Biomedical subjects

Steven H Reynolds

Publications and source records attributed to Steven H Reynolds.

13 recordsLinked to original sources

PubMed2006

Stem cells signal to the niche through the Notch pathway in the Drosophila ovary.

Stem cells are maintained and retain their capacity to continue dividing because of the influence of a niche. Although niches are important to maintain "stemness" in a wide variety of tissues, control of these niches is poorly understood. The Drosophila germline stem cells (GSCs) reside in a somatic cell niche. We show that Notch activation can induce the expression of niche-cell markers even in an adult fly; overexpression of Delta in the germline, or activated Notch in the somatic cells, results in extra niche cells, up to 10-fold over the normal number. In turn, these ectopic niche cells induce ectopic GSCs. Conversely, when GCSs do not produce functional Notch ligands, Delta and Serrate, the TGF-beta pathway is not activated in the GSCs, and they differentiate and subsequently leave the niche. Importantly, clonal analysis reveals that the receiving end of the Notch pathway is required in the somatic cells. These data show that a feedback loop exists between the stem cells and niche cells. Demonstration that stem cells can contribute to niche function has far-reaching consequences for stem cell therapies and may provide insight into how cancer can spread throughout an organism via populations of cancer stem cells.

Animals↗

PubMed2006

Increased gene copy number of the transcription factor E2F1 in malignant melanoma.

Translocations and unique chromosome break points in melanoma will aid in the identification of the genes that are important in the neoplastic process. We have previously shown a unique translocation in malignant melanoma cells der(12)t(12;20). The transcription factor E2F1 maps to 20q11. Increased expression of E2F has been associated with the autonomous growth of melanoma cells, however, the molecular basis has not yet been elucidated. To this end, we investigated E2F1 gene copy number and structure in human melanoma cell lines and metastatic melanoma cases. Fluorescent in situ hybridization (FISH) analysis using a specific E2F1 probe indicated increased E2F1 gene copies in melanoma cell lines compared to normal melanocytes. We also observed increased copies of the E2F1 gene in lymph node metastases of melanoma. In addition, Western blot analysis demonstrated increased E2F1 protein levels in 8 out of 9 melanoma cell lines relative to normal melanocytes. Inhibition of E2F1 expression with RNAi also reduced melanoma cell growth. Our results suggest that the release of E2F activity by elevated E2F1 gene copy numbers may play a functional role in melanoma growth.

Blotting, Western↗

PubMed2006

Welding fume exposure and associated inflammatory and hyperplastic changes in the lungs of tumor susceptible a/j mice.

It has been suggested that welding fume (WF) exposure increases lung cancer risk in welders. Epidemiology studies have failed to conclude that WF alone causes lung cancer and animal studies are lacking. We examined the course of inflammation, damage, and repair in the lungs of A/J mice, a lung tumor susceptible strain, caused by stainless steel WF. Mice were exposed by pharyngeal aspiration to 40 mg/kg of WF, silica, or saline. Bronchoalveolar lavage (BAL) was performed 24 hours, 1 and 16 weeks to assess lung injury and inflammation and histopathology was done 1, 8, 16, 24, and 48 weeks postexposure. Both exposures increased inflammatory cells, lactate dehydrogenase and albumin at 24 hr and 1 week. At 16 weeks, these parameters remained elevated in silica-exposed but not WF-exposed mice. Histopathologic evaluation at 1 week indicated that WF induced bronchiolar epithelial hyperplasia with associated cellular atypia, alveolar bronchiolo-alveolar hyperplasia (BAH) in peribronchiolar alveoli, and peribronchiolar lymphogranulomatous inflammation. Persistent changes included foci of histiocytic inflammation, fibrosis, atypical bronchiolar epithelial cells, and bronchiolar BAH. The principle changes in silica-exposed mice were histiocytic and suppurative inflammation, fibrosis, and alveolar BAH. Our findings that WF causes persistent bronchiolar and peribronchiolar epithelial changes, suggest a need for studies of bronchiolar changes after WF exposure.

Animals↗

PubMed2006

High-throughput TILLING for Arabidopsis.

Targeting induced local lesions in genomes (TILLING) is a general strategy for identifying induced point mutations that can be applied to almost any organism. In this chapter, we describe the basic methodology for high-throughput TILLING. Gene segments are amplified using fluorescently tagged primers, and products are denatured and reannealed to form heteroduplexes between the mutated sequence and its wild-type counterpart. These heteroduplexes are substrates for cleavage by the endonuclease CEL I. Following cleavage, products are analyzed on denaturing polyacrylamide gels using the LI-COR DNA analyzer system. High-throughput TILLING has been adopted by the Arabidopsis TILLING Project (ATP) to provide allelic series of point mutations for the general Arabidopsis community.

Arabidopsis↗

PubMed2004

Discovery of induced point mutations in maize genes by TILLING.

BACKGROUND: Going from a gene sequence to its function in the context of a whole organism requires a strategy for targeting mutations, referred to as reverse genetics. Reverse genetics is highly desirable in the modern genomics era; however, the most powerful methods are generally restricted to a few model organisms. Previously, we introduced a reverse-genetic strategy with the potential for general applicability to organisms that lack well-developed genetic tools. Our TILLING (Targeting Induced Local Lesions IN Genomes) method uses chemical mutagenesis followed by screening for single-base changes to discover induced mutations that alter protein function. TILLING was shown to be an effective reverse genetic strategy by the establishment of a high-throughput TILLING facility and the delivery of thousands of point mutations in hundreds of Arabidopsis genes to members of the plant biology community. RESULTS: We demonstrate that high-throughput TILLING is applicable to maize, an important crop plant with a large genome but with limited reverse-genetic resources currently available. We screened pools of DNA samples for mutations in 1-kb segments from 11 different genes, obtaining 17 independent induced mutations from a population of 750 pollen-mutagenized maize plants. One of the genes targeted was the DMT102 chromomethylase gene, for which we obtained an allelic series of three missense mutations that are predicted to be strongly deleterious. CONCLUSIONS: Our findings indicate that TILLING is a broadly applicable and efficient reverse-genetic strategy. We are establishing a public TILLING service for maize modeled on the existing Arabidopsis TILLING Project.

Ethyl Methanesulfonate↗

PubMed2004

DLC-1 operates as a tumor suppressor gene in human non-small cell lung carcinomas.

The deleted in liver cancer (DLC-1) gene at chromosome 8p21-22 is altered mainly by genomic deletion or aberrant promoter methylation in a large number of human cancers such as breast, liver, colon and prostate and is known to have an inhibitory effect on breast and liver tumor cell growth. Given the high frequency of deletion involving region 8p21-22 in human non-small cell lung carcinoma (NSCLC), we examined alterations of DLC-1 in a series of primary tumors and tumor cell lines and tested effects of DLC-1 on tumor cell growth. A significant decrease or absence of the DLC-1 mRNA expression was found in 95% of primary NSCLC (20/21) and 58% of NSCLC cell lines (11/19). Transcriptional silencing of DLC-1 was primarily associated with aberrant DNA methylation, rather than genomic deletion as 5-aza-2'-deoxycytidine induced reactivation of DLC-1 expression in 82% (9/11) NSCLC cell lines showing downregulated DLC-1. It was further evidenced by an aberrant DLC-1 promoter methylation pattern, which was detected by Southern blotting in 73% (8/11) of NSCLC cell lines with downregulation of the gene. The transfer of DLC-1 into three DLC-1 negative cell lines caused a significant inhibition in cell proliferation and/or a decrease in colony formation. Furthermore, stable transfer of DLC-1 abolished tumorigenicity in nude mice of two cell lines, suggesting that DLC-1 plays a role in NSCLC by acting as a bona fide new tumor suppressor gene.

Animals↗

PubMed2004

Mutational activation of the MAP3K8 protooncogene in lung cancer.

The MAP3K8 protooncogene (Cot/Tpl-2) activates the MAP kinase, SAP kinase, and NF-kappaB signaling pathways. MAP3K8 mutations occur in the rat homologue, but activating mutations have yet to be identified in primary human tumors. We have identified MAP3K8 as a transforming gene from a human lung adenocarcinoma and characterized a 3' end mutation in the cDNA. In addition, we confirmed that the mutation occurs in the original lung tumor, and we screened a series of lung cancer cell lines to determine whether the MAP3K8 mutation is a common occurrence in lung tumorigenesis. The oncogene was isolated and identified with the NIH3T3 nude mouse tumorigenicity assay and cDNA library screening. The gene was analyzed by polymerase chain reaction (PCR), single-strand conformational polymorphism (SSCP), and 3'RACE for mutations. The mutation was localized to MAP3K8 exon 8 and confirmed in the primary tumor DNA. Both wild-type and mutant MAP3K8 cDNAs transformed NIH3T3 cells, but the transforming activity of the mutant was much greater than that of the wild type. PCR-SSCP screening of cell line cDNAs identified one silent polymorphism in cell line SK-LU-1. Although we were unable to find additional activating mutations, these data support a role for MAP3K8 activity in cellular transformation, but suggest that mutational activation of the gene is a rare event in lung cancer.

Adenocarcinoma↗

PubMed2004

Efficient discovery of DNA polymorphisms in natural populations by Ecotilling.

We have adapted the mutation detection technology used in Targeting Induced Local Lesions in Genomes (TILLING) to the discovery of polymorphisms in natural populations. The genomic DNA of a queried individual is mixed with a reference DNA and used to amplify a target 1-kbp region of DNA with asymmetrically labeled fluorescent primers. After heating and annealing, heteroduplexes are nicked at mismatched sites by the endonuclease CEL I and cut strands are visualized using Li-cor gel analyzers. Putative polymorphisms detected in one fluorescence channel can be verified by appearance of the opposite cut strand in the other channel. We demonstrated the efficiency of this technology, called Ecotilling, by the discovery in 150+ individuals of 55 haplotypes in five genes, ranging from sequences differing by a single nucleotide polymorphism to those representing complex haplotypes. The discovered polymorphisms were confirmed by sequencing and included base-pair changes, small insertions and deletions, and variation in microsatellite repeat number. Ecotilling allows the rapid detection of variation in many individuals and is cost effective because only one individual for each haplotype needs to be sequenced. The technology is applicable to any organism including those that are heterozygous and polyploid.

DNA, Plant↗

PubMed2003

Spectrum of chemically induced mutations from a large-scale reverse-genetic screen in Arabidopsis.

Chemical mutagenesis has been the workhorse of traditional genetics, but it has not been possible to determine underlying rates or distributions of mutations from phenotypic screens. However, reverse-genetic screens can be used to provide an unbiased ascertainment of mutation statistics. Here we report a comprehensive analysis of approximately 1900 ethyl methanesulfonate (EMS)-induced mutations in 192 Arabidopsis thaliana target genes from a large-scale TILLING reverse-genetic project, about two orders of magnitude larger than previous such efforts. From this large data set, we are able to draw strong inferences about the occurrence and randomness of chemically induced mutations. We provide evidence that we have detected the large majority of mutations in the regions screened and confirm the robustness of the high-throughput TILLING method; therefore, any deviations from randomness can be attributed to selectional or mutational biases. Overall, we detect twice as many heterozygotes as homozygotes, as expected; however, for mutations that are predicted to truncate an encoded protein, we detect a ratio of 3.6:1, indicating selection against homozygous deleterious mutations. As expected for alkylation of guanine by EMS, >99% of mutations are G/C-to-A/T transitions. A nearest-neighbor bias around the mutated base pair suggests that mismatch repair counteracts alkylation damage.

Arabidopsis↗

PubMed2003

Large-scale discovery of induced point mutations with high-throughput TILLING.

TILLING (Targeting Induced Local Lesions in Genomes) is a general reverse-genetic strategy that provides an allelic series of induced point mutations in genes of interest. High-throughput TILLING allows the rapid and low-cost discovery of induced point mutations in populations of chemically mutagenized individuals. As chemical mutagenesis is widely applicable and mutation detection for TILLING is dependent only on sufficient yield of PCR products, TILLING can be applied to most organisms. We have developed TILLING as a service to the Arabidopsis community known as the Arabidopsis TILLING Project (ATP). Our goal is to rapidly deliver allelic series of ethylmethanesulfonate-induced mutations in target 1-kb loci requested by the international research community. In the first year of public operation, ATP has discovered, sequenced, and delivered >1000 mutations in >100 genes ordered by Arabidopsis researchers. The tools and methodologies described here can be adapted to create similar facilities for other organisms.

Alkylating Agents↗

PubMed2003

High-throughput TILLING for functional genomics.

Targeting-induced local lesions in genomes (TILLING) is a general strategy for identifying induced point mutations that can be applied to almost any organism. Here, we describe the basic methodology for high-throughput TILLING. Gene segments are amplified using fluorescently tagged primers, and products are denatured and reannealed to form heteroduplexes between the mutated sequence and its wild-type counterpart. These heteroduplexes are substrates for cleavage by the endonuclease CEL I. Following cleavage, products are analyzed on denaturing polyacrylamide gels using the LI-COR DNA analyzer system. High-throughput TILLING has been adopted by the Arabidopsis TILLING Project (ATP) to provide allelic series of point mutations for the general Arabidopsis community.

DNA, Plant↗

PubMed2002

Specific chromosomal aberrations in mouse lung adenocarcinoma cell lines detected by spectral karyotyping: a comparison with human lung adenocarcinoma.

Although adenocarcinoma is rapidly becoming the most common form of lung cancer in the United States, the difficulty in obtaining lung cancer families and representative samples of the various stages of adenocarcinoma progression has led to intense study of mouse models. As a powerful approach to delineating molecular changes, we have analyzed 15 early-passage mouse cell lines by spectral karyotyping. Entire copies of chromosomes 1, 2, 6, 12, 15, and 19 were gained, and entire copies of chromosomes 4, 7, 8, and 14 were lost. Significant gains of portions of chromosome 1 (93% of the tumor cell lines analyzed), chromosome 2 (53%), chromosome 6 (73%), chromosome 7 (80%), chromosome 12 (47%), and chromosome 15 (73%) and partial loss of chromosome 4 (87%), chromosome 7 (80%), chromosome 8 (53%), chromosome 10 (33%), and chromosome 14 (33%) were observed. Recurrent translocations included 10:del(10)(A1::C1), t(4;8)(C4;A1), and der (1;12)(C2;C2). The minimal regions of chromosomal alteration, 1G1, 2F1, 4C4, 6D, 7F1, 8B3, and 12C2, contain putative susceptibility genes for mouse lung adenocarcinoma. Chromosomal regions containing susceptibility genes linked to tumor size were frequently amplified, whereas regions with susceptibility loci linked to tumor multiplicity were deleted. Similar linkage groups are altered in human lung adenocarcinoma, implying that the mouse is a valid genetic model for the study of human lung adenocarcinoma susceptibility.

Adenocarcinoma↗

PubMedDate not supplied

Aberrant gene expression in human non small cell lung carcinoma cells exposed to demethylating agent 5-aza-2'-deoxycytidine.

The identification of genes undergoing genetic or epigenetic alterations and contributing to the development of cancer is critical to our understanding of the molecular mechanisms of carcinogenesis. A new approach in identifying alterations of genes that might be relevant to the process of tumor development was used in this study by examining the gene expression profile in human lung cancer cells exposed to 5-aza-2'-deoxycytidine (5-aza-dC). A cDNA array analysis was carried out on 5-aza-dC-treated and untreated non small cell lung cancer (NSCLC) cell line NCI-H522. Sixteen and 14 genes were upregulated and downregulated, respectively, by 5-aza-dC treatment. Among them, downregulation of tyrosine protein kinase ABL2 (ABL2) gene and upregulation of hint/protein kinase C inhibitor 1 (Hint/PKCI-1), DVL1, TIMP-1, and TRP-1 genes were found in expanded observations in two or three of five 5-aza-dC-treated NSCLC cell lines. Among these genes, we found that cDNA transfer of Hint/PKCI-1 resulted in a significant in vitro growth inhibition in two cell lines exhibiting 5-aza-dC-induced upregulation of Hint/PKCI-1 and significantly reduced in vivo tumorigenicity of one NSCLC cell line. Hint/PKCI-1, which is the only other characterized human histidine triad (HIT) nucleotide-binding protein in addition to tumor-suppressor gene FHIT, might be involved in lung carcinogenesis.

Antimetabolites, Antineoplastic↗