Promotion of the Self-renewal Capacity of Human Leukemia Cells by Sonic Hedgehog Protein

Materials and Methods

Cells and reagents. Four human AML cell lines were used: OCI/AML1, OCI/AML5 (established at the Ontario Cancer Institute, Canada), HL-60 (supplied by the Japanese Cancer Research Resources Bank), and NB4 (kindly provided by Dr. M. Lanotte) (10); and one B-lymphoma cell line, TMD8, established at our laboratory from cells of a patient with leukemic stage diffuse large B-cell lymphoma (11). OCI/AML1 cell growth is dependent on granulocyte colony-stimulating factor (G-CSF). The growth of OCI/AML5 is dependent on G-CSF or granulocyte-macrophage colony-stimulating factor. The other three cell lines are growth factor-independent. Recombinant mouse Shh was purchased from R & D Systems (Minneapolis, MN, USA).

Short-term growth assay. The effects of Shh on short-term growth were examined using a colorimetric assay (WST-1 assay). Cells (0.2–1×10⁴ cells/well) were cultured in 0.1 ml of 2% fetal calf serum-supplemented RPMI-1640 medium (Gibco, Invitrogen, USA) with increasing concentrations of Shh (0-200 ng/ml). A solution containing WST-1 and 1-methoxy-5-methylphenazinium methylsulfate (Dojindo Laboratories, Japan) was added after 3-5 days. The optical density was measured after incubation using an enzyme-linked immunosorbent assay reader to provide an indication of cell number. Student's t-test was used to determine the statistical significance of differences between controls and treated cells.

Colony assay. The effects of Shh on colony formation by leukemia cells were examined in methylcellulose culture. 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 Shh (200 ng/ml). After 7 days' culture, colonies containing more than 20 cells were counted under an inverted microscope.

Self-renewal capacity. The self-renewal capacity of leukemia cells was evaluated by the recovery of clonogenic cells after suspension culture, as described elsewhere (12). Using 24-well culture plates, 1×10⁵ cells were cultured in 1 ml of RPMI-1640 medium with or without Shh (200 ng/ml). Cells were harvested after 3 or 4 days, and the cell numbers were counted. The harvested cells were then plated on 0.1 ml of methylcellulose-containing RPMI-1640 medium without Shh and colony numbers were counted after 7 days' culture. The plating efficiency following suspension culture (PEs) was evaluated as the mean colony number/number of cells plated. The clonogenic cells recovered (CCR) were calculated by multiplying the number of harvested cells by the PE_s.

Cell morphology. To examine the effects of Shh on differentiation, cells cultured with Shh as above were observed under an inverted microscope. Cytospin preparations were created from harvested cells and stained with Wright-Giemsa.

Reverse transcription-polymerase chain reaction (RT-PCR). The effects of exogenous Shh stimulation on the expression of PTCH1, SMO, and SHH genes were examined using RT-PCR. Cells were cultured with or without 200 ng/ml Shh for 24 hours. Total RNA was extracted and first-strand cDNA was synthesized. PCR was performed using primer sets (Search LC, Germany), according to the manufacturer's protocols. PCR products were electrophoresed on an agarose gel, and stained with ethidium bromide.