Sam68 confers proliferative arrest in the absence of specific RNA binding
To study the effect of overexpression of Sam68 on cell function we attempted to generate NIH 3T3 cell lines stably overexpressing Sam68. However, we were unable to obtain any overexpressor clones, suggesting that Sam68 overexpression suppressed growth. We therefore established "tet-off" NIH 3T3-derived cell lines in which expression of wt or specific-RNA binding-defective Sam68 could be repressively controlled by doxycycline (Fig. 1A). We chose to study a mutant of Sam68 in which Gly178 in the KH domain is substituted by Glu (G178E) as a potential loss of function mutant, rather than deletion mutants, for the following reasons: 1) The G178E substitution mimics a mutation in the C. elegans gld-1 tumor suppressor gene which is sufficient for loss of Gld-1 function and tumor formation [28]. 2) We previously showed that Sam68G178E did not bind in vitro-selected, high affinity Sam68 RNA ligands, but bound to homopolymeric RNA, whereas KH domain deletion mutants did not bind to either ligand [9]. These two observations indicate that loss of high affinity, specific RNA binding is sufficient for loss of protein function. 3) KH domain deletion, but not the G178E substitution, compromises Sam68 self-interaction [8] and results in mislocalization of Sam68 within the nucleus [13, 14], which complicates the interpretation of data obtained with such proteins.
We first assessed the effect of Sam68 overexpression on cell proliferation: The cell lines were cultured at sub-confluence in the presence or absence of doxycycline and adherent cells were counted over 7 days (Fig 1B). The rate of proliferation of the tet-off wt or G178E Sam68-expressing cells was dramatically decreased in the absence of doxycycline. The number of cells overexpressing wt Sam68 actually decreased over 4 d, indicating Sam68-induced cell death. Since wt, but not G178E Sam68, provoked cell death, it was not possible to compare the relative effectiveness of the two proteins in retarding proliferation. Removal of doxycycline had no effect on the rate of proliferation of control cells that had been selected with an empty vector (neo; top panel).
It was previously reported that an RNA binding-defective, apparent, splice variant of Sam68 lacking part of the KH domain was able to compromise G1 phase progression, but that wt Sam68 could not [26]. Our results with wt Sam68 seemed at variance with those studies, so we examined the effect of overexpression of wt Sam68 or specific-RNA binding-defective Sam68G178E on the G1 to S phase transition. Following brief induction, or not, of Sam68 expression, cells were synchronized in mitosis by treatment with nocodazole and then released into G1 by drug washout. DNA synthesis was measured by BrdU incorporation (Fig. 2A). Twenty-four hours after release from mitosis most uninduced cells had incorporated BrdU and were in the subsequent G2/M or G1 phases, as determined by propidium iodide staining (data not shown). Overexpression of wt Sam68 reduced the number of BrdU positive cells from 89% to 24%, indicating G1 delay or arrest. Moreover, most of the BrdU positive cells were still in S phase, suggesting that the preceding G1 phase had been extended.
Overexpression of Sam68G178E also decreased the number of BrdU positive cells; from 87% to 53%. The lesser effect of this mutant may reflect an additional specific-RNA binding-dependent effect on G1 progression. Nonetheless, it is clear that this mutant severely inhibits proliferation and G1 passage. Immunoblotting 8 h after mitotic release showed that expression of wt or mutant Sam68 reduced the levels of cyclins D1 and E (Fig. 2A, top three panels, dox-lanes). [The clone expressing Sam68G178E appeared to express a higher level of cyclin D1 in the uninduced state (Fig. 2A, top panel, dox+ lanes), which may further explain the weaker ability of this mutant to inhibit G1 progression.]
To determine if the G1 cyclins were down-regulated by control of their RNA levels, we measured these levels by RT-PCR (Fig. 2B). Cyclin D1 transcript levels were reduced in cells overexpressing Sam68, most notably at 9 h after mitosis (late G1). However, levels of the related cyclin D2 RNA were unchanged by Sam68 overexpression. The normal increase in Cyclin E RNA level noted 9 h after release from mitosis in uninduced cells was abolished by induction of Sam68. These Sam68-induced changes in cyclin RNA levels are sufficient to explain the observed changes in cyclin protein levels.
To follow the consequences of cyclin D1 and E downregulation we compared the kinetics of the cyclin level changes with the activities of G1 cyclin-dependent kinases and the phosphorylation of Rb following release from mitosis or G0 into G1. Cells were synchronized in mitosis with nocodazole or in G0 by serum withdrawal and released by drug removal or serum replacement, respectively. Cyclin expression levels were determined by immunoblotting (Fig. 3A). In agreement with the RNA level changes (Fig. 2B), cyclin D1 levels were high in mitotic cells and stayed at the same levels in uninduced cells during G1 progression. Also in agreement, cyclin D1 expression was lower in mitotic cells overexpressing Sam68 and was barely detectable 5 and 10 h after release from mitosis. As expected, cyclin D1 levels were low in serum-starved cells and increased after 10 and 15 h of serum treatment of uninduced cells. This increase was reduced in cells overexpressing Sam68. Cyclin E was not detectably expressed in mitotic cells but expression in uninduced cells increased 5 and 10 h after release. This increase was substantially blocked by overexpressed Sam68. Sam68 overexpression had no noticeable effect on cyclin E levels in the serum-treated cells; this may be due to the much lower levels of Sam68 in the serum-depleted cells compared to the cells released from mitosis (Fig. 3A, top panel).
Cdk2 activity was indirectly determined by immunoblotting and examining the downward gel shift that accompanies its activation. Whereas there was a substantial increase in the level of downshifted Cdk2 10 h after release from mitosis in uninduced cells, this was abolished in the induced cells (Fig. 3A). There was also a small but noticeable decrease in Cdk2 activation in late G1 after serum stimulation, in spite of the unchanged cyclin E levels in these cells.
As expected from its effect on cyclin levels, Sam68 overexpression reduced phosphorylation of Rb (determined with an anti-phospho-Rb antibody) in cells treated with serum for 10 or 15 h, and completely abolished it in cells released from mitosis for 10 h (Fig. 3A, bottom panel). To confirm that the reduced Rb phosphorylation was due to decreased G1 Cdk activation, we measured the activities of Cdk2 and Cdk4 in immune complex kinase assays (Fig. 3B). Cyclin E-associated Cdk2 activity increased 6 and 9 h after exiting mitosis, and this was completely blocked by Sam68 overexpression (Fig. 3B, top panel). Immunoblotting of the anti-cyclin E immunoprecipitates with anti-Cdk2 confirmed that only the downshifted, activated form of Cdk2 was co-immunoprecipitated and that this form was greatly diminished in cells overexpressing Sam68 (Fig. 3B, third panel). Overexpression of Sam68 caused a less striking, but reproducible, decrease in Cdk4 activity after release from mitosis (53% activity at 9 h after mitosis in anti-Cdk4 immunoprecipitates from cells overexpressing Sam68 compared to uninduced cells; Fig. 3B, bottom panel). In summary, overexpression of Sam68 arrests or delays progression through G1. This is associated with reduced G1 cyclin levels and Rb phosphorylation and does not require specific RNA binding. The kinetics of cyclin reduction, which precedes S phase, are consistent with this being responsible for the decrease in Rb phosphorylation and consequent G1 arrest.
Sam68 induces apoptosis by a mechanism requiring specific RNA binding
As shown in Fig. 1B, overexpression of wt, but not specific-RNA binding-defective Sam68 caused cell death as well as proliferative arrest. In the absence of doxycycline a substantial number of Sam68 tet-off cells underwent nuclear condensation within 24 h, revealed by DAPI staining, indicative of apoptosis (Fig. 4A). As an alternative, more quantitative measure of apoptosis we used a cell-based fluorescence assay of caspase activation. Induction of Sam68 overexpression induced significant apoptosis (i.e., a large fraction of caspase-positive cells) 72 h after doxycycline removal (Fig. 4B). No caspase positive cells were detectable in cells expressing the RNA binding-defective G178E mutant. The increase in caspase-positive cells was matched by an increase in cells with sub-G1 DNA content, revealed by propidium iodide staining. These results show that wt, but not specific-RNA binding-defective, Sam68 strongly promotes apoptosis.
We were curious to see if any anti-cancer agents were able to enhance Sam68-induced apoptosis. Of the drugs screened, only trichostatin A (TSA) enhanced Sam68-driven apoptosis: In the absence of Sam68 overexpression, TSA (200 nM for 16 h) did not cause apoptosis. However, in cells overexpressing Sam68, TSA treatment increased the fraction of apoptotic cells, as determined by Annexin V-FITC binding, from 33% to 56% (Fig. 5). As expected, Sam68G178E did not induce apoptosis, and this was unaltered by TSA treatment.