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[Cancer Research 66, 9467-9473, October 1, 2006]
© 2006 American Association for Cancer Research

Cell, Tumor, and Stem Cell Biology

Regulation of hdm2 by Stress-Induced hdm2alt1 in Tumor and Nontumorigenic Cell Lines Correlating with p53 Stability

Chrisanne S. Dias, Yan Liu, Amy Yau, Lindsay Westrick and Susan C. Evans

Department of Chemistry and Biochemistry, Edison Biotechnology Institute, Ohio University, Athens, Ohio

Requests for reprints: Susan C. Evans, Department of Chemistry and Biochemistry, Konneker Research Laboratories, Ohio University, Building 25, The Ridges-Ohio University, Athens, OH 45701. Phone: 740-597-1319; Fax: 740-593-4795; E-mail: evanss1{at}ohio.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Alternative and aberrant splicing of hdm2 occurs in tumor and normal tissues. However, the factors that induce these splice variants and whether they are translated to protein products in vivo is unknown, making it difficult to decipher which of these hdm2 transcripts have a normal physiologic function or contribute to carcinogenesis. We investigated the conditions that induce this post-transcriptional modification of hdm2 in tumor and nontumorigenic cell lines. We showed that UV and {gamma} radiation as well as cisplatin treatment induced alternative splicing of hdm2, which resulted in a single splice variant, hdm2alt1, irrespective of the cell type. Interestingly, the mechanism of UV-induced splicing is independent of p53 status. Immunoanalysis revealed that, after UV radiation, HDM2ALT1 protein was expressed and interacted with HDM2 that correlated to increased p53 protein levels and its accumulation in the nucleus, whereas HDM2 localized more to the cytoplasm with a decrease in its RNA and protein level. We propose that stress-induced HDM2ALT1 regulates HDM2 at two levels, RNA and protein, further modulating the p53-HDM2 interaction or interactions of HDM2 with other cell cycle regulatory proteins. This kind of regulation may possibly restrict oncogenic functions of HDM2 and contribute to the many protective responses triggered by certain stress signals. Our data imply that HDM2ALT1 possesses a normal physiologic function in damaged cells, perhaps facilitating cellular defense. (Cancer Res 2006; 66(19): 9467-73)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
mdm2 was originally identified as an amplified gene on double minute chromosomes in spontaneously transformed 3T3 cells (1). Overexpression of mdm2 forms tumors in athymic mice and is capable of transforming NIH3T3 cells (2, 3). Its human counterpart, hdm2, encodes a 90-kDa (491 amino acids) nuclear phosphoprotein that is overexpressed in several types of human tumors (4). The primary function of the MDM2/HDM2 oncoprotein is the negative regulation of the tumor suppressor, p53 (5). This inhibitory function of HDM2 is mediated by either direct repression of transcriptional activity of p53 in the nucleus or promotion of its nuclear export and proteasomal degradation in the cytoplasm (6).

The mdm2 gene spans ~22 kb of genomic DNA consisting of 12 exons (7). The hdm2 gene spans ~33 kb of genomic DNA and also consists of 12 exons (8). Exons 3 to 12 contain the coding sequence of MDM2 and HDM2, whereas exons 1 and 2 are untranslated regions that regulate the rate of translation (9). More than 40 different mdm2 and hdm2 alternative and aberrant transcripts have been identified in tumors and normal tissues (10). However, there is limited knowledge of their expression patterns and putative functions in vivo.

Evans et al. (11) described the presence of three alternatively spliced hdm2 transcripts, in addition to the full-length hdm2 (FL-hdm2) transcript, in non–small cell lung carcinomas. All three forms lack most of the p53-binding domain, the nuclear localization signal, and the nuclear export signal but retain the zinc and RING finger domains. The smallest form detected, HDM2ALT1, also referred to as MDM2-B (12), containing only exons 3 and 12, binds to HDM2, consequently preventing its p53 inhibition, which leads to an increase in p53 activity in vitro (11).

Our study was initiated on the hypothesis that induction of alternative splicing of the hdm2 gene could be a response of cells to stress conditions to modulate HDM2 and p53 activities. In this report, we present evidence that cellular stress, in the form of UV and {gamma} radiation– and cisplatin-induced DNA damage, induces the alternative splicing of hdm2 to generate a single product, hdm2alt1, in tumor and nontumorigenic cell lines. The timing of hdm2alt1 induction varies with different cellular backgrounds. Interestingly, HDM2ALT1 protein interacts with HDM2 after UV radiation, correlating with an increase in p53 protein levels and its accumulation in the nucleus after cellular damage. The expression of hdm2alt1 also correlates with a decrease in HDM2 RNA and protein levels and its detection in the cytoplasm after the damage.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines and culture conditions. Human breast epithelial adenocarcinoma [MCF-7, wild-type (WT) p53] and colon epithelial carcinoma (RKO, WT p53) cell lines were purchased from American Type Culture Collection (ATCC; Rockville, MD). The bronchial epithelial carcinoma cell line (H1299, null for p53) was provided by Dr. Jack Roth (The University of Texas M. D. Anderson Cancer Center,. Houston, TX). The cell lines were cultured in DMEM (Invitrogen, Carlsbad, CA) supplemented with 10% bovine growth serum (Hyclone) and 1% penicillin/streptomycin (Invitrogen).

The human nontumorigenic immortalized cell lines, colon epithelial (FHC) and bronchial epithelial (NL20), were purchased from ATCC. These cells were cultured in DMEM:Ham's F-12 (1:1) medium with 2.5 mmol/L L-glutamine (Hyclone) supplemented with 10% fetal bovine serum (Hyclone), 1% penicillin/streptomycin (Invitrogen), 10 mg/L insulin (Invitrogen), 20 ng/mL epidermal growth factor (Invitrogen), 0.001 mg/mL transferrin (Invitrogen), and 500 ng/mL hydrocortisone (Sigma, St. Louis, MO). All cell lines were grown in a humidified environment of 5% CO2 at 37°C.

Radiation and drug treatment of cultured cells. Cells at 80% confluency were treated with UV radiation (254 nm) at doses of 2, 5, 10, or 30 J/m2 using a transilluminator (United Visual Products, Inc., Upland, CA) or with {gamma} radiation at doses of 4, 8, or 16 Gy using a 137Cs irradiator (J.L. Shepherd and Associates, San Fernando, CA). Cisplatin [cis-diamminedichloroplatinum(II); Sigma], a DNA-alkylating agent, was given to cultured cells at doses of 10, 50, or 80 µmol/L doses.

RNA isolation and nested reverse transcription-PCR. RNeasy total RNA extraction kit (Qiagen, Valencia, CA) was used to isolate total RNA from the tumor and nontumorigenic cell lines at different time points after UV and {gamma} radiation and cisplatin treatment as well as from nontreated cells. Contaminating DNA was removed from the RNA by treatment with RNase-free DNase (Qiagen) for 30 minutes during the RNA extraction procedure. Concentration of the RNA was determined spectrophotometrically, whereas the integrity and quality was examined by electrophoresis on 1.2% formaldehyde agarose gels. The total RNA was used for reverse transcription-PCR (RT-PCR) with the One-Step RT-PCR kit (Qiagen). A nested PCR approach was used with two primer sets spanning the complete coding region of the hdm2 mRNA to specifically detect any hdm2 splice variants.

The reverse transcription reaction was done with an external primer set [5'-CTGGGGAGTCTTGAGGGACC-3' (sense) and 5'-CAGGTTGTCTAAATTCCTAG-3' (antisense)]. This product was used for a second nested PCR with an internal primer set [5'-CGCGAAAACCCCGGGCAGGCAAATGTGCA-3' (sense) and 5'-CTCTTATAGACAGGTCAACTAG-3' (antisense)]. The products were analyzed on 1.5% agarose gels. As controls, plasmids containing FL-hdm2 or hdm2alt1 coding sequence were amplified using only the internal primer set. Bands were excised from the gel and DNA extracted with Ultrafree-MC centrifugal filter columns (Millipore, Billerica, MA). Extracted DNA was sequenced using the Big Dye Terminator cycle sequencer kit (Applied Biosystems, Foster City, CA) with the ABI Prism 310 genetic analyzer (Applied Biosystems) in the Department of Environmental and Plant Biology at Ohio University (Athens, Ohio).

Real-time RT-PCR. Total RNA (DNA free) was obtained as explained above. First-strand cDNA was synthesized using the iScript Select cDNA synthesis kit (Bio-Rad Laboratories, Hercules, CA). Briefly, 3 µg total RNA and iScript oligo(dT) primers were incubated at 65°C for 7 minutes and then immediately chilled on ice. Reverse transcriptase and iScript reaction mixture were added to a final volume of 20 µL. The reverse transcription reaction was carried out at 42°C for 90 minutes, and at the end of the reaction, the mixture was heated to 85°C for 5 minutes to inactivate the reverse transcriptase.

Experiments were done with nontreated cells and cells treated with 30 J/m2 UV radiation in three separate experiments. In each experiment, cDNAs were assayed in two quantitative PCR runs, yielding six determinations for each sample. All real-time quantitative PCRs were done in 25 µL reaction mixtures containing 3 µL of the cDNA preparation that were amplified with FL-hdm2-specific primers and a master mix (iQ SYBR Green Supermix, Bio-Rad Laboratories) in a thermocycler (iCycler iQ Real-time Detection System, Bio-Rad Laboratories). The PCR conditions were 95°C for 3 minutes and 40 cycles of 94°C for 30 seconds, 60°C for 1 minute, 72°C for 1 minute, and 95°C for 1 minute, followed by 80 cycles of 1°C increments from 55°C every second to determine the melting temperature of the product. The fluorescence threshold value was calculated using the iCycle iQ system software. The absolute number of FL-hdm2 transcripts was calculated by statistically comparing the fluorescence threshold values of the 30 J/m2-treated samples with the nontreated samples. To correct for RNA quality differences, we measured ß-actin mRNA levels in parallel with the same cDNA preparations and calculated the ratio of the absolute number of FL-hdm2 and ß-actin mRNA transcripts in each sample.

The FL-hdm2-specific primers span a region between exon 5 [5'-GAAAGAGCACAGGAAAATA-3'(sense)] and exon 9 [5'-AAAGGAAAGGGAAATACTA-3' (antisense)] that is absent in hdm2alt1 generating a product of 300 bp. The primer set was first analyzed for the best annealing temperature using the FL-hdm2 control plasmid in a gradient temperature PCR. hdm2alt1 plasmid was also used for PCR with the same primer set, serving as a negative control. ß-Actin-specific primers [5'-TGTGATGGTGGGAATGGGTCAG-3' (sense) and 5'-TTTGATGTCACGCACGATTTCC-3' (antisense)] were obtained from the real-time PCR database that generated a product of 300 bp. The products of all PCRs were analyzed on 1.5% agarose gels.

Immunofluorescence. Cells were grown on glass coverslips in complete medium and irradiated with 30 J/m2 UV radiation. After 24 hours, cells were washed in 1x PBS and fixed in 95% ethanol/5% acetic acid for 5 minutes at –20°C. Cells were incubated for 30 minutes at room temperature in PBS/2% chicken serum (Hyclone) and then for 1 hour at room temperature with antibodies N20, SMP14, or Bp53-12 (Santa Cruz Biotechnology, Santa Cruz, CA) diluted 1:400 in PBS/2% chicken serum. The N20 epitope spans 20 amino acids within the NH2 terminus of FL-HDM2 and HDM2ALT1. The SMP14 epitope spans amino acids 154 to 167 of FL-HDM2, which is absent in HDM2ALT1. The Bp53-12 epitope is near the NH2 terminus of p53. Cells were then incubated for 1 hour at room temperature in the appropriate Texas red–conjugated secondary antibody (Vector Laboratories, Burlingame, CA) diluted 1:500 in PBS/2% chicken serum. After thorough washing, cells were incubated in 4',6-diamidino-2-phenylindole (DAPI) for 30 minutes at room temperature. Finally, the coverslips were mounted onto slides in antifade mounting medium (Sigma). Photographs were taken using a Nikon (West Chester, OH) microscope equipped with a digital camera (SPOT, DC Imaging) at x400 magnification.

Immunoprecipitation and immunoblotting. Cells before and after 30 J/m2 UV radiation were lysed in buffer [50 mmol/L Tris-HCl (pH 7.6), 150 mmol/L NaCl, 0.05% SDS, 0.5% IGEPAL CA-630 (Sigma)] supplemented with a cocktail of protease inhibitors (Roche, Indianapolis, IN). For immunoprecipitation, equal amounts of lysate from MCF-7 cells were incubated with rabbit polyclonal H221 (Santa Cruz Biotechnology) overnight at 4°C and then with protein A-Sepharose (Sigma) for 1 hour at 4°C. H221 epitope spans amino acids 100 to 320 of FL-HDM2, thus not recognizing HDM2ALT1. The precipitate was separated by 10% SDS-PAGE and protein was electroblotted onto Immobilon-P membrane (Millipore). The blot was blocked in 5% milk/PBS-Tween 20 (0.1%) and probed with mouse monoclonal 4B11 (for HDM2-HDM2ALT1 interaction) diluted 1:200 in blocking buffer. The 4B11 (provided by Dr. Arnold Levine, Cancer Institute of New Jersey, New Brunswick, NJ) epitope spans amino acids 383 to 491 of FL-HDM2 and HDM2ALT1. As a control for the HDM2-HDM2ALT1 interaction, MCF-7 cells were transiently transfected with a plasmid (pCMV-HIS-ALT1) using LipofectAMINE 2000 (Invitrogen). The transfected cells were harvested for total protein 24 hours after transfection.

Equal volumes of the supernatants from the immunoprecipitation reaction were immunoblotted for actin with mouse monoclonal C2 (Santa Cruz Biotechnology) providing a loading control. Equal amounts of total extracts were also immunoblotted for total p53 using Bp53-12 and HDM2 using 4B11 with actin as the loading control. Appropriate horseradish peroxidase–conjugated secondary antibodies (Santa Cruz Biotechnology) diluted 1:20,000 in blocking buffer were used and proteins were detected by chemiluminescent enhanced chemiluminescence kit (Amersham, Piscataway, NJ).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
hdm2 is alternatively spliced after radiation- and cisplatin-induced damage. Using total RNA isolated from irradiated and cisplatin-treated cells and nontreated cells WT for p53 (tumor cell lines, MCF-7 and RKO; nontumorigenic cell lines, NL20 and FHC), we did nested RT-PCR with primers outside the coding region of hdm2. PCR analysis revealed the presence of a smaller variant transcript (~660 bp) in addition to the full-length transcript (~1550 bp; Figs. 1 and 2 ). DNA sequencing analysis of the smaller transcript verified it to be hdm2alt1, described previously by Evans et al. (11).


Figure 1
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  		Figure 1. Stress-induced alternative splicing of hdm2. Nested RT-PCR of total RNA isolated from RKO, MCF-7, and NL20 cells after UV radiation (10 and 30 J/m2; A), {gamma} radiation (4 and 8 Gy; B), and cisplatin treatment (80 µmol/L; C) of MCF-7 cells at different time points (numbers above each gel in hours) amplified with primer sets outside the coding region of hdm2. These were compared with untreated cells (0 hour). The products of the second round PCR were electrophoresed on 1.5% agarose gels. F and A, controls; FL-hdm2 and hdm2alt1, respectively, amplified from plasmids.

 

Figure 2
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  		Figure 2. UV-induced hdm2 alternative splicing is p53 independent. Nested RT-PCR of total RNA isolated from H1299 cells after UV radiation (10 and 30 J/m2; A) and {gamma} radiation (4, 8, and 16 Gy; B) at different time points (numbers above each gel in hours) amplified with primer sets outside the coding region of hdm2. These were compared with untreated cells (0 hour). The products of the second round PCR were electrophoresed on 1.5% agarose gels. F and A, controls; FL-hdm2 and hdm2alt1, respectively, amplified from plasmids.

 
After 30 J/m2 UV treatment, hdm2alt1 was detected between 6 and 48 hours in RKO and NL20 cells, whereas the expression of hdm2alt1 occurred as early as 4 hours and persisted throughout the time course of 98 hours in MCF-7 cells (Fig. 1A). Importantly, before UV treatment (0 hour), only FL-hdm2 transcript was detected in all three cell lines (Fig. 1A). Interestingly, hdm2alt1 was detected, albeit weakly, only at a single time point (6 hours) after 10 J/m2 UV radiation in the MCF-7 cells but at 18 and 24 hours in the NL20 cells after the same dose of UV radiation. At 48 hours, only FL-hdm2 was detected in both cell types. hdm2alt1 was not detected in any of the cell lines after 2 and 5 J/m2 UV radiation (data not shown). Similar splicing patterns to NL20 were observed in the other nontumorigenic cell line, FHC (data not shown).

MCF-7 and RKO cells did not express hdm2alt1 after 4 Gy {gamma} radiation, whereas it was weakly detected only at 24 hours with the same dose in the NL20 cells (Fig. 1B). After 8 Gy radiation, hdm2alt1 was detected in MCF-7 and NL20 cells at 24 hours and persisted until 72 hours. hdm2alt1 was not detected in RKO cells after 8 Gy radiation. Interestingly, in nonirradiated cells or with lower doses of UV (2 and 5 J/m2) and {gamma} (4 Gy) radiation, alternative splicing of hdm2 was not detected.

hdm2alt1 was detected between 24 and 72 hours in MCF-7 (Fig. 1C) and RKO cells (data not shown) after treatment with 80 µmol/L cisplatin. However, with 50 µmol/L cisplatin treatment, it was detected between 24 and 72 hours only in MCF-7 cells (data not shown). Alternative splicing was not detected in any of the WT p53 cell lines with 10 µmol/L cisplatin treatment (data not shown).

UV-induced hdm2 alternative splicing is independent of p53. Nested RT-PCR analysis was done with total RNA isolated from H1299 cells (null for p53) before and after UV and {gamma} radiation and cisplatin treatment. hdm2alt1 was expressed at 12, 18, and 24 hours after 30 J/m2 UV radiation, whereas it was detected only at 12 hours after 10 J/m2 (Fig. 2A). Only the FL-hdm2 transcript was detected before irradiation. hdm2alt1 was not detected after 2 and 5 J/m2 UV radiation (data not shown) or after 4, 8, and 16 Gy {gamma} radiation (Fig. 2B). In addition, cisplatin-induced damage (10, 50, or 80 µmol/L) did not induce hdm2alt1 in these cells (data not shown).

HDM2ALT1 interacts with HDM2 correlating with change in subcellular localization of HDM2 and p53 after damage. Immunoprecipitation of HDM2 with H221, which recognizes only FL-HDM2, followed by immunoblot analysis with 4B11, which recognizes both forms of HDM2, showed the interaction of HDM2ALT1 with HDM2 (Fig. 3 ). This interaction was observed after irradiation (30 J/m2) when hdm2alt1 transcripts were detected, whereas in nontreated cells, HDM2ALT1 protein is totally absent, supporting our RT-PCR results.


Figure 3
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  		Figure 3. HDM2ALT1 interacts with HDM2. Equal amounts (1 mg) of total cellular lysates of MCF-7 cells before (0 hour) and 24 hours after irradiation (30 J/m2) were immunoprecipitated with H221 and immunoblotted with 4B11 (see Materials and Methods). MCF-7 cells transiently transfected with an hdm2alt1 plasmid (pCMV-HIS-ALT1) was used as a control (T). Actin was used as the loading control.

 
To investigate the significance of this interaction, we analyzed the localization of HDM2 and p53 in cells before and after UV radiation (30 J/m2) using N20 antibody (data not shown), SMP14 antibody specific for FL-HDM2, and Bp53-12 antibody specific for p53 (Fig. 4 ). The subcellular localization of HDM2ALT1 could not be examined because an antibody that specifically recognizes HDM2ALT1 and does not cross-react with HDM2 is unavailable. Before irradiation, HDM2 was more localized to the nucleus, whereas p53 was at low levels and diffused between the cytoplasm and nucleus in MCF-7 and RKO cells (Fig. 4A and B). However, 24 hours after UV radiation, HDM2 was present in the cytoplasm, whereas p53 accumulated in the nucleus. In p53-null H1299 cells, HDM2 was also detected in the nucleus before radiation but the localization did not change significantly after radiation (Fig. 4C).


Figure 4
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  		Figure 4. Subcellular localization of HDM2 and p53. Immunofluorescence analysis before (0 hour) and 24 hours after UV radiation (30 J/m2) stained with SMP14 for HDM2 or Bp53-12 for p53 in MCF-7 (A), RKO (B), and H1299 cells (C; see Materials and Methods). Texas red–conjugated secondary antibodies were used for detection (converted to green). Cells were also stained with DAPI (blue) for localization of the nucleus and stains were merged using SPOT Advanced software. All pictures were photographed at x400 magnification.

 
hdm2alt1 expression correlates with decreased hdm2 RNA and protein expression and increased p53 levels after damage. As we observed persistent and strong expression of hdm2alt1 after 30 J/m2 UV radiation by conventional RT-PCR analysis, we did a real-time quantitative RT-PCR assay to examine the effect of hdm2alt1 induction on hdm2 gene expression before and after 30 J/m2 UV treatment. Intriguingly, we found a decrease in hdm2 RNA transcripts after UV radiation in MCF-7, NL-20 (Fig. 5A and B ), and H1299 cells (data not shown), corresponding with the detection of hdm2alt1 transcripts by conventional RT-PCR.


Figure 5
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  		Figure 5. FL-hdm2 RNA levels decrease after UV damage corresponding with hdm2alt1 induction. Equal amounts of total RNA (DNA free) of MCF-7 (A) and NL-20 (B) cells before and after 30 J/m2 UV radiation at different time points were used for real-time RT-PCR to quantitate hdm2 RNA transcripts. FL-hdm2-specific primers and iCycler iQ Real-time Detection System were used for the analysis. ß-Actin mRNA levels were also measured from the same cDNA preparations and used to normalize FL-hdm2 mRNA levels. The calculated mRNA levels of FL-hdm2 and ß-actin after irradiation were first compared with those of the nontreated samples and then expressed as a ratio of FL-hdm2 and ß-actin transcript levels. Each sample was analyzed in three separate experiments. In each experiment, cDNAs were assayed in two quantitative PCR runs, yielding six determinations for each sample that were used to calculate the SD.

 
We further elucidated the regulatory effects of hdm2alt1 expression on HDM2 and p53 by examining their protein levels before and after UV radiation–induced damage (30 J/m2). Immunoblot analysis of total HDM2 with 4B11 and total p53 with Bp53-12 antibodies indicated a decrease in HDM2 and an increase in p53 protein levels after irradiation in MCF-7 and NL-20 cells (Fig. 6A and B ). In the H1299 cells that are null for p53, we confirmed the absence of p53 by Western blot analysis and also detected a decrease in HDM2 protein levels after UV radiation (data not shown).


Figure 6
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  		Figure 6. HDM2 and p53 protein levels. Equal amounts of total cellular lysates of MCF-7 (A) and NL-20 (B) cells before (0 hour) and after 30 J/m2 UV radiation (6 and 24 hours) were analyzed for total HDM2 and p53 using 4B11 and Bp53-12 antibodies, respectively. Actin was used as the loading control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Cellular stressors, such as radiation, induce p53 transcriptional activity resulting in cell cycle arrest, DNA repair, or apoptosis through the transcription and expression of genes involved in these cell cycle regulatory processes (13). To elicit its transcriptional function, p53 must be stabilized and imported to the nucleus. HDM2, a transcriptional target of p53, forms a negative feedback loop that decreases the levels and stress-induced responses of p53 (14). Inhibiting the interaction of p53 with HDM2 contributes to the stabilization and activation of the cell cycle regulatory functions of p53. With intense cellular damage, different mechanisms have been adopted to decrease the p53-HDM2 interaction and increase p53 stability and activity, such as post-translational modifications and interaction with other cellular proteins (e.g., p14ARF and ribosomal proteins L5, L11, and L23; refs. 15–17). In this study, we propose a novel mechanism, the alternative splicing of hdm2, which may contribute to the disruption of p53-HDM2 interactions during cellular stress conditions.

Alternative splicing of hdm2 to a single form, hdm2alt1, was detected in tumor and nontumorigenic cells after radiation- and cisplatin-induced cellular damage. Intriguingly, only after higher doses of radiation (10-30 J/m2 and 8 Gy) and cisplatin (50 and 80 µmol/L) hdm2alt1 was detected, whereas, in nontreated cells or at lower doses of radiation (2 and 5 J/m2 and 4 Gy) and cisplatin (10 µmol/L), it was not detected. The timing of alternate splicing varied in the different cell lines. The presence of only one alternatively spliced variant of hdm2 after cellular damage indicates that this form may possess a physiologic role in protective response of cell to certain levels of stress.

The alternative splicing of hdm2 occurring only at early time points after 10 J/m2 UV treatment in the NL20 and H1299 cells may indicate that these cells are more sensitive to UV radiation but the damage caused might be readily repairable. After the damage is repaired, the cell reverses the alternative splicing regulation of hdm2 to restore only FL-hdm2. However, not all cell lines examined responded the same, indicating that different cellular backgrounds may have an effect on the splicing regulation of hdm2. The prolonged expression of hdm2alt1 in cells after high doses of radiation (30 J/m2 and 8 Gy) and cisplatin (80 µmol/L) may indicate that the damage caused is so severe that the cell chooses an alternative surveillance response through the alternative splicing of hdm2 to regulate HDM2 activity. This response can be correlated to the stabilization and activation of p53 through the down-regulation of HDM2. MCF-7 cells that lack p14ARF support this hypothesis. p14ARF is important in activating a p53 response to abnormal proliferation by binding and inactivating HDM2 (16, 18). Therefore, the prolonged expression of hdm2alt1 in these cells may be an alternative mechanism to down regulate HDM2 and activate p53 and its tumor-suppressive functions. However, the in vivo mechanism of HDM2ALT1 activity is not completely understood.

As shown by Evans et al. (11), HDM2ALT1 can bind HDM2 inhibiting its interaction with p53, thus increasing the activity of p53 in vitro. Our results from the protein studies in MCF-7 and NL-20 cells showed that p53 protein levels continuously increased through the time course after UV radiation. However, HDM2 protein levels decreased corresponding with a decrease in its RNA transcript levels after UV-induced cellular damage, contrary to its expected increase after induction of p53. These changes could be accounted for by the presence of HDM2ALT1.

We showed that endogenous HDM2ALT1 interacts with HDM2 after UV radiation and correlate this interaction with the observed increments in p53 protein levels. This is further correlated to the change in subcellular localization of HDM2 and p53 observed after the UV-induced damage, with p53 accumulating in the nucleus and HDM2 in the cytoplasm, indicating a decrease in p53-HDM2 interactions. These results support the mechanism that, after cytotoxic damage, HDM2ALT1 protein binds HDM2 and sequesters it in the cytoplasm resulting in p53 stabilization and activation.

Therefore, depending on its background, a cell responds to certain damage conditions by inducing alternative splicing of hdm2 that regulates HDM2 at two levels. First, the expression of hdm2alt1 RNA transcripts results in a decrease of the FL-hdm2 transcript levels that may prevent the expected steep increments in HDM2 protein after induction of p53 activity after irradiation. Second, after irradiation, the hdm2 that is translated is further inhibited by interaction with HDM2ALT1 that sequesters it in the cytoplasm away from p53 allowing p53 to do its transcriptional function in the nucleus.

In this study, we showed that UV-induced alternative splicing regulation of hdm2 is independent of p53 as shown in the H1299 cells that are null for p53. Thus, this post-transcriptional modification of hdm2 may have a p53-independent function that directly or indirectly regulates the response of cell to certain damage conditions. HDM2 regulates cell proliferation, cell cycle progression, and survival of tumor cells through p53-independent pathways by interacting with a variety of cell cycle checkpoint proteins (19, 20). Depending on the type of stress signals, a cell may induce the alternative splicing of hdm2 that further may interfere with these interactions and augment the response to stress. However, further investigation of this mechanism is required.

It is rather intriguing that 30 J/m2 UV radiation had the most profound effect on hdm2 splicing irrespective of the cell types. It would be interesting to identify the molecular mechanisms that are activated or inactivated by this particular dose of DNA/cellular damage in relation to the alternative splicing of hdm2. This knowledge along with our data may give us an insight to other damaging sources, possibly chemotherapeutic agents that could be used to regulate hdm2 activity via its alternative splicing.

Several mdm2/hdm2 splice variants, including hdm2alt1, are expressed in malignant cells and tissues (10). Some of these variants show transforming ability (12, 21, 22). However, many of the same alternatively and aberrantly spliced variants are detected in normal cells and tissues, indicating that these spliced forms might possess normal physiologic functions (23, 24). It is plausible that these spliced transcripts are expressed during transformation or may be remnants found during recuperation of a cell previously exposed to transforming signals; however, this hypothesis remains veiled. From our study, it seems highly conceivable that at least one spliced hdm2 variant, hdm2alt1, plays a role in directing the fate of a damaged cell toward apoptosis or DNA repair through the down-regulation of oncogenic function of hdm2. Regulation of response of a cell to damage by hdm2alt1 may be a contribution to the several other protective responses that are induced for maintaining homeostasis.


   Acknowledgments
 
Grant support: Ohio University Research Council Award and Pharmacia.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Martin Schmerr for his valuable suggestions and critical discussion and Dr. Arnold Levine for providing us with the mouse monoclonal antibody (4B11).

Received 8/30/05. Revised 7/15/06. Accepted 7/27/06.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Cahilly-Snyder L, Yang-Feng T, Francke U, George DL. Molecular analysis and chromosomal mapping of amplified genes isolated from a transformed mouse 3T3 cell line. Somat Cell Mol Genet 1987;13:235–44.[CrossRef][Medline]
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