Raji Padmanabhan

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.

Laser scanning and confocal microscopy of daunorubicin, doxorubicin, and rhodamine 123 in multidrug-resistant cells.

The multidrug-resistant gene (MDR1) encodes an energy-dependent drug efflux pump (P-glycoprotein) for many anti-cancer drugs. We have studied the intracellular distribution of rhodamine 123 (R123), daunorubicin (DN), and doxorubicin (DOX) in cells expressing a human MDR1 gene. The distribution of these fluorescent drugs was measured by laser scanning microscopy and confocal microscopy. We devised a new method for analysis of fluorescence line scan data to determine the intracellular distribution of fluorescent probes. This method and confocal microscopy showed that R123, DN, and DOX are localized to both plasma membrane and intracellular compartments in multidrug-resistant cells. When the cells are treated with verapamil, an inhibitor of the multidrug transporter, the amount of DOX, DN, and R123 associated with the cell rises. After inhibition, the relative distribution of DOX and DN between the cell surface and intracellular structures does not change dramatically. However, R123 tends to relocalize to intracellular sites from predominantly plasma membrane sites, indicating that this dye behaves differently than the anti-cancer drugs. These results show the subcellular distributions of R123, DN, and DOX in plasma membrane, cytoplasm, and intracellular membrane systems, but do not allow definitive distinctions among existing models of how P-glycoprotein affects the distribution of drugs.

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 [1], [2], [3], [4], [5], [6]. 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.

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. STEM Cells2012;30:2175–2187

ATP and GTP as alternative energy sources for vinblastine transport by P‐170 in KB‐V1 plasma membrane vesicles

Purified plasma membrane vesicles isolated from multidrug‐resistant human KB‐V1 cells accumulate [3H]vinblastine in an energy‐dependent manner. The accumulation of [3H]vinblastine in the presence of ATP is a saturable process. ATP can be replaced by other purine nucleotide triphosphates, of which GTP is the most efficient.

Magnetic affinity cell sorting to isolate transiently transfected cells, multidrug-resistant cells, somatic cell hybrids, and virally infected cells

Publisher Summary Many techniques have been developed to introduce foreign DNA into eukaryotic cells for stable expression of genes, transient analysis of gene products, and gene regulation. Several methods of transfection have been used, such as calcium phosphate coprecipitation, diethylaminoethyl (DEAE)-dextran, protoplast fusion, microinjection, electroporation, and lipofection. With the exception of DEAE-dextran transfection, which can be used only for transient assays, other techniques are employed to study both transiently and stably transfected cell populations. Isolation of stably transfected cells generally requires the expression of a dominant selectable marker gene, which often confers a drug resistance phenotype. Selection of stable transfectant is time consuming and at times may result in the selection of cells with rearrangements or the insertional mutagenesis of cellular genes. The chromatin structure at the integration sites may influence the expression of the gene of interest, so such studies may not be suitable for the analysis of gene expression.

Constitutive over-expression of transforming growth factor-alpha in rat liver epithelial cells leads to increased cell cycling without transformation

SummaryOver-expression of transforming growth factor-alpha (TGF-α) is consistently seen in spontaneous transformants of rat liver derived epithelial cells (RLE Φ13) and has been implicated in the transformation of other cultured cells. We have constitutively over-expressed TGF-α in RLE Φ13 cells, which are known to express epidermal growth factor receptors, to determine if TGF-α over-expression plays a role in transformation or differentiation, or both, of these cells. Early passage RLE Φ13 cells were infected with a replication-defective murine retrovirus that expresses both the full length coding sequence for human TGF-α and the neomycin-resistance gene. Integration of the transcriptionally active provirus and expression of TGF-α mRNA were confirmed. Neither morphologic transformation nor molecular evidence for differentiation was noted in TGF-α-producing clones. However, these clones did exhibit an accelerated growth rate, increased expression of several cell cycle related genes including mitotic cyclie B1, proliferating cell nuclear antigen, c-myc, and p53 as well as increased expression of the preneoplastic marker enzyme, glutathione-S-transferase. This suggests that over-expression of TGF-α results in increased cell cycling, and that subsequent events must be necessary for cellular transformation or differentiation or both.

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.

Transfer of DNA into Higher Eukaryotic Cells Using Recombinant Vectors Based on Simian Virus 40 and Chloramphenicol Acetyltransferase Genes

Over the past few years considerable progress has been made in the development of eukaryotic vectors. There have been reports describing improved methods for introduction of DNA into mammalian cells 1–3, more sensitive assays for vector function 4,5, and new selectable markers for stable transformation of tissue culture cells 6–8. In this article we present recent work from our laboratory relating to these areas.