Inhibition of c-Jun N-terminal kinase 2 expression suppresses growth and induces apoptosis of human tumor cells in a p53-dependent manner

c-Jun N-terminal kinase (JNK) plays a critical role in coordinating the cellular response to stress and has been implicated in regulating cell growth and transformation. To investigate the growth-regulatory functions of JNK1 and JNK2, we used specific antisense oligonucleotides (AS) to inhibit their expression. A survey of several human tumor cell lines revealed that JNKAS treatment markedly inhibited the growth of cells with mutant p53 status but not that of cells with normal p53 function. To further examine the influence of p53 on cell sensitivity to JNKAS treatment, we compared the responsiveness of RKO, MCF-7, and HCT116 cells with normal p53 function to that of RKO E6, MCF-7 E6, and HCT116 p53(-/-), which were rendered p53 deficient by different methods. Inhibition of JNK2 (and to a lesser extent JNK1) expression dramatically reduced the growth of p53-deficient cells but not that of their normal counterparts. JNK2AS-induced growth inhibition was correlated with significant apoptosis. JNK2AS treatment induced the expression of the cyclin-dependent kinase inhibitor p21(Cip1/Waf1) in parental MCF-7, RKO, and HCT116 cells but not in the p53-deficient derivatives. That p21(Cip1/Waf1) expression contributes to the survival of JNK2AS-treated cells was supported by additional experiments demonstrating that p21(Cip1/Waf1) deficiency in HCT116 cells also results in heightened sensitivity to JNKAS treatment. Our results indicate that perturbation of JNK2 expression adversely affects the growth of otherwise nonstressed cells. p53 and its downstream effector p21(Cip1/Waf1) are important in counteracting these detrimental effects and promoting cell survival.     

E2F1 inhibits MDM2 expression in a p53-dependent manner

MDM2 expression is down-regulated upon E2F1 over-expression, but the mechanism is not well defined. In the current study, we found that E2F1 inhibits MDM2 expression by suppressing its promoter activity. Although E2F1 binds to the MDM2 promoter, the inhibitory effect of E2F1 on the MDM2 promoter does not require the direct binding. We demonstrate that E2F1 inhibits MDM2 promoter activity in a p53-dependent manner. Knockdown of p53 in U2OS cells impairs the inhibitory effect of E2F1 on the MDM2 promoter. Consistent with this observation, E2F1 does not inhibit MDM2 promoter activity in p53-deficient H1299 cells, and the inhibition is restored when p53 is expressed exogenously. Both E2F1 and p53 are up-regulated after DNA damage stimulation. We show that such stimulation induces E2F1 to inhibit MDM2 promoter activity and promote p53 accumulation. Furthermore, inhibition of MDM2 by E2F1 promotes E2F1 induced apoptosis. These data suggest that E2F1 regulates the MDM2-p53 pathway by inhibiting p53 induced up-regulation of MDM2.     

LRH-1 drives colon cancer cell growth by repressing the expression of the CDKN1A gene in a p53-dependent manner

Liver receptor homologue 1 (LRH-1) is an orphan nuclear receptor that has been implicated in the progression of breast, pancreatic and colorectal cancer (CRC). To determine mechanisms underlying growth promotion by LRH-1 in CRC, we undertook global expression profiling following siRNA-mediated LRH-1 knockdown in HCT116 cells, which require LRH-1 for growth and in HT29 cells, in which LRH-1 does not regulate growth. Interestingly, expression of the cell cycle inhibitor p21 (CDKN1A) was regulated by LRH-1 in HCT116 cells. p21 regulation was not observed in HT29 cells, where p53 is mutated. p53 dependence for the regulation of p21 by LRH-1 was confirmed by p53 knockdown with siRNA, while LRH-1-regulation of p21 was not evident in HCT116 cells where p53 had been deleted. We demonstrate that LRH-1-mediated p21 regulation in HCT116 cells does not involve altered p53 protein or phosphorylation, and we show that LRH-1 inhibits p53 recruitment to the p21 promoter, likely through a mechanism involving chromatin remodelling. Our study suggests an important role for LRH-1 in the growth of CRC cells that retain wild-type p53.     

Proliferation of aneuploid human cells is limited by a p53-dependent mechanism

Most solid tumors are aneuploid, and it has been proposed that aneuploidy is the consequence of an elevated rate of chromosome missegregation in a process called chromosomal instability (CIN). However, the relationship of aneuploidy and CIN is unclear because the proliferation of cultured diploid cells is compromised by chromosome missegregation. The mechanism for this intolerance of nondiploid genomes is unknown. In this study, we show that in otherwise diploid human cells, chromosome missegregation causes a cell cycle delay with nuclear accumulation of the tumor suppressor p53 and the cyclin kinase inhibitor p21. Deletion of the p53 gene permits the accumulation of nondiploid cells such that CIN generates cells with aneuploid genomes that resemble many human tumors. Thus, the p53 pathway plays an important role in limiting the propagation of aneuploid human cells in culture to preserve the diploid karyotype of the population. These data fit with the concordance of aneuploidy and disruption of the p53 pathway in many tumors, but the presence of aneuploid cells in some normal human and mouse tissues indicates that there are known exceptions to the involvement of p53 in aneuploid cells and that tissue context may be important in how cells respond to aneuploidy.     

Caspase 2 is both required for p53-mediated apoptosis and downregulated by p53 in a p21-dependent manner

Upon treatment with some DNA damaging agents, human H1299 tumor-derived cells expressing inducible versions of wild-type or mutant p53 with inactive transactivation domain I (p53Q22/S23) undergo apoptosis. In cells expressing either version of p53, caspase 2 activation is required for release of cytochrome c and cell death. Furthermore, silencing of PIDD (a factor previously shown to be required for caspase 2 activation) by siRNA suppresses apoptosis by both wild-type p53 and p53Q22/S23. Despite the finding that caspase 2 is essential for DNA damage-facilitated, p53-mediated apoptosis, induction of wild-type p53 (with or without DNA damage) resulted in a reduction of caspase 2 mRNA and protein levels. In this study we sought to provide a mechanism for the negative regulation of caspase 2 by p53 as well as provide insight as to why p53 may repress a key mediator of p53-dependent apoptosis. Mechanistically, we show that DNA binding and/or transactivation domains of p53 are crucial for mediating transrepression. Further, expression of p21 (in p53-null cells inducibly expressing p21) is sufficient to mediate repression of caspase 2. Deletion of p21 or E2F-1 not only abrogated repression of caspase 2, but also stimulated the expression of caspase 2 above basal levels, implicating the requirement for an intact p21/Rb/E2F pathway in the down-regulation of caspase 2. As this p53/p21-dependent repression of caspase 2 can occur in the absence of DNA damage, caspase 2 repression does not simply seem to be a consequence of the apoptotic process. Down-regulation of caspase 2 levels by p53 may help to determine cell fate by preventing cell death when unnecessary.     

Expression of mouse Fbxw7 isoforms is regulated in a cell cycle- or p53-dependent manner

Fbxw7 is the F-box protein component of an SCF-type ubiquitin ligase that contributes to the ubiquitin-dependent degradation of cell cycle activators and oncoproteins. Three isoforms (alpha, beta, and gamma) of Fbxw7 are produced from mRNAs with distinct 5' exons. We have now investigated regulation of Fbxw7 expression in mouse tissues. Fbxw7alpha mRNA was present in all tissues examined, whereas Fbxw7beta mRNA was detected only in brain and testis, and Fbxw7gamma mRNA in heart and skeletal muscle. The amount of Fbxw7alpha mRNA was high during quiescence (G0 phase) in mouse embryonic fibroblasts (MEFs) and T cells, but it decreased markedly as these cells entered the cell cycle. The abundance of Fbxw7alpha mRNA was unaffected by cell irradiation or p53 status. In contrast, X-irradiation increased the amount of Fbxw7beta mRNA in wild-type MEFs but not in those from p53-deficient mice, suggesting that radiation-induced up-regulation of p53 leads to production of Fbxw7beta mRNA. Our results thus indicate that expression of Fbxw7 isoforms is differentially regulated in a cell cycle- or p53-dependent manner.     

Protoporphyrin IX interacts with wild-type p53 protein in vitro and induces cell death of human colon cancer cells in a p53-dependent and -independent manner

Photodynamic therapy (PDT) of cancer is an alternative treatment for tumors resistant to chemo- and radiotherapy. It induces cancer cell death mainly through generation of reactive oxygen species by a laser light-activated photosensitizer. It has been suggested that the p53 tumor suppressor protein sensitizes some human cancer cells to PDT. However, there is still no direct evidence for this. We have demonstrated here for the first time that the photosensitizer protoporphyrin IX (PpIX) binds to p53 and disrupts the interaction between p53 tumor suppressor protein and its negative regulator HDM2 in vitro and in cells. Moreover, HCT116 colon cancer cells exhibited a p53-dependent sensitivity to PpIX in a dose-dependent manner, as was demonstrated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and fluorescence-activated cell sorter (FACS) analysis of cell cycle profiles. We have also observed induction of p53 target pro-apoptotic genes, e.g. puma (p53-up-regulated modulator of apoptosis), and bak in PpIX-treated cells. In addition, p53-independent growth suppression by PpIX was detected in p53-negative cells. PDT treatment (2 J/cm2) of HCT116 cells induced p53-dependent activation of pro-apoptotic gene expression followed by growth suppression and induction of apoptosis.     

DNAJB1 stabilizes MDM2 and contributes to cancer cell proliferation in a p53-dependent manner

Both MDM2 and MDMX regulate p53, but these proteins play different roles in this process. To clarify the difference, we performed a yeast 2 hybrid (Y2H) screen using the MDM2 acidic domain as bait. DNAJB1 was found to specifically bind to MDM2, but not MDMX, in vitro and in vivo. Further investigation revealed that DNAJB1 stabilizes MDM2 at the post-translational level. The C-terminus of DNAJB1 is essential for its interaction with MDM2 and for MDM2 accumulation. MDM2 was degraded faster by a ubiquitin-mediated pathway when DNAJB1 was depleted. DNAJB1 inhibited the MDM2-mediated ubiquitination and degradation of p53 and contributed to p53 activation in cancer cells. Depletion of DNAJB1 in cancer cells inhibited activity of the p53 pathway, enhanced the activity of the Rb/E2F pathway, and promoted cancer cell growth in vitro and in vivo. This function was p53 dependent, and either human papillomavirus (HPV) E6 protein or siRNA against p53 was able to block the contribution caused by DNAJB1 depletion. In this study, we discovered a new MDM2 interacting protein, DNAJB1, and provided evidence to support its p53-dependent tumor suppressor function.     

p53 is associated with p34cdc2 in transformed cells.

The normal functioning of p53 is thought to involve p53 target proteins. We have previously identified a cellular 35 kd protein associated with p53 and now report evidence identifying this 35 kd protein as p34cdc2, product of the cell cycle control cdc2 gene. The association between p53 and p34cdc2 was detected in SV3T3 and T3T3 cell lines, both expressing the wild-type p53 phenotype, and in 3T3tx cells, expressing 'mutant' p53 phenotype. Binding of the mutant p53 phenotype with p34cdc2 was greatly reduced relative to wild-type. Complexes of p53-p34cdc2 may represent inactivation or activation of either component. The p34cdc2 kinase functions at cell cycle control points and is necessary for entry and passage through mitosis. It also operates in G1 and is involved in the commitment of cells into the proliferative cycle. Since we were unable to detect p53-p34cdc2 complexes in mitotic cells we propose that the interaction between p53 and p34cdc2 may be functional in cell growth control, possibly to promote or to suppress cell proliferation.     

The Peutz-Jegher gene product LKB1 is a mediator of p53-dependent cell death.

Here, we investigate the mechanism and function of LKB1, a Ser/Thr kinase mutated in Peutz-Jegher syndrome (PJS). We demonstrate that LKB1 physically associates with p53 and regulates specific p53-dependent apoptosis pathways. LKB1 protein is present in both the cytoplasm and nucleus of living cells and translocates to mitochondria during apoptosis. In vivo, LKB1 is highly upregulated in pyknotic intestinal epithelial cells. In contrast, polyps arising in Peutz-Jegher patients are devoid of LKB1 staining and have reduced numbers of apoptotic cells. We propose that a deficiency in apoptosis is a key factor in the formation of multiple benign intestinal polyps in PJS patients, and possibly for the subsequent development of malignant tumors in these patients.     

Induction of p53-dependent and mitochondria-mediated cell death pathway by dengue virus infection of human and animal cells

Previous studies have suggested that cells undergo apoptosis in response to dengue virus infection. However, the potential significance of dengue virus-induced apoptosis and the pathways are still not clearly defined. In this study, comparative analysis of dengue virus-induced apoptosis in BHK, H1299, HUH-7 and Vero cell lines was carried out. We show here that infection of BHK, HUH-7 and Vero cell lines with dengue type 1 virus (DEN1V) induces cell death typical of apoptosis. Virus-induced cell death was assayed by in situ terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, detection of oligonucleosomal DNA fragmentation, DNA content analysis and assay for the externalization of phosphatidylserine residues. Detailed study of dengue virus infection in HUH-7 cells showed activation of cell death via the mitochondrial pathway causing lowering of mitochondrial transmembrane potential (DeltaPsim) in HUH-7 cells. Interestingly, in the p53-deficient cell line, H1299, apoptosis was largely undetectable compared with the other cell lines used; suggesting that a p53- and mitochondria-mediated cell death pathway may play an important role in dengue virus-induced apoptosis.