An efficient strategy to identify early TPA-responsive genes during differentiation of HL-60 cells.
We have adopted a special experimental strategy to identify early responsive genes during 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced macrophage-like differentiation of human myeloid leukemia cells (HL-60). This was performed in cells that were synchronized by nocodazole and treated with TPA in the presence of a protein synthesis inhibitor, cycloheximide, to prevent activation of secondary targets and therefore increase the probability of early transcripts in total RNA pool. The expression alteration was analyzed by microarray and the selection criteria of candidate genes were adjusted by real-time PCR validation to increase its reliability. Finally, 56 genes were identified as early TPA-responsive genes in this multiscreening step approach. Furthermore, upregulation of three candidate genes (NFIL3, SKIL, and JMJD3) was shown to be dosage and time dependent with TPA treatment and was found to be directly regulated by TPA through PKC-dependent signaling. These results revealed that our screenings provide a useful and efficient approach to identify early TPA-responsive genes and these genes might involve the regulation of TPA-induced differentiation program of HL-60 cells as primary
Hu
Ling-Yueh
LY
Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan, ROC.
Tepper
Clifford G
CG
Lo
Su-Hao
SH
Lin
Wen-Chang
WC
D000970:Antineoplastic Agents / Q000494:pharmacology; D014408:Biomarkers, Tumor / Q000235:genetics / Q000378:metabolism*; D002273:Carcinogens / Q000494:pharmacology*; D002454:Cell Differentiation / Q000187:drug effects*; D003513:Cycloheximide / Q000494:pharmacology; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D018922:HL-60 Cells / Q000187:drug effects; D006801:Humans; D008264:Macrophages; D015739:Nocodazole / Q000494:pharmacology; D020411:Oligonucleotide Array Sequence Analysis; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
D000970:Antineoplastic Agents; D014408:Biomarkers, Tumor; D002273:Carcinogens; D011500:Protein Synthesis Inhibitors; D003513:Cycloheximide; D013755:Tetradecanoylphorbol Acetate; D015739:Nocodazole