Etoposide (VP-16) sensitizes p53-deficient human non-small cell lung cancer cells to caspase-7-mediated apoptosis

Human non-small-cell-lung-cancer (NSCLC) cells of _p53-null genotype were exposed to low-dosage topoisomearse II inhibitor etoposide (VP-16). The cellular proliferation rate could be effectively inhibited by VP-16 in dose-dependent manner. The effective drug concentration for growth inhibition could be as low as 0.5 M and the apoptotic phenotype became evident 48 h later. In H1299 cells, VP-16-induced cytotoxic effect was demonstrated associated with apoptosis that disappeared when restored with wild-type p53. Cell cycle analysis revealed that, upon VP-16 induction, cell death began with growth arrest by accumulating cells at the G₂-M phase. The cells at sub-G₁ phase increased at the expense of those at G₂-M transition state. To assess the regulation of cell cycle modulators, western blot analysis of H1299 cell lysates showed the release of apoptosis initiator, cytochrome c and apaf-1 hours following drug induction. The cleavage of downstream effectors, procaspase-9 and procaspase-7, but not procaspase-3, was accompanied with proteolysis of poly-(ADP-ribose) polymerase (PARP). VP-16-activated procaspase-7 cleavage was abrogated in cells with ectopically expressed p53.On the other hand, the inhibited procaspase-7 fragmentation by caspase-specific inhibitor reversed apoptotic phenotype caused by drug induction. Thus, VP-16-induced apoptotic cell death was contributed by caspase-7 activation in_p53-deficient human NSCLC cells.     

The tumour suppressor RASSF1A promotes MDM2 self-ubiquitination by disrupting the MDM2-DAXX-HAUSP complex

The tumour suppressor p53, which accumulates in response to DNA damage and induces cell-cycle arrest and apoptosis, has a key function in the maintenance of genome integrity. Under normal conditions, the antiproliferative effects of p53 are inhibited by MDM2, a ubiquitin ligase that promotes p53 ubiquitination and degradation. MDM2 is also self-ubiquitinated and degraded. Here, we show that the tumour suppressor RASSF1A regulates G(1)-S cell-cycle progression in a p53-dependent manner by promoting MDM2 self-ubiquitination and preventing p53 degradation. Importantly, RASSF1A associates with MDM2 and death-domain-associated protein (DAXX) in the nucleus, thereby disrupting the interactions between MDM2, DAXX, and the deubiquitinase, HAUSP, and enhancing the self-ubiquitin ligase activity of MDM2. Moreover, RASSF1A partially contributes to p53-dependent checkpoint activation at early time points in response to DNA damage. These findings reveal a new and important function for RASSF1A in regulating the p53-MDM2 pathway.     

Nutlin-3 induces apoptosis,disrupts viral latency and inhibits expression of angiopoietin-2 in Kaposi sarcoma tumor cells

Kaposi sarcoma (KS) tumors often contain a wild-type p53. However, the function of this tumor suppressor in KS tumor cells is inhibited by both MDM2 and latent nuclear antigen (LANA) of Kaposi sarcoma-associated herpes virus (KSHV). Here, we report that MDM2 antagonist Nutlin-3 efficiently reactivates p53 in telomerase-immortalized human umbilical vein endothelial cells (TIVE) that had been malignantly transformed by KSHV as well as in KS tumor cells. Reactivation of p53 results in a G₁ cell cycle arrest, leading to inhibition of proliferation and apoptosis. Nutlin-3 inhibits the growth of “KS-like” tumors resulting from xenografted TIVE-KSHV cells in nude mice. In addition, Nutlin-3 strongly inhibits expression of the pro-angiogenic and pro-inflammatory cytokine angiopoietin-2 (Ang-2). It also disrupts viral latency by inducing expression of KSHV lytic genes. these results suggest that Nutlin-3 might serve as a novel therapy for KS.Key words: Kaposi sarcoma (KS), nutlin-3, p53, cell cycle arrest, apoptosis, angiopoietin-2     

Nutlin-3 induces apoptosis,disrupts viral latency and inhibits expression of angiopoietin-2 in Kaposi sarcoma tumor cells

Kaposi sarcoma (KS) tumors often contain a wild-type p53. However, the function of this tumor suppressor in KS tumor cells is inhibited by both MDM2 and latent nuclear antigen (LANA) of Kaposi sarcoma-associated herpes virus (KSHV). Here, we report that MDM2 antagonist Nutlin-3 efficiently reactivates p53 in telomerase-immortalized human umbilical vein endothelial cells (TIVE) that had been malignantly transformed by KSHV as well as in KS tumor cells. Reactivation of p53 results in a G₁ cell cycle arrest, leading to inhibition of proliferation and apoptosis. Nutlin-3 inhibits the growth of “KS-like” tumors resulting from xenografted TIVE-KSHV cells in nude mice. In addition, Nutlin-3 strongly inhibits expression of the pro-angiogenic and pro-inflammatory cytokine angiopoietin-2 (Ang-2). It also disrupts viral latency by inducing expression of KSHV lytic genes. These results suggest that Nutlin-3 might serve as a novel therapy for KS.     

Novel Roles for P53 in the Genesis and Targeting of Tetraploid Cancer Cells

Tetraploid (4N) cells are considered important in cancer because they can display increased tumorigenicity, resistance to conventional therapies, and are believed to be precursors to whole chromosome aneuploidy. It is therefore important to determine how tetraploid cancer cells arise, and how to target them. P53 is a tumor suppressor protein and key regulator of tetraploidy. As part of the “tetraploidy checkpoint”, p53 inhibits tetraploid cell proliferation by promoting a G1-arrest in incipient tetraploid cells (referred to as a tetraploid G1 arrest). Nutlin-3a is a preclinical drug that stabilizes p53 by blocking the interaction between p53 and MDM2. In the current study, Nutlin-3a promoted a p53-dependent tetraploid G1 arrest in two diploid clones of the HCT116 colon cancer cell line. Both clones underwent endoreduplication after Nutlin removal, giving rise to stable tetraploid clones that showed increased resistance to ionizing radiation (IR) and cisplatin (CP)-induced apoptosis compared to their diploid precursors. These findings demonstrate that transient p53 activation by Nutlin can promote tetraploid cell formation from diploid precursors, and the resulting tetraploid cells are therapy (IR/CP) resistant. Importantly, the tetraploid clones selected after Nutlin treatment expressed approximately twice as much P53 and MDM2 mRNA as diploid precursors, expressed approximately twice as many p53-MDM2 protein complexes (by co-immunoprecipitation), and were more susceptible to p53-dependent apoptosis and growth arrest induced by Nutlin. Based on these findings, we propose that p53 plays novel roles in both the formation and targeting of tetraploid cells. Specifically, we propose that 1) transient p53 activation can promote a tetraploid-G1 arrest and, as a result, may inadvertently promote formation of therapy-resistant tetraploid cells, and 2) therapy-resistant tetraploid cells, by virtue of having higher P53 gene copy number and expressing twice as many p53-MDM2 complexes, are more sensitive to apoptosis and/or growth arrest by anti-cancer MDM2 antagonists (e.g. Nutlin).     

Persistent p21 Expression after Nutlin-3a Removal Is Associated with Senescence-like Arrest in 4N Cells

Nutlin-3a is a preclinical drug that stabilizes p53 by blocking the interaction between p53 and MDM2. In our previous study, Nutlin-3a promoted a tetraploid G₁ arrest in two p53 wild-type cell lines (HCT116 and U2OS), and both cell lines underwent endoreduplication after Nutlin-3a removal. Endoreduplication gave rise to stable tetraploid clones resistant to therapy-induced apoptosis. Prior knowledge of whether cells are susceptible to Nutlin-induced endoreduplication and therapy resistance could help direct Nutlin-3a-based therapies. In the present study, Nutlin-3a promoted a tetraploid G₁ arrest in multiple p53 wild-type cell lines. However, some cell lines underwent endoreduplication to relatively high extents after Nutlin-3a removal whereas other cell lines did not. The resistance to endoreduplication observed in some cell lines was associated with a prolonged 4N arrest after Nutlin-3a removal. Knockdown of either p53 or p21 immediately after Nutlin-3a removal could drive endoreduplication in otherwise resistant 4N cells. Finally, 4N-arrested cells retained persistent p21 expression; expressed senescence-associated β-galactosidase; displayed an enlarged, flattened phenotype; and underwent a proliferation block that lasted at least 2 weeks after Nutlin-3a removal. These findings demonstrate that transient Nutlin-3a treatment can promote an apparently permanent proliferative block in 4N cells of certain cell lines associated with persistent p21 expression and resistance to endoreduplication.     

MDMX promotes proteasomal turnover of p21 at G1 and early S phases independently of, but in cooperation with, MDM2

We have shown previously that MDM2 promotes the degradation of the cyclin-dependent kinase inhibitor p21 through a ubiquitin-independent proteolytic pathway. Here we report that the MDM2 analog, MDMX, also displays a similar activity. MDMX directly bound to p21 and mediated its proteasomal degradation. Although the MDMX effect was independent of MDM2, they synergistically promoted p21 degradation when coexpressed in cells. This degradation appears to be mediated by the 26S proteasome, as MDMX and p21 bound to S2, one of the subunits of the 19S component of the 26S proteasome, in vivo. Conversely, knockdown of MDMX induced the level of endogenous p21 proteins that no longer cofractionated with 26S proteasome, resulting in G₁ arrest. The level of p21 was low at early S phase but markedly induced by knocking down either MDMX or MDM2 in human cells. Ablation of p21 rescued the G₁ arrest caused by double depletion of MDM2 and MDMX in p53-null cells. These results demonstrate that MDMX and MDM2 independently and cooperatively regulate the proteasome-mediated degradation of p21 at the G₁ and early S phases.     

Induction of p53-, MDM2-, and WAF1/CIP1-like Molecules in Insect Cells by DNA-Damaging Agents

Cellular responses following DNA damage are ubiquitous in the biological world. In response to DNA damage, cell cycle checkpoints are activated, which delay cell cycle progression and most likely serve to allow time for repair. One important checkpoint in mammalian cells, activated in the G₁ phase of the cell cycle, is dependent on the p53 tumor suppressor gene product. While p53 is responsible for inducing G₁ arrest, the product of the MDM2 gene is believed to alleviate the arrest, allowing continuation of the cell cycle after a transient delay. Inasmuch as MDM2 and WAF1/CIP1 are transactivated by p53, while MDM2 binds to and modulates the activity of p53, a "feedback loop" is thus created. This pathway has been highly conserved in mammalian cells, but its presence outside of vertebrates is unknown. By using human MDM2 and WAF1/CIP1 cDNA probes, and monoclonal antibodies to p53 and Mdm2, we demonstrate in insect cell lines evidence for the existence of p53-, MDM2-, and WAF1/CIP1 -like molecules and a p53-regulated pathway following treatment by DNA-damaging agents.     

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.     

Concomitant Inhibition of MDM2 and Bcl-2 Protein Function Synergistically Induce Mitochondrial Apoptosis in AML

Disruption of Mdm2-p53 interaction activates p53 signaling, disrupts the balance ofantiapoptotic and proapoptotic Bcl-2 family proteins and induces apoptosis in acutemyeloid leukemia (AML). Overexpression of Bcl-2 may inhibit this effect. Thus,functional inactivation of antiapoptotic Bcl-2 proteins may enhance apoptogenic effects ofMdm2 inhibition. We here investigate the potential therapeutic utility of combinedtargeting of Mdm2 by Nutlin-3a and Bcl-2 by ABT-737, recently developed inhibitors ofprotein-protein interactions. Nutlin-3a and ABT-737 induced Bax conformational changeand mitochondrial apoptosis in AML cells in a strikingly synergistic fashion. Nutlin-3ainduced p53-mediated apoptosis predominantly in S and G₂/M cells, while cells in G₁ were protected through induction of p21. In contrast, ABT-737 induced apoptosis predominantly in G₁ , the cell cycle phase with the lowest Bcl-2 protein levels and Bcl-2/Bax ratios. In addition, Bcl-2 phosphorylation on Ser⁷⁰ was absent in G₁ but detectable in G₂/M, thus lower Bcl-2 levels and absence of Bcl-2 phosphorylation appeared to facilitate ABT-737-induced apoptosis of G₁ cells. The complementary effects of Nutlin-3a and ABT-737 in different cell cycle phases could, in part, account for their synergistic activity. Our data suggest that combined targeting of Mdm2 and Bcl-2 proteins could offer considerable therapeutic promise in AML.     

Retinoblastoma protein regulates the crosstalk between autophagy and apoptosis,and favors glioblastoma resistance to etoposide

Glioblastomas (GBMs) are devastating tumors of the central nervous system, with a poor prognosis of 1-year survival. This results from a high resistance of GBM tumor cells to current therapeutic options, including etoposide (VP-16). Understanding resistance mechanisms may thus open new therapeutic avenues. VP-16 is a topoisomerase inhibitor that causes replication fork stalling and, ultimately, the formation of DNA double-strand breaks and apoptotic cell death. Autophagy has been identified as a VP-16 treatment resistance mechanism in tumor cells. Retinoblastoma protein (RB) is a classical tumor suppressor owing to its role in G1/S cell cycle checkpoint, but recent data have shown RB participation in many other cellular functions, including, counterintuitively, negative regulation of apoptosis. As GBMs usually display an amplification of the EGFR signaling involving the RB protein pathway, we questioned whether RB might be involved in mechanisms of resistance of GBM cells to VP-16. We observed that RB silencing increased VP-16-induced DNA double-strand breaks and p53 activation. Moreover, RB knockdown increased VP-16-induced apoptosis in GBM cell lines and cancer stem cells, the latter being now recognized essential to resistance to treatments and recurrence. We also showed that VP-16 treatment induced autophagy, and that RB silencing impaired this process by inhibiting the fusion of autophagosomes with lysosomes. Taken together, our data suggest that RB silencing causes a blockage on the VP-16-induced autophagic flux, which is followed by apoptosis in GBM cell lines and in cancer stem cells. Therefore, we show here, for the first time, that RB represents a molecular link between autophagy and apoptosis, and a resistance marker in GBM, a discovery with potential importance for anticancer treatment.     

Calcium and S100B Regulation of p53-Dependent Cell Growth Arrest and Apoptosis

In glial C6 cells constitutively expressing wild-type p53, synthesis of the calcium-binding protein S100B is associated with cell density-dependent inhibition of growth and apoptosis in response to UV irradiation. A functional interaction between S100B and p53 was first demonstrated in p53-negative mouse embryo fibroblasts (MEF cells) by sequential transfection with the S100B and the temperature-sensitive p53Val135 genes. We show that in MEF cells expressing a low level of p53Val135, S100B cooperates with p53Val135 in triggering calcium-dependent cell growth arrest and cell death in response to UV irradiation at the nonpermissive temperature (37.5°C). Calcium-dependent growth arrest of MEF cells expressing S100B correlates with specific nuclear accumulation of the wild-type p53Val135 conformational species. S100B modulation of wild-type p53Val135 nuclear translocation and functions was confirmed with the rat embryo fibroblast (REF) cell line clone 6, which is transformed by oncogenic Ha-ras and overexpression of p53Val135. Ectopic expression of S100B in clone 6 cells restores contact inhibition of growth at 37.5°C, which also correlates with nuclear accumulation of the wild-type p53Val135 conformational species. Moreover, a calcium ionophore mediates a reversible G₁ arrest in S100B-expressing REF (S100B-REF) cells at 37.5°C that is phenotypically indistinguishable from p53-mediated G₁ arrest at the permissive temperature (32°C). S100B-REF cells proceeding from G₁ underwent apoptosis in response to UV irradiation. Our data support a model in which calcium signaling and S100B cooperate with the p53 pathways of cell growth inhibition and apoptosis.     

RASSF6 promotes p21Cip1/Waf1-dependent cell cycle arrest and apoptosis through activation of the JNK/SAPK pathway in clear cell renal cell carcinoma

Clear cell renal cell carcinoma (ccRCC) is a highly aggressive and common pathological subtype of renal cancer. This cancer is characterized by biallelic inactivation of the von Hippel–Lindau (VHL) tumor suppressor gene, which leads to the accumulation of hypoxia-inducible factors (HIFs). Although therapies targeted at HIFs can significantly improve survival, nearly all patients with advanced ccRCC eventually succumb to the disease. Thus, additional oncogenic events are thought to be involved in the development of ccRCC tumors. In this study, we investigated the role of RASSF6 in ccRCC. Downregulation of RASSF6 was commonly observed in primary tumors relative to matched adjacent normal tissues. Moreover, functional studies established that ectopic re-expression of RASSF6 in ccRCC cells inhibited cell proliferation, clonogenicity, and tumor growth in mice, whereas silencing of RASSF6 dramatically enhanced cell proliferation in vitro and in vivo. Mechanistic investigation suggested that RASSF6 triggers p21^Cip1/Waf1 accumulation to induce G₁ cell cycle arrest and promote apoptosis upon exposure to pro-apoptotic agents, and both of these mechanisms appear to be mediated by activated JNK signaling. Together, these findings suggest that RASSF6 may play a tumor suppressor role in the progression of ccRCC.     

A Fluorescence Polarization Assay for the Identification of Inhibitors of the p53–DM2 Protein–Protein Interaction

Improper function of the tumor suppressor protein p53 is a contributing factor in many human cancers. In normal cells, p53 acts to arrest the cell cycle in response to DNA damage or nucleotide depletion. One mechanism of regulating the amount of p53 in the cell is through the action of the Double Minute 2 protein, DM2 (also known as MDM2), which ubiquitinates p53 and targets it for proteosomal degradation. In a number of human cancers, the DM2 gene is amplified or overexpressed, leading to inadequate levels of p53 for cell cycle arrest or apoptosis. With the goal of restoring p53 function in cancers that overexpress DM2, we are developing inhibitors of the p53-DM2 protein-protein interaction that structurally mimic the N-terminal segment of p53 that binds to DM2. To assist this effort, we have devised a fluorescence polarization assay that quantifies the interaction between the N-terminal regions of both proteins in 384-well microtiter plates. Using this assay, we have demonstrated that a peptide with a nonhydrolyzable beta-amino acid substitution binds DM2 with an affinity comparable to a p53 peptide that is composed of only alpha-amino acids.     

Silencing of RASSF3 by DNA Hypermethylation Is Associated with Tumorigenesis in Somatotroph Adenomas

The pathogenic mechanisms underlying pituitary somatotroph adenoma formation, progression are poorly understood. To identify candidate tumor suppressor genes involved in pituitary somatotroph adenoma tumorigenesis, we used HG18 CpG plus Promoter Microarray in 27 human somatotroph adenomas and 4 normal human adenohypophyses. RASSF3 was found with frequent methylation of CpG island in its promoter region in somatotroph adenomas but rarely in adenohypophyses. This result was confirmed by pyrosequencing analysis. We also found that RASSF3 mRNA level correlated negatively to its gene promoter methylation level. RASSF3 hypermethylation and downregulation was also observed in rat GH3 and mouse GT1.1 somatotroph adenoma cell lines. 5-Aza-2′ deoxycytidine and trichostatin-A treatment induced RASSF3 promoter demethylation, and restored its expression in GH3 and GT1.1 cell lines. RASSF3 overexpression in GH3 and GT1.1 cells inhibited proliferation, induced apoptosis accompanied by increased Bax, p53, and caspase-3 protein and decreased Bcl-2 protein expression. We also found that the antitumor effect of RASSF3 was p53 dependent, and p53 knockdown blocked RASSF3-induced apoptosis and growth inhibition. Taken together, our results suggest that hypermethylation-induced RASSF3 silencing plays an important role in the tumorigenesis of pituitary somatotroph adenomas.     

MDM2 inhibitor Nutlin-3a suppresses proliferation and promotes apoptosis in osteosarcoma cells

Restoring p53 activity by inhibiting the interaction between p53 and the mouse double minutes clone 2 (MDM2) offers an attractive approach to cancer therapy. Nutlin-3a is a small-molecule inhibitor that inhibits MDM2 binding to p53 and subsequent p53-dependent DNA damage signaling. In this study, we determined the efficacy of Nutlin-3a in inducing p53-mediated cell death in osteosarcoma (OS) cell lines both in vivo and in vitro. Targeted disruption of the p53-MDM2 interaction by Nutlin-3a stabilizes p53 and selectively activates the p53 pathway only in OS cells with wild-type p53, resulting in a pronounced anti-proliferative and cytotoxic effect due to G1 cell cycle arrest and apoptosis both in vitro and in vivo. p53 dependence of these alternative outcomes of Nutlin-3a treatment was shown by the abrogation of these effects when p53 was knocked-down by small interfering RNA. These data suggest that the disruption of p53-MDM2 interaction by Nutlin-3a might be beneficial for OS patients with MDM2 amplification and wt p53 status.