Abstract
Expression of the cyclin-dependent kinase inhibitor gene p21 is induced after DNA damage and plays a role in cell survival. The exact mechanism of induction is not known, but enhancement of mRNA stability has recently been implicated as an important factor. To obtain further insight into the dynamics of p21 gene expression at the individual cell level, normal fibroblasts, GM1492 fibroblasts from a Bloom's syndrome patient, and U2OS osteosarcoma cells were UVC irradiated, fixed at different time points, and subjected to mRNA fluorescence in situ hybridization (FISH) and immunocytochemical staining. In mock-irradiated normal fibroblasts, a subfraction of cells revealed low levels of p21 mRNA synthesis. After UVC treatment, p21 transcripts accumulated over time in nuclear locations other than transcription foci. At 6 hr after irradiation, almost 50% of the cells displayed p21 mRNA in three different distribution patterns within the nuclei. The highest frequency of cells with cytoplasmic accumulation of p21 mRNA was seen at 17 hr after UVC treatment. We conclude that increased p21 gene transcription and possibly stabilization of newly synthesized p21 mRNA contribute to elevated levels of p21 protein after UVC irradiation.
T
The underlying mechanisms regulating p21 expression are still a point of debate. It has recently been reported that induction of p21 expression by UVC light is mediated through enhanced p21 mRNA stability rather than increased transcription (Gorospe et al. 1998). The Elav-type RNA-binding protein HuR was shown to play a role in enhancing p21 mRNA stability (Wang et al. 2000). However, to determine levels of p21 expression, these and related studies made use of Northern blotting and/or Western blotting techniques (see for example Lu and Lane 1993; Li et al. 1994; Bunz et al. 1998; McKay et al. 1998). These molecular techniques provide information about the average level of gene expression of the cells analyzed but cannot detect expression at the individual cell level. As a consequence, heterogeneity in expression levels or links between gene expression and other cell parameters cannot be studied by these methods. Furthermore, it has been shown that p21 protein levels do not necessarily correlate with p21 RNA levels (Erber et al. 1997). Therefore, it is anticipated that fluorescence in situ hybridization (FISH) for RNA offers the best approach to study the dynamics of p21 gene induction after UVC irradiation at the individual cell level.
The aim of this study was to monitor the kinetics of p21 gene expression by RNA FISH in individual human cells subjected to UVC irradiation. Here we report that exposure of cells to short-wavelength UVC irradiation leads to induction of p21 mRNA synthesis or, alternatively, to nuclear p21 mRNA stabilization. As early as 1 hr after UVC treatment, elevated levels of p21 transcripts are detected in one or two nuclear foci in a subfraction of the cells. Shortly thereafter, transcripts are observed throughout the nucleoplasm and eventually in high amounts in the cytoplasm of cells. Combined detection of p21 transcripts, together with speckle domains enriched for RNA processing factors and RNA polymerase II, suggests that actively transcribed p21 genes associate with speckles and that their transcripts accumulate in these compartments before being transported towards the cytoplasm.
Materials and Methods
Cell Culture and UVC Irradiation
Normal human diploid foreskin fibroblast strain VH10, skin fibroblast strain GM1492, derived from a Bloom's syndrome patient (Human Genetic Cell Repository; Camden, NJ), U2OS osteosarcoma cells, and HL-60 leukemia cells were grown in Dulbecco's modified Eagle's medium without phenol red, supplemented with 10% fetal bovine serum, L-glutamine, and penicillin/streptomycin (Life Technologies; Gaithersburg, MD) at 37C in a 5% CO2 atmosphere. The GM1492 cells do not express detectable levels of p53 and p53 levels are also not induced after UVC treatment (van Laar et al. 1995).
To standardize the conditions as much as possible, all adherent cells were grown for 2 or 3 days until 70% confluency on glass slides in Petri dishes before fixation or UVC treatment. For the UVC irradiation experiments using VH10 fibroblasts, cells were used between passage numbers 22 and 30 because later passages showed lower proliferative and transcriptional activity, as has also been observed for very sparse or completely confluent cells.
UVC irradiation was performed essentially as described (van Laar et al. 1995). Just before UVC irradiation of cells, the medium was removed and kept, and then the cells were washed twice with PBS at 37C. Irradiation was performed with a 30-W germicidal lamp (TUV; Philips Electronic Instrument, Eindhoven, The Netherlands) at a dose rate of 0.5 J.m−2.sec−1. Mock-treated cells were included in each experiment and were treated in the same way as the irradiated ones.
In Situ Hybridization and Immunocytochemistry
Cells were washed with PBS at 37C and fixed for 15 min in 4% formaldehyde, 5% acetic acid in PBS. Next, cells were briefly washed in PBS and stored in 70% ethanol at 4C until use (Dirks et al. 1993).
Plasmid probes containing the cDNA sequences for p21 (2.1 kb), collagenase IV (1.9 kb), β-actin (1.1 kb), and human elongation factor (1.5 kb) were labeled with digoxigenin-dUTP (Roche Diagnostics; Indianapolis, IN) by nick translation according to standard procedures. A plain pUC21 vector was used as negative control. Probes were dissolved in a hybridization mixture as described before (Dirks et al. 1993) at a final concentration of 4 ng/μl.
Poly(A) RNA detection was done with a 50-mer oligonucleotide (dT) probe. This probe was labeled with lissamine-dUTP (NEN Life Science Products; Boston, MA) using terminal deoxynucleotidyl transferase (Promega; Madison, WI) according to the manufacturers' instructions and dissolved in 10% formamide/4 × SSC at a concentration of 1 ng/μl.
The hybridization procedure was performed essentially as described previously (Dirks et al. 1993). Cells were treated with 0.1% pepsin, pH 2.0, and dehydrated. The time of pepsin treatment was optimized for each cell type. Ten μl of probe was applied to a slide and covered with a coverslip. Probe and target sequences were denatured simultaneously by placing the slides on an 80C metal plate for 2 min. Hybridization with the plasmid probe was done at 37C overnight in a moist chamber. After hybridization, slides were washed three times in 50% formamide, 2 × SSC, pH 7.0, for 5 min each at 37C, once in 2 × SSC for 3 min, and once in Tris-buffered saline (TBS: 100 mM Tris-HCl, 150 mM NaCl, pH 7.5) containing 0.05% Tween-20 (TNT) at room temperature (RT). Digoxigenin-labeled probe was detected with mouse anti-digoxigenin FITC (1:250) (Sigma; St Louis, MO) followed by goat anti-mouse Alexa488 (1:500) (Molecular Probes; Eugene, OR) or with sheep anti-digoxigenin FITC (Roche). All antibodies were diluted in TBS containing 0.5% (w/v) blocking reagent (Roche) (TNB) and were incubated in a moist chamber for 30 min at 37C. After each antibody incubation the slides were washed three times each for 5 min in TNT. The lissamine-labeled probe was visualized directly without further signal amplification.
For simultaneous detection of p21 mRNA and poly(A) RNA, slides were hybridized with the p21 probe first, washed as described above, and hybridized with the oligo (dT) 50 probe. After hybridization with the oligo (dT) 50 probe, slides were washed three times in 4 × SSC and embedded in Vectashield (Vector Labs; Burlingame, CA) containing 50 ng/ml 4′,6′-diamidino-2-phenyl indole (DAPI) as a DNA counterstain.
p53 and p21 protein expression was detected using the following antibodies: rabbit anti-human p53 clone FL393 (1:200) (Santa Cruz Biotechnology; Santa Cruz, CA), mouse anti-human p21 clone EA10 (1:50) and the secondary antibodies goat anti-rabbit Alexa594 (1:1000) and goat anti-mouse Alexa488 (1:500) (Molecular Probes). All antibodies were diluted in TNB and incubated for 40 min at RT. After each antibody incubation step, slides were washed three times for 5 min in TNT.
For detection of splicing factors, mouse anti-m3G antibody (Oncogene Science; Cambridge, MA), which reacts specifically with the 2,2,7-trimethyl guanosine cap of snRNAs (Reuter et al. 1984), was used as described (Dirks et al. 1997).
Microscopy and Photography
Slides were examined with a DM epifluorescence microscope (Leica) equipped with a 100-W mercury arc lamp and appropriate filter sets for FITC, Texas red, and DAPI single excitation. Digital images were captured with a cooled CCD camera (Photometrics) using a PL APO × 63/1.32 oil or a PL Fluotar × 100/1.3 oil objective. To prevent image shifts, multi-color images were taken by using double or triple excitation filters. By inserting appropriate filters in the excitation way, the different fluorochromes could be selectively excited and recorded without image shifts (Tanke et al. 1995). Image analysis was performed on a Macintosh computer.
To evaluate the percentage of cells displaying p21 RNA and the frequency of the different RNA expression patterns for p21, 100 cells were counted per slide. These evaluations were repeated at least on two slides of different irradiation experiments. Only cells with intact nuclei and cytoplasm and within an area of about 70% confluency were counted. The percentages given in the text and tables were taken from one representative experiment in which all experimental conditions were optimal.
Results
UVC Irradiation of VH10 Fibroblasts Results in Characteristic Expression Patterns of p21 RNA
After standard cell culture conditions and mRNA FISH using a digoxigenin-labeled cDNA probe for p21 RNA, p21 expression was detected in less than 15% of normal diploid VH10 skin fibroblasts. These cells showed one or two nuclear foci that were sometimes surrounded by a few small signals. Less than 2% of cells also revealed some cytoplasmic signals. The fluorescence intensities of the nuclear signals were clearly above background level, as revealed by control hybridization with the pUC21 vector sequence only. Other control experiments included hybridization of the p21 probe to HL60 and to U2OS osteosarcoma-derived cells. As expected, p53-deficient HL60 cells did not reveal any hybridization signals, whereas U2OS cells, containing wt p53, showed similar hybridization patterns as observed in VH10 cells.
To study the kinetics of induction of p21 gene expression in response to DNA damage, VH10 cells were exposed to 15 J/m2 UVC irradiation as described in Materials and Methods and fixed at 1, 3, 6, 17, or 24 hr after treatment. The first signs of p21 mRNA expression were observed as early as 1 hr after irradiation (Table 1). Approximately 25% of all cells showed p21 RNA in the nucleus. Among these cells, three different RNA distribution patterns could be recognized, all being sensitive for RNase treatment. About 24% of the cells showing nuclear p21 RNA contained one or two nuclear foci of p21 RNA accumulation, most likely representing the sites of transcription. The majority of these foci were situated at the periphery of a cell nucleus and a few of them had a track-like appearance extending into the nucleoplasm. In general, the sizes of these foci were larger than those observed in non-irradiated control cells. In addition, small fluorescent dots were observed surrounding the larger RNA foci that are indicative for RNA molecules being exported to the cytoplasm (Figures 1A and 1B). In 36% of the cells containing nuclear RNA foci, the small dots were found only in a restricted area of the nucleus. In the remaining portion of cells showing nuclear p21 RNA (40%), this RNA was distributed throughout the nucleoplasm, excluding nucleoli (Figures 1C and 1D). Because of the high numbers of small p21 RNA dots, the nuclear RNA foci, most likely representing synthesis sites of p21 RNA, could no longer be discriminated.
Frequency of p21 mRNA FISH patterns I-V in VH10 fibroblasts after UVC irradiation (15 J/m2) a
aNumbers are given in percent; mitotic and apoptotic cells are not included in the table. I, cells with one or two nuclear p21 RNA foci; II, cells with nuclear p21 RNA foci and small dots in a restricted area of the nucleus; III, cells with p21 RNA distributed throughout the nucleoplasm, excluding nucleoli; IV, cells with p21 RNA distributed in a speckle-like pattern in the nucleus; V, cells with elevated levels of p21 RNA in the cytoplasm.
At 3 hr after UVC treatment, the total percentage of p21 RNA-positive nuclei remained the same (25%) but the frequencies of these cells showing RNA distributed throughout the nucleus increased to 64% (Table 1). The proportion of cells showing only one or two nuclear foci decreased to 8%. Interestingly, p21 RNA was found to accumulate also in a speckle-like pattern in a subfraction (16%) of p21 RNA-positive cell nuclei (Figures 1E and 1F). At 6 hr after UVC treatment, with 52% of cells showing p21 RNA, the frequencies of these cells showing RNA distributed throughout the nucleus increased to 67%, while those showing p21 accumulated in speckle domains slightly increased (18%). The proportion of p21 RNA-positive cells showing transcription foci only further decreased to 4%. Cells showing elevated levels of cytoplasmic p21 RNA transcripts were found to appear only at 17 and 24 hr after UVC treatment, (respectively 17% and 10% of all cells; Figures 1G and 1H).
Mock-irradiated cells that were fixed at 1 and 24 hr
Nuclear and cytoplasmic distribution patterns of p21 RNA in VH10 cells fixed at 1 (
after mock treatment revealed no significant induction of p21 compared to non-irradiated cells. Comparable results to those described above were obtained with UVC-treated U2OS cells, but p21 RNA could not be detected in UVC-treated, p53-deficient GM1492 skin fibroblast cells.
To evaluate whether different dosages of UVC irradiation might influence the time of onset of p21 mRNA accumulation, VH10 cells were exposed to 5, 10, 15, or 20 J/m2 UVC and fixed at 30 min or 1 or 2 hr after irradiation. All different p21 RNA patterns observed after irradiation with 15 J/m2 were also seen after irradiation with the different dosages in approximally similar percentages (results not shown). Moreover, the amounts of transcripts observed did not significantly differ from those observed in cells exposed to 15 J/m2. In addition to p21 induction, we analyzed the induction of the collagenase IV gene, a delayed UV-responsive gene, in VH10 cells. Cells fixed at 3 and 7 hr after UVC treatment revealed no induction of collagenase IV expression, whereas cells fixed at 17 and 24 hr after irradiation did. Induction was shown by the presence of one or two transcription sites per nucleus, which were not observed at 3 and 7 hr after UVC treatment. An accumulation of collagenase IV mRNA in nuclear speckles was not observed (not shown). To investigate whether UVC irradiation would influence the expression patterns of housekeeping gene RNAs, UVC-treated and untreated cells were hybridized with probes specific for β-actin and human elongation factor mRNA. No differences in RNA expression patterns were observed, suggesting that UVC exposure does not lead to an induced expression of the two housekeeping genes studied (result not shown).
Early Onset of p21 mRNA Expression Is Reflected by the Association of p21 mRNA with Nuclear Speckles
Transcriptional activation of certain genes has been correlated with the recruitment of splicing factors from speckle domains towards the transcriptionally active genes (Baurén et al. 1996; Dirks et al. 1997). Alternatively, genes have been found associated with speckle domains when transcriptionally active and not associated when transcriptionally silent (Xing et al. 1995). To learn more about the nature of the nuclear p21 mRNA patterns, we performed a combined detection of p21 mRNA together with splicing factors and poly(A) sequences. Both splicing factors and poly(A) sequences can be used as markers to identify speckle domains (Huang et al. 1994). VH10 cells were irradiated with a dose of 15 J/m2, fixed at 1, 3, 17, or 24 hr after irradiation, and hybridized first with a probe for p21 mRNA and then with a probe for poly(A) RNA. A proportion of cells fixed at 1 hr after irradiation revealed one or two nuclear hybridization signals representing the p21 genes, which for the most part did not co-localize with the speckle domains stained with the poly(dT) probe. In only a small fraction (less than 2%) of the 25% cells displaying p21 RNA in the nucleus, the p21 genes were shown to be associated with a speckle domain (Figures 2A–2C). In another fraction of these cells showing a large fluorescent nuclear hybridization signal for p21 mRNA and often small fluorescent signals radiating from this site in all directions, the large fluorescent signal always overlapped with speckle domains (Figures 2D–2F). In cells fixed at 3 hr after irradiation, p21 mRNA was shown also to accumulate in speckle domains (Figures 2G–2I). At 17 and 24 hr after irradiation when p21 mRNA was shown to be present in the cytoplasm of cells, transcripts no longer accumulated in speckles (Figures 2J–2L). Identical results were obtained when UVC-treated cells were hybridized for p21 RNA detection and then subjected to immunocytochemical detection of splicing factors (result not shown).
Expression of p21 RNA Correlates with p21 Protein Expression
To examine how the p21 RNA expression patterns obtained by RNA FISH correlate with p21 and p53 protein expression, slides with VH10 cells were exposed to UVC irradiation and either stained with p21-and p53-specific antibodies or hybridized with the p21 mRNA-specific probe. At 3 hr after UVC treatment the number of p53 protein-positive cells was shown to increase from 39% on mock-treated slides to 69%, increasing to 86% p53-positive cells at 24 hr (Table 2). The numbers of p21-positive cells increased only on slides fixed at 17 and 24 hr after UVC treatment, from 10% after mock treatment to 18%. Cells that were positive for both p53 and p21 were observed in mock-treated cells (10%) and in cells that were fixed at 17 and 24 hr after UVC treatment (11% and 13%, respectively). Cells fixed at 1, 3, and 6 hr after UVC treatment did not reveal p21-positive cells.
At the time points at which the numbers of p53 protein-positive cells were shown to increase, the numbers of cells with nuclear p21 mRNA also increased. Strikingly, at 17 and 24 hr after UVC treatment, when the highest percentage of p21 protein-positive cells was observed, the highest number of cells with p21 mRNA in the cytoplasm was also found.
Similar dynamics of p53 and p21 protein expression were observed in U2OS cells, whereas GM1492 cells did not show nuclear positivity for p53 and p21.
Discussion
Using FISH to monitor p21 mRNA expression after UVC irradiation, we have shown in individual cells that p21 mRNA levels are induced by the DNA damage-causing agent UVC. Our observations suggest that even 1 hr after UVC treatment p21 mRNA synthesis is induced in a subset of cells, as reflected by the appearance of one or two intense fluorescent foci in the cell nuclei. These foci most likely represent the accumulation of p21 RNA transcripts at the site of transcription because, due to the small size of the p21 cDNA probe used in this study (2.1 kb) and to the limited sensitivity of the FISH technique, hybridization to the gene would only at best give rise to a very small fluorescent spot in a low percentage of cells. Furthermore, the dot-like or track-like signals proved to be sensitive to RNase treatment. Our interpretation of p21 RNA foci is consistent with other reports in which the transcriptional activity of specific genes has been visualized by RNA FISH (Xing et al. 1993; Dirks et al. 1997). Accumulation of transcripts next to a gene is believed to occur in situations in which a lot of RNA is synthesized by a given gene in a short time period (Dirks et al. 1999) or in which pre-mRNA is inefficiently processed (Custodio et al. 1999). Alternatively, the appearance of foci could be the result of stabilization of newly synthesized p21 mRNA. That RNA stabilization may lead to the accumulation of transcripts near their site of synthesis has, however, not been previously demonstrated. Nevertheless, regardless of which situation may be true, RNA processing may be completed within the RNA foci, after which mature transcripts are released from these sites to be transported towards the cytoplasm (Xing and Lawrence 1993). Indeed, we observed a considerable overlap between p21 RNA foci and splicing factors, although we cannot conclude whether splicing factors are recruited from speckle domains, which function as storage sites, to transcriptionally active p21 genes, or that p21 genes are associated with “existing” speckle domains. Both possibilities, however, are consistent with transcriptional activity of the p21 gene (Misteli and Spector 1998; Dirks et al. 1999; Smith et al. 1999).
p53 and p21 protein expression in VH10 cells after UVC irradiation (15 J/m2) a
aNumbers are given in percent; mitotic and apoptotic cells are not included in the table.
An additional indication for induction of high levels of p21 gene expression on UVC irradiation is our observation of p21 mRNA being present throughout the nucleoplasm in a subset of p21 RNA-positive cells. Similar nuclear distribution patterns of transcripts have been observed for other RNAs that are abundantly synthesized and have been interpreted as transport routes of RNAs towards the cytoplasm (Dirks et al. 1999). Interestingly, in another subset of UVC irradiated cells, p21 gene transcripts were also shown to reside in almost all speckle domains. This observation has thus far not been made for other endogenous gene products and indicates that abundantly synthesized or stabilized p21 transcripts are processed not only at or near their site of synthesis but also at other nuclear sites enriched for RNA processing factors. Although we have not tested whether intron sequences are still present within the speckle-localized p21 transcripts, Wang et al. (1991) provided evidence that intron sequences are required for binding to RNA splicing factors in speckle domains by microinjecting intron-containing and intron-lacking β-globin mRNA into cell nuclei. Furthermore, it has recently been shown that abundantly synthesized EBV transcripts are processed at nuclear sites distant from transcription (Melcák et al. 2000). Alternatively, p21 transcripts may accumulate in speckles as result of a UVC stress response and may be degraded at these sites. However, other endogenous RNA transcripts, including β-actin and human elongation factor mRNA, did not accumulate in speckles in response to UVC irradiation.
UVC irradiation of cells may lead to a burst of p21 gene transcription or enhanced p21 mRNA stability, after which transcripts are processed not only near the gene but also elsewhere in speckle domains. Indeed, prominent nuclear transcription sites were observed most frequently at 1 hr after UVC irradiation, whereas cells showing p21 RNA in speckles were observed at later time points. The relatively low percentage of cells showing the presence of p21 in speckles suggests that this stage reflects a transient situation, which means that once transcripts are spliced at these sites they are transported towards the cytoplasm.
Simultaneous detection of p21 DNA/RNA (
Recently, contradictory reports concerning the influence of UVC irradiation on the regulation of p21 expression appeared in the literature. For example, Wang et al. (1999) provided evidence that UVC radiation downregulates expression of p21, whereas others provided evidence for the opposite (see for example Loignon et al. 1997). These contradictory results can be partly explained by the use of different cell lines, different dosages and conditions for irradiation, or different methodologies to monitor p21 expression in these studies. Interestingly, on the basis of these studies different mechanisms have been proposed leading to a p53 and p21 response to UVC irradiation. Down-regulation of p21 expression has been correlated with inhibition of RNA polymerase II activity (Ljungman et al. 1999), whereas elevation of p21 expression has been correlated with enhanced mRNA stability (Gorospe et al. 1998; Wang et al. 2000). The results of our study are most consistent with enhanced p21 mRNA synthesis in at least a subset of cells after different dosages of UVC irradiation. However, it may also be true that an increase in nuclear and cytoplasmic p21 mRNA is a result of enhanced RNA stability. Taking into account the time points at which we observed the occurrence of p21 and p53 proteins in irradiated cells, we strongly support the model that, after UVC irradiation, p21 RNA expression is rapidly induced by p53 and that the delayed occurrence of p21 protein is the result of the time required to process p21 RNA in speckle domains and possibly the time that is required to further stabilize these transcripts in the cytoplasm of cells. Moreover, the observation that the proportion of cells containing detectable amount of p21 protein initially decreases suggests a temporary inhibition of p21 mRNA translation or increased p21 protein turnover.
At present, we have no clear explanation for our observation that p21 RNA is expressed only in a subset of VH10 and U2OS irradiated cells. Because we cannot follow the dynamics of this process in living cells, we cannot exclude the possibility that p21 expression is initiated at different time points in different cells after irradiation, giving rise to the heterogeneous expression pattern. In a series of preliminary experiments using markers for cell cycle stages, we have thus far not found a correlation between the onset of p21 RNA expression and a cell cycle stage. Another explanation for the absence of p21 RNA expression in a large population of cells after UVC treatment could be the inhibitory effect of UVC-induced DNA damage on transcription (McKay et al. 1998; Ljungman et al. 1999). Combined analysis of BrdU incorporation with expression of histone H3 RNA revealed that DNA synthesis and histone H3 transcription were blocked completely at 7 hr after UVC irradiation (unpublished observations). It has been indicated that this inhibition may even be the trigger by which p53 expression is induced (Ljungman et al. 1999). Because the p21 gene is relatively small (8.6 kb), the percentage of cells that will have pyrimidine dimers in both copies of the p21 gene after a dose of 15 J/m2 will be rather low (McKay et al. 1998). Nevertheless, the relatively low percentages of p21 RNA-positive cells at early time points (28% and 25% at respectively 1 and 3 hr) and the increase of p21 RNA-positive cells at 6 and 17 hr (respectively 52% and 57%) after UVC treatment correlates with the time needed to repair approximately 50% of a given transcriptionally active gene in a cell population (Venema et al. 1990). Our future experiments may provide a more detailed explanation for the observed heterogeneous expression patterns of p21 mRNA.
Footnotes
Acknowledgements
CMH was supported by FWF, Austria, project no. J1481-MED.
We thank Dr El-Deiry (Howard Hughes Medical Institute, University of Pennsylvania) for providing mouse anti-human p21 clone EA10, Dr Simons (Leiden University) for providing VH10 fibroblasts, and Dr Vogelstein (The Johns Hopkins University, Baltimore) for providing the p21 plasmid.