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J. Biol. Chem., Vol. 262, Issue 1, 234-238, Jan, 1987

Phorbol esters induce interleukin 2 mRNA in sensitive but not in resistant EL4 cells

JR Harrison, KR Lynch and JJ Sando

Phorbol ester sensitive EL4 cells become growth-inhibited and produce interleukin 2 when treated with phorbol-12, 13-dibutyrate. Resistant cells lack both responses. To determine whether the defect in phorbol ester-resistant EL4 cells occurs pre- or post-transcriptionally, a hybridization assay for interleukin 2 mRNA was developed using two synthetic oligonucleotides complementary to mouse interleukin 2 mRNA as probes. Both probes hybridized to a 1-kilobase band in RNA from phorbol ester-treated sensitive cells. This RNA was detectable within 3 h of phorbol ester administration, and accumulation peaked by 12 h. The 1- kilobase band was induced in a concentration-dependent manner by 4-beta- phorbol-12, 13-dibutyrate but not by the inactive analog, 4-alpha- phorbol-12, 13-dibutyrate. No bands hybridizing with the interleukin 2 probe were detected in RNA isolated from unstimulated cells or from phorbol ester-resistant EL4 cells at any time up to 24 h following phorbol ester stimulation. The accumulation of the RNA in sensitive cells was blocked when the protein synthesis inhibitors, cycloheximide (75 microM) or puromycin (90 microM) were added within 1 h of the addition of phorbol ester. If cycloheximide was added 2 or more h after phorbol ester treatment, superinduction of the 1-kilobase band was observed. These results indicate that the failure of phorbol ester- resistant EL4 cells to produce interleukin 2 is due to a defect proximal to interleukin 2 transcription and that the accumulation of interleukin 2 mRNA in phorbol ester-sensitive EL4 cells requires protein synthesis.
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