Molecular profiling of clinical tissue specimens: feasibility and applications.
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DNA, Complementary↗
Biomedical subjects
D B Krizman
Publications and source records attributed to D B Krizman.
Gene expression profiles in squamous cell carcinomas of the oral cavity: use of laser capture microdissection for the construction and analysis of stage-specific cDNA libraries.
Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer among men in the developed world affecting the oral cavity, salivary glands, larynx and pharynx. Utilizing tissue from patients with HNSCC, we sought to systematically identify and catalog genes expressed in HNSCC progression. Here, we demonstrate the successful use of laser capture microdissection for procuring pure populations of cells from patient tissue sets comprised of oral squamous cell carcinomas (OSCCs) and matching normal tissue. From the estimated 5000 cells procured for each sample, we were able to extract total RNA (14.7-18.6 ng) of sufficient quality to transcribe GAPDH by reverse transcriptase-polymerase chain reaction (RT-PCR). The RNA was used for the synthesis of blunt-ended, double-strand complementary DNAs (cDNAs) by oligo (dT)-mediated reverse transcription, followed by addition of linkers. Primers specific for these linkers with uracil deglycosylase-compatible ends were used to amplify these cDNAs by PCR and the product was subcloned into the pAMP10 cloning vector. Ninety-six clones from each of six libraries were randomly sequenced and results indicated that 76-96% of the inserts represent either anonymous expressed sequence tags (ESTs) (25-48%), known genes (9-29%) or novel sequences (27-51%), respectively, with very little redundancy. These results demonstrate that high quality, representative cDNA libraries can be generated from microdissected OSCC tissue. Furthermore, these finding suggest the existence of at least 132 novel genes expressed in our cDNA libraries, which may have a role in the pathogenesis of HNSCC, and may represent novel markers for early detection as well as targets for pharmacological intervention in this disease.
Aged↗
Molecular profiling of clinical tissues specimens: feasibility and applications.
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Databases, Factual↗
New approaches to molecular profiling of tissue samples.
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Alleles↗
Development of a prostate cDNA microarray and statistical gene expression analysis package.
A cDNA microarray comprising 5184 different cDNAs spotted onto nylon membrane filters was developed for prostate gene expression studies. The clones used for arraying were identified by cluster analysis of > 35 000 prostate cDNA library-derived expressed sequence tags (ESTs) present in the dbEST database maintained by the National Center for Biotechnology Information. Total RNA from two cell lines, prostate line 8.4 and melanoma line UACC903, was used to make radiolabeled probe for filter hybridizations. The absolute intensity of each individual cDNA spot was determined by phosphorimager scanning and evaluated by a bioinformatics package developed specifically for analysis of cDNA microarray experimentation. Results indicated 89% of the genes showed intensity levels above background in prostate cells compared with only 28% in melanoma cells. Replicate probe preparations yielded results with correlation values ranging from r = 0.90 to 0.93 and coefficient of variation ranging from 16 to 28%. Findings indicate that among others, the keratin 5 and vimentin genes were differentially expressed between these two divergent cell lines. Follow-up northern blot analysis verified these two expression changes, thereby demonstrating the reliability of this system. We report the development of a cDNA microarray system that is sensitive and reliable, demonstrates a low degree of variability, and is capable of determining verifiable gene expression differences between two distinct human cell lines. This system will prove useful for differential gene expression analysis in prostate-derived cells and tissue.
DNA, Complementary↗
The genetics of cancer--a 3D model.
Gene expression microarrays hold great promise for studies of human disease states. There are significant technical issues specific to utilizing clinical tissue samples which have yet to be rigorously addressed and completely overcome. Precise, quantitative measurement of gene expression profiles from specific cell populations is at hand, offering the scientific community the first comprehensive view of the in vivo molecular anatomy of normal cells and their diseased counterparts. Here, we propose a model for integrating-in three dimensions-expression data obtained using the microarray.
Databases, Factual↗
The Cancer Genome Anatomy Project: EST sequencing and the genetics of cancer progression.
As the process of tumor progression proceeds from the normal cellular state to a preneoplastic condition and finally to the fully invasive form, the molecular characteristics of the cell change as well. These characteristics can be considered a molecular fingerprint of the cell at each stage of progression and, analogous to fingerprinting a criminal, can be used as markers of the progression process. Based on this premise, the Cancer Genome Anatomy Project was initiated with the broad goal of determining the comprehensive molecular characterization of normal, premalignant, and malignant tumor cells, thus making a reality the identification of all major cellular mechanisms leading to tumor initiation and progression ([Strausberg, R.L., Dahl, C.A., and Klausner, R.D. (1997). "New opportunities for uncovering the molecular basis of cancer." Nat. Genet., 16: 415-516.], www.ncbi.nlm.nih.gov/ncicgap/). The expectation of determining the genetic fingerprints of cancer progression will allow for 1) correlation of disease progression with therapeutic outcome; 2) improved evaluation of disease treatment; 3) stimulation of novel approaches to prevention, detection, and therapy; and 4) enhanced diagnostic tools for clinical applications. Whereas acquiring the comprehensive molecular analysis of cancer progression may take years, results from initial, short-term goals are currently being realized and are proving very fruitful.
Databases, Factual↗
An improved method for construction of directionally cloned cDNA libraries from microdissected cells.
Here, we developed an improved method for constructing microdissected cDNA libraries, based on strand-switching properties of reverse transcriptase, followed by PCR amplification with primers to mediate unidirectional insert cloning. Using RNA from microdissected ovarian carcinoma cells, we constructed a cDNA library consisting of 1.3 x 10(6) unidirectional recombinants with an average insert size of 500 bp. Single-pass sequencing of 100 clones with the T7 primer revealed 89 inserts derived from known genes, anonymous expressed sequence tags (ESTs), or novel sequences. Among these clones were known genes and ESTs previously found in cDNA libraries from bulk ovarian tissue RNA, sequences seen for the first time in an ovarian-derived library, and novel sequences not previously seen in any cDNA library. These results demonstrate a methodology for constructing quality cDNA libraries that are cloned in a unidirectional fashion, are complex and diverse, and reflect the tissue of origin.
Actins↗
Microdissection, microchip arrays, and molecular analysis of tumor cells (primary and metastases).
Advances in biotechnology and bioinformatics are offering promise for new breakthroughs in gene discovery and elucidation of gene function. At present, many candidate genes related to cancer pathogenesis have been identified in several types of human cancer, yet frequently their function remains elusive. This is particularly true as it relates to the progression of human cancer. This landscape could change dramatically, however, as technological innovations and improvements continue to revolutionize these fields. High-throughput molecular approaches are emerging, which may become accurate, automated, and cost-effective. For example, DNA arrays on microchips are under development with numerous applications, including the ability to screen genes rapidly for mutations and to study patterns of gene expression on a large scale. Automated systems for microdissection and sequencing are also in their implementation stages. Commensurate with their integration and evolution, these information and technological tools have the potential to offer a more comprehensive understanding of multiple genetic and cellular alterations occurring during cancer initiation, development, and progression. Ultimately, this fundamental knowledge can provide strategies for intervention, prevention, and early diagnosis. This is a US government work. There are no restrictions on its use.
Base Sequence↗
Niemann-Pick C1 disease gene: homology to mediators of cholesterol homeostasis.
Niemann-Pick type C (NP-C) disease, a fatal neurovisceral disorder, is characterized by lysosomal accumulation of low density lipoprotein (LDL)-derived cholesterol. By positional cloning methods, a gene (NPC1) with insertion, deletion, and missense mutations has been identified in NP-C patients. Transfection of NP-C fibroblasts with wild-type NPC1 cDNA resulted in correction of their excessive lysosomal storage of LDL cholesterol, thereby defining the critical role of NPC1 in regulation of intracellular cholesterol trafficking. The 1278-amino acid NPC1 protein has sequence similarity to the morphogen receptor PATCHED and the putative sterol-sensing regions of SREBP cleavage-activating protein (SCAP) and 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase.
Amino Acid Sequence↗
Transcript mapping in a 46-kb sequenced region at the core of 12q13.3 amplification in human cancers.
We used a combination of sequence analysis and exon trapping in an effort to determine the complete transcript map for a cosmid (6E5) derived from 12q13.3, a region of DNA sequence amplification in human cancers. This cosmid, previously known to contain three genes (CDK4, SAS, and OS9), was sequenced, and that information was used for computer-assisted analysis. In addition, 6E5 was subjected to both internal and 3'-terminal exon-trapping protocols, and the results of these studies were used to guide cDNA cloning experiments. These studies demonstrate that this cosmid is derived from a remarkably gene-dense region and add two new transcripts (KIAA0167 and 6E5.2) to the list of sequences that are expressed in tumors bearing amplification of this region.
Amino Acid Sequence↗
Gene isolation by exon trapping.
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Cloning, Molecular↗
Construction of a representative cDNA library from prostatic intraepithelial neoplasia.
We report the construction of a plasmid-based cDNA library made from microdissected cells derived from prostatic intraepithelial neoplasia. Total RNA was extracted and converted to blunt-ended, double-stranded cDNA by oligo(dT)-mediated reverse transcription followed by linker addition. A linker-specific primer with UDG-compatible ends was used to amplify the cDNA and the resulting PCR product was subcloned. A total of 154 clones were sequenced and results indicated that 81.5% of the clones derived from either known genes, anonymous expressed sequence tags, or novel transcripts with very little redundancy of screened clones. These results demonstrate the feasibility of constructing complex representative cDNA libraries from specific microdissected cell populations that represent microscopic precursor stages of cancer progression. This method should facilitate identification of transcripts specifically expressed in cells of a distinct histological origin and tumorigenic stage.
Adenocarcinoma↗
Gene identification by 3' terminal exon trapping.
3' terminal exon trapping offers a powerful and efficient technology for rendering fragments of transcribed genes from large stretches of unsequenced, vertebrate, genomic DNA. The products from this methodology are the starting point for further studies of gene discovery and analysis. Future considerations for this technology include answering questions about the degree of efficiency from various trapping substrates and whether or not this approach can be scaled up for large-scale gene discovery such as individual chromosome or whole genomic approaches. With the onset of EST database searching, the final analysis becomes a computer assay that is consistent with the future of genetics as this field continues to move towards informational experimentation based on DNA sequence.
Animals↗
A YAC contig containing the reeler locus with preliminary characterization of candidate gene fragments.
The reeler mutation in the mouse maps to proximal chromosome 5 and defines a key gene involved in brain development and evolution. No gene product is known, and the locus is currently being characterized by positional cloning. YAC clones corresponding to the closest markers D5Mit61 and D5Mit72 have been isolated. Cloned extremities of the YAC inserts were used to construct a 1.1-Mb contig, a 700-kb fragment of which was shown to contain the reeler locus. The integrity of the contig was verified by physical mapping on genomic DNA. The classical allele of the reeler mutation was associated with a 150-kb deletion between D5Mit61 and D5Mit72, while no gross chromosomal anomaly was found in the Orleans allele. Candidate coding sequences were isolated to construct a preliminary transcriptional map of the reeler region. Cosmid clones mapping within the rl deletion revealed a large transcript of more than 11 kb, which was present in normal embryonic brain but barely detectable in homozygous rlOrl/rlOrl embryonic brain, suggesting strongly that it corresponds to the reeler transcript.
Animals↗
Identification of 3'-terminal exons from yeast artificial chromosomes.
We report an extension of 3'-terminal exon trapping technology to the identification of transcribed sequences from yeast artificial chromosomes (YACs). A 350-kb YAC containing mouse genomic DNA was gel-purified and used as the target DNA for the 3'-terminal exon trapping strategy. A novel direct ligation/transfection approach was employed to increase the efficiency of trapping 3'-terminal exons from recombinant vector-derived chimeric mRNA. The resulting RT-PCR product was then used to generate a plasmid library. Randomly chosen individual subclones from this library were sequenced, and the results indicate that 86% met sequence criteria characteristic of 3'-terminal exons, whereas 14% were background from identified sources. PCR mapping efforts suggest eight putative last exons present within this YAC, whereas RT-PCR studies demonstrate that three reside within valid expressed sequences.
Animals↗
Efficient selection of 3'-terminal exons from vertebrate DNA.
Identification of expressed sequences within genomic DNA is a hurdle in the characterization of complex genomes. We developed an exon trapping scheme that provides a positive selection for vertebrate 3'-terminal exons. A copy of the trapped exon sequence is obtained by RT/PCR amplification. The technique detects valid terminal exons without interference from partial exons or non-specific sequences, including simple human repeated sequences. Application to random human cosmids yielded one unique trapped terminal exon per cosmid on average. Because vertebrate terminal exons average 600-700 nucleotides in length, the technique provides transcribed sequences of sufficient length to assist further mapping efforts.
Animals↗
Correlation of increased levels of Ha-ras T24 protein with extent of loss of gap junction function in rat liver epithelial cells.
Although it is known that cells transformed by ras and other oncogenes show reduced gap junction function, to date there has been no investigation of the quantitative relationship between intracellular levels of ras oncoprotein and loss of cell-cell communication. Using the rat liver epithelial cell line MTR6, which carries a zinc-inducible metallothionein ras T24 (MTrasT24) fusion gene, we showed a direct correlation between the accumulation of ras T24 protein and the loss of dye transfer as measured by interactive laser cytometry. After stimulation with zinc sulfate, changes in both parameters were rapid and measurable by 24 h. Similarly, there was a dose-response relationship between loss of gap junction function and increase in ras T24 protein. Northern analysis of two gap junction proteins (connexins 43 and 32) showed no differences between cells that expressed high levels of ras and control cells. These data demonstrate that the degree of loss of gap junction function is dependent on the amount of increase in ras T24 protein levels, but the mechanism by which these changes are effected remains unclear.
Animals↗