Inhibition of the ABL kinase activity blocks the proliferation of BCR/ABL+ leukemic cells and induces apoptosis

Carlo Gambacorti-Passerini, Philipp Le Coutre, Luca Mologni, Mirco Fanelli, Carla Bertazzoli, Edoardo Marchesi, Massimo Di Nicola, Andrea Biondi, Gian Marco Corneo, Daniela Belotti, Enrico Pogliani, Nicholas B. Lydon

Research output: Contribution to journalArticle

282 Citations (Scopus)

Abstract

The BCR/ABL fusion protein transforms myeloid stem cells. Both chronic myelogenous leukemias (CML) and a subset of acute lymphoblastic leukemias (ALL) are associated with the expression BCR/ABL proteins. This knowledge has not yet been translated into any specific tool to control ABL drive neoplastic cells growth. CGP57148B is an ATP-competitive inhibitor of the ABL protein kinase; it has been shown to inhibit the kinase activity of ABL both in vitro and in vivo and to inhibit the growth of v-abl bcr/abl transfectants, as well as the in vitro formation of bone marrow (BM)-derived colonies in presence of growth factors in some CML patients. These studies were performed to investigate activity of CGP57148B on the spontaneous proliferation of both fresh and cultured, leukemic and normal BCR/ABL positive and negative cells, and to study its mechanism of action. Six cell lines derived from BCR/ABL+ leukemias (K562, BV173, KCL22, KU812, MC3, LAMA84), thirteen BCR/ABL negative lines, both neoplastic (KG1, SU-DHL-1, U937, Daudi, NB4, NB4.306) and derived from normal, cells (PHA blasts, LAK, fibroblasts, LCL, renal epithelial cells, endothelial cells, CD34+ cells), and 14 fresh leukemic samples were tested using a tritiated thymidine uptake assay. The in vivo phosphorylation of the BCR/ABL protein was evaluated by western blot, while apoptosis was detected by the annex V/propidium binding test. The induction of differentiation was assayed by immunofluorescence using multi antibodies. All six BCR/ABL+ lines showed a dose dependent inhibition of their spontaneous proliferative rate, which not accompanied by differentiation. The treatment caused, within minutes, dephosphorylation of the BCR/ABL protein, followed in 16-24 hours by a decrease in cycling cells and induction of apoptosis. No significant inhibition of DNA synthesis was observed in any BCR/ABL negative normal or neoplastic line at concentrations ≤3 μM, with the exception of fibroblasts and CD34 cells. Proliferation inhibition was observed also when using fresh samples obtained from two Ph+ ALL and 12 consecutive CML patients. Induction of apoptosis was observed in these samples too. The activity of CGP57148B can be monitored in ex vivo isolated or cultured cells using a simple and reproducible assay, without the need for exogenously added growth factors. This molecule possibly exerts its effects through inhibition of the kinase activity of BCR/ABL and the subsequent initiation of apoptosis, without inducing cell differentiation. Some normal cells are also affected. These data support the use of CGP57148B in initial clinical studies; possible toxic effects on BM and fibroblast- derived cells will have to be closely monitored. The in vivo monitoring of patients will have to be focused on the induction of apoptosis in leukemic cells.

Original languageEnglish
Pages (from-to)380-394
Number of pages15
JournalBlood cells, molecules & diseases
Volume23
Issue number3
DOIs
Publication statusPublished - Dec 1997

Fingerprint

Phosphotransferases
Apoptosis
Leukemia, Myelogenous, Chronic, BCR-ABL Positive
Fibroblasts
Precursor Cell Lymphoblastic Leukemia-Lymphoma
Intercellular Signaling Peptides and Proteins
Proteins
Bone Marrow
Myeloid Progenitor Cells
Propidium
Poisons
Physiologic Monitoring
Protein Kinase Inhibitors
Growth
Thymidine
Fluorescent Antibody Technique
Cell Differentiation
Cultured Cells
Leukemia
Endothelial Cells

Keywords

  • ALL
  • Apoptosis
  • BCR/ABL
  • CML
  • Tyrosine kinase inhibitors

ASJC Scopus subject areas

  • Molecular Biology
  • Molecular Medicine
  • Hematology

Cite this

Gambacorti-Passerini, C., Le Coutre, P., Mologni, L., Fanelli, M., Bertazzoli, C., Marchesi, E., ... Lydon, N. B. (1997). Inhibition of the ABL kinase activity blocks the proliferation of BCR/ABL+ leukemic cells and induces apoptosis. Blood cells, molecules & diseases, 23(3), 380-394. https://doi.org/10.1006/bcmd.1997.0155

Inhibition of the ABL kinase activity blocks the proliferation of BCR/ABL+ leukemic cells and induces apoptosis. / Gambacorti-Passerini, Carlo; Le Coutre, Philipp; Mologni, Luca; Fanelli, Mirco; Bertazzoli, Carla; Marchesi, Edoardo; Di Nicola, Massimo; Biondi, Andrea; Corneo, Gian Marco; Belotti, Daniela; Pogliani, Enrico; Lydon, Nicholas B.

In: Blood cells, molecules & diseases, Vol. 23, No. 3, 12.1997, p. 380-394.

Research output: Contribution to journalArticle

Gambacorti-Passerini, C, Le Coutre, P, Mologni, L, Fanelli, M, Bertazzoli, C, Marchesi, E, Di Nicola, M, Biondi, A, Corneo, GM, Belotti, D, Pogliani, E & Lydon, NB 1997, 'Inhibition of the ABL kinase activity blocks the proliferation of BCR/ABL+ leukemic cells and induces apoptosis', Blood cells, molecules & diseases, vol. 23, no. 3, pp. 380-394. https://doi.org/10.1006/bcmd.1997.0155
Gambacorti-Passerini, Carlo ; Le Coutre, Philipp ; Mologni, Luca ; Fanelli, Mirco ; Bertazzoli, Carla ; Marchesi, Edoardo ; Di Nicola, Massimo ; Biondi, Andrea ; Corneo, Gian Marco ; Belotti, Daniela ; Pogliani, Enrico ; Lydon, Nicholas B. / Inhibition of the ABL kinase activity blocks the proliferation of BCR/ABL+ leukemic cells and induces apoptosis. In: Blood cells, molecules & diseases. 1997 ; Vol. 23, No. 3. pp. 380-394.
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T1 - Inhibition of the ABL kinase activity blocks the proliferation of BCR/ABL+ leukemic cells and induces apoptosis

AU - Gambacorti-Passerini, Carlo

AU - Le Coutre, Philipp

AU - Mologni, Luca

AU - Fanelli, Mirco

AU - Bertazzoli, Carla

AU - Marchesi, Edoardo

AU - Di Nicola, Massimo

AU - Biondi, Andrea

AU - Corneo, Gian Marco

AU - Belotti, Daniela

AU - Pogliani, Enrico

AU - Lydon, Nicholas B.

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N2 - The BCR/ABL fusion protein transforms myeloid stem cells. Both chronic myelogenous leukemias (CML) and a subset of acute lymphoblastic leukemias (ALL) are associated with the expression BCR/ABL proteins. This knowledge has not yet been translated into any specific tool to control ABL drive neoplastic cells growth. CGP57148B is an ATP-competitive inhibitor of the ABL protein kinase; it has been shown to inhibit the kinase activity of ABL both in vitro and in vivo and to inhibit the growth of v-abl bcr/abl transfectants, as well as the in vitro formation of bone marrow (BM)-derived colonies in presence of growth factors in some CML patients. These studies were performed to investigate activity of CGP57148B on the spontaneous proliferation of both fresh and cultured, leukemic and normal BCR/ABL positive and negative cells, and to study its mechanism of action. Six cell lines derived from BCR/ABL+ leukemias (K562, BV173, KCL22, KU812, MC3, LAMA84), thirteen BCR/ABL negative lines, both neoplastic (KG1, SU-DHL-1, U937, Daudi, NB4, NB4.306) and derived from normal, cells (PHA blasts, LAK, fibroblasts, LCL, renal epithelial cells, endothelial cells, CD34+ cells), and 14 fresh leukemic samples were tested using a tritiated thymidine uptake assay. The in vivo phosphorylation of the BCR/ABL protein was evaluated by western blot, while apoptosis was detected by the annex V/propidium binding test. The induction of differentiation was assayed by immunofluorescence using multi antibodies. All six BCR/ABL+ lines showed a dose dependent inhibition of their spontaneous proliferative rate, which not accompanied by differentiation. The treatment caused, within minutes, dephosphorylation of the BCR/ABL protein, followed in 16-24 hours by a decrease in cycling cells and induction of apoptosis. No significant inhibition of DNA synthesis was observed in any BCR/ABL negative normal or neoplastic line at concentrations ≤3 μM, with the exception of fibroblasts and CD34 cells. Proliferation inhibition was observed also when using fresh samples obtained from two Ph+ ALL and 12 consecutive CML patients. Induction of apoptosis was observed in these samples too. The activity of CGP57148B can be monitored in ex vivo isolated or cultured cells using a simple and reproducible assay, without the need for exogenously added growth factors. This molecule possibly exerts its effects through inhibition of the kinase activity of BCR/ABL and the subsequent initiation of apoptosis, without inducing cell differentiation. Some normal cells are also affected. These data support the use of CGP57148B in initial clinical studies; possible toxic effects on BM and fibroblast- derived cells will have to be closely monitored. The in vivo monitoring of patients will have to be focused on the induction of apoptosis in leukemic cells.

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KW - CML

KW - Tyrosine kinase inhibitors

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