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

Convalescent plasma in patients admitted to hospital with COVID-19 (RECOVERY): a randomised controlled, open-label, platform trial

SummaryBackground Azithromycin has been proposed as a treatment for COVID-19 on the basis of its immunomodulatoryactions. We aimed to evaluate the safety and efficacy of azithromycin in patients admitted to hospital with COVID-19.Methods In this randomised, controlled, open-label, adaptive platform trial (Randomised Evaluation of COVID-19Therapy [RECOVERY]), several possible treatments were compared with usual care in patients admitted to hospitalwith COVID-19 in the UK. The trial is underway at 176 hospitals in the UK. Eligible and consenting patients wererandomly allocated to either usual standard of care alone or usual standard of care plus azithromycin 500 mg once perday by mouth or intravenously for 10 days or until discharge (or allocation to one of the other RECOVERY treatmentgroups). Patients were assigned via web-based simple (unstratified) randomisation with allocation concealment andwere twice as likely to be randomly assigned to usual care than to any of the active treatment groups. Participants andlocal study staff were not masked to the allocated treatment, but all others involved in the trial were masked to theoutcome data during the trial. The primary outcome was 28-day all-cause mortality, assessed in the intention-to-treatpopulation. The trial is registered with ISRCTN, 50189673, and ClinicalTrials.gov, NCT04381936.Findings Between April 7 and Nov 27, 2020, of 16 442 patients enrolled in the RECOVERY trial, 9433 (57%) wereeligible and 7763 were included in the assessment of azithromycin. The mean age of these study participants was65·3 years (SD 15·7) and approximately a third were women (2944 [38%] of 7763). 2582 patients were randomlyallocated to receive azithromycin and 5181 patients were randomly allocated to usual care alone. Overall,561 (22%) patients allocated to azithromycin and 1162 (22%) patients allocated to usual care died within 28 days(rate ratio 0·97, 95% CI 0·87–1·07; p=0·50). No significant difference was seen in duration of hospital stay (median10 days [IQR 5 to >28] vs 11 days [5 to >28]) or the proportion of patients discharged from hospital alive within 28 days(rate ratio 1·04, 95% CI 0·98–1·10; p=0·19). Among those not on invasive mechanical ventilation at baseline, nosignificant difference was seen in the proportion meeting the composite endpoint of invasive mechanical ventilationor death (risk ratio 0·95, 95% CI 0·87–1·03; p=0·24).Interpretation In patients admitted to hospital with COVID-19, azithromycin did not improve survival or otherprespecified clinical outcomes. Azithromycin use in patients admitted to hospital with COVID-19 should be restrictedto patients in whom there is a clear antimicrobial indication

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

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

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

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