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• W2014749419 abstract "Article1 May 1998free access The human oncoprotein MDM2 arrests the cell cycle: elimination of its cell-cycle-inhibitory function induces tumorigenesis Doris R. Brown Doris R. Brown Department of Microbiology, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX, 78284 USA Search for more papers by this author Charles A. Thomas Charles A. Thomas Department of Microbiology, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX, 78284 USA Search for more papers by this author Swati Palit Deb Corresponding Author Swati Palit Deb Department of Microbiology, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX, 78284 USA Search for more papers by this author Doris R. Brown Doris R. Brown Department of Microbiology, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX, 78284 USA Search for more papers by this author Charles A. Thomas Charles A. Thomas Department of Microbiology, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX, 78284 USA Search for more papers by this author Swati Palit Deb Corresponding Author Swati Palit Deb Department of Microbiology, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX, 78284 USA Search for more papers by this author Author Information Doris R. Brown1, Charles A. Thomas1 and Swati Palit Deb 1 1Department of Microbiology, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX, 78284 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (1998)17:2513-2525https://doi.org/10.1093/emboj/17.9.2513 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The human oncoprotein MDM2 (hMDM2) overexpresses in various human tumors. If amplified, the mdm2 gene can enhance the tumorigenic potential of murine cells. Here, we present evidence to show that the full-length human or mouse MDM2 expressed from their respective cDNA can inhibit the G0/G1–S phase transition of NIH 3T3 and normal human diploid cells. The protein harbors more than one cell-cycle-inhibitory domain that does not overlap with the p53-interaction domain. Deletion mutants of hMDM2 that lack the cell-cycle-inhibitory domains can be stably expressed in NIH 3T3 cells, enhancing their tumorigenic potential. The tumorigenic domain of hMDM2 overlaps with the p53-interaction domain. Some tumor-derived cells, such as Saos-2, H1299 or U-2OS, are relatively insensitive to the growth-inhibitory effects of hMDM2. These observations suggest that hMDM2 overexpression in response to oncogenic stimuli would induce growth arrest in normal cells. Elimination or inactivation of the hMDM2-induced G0/G1 arrest may contribute to one of the steps of tumorigenesis. Introduction The mouse double minute-2 (mdm2) gene is evolutionarily conserved among eukaryotes (Fakharzadeh et al., 1991), which suggests that the gene product (MDM2) serves an important function in eukaryotic cells. As is the case for many other oncogenes, the dysfunction of the mdm2 gene was discovered before its normal function. Originally, the gene was identified as an amplified and overexpressed gene in a spontaneously transformed derivative of mouse BALB/c cell line 3T3DM (Fakharzadeh et al., 1991). Since 3T3DM cells overexpress several splice variants of MDM2, it was not clear which form of the protein is capable of inducing tumorigenesis. Amplification of the entire mdm2 gene capable of expressing all the spliced forms was shown to enhance tumorigenic potential of murine cells (Fakharzadeh et al., 1991; Finlay, 1993). The human homolog of the mdm2 gene (hmdm2) is frequently overexpressed in many human cancers (Ladanyi et al., 1993; Leach et al., 1993; Sheikh et al., 1993; Cordon-Cardo et al., 1994; Florenes et al., 1994; Quesnel et al., 1994; Reifenberger et al., 1994; Gudas et al., 1995; Baunoch et al., 1996), suggesting that the genetic alteration may be one of the common causes of oncogenesis. Recently, Lundgren et al. (1997) reported that 16% of transgenic mice that overexpress MDM2 from a mini-gene containing the introns 7 and 8 of mdm2 inserted within its cDNA under a bovine β-lactoglobulin promoter develop breast tumor. These findings suggest that MDM2 has an oncogenic function. The mdm2 gene product was originally detected in a complex with the tumor suppressor p53 (Momand et al., 1992). Overexpression of hmdm2 gene was found in many cancer cells with wild-type p53 (Oliner et al., 1992; Leach et al., 1993). These findings led to the hypothesis that hMDM2 may induce oncogenesis by inactivating the tumor suppressor p53 (Oliner et al., 1992; Leach et al., 1993). Later studies reveal alteration in the expression of both p53 and hmdm2 genes (Cordon-Cardo et al., 1994), suggesting that abnormal expression of the two genes may confer an additive effect on cell growth. Besides p53, MDM2 also interacts with the retinoblastoma gene product pRb (Xiao et al., 1995), the transcription factors E2F1/DP1 (Martin et al., 1995), ribosomal L5 protein (Marechal et al., 1994), simian virus 40 (SV40) T antigen (Brown et al., 1993) and human TATA-binding protein (Leng et al., 1995a). Among all the interactions of MDM2 with cellular or viral proteins, the interaction of human or mouse MDM2 with p53 has been investigated the most. Our laboratory, as well as others, have shown that human or mouse MDM2 can interact with the tumor suppressor p53 in cell-free systems (Brown et al., 1993; Chen et al., 1993) or in the whole cell (Brown et al., 1993; Oliner et al., 1993; Haines et al., 1994; Leng et al., 1995b) and inhibit transactivation by wild-type p53. Work from our laboratory showed that the interaction of hMDM2 with p53 is needed for inhibition of p53-mediated transactivation (Brown et al., 1993), and only 127 amino acids (amino acids 28–154) of hMDM2, out of a total of 491, are sufficient to inhibit p53-directed transactivation (Leng et al., 1995b). MDM2 regulates several functions of p53. Human or mouse MDM2 regulates p53-mediated growth suppression and apoptosis (Chen et al., 1994, 1996; Haupt et al., 1996). Also, the p53-regulatory function of MDM2 is required for embryonic development (Jones et al., 1995; Montes de Oca Luna et al., 1995). Wild-type p53 induces MDM2 expression by recognizing a response element situated downstream of the first exon of the oncogene (Barak et al., 1993, 1994; Juven et al., 1993). Several laboratories (Perry et al., 1993; Chen et al., 1994; Price and Park, 1994; Bae et al., 1995) have shown that ionizing radiation induces MDM2 expression in a p53-dependent manner. Thus, the presence of an autoregulatory feedback loop has been suggested (Barak et al., 1993; Otto and Deppert, 1993; Picksley and Lane, 1993; Wu et al., 1993). At least five to seven MDM2-related polypeptides have been found in cultured mouse or human cells that overexpress MDM2 (Gudas et al., 1993; Olson et al., 1993; Haines et al., 1994; Maxwell, 1994; Sigalas et al., 1996). Two of these five forms (p90, the full length protein and p58, which harbors a deletion at the C-terminus) are capable of binding p53. The presence of alternately spliced forms of MDM2 that cannot bind p53 suggests p53-independent biochemical function(s) of hMDM2. It is not known which form(s) of MDM2 induce tumorigenesis. As stated above, several laboratories including ours have shown that full-length human or mouse MDM2 expressed from their respective cDNA can interact with p53 and inhibit p53-mediated transactivation (Momand et al., 1992; Brown et al., 1993; Oliner et al., 1993; Haines et al., 1994; Leng et al., 1995b). If hMDM2 induces tumorigenesis by inactivating the tumor suppressor p53, overexpression of full-length hMDM2 from its cDNA should enhance tumorigenic potential of NIH 3T3 cells. Also, the deletion mutants of hMDM2 that interact with p53 to inactivate its transcriptional activation should harbor the same property. To test this hypothesis, we overexpressed full-length hMDM2 or its deletion mutants from their respective cDNA in NIH 3T3 cells and analyzed the growth regulatory properties of the oncoprotein. Our results show that the full-length hMDM2 inhibits the G0/G1–S phase transition of the cell cycle and cannot confer growth advantage to NIH 3T3 cells. Some tumor-derived cells are partially insensitive to the hMDM2-mediated cell-cycle inhibition. Deletion of the cell-cycle-inhibitory domains of hMDM2 activates the tumorigenic potential of the oncoprotein. The cell-cycle-inhibitory function of hMDM2 is p53-independent, but the tumorigenic domain overlaps with its p53-interaction domain. Results Overexpression of full-length hMDM2 from its cDNA is disadvantageous to the growth of non-tumor cells Since full-length hMDM2 expressed from its cDNA can interact with p53 and inhibit p53-mediated transcriptional activation (Brown et al., 1993; Leng et al., 1995b), we wished to determine whether hMDM2 can enhance the tumorigenic potential of NIH 3T3 cells by inactivating p53. As a first step, we attempted to generate stable transfectants of NIH 3T3 cells that overexpress hMDM2. An hMDM2 expression plasmid harboring the hMDM2 cDNA (pCMVneo.hmdm2; Leng et al., 1995b) was transfected into NIH 3T3 cells. Forty-eight hours after transfection, half of the cells were analyzed for transient expression and the other half was selected to generate neomycin resistant colonies. The results (summarized in Table I) showed that although hMDM2 expressed transiently (Figure 1A) and G418-resistant colonies were generated, hMDM2 expression was not detected in the pooled G418-resistant stable transfectants or in isolated colonies. Similar results were found in several other cell lines that are not derived from tumors (Table I). Figure 1.(A) Transient expression of hMDM2 and/or hMDM2 del 491–221 in 10(3) (lanes 1–3), p53−/− MEF (lanes 4 and 5) and NIH 3T3 (lanes 6 and 7) cells detected by Western blot analysis. Stable transfectants isolated from the same experiments did not express either of these two proteins. ‘None’ lanes (3, 4 and 6) show Western blot analysis of vector transfected cell extracts. (B) Expression of full-length hMDM2 and hMDM2 del 491–221 in H1299 (lanes 1–4) and Saos-2 (lanes 5 and 6) stable transfectants detected by Western blot analysis. SM24 is a Saos-2, and HMW30 and HM10 are H1299 stable transfectants. SM24 and HMW30 express wild-type hMDM2 and HM10 expresses a C-terminal deletion mutant hMDM2 del 491–221. HC5 and SC4 are H1299 and Saos-2 cells, respectively, that are stably transfected with the vector. For both panels, migration of the mol. wt markers is shown on the left and migration of hMDM2 and hMDM2 del 491–221 is indicated by arrowheads. Download figure Download PowerPoint Table 1. Non-tumor cells do not tolerate stable overexpression of full-length hMDM2 Cells Plasmids transfected Transient hMDM2 expression Generation of antibiotic resistant colonies Antibiotic resistant colonies expressing hMDM2a Normal human keratinocyte pCMVneo.hmdm2 ND + − MEF (normal mouse embryo fibroblast) pHygb+ pCMVneo.hmdm2 + + − MEF (p53−/−) pHygb+ pCMVneo.hmd2 + + − pCMVneo.hmdm2 del 491–221 + + − 10(3) (p53−/− murine fibroblast) pCMVneo.hmdm2 + + − pCMVneo.hmdm2 del 491–221 + + − NIH 3T3 pCMVneo.hmdm2 + + − Saos-2 (osteosarcoma) pCMVneo.hmdm2 + + + H1299 (large cell lung carcinoma) pCMVneo.hmdm2 + + + pCMVneo.hmdm2 del 491–221 + + + 21PT (human breast tumor) pCMVneo.hmdm2 + + + pCMVneo.hmdm2 del 491–221 + + + ND: not done a At least 30 colonies were analyzed in each case. b Plasmid that confers hygromycin resistance to the cell (pHyg). Our attempts to generate stable transfectants of some tumor-derived cells overexpressing hMDM2, on the other hand, were successful. Tumor-derived cells, such as Saos-2 (Masuda et al., 1987), H1299 (Mitsudomi et al., 1992) and 21PT (Band et al., 1990), were transfected with the hMDM2 expression plasmid (pCMVneo.hmdm2; Leng et al., 1995b). G418-resistant colonies were selected and expanded. Western blot analysis of the cell extracts showed stable expression of hMDM2 in these cells (Figure 1B). These results suggest that overexpression of hMDM2 is not toxic to all of the cells. Also, MDM2 has been stably overexpressed in NIH 3T3 and primary rat embryo fibroblast (REF) cells by amplifying a cosmid harboring the entire mdm2 gene (Fakharzadeh et al., 1991; Finlay, 1993). The MDM2 overexpressing NIH 3T3 cells have been shown to form tumors in nude mice (Fakharzadeh et al., 1991). These results led us to speculate that MDM2 overexpressed from the cDNA may be functionally different from the oncoprotein overexpressed from the mouse genomic clone. The full-length hMDM2 overexpressed from the cDNA may cause growth disadvantage in cell lines that are not tumor derived. hMDM2 blocks the G0/G1–S phase transition of non-tumor cells To determine how hMDM2 modulates cell growth, we analyzed the effect of transiently overexpressed hMDM2 on cell-cycle progression of NIH 3T3 cells. NIH 3T3 cells were transfected with hMDM2 expression plasmid harboring the human cDNA (pCMV.hmdm2; Leng et al., 1995b). Cells were harvested and fixed 48 h after transfection. The hMDM2-overexpressing cells were identified by immunostaining with an anti-hMDM2 monoclonal antibody (2A10; Chen et al., 1993) and a fluorescein isothiocyanate (FITC)-conjugated secondary antibody. The cells were then stained with propidium iodide. The FITC-labeled and unlabeled cells were sorted (30 000 cells in each case) and analyzed for cell-cycle transition by flow cytometry (program PEAKS3.FTN) using a fluorescence-activated cell sorter (FAC Starplus, Becton Dickinson). Results of several (more than six) independent experiments using three different anti-hMDM2 antibodies showed that transient overexpression of full-length hMDM2 inhibits the G0/G1–S phase progression of the NIH 3T3 cells (Figure 2A). As shown in the figure, >90% of the FITC-labeled cells (shown by a broken line) were arrested at the G0/G1 phase, and only 8% of the cells were cycling (S+G2+M phases). On the other hand, 38% of unlabeled cells (solid line) were found in the S+G2+M phases. hMDM2 expression in the FITC-labeled cells was confirmed by Western blot analysis of the sorted FITC-labeled and unlabeled cells. Figure 2B (lane 2) shows that hMDM2 was expressed in the FITC-labeled cells. Figure 2.Overexpression of hMDM2 blocks the G0/G1–S phase transition of NIH 3T3 cells. pCMV.hmdm2- or pCMV.CD20-transfected cells were identified by immunostaining with their respective monoclonal antibody and an FITC-conjugated secondary antibody. (A) DNA content of the FITC-labeled (broken line) and unlabeled (solid line) cells were analyzed after propidium iodide staining. Compared with the unlabeled cells (38%), lower percentage (8%) of FITC-labeled cells progressed to the S+G2+M phases. (B) Transient expression of hMDM2 in sorted FITC-labeled cells detected by Western blot analysis. 30 000, 91% pure, FITC-labeled cells and 60 000, 99% pure, unlabeled cells were used for this purpose. Migration of the mol. wt markers is shown on the left and migration of hMDM2 is indicated by an arrowhead. (C) pCMV.CD20-transfected NIH 3T3 cells did not undergo G0/G1 arrest. Similar percentage of FITC-labeled (broken line) or unlabeled (solid line) cells progressed to the S+G2+M phases. Download figure Download PowerPoint To confirm that the hMDM2-mediated inhibition of the G0/G1–S phase transition is not an artifact due to transfection of plasmid DNA or non-specific overexpression of any protein, we transfected NIH 3T3 cells separately with three different plasmids. One of these plasmids (pCMV.CD20) expresses the surface marker CD20 under the control of cytomegalovirus (CMV) promoter, a second plasmid (pCMV.βgal) expresses the enzyme β-galactosidase under the control of CMV promoter, and the third plasmid (pSV.βgal) also expresses the enzyme β-galactosidase under the control of SV40 early promoter. Cells overexpressing CD20 or β-galactosidase were identified using their respective antibodies and an FITC-labeled secondary antibody. A monoclonal antibody against CD20 (Becton Dickinson) was used to identify pCMV.CD20 transfected cells, whereas cells overexpressing β-galactosidase were identified using a polyclonal antibody against β-galactosidase (5′ to 3′). Overexpression of the surface marker CD20 (Figure 2C) or the enzyme β-galactosidase (Figure 3A and B) did not significantly alter the phase distribution of transfected and untransfected cells, suggesting that the hMDM2-mediated inhibition is not due to overexpression of any protein. Figure 3.Human or mouse MDM2-mediated G0/G1 arrest is not a result of non-specific protein overexpression. (A) pCMV.β-gal, (B) pSV.β-gal, (C) pCMV.T or (D) pCMV.mdm2 expression plasmid was transfected into NIH 3T3 cells. Transfected cells were identified by immunostaining with the respective antibodies and an FITC-conjugated secondary antibody. DNA content of the FITC-labeled (broken line) and unlabeled (solid line) cells were analyzed after propidium iodide staining. The figure shows that overexpression of β-galactosidase from the CMV promoter (A) or from the SV40 early promoter (B) does not block the G0/G1–S phase transition of NIH 3T3 cells (similar percentage of FITC-labeled or unlabeled cells progressed to the S+G2+M phases). Overexpression of T antigen (C) accelerates the G0/G1–S phase transition of NIH 3T3 cells. Compared with the unlabeled cells (32%), higher percentage (72%) of FITC-labeled cells progressed to the S+G2+M phases, whereas mouse MDM2 (D) blocks the G0/G1–S phase transition (42.5% unlabeled and 9.9% labeled cells progressed to the S+G2+M phases). Download figure Download PowerPoint To determine whether our flow cytometric assay can recognize acceleration of the G1–S phase transition, we used the viral oncoprotein SV40 T antigen as a positive control. As in the case of hMDM2, SV40 large T antigen is a transforming protein that interacts with the tumor suppressor p53 (Lane and Crawford, 1979; Linzer and Levine, 1979) and inhibits p53-mediated transcriptional activation (reviewed in Levine, 1993; Lane 1994). To determine the effect T antigen overexpression on the G1–S phase transition of NIH 3T3 cells, a T antigen expression plasmid (pCMV.T; Deb et al., 1995) was transfected into NIH 3T3 cells. The T antigen-expressing cells were identified using a monoclonal antibody against T antigen (Pab419; Harlow et al., 1981), sorted and analyzed as described in the earlier experiment. Transient overexpression of T antigen from the CMV promoter enhanced the G0/G1–S phase transition of T antigen expressing cells as expected (Figure 3C). Although human and mouse MDM2 show high degree of sequence homology (Fakharzadeh et al., 1991; Oliner et al., 1992), overexpression of mouse MDM2 from the genomic clone was shown to enhance the tumorigenic potential of NIH 3T3 cells (Fakharzadeh et al., 1991; Finlay, 1993). Therefore, we tested whether the growth-inhibitory effect is specific for hMDM2. Overexpression of mouse MDM2 from an MDM2 expression plasmid (pCMV.mdm2; Haines et al., 1994) also led to the G0/G1 arrest of NIH 3T3 cells (Figure 3D), suggesting that the hMDM2-mediated growth arrest is not species specific. To determine whether hMDM2-mediated growth arrest is specific for NIH 3T3 cells, hMDM2 expression plasmid was transfected into two normal human diploid fibroblast cell lines, WI38 and MRC5. The untransfected and hMDM2 overexpressing cells were sorted and analyzed as described above. Transient overexpression of hMDM2 caused G0/G1 arrest in MRC5 and WI38 cells, suggesting that the effect is not specific for NIH 3T3 cells (Table IIA). As in the case of NIH 3T3 cells, overexpression of the surface marker CD20 from the pCMV.CD20 expression plasmid did not induce G0/G1 arrest (Table IIB). Table 2. WI38 MRC5 Unlabeled Cells FITC-labeled Cells Unlabeled Cells FITC-labeled Cells (A) hMDM2 arrests the G0/G1–S phase transition of normal human diploid cells %G0/G1 65.57 ± 0.37 89.65 ± 1.03 41.77 ± 0.20 79.64 ± 0.88 %S 17.15 ± 0.19 5.45 ± 0.25 35.42 ± 0.19 12.17 ± 0.34 %G2+M 20.28 ± 0.20 4.9 ± 0.24 22.81 ± 0.15 8.19 ± 0.28 (B) CD20 expression does not alter cell cycle distribution of normal human diploid cells %G0/G1 75.37 ± 0.59 77.90 ± 0.96 73.40 ± 0.63 75.13 ± 0.89 %S 6.75 ± 0.18 6.49 ± 0.28 12.13 ± 0.26 10.81 ± 0.34 %G2+M 17.53 ± 0.29 15.61 ± 0.43 14.47 ± 0.28 14.06 ± 0.38 hMDM2 blocks the G0/G1–S phase transition of NIH 3T3 cells at low or high levels Overexpression of MDM2 from the amplified mdm2 gene has been shown to induce tumorigenesis (Fakharzadeh et al., 1991; Finlay, 1993). Although the mdm2 gene was amplified to elevate the normal levels of the protein, in the genomic clone, MDM2 was expressed from its own promoter. Therefore, hMDM2-mediated G0/G1 arrest could be a result of high levels of hMDM2 overexpressed from a strong CMV promoter, while the protein may confer growth advantage at low levels. To test this possibility, we analyzed the growth-regulatory effects of hMDM2 at different levels of the protein expressed. We transiently transfected NIH 3T3 cells with 1, 5, 10 and 30 μg of an hMDM2 expression plasmid (pCMV.hmdm2; Leng et al., 1995b). To detect the G0/G1 arrest mediated by low levels of wild-type hMDM2, the cells were blocked at mitosis by treatment with nocodazole after transfection (Giunta and Carlo, 1995; Chen et al., 1996). Nocodazole arrests the cell cycle at mitosis. Therefore, the cells that have completed DNA replication (untransfected cells) will not undergo cell division in the presence of nocodazole, and accumulate at the G2/M phase. However, cells that will not be able to enter the S phase or complete DNA replication will not be able to reach G2/M phase. Thus, hMDM2 expressing cells should not show significant number of cells in the S and G2/M phases, even in the presence of nocodazole. The transfected cells were identified by immunostaining with a monoclonal antibody against hMDM2 (2A10, Chen et al., 1993) and an FITC-labeled secondary antibody, and then sorted. The intensity of the FITC fluorescence associated with the cells was measured at 530 nm using FACS (fluorescence activated cell sorter) and was plotted against cell numbers. The monoclonal antibody used for this experiment recognizes mouse as well as human MDM2 (Chen et al., 1993). Thus, the FITC fluorescence of the untransfected NIH 3T3 cells should be indicative of endogenous MDM2. The amounts of hMDM2 expressed in the transfected and untransfected cells were estimated from the intensity of fluorescence, and compared. The transfected and untransfected cells were also stained with propidium iodide and their DNA content determined. The result of this experiment is shown in Table III. When 1 μg hMDM2 expression plasmid was used for transfection, most of the transfected cells showed 1.7- to 1.95-fold higher intensity of green fluorescence than the untransfected cells, while the brightest transfected cells showed 2.5- to 3.7-fold higher fluorescence than the untransfected cells. These results suggest that the levels of hMDM2 expressed in these cells were 1.7- to 3.7-fold higher than the untransfected cells. The DNA content analysis showed that 1.7- to 3.7-fold higher expression of hMDM2 inhibited the G0/G1–S phase progression of cells (Table III). Table 3. Overexpression of full-length hMDM2 inhibits the G0/G1–S phase transition of NIH 3T3 cells at low or high levels Amount of pCMV.hmdm2 transfected (μg) Increase in FITC fluorescence in transfected cells Percent S+G2+M cells Unlabeled cells FITC-labeled cells 1 1.74- to 2.54-fold 48.42 ± 0.49 17.65 ± 0.42 5 5- to 6.6-fold 67.03 ± 0.58 30.34 ± 0.54 30 9.55- to 22.6-fold 55.48 ± 0.53 12.05 ± 0.35 1 1.95- to 3.7-fold 62.85 ± 0.57 25.99 ± 0.51 10 5.9- to 8.3-fold 58.1 ± 0.54 18.16 ± 0.43 Similarly, when we used 5 μg hMDM2 expression plasmid, most of the transfected cells expressed 5-fold higher levels of hMDM2, while the brightest cells showed 6.6-fold higher expression 6- to 8.3-fold. Similarly, 10 μg hMDM2 expression plasmid enhanced hMDM2 expression. When we used 30 μg expression plasmid, most of the cells expressed 9.5-fold higher levels of hMDM2, whereas the brightest cells showed 22.6-fold higher levels of hMDM2. Since an increase in the amount of expression plasmid from 5–30 μg showed an increase in the FITC fluorescence, the amount of antibody used was not limiting for cells expressing 5- to 10- fold higher levels of hMDM2. In all the cases, overexpression of hMDM2 resulted in G0/G1 arrest. The efficiency of inhibition increased with 10- to 20-fold increase in the levels of hMDM2, although not proportionally. These results indicate that 2- to 20-fold overexpression of hMDM2 from its full-length cDNA induces G0/G1 arrest in NIH 3T3 cells. Therefore, the hMDM2-mediated G0/G1 arrest could be physiologically relevant when hMDM2 is induced by a factor such as p53. Figure 4 shows the comparative levels of hMDM2 expression in NIH 3T3 cells transfected with 1 or 10 μg hMDM2 expression plasmid, as detected by Western analysis. Figure 4.Comparative Western blot analysis of hMDM2 expressed from 1 or 10 μg pCMV.hmdm2 in NIH 3T3 cells. (A) NIH 3T3 cells transfected with 1 or 10 μg pCMV.hmdm2 were sorted. 20 000 FITC-labeled (lane F) and unlabeled (lane U) cells were analyzed in each case. (B) hMDM2 expression from 1 μg pCMV.hmdm2 was confirmed by Western analysis of 40 000 FITC-labeled (lane F) and unlabeled (lane U) cells. The Western blot shown in (A) was developed for a shorter period of time than the blot shown in (B). Download figure Download PowerPoint Some tumor-derived cells are partially insensitive to hMDM2-mediated G0/G1 arrest Studies in our laboratory and elsewhere suggest that several tumor-derived cells can tolerate stable overexpression of hMDM2 from its cDNA (Figure 1B, lanes 4 and 5; Table I) (Chen et al., 1994). Therefore, we investigated how hMDM2 regulates the G0/G1–S phase transition of these cells. Since it was possible to generate hMDM2-overexpressing stable transfectants of H1299 or Saos-2 cells, these cells were transfected with the hMDM2 expression plasmid pCMV.hmdm2. The hMDM2-expressing cells were identified and analyzed by flow cytometry as described above. The results [Table IV (A)] show that overexpression of hMDM2 reduces the percentage of cycling Saos-2 cells from 54 to 32%, and H1299 cells from 57 to 47%. These results suggest that Saos-2 and H1299 cells are less sensitive to hMDM2-mediated G0/G1 arrest than NIH 3T3, WI38 or MRC5 cells, which show a 3- to 5-fold reduction in the number of cycling cells under similar conditions (Figure 2; Table II). Consistent with this observation, our stable transfection analysis indicates that H1299 and Saos-2 cells can stably overexpress hMDM2 (Table I; Figure 1B). These findings suggest that hMDM2 may stably overexpress in some genetically defective cells that are partially insensitive to the negative growth regulatory effects of hMDM2. Table 4. Saos2 H1299 Unlabeled cells FITC-labeled cells Unlabeled cells FITC-labeled cells %G0/G1 45.3 ± 0.18 67.7 ± 0.63 42.5 ± 0.52 52.48 ± 0.74 %S 38.2 ± 0.17 23.3 ± 0.37 43.37 ± 0.53 38.12 ± 0.63 %G2+M 16.5 ± 0.11 9.0 ± 0.23 14.13 ± 0.30 9.4 ± 0.31 pCMV.hmdm2 (1 μg) pCMV.hmdm2 (20 μg) Unlabeled cells FITC-labeled cells Unlabeled cells FITC-labeled cells %G0/G1 32.27 ± 0.41 29.34 ± 0.50 39.12 ± 0.45 74.17 ± 0.88 %S 45.70 ± 0.48 27.80 ± 0.49 37.40 ± 0.44 19.95 ± 0.45 %G2+M 22.63 ± 0.34 42.9 ± 0.61 23.48 ± 0.35 5.88 ± 0.25 It has been shown that MDM2 relieves p53 or Rb-mediated growth suppression in U-2OS cells (Chen et al., 1996; Xiao et al., 1995). Thus, the U-2OS cells may be completely resistant to hMDM2-mediated growth suppression. To test this possibility, we expressed high or low levels of hMDM2 in U-2OS cells. U-2OS cells were transfected with 1 or 20 μg hMDM2 expression plasmid (pCMV.hmdm2). Consistent with the results reported earlier (Chen et al., 1996; Xiao et al., 1995), U-2OS cells were tolerant to hMDM2 expressed from 1 μg pCMVhmdm2, and did show a slight increase in the G1–S phase transition. However, as in the case of Saos-2 and H1299, U-2OS cells do show G0/G1 arrest at high levels of hMDM2 expressed from 20 μg pCMVhmdm2 [Table IV (B)]. Thus, levels of hMDM2 required to induce growth suppression in tumor-derived cells are higher than that for untransformed cells. Both Saos-2 (Masuda et al., 1987) and H1299 (Mitsudomi et al., 1992) cells have a homozygous deletion in the p53 gene and do not express any p53 protein. Saos-2 cells express a C-terminal deletion mutant of the retinoblastoma susceptibility gene product (Rb). The protein is non-functional and does not translocate to the nucleus (Shew et al., 1990). H1299 cells express wild-type Rb (Mitsudomi et al., 1992). The U-2OS cell line contains wild-type p53 and Rb (Lee et al., 1987; Diller et al., 1990; Shew et al., 1990). Our data suggest that all three cell lines are partially resistant to hMDM2-mediated growth suppression. Therefore, hMDM2-mediated growth suppression is not dependent on the status of p53 or Rb. Growth-inhibitory domains of hMDM2 reside within amino acid residues 155 to 324 To identify the growth-inhibitory domain" @default.
• W2014749419 created "2016-06-24" @default.
• W2014749419 creator A5030731365 @default.
• W2014749419 creator A5034369343 @default.
• W2014749419 creator A5078205508 @default.
• W2014749419 date "1998-05-01" @default.
• W2014749419 modified "2023-09-24" @default.
• W2014749419 title "The human oncoprotein MDM2 arrests the cell cycle: elimination of its cell-cycle-inhibitory function induces tumorigenesis" @default.
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