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Biomedical subjects

C W Miller

Publications and source records attributed to C W Miller.

At least 37 records · Page 2Linked to original sources
PubMed2000

Allelotype analysis of the myelodysplastic syndrome.

Myelodysplastic syndromes (MDS) are a group of clonal hematologic disorders found predominantly in the elderly. The molecular mechanisms underlying the development of MDS remain obscure. In order to begin to identify tumor suppressor genes involved in these disorders, we performed a detailed microsatellite allelotype of chromosomal deletions associated with MDS. DNAs from both bone marrow and peripheral blood of 32 MDS patients were studied using 84 highly informative microsatellite markers on all autosomal arms, excluding the short arms of the acrocentric chromosomes. A high percentage of loss of heterozygosity (LOH) was identified on chromosome 5q (40% of informative cases), 7q (45%), 17p (23%) and 20q (20%), which corresponds to the most common cytogenetic abnormalities reported in MDS. In addition, a high incidence of LOH (> or =20%) was observed on chromosomal arms which had not been previously reported including 1p (36%), 1q (35%), and 18q (23%). This extensive allelotype analysis focuses attention on several novel genomic regions that probably contain novel tumor suppressor genes whose loss of function contributes to the development of MDS.

Adult↗
PubMed2000

Evaluation of the invader assay, a linear signal amplification method, for identification of mutations associated with resistance to rifampin and isoniazid in Mycobacterium tuberculosis.

We evaluated a recently described linear signal amplification method for sensitivity and specificity in detecting mutations associated with resistance to rifampin (RIF) and isoniazid (INH) in Mycobacterium tuberculosis. The assay utilizes the thermostable flap endonuclease Cleavase VIII, derived from Archaeoglobus fulgidus, which cleaves a structure formed by the hybridization of two overlapping oligonucleotide probes to a target nucleic acid strand. This method, termed the Invader assay, can discriminate single-base differences. Nine pairs of probes, encompassing five mutations in rpoB and katG that are associated with resistance to either RIF or INH, as well as the corresponding wild-type (drug-susceptible) alleles, were tested using amplified DNA. Fluorescent-labeled cleavage products, ranging from 4 to 13 nucleotides in length, depending on the genotype of the test sample, were separated by denaturing polyacrylamide (20 to 24%) gel electrophoresis and then detected by scanning. All nine alleles could be identified and differentiated on the basis of product size. Multiple mutations at a specific rpoB nucleotide in target PCR products could be identified, as could mutants that were present at > or =0.5% of the total population of target sequences. The Invader assay is a sensitive screen for some mutations associated with antituberculosis drug resistance in amplified gene regions.

Antitubercular Agents↗
PubMed1999

Rb independent inhibition of cell growth by p15(INK4B).

The INK4 cyclin dependent kinase inhibitors (CDKI), such as p15(INK4B) and p16(INK4A), block cell cycle progression from G to S phase. This is mediated by inhibition of phosphorylation of proteins, including the retinoblastoma susceptibility protein (Rb), by cyclin dependent kinases. Ectopic over-expression of the p16(INK4A) CDKI can inhibit growth of cell lines depending on Rb status. Cell lines lacking Rb, with few exceptions, are resistant to growth inhibition by p16(INK4A). The effects of ectopic over-expression of p15(INK4B) in cell lines with and without wild type Rb were examined by measuring cell recovery. Proliferation was inhibited in cells lacking Rb as well as in cells with wild type Rb expression. Experiments analyzing the effectiveness of chimeric p15(INK4B)/p16(INK4A) proteins indicated that the Rb independent growth inhibition required N-terminal residues of p15(INK4B). Linker insertion mutation of p15(INK4B) showed that the inhibition was dependent on intact ankyrin structures. Double staining flow cytometry found that the growth inhibition correlated with a decrease in cells in G2/M phases of the cell cycle. These findings are consistent with Rb independent inhibition of the progression from G1 to S caused by overexpression of p15(INK4B).

Carrier Proteins↗
PubMed1999

Loss of p73 gene expression in leukemias/lymphomas due to hypermethylation.

The p73 gene, a member of the p53 family, is a new candidate tumor suppressor gene. To investigate the possibility of genetic alteration of p73 in leukemia and lymphoma, we examined 55 cell lines and 39 patient samples together with 17 nonhematopoietic cancer cell lines. Gene expression of p73 was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) in cell lines (5 of 7 pre B/B-acute lymphoblastic leukemia [ALL], 13 of 21 T-ALL/lymphoblastic lymphomas [LBL], 9 of 10 B-non-Hodgkin's lymphomas [B-NHL], 8 of 9 acute myelogenous leukemias [AML], 2 of 2 T-NHL, 3 of 3 multiple myeloma), and in patient samples (16 of 23 pre B-ALL, 5 of 8 T-ALL/LBL, 5 of 8 B-NHL). PCR-single-strand conformation polymorphism (SSCP) of cDNAs showed no mutation in 43 p73-expressing cell lines within the regions that corresponded to the 5 mutational hotspots of the p53 gene. Neither homologous deletion nor rearrangement of the p73 gene were found by Southern blot analysis in any of the cell lines that lack expression of p73. In contrast to prior published data, analysis of a polymorphic site showed that the p73 gene was expressed biallelically in cell lines and normal peripheral blood. Notably, the p73-negative cell lines were hypermethylated at a CpG island in the 5' untranslated region of the p73 mRNA, and treatment of these cell lines with 5-azacytidine (5-AC), a demethylation reagent, induced p73 expression. Taken together, we found that a sizable proportion (32%) of ALL/B-NHL cell lines and primary tumors had negligible or limited expression of the p73 gene associated with hypermethylation of the gene. These findings suggest that silencing of the p73 gene by hypermethylation may contribute to development and/or progression of lymphoid neoplasms.

DNA Methylation↗
PubMed1999

Translocon-associated protein alpha transcripts are induced by granulocyte-macrophage colony-stimulating factor and exhibit complex alternative polyadenylation.

The cloning of full length cDNA for the translocon-associated protein alpha subunit, previously called signal sequence receptor alpha, is reported as a result of differential display experiments in search of genes induced by granulocyte-macrophage colony-stimulating factor. Its messenger RNA was more abundant in growing cells than in either factor-deprived cells or quiescent cells and comprised four species, each having microheterogeneity, as a result of complex alternative polyadenylation apparently dependent on arrays of non-canonical polyadenylation signals. Radiation hybrid mapping of the gene showed that the gene is on the short arm of chromosome 6.

Base Sequence↗
PubMed1999

Detection of free radicals in reperfused dog skin flaps using electron paramagnetic resonance spectroscopy: a pilot study.

This is a preliminary investigation of the usefulness of spin-trapping electron paramagnetic resonance (EPR) in detecting oxygen-derived free radicals after reperfusion of ischemic skin flaps. Fourteen island skin flaps approximately 12 cm long by 6 cm wide based on the caudal superficial epigastric vessels were isolated in seven dogs. Seven of the paired skin flaps were subjected to 4 hours of arterial and venous occlusion. The opposite skin flap on each dog served as a perfused control. Spin trapping-EPR was able to detect radical adducts in five of the seven reperfused skin flaps. Hyperfine splitting constants of the radical adducts from the blood of three of the flaps confirmed biologically derived free radical production. Contaminating EPR signals from free radicals created in the manufacture of plastics probably masked any biologically derived radical adducts in the remaining flaps. The authors conclude that EPR-spin trapping may be a valuable tool in the study of the importance of oxygen-derived free radicals in the failure of skin flaps subjected to transient ischemia.

Animals↗
PubMed1999

An application of the NCRP screening techniques to atmospheric radon releases from the former feed materials production center near Fernald, Ohio. National Council on Radiation Protection and Measurements.

The National Council on Radiation Protection and Measurements has published a series of screening models for releases of radionuclides to the environment. These models have been used to prioritize radionuclides being considered in environmental dose reconstructions. The NCRP atmospheric models are also accepted by the U.S. Nuclear Regulatory Commission for demonstrating compliance with the constraint on releases of airborne radioactive materials to the environment from licensees other than power reactors. This study tested the NCRP atmospheric techniques by comparing annual average predicted air concentrations of radon with measured radon concentrations at 14 locations 43 m to 598 m downwind of the former U.S. Department of Energy Feed Materials Production Center (FMPC) near Fernald, Ohio, for the period 2 July 1985 to 2 July 1986. Predictions were made using five different sets of meteorological data as input: (1) NCRP default values; (2) composite FMPC site data; (3) data from the Greater Cincinnati Airport; (4) data from the Dayton, Ohio, airport; and (5) data collected at Miami University, located near Oxford, Ohio. Following are the respective medians and ranges of the ratio of the predicted to observed annual radon air concentrations for each of these sources of meteorological data: (1) 5.2, 0.9-54; (2) 1.4, 0.1-8.2; (3) 0.7, 0.1-7.2; (4) 0.7, 0.1-8.4; and (5) 0.6, 0.1-10. The stated goal of the NCRP models is to predict doses that do not underpredict actual doses by greater than a factor of 10. In this comparison, all of the meteorological data produced air concentration predictions that meet this criteria. However, to ensure that final doses meet this criterion, one would need to carefully evaluate all assumptions used to calculate dose from each of these air concentrations.

Air Pollution, Radioactive↗
PubMed1998

Integration of SV40 in human osteosarcoma DNA.

Simian virus 40 (SV40) has been demonstrated in several types of tumors, including osteosarcoma, by polymerase chain reaction (PCR). We detected SV40 sequences by PCR, followed by hybridization, in nine of 35 osteosarcoma tumors and one of 11 osteosarcoma explants. PCR can detect fewer than one virus per cell but gives little detail of the gross structure and abundance of the virus. Analysis by Southern blotting of total DNA from ten osteosarcomas, positive for SV40 by PCR, found viral integration in half of these. Analysis showed integration of one to four copies per cell of rearranged SV40. No SV40 was detectable on blots of the remaining five SV40+ osteosarcomas, perhaps because of the lesser sensitivity of direct hybridization. Inactivation of the p53 and Rb tumor suppressors is a key activity of SV40 T-antigen. Unexpectedly, correlation of these findings with our prior studies indicated that five of ten osteosarcomas positive for SV40 DNA had mutations of p53, and two had deleted Rb. Apparently clonal integration with pre-existing alteration of a tumor suppressor gene, suggests that SV40 may play a role in the final conversion to malignant osteosarcoma.

Adolescent↗
PubMed1998

Using the Unger system to classify 386 long bone fractures in dogs.

A system already described by Unger and others was used to classify long bone fractures in dogs. The present paper reports experiences using the fracture classification system regarding its ease of use and the ability to analyse the data generated. Three hundred and eighty-six canine long bone fractures were classified from radiographs. Results were assessed by reviewing the medical records or by sending questionnaires to referring veterinarians. There were a few inconsistencies, particularly in classifying proximal ulnar fractures, but the system was easy to use and data retrieval was readily accomplished. Data from the system were used to compare the results of repairs of diaphyseal fractures of the radius/ulna, femur and tibia/fibula. A chi square analysis was used to determine significant differences between the outcome scores of the three fracture types. Fractures of the femoral diaphysis had a statistically poorer outcome than did diaphyseal fractures of the radius/ulna or tibia/fibula.

Animals↗
PubMed1997

HHV-8 (KSHV) does not establish latency in prostate cancer cell lines.

BACKGROUND: HHV-8 is a new herpesvirus found in lesions of Kaposi's sarcoma and some lymphoproliferative diseases. More recently, a report stated that normal prostate tissue also contains the virus. METHODS: The expression of HHV-8 was examined by a sensitive reverse-transcriptase PCR for the viral genes ORF 72, ORF 73, ORF 74, and ORF 75. In coculture experiments we attempted to infect 3 commonly studied prostate cancer cell lines using induced and uninduced lymphoid cell lines harboring HHV-8 (KS-1, BC-1, and BC-2). For induction of viral genes, butyrate and phorbol esters were used. RESULTS AND CONCLUSIONS: At baseline, prostate cancer cell lines LNCaP, DU-145, and PC-3 did not express viral gene products. Extensive coculture experiments were also negative. In no instance could latency for the virus be established. Our results argue against the involvement of HHV-8 in prostate cancer and for a limited tissue tropism of HHV-8.

B-Lymphocytes↗
PubMed1997

Localization of a polyunsaturated fatty acid response region in stearoyl-CoA desaturase gene 1.

Polyunsaturated fatty acids (PUFA) repress stearoyl-CoA desaturase gene 1 (SCD1) expression in liver and adipose tissues. We used HepG2 cells to localize genetic regulatory elements for PUFA in the SCD1 5'-flanking region. A chimeric reporter gene construct containing the 4.3 kb SCD1 putative promoter was transiently transfected into HepG2 cells, which were then treated with various fatty acids. We observed greater than 60% repression of transcription with 18:3n - 3 and 75% repression with 20:4n - 6 and 20:5n - 3. No significant change was seen with 18:0. Using smaller SCD1 chimeric constructs, we localized the genetic regulatory region to a 237 bp sequence within the SCD1 proximal promoter. DNA mobility shift analysis with HepG2 and mouse liver nuclear extracts demonstrated specific binding of nuclear proteins to this region. Mobility shift analysis with nuclear extract from 3T3-L1 adipocytes showed a similar pattern of protein binding. Competitive DNA mobility shift analysis identified a 60 bp region containing sites that specifically bind and compete for nuclear proteins. This region conferred responsiveness to PUFA when placed in a heterologous promoter. A homologous region in the stearoyl-CoA desaturase gene 2 (SCD2) promoter also mediated PUFA-specific repression in transfection experiments. These data suggest that a common transcriptional mechanism may exist in liver and adipose tissues for inhibition of lipogenesis by PUFA.

Adipocytes↗
PubMed1997

The p19INK4D cyclin dependent kinase inhibitor gene is altered in osteosarcoma.

Inhibition of cyclin dependent kinases (CDK) by cyclin dependent kinase inhibitors (CDKI) blocks cell cycle progression and inhibits cellular proliferation. The archetypical member of the INK4 CDKI family, p16INK4A (also called CDKN2), is a tumor suppressor frequently deleted or mutated in certain neoplasms and many cell lines. Because p19INK4D has strong structural and functional similarity to p16INK4A, we have assessed its role as a tumor suppressor. This was accomplished by screening the p19INK4D coding region for mutations, deletions and rearrangements in sarcomas and non-small cell lung cancers. Alterations of the p19INK4D gene were found in samples from five of 67 (7%) patients with osteosarcomas and none were found in other types of sarcomas or in lung cancers. Five osteosarcoma samples had Southern blot patterns consistent with gene rearrangement. These samples included a primary and recurrent osteosarcoma from the same patient; both with the same rearrangement. Four samples had SSCP patterns consistent with sequence alterations, sequencing determined that three were due to silent base changes and apparently polymorphisms. Sequencing the fourth shifted band revealed a one base insertion causing a frameshift beginning with codon 27. In summary, these studies found alterations affecting the p19INK4D gene in a small but significant number of osteosarcomas. Presumably, abnormalities of this gene contribute to the development of cancer of bone cells.

Carrier Proteins↗
PubMed1997

Localization of a negative thyroid hormone-response region in hepatic stearoyl-CoA desaturase gene 1.

The effect of thyroid hormone on stearoyl-CoA desaturase gene 1 (SCD1) expression was investigated in mouse liver. Daily injections of 15 micrograms triiodothyronine (T3)/100 g body weight to hypothyroid mice resulted in repression of SCD1 mRNA levels by more than 50% in 48 hours and up to 65% in 6 days. Transient co-transfections were performed with an expression vector for T3 receptor alpha (T3R alpha) in HepG2 cells using chimeric reporter gene constructs of the SCD1 5'-flanking region. Transcriptional repression of the SCD1 putative promoter was observed upon treatment with 100 nM T3 when cotransfected with T3R alpha, but not without cotransfection of receptor. Transient gene expression studies localized a T3 response region to a 70-bp sequence in the SCD1 putative promoter. Eliminating the TATA box and an AP-2 binding site, DNA mobility shift analysis demonstrated specific binding of in vivo nuclear protein from mouse liver nuclear extract to a 43-bp sequence. DNA mobility shift with purified T3R alpha confirmed the presence of a T3 receptor binding site in this thyroid hormone-responsive region. These data indicate that SCD1 contains a negative T3 response region in its proximal promoter.

Animals↗
PubMed1997

Regulation of hepatic stearoyl-CoA desaturase gene 1 by vitamin A.

The effect of vitamin A supplementation on stearoyl-CoA desaturase gene 1 expression in mouse liver was characterized. Normal BALB/c mice were fed 0.01% and 0.1% retinol palmitate as components of nonpurified diets. This treatment resulted in a 3-fold and a 7-fold induction of SCD1 mRNA levels, respectively, as determined by RNase protection analysis. Vitamin A-deficient animals were also fed diets containing 0.01% and 0.1% retinol palmitate, resulting in a similar pattern of SCD1 mRNA induction. Fatty acid synthase and beta-actin mRNA levels did not respond consistently or significantly to retinoic acid treatment. Dietary and hormonal studies were carried out to investigate the role of the retinoid X receptor in the regulation of SCD1 by type II steroid hormones. A receptor-saturating dose of thyroid hormone, triiodothyronine, repressed vitamin A-elevated SCD1 mRNA levels in vivo. Peroxisome proliferator-elevated SCD1 mRNA levels were unaffected by administration of thyroid hormone. This suggests that the retinoic acid receptor transcriptionally regulates SCD1 through a traditional mechanism of heterodimerization with the retinoid X receptor.

Actins↗
PubMed1997

Frequent loss of heterozygosity in the region of the D7S523 locus in advanced ovarian cancer.

Loss of heterozygosity (LOH) of the long arm of chromosome 7 occurs frequently in many types of primary cancers. We analyzed 22 primary ovarian cancers for LOH of chromosome arm 7q using a set of 16 microsatellite markers in order to determine the location of a putative tumor suppressor gene (TSG). Eleven samples (50%) showed LOH at least at one locus on chromosome arm 7q. We identified the smallest commonly deleted region to be at 7q31.1, which includes D7S523. LOH of chromosome arm 7q was more frequent in advanced stages (III-IV) (7/9, 78%) than in early stages (I-II) (4/13.31%) of ovarian cancer (P < 0.05). These data suggest that alteration of a TSG at 7q31.1 gene plays an important role in advanced ovarian cancer.

Chromosome Deletion↗
PubMed1997

Integrity of the 1,25-dihydroxyvitamin D3 receptor in bone, lung, and other cancers.

Differentiation and proliferation can be regulated in diverse cell types by 1,25-dihydroxyvitamin D3. These effects derive from modulation of gene expression mediated by the interaction of 1,25-dihydroxyvitamin D3 with the vitamin D receptor (VDR). The VDR is one of the nuclear hormone receptors. Because these transcription factors play a key role in growth control, some nuclear hormone receptors, such as the retinoic acid receptor alpha, can be disrupted in cancer. With these alterations in mind, we looked for alterations of the VDR gene in a variety of cancers, including 68 osteosarcomas, 23 other sarcomas, 34 non-small cell lung cancers, and 44 cell lines representing many tumor types. Gross integrity of the VDR gene was examined on Southern blots probed with the coding region of the VDR cDNA. The presence of point mutations targeting VDR exons 2-7 was assessed by polymerase chain reaction-single-strand conformation polymorphism analysis and direct DNA sequencing. Two alterations were detected; direct DNA sequencing of these samples revealed one silent mutation in codon 79 and a base change in intron 3. These results suggest that mutations and rearrangement of the VDR do not play a role in the cancers studied.

Blotting, Southern↗