scholarly journals Growth-inhibitory Activity and Downregulation of the Class II Tumor-suppressor Gene H-rev107 in Tumor Cell Lines and Experimental Tumors

1997 ◽  
Vol 136 (4) ◽  
pp. 935-944 ◽  
Author(s):  
Christine Sers ◽  
Urban Emmenegger ◽  
Knut Husmann ◽  
Katharina Bucher ◽  
Ann-Catherine Andres ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Cell Lines ◽  
Nude Mice ◽  
Class Ii ◽  
Tumor Formation ◽  
Pancreatic Tumors ◽  
Rat Tissues ◽  
Transformed Cell Lines

The H-rev107 gene is a new class II tumor suppressor, as defined by its reversible downregulation and growth-inhibiting capacity in HRAS transformed cell lines. Overexpression of the H-rev107 cDNA in HRAS-transformed ANR4 hepatoma cells or in FE-8 fibroblasts resulted in 75% reduction of colony formation. Cell populations of H-rev107 transfectants showed an attenuated tumor formation in nude mice. Cells explanted from tumors or maintained in cell culture for an extended period of time no longer exhibited detectable levels of the H-rev107 protein, suggesting strong selection against H-rev107 expression in vitro and in vivo. Expression of the truncated form of H-rev107 lacking the COOH-terminal membrane associated domain of 25 amino acids, had a weaker inhibitory effect on proliferation in vitro and was unable to attenuate tumor growth in nude mice. The H-rev107 mRNA is expressed in most adult rat tissues, and immunohistochemical analysis showed expression of the protein in differentiated epithelial cells of stomach, of colon and small intestine, in kidney, bladder, esophagus, and in tracheal and bronchial epithelium. H-rev107 gene transcription is downregulated in rat cell lines derived from liver, kidney, and pancreatic tumors and also in experimental mammary tumors expressing a RAS transgene. In colon carcinoma cell lines only minute amounts of protein were detectable. Thus, downregulation of H-rev107 expression may occur at the level of mRNA or protein.

The Role Of BIN1 Tumor Suppressor Isoforms In Regulation Of Proliferation, Apoptosis and Tumor Formation Of Human Cutanous T-Cell Lymphoma Cells In Vitro and In Vivo

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2517-2517
Author(s):  
Sharmin Esmailzadeh ◽  
Youwen Zhou ◽  
Xiaoyan Jiang
Keyword(s):  
T Cell ◽  
Tumor Suppressor ◽  
Cell Lines ◽  
Tumor Formation ◽  
Post Injection ◽  
Apoptosis Pathway ◽  
Sézary Cells

Abstract Cutaneous T-cell lymphomas (CTCLs) represent a group of lymphoproliferative disorders that are characterized by homing of malignant T-cells to the surface of skin. There are two main types of CTCL: Mycosis Fungoides (MF) and its leukemic variant Sezary Syndrome (SS), which together represent about 65-70% of all CTCL cases. The precise genetic pathogenesis of these diseases remains largely undetermined. Recently, our research group has demonstrated that AHI-1 (Abelson Helper Integration site-1) oncogene is involved in CTCL. Expression of AHI-1 is increased in human leukemia cell lines, with marked upregulation (up to 40 fold) in CTCL lines (Hut78 and Hut102). Moreover, in FACS-purified CD4+CD7- Sezary cells from patients with Sezary Syndrome, AHI-1 expression is higher at both the RNA and protein levels compared to normal CD4+ cells. Furthermore, stable suppression of endogenous AHI-1 in Hut78 cells using small interfering RNA, normalizes their transforming activity both in vitro and in vivo. Thus, lymphomagenic activity of Hut78 cells is partially dependent on the expression of AHI-1. Interestingly, BIN1 (Bridging integrator 1) was identified through microarray analysis as one of the genes that may be involved in AHI-1-mediated leukemic transformation in CTCL cells. BIN1 is a nucleocytosolic adaptor protein with more than ten isoforms; some isoforms, including the BIN1 isoform (+10, +13), act as tumor suppressors, whereas the BIN1 (+12A) behaves as a cancer-related isoform in solid tumor models. However, the role of BIN1 in regulation of normal hematopoiesis and lymphomagenesis remains unknown. We have recently demonstrated that transcript levels of BIN1 isoforms are significantly lower in patients with MF or SS compared to controls. Four isoforms of BIN1 have been identified in Hut78 and primary CD4+CD7- Sezary cells. To investigate the role of BIN1 in CTCL, the BIN1 isoforms (+10, +13) and BIN1 (+12A) lentiviral constructs were transduced into two CTCL cell lines, Hut78 and HH cells. Overexpression of BIN1 isoforms led to a significant reduction in cell proliferation, as assessed by colony forming cell assays and 3H-Thymidine uptake assays (2-3 fold, p<0.05). Furthermore, a significant increase in spontaneous and specific apoptosis was observed in BIN1-transduced cells, with and without exogenous FAS-ligand (2-3 fold, p<0.05). Interestingly, a significant reduction in protein expression of c-FLIP (inhibitor of the FAS-mediated apoptosis pathway) and upregulation of downstream cleaved caspase-8 and caspase-3 was demonstrated in BIN1-transduced cells, suggesting that BIN1 isoforms induce apoptosis by downregulating the expression of c-FLIP, which leads to activation of the FAS-mediated apoptosis pathway. These findings show anti-proliferative and pro-apoptotic roles for BIN1 isoforms in human CTCL cells. In addition, subcellular fractionation and confocal microscopy further indicated that both the BIN1 (+10, +13) and BIN1 (+12A) isoforms are mainly located in the nucleus in Hut78 and HH cells. Furthermore, to investigate the effects of overexpression of BIN1 isoforms on the ability to induce tumors in vivo, we tested their leukemogenic potential by injecting transduced HH cells into non-obese diabetic/severe-combined immunodeficiency (NOD/SCID) mice. Mice injected subcutaneously with either parental HH or control empty vector cells (2 x 107/per mouse), showed local tumor formation in 6 of 6 mice within 4 days post-injection. The local tumors enlarged progressively and were often 1.5–2 cm in diameter by 3 weeks after injection. In contrast, no local tumors formed in mice given injections of equal numbers of BIN1-transduced HH cells after 14 days, in 12 out of 12 mice. Tumor formation was only observed in BIN1-transduced HH cells after 3 weeks post-injection. However, the local tumors were significantly smaller in BIN1-transduced HH cells compared to controls (∼4-fold). These findings further indicate that the two BIN1 isoforms have tumor suppressor activities in NOD/SCID mice and can significantly delay tumor formation and reduce tumor size in vivo. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Cellular tumorigenicity in nude mice. Test of associations among loss of cell-surface fibronectin, anchorage independence, and tumor-forming ability.

1979 ◽  
Vol 82 (1) ◽  
pp. 1-16 ◽  
Author(s):  
P Kahn ◽  
S I Shin
Keyword(s):  
Cell Surface ◽  
Cell Lines ◽  
Nude Mice ◽  
Diploid Cell ◽  
Tumor Formation ◽  
Surface Glycoprotein ◽  
Cell Surface Glycoprotein ◽  
Chicken Cell

Fibronectin (FN; also called large external transformation-sensitive [LETS] protein or cell-surface protein [CSP]) is a large cell-surface glycoprotein that is frequently observed to be either absent or greatly reduced on the surfaces of malignant cells grown in vitro. Because FN may be a useful molecular marker of cellular malignancy, we have carried out an extensive screening to test the specific association among the degree of expression of FN, anchorage-independent growth, and tumorigenicity in the athymic nude mouse. A variety of diploid cell strains and established cell lines were tested for the expression of surface FN by indirect immunofluorescence using rabbit antisera against human cold insoluble globulin, rodent plasma FN, or chicken cell-surface FN. Concomitantly, the cells were assayed for tumor formation in nude mice and for the ability to form colonies in methylcellulose. Tumorigenic cells often showed very low surface fluorescence, confirming earlier reports. However, many highly tumorigenic fibroblast lines from several species stained strongly with all three antisera. In contrast, the anchorage-independent phenotype was nearly always associated with tumorigenicity in approximately 35 cell lines examined in this study. In another series of experiments, FN-positive but anchorage-independent cells were grown as tumors in nude mice and then reintroduced into culture. In five of the six tumor-derived cell lines, cell-surface FN was not significantly reduced; one such cell line showed very little surface FN. Our data thus indicate that the loss of cell-surface FN is not a necessary step in the process of malignant transformation and that the growth of FN-positive cells as tumors does not require a prior selection in vivo for FN-negative subpopulations.

CNS-9, a Novel Specific Inhibitor of ABL Tyrosine Kinase Overcomes Resistance Mechanism of Imatinib.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 761-761 ◽  
Author(s):  
Shinya Kimura ◽  
Hidekazu Segawa ◽  
Junya Kuroda ◽  
Takeshi Yuasa ◽  
Taira Maekawa
Keyword(s):  
Tyrosine Kinase ◽  
Growth Inhibition ◽  
Cell Lines ◽  
Nude Mice ◽  
Philadelphia Chromosome ◽  
Specific Inhibitor ◽  
Vitro Assay ◽  
Abl Tyrosine Kinase

Abstract Imatinib mesylate (also known as STI-571 and Gleevec) has drastically changed the treatment of Philadelphia chromosome positive (Ph+) leukemias. However, the resistance to imatinib has frequently been reported, particularly in patients with advanced-stage disease. A novel orally bioavailable inhibitor of the ABL tyrosine kinase (TK) named CNS-9 was developed from the 2-(phenylamino)pyrimidine class to overcome resistance mechanisms of imatinib. Inhibition of TK phosphorylation (IC50) on wild type (wt) BCR/ABL in 293T cell line by CNS-9 was 22nM, which was 2-log more potent than imatinib. Importantly, CNS-9 inhibited TK phosphorylation of E255K mutant BCR/ABL with IC50 of 98nM, while imatinib could not inhibit it with clinically relevant concentration. The T315I mutant BCR/ABL protein was resistant to CNS-9 and imatinib. CNS-9 also inhibited TK phosphorylation of platelet-derived growth factor receptor (PDGFR) or c-Kit pathways at the very similar observed IC50s when compared with imatinib, in spite of significant higher potency against ABL. The ability of CNS-9 in vitro to inhibit 101 TK molecules was assayed by KinaseProfilerTM (Upstate), showing also more specific inhibitory activity against ABL than imatinib. The growth of BCR/ABL-positive cell lines K562, KU812, BaF3 harboring wt BCR/ABL (BaF3/wt) and E255K (BaF3/E255K) was inhibited by CNS-9 with IC50 of 5, 3, 17, and 110nM, respectively (Table 1). Generally, CNS-9 was 20 to 30-fold more potent on the growth inhibition than imatinib in these same cell lines. We next investigated the in vivo effect on the leukemic growth inhibition of CNS-9. Nude mice were injected subcutaneously with 3x107 KU812 (wt BCR/ABL) on Day 0. CNS-9 or imatinib were orally administrated twice a day from Day 7 to Day 18. The dosages of CNS-9 and imatinib, which inhibited completely tumor growth were 20mg/kg/day and 200mg/kg/day, respectively, indicating that CNS-9 is 10-fold potent than imatinib in vivo. To examine the in vivo effect of CNS-9 against mutant BCR/ABL, BaF3/wt, BaF3/E255K or BaF3/T315I were engrafted to nude mice and treated with CNS-9 or imatinib. CNS-9 was also 10-fold potent than imatinib against BaF3/wt. Intriguingly, mice harboring BaF3/wt or BaF3/E255K showed significantly prolonged survival when treated with CNS-9. Consistent with in vitro assay, CNS-9 had no effect on T315I, and imatinib was not effective against both E255K and T315I. In conclusion, CNS-9 is substantially more inhibitory and more specifically than imatinib toward BCR/ABL-dependent cell growth both in vitro and in vivo Moreover, CNS-9 may be effective for leukemia patients whose leukemic cells harbor E255K mutant. The efficacy and safety of CNS-9 for Ph+ leukemias should be verified in early phase clinical trials. The IC50s values of leukemic cell lines for CNS-9 and imatinib CNS-9 (nM) imatinib (nM) K562 p210 wt BCR/ABL 5 130 KU812 p210 wt BCR/ABL 3 67 U937 BCR/ABL (−) >1000 >1000 BaF3 p190 wt BCR/ABL 17 360 BaF3 p190 E255K BCR/ABL 110 >1000 BaF3 p190 T315I BCR/ABL >1000 >1000

Purified, Fermented, Extract of Triticum Aestivum Has Significant Independent Lymphomacidal Activity and Augments the Activity of Rituximab

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2857-2857
Author(s):  
Laura Newell ◽  
Joseph Tuscano ◽  
Robert o'Donnell ◽  
Yunpeng Ma
Keyword(s):  
Triticum Aestivum ◽  
T Cells ◽  
Cell Death ◽  
Cell Lines ◽  
Nude Mice ◽  
Mechanism Of Action ◽  
Wheat Germ ◽  
The United States

Abstract Abstract 2857 Background: Non-Hodgkin's lymphoma (NHL) affects over 400,000 people in the United States and its incidence increases with age. Treatment options include cytotoxic chemotherapy, which is often poorly tolerated by elderly patients, and monoclonal antibody (mAb) therapy. Nearly 70% of NHL patients eventually die of the disease. Development of effective alternate treatments with favorable toxicity profiles is necessary. Fermented wheat germ extract (FWGE) has shown anticancer potential in laboratory animals as well as in some small clinical studies; it is produced under GMP conditions in Europe and sold as Avemar™. The mechanism of action of FWGE is unclear, but is thought to involve metabolic pathways involved in tumor cell death. We examined the effects of FWGE on NHL and found significant lymphomacidal activity using in vitro and in vivo assays. We then further purified and characterized the active components of FWGE in order to develop a more potent form and to understand the mechanism of action, physiologic, and immunologic properties. Methods: FWGE was produced by fermenting purified wheat germ (Triticum aestivum) with Baker's yeast. The FWGE was further purified by removing insoluble material, precipitating proteins, freeze drying, fractionating with Sepharose and Sephadex columns, and then dialyzing to remove small molecules. The resultant fermented wheat germ proteins (FWGP) were assessed for in vitro cytotoxicity and pro-apoptotic activity using a panel of NHL cell lines. In vivo lymphomacidal activity was assessed in nude mice bearing Raji lymphoma xenografts. Mice were treated with increasing daily doses of FWGE by gastric lavage and compared to untreated controls as well as the commercially available fermented wheat germ product, Avemar. Results: In vitro killing assays with FWGE (regardless of the source) demonstrated lymphomacidal properties in three NHL cell lines (Jurkat, Raji, and Ramos). Pre-treatment of FWGE with heat or proteinase K reduced the lymphomacidal activity, suggesting that the active component was a protein. Nude mice bearing Raji lymphoma xenografts treated with FWGE confirmed the lymphomacidal properties of FGWE; there was no detectable toxicity as assessed by observation, mouse weight, or blood counts. The purified low molecular weight proteins (FWGP) also demonstrated lymphomacidal properties by cytotoxicity assays and murine NHL models, but at 1/1000th of the original dose. When FWGP was combined with rituximab, there was enhanced in vitro lymphomacidal activity, with over a 4000-fold reduction in the IC50. FWGP-induced NHL cell death was mediated by caspase-3-dependent apoptosis. FWGP augmented the host immune effector mechanisms, including ADCC and CDC, along with potent activation of NK-T cells (CD3/69/16), CD4+ T-cells and monocytes. Conclusions: FWGE can be easily produced and has cytotoxic effects in in vitro assays and in vivo. The purified FWGP are quantifiable, and are 10–1000 times more potent than FWGE. The mechanism of FWGP activity is based on direct pro-apoptotic effects as well as augmentation of host immune mediators. FWGP has activity against various subtypes of NHL. Studies are ongoing to further characterize the immune effects and anti-cancer properties of FWGP, as is planning for a human clinical trial +/− rituximab in patients with NHL. Disclosure: No relevant conflicts of interest to declare.

Jak1 deficiency leads to enhanced Abelson-induced B-cell tumor formation

Blood ◽  
2003 ◽  
Vol 101 (12) ◽  
pp. 4937-4943 ◽  
Author(s):  
Veronika Sexl ◽  
Boris Kovacic ◽  
Roland Piekorz ◽  
Richard Moriggl ◽  
Dagmar Stoiber ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Interferon Γ ◽  
Tumor Formation ◽  
Scid Mice ◽  
Janus Kinase ◽  
Interferon Α ◽  
Ifn Γ ◽  
Transformed Cell Lines

AbstractThe Janus kinase Jak1 has been implicated in tumor formation by the Abelson oncogene. In this study we show that loss of Jak1 does not affect in vitro transformation by v-abl as defined by the ability to induce cytokine-independent B-cell colony formation or establishment of B-cell lines. However, Jak1-deficient, v-abl–transformed cell lines were more tumorgenic than wild-type cells when transplanted subcutaneously into severe combined immunodeficient (SCID) mice or injected intravenously into nude mice. Jak1 deficiency was associated with a loss in the ability of interferon-γ (IFN-γ)to induce growth arrest and/or apoptosis of v-abl–transformed pre-B cells or tumor growth in SCID mice. Moreover, IFN-γ mRNA could be detected in growing tumors, and tumor cells explanted from SCID mice had lost the ability to respond to IFN-γ in 9 of 20 cases, whereas the response to interferon-α (IFN-α) remained intact. Importantly, a similar increase in tumorgenicity was observed when IFN-γ–deficient cells were injected into SCID mice, identifying the tumor cell itself as the main source of IFN-γ. These findings demonstrate that Jak1, rather than promoting tumorgenesis as previously proposed, is critical in mediating an intrinsic IFN-γ–dependent tumor surveillance.

scholarly journals Mutagenesis of Adeno-Associated Virus Type 2 Capsid Protein VP1 Uncovers New Roles for Basic Amino Acids in Trafficking and Cell-Specific Transduction

2010 ◽  
Vol 84 (17) ◽  
pp. 8888-8902 ◽  
Author(s):  
Jarrod S. Johnson ◽  
Chengwen Li ◽  
Nina DiPrimio ◽  
Marc S. Weinberg ◽  
Thomas J. McCown ◽  
...  
Keyword(s):  
Cell Lines ◽  
Class Ii ◽  
Adeno Associated Virus ◽  
Hek 293 ◽  
Nuclear Localization Signals ◽  
Transformed Cell ◽  
Correct Ratio ◽  
Capsid Protein Vp1 ◽  
Transformed Cell Lines

ABSTRACT The N termini of the capsid proteins VP1 and VP2 of adeno-associated virus (AAV) play important roles in subcellular steps of infection and contain motifs that are highly homologous to a phospholipase A2 (PLA2) domain and nuclear localization signals (NLSs). To more clearly understand how virion components influence infection, we have generated mutations in these regions and examined their effects on subcellular trafficking, capsid stability, transduction, and sensitivity to pharmacological enhancement. All mutants tested assembled into capsids; retained the correct ratio of VP1, VP2, and VP3; packaged DNA similarly to recombinant AAV2 (rAAV2); and displayed similar stability profiles when heat denatured. Confocal microscopy demonstrated that these mutants trafficked through a perinuclear region in the vicinity of the Golgi apparatus, with a subset of mutants displaying more-diffuse localization consistent with an NLS-deficient phenotype. When tested for viral transduction, two mutant classes emerged. Class I (BR1−, BR2−, and BR2+K) displayed partial transduction, whereas class II (VP3only, 75HD/AN, BR3−, and BR3+K) were severely defective. Surprisingly, one class II mutant (BR3+K) trafficked identically to rAAV2 and accumulated in the nucleolus, a step recently described by our laboratory that occurs with wild-type infection. The BR3+K mutant, containing an alanine-to-lysine substitution in the third basic region of VP1, was 10- to 100-fold-less infectious than rAAV2 in transformed cell lines (such as HEK-293, HeLa, and CV1-T cells), but in contrast, it was indistinguishable from rAAV2 in several nontransformed cell lines, as well as in tissues (liver, brain, and muscle) in vivo. Complementation studies with pharmacological adjuvants or adenovirus coinfection suggested that additional positive charges in NLS regions restrict mobilization in the nucleus and limit transduction in a transformed-cell-specific fashion. Remarkably, besides displaying cell-type-specific transduction, this is the first description of a capsid mutant indicating that nuclear entry is not sufficient for AAV-mediated transduction and suggests that additional steps (i.e., subnuclear mobilization or uncoating) limit successful AAV infection.

scholarly journals NOX1 Inhibition Attenuates the Development of a Pro-Tumorigenic Environment in Experimental Hepatocellular Carcinoma

2020 ◽  
Author(s):  
Astrid Vandierendonck ◽  
Helena Degroote ◽  
Bart Vanderborght ◽  
Lindsey Devisscher ◽  
Hans Van Vlierberghe
Keyword(s):  
Cell Lines ◽  
Poor Prognosis ◽  
Macrophage Polarization ◽  
Tumor Formation ◽  
Advanced Hcc ◽  
Treatment Regimens ◽  
Metavir Score ◽  
Systemic Treatments

Abstract Background The poor prognosis of advanced HCC and limited efficacy of current systemic treatments emphasize the need for new or combined targeted therapies. The development of HCC is a multistage process in which liver injury appears in a complex microenvironment associated with oxidative stress. NOX enzymes are the main source of ROS during hepatocarcinogenesis and NOX1 in particular has shown correlation with poor prognosis of HCC patients. This study evaluates the effect of pharmacological NOX1 inhibition on the development and progression of HCC and its effect on the tumor microenvironment.Methods The in vitro cytotoxic effects of the NOX1 inhibitor GKT771 (Genkyotex) on human Huh7 and Hep3B and murine Hepa1-6 HCC cell lines, and murine macrophages were evaluated via MTT, LDH activity and CaspGlo® assays. In order to induce in vivo HCC, male SV129 wild-type mice received weekly IP injections of diethylnitrosamine (DEN) (35 mg/kg) for 20-25 weeks. Mice were treated with vehicle or GKT771 (30 mg/kg) via oral gavage. Treatment duration and frequency (daily or twice daily) varied in the preventive and therapeutic studies. mRNA transcript levels in the tumor and liver tissue were determined by RT-qPCR. Fibrosis was visualized by Sirius Red staining and quantified by the Metavir​ score. Results A concentration-dependent reduction in cellular activity of the human HCC cell lines without cytotoxicity was observed. GKT771 treatment reduced LPS-induced pro-inflammatory bone-marrow derived macrophage polarization. DEN injections resulted in 100% tumor formation and the induction of HCC markers which could be reduced by twice daily dosing of GKT771 at early onset of advanced HCC. DEN-induced HCC resulted in an upregulation of pro-inflammatory, angiogenic and fibrotic markers which was less pronounced in GKT771 treated mice in all treatment regimens. In line, liver fibrosis was induced in HCC mice and this to a lesser extend upon GKT771 treatment.Conclusion NOX1 inhibition showed to be safe and well tolerated and was able to attenuate the induction of a pro-inflammatory, angiogenic and pro-fibrotic microenvironment suggesting that this might be a promising adjuvant therapeutic strategy in the treatment of advanced HCC.

scholarly journals Regulation of the CDK-related protein kinase PCTAIRE-1 and its possible role in neurite outgrowth in Neuro-2A cells

2002 ◽  
Vol 115 (17) ◽  
pp. 3479-3490 ◽  
Author(s):  
Ralph Graeser ◽  
Julian Gannon ◽  
Randy Y. C. Poon ◽  
Thierry Dubois ◽  
Alastair Aitken ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Neurite Outgrowth ◽  
Cell Lines ◽  
Mammalian Cells ◽  
Neuroblastoma Cell ◽  
Brain Extract ◽  
Related Protein ◽  
Transformed Cell Lines

PCTAIRE-1 is a CDK-related protein kinase found in terminally differentiated cells in brain and testis, and in many immortalised and transformed cell lines. Bacterially expressed PCTAIRE is completely inactive as a protein kinase, but is a very good substrate for protein kinase A (PKA),which phosphorylates a total of four sites in the N-terminus of PCTAIRE-1. Phosphorylation of one of these sites, Ser119, generates a 14-3-3 binding site, which is functional in vitro as well as in vivo. Mutation of another PKA site, Ser153, to an alanine residue generated an activated kinase in transfected mammalian cells. This activity was comparable to that of CDK5 activated by a bacterially expressed, truncated version of p35nck,p21. Gel filtration analysis of a brain extract suggested that monomeric PCTAIRE-1 was the active species, implying that PCTAIRE-1 may not be a true CDK, in that it does not require a partner (cyclin-like) subunit for kinase activity. Finally, we found that various forms of PCTAIRE-1 transfected into neuroblastoma cell lines could either promote or inhibit neurite outgrowth,suggesting a potential role for the PCTAIRE-1 gene product in the control of neurite outgrowth.

DDRE-33. PRECLINICAL THERAPEUTIC EFFICACY OF THE NOVEL BLOOD BRAIN BARRIER PENETRANT ATR INHIBITOR LR02 IN GLIOBLASTOMA

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi81-vi81
Author(s):  
Dimpy Koul ◽  
Veerakumar Balasubramaniyan ◽  
Xiaolong Li ◽  
Sabbir Khan ◽  
Davide Guggi ◽  
...  
Keyword(s):  
Cell Lines ◽  
Cellular Response ◽  
Methylation Status ◽  
Chemotherapy Resistance ◽  
Tumor Formation ◽  
Orthotopic Model ◽  
Ic50 Values ◽  
Orally Bioavailable

Abstract Glioblastoma (GBM) remains an incurable tumor with median overall survival of 15 months despite radiation and alkylating temozolomide (TMZ) chemotherapy. DNA damage response (DDR) pathways are among the most important key players of oncogenic mutations associated with resistance to both chemotherapy and radiation in GBM. The high frequency of alterations in DDR pathways in GBM suggests that its inhibition by DDR inhibitors may render GBM cells more susceptible to DNA damaging interventions. Here, we report the preclinical in vitro and in vivo activity of a novel, orally bioavailable Ataxia-telangiectasia mutated serine/threonine protein kinase and Rad3-related (ATR) inhibitor LR02 (Laevoroc Oncology) in a panel of 15 well-characterized glioma stem-like cells (GSCs). Effects on cell proliferation, survival and tumor formation were analyzed following treatment with LR02. Growth inhibition was time- and dose-dependent with a 3-day exposure resulting in a growth inhibitory IC50 (gIC50) in the low nM range in all the glioblastoma cell lines tested. LR02 inhibited growth of GSCs at IC50 values ranging from 500nmol/L to-~2umol/L. Additional studies showed that temozolomide sensitized GSC to LR02. Importantly, we demonstrate that MGMT promotor methylation status was associated with cellular response to LR02 treatment with preferential inhibition of cell growth in MGMT promotor methylated (MGMT deficient) cell lines. LR02 showed efficacy and survival benefit in a GSC262 (MGMT methylated) orthotopic model of GBM. Further administration of LR02 further enhanced the in vivo antitumor efficacy of temozolomide (TMZ) against GBM using the GSC262 model demonstrating that ATR inhibitor LR02 may enhance alkylating agent-mediated cytotoxicity and provide a novel treatment combination for GBM patients. Our present findings establish that the ATR inhibitor LR02 can specifically be used in tumors with MGMT deficiency when combined with alkylating chemotherapy. Further studies are ongoing to evaluate the potential of LR02 to overcome radiation and chemotherapy resistance in glioblastoma.

MiR-202 inhibits the proliferation and invasion of colorectal cancer by targeting UHRF1

2019 ◽  
Vol 51 (6) ◽  
pp. 598-606 ◽  
Author(s):  
Yilin Lin ◽  
Zhihua Chen ◽  
Suyong Lin ◽  
Yan Zheng ◽  
Yisu Liu ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Cell Lines ◽  
Gene Prediction ◽  
Ring Finger ◽  
Tumor Formation ◽  
Luciferase Assay ◽  
Control Group ◽  
Proliferation And Invasion

Abstract The purpose of this study was to investigate the expression of microRNA-202 (miR-202) and its role in colorectal cancer (CRC) in vivo and in vitro. We examined the expression of miR-202 in CRC tissues by quantitative real-time PCR (qRT-PCR) assay. Lentiviral vectors were constructed to overexpress or inhibit the expression of miR-202 in the CRC cell lines HCT116 and SW480 to determine its effects on cell invasion and proliferation. We found that overexpression of miR-202 significantly inhibited the proliferation and invasion of HCT116 cells. MiRNA target gene prediction, dual luciferase assay, and western blot analysis demonstrated that miR-202 regulated ubiquitin-like with PHD and RING finger domain 1 (UHRF1) expression in both cell lines. The effect of miR-202 on cell proliferation and invasion was partially reversed by activating the expression of UHRF1. Furthermore, miR-202 induced tumor formation in HCT116 xenograft BALB/c nude mice. Mice vaccinated with miR-202-overexpressing cells had smaller tumors and lower UHRF1 expression than the control group. These results indicate the possibility that miR-202 is under-expressed in CRC tissues, and that miR-202 inhibits the proliferation and invasion of CRC via targeting UHRF1. MiR-202 is a potential therapeutic target for CRC.

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