|Home | About | Journals | Submit | Contact Us | Français|
The TP53 tumor suppressor gene is the most frequently inactivated gene in human cancer identified to date. However, TP53 mutations are rare in human mesotheliomas, as well as in many other types of cancer, suggesting that aberrant TP53 function may be due to alterations in its regulatory pathways. Mouse Double Minute 4 (MDM4) has been shown to be a key regulator of TP53 activity, both independently as well as in concert with its structural homolog, Mouse Double Minute 2 (MDM2). The purpose of this study was to characterize the effects of MDM4 suppression on TP53 and other proteins involved in cell cycle control before and after ultraviolet (UV) exposure in MeT5a cells, a nonmalignant human mesothelial line. Short hairpin RNA (shRNA) was used to investigate the impact of MDM4 on TP53 function and cellular transcription. Suppression of MDM4 was confirmed by Western blot. MDM4 suppressed cells were analyzed for cell cycle changes with and without exposure to UV. Changes in cell growth as well as differences in the regulation of direct transcriptional targets of TP53, CDKN1A (cyclin-dependent kinase 1α, p21) and BAX, suggest a shift from cell cycle arrest to apoptosis upon increasing UV exposure. These results demonstrate the importance of MDM4 in cell cycle regulation as well as a possible role in the pathogenesis of mesothelioma-type cancers.
Regulation of the four distinct phases of the eukaryotic cell cycle is highly complicated and tightly controlled. Balancing the need for cell death signaling with strong proliferation or cell cycle arrest is of primary importance for maintaining genetic integrity and preventing cancer. There are highly conserved molecular linkages between these different signaling pathways that are affected when aberrant activity occurs in one or more of the links (Maddika et al., 2007). The TP53 tumor suppressor gene is of central importance in the regulation of cell proliferation or cell death following DNA damage.
Genetic conservation and regulation of the TP53 tumor suppressor gene is arguably the most important factor involved in the development of human cancer. While the TP53 gene is mutated in a wide array of cancers, a significant number of patients develop cancer with no detectable mutations in the gene (Danovi et al., 2004). This suggests that genetic or functional aberrations in genes that regulate TP53 are a factor in human cancers that retain wild type TP53.
Mouse double minute 4 (MDM4) and its structural homolog, mouse double minute 2 (MDM2), are two negative regulators of TP53 (Brooks and Gu, 2006; Marine and Jochemsen, 2005) and have recently been implicated as amplification targets in wild type TP53 bladder cancers (Veerakumarasivam et al., 2008) and papillary thyroid carcinomas (Prodosmo et al., 2008). MDM2 is an E3 ubiquitin ligase that targets TP53 for degradation (Michael and Oren, 2002). The exact role that MDM4 plays in the regulation of TP53 is not completely understood. MDM4 has been shown to inhibit the transactivating activity of TP53 in transient overexpression studies (Mancini et al., 2004). Furthermore, Stad et al., (2000) proposed a model in which MDM4 interacts with intracellular TP53 reserves, maintaining it in the inactive state until needed. Studies looking at the expression levels of both MDM2 (Momand et al., 2000) and MDM4 (Riemenschneider et al., 2003) have documented an increase in the protein levels in a significant percentage of cancer patients.
The TP53 tumor suppressor gene is a direct transcription factor for CDKN1A and Bcl2-associated × protein (BAX), both of which are involved in cell cycle control. The CDKN1A protein is a member of the Cip1/Waf1/Kip1-2-family and is known to contribute to cell cycle arrest when upregulated upon TP53 activation (El-Deiry et al., 1993; Harper et al., 1993). The BAX protein is a member of the BCL-2 family and is primarily associated with acceleration of apoptosis (Miyashita and Reed, 1995). Studies have shown that MDM4-null embryos die at 7.5 days postcoitum (dpc) due to cell cycle arrest (Chavez-Reyes et al., 2003). However, concomitant deletion of the CDKN1A gene in an MDM4-null embryo switched the cause of death from cell-cycle arrest to apoptosis, suggesting a primary role for the CDKN1A gene in the absence of MDM4. Upregulation of proapoptotic proteins, such as BAX, is a likely cause of the shift from cell cycle arrest to apoptosis following CDKN1A deletion in these mice. In this study, we report a transition from cell cycle arrest following ultraviolet (UV) irradiation in an MDM4 suppressed human mesothelial cell line to apoptosis with increasing UV exposure.
Three short hairpin RNA (shRNA) bacterial glycerol stocks, each targeting a different region of the coding region of MDM4 (Sigma Aldrich; St. Louis, MO) were used. Following plasmid DNA preparation, 293FT cells were transfected with each shRNA plasmid with Lentivral ViraPower Packaging Mix (Invitrogen; Carlsbad, CA). MeT5a cells were transduced with each shRNA lentiviral stock. Stably transduced cell lines were grown using puromycin selection and individual clones isolated.
Cells were harvested in a PBS-TDS lysis buffer and protein levels measured using the BioRad Protein DC assay. Samples were loaded onto a 4-12 % Bis-Tris Nupage gel and then transferred onto a PVDF membrane. Membranes were blocked and the primary antibody added (MDM2 Calbiochem; San Diego CA, MDM4 Santa Cruz Biotechnology; Santa Cruz, CA, TP53 Santa Cruz Biotechnology; CDKN1A Santa Cruz Biotechnology; BAX Trevigen, Inc.; Gaithersburg, MD, ATM Abcam; Cambridge, MA). Following incubation with the primary antibody, membranes were washed with TBST and a goat anti-mouse or goat anti-rabbit secondary antibody was added. Membranes were washed with TBST prior to chemiluminescence detection using the immobilon detection kit. Blots were stripped and re-probed using an anti-β actin or tubulin antibody (Cell Signaling Technology; Danvers, MA).
Cells were seeded in 6-well plates at a density of 10,000 cells/well and allowed to attach overnight. Cells were trypsinized, centrifuged, and resuspended in 1 ml media on days 1, 3, 6, 9, and 15 then counted using a hemacytometer.
ShRNA-Control and MDM4 suppressed MeT5a cells were seeded at a density of 20,000 cells/well in a 96-well plate and allowed to attach overnight. Cells were then exposed to 0, 2.5, or 5 J/m2 UV radiation and allowed to incubate for 24 hours. UV irradiation was performed using a Stratagene Stratalinker (LaJolla, CA) using 254nm UV. Apoptosis was then measured using the Titertacs assay (Trevigen, Inc.; Gaithersburg, MD) according to manufacturers protocol.
ShRNA-Control and MDM4 suppressed MeT5a cells were seeded at a density of 10,000 cells/well in a 96-well plate and allowed to attach overnight. Cells were then exposed to 0 or 2.5 J/m2 UV radiation. BrdU labeling reagent was added and cells were allowed to incubate for 24 h. The BrdU uptake assay was completed following the manufacturers protocol (Calbiochem; San Diego, CA).
Total RNA was isolated from MDM4-3859 suppressed or shRNA-Control MeT5a cells exposed to 2.5 J/cm2 after 4 hours using the standard Trizol Reagent (Invitrogen; Carlsbad, CA) according to manufacturer's protocol. A further RNA cleanup step was performed using an RNeasy (Qiagen; Valencia, CA) to remove contaminants. RNA quantity and quality were assessed using a Nanodrop Spectrophotometer (Nanodrop Technologies; Wilmington, DE) and Agilent Bioanalyzer (Agilent Technologies; Santa Clara, CA). Reverse transcription and cDNA labeling were performed using 8 μg of total RNA according to the Superscript Plus Direct cDNA Labeling Kit (Invitrogen; Carlsbad, CA). Labeling efficiency was determined using a Nanodrop Spectrophotometer. Labeled cDNA was hybridized overnight at 42 °C to in-house spotted arrays of oligonucleotides produced by MWG Biotech, Inc. (Des Moines, IA). Then, 9850 oligos designed to detect expression of human genes with a known function or clearly defined protein domains were spotted in duplicate on epoxysilane slides (Erie Sci.; Portsmouth, NH). Arrays were scanned and analyzed using an Axon GenePix 4000B microarray scanner and Axon GenePix Pro 5.1 software (Molecular Devices; Downingtown, PA). Further analyses were performed using Iobion GeneTraffic Duo (Stratagene; La Jolla, CA), GoMiner (NCBI), and PathwayStudio (Ariadne Genomics; Rockville, MD).
The role of the MDM4 protein in nonmalignant mesothelial cell growth and phenotype has been largely unexplored. Using shRNA, we achieved suppression of MDM4 ranging from 50-70 % in MeT5a cells using 3 different shRNA constructs (Figure 1). MDM4 is known to interact with and regulate the function of the TP53 tumor suppressor protein. The TP53 protein is known to be a primary regulator of the cell cycle, acting at cell cycle checkpoints to halt cell growth by initiating cell cycle arrest, or conversely signal termination of the cell via apoptosis. Alternatively, removal of TP53 function, either by normal regulation or through aberrant pathways, can signal for the cell to continue through the cell cycle and result in mitosis.
We investigated whether the loss of MDM4 would have an overall impact on the rate of cell growth in non-stressed MeT5a cells. Figure 2 shows that both the rate of cell growth and the growth curve Vmax were significantly reduced in cells with suppressed MDM4 synthesis. This remained true in MDM4-suppressed cells, regardless of the amount of suppression. There was not a significant difference in growth between cells with 50 % or 70 % loss of MDM4.
It is known that exposure to UV irradiation can result in an increase in TP53 activity, often resulting in the induction of apoptosis (Qin et al., 2002). We tested the impact of MDM4 suppression on the ability of these cells to survive UV irradiation by measuring apoptosis using the Titertacs assay. There was a significant increase in the number of cells undergoing apoptosis after 5 Joules/meter2 (J/m2) UV exposure in the cells with 60 % and 70 % MDM4 suppression after 24 h (Figure 3). Interestingly, this effect was not seen with an exposure of 2.5 J/m2. Since we observed a decrease in cell growth after MDM4 suppression in non-stressed cells, we hypothesized that the cell cycle was still being negatively regulated after exposure to 2.5 J/m2, but the mechanism was not through apoptosis. We measured BrdU uptake to test the possibility that, at lower UV exposure, the MDM4-suppressed cells were going into cell cycle arrest to a greater degree than shRNA-Control cells. There was a significant decrease in the amount of BrdU uptake in the 70 % MDM4-suppressed cells exposed to 2.5 J/m2 after 24 h (Figure 4) as compared to the shRNA-Control cells, suggesting a change in TP53 signaling at higher doses of UV exposure in MDM4-suppressed cells.
A microarray analysis of the MDM4-suppressed versus shRNA-Control cells was done after UV exposure in order to determine possible pathways involved in the changes in cell cycle regulation. Increases in the expression of ataxia telangiectasia mutated gene (ATM), B-cell lymphoma 3 (BCL-3), BCL-2, CDKN1B (p27) and CDKN1C (p57) were all observed after exposure to 2.5 J/m2 UV while the expression of CDKN1A was reduced in MDM4 suppressed versus shRNA-Control cells (Table 1). ATM is upregulated in response to UV and is responsible for TP53 phosphorylation, which effectively stabilizes the protein (Barlow et al., 1997). An increase in ATM in MDM4-suppressed cells when compared to shRNA-Control cells could play a significant role in increasing the overall activity of TP53. We confirmed that ATM is upregulated at the protein level in MDM4-suppressed cells when compared to shRNA-Control cells (Figure 5). Mutations in the ATM gene are linked to loss of cell cycle control checkpoints and increases in the incidence of cancer (Pereg et al., 2005). It is possible that the changes in cell cycle regulation observed in this study are partially due to changes in the regulation of the ATM gene. In addition to ATM, an increase in BCL-3 transcripts was observed by microarray analysis. BCL-3 is transiently upregulated after DNA damage and loss of BCL-3 leads to enhanced TP53 activity (Kashatus et al., 2006).
Amplification of the MDM4 gene has been shown to inhibit TP53 activity (Francoz et al., 2006) and here we show evidence supporting an increase in TP53 protein expression with MDM4 suppression (Figures 6 and and7).7). Perhaps upregulation of BCL-3 is a cellular attempt to “balance” the increased activity of TP53 due to MDM4 loss and may ultimately contribute to whether the cell remains in cell cycle arrest or is pushed into apoptosis. Furthermore, microarray analysis shows an increase in BCL-2. BCL-2 is an anti-apoptotic protein. Therefore, an increase in BCL-2 at 4 hours would support no increase in apoptosis at 24 h after 2.5 J/m2 of UV as reported here. The p27Kip1 and p57Kip1 proteins are members of the Kip/Cip family of cyclin-dependent kinase inhibitors, similar to the CDKN1A. Both the p27kip1 (Nickeleit et al., 2007) and p57kip1 (Puhalla et al., 2007) proteins are involved in cell cycle regulation and are implicated in various cancers.
There are many possible changes in the cell machinery that would result in a shift from cell cycle arrest to apoptosis with increasing UV exposure. We measured changes in the expression of various proteins that are controlled by TP53 and are important for regulating the cell cycle. An initial evaluation of TP53 itself showed an increase in TP53 protein that correlated with MDM4-suppression in non-stressed cells. There was an increase in the amount of detectable TP53 protein 24 hours following UV exposure in both the MDM4-suppressed and shRNA-Control cells; however the increase was significantly higher in the MDM4-suppressed cells (Figure 6a).
MDM2 is a protein homolog of MDM4 that is known to regulate TP53 by targeting it for degradation through the ubiquitin-proteasome pathway. There is evidence to suggest that MDM2 and MDM4 can act in concert as well as independently in order to fine-tune the regulation of TP53. Therefore, we looked for changes in the expression of MDM2 in response to MDM4-suppression in both UV-exposed and non-exposed cells. Overall, there were no significant changes in the expression of MDM2 under these conditions (Figure 6b), suggesting that loss of MDM4 has no impact on the regulation of TP53 by MDM2.
The TP53 tumor suppressor gene regulates the cell cycle by inducing cell cycle arrest as well as apoptosis, depending on circumstances. The CDKN1A protein can be upregulated in response to an increase in TP53 activity and is a key component for cell cycle arrest. There were no differences in the basal levels of the CDKN1A protein in MDM4-suppressed cells versus shRNA-Controls. In support of the microarray data, expression of CDKN1A can no longer be detected 24 hours after UV exposure (data not shown). We were interested to see if the proteins involved in apoptosis were differentially regulated with increasing exposure to UV irradiation and whether these changes would correlate with the apoptosis results. We measured the pro-apoptotic BAX protein at 1-hour post-UV exposure at 2.5 J/m2. An increase in the BAX protein was observed in the MDM4-suppressed cells versus shRNA-Control cells both basally and following 2.5 J/m2 UV irradiation, although the increase was significant following UV exposure only (Figure 7 a and b). This supports the hypothesis that MDM4 is involved in a transition from cell cycle arrest to apoptosis signaling with increasing UV exposure. Furthermore, there was a detectable increase in TP53 levels in the MDM4-suppressed cells versus shRNA-Controls 1 hour following UV irradiation (Figure 7 c and d).
Loss of MDM4 results in a lethal embryonic phenotype 7.5 dpc due to improper regulation of the TP53 tumor suppressor gene and increased cell cycle arrest (Parant et al., 2001). There is a concomitant increase in the CDKN1A protein with loss of MDM4 in these mice. However, deletion of the CDKN1A gene in MDM4 null mice does not “rescue” the embryo but rather switches the cause of death from cell cycle arrest to apoptosis. In MeT5a cells with suppressed MDM4 protein levels we see similar results; however complete cell death does not occur until the cells are exposed to some type of “stress”. In this study, we use UV irradiation to induce DNA damage and cause cellular stress. Interestingly, the amount of UV exposure the MDM4-suppressed cells receive determines whether they remain in cell cycle arrest or undergo apoptosis.
Loss of MDM4 in mouse embryonic fibroblasts (MEF) results in a decreased rate of proliferation similar to the results observed here (Steinman et al., 2004). The cyclin-dependent kinase, CDKN1A, was upregulated in MDM4 null MEF, and evidence suggests that CDKN1A may act as a downstream mediator of MDM4. Therefore, the complete loss of the CDKN1A protein following exposure to UV irradiation could have a significant influence on whether the cells are programmed for cell death or remain in cell cycle arrest. In contrast, Jin et al., (2008) showed that MDM4 has a direct regulatory effect on CDKN1A in three separate cell lines, including HEK, H1299 and MEF cells. According to these authors, MDM4 binding to CDKN1A helps facilitate its degradation. These apparent contradictions will need to be further studied for clarification. However, regulation of the CDKN1A gene appears to be cell-type and dose-dependent as well as being affected by the medium conditions used during exposure (Itoh and Linn, 2005). We can no longer detect the presence of the CDKN1A protein 24 h after UV exposure under our conditions. It is possible that under different conditions the expression profile of the CDKN1A protein would remain unchanged or even increase.
Microarray analysis of UV exposed MDM4-suppressed cells showed several possible pathways that could be involved in the “switch” from cell cycle arrest to apoptosis. Increases in ATM, BCL-3, BCL-2, CDKN1B (p27) and CDKN1C (p57) were all observed after exposure to 2.5 J/m2 UV in MDM4-suppressed versus shRNA-Control cells. Changes in ATM in particular could play an important role in the function of MDM4 in communicating to the cell to undergo apoptosis, maintain cell cycle arrest, continue proliferating, or initiate carcinogenesis.
We are pleased to acknowledge the assistance of Shane T. Heivly in the generation of the shRNA-Control and MDM4 suppressed cells.
Grant Support: This manuscript was possible due to Grant Number RR017670 from the National Center for Research Resources (NCRR), a component of the National Institutes of Health (NIH), an EPSCoR fellowship to A.K.E. (NSF-0346458) and the UM Small Grants Program (MBS). This manuscript was also supported by the efforts of the Microarray Core Facility sponsored by the Center for Environmental Health Sciences at the University of Montana.
Melisa Bunderson-Schelvan, The University of Montana, Center for Environmental Health Sciences.
Amy K. Erbe, Dept. of Biochemistry, University of Wisconsin, Madison.
Corbin Schwanke, The University of Montana, Center for Environmental Health Sciences.
Mark A. Pershouse, The University of Montana, Center for Environmental Health Sciences, 32 Campus Drive, Missoula, MT 59812, Phone: (406) 243-4769; Fax: (406) 243-5228.