scholarly journals Measurement and models accounting for cell death capture hidden variation in compound response

2019 ◽  
Author(s):  
Song Yi Bae ◽  
Ning Guan ◽  
Rui Yan ◽  
Katrina Warner ◽  
Aaron S Meyer
Keyword(s):  
Cell Death ◽  
Cell Growth ◽  
Apoptotic Cell ◽  
Resistance Mechanisms ◽  
Cell Number ◽  
Live Cell ◽  
Response Analysis ◽  
List Type ◽  
Pharmacologic Response ◽  
Hidden Variation

AbstractCancer cell sensitivity or resistance is almost universally quantified through a direct or surrogate measure of cell number. However, compound responses can occur through many distinct phenotypic outcomes including changes in cell growth, apoptosis, and non-apoptotic cell death. These outcomes have distinct effects on the tumor microenvironment, immune responses, and resistance mechanisms. Here, we show that quantifying cell viability alone is insufficient to distinguish between these compound responses. Using an alternative assay and drug response analysis amenable to high-throughput measurement, we find that compounds with identical viability outcomes can have very different effects on cell growth and death. Moreover, compound pairs with additive cell growth and death effects can appear synergistic when only assessed by viability. Overall, these results demonstrate an approach to incorporating measurements of cell death when characterizing a pharmacologic response.Summary PointsMeasurements of solely live cell numbers mask important differences in compound effects.Additive effects on growth and death rates can appear synergistic when analyzed solely via live cell number.Automated imaging can provide reasonable throughput to analyze cell response in terms of cell growth and death, and endpoint analysis is similarly informative.

Diagnostische Bedeutung zirkulierender DNA- Fragmente in der Onkologie. Diagnostic importance of circulating DNA fragments in oncology

2005 ◽  
Vol 29 (2) ◽  
Author(s):  
Stefan Holdenrieder ◽  
Joachim von Pawel ◽  
Andreas Schalhorn ◽  
Petra Stieber
Keyword(s):  
Cell Death ◽  
Apoptotic Cell ◽  
Therapy Response ◽  
Cell Number ◽  
Circulating Dna ◽  
Malignant Diseases ◽  
Qualitative And Quantitative ◽  
Nucleosomal Dna ◽  
Quantitative Changes ◽  
Tissue Specimens

AbstractThe past decade witnessed an increasing interest in assessing circulating DNA in the plasma and serum of patients with malignant and non-malignant diseases. This might be due to the availability of new and sensitive methods for the determination of qualitative and quantitative changes in circulating DNA. As, previously, tumor-specific mutations or epigenetic modifications have been detected predominantly in tissue specimens, the appealing possibility to use less invasive though specific methods for tumor diagnosis was a noticeable incentive for the exploration of circulating DNA.A considerable part of the circulating DNA, which is mostly present in serum and plasma as nucleosomal DNA, is released during apoptotic cell death. Because the rate of apoptosis is deregulated in many pathological situations such as degenerative, traumatic, ischemic, inflammatory, and malignant diseases, and because many cytotoxic therapies aim at reducing the cancer cell number by apoptosis, the cell death product “circulating DNA” might serve as an attractive and appropriate biochemical correlative.In this review, the physiological and pathophysiological background of the arrangement of DNA as nucleosomes and of its release into circulation is shown. Further, the metabolism of circulating DNA in plasma and serum and its role in the pathogenesis of various diseases is discussed. Finally, the diagnostic relevance of qualitative and quantitative changes in circulating DNA for screening, differential diagnosis, prognosis, monitoring of systemic therapies, early prediction of therapy response and detection of recurrence in malignant diseases is reviewed. Concluding, some methodical considerations regarding the measurement of circulating DNA are given.

scholarly journals Arginine deprivation therapy induces apoptotic cell death in melanoma brain metastasis

2021 ◽  
Vol 23 (Supplement_4) ◽  
pp. iv24-iv24
Author(s):  
Aithne Atkinson ◽  
Nelofer Syed
Keyword(s):  
Cell Death ◽  
Brain Metastasis ◽  
Metastatic Melanoma ◽  
Caspase 3 ◽  
Apoptotic Cell ◽  
Apoptotic Cell Death ◽  
Live Cell ◽  
Melanoma Brain Metastasis ◽  
Arginine Deprivation ◽  
2D And 3D

Abstract Aims The development of melanoma brain metastasis (MBM) occurs in ~50% of metastatic melanoma cases, and significantly worsens prognosis to a median survival of 12.8 months. Melanoma is often reported as an arginine auxotroph due to transcriptional silencing of argininosuccinate synthase 1 (ASS1). Arginine deiminase (ADI) is a non-mammalian enzyme which depletes blood arginine by converting it to citrulline and ammonia, and in its pegylated form ADI shows clinical efficacy in the treatment of a number of cancers via exploiting tumour arginine auxotrophy, resulting in targeted arginine deprivation of tumour cells. While cutaneous melanoma is the prototype cancer for this therapy, studies to date have excluded central nervous system metastasis. We have demonstrated that patient derived primary MBM models are sensitive to arginine deprivation in vitro, confirmed suitable clinical biomarkers of sensitivity, and established the mechanism of tumour cell specific cytotoxicity. Method Patient derived primary cultures of MBM were established and subject to treatment with arginine deprivation. Gene expression and methylation analysis was examined by RT-qPCR, western blot, Illumina mRNA sequencing and Illumina methylated DNA immunoprecipitation-sequencing (MeDIP-seq) on ADI treated and untreated samples. Cell death, cytotoxicity induction and caspase-3 and-7 recruitment was analysed using an Incucyte S3 live-cell imager, by fluorescently labelling cells with Incucyte Cytolight Red Rapid dye, Cytotox Green dye and Caspase-3/7 Green dye, and imaging cells every 2 hours over the course of 2 weeks. 3D spheroid growth and invasion was measured by culturing cells as tumour spheroids before treating with ADI, and imaging spheroids every 2 hours for 2 weeks using an Incucyte S3 live-cell imager. Nuclear leakage and mitochondrial morphology was observed by fluorescently staining treated and untreated cells with DAPI and MitoTracker Red, and imaging on a Leica DMi8 confocal microscope. Results Primary MBMs differentially express ASS1 at substantially lower levels than non-cancerous melanocytes, however some models are capable of upregulating ASS1 following confrontation with arginine deprivation. Despite this, long-term sensitivity of primary MBMs to arginine deprivation was observed in both 2D and 3D models. In addition, arginine deprivation was seen to inhibit MBM invasion in a 3D model – an important feature in MBM pathogenesis. Initially, autophagy was induced in arginine deprived MBM, however in all models the induction of cytotoxicity correlated with recruitment of caspase-3 and -7, and intrinsic apoptotic cell death confirmed. Nuclear leakage, and eventually complete nuclear destruction was observed, in addition to mitochondrial fragmentation. Conclusion Arginine deprivation is highly effective in reducing 2D and 3D MBM growth, as well as limiting invasion. While apoptotic cell death was observed in all models, the initial induction of autophagy could pose threat of resistance development in a clinical setting, and so combinational therapies with autophagic inhibitors and/or additional apoptotic inducers should be investigated. It is unclear whether nuclear leakage and mitochondrial degradation are the cause or product of apoptosis. Considering the strong clinical evidence for the use of arginine deprivation in non-CNS metastatic melanoma and the results of this study, arginine deprivation is a highly suitable treatment for pre-surgical MBM to limit invasion and increase resection, and for post-surgical continuation.

Novel Therapeutic Approach to Overcome Both Bortezomib- and Lenalidomide-Resistant Multiple Myeloma By Targeting TOPK/PBK

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1763-1763
Author(s):  
Takayuki Tabayashi ◽  
Yasuyuki Takahashi ◽  
Yuta Kimura ◽  
Tatsuki Tomikawa ◽  
Tomoe Nemoto-Anan ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Death ◽  
Protein Kinase ◽  
Cell Growth ◽  
Research Funding ◽  
Apoptotic Cell ◽  
Mapk Signaling ◽  
Apoptotic Cell Death ◽  
Dependent Manner ◽  
Dose Dependent

Abstract Multiple myeloma (MM) is a neoplasm of plasma cells that often remains fatal despite the use of high-dose chemotherapy with hematopoietic stem cell transplantation. In the clinical setting, the introduction of novel agents, such as proteasome inhibitors and immunomodulatory drugs, has improved the clinical outcomes of both patients with newly diagnosed MM and patients with advanced MM. However, most patients eventually relapse and develop drug resistance. T-LAK cell-originated protein kinase (TOPK), also known as PDZ-binding kinase (PBK), is a mitogen-activated protein kinase kinase (MAPKK)-like serine/threonine kinase that plays a critical role in many cellular functions, such as cell proliferation, apoptotic cell death, and inflammation, in normal tissues. Because the expression of TOPK is up-regulated during mitosis and is activated by the Cdk1/cyclin B1 complex, TOPK is thought to have a role in cytokinesis. While the expression of TOPK is very low in most normal human tissues except for testis and placenta, it is overexpressed in various malignant neoplasms, indicating its crucial role in tumorigenesis. Phosphorylation of TOPK leads to the activation of the MAPK signaling pathway including p38 and Ras extracellular signal-regulated kinase (ERK). Moreover, TOPK interacts with p53 tumor suppressor protein and inhibits its function. Ribosomal protein S6 kinase (RSK2) is a downstream target of the ERK/MAPK signaling cascade and it has a pivotal role in cell survival and proliferation. Recent studies suggest that RSK2 inhibition induces apoptotic cell death and sensitizes MM cells to lenalidomide. Suppression of p53 function is also involved in MM progression. Taken together, these data suggest that TOPK might be an attractive target for new therapeutic agents against this incurable hematological malignancy. HI-TOPK-032, which is a potent and specific inhibitor of TOPK, occupies the ATP-binding site of TOPK and thereby suppresses TOPK kinase activity. In the present study, we investigated the role of TOPK/PBK in MM as a potential therapeutic target by using HI-TOPK-032. MTSand trypan blue dye exclusion assays showed that HI-TOPK-032 inhibited the proliferation of various MM cell lines, including U266, RPMI8226, MM1.S, OPM-2, and KMS-11, in a dose- (0 to 10 mM) and time- (0 to 72 h) dependent manner. To examine the mechanisms behind the growth inhibition effect induced by HI-TOPK-032, assays for apoptotic cell death were performed; these assays demonstrated that HI-TOPK-032 induced both early and late apoptosis in MM cells. To investigate the molecular mechanisms of HI-TOPK-032-induced cell death in MM cells, the expression of various cell death-associated proteins and down-stream molecules of TOPK was examined. Western blotting analysis showed that HI-TOPK-032 arrested cell growth and induced apoptotic cell death in MM cells in a dose-dependent manner by reducing t he phosphorylation of ERK and RSK2, thereby reducing the expression of the target molecules of RSK2, i.e., MCL1 and c-Myc. Moreover, HI-TOPK-032 induced p53 expression in a dose-dependent manner. We next examined the effects of HI-TOPK-032 on bortezomib (BTZ)-resistant MM cells, which represent an urgent issue in clinics and for which a therapeutic solution is important. Interestingly, HI-TOPK-032 inhibited the proliferation of both BTZ-sensitive wild-type KMS cells and BTZ-resistant KMS cells, suggesting that BTZ resistance can be overcome by targeting TOPK. Because our results showed that HI-TOPK-032 reduced the phosphorylation of RSK2, and previous studies have suggested that RSK2 inhibition sensitized MM cells to lenalidomide, we next studied the effects of HI-TOPK-032 in combination with lenalidomide on MM cell growth. HI-TOPK-032 and lenalidomide synergisticallyinduced growth arrest in not only lenalidomide-sensitive MM cells, but also in lenalidomide-resistant cells. To determine whether HI-TOPK-032 can re-sensitize BTZ-resistant cells to the anti-MM activity of BTZ, the effects of the combination of HI-TOPK-032 and BTZ were tested using an MTS assay. Interestingly, HI-TOPK-032 was able to re-sensitize BTZ-resistant MM cells to BTZ. These results indicate that the inhibition of TOPK may serve as an attractive therapeutic option for both patients with BTZ- or lenalidomide-resistant MM. In conclusion, these data suggest that TOPK/PBK can be a promising molecular target for the treatment of MM. Disclosures Kizaki: Nippon Shinyaku Co., Ltd.: Research Funding; Ono Phranacutical Co., Ltd.: Consultancy; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Chugai Phrarmaceutical Co., Ltd.: Research Funding.

scholarly journals Direct Induction of Autophagy by Atg1 Inhibits Cell Growth and Induces Apoptotic Cell Death

2007 ◽  
Vol 17 (1) ◽  
pp. 1-11 ◽  
Author(s):  
Ryan C. Scott ◽  
Gábor Juhász ◽  
Thomas P. Neufeld
Keyword(s):  
Cell Death ◽  
Cell Growth ◽  
Apoptotic Cell ◽  
Apoptotic Cell Death ◽  
Direct Induction

CSIG-22. Cd95l/Cd95 GENE DELETION DECREASES MOUSE GLIOMA TUMORIGENESIS THROUGH THE INHIBITION OF NON-CANONICAL CD95L/CD95-MEDIATED CELL GROWTH AND IMMUNOSUPPRESSION

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi37-vi38
Author(s):  
Clara Quijano-Rubio ◽  
Michael Weller
Keyword(s):  
Cell Death ◽  
Cell Growth ◽  
Cell Lines ◽  
Cancer Cell ◽  
Apoptotic Cell ◽  
Immune Effector ◽  
Immunodeficient Mice ◽  
Murine Glioma

Abstract CD95 is a transmembrane receptor with potential to promote both cell death and growth. Initially described to trigger apoptosis upon ligand (CD95L) engagement, CD95 may also prompt cell proliferation, invasion and stemness. CD95 stimulation to induce cancer cell apoptosis has been proved clinically impracticable. However, in tumors expressing both CD95 and CD95L, strategically inhibiting CD95-CD95L interactions to simultaneously block cancer cell growth and apoptotic cell death in tumor microenvironment components, including CD95-expressing antitumor immune effector cells, may represent an alternative therapeutic strategy. Here we characterized the expression of CD95 and CD95L in murine glioma models in vitro and in vivo. To fully disrupt CD95-CD95L interactions, we deleted Cd95 or Cd95l by CRISPR-Cas9-mediated knockout (KO) and assessed the consequences on cell growth and tumorigenicity in immunocompetent and immunocompromised mice. CD95 expression was identified in selected murine glioma cell lines. In vitro, expression of the canonical, membrane-bound, form of CD95L was not detected but cell lines expressed a shorter non-canonical, soluble, Cd95l variant. Tumors generated upon implantation of the same cells in vivo expressed both Cd95l variants. Upon Cd95l KO, all investigated cell lines exhibited reduced growth in vitro. Cell growth reduction upon Cd95 KO in SMA-497 murine glioma cells was rescued upon Cd95 re-transfection, validating CD95 specificity of the phenotype. Cd95-overexpression in Cd95-expressing cells did not increase growth. In vivo, Cd95 or Cd95l KO cell implantation in syngeneic mice generated smaller tumors than wildtype cells, resulting in prolonged survival. While 40% Cd95l KO cell-implanted immunocompetent mice did not develop tumors, all immunodeficient mice did. Altogether, these data reveal a growth-promoting role of non-canonical CD95L-CD95 interactions in murine gliomas, which blockade through gene KO results in decreased tumorigenicity. Furthermore, our data suggest the contribution of CD95L-mediated immunosuppression to the reduction of Cd95l KO-associated tumorigenicity.

GRN163L, a Novel and Potent Telomerase Inhibitor, Inhibits Myeloma Cell Growth In Vitro and In Vivo.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 639-639
Author(s):  
Masood A. Shammas ◽  
Hemanta Koley ◽  
Pierfrancesco Tassone ◽  
Paola Neri ◽  
Alexei Protopopov ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Growth ◽  
Apoptotic Cell ◽  
Myeloma Cell ◽  
Telomerase Activity ◽  
Hsp90 Inhibitor ◽  
Telomere Shortening ◽  
Myeloma Cells

Abstract Telomerase activity is either low or completely absent in most normal somatic cells; while it is elevated in most cancer cells providing unlimited proliferative potential by preventing telomere shortening. The inhibitors of telomerase, therefore, induce telomere shortening leading to apoptotic cell death in tumor cells while having little or no effect on normal diploid cells. We have evaluated the in vitro and in vivo efficacy of thio-phosphoramidate oligonucleotide specifically targeting the RNA component of telomerase (GRN163L) with demonstrated nuclear uptake by >99% cells without the transfection enhancer. Delivery of GRN163L (1 μM) to MM cells (INA6 and ARP) was specifically associated with complete loss of telomerase activity as early as 6 hrs following exposure and was accompanied by a reduction in myeloma cell growth and survival. Treatment of INA6 cells with GRN163L for three weeks induced 96±4% and 100% cell death at 0.5 and 1 μM concentrations, respectively. ARP cells, which express higher levels of telomerase activity and have longer telomeres, showed 67±4% cell death at 5 weeks with 0.5 μM inhibitor and 82±3% and 100% cell death at 4 and 5 weeks, respectively, with 2 μM GRN163L. The apoptotic cell death was confirmed in 51% INA6 cells at two weeks and in >80% ARP cells at four weeks. Apoptosis was associated with reduction in mean Telomere Fluorescence Intensity (TFI) on interphase chromosomes from 87.1±6.2 in control oligo treated INA6 cells to 36.2±2 (2.4 fold) in GRN163L treated cells. Moreover, GRN163L treatment was also associated with a similar reduction in number of chromosomes with detectable telomeres, indicating development of telomere-free ends. We have confirmed in vivo efficacy of GRN163L in a SCID-hu murine model of multiple myeloma. Following growth of GFP-transduced myeloma cells in the fetal bone chip introduced into the mice, GRN163L was injected on alternate days. In two independent experiments significant reduction in tumor cell growth, as measured by reduction in human myeloma related protein, and better survival than mice injected with control oligo was observed. We have now evaluated efficacy of combination of GRN163L with other novel agents. We have observed synergistic activity with Hsp90 inhibitor 17AAG on myeloma cell death. Addition of 17AAG (0.05 μM) to myeloma cells pre-treated with GRN163L (1 μM) for one week led to complete growth arrest within four days compared to continued growth of cells not pre-treated with GRN-163. These data provide the preclinical rationale for clinical evaluation of GRN163L in myeloma and in combination with Hsp90 inhibitor.

Inhibition of B16 Mouse Melanoma Cell Growth and Induction of Apoptotic Cell Death with 8-Chloroadenosine-3′,5′-monophosphate and Tiazofurin

2004 ◽  
Vol 1030 (1) ◽  
pp. 384-392 ◽  
Author(s):  
LELA B. KORIĆANAC ◽  
DANIJELA V. TODOROVIĆ ◽  
NATAŠA M. POPOVIĆ ◽  
MIROSLAV A. DEMAJO ◽  
SABERA D. RUŽDIJIĆ ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Growth ◽  
Melanoma Cell ◽  
Apoptotic Cell ◽  
Apoptotic Cell Death ◽  
Mouse Melanoma ◽  
Mouse Melanoma Cell ◽  
B16 Mouse Melanoma
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