Flow cytometric analysis identifies changes in S and M phases as novel cell cycle alterations induced by the splicing inhibitor isoginkgetin

The spliceosome is a large ribonucleoprotein complex that catalyzes the removal of introns from RNA polymerase II-transcribed RNAs. Spliceosome assembly occurs in a stepwise manner through specific intermediates referred to as pre-spliceosome complexes E, A, B, B* and C. It has been reported that small molecule inhibitors of the spliceosome that target the SF3B1 protein component of complex A lead to the accumulation of cells in the G1 and G2/M phases of the cell cycle. Here we performed a comprehensive flow cytometry analysis of the effects of isoginkgetin (IGG), a natural compound that interferes with spliceosome assembly at a later step, complex B formation. We found that IGG slowed cell cycle progression in multiple phases of the cell cycle (G1, S and G2) but not M phase. This pattern was somewhat similar to but distinguishable from changes associated with an SF3B1 inhibitor, pladienolide B (PB). Both drugs led to a significant decrease in nascent DNA synthesis in S phase, indicative of an S phase arrest. However, IGG led to a much more prominent S phase arrest than PB while PB exhibited a more pronounced G1 arrest that decreased the proportion of cells in S phase as well. We also found that both drugs led to a comparable decrease in the proportion of cells in M phase. This work indicates that spliceosome inhibitors affect multiple phases of the cell cycle and that some of these effects vary in an agent-specific manner despite the fact t

A temperature sensitive variant of p53 drives p53-dependent MicroRNA expression without evidence of widespread post-transcriptional gene silencing

The p53 tumour suppressor is a transcription factor that can regulate the expression of numerous ge

Similar effects on cell growth but differences in cell cycle distribution in IGG treated HCT116 and p53KO cells.

<p>HCT116 and p53KO cells were incubated in 15 μM IGG, 30 μM IGG or vehicle control for either 8 or 24 hours. One-parameter flow cytometric analysis of PI stained cells was used to determine cell cycle distributions (G₁, S and G₂/M) based on DNA content, using Modfit 4.1 cell cycle analysis software. (A) Representative histograms are presented for samples collected 24 hours following exposure to 30 μM IGG and its vehicle control. (B and C) The compiled cell cycle results from similar analysis of HCT116 cells collected at 8 (B) and 24 (C) hours following treatment with the indicated concentration of IGG (open symbols) and the corresponding vehicle controls (closed symbols). (D) HCT116 and p53KO (left and right panels, respectively) were incubated in growth medium alone (circles), DMSO (triangles) or IGG (inverted triangles) along with BrdU for the indicated period. The proportion of cells incorporating BrdU at each time point was estimated by flow cytometry. (E and F) p53KO cells were incubated in IGG for either 8 (D) or 24 hours (E) at the indicated concentration (open symbols) or with an equivalent volume of DMSO (closed symbols). Each value in B through F represents the mean (+/- SEM) determined from a minimum of 3 independent experiments. *** indicates that the value is significant different (P<0.001) from controls (DMSO and no drug) by one way ANOVA followed by Tukey multiple comparisons test.</p

Markers of M phase are decreased in response to IGG.

<p>(A) HCT116 cells were incubated in IGG for 24 hours. Protein lysates were collected and analyzed by immunoblot using antibodies raised against the indicated proteins. (B) p53 KO cells were treated as described in (A) except that colcemid, a microtubule-depolymerizing agent, was included in lane 6. HCT116 (C) and p53KO (D) cells were exposed to DMSO, IGG or colcemid for 24 hours and the M phase-specific phosphorylation of histone H3 was detected using a phosphospecific antibody coupled with flow cytometry. ‘M’ denotes the mitotic population. The proportion of phospho-H3 positive cells was determined from 3 independent experiments (E and F). *, ** and *** denote that the values were significantly different (P 0.05, 0.01, 0.001, respectively) by one way ANOVA followed by a Tukey multiple comparisons test. Statistical analysis of colcemid, the positive control, was not included for clarity.</p

The spliceosome inhibitors isoginkgetin and pladienolide B induce ATF3-dependent cell death.

The spliceosome assembles on pre-mRNA in a stepwise manner through five successive pre-spliceosome complexes. The spliceosome functions to remove introns from pre-mRNAs to generate mature mRNAs that encode functional proteins. Many small molecule inhibitors of the spliceosome have been identified and they are cytotoxic. However, little is known about genetic determinants of cell sensitivity. Activating transcription factor 3 (ATF3) is a transcription factor that can stimulate apoptotic cell death in response to a variety of cellular stresses. Here, we used a genetic approach to determine if ATF3 was important in determining the sensitivity of mouse embryonic fibroblasts (MEFs) to two splicing inhibitors: pladienolide B (PB) and isoginkgetin (IGG), that target different pre-spliceosome complexes. Both compounds led to increased ATF3 expression and apoptosis in control MEFs while ATF3 null cells were significantly protected from the cytotoxic effects of these drugs. Similarly, ATF3 was induced in response to IGG and PB in the two human tumour cell lines tested while knockdown of ATF3 protected cells from both drugs. Taken together, ATF3 appears to contribute to the cytotoxicity elicited by these spliceosome inhibitors in both murine and human cells

Effects of PB on S and M phases of the cell cycle.

<p>HCT116 and p53KO cells were exposed to either 0 or 25 nM PB for 24 hours. HCT116 (A) and p53KO cells (B) were BrdU labelled for 1 hour prior to collection. BrdU incorporation was then assessed by two-parameter flow cytometric analysis. (C) The proportion of cells incorporating BrdU was determined and this is expressed relative to untreated control cells. (D) Phospho-H3 was detected as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0191178#pone.0191178.g002" target="_blank">Fig 2</a>. Each value in (C) and (D) represents the mean (+/- SEM) determined from 3 independent experiments. In (C), the mean values for both cell lines at 10 and 25 nM PB were significantly different from 1 by single sample T test (P < 0.05). In (D), ** indicates that the values were significantly different (P<0.01) by Student T test.</p

IGG induces S phase arrest in HCT116 and p53KO cells.

<p>Two-parameter flow cytometry analysis of BrdU incorporation and DNA content was performed following a 24-hour exposure of HCT116 (A) and p53 KO (B) cells to 30 μM IGG. (C) The proportion of BrdU positive cells was determined and expressed relative to untreated controls. (D) Relative replication is an estimate of the efficiency of DNA replication in the BrdU positive population of cells. (E) Cell cycle distribution of no drug- (ND), vehicle control- (DMSO) and 30 IGG-treated cells was estimated from dot plots like those presented in A and B. (F) HCT116 cells were exposed to either DMSO for 48 hours (DMSO) or IGG for 24 hours (dashed line) followed by either an additional 24 hours in IGG or fresh medium with DMSO (REV). Data obtained from multiple experiments were expressed as the mean percentage of BrdU positive cells. Each value in C-F represents the mean (+/- SEM) determined from 4, 4, 6 and 3 independent experiments, respectively. In C and D, ** indicates that the values were significantly different (P < 0.01) by two way ANOVA followed by Bonferroni post hoc tests. In E and F, * and ** denote that the value was significantly different (P < 0.05 or P< 0.01) by one-way ANOVA followed by Tukey multiple comparisons test.</p

The p53 protein induces stable miRNAs that have the potential to modify subsequent p53 responses

The p53 tumour suppressor is a transcription factor that can increase the expression of mRNAs and microRNAs (miRNAs). HT29-tsp53 cells expressing a temperature sensitive variant of p53 have provided a useful model to rapidly and reversibly control p53 activity. In this model, the majority of p53-responsive mRNAs were upregulated rapidly but they were short-lived leading to rapid decay of the p53 response at the restrictive temperature. Here we used oligonucleotide microarrays and reverse transcriptase PCR to show that p53-induced miRNAs exhibited a distinct temporal pattern of expression. Whereas p53-induced miRNAs like miR-143-3p, miR-145-5p, miR-34a-5p and miR-139-5p increased as fast as mRNAs, they were extremely stable persisting long after p53 induced mRNAs and even their corresponding primary miRNAs had decayed to baseline levels. Three p53-induced mRNAs (MDM2, BTG2 and CDKN1A) are experimentally verified targets of one or more of these specific miRNAs so we hypothesized that the sustained expression of p53-induced miRNAs could be explained by a post-transcriptional feedback loop. Activation of consecutive p53 responses separated by a period of recovery led to the selective attenuation of a subset of p53 regulated mRNAs corresponding to those targeted by one or more of the p53-responsive miRNAs. Our results indicate that the long term expression of p53 responsive miRNAs leads to an excess of miRNAs during the second response and this likely prevents the induction of MDM2, BTG2 and CDKN1A mRNA and/or protein. These observations are likely to have important implications for daily cancer therapies that activate p53 in normal tissues and/or tumour cells

The expression of CDK4 and CDK6 at the permissive temperature.

<p>(A) Total RNA was isolated from control (open symbols) and HT29tsp53 (closed symbols) cells at the indicated time and the expression of a variety of mRNAs was determined. Expression in each sample was normalized to the expression of the same transcript in samples collected immediately before the temperature shift. (Each point represents the mean (± SEM) determined from a minimum of 3 independent experiments. (B) Signal intensity of individual probesets from microarray analysis at the restrictive temperature (X axis) is compared to the signal intensity at the permissive temperature (y axis). The mean fold change (± SEM and n) for each transcript is inset in the corresponding panel. (C) Immunoblot analysis of the indicated protein derived from whole cell lysates collected at the indicated time at the permissive temperature. Similar results were obtained in 3 independent experiments. In A, B and C, CDKN1A (p21 ^<b>WAF</b>) and/or MDM2 serve as positive controls for the activation of the p53 transcriptional response.</p

The p53-dependent induction of miRNAs.

<p>(A) Small RNAs were collected from HT29-tsp53 cells following incubation for 16 hours at the permissive temperature. RNA samples obtained from 2 independent experiments were labelled and hybridized to oligonucleotide microarrays. Individual miRNAs meeting our statistical cut off (P ≤ 0.01) are presented. Values reflect fold change in expression at the permissive compared to the restrictive temperature. The miRNAs are arranged in order of expression from most highly induced on the left to least well-induced on the right. Open bars denote passenger strand miRNAs while solid bars represent guide strand miRNAs. ‘*’ indicates that the increased expression of the miRNA was confirmed by qRT-PCR. The ‘X’ denotes the only miRNA that we could not confirm by qRT-PCR. (B) qRT-PCR was performed using similar samples derived from vector control (HT29-neo) and HT29-tsp53 cells. Each value in (B) represents the mean (± SEM) determined from a minimum of 3 independent experiments.</p