(Received for publication, April 11, 1995; and in revised form, June 12, 1995)
From the
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.
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, ()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.
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
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.
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 [H]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-(
AU)IL3 and
class II R56VT Mx-(
AU)IL3.
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 -actin levels, and
plotted versus the time of Act-D treatment (bottom)
taking the time 0 min value as 100%.
, IL3 class I V2D1;
,
IL3 class II V4D6;
, IL6 class II V4D6;
, IL6 class I V2D1;
, c-myc class I V2D1;
, c-myc class II
V4D6.
Figure 3:
Mx-IL3 and Mx-(AU)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-(
AU)IL3 (left) and class II R56VT Mx-(
AU)IL3 (right). C, decay of IL3 reporter transcripts.
, Mx-(
AU)IL3 class I V2D1;
, Mx-(
AU)IL3 class
II R56VT;
, Mx-IL3 class I V2D1;
, 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.
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.''
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. , PB-3c Mx-(AUMYC)IL3;
,
PB-3c Mx-(AUFOS)IL3;
, PB-3c Mx-(AU3)IL3;
, PB-3c
Mx-(AU6)IL3.
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 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(AU) 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
-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.