Abstract
The clinical phenotype of RPS19-mutated and non-mutated DBA is indistinguishable, suggesting a common function for all DBA genes. The recent identification of mutations affecting other ribosomal protein genes in DBA provides powerful support for this common function being ribosomal biogenesis. However, the molecular basis for the characteristic steroid responsive red cell aplasia of DBA remains elusive. Using a 2-phase culture system, in which the phases of erythropoietin dependence are separated, we have demonstrated a consistent and profound deficiency in EPO/SCF-induced in vitro expansion of peripheral blood erythroid progenitor cells in DBA. To further localise the defect in DBA we have studied early cell cycle, proliferation and transcriptional events of enriched erythroid cells on transfer to phase II. Phase II cultures were established in 10μM BrdU for bivariate analysis of BrdU uptake and cell cycle status by FACS. Propidium iodide staining showed a reduction of cells in G0/G1 from 67.09±4.5% at t=0 to 58.44±3.8% at 24hrs post EPO in control cultures (n=6). Cumulative BrdU uptake after 4 hours of exposure to EPO showed 51.5±8.5% of cells to have entered S phase in control (n=4) and 57.5±3.5% in DBA (n=2). Erythroid progenitors at the end of phase I in DBA are thus responsive to the EPO-induced transition to phase II, with apparently normal kinetics of entry into the first cell cycle, consistent with the previously observed similarity in phase II colony numbers in control and DBA cultures. EPO-dose dependence was not apparent in the first cycle, with BrdU uptake similar at limiting (0.2U/ml) and saturating (2U/ml) EPO (65.4 vs 66.1%) at 9hrs post EPO, again consistent with observation of colony numbers. However, by 72hrs post EPO, the proportion of cells in S phase was lower in DBA cultures (n=3) than controls (n=6) (31.16±1.99% vs 38.4±1.05%; p<0.05), with proportionately more in G0/G1 (59.88±3.6 vs 50.78±2.4%). MTS assay showed a detectable onset of proliferation from day 3 in both control and DBA cultures, with a lower proliferation rate in DBA. In cultures supplemented with 10−7 M dexamethasone, there was a higher proliferation rate in both control and DBA cultures. RNA extracted from harvested pre- and at 24 hours post-EPO exposure was subjected to one round of linear amplification and labelled with Cy3/Cy5 for hybridisation to an in-house microarray comprising 395 genes. There was little change in the transcriptional profile at 24 hrs post-EPO in comparison with pre-EPO exposure, or between control and DBA cultures. The time course of expression of selected genes was studied by quantitative RT-PCR at 0–96hrs following EPO exposure. In both control and DBA cultures, there was no change in the level of expression of the selected genes at 24hrs, consistent with microarray data. An increase in expression of α-globin mRNA was first apparent at 48hrs post-EPO, observed in both DBA and control cultures. Erythroid cells at the end of the pre-EPO phase I are thus predicted to be quiescent until exposure to EPO on transfer to phase II, with EPO acting in an initial permissive role, enabling cells to re-enter cycle, followed by synchronous EPO-dose dependent proliferation coincident with active globin synthesis, when the erythroid defect in DBA becomes apparent. We propose that the 2-phase culture thus imposes significant translational stress, analogous to starvation/refeeding in hepatocytes, exposing a proliferation/cell cycle defect to explain our observation of a consistent severe in vitro erythroid defect in DBA irrespective of clinical severity.
Transcription of the histone H5 gene is not S-phase regulated.