P53-deficient H1299 Cells Research Articles

Arsenite-induced cytotoxicity is regulated by poly-ADP ribose polymerase 1 activation and parthanatos in p53-deficient H1299 cells: The roles of autophagy and p53

Arsenic is a double-edged sword metalloid since it is both an environmental carcinogen and a chemopreventive agent. Arsenic cytotoxicity can be dependent or independent of the tumor suppressor p53. However, the effects and the underlying molecular mechanisms of arsenic cytotoxicity in p53-deficient cells are still unclear. Here, we report a distinctive cell death mode via PARP-1 activation by arsenic in p53-deficient H1299 cells. H1299 (p53−/−) cells showed higher sensitivity to sodium arsenite (NaAR) than H460 (p53+/+) cells. H460 cells induced canonical apoptosis through caspase-dependent poly-ADP ribose polymerase 1 (PARP-1) cleavage and induced the expression of phospho-p53 and p21. However, H1299 cells induced poly-ADP-ribose (PAR) polymer accumulation and caspase-independent parthanatos, which was inhibited by 3-aminobenzamide (AB) and nicotinamide (NAM). Fractionation studies revealed the mitochondrial translocation of PAR polymers and nuclear translocation of the apoptosis-inducing factor (AIF). Although the exposure of NaAR to p53-overexpressing H1299 cells increased the PAR polymer levels, it inhibited parthanatos by inducing p21 and phospho-p53 expression. LC3-II and p62 accumulated in a NaAR dose- and exposure time-dependent manner, and this accumulation was further enhanced by autophagy inhibition, indicating that arsenic inhibits autophagic flux. p53 overexpression led to a decrease in the p62 levels, an increase in the LC3-II levels, and reduced parthanatos, indicating that arsenic induces p53-dependent functional autophagy. These results show that the NaAR-induced cytotoxicity in p53-deficient H1299 cells is regulated by PARP-1 activation-mediated parthanatos, which is promoted by autophagy inhibition. This suggests that PARP-1 activation could be used as an effective therapeutic approach for arsenic toxicity in p53-deficient cells.

CD44+ and CD133+ Non-Small Cell Lung Cancer Cells Exhibit DNA Damage Response Pathways and Dormant Polyploid Giant Cancer Cell Enrichment Relating to Their p53 Status.

Cancer stem cells (CSCs) play a critical role in the initiation, progression and therapy relapse of many cancers including non-small cell lung cancer (NSCLC). Here, we aimed to address the question of whether the FACS-sorted CSC-like (CD44 + &CD133 +) vs. non-CSC (CD44−/CD133− isogenic subpopulations of p53wt A549 and p53null H1299 cells differ in terms of DNA-damage signaling and the appearance of “dormant” features, including polyploidy, which are early markers (predictors) of their sensitivity to genotoxic stress. X-ray irradiation (IR) at 5 Gy provoked significantly higher levels of the ATR-Chk1/Chk2-pathway activity in CD44−/CD133− and CD133+ subpopulations of H1299 cells compared to the respective subpopulations of A549 cells, which only excited ATR-Chk2 activation as demonstrated by the Multiplex DNA-Damage/Genotoxicity profiling. The CD44+ subpopulations did not demonstrate IR-induced activation of ATR, while significantly augmenting only Chk2 and Chk1/2 in the A549- and H1299-derived cells, respectively. Compared to the A549 cells, all the subpopulations of H1299 cells established an increased IR-induced expression of the γH2AX DNA-repair protein. The CD44−/CD133− and CD133+ subpopulations of the A549 cells revealed IR-induced activation of ATR-p53-p21 cell dormancy signaling-mediated pathway, while none of the CD44+ subpopulations of either cell line possessed any signs of such activity. Our data indicated, for the first time, the transcription factor MITF–FAM3C axis operative in p53-deficient H1299 cells, specifically their CD44+ and CD133+ populations, in response to IR, which warrants further investigation. The p21-mediated quiescence is likely the predominant surviving pathway in CD44−/CD133− and CD133+ populations of A549 cells as indicated by single-cell high-content imaging and analysis of Ki67- and EdU-coupled fluorescence after IR stress. SA-beta-galhistology revealed that cellular-stress-induced premature senescence (SIPS) likely has a significant influence on the temporary dormant state of H1299 cells. For the first time, we demonstrated polyploid giant and/or multinucleated cancer-cell (PGCC/MGCC) fractions mainly featuring the progressively augmenting Ki67low phenotype in CD44+ and CD133+ A549 cells at 24–48 h after IR. In contrast, the Ki67high phenotype enrichment in the same fractions of all the sorted H1299 cells suggested an increase in their cycling/heterochromatin reorganization activity after IR stress. Our results proposed that entering the “quiescence” state rather than p21-mediated SIPS may play a significant role in the survival of p53wt CSC-like NSCLC cells after IR. The results obtained are important for the selection of therapeutic schemes for the treatment of patients with NSCLC, depending on the functioning of the p53 system in tumor cells.

Induction of APOBEC3C Facilitates the Genotoxic Stress-Mediated Cytotoxicity of Artesunate.

The artemisinin class of anticancer compounds is well known for oxidative DNA-damage-mediated growth arrest, followed by cell death. However, the nature of this genotoxic stress for cancer therapeutics remains elusive. Here we show that artesunate (Art), a water-soluble artemisinin analog, triggers inducible anticancer responses directly implicated in the DNA-damage-intended therapy. We observed that the level of the antiviral enzyme APOBEC3C (apolipoprotein-B mRNA-editing catalytic polypeptide-like 3C (A3C)) preferentially increased upon the treatment with Art against tumor xenografts of p53-deficient H1299 cells. Using gain-of-function experiments, A3C could improve the therapeutic efficacy of Art, as determined by cell proliferation and colony formation assays. Furthermore, elevated A3C provoked a minor accumulation of γH2AX foci and the phosphorylation of RPA32 and Chk1, which strongly sensitized H1299 cells to Art. The employment of A3C also caused an increase in the synergistic interaction between Art and Chk1 inhibition. Besides, A3C overexpression delayed the cell cycle at the S phase, accompanied by attenuated G2/M arrest in the presence of Art. The enzymatic inactivation of A3C by the mutation of zinc-coordinating residues (C97S and C100S) indicated that A3C sensitized Art in a deaminase-dependent manner. Furthermore, we showed that using small interfering RNA against A3C can induce the chemoresistance of Art. These studies combine to suggest that upregulated A3C is involved in the Art-induced DNA damage response as a consequent event to improve the overall cytotoxic responses of Art.

Nutlin sensitizes lung carcinoma cells to interferon-alpha treatment in MDM2-dependent but p53-independent manner

As an anticancer therapeutic, Interferon-alpha (IFNα) is used to treat a number of malignancies. However, the application of IFNα is restricted mostly due to its high toxicity. Therefore, novel combination therapeutic regimens are required to decrease the toxicity of IFNα and enhance its efficacy. Here we show that the treatment of p53-deficient human non-small lung carcinoma H1299 cells with IFNα in combination with an inhibitor of MDM2, Nutlin-3a, synergistically affects the proliferation of cancer cells. Importantly, Nutlin-3a was able to reduce the effective dose of IFNα about 3.4 times. Strikingly, this phenomenon is p53-independent, because H1299 cells lack p53, but is highly dependent on MDM2 because its ablation makes tumor cells completely insensitive to IFNα alone or in combination with Nutlin-3a. On the contrary, overexpression of MDM2 makes H1299 cells more susceptible to both IFNα and IFNα/Nutlin-3a treatments. Mechanistically, treatment with combination of IFNα and Nutlin-3a attenuates cyclin D1/CDK4 on the protein level and hence blocks cell cycle progression. This mechanism may be responsible, at least in part, for the anti-proliferative effects on H1299 cells observed.Our data suggest that the expression of MDM2 confers sensitivity of cancer cells to IFNα/Nutlin-3a treatment. Moreover, our data also confirm positive effect of Nutlin even on p53-deficient neoplasms.

Xanthatin triggers Chk1-mediated DNA damage response and destabilizes Cdc25C via lysosomal degradation in lung cancer cells

Previous studies had shown that xanthatin, a natural xanthanolide sesquiterpene lactone, could induce mitotic arrest and apoptosis in non-small cell lung cancer (NSCLC) cells. Here, we examined whether the DNA damage response (DDR) could be a primary cytotoxic event underlying xanthatin-mediated anti-tumor activity. Using EdU incorporation assay in combination with novel imaging flow cytometry, our data indicated that xanthatin suppressed DNA replication, prevented cells from G2/M entry and increased the spot count of γH2AX nuclear foci. Given that checkpoint kinase 1 (Chk1) represents a core component in DDR-mediated cell cycle transition and the phosphorylation on Ser-345 is essential for kinase activation and function, we surprisingly found xanthatin distinctly modulated Ser-345 phosphorylation of Chk1 in A549 and H1299 cells. Further investigation on Cdc25C/CDK1/CyclinB1 signaling cascade in the absence or presence of pharmacological DDR inhibitors showed that xanthatin directly destabilized the protein levels of Cdc25C, and recovery of p53 expression in p53-deficient H1299 cells further intensified xanthatin-mediated inhibition of Cdc25C, suggesting p53-dependent regulation of Cdc25C in a DDR machinery. Moreover, exogenous expression of Cdc25C was also substantially repressed by xanthatin and partially impaired xanthatin-induced G2 arrest. In addition, xanthatin could induce accumulation of ubiquitinated Cdc25C without undergoing further proteasomal degradation. However, an alternative lysosomal proteolysis of Cdc25C was observed. Interestingly, lysosome-like vesicles were produced upon xanthatin treatment, accompanied by rapid accumulation of lysosomal associated membrane protein LAPM-1. Furthermore, vacuolar proton (V)-ATPases inhibitor bafilomycin A1 and lysosomal proteases inhibitor leupeptin could remarkably overturn the levels of Cdc25C in xanthatin-treated H1299 cells. Altogether, these data provide insight into how xanthatin can be effectively targeted DDR molecules towards certain tumors.

53BP1 contributes to regulation of autophagic clearance of mitochondria

Autophagy, the primary recycling pathway within cells, plays a critical role in mitochondrial quality control under normal growth conditions and in the cellular response to stress. Here we provide evidence that 53BP1, a DNA damage response protein, is involved in regulating mitochondrial clearance from the cell via a type of autophagy termed mitophagy. We found that when either human or mouse cells were 53BP1-deficient, there was an increase in mitochondrial abnormalities, as observed through staining intensity, aggregation, and increased mass. Moreover, a 53BP1-depleted cell population included an increased number of cells with a high mitochondrial membrane potential (ΔΨm) relative to controls, suggesting that the loss of 53BP1 prevents initiation of mitophagy thereby leading to the accumulation of damaged mitochondria. Indeed, both 53BP1 and the mitophagy-associated protein LC3 translocated to mitochondria in response to damage induced by the mitochondrial uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP). The recruitment of parkin, an E3-ubiquitin ligase, to mitochondria in response to CCCP treatment was significantly decreased in 53BP1-deficient cells. And lastly, using p53-deficient H1299 cells, we confirmed that the role of 53BP1 in mitophagy is independent of p53. These data support a model in which 53BP1 plays an important role in modulating mitochondrial homeostasis and in the clearance of damaged mitochondria.

Abstract 4490: Selinexor (KPT-330) radio-sensitizes non-small cell lung cancer cells in vitro and in vivo

Abstract The primary nuclear export protein, Exportin 1 (XPO1/CRM1), is overexpressed in most cancers and this overexpression is frequently correlated with poor prognosis. Selective Inhibitors of Nuclear Export (SINE) compounds are a family of small-molecule bioavailable drugs that bind covalently to XPO1 to inhibit nuclear export. This results in nuclear retention of major tumor suppressor proteins, such as p53, pRB, FOXO3A and other critical proteins in cancer biology, which leads to selective cancer cell death. Selinexor is the most advanced SINE with >500 hematological and solid tumor cancer patients treated to date in a number of Phase I/II clinical trials. Blocking XPO1 function also blocks nuclear transport of key DNA damage repair (DDR) proteins and we therefore hypothesized that combination of selinexor with radiation therapy (RT) would lead to synergistic anti-tumor activity. Radiation-chemotherapy combination is an established therapeutic option for patients with medically inoperable non-small cell lung cancer (NSCLC), but its use is limited due to tolerability and adverse effects. Moreover, this treatment strategy typically has short-term benefits and the patients relapse with progressive disease. Here we report on studies designed to test whether selinexor could interfere with DDR and synergize with radiation to enhance anti-tumor potency relative to either treatment alone. Cell cycle analysis and clonogenic assays were conducted in the presence or absence of selinexor in combination with escalating doses of RT on the radiation-sensitive and radiation-insensitive H1299 and A549 cell lines, respectively. Interestingly, Selinexor-treatment induced G1 cell cycle arrest in the p53-deficient cell line H1299 and a prominent G2 cell cycle arrest in p53-wild type A549 cells 24 hrs post treatment. Furthermore, clonogenic assays revealed that selinexor + RT treatment act synergistically to produce a dose-dependent growth inhibition in both cell lines. In vivo combination treatment in the A549 NSCLC xenograft model with low doses of selinexor and RT displayed a strong synergistic effect for reduction of tumor volume. In addition, microscopic and immunohistochemical analysis of the resected tumors showed an overall reduction in tumor cell numbers, increased fibrosis and induction of apoptosis in the selinexor-RT treated tumors compared to controls. Finally, mechanistic studies revealed that selinexor does not induce DNA damage like RT, but downregulates CHK1, RAD51 and other DDR proteins. Together, our results suggest that selinexor treatment sensitizes cells to RT by preventing single-stranded DNA break repair via downregulation of DDR protein expression. In contrast, selinexor induces cell cycle arrest at G1 in p53 deficient H1299 cells, allowing DNA damage accumulation and induction of apoptosis. These results provide a rationale basis for combining selinexor with RT in clinical trials studies. Citation Format: Tami Rashal, Sivan Elloul, Marsha Crochiere, Trinayan Kashyap, William Senapedis, Ryan George, Sharon Friedlander, Maya Ilouze, Yosef Landesman, Robert Carlson, Nir Peled, Michael Kauffman, Sharon Shacham, Yaacov Lawrence. Selinexor (KPT-330) radio-sensitizes non-small cell lung cancer cells in vitro and in vivo. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4490. doi:10.1158/1538-7445.AM2015-4490

Comparative Evaluation of Cytotoxic and Apoptogenic Effects of Several Coumarins on Human Cancer Cell Lines: Osthole Induces Apoptosis in p53-Deficient H1299 Cells.

Natural products are excellent resources for finding lead structures for the development of chemotherapeutic agents. Coumarins are a class of natural compounds found in a variety of plants. In this study, we evaluated the cytotoxic potential of coumarins isolated from Prangos ferulacea (L.) Lindl. in PC3, SKNMC, and H1299 (p53 null) human carcinoma cell lines. Osthole proved to be an outstanding potent cytotoxic agent especially against PC3 cells. Isoimperatorin exhibited moderate inhibitory effect against SKNMC and PC3 cell lines. Oxypeucedanin and braylin did not display any cytotoxic activity. In the next set of experiments, the apoptotic potentials of osthole and isoimperatorin were investigated. Induction of apoptosis by isoimperatorin was accompanied by an increase in activation of caspase-3, -8, and -9 in SKNMC cells and caspase-3 and -9 in PC3 cells. Moreover, isoimperatorin induced apoptosis by upregulating Bax and Smac/DIABLO genes in PC3 and SKNMC cells. Osthole induced apoptosis by downregulating antiapoptotic Bcl-2 in only PC3 cells and upregulating the proapoptotic genes Bax and Smac/DIABLO in PC3, SKNMC, and H1299 cells. The effects of osthole on H1299 cells are important because the loss of p53 has been associated with poor clinical prognosis in cancer treatment.

Ligularia fischeri regulates lung cancer cell proliferation and migration through down-regulation of epidermal growth factor receptor and integrin β1 expression

In the present study, we report the effect and molecular mechanism of Ligularia fischeri (LF) on proliferation and migration in human lung cancer cells. LF-mediated inhibition of cell proliferation in p53 wild-type A549 and p53-deficient H1299 cells is accompanied by reduced expression of cell cycle-related proteins such as cyclin-dependent kinases and cyclins, resulting in pRb hypophosphorylation and G1 phase cell cycle arrest. In contrast, LF inhibits cell migration in A549 cells, but not in H1299 cells. These regulatory effects of LF on cell proliferation and migration are associated with inactivation of mitogenic signaling pathways such as ERK, Akt and p70S6K, and down-regulation of epidermal growth factor receptor and integrin β1 expression. Collectively, these findings suggest further development and evaluation of LF for the prevention and treatment of lung cancer with mutated p53 as well as wild-type p53.

Methyl methanesulfonate induces apoptosis in p53-deficient H1299 and Hep3B cells through a caspase 2- and mitochondria-associated pathway

Methyl methanesulfonate (MMS) has been shown to induce apoptosis in various cell types through p53-dependent pathways. Nevertheless, pharmacological and genetic blockade of p53 functions results in similar or delayed sensitivity to MMS treatment, suggesting the presence of p53-independent apoptotic mechanisms. To understand the p53-independent mechanisms that are engaged during MMS-induced apoptosis, we established MMS-induced apoptotic cell models using p53-deficient H1299 and Hep3B cells. Our results demonstrated that MMS at concentrations of 50, 100, 200, 400 and 800μM induced the formation of gammaH2AX foci, and that at higher concentrations, 400 and 800μM, MMS treatment led to apoptosis in the two cell lines. This apoptotic cell death was concurrent with the loss of mitochondrial membrane potential, nuclear-cytosolic translocation of active caspase 2, release of cytochrome c from mitochondria, and the cleavage of caspase 9, caspase 3 and PARP. However, MMS-induced DNA damage failed to stabilize the p53 family members TAp73 and DNp73. These results demonstrated a p53- and p73-independent mechanism for MMS-induced apoptosis that involves the nuclear-cytosolic translocation of active caspase 2 as well as the mitochondria-mediated pathway.

P53 modulates homologous recombination at I-SceI-induced double-strand breaks through cell-cycle regulation

Inhibition of homologous recombination (HR) is believed to be a transactivation-independent function of p53 that protects from genetic instability. Misrepair by HR can lead to genetic alterations such as translocations, duplications, insertions and loss of heterozygosity, which all bear the risk of driving oncogenic transformation. Regulation of HR by wild-type p53 (wtp53) should prevent these genomic rearrangements. Mutation of p53 is a frequent event during carcinogenesis. In particular, dominant-negative mutants inhibiting wtp53 expressed from the unperturbed allel can drive oncogenic transformation by disrupting the p53-dependent anticancer barrier. Here, we asked whether the hot spot mutants R175H and R273H relax HR control in p53-proficient cells. Utilizing an I-SceI-based reporter assay, we observed a moderate (1.5 × ) stimulation of HR upon expression of the mutant proteins in p53-proficient CV-1, but not in p53-deficient H1299 cells. Importantly, the stimulatory effect was exactly paralleled by an increase in the number of HR competent S- and G2-phase cells, which can well explain the enhanced recombination frequencies. Furthermore, the impact on HR exerted by the transactivation domain double-mutant L22Q/W23S and mutant R273P, both of which were reported to regulate HR independently of G1-arrest execution, is also exactly mirrored by cell-cycle behavior. These results are in contrast to previous concepts stating that the transactivation-independent impact of p53 on HR is a general phenomenon valid for replication-associated and also for directly induced double-strand break. Our data strongly suggest that the latter is largely mediated by cell-cycle regulation, a classical transactivation-dependent function of p53.

Ginsenoside Rp1 Inhibits Proliferation and Migration of Human Lung Cancer Cells

Ginsenoside Rp1 (G-Rp1) is a novel ginseng saponin derivative with anti-tumor activity. However, the biochemical and molecular mechanisms of G-Rp1 on anti-tumor activity are not fully understood. In the present study, we report that G-Rp1 inhibits lung cancer cell proliferation, migration and adhesion in p53 wild-type A549 and p53-defi cient H1299 cells. Anti-proliferative activity of G-Rp1 in lung cancer cells is mediated by enhanced nuclear localization of cyclin-dependent kinase inhibitors including <TEX>$p27^{Kip1}$</TEX> and <TEX>$p21^{WAF1/Cip1}$</TEX>, and subsequent inhibition of pRb phosphorylation. We also show that these anti-tumor activities of G-Rp1 in both A549 and H1299 cells appear to be mediated by suppression of mitogenic signaling pathways such as ERK, Akt and <TEX>$p70^{S6K}$</TEX>. Taken together, these findings suggest further development and evaluation of G-Rp1 for the treatment of lung cancers with mutated p53 as well as wild-type p53.

Sepiapterin regulates cell proliferation and migration: its association with integrin α3β1 and p53 in human lung cancer cells

Tetrahydrobiopterin (BH4) has been known to be an essential cofactor for nitric oxide synthase as well as the aromatic amino acid hydroxylases, which are involved in regulation of cellular fates including proliferation, migration and differentiation. In the present study, we report that sepiapterin, a stable form of BH4 precursor, modulates proliferation and migration in human lung cancer cells. Sepiapterin induction of cell proliferation in p53 wild-type A549 cells, but not in p53-deficient H1299 cells, is accompanied by enhanced expression of cell cycle-related proteins such as cyclin-dependent kinase 4 (Cdk4), cyclin D and cyclin E, and reduced expression of Cdk inhibitor p21WAF1/Cip1, demonstrating that sepiapterin-induced mitogenic responses might be associated with p53 expression status in lung cancer cells. In addition, sepiapterin enhances cell migration in A549 cells, but not H1299 cells. Finally, we show that sepiapterin induces A549 cell proliferation and migration through the activation of Akt and p70S6K signaling pathways, as evidenced by using Akt and p70S6K inhibitor. Collectively, these findings indicate that sepiapterin might play differential roles in regulation of cellular fates, depending on the status of p53 expression in lung cancer.

RNA content in the nucleolus alters p53 acetylation via MYBBP1A

A number of external and internal insults disrupt nucleolar structure, and the resulting nucleolar stress stabilizes and activates p53. We show here that nucleolar disruption induces acetylation and accumulation of p53 without phosphorylation. We identified three nucleolar proteins, MYBBP1A, RPL5, and RPL11, involved in p53 acetylation and accumulation. MYBBP1A was tethered to the nucleolus through nucleolar RNA. When rRNA transcription was suppressed by nucleolar stress, MYBBP1A translocated to the nucleoplasm and facilitated p53-p300 interaction to enhance p53 acetylation. We also found that RPL5 and RPL11 were required for rRNA export from the nucleolus. Depletion of RPL5 or RPL11 blocked rRNA export and counteracted reduction of nucleolar RNA levels caused by inhibition of rRNA transcription. As a result, RPL5 or RPL11 depletion inhibited MYBBP1A translocation and p53 activation. Our observations indicated that a dynamic equilibrium between RNA generation and export regulated nucleolar RNA content. Perturbation of this balance by nucleolar stress altered the nucleolar RNA content and modulated p53 activity.

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.

450 Leptin and estrogen receptor expression in breast cancer patients with different clinical characteristics

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.

The effect of cellular environment and p53 status on the mode of action of the platinum derivative LA-12

In this study, we characterized the effects of LA-12 on tumor cell lines possessing wild type p53 and on p53-deficient/mutant cell lines and the results were compared to those obtained using cisplatin. We have determined changes of p53 levels, of its transcriptional activity, of its posttranscriptional modifications and the effect of the treatment on the cell cycle, on the induction of apoptosis and on gene expression. LA-12 induces weak accumulation of both transcriptionally active p53 tumor suppressor and of p21(WAF1/CIP1) protein. LA-12 and cisplatin also significantly differ in their effects on apoptosis and cell cycle and on gene expression spectra in studied cell lines. LA-12 induces higher apoptosis levels in comparison with those induced by cisplatin, especially in p53-deficient H1299 cells and in MCF-7DD cells with transcriptionally inactive p53. We suggest that LA-12-mediated apoptosis is not fully dependent on p53. This confirms the therapeutic potential of LA-12 as a more potent cytostatic agent for both tumor cells expressing wild type p53 and for p53-deficient or mutant cells.

Regulation of Plasminogen Activator Inhibitor-1 Expression by Tumor Suppressor Protein p53

H1299 lung carcinoma cells lacking p53 (p53-/-) express minimal amounts of plasminogen activator inhibitor-1 (PAI-1) protein as well as mRNA. p53(-/-) cells express highly unstable PAI-1 mRNA. Transfection of p53 in p53(-/-) cells enhanced PAI-1 expression and stabilized PAI-1 mRNA. On the contrary, inhibition of p53 expression by RNA silencing in non-malignant human lung epithelial (Beas2B) cells decreased basal as well as urokinase-type plasminogen activator-induced PAI-1 expression because of accelerated degradation of PAI-1 mRNA. Purified p53 protein specifically binds to the PAI-1 mRNA 3'-un-translated region (UTR), and endogenous PAI-1 mRNA forms an immune complex with p53. Treatment of purified p53 protein with anti-p53 antibody abolished p53 binding to the 3'-UTR of PAI-1 mRNA. The p53 binding region maps to a 70-nucleotide PAI-1 mRNA 3'-UTR sequence, and insertion of the p53-binding sequence into beta-globin mRNA destabilized the chimeric transcript. Deletion experiments indicate that the carboxyl-terminal region (amino acid residues 296-393) of p53 protein interacts with PAI-1 mRNA. These observations demonstrate a novel role for p53 as an mRNA-binding protein that regulates increased PAI-1 expression and stabilization of PAI-1 mRNA in human lung epithelial and carcinoma cells.

Histone methyltransferase protein SETD2 interacts with p53 and selectively regulates its downstream genes

SETD2 (SET domain containing protein 2) is a histone H3K36 trimethyltransferase protein that associates with hyperphosphorylated RNA polymerase II and involves in transcriptional elongation. However, whether and how SETD2 is implicated in the specific regulation of gene transcription remains unknown. Here we show that SETD2 could interact with p53 and selectively regulate the transcription factor activity of p53. The interaction was dependent of C-terminal region of SETD2, which contains the SET and WW domains, and the N-terminal transactivation domain (residues 1–45) of p53. Overexpression of SETD2 upregulated the expression levels of a subset of p53 targets including puma, noxa, p53AIP1, fas, p21, tsp1, huntingtin, but downregulated that of hdm2. In contrast, it had no significant effect on those of 14-3-3σ, gadd45 and pig3. Consistently, knockdown of endogenous SETD2 expression by RNA interference resulted in converse effects as expected. In p53-deficient H1299 cells, SETD2 lost the ability to regulate these gene expression except hdm2, indicating the dependence of p53. Furthermore, we demonstrated that SETD2 downregulated hdm2 expression by targeting its P2 promoter and then enhanced p53 protein stability. Collectively, these findings suggest that the histone methyltransferase SETD2 could selectively regulate the transcription of subset genes via cooperation with the transcription factor p53.

A newly identified dependence receptor UNC5H4 is induced during DNA damage-mediated apoptosis and transcriptional target of tumor suppressor p53

UNC5H4 is a netrin-1 receptor UNC5H family member. In this study, we found that UNC5H4 is a direct transcriptional target of p53. During adriamycin (ADR)-mediated apoptosis, UNC5H4 was significantly induced in p53-proficient U2OS cells but not in p53-deficient H1299 cells. Enforced expression of p53 induced UNC5H4. Consistent with these results, siRNA-mediated knockdown of p53 in U2OS cells attenuated ADR-dependent induction of UNC5H4. Indeed, we found four putative p53-responsive elements within intron 1 of UNC5H4 gene. Luciferase reporter assay and ChIP analysis demonstrated that, among them, two tandem elements respond to exogenous p53 which is efficiently recruited onto them. Furthermore, enforced expression of UNC5H4 remarkably reduced number of drug-resistant colonies in p53-proficient cells but not in p53-deficient cells, suggesting that UNC5H4-induced apoptosis is dependent on p53 status. siRNA-mediated knockdown of UNC5H4 rendered U2OS cells resistant to ADR. Collectively, our present results suggest that UNC5H4 amplifies p53-dependent apoptotic response.