Replication Study: Coding-independent regulation of the tumor suppressor PTEN by competing endogenous mRNAs

As part of the Reproducibility Project: Cancer Biology, we published a Registered Report (Phelps et al., 2016) that described how we intended to replicate selected experiments from the paper ‘Coding-independent regulation of the tumor suppressor PTEN by competing endogenous mRNAs’ (Tay et al., 2011). Here, we report the results. We found depletion of putative PTEN competing endogenous mRNAs (ceRNAs) in DU145 cells did not impact PTEN 3’UTR regulation using a reporter, while the original study reported decreased activity when SERINC1, VAPA, and CNOT6L were depleted (Figure 3C; Tay et al., 2011). Using the same reporter, we found decreased activity when ceRNA 3’UTRs were overexpressed, while the original study reported increased activity (Figure 3D; Tay et al., 2011). In HCT116 cells, ceRNA depletion resulted in decreased PTEN protein levels, a result similar to the findings reported in the original study (Figure 3G,H; Tay et al., 2011); however, while the original study reported an attenuated ceRNA effect in microRNA deficient (DicerEx5) HCT116 cells, we observed increased PTEN protein levels. Further, we found depletion of the ceRNAs VAPA or CNOT6L did not statistically impact DU145, wild-type HCT116, or DicerEx5 HCT116 cell proliferation. The original study reported increased DU145 and wild-type HCT116 cell proliferation when these ceRNAs were depleted, which was attenuated in the DicerEx5 HCT116 cells (Figure 5B; Tay et al., 2011). Differences between the original study and this replication attempt, such as variance between biological repeats, are factors that might have influenced the results. Finally, we report meta-analyses for each result.

Download Full-text

Replication Study: RAF inhibitors prime wild-type RAF to activate the MAPK pathway and enhance growth

10.1101/2021.11.30.470372 ◽

2021 ◽

Author(s):

Steven Pelech ◽

Curtis Gallagher ◽

Catherine Sutter ◽

Lambert Yue ◽

John Kerwin ◽

...

Keyword(s):

Cell Proliferation ◽

Cancer Biology ◽

Mapk Pathway ◽

Mek Inhibitor ◽

Original Study ◽

Braf V600e ◽

Cancer Cell Proliferation ◽

Wild Type ◽

Differential Binding ◽

Meta Analyses

As part of the Reproducibility Project: Cancer Biology, we published a Registered Report (Bhargava et al., 2016) that described how we intended to replicate selected experiments from the paper "RAF inhibitors prime wild-type RAF to activate the MAPK pathway and enhance growth" (Hatzivassiliou et al., 2010). Here we report the results. We found two unrelated RAF inhibitors, PLX4720 or GDC-0879, selectively inhibited BRAF(V600E) cell proliferation, while the MEK inhibitor, PD0325901, inhibited BRAF(V600E), wild-type RAF/RAS, and mutant RAS cancer cell proliferation, similar to the original study (Figure 1A; Hatzivassiliou et al., 2010). We found knockdown of CRAF, but not BRAF, in mutant RAS cells attenuated the phospho-MEK induction observed after PLX4720 treatment, similar to the original study (Figure 2B; Hatzivassiliou et al., 2010). The original study reported analogous results with GDC-0879, which was not observed in this replication, although unexpected control results confound the interpretation. We also attempted a replication of an assay with recombinant proteins to test the differential effect of RAF inhibitors on BRAF-CRAF heterodimerization (Figure 4A; Hatzivassiliou et al., 2010). Although we were unable to conduct the experiment as planned, we observed differential binding of BRAF by RAF inhibitors; however, it was between BRAF and beads, independent of CRAF. While these data were unable to address whether, under the conditions of the original study, the same observations could be observed, we discuss key differences between the original study and this replication that are important to consider for further experiments. Finally, where possible, we report meta-analyses for each result.

Download Full-text

Registered report: A coding-independent function of gene and pseudogene mRNAs regulates tumour biology

eLife ◽

10.7554/elife.08245 ◽

2015 ◽

Vol 4 ◽

Cited By ~ 4

Author(s):

Israr Khan ◽

John Kerwin ◽

Kate Owen ◽

Erin Griner ◽

Keyword(s):

Cell Proliferation ◽

Tumor Cell ◽

Cancer Biology ◽

Open Science ◽

Tumor Cell Proliferation ◽

Independent Function ◽

Pten Protein ◽

Protein Levels ◽

High Profile ◽

Tumour Biology

The Reproducibility Project: Cancer Biology seeks to address growing concerns about reproducibility in scientific research by conducting replications of selected experiments from a number of high-profile papers in the field of cancer biology. The papers, which were published between 2010 and 2012, were selected on the basis of citations and Altmetric scores (<xref ref-type="bibr" rid="bib9">Errington et al., 2014</xref>). This Registered report describes the proposed replication plan of key experiments from ‘A coding-independent function of gene and pseudogene mRNAs regulates tumour biology’ by <xref ref-type="bibr" rid="bib26">Poliseno et al. (2010)</xref>, published in Nature in 2010. The key experiments to be replicated are reported in Figures 1D, 2F-H, and 4A. In these experiments, Poliseno and colleagues report microRNAs miR-19b and miR-20a transcriptionally suppress both PTEN and PTENP1 in prostate cancer cells (Figure 1D; <xref ref-type="bibr" rid="bib26">Poliseno et al., 2010</xref>). Decreased expression of PTEN and/or PTENP1 resulted in downregulated PTEN protein levels (Figure 2H), downregulation of both mRNAs (Figure 2G), and increased tumor cell proliferation (Figure 2F; <xref ref-type="bibr" rid="bib26">Poliseno et al., 2010</xref>). Furthermore, overexpression of the PTEN 3′ UTR enhanced PTENP1 mRNA abundance limiting tumor cell proliferation, providing additional evidence for the co-regulation of PTEN and PTENP1 (Figure 4A; <xref ref-type="bibr" rid="bib26">Poliseno et al., 2010</xref>). The Reproducibility Project: Cancer Biology is collaboration between the Center for Open Science and Science Exchange, and the results of the replications will be published in eLife.

Download Full-text

Replication Study: The common feature of leukemia-associated IDH1 and IDH2 mutations is a neomorphic enzyme activity converting alpha-ketoglutarate to 2-hydroxyglutarate

eLife ◽

10.7554/elife.26030 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 9

Author(s):

Megan Reed Showalter ◽

Jason Hatakeyama ◽

Tomas Cajka ◽

Kacey VanderVorst ◽

Kermit L Carraway ◽

...

Keyword(s):

Enzyme Activity ◽

Vector Control ◽

Cancer Biology ◽

Original Study ◽

Idh Mutation ◽

Wild Type ◽

The Common ◽

Idh1 And Idh2 Mutations ◽

Mutant Idh1 ◽

Meta Analyses

In 2016, as part of the Reproducibility Project: Cancer Biology, we published a Registered Report (<xref ref-type="bibr" rid="bib14">Fiehn et al., 2016</xref>), that described how we intended to replicate selected experiments from the paper "The common feature of leukemia-associated IDH1 and IDH2 mutations is a neomorphic enzyme activity converting alpha-ketoglutarate to 2-hydroxyglutarate" (Ward et al., 2010). Here, we report the results of those experiments. We found that cells expressing R172K mutant IDH2 did not display isocitrate-dependent NADPH production above vector control levels, in contrast to the increased production observed with wild-type IDH2. Conversely, expression of R172K mutant IDH2 resulted in increased alpha-ketoglutarate-dependent consumption of NADPH compared to wild-type IDH2 or vector control. These results are similar to those reported in the original study (Figure 2; Ward et al., 2010). Further, expression of R172K mutant IDH2 resulted in increased 2HG levels within cells compared to the background levels observed in wild-type IDH2 and vector control, similar to the original study (Figure 3D; Ward et al., 2010). In primary human AML samples, the 2HG levels observed in samples with mutant IDH1 or IDH2 status were higher than those observed in samples without an IDH mutation, similar to what was observed in the original study (Figure 5C; Ward et al., 2010). Finally, we report meta-analyses for each result.

Download Full-text

Replication Study: Coadministration of a tumor-penetrating peptide enhances the efficacy of cancer drugs

eLife ◽

10.7554/elife.17584 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 34

Author(s):

Christine Mantis ◽

Irawati Kandela ◽

Fraser Aird ◽

Keyword(s):

Cancer Biology ◽

Xenograft Model ◽

Original Study ◽

Cancer Drug ◽

Tunel Staining ◽

Positive Staining ◽

Cancer Drugs ◽

Tumor Weight ◽

Significant Difference ◽

Meta Analyses

In 2015, as part of the Reproducibility Project: Cancer Biology, we published a Registered Report (Kandela et al., 2015) that described how we intended to replicate selected experiments from the paper “Coadministration of a tumor-penetrating peptide enhances the efficacy of cancer drugs“ (Sugahara et al., 2010). Here we report the results of those experiments. We found that coadministration with iRGD peptide did not have an impact on permeability of the chemotherapeutic agent doxorubicin (DOX) in a xenograft model of prostate cancer, whereas the original study reported that it increased the penetrance of this cancer drug (Figure 2B; Sugahara et al., 2010). Further, in mice bearing orthotopic 22Rv1 human prostate tumors, we did not find a statistically significant difference in tumor weight for mice treated with DOX and iRGD compared to DOX alone, whereas the original study reported a decrease in tumor weight when DOX was coadministered with iRGD (Figure 2C; Sugahara et al., 2010). In addition, we did not find a statistically significant difference in TUNEL staining in tumor tissue between mice treated with DOX and iRGD compared to DOX alone, while the original study reported an increase in TUNEL positive staining with iRGD coadministration (Figure 2D; Sugahara et al., 2010). Similar to the original study (Supplemental Figure 9A; Sugahara et al., 2010), we did not observe an impact on mouse body weight with DOX and iRGD treatment. Finally, we report meta-analyses for each result.

Download Full-text

Anticancer Effects of the Marine Sponge Lipastrotethya sp. Extract on Wild-Type and p53 Knockout HCT116 Cells

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2017/7174858 ◽

2017 ◽

Vol 2017 ◽

pp. 1-6 ◽

Cited By ~ 2

Author(s):

Kiheon Choi ◽

Hyun Kyung Lim ◽

Sung Ryong Oh ◽

Woo-Hyun Chung ◽

Joohee Jung

Keyword(s):

Target Genes ◽

Apoptotic Cell ◽

Marine Sponges ◽

Wild Type ◽

Anticancer Effect ◽

Protein Levels ◽

Ocean Science ◽

Autophagy Induction ◽

Wide Range ◽

Hct116 Cells

Interest in marine bioresources is increasing in the drug development sector. In particular, marine sponges produce a wide range of unique metabolites that enable them to survive in challenging environments, which makes them attractive sources of candidate pharmaceuticals. In previous study, we investigated over 40 marine specimens collected in Micronesia and provided by the Korean Institute of Ocean Science and Technology, for their antiproliferative effects on various cancer cell lines, and Lipastrotethya sp. extract (LSSE) was found to have a marked antiproliferative effect. In the present study, we investigated the mechanism responsible for its anticancer effect on wild-type p53 (WT) or p53 knockout (KO) HCT116 cells. LSSE inhibited cell viability and induced apoptotic cell death more so in HCT116 p53 KO cells than the WT. HCT116 WT cells treated with LSSE underwent apoptosis associated with the induction of p53 and its target genes. On the other hand, in HCT116 p53 KO cells, LSSE reduced mTOR and Bcl-2 and increased Beclin-1 and LC3-II protein levels, suggesting autophagy induction. These results indicate that the mechanisms responsible for the anticancer effect of LSSE depend on p53 status.

Download Full-text

Dual Targeting of Y-Box Binding Protein-1 and Akt Inhibits Proliferation and Enhances the Chemosensitivity of Colorectal Cancer Cells

Cancers ◽

10.3390/cancers11040562 ◽

2019 ◽

Vol 11 (4) ◽

pp. 562 ◽

Cited By ~ 7

Author(s):

Maier ◽

Attenberger ◽

Tiwari ◽

Lettau ◽

Rebholz ◽

...

Keyword(s):

Cell Proliferation ◽

Binding Protein ◽

Wild Type ◽

Endogenous Expression ◽

Dual Targeting ◽

Mutational Status ◽

Conditional Expression ◽

Ribosomal S6 Kinase ◽

Induced Apoptosis ◽

Hct116 Cells

KRAS-mutated colorectal cancers (CRCs) are resistant to cetuximab treatment. The multifunctional Y-box binding protein 1 (YB-1) is overexpressed in CRC and is associated with chemoresistance. In this study, the effects of oncogenic mutated KRAS(G12V) and KRAS(G13D) on YB-1 phosphorylation were investigated in CRC cells. The effects of the inhibition of p90 ribosomal S6 kinase (RSK) on YB-1 phosphorylation, cell proliferation and survival were tested with and without treatment with 5-fluorouracil using pharmacological inhibitors and siRNA. YB-1 phosphorylation status and subcellular distribution in CRC patient tissues were determined by immunofluorescence staining and confocal microscopy. Endogenous expression of mutated KRAS(G13D) and conditional expression of KRAS(G12V) significantly stimulated YB-1 phosphorylation via RSK and were associated with cetuximab resistance. Inhibition of YB-1 by targeting RSK stimulated the Akt signaling pathway, and this stimulation occurred independently of KRAS mutational status. Akt activation interfered with the antiproliferative effect of the RSK inhibitor. Consequently, dual targeting of RSK and Akt efficiently inhibited cell proliferation in KRAS(G13D)-mutated HCT116 and KRAS wild-type SW48 cells. Treatment with 5-fluorouracil (5-FU) significantly enhanced YB-1 phosphorylation in KRAS(G13D)-mutated HCT116 cells but not in KRAS wild-type SW48 cells. Dual targeting of Akt and RSK sensitized HCT116 cells to 5-FU by stimulating 5-FU-induced apoptosis and inhibiting repair of 5-FU-induced DNA damage. YB-1 was highly phosphorylated in CRC patient tumor tissues and was mainly localized in the nucleus. Together, dual targeting of RSK and Akt may be an alternative molecular targeting approach to cetuximab for treating CRC in which YB-1 is highly phosphorylated.

Download Full-text

Replication Study: Inhibition of BET recruitment to chromatin as an effective treatment for MLL-fusion leukaemia

eLife ◽

10.7554/elife.25306 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 8

Author(s):

Xiaochuan Shan ◽

Juan Jose Fung ◽

Alan Kosaka ◽

Gwenn Danet-Desnoyers ◽

Keyword(s):

Growth Inhibition ◽

Effective Treatment ◽

Cell Line ◽

Cancer Biology ◽

Vehicle Control ◽

Original Study ◽

Selective Growth ◽

Significant Difference ◽

K 562 Cells ◽

Meta Analyses

In 2015, as part of the Reproducibility Project: Cancer Biology, we published a Registered Report (Fung et al., 2015), that described how we intended to replicate selected experiments from the paper "Inhibition of BET recruitment to chromatin as an effective treatment for MLL-fusion leukaemia" (Dawson et al., 2011). Here, we report the results of those experiments. We found treatment of MLL-fusion leukaemia cells (MV4;11 cell line) with the BET bromodomain inhibitor I-BET151 resulted in selective growth inhibition, whereas treatment of leukaemia cells harboring a different oncogenic driver (K-562 cell line) did not result in selective growth inhibition; this is similar to the findings reported in the original study (Figure 2A and Supplementary Figure 11A,B; Dawson et al., 2011). Further, I-BET151 resulted in a statistically significant decrease in BCL2 expression in MV4;11 cells, but not in K-562 cells; again this is similar to the findings reported in the original study (Figure 3D; Dawson et al., 2011). We did not find a statistically significant difference in survival when testing I-BET151 efficacy in a disseminated xenograft MLL mouse model, whereas the original study reported increased survival in I-BET151 treated mice compared to vehicle control (Figure 4B,D; Dawson et al., 2011). Differences between the original study and this replication attempt, such as different conditioning regimens and I-BET151 doses, are factors that might have influenced the outcome. We also found I-BET151 treatment resulted in a lower median disease burden compared to vehicle control in all tissues analyzed, similar to the example reported in the original study (Supplementary Figure 16A; Dawson et al., 2011). Finally, we report meta-analyses for each result.

Download Full-text

Replication Study: Melanoma genome sequencing reveals frequent PREX2 mutations

eLife ◽

10.7554/elife.21634 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 19

Author(s):

Stephen K Horrigan ◽

Pascal Courville ◽

Darryl Sampey ◽

Faren Zhou ◽

Steve Cai ◽

...

Keyword(s):

Genome Sequencing ◽

Cancer Biology ◽

Tumor Formation ◽

Original Study ◽

Free Survival ◽

Tumor Onset ◽

Study Evaluation ◽

Significant Difference ◽

Meta Analyses

In 2015, as part of the Reproducibility Project: Cancer Biology, we published a Registered Report (Chroscinski et al., 2014) that described how we intended to replicate selected experiments from the paper "Melanoma genome sequencing reveals frequent PREX2 mutations" (Berger et al., 2012). Here we report the results of those experiments. We regenerated cells stably expressing ectopic wild-type and mutant phosphatidylinositol-3,4,5-trisphosphate-dependent Rac exchange factor 2 (PREX2) using the same immortalized human NRASG12D melanocytes as the original study. Evaluation of PREX2 expression in these newly generated stable cells revealed varying levels of expression among the PREX2 isoforms, which was also observed in the stable cells made in the original study (Figure S6A; Berger et al., 2012). Additionally, ectopically expressed PREX2 was found to be at least 5 times above endogenous PREX2 expression. The monitoring of tumor formation of these stable cells in vivo resulted in no statistically significant difference in tumor-free survival driven by PREX2 variants, whereas the original study reported that these PREX2 mutations increased the rate of tumor incidence compared to controls (Figure 3B and S6B; Berger et al., 2012). Surprisingly, the median tumor-free survival was 1 week in this replication attempt, while 70% of the control mice were reported to be tumor-free after 9 weeks in the original study. The rapid tumor onset observed in this replication attempt, compared to the original study, makes the detection of accelerated tumor growth in PREX2 expressing NRASG12D melanocytes extremely difficult. Finally, we report meta-analyses for each result.

Download Full-text

Effect of panitumumab on autophagy in colon cancer

Journal of Clinical Oncology ◽

10.1200/jco.2009.27.15_suppl.e22151 ◽

2009 ◽

Vol 27 (15_suppl) ◽

pp. e22151-e22151

Author(s):

H. P. Kalofonos ◽

A. Antonacopoulou ◽

P. Matsouka ◽

E. Giannopoulou

Keyword(s):

Cell Cycle ◽

Colon Cancer ◽

Cell Proliferation ◽

Redox Status ◽

Colon Cancer Cell ◽

Wild Type ◽

Kras Mutations ◽

Protein Levels ◽

Cycle Arrest ◽

Ht 29

e22151 Background: Panitumumab, a human monoclonal antibody raised against EGFR, has been approved by the Food and Drug Administration (FDA) and the European Medicines Agency (EMEA) for the treatment of patients with EGFR-expressing mCRC and wild type kras. The ratio of reduced/oxidised form of glutathione (GSH/GSSG) is an indicator of the redox status in cells. The aim of the current study was to investigate the effect of panitumumab on the redox status of colon cancer cell lines Caco-2, DLD-1 and HT-29 regarding proliferation, apoptosis, necrosis, cell cycle arrest and autophagy. Methods: Cell proliferation was measured by MTT assay. Apoptosis and necrosis were detected by annexin v/propidium iodide assay. Cell cycle arrest was estimated by propidium iodide assay. Autophagy was detected by immunobloting and GSH levels were measured by spectrophotometrical analysis. kras mutations were detected by sequencing analysis. Results: Caco-2, DLD-1 and HT-29 cell lines differ in the expression levels of EGFR and HER-2. Kras mutation analysis in previous studies and in the current study showed that DLD-1 cells express mutated kras while Caco-2 and HT-29 cells express wild type of kras. Panitumumab decreased proliferation only in DLD-1 cells 48 h after its application besides the mutated kras. However, panitumumab did not affect DLD-1 cell apoptosis, necrosis or cell cycle progression 24 and 48 h after cells treatment. Interestingly, panitumumab increased protein levels of beclin 1, an indicator of autophagy, 24 h after its addition in cells. Moreover, an increase in GSH levels was noted 48 h after cells treatment with panitumumab. Conclusions: This is the first study to show that panitumumab, an EGFR inhibitor, affects colon cancer cell proliferation independently of kras mutations and EGFR protein levels through the induction of autophagy. The inhibition in cell proliferation was followed by an increase in GSH levels reflecting an imbalance on the redox status of cells. No significant financial relationships to disclose.

Download Full-text

Characterization of the Role of the Tumor Suppressor BIN1 as a Potential Mediator of the AHI-1 Oncogene in Human Cutaneous T-Cell Lymphomas

Blood ◽

10.1182/blood.v118.21.5229.5229 ◽

2011 ◽

Vol 118 (21) ◽

pp. 5229-5229

Author(s):

Sharmin Esmailzadeh ◽

Helena Wang ◽

Xiaoyan Jiang

Keyword(s):

Cell Proliferation ◽

T Cell ◽

Tumor Suppressor ◽

High Expression ◽

Sézary Syndrome ◽

Protein Levels ◽

Viable Cells ◽

T Cell Lymphomas ◽

Sézary Cells

Abstract Abstract 5229 Cutaneous T-cell lymphomas (CTCLs) represent a group of lymphoproliferative disorders that are characterized by the homing of malignant T-cells to the surface of skin. There are two main types of CTCL: Mycosis Fungoides (MF) and its leukemic variant Sezary Syndrome (SS), which together represent ∼65–70% of all CTCL cases. The precise genetic pathogenesis of these diseases remains largely undetermined. Recently, our research group has demonstrated that AHI-1 (Abelson Helper Integration site-1) oncogene is involved in CTCL. AHI-1 is often the target of provirus insertional mutagenesis in a number of murine leukemias and lymphomas. High expression of AHI-1 is also observed in human leukemia cell lines, with marked upregulation (up to 40 fold) in CTCL lines (Hut78 and Hut102). Moreover, in FACS-purified CD4+CD7− Sezary cells from patients with Sezary Syndrome, AHI-1 has higher expression at both the RNA and protein levels compared to normal CD4+ cells. Furthermore, stable suppression of endogenous AHI-1 in Hut78 cells using small interfering RNA (AHI-1/sh4), reduces autocrine production of interleukin-2 (IL-2), IL-4 and tumor necrosis factor-alpha, and normalizes their transforming activity both in vitro and in vivo. Thus, lymphomagenic activity of Hut78 cells is partially dependent on the expression of AHI-1. Several differentially expressed genes that may play critical roles in AHI-1-mediated leukemic transformation in Hut78 cells have recently been identified through microarray analysis. One candidate is BIN1 (Bridging integrator 1), a tumor suppressor gene which is inactivated or deleted in various cancers. BIN1 protein interacts directly with c-MYC oncogene and inhibits c-MYC–mediated transactivation and transformation. Overexpression of BIN1 increases cell death and decreases cell proliferation in transformed cells. Furthermore, a recent study demonstrated that BIN1 sustains cancer cell sensitivity to some chemotherapy drugs. However, the role of BIN1 in regulation of normal hematopoiesis and lymphomagenesis remains unknown. In our study, BIN1 was shown to be up-regulated at both RNA and protein levels in AHI-1/sh4 cells compared to control Hut78 cells and these data were further confirmed at the mRNA level in primary CD4+CD7− SS cells. It has also been demonstrated that overexpression or suppression of AHI-1 mediate expression changes of BIN1, suggesting that the BIN1 is a potential co-operator of the AHI-1 oncogene. To investigate the tumor suppressor activity of BIN1 in Sezary cells and its potential molecular connection to AHI-1, full-length BIN1 was overexpressed in Hut78 (BIN1/Hut78) and AHI-1/sh4 cells using a lentiviral vector. Increased transcript levels and protein expression of BIN1 were confirmed in transduced cells compared to controls by Q-RT-PCR and Western analysis, respectively. Interestingly, significant reduction in cell proliferation was observed in BIN1/Hut78 cells compared to controls using the 3H- Thymidine uptake assay (>20% reduction in radioactive signal, p= 0.01). These results were further confirmed by using the colony forming cell (CFC) assay and observing a 40% reduction in colony numbers in BIN1/Hut78 cells compared to controls. Furthermore, a significant increase in the number of apoptotic cells was observed in BIN1/Hut78 cells compared to controls after culturing the cells for 48 hours in serum-free conditions using 7-amino-actinomycin D and PE-conjugated AnnexinV antibody staining (∼17% increase, p=0.03). No significant difference was observed in BIN1-transduced AHI-1/sh4 cells compared to controls, possibly due to high expression of endogenous level of BIN1 in these cells. To further investigate the effect of BIN1 on chemoresistance, Hut78 and BIN1/Hut78 cells were treated with different dosages of chemotherapeutic drugs (e.g. Etoposide) and the number of viable cells was counted after specific time points using the Trypan blue exclusion assay. Significant reduction in the number of viable cells was observed in BIN1/Hut78 cells compared to controls after 24 hours of drug treatment (∼20% reduction in cell viability, p=0.03). These findings suggest anti-proliferative and pro-apoptotic roles for BIN1 in human CTCL cells, and that restoration of BIN1 could potentially mediate the chemoresistance of these cells. Disclosures: No relevant conflicts of interest to declare.

Download Full-text