Abstract
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 targets.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 179-189 |
| Number of pages | 11 |
| Journal | Gene Expression |
| Volume | 13 |
| Issue number | 3 |
| DOIs | |
| State | Published - 2006 |
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Keywords
- Cycloheximide
- Differentiation
- Early responsive gene
- Microarray
- TPA
ASJC Scopus subject areas
- Genetics
Cite this
An efficient strategy to identify early TPA-responsive genes during differentiation of HL-60 cells. / Hu, Ling Yueh; Tepper, Clifford G; Lo, Su Hao; Lin, Wen Chang.
In: Gene Expression, Vol. 13, No. 3, 2006, p. 179-189.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - An efficient strategy to identify early TPA-responsive genes during differentiation of HL-60 cells
AU - Hu, Ling Yueh
AU - Tepper, Clifford G
AU - Lo, Su Hao
AU - Lin, Wen Chang
PY - 2006
Y1 - 2006
N2 - 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 targets.
AB - 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 targets.
KW - Cycloheximide
KW - Differentiation
KW - Early responsive gene
KW - Microarray
KW - TPA
UR - http://www.scopus.com/inward/record.url?scp=33947375476&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=33947375476&partnerID=8YFLogxK
U2 - 10.3727/000000006783991791
DO - 10.3727/000000006783991791
M3 - Article
C2 - 17193924
AN - SCOPUS:33947375476
VL - 13
SP - 179
EP - 189
JO - Gene Expression
JF - Gene Expression
SN - 1052-2166
IS - 3
ER -