Elsevier

Toxicology Letters

Volume 199, Issue 3, 15 December 2010, Pages 317-322
Toxicology Letters

Development of an in vitro model for the simultaneous study of the efficacy and hematotoxicity of antileukemic compounds

https://doi.org/10.1016/j.toxlet.2010.09.014Get rights and content

Abstract

Hematopoietic system displays a wide spectrum of cell populations hierarchically organized in the bone marrow. Homeostasis in this system requires equilibrium between the self-renewal of the stem cells and their capacity of differentiation. Any failure on this equilibrium could lead to fatal consequences, such as the development of leukemia. Due to its rapid rate of renewal, hematopoietic tissue is a major target for antitumoral compounds and often becomes a dose limiting factor in the development of antineoplastics. Our aim was to develop an in vitro model for predicting the efficacy of antitumoral compounds on leukemic cells and their toxic effects on the healthy hematopoietic cells.

The mouse myelomonocytic leukemia WEHI-3b was transduced with a lentiviral vector for expressing the green fluorescence protein. Mixed semisolid clonogenic cultures of transduced WEHI-3b and murine bone marrow cells were exposed to five pharmaceuticals: daunorubicin (positive control), atropine sulphate (negative control) and three in different stages of clinical development (trabectedin, Zalypsis® and PM01183). Colonies of leukemic cells were distinguishable from healthy CFU-GM under fluorescence microscope. The sensitivity of leukemic cells to daunorubicin, trabectedin, Zalypsis® and PM01183 was higher compared to healthy cells. The effect of a non-antitumoral compound, atropine sulphate, was the same on both populations. Our results show that this in vitro model is a valuable tool for studying the effect of antitumoral compounds in both tumoral and normal hematopoietic cells under the same toxic microenvironment and could safe time and facilitate the reduction of the number of animals used in preclinical development of pharmaceuticals.

Introduction

Hematopoietic system displays a wide spectrum of cell populations whose constant proliferation and differentiation in the bone marrow (BM) give rise to erythroid, granulocytic, macrophagic, megakaryocytic and lymphoid blood cells. BM is an extremely complex tissue which is regulated by an equally complex set of hematopoietic growth factors and by specific interactions with the stromal cells of the hematopoietic environment. Hematopoietic tissue is hierarchically organized, a small number of stem cells gives rise to a whole variety of morphologically and functionally differentiated cells. The stem cell population is the fundamental base from which all the mayor blood lines are derived. The main property of this population is its ability to maintain its own number. Committed progenitors are derived from the stem cells and are committed to a particular line of cell development. They are transit cells that amplify the population prior to its production of functional maturation stages (Sachs, 1987, Lord and Testa, 1988, Morrison et al., 1995). Homeostasis in the hematopoietic system requires equilibrium between the self-renewal of the stem cells and their capacity of differentiation. Any failure on this equilibrium could lead to fatal consequences. One of these consequences is the development of leukemias (Zeleznik-Le et al., 1995, Bonnet and Dick, 1997).

The objectives of toxicology studies are the identification of potentially dangerous toxicants, so that human exposure can be prevented or controlled, and the provision of relevant information for undertaking risk-benefit analyses and for conducting clinical trials. Toxicants can cause hematotoxicity by interfering with mature blood cell, committed progenitors or stem cells functions or survival. Following cytotoxic insult, for instance after chemotherapy, neutropenia and trombocytopenia are often non-desirable clinical effects which make the hematopoietic system to be the limiting factor. For the preclinical development of antineoplastics, in vivo toxicology studies are generally conducted in at least two different species, employing various dosing schedules. These in vivo data are used to derive the dose for the first human exposure, which is usually at least 10 times lower than the maximum tolerated dose (MTD) in animals (DeGeorge et al., 1998, Sistare and DeGeorge, 2007).

Any in vitro test which can refine these safety margins, by reducing the toxicological uncertainties underlying laboratory animal/human extrapolations, would be of great benefit, since it would provide a more scientific and rational basis for calculating clinical dosages and for setting human exposure limits.

During the first stages of the preclinical development of an antitumoral, in order to get the first hints of effect on the hematopoietic tissue, studies on established hematopoietic cell lines are generally performed (HL-60, K562, etc.). Later on, in vivo hematological parameters are determined in different animal models. On the other hand, the effect of the compounds on tumoral cells is also studied, first with cell lines and then in animal models bearing artificial tumors. There are not steps in the process in which tumoral and normal cells are studied in the same model and under the same microenvironmental parameters. This is especially important in the case of leukemic cells which share a lot of characteristics with the healthy hematopoietic cells (Rosmarin et al., 2005, Misaghian et al., 2009).

In the present study, a new in vitro model is described. We have developed an in vitro assay in which, leukemic and normal hematopoietic progenitor cells, can growth simultaneously and be distinguished from each other. Focusing on a model for acute myeloid leukemia (AML), we have used WEHI-3b cells, as the leukemic population, and Colony Forming Units of Granulocytes and Macrophages (CFU-GM) progenitors from healthy murine BM. By means of this assay, the efficacy of antileukemic compounds on leukemic cells and, their toxic effect on normal hematopoietic progenitors, can be study in the same in vitro system, thereby allowing the growth under the same cytotoxic pressure. In order to facilitate the identification of leukemic colonies, WEHI-3b cells have been transduced with a lentiviral vector (LV) for expressing the green fluorescence protein. Colonies of transduced WEHI-3b cells were distinguishable from healthy CFU-GM colonies of murine BM in semisolid cultures under fluorescence microscopy. The use of this assay in early stages of the preclinical development could avoid surprises in later stages when a lot of animals, money and time could be misspent.

Section snippets

Mice

Twelve to 14 week-old Balb/C mice were used as donors of bone marrow cells (BMC). Breeding pairs, originally obtained from the Jackson Laboratory (Bar Harbor, ME) were bred at the Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT) Animal Facility (Madrid, Spain; Registration No. 28079-21 A) and allowed food and water ad libitum. After euthanasia by carbon monoxide, femurs were aseptically removed, and the marrow was flushed from the central canal with Iscove's

Gene marking of WEHI-3b cells with EGFP

Lentiviral vectors (LVs) carrying the EGFP (Enhanced Green Fluorescent Protein) gene (as a marker gene), under the control of the different promoters, were used for the transduction of WEHI-3b cells. The spleen focus-forming virus (SFFV) promoter was clearly the strongest with a Mean Fluorescence Intensity (MFI) of 210, although this LV was the least efficient transducing WEHI-3b cells (11.7% of EGFP+ cells). The highest percentage of transduced cells was obtained with the cytomegalovirus

Discussion

Drug development is a costly process both because of time reasons and financial reasons. A large proportion of these compounds (approximately 90%), fail due to toxicity (Davies et al., 2008). To decrease costs in the drug discovery process, the pharmaceutical industry has invested in high throughput automated technologies which has brought a lot of advantages. However, in the last years, there has been an increasing demand of new cell based assays (Brinker and Caldwell, 2008). Taking into

Conflict of interest statement

Sandra Martínez: PharmaMar employee. The other authors reported no potential conflicts of interest.

Funding information

This work was supported by the Spanish Ministry of Science and Innovation (Grant No. BIO2004-01727) and a CIEMAT Training Grant for the Division of Hematopoiesis (A.V.).

Acknowledgements

The authors would like to express their appreciation and thanks to Jesus Martinez and Edilia de Almeida for careful maintenance of the animals, Sergio Garcia for excellent technical assistance and I. Ormán for expert assistance with the flow cytometry.

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