Analysis Of DNA Content Research Articles

Correlation of the HPV detection, protein expression and DNA content in cutaneous pre-invasive and invasive carcinoma among Croatian patients

Development of cutaneous carcinomas has been associated with HPV infection. There have been various reports on p16, p53 and pRb expression in cutaneous carcinomas and on its linkage to HPV status. Association of protein expression and HPV infection with DNA content is not clear. The aim of this study was to determine a possible correlation between HPV type, protein expression and DNA content in both pre-invasive and invasive squamous cell carcinoma, as well as differences between studied groups in these parameters. Sections of formalin fixed paraffin-embedded tumor tissue from 54 cases of Morbus Bowen (preinvasive cutaneous carcinoma) and 41 cases of invasive squamous cell carcinoma of the skin were subjected to HPV genotyping using Lipa (Line imuno probe assay), immunohistochemical staining for p16(INK4A), p53, pRb and prepared for flow cytometry DNA content analysis. Obtained data were analyzed in SPSS using Chi square test. Only p16 expression showed statistically significant differences in studied groups. Statistically significant correlations were found only in MB between parameters HPV-p53, p53-pRb and p53-p16. Our results suggest different virus-induced pathobiology pathways for different cutaneous carcinoma groups.

Pancreatic Cancer is Associated with Peripheral Leukocyte Oxidative DNA Damage

Background:DNA damage accumulation has been linked to the cancer phenotype. The purpose of this study was to compare the levels of DNA base 8-hydroxy-2’-deoxyguanosine (8-OHdG) and C-reactive protein (CRP) inflammatory markers in healthy controls and pancreatic cancer patients from a hospital-based case-control study.Materials and Methods:Fifty-five pancreatic cancer patients and 55 healthy controls were enrolled from a pool of patients referred to the Endoscopic Ultrasound (EUS) center. Analysis of DNA content of peripheral blood cells was conducted for 8-OHdG with the 32P-postlabelling assay. Serum CRP levels were measured by high-sensitivity assays and demographic data for comparison were collected from individual medical records.Results:The group of cases showed significant increased median (IQR) 8-OHdG DNA adducts/106 nucleotides and CRP compared to the controls (208.8 (138.0-340.8) vs 121.8 (57.7-194.8) RAL value; P<0.001) and (3.5 (1.5-8.6) vs 0.5 (0.2-1.5) mg/L P<0.001). A number of conditional regression models confirmed associations of pancreatic cancer with oxidative DNA damage in peripheral leukocytes.Conclusions:Our findings suggest the importance of leukocyte 8-OHdG adducts as an indicator for systemic oxidative DNA damage in pancreatic cancer patients. In addition to increase in the CRP inflammatory marker, this supports the impact of inflammation in the occurrence of pancreatic cancer as well as inflammatory responses during cancer development.

High-resolution DNA content analysis of microbiopsy samples in oral lichen planus.

DNA aneuploidy has been reported to be a predictor of poor prognosis in both premalignant and malignant lesions. In oral lichen planus (OLP), this hypothesis remains to be proved. This study aimed to determine the rate of occurrence of DNA aneuploidy in patients with OLP by high-resolution DNA flow cytometry. Patients with OLP were consecutively enrolled. Tissue samples were subdivided for formalin fixation and routine histological assessment and for immediate storage at -20°C for later DNA ploidy analysis, which was performed by DAPI staining of the extracted nuclei and excitation with a UV lamp. The DNA aneuploid sublines were characterized by the DNA Index. A DNA aneuploid status was observed in two of 77 patients with OLP (2.6%). When considering the clinical aspect of the OLP lesions, both DNA aneuploid cases had a reticular clinical aspect. DNA aneuploidy is an uncommon event in OLP and less frequent compared to other non-dysplastic and non-OLP oral potentially malignant disorders. The extremely low rate of DNA aneuploidy could represent an occasional finding or reflect the low rate of malignant transformation observed in patients with OLP even if the real prognostic value of DNA ploidy analysis in patients with OLP remains to be confirmed.

The In Vitro Effect of Acidic-Pepsin on Nuclear Factor KappaB Activation and Its Related Oncogenic Effect on Normal Human Hypopharyngeal Cells.

BackgroundExtra-esophageal carcinogenesis has been widely discussed in relation to the chronic effects of laryngopharyngeal reflux and most prominently with pepsin historically central to this discussion. With refluxate known to include gastric (pepsin) and duodenal (bile) fluids, we recently demonstrated the mechanistic role of NF-κB in mediating the preneoplastic effects of acidic-bile. However, the role of pepsin in promoting hypopharyngeal premalignant events remains historically unclear. Here, we investigate the in vitro effect of acidic-pepsin on the NF-κB oncogenic pathway to better define its potential role in hypopharyngeal neoplasia.MethodsHuman hypopharyngeal primary cells (HHPC) and keratinocytes (HHK) were repetitively exposed to physiologic pepsin concentrations (0.1 mg/ml) at pH 4.0, 5.0 and 7.0. Cellular localization of phospho-NF-κB and bcl-2 was determined using immunofluorescence and western blotting. NF-κB transcriptional activity was tested by luc reporter and qPCR. Analysis of DNA content of pepsin treated HHK and HHPC was performed using Fluorescence-activated-cell sorting assay. To explore a possible dose related effect, pepsin concentration was reduced from 0.1 to 0.05 and 0.01 mg/ml.ResultsAt physiologic concentration, acidic-pepsin (0.1 mg/ml at pH 4.0) is lethal to most normal hypopharyngeal cells. However, in surviving cells, no NF-κB transcriptional activity is noted. Acidic-pepsin fails to activate the NF-κB or bcl-2, TNF-α, EGFR, STAT3, and wnt5α but increases the Tp53 mRNAs, in both HHPC and HHK. Weakly acidic-pepsin (pH 5.0) and neutral-pepsin (pH 7.0) induce mild activation of NF-κB with increase in TNF-α mRNAs, without oncogenic transcriptional activity. Lower concentrations of pepsin at varying pH do not produce NF-κB activity or transcriptional activation of the analyzed genes.ConclusionOur findings in vitro do not support the role of acidic-pepsin in NF-κB related hypopharyngeal carcinogenesis.

Synthesis and Antiproliferative Activity of New pyrazolo[3,4-c]pyridines

Several pyrazolopyridines possess promising pharmacological activities, mainly attributed to their antagonistic nature towards the natural purines in many biological processes. Cytotoxicity and anticancer potential of this class of compounds is mainly related to induction of apoptotic cell death and inhibition of protein kinases. This prompted us to design, synthesize and study the antiproliferative activity of a number of new 3,7-disubstituted pyrazolo[3,4-c] pyridines. 2-amino-3-nitro-4-picoline was suitably modified and ring closed to provide 7-chloropyrazolo[3,4-c]pyridine. Iodination of this derivative was followed by the insertion of 3-aryl substituents via Suzuki coupling and aromatic nucleophilic substitution of the 7-chloro group by the appropriate amines. The antiproliferative activity of the target compounds was evaluated against A2058 melanoma, DU145 and PC3 prostate cancer cell lines. Flow cytometric analysis of DNA content for selected compounds was performed, after incubation of exponentially growing PC-3 cells. Eighteen new pyrazolopyridines have been synthesized and fully characterized. Among them, the majority of the 3-(3-fluorophenyl) derivatives exhibit interesting antiproliferative activity, with IC50 values in the range of 3.0-16.0 μM and present reasonable SARs. Cell-cycle perturbations revealed that the most active derivative 12a blocks cells at the S phase. 3-(3-Fluorophenyl)-7-(4-methylpiperazin-1-yl)-1H-pyrazolo[3,4-c]pyridine (12a) and the corresponding 1-(4-methoxybenzyl)- analogue (11a) possess interesting antiproliferative activity against all cell lines tested. Derivatives bearing this substitution pattern can be considered as useful leads for further biological evaluation.

Pilot-Scale Production of Megakaryocytes/Platelets from Cord Blood CD34+ Cells in a Bottle Turning Device Culture System

Due to a donor-platelet shortage, ex vivo generated megakaryocytes/platelets have emerged as an effective substitute to alleviate thrombocytopenia. We have previously reported a "two-stage culture system" for producing megakaryocytes/platelets from CD34+ cellsand demonstrated their safety and efficacy in myeloablative murine and non-human primate models. Here, we present pilot-scale production of megakaryocytes/platelets in a bottle turning device culture system that significantly increases the yield of functional megakaryocytes/platelets.Enrichedcord blood CD34+ cells were first cultured in modified IMDM basal medium (serum-free) with the addition of stem cell factor (SCF), Flt-3 ligand (FL), thrombopoietin (TPO), and interleukin (IL)-3 in a T-25 flask. After 6 days of expansion, cells were transferred into a 2 L culture bottle with 600 ml medium in a bottle turning device at the density of 1.5×105/mL for additional 7 days. The culture medium was modified IMDM supplemented with SCF, TPO, IL-3, IL-6, and granulocyte-macrophage colony-stimulating factor (GM-CSF). At various time points, cell numbers were counted with a hemocytometer and expression of CD34, CD41a, and CD42b surface markers was monitored by flow cytometry. Resulting megakaryocytes were further confirmed by morphological examination and DNA ploidy analysis. Expression of relevant oncogenes was measured by quantitative real-time polymerase chain reaction (qPCR) for evaluating potential tumorigenicity. Moreover, these megakaryocytes were transplanted into sub-lethally irradiated NOD/SCID mice (1×106 CD41a+/CD42b+ megakaryocytes per mouse) to assess human platelet-releasing potential and function in vivo.The initial cell number was 1×106 with CD34 purity of 93.4% ±2.8%. For 6-day culture, the absolute number of total cells and CD34+ cells reached 9.3×107±1.8×107 and 4.0×107±5.6×106, respectively, which were calculated into a 92.9±18.1 and 42.8±5.2-fold increase, respectively. For additional 7-day culture in the turning bottles, 2.2×1010±3.2×109 cells were obtained with the proportions of CD41a+ and CD42b+ cells at 80.1%±7.4% and 63.6%±5.9%, respectively. Approximately 2.0×104 megakaryocytes/platelets were generated from each input CD34+ cell. These megakaryocytes were morphologically discernible as they were much larger than starting CD34+ cells with apparent lobular nuclei and numerous α-granules. DNA content analysis revealed that about 25.4%±1.3% of megakaryocytes exhibited a polyploidy level of >8N. Moreover, about 10% megakaryocytes have formed pro-platelet fragments and released platelets in cultures. Compared to initial CD34+ cells, generated megakaryocytes showed similar levels of expression of proto-oncogenes including c-Myb, N-Ras, and K-Ras whereas c-Myc, bmi-1, cyclin B, and hTERT expression was decreased, strongly suggesting that ex vivogenerated megakaryocytes are unlikely tumorigenic. Releasing of human platelets by injected megakaryocytes were detected in mouse peripheral blood with a percentage of 0.5%±0.1% as early as 30 min following transplantation, after which the percentage of human platelets increased rapidly with a peak level of 3.2%±0.6% at about 4-6 h post-transplantation. The half-life of released platelets in mice was about 10 h and about 23±6 human platelets were released per megakaryocyte in the transplanted mice. Furthermore, these platelets were activated, expressing CD62P after stimulation by ADP, suggesting that released human platelets in mice are functional.Combined, we have established a bottle turning device culture system for pilot-scale production of megakaryocytes/platelets ex vivo. One unit of cord blood (80 ml) with 2×106 CD34+ cells can generate 4×1010 megakaryocytes/platelets that are sufficient for treating 130 patients (with an average weight of 60 kg and infusion cell number 5×106cells/kg). In conclusion, our new culture system is capable of manufacturing GMP-grade megakaryocytes/platelets sufficient for various clinical applications. DisclosuresRen:Biopharmagen Corp: Employment. Jiang:Biopharmagen Corp: Consultancy.

Discovery of BPR1K871, a quinazoline based, multi-kinase inhibitor for the treatment of AML and solid tumors: Rational design, synthesis, in vitro and in vivo evaluation.

The design and synthesis of a quinazoline-based, multi-kinase inhibitor for the treatment of acute myeloid leukemia (AML) and other malignancies is reported. Based on the previously reported furanopyrimidine 3, quinazoline core containing lead 4 was synthesized and found to impart dual FLT3/AURKA inhibition (IC50 = 127/5 nM), as well as improved physicochemical properties. A detailed structure-activity relationship study of the lead 4 allowed FLT3 and AURKA inhibition to be finely tuned, resulting in AURKA selective (5 and 7; 100-fold selective over FLT3), FLT3 selective (13; 30-fold selective over AURKA) and dual FLT3/AURKA selective (BPR1K871; IC50 = 19/22 nM) agents. BPR1K871 showed potent anti-proliferative activities in MOLM-13 and MV4-11 AML cells (EC50 ∼ 5 nM). Moreover, kinase profiling and cell-line profiling revealed BPR1K871 to be a potential multi-kinase inhibitor. Functional studies using western blot and DNA content analysis in MV4-11 and HCT-116 cell lines revealed FLT3 and AURKA/B target modulation inside the cells. In vivo efficacy in AML xenograft models (MOLM-13 and MV4-11), as well as in solid tumor models (COLO205 and Mia-PaCa2), led to the selection of BPR1K871 as a preclinical development candidate for anti-cancer therapy. Further detailed studies could help to investigate the full potential of BPR1K871 as a multi-kinase inhibitor.

Analysis of relative nuclear DNA content in carnation (Dianthus caryophyllus) accessions reveals ploidy levels by flow cytometry

Analysis of relative nuclear DNA content in carnation (Dianthus caryophyllus) accessions reveals ploidy levels by flow cytometry

Flow Cytometry Analysis of Cell Cycle and Specific Cell Synchronization with Butyrate.

Synchronized cells have been invaluable in many kinds of cell cycle and cell proliferation studies. Butyrate induces cell cycle arrest and apoptosis in MDBK cells. We explore the possibility of using butyrate-blocked cells to obtain synchronized cells and we characterize the properties of butyrate-induced cell cycle arrest. The site of growth inhibition and cell cycle arrest was analyzed using 5-bromo-2'-deoxyuridine (BrdU) incorporation and flow cytometry analyses. Exposure of MDBK cells to 10 mM butyrate caused growth inhibition and cell cycle arrest in a reversible manner. Butyrate affected the cell cycle at a specific point both immediately after mitosis and at a very early stage of the G1 phase. After release from butyrate arrest, MDBK cells underwent synchronous cycles of DNA synthesis and transited through the S phase. It takes at least 8 h for butyrate-induced G1-synchronized cells to begin the progression into the S phase. One cycle of cell division for MDBK cells is about 20 h. By combining BrdU incorporation and DNA content analysis, not only can the overlapping of different cell populations be eliminated, but the frequency and nature of individual cells that have synthesized DNA can be determined.

УРОВНИ ПЛОИДНОСТИ И ОТНОСИТЕЛЬНОГО СОДЕРЖАНИЯ ДНК В КУЛЬТУРЕ КЛЕТОК И ТКАНЕЙ РАСТЕНИЙ IN VITRO

&lt;p&gt;We presented results of variations in the ploidy level and the genome size of the &lt;em&gt;R. acetosa&lt;/em&gt; regenerants. These regenerants was obtained by indirect and direct morphogenesis in in vitro culture. Explants were prepared from seedlings on the three-leaf stage of plant development. More than 100 explants were used to stimulate the indirect and direct morphogenesis. Mesophilic explants were cultured on the MS nutrient medium containing auxin to callus proliferation (2 mg/L NAA, 1 mg/L BA). Cultivation of the callus was maintained for 4 weeks followed by an indirect morphogenes. Indirect morphogenesis stimulated on the MS medium with cytokinin and gibberellic acid predominance (0.5 mg/L BA, 0.2 mg/L GA3). Direct stimulate morphogenesis from the apical meristem of seedlings on nutrient media with a predominance of cytokinins (1 mg/L BA, 0.25 mg/L NAA). Rhizogenesis have stimulated by transferring of the regenerants to the ½MS medium supplemented with 0.2 mg/L of NAA. Research of a ploidy level and genome size was performed by flow cytometry used propidium iodide staining with &lt;em&gt;Vicia faba&lt;/em&gt; cv “Innovec” (2C=26.90 pg) as internal DNA standard. We calculated the relative DNA content (2C) for &lt;em&gt;R. acetosa&lt;/em&gt; equal to 6,98 pg. Cytogenetical analisis showed that the maximum genome size variation recorded for regenerants obtained through the indirect morphogenesis. Variations in the genome size of the regenerants obtained by direct morphogenesis deviates from the control group to 0.30 pg (2С=7.28 pg) and after indirect morphogenesis to 1.04 pg (2С=8.2 pg). Cytogenetical analysis of the regenerated plants showed the presence of different somatic chromosome numbers ranging from 2n = 14 to 2n = 28. The relative DNA content of tetraploid forms was 11.87 pg. In our study was shown, that the most effective method of plant conservation in the &lt;em&gt;in vitro&lt;/em&gt; culture is a direct morphogenesis. Analysis of the relative nuclear DNA content and chromosome counts of regenerants obtained by indirect morphogenesis from the callus cultures showed significant variations in the DNA content, as well as the appearance of polyploid forms. Therefore, long-term cultivation of callus cultures increases the probability of genomic aberrations, which reduces the stability of the plant genome.&lt;/p&gt;

Hedgehog Controls Quiescence and Activation of Neural Stem Cells in the Adult Ventricular-Subventricular Zone.

SummaryIdentifying the mechanisms controlling quiescence and activation of neural stem cells (NSCs) is crucial for understanding brain repair. Here, we demonstrate that Hedgehog (Hh) signaling actively regulates different pools of quiescent and proliferative NSCs in the adult ventricular-subventricular zone (V-SVZ), one of the main brain neurogenic niches. Specific deletion of the Hh receptor Patched in NSCs during adulthood upregulated Hh signaling in quiescent NSCs, progressively leading to a large accumulation of these cells in the V-SVZ. The pool of non-neurogenic astrocytes was not modified, whereas the activated NSC pool increased after a short period, before progressively becoming exhausted. We also showed that Sonic Hedgehog regulates proliferation of activated NSCs in vivo and shortens both their G1 and S-G2/M phases in culture. These data demonstrate that Hh orchestrates the balance between quiescent and activated NSCs, with important implications for understanding adult neurogenesis under normal homeostatic conditions or during injury.

The Effectiveness of Natural Diarylheptanoids against Trypanosoma cruzi: Cytotoxicity, Ultrastructural Alterations and Molecular Modeling Studies.

Curcumin (CUR) is the major constituent of the rhizomes of Curcuma longa and has been widely investigated for its chemotherapeutic properties. The well-known activity of CUR against Leishmania sp., Trypanosoma brucei and Plasmodium falciparum led us to investigate its activity against Trypanosoma cruzi. In this work, we tested the cytotoxic effects of CUR and other natural curcuminoids on different forms of T. cruzi, as well as the ultrastructural changes induced in epimastigote form of the parasite. CUR was verified as the curcuminoid with more significant trypanocidal properties (IC50 10.13 μM on epimastigotes). Demethoxycurcumin (DMC) was equipotent to CUR (IC50 11.07 μM), but bisdemethoxycurcumin (BDMC) was less active (IC50 45.33 μM) and cyclocurcumin (CC) was inactive. In the experiment with infected murine peritoneal macrophages all diarylheptanoids were more active than the control in the inhibition of the trypomastigotes release. The electron microscopy images showed ultrastructural changes associated with the cytoskeleton of the parasite, indicating tubulin as possible target of CUR in T. cruzi. The results obtained by flow cytometry analysis of DNA content of the parasites treated with natural curcuminoids suggested a mechanism of action on microtubules related to the paclitaxel`s mode of action. To better understand the mechanism of action highlighted by electron microscopy and flow cytometry experiments we performed the molecular docking of natural curcuminoids on tubulin of T. cruzi in a homology model and the results obtained showed that the observed interactions are in accordance with the IC50 values found, since there CUR and DMC perform similar interactions at the binding site on tubulin while BDMC do not realize a hydrogen bond with Lys163 residue due to the absence of methoxyl groups. These results indicate that trypanocidal properties of CUR may be related to the cytoskeletal alterations.

Ribosomal DNA ITS1, 5.8S and ITS2 secondary structure, nuclear DNA content and phytochemical analyses reveal distinctive characteristics of four subclades of Protasparagus

AbstractThe use of ribosomal DNA (rDNA) internal transcribed spacer (ITS) primary sequence based phylogeny is a conventional practice to estimate the evolutionary interspecies relationship. However, analysis of the functional folding patterns and higher order secondary structures of ITS regions can provide additional important information regarding species relatedness and interspecies variations. In the present study, we provide the first detailed information on the rDNA ITS secondary structure diversity in the four subclades of the subgenus Protasparagus. Several angiospermic conserved motifs were identified in each of the ITS1, 5.8S and ITS2 secondary structures of the studied taxon. Topological comparison of the ITS1 secondary structures showed variations in the helix‐ IV regions. Moreover, presence of unique sequence motifs and differences in the internal loop structures were found to be subclade specific. The present study suggests that comprehensive analysis of the ITS1, 5.8S and ITS2 structural elements including helices, loops and bulges can be used as an important tool for species delimitation. The present study investigated the evolution of the secondary structure of ITS marker (its phylogenetic utility), genome size, base chromosome number and phytochemicals, and identified a putative polyploid event shared by a number of Protasparagus species. The phytochemical analysis of two important active compounds, i.e., shatavarin‐IV and sarsasapogenin, also reveals their presence in all the studied taxa constitutively even at the subclade level.

In Vitro and in Vivo Densitometric Analysis of DNA Content and Chromatin Texture in Nuclei of Tumor Cells under the Influence of a Nano composite and Magnetic Field

DNA content and chromatin texture of interphase nuclei in MCF-7 and Guerin carcinoma cells that are sensitive or resistant to chemotherapeutic agents i n the setting of the treatment with cisplatin, nanocomposite and static magnetic field have been a nalyzed. The results obtained indicate that chemotherapy agents and physical factors both in vi tro and in vivo cause the increase in the DNA conte nt. Tumor exposure to chemotherapy agents, nanocomposite and magnetic field causes the increase in the percentage of heterochromatin in nuclei of resistan t tumor cells compared with sensitive ones, especia lly under the influence of combination of nanocomposite with static magnetic field. Probably it suggest th e specific effects of combination of nanocomposite wi th magnetic field on cell chromatin depending on th e changes in the strength of the DNA-histone interact ions.

English

Banana Germplasm Collection serves as a source of useful genes for banana breeding. However, insufficient and/or inaccurate information on the ploidy level of the germplasm renders its utilization in breeding difficult. The objective of this study was to determine and validate the ploidy level of 120 banana accessions in the ex situ germplasm collection centre for the East and Central Africa, located in Mbarara, Uganda. Flow cytometric analysis of the nuclear DNA content was used to determine the ploidy level of the accessions. Results indicate that accessions: Bura, Diana, Kambani-Rungwe, Paji and Pagatau, and Rungwe that were previously classified as diploids are actually triploids, whereas Selangor previously known to be a diploid is a tetraploid. Accessions such as Galeo, Mwitupemba and Ntindi 1 that were previously classified as triploids were found diploids. GT, FHIA 25 and Muzungu Mwekundu that were considered as tetraploids, were found triploids. The information generated will guide correct placement of these accessions in the regional germplasm collection centre for the East and Central Africa and their utilization in banana breeding. &nbsp; Key words: Banana germplasm, breeding ex situ germplasm collection, flow cytometry, ploidy.

Abstract 2063: Improved sensitivity and in vitro efficacy of RGD grafted PEGylated gemcitabine liposomes in RRM1 siRNA pretreated cancer cells

Abstract Aim of the study was to prepare and characterize RGD grafted PEGylated liposomes of gemcitabine (PLGs) and to evaluate its cellular uptake, in vitro anti-proliferative activity and apoptotic effect in siRNA pretreated lung cancer cells. PLGs were prepared by thin film hydration method and optimized for particle size, zeta potential and entrapment efficiency. Functionalization of liposomes was done by coupling reaction between DSPE mPEG and cRGD peptide by maleimide based reaction. Similarly, RGD grafted RRM1 siRNA liposomes were also formulated and evaluated. MTT assay was done to determine IC50 values of RGD grafted PLGs in A549 and H1299 cancer cells which were pre-treated with RGD grafted RRM1 siRNA loaded liposomes at a concentration of 50 pM of siRNA for gene silencing. DNA content analysis was done by flow cytometry using rhodamine in A549. The mode of cell death at different time and concentration was determined by FITC-AnnexineV assay in A549 cells and confocal microscopy was performed to assess the potential of RGD grafting on cellular uptake. RGD conjugated and unconjugated PLGs were found Nano sized and had negative zeta potential with entrapment of 65%. H1299 cell line showed more amount of viable cells after 48 hr as compared to A549 cells in RGD conjugated PLGs. siRNA pre-treated PLGs exposed cells showed significantly less IC50 values as compared to cells without siRNA pretreatment and non-grafted liposomes. The results showed that the RGD conjugated liposomes at the concentration of 7nM showed 46% G1 phase arrest in siRNA pretreated cells as compared to 22% G1 phase arrest without prior siRNA treatment at 16hr. Two types of mode of cell death were found during the FITC-Annexine V assay. At 24 hr, the treatment with RGD grafted PLGs resulted in 17% &amp; 4.4% necrotic &amp; apoptotic cell death respectively. While at equivalent drug concentration, the PLGs and drug solution showed 5.3% &amp; 32.2% and 4.2% &amp; 29.6% necrotic &amp; apoptotic cell death respectively. Furthermore, the apoptosis was found to be time and concentration dependent. Results substantiate the sensitization effect by pre-exposure of siRNA in liposomal forms at Pico molar concentration along-with phagocytosis as mechanism of uptake of RGD-grafted liposomes. To conclude, prior silencing of the resistance imparting gene can manifest the effect of therapy by conferring improved sensitivity in cancer cell lines. The effect can further be augmented by employing receptor targeting peptides such as RGD. Hence, Nano-constructs of chemotherapeutic drugs conjugated with RGD can effectively target lung cancer cells and pretreatment of RRM1 siRNA can probably reduce the limitation of drug resistance associated with lung cancer chemotherapy. Citation Format: Rohan A. Lalani, Priyanka Bhatt, Mohan Rathi, Ambikanandan Misra. Improved sensitivity and in vitro efficacy of RGD grafted PEGylated gemcitabine liposomes in RRM1 siRNA pretreated cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2063.

Abstract 1894: Understanding the role of microRNA-206 in breast cancer

Abstract MicroRNAs are short non-coding RNA sequences that that can modify genes in post transcriptional manner and are often de-regulated in cancer. Cancer stem cells, a subset of cancer cells with stem cell properties, are considered the root of tumorigenesis and seeds of metastasis. In this study we are interested in elucidating the role of miR-206 in breast cancer stem cell-mediated tumorigenesis and metastasis. We have observed that microRNA-206 (miR-206) regulate the metastatic traits of breast cancer cells, both in vitro and in vivo. Our studies have elucidated a signaling cascade that culminates in the silencing of cytokine (such as interleukin) and integrin expression. Knockdown of the interleukin and integrin genes by siRNAs mimics the phenotype of miR-206 expression in breast cancer cells. DNA content analysis using propidium iodide staining revealed that knockdown of miR-206 targets as well as over-expression of miR-206 in MDA-MB-231 cells regulates cell cycle arrest. Our results indicate that miR-206 mediates its regulatory effects through inhibiting multiple targets. MiR-206 may serve as a new therapeutic candidate for the future treatment of breast cancer. Citation Format: Valery Adorno-Cruz, Huiping Liu. Understanding the role of microRNA-206 in breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1894.

Abstract 3309: Novel small molecule ONC201 induces cell death and targets chemotherapy-resistant cancer stem-like cells in triple negative breast cancer

Abstract Breast cancer is the most commonly diagnosed cancer in US women today. Triple negative breast cancer (TNBC) accounts for 15% of all breast cancers that are diagnosed and has a poor prognosis. TNBC has a high recurrence rate following treatment with chemotherapy and radiation compared to other breast cancer subtypes. Many tumors, including those of the breast, contain a population of stem-like cells known as cancer stem cells (CSCs). The CSC population is capable of self-renewal and has been shown to be resistant to chemotherapy and radiation. These observations suggest that the CSCs may be responsible for tumor relapse after initial response to therapy. We previously identified a small molecule inducer of the TRAIL pathway, ONC201/TIC10. This compound functions through the ATF4/CHOP pathway to upregulate DR5 (Kline et. al., in press, 2015), and through dual inhibition of Akt/ERK signaling to upregulate TRAIL (Allen et. al Science Translational Medicine, 2013). ONC201 recently completed its first-in-human clinical trial in advanced solid tumors that defined its safety, pharmacokinetics and recommended phase II dose (Stein et al., Abstract C138, 2015 AACR-NCI-EORTC meeting). ONC201 is being tested in multiple phase I/II clinical trials (NCT02250781, NCT02324621, NCT02420795, NCT02392572, NCT02609230, NCT02525692, NCT02038699). We have shown that ONC201 is able to decrease formation of colonospheres as well as deplete colorectal CSC markers in vitro. We have also showed that treatment with ONC201 prevents colorectal CSCs from initiating xenograft tumor growth in mice. (Prabhu et. al Cancer Research., 2015). The goal of this work was to investigate the efficacy of ONC201 in triple negative breast cancer cells, as well as to determine if ONC201 was able to target breast CSCs. We used cell viability assays to determine that the IC50 values for 6 TNBC cell types are in the low micromolar range, doses which are achievable based on human pharmacokinetic data. Propidium iodide staining and analysis of SubG1 DNA content indicates that ONC201 induces apoptosis in TNBC cells. Levels of TRAIL receptor DR5 are increased on the cell surface in TNBC cells following ONC201 treatment. In addition we show that ONC201 is well tolerated and efficacious in vivo against the MDA-MB-231 TNBC xenograft model. We have observed that ONC201 depletes Aldefluor+ TNBC cells in vitro. ONC201 inhibits formation of mammospheres from TNBC CSC-like cells while paclitaxel, a chemotherapeutic drug used to treat TNBC, does not. Our findings suggest that ONC201 exerts cytotoxic effects against TNBC cell types and that ONC201 may target paclitaxel resistant CSCs. Our work contributes to the development of a preclinical rationale for the use of ONC201 as a novel therapy for triple negative breast cancers. Citation Format: Marie D. Baumeister, Jessica Wagner, Varun V. Prabhu, Christina LB Kline, Bora Lim, Josh E. Allen, David T. Dicker, Wafik S. El-Deiry. Novel small molecule ONC201 induces cell death and targets chemotherapy-resistant cancer stem-like cells in triple negative breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3309.

Abstract 4745: Repurposing of valproic acid and simvastatin combination as anticancer agents in prostate cancer: synergistic interaction with docetaxel and suppression of docetaxel resistance

Abstract Although docetaxel (DTX) remains a standard of care for advanced prostate cancer (PCa), limited long-term responses, side effects and resistant disease suggested the need of novel combination strategies. Increased expression of histone deacetylases (HDAC) and alteration of the mevalonate pathway (MVP) are common aberrations in PCa. In this study, we analyzed the antitumor effect of DTX in combination with valproic acid (VPA), an anticonvulsant with HDAC inhibitory activity, and simvastatin (SIM), a cholesterol-lowering drug inhibiting the rate-limiting enzyme of MVP HMG-CoA reductase, on androgen-dependent 22RV1 and LNCAP and androgen-independent PC3 and DU145 PCa cells, as well as on the highly aggressive SIM-resistant DU145R80 subline developed in our laboratory from DU145 cells (Milone MR et al. Cell Death Dis. 2013; Milone MR et al. Oncotarget 2014). We first showed a potent synergistic anti-proliferative effect of VPA/SIM combination, assessed by calculating combination index (CI) according to the method of Chou and Talalay, on all cell lines, including SIM-resistant cells, whatever schedule of administration (simultaneous vs sequential) we used, confirming our previous data on the combination between the HDAC inhibitor (HDACi) panobinostat and zoledronic acid, the latter also targeting the MVP pathway (Bruzzese F. et al, Cell Death Dis. 2013). Notably, exposure to triple combinations (VPA/SIM/DTX) resulted in a further strong synergistic anti-proliferative effect, with sequential exposure with 24h delay between VPA/SIM and DTX as the best schedule. The synergistic interaction of VPA/SIM and DTX combination involved apoptotic effect, measured by FACS analysis of sub-diploid DNA content and caspase 3/7 cleavage, and DNA damage induction, assessed by γH2AX expression. Synergistic effect of VPA/SIM and DTX combination was confirmed by soft agar clonogenic assay and by 3D culture on self-assembled PCa spheroids. Significantly, VPA/SIM combination was also able to revert DTX-resistance in DTX-resistant PC3 and DU145 sublines developed in our laboratory from the parental cells. All together these findings suggested that the combination of two safe generic drugs such as VPA and SIM can improve DTX efficacy, representing a novel therapeutic approach that warrant clinical investigation in advanced PCa patients. Our study also suggests a new strategy to overcome resistance to standard taxane-based therapy in PCa patients. Citation Format: Federica Iannelli, Rita Lombardi, Biagio Pucci, Maria Rita Milone, Chiara Ciardiello, Alessandra Leone, Elena Di Gennaro, Alfredo Budillon, Francesca Bruzzese. Repurposing of valproic acid and simvastatin combination as anticancer agents in prostate cancer: synergistic interaction with docetaxel and suppression of docetaxel resistance. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4745.

High-throughput analysis of the satellitome illuminates satellite DNA evolution.

Satellite DNA (satDNA) is a major component yet the great unknown of eukaryote genomes and clearly underrepresented in genome sequencing projects. Here we show the high-throughput analysis of satellite DNA content in the migratory locust by means of the bioinformatic analysis of Illumina reads with the RepeatExplorer and RepeatMasker programs. This unveiled 62 satDNA families and we propose the term “satellitome” for the whole collection of different satDNA families in a genome. The finding that satDNAs were present in many contigs of the migratory locust draft genome indicates that they show many genomic locations invisible by fluorescent in situ hybridization (FISH). The cytological pattern of five satellites showing common descent (belonging to the SF3 superfamily) suggests that non-clustered satDNAs can become into clustered through local amplification at any of the many genomic loci resulting from previous dissemination of short satDNA arrays. The fact that all kinds of satDNA (micro- mini- and satellites) can show the non-clustered and clustered states suggests that all these elements are mostly similar, except for repeat length. Finally, the presence of VNTRs in bacteria, showing similar properties to non-clustered satDNAs in eukaryotes, suggests that this kind of tandem repeats show common properties in all living beings.