ENHANCED CHEMOSENSITIVITY IN ACUTE MYELOID-LEUKEMIA BY HEMATOPOIETIC GROWTH-FACTORS - A COMPARISON OF THE MTT ASSAY WITH A CLONOGENIC-ASSAY

The MTT assay, a colorimetric assay, is found to be suitable for chemo sensitivity testing. Recently, it has been suggested that hematopoieti c growth factors (HGF) may enhance the effects of cytostatic drugs in acute myeloid leukemia (AML). We therefore studied the effects of gran ulocyte colony-stimulating factor (G-CSF), interleukin 3 (IL-3), and g ranulocyte-macrophage colony-stimulating factor (GM-CSF) combined with cytosine arabinoside (Ara-C), daunorubicin (DNR), mitoxantrone (MXT), or etoposide (VP-1 6) by using the MTT assay. The results were compar ed with in vitro clonogenic assays. Briefly, AML cells of nine patient s were incubated in the presence or absence of G-CSF, IL-3, or GM-CSF under serum-free conditions for 24 hours. Next, for the MTT assay, Ara -C (final dilution range: 0.0024-240 mug/ml), DNR (final dilution rang e: 0.05-3.2 mug/ml), MXT (final dilution range: 0.05-3.2 mug/ml), or V P-16 (final dilution range: 0.1-100 mug/ml) were added and incubated f or 48 hours. Cell survival was determined and IC75 values (75% reducti on as compared to control cultures) were calculated. For clonogenic as says, the three lowest drug concentrations were used. After 48 hours, the clonogenic response was determined in serum-free, semi-solid cultu res with G-CSF, IL-3, or GM-CSF. The results obtained by the MTT assay showed no significant enhancement of cytotoxicity by HGF on cytostati c drug preincubated cells compared to cytostatic drugs alone. The resu lts obtained by the clonogenic assays showed increased cytotoxicity of Ara-C combined with G-CSF, IL-3, or GM-CSF. The median IC75 values of Ara-C decreased from 0.056 to 0.0168 mug/ml with G-CSF (p = 0.01), fr om 0.108 to 0.0168 mug/ml with IL-3 (p = 0.004) and from 0.1 2 to 0.02 04 mug/ml for GM-CSF (p = 0.02). Only moderate enhanced cytotoxicity w as observed when VP-1 6 was combined with IL-3 (p = 0.036) or GM-CSF ( p = 0.036), but not with G-CSF. No enhanced cytotoxicity of DNR and MX T to clonogenic AML cells was found when these agents were combined wi th HGF stimulation. The results indicate that the MTT assay underestim ates HGF enhanced cytotoxicity of Ara-C or VP-16 to clonogenic cells. Therefore, the assay is not useful for accurately detecting difference s of clonogenic response due to the proliferative status of cells. In this paper, the potential explanations for the failure of the MTT assa y are discussed.