©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Interleukin-3 mRNA Stabilization by a trans-Acting Mechanism in Autocrine Tumors Lacking Interleukin-3 Gene Rearrangements (*)

(Received for publication, April 11, 1995; and in revised form, June 12, 1995)

Hans H. Hirsch (§) Asha P. K. Nair Verena Backenstoss Christoph Moroni

From the Institute for Medical Microbiology of the University of Basel, Petersplatz 10, CH-4003 Basel, Switzerland

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

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 (t > 3 h), whereas rapid decay of IL3 transcripts (t < 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.


INTRODUCTION

Escape from proliferation control is a central feature of tumorigenesis. In autocrine tumors, growth autonomy is accomplished by the unregulated production of self-stimulating mitogens(1, 2) . Where identified in experimental or clinical hemopoietic malignancies, aberrant growth factor expression mostly involved rearrangements of the respective genes. Thus, a t (5;14) chromosomal translocation in a human B-cell leukemia placed the IL3 gene in the vicinity of the immunoglobulin heavy chain enhancer(3, 4) . In murine malignancies, activation of IL3, (^1)GM-CSF, CSF-1, interleukin-5, and interleukin-6 (IL6) by insertion of retroviral elements has been described(5, 6, 7, 8, 9, 10, 11, 12) . In most of these cis-acting alterations, transcriptional activation of the growth factor locus has either been shown or implicated. Given the importance of post-transcriptional regulation for cytokine expression (13-15, for reviews see (16, 17, 18, 19) ), it is conceivable that perturbance of post-transcriptional control mechanisms might also play a role in oncogenesis. Indeed, constitutive growth factor expression associated with stable transcripts has been described in leukemic cell lines(20, 21, 22) . Consistent with the role of the AU-rich elements (ARE) as mRNA instability determinants(13, 23) , the stabilizing alterations involved truncations of ARE from the 3`-UTR of the respective growth factor transcripts by insertion of endogenous retroviral elements(20, 21) . On the other hand, stabilization of the GM-CSF mRNA by a trans-acting mechanism has been described in a c-myc-transduced monocytic tumor line. In this tumor line, heterologous transcripts fused to the 3`-UTR of GM-CSF were stable, whereas the 3`-UTRs of the protooncogenes c-fos and c-myc were still effective to direct rapid transcript degradation(22) .

We are characterizing a murine tumor model in which v-Ha-ras-transduced IL3-dependent PB-3c cells progress in vivo to two different classes of tumors with autocrine IL3 production(11, 24) . Class I tumor lines lack detectable rearrangements of the IL3 gene. Nuclear transcription run-on data showed that IL3 mRNA expression in class I tumor lines did not involve a transcriptional mechanism. The mechanism appeared to be recessive because down-regulation of autocrine IL3 expression, reversion to IL3 dependence, and inhibition of tumor cell growth was observed following somatic cell fusion to IL3-dependent PB-3c cells(11, 25) . More recently, we found that autocrine proliferation of class I tumor cells could also be inhibited by treatment with the immunosuppressant cyclosporin-A, which appeared to act by promoting the degradation of tumor-expressed IL3 transcripts (26) . In contrast, autocrine class II tumor lines are characterized by IL3 gene rearrangements due to the insertion of endogenous retroviral elements (intracisternal A-particles), enhanced IL3 transcription rates, and maintained IL3 expression in somatic cell hybrids(11) . We now provide evidence that class I and class II tumor lines differ in the post-transcriptional regulation of IL3 mRNA. Autocrine IL3 expression in class I tumor lines involves IL3 transcript stabilization by a trans-acting mechanism that is not operative in autocrine class II tumor lines, or in the IL3-dependent precursor cells.


MATERIALS AND METHODS

Cells and Tissue Culture

PB-3c is a cloned IL3-dependent mast cell line derived from murine DBA/2 bone marrow(27) . The IL3 autocrine tumor lines V2D1, 15V4T2, V4D6, and R56VT have been described previously(11, 24) . 15V4 and R56V were obtained by v-Ha-ras transduction of the IL3-dependent PB-3c clone 15 and R56, respectively(11, 28) . The lines are cultured in Iscove's modified Dulbecco's medium supplemented with 10% fetal calf serum (Life Technologies, Inc.), 100 units/ml penicillin, 100 mg/liter steptomycin, and 50 µM of 2-mercaptoethanol and is referred to as IMDM/10% FCS. For the culture of IL3-dependent lines, conditioned medium from the X63-mIL3 line (29) was added in saturating amounts, typically 1% (referred to as IMDM/10% FCS/IL3).

IL3 Reporter Constructs and Electroporation

In the Mx-IL3 construct(30) , the 0.6-kilobase pair HindIII/SacI fragment of the Moloney murine leukemia virus long terminal repeat enhancer drives the 2.5-kilobase pair ApaI/SpeI fragment of the mouse genomic IL3 gene (31) starting 25 nucleotides upstream of the TATA box and ending 256 bp downstream of the polyadenylation site. This plasmid construct contains also the hygromycin-B phosphotransferase gene hph(32) under the control of the SV40 regulatory regions to allow selection of stable transfectants and serves as control for a stable mRNA. In the Mx-(DeltaAU)IL3, the 216-bp NcoI/StyI fragment in the 3`-UTR of IL3 was deleted (see Fig. 2)(30) . In the AUIL3, AUFOS, and AUMYC derivatives of Mx-IL3, the NcoI/StyI fragment was replaced with the respective sequences indicated in Fig. 2as follows. First the annealed sense and antisense oligonucleotides were phosphorylated, cloned into the SmaI site of KS-Bluescript vector and sequenced, then cut out as NcoI/StyI fragment, ligated into the corresponding sites of the 568-bp BglII fragment of IL3 present in the pSP72 vector, and finally inserted into the Mx-IL3 gene as BglII fragment. The Mx-(AU6)IL3 and the Mx-(AU3)IL3 were obtained by polymerase chain reaction using the sense primers M820 5`-CCCATGGCTATTTATTTATGTATTTATGT-3` or M821 5`-CCCATGGTGTATTTATTTATTTATTGCC-3`, respectively, with the antisense primer M822 5`-GATACATGTTGCATGCTGTGT-3` on Mx-AUIL3 plasmid DNA as template. The amplicon was cut with NcoI and SphI and cloned into the correspondingly cut BglII-fragment of IL3 (in pSP72), which was inserted into the IL3 gene. Sequence and orientation was verified in all final constructs by dideoxysequencing (U. S. Biochemical Corp.).


Figure 2: Summary of the transfection experiments with the IL3 reporter constructs and the respective mRNA half-life. For constitutive expression, the Moloney murine leukemia virus long terminal repeat (MoMuLVLTR) enhancer is set in front of the TATA box of the genomic IL3 reporter gene, which is marked by two silent point mutations (circle with cross-hairs) in exon 3. Expression of the transfected Mx-IL3 reporter gene is monitored by RNase A/T1 protection using the exon 1-5 probe, which yields two fragments of 209 and 156 nt, whereas a fragment of 368 nt is protected by the endogenous IL3 transcript. Deletion or replacement of the 220-bp NcoI/StyI inserts in the 3`-UTR with the indicated fragments is described under ``Materials and Methods.''



For electroporation using the gene pulser (Bio-Rad), 1 10^7 of the indicated cell lines were incubated in IMDM/10% FCS for 2 h before washing twice with ice-cold phosphate-buffered saline. After resuspension in 0.8 ml of phosphate-buffered saline, the cells were mixed with 15 µg of the plasmid (linearized with Asp) and incubated for 10 min on ice in the electroporation cuvette (0.4-µm electrode, Bio-Rad). After pulsing with 300 V and 960 microfarad, the cells were again put on ice for 10 min and then resuspended in prewarmed IMDM/10% FCS/IL3. After 48 h, selection of stable transfectants was started by adding 1 mg/ml of hygromycin-B (Calbiochem) to culture medium.

RNA Extraction and Northern Blotting

Cells were resuspended in fresh prewarmed IMDM/10% FCS at 1 10^6 cells/ml for 2 h. Total cytoplasmic RNA was extracted according to Gough (33) either directly (time point, 0 min) or at the times indicated after addition of 5 mg/liter Act-D. Poly(A)-enriched RNA was obtained as described previously(11) . RNA was subjected to electrophoretic separation in 1.1% (w/v) agarose gel containing 0.66 M formaldehyde in MOPS buffer, pH 5.9, according to Thomas(34) , blotted onto nitrocellulose filters (BA-S85, Schleicher & Schuell). Prehybrization, hybridization, and washing were done as described previously(11) . The IL3 exon 1-5 probe was generated from a SP6 vector containing a 368-bp IL3 cDNA fragment kindly provided by N. Gough using an in vitro transcription kit (Boehringer Mannheim). The chicken beta-actin probe (570-bp PstI fragment), a gift from Y. Nagamine, was labeled using a random priming kit (Boehringer Mannheim). The c-myc probe was generated by random priming from a 2.8-kilobase pair fragment (35) spanning from exon 2 to exon 3. The IL6 cDNA EcoRI fragment, a gift of J. Van Snick, was labeled by random priming.

RNase A/T1 Protection Assay

The RNase A/T1 protection assay was done as described previously (30) hybridizing 5 or 10 µg of total RNA for 16 h with an excess of labeled antisense probe. Antisense RNA probes labeled with [alpha-P]GTP were synthesized from SP6 promoters using the SP6 transcription kit (Boehringer Mannheim), and full-length transcripts were purified by polyacrylamide gel electrophoresis. The exon 1-5 probe was prepared as indicated above. The exon 1 template contained the genomic 274-bp ApaI/Nae1 fragment cut with ApaI, the exon 5 template the 627-bp XbaI/Spe1 fragment cut with XbaI, both of which are derived from the 8.5-kilobase pair EcoRI genomic IL3 fragment (31) and cloned into the pGEM3Z vector. The template for hygromycin resistance gene hph mRNA (26) was a 96-bp EcoRI/PstI fragment in the pGEM vector cut with EcoRI.

Quantitation of mRNA Levels

For quantitation, the activity of the specific mRNA hybridization signals for IL3, c-myc, hph, and beta-actin were determined after exposure to a storage phosphor screen and analysis in a PhosphorImager by the Image Quant program (Molecular Dynamics, Sunnyvale, CA). For decay measurements, the time point 0 min values of the beta-actin- or hph-normalized IL3, c-myc, and IL6 signals were taken as 100%, and the averaged values of two independent experiments were plotted versus the time of Act-D exposure.

IL3 Production in Culture Supernatants

The cells were seeded at 2 10^5/ml in IMDM/10% FCS after three rounds of washing and incubated for 24 h. The culture supernatants were collected following centrifugation and passed through 0.45-µm filters (Acrodisc). Supernatant dilutions in IMDM/10% FCS were done in triplicate in microtiter plates. After 24 h of incubation, mitogenic activity was tested by [^3H]thymidine incorporation of IL3-dependent PB-3c cells (5 10^4 cells/100 µl) during 6 h. The mitogenic activity was inhibitable by the addition of the anti-IL3 antibody from the rat anti-mouse IL3 hybridoma ATCC HB10652 ( (36) and data not shown). Comparison of the 24-h IL3 production of cell lines after 1 h of actinomycin-D exposure was done as described in the legend to Fig. 5.


Figure 5: IL3 production of Mx-IL3-transfected tumor lines with or without Act-D treatment. Cells were treated as outlined in the flow chart, and the mitogenic activity in the diluted supernatants was assayed by [^3H]thymidine incorporation of IL3-dependent PB-3c cells as described under ``Materials and Methods.'' The effect of Act-D treatment on IL3 production is indicated in percentages on top of the bars. A, class I V2D1 Mx-IL3, class II R56VT Mx-IL3, and untransfected V2D1; B, class I V2D1 Mx-(DeltaAU)IL3 and class II R56VT Mx-(DeltaAU)IL3.




RESULTS

IL3 Transcripts Are Stable in Class I Tumor Lines but Not in Class II Tumor Lines

We have previously reported that autocrine IL3 expression in tumors derived from v-Ha-ras-expressing PB-3c cells involved two different mechanisms: transcriptional activation by insertion of an intracisternal A-particle in class II tumor lines and an unknown post-transcriptional mechanism in class I tumor lines that lack IL3 gene rearrangements(11) . To further characterize class I and class II tumors, we have now compared the rates of IL3 mRNA decay in both tumor classes after inhibition of transcription with Act-D. The analysis of mRNA decay was limited to 3 h throughout this study to limit the side effects of Act-D. As shown by Northern blotting (Fig. 1, top), IL3 mRNA was stable in the class I tumor line V2D1 over the 3 h of Act-D treatment, whereas IL3 mRNA levels decreased in the class II tumor line V4D6 over the same time period. Unlike the IL3 transcripts, IL6 and c-myc transcript levels decreased in both tumor classes ( Fig. 1and data not shown). Rehybridization for beta-actin provided the reference of a stable mRNA (Fig. 1, top). Analysis of two other tumor lines 15V4T2 (class I) and R56VT (class II) showed a corresponding pattern for each class (data not shown). The specific mRNA signals were quantitated by storage phosphor screens, normalized to beta-actin expression, and plotted versus the time of Act-D treatment (Fig. 1, bottom). As reported for other transcripts(17, 37) , a biphasic decay pattern of the normalized IL3 levels was observed in class II tumor lines. After 1 h of Act-D treatment, IL3 mRNA decay leveled off at around 30% of the time point 0 min value. For the initial phase, a half-life of about 0.5 h was determined for IL3 mRNA in the class II tumor lines V4D6 and R56VT. In contrast, IL3 mRNA was stable (t > 3 h) in the class I tumor lines V2D1 and 15V4T2. In all tumor lines, c-myc transcripts showed rapid decay rates with a half-life of less than 0.6 h. IL6 mRNA decay was not impaired in the class I tumor V2D1 decaying as rapidly as in the class II tumor line V4D6 (t < 0.5 h) (Fig. 1, bottom, and data not shown). We conclude from these data that autocrine IL3 expression in the class I tumors V2D1 and 15V4T2 involves a post-transcriptional alteration at the level of mRNA stability that is not found in the class II tumor lines V4D6 and R56VT.


Figure 1: IL3 mRNA levels in IL3 autocrine tumor lines after Act-D treatment. Poly(A) RNA was prepared from total cytoplasmic RNA extracted before or at the indicated times after Act-D addition from the indicated tumor lines and analyzed by Northern blotting with the indicated cDNA probes (top) as described under ``Materials and Methods.'' The signals were quantitated, normalized to beta-actin levels, and plotted versus the time of Act-D treatment (bottom) taking the time 0 min value as 100%. , IL3 class I V2D1; up triangle, filled, IL3 class II V4D6; bullet, IL6 class II V4D6; , IL6 class I V2D1; , c-myc class I V2D1; black square, c-myc class II V4D6.



No Evidence for Altered IL3 Transcripts in Class I Tumor Cells

The rapid degradation of short-lived transcripts has been shown to require specific cis-acting nucleotide sequences that function as instability determinants(13, 17) . With respect to IL3, the deletion of the entire ARE or specific point mutations in AUUUA motifs have been shown to abolish rapid IL3 mRNA degradation and result in stable transcripts(30) . We therefore analyzed the IL3 transcripts for alterations by RNase A/T1 protection assay using three probes that were chosen to cover the entire IL3 transcript from the 5`-UTR to the polyadenylation site in the 3`-UTR. In the class I tumor lines V2D1 and 15V4T2, the protected fragments corresponded to the sizes of 192, 368, and 359 nt as expected for unaltered transcripts (data not shown). The absence of point mutations in the ARE was confirmed directly by dideoxy sequencing of the IL3 cDNA obtained by reverse transcriptase polymerase chain reaction from the class I tumor line V2D1 (data not shown). Thus, cis-acting alterations were not very likely to account for the stability of IL3 transcripts found in the class I tumor lines V2D1 and 15V4T2.

Evidence for IL3 mRNA Stabilization by a trans-Acting Mechanism in Class I Tumor Cells

To identify differences in the trans-acting regulation of IL3 mRNA stability, an exogenous IL3 reporter gene termed Mx-IL3 (30) was transfected into both tumor classes. For high constitutive expression of the Mx-IL3, the genomic IL3 transcription unit was driven by the Moloney leukemia virus long terminal repeat enhancer (depicted in the upper part of Fig. 2). Due to two silent point mutations in exon 3 (indicated as focus in Fig. 2), Mx-IL3 expression levels could be distinguished as two bands of 206 and 156 nt from the endogenous transcript of 368 nt using the exon 1-5 probe in an RNase A/T1 protection assay (Fig. 3A). The hph probe for the hygromycin-B resistance gene was included as loading control. As shown in the Act-D experiment (Fig. 3A), Mx-IL3 specific transcripts decayed rapidly in the transfected class II tumor line R56VT. In contrast, Mx-IL3 mRNA levels were stable in the class I tumor line V2D1 (Fig. 3A). By quantitation of the Mx-IL3 mRNA expression levels and normalization to the hph loading control, the half-life of the Mx-IL3 mRNA was calculated by linear regression to be around 0.5 h in the class II tumor line R56VT but greater than 3 h in the class I tumor V2D1 (Fig. 3C). The data indicate that IL3 mRNA stabilization in class I tumor cells involves a trans-acting mechanism that counters the ARE instability function.


Figure 3: Mx-IL3 and Mx-(DeltaAU)IL3 decay in tumor cells after Act-D treatment. Total RNA was extracted from the indicated cell lines before or at the indicated times after Act-D treatment and analyzed by RNase A/T1 protection assay using the exon 1-5 and the hph probe as described under ``Materials and Methods.'' The expression levels of the IL3 reporter transcripts were quantitated by storage phosphor technique, and the values plotted after normalization to the respective hph signal. To plot the mRNA levels remaining, the normalized time point 0 min values were taken as 100%. A, class I V2D1 Mx-IL3 (left) and class II R56VT Mx-IL3 (right). B, class I V2D1 Mx-(DeltaAU)IL3 (left) and class II R56VT Mx-(DeltaAU)IL3 (right). C, decay of IL3 reporter transcripts. , Mx-(AU)IL3 class I V2D1; black square, Mx-(AU)IL3 class II R56VT; , Mx-IL3 class I V2D1; up triangle, filled, Mx-IL3 class II R56VT. D, relative expression levels of IL3 reporter transcripts at 0 min. Thick hatching lines, Mx-IL3; thin hatching lines, Mx-(AU)IL3.



ARE Deletion Increases the Steady-state Levels of the IL3 Transcripts in Both Tumor Classes

Because ARE deletion rendered IL3 reporter transcripts stable in untransformed PB-3c cells(30) , it seemed possible that the trans-acting stabilization in class I tumor cells represented a defect in factor(s) recognizing the ARE instability determinant. If that was the case, ARE deletion from the reporter transcripts would be predicted to remain without effect in class I tumor cells but should abrogate the differences to the class II tumor cells. We therefore transfected Mx-(DeltaAU)IL3 reporter constructs into both tumor classes (see Fig. 2). As expected, the (DeltaAU)IL3 transcripts were now stable in the class II tumor cells (Fig. 3C). Little difference in the stability of the Mx-IL3 and the Mx-(DeltaAU)IL3 transcripts were detected in the class I tumor line V2D1 over the 3 h of the Act-D experiment. In both tumor classes, however, the (DeltaAU)IL3 reporter transcripts were present at higher levels relative to the hph reference transcripts or the endogenous IL3 transcripts (Fig. 3B). Previous studies in PB-3c cells have shown that the steady-state levels of the Mx-IL3 mRNA correlate with increasing transcript stability(30) . To estimate the steady-state levels, we determined the ratio of the exogenous IL3 signal (lower band of 156 nt) to the hph signal at time point 0 min (Fig. 3D). The results show that the Mx-IL3 levels are about 5-fold higher in the class I tumor cells than in the class II tumor cells. The levels of the reporter transcripts lacking the ARE were found to be 4-fold higher in class I tumor cells but 20-fold higher in class II tumor cells, thereby reaching nearly equal levels (Fig. 3D). Thus, IL3 mRNA accumulation could be increased by ARE deletion not only in the class II but also in the class I tumor cells. The data suggest that the trans-acting mechanism operating in class I tumor cells is not equivalent to ARE deletion.

IL3 mRNA Stabilization Is Linked to Tumor Progression

We also compared Mx-IL3 transfectants of the v-Ha-ras expressing PB-3c clone 15V4 with the clonal class I tumor derivative 15V4T2 (Fig. 4). The results showed that the exogenous Mx-IL3 transcripts are stable in the class I tumor line 15V4T2 but decay rapidly in the v-Ha-ras-expressing precursor line 15V4 Mx-IL3. Due to the weak expression levels, the endogenous transcript in 15V4T2 is not apparent on this exposure. Rapid decay was also observed in the v-Ha-ras-transduced precursor clone R56V as well as in the untransformed PB-3c cells (data not shown). The comparison between precursor and tumor lines suggests that the stabilization of IL3 transcripts is linked to the autocrine class I tumor stage.


Figure 4: Mx-IL3 mRNA decay in the v-Ha-ras-expressing precursor 15V4 (left) and the clonal tumor derivative 15V4T2 (right). Total RNA was extracted from the indicated cell lines before or after the indicated times of Act-D treatment and analyzed by RNase A/T1 protection assay using the exon 1-5 and the hph probe as described under ``Materials and Methods.''



IL3 Production Correlates with Transcript Stability in Class I Tumor Cells

As evident from Fig. 3A, a substantial difference in Mx-IL3 mRNA levels of the transfected class I and class II tumor lines could be observed after 1 h of Act-D treatment. To investigate whether differences of IL3 mRNA stability in tumor cells would be translated into corresponding amounts of protein, we compared the 24 h IL3 production of Mx-IL3 transfected tumor cell lines with or without 1 h of preincubation with Act-D (Fig. 5A). Because Act-D inhibits transcription by intercalation of double-stranded DNA, its effect is essentially irreversible. Before seeding the cells in IL3-free medium, unbound Act-D was removed from the cell cultures by washing. The cell number was determined before and after each protocol, confirming that both lines had doubled only when not exposed to Act-D. As shown for two dilutions of the culture supernatants, the class I V2D1 Mx-IL3 cells secreted about 4-fold more IL3 than the class II R56VT Mx-IL3 cells. After Act-D exposure, the average 24-h production of IL3 is reduced to about 17% in the class I tumor supernatants but to 4% in the class II tumor supernatants (Fig. 5A). Comparison with the untransfected tumor line V2D1 indicates that the contribution of the endogenous IL3 is negligible at these dilutions. Similar results were obtained examining the 5 h production, albeit at a lower level (data not shown). Thus, the abundance of transcripts without and with Act-D treatment (Fig. 3) correlates with the amount of IL3 produced. These results support the notion that the increased stability of IL3 transcripts in class I tumor cells is a relevant mechanism for the autocrine IL3 loop.

Deletion of the ARE Increases IL3 Production of Class I Tumor Cells

When class I and class II tumor lines transfected with the ARE-deleted IL3 construct Mx-(DeltaAU)IL3 were analyzed, both tumor classes produced nearly equal amounts but at a 30-fold higher level (Fig. 5B). Pretreatment with Act-D lowered the IL3 production of the transfected class I and class II tumor cells to about 50 and 30%, respectively. Thus, the deletion of the ARE instability determinant from the IL3 transcripts had not only abolished the difference between the two tumor classes with respect to the overall IL3 production but also reduced the effect of transcriptional inhibition by Act-D on the IL3 yield as expected for stable transcripts.

The AU-rich Elements of c-fos and c-myc Are Not Sufficient to Mediate Rapid Decay of IL3 Transcripts

The insertion of ARE instability determinants derived from the 3`UTR of GM-CSF, c-fos, or c-myc has been shown to mediate destabilization of a heterologous stable transcripts(13, 17, 22, 37, 38, 39) . We thought of applying a similar approach to the characterization of class I alteration by targeting the stable Mx-(DeltaAU)IL3 reporter transcripts to the c-myc or the c-fos degradation pathway. Therefore, we inserted the heterologous ARE of c-myc or c-fos as 78-nt fragments in place of the autologous IL3 ARE (Fig. 2). The validity of this approach was tested in PB-3c cells by analyzing the decay of IL3(DeltaAU) transcripts with reinserted autologous ARE fragments of 57, 39, or 19 nt (Fig. 2). Rapid decay of transcripts with a half-life of less than 30 min was observed for Mx-AUIL3 (data not shown) or Mx-(AU6)IL3 containing six AUUUA motifs (Fig. 6). For Mx-(AU3)IL3 containing only the three downstream AUUUA-motifs of the ARE, transcript decay was slowed down showing a half-life of approximately 85 min as revealed by normalization to hph mRNA levels (Fig. 6B). In contrast, reinsertion of ARE derived from c-fos or c-myc resulted in stable transcripts with a half-life of more than 3 h (Fig. 6, A and B). The lack of rapid degradation upon reinsertion of the c-fos and c-myc ARE into the IL3 transcript was confirmed in the class II tumor line R56VT (data not shown). Thus, this approach did not allow characterization of the specificity of the class I alteration. However, the data emphasize the role of the clustered AUUUA motifs for IL3 transcript degradation in PB-3c cells and indicate that the degradation pathways for c-myc or c-fos are not efficiently targeted in PB-3c cells by the respective AU elements alone in the context of a constitutively expressed IL3 transcript.


Figure 6: Decay of IL3 reporter transcripts with replaced ARE in the untransformed PB-3c cell line. A, RNase A/T1 protection assay. Total RNA was extracted from the indicated cells before or at the indicated times after actinomycin-D treatment and analyzed by RNase A/T1 protection assay using the exon 1-5 probe and the hph probe as described under ``Materials and Methods.'' B, reporter transcript decay. IL3 mRNA levels and hph mRNA levels were quantitated and plotted as described in the legend of Fig. 3. black square, PB-3c Mx-(AUMYC)IL3; , PB-3c Mx-(AUFOS)IL3; up triangle, filled, PB-3c Mx-(AU3)IL3; , PB-3c Mx-(AU6)IL3.




DISCUSSION

The present report shows that autocrine IL3 expression in class I tumor cells involves stabilization of IL3 transcripts that is not found in class II tumor cells. The mechanism appears to be active in trans because IL3 transcripts are stabilized despite the presence of intact ARE instability determinants. This conclusion is based on (i) the prolonged metabolic stability of IL3 mRNA in class I tumor cells compared to class II tumor cells (Fig. 1); (ii) the absence of cis-acting alterations in RNase protection and ARE sequence analysis (data not shown); and (iii) the stability of a Moloney murine leukemia virus-driven exogenous IL3 transcript in class I V2D1 tumor cells (t > 3 h) but not in the class II tumor R56VT (t of leq 0.6 h) (Fig. 3, summarized in Fig. 2). After 1 h of Act-D exposure, the abundance of IL3 mRNA signals differed considerably. To assess whether or not the IL3 transcripts stabilized in class I tumor cells represent functional mRNA, we have determined the mitogenic IL3 activity in 24 h culture supernatants of class I and class II Mx-IL3 transfectants with or without 1 h of Act-D pretreatment (Fig. 5). The results show that the Mx-IL3 transfected class I V2D1 cells produce 4-fold higher and following Act-D exposure about 16-fold higher mitogenic IL3 activity than the Mx-IL3 transfected class II tumor line. The differences between the tumor classes with respect to IL3 mRNA stability and IL3 production disappeared upon deletion of the ARE from the transfected IL3 reporter gene ( Fig. 3and Fig. 5). The data support the view that ARE-mediated transcript decay is at least partially inactivated in class I tumor cells and suggest that IL3 mRNA stabilization is of relevance to the autocrine IL3 loop in class I tumor cells. In contrast, an increased rate in IL3 gene transcription as found in class II tumor lines (11) or as constructed experimentally in Mx-IL3 transfected PB-3c (30) appears to be sufficient to counterbalance rapid transcript degradation in order to maintain steady-state expression levels required for a tumorigenic autocrine loop. Thus, post-transcriptional (class I) or transcriptional up-regulation (class II) appear to occur as alternative mechanisms in the autocrine tumor formation of v-Ha-ras expressing PB-3c mast cells.

Increased stability of growth factor transcripts as an oncogenic event has been discussed in a detailed study by Schuler and Cole on the c-myc-transduced monocytic tumor line 2.3(22, 40) . Akin to IL3 in class I tumor lines, constitutive GM-CSF expression in the 2.3 tumor line involved increased stability of the transcript (t of 2.4 h) in the absence of detectable gene alterations by Southern blot. The hypothesis of a trans-acting alteration was supported by the observation that transcripts of a transfected neomycin cDNA fused to the 3`-UTR of GM-CSF were stable in this 2.3 tumor but not in two other cell lines. In contrast, reporter transcript destabilization mediated by the entire 3`-UTR of the protooncogenes c-fos or c-myc was still functional. These experiments clearly indicated that GM-CSF mRNA and protooncogene decay were differently regulated in the 2.3 tumor line and could be put into effect by the respective 3`-UTR in cis.

Our present study on the v-Ha-ras-dependent tumor formation of PB-3c mast cells independently confirms the hitherto unique observation by Schuler and Cole (22) that growth factor mRNA stabilization in trans may be an oncogenic target in autocrine tumor formation. Furthermore, with the precursor cells at hand, we observed that IL3 mRNA stabilization did not occur in the untransformed PB-3c cells nor in the v-Ha-ras-expressing tumorigenic precursor cells 15V4 and R56V but in the class I tumor lines 15V4T2 and V2D1 ( Fig. 3and 4 and data not shown). Thus, we suggest that IL3 mRNA stabilization is a property acquired as a late step in the PB-3c tumor model.

No significant differences between class I and class II tumor lines could be detected for the decay rates of the short-lived c-myc and IL6 transcripts showing half-lives of less than 0.6 h (Fig. 1). Although c-myc mRNA degradation is known to be mediated by more than one instability determinant(17, 22) , these data support the view that class I tumor cells are not defective in a more general mechanism of cytoplasmic mRNA turnover.

To evaluate the specificity of IL3 mRNA stabilization in class I tumor lines, we have tried to target the stable IL3(DeltaAU) transcripts to the c-fos or the c-myc degradation pathway in PB-3c cells by inserting the respective ARE (Fig. 6). In contrast to the rapid degradation conferrable onto stable reporter transcripts like beta-globin in fibroblasts(17) , we found that IL3 reporter transcripts containing the c-fos or the c-myc ARE were already rather stable in the untransformed PB-3c (summarized in Fig. 2). However, rapid degradation of IL3 reporter transcripts in PB-3c could be restored by the IL3 ARE of 57 or 39 nt containing six AUUUA motifs (Fig. 6). A short sequence of 19 nt containing three AUUUA motifs showed an intermediate IL3 decay rate with a half-life of around 85 min. This indicates that despite some similarity, the AREs of IL3, c-fos, and c-myc are not functionally equivalent in the context of the IL3 transcripts and the cell system used. Inspection of the inserted ARE suggests that the structure and/or number of three AUUUA motifs present in a cluster might be critical (Fig. 2), as suggested by a detailed mutational analysis of AUUUA-motifs in the ARE in IL3(30) . A recent study on the ARE of GM-CSF indicated that rapid degradation (t < 1 h) may in fact require repeated copies of a slightly larger UUAUUUA(U/A)(U/A) determinant(23, 41) .

At present, the molecular basis of the IL3 mRNA stabilization in class I tumor lines is not known. Our recent observation of IL3 transcript destabilization and the reversion to IL3 dependence of class I tumor by treatment with the immunosuppressant cyclosporin-A suggests involvement of an immunophilin-targeted step(26) . Given the role of the six AUUUA repeats as necessary cis-elements for IL3 mRNA decay (Fig. 6) (30) and the loss of autocrine IL3 expression in somatic cell hybrids(11, 25) , the simplest model would be that trans-acting factor(s) involved in recognition of the ARE instability determinant are inactive in class I tumor cells. This model is challenged by the increase of IL3 mRNA and IL3 protein expressed in class I tumor cells when the ARE is altered or deleted from IL3 reporter transcripts (Fig. 3D and 5B and data not shown). One explanation for this might be that ARE-mediated degradation of IL3 transcripts in class I tumor cells is not completely inactive. On the other hand, IL3 mRNA stabilization in class I tumors might be mediated by sequences other than the ARE, as discussed for GM-CSF mRNA stabilization in a lung cancer line(42) . In addition, the deletion of the ARE instability determinant might contribute to IL3 production by removing other restrictions of gene expression, for example at the level of translation(19, 43, 44, 45) . These intriguing possibilities will have to be resolved in further studies.


FOOTNOTES

*
This work was supported by Grant 31-40816.94 of the Schweizerische Nationalfonds zur Förderung der Wissenschaftlichen Forschung. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed.

(^1)
The abbreviations used are: IL, interleukin; GM, granulocyte macrophage; CSF, colony-stimulating factor; ARE, AU-rich element(s); UTR, untranslated region; IMDM, Iscove's modified Dulbecco's medium; FCS, fetal calf serum; bp, base pair; Act-D, actinomycin-D; MOPS, 4-morpholinepropanesulfonic acid; nt, nucleotide(s).


ACKNOWLEDGEMENTS

We thank our colleagues Drs. J. Garcia-Sanz, S. Hahn, and Y. Nagamine for helpful discussions.


REFERENCES

  1. Sporn, M. B., and Roberts, A. B. (1985) Nature 313,745-747 [Medline] [Order article via Infotrieve]
  2. Lang, R. A., and Burgess, A. W. (1990) Immunol. Today 11,244-249 [CrossRef][Medline] [Order article via Infotrieve]
  3. Meeker, T., Hardy, D., Willman, C., Hogan, T., and Abrams, J. (1990) Blood 76,285-289 [Abstract]
  4. Grimaldi, J. C., and Meeker, T. (1989) Blood 73,2081-2085 [Abstract]
  5. Dührsen, U., Stahl, J., and Gough, N. M. (1990) EMBO J. 9,1087-1096 [Abstract]
  6. Stocking, C., Löliger, C., Kawai, M., Suciu, S., Gough, N., and Ostertag, W. (1988) Cell 53,869-879 [Medline] [Order article via Infotrieve]
  7. Ymer, S., Tucker, W. Q. J., Sanderson, C. J., Hapel, A., Campbell, H. D., and Young, I. (1985) Nature 317,255-258 [Medline] [Order article via Infotrieve]
  8. Tohyama, K., Lee, K. H., Tashiro, K., Kinashi, T., and Honjo, T. (1990) EMBO J. 9,1823-1830 [Abstract]
  9. Blankenstein, T., Qin, Z., Li, W., and Diamantstein, T. (1990) J. Exp. Med. 171,965-970 [Abstract]
  10. Leslie, K., Lee, F., and Schrader, J. W. (1991) Mol. Cell. Biol. 11,5562-5570 [Medline] [Order article via Infotrieve]
  11. Hirsch, H. H., Nair, A. P. K., and Moroni, C. (1993) J. Exp. Med. 178,403-411 [Abstract]
  12. Baumbach, W. R., Stanley, R., and Cole, M. D. (1987) Mol. Cell. Biol. 7,664-671 [Medline] [Order article via Infotrieve]
  13. Shaw, G., and Kamen, R. (1986) Cell 46,659-667 [Medline] [Order article via Infotrieve]
  14. Wodnar-Filipowicz, A., and Moroni, C. (1990) Proc. Natl. Acad. Sci. U. S. A. 87,777-781 [Abstract]
  15. Lindsten, T., June, C. H., Ledbetter, J. A., Stella, G., and Thompson, G. B. (1989) Science 244,339-343 [Medline] [Order article via Infotrieve]
  16. Carter, B. Z., and Malter, J. S. (1991) Lab. Invest. 65,610-621 [Medline] [Order article via Infotrieve]
  17. Greenberg, M. E., and Belasco, J. G. (1993) in Control of Messenger RNA Stability (Belasco, J., and Brawerman, G., eds) pp. 199-218, Academic Press, San Diego, CA
  18. Hentze, M. W. (1991) Biochim. Biophys. Acta 1090,281-292 [Medline] [Order article via Infotrieve]
  19. Sachs, A. B. (1993) Cell 74,413-421 [Medline] [Order article via Infotrieve]
  20. Algate, P. A., and McCubrey, J. A. (1993) Oncogene 8,1221-1232 [Medline] [Order article via Infotrieve]
  21. Henics, T., Sanfridson, A., Hamilton, B. J., Nagy, E., and Rigby, W. F. C. (1994) J. Biol. Chem. 269,5377-5383 [Abstract/Free Full Text]
  22. Schuler, G. D., and Cole, M. D. (1988) Cell 55,1115-1122 [Medline] [Order article via Infotrieve]
  23. Caput, D., Beutler, B., Hartog, K., Thayer, R., Brown-Shimer, S., and Cerami, A. (1986) Proc. Natl. Acad. Sci. U. S. A. 83,1670-1674 [Abstract]
  24. Nair, A. P. K., Diamantis, I. D., Conscience, J. -F., Kindler, V., Hofer, P., and Moroni, C. (1989) Mol. Cell. Biol. 9,1183-1190 [Medline] [Order article via Infotrieve]
  25. Diamantis, I. D., Nair, A. P. K., Hirsch, H. H., and Moroni, C. (1989) Proc. Natl. Acad. Sci. U. S. A. 86,9299-9302 [Abstract]
  26. Nair, A. P. K., Hahn, S., Banholzer, R., Hirsch, H. H., and Moroni, C. (1994) Nature 369,239-242 [CrossRef][Medline] [Order article via Infotrieve]
  27. Ball, P. E., Conroy, M. C., Heusser, C., Davis, J. M., and Conscience, J. F. (1983) Differentiation 24,74-78 [Medline] [Order article via Infotrieve]
  28. Nair, A. P. K., Hirsch, H. H., and Moroni, C. (1992) Oncogene 7,1963-1972 [Medline] [Order article via Infotrieve]
  29. Karasuyama, H., and Melchers, F. (1988) Eur. J. Immunol. 18,97-104 [Medline] [Order article via Infotrieve]
  30. Stoecklin, G., Hahn, S., and Moroni, C. (1994) J. Biol. Chem. 269,28591-28597 [Abstract/Free Full Text]
  31. Campbell, H. D., Ymer, S., Fung, M., and Young, I. G. (1985) Eur. J. Biochem. 150,297-304 [Abstract]
  32. Blochlinger, K., and Diggelmann, H. (1984) Mol. Cell. Biol. 4,2929-2931 [Medline] [Order article via Infotrieve]
  33. Gough, N. M. (1988) Anal. Biochem. 173,93-95 [Medline] [Order article via Infotrieve]
  34. Thomas, P. (1980) Proc. Natl. Acad. Sci. U. S. A. 77,5201-5205 [Abstract]
  35. Stanton, L. W., Watt, R., and Marcu, K. (1983) Nature 303,401-406 [Medline] [Order article via Infotrieve]
  36. Abrams, J. S., and Pearce, M. K. (1988) J. Immunol. 140,131-137 [Abstract/Free Full Text]
  37. Shyu, A.-B., Belasco, J. G., and Greenberg, M. E. (1991) Genes & Dev. 5,221-231
  38. Kabnick, K. S., and Housman, D. E. (1988) Mol. Cell. Biol. 8,3244-3250 [Medline] [Order article via Infotrieve]
  39. Wilson, T., and Treismann, R. (1988) Nature 336,396-399 [CrossRef][Medline] [Order article via Infotrieve]
  40. Baumbach, W. R., Keath, E. J., and Cole, M. D. (1986) J. Virol. 59,276-283 [Medline] [Order article via Infotrieve]
  41. Lagnado, C. A., Brown, C. Y., and Goodall, G. J. (1994) Mol. Cell. Biol. 14,7984-7995 [Abstract]
  42. Ross, H. J., Sato, N., Ueyama, Y., and Koeffler, H. P. (1991) Blood 77,1787-1795 [Abstract]
  43. Kruys, V., Marinx, O., Shaw, G., Deschamps, J., and Huez, G. (1989) Science 245,852-855 [Medline] [Order article via Infotrieve]
  44. Han, J., Brown, T., and Beutler, B. (1990) J. Exp. Med. 171,465-475 [Abstract]
  45. Grafi, G., Sela, I., and Galili, G. (1993) Mol. Cell. Biol. 13,3487-3493 [Abstract]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.