Relative Number Of Viable Cells Research Articles

Roles of Ribosomal S6 Kinases in Acute Leukemia and Normal Hematopoiesis

Ribosomal S6 kinases (RSKs) are serine-threonine kinases that are activated by the Ras/mitogen activated protein kinase signaling pathways and regulate cell proliferation and survival. There are 4 isoforms of RSK (RSK1, 2, 3, 4), and there is evidence that the isoforms mediate tissue-specific cellular functions. We hypothesized that RSK isoforms have distinct roles in hematopoiesisand evaluated RSK as a target for the treatment of acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). To investigate isoform expression in hematopoiesis, we sorted cord blood mononuclear cells into hematopoietic progenitor stages and quantified RSK isoform mRNA by RT-qPCR. RSK1 expression remained consistent throughout hematopoiesis, whereas RSK2 expression decreased in the more differentiated populations. RSK3 was elevated in the lymphoid-primed multipotential progenitor and common myeloid progenitor stages and was greatly reduced in the more mature megakaryocyte-erythroid progenitor population. These results suggest that RSK proteins have isoform-specific roles in hematopoiesis. To analyze the importance of each isoform on hematopoiesis, we transduced cord blood CD34+ cells with lentiviral constructs expressing isoform-specific or control shRNAs. After liquid culture for 5 days, the cells were plated in methylcellulose media containing SCF, IL-3, GM-CSF, and EPO to determine the colony-forming potential. RSK1 and RSK2 knockdown decreased the colony-forming unit - erythroid colonies and increased the granulocyte/macrophage colony numbers. RSK3 knockdown increased the burst-forming unit - erythroid colonies and total colony numbers. After liquid culture days 8, 11, and 14, the erythroid and myeloid populations were analyzed by flow cytometry. RSK1 knockdown increased the CD11b+ and CD14+ myeloid populations and decreased the CD71+ erythroid population. RSK3 knockdown increased the erythroid population and decreased the myeloid populations. These data demonstrate that RSK1 and RSK3 have opposing effects on hematopoietic lineage determination. Overexpression of RSK proteins leads to aberrant cell survival and proliferation in many cancers including leukemia. We have previously reported that RSK and phosphorylated RSK are elevated in pediatric AML and are associated with a worse prognosis. There are only two orally bioavailable RSK inhibitors that have moved to phase I clinical trials: TAS0612 and PMD-026. TAS0612 is a multi-kinase inhibitor of RSK, AKT, and S6K. PMD-026 has high specificity to all four RSK isoforms. We evaluated the effects of both compounds in AML and ALL. To determine the effects of the RSK inhibitors on AML, we treated AML cell lines and patient samples with a range of doses of PMD-026, TAS0612, or 0.1% DMSO. After 3 days, we performed CellTiter Glo assays to determine the relative viable cell number and calculate the concentration of drug that inhibits cell growth by 50% (IC50). TAS0612 had an IC50 of 0.096-0.64 µM in AML cell lines Molm13, U937, AML193, and MV4;11. To test the cytotoxicity of TAS0612 on normal hematopoiesis, we plated healthy bone marrow mononuclear cells with TAS0612 in methylcellulose media. Colony formation was not adversely affected by TAS0612 up to 1 µM. PMD-026 was effective in Molm13, Kasumi-1, and MV4;11 with an IC50 of 0.34-0.55 µM and in AML patient samples 3123 and 4192 with an IC50 of 1.5-2.3 µM. PMD-026 did not affect healthy bone marrow colony formation up to 5 µM for a 2.5- to 10-fold therapeutic window. Furthermore, we tested the effects of RSK inhibitors in ALL. While PMD-026 was not effective, we found that B-ALL cell lines (REH, Nalm-6) and T-ALL cell lines (Loucy, Jurkat) were sensitive to RSK inhibition by TAS0612 with an IC50 of 0.056-0.19 µM. Western blot analysis of RSK protein revealed that RSK1, RSK2, and phospho-RSK proteins are elevated in ALL cell lines compared to healthy peripheral blood mononuclear cells. We are investigating the role of RSK and RSK isoforms in ALL. We found that RSK isoform expression is modified during hematopoiesis. Isoform expression influences early-fate decisions to differentiate into erythroid and myeloid lineages. RSK inhibitors PMD-026 and TAS0612 are effective at targeting AML and ALL cells with limited cytotoxicity to healthy cells, making them promising candidates for translation into clinical trials for AML and ALL.

Abstract 1046: Combination of CDK 4/6 inhibitor Palbociclib with Rapamycin synergistically inhibits the growth of hepatocellular carcinoma cells

Abstract Introduction: Aberrant cell-cycle regulation is common in cancer, making it an attractive therapeutic target. The cell cycle inhibitor Palbociclib has been shown to have a cytostatic effect on HCC cells via the induction of quiescence and senescence, though it has limited cytocidal effect. We hypothesized that arrested cells upregulate mTOR signaling to stay metabolically active and investigated the effect of combining Palbociclib with the mTOR inhibitor Rapamycin on HCC cell lines in-vitro. Methods: We employed single sample gene set enrichment analysis (ssGSEA) of 10 paired RNA-seq and 15 paired proteomic data sets as well as 371 HCC tumors from TCGA to identify commonly altered molecular markers in HCC, including cell cycle related genes. Subsequently, we studied the effect of Palbociclib and Rapamycin on two HCC cell lines, SNU398 and HuH7. We treated the cell lines with increasing doses of Palbociclib and used cell counting to assess growth inhibition, as well as β-galactosidase activity assay to assess induction of senescence. We then treated both cell lines with Palbociclib for ten days followed by Palbociclib plus Rapamycin for 4 days and performed an MTT assay to assess relative viable cell number. We also did a fixed ratio treatment with Palbociclib and Rapamycin, followed by calculation of combination index to determine the nature of their interaction. Finally, we performed Western Blot analysis for various proteins of the cell cycle and mTOR pathways to elucidate the effects of combination treatment. Results: ssGSEA revealed significant positive enrichment of several cell cycle and mitosis related gene sets. Palbociclib treatment showed a dose-dependent inhibition of growth as well as increase in senescence associated β-galactosidase activity for both SNU398 and HuH7 cell lines. Addition of Rapamycin demonstrated a dose-dependent potentiation of Palbociclib induced growth inhibition, and fixed ratio treatment with the two drugs established a synergistic effect as indicated by a combination index of less than 1. Western blot analysis showed increased levels of intrinsic CDK inhibitors p16, p15, p21 and p27 with Palbociclib treatment. Western blot analysis of proteins of mTOR pathway revealed that Palbociclib treatment caused an increase in levels of p-mTOR as well as its downstream signaling proteins p-4eBP1, p-p70 and p-s6, and addition of Rapamycin suppressed their levels. Conclusion: HCC cells over-express genes and proteins that drive cell cycle progression. Palbociclib induces senescence in hepatocellular carcinoma cell lines along with an associated upregulation in mTOR signaling, and combination with Rapamycin has a synergistic effect on growth inhibition in-vitro possibly by eliminating senescent cells. Citation Format: Ankur Tiwari, Araceli Huerta, Hakim Bouamar, Debodipta Das, Francisco G. Cigarroa, LuZhe Sun. Combination of CDK 4/6 inhibitor Palbociclib with Rapamycin synergistically inhibits the growth of hepatocellular carcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1046.

Anticancer Activity of Fucoidan via Apoptosis and Cell Cycle Arrest on Cholangiocarcinoma Cell.

Objective:Many previous studies reported that fucoidan has antitumor activities. The objective of the present study was to determine the cytotoxic effects and related mechanisms of cell death induced by fucoidan extracted from Fucus vesiculosus on CL-6 cholangiocarcinoma cell. Methods:CL-6 and OUMS cells were treated with 0, 100, 200, and 300 μg/mL of fucoidan. MTT assay was used to determine cytotoxicity. Flow cytometry-based assay was used to examine the distribution of apoptosis and cell cycle. The changes in nuclear morphology were determined using Hoechst 33,342 staining. Mitochondrial membrane potential (ΔΨm) was evaluated using the JC-1 kit. The apoptotic, anti-apoptotic, and cell cycle-related proteins study were examined by Western blot analysis. Results:The relative viable cell number of treated CL-6 cells was decreased but no effect was observed in OUMS normal cells. Furthermore, treated cells were arrested in the G0/G1 phase with down-regulation of cyclin D1 and CDK4. Annexin V/PI staining with flow cytometry analysis suggested that fucoidan could induce apoptosis in CL-6 cells. Western blot study revealed the up-regulation of apoptotic markers including Bax, cleaved PARP, cleaved caspase-3, but down-regulation of anti-apoptotic markers, cl-2. Moreover, fucoidan could induce nuclear fragmentation and chromatin condensation with alteration of ΔΨm. Conclusion:Fucoidan exerts antitumor properties against CL-6 cholangiocarcinoma cells illustrated by the induction of apoptosis and cell cycle arrest.

Features of local immunity in women with endometriosis and genital infection

Introduction. External genital endometriosis is an inflammatory estrogen-dependent disease characterized by implantation and proliferation of endometrial tissue outside the uterus, accompanied by increased production of proinflammatory cytokines, prostaglandins, components of the complement, hydrolytic enzymes, increased angiogenesis and anomalies of ectopic endometrium. According to implantation theory, external genital endometriosis develops from viable endometrial cells transferred retrogradely through the fallopian tubes to the abdominal cavity during menstruation, while a disturbed local immunity is an important factor in its pathogenesis. Genital pathogens may be involved in the formation of the immune environment of the peritoneal cavity in women with endometriosis.Purpose. To study the peculiarities of local immunity in women with external genital endometriosis and genital infection pathogens.Materials and methods. 159 women with external genital endometriosis were examined. The total number of leukocytes, the absolute and relative number of viable cells, counts of neutrophils, macrophages and their functional activity, the level of IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IFNα, IFNγ, TNFα in the peritoneal fluid were evaluated. The study of local immunity was performed in women with endometriosis, stage 1—2 and 3—4, depending on detected genital pathogens. Chlamydia trachomatis, Ureaplasma spp., Mycoplasma genitalium, HSV1,2/CMV, high carcinogenic risk HPV were analyzed by using PCR in samples collected from the endometrium, peritoneal fluid, and endometrioid heterotopies. Statistical processing was performed by using the IBM SPSS Statistics Version 22.2 statistical analysis software package.Results. In the presence of HPV and Ureaplasma spp. in women with endometriosis, stage 1—2, the decreased functional activity of peritoneal neutrophils and macrophages was found. At 3—4 stage, a correlation analysis revealed that detected HPV and Ureaplasma spp. obtained increased both pro-inflammatory and anti-inflammatory cytokines in the peritoneum. However, the higher activity of Th-2 cells in the peritoneal fluid secreting IL-4 and IL-10 and suppressing cellular immunity, was observed in HPV-positive samples. In addition, HPV also correlated with a decreased IL-2 and IL-4 levels.Conclusions. The most prominent changes in the immunological parameters from peritoneal fluid samples were observed in case of detected genital infection pathogens, particularly HPV. Thus, immune disturbances emerged upon bacterial and viral pathogen detection may contribute to the implantation of endometrial cells in the pelvic organs and disease progression.

Synergistic Effects of Photo-Irradiation and Curcumin-Chitosan/Alginate Nanoparticles on Tumor Necrosis Factor-Alpha-Induced Psoriasis-Like Proliferation of Keratinocytes.

Psoriasis is a chronic inflammatory skin disease characterized by hyperproliferation of the epidermal cells and is clinically presented as thick, bright red to pink plaques with a silvery scale. Photodynamic therapy (PDT) using visible light has become of increasing interest in the treatment of inflammatory skin diseases. In this study, we demonstrate that a combination of curcumin-loaded chitosan/alginate nanoparticles (Cur-CS/Alg NPs) and blue light emitting diodes (LED) light irradiation effectively suppressed the hyperproliferation of tumor necrosis factor-alpha (TNF-α)-induced cultured human kerlatinocyte (HaCaT) cells. The Cur-CS/Alg NPs were fabricated by emulsification of curcumin in aqueous sodium alginate solution and ionotropic gelation with calcium chloride and chitosan using an optimized formulation derived from a Box-Behnken design. The fabricated Cur-CS/Alg NPs were characterized for their particle size, zeta potential, encapsulation efficiency, and loading capacity. The surrogate 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, to measure the relative number of viable cells, showed that the CS/Alg NPs were nontoxic to normal HaCaT cells, while 0.05 µg/mL and 0.1 µg/mL of free curcumin and Cur-CS/Alg NPs inhibited the hyperproliferation of HaCaT cells induced by TNF-α. However, the Cur-CS/Alg NPs demonstrated a stronger effect than the free curcumin, especially when combined with blue light irradiation (10 J/cm2) from an LED-based illumination device. Therefore, the Cur-CS/Alg NPs with blue LED light could be potentially developed into an effective PDT system for the treatment of psoriasis.

Antibacterial activity absence UV irradiation of Ag, TiO2 and ZnO NPs prepared by sparking method

Abstract Silver (Ag) Titanium dioxide (TiO2) and Zinc Oxide (ZnO) nanoparticles (NPs) successfully prepared into distilled water using sparking method. An antibacterial activity against Escherichia coli (E. coli) was typically studied by applying the spread plate test. The bacterial activity was estimated by a relative number of viable cells which form colonies on the nutrient agar plates. From the results, the particle sizes linearly decreased with increasing of the melting temperature of each metal tips. Moreover, the deposition rate of NPs in the water depends on the sparking time and melting temperature. The antibacterial activity absence UV irradiation of Ag and TiO2 completely inhibited after 1 h. Although in the dark, TiO2 can be damage and disorganize the cell membrane due to the nanoparticles in the sizes less than 20 nm can diffuse into the cell membrane of E. coli. The cells were then deformed and intracellular structures were disorganized.

N-acetylglucosaminyltransferase V modulates radiosensitivity and migration of small cell lung cancer through epithelial-mesenchymal transition.

N-acetylglucosaminyltransferase V (Gnt-V) has been linked to the migration of various human cancers. Recently we have found that inhibition of Gnt-V increases the radiosensitivity of cancer cells. However, the mechanisms by which Gnt-V mediates radiosensitivity and migration, especially in small cell lung cancer (SCLC) remain unknown. In our study, two SCLC cell lines (H1688 and H146) were used to investigate whether Gnt-V modulated the radiosensitivity and migration of SCLC cells through the epithelial-mesenchymal transition (EMT). The results showed that the expression of Gnt-V correlated with the N stage in patients with SCLC. Overexpression of Gnt-V led to a further increase in the relative viable cell number and survival fraction with a decrease in apoptosis rate and Bax/Bcl-2 ratio, when the cells were treated with irradiation. By contrast, knockdown of Gnt-V with irradiation resulted in a further decrease in the relative viable cell number and survival fraction but an increase in apoptosis rate and Bax/Bcl-2 ratio. Cells expressing high levels of Gnt-V increased migration whereas low levels of Gnt-V suppressed cell migration. Besides, the transient knockdown of ZEB2 led to an increase in radiosensitivity and an inhibition in the migration of SCLC cells. Furthermore, Gnt-V was negatively correlated with E-cadherin expression but positively correlated with N-cadherin, vimentin and ZEB2 expression. Finally, an in vivo study revealed that upregulation of Gnt-V caused tumour growth more quickly, as well as the expression of EMT-related markers (N-cadherin, vimentin and ZEB2). Taken together, the study suggested that an elevation of Gnt-V could lead to the radiosensitivity and migration of SCLC cells by inducing EMT, thereby highlighting Gnt-V as a potential therapeutic target for the prevention of EMT-associated tumour radioresistance and migration.

12 Next generation sequencing (NGS) guided therapy prediction for the treatment of glioblastoma multiforme (GBM)

also allows for serial imaging and tissue collection over time. The goal of the current study was to investigate the antineoplastic effects of auranofin in a panel of canine lymphoma-derived cell lines, and to identify potential biomarkers of drug activity for future translational studies in spontaneous canine LSA. Materials and Methods: 4 canine LSA-derived cell lines were incubated in varying concentrations of auranofin +/− bortezomib, elesclomol, or the conventional antineoplastic agents doxorubicin (DOX), CCNU and vincristine (VCR) for 24−72 hours. Relative viable cell number was assessed using MTS. Cell cycle distribution and apoptosis induction were evaluated cytometrically with propidium iodide staining and Annexin V/PI staining respectively. Induction of ROS was assessed using the redoxsensitive fluorescent dye CM-H2DCFDA. Results: Auranofin induced dose-dependent antiproliferative effects in all canine LSA cell lines, with 50% inhibitory concentrations between 0.1 and 1 uM. These are similar to those observed in human hematopoietic tumor cells, and well within clinically achievable serum concentrations. Doseand time-dependent apoptosis induction was observed, and additive to synergistic antiproliferative/propaptotic activity was observed with bortezomib and elesclomol, but not DOX, CCNU or VCR. Doseand timedependent induction of ROS was observed following auranofin treatment. Conclusions: Auranofin-associated antitumor activity is observed in vitro in canine LSA cells, at pharmacologically achievable concentrations. This is similar to activity observed in human LSA and leukemia. Cooperative activity was observed with the targeted agents bortezomib and elesclomol. These data support translational evaluation of auranofin in canine LSA as a model for the human disease.

Effects of zoledronic acid on osteoblasts in three-dimensional culture

Background/purposeBisphosphonates (BPs) are synthetic drugs with antitumor and bone antiresorptive activities. The use of BPs is suspected to favor the emergence of osteonecrosis of the jaw (ONJ), a putative side effect whose pathogenesis remains unclear. Thus, we aimed to get insights about the impact of BPs on osteoblasts functions, using a three-dimensional (3D) culture model, which is suggested to be more similar to the in vivo tissues than monolayers. Materials and methodsEffects of low (0.1 μM) and high (10 μM) concentrations of zoledronic acid were investigated on osteoblasts (hFOB 1.19), cultured as multicellular spheroids (MCS). Proliferation, apoptosis, spheroid growth kinetics, and morphology were studied using the 3-(4,5-dimethyl-thiazoyl-2yl) 2,5-diphenyl-tetrazolium bromide (MTT) and acid phosphatase (APH) assays, caspase 3 Western-blotting, phase contrast imaging and scanning electron microscopy (SEM). ResultsProliferation, apoptosis, and spheroid morphology showed that 10 μM zoledronic acid (ZA) induced a significant reduction in the relative viable cell number, correlated with morphological alterations of spheroids, and induction of apoptosis. A lower ZA concentration (0.1 μM) promoted cell proliferation without affecting growth kinetics or spheroid morphology. ConclusionZA sensitivity of osteoblasts depends on concentration and experimental models. The dual dose-dependent effects of ZA on osteoblasts cultured as spheroids, thereby promoting or inhibiting cell proliferation, may provide opportunities in tissue engineering. At last, the hFOB spheroid culture system represents a valuable model for the exploration of the molecular basis of BPs action on osteoblasts and for the development and evaluation of implantable biomaterials in bone sites.

MicroRNA-31 Activates the RAS Pathway and Functions as an Oncogenic MicroRNA in Human Colorectal Cancer by Repressing RAS p21 GTPase Activating Protein 1 (RASA1)

MicroRNAs (miRNAs) are known to play a vital role in colorectal cancer. We found a widespread disruption in miRNA expression during colorectal tumorigenesis using microarray and quantitative RT-PCR analysis; of the 161 miRNAs altered in colorectal cancer compared with normal adjacent tissue samples, miR-31 was the most significantly dysregulated. We identified candidate targets of miR-31 using bioinformatics approaches and validated RAS p21 GTPase activating protein 1 (RASA1) as a direct target. First, we found an inverse correlation between miR-31 and RASA1 protein levels in vivo. Second, in vitro evidence demonstrated that RASA1 expression was significantly decreased by treatment with pre-miR-31-LV, whereas anti-miR-31-LV treatment increased RASA1 protein levels. Third, a luciferase reporter assay confirmed that miR-31 directly recognizes a specific location within the 3'-untranslated region of RASA1 transcripts. Furthermore, the biological consequences of miR-31 targeting RASA1 were examined by the cell proliferation assay in vitro and by the immunodeficient mouse xenograft tumor model in vivo. Taken together, our results demonstrate for the first time that miR-31 plays a significant role in activating the RAS signaling pathway through the inhibition of RASA1 translation, thereby improving colorectal cancer cell growth and stimulating tumorigenesis.

Iron Deprivation Impairs Proliferation of CALM-AF10 leukemia Cells in Vitro and in Vivo

Abstract 1386 Background:The CALM-AF10 fusion protein arises from the t(10;11) chromosomal translocation and is found in 8–10% of T-cell acute lymphoblastic leukemias and a smaller percentage of acute myeloid leukemias; these hematopoietic malignancies are associated with a poor clinical outcome. The precise contributions of the Clathrin Assembly Lymphoid Myeloid leukemia (CALM) protein to leukemogenesis remain uncertain. CALM plays a role in clathrin-dependent endocytosis, which mediates the entry of growth factor receptors and nutrients into cells and is essential for the internalization of iron-bound transferrin. We have previously shown that Calm-deficient (Calm−/−) mouse fibroblasts derived from fit1 mice are iron deficient, and are more sensitive to treatment with iron chelators. Objective:Since CALM-AF10 leukemia cells are haploinsufficient for CALM, we hypothesize that reduced levels of CALM in CALM-AF10 leukemia cells cause a relative iron deficiency, and result in enhanced sensitivity to iron deprivation. Design/Methods:Fibroblasts and fetal liver hematopoietic progenitors (HP) were derived from Calm−/−, Calm+/− or Calm+/+ E14 embryos. The proliferation rates of non-immortalized fibroblasts were compared in the presence and absence of supplemental iron (ferric ammonium citrate (FAC)) or treatment with an iron chelator (deferoxamine (DFO)). Surface transferrin receptor expression was quantified by flow cytometry. Primary HP cells were cultured in the presence or absence of DFO and viable cell numbers were determined. To examine the anti-leukemic effect of iron deprivation in vivo, C57BL/6J-Tyrc-2J mice were fed a low-iron diet, transplanted with CALM-AF10-transduced Calm+/− leukemia cells and leukemia latency was determined. Results:Heterozygous Calm+/− fibroblasts exhibit CALM protein levels that are intermediate between their wildtype (Calm+/+) and deficient (Calm−/−) counterparts. Calm+/− cells display a slower rate of proliferation in vitro (50% reduction of viable cells) compared to their wildtype fibroblasts, and this growth deficiency can be corrected by iron supplementation with 50 mM FAC. The presence of reduced intracellular iron levels in Calm+/− fibroblasts was manifested by increased transferrin receptor expression relative to wildtype cells. In vitro, Calm+/− HP cells showed greater sensitivity to iron chelation by DFO (5 mM) than Calm+/+ controls: the relative number of viable cells in the presence of DFO was 20% lower in Calm+/− compared to Calm+/− HP cells. To assess the impact of iron deprivation in vivo, bone marrow cells from Calm+/−CALM-AF10 leukemic mice were injected into secondary recipient mice preconditioned by having received a low-iron diet for 8 weeks (n=9). The onset of leukemia in transplanted mice maintained on the iron-deficient diet was considerably delayed (median survival 93 days versus 63 days, p=0.013) relative to age- and gender-matched control mice fed a normal diet (n=9). Conclusions:We have shown that reduced CALM expression impairs iron import and consequently limits the rate of cell proliferation. Both in vitro and in vivo results suggest that CALM haploinsufficient CALM-AF10 leukemias are particularly sensitive to iron deprivation. This raises the possibility that iron chelation may be a previously unappreciated treatment option for patients with aggressive CALM-rearranged leukemias. We are currently studying the impact of iron chelation in murine CALM-AF10 in vivo leukemia models. Disclosures:No relevant conflicts of interest to declare.

Abstract A56: Canine Mast Cell Tumor and Toceranib Phosphate (Palladia™): Development of a Spontaneous Canine Model of Kinase Inhibitor Resistance

Abstract Mast cell tumors (MCTs) are one of the most common skin tumors in dogs, accounting for up to 21% of all canine cutaneous tumors, and exhibit extremely variable biologic behavior. Mutations in the juxtamembrane, kinase and ligand binding domains of the c-kit proto-oncogene have been associated with the tumorigenesis of canine MCTs, resulting in growth factor-independent and constitutive phosphorylation of the Kit receptor tyrosine kinase (RTK). Approximately one-third of canine MCTs carry a c-kit mutation. As such, small molecule inhibitors of Kit are an attractive therapeutic strategy for MCTs in dogs. Toceranib (TOC) phosphate (Palladia) is one such RTK inhibitor of Kit that has biological activity against canine MCTs. Approximately 40% of dogs experience an objective response to TOC while the remaining 60% have intrinsic resistance. Two-thirds of the responders are positive for an activating mutation in c-kit. The time to tumor progression in all responders is 18 weeks; therefore, essentially all dogs with MCTs either have intrinsic TOC resistance or eventually develop resistance. Therefore, there is a need to identify distinctive clinical and molecular features of resistance in this population. The canine C2 mastocytoma cell line, established from a spontaneously occurring MCT, contains the previously described c-kit internal tandem duplication in the juxtamembrane domain involving exon 11. TOC-resistant C2 cells were selected by growing cells in concentrations of TOC ranging from 0.02 uM to 0.3 uM and increasing in 0.025–0.05 uM increments. Three resistant sublines were established over a period of 7 months. All three resistant sublines as well as the original naïve C2 cells were grown in the presence of increasing concentrations of TOC, 3 other KIT kinase inhibitors, or the cytotoxic agents vinblastine (VBL) or lomustine (CCNU) for 72 hours, followed by determination of relative viable cell number using a fluorometric bioreductive assay (Alamar Blue). To evaluate drug effects on Kit autophosphorylation, parental C2 cells and the resistant sublines were incubated for 24 hours with increasing concentrations of TOC (0 nM–100 nM) and phosphorylated and total Kit were analyzed by western blot. Western analysis of the C2 line demonstrated constitutive tyrosine phosphorylation of Kit in the absence of exogenous ligand. TOC inhibited Kit phosphorylation in a dose-dependent manner. Proliferation of the naïve C2 cells was inhibited by TOC in a dose-dependent manner with an IC50 of <10 nM. In contrast, all three resistant sublines were resistant to inhibition by TOC (IC50 > 1,000 nM). Interestingly, sensitivity to the other 3 Kit RTK inhibitors was variable between the sublines, and all 3 sublines retained sensitivity to the cytotoxic agents VBL and CCNU. To determine whether this lack of growth inhibition was due to a lack of inhibition of autophosphorylation by TOC, the resistant sublines were incubated with increasing concentrations of TOC for 24 hours and western analysis for phophorylated and total Kit was performed. Phosphorylation of the Kit receptor was less inhibited in all three resistant sublines compared to the TOC-sensitive C2 cells, suggesting that acquisition of secondary mutations in the c-kit gene is likely responsible for the observed TOC resistance. Canine MCTs and their intrinsic or acquired resistance to the TKI TOC (Palladia) demonstrate an excellent in vivo model of a spontaneous, Kit driven neoplastic disease. This study demonstrates the development of a canine model to study drug-resistant tumor clones with a c-kit activating mutation. This model may be better utilized to study the molecular basis of and strategies to overcome drug resistance.

Cold preservation of rat osteochondral tissues in two types of solid organ preservation solution, culture medium and saline

We compared Dulbecco's modified Eagle's medium (DMEM), saline, Euro-Collins (EC) solution and University of Wisconsin (UW) solution to determine which was best for cold preservation of rat osteochondral tissues (OCTs). After 7 days' cold preservation, OCTs kept in UW solution had the highest relative viable cell number by the tetrazolium assay and the lowest activity of lactate dehydrogenase released from damaged cells. Histological evaluation revealed chondrocyte deformity, such as shrunken cytoplasm and pyknotic nuclei, particularly in the deeper layer of articular cartilage after preservation in saline and EC solution and predominantly in all layers if preserved in DMEM. In contrast, chondrocyte morphology in all layers of the articular cartilage preserved in UW solution was relatively unchanged and remained similar to fresh OCTs. It is therefore concluded that UW solution is the most suitable for cold preservation of rat OCTs as well as solid organs.

Poster 031: Evaluation of the Type of Cell Death Induced by Mitomycin C in Human Oral Squamous Cell Carcinoma Cell Lines

Poster 031: Evaluation of the Type of Cell Death Induced by Mitomycin C in Human Oral Squamous Cell Carcinoma Cell Lines

Rapid evaluation of the efficacy of microbial cell removal from fabrics

The efficacy of microbial cell removal (EMR) from fabrics is a practically important indicator for the evaluation of cleansers and detergents. EMR is expressed quantitatively by the relative number of viable cells remaining on a fabric swatch after the treatment with these reagents. In order to count the viable cells on the swatch directly and rapidly, we have developed a unique microscopic imaging system with an ultra-deep focusing range. Standard swatches of cotton fabric were inoculated with microorganisms such as Pseudomonas fluorescence, Staphylococcus aureus, or Candida albicans. After the incubation on an agar medium, each swatch was treated with a fluorescent glucose, 2-[N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl) amino]-2-deoxyglucose, to stain only viable cells. The images of every cell distributed within the surface layer with no greater than 130 microm thickness could be integrated into one image. Thus visualized cells could be counted automatically by a novel imaging program. Using a pair of cotton swatches (0.5 x 1.0 cm(2)) inoculated with C. albicans, EMR was evaluated quantitatively. Before washing, the total number of viable cells found on the observation area (3.8 x 10(-4 )cm(2)) was 288 cells. After washing with a test detergent, no cell (<1) was detected. For this case, EMR was given by the formula: log(288/<1)=greater than 2.5. The imaging and cell count of a test fabric could be performed within 1 h.

A non-radioactive complement-dependent cytotoxicity assay for anti-CD20 monoclonal antibody

A simple and non-radioactive complement-dependent cytotoxicity assay was developed to determine the relative potency of an anti-CD20 mAb, IDEC-C2B8. The assay measures the relative number of viable cells based on the uptake and metabolism of the redox dye, Alamar blue. A linear relationship between the relative fluorescence unit generated and the number of viable cells was demonstrated. The assay is simple, has high throughput (performed in 96-well microtiter plates), and shows reproducible dose–response curves in the concentration range of 0.02–3.3 μg/ml. With intra-assay variability of 5–12%, interassay variability of 6–10% and spike recoveries of 101–109%, the assay has high precision and accuracy. Specificity was demonstrated by the lack of activity of immunoglobulins that do not bind CD20, or anti-CD20 antibody isotype (gamma 4) which does not bind complement. The assay is able to detect degradative changes in the molecule caused by heat, light and proteolytic treatments, suggesting its use as a stability-indicating method. Finally, the Alamar blue method compared favorably with other more conventional methods used to assess cell viability. The assay has the desired properties for use as a potency assay for quality control testing of anti-CD20 mAb.

A rapid fluorometric assay to measure neuronal survival in vitro

We report the development and characterization of a rapid fluorometric microassay suitable for quantifying neuronal cell survival. The method can be used in two formats: (1) a time course analysis of survival response or (2) as a simple endpoint assay for the assessment of neuronal survival promoted by a variety of reagents. The assay uses calcein AM, a non-fluorescent, electrically neutral, non-polar analogue of fluorescein diacetate, which passively crosses cell membranes and is cleaved to a fluorescent derivative by non-specific intracellular esterases. Once cleaved in viable cells, the resultant fluorescent salts are retained by intact cell membranes. The relative number of viable cells under various conditions can be quantified by measuring the emitted fluorescence. Described herein are the conditions that allow the determination of low viable neuronal cell numbers (10 2-10 3 cells/cm 2).