Dimethylsulfoxide (DMSO), hexamethylene-bis-acetamide (HMBA), benzidine dihydrocloride, vanadyl ribonucleotide complexes (VRC) and proteinase K was purchased from Sigma (St. Louis, MO, USA). [γ-³²P]-dCTP (111 Tbq/mmol) was obtained from Izotop, Institute of Isotopes Co., Ltd., Budapest, Hungary, whereas the DNA ³²P-labeling system kit was purchased from Invitrogen (Red Prime DNA Labeling System). The rabbit anti-RPS5 polyclonal antibody (pAb) raised against C-terminal oligopeptide of RPS5 was kindly provided by Dr Shuetsu Fukushi (R&D Center, BioMedical Laboratories, Matoba, Kawagoe, Saitama, Japan). The rat anti-mouse MYC, CDK2, CDK4, CDK6 and Gata-1 mAbs were purchased from Invitrogen, Cell Signaling, Transduction Bioscience (BD) and Santa Cruz. Also, goat anti-mouse IgG and goat anti-rabbit IgG were obtained from Santa Cruz, respectively.

Parental MEL cells were maintained in Dulbecco’s modified Eagle’s medium as previously published (22). MEL cell cultures were exposed to DMSO (1.5% v/v) and/or HMBA (5 mM) as indicated under individual figure. At certain time intervals upon exposure, the accumulation of differentiated (haemoglobin-producing; Bz⁺ cells) cells was assessed cytochemically with benzidine-H₂O₂ solution as previously described (16). Also the haemoglobin content within the cells was determined spectrophotometrically as described elsewhere (23).

MEL-C14 cells generated by stable transfection of MEL cells with the full length mouse RPS5 cDNA to express recombinant RPS5-Myc-His protein has been previously described (22). Stable transfection of MEL cells with the full length mouse antisense RPS5 cDNA was carried out as briefly described below: logarithmically growing MEL cells were transfected with the recombinant vector pcDNA3.1(+) carrying the full-length mouse RPS5 cDNA (715 bp in length). Such construct was generated through the ligation of the EcoRI/XhoI fragment derived from the original pBluescript SK +/− plasmid that carry the full length of RPS5 cDNA cloned in our laboratory (16) (GenBank accession no. Y12431) into the respective EcoRI/XhoI sites of pcDNA3.1(+) vector. The RPS5 DNA fragment was inserted into the recombinant pcDNA3.1(+) vector (Invitrogen Life Technologies, USA) in antisense orientation with respect to CMV promoter to generate the construct pcDNA3.1(+)-anti-RPS5. Subsequent stable transfection of MEL cells was performed by using Lipofectamine-2000™ reagent (Invitrogen Life Technologies) and 1 μg plasmid DNA of the recombinant construct pcDNA3.1(+)-anti-RPS5 according to the accompanying manufacturer’s protocol. Stably transfected cells were then selected with G418, (Gibco BRL, Gaithersburg, MD, USA; 0.6 mg/ml) added in the culture medium. G418-resistant cells outgrown from the transfected pcDNA3.1(+)-anti-RPS5 MEL culture allowed us to establish a culture designated as MEL-antisenseRPS5 and used then throughout this study. After the establishment of MEL-antisenseRPS5 culture, G418 (0.2–0.25 mg/ml) was used throughout all experiments described in this study.

Total cytoplasmic RNA isolated from control and/or inducer-treated cells at various time intervals upon incubation in culture was subjected to northern blot hybridization analysis to assess the steady-state levels of RNA transcripts encoded the RPS5 and β^major globin genes, as previously described (22,29).

For RT-PCR analysis, total cytoplasmic RNA (0.2–0.5 μg) isolated from various MEL cell cultures was used for RT-PCR. The PCR experiments were performed by using the RobusT™ I kit (Finnzymes). In detail, the primers used were: 5′-GCGGGATCCATGACTGAGTGGGAAGCA-3′ (forward) and 5′-GCGGAATTCTCAGCGGTTAGACTTGGC-3′ (reverse) specifically designed to allow the detection of mRNA level of endogenous RPS5 (441 bp) as previously published (22); 5′-GGACTTCGAGCAAGAGATGG-3′ (forward) and 5′-AGC ACTGTGTTGGCGTACAG-3′ (reverse) for β-actin (234 bp); 5′-GACCTCGACTACGACTCCGTAC-3′ (forward) and 5′-CCACTGAGGGGTCAATGCAC-3′ (reverse) for c-myc (547 bp); 5′-CTGCTGGTTGTCTACCCTTGG-3′ (forward) and 5′-CCTGAAGTTCTCAGGATCCAC-3′ (reverse) for β^major globin (222 bp).

All siRNAs used including a negative control siRNA [negative control siRNA (#1022076)] designed with no significant homology to any sequence in the mouse genome (scrambled siRNA) along with two predesigned siRNAs directed against mouse RPS5 (NM_009095) (Mm_Rps5_4 #SI01407322; Mm_Rps5_4 #SI01407336) were obtained from Qiagen. Based on extensive preliminary data (not shown), however, only Mm_Rps5_4 was capable of specifically downregulating the expression of RPS5 gene in parental MEL cells and, therefore, was used throughout this study. Scrambled and Mm_Rps5_4 siRNAs were transfected in MEL cells using HiPerFect reagent (Qiagen). The oligo-nucleotide sequence for siRNAs included in Mm_Rps5_4 were: sense, r(GCGCUUCCGCAAAGCACAA)dTdT; antisense, r(UUGUGCUUUGCGGAAGCGC)dTdT), whereas in scrambled siRNA were sense, UUCUCCGAACGUGUCACGUdTdT; antisense, ACGUGACACGUUCGGAGAAdTdT. The procedure briefly was as follows. The day before transfection, MEL cells were seeded at a density of 0.4–1.6×10⁶ cells in 60-mm dishes with 4 ml of DMEM containing serum and antibiotics. On the day of transfection either RPS5 specific or scrambled siRNA were diluted in culture to give a final concentration of 120 nM. The transfection of siRNAs was facilitated by adding 20 μl of HiPerFect reagent buffer according to the manufacturer’s protocol and the cells were further incubated under normal growth conditions before silencing of the RPS5 gene to be monitored by RT-PCR analysis.

Samples of parental MEL cells as well from those exposed either to specific RPS5 (MEL-RPS5siRNA) or scrambled (MEL-scrambled-siRNA) siRNAs before harvested from culture at time intervals as shown in each figure, were subjected to flow cytometry DNA analysis and assessment of apoptosis, as previously published (22,24).

Stable transfection of MEL cells with the mouse antisense RPS5 cDNA allowed us to assess the proliferation and differentiation behavior of such cells in culture. As shown in Fig. 1B, MEL-antisenseRPS5 cells failed to reach the maximum differentiation level upon exposure to chemical inducer DMSO as compared to parental MEL cells, although comparatively no significant change on their cell growth capacity was observed (Fig. 1A). In particular, parental MEL cells exhibited high levels of Bz⁺ (haemoglobin-producing) cells after 96-h exposure to DMSO (>80%) as expected (22). On the contrary, MEL-antisenseRPS5 culture has shown a delay of 18–24 h in the initiation of differentiation and the proportion of Bz⁺ cells was ~50% (Fig. 1B). These data suggest that stable transfection of MEL cells with antisense RPS5 cDNA delayed the onset of differentiation by reducing the number of terminally differentiated cells accumulated in culture.

By being aware on how the commitment to erythroid maturation proceeds in MEL cells, a more thorough kinetic analysis was performed in cultures to better understand the behavior of MEL-antisenseRPS5. In particular it has been reported earlier that in parallel to haemoglobin synthesis, commitment of inducer-treated cells to terminal erythroid maturation leads to limitation of proliferation potential of MEL cells up to 4–5 cellular divisions (13,14). This event becomes quite clear upon dilution of cultures of inducer-treated cells. Such dilution approach has been successfully applied in our work in MEL-C14 culture overexpressing the recombinant RPS5-Myc-His protein (22). Inducer-treated cells, fail to grow and divide continuously in contrast to uncommitted-undifferentiated MEL cells, which exhibit an unlimited proliferation potential. To this end, MEL-antisenseRPS5 cells were exposed continuously to inducer DMSO for either 96 or 72 h, before the dilution of cultures with fresh medium to permit cells to proliferate for further 3 days in the absence (Fig. 1C and D) or 2 days in the presence of DMSO (Fig. 1E and F), respectively. Under these conditions, cells are capable of exhibiting their maximum proliferation potential by executing the erythroid maturation program in the absence of the inducer. As shown in Fig. 1C, parental MEL cells initially growing for 96 h in the presence of DMSO continued in the absence of DMSO for additional 72 h, they reached their plateau phase of growth and maintained at that level despite the dilution (1:10) of the culture with fresh medium. The latter result is attributed to the differentiation-dependent restriction of proliferation potential of DMSO-treated cells (13,14,22), since they achieved high level of differentiated cells (>80%) after 96 h in culture (Fig. 1D). However, the proportion of differentiated cells decreased thereafter upon dilution as expected, since the committed cells can go on only for 4–5 divisions, as mentioned above. On the contrary, MEL-antisenseRPS5 cells did not exceed the level of 50% of differentiated cells under the same culture conditions (Fig. 1D). Moreover in this culture, cellular growth increased after the 6th day (Fig. 1C), since the higher number of non-committed cells (~50%) continued to grow in the absence of DMSO, as expected. These data suggest that in MEL-antisenseRPS5 culture fewer cells enter the differentiation program at a slower rate. Such observation proposes that the plateau phase of growth seen in antisense-RPS5-transfected MEL cells before dilution has to be attributed to cell-cell contact inhibition rather than differentiation-dependent restriction of cell proliferation potential, as seen in parental MEL cells. To confirm this observation, the same experiment was repeated but allowing cells to grow in the presence of DMSO even after dilution of cultures. Interestingly enough, in that case the MEL-antisenseRPS5 cells reached the same high level of differentiation (>80%) after 5 days in culture compared to parental MEL, as illustrated in Fig. 1E and F. These results suggest that the onset of differentiation has been significantly delayed by the transfection of antisense-RPS5 cDNA and the presence of inducer is required throughout the entire exposure period for MEL cells to complete their erythroid maturation program. At this point it has to be noted that such behavior is unique for MEL-antisenseRPS5 culture, since both parental MEL and MEL-C14 did not require the presence of inducer after initiation of commitment to execute the differentiation program, as previously published (22).

To assess any changes in the steady-state level of RPS5 RNA transcripts in MEL-antisenseRPS5 cells in proliferating and/or differentiating cultures, isolated cytoplasmic RNA was subjected to northern blot hybridization analysis using β^major globin gene as internal control. As shown in Fig. 2A, the level of RPS5 RNA transcripts in the cytoplasm remained constant in MEL-antisenseRPS5 cells growing in culture in the absence of DMSO. Similar data were obtained even after 96-h exposure to DMSO (Fig. 2C). On the contrary, the gradual cytoplasmic accumulation of β^major globin RNA transcripts was evident in differentiating cells only after 48–72 h (Fig. 2D). The latter, represents a difference in what was seen in differentiating parental MEL cells where a substantial decrease of RPS5 mRNA after 24 h and a significant accumulation of β^major mRNA after 36 h has been detected (16,22). These marginal differences seen in the expression level of both RPS5 and β^major globin genes in differentiating MEL-antisenseRPS5 cells seems more likely to be related to their limited commitment potential to erythroid maturation, as shown in Fig. 1.

By knowing that commitment of MEL cells to erythroid maturation is correlated with cell cycle changes, the assessment of the expression profile of endogenous RPS5 and CDKs (CDK2, CDK4 and CDK6) upon differentiation of MEL-antisenseRPS5 cells was investigated. During the course of differentiation, the Gata-1 protein level was also assessed as an internal control for MEL cells, as previously described (22). Cell lysates prepared from differentiating MEL-antisenseRPS5 cultures and subjected to immunoblotting revealed that the RPS5 protein level decreased quite early after 24 h (Fig. 2F). This is interesting, since in parental MEL cells the decrease of RPS5 protein is detected only after 72 h (22). Regarding the assessment of CDKs, the level of CDK2 and CDK4 proteins remained almost constant, whereas that of CDK6 decreased very early ~2 h after the induction of differentiation (Fig. 2G–I). These differences seen in the expression level of CDKs seems to be correlated with the differentiation potential of MEL-antisenseRPS5 cells, since the expression profile of Gata-1 remained almost constant throughout the course of the experiment (Fig. 2J). The expression profile of CDKs detected at late stages of differentiating MEL cells coincide with previous observations (22,25–28).

In an effort to rule out the possibility that the delay observed in the onset of differentiation of MEL-antisenseRPS5 cells could be attributed to inducer DMSO, the chemical inducer hexamethylene-bis-acetamide (HMBA) has been also included for further analysis (13,14). Based on the experimental data obtained, a decrease in the onset of differentiation was also recorded since the number of differentiating MEL-antisenseRPS5 cells accumulated in culture after 96 h is less (~40%) as compared to that seen in parental MEL cells (>80%) (Fig. 3E). Interestingly, this effect was more pronounced even by comparing the number of differentiating cells seen in MEL-C14 cells (~60%). The latter culture, as reported earlier, also exhibits a delay in the initiation and the onset of differentiation upon treatment with HMBA attributed to the overexpression of recombinant RPS5-Myc-His protein (22). Moreover, the amount of haemoglobin accumulated in differentiating MEL-antisenseRPS5 is lower than that measured in parental MEL and MEL-C14 cultures (Fig. 3F). In addition, cell growth is decreased dramatically in MEL-antisenseRPS5 culture upon exposure to HMBA similarly to MEL-C14 (Fig. 3C), whereas the number of non-viable cells is much higher in MEL-antisenseRPS5 (~85%) compared to both parental MEL (~25%) and MEL-C14 (~45%) cells (Fig. 3D). However, these differences in cell growth capacity and viability recorded in cultures is more likely to be attributed to their differentiation potential, since the untreated-control cells behave similarly and achieve comparable high numbers of proliferating and viable cells after 96 h (Fig. 3A and B). This notion is further supported by examining the morphology of cells in culture as well as assessing the gene expression profile of β-actin (housekeeping gene), RPS5 (gene of interest) and β^major globin (a developmentally regulated gene) upon induction of differentiation of MEL-antisenseRPS5. Indeed, the morphology of untreated MEL-antisenseRPS5 exhibit an altered phenotype with a larger size that, however, upon 96-h exposure to inducing agent HMBA is further exacerbated by giving smaller although abnormally differentiated cells (distorted nucleolus and nucleus) (Fig. 4). Moreover, a dramatic decrease in mRNA levels for all three genes was observed after 48–72-h exposure of cells to HMBA (data not shown), whereas no significant alteration was seen for RNA transcripts of β-actin, β^major globin and c-myc (an oncogene with crucial role in the differentiation potential of MEL cells) in control-untreated cells continuously growing in culture even for 120 h. Such data collectively propose a correlation between antisense-RPS5 transfection and the decrease in the onset of differentiation seen in MEL-antisenseRPS5 cell culture, since dismantling of the differentiation program is observed very early regardless of the nature of the chemical inducer (DMSO and/or HMBA) employed.

In order to more thoroughly investigate the correlation of RPS5 gene expression with the capacity of MEL cells to initiate their erythroid maturation program in vitro, we applied the RNA interference (RNAi) methodology to specifically downregulate the expression of RPS5 in this culture. As shown in Fig. 5, by using specific siRNAs the silencing of RPS5 gene in MEL cells was achieved in both mRNA and protein level after 24 and 36 h, respectively (Fig. 5A and C). Interestingly, this effect was evident even after 72–96-h continuous exposure to specific siRNAs for the RPS5 gene and not for the scrambled siRNAs used as control (data not shown). The successful RNAi-mediated silencing of RPS5 gene in MEL cells prompted us to assess the capacity of such culture to differentiate in vitro upon exposure to the chemical inducer HMBA. The experimental design shown in Fig. 6 allowed us to verify for each experiment the successful RPS5 gene silencing and also to assess the differentiation capacity of such a culture (Fig. 6A). In particular, the cells were initially exposed to suitable siRNAs (scrambled and/or RPS5-specific) for 24 h before the addition of HMBA in culture. After an additional period of 24 h, the silencing of RPS5 gene expression was verified by RT-PCR, as shown in Fig. 6B. The RPS5 protein level was not detectable after 96-h exposure to RPS5-specific siRNAs, in contrast to cultures exposed to scrambled siRNAs (Fig. 6D). Importantly however, the monitoring of differentiation potential of these cultures for the entire 96-h exposure period to inducer HMBA indicated that only MEL cells exposed to RPS5-specific siRNAs failed to initiate their erythroid maturation program in vitro (Fig. 6F). The number of differentiating cells (cells producing haemoglobin) accumulated after 96 h in culture exposed to specific RPS5 siRNAs is very low (~15%), whereas MEL cells exposed to scrambled siRNAs reach a level comparable to parental ones (>85%). In parallel, assessment of trypan-blue positive cells in these cultures revealed that in both conditions the number of dead cells marginally exceed 20% after 72-h exposure to HMBA; however, after 96 h in culture only MEL cells exposed to RPS5-specific siRNAs exhibited a high proportion (80%) of dead cells (Fig. 6G).

MEL cultures exposed to RPS5-specific siRNAs and being induced to differentiate by HMBA exhibit different cellular morphology. In particular, parental and scrambled siRNA-treated MEL cells showed the typical erythroid maturation phenotype as expected after 72–96 h. However, differentiating cells exposed to RPS5-specific siRNAs present a phenotype more resembling that of undifferentiated cells where nucleolus and nucleus are distorted (data not shown). The latter, is in accordance with the inability of such a culture to initiate commitment of erythroid maturation and accumulate high proportion of haemoglobin-producing cells, as presented above. In order to clarify such differentiation behavior of cells, the assessment of the expression level of RPS5, c-myc, β^major globin and β-actin genes was further investigated. Upon induction of MEL cell differentiation with HMBA, parental MEL, MEL-C14 and MEL-scrambled siRNA-treated cells exhibited decreased level of RPS5 (data not shown). On the contrary, the steady-state level of RPS5 RNA transcripts in MEL-siRNA RPS5 culture was much lower from the beginning and throughout the entire exposure period, as expected. The steady-state mRNA level of c-myc in parental MEL showed an early initial increase at 6 h before a final decline after 48 h, in agreement with a previous report (29). In MEL-C14 culture, the steady-state c-myc RNA transcripts indicated a biphasic profile, where an initial downregulation was observed very early (3 h) followed by an increase after 24 h before the final decrease afterwards (data not shown). Moreover, in both MEL-scrambled siRNA and MEL-siRNA RPS5 cultures, the low expression level of c-myc at 24–48 h was followed by a significant increase at 72 h before a final decrease later on. In parallel, the level of β^major globin gene expression in differentiating parental MEL, MEL-C14 and MEL-scrambled siRNAs cells showed a time-dependent increase, as expected, with the higher level seen after 96 h in all cultures. Importantly, however, the level of β^major globin gene expression in differentiating MEL-RPS5 siRNA exhibited no substantial change by being kept at low levels even after 96 h as compared to all other cultures (data not shown). Furthermore, the level of β-actin mRNA did not show any significant change either between the four cultures or during the course of differentiation.

The commitment of MEL cells to erythroid maturation is associated with early accumulation of cells in G₀/G₁ cell cycle phase arrest. In order to assess that propensity of MEL cells in both RPS5-specific siRNAs- and/or scrambled siRNAs-treated cultures, the accumulation of cells to G₀/G₁ was evaluated upon differentiation in vitro. No substantial difference between the two cultures was recorded in the 24–72-h period tested (data not shown). However, a difference in the differentiation potential in the two cell cultures was previously seen and discussed regarding the inability to commit only in cells treated with RPS5-specific siRNAs. We reasoned additionally to assess the accumulation of apoptotic cells upon induction of differentiation with HMBA. Indeed, as shown in Fig. 7, by using flow cytometry to assess Annexin V-positive cells, we were able to evaluate the kinetics of apoptotic cells accumulated in these cultures for the entire 24–96-h period. MEL-RPS5siRNA culture from the beginning of the induction of differentiation showed much lower proportion of cells exhibiting a pre-apoptotic or apoptotic phenotype. Interestingly, MEL cells treated with scrambled siRNAs accumulate ~32% of both pre-apoptotic and apoptotic cells after 96 h, whereas specific RPS5 siRNAs-treated cells only ~17% during the same period.

The fact that the modulated gene expression level of RPS5 is associated with an altered differentiation capacity of MEL cell cultures in vitro (MEL-C14, MEL-antisenseRPS5 and MEL-RPS5 siRNA treated cells) has clearly proposed a correlation between MEL cell differentiation potential and RPS5 gene expression. In order to more precisely inter-correlate such connection, an attempt was made to restore the normal RPS5 expression levels in MEL cultures that previously exhibited an RNAi-mediated RPS5 gene silencing and then reversely assess their capacity to commit to erythroid maturation. In doing so, the experiment shown in Fig. 8 was designed. MEL cells were exposed for 48 h with either scrambled- or RPS5-specific siRNAs, before the replenishment of cultures with fresh medium containing only the inducer HMBA, no siRNAs (shown in Fig. 8A). MEL cells cultured in the presence of specific RPS5 siRNAs for 48 h, were washed out and changed into fresh medium with the addition of inducer, restored their RPS5 gene expression levels within 24 h (Fig. 8B), whereas the level of β-actin mRNA used as control remained unchanged (Fig. 8C). However, such MEL cells exhibit their maximum differentiation potential comparable to that seen for both parental MEL and scrambled-siRNAs treated MEL cultures (Fig. 8F). Similarly, the kinetics of cellular proliferation in both terms of growth (Fig. 8D) and trypan-blue positive (dead) cells (Fig. 8E) are indistinguishable between the three differentiating MEL cultures. Overall, such data propose a close relationship between the recorded RPS5 gene expression level in MEL cells and their capacity to initiate the in vitro differentiation program to erythroid maturation.