Prolonged Cell Cycle Research Articles

GABA-Alleviated Oxidative Injury Induced by Salinity, Osmotic Stress and their Combination by Regulating Cellular and Molecular Signals in Rice.

This study was conducted in order to determine the effect of priming with γ-aminobutyric acid (GABA) at 0.5 mM on rice (Oryza sativa L.) seed germination under osmotic stress (OS) induced by polyethylene glycol (30 g/L PEG 6000); and salinity stress (S, 150 mM NaCl) and their combination (OS+S). Priming with GABA significantly alleviated the detrimental effects of OS, S and OS+S on seed germination and seedling growth. The photosynthetic system and water relation parameters were improved by GABA under stress. Priming treatment significantly increased the GABA content, sugars, protein, starch and glutathione reductase. GABA priming significantly reduced Na+ concentrations, proline, free radical and malonaldehyde and also significantly increased K+ concentration under the stress condition. Additionally, the activities of antioxidant enzymes, phenolic metabolism-related enzymes, detoxification-related enzymes and their transcription levels were improved by GABA priming under stress. In the GABA primed-plants, salinity stress alone resulted in an obvious increase in the expression level of Calcineurin B-like Protein-interacting protein Kinases (CIPKs) genes such as OsCIPK01, OsCIPK03, OsCIPK08 and OsCIPK15, and osmotic stress alone resulted in obvious increase in the expression of OsCIPK02, OsCIPK07 and OsCIPK09; and OS+S resulted in a significant up-regulation of OsCIPK12 and OsCIPK17. The results showed that salinity, osmotic stresses and their combination induced changes in cell ultra-morphology and cell cycle progression resulting in prolonged cell cycle development duration and inhibitory effects on rice seedlings growth. Hence, our findings suggested that the high tolerance to OS+S is closely associated with the capability of GABA priming to control the reactive oxygen species (ROS) level by inducing antioxidant enzymes, secondary metabolism and their transcription level. This knowledge provides new evidence for better understanding molecular mechanisms of GABA-regulating salinity and osmotic-combined stress tolerance during rice seed germination and development.

Glutamine synthetase facilitates cancer cells to recover from irradiation-induced G2/M arrest

ABSTRACTResistance to radiation of cancer cells can be either intrinsic or acquired, leading to treatment failure. In response to DNA damage caused by IR, cancer cells are arrested in cell cycle showing limited proliferation and increased apoptosis. However, radiation-resistant cells are able to overcome the cell cycle block and proceed to proliferation, for which the detailed mechanism remains to be elucidated. In the present study, we showed that radioresistant cells exhibited a recoverable G2/M phase during prolonged cell cycle and manifested lower apoptosis rate and more colony formation. RNA-seq analysis revealed that glutamine synthetase (GS, GLUL) gene was highly expressed in radioresistant cancer cells in comparison with the parental cells, which was in accordance with the G2/M arrest after ionizing radiation. Knocking out of GS in radioresistant cells resulted in a delayed G2/M recovery and lowered proliferation rate after ionizing radiation treatment, which was accompanied with increased inhibitory phosphorylation of CDK1 at Y15 and downregulated Cdc25B, a dual specific phosphatase of CDK1. Moreover, there was an enhanced complex formation of CDK1 and Cyclin B1 when the cells were rescued by re-introducing GS. In vivo, knocking down of GS significantly sensitized CNE2-R xenografts to RT in mice. In this study, we demonstrate a novel role of glutamine synthetase independent of metabolic function in promoting recovery from G2/M arrest caused by ionizing radiation, thus, causing cancer cell resistance to radiotherapy.

The RNA-binding protein DND1 acts sequentially as a negative regulator of pluripotency and a positive regulator of epigenetic modifiers required for germ cell reprogramming.

The adult spermatogonial stem cell population arises from pluripotent primordial germ cells (PGCs) that enter the fetal testis around embryonic day (E)10.5. PGCs undergo rapid mitotic proliferation, then enter prolonged cell cycle arrest (G1/G0), during which they transition to pro-spermatogonia. In mice homozygous for the Ter mutation in the RNA-binding protein Dnd1 (Dnd1Ter/Ter ), many male germ cells (MGCs) fail to enter G1/G0 and instead form teratomas: tumors containing many embryonic cell types. To investigate the origin of these tumors, we sequenced the MGC transcriptome in Dnd1Ter/Ter mutants at E12.5, E13.5 and E14.5, immediately prior to teratoma formation, and correlated this information with DO-RIP-Seq-identified DND1 direct targets. Consistent with previous results, we found DND1 controls downregulation of many genes associated with pluripotency and active cell cycle, including mTor, Hippo and Bmp/Nodal signaling pathway elements. However, DND1 targets also include genes associated with male differentiation, including a large group of chromatin regulators activated in wild-type but not mutant MGCs during the E13.5 and E14.5 transition. Results suggest multiple DND1 functions and link DND1 to initiation of epigenetic modifications in MGCs.

Radiation-induced synthetic lethality: combination of poly(ADP-ribose) polymerase and RAD51 inhibitors to sensitize cells to proton irradiation

ABSTRACTAlthough improvements in radiation therapy were made over the years, radioresistance is still a major challenge. Cancer cells are often deficient for DNA repair response, a feature that is currently exploited as a new anti-cancer strategy. In this context, combination of inhibitors targeting complementary pathways is of interest to sensitize cells to radiation. In this work, we used PARP (Olaparib) and RAD51 (B02) inhibitors to radiosensitize cancer cells to proton and X-ray radiation. More particularly, Olaparib and B02 were used at concentration leading to limited cytotoxic (alone or in combination) but increasing cell death when the cells were irradiated. We showed that, although at limited concentration, Olaparib and B02 were able to radiosensitize different cancer cell lines, i.e. lung and pancreatic cancer cells. Antagonistic, additive or synergistic effects were observed and correlated to cell proliferation rate. The inhibitors enhanced persistent DNA damage, delayed apoptosis, prolonged cell cycle arrest and senescence upon irradiation. These results demonstrated that radiation-induced synthetic lethality might widen the therapeutic window, hence extending the use of PARP inhibitors to patients without BRCAness.

A phase I trial of palbociclib plus bortezomib in previously treated mantle cell lymphoma

In mantle cell lymphoma (MCL), cyclin D1 combines with CDK4/6 to phosphorylate Rb, releasing a break on the G1 to S phase cell cycle. Palbociclib is a specific, potent, oral inhibitor of CDK4/6 capable of inducing a complete, prolonged G1 cell cycle arrest (pG1) in Rb+ MCL cells. The proteasome inhibitor bortezomib is approved by the US Food and Drug Administration for treatment of mantle cell lymphoma. Palbociclib-induced pG1 appears to sensitize MCL cells to killing by low-dose bortezomib, potentially improving its activity and tolerability. We conducted a phase 1 trial of palbociclib plus bortezomib in patients with previously treated MCL (NCT01111188). Patients received palbociclib at 75 mg (dose level 1), 100 mg (dose level 2), or 125 mg (dose levels 3 and 4) on days 1–12 of each 21-day cycle in addition to intravenous bortezomib 1.0 mg/m2 (dose levels 1, 2, 3) or 1.3 mg/m2 (dose level 4) on days 8, 11, 15 and 18. A total of 19 patients with a median age of 64 and an average of 2 prior therapies were enrolled. Two subjects experienced dose limiting toxicity (DLT): thrombocytopenia (dose level 1) and neutropenia (dose level 3). Although no DLTs were seen at dose level 4, all patients required dose delays during cycle 2 due to cytopenias, and the study team decided to stop the trial. Four of 19 patients achieved a clinical response, including one patient with a complete response. Three patients received treatment for more than one year, including one patient receiving single-agent palbociclib for more than 6 years. The combination of palbociclib 125 mg on days 1–12 plus bortezomib 1.0 mg/m2 on days 8, 11, 15, and 18 of a 21-day cycle is feasible and active in previously treated MCL, with the primary toxicity being myelosuppression. The regimen may be worthy of further evaluation in patients with non-blastoid MCL following failure of other newer agents.

Pigment epithelium-derived factor inhibits adipogenesis in 3T3-L1 adipocytes and protects against high-fat diet-induced obesity and metabolic disorders in mice

Obesity is a major cause of metabolic syndrome and type II diabetes, and it presents with metabolic disorders, such as hyperglycemia, hyperlipidemia, and insulin resistance. Pigment epithelium-derived factor (PEDF), a protein isolated from retinal pigment epithelial cells, has multiple functions, including neuronal protection, antineoplastic effects, and anti-inflammatory activity. The aim of this study is to investigate the antiobesity effects of PEDF. The antiobesity effects of PEDF on fat accumulation, inflammation, energy expenditure, insulin resistance, and obesity-related physiological parameters and protein levels were assessed in high-fat diet (HFD)-induced obese mice in vivo and in 3T3-L1 adipocytes, palmitate (PA)-treated HepG2 cells, and C2C12 myotubes in vitro. In an in vivo assay, PEDF effectively decreased body weight gain, white adipose tissue mass, and inflammation and improved insulin resistance, dyslipidemia, and hyperglycemia in HFD-induced mice. In liver tissue, PEDF decreased lipid accumulation and fibrosis. In an in vitro assay, PEDF diminished the differentiation of 3T3-L1 preadipocytes. We also determined that PEDF promoted lipolysis and prolonged cell cycle progression, through the mTOR-S6K pathway and downstream transcription factors, such as peroxisome proliferator-activated receptor gamma, CCAAT/enhancer-binding protein α (CEBP-α), and CEBP-β. In addition, PEDF decreased reactive oxygen species production in PA-induced HepG2 cells and improved glucose uptake ability in PA-induced HepG2 cells and C2C12 myotubes. In the present study, PEDF protected against HFD-induced obesity and metabolic disorders in mice, inhibited adipogenesis, and improved insulin resistance. These results provide a new potential treatment for obesity in the future.

Synthesis and biological evaluation of amino acid derivatives containing chrysin that induce apoptosis

A series of chrysin amino acid derivatives were synthesized to evaluate for their antiproliferative activities against several cancer cell lines. Among the compounds tested, N-(2-((5-hydroxy-4-oxo-2-phenyl-4H-chromen-7-yl)oxy)octanoyl)-L-leucine methyl ester(5d) presented a good anti-proliferative activity in MDA-MB-231 and MCF-7 cells. Flow cytometry analysis showed that 5d induced apoptosis and prolonged cell cycle progression in MDA-MB-231 and MCF-7 cells. Western blot analysis showed that 5d significantly inhibited Akt phosphorylation (Ser473) in MDA-MB-231 and MCF-7 cells. In addition, 5d treatment markedly downregulated Bcl-2 and upregulated Bax in a dose-dependent manner. In vitro caspase activation assay showed that 5d induced apoptosis of MDA-MB-231 cells by enhancing caspase 3/7 activity. The regulatory effect of 5d on apoptosis of MDA-MB-231 and MCF-7 cells may be induced by mitochondrial apoptosis pathway. This study is of great significance for designing and developing more effective chrysin amino acid derivatives.

A phase 1 trial of ibrutinib plus palbociclib in previously treated mantle cell lymphoma

AbstractSingle-agent ibrutinib is active in patients with previously treated mantle cell lymphoma (MCL); however, nearly half of all patients experience treatment failure during the first year. We previously demonstrated that prolonged early G1 cell cycle arrest induced by the oral, specific CDK4/6 inhibitor palbociclib can overcome ibrutinib resistance in primary human MCL cells and MCL cell lines expressing wild-type Bruton's tyrosine kinase (BTK). Therefore, we conducted a phase 1 trial to evaluate the dosing, safety, and preliminary activity of palbociclib plus ibrutinib in patients with previously treated mantle cell lymphoma. From August 2014 to June 2016, a total of 27 patients (21 men, 6 women) were enrolled. The maximum tolerated doses were ibrutinib 560 mg daily plus palbociclib 100 mg on days 1 to 21 of each 28-day cycle. The dose-limiting toxicity was grade 3 rash. The most common grade 3 to 4 toxicities included neutropenia (41%), thrombocytopenia (30%), hypertension (15%), febrile neutropenia (15%), and lung infection (11%). The overall and complete response rates were 67% and 37%, and with a median follow-up of 25.6 months, the 2-year progression-free survival was 59.4% and the 2-year response duration was 69.8%. A phase 2 multicenter clinical trial to further characterize efficacy is now ongoing. The current trial was registered at www.clinicaltrials.gov as #NCT02159755.

Abstract PD2-10: Treatment with abemaciclib modulates the immune response in gene expression analysis of the neoMONARCH neoadjuvant study of abemaciclib in postmenopausal women with HR+, HER2 negative breast cancer

Abstract Background: Abemaciclib is a selective inhibitor of CDK4 & 6 approved on a continuous dosing schedule for the treatment of HR+, HER2- metastatic breast cancer (MBC) patients (pts) in combination with endocrine therapy or as monotherapy. Recent studies have demonstrated the potential for CDK4 & 6 inhibitors, including abemaciclib, to promote anti-tumor immunity. Schaer et al., (Cell Reports 2018) showed that abemaciclib monotherapy results in upregulation of antigen presentation on tumor cells and increases T-cell activation. These activities synergized with anti-PD-L1 therapy to further enhance immune activation leading to complete tumor rejection in murine tumor models (Schaer et al., Cell Reports 2018). In this exploratory analysis, we evaluated the early and late immune-modulating effects of abemaciclib in the neoadjuvant study neoMONARCH (NCT02441946). Methods: NeoMONARCH is a Phase II trial in women with stage I-IIIB HR+, HER2- BC evaluating neoadjuvant treatment with 2 weeks of abemaciclib, alone or in combination with anastrozole (abemaciclib+ANZ), or ANZ alone. All patients received 14 weeks of abemaciclib +ANZ after the first 2 weeks of treatment. Serial biopsies were collected at 3 time points: Baseline (BL) - prior to treatment, Early - after 2 weeks of therapy with abemaciclib, ANZ, or abemaciclib+ANZ, and Late – after 2 weeks of initial therapy followed by 14 weeks of abemaciclib+ANZ. RNA was extracted from formalin fixed paraffin embedded (FFPE) tumor biopsies at each time point and subjected to whole transcriptome RNA sequencing. The curated data were subjected to statistical analysis using ANOVA tests followed by pathway analysis using Ingenuity Pathway Analysis (IPA) and Gene Set Enrichment Analysis (GSEA). Results: Consistent with the known activity of abemaciclib to inhibit the cell cycle, we observed at the early and late time points a significant treatment induced downregulation of genes related to mitotic spindle organization, replication stress response, G2M checkpoint, and E2F targets. Abemaciclib treatment for 2 weeks, alone or in combination with ANZ, followed by 14 weeks of combination therapy was associated with upregulation of gene expression signatures related to T-cell immune response and antigen presentation. Importantly, this phenomenon was notobserved with 2 weeks of ANZ treatment alone followed by 14 weeks of combination therapy. Conclusion: These data lend support that continuous inhibition of CDK4 & 6 signaling by abemaciclib treatment leads to prolonged cell cycle arrest resulting in tumor cell apoptosis & senescence, which then leads to an enhanced immune activation. Citation Format: Hurvitz S, Martin M, Press M, Wijayawardana S, Brahmachary M, Ebert PJ, Young S, Jansen V, Slamon D. Treatment with abemaciclib modulates the immune response in gene expression analysis of the neoMONARCH neoadjuvant study of abemaciclib in postmenopausal women with HR+, HER2 negative breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr PD2-10.

JNK-dependent cell cycle stalling in G2 promotes survival and senescence-like phenotypes in tissue stress.

The restoration of homeostasis after tissue damage relies on proper spatial-temporal control of damage-induced apoptosis and compensatory proliferation. In Drosophila imaginal discs these processes are coordinated by the stress response pathway JNK. We demonstrate that JNK signaling induces a dose-dependent extension of G2 in tissue damage and tumors, resulting in either transient stalling or a prolonged but reversible cell cycle arrest. G2-stalling is mediated by downregulation of the G2/M-specific phosphatase String(Stg)/Cdc25. Ectopic expression of stg is sufficient to suppress G2-stalling and reveals roles for stalling in survival, proliferation and paracrine signaling. G2-stalling protects cells from JNK-induced apoptosis, but under chronic conditions, reduces proliferative potential of JNK-signaling cells while promoting non-autonomous proliferation. Thus, transient cell cycle stalling in G2 has key roles in wound healing but becomes detrimental upon chronic JNK overstimulation, with important implications for chronic wound healing pathologies or tumorigenic transformation.

CCNA2 Ablation in Aged Mice Results in Abnormal rRNA Granule Accumulation in Hippocampus

Mounting evidence in the literature suggests that RNA-RNA binding protein aggregations can disturb neuronal homeostasis and lead to symptoms associated with normal aging as well as dementia. The specific ablation of cyclin A2 in adult neurons results in neuronal polyribosome aggregations and learning and memory deficits. Detailed histologic and ultrastructural assays of aged mice revealed that post-mitotic hippocampal pyramidal neurons maintain cyclin A2 expression and that proliferative cells in the dentate subgranular zone express cyclin A2. Cyclin A2 loss early during neural development inhibited hippocampal development through canonical/cell-cycle mechanisms, including prolonged cell cycle timing in embryonic hippocampal progenitor cells. However, in mature neurons, cyclin A2 colocalized with dendritic rRNA. Cyclin A2 ablation in adult hippocampus resulted in decreased synaptic density in the hippocampus as well as in accumulation of rRNA granules in dendrite shafts. We conclude that cyclin A2 functions in a noncanonical/non-cell cycle regulatory role to maintain adult pyramidal neuron ribostasis.

Loss of miR-199b Impairs Active Cycling of Hematopoietic Stem Cells during Steady-State Hematopoiesis

Several recent studies have showed that dysregulation of microRNA (miRNA) expression in hematopoietic stem cells (HSC) can affect self-renewal of HSCs, and indicated a role for miRNAs in development of acute myeloid leukemia (AML). We and others have reported a significant down-regulation of miR-199b in AML patients. Recently we found that miR-199b is enriched in long-term hematopoietic stem cells (LT-HSC), suggesting that miR199-b may regulate HSCs function. Therefore, to understand the physiologic role of miR-199 in hematopoiesis during homeostasis, we evaluated various hematopoietic stem and progenitor cells (HSPC) populations in mice harboring genetic deletion of miR199b-5p using CRISPR/Cas method. We found that ablation of miR199b resulted in markedly increased frequencies of primitive HSC and MPPs, and analyses of distribution pattern in myeloid progenitor populations showed reduced numbers of common myeloid progenitors (CMPs) biased toward granulocyte-monocyte (GMPs) linage with no changes in megakaryocytic-erythroid progenitors (MEPs). The elevated numbers of HSC and MPPs may indicate that increased proportion of HSC population is actively cycling, thus we analyzed LSK populations for expression of proliferation marker Ki67 along with DNA staining. We found that miR-199b deletion reduces proportion of primitive HSC and MPPs in cell-cycle, which may affect HSC cell self-renewal. Futher cell-cycle analyses revealed that miR-199b null HSCs leave G0 faster to accumulate in G1, but rather do not progress into mitosis, which was recovered upon 5-fluorouracil-induced cytokine burst. These results indicate that loss of miR-199b increases cell cycle duration. To verify that the absence of miR-199b influences proliferation of HSCs we pulsed miR-199b KO and WT mice with BrdU for 16 hours. We found the difference in the cell cycle distribution between HSCs and progenitors, namely reduction of BrdU-positive HSC and MPPs and progression of GMP compartment. These results show that miR-199b deletion decreases HSC active cell cycle by prolonging cell cycle transition during steady-state hematopoiesis and promotes proliferation of myeloid cells. Because quiescent cells only become susceptible to 5-FU during hematopoietic stress, driving them into cycle, we injected 5-FU into miR-199 KO and WT mice once per week until hematopoietic failure occurred. We found that miR199-b KO mice died soon after two subsequent injections, most likely due to the faster HSC exhaust as compared to WT mice. These results show that loss of miR199b produces HSC with reduced quiescence and prolonged cell cycle, however upon stress these cells progress into cell cycle, making them more susceptible for 5-FU treatment. These results demonstrate that miR-199b intrinsically regulates active cycling of HSCs. CFU-S assays showed that miR-199b KO donors showed decreased colonies in spleen, suggesting that miR-199b deletion affects short-term repopulation. In long-term repopulation assay, we observed a significant reduction of HSCs compartment, but elevated numbers of MPPs in host mice transplanted with BM from miR-199 KO mice. This data indicates that loss of miR-199b causes defects in HSC self-renewal and alters HSCs reconstitution potential. To identify potential miR-199b targets in HSCs under steady-state hematopoiesis, we performed a gene profiling in SLAM-HSCs. mRNA levels of several putative miR-199b targets were markedly elevated in miR-199b KO HSCs. These genes are known to be involved in cell adhesion, cell cycle, transcription regulation and chromatin remodeling including Klf12, Tox3 and Cdk18. Our findings reveal a novel functional role for miR-199b in governing HSC maintenance. DisclosuresNo relevant conflicts of interest to declare.

Haploinsufficiency of Insm1 Impairs Postnatal Baseline β-Cell Mass.

Baseline β-cell mass is established during the early postnatal period when β-cells expand. In this study, we show that heterozygous ablation of Insm1 decreases baseline β-cell mass and subsequently impairs glucose tolerance. When exposed to a high-fat diet or on an ob/ob background, glucose intolerance was more severe in Insm1+/lacZ mice compared with Insm1+/+ mice, although no further decrease in the β-cell mass was detected. In islets of early postnatal Insm1+/lacZ mice, the cell cycle was prolonged in β-cells due to downregulation of the cell cycle gene Ccnd1 Although Insm1 had a low affinity for the Ccnd1 promoter compared with other binding sites, binding affinity was strongly dependent on Insm1 levels. We observed dramatically decreased binding of Insm1 to the Ccnd1 promoter after downregulation of Insm1 expression. Furthermore, downregulation of Ccnd1 resulted in a prolonged cell cycle, and overexpression of Ccnd1 rescued cell cycle abnormalities observed in Insm1-deficient β-cells. We conclude that decreases in Insm1 interfere with β-cell specification during the early postnatal period and impair glucose homeostasis during metabolic stress in adults. Insm1 levels are therefore a factor that can influence the development of diabetes.

FTO regulates adipogenesis by controlling cell cycle progression via m6A-YTHDF2 dependent mechanism

N6-methyladenosine (m6A) is the most prevalent internal mRNA modification in eukaryotes. Loss of m6A demethylase FTO increases m6A levels and inhibits adipogenesis of preadipocytes. However, its underlying mechanism remains elusive. Here, we demonstrated that silencing FTO inhibited adipogenesis of preadipocytes through impairing cell cycle progression at the early stage of adipogenesis. FTO knockdown markedly decreased the expression of CCNA2 and CDK2, crucial cell cycle regulators, leading to delayed entry of MDI-induced cells into G2 phase. Furthermore, the m6A levels of CCNA2 and CDK2 mRNA were significantly upregulated following FTO knockdown. m6A-binding protein YTHDF2 recognized and decayed methylated mRNAs of CCNA2 and CDK2, leading to decreased protein expression, thereby prolonging cell cycle progression and suppressing adipogenesis. Our work unravels that FTO regulates adipogenesis by controlling cell cycle progression in an m6A-YTHDF2 dependent manner, which provides insights into critical roles of m6A methylation in adipogenesis.

Transmissible gastroenteritis virus N protein causes endoplasmic reticulum stress, up-regulates interleukin-8 expression and its subcellular localization in the porcine intestinal epithelial cell

This essay focuses on transmissible gastroenteritis virus (TGEV), which is an enteropathogenic virus related to contagious and acute diseases in suckling piglets. Previous literature suggests that the TGEV nucleocapsid protein (N) plays a significant role in viral transcriptional process, however, there is a need to examine other functions of TGEV N protein in the porcine intestinal epithelial cell (IEC) which is the target cell of TGEV. In the present study, we investigated the degradation, subcellular localisation, and function of TGEV N protein by examining its effects on cycle progression, endoplasmic reticulum (ER) stress, interleukin-8 (IL-8) expression, and cell survival. The results showed that TGEV N protein localised in the cytoplasm, inhibited IEC growth, prolonged the S-phase cell cycle by down-regulating cell cycle protein cyclin A, and was mainly degraded through the proteasome pathway. Moreover, TGEV N protein induced ER stress and activated NF-κB, which was responsible for the up-regulation of IL-8 and Bcl-2 expression. This report mainly considers the functions of TGEV N protein in IEC. To be specific, in IEC, TGEV N protein induces cell cycle prolongation at the S-phase, ER stress and up-regulates IL-8 expression. These results provide a better understanding of the functions and structural mechanisms of TGEV N protein.

The acid-base transport proteins NHE1 and NBCn1 regulate cell cycle progression in human breast cancer cells

ABSTRACTPrecise acid-base homeostasis is essential for maintaining normal cell proliferation and growth. Conversely, dysregulated acid-base homeostasis, with increased acid extrusion and marked extracellular acidification, is an enabling feature of solid tumors, yet the mechanisms through which intra- and extracellular pH (pHi, pHe) impact proliferation and growth are incompletely understood. The aim of this study was to determine the impact of pH, and specifically of the Na+/H+ exchanger NHE1 and Na+, HCO3− transporter NBCn1, on cell cycle progression and its regulators in human breast cancer cells. Reduction of pHe to 6.5, a common condition in tumors, significantly delayed cell cycle progression in MCF-7 human breast cancer cells. The NHE1 protein level peaked in S phase and that of NBCn1 in G2/M. Steady state pHi changed through the cell cycle, from 7.1 in early S phase to 6.8 in G2, recovering again in M phase. This pattern, as well as net acid extrusion capacity, was dependent on NHE1 and NBCn1. Accordingly, knockdown of either NHE1 or NBCn1 reduced proliferation, prolonged cell cycle progression in a manner involving S phase prolongation and delayed G2/M transition, and altered the expression pattern and phosphorylation of cell cycle regulatory proteins. Our work demonstrates, for the first time, that both NHE1 and NBCn1 regulate cell cycle progression in breast cancer cells, and we propose that this involves cell cycle phase-specific pHi regulation by the two transporters.

The Impact of Preexisting Chronic Kidney Disease on the Severity and Recovery of Acute Kidney Injury

Background: Recent observational studies have shown that in chronic kidney disease (CKD) patients, a significantly smaller percentage of patients with an episode of acute kidney injury (AKI) have full recovery of renal function compared to those without CKD. However, precise mechanisms involved in the incomplete repair after AKI with preexisting CKD have not been completely ascertained. Here, we assessed the impact of preexisting CKD on the severity and recovery of AKI in a mouse model of 5/6 nephrectomy. Methods: Male CD-1 mice underwent 5/6 nephrectomy (Nx). Six weeks post surgery, ischemia reperfusion injury (IRI) or a sham operation was performed and functional, histological, and various molecular parameters were compared between them. Results: Serum creatinine level on day 1 after IRI was comparable between control and Nx mice. However, serum creatinine remained significantly higher throughout the recovery phase in Nx mice compared to control mice. mRNA and protein expression of the cell cycle regulatory proteins were persistently elevated in Nx mice and this was associated with significantly increased levels of the G1 cell cycle arrest markers. Treatment with a p53 inhibitor following IRI resulted in not only decreased expression of G1 arrest markers but also decreased fibrosis, suggesting that prolonged epithelial G1 cell cycle arrest might be partially responsible for impaired recovery from superimposed AKI on CKD. Conclusion: Taken together, reduced nephron mass have a negative effect on the repair process that is partially mediated by the disruption of the cell cycle regulation.

Sulfasalazine, an inhibitor of the cystine-glutamate antiporter, reduces DNA damage repair and enhances radiosensitivity in murine B16F10 melanoma.

The sodium-independent cystine-glutamate antiporter plays an important role in extracellular cystine uptake. It comprises the transmembrane protein, xCT and its chaperone, CD98. Because glutathione is only weakly cell membrane permeable, cellular uptake of its precursor, cystine, is known to be a key step in glutathione synthesis. Moreover, it has been reported that xCT expression affects the progression of tumors and their resistance to therapy. Sulfasalazine is an inhibitor of xCT that is known to increase cellular oxidative stress, giving it anti-tumor potential. Here, we describe a radio-sensitizing effect of sulfasalazine using a B16F10 melanoma model. Sulfasalazine decreased glutathione concentrations and resistance to H2O2 in B16F10 melanoma cells, but not in mouse embryonic fibroblasts. It synergistically enhanced the cyto-killing effect of X-irradiation in B16F10 cells. It inhibited cellular DNA damage repair and prolonged cell cycle arrest after X-irradiation. Furthermore, in an in vivo transplanted melanoma model, sulfasalazine decreased intratumoral glutathione content, leading to enhanced susceptibility to radiation therapy. These results suggest the possibility of using SAS to augment the treatment of radio-resistant cancers.

Investigating the consequences of asymmetric endoplasmic reticulum inheritance in Saccharomyces cerevisiae under stress using a combination of single cell measurements and mathematical modelling

Adaptation allows organisms to maintain a constant internal environment, which is optimised for growth. The unfolded protein response (UPR) is an example of a feedback loop that maintains endoplasmic reticulum (ER) homeostasis, and is characteristic of how adaptation is often mediated by transcriptional networks. The more recent discovery of asymmetric division in maintaining ER homeostasis, however, is an example of how alternative non-transcriptional pathways can exist, but are overlooked by gold standard transcriptomic or proteomic population-based assays. In this study, we have used a combination of fluorescent reporters, flow cytometry and mathematical modelling to explore the relative roles of asymmetric cell division and the UPR in maintaining ER homeostasis. Under low ER stress, asymmetric division leaves daughter cells with an ER deficiency, necessitating activation of the UPR and prolonged cell cycle during which they can recover ER functionality before growth. Mathematical analysis of and simulation results from our mathematical model reinforce the experimental observations that low ER stress primarily impacts the growth rate of the daughter cells. These results demonstrate the interplay between homeostatic pathways and the importance of exploring sub-population dynamics to understand population adaptation to quantitatively different stresses.

Abstract A141: Preclinical evaluation of a new ROS1 inhibitor to overcome resistance to EGFR therapy in glioblastoma

Abstract Glioblastoma (GBM) is the most aggressive brain tumor in adults and remains incurable despite multimodal intensive treatment regimens. The majority of GBM tumors show a mutated or overexpressed epidermal growth factor receptor (EGFR); however, tumors treated with EGFR inhibitors such as gefitinib or erlotinib inevitably recur. This recurrence highlights the need to identify signaling pathways involved in GBM resistance to EGFR therapy that may serve as targets for intervention. In our recently published work, we found that activation of the ROS1 pathway is a primary mechanism by which glioma cells become resistant to EGFR-targeted therapy. ROS1 is a proto-oncogene receptor tyrosine kinase activated by chromosomal rearrangement in several human cancers, including non-small cell lung cancer (NSCLC), cholangiocarcinoma, gastric cancer, ovarian cancer, and glioblastoma. We therefore designed, synthesized, and screened a series of new ROS1 inhibitors. The lead compound, ROS-Ic, is very potent (IC50 = 24nM) and highly specific to ROS1 protein. Treatment of gefitinib-resistant GBM cells with ROS1-lc sensitized the resistant cells to low concentrations of gefitinib, resulting in apoptosis following a prolonged S-phase cell cycle arrest. When compared to crizotinib, an FDA-approved small-molecule tyrosine kinase inhibitor currently used to treat ALK- and ROS1+ NSCLC patients, ROS-Ic showed superior cytotoxic activity in vitro towards ROS1-positive GBM cells. Crizotinib resistance is thought to be due to abolished crizotinib binding by ROS1 mutations in crizotinib binding pocket. Interestingly, the binding of our leading compound ROS1-Ic was not affected by these mutations and crizotinib-resistant cells harboring these mutations were still sensitive to ROS1-Ic, highlighting the potential therapeutic value of this compound. Citation Format: Hashim M. Aljohani, Mohamed Aittaleb, Byung Sun Park, So Ha Lee, El Mustapha Bahassi. Preclinical evaluation of a new ROS1 inhibitor to overcome resistance to EGFR therapy in glioblastoma [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A141.