alpha or a combination of both molecules and then plated on semi-solid cultures for colony-forming cell assay. IFN alpha was found to inhibit preferentially the generation of late progenitors. ATRA at a concentration of 10(-8) M was found strongly to inhibit CFU-M colonies. Addition of ATRA to IFN alpha dramatically potentiated the inhibitory effects of INF alpha on CFU-GM growth. In the presence of both molecules the inhibition of day 14 CFU-GM from CD34+ cells was lowered to 27 +/- 4% of control. CFU-M colonies were completely inhibited. RT-PCR analysis of the colonies resulting from the action of the combination IFN alpha plus ATRA showed the presence of an increased number of BCR-ABL-negative colonies relatively to what was observed with IFN alpha alone. FISH analysis showed a higher percentage of Ph-negative cells in the ATRA plus IFN alpha-treated samples, confirming PCR experiments. These results indicate that, in vitro, the combination of IFN alpha and ATRA effectively inhibits CFU-GM colony formation in CML and suggest that it has a potential interest for the treatment of CML.