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Biomedical subjects

D Busbee

Publications and source records attributed to D Busbee.

At least 19 recordsLinked to original sources
PubMed2006

Altered gene expression in human cells treated with the insecticide diazinon: correlation with decreased DNA excision repair capacity.

Many industrial and agricultural chemicals have steroid hormone agonist or antagonist activities and disrupt hormone-regulated gene expression. The widely-used agricultural insecticide, diazinon, was evaluated using MCF-7 cells - a breast cancer-derived, estrogen-dependent, human cell line - to examine the capacity of this chemical to alter steroid hormone-regulated gene expression. MCF-7 cells were treated with 30, 50, or 67 ppm of diazinon, and gene expression in treated cells was measured as mRNA levels in the cells compared to mRNA levels in untreated or estrogen-treated cells. DNA microarray analysis showed significant up- or down-regulation of a number of genes in treated cells compared to untreated cells. Of the 600 human genes on the chip utilized, specific genes with related functions were selected for additional consideration. Real time quantitative PCR (qrtPCR) completed to corroborate mRNA levels as a measure of specific gene expression, confirmed results obtained from analysis of the micro-array data. The data show that ERCC5, encoding Xeroderma pigmentosum protein G (XPG), essential for DNA excision repair, and ribonucleotide reductase subunit M1 (RNRM1), encoding a gene necessary for providing the nucleotides needed for DNA repair, were down-regulated in cells treated with diazinon. These studies were designed to provide base-line data on the gene expression-altering capacity of a specific agricultural chemical, diazinon, and allow assessment of some of the potentially deleterious effects associated with exposure of human cells to diazinon.

Cell Line, Tumor↗
PubMed2006

Alteration of gene expression in human cells treated with the agricultural chemical diazinon: possible interaction in fetal development.

Agricultural chemicals frequently alter human health or development, typically because they have endocrine agonist or antagonist activities and alter hormone-regulation of gene expression. The insecticide, diazinon, was evaluated for gene expression disrupting activity using MCF-7 cells, an estrogen-dependent human cell line, to examine the capacity of the insecticide to disrupt gene expression essential for morphological development, immune system development or function, and/or central nervous system development and function. MCF-7 cells were treated with 30, 50 or 67 ppm diazinon, and gene expression was measured in treated cells compared to expression in untreated or estrogen-treated cells. DNA microarray analysis of diazinon-treated cells showed significant up- or down-regulation of a large number of genes compared to untreated cells. Of the 600 human genes on the Phase 1 chip utilized for these studies, two specific genes--calreticulin and TGF-beta3--were selected for corroboration using quantitative real time PCR (qrtPCR). qrtPCR, completed to assess gene expression levels for calreticulin and TGFbeta3, confirmed results showing significant up-regulation of these two genes obtained from the microarray data. These studies were designed to provide baseline data on the gene expression-altering capacity of a specific chemical, diazinon, and allow a partial assessment of the potentially deleterious effects associated with exposure of human cells to this chemical. Currently, it is not known whether results from cells in vitro can be extrapolated to human health consequences of chemical exposure.

Agrochemicals↗
PubMed2004

Generation and partial characterization of a transformed cetacean cell line.

A primary epithelial cell line, DK1, established from renal tissue of a spontaneously aborted female Atlantic bottlenose dolphin was transfected with linearized pSV3.neo, an SV40 virus-derived plasmid encoding large tumor antigen (Tag). Transfected cells were grown in cetacean culture medium supplemented with 400 microg/ml geneticin (G418), and individual clones were selected using cloning rings. DKN1 was the first clone to be evaluated for future research use, and has been continuously cultured for 8 years. Intracellular cytokeratin and the expression of Tag were determined in DKN1, and cell growth was evaluated under different concentrations of l-glutamine, glutathione, and N-acetylcysteine. DKN1 cells did not require high levels of l-glutamine as previously reported for cetacean cells, and addition of antioxidants at the concentrations used in this study (2.0mM) decreased the rate of cell division. These data suggest strongly that these immortalized bottlenose dolphin epithelial cells have different levels of, and requirements for, glutathione than would be considered normal for terrestrial mammalian cells, do not require high levels of l-glutamine as previously suggested for dolphin cells, and exhibit decreased levels of cell growth and viability in high levels of the antioxidant GSH and its precursor, NAC.

Acetylcysteine↗
PubMed2004

Altered gene expression in human cells induced by the agricultural chemical Enable.

Steroid hormones bind to highly specific nuclear receptors, regulating gene expression that results in normal fetal growth and development and/or in normal adult physiological function. Many industrial and agricultural chemicals may bind one or more nuclear receptors, acting as mimics of steroid hormones, and are called endocrine disruptive chemicals (EDC) because they alter the expression of endocrine-regulated genes. A widely used fungicide, Enable (fenbuconazole), was evaluated to examine its capacity to alter endocrine-regulated gene expression. Cells of an oestrogen-dependent human breast cancer-derived line, MCF-7, were treated with a range, 0.033-3.3 ppb (ng/mL), of Enable, and gene expression was compared to that of untreated cells. Microarray analysis using a chip with 600 gene spots showed downregulation of eight genes and upregulation of 34 genes in cells treated with 3.3 ppb of Enable, compared to untreated cells. Specific genes were selected for consideration. Real-time PCR confirmed results obtained from analysis of the microarray data for the genes phenol sulphotransferase (PST), intercellular adhesion molecule-1 (ICAM-1), transforming growth factor beta-3 (TGF beta-3) and calreticulin. These studies were designed to provide base-line data on the gene expression-altering capacity of a specific chemical at a low dose, and will allow assessment of the possible deleterious effects that may be caused in human cells by exposure to the agricultural chemical Enable.

Agriculture↗
PubMed2000

Activity of DNA polymerase alpha in aging human fibroblasts.

During the past decade intense investigation has focused on cellular aging with the expectation of discovering factors that regulate the replication complex and contribute to the onset and progression of cellular aging. The most striking feature of cellular aging is the failure of sensing diploid cells to enter or complete S phase of the cell cycle. The G1/S phase transition is an initial critical step in the regulation of proliferation in eukaryotic cells, and significant advances have been made toward understanding the basic mechanisms of aging by identifying components of the macromolecular assemblies participating in the G1/S transition. These studies have identified multiple DNA polymerases and their accessory factors, and have provided important strategies for investigating the molecular events that contribute to aging processes. DNA replication, repair and recombination in eukaryotic cells require the action of a variety of DNA polymerases, at least six of which are known, alpha, beta, gamma, delta, epsilon, and zeta. Among them the highly conserved DNA polymerase alpha-primase (pol alpha-primase) is the only enzyme capable of initiating DNA replication at chromosomal origin sites and at sites of initiation of discontinuous synthesis of Okazaki fragments on the lagging side of the replication fork. Numerous protein factors that play strategic roles in DNA replication have been identified and the understanding of their regulation has been an important step for identifying the elements that are involved in, and possibly necessary for, governing cellular senescence and aging. In this review we summarize the current information regarding DNA pol alpha modulation during aging. We focus in particular on the coordinated actions of DNA pol alpha in the presence of other cellular proteins involved in the replication complex in the hope that understanding pol alpha interactions with components of the replication complex may provide insight into the mechanisms by which aging and age-related diseases occur.

Aging↗
PubMed2000

A rapid and sensitive reporter gene that uses green fluorescent protein expression to detect chemicals with estrogenic activity.

A reporter gene sequence was constructed within a eukaryotic expression vector. The altered plasmid contained 2 sequential estrogen response elements (ERE) coupled to a human phosphoglycerate kinase (PGK) promoter inserted upstream from a cDNA sequence encoding enhanced green fluorescent protein (GFP) with a 3'-polyadenylation signal. The plasmid was linearized and transfected into MCF-7 cells, a human breast cancer-derived line that expresses the estrogen receptor (ER). No selectable marker was present in the plasmid, requiring stably transfected cells to be selected by fluorescence-activated cell sorting based on GFP expression after the cells were treated with 10(-9) M 17beta-estradiol (E2). Stably transfected MCF-7 cells (MCF7-ERE) exhibited 2000-3000 times more fluorescence at 488 nm excitation and 512 nm emission than non-transfected cells. MCF7-ERE cells exhibited a linear increase in GFP expression induced over a range of 10(-12) M E2, a concentration giving 2 times the background expression, to maximal expression at 3 x 0(-10) M E2. From the maximal level, GFP expression plateaued, and then declined when E2 was increased to the highest concentration tested, 10(-7) M. 4-Hydroxytamoxifen (TFN-OH) treatment of cells produced a dose-dependent inhibition of E2-induced GFP expression, indicating the interaction of ER in the regulation of GFP gene expression. A series of estrogenic chemicals were evaluated for their capacity to induce GFP expression in MCF7-ERE cells, showing induced expression of GFP at concentrations 2-4 log units higher than the E2 concentration giving maximal GFP expression. The ERE-PGK-GFP reporter gene system is capable of rapid GFP expression in the presence of low concentrations of E2, and of quantifying estrogenicity of chemicals compared with a standard curve of the natural ligand, 17beta-estradiol.

Cell Division↗
PubMed1997

A DNA polymerase alpha accessory protein exhibits structural and functional similarities to SV40 large tumor antigen.

Untransformed cells have been proposed to require a protein homologous to SV40 large tumor antigen (TAg) which functions as a component of the replicase complex during the initiation of DNA synthesis. By definition, this should be a phosphoprotein which interacts with the retinoblastoma protein (pRb) in G0 or early G1, and is capable of binding to and potentiating the activity of DNA polymerase alpha (pol alpha). This protein should also be an ATP-dependent helicase which interacts with the single-stranded DNA (ssDNA) binding protein, RP-A. Because of these requirements, a TAg homologous protein could be expected to contain epitopes with amino acid sequences similar to those of TAg at critical functional sites, such as ATP, pRb and pol alpha binding sites. TAg and a putative cellular homolog of TAg, DNA pol alpha accessory protein (alpha AP), were compared for pRb and pol alpha interaction, and for immunological identity. The analyses utilized immunoaffinity-purified TAg and pRb from a baculovirus expression system, and DNA pol alpha/primase and alpha AP chromatographically isolated from a mouse lymphocytic leukemia cell line. Monoclonal antibodies specific for the pol alpha or pRb binding sites on TAg interacted with alpha AP strongly enough to be employed for immunoaffinity purification of alpha AP. Anti-pRb and anti-TAg reciprocally coimmunoprecipitated pRb bound to TAg and pRb bound to alpha AP. The functional consequences of pol alpha interaction with TAg or alpha AP in the presence or absence of pRb was determined using pol alpha nucleotide incorporation assays. alpha AP exhibited the capacity to stimulate pol alpha activity, a capacity which was diminished in the presence of pRb. Lastly, TAg and alpha AP independently co-purified with pol alpha through a multi-step chromatographic protocol. These data indicate that a pol alpha accessory protein, alpha AP, exhibits functional and immunological similarities to SV40 TAg, suggest that alpha AP is involved in regulation of the initiation of DNA synthesis, and support the proposal that alpha AP may be a normal cell protein homologous to SV40 large T antigen.

Animals↗
PubMed1996

An accessory protein enhances both DNA binding and activity of DNA polymerase alpha isolated from normal, but not transformed, human fibroblasts.

DNA polymerase alpha/primase (pol alpha) isolated from fibroblasts established from a 66-year-old human donor (GM3529) exhibited decreased specific activity compared with pol alpha from either fetal-derived fibroblasts (WI38), or pSV3.neo-transformed GM3529 fibroblasts. The pol alpha specific activity decrease was correlated with a decreased proliferative capacity frequently seen in cells from aged donors. Pol alpha isolated from pSV3.neo-transformed GM3529 cells (GM3529T) exhibited a single isoform with about 10-fold higher specific activity than pol alpha from GM3529 cells. GM3529T pol alpha was immunoreactive with both anti-pol alpha and anti-SV40 large tumor antigen. Polymerases from GM3529 and GM3529T cells were treated with a pol alpha accessory protein, alpha AP, isolated from L1210 cells. Pol alpha from GM3529T cells showed no increase in activity in the presence of alpha AP, while pol alpha isolated from GM3529 cells exhibited about an 8-fold increase in activity after treatment with alpha AP. Double stranded SV40 DNA containing multiple ori sequences exhibited a greater decrease in electrophoretic mobility in the presence of GM3529T pol alpha than when treated with GM3529 pol alpha. In the presence of pol alpha from either GM35229 or GM3529T cells SV40 dsDNA exhibited a decrease in electrophoretic mobility, and in each instance addition of alpha AP resulted in an even greater decrease in DNA mobility. These data indicate that alpha AP increased pol alpha binding to SV40 dsDNA, or that alpha AP bound the DNA in addition to previously bound pol alpha. GM3529 pol alpha also bound non-specific, non-SV40, dsDNA, whereas GM3529T pol alpha with associated TAg did not bind the non-viral dsDNA unless alpha AP was added to the preparation. While not all human diploid fibroblast cell lines derived from aged human donors necessarily exhibit decreased proliferative capacity compared with cells from young donors, decreased specific activity associated with a decline in cellular DNA synthesis is typical of pol alpha from cells derived from aged human donors. We suggest that a decrease in endogenous alpha AP interaction with pol alpha may account, in part, for the loss of DNA binding affinity and specific activity of pol alpha from GM3529 cells derived from an aged donor.

Aged↗
PubMed1993

Immunofluorescent evaluation of DNA repair synthesis using interactive laser cytometry.

An improved differential fluorescence analysis technique which employs scanning laser cytometry for the detection of two DNA binding fluorochromes was used to quantitate both total DNA and cellular incorporation of the base analogue, bromodeoxyuridine (BrdUrd), into UV- or methyl methane sulphonate (MMS)-treated DNA. In this procedure DNA containing BrdUrd was partially denatured and immunolabeled by binding anti-BrdUrd monoclonal IgG to incorporated BrdUrd. Anti-BrdUrd was detected with a secondary fluorochrome-labeled polyclonal anti-IgG. Cells were counterstained with propidium iodide to allow the determination of total DNA. Fluorescence was determined using the Meridian ACAS 570 Scanning Laser Cytometer. The computer-analyzed images were generated from monodisperse populations of UV- or MMS-treated cells, allowing the evaluation of DNA synthesis associated with excision repair and of total DNA content in single cells. These data were compared with 3H-thymidine incorporation occurring as a function of excision repair. The analysis of DNA synthesis with this technique is consistent, relatively simple, and rapid with excellent sensitivity and provides a viable method for determining cycling vs. non-cycling cells, total cellular DNA, and excision repair-associated DNA synthesis of individual cells within heterogeneous cell populations.

Bromodeoxyuridine↗
PubMed1993

Age-related changes in expression and activity of DNA polymerase alpha: some effects of dietary restriction.

DNA polymerase alpha (pol alpha) purified from human diploid fibroblasts (HDF) and from livers of C57BL/6N mice showed age-related decreases in: (1) mRNA levels; (2) the amount of enzyme isolated per cell; and (3) enzyme activity (HDF); as well as: a) the amount of enzyme isolated; b) the specific activity; and c) the enzyme fidelity (liver). Hepatic pol alpha from dietary restricted (DR) mice exhibited less of a decline in specific activity and copied synthetic DNA templates with relatively higher fidelity than did enzymes from animals fed ad libitum (AL). Pol alpha from fetal-derived HDF exhibited increased expression compared with aged donor-derived HDF, with both fetal and old cell pol alpha in normal cells being expressed at lower levels than in their transformed cell corollaries. Treatment of human pol alpha from aged donor-derived HDF with a pol alpha accessory protein isolated from log phase murine cells resulted in increased pol alpha binding of DNA and increased pol alpha activity. However, highly active pol alpha isolated from fetal-derived or transformed HDF, or from transformed murine cells, showed little or no activity enhancement in the presence of accessory protein. These data indicate that, as a function of increased age, there is a decrease in pol alpha expression and specific activity in HDF, as well as decreases in specific activity and fidelity of pol alpha in essentially amitotic murine hepatic tissues. Dietary restriction impedes the age-related declines in both activity and fidelity of hepatic pol alpha in mice. The data further indicate that transformation of slowly dividing HDF is associated with increased expression of pol alpha, but suggest that increased expression alone is not sufficient to explain the difference in polymerase activity levels between parental and transformed HDF. Lastly, the data suggest that interaction of pol alpha with an essential accessory protein may be altered as a function of age, an alteration that appears to be correlated with the decline in pol alpha DNA binding and specific activity.

Aging↗
PubMed1992

Enhanced unscheduled DNA synthesis by secondary cultures of lung cells established from calorically restricted aged rats.

Unscheduled DNA synthesis (UDS) induced by two exposure levels of ultraviolet light (UV) or two concentrations of methyl methane sulfonate (MMS) was evaluated in secondary cultures of lung fibroblasts established from weanling, 11-month-old and 31-month-old female Fischer 344 rats fed ad libitum (AL) or calorically restricted (CR) diets. [3H]Thymidine incorporation as a function of UDS was highest for weanling-derived cells treated with either UV or MMS, declining consistently with increased age between cells from weanling, 11-month-old and 31-month-old animals. [3H]Thymidine incorporation as a function of UDS in cells from 11-month-old AL vs. CR rats differed only at the highest UV exposure level. In contrast, cells derived from 31-month-old CR rats exhibited UDS levels which were at least twice as high at each UV treatment level as UDS levels of cells derived from the same age AL rats. Cells from both old AL and old CR rats were shown to initiate DNA excision repair at about the same rate. Cells from CR rats, however, repaired DNA damage at an accelerated rate and completed excision repair while repair in cells from AL animals was slower and apparently did not proceed to completion. Data from this study indicate that cells from young and old AL and CR animals initiate excision repair, but demonstrate an age-related loss of UV- or MMS-stimulated [3H]thymidine incorporation in cells derived from AL animals. Cells derived from CR animals did not exhibit that age-related loss of UDS activity; rather, they showed an enhanced UDS response to DNA damage and appeared to complete ligation as the final step in excision repair. The data suggest that caloric restriction of a cell donor animal not only delays the age-associated decrease in in vitro DNA excision repair capacity in cells from that animal, but may actually enhance repair capacity.

Aging↗
PubMed1991

Effects of aging and dietary restriction on DNA polymerase expression in mice.

DNA polymerase alpha was isolated from livers of 6-month-, 16-month-, or 26-month-old mice fed ad libitum, or calorically restricted. The enzymes differed in chromatographic characteristics, binding affinity for DNA, and activity, with both total activity and specific activity of DNA polymerase alpha decreasing as a function of age. A positive correlation was observed between polymerase alpha specific activity and the affinity of enzyme binding to activated DNA template-primer. The age-associated decline in enzyme activity was modified by dietary restriction, with measurably higher activity seen for polymerases from dietary restricted animals compared with ad libitum animals of all ages. The data suggest that dietary restriction could act to delay the age-associated decrease in cellular capacity for DNA synthesis, which may play a significant role in prolonging the onset of age-related diseases in which decreased DNA synthesis is a potential component.

Aging↗
PubMed1991

Decreased mortality of Norman murine sarcoma in mice treated with the immunomodulator, Acemannan.

An extract from the parenchyma of Aloe barbadensis Miller shown to contain long chain polydispersed beta (1,4)-linked mannan polymers with random O-acetyl groups (acemannan, Carrisyn) was found to initiate the phagocyte production of monokines that supported antibody dependent cellular cytotoxicity and stimulated blastogenesis in thymocytes. Acemannan, in both enriched and highly purified forms, was administered intraperitoneally to female CFW mice into which murine sarcoma cells had been subcutaneously implanted. The rapidly growing, highly malignant and invasive sarcoma grew in 100% of implanted control animals, resulting in mortality in 20 to 46 days, dependent on the number of cells implanted. Approximately 40% of animals treated with acemannan at the time of tumor cell implantation (1.5 x 10(6) cells) survived. Tumors in acemannan-treated animals exhibited vascular congestion, edema, polymorphonuclear leukocyte infiltration, and central necrosing foci with hemorrhage and peripheral fibrosis. The data indicate that in vivo treatment of peritoneal macrophages stimulates the macrophage production of monokines, including interleukin-1 and tumor necrosis factor. The data further indicate that sarcomas in animals treated i.p. with acemannan at the time of tumor cell implantation were infiltrated by immune system cells, became necrotic, and regressed. The combined data suggest that acemannan-stimulated synthesis of monokines resulted in the initiation of immune attack, necrosis, and regression of implanted sarcomas in mice.

Adjuvants, Immunologic↗
PubMed1990

Mitogen-activation of spleen cells in aged animals is potentiated by dietary restriction: a preliminary report.

Dietary restriction has been reported to be associated with increased life span and increased DNA repair capacity in both male and female rats and mice. We examined dietary restriction effects on immune system function and prolongation of life span in specific pathogen free rats and mice. In this preliminary report the authors show that dietary restriction is correlated with a dramatic increase in the lifespan of both male and female rats and mice in a pathogen free environment, and in the capacity of cultured splenocytes from those animals to initiate blastogenesis in response to antigenic stimulation.

Aging↗