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First published online July 12, 2005
doi: 10.1242/10.1242/jcs.02448

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Roles of heterogeneous nuclear ribonucleoproteins A and B in cell proliferation

¹ Department of Biochemistry and Molecular Biology, University of Queensland, St Lucia Campus, QLD 4072, Australia
² Epithelial Pathobiology Group, Centre for Immunology and Cancer Research, University of Queensland, Woolloongabba, QLD 4102, Australia

View larger version (80K): [in a new window]   Fig. 1. Expression of main hnRNP A/B proteins at different cell cycle stages. (A) Colo16 and HaCaT cells were synchronized by double thymidine block and DNA contents were analysed by flow cytometry to determine when S, G2, M and G1 phases occur. (B) Specificities of antibodies against hnRNPs A1, A2, A3 and B1 were confirmed by western blotting. Total proteins isolated from HaCaT cells were separated on SDS/12% polyacrylamide gels, blotted onto PVDF membrane, probed with antibodies against hnRNP A1, A2, A3, or B1 and detected with ECL-Plus. (C) The synchronized cells were collected at different times after the release of second thymidine block with the sampling times indicated at the top of lanes. For Colo16 cells, S, G2, M and G1 phase started at 0, 7, 8 and 10 hours after synchronization, respectively, whereas HaCaT cells entered G2, M and G1 phases 1 hour later. Levels of hnRNP A/B proteins were determined by western blotting following the same protocol as above. The membranes were then stripped, and re-probed with a mouse anti-GAPDH antibody to ensure a same loading ratio of samples. Band intensities were measured by densitometry to assess the relative abundance of the target protein against GAPDH, which was indicated under the lanes. The experiments were performed in duplicate.

View larger version (72K): [in a new window]   Fig. 2. Distribution of hnRNPs A2 and B1 in Colo16 cells at different cell cycle stages. Cells collected at S, G2, M and G1 phases were immunostained with a rabbit hnRNP A2 antibody, which also recognizes the B1 isoform, and visualized using a FITC-conjugated donkey secondary antibody. A monoclonal -tubulin antibody raised in mouse was used to counterstain microtubules with a Cy3-conjugated goat secondary antibody (red in merged images). DAPI was used to stain DNA (blue). Bar, 10 µm.

View larger version (49K): [in a new window]   Fig. 3. Real-time PCR revealed stable levels of hnRNP A/B mRNAs during the cell cycle processes. Cells at S, G2, M and G1 phases were collected at 0, 7, 8 and 10 hours after synchronization for Colo16 cells, and 0, 8, 9 and 11 hours for HaCaT cells. The relative abundance of hnRNP A1, A2, B1 and A3 mRNAs was determined using ß-actin as a control. The means±s.e.m. were calculated from six replicates. Data were processed by ANOVA.

View larger version (89K): [in a new window]   Fig. 4. Expression of hnRNP A1, A2, B1 and A3 in different cells. Western analysis of total protein extracts with rabbit polyclonal antibodies raised against the A1, A2, B1 and A3 peptides, using a mouse monoclonal GAPDH antibody as a loading control. The keratinocytes were isolated from normal foreskin. Colo16 and SCC25 are squamous cancer cell lines originated from tumours on foreleg and tongue, respectively. HeLa is a cervical cancer cell line and A549 was cloned from lung cancer. MCF10a is an immortalized non-tumorigenic breast cell line, and MCF7 and T-47D are two breast cancer cell lines. RPMEI is an immortalized prostate cell line and LNCAP was cloned from human prostate cancer.

View larger version (34K): [in a new window]   Fig. 5. hnRNP A/B mRNA levels in different cells. Real-time PCR was carried out to quantify the amount of A1, A2, B1 and A3 mRNAs in the selected cell lines using ß-actin as a control. The relative abundance of individual mRNA, shown above the columns, was calculated as the ratio between the targeted and ß-actin mRNAs. Means±s.e.m. of three experiments, each with two replicates, were calculated for each type of cell. Col, Colo16; HeL, HeLa; Ker, keratinocytes.

View larger version (80K): [in a new window]   Fig. 6. shRNA suppression of hnRNPs A1, A2 and A3 in Colo16 cells. Colo16 cells were seeded in six-well plates (360,000 cells/well) and transfected 20 hours later. Control samples were treated with lipofectamine only. 72 hours after transfection, cells were collected and lysed. Total proteins were then separated on SDS-PAGE, transferred onto PVDF membrane and probed with antibodies against A1, A2 or A3 using GAPDH as a loading control.

View larger version (41K): [in a new window]   Fig. 7. Impact of shRNAs against hnRNPs A1, A2 and A3 on cell growth. Colo16 cells were seeded into six-well plates (360,000 cells/well) 20 hours before transfection. The number of cells in each well was counted 72 hours after transfection. Values are means±s.e.m. of eight replicates. Significance differences **P<0.05 and ***P<0.001 were found using Student's t-test compared to counts in cells transfected with empty vector.

View larger version (23K): [in a new window]   Fig. 8. Suppression of hnRNP A2 alone slows cell growth. Colo16 cells were collected 48 hours after transfection, counted and re-plated into 96-well plates (1500 cells/well, six replicates for each treatment). From 3 hours after re-plating, cells were sampled at 6-hour intervals to perform MTT proliferation assays. The absorbance at 570 nm was then logarithmically plotted against the time after replating. The experiment was repeated three times., lipofectamine;, pS;, pS-Luc; x, pS-A2.

View larger version (52K): [in a new window]   Fig. 9. Effect of A2 suppression by RNAi on the expression of p53 and BRCA1. (A) Western blotting for p53 after treating Colo16 cells with pS, pS-Luc and pS-A2. Total protein isolated from cells collected 72 hours post-transfection was separated on SDS/12% polyacrylamide gel, transferred onto PVDF and probed with a monoclonal mouse antibody against p53. The band intensities of p53 and hnRNP A2 shown in the graph were normalized to that of GAPDH. The p53 data are the means of four replicates. (B) Western blot analysis of BRCA1 after treating Colo16 with pS, pS-Luc and pS-A2. Total proteins isolated from cells harvested 72 hours post-transfection were separated on SDS/5% polyacrylamide gel, transferred onto PVDF and probed with a monoclonal mouse anti-BRCA1 antibody. Levels of BRCA1 protein, averaged from three replicates were calculated as the ratio of BRCA1 to GAPDH protein. A significant difference (*P<0.05) in protein levels in the pS-A2 transformed cells was observed compared to levels in control cells. (C) Northern blot analysis was carried out to determine the amount of BRCA1 mRNA in Colo16 cells collected 72 hours after transfection with pS, pS-Luc or pS-A2. The results were quantified by densitometry and the relative amount of BRCA1 was calculated using ß-actin as a control. Results are the means±s.e.m. of four experiments. A significant difference (**P<0.05) in BRCA1 level was observed in pS-A2 transfected cells compared to levels in control cells.

View larger version (30K): [in a new window]   Fig. 10. Effect of A2 suppression by RNAi on the expression of p21. (A) Gene expression of p21 in Colo16 cells was determined by northern blotting using cells harvested 72 hours after transfection to analyse the effect of A2 suppression on p21. A densitometric analysis was performed to calculate the relative abundance of p21 mRNA. Results are the means±s.e.m. of three replicates. A significant difference (*P<0.01) in p21 mRNA level was observed in pS-A2 transfected cells compared to levels in control cells. (B) Total RNAs isolated from cells collected 72 hours after transfection were reverse-transcribed, and the abundance of hnRNP A2 and p21 mRNAs were determined by real-time PCR. The relative abundance, the mean (±s.e.m.) of three replicates, was calculated as the ratio between A2 or p21 mRNA and ß-actin. A significant difference (**P<0.05) in relative abundance of p21 mRNA was observed in pS-A2 transfected cells compared with levels in control cells. (C) Western blot analysis of p21 after suppressing hnRNP A2. Total proteins isolated from cells harvested 72 hours post-transfection were separated on SDS/12% polyacrylamide gel, transferred onto PVDF and probed with a monoclonal mouse anti-p21 antibody. The mean levels (±s.e.m.) of p21 shown in the graph were taken from duplicate experiments.

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