Impact of the acidic environment on gene expression and functional parameters of tumors in vitro and in vivo

Abstract Background The low extracellular pH (pHe) of tumors resulting from glycolytic metabolism is a stress factor for the cells independent from concomitant hypoxia. The aim of the study was to analyze the impact of acidic pHe on gene expression on mRNA and protein level in two experimental tumor lines in vitro and in vivo and were compared to hypoxic conditions as well as combined acidosis+hypoxia. Methods Gene expression was analyzed in AT1 prostate and Walker-256 mammary carcinoma of the rat by Next Generation Sequencing (NGS), qPCR and Western blot. In addition, the impact of acidosis on tumor cell migration, adhesion, proliferation, cell death and mitochondrial activity was analyzed. Results NGS analyses revealed that 147 genes were uniformly regulated in both cell lines (in vitro) and 79 genes in both experimental tumors after 24 h at low pH. A subset of 25 genes was re-evaluated by qPCR and Western blot. Low pH consistently upregulated Aox1, Gls2, Gstp1, Ikbke, Per3, Pink1, Tlr5, Txnip, Ypel3 or downregulated Acat2, Brip1, Clspn, Dnajc25, Ercc6l, Mmd, Rif1, Zmpste24 whereas hypoxia alone led to a downregulation of most of the genes. Direct incubation at low pH reduced tumor cell adhesion whereas acidic pre-incubation increased the adhesive potential. In both tumor lines acidosis induced a G1-arrest (in vivo) of the cell cycle and a strong increase in necrotic cell death (but not in apoptosis). The mitochondrial O2 consumption increased gradually with decreasing pH. Conclusions These data show that acidic pHe in tumors plays an important role for gene expression independently from hypoxia. In parallel, acidosis modulates functional properties of tumors relevant for their malignant potential and which might be the result of pH-dependent gene expression.

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Effects of Vitrification on the Blastocyst Gene Expression Profile in a Porcine Model

International Journal of Molecular Sciences ◽

10.3390/ijms22031222 ◽

2021 ◽

Vol 22 (3) ◽

pp. 1222

Author(s):

Cristina Cuello ◽

Cristina A. Martinez ◽

Josep M. Cambra ◽

Inmaculada Parrilla ◽

Heriberto Rodriguez-Martinez ◽

...

Keyword(s):

Gene Expression ◽

Gene Expression Analysis ◽

Survival Rates ◽

Control Group ◽

P Value ◽

Transcriptome Profile ◽

Embryo Viability ◽

The Impact

This study was designed to investigate the impact of vitrification on the transcriptome profile of blastocysts using a porcine (Sus scrofa) model and a microarray approach. Blastocysts were collected from weaned sows (n = 13). A total of 60 blastocysts were vitrified (treatment group). After warming, vitrified embryos were cultured in vitro for 24 h. Non-vitrified blastocysts (n = 40) were used as controls. After the in vitro culture period, the embryo viability was morphologically assessed. A total of 30 viable embryos per group (three pools of 10 from 4 different donors each) were subjected to gene expression analysis. A fold change cut-off of ±1.5 and a restrictive threshold at p-value < 0.05 were used to distinguish differentially expressed genes (DEGs). The survival rates of vitrified/warmed blastocysts were similar to those of the control (nearly 100%, n.s.). A total of 205 (112 upregulated and 93 downregulated) were identified in the vitrified blastocysts compared to the control group. The vitrification/warming impact was moderate, and it was mainly related to the pathways of cell cycle, cellular senescence, gap junction, and signaling for TFGβ, p53, Fox, and MAPK. In conclusion, vitrification modified the transcriptome of in vivo-derived porcine blastocysts, resulting in minor gene expression changes.

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Effect of Auraptene on angiogenesis in Xenograft model of breast cancer

Hormone Molecular Biology and Clinical Investigation ◽

10.1515/hmbci-2021-0056 ◽

2021 ◽

Vol 0 (0) ◽

Author(s):

Mohammad Reza Shiran ◽

Elham Mahmoudian ◽

Abolghasem Ajami ◽

Seyed Mostafa Hosseini ◽

Ayjamal Khojasteh ◽

...

Keyword(s):

Breast Cancer ◽

Gene Expression ◽

Animal Model ◽

Protein Expression ◽

Western Blot ◽

Xenograft Model ◽

Dependent Manner ◽

Concentration Dependent Manner

Abstract Objectives Angiogenesis is the most important challenge in breast cancer treatment. Recently, scientists become interesting in rare natural products and intensive researches was performed to identify their pharmacological profile. Auraptene shows helpful effects such as cancer chemo-preventive, anti-inflammatory, anti-oxidant, immuno-modulatory. In this regard, we investigated the anti-angiogenesis effect of Auraptene in in-vitro and in-vivo model of breast cancer. Methods In this study, 4T, MDA-MB-231 and HUVEC cell lines were used. The proliferation study was done by MTT assay. For tube formation assay, 250 matrigel, 1 × 104 HUVEC treated with Auraptene, 20 ng/mL EGF, 20 ng/mL bFGF and 20 ng/mL VEGF were used. Gene expression of important gene related to angiogenesis in animal model of breast cancer was investigated by Real-time PCR. Protein expression of VCAM-1 and TNFR-1 gene related to angiogenesis in animal model of breast cancer was investigated by western-blot. Results Auraptene treatment led to reduction in cell viability of MDA-MB-231 in a concentration-dependent manner. Also, we observed change in the number of tubes or branches formed by cells incubated with 40 and 80 μM Auraptene. Auraptene effect the gene expression of important gene related to angiogenesis (VEGF, VEGFR2, COX2, IFNɣ). Moreover, the western blot data exhibited that Auraptene effect the protein expression of VCAM-1 and TNFR-1. Conclusions Overall, this study shows that Auraptene significantly suppressed angiogenesis via down-regulation of VEGF, VEGFR2, VCAM-1, TNFR-1, COX-2 and up-regulation of IFNγ.

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A Novel Panel of Rabbit Monoclonal Antibodies and Their Diverse Applications Including Inhibition of Clostridium perfringens Epsilon Toxin Oligomerization

Antibodies ◽

10.3390/antib7040037 ◽

2018 ◽

Vol 7 (4) ◽

pp. 37 ◽

Cited By ~ 3

Author(s):

Jennifer Linden ◽

Kiel Telesford ◽

Samantha Shetty ◽

Paige Winokour ◽

Sylvia Haigh ◽

...

Keyword(s):

Flow Cytometry ◽

Cell Death ◽

Monoclonal Antibodies ◽

Western Blot ◽

Clostridium Perfringens ◽

Specificity And Sensitivity ◽

Epsilon Toxin ◽

Blocking Antibodies

The pore-forming epsilon toxin (ETX) produced by Clostridium perfringens is among the most lethal bacterial toxins known. Sensitive antibody-based reagents are needed to detect toxin, distinguish mechanisms of cell death, and prevent ETX toxicity. Using B-cell immuno-panning and cloning techniques, seven ETX-specific monoclonal antibodies were generated from immunized rabbits. ETX specificity and sensitivity were evaluated via western blot, ELISA, immunocytochemistry (ICC), and flow cytometry. ETX-neutralizing function was evaluated both in vitro and in vivo. All antibodies recognized both purified ETX and epsilon protoxin via western blot with two capable of detecting the ETX-oligomer complex. Four antibodies detected ETX via ELISA and three detected ETX bound to cells via ICC or flow cytometry. Several antibodies prevented ETX-induced cell death by either preventing ETX binding or by blocking ETX oligomerization. Antibodies that blocked ETX oligomerization inhibited ETX endocytosis and cellular vacuolation. Importantly, one of the oligomerization-blocking antibodies was able to protect against ETX-induced death post-ETX exposure in vitro and in vivo. Here we describe the production of a panel of rabbit monoclonal anti-ETX antibodies and their use in various biological assays. Antibodies possessing differential specificity to ETX in particular conformations will aid in the mechanistic studies of ETX cytotoxicity, while those with ETX-neutralizing function may be useful in preventing ETX-mediated mortality.

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3 IN VITRO MATURATION ALTERS GENE EXPRESSION IN MOUSE OOCYTES

Reproduction Fertility and Development ◽

10.1071/rdv20n1ab3 ◽

2008 ◽

Vol 20 (1) ◽

pp. 82

Author(s):

M. Paczkowski ◽

C. Bidwell ◽

D. Spurlock ◽

J. Waddell ◽

R. L. Krisher

Keyword(s):

Gene Expression ◽

Cell Death ◽

Dna Methyltransferase ◽

In Vitro Maturation ◽

Fold Increase ◽

Developmental Competence ◽

Differentially Expressed ◽

Dna Methyltransferase 1

The in vitro culture environment significantly impacts nuclear maturation, fertilization, embryonic development, and epigenetic competence; however, our knowledge of the effects of in vitro maturation on oocyte developmental competence, and specifically cytoplasmic maturation, is limited. The objective of this experiment was to identify alterations in the transcriptome of oocytes matured in vitro compared to those matured in vivo that correlate to developmental competence. Immature oocytes were collected from Day 26 and 7-8-week-old B6D2F1 mice 48 h post-pregnant mare serum gonadotropin (PMSG) administration and matured for 16 h in Gmat supplemented with 0.5 mm citric acid, 0.5 mm cysteamine, 100 ng mL–1 epidermal growth factor (EGF), 0.05% insulin-transferrin-selenium (ITS; v/v), 0.01% recombumin (v/v) and 2 mg mL–1 fetuin. In vivo-matured oocytes from females of the same ages were collected from the oviducts 62 h post-PMSG and 14 h post-hCG and mating to vasectomized males. In vivo- and in vitro-matured oocytes were identified visually by the presence of the first polar body. Mature oocytes were pooled into three groups of 150 oocytes per treatment and lysed; poly A+ RNA was extracted. Samples were processed through two cycles of linear amplification and hybridized to the GeneChip� Mouse Genome 430 2.0 Array (Affymetrix, Inc., Santa Clara, CA, USA), with three arrays per treatment. Microarray data were sorted and filtered to include genes that were classified as having two present calls per treatment. The data were then normalized to the chip median and analyzed using a one-way analysis of variance; the level of significance was calculated at P < 0.01. In total, 2.17% (482/22170) and 1.61% (358/22170) of genes were differentially expressed between in vitro- and in vivo-matured oocytes in Day 26 and 7–8-week-old mice, respectively. However, 72.82% (351/482) and 67.87% (243/358) of differentially expressed genes had increased abundance in the in vitro- and in vivo-matured oocytes, respectively. Transcripts involved in gene expression, cellular growth and proliferation, and cellular development were increased in in vivo-matured oocytes from both age groups compared to those matured in vitro. Cell death was one of the higher ranking functional groups increased in the 7–8-week-old in vitro-matured oocytes compared to the 7–8-week-old in vivo-matured oocytes. Specific genes altered by in vitro maturation conditions in Day 26 oocytes were DNA methyltransferase 1 (>7-fold increase in vivo), caspase 8 (>4-fold increase in vivo), and eukaryotic translation initiation factor 1B (>4-fold increase in vivo). DNA methyltransferase 1 and ubiquitin-conjugating enzyme E2T were significantly increased in in vivo-matured 7–8-week-old oocytes (>3-fold and >5-fold, respectively). These results indicate that gene expression is altered in oocytes matured in vitro compared to those matured in vivo. Based on the functional annotations of genes differentially expressed, dysregulation of gene expression in the oocyte resulting in altered DNA methylation and an up-regulation in cell death pathways are potential developmental mechanisms influenced by in vitro culture conditions that correlate to reduced embryonic developmental potential.

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Dominant-negative ATF5 rapidly depletes survivin in tumor cells

Cell Death and Disease ◽

10.1038/s41419-019-1872-y ◽

2019 ◽

Vol 10 (10) ◽

Author(s):

Xiaotian Sun ◽

James M. Angelastro ◽

David Merino ◽

Qing Zhou ◽

Markus D. Siegelin ◽

...

Keyword(s):

Cell Death ◽

Tumor Cell ◽

Cancer Cells ◽

Dominant Negative ◽

Tumor Cell Death ◽

Cell Penetrating ◽

Selective Death ◽

Tumor Types

Abstract Survivin (BIRC5, product of the BIRC5 gene) is highly expressed in many tumor types and has been widely identified as a potential target for cancer therapy. However, effective anti-survivin drugs remain to be developed. Here we report that both vector-delivered and cell-penetrating dominant-negative (dn) forms of the transcription factor ATF5 that promote selective death of cancer cells in vitro and in vivo cause survivin depletion in tumor cell lines of varying origins. dn-ATF5 decreases levels of both survivin mRNA and protein. The depletion of survivin protein appears to be driven at least in part by enhanced proteasomal turnover and depletion of the deubiquitinase USP9X. Survivin loss is rapid and precedes the onset of cell death triggered by dn-ATF5. Although survivin downregulation is sufficient to drive tumor cell death, survivin over-expression does not rescue cancer cells from dn-ATF5-promoted apoptosis. This indicates that dn-ATF5 kills malignant cells by multiple mechanisms that include, but are not limited to, survivin depletion. Cell-penetrating forms of dn-ATF5 are currently being developed for potential therapeutic use and the present findings suggest that they may pose an advantage over treatments that target only survivin.

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Modulation of the extrinsic cell death signaling pathway by viral Flip induces acute-death mediated liver failure

Cell Death and Disease ◽

10.1038/s41419-019-2115-y ◽

2019 ◽

Vol 10 (12) ◽

Cited By ~ 1

Author(s):

Miriam Bittel ◽

Andreas E. Kremer ◽

Michael Stürzl ◽

Stefan Wirtz ◽

Iris Stolzer ◽

...

Keyword(s):

Cell Death ◽

Viral Infections ◽

Tissue Injury ◽

Multiple Organ ◽

Severe Liver ◽

Transgenic Expression ◽

Effector Caspase ◽

The Impact

AbstractDuring viral infections viruses express molecules that interfere with the host-cell death machinery and thus inhibit cell death responses. For example the viral FLIP (vFLIP) encoded by Kaposi’s sarcoma-associated herpesvirus interacts and inhibits the central cell death effector, Caspase-8. In order to analyze the impact of anti-apoptotic viral proteins, like vFlip, on liver physiology in vivo, mice expressing vFlip constitutively in hepatocytes (vFlipAlbCre+) were generated. Transgenic expression of vFlip caused severe liver tissue injury accompanied by massive hepatocellular necrosis and inflammation that finally culminated in early postnatal death of mice. On a molecular level, hepatocellular death was mediated by RIPK1-MLKL necroptosis driven by an autocrine TNF production. The loss of hepatocytes was accompanied by impaired bile acid production and disruption of the bile duct structure with impact on the liver-gut axis. Notably, embryonic development and tissue homeostasis were unaffected by vFlip expression. In summary our data uncovered that transgenic expression of vFlip can cause severe liver injury in mice, culminating in multiple organ insufficiency and death. These results demonstrate that viral cell death regulatory molecules exhibit different facets of activities beyond the inhibition of cell death that may merit more sophisticated in vitro and in vivo analysis.

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Expression and subcellular localization of BNIP3 in hypoxic hepatocytes and liver stress

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.90526.2008 ◽

2009 ◽

Vol 296 (3) ◽

pp. G499-G509 ◽

Cited By ~ 21

Author(s):

Mallikarjuna R. Metukuri ◽

Donna Beer-Stolz ◽

Rajaie A. Namas ◽

Rajeev Dhupar ◽

Andres Torres ◽

...

Keyword(s):

Cell Death ◽

Western Blot ◽

Blot Analysis ◽

Western Blot Analysis ◽

Ischemia Reperfusion ◽

Redox Stress ◽

No Donor ◽

Interacting Protein

We have previously demonstrated that the Bcl-2/adenovirus EIB 19-kDa interacting protein 3 (BNIP3), a cell death-related member of the Bcl-2 family, is upregulated in vitro and in vivo in both experimental and clinical settings of redox stress and that nitric oxide (NO) downregulates its expression. In this study we sought to examine the expression and localization of BNIP3 in murine hepatocytes and in a murine model of hemorrhagic shock (HS) and ischemia-reperfusion (I/R). Freshly isolated mouse hepatocytes were exposed to 1% hypoxia for 6 h followed by reoxygenation for 18 h, and protein was isolated for Western blot analysis. Hepatocytes grown on coverslips were fixed for localization studies. Similarly, livers from surgically cannulated C57Bl/6 mice and from mice cannulated and subjected to 1–4 h of HS were processed for protein isolation and Western blot analysis. In hepatocytes, BNIP3 was expressed constitutively but was upregulated under hypoxic conditions, and this upregulation was countered by treatment with a NO donor. Surprisingly, BNIP3 was localized in the nucleus of normoxic hepatocytes, in the cytoplasm following hypoxia, and again in the nucleus following reoxygenation. Upregulation of BNIP3 partially required p38 MAPK activation. BNIP3 contributed to hypoxic injury in hepatocytes, since this injury was diminished by knockdown of BNIP3 mRNA. Hepatic BNIP3 was also upregulated in two different models of liver stress in vivo, suggesting that a multitude of inflammatory stresses can lead to the modulation of BNIP3. In turn, the upregulation of BNIP3 appears to be one mechanism of hepatocyte cell death and liver damage in these settings.

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Green tea polyphenols block the anticancer effects of bortezomib and other boronic acid–based proteasome inhibitors

Blood ◽

10.1182/blood-2008-07-171389 ◽

2009 ◽

Vol 113 (23) ◽

pp. 5927-5937 ◽

Cited By ~ 158

Author(s):

Encouse B. Golden ◽

Philip Y. Lam ◽

Adel Kardosh ◽

Kevin J. Gaffney ◽

Enrique Cadenas ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Death ◽

Tumor Cell ◽

Green Tea ◽

Boronic Acid ◽

Proteasome Inhibitors ◽

Green Tea Polyphenols ◽

Tumor Cell Death

Abstract The anticancer potency of green tea and its individual components is being intensely investigated, and some cancer patients already self-medicate with this “miracle herb” in hopes of augmenting the anticancer outcome of their chemotherapy. Bortezomib (BZM) is a proteasome inhibitor in clinical use for multiple myeloma. Here, we investigated whether the combination of these compounds would yield increased antitumor efficacy in multiple myeloma and glioblastoma cell lines in vitro and in vivo. Unexpectedly, we discovered that various green tea constituents, in particular (-)-epigallocatechin gallate (EGCG) and other polyphenols with 1,2-benzenediol moieties, effectively prevented tumor cell death induced by BZM in vitro and in vivo. This pronounced antagonistic function of EGCG was evident only with boronic acid–based proteasome inhibitors (BZM, MG-262, PS-IX), but not with several non–boronic acid proteasome inhibitors (MG-132, PS-I, nelfinavir). EGCG directly reacted with BZM and blocked its proteasome inhibitory function; as a consequence, BZM could not trigger endoplasmic reticulum stress or caspase-7 activation, and did not induce tumor cell death. Taken together, our results indicate that green tea polyphenols may have the potential to negate the therapeutic efficacy of BZM and suggest that consumption of green tea products may be contraindicated during cancer therapy with BZM.

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Tumorigenicity of simian virus 40-hepatocyte cell lines: effect of in vitro and in vivo passage on expression of liver-specific genes and oncogenes.

Molecular and Cellular Biology ◽

10.1128/mcb.8.10.4492 ◽

1988 ◽

Vol 8 (10) ◽

pp. 4492-4501 ◽

Cited By ~ 36

Author(s):

C D Woodworth ◽

J W Kreider ◽

L Mengel ◽

T Miller ◽

Y L Meng ◽

...

Keyword(s):

Gene Expression ◽

Tumor Cell ◽

Cell Lines ◽

Phosphoenolpyruvate Carboxykinase ◽

Simian Virus 40 ◽

Simian Virus ◽

Tumor Cell Lines ◽

Glutathione S Transferase

Five simian virus 40 (SV40)-hepatocyte cell lines were examined for tumorigenicity and the effect of in vitro passage on the expression of four liver-specific genes (albumin, transferrin, alpha 1-antitrypsin, and phosphoenolpyruvate carboxykinase), two oncogenes (c-Ha-ras and c-raf), and two genes associated with hepatocarcinogenesis (alpha-fetoprotein and placental-type glutathione-S-transferase). At low passage (12 to 22), all five cell lines expressed the four liver-specific genes at levels similar to those in the liver and were not tumorigenic or were weakly tumorigenic. At high passage (33 to 61), the cell lines formed carcinomas, and four out of five cell lines produced primary tumors that metastasized. At least two cell lines produced well-differentiated hepatocellular carcinomas that expressed liver-specific RNAs. Levels of expression of liver-specific genes changed with time in culture. Some of the changes in liver-specific gene expression in the tumor tissue (such as for the phosphoenolpyruvate carboxykinase gene) paralleled those that occurred with in vitro passage, while other changes (such as for the albumin gene) did not parallel those that occurred with in vitro passage. Correlations between enhanced expression of c-Ha-ras and tumorigenic potential and between the process of SV40 immortalization and induced expression of c-raf and glutathione-S-transferase-P were observed. Induction of alpha-fetoprotein was detected with in vitro and in vivo passage only in the CWSV14 cell line and was paralleled by diminished albumin expression. In conclusion, we developed a model system with five SV40-hepatocyte cell lines, tumors induced by them, and tumor cell lines to examine changes in gene expression that accompany the progression from a normal cell to a hepatocellular carcinoma. Because the SV40-hepatocyte cell lines and tumor cell lines remain highly differentiated and vary in the magnitude of expression of specific genes, they can be used to study the molecular mechanisms regulating gene expression, in particular those regulating specific genes associated with differentiation.

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The Vent-Like Homeobox Gene VENTX Promotes Human Myeloid Development and Is Highly Expressed In Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v116.21.739.739 ◽

2010 ◽

Vol 116 (21) ◽

pp. 739-739

Author(s):

Vijay P. S. Rawat ◽

Natalia Arseni ◽

Farid Ahmed ◽

Medhanie A. Mulaw ◽

Silvia Thoene ◽

...

Keyword(s):

Gene Expression ◽

Progenitor Cells ◽

Cell Lines ◽

Myeloid Cells ◽

Lymphoid Cells ◽

Hematopoietic Development ◽

Cd34 Cells ◽

The Impact

Abstract Abstract 739 Recent studies suggest that a variety of regulatory molecules active in embryonic development such as clustered and non-clustered homeobox genes play an important role in normal and malignant hematopoiesis. Since it was shown that the Xvent-2 homeobox gene is part of the BMP-4 signalling pathway in Xenopus, it is of particular interest to examine the expression profile and function of its only recently discovered human homologue VENTX in hematopoietic development. Expression of the VENTX gene was analyzed in normal human hematopoiesis and AML patients samples by microarray and qPCR. To test the impact of the constitutive expression of VENTX on human progenitor cells, CD34+ cord blood (CB) cells were retrovirally transduced with VENTX or the empty control vector and analyzed using in vitro and in vivo assays. So far we and others have not been able to identify a murine Xenopus xvent gene homologue. However, we were able to document the expression of this gene by qPCR in human lineage positive hematopoietic subpopulations. Amongst committed progenitors VENTX was significantly 13-fold higher expressed in CD33+ BM myeloid cells (4/4 positive) compared to CD19+ BM lymphoid cells (5/7 positive, p=0.01). Of note, expression of VENTX was negligible in normal CD34+/CD38− but detectable in CD34+ BM human progenitor cells. In contrast to this, leukemic CD34+/CD38− from AML patients (n=3) with translocation t(8,21) showed significantly elevated expression levels compared to normal CD34+ BM cells (n=5) (50-fold higher; p≤0.0001). Furthermore, patients with normal karyotype NPM1c+/FLT3-LM− (n=9), NPM1c−/FLT3-LM+ (n=8) or patients with t(8;21) (n=9) had an >100-fold higher expression of VENTX compared to normal CD34+ BM cells and a 5- to 7.8-fold higher expression compared to BM MNCs. Importantly, lentivirus-mediated long-term silencing of VENTX in human AML cell lines (mRNA knockdown between 58% and 75%) led to a significant, reduction in cell number compared to the non-silencing control construct (>79% after 120h). Suggesting that growth of human leukemic cell lines depends on VENTX expression in vitro. As we observed that VENTX is aberrantly expressed in leukemic CD34+ cells with negligible expression in normal counterparts, we assessed the impact of forced VENTX gene expression in normal CD34+ human progenitor cells on the transcription program. Gene expression and pathway analysis demonstrated that in normal CD34+ cells enforced expression of VENTX initiates genes associated with myeloid development (CD11b, CD125, CD9,CD14 and M-CSF), and downregulates genes involved in early lymphoid development (IL-7, IL-9R, LEF1/TCF and C-JUN) and erythroid development such as EPOR, CD35 and CD36. We then tested whether enforced expression of VENTX in CD34+ cells is able to alter the hematopoietic development of early human progenitors as indicated by gene expression and pathway analyses. Functional analyses confirmed that aberrant expression of VENTX in normal CD34+ human progenitor cells induced a significant increase in the number of myeloid colonies compared to the GFP control with 48 ± 6.5 compared to 28.9 ± 4.8 CFU-G per 1000 initially plated CD34+ cells (n=11; p=0.03) and complete block in erythroid colony formation with an 81% reduction of the number of BFU-E compared to the control (n=11; p<0.003). In a feeder dependent co-culture system, VENTX impaired the development of B-lymphoid cells. In the NOD/SCID xenograft model, VENTX expression in CD34+ CB cells promoted generation of myeloid cells with an over 5-fold and 2.5-fold increase in the proportion of human CD15+ and CD33+ primitive myeloid cells compared to the GFP control (n=5, p=0.01). Summary: Overexpression of VENTX perturbs normal hematopoietic development, promotes generation of myeloid cells and impairs generation of lymphoid cells in vitro and in vivo. Whereas VENTX depletion in human AML cell lines impaired their growth.Taken together, these data extend our insights into the function of human embryonic mesodermal factors in human hematopoiesis and indicate a role of VENTX in normal and malignant myelopoiesis. Disclosures: No relevant conflicts of interest to declare.

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