Interleukin-3 mRNA Stabilization by a trans-Acting Mechanism in Autocrine Tumors Lacking Interleukin-3 Gene Rearrangements

Abstract

Tumors obtained from v-Ha-ras-transformed PB-3c cells are characterized by autocrine interleukin-3 (IL3) expression, which occurs either without (class I tumors) or with enhanced transcription (class II tumors). To address possible post-transcriptional mechanisms of IL3 expression, IL3 mRNA stability was examined in both tumor classes. Increased stability of IL3 mRNA was detected in class I tumor lines (t1/2 > 3 h), whereas rapid decay of IL3 transcripts (t1/2 < 0.5 h) was found in class II tumor lines. In both tumor classes, the c-myc and interleukin-6 transcripts were short-lived. Transcripts of a constitutively expressed IL3 reporter gene were stable in class I tumor cells but unstable in class II tumor cells, suggesting that IL3 mRNA stabilization involved a trans-acting mechanism. Rapid decay of IL3 reporter transcripts was observed in untransformed PB-3c as well as in v-Ha-ras expressing precursor cells linking transcript stabilization to the tumor stage. Reporter transcript stabilization in class I tumor cells correlated with increased IL3 production. Deletion of the AU-rich element from the IL3 reporter gene further augmented IL3 mRNA levels as well as IL3 production, suggesting that the stabilizing mechanism in class I tumor cells is not equivalent to AU-rich element deletion.

A novel mechanism of tumor suppression by destabilizing AU-rich growth factor mRNA

“…In addition, mutation of an ARE in cis, or loss of a trans-acting decay effector may lead to oncogenic mRNA stabilization, and evidence for both mechanisms has been provided in experimental hemopoietic tumor systems by us and others (Schuler and Cole, 1988;Algate and McCubrey, 1993;Nair et al, 1994;Hirsch et al, 1995). In the tumor model used for this study, mRNA stabilization occurs in the IL-3-dependent mast cell line PB-3c upon transformation with the v-H-ras oncogene.…”

“…We chose the prototypic tumor line V2D1 characteristic for expressing abnormally stable IL-3 mRNA (Nair et al, 1994;Hirsch et al, 1995), introduced TTP wt and TTP C139R by electroporation, and selected four subclones each ( Figure 3a, lanes 2-5 and 6-9). Northern blots performed after 4 weeks ( Figure 3b) indicated that IL-3 mRNA levels were reduced in the TTP wt clones (lanes 2-5) compared to the TTP C139R clones (lanes 6-9).…”

“…Following in vivo inoculation, however, IL-3-secreting autocrine tumors emerge after a long latency . Interestingly, many thus generated tumors lines are characterized by abnormally stable IL-3 transcripts (Nair et al, 1994;Hirsch et al, 1995). The presumed oncogenic mutation acts in trans, since ARE-containing reporter transcripts were also stable in the tumor, but labile in the precursor cells.…”

Search for oncogenic regulators in an autocrine tumor model using differential display PCR: Identification of novel candidate genes including the calcium channel mtrp6

“…In the majority of the tumors (class I tumors), the autocrine IL-3 loop is activated by a recessive step and involves posttranscriptional stabilization of the normally short-lived IL-3 mRNA. This stabilization results from a defect in an unknown transacting factor, as rapid decay can be restored by cell fusion with precursor cell lines Hirsch et al, 1995). Accordingly, somatic cell hybrids are IL-3 dependent, mRNA decay is rapid and tumorigenicity is suppressed.…”

“…In order to create a situation with minimal genetic variability, we ®rst recloned PB-3c and V2D1, and, for control, performed a cell fusion experiment ( Figure 1a). We then ensured that the selected clones PB-3c-20, V2D1-2 and the newly generated somatic cell hybrid PB-3c-20/ V2D1-2 full®lled the criteria of the PB-3c tumor model prototypes: exogenous IL-3 dependency in the precursor line, autocrine IL-3 production in the tumor line and suppression of autocrine IL-3 production by cell fusion Hirsch et al, 1995). The data presented in Figure 1b show that these criteria were met by the selected subclones.…”