Little is known of the genetic architecture of cancer at the subclonal and single-cell level or in the cells responsible for cancer clone maintenance and propagation. Here we have examined this issue in childhood acute lymphoblastic leukaemia in which the ETV6-RUNX1 gene fusion is an early or initiating genetic lesion followed by a modest number of recurrent or 'driver' copy number alterations. By multiplexing fluorescence in situ hybridization probes for these mutations, up to eight genetic abnormalities can be detected in single cells, a genetic signature of subclones identified and a composite picture of subclonal architecture and putative ancestral trees assembled. Subclones in acute lymphoblastic leukaemia have variegated genetics and complex, nonlinear or branching evolutionary histories. Copy number alterations are independently and reiteratively acquired in subclones of individual patients, and in no preferential order. Clonal architecture is dynamic and is subject to change in the lead-up to a diagnosis and in relapse. Leukaemia propagating cells, assayed by serial transplantation in NOD/SCID IL2Rγ(null) mice, are also genetically variegated, mirroring subclonal patterns, and vary in competitive regenerative capacity in vivo. These data have implications for cancer genomics and for the targeted therapy of cancer.

The objectives of our studies are to characterize the ability of dietary soybean components to inhibit the growth of prostate cancer in mice and alter tumor biomarkers associated with angiogenesis. Soy isoflavones (genistein or daidzein) or soy phytochemical concentrate inhibit the growth of prostate cancer cells LNCaP, DU 145 and PC-3 in vitro, but only at supraphysiologic concentrations, i.e., 50% inhibitory concentration (IC(50)) > 50 micromol/L. G2-M arrest and DNA fragmentation consistent with apoptosis of prostate cancer cells are also observed at concentrations causing growth inhibition. In contrast, the in vitro proliferation of vascular endothelial cells was inhibited by soy phytochemcials at much lower concentrations. We evaluated the ability of dietary soy phytochemical concentrate and soy protein isolate to inhibit the growth of the LNCaP human prostate cancer in severe combined immune-deficient mice. Mice inoculated subcutaneously with LNCaP cells (2 x 10(6)) were randomly assigned to one of the six dietary groups based on the AIN-76A formulation for 3 wk. A 2 x 3 factorial design was employed with two protein sources (20%, casein vs. soy protein) and three levels of soy phytochemical concentrate (0, 0.2 and 1.0% of the diet). Soy components did not alter body weight gain or food intake. Compared with casein-fed controls, the tumor volumes after 3 wk were reduced by 11% (P = 0.45) by soy protein, 19% (P = 0.17) by 0.2% soy phytochemical concentrate, 28% by soy protein with 0.2% soy phytochemical concentrate (P < 0.05), 30% by 1.0% soy phytochemical concentrate (P < 0.05) and 40% by soy protein with 1.0% soy phytochemical concentrate (P < 0.005). Histologic examination of tumor tissue showed that consumption of soy products significantly reduced tumor cell proliferation, increased apoptosis and reduced microvessel density. The angiogenic protein insulin-like growth factor-I was reduced in the circulation of mice fed soy protein and phytochemical concentrate. Our data suggest that dietary soy products may inhibit experimental prostate tumor growth through a combination of direct effects on tumor cells and indirect effects on tumor neovasculature.

For analysis of multidrug resistance, a major barrier to effective cancer chemotherapy, we profiled mRNA expression of the 48 known human ABC transporters in 60 diverse cancer cell lines (the NCI-60) used by the National Cancer Institute to screen for anticancer activity. The use of real-time RT-PCR avoided artifacts commonly encountered with microarray technologies. By correlating the results with the growth inhibitory profiles of 1,429 candidate anticancer drugs tested against the cells, we identified which transporters are more likely than others to confer resistance to which agents. Unexpectedly, we also found and validated compounds whose activity is potentiated, rather than antagonized, by the MDR1 multidrug transporter. Such compounds may serve as leads for development.

MRP8 (ABCC11) is a recently identified cDNA that has been assigned to the multidrug resistance-associated protein (MRP) family of ATP-binding cassette transporters, but its functional characteristics have not been determined. Here we examine the functional properties of the protein using transfected LLC-PK1 cells. It is shown that ectopic expression of MRP8 reduces basal intracellular levels of cAMP and cGMP and enhances cellular extrusion of cyclic nucleotides in the presence or absence of stimulation with forskolin or SIN-1A. Analysis of the sensitivity of MRP8-overexpressing cells revealed that they are resistant to a range of clinically relevant nucleotide analogs, including the anticancer fluoropyrimidines 5-fluorouracil (ϳ3-fold), 5-fluoro-2-deoxyuridine (ϳ5-fold), and 5-fluoro-5-deoxyuridine (ϳ3-fold), the anti-human immunodeficiency virus agent 2,3-dideoxycytidine (ϳ6-fold) and the anti-hepatitis B agent 9-(2-phosphonylmethoxynyl)adenine (PMEA) (ϳ5-fold). By contrast, increased resistance was not observed for several natural product chemotherapeutic agents. In accord with the notion that MRP8 functions as a drug efflux pump for nucleotide analogs, MRP8-transfected cells exhibited reduced accumulation and increased efflux of radiolabeled PMEA. In addition, it is shown by the use of in vitro transport assays that MRP8 is able to confer resistance to fluoropyrimidines by mediating the MgATP-dependent transport of 5-fluoro-2-deoxyuridine monophosphate, the cytotoxic intracellular metabolite of this class of agents, but not of 5-fluorouracil or 5-fluoro-2-deoxyuridine. We conclude that MRP8 is an amphipathic anion transporter that is able to efflux cAMP and cGMP and to function as a resistance factor for commonly employed purine and pyrimidine nucleotide analogs.Cellular extrusion of cyclic nucleotides has been described in prokaryotic and eukaryotic cells (1-4). This process provides extracellular cAMP involved in intercellular signaling, as determined for Dictyostelium discoideum, in which cAMP effluxed by solitary amoebae under low nutrient conditions mediates cellular aggregation and differentiation, and has also been proposed as a potential mechanism that may contribute to the attenuation of intracellular signaling mediated by these second messengers (5). Investigations employing cultured cells and membrane vesicle preparations have established that cyclic nucleotide efflux is energy-dependent, and the susceptibility of this process to inhibition by antagonists of organic anion pumps indicates that it is mediated by amphipathic anion transporters (2, 3, 6 -16). Recently, insights into the identities of the cellular components that mediate cyclic nucleotide efflux have come from studies of the MRP 1 family of ABC transporters. MRP4 and MRP5, two members of this extended family of amphipathic anion transporters (17), have been determined to be competent in the transport of cyclic nucleotides (18 -20). By contrast, other characterized MRP family members are able to transport a variety of lipophilic anions, ...

SummaryWe used the paradigmatic GATA-PU.1 axis to explore, at the systems level, dynamic relationships between transcription factor (TF) binding and global gene expression programs as multipotent cells differentiate. We combined global ChIP-seq of GATA1, GATA2, and PU.1 with expression profiling during differentiation to erythroid and neutrophil lineages. Our analysis reveals (1) differential complexity of sequence motifs bound by GATA1, GATA2, and PU.1; (2) the scope and interplay of GATA1 and GATA2 programs within, and during transitions between, different cell compartments, and the extent of their hard-wiring by DNA motifs; (3) the potential to predict gene expression trajectories based on global associations between TF-binding data and target gene expression; and (4) how dynamic modeling of DNA-binding and gene expression data can be used to infer regulatory logic of TF circuitry. This rubric exemplifies the utility of this cross-platform resource for deconvoluting the complexity of transcriptional programs controlling stem/progenitor cell fate in hematopoiesis.