Hm Goselink

Long–term culture of primary human lymphoblastic leukemia cells in the absence of serum or hematopoietic growth factors

OBJECTIVE B-lineage acute lymphoblastic leukemia (ALL) and chronic myeloid leukemia in lymphatic blastic phase in adults have poor prognoses despite intensive chemotherapy. Novel targeted treatment modalities emerge, but their evaluation requires relevant in vitro models of lymphoblastic leukemia. Presently available cell lines do not fully represent this heterogeneous disease. Available in vitro culturing protocols do not support long-term proliferation of primary cells. We therefore aimed to develop a culture system that allows long-term proliferation of primary human B-lineage lymphoblastic leukemia. MATERIALS AND METHODS Primary lymphoblastic leukemia cells were cultured in a defined serum-free medium, in the absence or presence of human hematopoietic growth factors or serum. RESULTS In the defined serum-free medium, cells from 12 of 34 cases immediately proliferated in vitro. In the absence of hematopoietic growth factors and serum these cases proliferated for more than 1 year without signs of exhaustion. The culturing system supported different subtypes of lymphoblastic leukemia. Two chronic myeloid leukemia in lymphatic blastic phase, four bcr/abl-positive ALL, one etv6/abl-positive ALL, 2 e2a-pbx1-positive ALL, and one t(9;11)-positive ALL could be long-term expanded, as well as two ALL that displayed nontypical cytogenetics. Not all bcr/abl- or e2a-pbx1-positive ALL proliferated in vitro, demonstrating heterogeneity within these subtypes. The proliferating bcr/abl- and etv6/abl-positive cells displayed sensitivity to imatinib, demonstrating that their proliferation depended on the activity of these oncoproteins. CONCLUSION The serum-free culturing system may be a valuable instrument in the study of ALL cell biology, as well as in the evaluation of novel targeted therapeutics.

BH3 Inhibitor Sensitivity and Bcl-2 Dependence in Primary Acute Lymphoblastic Leukemia Cells

BH3 mimetic drugs may be useful to treat acute lymphoblastic leukemia (ALL) but the sensitivity of primary tumor cells has not been fully evaluated. Here, B-lineage ALL cell cultures derived from a set of primary tumors were studied with respect to sensitivity to the BH3 mimetics ABT-263 and ABT-199 and to Bcl-2 dependence and function. These ALL cells each expressed high levels of Bcl-2 and exhibited great sensitivity to ABT-263 and ABT-199, which induced rapid apoptotic cell death. BH3 profiling indicated that the ALL cultures were Bcl-2 dependent. Coimmunoprecipitation studies revealed a multifaceted role for Bcl-2 in binding proapoptotic partners including Bax, Bak, Bik, and Bim. ABT-263 disrupted Bcl-2:Bim interaction in cells. Mcl-1 overexpression rendered ALL cells resistant to ABT-263 and ABT-199, with Mcl-1 assuming the role of Bcl-2 in binding Bim. Freshly isolated pediatric ALL blasts also expressed high levels of Bcl-2 and exhibited high sensitivity to Bcl-2 inhibition by the BH3 mimetic compounds. Overall, our results showed that primary ALL cultures were both more sensitive to BH3 mimetics and more uniform in their response than established ALL cell lines that have been evaluated previously. Furthermore, the primary cell model characterized here offers a powerful system for preclinical testing of novel drugs and drug combinations to treat ALL.

Tumor necrosis factor alpha (Tnf-α) Production by acute myeloid leukemic (Aml) Blasts results in impaired proliferation of normal hematopoietic progenitor cells (Hpc)

Abstract Previously, we demonstrated that the production of proteinases by normal myeloid precursor cells was responsible for impaired proliferation of normal HPC in the presence of hematopoietic growth factors (HGF) in a liquid serum free medium culture consisting of IMDM, human serum albumin, cholesterol, transferrin and insulin. Addition of the serine proteinase inhibitor α-1 PI restored the proliferation. However, AML blasts co-cultured with normal HPC in the presence of α-1 PI, using micro-membrane-transwells (poresize 0.45 μm) in the absence of cell-cell contact, were still capable of suppressing the proliferation of normal HPC up to 60 ± 7% (n=17). AML blasts from all FAB classifications, but most profoundly AML-M4 blasts released a factor capable of strong inhibition of normal HPC (87 ± 2% (n=4)). TNF-α has been shown to be produced in high amounts by AML blasts, has previously shown to have anti-proliferative features and is a mediator in the pathways of cellular apoptosis. Therefore, we studied the effect of anti-TNF-α for its ability to neutralize the inhibitory effect of AML cells on in vitro proliferation of normal HPC in these serum fee co-cultures. Addition of anti-TNF-a completely restored the proliferation of normal CD34 + HPC in response to HGF. In addition, anti-TNF-α increased the proliferation of the AML blasts int he presence of HGF by 166 ± 43% (n=18) independent of their FAB classification. These results may indicate that treatment with anti-TNF-antibodies may increase the proliferative capacity of residual normal HPC in patients with AML, but may also lead to increased proliferation of the leukemic cells.