Biomedical subjects

D R Crawford

Publications and source records attributed to D R Crawford.

At least 19 recordsLinked to original sources

PubMed2001

Maf genes are involved in multiple stress response in human.

The Maf protein family consists of eight transcription factors containing a basic-leucine zipper (bZIP) domain. We have previously reported that the mRNA to one of these members, mafG/adapt66, is induced by oxidative stress in hamster HA-1 cells. It has subsequently been reported that mafG is induced bystress that activates the expression of genes under the control of the antioxidant/electrophile response element (ARE/EpRE), and that small Maf proteins are present in ARE/EpRE-protein complexes. Here we extend these studies to assess the effects of various types of stress on maf mRNA induction in human cells. The oxidative stressor cadmium, and the heavy metals cadmium, zinc, and arsenite induced mafG RNA levels within two hours, and maximally at five hours for cadmium and zinc. This induction was observed for multiple transcripts including two not normally associated with mafG, suggesting that these stress agents induced the expression of other related maf family RNAs. Modest induction of mafG mRNA was also observed with heat shock but not calcium elevation. These results suggest that mafG is a human stress-response gene induced by multiple stress, and that several maf (proto-)oncogene members play an important role in cellular stress response.

Animals↗

PubMed2000

adapt78 protects cells against stress damage and suppresses cell growth.

We have previously identified several genes whose RNA products are induced in HA-1 hamster cells under conditions where a cytoprotective adaptive response is observed. One of these genes, designated adapt78, was found to have a human homolog with some homology to glucose-regulated protein 78 (Grp78). We subsequently determined that adapt78 and grp78 mRNAs are induced by the same stress agents and conclude that adapt78 is a stress-response gene and putative new member of the grp stress gene family. Here we extend these studies to assess the effect of overexpressing adapt78 on stress protection and growth arrest. HA-1 cells stably transfected with adapt78 cDNA were found to exhibit significantly reduced calcium- and hydrogen peroxide-mediated cytotoxicity as compared with control transfectants. In addition, adapt78 stable overexpressors exhibited significantly reduced cell growth. Both cytoprotection and growth arrest accompanied only modest overexpression of adapt78. Flow cytometry revealed that the growth arrest occurred in G(1)-phase. Immunoflourescent analysis revealed that Adapt78 protein exhibits significant perinuclear staining suggestive of endoplasmic reticulum localization in addition to cytoplasmic localization. These data indicate that adapt78 is both cytoprotective and growth suppressive and that these effects may be mediated by Adapt78 protein at the endoplasmic reticulum.

Adaptation, Physiological↗

PubMed2000

Polynucleotide degradation during early stage response to oxidative stress is specific to mitochondria.

Oxidative stress is known to modulate RNA expression in both prokaryotic and eukaryotic cells. We have previously determined that a preferential and calcium-dependent downregulation of mitochondrial RNA occurs in HA-1 hamster fibroblasts in response to hydrogen peroxide, and that this is accompanied by the degradation of mitochondrial genomic DNA. Here we extended these studies to determine whether downregulation is specific to transcripts derived from mitochondrial-encoded genes; to determine whether genomic DNA degradation occurs in the nucleus; and to compare overall polynucleotide stress response with cellular growth arrest and apoptosis. We observed that nuclear genome-encoded mRNAs whose protein products are targeted for the electron transport chain of mitochondria were not degraded. Furthermore, early stage degradation of genomic DNA, assessed within the first 5 h of peroxide exposure, was specific to mitochondria, as nuclear genomic DNA was not degraded under the same treatment conditions. These differential degradations occurred under conditions where extensive growth-arrest and moderate apoptosis were observed, and were accompanied by significant induction of the growth arrest mRNAs gadd45, gadd153, and adapt15/gadd7. Combined, these results indicate that there is a general degradation of mitochondrial- but not nuclear-polynucleotides during early stage response of HA-1 fibroblasts to oxidative stress.

Animals↗

PubMed2000

Control selection for RNA quantitation.

The study of mammalian gene expression is often carried out at the level of mRNA. In such analyses, one usually measures the amount of an mRNA of interest under different conditions such as stress, growth, development, cell and tissue localization or as part of an evaluation of the effects of gene transfection. A variety of techniques exist to measure gene expression and most commonly involve Northern hybridization analysis, ribonuclease protection or RT-PCR. Common to all of these assays is the inclusion of a so-called loading or internal control (i.e., analysis of an mRNA that does not change in relative abundance during the course of treatments). Here, we discuss the uses and pitfalls of the most popular of these controls, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and beta-actin, with special emphasis on precautions associated with the use of GAPDH.

Actins↗

PubMed1999

adapt78, a stress-inducible mRNA, is related to the glucose-regulated protein family of genes.

We have recently reported a new oxidant- and calcium-inducible mRNA, adapt78, from hamster HA-1 cells. The adapt78 mRNA is induced in HA-1 cells under conditions where a protective adaptive response is observed and contains a translatable open reading frame whose protein product shows strong homology to a human sequence. Computer analysis of the predicted Adapt78 protein sequence also revealed a stretch of amino acids homologous to a portion of the glucose-regulated protein78 (Grp78). Based on this homology, we tested the hypothesis that adapt78 may be a new member of the grp gene family. Toward this, we assessed the modulation of adapt78 mRNA by stress agents known to induce grp78. In HA-1 cells, adapt78 mRNA was induced by the calcium ionophore A23187, 2-deoxyglucose, brefeldin A, tunicamycin, thapsigargin, and cyclopiazonic acid, with thapsigargin being the most potent inducer (7.3-fold). As expected, grp78 mRNA was also induced by these agents in our model system. In contrast, heat shock treatment produced little if any modulation of either grp78 or adapt78. Differences were also observed, as adapt78 mRNA but not grp78 mRNA was induced by 160 microM hydrogen peroxide, and adapt78 demonstrated earlier induction kinetics for certain agents compared with grp78. adapt78 mRNA was also found to be induced in several different human cell lines. A23187 had the strongest effect on adapt78 mRNA levels in human cells, inducing greater than 20-fold in all human cell cultures tested. Furthermore, in vitro transcription translation of human adapt78 cDNA produced an Adapt78 protein product. We conclude that adapt78 may be a new member of the grp family of genes and may represent an early response grp that complements the actions of grp78 and grp94.

Amino Acid Sequence↗

PubMed1999

CREB binding protein coordinates the function of multiple transcription factors including nuclear factor I to regulate phosphoenolpyruvate carboxykinase (GTP) gene transcription.

Nuclear factor I (NFI) binds to a region of the phosphoenolpyruvate carboxykinase (GTP) (PEPCK) gene promoter adjacent to the cAMP regulatory element (CRE) and inhibits the induction of transcription from the gene promoter caused by the catalytic subunit of protein kinase A. In vivo footprinting studies demonstrated that both the CRE and the NFI-binding site are occupied by transcription factors, regardless of the presence of factors that stimulate (dibutyryl cAMP or dexamethasone) or inhibit (insulin) transcription from the PEPCK gene promoter. The NFI effects on transcription from the PEPCK gene promoter were observed even in the absence of the NFI binding site, suggesting the possibility of other weaker binding sites on the promoter or an interaction of NFI with a transcriptional co-activator. A mammalian two-hybrid system was used to demonstrate direct interaction between the transactivation domain of NFI-C and the CREB binding domain of the CREB-binding protein (CBP). Overexpression of a gene fragment encoding the CREB binding domain of CBP stimulates transcription from the PEPCK gene promoter. The inhibitory effect of NFI on transcription of the PEPCK gene induced by the catalytic subunit of protein kinase A appears to be the result of an interaction between NFI and the CREB-binding protein in which NFI competes with CREB for binding to the CREB-binding site on CBP. In contrast, glucocorticoids and thyroid hormone use the steroid hormone receptor binding domain of CBP to stimulate transcription from the PEPCK gene promoter. NFI-A combines with dexamethasone or thyroid hormone in an additive manner to stimulate PEPCK gene transcription. We conclude that CBP coordinates the action of the multiple factors known to control transcription of the PEPCK gene.

Binding Sites↗

PubMed1998

Nuclear factor I regulates expression of the gene for phosphoenolpyruvate carboxykinase (GTP).

Nuclear factor-I (NFI) binds to the phosphoenolpyruvate carboxykinase (GTP) (PEPCK) gene promoter immediately 5' to the cAMP regulatory element (CRE). This suggests an interaction between NFI and factors that bind the CRE. Of the four NFI isoforms expressed in mammalian tissues, NFI-A and -B stimulate basal transcription from the PEPCK gene promoter in HepG2 cells, while NFI-C and -X are slightly inhibitory. All four NFI isoforms abrogate the 20-fold protein kinase Ac (PKAc)-mediated induction of transcription from the PEPCK gene promoter. Normal PKAc-mediated induction was noted when the CRE was moved 10 base pairs 3' of its original location. However if the CRE was moved 5 base pairs 3', placing it out of phase with the other elements in the promoter, or moved 5' to -285 (the P3(I) site in the promoter), some PKA-mediated stimulation was lost. The NFI-C isoform effectively inhibited PKAc induction regardless of the relative positions of the CRE and the NFI binding sites. NFI-C also abrogated cAMP regulatory element-binding protein (CREB)-induced activity of wild type and mutant PEPCK promoters. There was some cooperativity in the binding of CREB and NFI to their respective binding sites but this did not appear to be physiologically important.

Animals↗

PubMed1998

Oxidative stress causes a general, calcium-dependent degradation of mitochondrial polynucleotides.

Oxidative stress has many effects on biological cells, including the modulation of gene expression. Reactive oxygen species are known to up-regulate and down-regulate RNA expression in prokaryotic and eukaryotic cells. We have previously reported that a preferential and calcium-dependent down-regulation of mitochondrial RNAs occurs when HA-1 hamster fibroblasts are exposed to hydrogen peroxide. Here we extend these studies to determine whether this down-regulation is specific to mitochondria RNA or involves general polynucleotide degradation. Degradation and associated decreases in the levels of 16S mitochondrial rRNA following exposure of cells to 400 microM hydrogen peroxide were found to be dependent on calcium at 2 and 5 h. Degradation of mitochondrial genomic DNA was also observed following peroxide exposure, and occurred at similar time points as for mitochondrial RNA degradation. As with mitochondrial RNA degradation, this mitochondrial genomic DNA degradation was dependent on calcium. These results indicate that there is a general, calcium-dependent degradation of mitochondrial polynucleotides following exposure of HA-1 fibroblasts to oxidative stress, and suggest that a dramatic shut-down in mitochondrial biosynthesis is an early-stage response to oxidative stress.

Animals↗

PubMed1997

Hamster adapt78 mRNA is a Down syndrome critical region homologue that is inducible by oxidative stress.

We are using the technique of mRNA differential display to identify RNAs that may be important in protecting cells against the damaging effects of oxidative stress. For these studies, we utilize a so-called "adaptive response" model system in which hamster HA-1 cells respond to a minimally toxic "pretreatment" dose of hydrogen peroxide by synthesizing RNAs and proteins that protect them against subsequent exposure to a highly cytotoxic concentration of hydrogen peroxide. Using this approach, we have recently reported several novel RNAs whose levels are increased under conditions of adaptive response. Here we report a new RNA, designated adapt78, whose steady-state level is significantly induced by a pretreatment dose of hydrogen peroxide. adapt78 mRNA was calculated to be 2.35 kb in size and inducible by the standard pretreatment dose of 4 micromol H2O2/10(7) cells. It was induced as early as 90 min after peroxide exposure and maximally at 5 h. Induction was strongly dependent upon calcium. Cloning and sequencing revealed a large predicted open reading frame of 197 amino acids. In vitro transcription and translation generated a protein of 25,000 Da. GenBank homology analysis revealed that much of adapt78 is strongly homologous to a sequence that has been mapped to the Down syndrome critical region (Fuentes et al., Hum. Mol. Genet. 4, 1935-1944, 1995). However, both the 5' and the 3' ends of adapt78 show no homology to any previously reported complete sequence. adapt78 represents a new oxidant-inducible RNA and marker of cellular oxidative stress and may provide new insight into our understanding of oxidant-related disorders and neural degeneration.

Amino Acid Sequence↗

PubMed1997

Modulation of a cardiogenic shock inducible RNA by chemical stress: adapt73/PigHep3.

BACKGROUND: Understanding how mammalian cells respond to stress is important in the study, detection, and therapy of stress-related disorders. We have been studying cellular stress response in hamster HA-1 cells by using an adaptive response model. HA-1 cells respond to a minimally toxic "pretreatment" dose of hydrogen peroxide by synthesizing RNAs and proteins that protect them against subsequent exposure to a higher cytotoxic concentration of peroxide. The purpose of our studies is to identify and partially characterize any mRNA whose steady state level is significantly modulated during adaptation. METHODS: HA-1 cells were exposed to a pretreatment dose of hydrogen peroxide and RNA extracted. The differential display technique was used to identify modulated mRNAs. The effects of calcium ionophore A23187 and cis (II)-platinum on the modulation of mRNA from HA-1 cells and A23187 on the modulation of mRNAs from human IMR-90 cells were also determined. RESULTS: One of the RNAs induced by a pretreatment concentration of hydrogen peroxide was designated adapt73. The size of the induced adapt73 RNA was determined to be 2.1 kb. Induction of adapt73 was maximal 5 hours after peroxide treatment, but elevated levels were still obvious at 10 hours. This induction was not specific to oxidative stress, because other stress agents including as (II)-platinum and especially calcium ionophore A23187 also induced adapt73 mRNA levels. Partial sequencing of adapt73 and a subsequent GenBank homology search revealed extensive homology to a novel RNA from pig, designated PigHep3, that was identified as a cardiogenic shock response gene from liver in pigs that were undergoing resuscitation after circulatory shock. Homology to a completely sequenced but uncharacterized human homolog was also found. Using a partially sequenced expressed sequence tag (EST) human clone to adapt73, we probed Northern blots containing RNA from IMR-90 human fibroblasts treated with A23187. A strongly induced human adapt73 mRNA homolog was observed, almost identical in size to its hamster homolog. In vitro transcription and translation of the human EST clone revealed a translatable Adapt73 protein product. CONCLUSIONS: These data indicate that adapt73/PigHep3 RNA can be induced by multiple chemical stress, that these inductions occur under protective or adaptive response conditions, that there is an inducible human homolog to adapt73, and suggest that adapt73 may be an important physiologic mediator of organ and cellular shock response in mammals.

Animals↗

PubMed1997

Down-regulation of mammalian mitochondrial RNAs during oxidative stress.

We have identified an RNA species that appears to be induced by oxidative stress in hamster HA-1 fibroblasts using the differential display technique, but instead is found to be degraded when evaluated by Northern blot hybridization. Cloning and subsequent sequencing identified the partially degraded RNA as 16S ribosomal RNA (rRNA), a major component of mitochondrial ribosomes. Degradation, and associated decreases in the levels of the mature- and precursor-species of 16S rRNA, appear to be dependent upon calcium, but not cytoplasmic protein synthesis nor nuclear transcription. Other decreased mitochondrial RNAs were also identified, including 12S rRNA, NADH dehydrogenase subunit 6, ATPase subunit 6, and cytochrome oxidase subunits I and III. A significant part of many, if not all, of these RNA decreases was due to degradation. As compared with 16S rRNA, significantly less degradation was observed for cytoplasmic 28S/18S rRNAs, even at very high peroxide concentration. Analysis of 21 cytoplasmic mRNAs revealed little or no decrease in mature band signal in response to peroxide, and several cytoplasmic mRNAs were actually up-regulated. Thus, a preferential down-regulation of mitochondrial RNAs occurs in HA-1 fibroblasts in response to hydrogen peroxide. Subcellular fractionation analysis, using 16S rRNA degradation as a gauge, indicates that this down-regulation is specific to mitochondria. The down-regulation of mitochondrial RNAs may represent a general mechanism by which cells protect themselves against oxidative stress.

Adenosine Triphosphatases↗

PubMed1997

16S mitochondrial ribosomal RNA degradation is associated with apoptosis.

The use of mitochondrial RNA as an indicator of apoptosis was investigated. Exposure of HA-1 fibroblastic cells to 10 micromol H(2)O(2) per 10(7) cells induced nuclear fragmentation, cell shrinkage, and internucleosomal DNA fragmentation, all characteristics of apoptosis. RNA extracted from control and apoptotic cultures, and analyzed by Northern blot hybridization, revealed a significant increase in the degradation of mitochondrial 16S ribosomal RNA (rRNA) that was associated with apoptosis. Conversely, minimal, if any, degradation of glyceraldehyde-3-phosphate dehydrogenase or actin mRNAs was observed. Similar results were obtained for HA-1 cells treated with the protein kinase inhibitor staurosporine, and for HT-2 T-lymphocytes induced to undergo apoptosis by interleukin-2 withdrawal. In addition, 16S rRNA degradation was an early event that was discernable well before chromatin condensation in hydrogen peroxide-treated HA-1 cells. These observations suggest that degradation of mitochondrial 16S ribosomal RNA is a new marker of mammalian cell apoptosis.

Animals↗

PubMed1997

The promoter regulatory regions of the genes for the cytosolic form of phosphoenolpyruvate carboxykinase (GTP) from the chicken and the rat have different species-specific roles in gluconeogenesis.

Hepatic expression of the gene for phosphoenolpyruvate carboxykinase (GTP) (PEPCK-C) (EC 4.1.1.32) in birds occurs prior to birth and decreases to negligible levels before hatching, whereas in mammals the gene for PEPCK-C in the liver is expressed at birth and is active throughout the life of the animal. The administration of cyclic AMP to adult chickens results in the induction of transcription of the gene for PEPCK-C and the transient accumulation of PEPCK-C mRNA in the liver. DNase I footprint analysis of 330 bp of the avian PEPCK-C promoter immediately 5' of the start-site of transcription indicated the presence of several protein binding domains, purified CAAT/enhancer binding protein alpha, cAMP regulatory element binding protein and nuclear factor-1 bound to these regions of the promoter. Sequences corresponding to an hepatic nuclear factor-1 binding domain and to the insulin response sequence, previously identified in the rat PEPCK-C promoter, were also found in the chicken PEPCK-C promoter. Co-transfection of an expression vector for CAAT/enhancer binding protein alpha or CAAT/enhancer binding protein beta markedly stimulated transcription from both the chicken and rat PEPCK-C promoters in human hepatoma cells. Sequences involved in the regulation of gene transcription by cyclic AMP and insulin were found to reside between -210 and +1 of the avian PEPCK-C promoter. In general, transcription from the avian promoter was more sensitive to inhibition by insulin than was noted for the rat PEPCK-C promoter, which may explain in part the lack of expression of the gene for PEPCK-C in the livers of adult birds.

Animals↗

PubMed1996

adapt33, a novel oxidant-inducible RNA from hamster HA-1 cells.

We have recently chosen to undertake a comprehensive evaluation of the modulation of gene expression by oxidative stress, using mRNA as a marker. Our model system is HA-1 hamster fibroblasts, using conditions under which we observe an adaptive response. Under these conditions, the HA-1 cells respond to a minimally toxic "pretreatment" dose of hydrogen peroxide by synthesizing RNAs and proteins that protect them against subsequent exposure to a highly cytotoxic concentration of hydrogen peroxide. Using the differential display technique to screen for modulated RNAs, we have recently reported an RNA species, adapt15/gadd7, whose steady-state level is significantly induced by a pretreatment dose of hydrogen peroxide (D. R. Crawford, G. P. Schools, S. L. Salmon, and K. J. A. Davies (1996) Arch. Biochem. Biophys. 325, 256-264). Here we report a second induced mRNA, designated adapt33. Two homologous adapt33 mRNAs were revealed by Northern blot hybridization. Both of these species were inducible by hydrogen peroxide, and they were sized at 1.46 and 0.99 kb. These inductions appeared to be dependent upon calcium, occurred as early as 90 min, and were maximal at 5 h. Cell fractionation revealed that a significant proportion of adapt33 RNA is associated with active translation. adapt33 is a novel sequence, as determined by cloning, sequencing, and GenBank analysis. adapt33 represents a new oxidant-inducible RNA and marker of cellular oxidative stress and a potential aid in the study, detection, and possible therapy of oxidant-related disorders.

Adaptation, Physiological↗

PubMed1996

Oxidant-inducible adapt 15 RNA is associated with growth arrest- and DNA damage-inducible gadd153 and gadd45.

We have recently described a novel RNA, designated adapt15, which is strongly induced by a pretreatment concentration of hydrogen peroxide in hamster HA-1 fibroblasts under conditions where a protective "adaptive response" occurs. adapt15 may therefore be involved in protecting cells against the damaging effects of oxidative stress. Since two other known sequences, gadd45 and gadd153m were also found to be induced under our pretreatment conditions and are known to be growth arrest and DNA damage inducible, we decided to also assess the possible association of adapt15 with growth arrest and DNA damage. We found that, like gadd45 and gadd153, the levels of adapt15 RNA were low during proliferation, but high during density saturation- and low serum (G0)-growth arrests. Exposure of HA-1 cells to DNA-damaging agents revealed significant induction of adapt15 RNA by methylmeth- anesulfonate and cis-platinum but not X-irradiation. Near identical responses were also observed for gadd45 and gadd153 RNAs, suggesting coordinate regulation of adapt15, gadd45, and gadd153. All three RNAs were also increased relative to control following heat shock, a nongenotoxic treatment. Finally, the induction of adapt15 by hydrogen peroxide was strongly dependent upon calcium, a hallmark of gadd153 induction. The coordinate inductions of adapt15, gadd45, and gadd153 by multiple agents, and their induction by an adaptive- but not by a nonadaptive-response level of hydrogen peroxide, suggest these RNAs may act in concert to protect cells against the damaging effects of oxidative stress.

Adaptation, Biological↗

PubMed1996

Hydrogen peroxide induces the expression of adapt15, a novel RNA associated with polysomes in hamster HA-1 cells.

In mammalian cells, hydrogen peroxide is known to induce the expression of several genes thought to be important in protection against oxidative stress. Using mRNA differential display, we have identified a novel RNA in hamster HA1 cells that is strongly induced by hydrogen peroxide under conditions where a protective adaptive response occurs. This induction was observed between 1 and 18 h after peroxide treatment, and at an H202 concentration that caused little or no cytotoxicity. Northern blot analysis revealed that this major inducible RNA species, termed adapt15, is 950 bases in size. Two versions of this RNA were found by sequence analysis, differing only by a short trinucleotide stretch. Despite polyadenylation, no large open reading frame was observed. Fractionation studies, however, indicate that adapt15 RNA is primarily located in the cytoplasm, and a significant percentage of it is associated with active translation. adapt15 RNA may act at the level of translation to protect cells against the damaging effect of oxidative stress.

Adaptation, Physiological↗

PubMed1996

Oxidative stress induces the levels of a MafG homolog in hamster HA-1 cells.

The Maf family encodes nuclear proteins that recognize AP-1-like response elements. MafB, MafK, MafF, and MafG, are all able to heterodimerize with each other, c-fos, and erythroid cell-specific transcription factor NF-E2, to affect the transcription of target genes. Using the technique of differential display, we recently identified a new oxidant-inducible mRNA, designated adapt66, in HA-1 hamster fibroblasts. Cloning, partial sequencing, and GenBank analysis of adapt66 revealed strong homology to chicken mafG, a newly identified member of the maf oncogene family. The mafG homolog/adapt66 mRNA induction appeared to be dependent upon calcium; occurred as early as 90 minutes following exposure of HA-1 cells to hydrogen peroxide; and peaked between 5 and 10 hours after peroxide treatment. It has previously been demonstrated that several cellular transcription factors, including Fos, can be induced by oxidative stress. The induction of the DNA binding sequence mafG homolog/adapt66 by hydrogen peroxide, and it's known interaction with c-fos, may represent important mechanisms by which oxidative stress can modulate gene expression.

Animals↗

PubMed1996

Tumor markers. An update.

Cancer remains the second leading cause of death in developed countries, and the incidence of certain tumors is increasing despite emphasis on prevention and screening. Tumor markers are biologic or biochemical substances that are produced by tumor cells and then secreted into the circulation in detectable amounts. This article covers some of the more common tumor markers currently being utilized for diagnostic and prognostic purposes.

Biomarkers, Tumor↗