Effect of BMP4 on the Growth and Clonogenicity of Human Leukemia and Lymphoma Cells

Materials and Methods

Cells and reagents. Twelve human leukemia and lymphoma cell lines derived from acute T-lymphoblastic leukemia (T-ALL), B-cell malignant lymphoma (B-ML), chronic lymphocytic leukemia (B-CLL), acute myeloblastic leukemia (AML), and chronic myelogenous leukemia (CML) were used (Table I). T-ALL cell lines were provided as a gift from Dr. A. Harashima and Dr. K. Orita (Fujisaki Cell Center, Okayama, Japan). The TMD cell lines were established in our laboratory. NB4 (11) was kindly provided by Dr. M. Lanotte (Hôpital Saint-Louis, Paris, France). The OCI/AML cell lines were established at the Ontario Cancer Institute (Toronto, Canada). AA (12) was established by Dr. A. Arai (Tokyo Medical and Dental University, Japan) from acute pure erythroid leukemia cells. The other cell lines were supplied by the Japanese Cancer Research Resources Bank (Tokyo, Japan). Recombinant human BMP4 was purchased from R&D Systems (Minneapolis, MN, USA).

Short-term growth assay. The effects of BMP4 on short-term growth were examined using a colorimetric assay (WST-1 assay). The cells (0.2-1×10⁴ cells/well) were cultured in 0.1 ml of RPMI-1640 medium (GIBCO, Invitrogen, Grand Island, NY, USA) supplemented with 10% fetal calf serum, with or without BMP4. After 5 days of culture, a solution of WST-1 and 1-methoxy-5-methylphenazinium methylsulfate (Dojindo Laboratories, Kumamoto, Japan) was added to the cells. The optical density was measured using an enzyme-linked immunosorbent assay reader, to provide an indication of the cell number. The Student's t-test was used to determine the statistical significance of the differences between the controls and the BMP4-treated cells.

Colony assay. The effects of BMP4 on colony formation were examined. Six cell lines that form distinct colonies were used. The cells were plated at 0.2-1×10³ cells/well in 96-well culture plates, in 0.1 ml of RPMI-1640 medium containing 0.8% methylcellulose, with or without BMP4. After 7 days of culture, colonies containing more than 20 cells were counted under an inverted microscope.

Cell morphological analysis. To examine the effects of BMP4 on differentiation, cells cultured with or without BMP4 were observed under an inverted microscope. Cytospin preparations were created from the harvested cells, stained with Wright, and observed under a microscope.

Reverse transcription-polymerase chain reaction (RT-PCR). The expressions of the genes for BMP4 and its receptors were examined using qualitative RT-PCR. Total RNA was extracted, and first-strand cDNA was synthesized. PCR was performed using the primers shown in Table II. The products were electrophorised on an agarose gel and stained with ethidium bromide. The effects of BMP4 stimulation on gene expression were examined with quantitative RT-PCR using a FastStart DNA Master SYBR Green I kit and a LightCycler (Roche Diagnostics, Mannheim, Germany), according to the manufacturer's protocols. We used QuantiTect primers (QIAGEN, Hilden, Germany) for inhibitor of DNA binding 1 (ID1) and ID2 genes, which are down-stream target genes of BMP signaling (9), and LightCycler primer sets for the β-actin gene, used as a control, and for the Notch-related genes.

Immunoblot analysis. In order to confirm that BMP4 treatment mediated signaling transduction in these cell lines, we performed immunoblot analysis to examine the phosphorylation of the SMAD1/5/8 complex. Cells cultured for 24 hours, with or without 50 ng/ml BMP4, were harvested and lysed. The lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and were immunoblotted with antibodies against SMAD1, phosphorylated SMAD1/5/8 (Cell Signaling Technology, Danvers, MA, USA), and α-tubulin (Abcam, Cambridge, MA, USA), used as a loading control.