Redefining the relevance of established cancer cell lines to the study of mechanisms of clinical anti-cancer drug resistance.

Although in vitro models have been a cornerstone of anti-cancer drug development, their direct applicability to clinical cancer research has been uncertain. Using a state-of-the-art Taqman-based quantitative RT-PCR assay, we investigated the multidrug resistance (MDR) transcriptome of six cancer types, in established cancer cell lines (grown in monolayer, 3D scaffold, or in xenograft) and clinical samples, either containing >75% tumor cells or microdissected. The MDR transcriptome was determined a priori based on an extensive curation of the literature published during the last three decades, which led to the enumeration of 380 genes. No correlation was found between clinical samples and established cancer cell lines. As expected, we found up-regulation of genes that would facilitate survival across all cultured cancer cell lines evaluated. More troubling, however, were data showing that all of the cell lines, grown either in vitro or in vivo, bear more resemblance to each other, regardless of the tissue of origin, than to the clinical samples they are supposed to model. Although cultured cells can be used to study many aspects of cancer biology and response of cells to drugs, this study emphasizes the necessity for new in vitro cancer models and the use of primary tumor models in which gene expression can be manipulated and small molecules tested in a setting that more closely mimics the in vivo cancer microenvironment so as to avoid radical changes in gene expression profiles brought on by extended periods of cell culture.

Regulation and expression of the ATP-binding cassette transporter ABCG2 in human embryonic stem cells.

The expression and function of several multidrug transporters (including ABCB1 and ABCG2) have been studied in human cancer cells and in mouse and human adult stem cells. However, the expression of ABCG2 in human embryonic stem cells (hESCs) remains unclear. Limited and contradictory results in the literature from two research groups have raised questions regarding its expression and function. In this study, we used quantitative real-time PCR, Northern blots, whole genome RNA sequencing, Western blots, and immunofluorescence microscopy to study ABCG2 expression in hESCs. We found that full-length ABCG2 mRNA transcripts are expressed in undifferentiated hESC lines. However, ABCG2 protein was undetectable even under embryoid body differentiation or cytotoxic drug induction. Moreover, surface ABCG2 protein was coexpressed with the differentiation marker stage-specific embryonic antigen-1 of hESCs, following constant BMP-4 signaling at days 4 and 6. This expression was tightly correlated with the downregulation of two microRNAs (miRNAs) (i.e., hsa-miR-519c and hsa-miR-520h). Transfection of miRNA mimics and inhibitors of these two miRNAs confirmed their direct involvement in the regulation ABCG2 translation. Our findings clarify the controversy regarding the expression of the ABCG2 gene and also provide new insights into translational control of the expression of membrane transporter mRNAs by miRNAs in hESCs.

Host gene expression modulated by Zika virus infection of human-293 cells.

The HEK-293 cell line was created in 1977 by transformation of primary human embryonic kidney cells with sheared adenovirus type 5 DNA. A previous study determined that the HEK-293 cells have neuronal markers rather than kidney markers. In this study, we tested the hypothesis whether Zika virus (ZIKV), a neurotropic virus, is able to infect and replicate in the HEK-293 cells. We show that the HEK-293 cells infected with ZIKV support viral replication as shown by indirect immunofluorescence (IFA) and quantitative reverse transcriptase-PCR (qRT-PCR). We performed RNA-seq analysis on the ZIKV-infected and the control uninfected HEK-293 cells and find 659 genes that are differentially transcribed in ZIKV-infected HEK-293 cells as compared to uninfected cells. The results show that the top 10 differentially transcribed and upregulated genes are involved in antiviral and inflammatory responses. Seven upregulated genes, IFNL1, DDX58, CXCL10, ISG15, KCNJ15, IFNIH1, and IFIT2, were validated by qRT-PCR. Altogether, our findings show that ZIKV infection alters host gene expression by affecting their antiviral and inflammatory responses.

Efficient serum-free derivation of oligodendrocyte precursors from neural stem cell-enriched cultures.

Oligodendrocytes derived in the laboratory from stem cells have been proposed as a treatment for acute and chronic injury to the central nervous system. Platelet-derived growth factor (PDGF) receptor alpha (PDGFRalpha) signaling is known to regulate oligodendrocyte precursor cell numbers both during development and adulthood. Here, we analyze the effects of PDGFRalpha signaling on central nervous system (CNS) stem cell-enriched cultures. We find that AC133 selection for CNS progenitors acutely isolated from the fetal cortex enriches for PDGF-AA-responsive cells. PDGF-AA treatment of fibroblast growth factor 2-expanded CNS stem cell-enriched cultures increases nestin(+) cell number, viability, proliferation, and glycolytic rate. We show that a brief exposure to PDGF-AA rapidly and efficiently permits the derivation of O4(+) oligodendrocyte-lineage cells from CNS stem cell-enriched cultures. The derivation of oligodendrocyte-lineage cells demonstrated here may support the effective use of stem cells in understanding fate choice mechanisms and the development of new therapies targeting this cell type.

Complementary roles for histone deacetylases 1, 2, and 3 in differentiation of pluripotent stem cells.

In eukaryotic cells, covalent modifications to core histones contribute to the establishment and maintenance of cellular phenotype via regulation of gene expression. Histone acetyltransferases (HATs) cooperate with histone deacetylases (HDACs) to establish and maintain specific patterns of histone acetylation. HDAC inhibitors can cause pluripotent stem cells to cease proliferating and enter terminal differentiation pathways in culture. To better define the roles of individual HDACs in stem cell differentiation, we have constructed "dominant-negative" stem cell lines expressing mutant, Flag-tagged HDACs with reduced enzymatic activity. Replacement of a single residue (His-->Ala) in the catalytic center reduced the activity of HDACs 1 and 2 by 80%, and abolished HDAC3 activity; the mutant HDACs were expressed at similar levels and in the same multiprotein complexes as wild-type HDACs. Hexamethylene bisacetamide-induced MEL cell differentiation was potentiated by the individual mutant HDACs, but only to 2%, versus 60% for an HDAC inhibitor, sodium butyrate, suggesting that inhibition of multiple HDACs is required for full potentiation. Cultured E14.5 cortical stem cells differentiate to neurons, astrocytes, and oligodendrocytes upon withdrawal of basic fibroblast growth factor. Transduction of stem cells with mutant HDACs 1, 2, or 3 shifted cell fate choice toward oligodendrocytes. Mutant HDAC2 also increased differentiation to astrocytes, while mutant HDAC1 reduced differentiation to neurons by 50%. These results indicate that HDAC activity inhibits differentiation to oligodendrocytes, and that HDAC2 activity specifically inhibits differentiation to astrocytes, while HDAC1 activity is required for differentiation to neurons.

Lost in Translation: Regulation of ABCG2 Expression in Human Embryonic Stem Cells.

The expression and function of the ATP-binding cassette (ABC) transporter ABCG2 have been studied for two decades in both adult and cancer stem cells. However, this important ABC transporter has not been well characterized in human embryonic stem cells (hESCs). Studies designed to understand the role of ABCG2 in hESCs are still in their initial stages. Several recent reports on expression patterns of the ABCG2 gene in hESCs contain contradictory results at both the mRNA and protein levels. In this review, we provide possible explanations for these discrepancies in ABCG2 expression patterns. We discuss micro-RNA-mediated regulatory roles in controlling ABCG2 mRNA stability and translation, which are associated with hESC pluripotency and differentiation.

Angiogenic factors stimulate growth of adult neural stem cells.

BACKGROUND: The ability to grow a uniform cell type from the adult central nervous system (CNS) is valuable for developing cell therapies and new strategies for drug discovery. The adult mammalian brain is a source of neural stem cells (NSC) found in both neurogenic and non-neurogenic zones but difficulties in culturing these hinders their use as research tools. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that NSCs can be efficiently grown in adherent cell cultures when angiogenic signals are included in the medium. These signals include both anti-angiogenic factors (the soluble form of the Notch receptor ligand, Dll4) and pro-angiogenic factors (the Tie-2 receptor ligand, Angiopoietin 2). These treatments support the self renewal state of cultured NSCs and expression of the transcription factor Hes3, which also identifies the cancer stem cell population in human tumors. In an organotypic slice model, angiogenic factors maintain vascular structure and increase the density of dopamine neuron processes. CONCLUSIONS/SIGNIFICANCE: We demonstrate new properties of adult NSCs and a method to generate efficient adult NSC cultures from various central nervous system areas. These findings will help establish cellular models relevant to cancer and regeneration.