Altered cellular responses by varying expression of a ribosomal protein gene: sequential coordination of enhancement and suppression of ribosomal protein S3a gene expression induces apoptosis - PubMed (original) (raw)

Altered cellular responses by varying expression of a ribosomal protein gene: sequential coordination of enhancement and suppression of ribosomal protein S3a gene expression induces apoptosis

H Naora et al. J Cell Biol. 1998.

Abstract

A growing body of evidence indicates that individual ribosomal proteins and changes in their expression, participate in, and modulate, a variety of cellular activities. Our earlier studies have found that apoptosis could be induced by inhibiting expression of ribosomal protein S3a (RPS3a) in many tumor cells which constitutively express RPS3a at levels much higher than in normal cells. This study aimed to investigate cellular responses to enhancement of RPS3a expression, and whether apoptosis could be induced by sequential alterations in RPS3a expression involving enhancement from an initially low constitutive level, followed by suppression. Stably transfected NIH 3T3- derived cell lines were established in which exogenous RPS3a expression could be readily manipulated. Enhancement of RPS3a expression appeared to induce transformation as assessed by well-established criteria such as foci formation and anchorage-independent growth in vitro, and formation of tumors in nude mice. These properties were compared with those observed in ras-transformed NIH 3T3 cells. Apparent transformation occurred only when enhanced RPS3a-expressing cells were in close cell-cell contact. Suppression of enhanced RPS3a expression was observed to induce apoptosis as assessed by various morphological and biochemical characteristics including cell shrinkage, membrane blebbing, chromatin condensation, nuclear and cell fragmentation, phosphatidylserine externalization, and internucleosomal DNA fragmentation. This induction of apoptosis was not specific to apparently transformed cells, as cells at low confluence, which likewise expressed RPS3a at enhanced levels but exhibited no morphological transformation, underwent apoptosis when RPS3a expression was inhibited. These results support a role for RPS3a in the apoptotic process, but not as an oncoprotein per se.

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Figures

Figure 1

Figure 1

Schematic diagram of plasmid pMSG-RPS3a. A human RPS3a cDNA, containing the complete protein coding region plus 5′ and 3′ UTRs and expressed under the control of the MMTV LTR, and the SV-40 early promoter–driven gpt gene, are present in the same orientation. The 950-bp EcoRI fragment containing the RPS3a cDNA and the 950-bp HindIII–AatII fragment of the gpt gene were used as probes for Southern and Northern blot analyses.

Figure 2

Figure 2

Growth curves of P-12 and S-12 cells. P-12 (•) and S-12 (○) cells were plated at 0.7 × 103 cells per well and cultured in gpt medium. S-12 cells were likewise plated and cultured in Dex medium (▵). The total cell number per well was determined every 2 d thereafter. Data represent the mean ± SD of duplicate or triplicate wells. Culture medium was removed on day 4, and detached cells collected, resuspended in fresh medium, and then transferred to the original well for continuation of culture. Values observed for P-12 and S-12 cells cultured in gpt medium were very similar at a given time point, and these values are plotted to the right and left sides of the appropriate time point. The growth curve of P-12 cells cultured in Dex medium (not shown) was almost identical to that of P-12 cells cultured in gpt medium.

Figure 3

Figure 3

Determination of stable integration by Southern blot analysis of genomic DNA. (a) SmaI- and EcoRV-digested DNA of parental NIH 3T3 cells and P-7 and P-12 transfectants, and (b) EcoRV-digested DNA of NIH 3T3 and S-8 and S-12 transfectants, were hybridized with the gpt probe. (c) HindIII-digested DNA of NIH 3T3, S-8, and S-12 cells, and (d) EcoRV-digested DNA of NIH 3T3 and S-12 cells, were hybridized with the RPS3a cDNA probe. The fragments derived from transfected RPS3a/gpt sequences are shown by ◂. Sizes of HindIII- or PstI-digested λ DNA fragments (in kb) are shown at the left of each panel.

Figure 4

Figure 4

Northern blot analysis of RPS3a expression. RNA was isolated from parental NIH 3T3 cells cultured in normal medium (lane 1), and from P-12 and S-12 cells cultured in gpt medium (lanes 2 and 5), switched to Dex medium for 2 d (lanes 3 and 6), and then switched back to gpt medium for 1 d (lanes 4 and 7). 8 μg of RNA was electrophoresed on a 1.6% agarose gel and used to prepare a Northern blot that was hybridized with the RPS3a cDNA probe. Endogenous RPS3a mRNAs were detected in all samples. The longer exogenous RPS3a transcripts were induced in S-12 cells switched from gpt to Dex medium (lane 6), and then inhibited when cells were switched back to gpt medium (lane 7). Equalization of RNA content was checked by visualizing 28S and 18S rRNAs on the ethidium bromide–stained RNA gel.

Figure 5

Figure 5

Alterations in morphology of S-12 (a–e) and P-12 (f–j) cells. Cells were cultured in gpt medium (a and f), switched to Dex medium for 1 d (b and g), cultured in fresh Dex medium for an additional day (c and h), and then switched back to gpt medium for 1 d (d and i, and e and j at lower magnification). S-12 cells began to pile up when switched from gpt to Dex medium (b). A typical transformed focus formed when culture was continued in Dex medium is shown in c. Switching S-12 cells from Dex to gpt medium resulted in massive detachment from the substratum and loss of viability (d and e). In contrast, the same alterations in culture conditions did not significantly change the morphology of P-12 cells. Bar in f: (a–d, f, and g–i) 50 μm; bar in j: (j and e) 150 μm.

Figure 6

Figure 6

Foci formation in P-12 and S-12 cultures. P-12 (a and b) and S-12 (c and d) cells were plated at 1.5 × 105 cells in six-well plates and cultured in either gpt or Dex medium until growth reached ∼70–80% confluence. Cells were fixed and stained with Giemsa. Note that darkly and weakly stained foci were widely observed only when S-12 cells were cultured in Dex medium (d). Bar, 1 mm.

Figure 7

Figure 7

Growth of P-12 and S-12 cells in poly(HEMA)-coated wells. P-12 and S-12 cells were plated at 3 × 103 cells in poly(HEMA)-coated 96-well plates, and maintained in gpt or Dex medium for 4 d. Growth of S-12 cells was observed in Dex medium (d) but not in gpt medium (c). P-12 cells formed aggregates and were unable to grow in gpt (a) or Dex (b) medium. Bar, 50 μm.

Figure 8

Figure 8

Morphology of apoptotic and nonviable S-12 cells. S-12 cells, precultured in gpt medium, were incubated in Dex medium for 2 d and then switched back to gpt medium for 1 d. In A, cells that became detached were collected from the culture medium and examined by phase contrast microscopy (a), which revealed rounded morphology and blebbing (arrow), and by Giemsa staining, which revealed shrinkage and chromatin condensation (b) and nuclear fragmentation (c). In B, cells which remained attached were harvested and pooled with detached cells, and then treated with Annexin V–FITC reagent and examined by fluorescence (a and c) and differential interference contrast (b and d) microscopy. Approximately half of the total cell population showed Annexin V–binding activity, with some cells being strongly positive and others exhibiting weak or localized fluorescence, the former showing marked ruffling on their surface (d). Bars in A: (a) 20 μm; (b and c) 10 μm. Bars in B: (a and b) 25 μm; (c and d) 10 μm.

Figure 8

Figure 8

Morphology of apoptotic and nonviable S-12 cells. S-12 cells, precultured in gpt medium, were incubated in Dex medium for 2 d and then switched back to gpt medium for 1 d. In A, cells that became detached were collected from the culture medium and examined by phase contrast microscopy (a), which revealed rounded morphology and blebbing (arrow), and by Giemsa staining, which revealed shrinkage and chromatin condensation (b) and nuclear fragmentation (c). In B, cells which remained attached were harvested and pooled with detached cells, and then treated with Annexin V–FITC reagent and examined by fluorescence (a and c) and differential interference contrast (b and d) microscopy. Approximately half of the total cell population showed Annexin V–binding activity, with some cells being strongly positive and others exhibiting weak or localized fluorescence, the former showing marked ruffling on their surface (d). Bars in A: (a) 20 μm; (b and c) 10 μm. Bars in B: (a and b) 25 μm; (c and d) 10 μm.

Figure 9

Figure 9

Analysis of genomic DNA fragmentation in P-12 and S-12 cells. Genomic DNA was isolated from parental NIH 3T3 cells cultured in normal medium (lane 2), and from P-12 and S-12 cells cultured in gpt medium (lanes 3 and 6), switched to Dex medium for 2 d (lanes 4 and 7), and then switched back to gpt medium for 1 d (lanes 5 and 8). Genomic DNA was isolated from detached cells recovered from the culture medium pooled with attached cells. 10 μg of DNA was electrophoresed on a 0.8% agarose gel, together with PstI- digested λ DNA as size markers (lane 1; sizes of various fragments in kb shown at left).

Figure 10

Figure 10

Effects of Act D on DNA fragmentation (A) and morphology (B) of P-12 and S-12 cells. In A, genomic DNA was isolated from P-12 and S-12 cells incubated for 2 d in Dex medium (lanes 3 and 6), and for a further 6 h with the addition of Act D (1 μg/ml) (lanes 2 and 5). Genomic DNA was also isolated from P-12 and S-12 cells, incubated in gpt medium with the addition of Act D for 6 h (lanes 4 and 7), and from S-12 cells switched to gpt medium for 1 d after incubation in Dex medium for 2 d (lane 8). Genomic DNA was isolated from detached cells recovered from the culture medium pooled with attached cells, and electrophoresed as described in Fig. 9 with PstI-digested λ DNA (lane 1). In B, P-12 (a) and S-12 (b) cells were incubated in Dex medium for 2 d, followed by incubation for a further 24 h with the addition of Act D (1 μg/ml). Bar, 50 μm.

Figure 10

Figure 10

Effects of Act D on DNA fragmentation (A) and morphology (B) of P-12 and S-12 cells. In A, genomic DNA was isolated from P-12 and S-12 cells incubated for 2 d in Dex medium (lanes 3 and 6), and for a further 6 h with the addition of Act D (1 μg/ml) (lanes 2 and 5). Genomic DNA was also isolated from P-12 and S-12 cells, incubated in gpt medium with the addition of Act D for 6 h (lanes 4 and 7), and from S-12 cells switched to gpt medium for 1 d after incubation in Dex medium for 2 d (lane 8). Genomic DNA was isolated from detached cells recovered from the culture medium pooled with attached cells, and electrophoresed as described in Fig. 9 with PstI-digested λ DNA (lane 1). In B, P-12 (a) and S-12 (b) cells were incubated in Dex medium for 2 d, followed by incubation for a further 24 h with the addition of Act D (1 μg/ml). Bar, 50 μm.

Figure 11

Figure 11

Effects of cell density on morphological transformation and apoptosis. S-12 cells, precultured in gpt medium, were incubated at low and at high cell densities in Dex medium for 2 d. In C, cells at high density grew in multicellular layers and exhibited foci formation, whereas cells at low density remained as a monolayer and exhibited no morphological transformation. Exogenous RPS3a expression levels in these cells were quantified by PhosphorImager analysis of hybridization signals of exogenous RPS3a transcripts. In A, the level of exogenous RPS3a expression in cells at high density is expressed relative to the level in cells at low density (open bar). S-12 cells incubated at low and at high cell densities were then switched from Dex to gpt medium for 1 d. In B, the resulting cell death is expressed in terms of percentages (mean ± SD of duplicate wells) of apoptotic and nonviable cells in the total cell population. Filled and open sections represent the fraction of apoptotic and nonviable cells composed of detached cells collected from the culture medium, and of cells collected after trypsinization, respectively. The extent of inhibition of exogenous RPS3a expression, induced when cells were switched from Dex to gpt medium, was quantified by PhosphorImager analysis, and is shown in A (dotted bar). Data in A represents the mean ± SD of two experiments. Bar, 200 μm.

Figure 11

Figure 11

Effects of cell density on morphological transformation and apoptosis. S-12 cells, precultured in gpt medium, were incubated at low and at high cell densities in Dex medium for 2 d. In C, cells at high density grew in multicellular layers and exhibited foci formation, whereas cells at low density remained as a monolayer and exhibited no morphological transformation. Exogenous RPS3a expression levels in these cells were quantified by PhosphorImager analysis of hybridization signals of exogenous RPS3a transcripts. In A, the level of exogenous RPS3a expression in cells at high density is expressed relative to the level in cells at low density (open bar). S-12 cells incubated at low and at high cell densities were then switched from Dex to gpt medium for 1 d. In B, the resulting cell death is expressed in terms of percentages (mean ± SD of duplicate wells) of apoptotic and nonviable cells in the total cell population. Filled and open sections represent the fraction of apoptotic and nonviable cells composed of detached cells collected from the culture medium, and of cells collected after trypsinization, respectively. The extent of inhibition of exogenous RPS3a expression, induced when cells were switched from Dex to gpt medium, was quantified by PhosphorImager analysis, and is shown in A (dotted bar). Data in A represents the mean ± SD of two experiments. Bar, 200 μm.

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