Differential adhesion molecule expression during murine embryonic stem cell commitment to the hematopoietic and endothelial lineages.

Mouse embryonic stem cells (ESC) make cell fate decisions based on intrinsic and extrinsic factors. The decision of ESC to differentiate to multiple lineages in vitro occurs during the formation of embryoid bodies (EB) and is influenced by cell-environment interactions. However, molecular mechanisms underlying cell-environmental modulation of ESC fate decisions are incompletely understood. Since adhesion molecules (AM) influence proliferation and differentiation in developing and adult tissues, we hypothesized that specific AM interactions influence ESC commitment toward hematopoietic and endothelial lineages. Expression of AM in the adherens, tight and gap junction pathways in ESC subpopulations were quantified. E-cadherin (E-cad), Claudin-4 (Cldn4), Connexin-43 (Cx43), Zona Occludens-1 (ZO-1) and Zona Occludens-2 (ZO-2) transcript levels were differentially expressed during early stages of hematopoietic/endothelial commitment. Stable ESC lines were generated with reduced expression of E-cad, Cldn4, Cx43, ZO-1 and ZO-2 using shRNA technology. Functional and phenotypic consequences of modulating AM expression were assessed using hematopoietic colony forming assays, endothelial sprouting assays and surface protein expression. A decrease in E-cad, Cldn4, Cx43 and ZO-1 expression was associated with less commitment to the hematopoietic lineage and increased endothelial differentiation as evidenced by functional and phenotypic analysis. A reduction in ZO-2 expression did not influence endothelial differentiation, but decreased hematopoietic commitment two-fold. These data indicate that a subset of AM influence ESC decisions to commit to endothelial and hematopoietic lineages. Furthermore, differentially expressed AM may provide novel markers to delineate early stages of ESC commitment to hematopoietic/endothelial lineages.

AG-013736, a novel inhibitor of VEGF receptor tyrosine kinases, inhibits breast cancer growth and decreases vascular permeability as detected by dynamic contrast-enhanced magnetic resonance imaging.

Dynamic contrast-enhanced MRI (DCE-MRI) was used to noninvasively evaluate the effects of AG-03736, a novel inhibitor of vascular endothelial growth factor (VEGF) receptor tyrosine kinases, on tumor microvasculature in a breast cancer model. First, a dose response study was undertaken to determine the responsiveness of the BT474 human breast cancer xenograft to AG-013736. Then, DCE-MRI was used to study the effects of a 7-day treatment regimen on tumor growth and microvasculature. Two DCE-MRI protocols were evaluated: (1) a high molecular weight (MW) contrast agent (albumin-(GdDTPA)(30)) with pharmacokinetic analysis of the contrast uptake curve and (2) a low MW contrast agent (GdDTPA) with a clinically utilized empirical parametric analysis of the contrast uptake curve, the signal enhancement ratio (SER). AG-013736 significantly inhibited growth of breast tumors in vivo at all doses studied (10-100 mg/kg) and disrupted tumor microvasculature as assessed by DCE-MRI. Tumor endothelial transfer constant (K(ps)) measured with albumin-(GdDTPA)(30) decreased from 0.034+/-0.005 to 0.003+/-0.001 ml min(-1) 100 ml(-1) tissue (P<.0022) posttreatment. No treatment-related change in tumor fractional plasma volume (fPV) was detected. Similarly, in the group of mice studied with GdDTPA DCE-MRI, AG-013736-induced decreases in tumor SER measures were observed. Additionally, our data suggest that 3D MRI-based volume measurements are more sensitive than caliper measurements for detecting small changes in tumor volume. Histological staining revealed decreases in tumor cellularity and microvessel density with treatment. These data demonstrate that both high and low MW DCE-MRI protocols can detect AG-013736-induced changes in tumor microvasculature. Furthermore, the correlative relationship between microvasculature changes and tumor growth inhibition supports DCE-MRI methods as a biomarker of VEGF receptor target inhibition with potential clinical utility.

Quantitative proteome analysis of breast cancer cell lines using 18O-labeling and an accurate mass and time tag strategy.

Proteome comparison of cell lines derived from cancer and normal breast epithelium provide opportunities to identify differentially expressed proteins and pathways associated with specific phenotypes. We employed 16O/18O peptide labeling, FT-ICR MS, and an accurate mass and time (AMT) tag strategy to simultaneously compare the relative abundance of hundreds of proteins in non-cancer and cancer cell lines derived from breast tissue. A cell line reference panel allowed relative protein abundance comparisons among multiple cell lines and across multiple experiments. A peptide database generated from multidimensional LC separations and MS/MS analysis was used for subsequent AMT tag-based peptide identifications. This peptide database represented a total of 2299 proteins, including 514 that were quantified in five cell lines using the AMT tag and 16O/18O strategies. Eighty-six proteins showed at least a threefold protein abundance change between cancer and non-cancer cell lines. Hierarchical clustering of protein abundance ratios revealed that several groups of proteins were differentially expressed between the cancer cell lines.

Comparison of normal and breast cancer cell lines using proteome, genome, and interactome data.

Normal and cancer cell line proteomes were profiled using high throughput mass spectrometry techniques. Application of protein-level and peptide-level sample fractionation combined with LC-MS/MS analysis enabled identification of 2235 unmodified proteins representing a broad range of functional and compartmental classes. An iterative multistep search strategy was used to identify post-translational modifications, revealing several proteins that are preferentially modified in cancer cells. Information regarding both unmodified and modified protein forms was combined with publicly available gene expression and protein-protein interaction data. The resulting integrated dataset revealed several functionally related proteins that are differentially regulated between normal and cancer cell lines.

Heterogeneity in the angiogenic response of a BT474 human breast cancer to a novel vascular endothelial growth factor-receptor tyrosine kinase inhibitor: assessment by voxel analysis of dynamic contrast-enhanced MRI.

PURPOSE: To investigate the heterogeneity in the angiogenic response of a human breast cancer xenograft to a novel vascular endothelial growth factor (VEGF)-receptor tyrosine kinase inhibitor, AG-013736, using dynamic contrast-enhanced MR imaging (DCE-MRI). MATERIALS AND METHODS: Changes in pharmacokinetic parameters in a seven-day interval were compared between AG-treated and control groups, using Gd-DTPA and albumin-(Gd-DTPA)30. A voxel-by-voxel analysis was performed to produce parametric spatial pharmacokinetic parametric maps and histograms. Histogram segmentation was used to quantify the heterogeneity in tumor response to therapy, and compared with conventional descriptive measures of distribution in terms of their capacity to separate control from AG-treated tumors. RESULTS: The albumin-(Gd-DTPA)30 endothelial transfer constant, Kps, showed changes with AG-013736 treatment and tumor growth. The changes were highly heterogeneous for individual segments of the histogram with different Kps values, and the overall patterns in which the frequency distribution changed differed significantly between the two groups. A change in the number of voxels with Kps ranging from 0.03 to 0.14 mL/min/(100 mL tissue) was the most sensitive variable for separating control from AG-treated tumors (P = 0.0008). Parametric maps of the kinetic parameters also showed spatial heterogeneity in tumor response to treatment. The Kps maps depicted rapid development of central necrosis as a result of AG-013736 treatment. Maps of v(p) demonstrated a marked increase at peripheral regions of necrotic areas. Similar trends were noted in the Gd-DTPA rate constant Ktrans distribution. CONCLUSION: This study demonstrates the value of histogram analysis of maps of pharmacokinetic parameters for assessing heterogeneity in tumor response to antiangiogenic therapy. Changes in the number of voxels within certain segments of the Kps histogram were the most sensitive variable for separating control from AG-treated tumors.

Proteome and transcriptome analysis of retinoic acid-induced differentiation of human acute promyelocytic leukemia cells, NB4.

Acute promyelocytic leukemia (APL) is characterized by a translocation t(15:17) that fuses the retinoic acid receptor gene with the promyelocytic gene, which blocks differentiation to normal granulocytes. NB4 cells, derived from human acute promyelocytic leukemia, display this genotype and phenotype. All trans-retinoic acid (ATRA) induces differentiation of NB4 cell cultures in vitro and APL in vivo, although resistance to differentiation therapy frequently develops. To identify genes involved in differentiation, we compared gene expression at the mRNA and protein levels using microarray analyses and two-dimensional (2D) difference gel electrophoresis (DIGE), plus MALDI-TOF-TOF mass spectrometry. Differentially expressed transcripts were identified using oligonucleotide-based microarrays with targets representing almost 14 000 genes. Real time PCR was performed on a subset of genes whose products were shown to be differentially expressed using proteomic and/or genomic approaches. Our analyses identified 46 genes that were differentially expressed in NB4 +/- ATRA; 22 were identified using 2D-DIGE and 24 using microarray analysis. All but four of these genes were expressed at higher levels in differentiated cells, and several controlled cell structure (internal and cytoskelatal) or signal transduction. We observed that proteome analysis with DIGE and silver-stained 2D gel electrophoresis analyses revealed significant differences between the two measurement approaches. Furthermore, our data showed significant discordance between gene expression at the protein and transcript levels.

Differential protein expression in MCF7 breast cancer cells transfected with ErbB2, neomycin resistance and luciferase plus yellow fluorescent protein.

Gene transfection is frequently used to explore the molecular and phenotypic consequences of introduced genes. Breast cancer cell lines transfected with genes for growth factor receptors, intracellular signaling molecules or genes that generate luminescent signals are widely used in basic science and preclinical studies. Typically, a target gene of interest is co-transfected with selectable markers that are generally assumed to be innocuous. Perturbations of the cellular genome by transfected sequences may induce subtle and/or unexpected modulations in protein expression, only some of which may be attributable to the target gene of interest. In this study, we show that neomycin resistant MCF7 cells (MCF7 Neo(r)) proliferate twice as rapidly in nude mice as do the untransfected parent cells, but show similar growth rates in vitro. MCF7 transfected with the ErbB2 gene shows minimal alteration in growth rate in vitro, and approximately a threefold increased growth rate in vivo. MCF7 cells that express luciferase and yellow fluorescent protein proliferate slowly in vitro and show essentially no growth in vivo suggesting that overexpression of these tracking proteins adversely affects cellular proliferative capacity. The molecular basis for alterations in proliferative capacity of the transfected sub-lines is poorly understood. We performed two-dimensional gel electrophoresis (2-DE) to compare relative protein expression among the cell lines. Relative to the parental MCF7, transfected cell lines displayed numerous differentially expressed proteins (69 to 149), relative to parental MCF7. Twenty-one of these differentially expressed proteins were identified by mass spectrometry, and included metabolic, structural, and signaling proteins. Possible roles of differentially expressed proteins in altering cellular proliferation are discussed.

Progenitor cell involvement is predictive of response to induction chemotherapy in paediatric acute myeloid leukaemia.

In acute myeloid leukaemia (AML), involvement of early progenitor cells may predict poor response to induction chemotherapy. We evaluated the involvement of early progenitor cells in two AML subtypes with a favourable prognosis [t(8;21) and t(15;17)], and a subtype with poor prognosis (monosomy 7). CD34+CD33- cells were isolated by fluorescence-activated cell sorting, grown in liquid medium followed by culture in semi-solid medium, and the colonies that were formed were analysed for the identifiable genetic markers. Two of 136 colonies from six t(8;21) AML patients expressed the AML1-ETO transcript, and all six patients achieved remission after induction. None of 192 colonies from five t(15;17) AML patients expressed the RARalpha-PML transcript and all achieved remission. In contrast, in three of 10 cases of monosomy 7 AML, colonies were positive for monosomy 7, and all three patients failed to enter remission. However, five of six evaluable patients with colonies negative for monosomy 7 entered remission. These data support the hypothesis that leukaemic involvement of early progenitor cells affects the response to induction chemotherapy.