Study On Reversal Of STI571 Resistance In Chronic Myelogenous Leukemia Cells By TPA In Vitro

Background and ObjectSTI571 can specific inhibit the hyperplasia and induce the apoptosis of Ph⁺ cells because it can inhibit the protein tyrosine kinase (PTK) activity of ABL gene in BCR-ABL fusion gene as a PTK inhibitor. It was approved that it can be used to treat the patients with chronic myelogenous leukemia (CML) in blast crisis, accelerated phase, or in chronic phase after treat failed with interferon-α(IFN-α) as treatment program through peroral administration (P.O.) by Food Drug Administration (FDA) of USA in May 2001, furthermore, it got an approval to used for each period of CML patients in May 2003. It was used more commonly in China in recent years. Now it is recommended for front-line therapy of this disease. However, it was observed that there were quite a part of CML patients who were sensitive to the STI571 at the beginning appearance resistant soon; even if in chronic phase, there were also 45%～50% of patients cannot achieve the genetic remission in the first 9 months with treatment, it is that appeared the genetic resistant. The latest study have demonstrated that the reason of this phenomenon may be the over magnification and express of Bcr/Abl gene in Bcr/Abl positive cells; furthermore, it also concerned with the over expression of the multi-drug resistant (MDR) gene and its product P-Glycoprotein. Nevertheless, these points of view may be cannot explain all the STI571 resistant phenomenon clinically. There were more and more evidences prompt that the CML origins from its leukemia stem cell (LSC) and the relative ripe daughter cells derived from it is the majority of leukemia cells, STI571 can inhibit the growth of these leukemic cells conspicuously, but the CML leukemia stem cell may be resistant to the STI571 partly even completely.12-O- tetradecanoylphorbol- 13-acetate (TPA) is an activator of protein kinase C (PKC) extracted from the croton oil, it is one of the most powerful inducers of differentiation, at the same time, also is a powerful tumor promoter. It has been widely used in the induction differentiation experimental study since Huberman discovered the human promyelocyte can be differentiated by the TPA in 1979, it is able to induce many kinds of cell of leukemic cell strain like HL-60,HEL and so on, moreover, promote the cell above-mentioned except the progenitor megakaryocyte leukemia cell strain differentiating toward the monocyte/macrophage, and appear some phenotype and function as well as some zymologic change. Some literature reported that TPA had the function of differentiation of primary leukemic cell showed the main action to differentiate toward the monocyte/macrophage, as well, different typing of patients showed the different reaction grade to the induction of TPA and there were differences between the different individual in the same type. TPA has the effect of induction differentiate not only on mouse leukemic cell lines, but also on human primary leukemic cells at low concentration (10^-6mol/L～10^-8mol/L) in vitro, in addition, it has been demonstrated on clinical that it have certain effect on leukemia refractoriness and relapse.Recently, we separated the cell colony carrying Bcr/Abl fusion gene and express Flk1⁺CD34^- from the bone marrow of CML patients, conformed that it could be induced toward hematopoietic cell and endothelial cell in the mono cell level in vitro, and the differentiated cells all carried Bcr/Abl fusion gene. This prompts us that the Flk1⁺CD34^- cell have the characteristic of hemangioblasts we separated possibly is the stem cell of CML. In this study, we initially discussed the sensitivity of STI571 to the CML stem/progenitor cell in the different differentiation stage used the Flk1⁺CD34^- cell, in order to further understand the influences of STI571 to the induction and proliferation of leukemic stem/progenitor cell carry Bcr/Abl fusion gene at primary and different differentiation stage of CML patients in vivo, thus can illuminate the mechanism of CML patients relapsed resistant to the STI571 after experienced section of hematology remission even complete remission in the cytogenetics level; moreover, using TPA unite STI571 overcomes the CML patients resistant to the STI571, thus provide the theory basis for clinical uses TPA unite STI571 to overcome CML patients to bear the medicine phenomenon to STI571.Methods and Subjects1. Patients selectionFifteen patients with newly diagnosed CML (all male, aged 19-65 years) were recruited in this study. All were Ph⁺ patients with CML in chronic phase as revealed by bone marrow histology and cytogenetic analysis. None were treated with hydroxyurea or interferon before (Table 1).2. Isolation and culture of bone marrow-derived Flk1⁺CD31^-CD34^- cells from patients with CMLMononuclear cells separated by a Ficoll-Paque gradient centrifugation (specific gravity 1.077g/mL) from bone marrow of patients with CML were depleted of CD45⁺, GlyA⁺ and CD34⁺ cells by micromagnetic beads, and then cultured by limiting dilution. Three single colonies were harvested, expanded and characterized.3. Assement of drug resistance of Bcr/Abl⁺Flk1⁺CD31^- CD34CML stem cells Bone marrow-derived, malignant, BCR/ABL-positive, Flk1⁺CD31CD34^- cellswith hemangioblastic characteristics from CML patients were grown in Methocult GF+media with or without STI571. Then the characterization of the proliferation or apoptosis of the quiescent (G₀) and cycling (G₁ and S/G₂+M) subsets of FACS-purified Bcr/Abl⁺Flk1⁺CD31^-CD34^- cells to STI571, TPA, or STI571 + TPA was examined respectively.Results1. Exposure to 5μM STI571 (the concentration of STI571 usually achieved in patients is 1-2μM for 96 hours) in vitro for 96 hours inhibited Bcr/Ab⁺ committed progenitors (colony-forming cells [CFCs]). No evident suppression of primitive, Bcr/Abl-positive and Flk1⁺CD34^- cells were observed.2. The resistance of the quiescent (Go) Bcr/Abl⁺Flk1⁺CD31^-CD34^- cells was more stronger than that of cycling (G₁ and S/G₂+M) ones; Coexposure of Bcr/Abl⁺Flk1⁺CD31^-CD34^- cells to TPA in combination with STI571 could inhibit Bcr/Abl⁺Flk1⁺CD31^-CD34^-cells form proliferating and promote STI571-mediated apoptosis.Conclusion1. Inhibiting BCR/ABL tyrosine kinase activity by imatinib mesylate might not eliminate malignant primitive progenitors in CML patients and such in vivo CML primitive stem cells which are insensitivity to STI571 might translate into disease relapse after prolonged therapy.2. Coexposure of Bcr/Abl⁺Flk1⁺CD31^-CD34^- cells to TPA in combination with STI571 can enhance the effect of imatinib mesylate on CML cells, especially by specifically targeting the primitive quiescent leukemic elements (the G₀ subsets). A protocol for treating chronic-phase CML patients with STI571 that incorporates TPA exposure may offer a novel strategy for obtaining improved responses in vivo.