P53-deficient Mouse Embryonic Fibroblasts Research Articles

Dependence of p53-deficient cells on the DHX9 DExH-box helicase.

DHX9 is a DExH-box helicase family member with key regulatory roles in a broad range of cellular processes. It participates at multiple levels of gene regulation, including DNA replication, transcription, translation, RNA transport, and microRNA processing. It has been implicated in tumorigenesis and recent evidence suggests that it may be a promising chemotherapeutic target. Previous studies have determined that DHX9 suppression elicits an apoptotic or senescence response by activating p53 signaling. Here, we show that DHX9 inhibition can also have deleterious effects in cells lacking functional p53. Loss of DHX9 led to increased cell death in p53-deficient mouse lymphomas and HCT116 human colon cancer cells, and G0/G1 cell cycle arrest in p53-deficient mouse embryonic fibroblasts. Analysis of mRNA levels for p53 transcriptional targets showed that a subset of p53 targets in the p53-null lymphomas and HCT116 cells were activated despite the absence of functional p53. This implies an alternative pathway of DHX9-mediated activation of cell death and cell cycle arrest in p53-deficient cells and supports the feasibility of targeting DHX9 in p53-deficient tumors.

Loss of p53 in stromal fibroblasts enhances tumor cell proliferation through nitric-oxide-mediated cyclooxygenase 2 activation

Overexpression of cyclooxygenase 2 (COX-2) by stromal fibroblasts plays a critical role in the early stage of carcinogenesis. COX-2 expression is thought to be positively or negatively regulated by inflammatory chemical mediators or tumor suppressors. In this study, the contributions of inducible nitric oxide synthase (iNOS) and p53 to COX-2 expression were examined using mouse embryonic fibroblasts (MEFs) from wild-type, p53-deficient, iNOS-deficient, and p53/iNOS-deficient mice. These MEFs were treated with 1 μg/mL of lipopolysaccharide and 100 IU/mL of interferon gamma for up to 72 h. iNOS and COX-2 expression were analyzed by Western blotting. iNOS was induced earlier (16 h) in p53-deficient MEFs than in wild-type MEFs (48 h). Elevated expression of COX-2 was sustained for a longer duration in the p53-deficient MEFs. In contrast, COX-2 expression was reduced earlier in the iNOS-deficient MEFs. Addition of an exogenous NO donor (0.8 mM of S-nitroso-l-glutathione) to the iNOS-deficient MEFs augmented COX-2 expression. Co-culture with stimulated p53-deficient MEFs promoted cell proliferation of mouse rectal polyploid carcinoma CMT93 cells, but treatment with a COX-2-specific inhibitor counteracted this effect. These results suggest that loss of function of the p53 gene in stromal fibroblasts enhances COX-2 expression by enhancing iNOS expression and the resultant production of NO, contributing to the promotion of tumor growth.

Hexokinase 2-Mediated Warburg Effect Is Required for PTEN- and p53-Deficiency-Driven Prostate Cancer Growth

Accumulating evidence suggests that codeletion of the tumor suppressor genes Pten and p53 plays a crucial role in the development of castration-resistant prostate cancer in vivo. However, the molecular mechanism underlying Pten-/p53-deficiency-driven prostate tumorigenesis remains incompletely understood. Building upon insights gained from our studies with Pten-/p53-deficient mouse embryonic fibroblasts (MEFs), we report here that hexokinase 2 (HK2) is selectively upregulated by the combined loss of Pten and p53 in prostate cancer cells. Mechanistically, Pten deletion increases HK2 mRNA translation through the activation of the AKT-mTORC1-4EBP1 axis, and p53 loss enhances HK2 mRNA stability through the inhibition of miR143 biogenesis. Genetic studies demonstrate that HK2-mediated aerobic glycolysis, known as the Warburg effect, is required for Pten-/p53-deficiency-driven tumor growth in xenograft mouse models of prostate cancer. Our findings suggest that HK2 might be a therapeutic target for prostate cancer patients carrying Pten and p53 mutations.

Abstract B53: Induction of cells with cancer stem cell properties from p53-deficient somatic cells.

Abstract The tumor suppressor p53 prevents the initiation of tumor formation by inducing cell cycle arrest, senescence and apoptosis during the process of oncogenesis. Although p53 deficiency promotes the efficiency of somatic cell reprogramming to a pluripotent state upon the transduction of four reprogramming genes Oct4, Sox2, Klf4, and c-Myc, it often gives rise to induced pluripotent stem cells (iPSC) with cancer-like features. We attempted to generate cells with cancer stem cell (CSC) properties from p53-deficient mouse embryonic fibroblasts (MEFs) in vitro. The introduction of iPS reprogramming factors into p53-/- MEFs, followed by partial differentiation via embryoid body formation, transformed the bulk of cells into tumorigenic cells with CSC features. These transformed cells displayed a malignant phenotype and formed tumors of multiple lineages when injected into immunocompromised mice. Immunohistochemical analysis revealed the presence of epithelial (E-cadherin), mesenchymal (Vimentin) and myogenic (Desmin) positive cells in the tumor tissues. These transformed CSC-like cells can be maintained in regular cell culture media in vitro with 10% fetal bovine serum, permitting easy manipulation and amenability to large-scale drug screening. Our current approach could therefore provide a valuable basis for the further analysis of CSC characteristics and drug development strategies designed to target CSCs. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B53. Citation Format: Mayuko Nishi, Akihide Ryo. Induction of cells with cancer stem cell properties from p53-deficient somatic cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B53.

Abstract B30: Identification of new regulator in p53-mediated cellular senescence

Abstract The downstream events and target genes of p53 in senescence responses are not fully understood. Here, we identify a novel regulator, A-core gene, in p53-mediated cellular senescence. Overexpression of A-core in p53-deficient mouse embryonic fibroblasts (MEFs) accelerates senescence, whereas A-core gene knockdown by small interfering RNA (siRNA) leads to escape from p53-mediated senescence in p53-expressing MEFs. A-core overexpression also increased intracellular levels of reactive oxygen species (ROS). The ROS inhibitor N-acetyl-L-cysteine rescued A-core overexpression-induced senescence. Surprisingly, the elevated levels of ROS that accompanied A-core overexpression were paralleled by an increase in p21 expression. siRNA-mediated knockdown of p21 in A-core overexpressing MEFs abrogated the A-core-dependent increase in ROS levels, indicating that A-core acts through p21 induction and subsequent ROS elevation to trigger senescence. In addition, expression of the cell proliferation-related proteins cyclin E and B was decreased after A-core expression. Collectively, these results suggest that A-core is a downstream target of p53 that is sufficient to induce p21 expression and ROS production, and is necessary for p53-mediated senescence. Citation Format: Jae-Sik Shin, Seung-Woo Hong, Dae-Hee Lee, Jai-Hee Moon, Jeong Eun Kim, Yong Sang Hong, Kyu-pyo Kim, Jae-Lyun Lee, Eun Kyung Choi, Jung Shin Lee, Dong-Hoon Jin, Tae Won Kim. Identification of new regulator in p53-mediated cellular senescence. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr B30.

The Roles of Telomerase in the Generation of Polyploidy during Neoplastic Cell Growth

Polyploidy contributes to extensive intratumor genomic heterogeneity that characterizes advanced malignancies and is thought to limit the efficiency of current cancer therapies. It has been shown that telomere deprotection in p53-deficient mouse embryonic fibroblasts leads to high rates of polyploidization. We now show that tumor genome evolution through whole-genome duplication occurs in ∼15% of the karyotyped human neoplasms and correlates with disease progression. In a panel of human cancer and transformed cell lines representing the two known types of genomic instability (chromosomal and microsatellite), as well as the two known pathways of telomere maintenance in cancer (telomerase activity and alternative lengthening of telomeres), telomere dysfunction-driven polyploidization occurred independently of the mutational status of p53. Depending on the preexisting context of telomere maintenance, telomerase activity and its major components, human telomerase reverse transcriptase (hTERT) and human telomerase RNA component (hTERC), exert both reverse transcriptase-related (canonical) and noncanonical functions to affect tumor genome evolution through suppression or induction of polyploidization. These new findings provide a more complete mechanistic understanding of cancer progression that may, in the future, lead to novel therapeutic interventions.

Unmet Expectations: miR-34 Plays No Role in p53-Mediated Tumor Suppression In Vivo

Unmet Expectations: miR-34 Plays No Role in p53-Mediated Tumor Suppression In Vivo

Reversible induction of PARP1 degradation by p53-inducible cis-imidazoline compounds

PARP1 is an important enzyme involved in various patho-physiological phenomena such as ischemia/reperfusion (I/R) injury, which occurs when blood flow is restored after cerebral infarction, myocardial infarction and transplantation of various organs. I/R-induced PARP1 over-activation is mediated by production of reactive oxygen species and is involved in NF-κB transactivation. For these reasons, PARP1 is an attractive target for strategies to protect against I/R injury. We previously reported that an MDM2 inhibitor Nutlin3a, a cis-imidazoline compound, induces PARP1 degradation in a p53 and proteasome-dependent manner. In this study, we evaluated the effect of Nutlin3a analogs, Nutlin3b and Caylin2, on PARP1 degradation. Like Nutlin3a, Caylin2, but not Nutlin3b, induced PARP1 degradation in both 3T3-L1 and 3T3-F442A. This result occurred almost in parallel with p53 accumulation. Furthermore Caylin2-induced PARP1 degradation was not observed in p53 deficient mouse embryonic fibroblasts or in the presence of the proteasome inhibitor MG132. These results suggest that Caylin2 induces PARP1 degradation by the same mechanism as Nutlin3a. Finally, we showed that Nutlin3a or Caylin2 treatment induces reversible PARP1 down-regulation without an inflammatory response. For protection against I/R injury, our results support the usability of the p53 inducible cis-imidazoline compounds, Nutlin3a and its analogs, as PARP1 inhibitors.

Downregulation of Mdm2 and Mdm4 enhances viral gene expression during adenovirus infection

Successful viral replication entails elimination or bypass of host antiviral mechanisms. Here, we show that shRNA-mediated knockdown of murine double minute (Mdm2) and its paralog Mdm4 enhanced the expression of early and late viral gene products during adenovirus (HAdV) infection. Remarkably, whereas the expression of HAdV genes was low in p53-deficient mouse embryonic fibroblasts (p53KO MEFs), the HAdV early gene products were efficiently expressed in Mdm2/p53 double-knockout (DKO) and Mdm4/p53 DKO MEFs, and viral capsid proteins were produced in Mdm2/p53 DKO MEFs. Thus, Mdm2 and Mdm4 seem to have potent antiviral property. In cells infected with wt HAdV or a mutant virus lacking the E1B-55K gene (dl1520), both Mdm2 and Mdm4 were rapidly depleted, whereas replication-deficient mutant viruses (Ad-GFP) or ΔpTP with deletions within the coding sequence of preterminal binding protein failed to induce their downregulation. Reduced expression of Mdm2 and Mdm4 was not due to general shutoff of host protein synthesis. Additionally, expression of a dominant-negative mutant of Cul5 did not affect Mdm2/Mdm4 downregulation. Thus, viral replication but not the presence of E1B-55K is required for Mdm2/Mdm4 degradation. Surprisingly, treatment of HAdV-infected cells with proteasome inhibitor MG132 only partially restored the protein levels of Mdm2 and Mdm4, suggesting that they may also be downregulated through an additional mechanism independent of proteasome. Interestingly, cyclin D1 and p21 appear to be downregulated similarly during HAdV infection. Collectively, our work provides the first biochemical evidence for antiviral function of Mdm2 and Mdm4 and that viruses employ efficient countermeasure to ensure viral replication.

Activation of Interferon-Stimulated Genes by γ-Ray Irradiation Independently of the Ataxia Telangiectasia Mutated–p53 Pathway

The ataxia telangiectasia mutated (ATM)-p53 pathway is a well-known main signal transduction pathway for cellular responses, which is activated by γ-ray irradiation. Microarray analysis showed changes in the expressions of IFN-stimulated genes (ISG) in γ-ray-irradiated Balb/cA/Atm-deficient mouse embryonic fibroblasts (MEF) (ATM-KO), indicating that another pathway for cellular responses besides the ATM-p53 pathway was activated by γ-ray irradiation. The basal expression levels of Irf7 and Stat1 in ATM-KO and p53-deficient MEFs (p53-KO) were higher than those in Atm-wild-type MEFs (ATM-WT) and p53-wild-type MEFs (p53-WT), respectively. Irradiation stimulated the expressions of Irf7 and Stat1 in ATM-KO, p53-KO, ATM-WT, and p53-WT, indicating that upregulation of Irf7 and Stat1 expressions by irradiation did not depend on the ATM-p53 pathway. When conditioned medium (CM) obtained from irradiated ATM-WT or ATM-KO was added to nonirradiated MEFs, the expressions of Irf7 and Stat1 increased. We predicted that gene activation in nonirradiated MEFs was caused by IFN-α/β. Unexpectedly, significant amount of IFN-α/β could not be detected in the CM from irradiated ATM-WT or ATM-KO. Meanwhile, increased expression of Ccl5 (RANTES) protein was detected in the CM from irradiated MEFs. These results indicate that ISGs were activated by γ-ray irradiation independently of the ATM-p53 pathway and gene activation was caused by radiation-induced soluble factors.

Pierce1, a Novel p53 Target Gene Contributing to the Ultraviolet-Induced DNA Damage Response

Retinoblastoma (Rb) and p53 genes are mutated or inactivated in most human cancers and mutually regulate each other. Recently, we reported that expression of diverse genes was altered in Rb-deficient mouse embryonic fibroblasts (MEF). In this study, we found that Pierce1, a novel transcript upregulated in Rb-deficient MEFs, is a transcriptional target of p53. Although Pierce1 promoter did not respond to the ectopic expression of E2F1, it was strongly activated by p53 via 2 cis-elements. Consistently, the expression of Pierce1 was induced by genotoxic stresses that activate p53 but was not detected in p53-deficient MEFs. Pierce1 was posttranslationally stabilized by ultraviolet C (UVC) irradiation, and UVC-activated ATR (ataxia telangiectasia-mutated and Rad3-related) signaling suppressed proteosomal degradation of Pierce1 protein. Furthermore, knockdown of Pierce1 compromised the checkpoint response of wild-type MEFs to UVC irradiation, accompanying the diminished expression of p53 target genes. Together, our data suggest that Pierce1 is an important p53 target gene contributing to normal DNA damage response and may play crucial roles in maintaining genomic integrity against genotoxic stresses, including UVC irradiation.

Topotecan-Triggered Degradation of Topoisomerase I Is p53-Dependent and Impacts Cell Survival

The anticancer drug topotecan belongs to the group of topoisomerase I (topo I) inhibitors. In the presence of topotecan, topo I cleaves the DNA but is unable to religate the single-strand break. This leads to stabilization of topo I-DNA-bound complexes and the accumulation of DNA strand breaks that may interfere with DNA replication. The molecular mechanism of controlling the repair of topo I-DNA covalent complexes and its impact on sensitivity of cells to topotecan is largely unknown. Here, we used mouse embryonic fibroblasts expressing wild-type p53 and deficient in p53, in order to elucidate the role of p53 in topotecan-induced cell death. We show that p53-deficient mouse embryonic fibroblasts are significantly more sensitive to topotecan than wild-type cells, displaying a higher frequency of topotecan-induced apoptosis and DNA strand breaks. Treatment of p53 wild-type cells with pifithrin-alpha, an inhibitor of the trans-activating activity of p53, caused reversal of the phenotype, making wild-type cells more sensitive to topotecan. Upon topotecan treatment, topo I was degraded in wild-type but not in p53-deficient cells. Topo I degradation was attenuated by the proteosomal inhibitor MG132. Similar data were obtained with human glioblastoma cells. U138 cells (p53 mutated) were significantly more sensitive to topotecan than U87 cells (p53 wild-type). Furthermore, U87 cells showed significant degradation of topo I upon topotecan treatment, whereas in U138 cells, this response was abrogated. Topo I degradation was again attenuated by pifithrin-alpha. The data suggests that p53 causes resistance of cells to topo I inhibitors due to stimulation of topotecan-triggered topo I degradation which may impact topotecan-based cancer therapy.

TNFα induces sequential activation of Cdc42- and p38/p53-dependent pathways that antagonistically regulate filopodia formation

Cell migration is an essential function in various physiological processes, including tissue repair and tumour invasion. Repair of tissue damage requires the recruitment of fibroblasts to sites of tissue injury, which is mediated in part by the cytokine tumour necrosis factor alpha (TNFalpha). As dynamic rearrangements of actin cytoskeleton control cell locomotion, this implicates that TNFalpha is a potent coordinator of cellular actin changes. We have investigated the role of TNFalpha in regulating the cortical actin-containing structures essential for cell locomotion called filopodia. Kinetic analysis of TNFalpha-treated mouse embryonic fibroblasts (MEFs) revealed a dual effect on filopodia formation: a rapid and transient induction mediated by Cdc42 GTPase that is then counteracted by a subsequent sustained inhibition requiring activation of the mitogen-activated protein kinase p38 but not Cdc42 activity. This inhibition also involves the tumour suppressor p53, given that it is activated in response to TNFalpha following the same time course as the decrease of filopodia formation. This functional activation of p53, measured by transcription induction of its target p21WAF1(p21), is also associated with p38 kinase-dependent phosphorylation of p53 at serine 18. Furthermore, TNFalpha did not inhibit filopodia formation in MEFs treated with the transcription inhibitor actinomycin D, in p53-deficient MEFs, or MEFs expressing p53 mutants H273 or H175, which supports a role for the transcriptional activity of p53 in mediating TNFalpha-dependent filopodia inhibition. Our data delineate a novel inhibitory pathway in which TNFalpha prevents filopodia formation and cell migration through the activation of the mitogen-activated protein kinase (MAPK) p38, which in turn activates p53. This shows that TNFalpha on its own initiates antagonistic signals that modulate events linked to cell migration.

Cell growth- and P53-dependent transcriptional activity of the midkine promoter confers suicide gene expression in tumor cells

Midkine (MK) is preferentially expressed in a number of human tumors, while the expression in adult normal tissues is restricted. Previous studies showed that a 2.3-kb regulatory region of the human MK gene could selectively activate a linked suicide gene in tumors. In this study, we explored the minimal promoter region using genomic fragments deleted from the 5'-upstream side and analyzed the mechanism of the preferential activation in tumor cells. Luciferase assays showed that the 0.3-kb fragment from the transcription start site contained a cis-acting element(s) for the promoter activity. Expression of the herpes simplex virus-thymidine kinase gene under the control of the MK promoter followed by ganciclovir administration produced antitumor effects in vivo. Transfection of the wild-type p53 gene into the immortalized fibroblasts bearing mutated p53 and tumor cell lines, which induced cell cycle arrest, decreased the MK promoter-mediated transcription more effectively than the SV40 or the cytomegalovirus promoter-mediated transcription. The P53-mediated downregulation of the MK promoter activity was stronger in p53-defective tumors than in wild-type p53-bearing tumors. Moreover, the MK promoter-mediated luciferase activity was greater in p53-deficient mouse embryonic fibroblasts than in those bearing wild-type p53 gene. The transcriptional activity of the MK promoter could be regulated by cell growth and in part P53-dependent pathways.

P53-independent apoptosis is induced by the p19 ARF tumor suppressor

p19 ARF is a potent tumor suppressor. By inactivating Mdm2, p19 ARF upregulates p53 activities to induce cell cycle arrest and sensitize cells to apoptosis in the presence of collateral signals. It has also been demonstrated that cell cycle arrest is induced by overexpressed p19 ARF in p53-deficient mouse embryonic fibroblasts, only in the absence of the Mdm2 gene. Here, we show that apoptosis can be induced without additional apoptosis signals by expression of p19 ARF using an adenovirus-mediated expression system in p53-intact cell lines as well as p53-deficient cell lines. Also, in primary mouse embryonic fibroblasts (MEFs) lacking p53/ARF, p53-independent apoptosis is induced irrespective of Mdm2 status by expression of p19 ARF. In agreement, p19 ARF-mediated apoptosis in U2OS cells, but not in Saos2 cells, was attenuated by coexpression of Mdm2. We thus conclude that there is a p53-independent pathway for p19 ARF-induced apoptosis that is insensitive to inhibition by Mdm2.

A p53-dependent Apoptosis Perpetrator

The activation of p53 can lead to cell-cycle arrest or apoptosis. Attardi et al . used subtractive hybridization to identify genes involved in the p53-dependent apoptosis pathway. They identified a gene called p53 apoptosis effector related to PMP-22 (PERP) that is expressed in p53-mediated apoptosis of mouse embryonic fibroblasts (MEFs), but not expressed in p53-deficient MEFs. Transfection of luciferase vectors containing the PERP promoter into wild-type and p53-deficient MEFs revealed that the PERP promoter was activated in a p53-dependent manner, through p53 response elements. Fluorescence assays indicated that PERP localizes to the Golgi complex and, to a lesser extent, the plasma membrane. In the absence of p53, overexpression of PERP led to apoptosis of MEFs. Expression of Bcl-2 inhibited apoptosis induced by overexpressed PERP without affecting the amount of PERP expressed. Thus, p53-induced expression of PERP may be sufficient to cause apoptosis. PERP shares sequence similarity to the PMP-22/gas3 family of tetraspanins, a group of plasma membrane proteins whose mutations are thought to lead to human neuropathies such as Charcot-Marie-Tooth. Attardi, L.D., Reczek, E.E., Cosmas, C., Demicco, E.G., McCurrach, M.E., Lowe, S.W., and Jacks, T. (2000) PERP, an apoptosis-associated target of p53, is a novel member of the PMP-22/gas3 family. Genes Dev . 14 : 704 -718. [Abstract] [Full Text]

Abrogation of oncogene-associated apoptosis allows transformation of p53-deficient cells.

p53-deficient mouse embryonic fibroblasts were used to establish a direct mechanism of tumor suppression by p53 involving the destruction of oncogene-expressing cells by apoptosis. The absence of p53 enhanced cell growth, appeared sufficient for immortalization, and allowed a single oncogene [adenovirus early region 1A (E1A)] to transform cells to a tumorigenic state. p53 suppressed transformation of E1A-expressing cells by apoptosis. Apoptosis was associated with p53 stabilization and was triggered by environmental signals that normally suppress cell growth. Absence of even a single p53 allele significantly enhanced cell growth and survival. Although abrogation of apoptosis allowed transformation by E1A alone, escape from apoptosis susceptibility was not a prerequisite for tumor growth. Consequently, p53 mutation could enhance the survival of malignant cells expressing oncogenes activated early in tumor progression.

P53-dependent apoptosis modulates the cytotoxicity of anticancer agents

Although the primary cellular targets of many anticancer agents have been identified, less is known about the processes leading to the selective cell death of cancer cells or the molecular basis of drug resistance. p53-deficient mouse embryonic fibroblasts were used to examine systematically the requirement for p53 in cellular sensitivity and resistance to a diverse group of anticancer agents. These results demonstrate that an oncogene, specifically the adenovirus E1A gene, can sensitize fibroblasts to apoptosis induced by ionizing radiation, 5-fluorouracil, etoposide, and adriamycin. Furthermore, the p53 tumor suppressor is required for efficient execution of the death program. These data reinforce the notion that the cytotoxic action of many anticancer agents involves processes subsequent to the interaction between drug and cellular target and indicate that divergent stimuli can activate a common cell death program. Consequently, the involvement of p53 in the apoptotic response suggests a mechanism whereby tumor cells can acquire cross-resistance to anticancer agents.