P53-mediated Cell Cycle Arrest Research Articles

Understanding the complexity of p53 in a new era of tumor suppression.

p53 was discovered 45 years ago as an SV40 large T antigen binding protein, coded by the most frequently mutated TP53 gene in human cancers. As a transcription factor, p53 is tightly regulated by a rich network of post-translational modifications to execute its diverse functions in tumor suppression. Although early studies established p53-mediated cell-cycle arrest, apoptosis, and senescence as the classic barriers in cancer development, a growing number of new functions of p53 have been discovered and the scope of p53-mediated anti-tumor activity is largely expanded. Here, we review the complexity of different layers of p53 regulation, and the recent advance of the p53 pathway in metabolism, ferroptosis, immunity, and others that contribute to tumor suppression. We also discuss the challenge regarding how to activate p53 function specifically effective in inhibiting tumor growth without harming normal homeostasis for cancer therapy.

Abstract 7577: A new cancer therapy strategy: Synthetic lethality induced by inhibition of mTOR and cMYC and JNK activation

Abstract Cancer cells are characterized by aberrant signal transduction and cell cycle control pathways that lead to unchecked cell proliferation. Uncontrolled cancer cell proliferation, as well as immune suppressive macrophage proliferation, contribute to tumor growth and spread. Effort to suppress tumor cell growth using Inhibitors of PI3K or MEK signaling pathways can initially inhibit tumor growth, but resistance develops due to upregulation of the alternative pathway. We identified a novel series of allosteric tyrosine kinase receptor inhibitors that induce synthetic lethality in AML, lung carcinoma, pancreatic carcinoma, and other tumor cells and in proliferating macrophages in vitro and in vivo by simultaneously inhibiting cMyc and Tor and activating the stress activated kinase JNK, leading to p53 mediated cell cycle arrest. Select kinase inhibitors with a unique positioning of a single side chain inhibited CDK8/19 and thereby Myc while stimulating phosphorylation and activation of JNK and p38. JNK activation promoted sustained ATF-2 and cJun-mediated p53 and Chk1/Chk2 activation, thereby promoting cell cycle arrest in G2/M and ultimately, apoptosis. These inhibitors potently suppressed tumor growth in vivo by halting both tumor cell and macrophage proliferation in G2/M. They synergized with anti-PD-1 and anti-CTLA-4 and led to tumor eradication and immunological memory formation. These studies identify novel allosteric inhibitors with potent synthetically lethal anti-tumor activity. Citation Format: Hui Chen, Jason Rivera, Marc Paradise, Zihan Xu, Giuliana Mognol, Judith Varner. A new cancer therapy strategy: Synthetic lethality induced by inhibition of mTOR and cMYC and JNK activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7577.

CREB3L1/OASIS: cell cycle regulator and tumor suppressor.

Cell cycle checkpoints detect DNA errors, eventually arresting the cell cycle to promote DNA repair. Failure of such cell cycle arrest causes aberrant cell proliferation, promoting the pathogenesis of multiple diseases, including cancer. Endoplasmic reticulum (ER) stress transducers activate the unfolded protein response, which not only deals with unfolded proteins in ER lumen but also orchestrates diverse physiological phenomena suchascell differentiation and lipid metabolism. Among ER stress transducers,cyclic AMP-responsive element-binding protein 3-like protein 1 (CREB3L1) [also known as old astrocyte specifically induced substance (OASIS)] is an ER-resident transmembrane transcription factor. This molecule is cleaved by regulated intramembrane proteolysis, followed by activation as a transcription factor. OASIS is preferentially expressed in specific cells, including astrocytes and osteoblasts, to regulate their differentiation. In accordance with its name, OASIS was originally identified as being upregulated in long-term-cultured astrocytes undergoing cell cycle arrest because of replicative stress. In the context of cell cycle regulation, previously unknown physiological roles of OASIS have been discovered. OASIS is activated as a transcription factor in response to DNA damage to induce p21-mediated cell cycle arrest. Although p21 is directly induced by the master regulator of the cell cycle, p53, no crosstalk occurs between p21 induction by OASIS or p53. Here, we summarize previously unknown cell cycle regulation by ER-resident transcription factor OASIS, particularly focusing on commonalities and differences in cell cycle arrest between OASIS and p53. This review also mentions tumorigenesis caused by OASIS dysfunctions, and OASIS's potential as a tumor suppressor and therapeutic target.

A dual role of RBM42 in modulating splicing and translation of CDKN1A/p21 during DNA damage response

p53-mediated cell cycle arrest during DNA damage is dependent on the induction of p21 protein, encoded by the CDKN1A gene. p21 inhibits cyclin-dependent kinases required for cell cycle progression to guarantee accurate repair of DNA lesions. Hence, fine-tuning of p21 levels is crucial to preserve genomic stability. Currently, the multilayered regulation of p21 levels during DNA damage is not fully understood. Herein, we identify the human RNA binding motif protein 42 (RBM42) as a regulator of p21 levels during DNA damage. Genome-wide transcriptome and interactome analysis reveals that RBM42 alters the expression of p53-regulated genes during DNA damage. Specifically, we demonstrate that RBM42 facilitates CDKN1A splicing by counteracting the splicing inhibitory effect of RBM4 protein. Unexpectedly, we also show that RBM42, underpins translation of various splicing targets, including CDKN1A. Concordantly, transcriptome-wide mapping of RBM42-RNA interactions using eCLIP further substantiates the dual function of RBM42 in regulating splicing and translation of its target genes, including CDKN1A. Collectively, our data show that RBM42 couples splicing and translation machineries to fine-tune gene expression during DNA damage response.

Effect of IFN‑γ encapsulated liposomes on major signal transduction pathways in the lymphocytes of patients with lung cancer.

Globally, lung cancer affected 2.2 million individuals and caused 1.8 million deaths in 2021. Lung cancer is caused by smoking, genetics and other factors. IFN-γ has anticancer activity. However, the mechanism by which IFN-γ has an effect on lung cancer is not fully understood. The present study aimed to assess the effect of IFN-γ on the peripheral lymphocytes of patients with lung cancer compared with healthy controls. The efficacy of IFN-γ against oxidative stress was assessed using a comet repair assay and the effects of IFN-γ on p53, PARP1 and OGG1 genes and protein levels in lymphocytes was evaluated by RT-qPCR and western blotting. DNA damage was significantly reduced in the lymphocytes of patients treated with IFN-γ. However, there was no effect in the cells of healthy individuals after treatment with naked IFN-γ [IFN-γ (N)] and liposomal IFN-γ [IFN-γ (L)]. Following treatment with IFN-γ (N) and IFN-γ (L), the p53, PARP1 and OGG1 protein and gene expression levels were significantly increased (P<0.001). It has been suggested that IFN-γ may induce p53-mediated cell cycle arrest and DNA repair in patients. These findings supported the idea that IFN-γ (N) and IFN-γ (L) may serve a significant role in the treatment of lung cancer, via cell cycle arrest of cancer cells and repair mechanisms.

Tumor suppressor Parkin induces p53-mediated cell cycle arrest in human lung and colorectal cancer cells

Tumor suppressor Parkin induces p53-mediated cell cycle arrest in human lung and colorectal cancer cells

Ebracteolatain A Inhibits Lung Cancer Growth via p21 and p27 Mediated Cell-Cycle Arrest

Objective: Lung cancer is a refractory tumor with a poor five-year survival rate. Ebracteolatain A (EA), a key acetyl-phloroglucinol derived from the traditional Chinese herb Langdu, has been reported to have anti-tumor activity in human breast cancer. However, the therapeutic efficacy and underlying mechanism of lung cancer remain to be elucidated. In the present study, we intended to evaluate whether EA exhibits anti-lung cancer activity and elucidate the underlying mechanisms. Methods: ATPlite and clonogenic survival assays were used to assess the inhibiting effect of EA on two lung cancer cell lines in vitro. FACS was conducted to determine the cell-cycle profiling of lung cancer cells upon treatment with EA. Western blot was performed to detect the level of related proteins in EA-treated lung cancer cells. RNA silencing and other experiments were used to identify the molecular mechanism of EA-triggered cell-cycle arrest. Furthermore, a xenograft tumor model was constructed to evaluate the therapeutic effect and potential mechanism of EA in vivo in mice. Results: EA significantly inhibited the growth of lung cancer cells both in vitro and in vivo. Mechanistically, EA-induced G1 phase cell-cycle arrest is mediated by the accumulation of p21 and p27. Conclusion: Our findings revealed the anti-lung cancer effect of EA and the mechanisms, providing a solid scientific basis for EA as an attractive choice for lung cancer therapy.

USP7 - a crucial regulator of cancer hallmarks.

Over the course of three decades of study, the deubiquitinase Herpesvirus associated Ubiquitin-Specific Protease/Ubiquitin-Specific Protease 7 (HAUSP/USP7) has gradually come to be recognized as a crucially important molecule in cellular physiology. The fact that USP7 is overexpressed in a number of cancers, including breast, prostate, colorectal, and lung cancers, supports the idea that USP7 is also an important regulator of tumorigenesis. In this review, we discuss USP7's function in relation to the cancer hallmarks described by Hanahan and Weinberg. This post-translational modifier can support increased proliferation, block unfavorable growth signals, stop cell death, and support an unstable cellular genome by manipulating key players in the pertinent signalling circuit. It is interesting to note that USP7 also aids in the stabilization of molecules that support angiogenesis and metastasis. Targeting USP7 has now emerged as a crucial component of USP7 research because pharmacological inhibition of USP7 supports p53-mediated cell cycle arrest and apoptosis. Efficacious USP7 inhibition is currently being investigated in both synthetic and natural compounds, but issues with selectivity and a lack of co-crystal structure have hindered USP7 inhibition from being tested in clinical settings. Moreover, the development of new, more effective USP7 inhibitors and their encouraging implications by numerous groups give us a glimmer of hope for USP7-targeting medications as effective substitutes for hazardous cancer chemotherapeutics.

Apoptosis-modulatory miR-361-3p as a novel treatment target in endocrine-responsive and endocrine-resistant breast cancer.

Breast cancer (BC) is the most diagnosed cancer in women worldwide. In estrogen receptor (ER)-positive disease, anti-estrogens and aromatase inhibitors (AI) improve patient survival; however, many patients develop resistance. Dysregulation of apoptosis is a common resistance mechanism; thus, agents that can reinstate the activity of apoptotic pathways represent promising therapeutics for advanced drug-resistant disease. Emerging targets in this scenario include microRNAs (miRs). To identify miRs modulating apoptosis in drug-responsive and -resistant BC, a high-throughput miR inhibitor screen was performed, followed by high-content screening microscopy for apoptotic markers. Validation demonstrated that miR-361-3p inhibitor significantly increases early apoptosis and reduces proliferation of drug-responsive (MCF7), plus AI-/antiestrogen-resistant derivatives (LTED, TamR, FulvR), and ER- cells (MDA-MB-231). Importantly, proliferation-inhibitory effects were observed in vivo in a xenograft model, indicating the potential clinical application of miR-361-3p inhibition. RNA-seq of tumour xenografts identified FANCA as a direct miR-361-3p target, and validation suggested miR-361-3p inhibitor effects might be mediated in part through FANCA modulation. Moreover, miR-361-3p inhibition resulted in p53-mediated G1 cell cycle arrest through activation of p21 and reduced BC invasion. Analysis of publicly available datasets showed miR-361-3p expression is significantly higher in primary breast tumours vspaired normal tissue and is associated with decreased overall survival. In addition, miR-361-3p inhibitor treatment of BC patient explants decreased levels of miR-361-3p and proliferation marker, Ki67. Finally, miR-361-3p inhibitor showed synergistic effects on BC growth when combined with PARP inhibitor, Olaparib. Together, these studies identify miR-361-3p inhibitor as a potential new treatment for drug-responsive and -resistant advanced BC.

Senescence-associated exosomes transfer miRNA-induced fibrosis to neighboring cells.

Radiation-induced fibrosis is a common side effect of radiotherapy, which is the most common treatment for cancer. However, radiation also causes p53-mediated cell cycle arrest, prolonged expression of p21, and the development of senescence in normal cells that reside in irradiated tissues. Bone marrow-derived mesenchymal stem cells (MSCs) accumulate in primary tumor sites because of their natural tropism for inflammatory and fibrotic tissues. MSCs are extremely sensitive to low doses of ionizing radiation and acquire senescence as a result of bystander radiation effects. Senescent cells remain metabolically active but develop a potent senescence-associated secretory phenotype (SASP) that correlates to hyperactive secretion of cytokines, pro-fibrotic growth factors, and exosomes (EXOs). Integrative pathway analysis highlighted that radiation-induced senescence significantly enriched cell-cycle, extracellular matrix, transforming growth factor-β (TGF-β) signaling, and vesicle-mediated transport genes in MSCs. EXOs are cell-secreted nanovesicles (a subclass of small extracellular vesicles) that contain biomaterials-proteins, RNAs, microRNAs (miRNAs)-that are critical in cell-cell communication. miRNA content analysis of secreted EXOs further revealed that radiation-induced senescence uniquely altered miRNA profiles. In fact, several of the standout miRNAs directly targeted TGF-β or downstream genes. To examine bystander effects of radiation-induced senescence, we further treated normal MSCs with senescence-associated EXOs (SA-EXOs). These modulated genes related to TGF-β pathway and elevated both alpha smooth muscle actin (protein increased in senescent, activated cells) and Ki-67 (proliferative marker) expression in SA-EXO treated MSCs compared to untreated MSCs. We revealed SA-EXOs possess unique miRNA content that influence myofibroblast phenotypes via TGF-β pathway activation. This highlights that SA-EXOs are potent SASP factors that play a large role in cancer-related fibrosis.

P53 mutation and deletion contribute to tumor immune evasion.

TP53 (or p53) is widely accepted to be a tumor suppressor. Upon various cellular stresses, p53 mediates cell cycle arrest and apoptosis to maintain genomic stability. p53 is also discovered to suppress tumor growth through regulating metabolism and ferroptosis. However, p53 is always lost or mutated in human and the loss or mutation of p53 is related to a high risk of tumors. Although the link between p53 and cancer has been well established, how the different p53 status of tumor cells help themselves evade immune response remains largely elusive. Understanding the molecular mechanisms of different status of p53 and tumor immune evasion can help optimize the currently used therapies. In this context, we discussed the how the antigen presentation and tumor antigen expression mode altered and described how the tumor cells shape a suppressive tumor immune microenvironment to facilitate its proliferation and metastasis.

Cell Cycle and Senescence Regulation by Podocyte Histone Deacetylase 1 and 2.

The loss of integrity of the glomerular filtration barrier results in proteinuria that is often attributed to podocyte loss. Yet how damaged podocytes are lost remains unknown. Germline loss of murine podocyte-associated Hdac1 and Hdac2 ( Hdac1/2 ) results in proteinuria and collapsing glomerulopathy due to sustained double-stranded DNA damage. Hdac1/2 deletion induces loss of podocyte quiescence, cell cycle entry, arrest in G1, and podocyte senescence, observed both in vivo and in vitro . Through the senescence secretory associated phenotype, podocytes secrete proteins that contribute to their detachment. These results solidify the role of HDACs in cell cycle regulation and senescence, providing important clues in our understanding of how podocytes are lost following injury. Intact expression of podocyte histone deacetylases (HDAC) during development is essential for maintaining a normal glomerular filtration barrier because of its role in modulating DNA damage and preventing premature senescence. Germline podocyte-specific Hdac1 and 2 ( Hdac1 / 2 ) double-knockout mice were generated to examine the importance of these enzymes during development. Podocyte-specific loss of Hdac1 / 2 in mice resulted in severe proteinuria, kidney failure, and collapsing glomerulopathy. Hdac1 / 2 -deprived podocytes exhibited classic characteristics of senescence, such as senescence-associated β-galactosidase activity and lipofuscin aggregates. In addition, DNA damage, likely caused by epigenetic alterations such as open chromatin conformation, not only resulted in podocyte cell-cycle entry as shown in vivo by Ki67 expression and by FUCCI-2aR mice, but also in p21-mediated cell-cycle arrest. Through the senescence secretory associated phenotype, the damaged podocytes secreted proinflammatory cytokines, growth factors, and matrix metalloproteinases, resulting in subsequent podocyte detachment and loss, evidenced by senescent podocytes in urine. Hdac1 / 2 plays an essential role during development. Loss of these genes in double knockout mice leads to sustained DNA damage and podocyte senescence and loss.

Synergistic effects of PI3K inhibition and pioglitazone against acute promyelocytic leukemia cells.

Although pioglitazone, a well-known anti-diabetic agent, has recently established itself as a pillar of cancer treatment, its therapeutic value could be attenuated by the aberrant activation of the PI3K/Akt pathway. To evaluate whether the PI3K/Akt suppression in leukemic cells could potentiate the anti-leukemic effects of pioglitazone. To assess the anti-leukemic effects of PI3K/Akt inhibitors on anti-leukemic effects of pioglitazone, we used MTT and trypan blue assays. Flow cytometric analysis and qRT-PCR were also applied to evaluate cell cycle and apoptosis. The resulting data revealed that upon PPARγ stimulation in different leukemic cell lines using pioglitazone, the survival and the proliferative capacity of the cells were significantly halted. Then, we evaluated the impact of the PI3K/Akt axis on the effectiveness of the drug in the most sensitive leukemic cell line; NB4 cells. Our results showed that treatment of NB4 cells with the PI3K inhibitors increased the sensitivity of leukemic cells to pioglitazone to the degree that even lower concentrations of the agent succeeded to induce apoptotic as well as the anti-proliferative effects. Moreover, it seems that PI3K inhibition could potentiate the anti-leukemic effect of pioglitazone through induction of p21-mediated sub-G1 cell cycle arrest and altering the balance between the pro-and anti-apoptotic genes. This study sheds light on the significance of the PI3K/Akt pathway in APL cell sensitivity to pioglitazone and proposed that the presence of the PI3K inhibitor in the therapeutic regimen containing pioglitazone could be promising in the treatment of this malignancy.

Elucidating the Mechanism of Action (MOA) of Navtemadlin, an MDM2 Inhibitor, and Its Synergy with Gilteritinib in Myeloid Malignancies

Introduction: Murine double minute 2 (MDM2) is the primary negative regulator of the tumor suppressor protein, p53. Navtemadlin (KRT-232), a potent, selective, orally available MDM2 inhibitor, is being evaluated in the phase 3 BOREAS trial of patients with relapsed/refractory myelofibrosis (MF; NCT03662126) and in phase 1b/2 trials in various tumor types. In preclinical studies, navtemadlin has overcome MDM2 dysregulation and has restored p53 activity to drive apoptosis in TP53WT malignancies. The primary determinant of tumor cell sensitivity to navtemadlin is TP53 mutation status. Because MDM2 is often overexpressed and p53 mutations are less common in treatment-naïve acute myeloid leukemia (AML) and MF, navtemadlin represents a rational therapeutic candidate for these malignancies (Prokocimer et al. Blood. 2017). The present study aims to elucidate the effects of navtemadlin on proteins downstream of p53 and synergy of navtemadlin with the FLT3 inhibitor gilteritinib in myeloid malignancies. Methods: Cell viability was evaluated in three TP53WT myeloid cell lines, MOLM-13 (FLT3 AML), MV-4-11, and UKE-1, and one TP53MUT myeloid cell line, HEL-92, treated with 1-10 µM navtemadlin for 72 h. Growth inhibition was determined using the ATP-based Cell Titer Glo assay. The treatment effect on cell cycle progression, BCL-2 family proteins, and apoptosis was determined by flow cytometry. The effects of navtemadlin (0.5-1 µM) + gilteritinib (0.5-1 µM) were assessed in MOLM-13 and MV-4-11 (TP53WT and TP53MUT clones) and visualized using Combenefit. A MOLM-13 TP53WT mouse xenograft model was used to assess in vivo activity. Engrafted mice were randomly assigned to one of five treatment groups (n=10 each): vehicle control, 30 mg/kg gilteritinib, 50 mg/kg navtemadlin, 50 mg/kg navtemadlin + 30 mg/kg gilteritinib, or 100 mg/kg navtemadlin. Mice were treated orally once a day until the mice met early removal criteria or the study was terminated. Survival was measured by the percentage of mice in each group that remained alive at the end of the study. Results: Navtemadlin exposure led to dose-dependent growth inhibition in the TP53WT myeloid cell lines MOLM13, MV-4-11, and UKE-1 with an EC90 of 2-6 µM. Conversely, no growth inhibition was observed in the TP53MUT myeloid cell line HEL-92, up to 10 µM of navtemadlin. Assessment of cell cycle progression following navtemadlin exposure showed the induction of threshold-dependent apoptosis in the TP53WT myeloid cell lines, but not in the TP53MUT myeloid cell line. The threshold at which a switch from p21-mediated cell-cycle arrest to p53-directed apoptosis occurs is illustrated in Figure 1a. Navtemadlin-induced apoptosis was indicated by a release of cytochrome c and increase in processed caspase-3. Clinically, a steady-state Cmax of 2-6 µM is achieved at therapeutically active and relevant doses of navtemadlin ≥180 mg given once a day on a 5-day or 7-day schedule in clinical trials, leading to maximum tumor cell death. To gain better insight into the contribution of BCL-2 family members alterations as an MOA for navtemadlin-induced apoptosis, expression levels of BCL-2 family members were evaluated in TP53WT myeloid cell lines. Navtemadlin increased levels of the effector pro-apoptotic proteins Bak, Bax, and Bim in leukemia cells. Synergy was observed in vitro for navtemadlin + gilteritinib in TP53WT, but not TP53MUT myeloid cell lines. Treatment with 100 mg/kg navtemadlin monotherapy and 50 mg/kg navtemadlin + gilteritinib combination led to prolonged survival in mice harboring MOLM-13 TP53WT xenografts, with all mice still alive at day 113 when the study was terminated (Figure 1b). In contrast, median survival times for the vehicle, 50 mg/kg navtemadlin monotherapy, and gilteritinib monotherapy groups were 18, 53, and 65 days, respectively. Conclusion: Navtemadlin induces potent, threshold-dependent apoptosis in cell culture models of TP53WT myeloid malignancies at clinically relevant concentrations. Navtemadlin-induced p53 activity initiates apoptosis by activating the caspase cascade via cytochrome c release. Navtemadlin was shown to induce the expression of pro-apoptotic BCL-2 family proteins, key mediators in driving cell death. These pro-apoptotic effects translate to robust antitumor activity in xenograft models of TP53WT myeloid malignancies, with the navtemadlin + gilteritinib combination demonstrating synergy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Quantitation of navtemadlin in human plasma and brain tissue by liquid chromatography-tandem mass spectrometry.

Navtemadlin is an orally bioavailable small molecule that blocks the protein-protein interaction between murine double minute 2 protein (MDM2) and the tumor suppressor protein p53, leading to p53-mediated cell cycle arrest and apoptosis. It is being evaluated in clinical trials for a variety of malignancies, both as a single agent and in combination regimens. A sensitive, robust LC-tandem mass spectrometry (LC-MS/MS) method was developed to quantitate navtemadlin in plasma, and this method was also validated using brain tissue homogenate. Sample preparation involved protein precipitation of plasma or brain tissue homogenate using acetonitrile. Navtemadlin, navtemadlin glucuronide, and the internal standard, D6 -navtemadlin, were separated from microsomal incubation extracts using gradient elution and a ZORBAX XDB C18 column. Analytes were detected using a SCIEX 5500 triple quadrupole mass spectrometer in positive electrospray ionization mode. The assay range of 1-1000 ng/mL was shown to be accurate (96.1-102.0% and 95.7-104%) and precise (coefficient of variation ≤ 10.6% and ≤ 6.6%) in plasma and brain tissue homogenate, respectively. An 8000 ng/mL navtemadlin sample diluted 1:10 (v/v) with plasma was also accurately quantitated. Navtemadlin has been stable in frozen plasma at -70°C for at least 20 months. This validated LC-MS/MS method was applied to determine navtemadlin concentrations in plasma and brain tissue samples from two separate patients receiving 120 mg/day navtemadlin on protocol ABTC1604.

High cysteine concentrations in cell culture media lead to oxidative stress and reduced bioprocess performance of recombinant CHO cells.

Cysteine is considered an essential amino acid in the cultivation of Chinese hamster ovary (CHO) cells. An optimized cysteine supply during fed-batch cultivation supports the protein production capacity of recombinant CHO cell lines. However, we observed that CHO production cell lines seeded at low cell densities in chemically defined media enriched with cysteine greater than 2.5mm resulted in markedly reduced cell growth during passaging, hampering seed train performance and scale-up. To investigate the underlying mechanism, seeding cell densities and initial cysteine concentrations ranging from low to high cysteine concentrations were varied followed by an analysis of cell culture performance. Additionally, cell cycle analysis, intracellular quantification of reactive oxygen species (ROS) as well as transcriptomic analyses by next-generation sequencing were carried out. Our results demonstrate that CHO cells seeded at low cell densities at high initial cysteine concentrations encountered increased oxidative stress leading to a p21-mediated cell cycle arrest in the G1/S phase. The resulting oxidative stress caused redox imbalance in the endoplasmic reticulum and activation of the unfolded protein response as well as the major antioxidant nuclear factor-like 2 response pathways. Potential signature genes related to oxidative stress and the inhibition of the pentose phosphate pathway were identified in the study. Finally, the study presents that seeding cells at a higher concentration counteract oxidative stress in cysteine-enriched cell culture media.

The p53 Transactivation Domain 1-Dependent Response to Acute DNA Damage in Endothelial Cells Protects against Radiation-Induced Cardiac Injury.

Thoracic radiation therapy can cause endothelial injury in the heart, leading to cardiac dysfunction and heart failure. Although it has been demonstrated that the tumor suppressor p53 functions in endothelial cells to prevent the development of radiation-induced myocardial injury, the key mechanism(s) by which p53 regulates the radiosensitivity of cardiac endothelial cells is not completely understood. Here, we utilized genetically engineered mice that express mutations in p53 transactivation domain 1 (TAD1) (p5325,26) or mutations in p53 TAD1 and TAD2 (p5325,26,53,54) specifically in endothelial cells to study the p53 transcriptional program that protects cardiac endothelial cells from ionizing radiation in vivo. p5325,26,53,54 loses the ability to drive transactivation of p53 target genes after irradiation while p5325,26 can induce transcription of a group of non-canonical p53 target genes, but not the majority of classic radiation-induced p53 targets critical for p53-mediated cell cycle arrest and apoptosis. After 12 Gy whole-heart irradiation, we found that both p5325,26 and p5325,26,53,54 sensitized mice to radiation-induced cardiac injury, in contrast to wild-type p53. Histopathological examination suggested that mutation of TAD1 contributes to myocardial necrosis after whole-heart irradiation, while mutation of both TAD1 and TAD2 abolishes the ability of p53 to prevent radiation-induced heart disease. Taken together, our results show that the transcriptional program downstream of p53 TAD1, which activates the acute DNA damage response after irradiation, is necessary to protect cardiac endothelial cells from radiation injury in vivo.

Fenbendazole Suppresses Growth and Induces Apoptosis of Actively Growing H4IIE Hepatocellular Carcinoma Cells &lt;i&gt;via&lt;/i&gt; p21-Mediated Cell-Cycle Arrest

Bendimidazole anthelmintics (BAs) have gained interest for their anticancer activity. The anticancer activity is mediated via multiple intracellular changes, which are not consistent under different conditions even in the same cells. We investigated the anticancer activity of fenbendazole (FZ, one of BAs) under two different growth conditions. The growth rate of H4IIE cells was dose-dependently decreased by FZ only in actively growing cells but not in fully confluent quiescent cells. Apoptosis-associated changes were also induced by FZ in actively growing cells. Markers of autophagy were not changed by FZ. The number of cells was markedly increased in sub-G1 phase but decreased in S- and G2/M phases by FZ. FZ up-regulated p21 (an inhibitor of cyclin-CDK) but suppressed the expression of cell cycle-promoting proteins (cyclin D1 and cyclin B1). FZ did not affect integrin αV or n-cadherin expression as well as cell migration. Glycolytic changes (glucose consumption and lactate production) and the generation of reactive oxygen species (ROS) were not affected by FZ. Although the activity of mitogen-activated protein kinases (MAPKs) was altered by FZ, the inhibition of MAPKs did not affect the pro-apoptotic activity of FZ. Taken together, FZ selectively suppressed the growth of cells via p21-mediated cell cycle arrest at G1/S and G2/M, and resulted in apoptosis only in actively growing cells but not in quiescent cells. Glucose metabolism, ROS generation, and MAPKs are unlikely targets of FZ at least in H4IIE rat hepatocellular carcinoma cells used in this study.

Novel imidazo[1,2-a]pyridine derivatives induce apoptosis and cell cycle arrest in non-small cell lung cancer by activating NADPH oxidase mediated oxidative stress

AimsImidazo[1,2-a]pyridine-based analogues have recently gained significant interest because of their wide spectrum of biological activities including anti-cancer potential, however the development of targeted therapeutic candidates against non-small cell lung cancer (NSCLC) is of utmost need due to its high prevalence and poor prognosis. Herein, we have aimed to synthesized novel imidazo [1,2-a] pyridine derivatives (IMPA) by coupling with 2-amino-4H-pyran to enhance bioactivity against NSCLC. Main methodsWe have designed and synthesized a series of fifteen novel imidazo [1,2-a] pyridine derivatives through molecular hybridization and studied their anti-cancer activity against in-vitro lung adenocarcinoma and 3D multicellular lung tumor spheroids. Key findingsIMPA-2, IMPA-5, IMPA-6, IMPA-8, and IMPA-12 markedly induced cytotoxicity by notably increased NADPH oxidase (NOX) activity, which results in the induction of ROS-mediated apoptosis in A549 lung cancer cells. It caused impairment of mitochondrial membrane potential by increasing pro-apoptotic BAX, and BAK1 expressions, and decreasing anti-apoptotic BCL2 expression, along with the induction of caspase-9/3 activation, however, these attributes were compromised in presence of N-acetyl-L-cysteine (NAC), a free radical scavenger. Increased ROS production by IMPAs also promotes p53 mediated cell cycle arrest through the inactivation of p38MAPK. Reduction of tumor size in IMPAs-treated 3D multicellular lung tumor spheroids gave further validation. SignificanceBeside cytotoxicity, IMPAs also inhibit lung cancer cell invasion and migration, suggesting their applicability in metastatic lung cancer. Therefore, IMPA derivatives could be used as potential anti-cancer agents in treating non-small cell lung cancer.

Non-coding small nucleolar RNA SNORD17 promotes the progression of hepatocellular carcinoma through a positive feedback loop upon p53 inactivation.

Recent evidence suggests that small nucleolar RNAs (snoRNAs) are involved in the progression of various cancers, but their precise roles in hepatocellular carcinoma (HCC) remain largely unclear. Here, we report that SNORD17 promotes the progression of HCC through a positive feedback loop with p53. HCC-related microarray datasets from the Gene Expression Omnibus (GEO) database and clinical HCC samples were used to identify clinically relevant snoRNAs in HCC. SNORD17 was found upregulated in HCC tissues compared with normal liver tissues, and the higher expression of SNORD17 predicted poor outcomes in patients with HCC, especially in those with wild-type p53. SNORD17 promoted the growth and tumorigenicity of HCC cells in vitro and in vivo by inhibiting p53-mediated cell cycle arrest and apoptosis. Mechanistically, SNORD17 anchored nucleophosmin 1 (NPM1) and MYB binding protein 1a (MYBBP1A) in the nucleolus by binding them simultaneously. Loss of SNORD17 promoted the translocation of NPM1 and MYBBP1A into the nucleoplasm, leading to NPM1/MDM2-mediated stability and MYBBP1A/p300-mediated activation of p53. Interestingly, p300-mediated acetylation of p53 inhibited SNORD17 expression by binding to the promoter of SNORD17 in turn, forming a positive feedback loop between SNORD17 and p53. Administration of SNORD17 antisense oligonucleotides (ASOs) significantly suppressed the growth of xenograft tumors in mice. In summary, this study suggests that SNORD17 drives cancer progression by constitutively inhibiting p53 signaling in HCC and may represent a potential therapeutic target for HCC.