Down-regulation of the desmosomal cadherin desmocollin 3 in human breast cancer.

In previous studies using cDNA microarray analysis, we have identified an expressed sequence tag which is consistently down-regulated in six human breast tumor cell lines. In the current study, we have determined this tag to be part of the mRNA sequence of human desmocollin 3, a member of the cadherin superfamily of proteins and an integral component of desmosomes. Desmosomes are sites of adhesion between adjacent cells in layers of epithelia, as well as in some non-epithelial tissues, and play an important role in the maintenance of tissue structure. Northern analysis, quantitative real-time polymerase chain reaction assay and Western blot analysis showed that desmocollin 3 is present in normal and immortalized human mammary epithelial cells, but consistently exhibits a significant, and often complete, down-regulation in breast cancer cell lines and primary breast tumors, both at the mRNA and protein levels.

Human prostate cancer and benign prostatic hyperplasia: molecular dissection by gene expression profiling.

Critical aspects of the biology and molecular basis for prostate malignancy remain poorly understood. To reveal fundamental differences between benign and malignant growth of prostate cells, we performed gene expression profiling of primary human prostate cancer and benign prostatic hyperplasia (BPH) using cDNA microarrays consisting of 6500 human genes. Frozen prostate specimens were processed to facilitate extraction of RNA from regions of tissue enriched in either benign or malignant epithelial cell growth within a given specimen. Gene expression in each of the 16 prostate cancer and nine BPH specimens was compared with a common reference to generate normalized measures for each gene across all of the samples. Using an analysis of complete pairwise comparisons of expression profiles among all of the samples, we observed clearly discernable patterns of overall gene expression that differentiated prostate cancer from BPH. Further analysis of the data identified 210 genes with statistically significant differences in expression between prostate cancer and BPH. These genes include many not recognized previously as differentially expressed in prostate cancer and BPH, including hepsin, which codes for a transmembrane serine protease. This study reveals for the first time that significant and widespread differences in gene expression patterns exist between benign and malignant growth of the prostate gland. Gene expression analysis of prostate tissues should help to disclose the molecular mechanisms underlying prostate malignant growth and identify molecular markers for diagnostic, prognostic, and therapeutic use.

Comprehensive copy number and gene expression profiling of the 17q23 amplicon in human breast cancer.

The biological significance of DNA amplification in cancer is thought to be due to the selection of increased expression of a single or few important genes. However, systematic surveys of the copy number and expression of all genes within an amplified region of the genome have not been performed. Here we have used a combination of molecular, genomic, and microarray technologies to identify target genes for 17q23, a common region of amplification in breast cancers with poor prognosis. Construction of a 4-Mb genomic contig made it possible to define two common regions of amplification in breast cancer cell lines. Analysis of 184 primary breast tumors by fluorescence in situ hybridization on tissue microarrays validated these results with the highest amplification frequency (12.5%) observed for the distal region. Based on GeneMap'99 information, 17 known genes and 26 expressed sequence tags were localized to the contig. Analysis of genomic sequence identified 77 additional transcripts. A comprehensive analysis of expression levels of these transcripts in six breast cancer cell lines was carried out by using complementary DNA microarrays. The expression patterns varied from one cell line to another, and several overexpressed genes were identified. Of these, RPS6KB1, MUL, APPBP2, and TRAP240 as well as one uncharacterized expressed sequence tag were located in the two common amplified regions. In summary, comprehensive analysis of the 17q23 amplicon revealed a limited number of highly expressed genes that may contribute to the more aggressive clinical course observed in breast cancer patients with 17q23-amplified tumors.

General nonlinear framework for the analysis of gene interaction via multivariate expression arrays.

A cDNA microarray is a complex biochemical-optical system whose purpose is the simultaneous measurement of gene expression for thousands of genes. In this paper we propose a general statistical approach to finding associations between the expression patterns of genes via the coefficient of determination. This coefficient measures the degree to which the transcriptional levels of an observed gene set can be used to improve the prediction of the transcriptional state of a target gene relative to the best possible prediction in the absence of observations. The method allows incorporation of knowledge of other conditions relevant to the prediction, such as the application of particular stimuli or the presence of inactivating gene mutations, as predictive elements affecting the expression level of a given gene. Various aspects of the method are discussed: prediction quantification, unconstrained prediction, constrained prediction using ternary perceptrons, and design of predictors given small numbers of replicated microarrays. The method is applied to a set of genes undergoing genotoxic stress for validation according to the manner in which it points toward previously known and unknown relationships. The entire procedure is supported by software that can be applied to large gene sets, has a number of facilities to simplify data analysis, and provides graphics for visualizing experimental data, multiple gene interaction, and prediction logic.

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.

Detecting activation of ribosomal protein S6 kinase by complementary DNA and tissue microarray analysis.

BACKGROUND: Studies by comparative genomic hybridization (CGH) have shown that chromosomal region 17q23 is amplified in up to 20% of primary breast cancers. We used microarray analyses to measure the expression levels of genes in this region and to explore their prognostic importance. METHODS: A microarray that contained 4209 complementary DNA (cDNA) clones was used to identify genes that are overexpressed in the MCF-7 breast cancer cell line as compared with normal mammary tissue. Fluorescence in situ hybridization was used to analyze the copy number of one overexpressed gene, ribosomal protein S6 kinase (S6K), and to localize it to the 17q23 region. Northern and western blot analyses were used to measure S6K gene and protein expression, and an enzymatic assay was used to measure S6K activity. Tumor tissue microarray analysis was used to study amplification of S6K and the HER-2 oncogene, another 17q-linked gene, and the relationship between amplification and prognosis was analyzed. The Kaplan-Meier method was used for data analysis, and the log-rank test was used for statistical analysis. All P values are two-sided. RESULTS: S6K was amplified and highly overexpressed in MCF-7 cells relative to normal mammary epithelium, and protein expression and enzyme activity were increased. S6K was amplified in 59 (8.8%) of 668 primary breast tumors, and a statistically significant association between amplification and poor prognosis (P =.0021) was observed. Amplification of both S6K and HER-2 implied particularly poor survival (P =.0001). CONCLUSIONS: The combination of CGH information with cDNA and tissue microarray analyses can be used to identify amplified and overexpressed genes and to evaluate the clinical implications of such genes and genomic rearrangements. S6K is likely to be one of the genes at 17q23 that is amplified during oncogenesis and may adversely affect the prognosis of patients with this amplification.

Identification of tumor-suppressor genes using human melanoma cell lines UACC903, UACC903(+6), and SRS3 by comparison of expression profiles.

The development and progression of cancer are believed to be due to multiple genetic alterations resulting in complex changes in expression of many genes. The parental malignant melanoma cell line UACC903 displays anchorage-independent growth, and the chromosome 6-suppressed subline UACC903(+6) displays anchorage-dependent growth. The anchorage-independent revertant cell line SRS3 derived from UACC903(+6) by retroviral transduction resembles the phenotype of UACC903. In this study, we first compared the expression profiles of 3317 genes between these three cell lines in pairs by cDNA microarrays, resulting in identification of genes with known suppressor activities. We then demonstrated connexin 43 (Cx43)-suppressing anchorage-independent growth of UACC903 on overexpression. Of 3317 genes with informative expression detected by cDNA microarray, 321 (9.68%) showed expression changes between at least one pair of the three cell lines. Notably, 12 genes displayed higher levels of expression in UACC903(+6) than in both UACC903 and SRS3, providing candidates for further identification of melanoma-suppressor genes. Genes encoding Cx43 (suppressor activity), monocyte chemotactic protein 1 (suppressor activity), and cysteine proteinase P32alpha (apoptotic activity) were all upregulated in UACC903(+6), in contrast to both UACC903 and SRS3. Transfection of Cx43, encoded on human chromosome 6q21-q23, a region frequently altered in malignant melanoma, resulted in its overexpression and the suppression of anchorage-independent growth of UACC903. Thus, our result proves the principle that the combination of the ability to alter cellular phenotype by successive genetic alterations and the ability to examine the global expression profiles facilitates the identification of tumor suppressor genes. Mol. Carcinog. 28:119-127, 2000.

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.

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.

cDNA microarrays detect activation of a myogenic transcription program by the PAX3-FKHR fusion oncogene.

Alveolar rhabdomyosarcoma is an aggressive pediatric cancer of striated muscle characterized in 60% of cases by a t(2;13)(q35;q14). This results in the fusion of PAX3, a developmental transcription factor required for limb myogenesis, with FKHR, a member of the forkhead family of transcription factors. The resultant PAX3-FKHR gene possesses transforming properties; however, the effects of this chimeric oncogene on gene expression are largely unknown. To investigate the actions of these transcription factors, both Pax3 and PAX3-FKHR were introduced into NIH 3T3 cells, and the resultant gene expression changes were analyzed with a murine cDNA microarray containing 2,225 elements. We found that PAX3-FKHR but not PAX3 activated a myogenic transcription program including the induction of transcription factors MyoD, Myogenin, Six1, and Slug as well as a battery of genes involved in several aspects of muscle function. Notable among this group were the growth factor gene Igf2 and its binding protein Igfbp5. Relevance of this model was suggested by verification that three of these genes (IGFBP5, HSIX1, and Slug) were also expressed in alveolar rhabdomyosarcoma cell lines. This study utilizes cDNA microarrays to elucidate the pattern of gene expression induced by an oncogenic transcription factor and demonstrates the profound myogenic properties of PAX3-FKHR in NIH 3T3 cells.

DNA microarray technology: the anticipated impact on the study of human disease.

One can imagine that, one day, there will be a general requirement that relevant array data be deposited, at the time of publication of manuscripts in which they are described, into a single site made available for the storage and analysis of array data (modeled after the GenBank submission requirements for DNA sequence information). With this system in place, one can anticipate a time when data from thousands of gene expression experiments will be available for meta-analysis, which has the potential to balance out artifacts from many individual studies, thus leading to more robust results and subtle conclusions. This will require that data adhere to some type of uniform structure and format that would ideally be independent of the particular expression technology used to generate it. The pros and cons of various publication modalities for these large electronic data sets have been discussed elsewhere [12], but, practical difficulties aside, general depositing must occur for this technology to reach the broadest range of investigators. Finally, as mentioned at the beginning of this review, it is unfortunate that this important research tool remains largely restricted to a few laboratories that have developed expertise in this area and to a growing number of commercial interests. Ultimately the real value of microarray technology will only be realized when this approach is generally available. It is hoped that issues including platforms, instrumentation, clone availability, and patents [20] will be resolved shortly, making this technology accessible to the broadest range of scientists at the earliest possible moment.

Expression profiling in cancer using cDNA microarrays.

Currently there are over 1,000,000 human expressed sequence tag (EST) sequences available on the public database, representing perhaps 50-90% of all human genes. The cDNA microarray technique is a recently developed tool that exploits this wealth of information for the analysis of gene expression. In this method, DNA probes representing cDNA clones are arrayed onto a glass slide and interrogated with fluorescently labeled cDNA targets. The power of the technology is the ability to perform a genome-wide expression profile of thousands of genes in one experiment. In our review we describe the principles of the microarray technology as applied to cancer research, summarize the literature on its use so far, and speculate on the future application of this powerful technique.

Data management and analysis for gene expression arrays.

Microarray technology makes it possible to simultaneously study the expression of thousands of genes during a single experiment. We have developed an information system, ArrayDB, to manage and analyse large-scale expression data. The underlying relational database was designed to allow flexibility in the nature and structure of data input and also in the generation of standard or customized reports through a web-browser interface. ArrayDB provides varied options for data retrieval and analysis tools that should facilitate the interpretation of complex hybridization results. A sampling of ArrayDB storage, retrieval and analysis capabilities is available (www.nhgri.nih.gov/DIR/LCG/15K/HTML/ ), along with information on a set of approximately 15,000 genes used to fabricate several widely used microarrays. Information stored in ArrayDB is used to provide integrated gene expression reports by linking array target sequences with NCBI's Entrez retrieval system, UniGene and KEGG pathway views. The integration of external information resources is essential in interpreting intrinsic patterns and relationships in large-scale gene expression data.

A 2.8-Mb clone contig of the multiple endocrine neoplasia type 1 (MEN1) region at 11q13.

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder that results in parathyroid, anterior pituitary, and pancreatic and duodenal endocrine tumors in affected individuals. The MEN1 locus is tightly linked to the marker PYGM on chromosome 11q13, and linkage analysis has placed the MEN1 gene within a 2-Mb interval flanked by D11S1883 and D11S449. As a step toward cloning the MEN1 gene, we have constructed a 2.8-Mb clone contig consisting of YAC and bacterial clones (PAC, BAC, and P1) for the D11S480 to D11S913 region. The bacterial clones alone represent nearly all of the 2.8-Mb contig. The contig was assembled based on a high-density STS-content analysis of 79 genomic clones (YAC, PAC, BAC, and P1) with 118 STSs. The STSs included 22 polymorphic markers and 20 transcripts, with the remainder primarily derived from the end sequences of the genomic clones. An independent cosmid contig for the 1-Mb PYGM-SEA region was also generated. Support for correctness of the 2.8-Mb contig map comes from an independent ordering of the clones by fiber-FISH. This sequence-ready contig will be a useful resource for positional cloning of MEN1 and other disease genes whose loci fall within this region.

Functional human CFTR produced by stable Chinese hamster ovary cell lines derived using yeast artificial chromosomes.

The cystic fibrosis transmembrane conductance regulator gene (CFTR) encodes a transmembrane protein (CFTR) which functions in part as a cyclic adenosine monophosphate (cAMP)-regulated chloride channel. CFTR expression is controlled temporally and cell specifically by mechanisms that are poorly understood. Insight into CFTR regulation could be facilitated by the successful introduction of the entire 230 kb human CFTR and adjacent sequences into mammalian cells. To this end, we have introduced two different CFTR-containing yeast artificial chromosomes (YACs) (320 and 620 kb) into Chinese hamster ovary-K1 (CHO) cells. Clonal cell lines containing human CFTR were identified by PCR, and the genetic and functional analyses of one clone containing each YAC are described. Integration of the human CFTR-containing YACs into the CHO genome at a unique site in each cell line was demonstrated by fluorescence in situ hybridization (FISH). Southern blot analysis suggested that on the order of one copy of human CFTR was integrated per CHO cell genome. Fiber-FISH and restriction analysis suggested that CFTR remained grossly intact. Northern analysis showed full-length, human CFTR mRNA. Immunoprecipitation followed by phosphorylation with protein kinase demonstrated mature, glycosylated CFTR. Finally, chloride secretion in response to cAMP indicated the functional nature of the human CFTR. This study provides several novel results including: (i) functional human CFTR can be expressed from these YACs; (ii) CHO cells are a permissive environment for expression of human CFTR; (iii) the level of human CFTR expression in CHO cells is unexpectedly high given the lack of endogenous CFTR production; and (iv) the suggestion by Fiber-FISH of CFTR integrity correlates with functional gene expression. These YACs and the cell lines derived from them should be useful tools for the study of CFTR expression.

Use of a cDNA microarray to analyse gene expression patterns in human cancer.

The development and progression of cancer and the experimental reversal of tumorigenicity are accompanied by complex changes in patterns of gene expression. Microarrays of cDNA provide a powerful tool for studying these complex phenomena. The tumorigenic properties of a human melanoma cell line, UACC-903, can be suppressed by introduction of a normal human chromosome 6, resulting in a reduction of growth rate, restoration of contact inhibition, and suppression of both soft agar clonogenicity and tumorigenicity in nude mice. We used a high density microarray of 1,161 DNA elements to search for differences in gene expression associated with tumour suppression in this system. Fluorescent probes for hybridization were derived from two sources of cellular mRNA [UACC-903 and UACC-903(+6)] which were labelled with different fluors to provide a direct and internally controlled comparison of the mRNA levels corresponding to each arrayed gene. The fluorescence signals representing hybridization to each arrayed gene were analysed to determine the relative abundance in the two samples of mRNAs corresponding to each gene. Previously unrecognized alterations in the expression of specific genes provide leads for further investigation of the genetic basis of the tumorigenic phenotype of these cells.

Direct visualization of the clonal progression of primary cutaneous melanoma: application of tissue microdissection and comparative genomic hybridization.

Human cutaneous malignant melanoma progresses through a series of well defined clinical and histopathological stages. It has been assumed that the neoplastic progression of this disease advances from a common acquired nevus or dysplastic nevus through the primary radial growth phase (RGP), primary vertical growth phase (VGP), and finally to distant metastasis. However, it has never been directly shown that VGP is clonally derived from RGP. Furthermore, it has not been possible previously to conduct a detailed genetic analysis on pure tumor cells from archival material because the lesions are a heterogeneous mixture of normal and neoplastic cells, and the entire specimen must be excised and fixed for clinical diagnosis. This report describes a new approach designed to identify DNA copy number changes in tumor cells from a series of progressive primary stages of cutaneous melanoma archival biopsies. Under direct high-power visualization, cells are procured with a sterile needle from highly specific areas of the tissue section. DNA is extracted from microdissected cells (normal, RGP, and VGP), PCR amplified, fluorescently labeled, and examined by comparative genomic hybridization to determine DNA copy number changes. Data obtained from three representative cases suggest a clonal derivation of VGP cells from RGP. This approach could be useful in identifying the sequence of genetic changes in progressive cutaneous melanoma stages.

Chromosome microdissection identifies cryptic sites of DNA sequence amplification in human ovarian carcinoma.

DNA sequence amplification contributes to the multistep process of carcinogenesis, and overexpression of amplified genes has been shown to contribute to the malignant phenotype. Cytogenetic analyses of human tumor cells, including ovarian malignancies, frequently show cytological evidence of DNA amplification in the form of double minutes and homogeneously staining regions. In this report, we have combined the techniques of chromosome microdissection and fluorescence in situ hybridization (P. S. Meltzer et al., Nat. Genet., 1: 24-28, 1992) to identify the composition and chromosomal origin of seven homogeneously staining regions from seven cases of ovarian cancer. Twelve specific chromosome band regions were identified as amplified including 11q, 12p, 16p, 19p, and 19q. These results provide important insights into the organization of amplified sequences within ovarian malignancies and add further to our recognition of regions likely to harbor genes important to the development or progression of ovarian cancer.

Expression, isolation and properties of Fur (ferric uptake regulation) protein of Escherichia coli K 12.

The cloned fur (ferric uptake regulation) gene of Escherichia coli K12 was ligated to an expression vector which was inducible with nalidixic acid. The Fur protein was isolated in a single step by immobilized metal-ion-affinity chromatography over zinc iminodiacetate agarose. The amino acid composition of the isolated protein agreed with that predicted from the gene sequence and indicated post-transcriptional removal of the N-terminal methionine residue. All four cysteines were shown to be present as thiols. Proteolysis with trypsin and chymotrypsin yielded large fragments identifiable on polyacrylamide gel electrophoresis. Various divalent metal ions were found by a nitrocellulose filter binding assay to effect non-specific interaction of the Fur dimer with DNA with a dissociation constant of 7 x 10(-12) M. A much smaller value, 2.5 x 10(-17) M, was measured by gel mobility retardation assay for binding of Fur to a DNA fragment containing the operator sequences of the aerobactin promoter.