Cell Cycle Arrest In Response Research Articles

Cellular senescence impairs tendon extracellular matrix remodeling in response to mechanical unloading.

Musculoskeletal injuries, including tendinopathies, present a significant clinical burden for aging populations. While the biological drivers of age-related declines in tendon function are poorly understood, it is well accepted that dysregulation of extracellular matrix (ECM) remodeling plays a role in chronic tendon degeneration. Senescent cells, which have been associated with multiple degenerative pathologies in musculoskeletal tissues, secrete a highly pro-inflammatory senescence-associated secretory phenotype (SASP) that has potential to promote ECM breakdown. However, the role of senescent cells in the dysregulation of tendon ECM homeostasis is largely unknown. To assess this directly, we developed an invitro model of induced cellular senescence in murine tendon explants. This novel technique enables us to study the isolated interactions of senescent cells and their native ECM without interference from age-related systemic changes. We document multiple biomarkers of cellular senescence in induced tendon explants including cell cycle arrest, apoptosis resistance, and sustained inflammatory responses. We then utilize this invitro senescence model to compare the ECM remodeling response of young, naturally aged, and induced-senescent tendons to an altered mechanical stimulus. We found that both senescence and aging independently led to alterations in ECM-related gene expression, reductions in protein synthesis, and tissue compositional changes. Furthermore, MMP activity was sustained, thus shifting the remodeling balance of aged and induced-senescent tissues towards degradation over production. Together, this demonstrates that cellular senescence plays a role in the altered mechano-response of aged tendons and likely contributes to poor clinical outcomes in aging populations.

Advanced Design, Synthesis, and Evaluation of Highly Selective Wee1 Inhibitors: Enhancing Pharmacokinetics and Antitumor Efficacy.

Wee1 is a kinase that regulates cell cycle arrest in response to DNA damage. Wee1 inhibition is a potential strategy to suppress the growth of tumors with defective p53 or DNA repair pathways. However, the development of Wee1 inhibitors faces some challenges. AZD1775, the first-in-class Wee1 inhibitor, has poor kinase selectivity and dose-limiting toxicity. Here, we report the discovery of 12h, a highly selective and potent Wee1 inhibitor with a favorable pharmacokinetic profile. 12h showed strong antiproliferative effects against Lovo cells, a colorectal cancer cell line, both in vitro and in vivo. Moreover, 12h showed a clean kinase profile and effectively induced cell apoptosis. Our results suggest that 12h is a promising drug candidate for further development as a novel anticancer agent.

Abstract 5969: VRK1 can be a therapeutic target in small cell neuroendocrine carcinoma of the uterine cervix

Abstract Objective: Small cell neuroendocrine carcinoma of the uterine cervix (SCNEC) is extremely rare but highly aggressive and its prognosis is usually poor. Vaccinia-related kinase (VRK)1 is a serine/threonine protein kinase that is involved in cell proliferation by regulating cell-cycle progression and inducing cell-cycle arrest in response to stress signals. VRK2 belongs to the same family as VRK1, and they are paralogs. VRK1 has been reported as a potential therapeutic target in nervous system tumors with low VRK2 expression. Previously, we identified high expression of VRK1 in SCNEC through proteomic analysis of isolated organoids. The objective of the present study is to investigate the effect of VRK1 on the proliferation of SCNEC and its potential as a therapeutic target. Methods: The mRNA and protein expression of VRK1 and VRK2 in ten organoids and two cell lines derived from SCNEC were examined and compared with cervical cancer of other histological subtypes by RNA microarray and western blot analysis. The effect of VRK1 on tumor growth in vitro and in vivo (xenograft model) were evaluated by shRNA-mediated knockdown of VRK1 (shVRK1). RNA-seq was performed from the xenograft tumors. The effects of shVRK1 on cell proliferation were evaluated under various levels of oxidative stress induced by H2O2. Results: VRK1 expression was higher in organoids and cell lines derived from SCNEC than those derived from squamous cell carcinoma and adenocarcinoma, while VRK2 expression was lower in SCNEC. Tumor growth was suppressed significantly by shVRK1 in vivo (mean weight: cell line 1: 0.62 vs 0.06g, p<0.05, 2: 1.79 vs 0.58g, p<0.01). Gene ontology analysis of RNA-seq data suggested that VRK1 is related to cellular responses to stress. Proliferation was slightly suppressed by shVRK1 under normal conditions; however, it was significantly enhanced under oxidative stress (Cell line 1: in shVRK1, -14.0% without oxidative stress vs -54.9% under H2O2 750μM, p<0.01, Cell line 2: similar to Cell line 1). Conclusion: SCNEC exhibited high VRK1 and low VRK2 expression, and the tumor suppressive effect of shVRK1 was more significant in vivo than in vitro. VRK1 may play a role in the tumor microenvironment, including oxidative stress, and is a potential therapeutic target for SCNEC. Citation Format: Mariya Kobayashi, Satoshi Nakagawa, Mizuki Kanda, Yuji Kamei, Masuda Tatsuo, Mamoru Kakuda, Kosuke Hiramatsu, Toshihiro Kimura, Yutaka Ueda, Takayuki Enomoto, Masahiro Inoue, Tadashi Kimura. VRK1 can be a therapeutic target in small cell neuroendocrine carcinoma of the uterine cervix [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 5969.

Dihydroartemisinin eliminates senescent cells by promoting autophagy-dependent ferroptosis via AMPK/mTOR signaling pathway.

Cellular senescence is an irreversible cell-cycle arrest in response to a variety of cellular stresses, which contribute to the pathogenesis of a variety of age-related degenerative diseases. However, effective antisenescence strategies are still lacking. Drugs that selectively target senescent cells represent an intriguing therapeutic strategy to delay aging and age-related diseases. Thus, we thought to investigate the effects of dihydroartemisinin (DHA) on senescent cells and elucidated its mechanisms underlying aging. Stress-induced premature senescence (SIPS) model was built in NIH3T3 cells using H2O2 and evaluated by β-galactosidasestaining. Cells were exposed to DHA and subjected to cellular activity assays including viability, ferroptosis, and autophagy. The number of microtubule-associated protein light-chain 3puncta was detected by immunofluorescence staining. The iron content was assessed by spectrophotometer and intracellular reactive oxygen species (ROS) was measured by fluorescent probe dichlorodihydrofluorescein diacetate. We found that DHA triggered senescent cell death via ferroptosis. DHA accelerated ferritin degradation via promoting autophagy, increasing the iron contents, promoting ROS accumulation, thus leading to ferroptotic cell death in SIPS cells. In addition, autophagy inhibitor BafA1 preconditioning inhibited ferroptosis induced by DHA. Moreover, Atg5 silencing and autophagy inhibitor BafA1 preconditioning inhibited ferroptosis induced by DHA. We also revealed that the expression of p-AMP-activated protein kinase (AMPK)and p-mammalian target of rapamycin (mTOR) in senescent cells was downregulated. These results suggested that DHA may be a promising drug candidate for clearing senescent cells by inducing autophagy-dependent ferroptosis via AMPK/mTOR signaling pathway.

Targeting Mutant KRAS with Novel Mutant-Specific Pharmacological Inhibitors: Activity Against Myeloma and Other Lymphoid Malignant Cells and Molecular Mechanisms of Resistance

Oncogenic RAS mutations are frequent genomic drivers in hematologic neoplasias such as multiple myeloma (MM) but are traditionally considered non-“druggable”. Novel specific inhibitors of G12C and G12D KRAS mutants are clinically active in solid tumors, but their impact on KRAS-mutant lymphoid malignancies has not been explored. Here, we examined the activity of these inhibitors against G12C/D KRAS-mutant cells from MM, B-ALL and T-ALL; the functional sequelae of pharmacological KRAS inhibition; and the potential mechanisms of resistance to these agents. We assessed the activity of novel G12C- (MRTX1257) and G12D- (MRTX1133) specific pharmacological KRAS inhibitors against 2 MM lines with G12C (KHM1B, XG7) and 2 with G12D (KARPAS620, KP6) KRAS mutations; as well as the G12D mutant KOPN-8 (B-ALL) and CCRF-CEM (T-ALL) lines. KRAS inhibitors were tested at concentration ranges achievable in patients with solid tumors. Potent and mutant-specific activity was achieved with both of these KRAS inhibitors in their respective lines, with IC50 in the range of 5-150 nM after 3-7d treatment. This level of in vitro activity was similar to KRAS-mutant lines (e.g., in our in vitro studies and also in the literature) from solid tumors that are clinically responsive to these agents. Furthermore, the KRAS inhibitors effectively abrogated downstream MAPK and AKT pathway activation (e.g. phosphorylation of ERK or AKT by immunoblotting). MRTX1133 led to potent induction of cell death of KARPAS620, KP6 and KOPN8 cells (flow cytometry for Annexin V/PI staining) while MRTX1257 induced cell death in KHM1B and had a predominantly cytostatic effect on XG7 cells. Combinations of MRTX1257 with clinically established anti-MM drugs (incl. melphalan, bortezomib, pomalidomide, trametinib) caused in some cases supra-additive effects, but - more importantly - showed no antagonism with any combination. RNA sequencing revealed that KRAS inhibitor treatment downregulated RAS effector genes (e.g., ETV4, ETV5), and negative regulators of RAS signaling (e.g., DUSP6, SPRY4 reflecting a RAS-driven negative feedback loop controlling expression of these genes), and molecules involved in cell cycle regulation (e.g., D-type cyclin), anti-apoptosis, and DNA replication/repair. Conversely, KRAS inhibition upregulated genes involved in cell cycle arrest and stress responses. Importantly, we performed genome-scale CRISPR activation studies in 4 MM lines with G12C/G12D mutant KRAS to identify genes whose gain-of-function (GOF) enhances or suppresses MM cell response to the respective KRAS inhibitors. Top “hits” from these screens were validated with individual sgRNAs. CRISPR activation of mutant KRAS itself promotes MM cell escape from treatment, likely due to stoichiometric reasons and decreased target saturation at a given drug concentration. Notably, CRISPR activation of positive regulators of RAS-MAPK signaling (e.g., SHOC2), and upstream surface receptors (e.g., EGFR) were among top genes mediating KRAS inhibitor resistance. Beyond KRAS itself, these top “hit” genes are typically not recurrently mutated, amplified, differentially expressed (e.g., compared with normal plasma cells) in MM and are not associated with “high-risk” clinical behavior. Some (e.g., EGFR) of the genes identified from these CRISPR studies are not typically expressed in MM cells at diagnosis, thus these results outline how ectopic expression (e.g., EGFR) or overexpression of these “hit” genes could represent candidate biomarkers for decreased response or early relapse in future clinical studies of KRAS inhibitors in KRAS-mutant MM. Ongoing studies are addressing how these genomic perturbations may affect the in vivo response of KRAS-mutant cells to KRAS inhibitors alone or in combinations with established therapies. Our study documents that novel KRAS G12C and G12D inhibitors exhibit potent and mutant-specific activity against MM and other lymphoid malignant cells at dose levels that are clinically achievable in solid tumor patients. This study also provides functional evidence about genes and molecular pathways that regulate MM cell sensitivity vs. resistance to KRAS inhibitors. These results create a framework for ongoing and future efforts to translate the use of KRAS inhibitors into clinical studies for MM and other lymphoid malignancies.

RBIO-02. NOVEL, QUIESCENT SUBPOPULATION EMERGES AFTER LOW-DOSE IRRADIATION OF GLIOMA STEM CELLS

Abstract Glioblastoma (GBM) tumors contain a subset of highly radiation resistant cells referred to as glioma stem cells (GSCs). We used single-cell RNA-seq (scRNA-seq) to characterize the transcriptional states that increase in size following GSC irradiation to further our understanding of the acute responses to radiotherapy. We treated two GBM patient-derived xenograft-derived GSC lines to low-dose irradiation (4 Gy). We then conducted scRNA-seq before irradiation and three days and one-week post-irradiation. As expected, the number of GSCs undergoing division through the cell cycle was drastically reduced relative to control conditions, and a subpopulation of GSCs with DNA damage response markers emerged after irradiation. Interestingly, we observed a novel, distinct subpopulation that was common between GSC lines, expanded after irradiation, and featured marker genes related to cell cycle arrest in response to external stressors. Gene regulatory network analysis identified transcription factors (TFs) likely to regulate these marker genes and potentially the dynamics of this subpopulation. The TFs were also related to cell cycle suppression and the overall expression state of these cells was similar to the quiescent Neural G0 state, indicating that our novel subpopulation may contain quiescent GSCs. In upcoming studies, we plan to perturb these TFs and marker genes to determine the importance of these genes in the GSC response to irradiation. Further exploration of this novel quiescent subpopulation may lead to new druggable targets that sensitize GBM tumors to radiotherapy.

Impact of Aging and Cellular Senescence in the Pathophysiology of Preeclampsia.

The incidence of hypertensive disorders of pregnancy is increasing, which may be due to several factors, including an increased age at pregnancy and more comorbid health conditions during reproductive years. Preeclampsia, the most severe hypertensive disorder of pregnancy, has been associated with an increased risk of future disease, including cardiovascular and kidney diseases. Cellular senescence, the process of cell cycle arrest in response to many physiologic and maladaptive stimuli, may play an important role in the pathogenesis of preeclampsia and provide a mechanistic link to future disease. In this article, we will discuss the pathophysiology of preeclampsia, the many mechanisms of cellular senescence, evidence for the involvement of senescence in the development of preeclampsia, as well as evidence that cellular senescence may link preeclampsia to the risk of future disease. Lastly, we will explore how a better understanding of the role of cellular senescence in preeclampsia may lead to therapeutic trials. © 2023 American Physiological Society. Compr Physiol 13:5077-5114, 2023.

Senescent Cells: Dual Implications on the Retinal Vascular System.

Cellular senescence, a state of permanent cell cycle arrest in response to endogenous and exogenous stimuli, triggers a series of gradual alterations in structure, metabolism, and function, as well as inflammatory gene expression that nurtures a low-grade proinflammatory milieu in human tissue. A growing body of evidence indicates an accumulation of senescent neurons and blood vessels in response to stress and aging in the retina. Prolonged accumulation of senescent cells and long-term activation of stress signaling responses may lead to multiple chronic diseases, tissue dysfunction, and age-related pathologies by exposing neighboring cells to the heightened pathological senescence-associated secretory phenotype (SASP). However, the ultimate impacts of cellular senescence on the retinal vasculopathies and retinal vascular development remain ill-defined. In this review, we first summarize the molecular players and fundamental mechanisms driving cellular senescence, as well as the beneficial implications of senescent cells in driving vital physiological processes such as embryogenesis, wound healing, and tissue regeneration. Then, the dual implications of senescent cells on the growth, hemostasis, and remodeling of retinal blood vessels are described to document how senescent cells contribute to both retinal vascular development and the severity of proliferative retinopathies. Finally, we discuss the two main senotherapeutic strategies-senolytics and senomorphics-that are being considered to safely interfere with the detrimental effects of cellular senescence.

Targeting MDM2-p53 Axis through Drug Repurposing for Cancer Therapy: A Multidisciplinary Approach.

Cancer remains a major cause of morbidity and mortality worldwide, and while current therapies, such as chemotherapy, immunotherapy, and cell therapy, have been effective in many patients, the development of novel therapeutic options remains an urgent priority. Mouse double minute 2 (MDM2) is a key regulator of the tumor suppressor protein p53, which plays a critical role in regulating cellular growth, apoptosis, and DNA repair. Consequently, MDM2 has been the subject of extensive research aimed at developing novel cancer therapies. In this study, we employed a machine learning-based approach to establish a quantitative structure-activity relationship model capable of predicting the potential in vitro efficacy of small molecules as MDM2 inhibitors. Our model was used to screen 5883 FDA-approved drugs, resulting in the identification of promising hits that were subsequently evaluated using molecular docking and molecular dynamics simulations. Two antihistamine drugs, cetirizine (CZ) and rupatadine (RP), exhibited particularly favorable results in the initial in silico analyses. To further assess their potential use as the activators of the p53 pathway, we investigated the antiproliferative capability of the abovementioned drugs on human glioblastoma and neuroblastoma cell lines. Both the compounds exhibited significant antiproliferative effects on the abovementioned cell lines in a dose-dependent manner. The half-maximal inhibitory concentration (IC50) of CZ was found to be 697.87 and 941.37 μM on U87 and SH-SY5Y cell lines, respectively, while the IC50 of RP was found to be 524.28 and 617.07 μM on the same cell lines, respectively. Further investigation by quantitative reverse transcriptase polymerase chain reaction analysis revealed that the CZ-treated cell lines upregulate the expression of the p53-regulated genes involved in cell cycle arrest, apoptosis, and DNA damage response compared to their respective vehicle controls. These findings suggest that CZ activates the p53 pathway by inhibiting MDM2. Our results provide compelling preclinical evidence supporting the potential use of CZ as a modulator of the MDM2-p53 axis and its plausible repurposing for cancer treatment.

N-terminal intrinsic disorder is an ancestral feature of Gγ subunits that influences the balance between different Gβγ signaling axes in yeast

Activated G protein-coupled receptors promote the dissociation of heterotrimeric G proteins into Gα and Gβγ subunits that bind to effector proteins to drive intracellular signaling responses. In yeast, Gβγ subunits coordinate the simultaneous activation of multiple signaling axes in response to mating pheromones, including MAP kinase (MAPK)-dependent transcription, cell polarization, and cell cycle arrest responses. The Gγ subunit in this complex contains an N-terminal intrinsically disordered region that governs Gβγ-dependent signal transduction in yeast and mammals. Here, we demonstrate that N-terminal intrinsic disorder is likely an ancestral feature that has been conserved across different Gγ subtypes and organisms. To understand the functional contribution of structural disorder in this region, we introduced precise point mutations that produce a stepwise disorder-to-order transition in the N-terminal tail of the canonical yeast Gγ subunit, Ste18. Mutant tail structures were confirmed using circular dichroism and molecular dynamics and then substituted for the wildtype gene in yeast. We find that increasing the number of helix-stabilizing mutations, but not isometric mutation controls, has a negative and proteasome-independent effect on Ste18 protein levels as well as a differential effect on pheromone-induced levels of active MAPK/Fus3, but not MAPK/Kss1. When expressed at wildtype levels, we further show that mutants with an alpha-helical N terminus exhibit a counterintuitive shift in Gβγ signaling that reduces active MAPK/Fus3 levels whilst increasing cell polarization and cell cycle arrest. These data reveal a role for Gγ subunit intrinsically disordered regions in governing the balance between multiple Gβγ signaling axes.

Senescence: a double-edged sword in beta-cell health and failure?

Cellular senescence is a complex process marked by permanent cell-cycle arrest in response to a variety of stressors, and acts as a safeguard against the proliferation of damaged cells. Senescence is not only a key process underlying aging and development of many diseases, but has also been shown to play a vital role in embryogenesis as well as tissue regeneration and repair. In context of the pancreatic beta-cells, that are essential for maintaining glucose homeostasis, replicative senescence is responsible for the age-related decline in regenerative capacity. Stress induced premature senescence is also a key early event underlying beta-cell failure in both type 1 and type 2 diabetes. Targeting senescence has therefore emerged as a promising therapeutic avenue for diabetes. However, the molecular mechanisms that mediate the induction of beta-cell senescence in response to various stressors remain unclear. Nor do we know if senescence plays any role during beta-cell growth and development. In this perspective, we discuss the significance of senescence in beta-cell homeostasis and pathology and highlight emerging directions in this area that warrant our attention.

Morusinol extracted from Morus alba induces cell cycle arrest and apoptosis via inhibition of DNA damage response in melanoma by CHK1 degradation through the ubiquitin-proteasome pathway

BackgroudFlavonoids have a variety of biological activities, such as anti-inflammation, anti-tumor, anti-thrombosis and so on. Morusinol, as a novel isoprene flavonoid extracted from Morus alba root barks, has the effects of anti-arterial thrombosis and anti-inflammatory in previous studies. However, the anti-cancer mechanism of morusinol remains unclear. PurposeIn present study, we mainly studied the anti-tumor effect of morusinol and its mode of action in melanoma. MethodsThe anti-cancer effect of morusinol on melanoma were evaluated by using the MTT, EdU, plate clone formation and soft agar assay. Flow cytometry was used for detecting cell cycle and apoptosis. The ɣ-H2AX immunofluorescence and the alkaline comet assay were used to detect DNA damage and the Western blotting analysis was used to investigate the expressions of DNA-damage related proteins. Ubiquitination and turnover of CHK1 were also detected by using the immunoprecipitation assay. The cell line-derived xenograft (CDX) mouse models were used in vivo to evaluate the effect of morusinol on tumorigenicity. ResultsWe demonstrated that morusinol not only had the ability to inhibit cell proliferation, but also induced cell cycle arrest at G0/G1 phase, caspase-dependent apoptosis and DNA damage in human melanoma cells. In addition, morusinol effectively inhibited the growth of melanoma xenografts in vivo. More strikingly, CHK1, which played an important role in maintaining the integrity of cell cycle, genomic stability and cell viability, was down-regulated in a dose- and time-dependent manner after morusinol treatment. Further research showed that CHK1 was degraded by the ubiquitin-proteasome pathway. Whereafter, morusinol-induced cell cycle arrest, apoptosis and DNA damage were partially salvaged by overexpressing CHK1 in melanoma cell lines. Herein, further experiments demonstrated that morusinol increased the sensitivity of dacarbazine (DTIC) to chemotherapy for melanoma in vitro and in vivo. ConclusionMorusinol induces CHK1 degradation through the ubiquitin-proteasome pathway, thereby inducing cell cycle arrest, apoptosis and DNA damage response in melanoma. Our study firstly provided a theoretical basis for morusinol to be a candidate drug for clinical treatment of cancer, such as melanoma, alone or combinated with dacarbazine.

Loss of RanGAP1 drives chromosome instability and rapid tumorigenesis of osteosarcoma.

Chromothripsis is a catastrophic event of chromosomal instability that involves intensive fragmentation and rearrangements within localized chromosomal regions. However, its cause remains unclear. Here, we show that reduction and inactivation of Ran GTPase-activating protein 1 (RanGAP1) commonly occur in human osteosarcoma, which is associated with a high rate of chromothripsis. In rapidly expanding mouse osteoprogenitors, RanGAP1 deficiency causes chromothripsis in chr1q, instant inactivation of Rb1 and degradation of p53, consequent failure in DNA damage repair, and ultrafast osteosarcoma tumorigenesis. During mitosis, RanGAP1 anchors to the kinetochore, where it recruits PP1-γ to counteract the activity of the spindle-assembly checkpoint (SAC) and prevents TOP2A degradation, thus safeguarding chromatid decatenation. Loss of RanGAP1 causes SAC hyperactivation and chromatid decatenation failure. These findings demonstrate that RanGAP1 maintains mitotic chromosome integrity and that RanGAP1 loss drives tumorigenesis through its direct effects on SAC and decatenation and secondary effects on DNA damage surveillance.

MDM2 antagonists promote CRISPR/Cas9-mediated precise genome editing in sheep primary cells

CRISPR-Cas9-mediated genome editing in sheep is of great use in both agricultural and biomedical applications. While targeted gene knockout by CRISPR-Cas9 through non-homologous end joining (NHEJ) has worked efficiently, the knockin efficiency via homology-directed repair (HDR) remains lower, which severely hampers the application of precise genome editing in sheep. Here, in sheep fetal fibroblasts (SFFs), we optimized several key parameters that affect HDR, including homology arm (HA) length and the amount of double-stranded DNA (dsDNA) repair template; we also observed synchronization of SFFs in G2/M phase could increase HDR efficiency. Besides, we identified three potent small molecules, RITA, Nutlin3, and CTX1, inhibitors of p53-MDM2 interaction, that caused activation of the p53 pathway, resulting in distinct G2/M cell-cycle arrest in response to DNA damage and improved CRISPR-Cas9-mediated HDR efficiency by 1.43- to 4.28-fold in SFFs. Furthermore, we demonstrated that genetic knockout of p53 could inhibit HDR in SFFs by suppressing the expression of several key factors involved in the HDR pathway, such as BRCA1 and RAD51. Overall, this study offersan optimized strategy for the usage of dsDNA repair template, more importantly, the application of MDM2 antagonists provides a simple and efficient strategy to promote CRISPR/Cas9-mediated precise genome editing in sheep primary cells.

β-Galactosidase-Activatable Nile Blue-Based NIR Senoprobe for the Real-Time Detection of Cellular Senescence.

Cellular senescence is a stable cell cycle arrest in response to stress or other damage stimuli to maintain tissue homeostasis. However, the accumulation of senescent cells can lead to the progression of various senescence-related disorders. In this paper, we describe the development of a β-galactosidase-activatable near-infrared (NIR) senoprobe, NBGal, for the detection of senescent cells based on the use of the FDA-approved Nile blue (NB) fluorophore. NBGal was validated in chemotherapeutic-induced senescence cancer models in vitro using SK-Mel 103 and 4T1 cell lines. In vivo monitoring of cellular senescence was evaluated in orthotopic triple-negative breast cancer-bearing mice treated with palbociclib to induce senescence. In all cases, NBGal exhibited a selective tracking of senescent cells mainly ascribed to the overexpressed β-galactosidase enzyme responsible for hydrolyzing the NBGal probe generating the highly emissive NB fluorophore. In this way, NBGal has proven to be a qualitative, rapid, and minimally invasive probe that allows the direct detection of senescent cells in vivo.

Mechanisms of RNA and Protein Quality Control and Their Roles in Cellular Senescence and Age-Related Diseases.

Cellular senescence, a hallmark of aging, is defined as irreversible cell cycle arrest in response to various stimuli. It plays both beneficial and detrimental roles in cellular homeostasis and diseases. Quality control (QC) is important for the proper maintenance of cellular homeostasis. The QC machineries regulate the integrity of RNA and protein by repairing or degrading them, and are dysregulated during cellular senescence. QC dysfunction also contributes to multiple age-related diseases, including cancers and neurodegenerative, muscle, and cardiovascular diseases. In this review, we describe the characters of cellular senescence, discuss the major mechanisms of RNA and protein QC in cellular senescence and aging, and comprehensively describe the involvement of these QC machineries in age-related diseases. There are many open questions regarding RNA and protein QC in cellular senescence and aging. We believe that a better understanding of these topics could propel the development of new strategies for addressing age-related diseases.

Radiotherapy Induces Innate Immune Responses in Patients Treated for Prostate Cancers.

Radiotherapy is a curative therapeutic modality used to treat cancers as a single agent or in combination with surgery and chemotherapy. Advanced radiotherapy technologies enable treatment with large fractions and highly conformal radiation doses to effect free-radical damage to cellular DNA leading to cell-cycle arrest, cell death, and innate immune response (IIR) stimulation. To understand systemic clinical responses after radiation exposure, proteomic and metabolomic analyses were performed on plasma obtained from patients with cancer at intervals after prostate stereotactic body radiotherapy. Pathway and multivariate analyses were used to delineate molecular alterations following radiotherapy and its correlation with clinical outcomes. DNA damage response increased within the first hour after treatment and returned to baseline by 1 month. IIR signaling also increased within 1 hour of treatment but persisted for up to 3 months thereafter. Furthermore, robust IIR and metabolite elevations, consistent with an early proinflammatory M1-mediated innate immune activation, were observed in patients in remission, whereas patients experiencing prostate serum antigen-determined disease progression demonstrated less robust immune responses and M2-mediated metabolite elevations. To our knowledge, these data are the first report of longitudinal proteomic and metabolomic molecular responses in patients after radiotherapy for cancers. The data supports innate immune activation as a critical clinical response of patients receiving radiotherapy for prostate cancer. Furthermore, we propose that the observed IIR may be generalized to the treatment of other cancer types, potentially informing multidisciplinary therapeutic strategies for cancer treatment.

Unaffected Li-Fraumeni Syndrome Carrier Parent Demonstrates Allele-Specific mRNA Stabilization of Wild-Type TP53 Compared to Affected Offspring

Li-Fraumeni Syndrome (LFS) is an autosomal dominant disorder where an oncogenic TP53 germline mutation is inherited by offspring of a carrier parent. p53 is a key tumor suppressor regulating cell cycle arrest in response to DNA damage. Unexpectedly, some mutant TP53 carriers remain unaffected, while their children develop cancer early in life. To begin unravelling this paradox, the response of dermal fibroblasts (dFb) isolated from a child with LFS was compared to those from her unaffected father after UV exposure. Phospho-Chk1[S345], a key activator of cell cycle arrest, was increased by UV induction in the LFS patient compared to their unaffected parent dFb. This result, along with previous findings of reduced CDKN1A/p21 UV induction in affected dFb, suggest that cell cycle dysregulation may contribute to cancer onset in the affected LFS subject but not the unaffected parent. Mutant p53 protein and its promoter binding affinity were also higher in dFb from the LFS patient compared to their unaffected parent. These results were as predicted based on decreased mutant TP53 allele-specific mRNA expression previously found in unaffected dFb. Investigation of the potential mechanism regulating this TP53 allele-specific expression found that, while epigenetic promoter methylation was not detectable, TP53 wild-type mRNA was specifically stabilized in the unaffected dFb. Hence, the allele-specific stabilization of wild-type TP53 mRNA may allow an unaffected parent to counteract genotoxic stress by means more characteristic of homozygous wild-type TP53 individuals than their affected offspring, providing protection from the oncogenesis associated with LFS.

Molecular Mechanisms to Target Cellular Senescence in Aging and Disease

Cellular senescence is a state of irreversible cell cycle arrest in response to several stressors, including DNA damage, increased cellular oxidative stress, telomere shortening, oncogene activation, and a deep epigenetic remodeling [...].

FtsZ phosphorylation brings about growth arrest upon DNA damage in Deinococcus radiodurans.

The polymerization/depolymerization dynamics of FtsZ play a pivotal role in cell division in the majority of the bacteria. Deinococcus radiodurans, a radiation-resistant bacterium, shows an arrest of growth in response to DNA damage with no change in the level of FtsZ. This bacterium does not deploy LexA/RecA type of DNA damage response and cell cycle regulation, and its genome does not encode SulA homologues of Escherichia coli, which attenuate FtsZ functions in response to DNA damage in other bacteria. A radiation-responsive Ser/Thr quinoprotein kinase (RqkA), characterized for its role in radiation resistance in this bacterium, could phosphorylate several cognate proteins, including FtsZ (drFtsZ) at Serine 235 (S235) and Serine 335 (S335) residues. Here, we reported the detailed characterization of S235 and S335 phosphorylation effects in the regulation of drFtsZ functions and demonstrated that the phospho-mimetic replacements of these residues in drFtsZ had grossly affected its functions that could result in cell cycle arrest in response to DNA damage in D. radiodurans. Interestingly, the phospho-ablative replacements were found to be nearly similar to drFtsZ, whereas the phospho-mimetic mutant lost the wild-type protein's signature characteristics, including its dynamics under normal conditions. The kinetics of post-bleaching recovery for drFtsZ and phospho-mimetic mutant were nearly similar at 2h post-irradiation recovery but were found to be different under normal conditions. These results highlighted the role of S/T phosphorylation in the regulation of drFtsZ functions and cell cycle arrest in response to DNA damage, which is demonstrated for the first time, in any bacteria.