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Biomedical subjects

P S Meltzer

Publications and source records attributed to P S Meltzer.

At least 19 recordsLinked to original sources
PubMed2002

Human AML cells in NOD/SCID mice: engraftment potential and gene expression.

Most cases of human acute myeloid leukemia (AML) engraft in irradiated non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice. Intravenous transfer of as few as 10(5) human AML cells resulted in engraftment. Cases with poor prognosis clinical features, including FLT3 mutations, tended to engraft efficiently. Nevertheless, AML cells obtained from patients at relapse did not engraft more efficiently than cells obtained from the same patients at initial diagnosis. One passage of human AML cells in NOD/SCID mice did not appear to select for increased virulence, as measured by serial transplantation efficiency. Finally, cDNA microarray analyses indicated that approximately 95% of genes were expressed at similar levels in human AML cells immunopurified after growth in mice, as compared to cells assessed directly from patients. Thus, the growth of human AML cells in NOD/SCID mice could yield large numbers of human AML cells for direct experimental use and could also function as a renewable, potentially unlimited source of leukemia cells, via serial transplantation.

Acute Disease↗
PubMed2001

Gastrointestinal stromal tumors with KIT mutations exhibit a remarkably homogeneous gene expression profile.

Gastrointestinal stromal tumors (GISTs), the most common mesenchymal tumors of the digestive tract, are believed to arise from the interstitial cells of Cajal. GISTs are characterized by mutations in the proto-oncogene KIT that lead to constitutive activation of its tyrosine kinase activity. The tyrosine kinase inhibitor STI571, active against the BCR-ABL fusion protein in chronic myeloid leukemia, was recently shown to be highly effective in GISTs. We used 13,826-element cDNA microarrays to define the expression patterns of 13 KIT mutation-positive GISTs and compared them with the expression profiles of a group of spindle cell tumors from locations outside the gastrointestinal tract. Our results showed a remarkably distinct and uniform expression profile for all of the GISTs. In particular, hierarchical clustering of a subset of 113 cDNAs placed all of the GIST samples into one branch, with a Pearson correlation >0.91. This homogeneity suggests that the molecular pathogenesis of a GIST results from expansion of a clone that has acquired an activating mutation in KIT without the extreme genetic instability found in the common epithelial cancers. The results provide insight into the histogenesis of GIST and the clinical behavior of this therapeutically responsive tumor.

Adult↗
PubMed2001

Disease fingerprinting with cDNA microarrays reveals distinct gene expression profiles in lethal type 1 and type 2 cytokine-mediated inflammatory reactions.

Development of polarized immune responses controls resistance and susceptibility to many microorganisms. However, studies of several infectious, allergic, and autoimmune diseases have shown that chronic type-1 and type-2 cytokine responses can also cause significant morbidity and mortality if left unchecked. We used mouse cDNA microarrays to molecularly phenotype the gene expression patterns that characterize two disparate but equally lethal forms of liver pathology that develop in Schistosoma mansoni infected mice polarized for type-1 and type-2 cytokine responses. Hierarchical clustering analysis identified at least three groups of genes associated with a polarized type-2 response and two linked with an extreme type-1 cytokine phenotype. Predictions about liver fibrosis, apoptosis, and granulocyte recruitment and activation generated by the microarray studies were confirmed later by traditional biological assays. The data show that cDNA microarrays are useful not only for determining coordinated gene expression profiles but are also highly effective for molecularly "fingerprinting" diseased tissues. Moreover, they illustrate the potential of genome-wide approaches for generating comprehensive views on the molecular and biochemical mechanisms regulating infectious disease pathogenesis.

Animals↗
PubMed2001

Cooperative interactions of laminin 5 gamma2 chain, matrix metalloproteinase-2, and membrane type-1-matrix/metalloproteinase are required for mimicry of embryonic vasculogenesis by aggressive melanoma.

Vasculogenic mimicry describes a process where aggressive tumor cells in three-dimensional matrices mimic embryonic vasculogenesis by forming extracellular matrix (ECM)-rich, patterned tubular networks. Microarray gene chip analyses revealed significant increases in the expression of laminin 5 (Ln-5, gamma2 chain) and matrix metalloproteinases (MMP)-1, -2, -9, and MT1-MMP (MMP-14) in aggressive compared with poorly aggressive melanoma cells. These components colocalized with developing patterned networks and antisense oligonucleotides to the Ln-5 gamma2 chain (but not sense oligonucleotides), and antibodies to MMP-2 or MT1-MMP (but not MMP-9) inhibited the formation of these networks. Cultures which did not receive antibodies to either MMPs-2 or -14 contained the Ln-5 gamma2 chain promigratory cleavage fragments. Poorly aggressive melanoma cells seeded on collagen I matrices preconditioned by the aggressive cells formed tubular networks along the Ln-5 gamma2 chain-enriched tracks deposited by the aggressive cells. These results suggest that increased expression of MMP-2 and MT1-MMP, along with matrix deposition of the Ln-5 gamma2 chain and/or its cleavage fragments, are required for vasculogenic mimicry by aggressive melanoma cells. Furthermore, the apparent recapitulation of laminin-rich, patterned networks observed in aggressive melanoma patients' tissue sections by aggressive melanoma tumor cells in three-dimensional culture may also serve as a model to help identify specific molecular targets which could function as templates for the coordinated migration of aggressive tumor cells and their proteolytic remodeling of the ECM and may have profound implications for the development of novel therapies directed at the ECM to alter tumor progression.

Cell Adhesion Molecules↗
PubMed2001

Estrogen receptor status in breast cancer is associated with remarkably distinct gene expression patterns.

To investigate the phenotype associated with estrogen receptor alpha (ER) expression in breast carcinoma, gene expression profiles of 58 node-negative breast carcinomas discordant for ER status were determined using DNA microarray technology. Using artificial neural networks as well as standard hierarchical clustering techniques, the tumors could be classified according to ER status, and a list of genes which discriminate tumors according to ER status was generated. The artificial neural networks could accurately predict ER status even when excluding top discriminator genes, including ER itself. By reference to the serial analysis of gene expression database, we found that only a small proportion of the 100 most important ER discriminator genes were also regulated by estradiol in MCF-7 cells. The results provide evidence that ER+ and ER- tumors display remarkably different gene-expression phenotypes not solely explained by differences in estrogen responsiveness.

Breast Neoplasms↗
PubMed2001

Expression and functional significance of VE-cadherin in aggressive human melanoma cells: role in vasculogenic mimicry.

We recently have introduced the term vasculogenic mimicry to describe the unique ability of aggressive melanoma tumor cells to form tubular structures and patterned networks in three-dimensional culture, which "mimics" embryonic vasculogenic networks formed by differentiating endothelial cells. In the current study, we address the biological significance of several endothelial-associated molecules (revealed by microarray analysis) with respect to expression and function in highly aggressive and poorly aggressive human cutaneous melanoma cell lines (established from the same patient). In a comparative analysis, CD31 was not expressed by any of the melanoma cell lines, whereas TIE-1 (tyrosine kinase with Ig and epidermal growth factor homology domains-1) was strongly expressed in the highly aggressive tumor cells with a low level of expression in one of the poorly aggressive cell lines. Vascular endothelial (VE)-cadherin was exclusively expressed by highly aggressive melanoma cells and was undetectable in the poorly aggressive tumor cells, suggesting the possibility of a vasculogenic switch. Down-regulation of VE-cadherin expression in the aggressive melanoma cells abrogated their ability to form vasculogenic networks and directly tested the hypothesis that VE-cadherin is critical in melanoma vasculogenic mimicry. These results highlight the plasticity of aggressive melanoma cells and call into question their possible genetic reversion to an embryonic phenotype. This finding could pose a significant clinical challenge in targeting tumor cells that may masquerade as circulating endothelial cells or other embryonic-like stem cells.

Antigens, CD↗
PubMed2001

Detection of antisense and ribozyme accessible sites on native mRNAs: application to NCOA3 mRNA.

The efficacies of antisense oligonucleotides and ribozymes are greatly dependent on the accessibility of their mRNA targets. Target site accessibility is affected by both RNA structure and the proteins associated along the length of the RNA. To mimic the native state of mRNA for site identification, we have previously used endogenous mRNAs in cellular extracts as targets for defined sequence oligodeoxynucleotides (ODNs) designed to identify both antisense pairing and potential ribozyme cleavage sites. The rationale for this approach is that the specific pairing of an ODN with a mRNA forms a DNA:RNA hybrid that is cleaved by the endogenous RNaseH in the cell extract. To extend the usefulness of this basic approach, we report here the use of semi-random ODN libraries to identify hammerhead ribozyme cleavage sites. Thus, the most accessible sites for antisense and ribozyme base pairing are selected by this approach. A novel feature of the approach described here is the use of terminal transferase-dependent PCR (TDPCR) after reverse transcription to estimate the cleavage efficiency and to precisely determine the RNaseH and ribozyme cleavage sites on mRNAs in cell extracts following treatment with ODN or ribozyme libraries. As a model system, we have targeted the NCOA3 (also known as AIB-1) mRNA in cell extracts. The NCOA3 mRNA encodes a nuclear receptor co-activator that is amplified and over-expressed in a high proportion of breast and ovarian cancers. A highly accessible site on this mRNA was identified, and a ribozyme targeted to this site was demonstrated to effectively downregulate NCOA3 function in cells.

Base Pairing↗
PubMed2001

Application of molecular cytogenetic techniques in a case study of human cutaneous metastatic melanoma.

Consistent structural chromosome rearrangements have rarely been identified in adult solid tumors. The introduction of advanced molecular cytogenetic techniques has provided new ways of analyzing highly complex karyotypes commonly encountered in these malignancies. This study describes a detailed molecular cytogenetic analysis of a sporadic human cutaneous melanoma biopsy, M92-047, using a combination of G-banding, fluorescence in situ hybridization (FISH), chromosome microdissection, and comparative genomic hybridization (CGH). G-banding revealed that this tumor was composed primarily of closely related near-diploid and near-tetraploid cell subpopulations containing several clonal numerical and structural chromosome alterations. Fluorescence in situ hybridization using whole chromosome painting probes and chromosome arm painting probes, was employed to verify the rearranged chromosomes; dic(1;4), der(8)t(1;8), and der(15)t(6;15), whereas marker chromosomes dic(8;1;16), der(12)t(9;12), and der(17)t(13;17) were discerned by chromosome microdissection and subsequent reverse in situ hybridization (rev ish) analysis. Comparative genomic hybridization illustrated DNA copy number changes in good agreement with the karyotypic analysis. Although this line exhibits recurrent alterations representative of melanoma, two unique breakpoints--1p13 and 8p21--were identified in two different rearranged chromosomes, suggesting potentially important regions for further dissection by molecular genetic techniques. This report demonstrates the advantages of combining multiple techniques in order to obtain a detailed description of cytogenetic changes in melanoma.

Aged↗
PubMed2001

Spotting the target: microarrays for disease gene discovery.

Microarray technologies enable genome-scale expression measurements. Already proved to be of value for the functional analysis of individual genes and biological processes, the application of expression profiling to disease gene discovery is now growing in importance and practicality.

Animals↗
PubMed2001

Association of steroid receptor coactivator AIB1 with estrogen receptor-alpha in breast cancer cells.

The steroid receptor coactivator AIB1 (amplified in breast cancer-1) is a transcriptional coactivator which has been found to be amplified in breast cancer. We have now investigated the role of the AIB1 protein in breast cancer cell lines. Although detectable levels of AIB1 were present in most cell lines, high levels of AIB1 expression were observed only in the ER-positive cell lines MCF-7 and BT-474 by western blot analysis. Newly developed monoclonal antibodies (mAbs) were used in several assays to show an association between AIBI and estrogen receptor-alpha (ER). AIB1 and ER co-localized to the nucleus of ER positive cell lines as shown by immunofluorescence microscopy, and a functional association of native AIB1 and ER in MCF-7 nuclear extracts was shown by EMSA. Recombinant ER also recruited AIB1 protein from nuclear extracts, shown by EMSA and by precipitation of ER-complex proteins bound to a biotinylated-ERE DNA target. Additionally, anti-AIB1 mAbs were able to immunoprecipitate ER from nuclear extracts of chemically cross-linked cells but not from uncross-linked cells. Both immunoprecipitation and oligonucleotide precipitation studies demonstrated the presence of p300 and CBP as part of the ER transcriptional complex. These results suggest that AIB1 and ER do associate physically in ER-positive breast cancer cell lines. We propose that in AIB1 amplified breast cancers, a heightened AIB1/ER association may play a crucial role in the progression of these tumors.

Breast Neoplasms↗
PubMed2001

Classification and diagnostic prediction of cancers using gene expression profiling and artificial neural networks.

The purpose of this study was to develop a method of classifying cancers to specific diagnostic categories based on their gene expression signatures using artificial neural networks (ANNs). We trained the ANNs using the small, round blue-cell tumors (SRBCTs) as a model. These cancers belong to four distinct diagnostic categories and often present diagnostic dilemmas in clinical practice. The ANNs correctly classified all samples and identified the genes most relevant to the classification. Expression of several of these genes has been reported in SRBCTs, but most have not been associated with these cancers. To test the ability of the trained ANN models to recognize SRBCTs, we analyzed additional blinded samples that were not previously used for the training procedure, and correctly classified them in all cases. This study demonstrates the potential applications of these methods for tumor diagnosis and the identification of candidate targets for therapy.

Burkitt Lymphoma↗
PubMed2000

Endogenously expressed estrogen receptor and coactivator AIB1 interact in MCF-7 human breast cancer cells.

Coactivators are believed to mediate estrogen-induced gene responses via interaction with estrogen receptors (ER). Currently, a major challenge is to determine the importance of each coactivator in a specific cell type and promoter context in response to a particular ligand. The potential of ER to interact with a growing list of coactivators has been shown in a variety of in vitro and gene transfer assays, yet very few data have demonstrated the interaction of endogenous coactivators with ER in intact cells. We report here a ligand-specific interaction of endogenous human ER (hER) and the AIB1 coactivator in MCF-7 human breast cancer cells by using immunoprecipitation analyses. Complexes between endogenously expressed hER and AIB1 were detected in estradiol-treated cells and to a much lesser extent in cells treated with the partial agonist, monohydroxytamoxifen. We were unable to detect an hER-SRC-1 complex in our immunoprecipitations from MCF-7 cells. The in vitro-binding affinity for mouse ER interaction with AIB1 was estimated to be 40-120 nM. We conclude that AIB1 is a major coactivator for hER in MCF-7 human breast cancer cells.

Animals↗
PubMed2000

Monitoring the expression profiles of doxorubicin-induced and doxorubicin-resistant cancer cells by cDNA microarray.

Drug resistance in cancer is a major obstacle to successful chemotherapy. Cancer cells exposed to antitumor drugs may be directly induced to express a subset of genes that could confer resistance, thus allowing some cells to escape killing and form the relapsed resistant tumor. Alternatively, some cancer cells may be expressing an array of genes that could confer intrinsic resistance, and exposure to cytotoxic drugs select for the survival of these cells that form the relapsed tumor. We have used cDNA microarray to monitor the expression profiles of MCF-7 cells that are either transiently treated with doxorubicin or selected for resistance to doxorubicin. Our results showed that transient treatment with doxorubicin altered the expression of a diverse group of genes in a time-dependent manner. A subset of the induced genes was also found to be constitutively overexpressed in cells selected for resistance to doxorubicin. This distinct set of overlapping genes may represent the signature profile of doxorubicin-induced gene expression and resistance in cancer cells. Our studies demonstrate the feasibility of obtaining potential molecular profile or fingerprint of anticancer drugs in cancer cells by cDNA microarray, which might yield further insights into the mechanisms of drug resistance and suggest alternative methods of treatment.

Antineoplastic Agents↗
PubMed2000

Detection of gene amplification by genomic hybridization to cDNA microarrays.

Gene amplification is one of the major mechanisms of oncogene activation in tumorigenesis. To facilitate the identification of genes mapping to amplified regions, we have used a technique based on the hybridization of total genomic DNA to cDNA microarrays. To aid detection of the weak signals generated in this complex hybridization, we have used a tyramide-based technique that allows amplification of a fluorescent signal up to 1000-fold. Dilution experiment suggests that amplifications of 5-fold and higher can be detected by this approach. The technique was validated using cancer cell lines with several known gene amplifications, such as those affecting MYC, MYCN, ERBB2, and CDK4. In addition to the detection of the known amplifications, we identified a novel amplified gene, ZNF133, in the neuroblastoma cell line NGP. Hybridization of NGP cDNA on an identical array also revealed over expression of ZNF133. Parallel analysis of genomic DNA for copy number and cDNA for expression now provides rapid approach to the identification of amplified genes and chromosomal regions in tumor cells.

Cyclin-Dependent Kinase 4↗
PubMed2000

Identification of a larger than 3 Mb deletion including JAG1 in an Alagille syndrome patient with a translocation t(3;20)(q13.3;p12.2).

Alagille syndrome (AGS) is an autosomal dominant, developmental disorder affecting multiple organ systems including liver, heart, vertebrae, eye and face. Recurrent deletions of the 20p12 region led to the localization, and ultimately to the identification of mutations in the Jagged1 gene (JAG1) in AGS patients. A translocation t(3;20)(q13.3;p12.2) in an AGS patient was characterized using fluorescent in situ hybridization (FISH). The involvement of 3q and 20p in this translocation was demonstrated using probes for 3q and 20p. Three overlapping YAC clones, 940D11, 953A2, and 675G11 extending to nearly 4 Mb including the JAG1 were used as probes for FISH analysis to define the translocation breakpoint. The translocated chromosome was found to have a deletion of more than 3 Mb including the entire JAG1 gene. The observation of an accompanying large deletion, revealed by molecular characterization of the t(3;20) translocation, is similar to the only other translocation reported in an AGS patient; a t(2;20) translocation was also found to have a large deletion of the JAG1 region at 20p12.

Alagille Syndrome↗
PubMed2000

Positional cloning utilizing genomic DNA microarrays: the Niemann-Pick type C gene as a model system.

A major obstacle in positional cloning is identifying the specific mutated gene from within a large physical contig. Here we describe the application of DNA microarray technology to a defined genomic region (physical map) to identify: (i) exons without a priori sequence data and (ii) the disease gene based on differential gene expression in a recessive disorder. The feasibility was tested using resources from the positional cloning of the Neimann-Pick Type C (NP-C) disease gene, NPC1. To identify NPC1 exons and optimize the technology, an array was generated from genomic fragments of the 110-kb bacterial artificial chromosome, 108N2, which encodes NPC1. First, as a test case for blindly identifying exons, fluorescently labeled NPC1 cDNA identified 108N2 fragments that contained NPC1 exons, many of which also contained intronic sequences and could be used to determine part of the NPC1 genomic structure. Second, to demonstrate that the NPC1 disease gene could be identified based upon differential gene expression, subarrays of 108N2 fragments were hybridized with fluorescently labeled cDNA probes generated from total RNA from hamster cell lines differentially expressing NPC1. A probe derived from the NP-C cell line CT60 did not detect NPC1 exons or other genomic fragments from 108N2. In contrast, several NPC1 exons were detected by a probe generated from the non-NP-C cell line 911D5A13, which was derived from CT60, and expressed NPC1 as a consequence of stable transduction with a YAC that contains NPC1 and encompasses 108N2. Thus, the array technology identified NPC1 as a candidate gene based on a physical contig and differential NPC1 expression between NP-C and non-NP-C cells. This technique should facilitate gene identification when a physical contig exists for a region of interest and mutations result in changes in the mRNA level of the disease gene or portions thereof.

Animals↗
PubMed2000

Activating protein-1, nuclear factor-kappaB, and serum response factor as novel target molecules of the cancer-amplified transcription coactivator ASC-2.

ASC-2 was recently discovered as a cancer-amplified transcription coactivator molecule of nuclear receptors, which interacts with multifunctional transcription integrators steroid receptor coactivator-1 (SRC-1) and CREB-binding protein (CBP)/p300. Herein, we report the identification of three mitogenic transcription factors as novel target molecules of ASC-2. First, the C-terminal transactivation domain of serum response factor (SRF) was identified among a series of ASC-2-interacting proteins from the yeast two-hybrid screening. Second, ASC-2 specifically interacted with the activating protein-1 (AP-1) components c-Jun and c-Fos as well as the nuclear factor-kappaB (NFkappaB) components p50 and p65, as demonstrated by the glutathione S-transferase pull-down assays as well as the yeast two-hybrid tests. In cotransfection of mammalian cells, ASC-2 potentiated transactivations by SRF, AP-1, and NFkappaB in a dose-dependent manner, either alone or in conjunction with SRC-1 and p300. In addition, ASC-2 efficiently relieved the previously described transrepression between nuclear receptors and either AP-1 or NFkappaB. Overall, these results suggest that the nuclear receptor coactivator ASC-2 also mediates transactivations by SRF, AP-1, and NFkappaB, which may contribute to the putative, ASC-2-mediated tumorigenesis.

3T3 Cells↗