Lymphoblastoid cell lines from Diamond Blackfan anaemia patients exhibit a full ribosomal stress phenotype that is rescued by gene therapy

Abstract

Diamond Blackfan anaemia (DBA) is a congenital bone marrow failure syndrome characterised by selective red cell hypoplasia. DBA is most often due to heterozygous mutations in ribosomal protein (RP) genes that lead to defects in ribosome biogenesis and function and result in ribosomal stress and p53 activation. The molecular mechanisms underlying this pathology are still poorly understood and studies on patient erythroid cells are hampered by their paucity. Here we report that RP-mutated lymphoblastoid cell lines (LCLs) established from DBA patients show defective rRNA processing and ribosomal stress features such as reduced proliferation, decreased protein synthesis, and activation of p53 and its target p21. These phenotypic alterations were corrected by gene complementation. Our data indicate that DBA LCLs could be a useful model for molecular and pharmacological investigations.

Figure 1

Characterisation of the pathological phenotype of LCLs from DBA patients. (A) Proliferation curve obtained by cell count. Growth rate in DBA LCLs was significantly lower than in controls after three days (p = 0.0043). Blue: controls, red: DBA patients. (B) Representative cell cycles of control and DBA cells; cells were fixed, stained with propidium iodide and subjected to flow cytometry. M1: subG1; M2: G0/G1; M3: S/G2/M. (C) Box plots reporting the mean percentages of cells in subG1 and S/G2/M phases. DBA patients show a higher proportion of cells in subG1 and a lower proportion of cells in S/G2/M phase, compared to controls. Ten control and 11 DBA LCLs were examined. Horizontal line represents median. **p value ≤ 0.01, ***p value ≤ 0.001. (D) General protein synthesis is decreased in DBA cells, as assessed by incorporation of [³⁵S]methionine/cysteine. Horizontal line represents median. *p value ≤ 0.05. (E) Western blot shows an increase in the levels of p53, p21, MDM2 and phosphoAMPK (Thr172), whereas the level of total AMPK is unchanged. (F) Densitometric analysis on western blot data from three separate experiments. Horizontal line represents median. (G) Expression of p21 transcript, measured by qRT-PCR, is increased in DBA patients. Horizontal line represents median.

Figure 2

The phenotype due to RPS19 mutations can be rescued by the expression of a RPS19 transgene. (A) Northern blot: normal maturation of rRNA is restored in RPS19 mutated patients after expression of an exogenous RPS19 cDNA. The probe labels 18S precursors. (B) Proliferation curve: the expression of RPS19 cDNA rescues the growth defect in RPS19-mutated patients. (C) Representative cell cycles in S19–2 LCL after transduction with LV-RPS19: percentage of subG1 cells was decreased and percentage of S/G2/M cells was increased. LV:LV-RPS19. (D) Protein synthesis, measured by incorporation of [³⁵S]methionine/cysteine, is improved in patient cells after transduction with LV-RPS19. (E) Western blot shows a normalisation in the levels of p53, p21, MDM2 and phospho-AMPK. (F) Densitometric data calculated on three western blot experiments. (G) Level of p21 transcript in control and RPS19-mutated LCLs: transduction with LV-RPS19 reduced p21 in DBA patients.

Figure 3

A RPL5 transgene ameliorates the pathological phenotype of a RPL5-mutated LCL. (A) Deficient rRNA maturation in RPL5-mutated cells is partially rescued by transduction with LV-RPL5. Normal 28S/18S ratio ranges between 1.9 and 2.2 in our experimental model. RPL5-mutated cells have a reduced 28S/18S ratio due to deficient 28S maturation, together with a prominent 32S pre-RNA peak. Expression of RPL5 after transduction with LV-RPL5 can improve the production of mature 28S rRNA. (B) Protein synthesis, measured by incorporation of [³⁵S]methionine/cysteine, is improved after transduction with LV-RPL5 in patient cells, but decreased in control cells. LV:LV-RPL5. (C) p53 level is slightly decreased in RPL5-mutated cells after RPL5 gene transfer, but increased in control cells. LV:LV-RPL5. Original uncropped blots are shown in Supplementary Information. (D) Quantitative RT-PCR showing expression of p21:transduction with LV-RPL5 increases p21 expression in control cells, and slightly decreases p21 level in RPL5-mutated cells. LV:LV-RPL5.

Figure 4

Scheme depicting the interplay among p53, MDM2 and RPs. In normal cells, MDM2 targets p53 for degradation by the proteasome. In DBA cells, the deficiency of a RP perturbs ribosome assembly and causes the accumulation of ribosome-free RPs, such as RPL5 and RPL11, that bind MDM2 and inhibit its activity on p53. Once stabilised, p53 can activate its downstream effectors.