DNA Damage-inducible Gene Research Articles

Nuclear factor 1 is a negative regulator of gadd153 gene expression in vascular smooth muscle cells.

Growth arrest and DNA damage inducible gene 153 (gadd153) is expressed at very low levels in growing cells but is markedly induced in response to cellular stresses, including glucose deprivation, exposure to genotoxic agents, and other growth-arresting situations. Forced expression of GADD153 can induce cell cycle arrest and/or apoptosis in many types of cells. Recently, we reported that GADD153 was induced in vascular smooth muscle cells (VSMCs) in neointimal lesions of balloon-injured carotid arteries. To investigate the underlying molecular mechanisms of gadd153 gene expression in VSMCs, we isolated and characterized a promoter region of the rat gadd153 gene. Sequence alignments of this region revealed 1 TATA-like sequence and several well-known cis elements. The 5'-deletion analysis for this region showed that a domain spanning -447 through -368 drastically reduced the promoter activity to almost equal levels of promoterless control. Because this domain contained a consensus sequence for the nuclear factor 1 family of proteins (NF1), DNA-binding studies were performed by use of 2 types of NF1 consensus probes. Both probes were specifically shifted by nuclear extracts from proliferating VSMCs and were supershifted by antiserum against CCAAT transcription factor/NF1. In addition, promoter activity of a mutant luciferase vector, which was generated by a point mutation at the NF1 binding motif of the gadd153 gene, was 14-fold higher than that of a wild-type one. These results suggest that gadd153 gene expression in VSMCs is negatively regulated by an NF1-binding motif, and NF1 may act as an antiapoptotic factor by continuously suppressing gadd153 gene expression in growing VSMCs.

Role of GADD153 (growth arrest- and DNA damage-inducible gene 153) in vascular smooth muscle cell apoptosis

GADD153 (growth arrest- and DNA damage-inducible gene 153) is expressed at very low levels in growing cells, but is markedly induced in response to a variety of cellular stresses, including glucose deprivation, exposure to genotoxic agents and other growth-arresting situations. Forced expression of GADD153 induces cell cycle arrest in many types of cells. It is also reported that GADD153 is directly associated with apoptosis. Recently we have reported that platelet-derived growth factor (PDGF)-BB induces apoptosis in cultured vascular smooth muscle cells (VSMC), but only when 100% confluency is reached. These results suggested that cell-cell contact inhibition (cell growth arrest) may be a critical factor for induction of VSMC apoptosis by PDGF-BB. In the present study, we explored the role of GADD153, one of a number of growth-arrest-related gene products, in the molecular mechanisms of VSMC apoptosis in vitro and in vivo. GADD153 was markedly induced at both the mRNA and protein levels, in parallel with the induction of VSMC apoptosis, after treatment with PDGF-BB. Moreover, overexpression of GADD153 in VSMC significantly reduced cell viability and induced apoptosis. In the carotid artery balloon injury model in rats, GADD153 protein was expressed in apoptotic VSMC which were positively stained by in situ DNA labelling. These results demonstrate an important role for GADD153 in the molecular mechanisms of VSMC apoptosis.

NF1 Negatively Regulates Gadd153 Expression in Vascular Smooth Muscle Cells.

P67 OBJECTIVES: Vascular remodeling is determined by the balance of proliferation and apoptosis in vascular smooth muscle cells (VSMC). Endoplasmic reticulum (ER) stress has been reported as an inducible factor of apoptosis. We have recently reported that growth arrest and DNA damage inducible gene (Gadd)153, which is known to be upregulated by ER stress, plays a pivotal role in the regulation of apoptosis in VSMC. The aim of this study is to characterize a promoter region of the Gadd153 gene for further elucidation of underlying mechanisms of VSMC apoptosis mediated by Gadd153. METHODS: Promoter region was isolated by screening of a rat genomic library. Transcription start sites were determined by primer extension analysis. Progressive 5′-deletions were prepared and used for the construction of luciferase-fusion vectors. For all experiments, VSMC were maintaining in the presence of 10% FCS to make them a proliferative state. Promoter activity was measured by luciferase assay, and DNA-binding studies were performed by electromobility shift and supershift assays. RESULTS: A 1500-bp fragment containing a Gadd153 promoter region was obtained, and its sequence alignment was determined. Primer extension analysis revealed that there were two major transcription start sites. Further, one TATA-like box was found within 30 bp from the transcription start sites. Although the 5′-deletion that was deleted the sequence spanning -1500 through -400 showed the highest promoter activity in VSMC, the presence of sequence spanning -447 and -400 reduced promoter activity drastically to the almost equal level of promoter less control. This domain between -447 and -400 contained a well-known consensus element, NF1 binding site, located at positions between -447 and -417. DNA-binding studies indicated that nuclear extracts from VSMC specifically produced a retarded band that was supershifted by addition of antibodies against NF1. CONCLUSION: In proliferative VSMC, basal transcriptional activity of the Gadd153 gene was regulated mainly by a nuclear factor NF1 via a negative regulatory element located at positions between -447 and -417, suggesting that NF1 may act as an anti-apoptotic factor of VSMC.

NF1 Negatively Regulates Gadd153 Expression in Vascular Smooth Muscle Cells.

P67 OBJECTIVES: Vascular remodeling is determined by the balance of proliferation and apoptosis in vascular smooth muscle cells (VSMC). Endoplasmic reticulum (ER) stress has been reported as an inducible factor of apoptosis. We have recently reported that growth arrest and DNA damage inducible gene (Gadd)153, which is known to be upregulated by ER stress, plays a pivotal role in the regulation of apoptosis in VSMC. The aim of this study is to characterize a promoter region of the Gadd153 gene for further elucidation of underlying mechanisms of VSMC apoptosis mediated by Gadd153. METHODS: Promoter region was isolated by screening of a rat genomic library. Transcription start sites were determined by primer extension analysis. Progressive 5′-deletions were prepared and used for the construction of luciferase-fusion vectors. For all experiments, VSMC were maintaining in the presence of 10% FCS to make them a proliferative state. Promoter activity was measured by luciferase assay, and DNA-binding studies were performed by electromobility shift and supershift assays. RESULTS: A 1500-bp fragment containing a Gadd153 promoter region was obtained, and its sequence alignment was determined. Primer extension analysis revealed that there were two major transcription start sites. Further, one TATA-like box was found within 30 bp from the transcription start sites. Although the 5′-deletion that was deleted the sequence spanning -1500 through -400 showed the highest promoter activity in VSMC, the presence of sequence spanning -447 and -400 reduced promoter activity drastically to the almost equal level of promoter less control. This domain between -447 and -400 contained a well-known consensus element, NF1 binding site, located at positions between -447 and -417. DNA-binding studies indicated that nuclear extracts from VSMC specifically produced a retarded band that was supershifted by addition of antibodies against NF1. CONCLUSION: In proliferative VSMC, basal transcriptional activity of the Gadd153 gene was regulated mainly by a nuclear factor NF1 via a negative regulatory element located at positions between -447 and -417, suggesting that NF1 may act as an anti-apoptotic factor of VSMC.

Sequence-Specific Transcriptional Corepressor Function for BRCA1 through a Novel Zinc Finger Protein, ZBRK1

BRCA1 has been implicated in the transcriptional regulation of DNA damage–inducible genes that function in cell cycle arrest. To explore the mechanistic basis for this regulation, a novel human gene, ZBRK1, which encodes a 60 kDa protein with an N-terminal KRAB domain and eight central zinc fingers, was identified by virtue of its interaction with BRCA1 in vitro and in vivo. ZBRK1 binds to a specific sequence, GGGxxxCAGxxxTTT, within GADD45 intron 3 that supports the assembly of a nuclear complex minimally containing both ZBRK1 and BRCA1. ZBRK1 represses transcription through this recognition sequence in a BRCA1-dependent manner. These results thus reveal a novel corepressor function for BRCA1 and provide a mechanistic basis for the biological activity of BRCA1 through sequence-specific transcriptional regulation.

Inactivation of mouse Hus1 results in genomic instability and impaired responses to genotoxic stress

The eukaryotic cell cycle is overseen by regulatory mechanisms, termed checkpoints, that respond to DNA damage, mitotic spindle defects, and errors in the ordering of cell cycle events. The DNA replication and DNA damage cell cycle checkpoints of the fission yeast Schizosaccharomyces pombe require the hus1(+) (hydroxyurea sensitive) gene. To determine the role of the mouse homolog of hus1(+) in murine development and cell cycle checkpoint function, we produced a targeted disruption of mouse Hus1. Inactivation of Hus1 results in mid-gestational embryonic lethality due to widespread apoptosis and defective development of essential extra-embryonic tissues. DNA damage-inducible genes are up-regulated in Hus1-deficient embryos, and primary cells from Hus1-null embryos contain increased spontaneous chromosomal abnormalities, suggesting that loss of Hus1 leads to an accumulation of genome damage. Embryonic fibroblasts lacking Hus1 fail to proliferate in vitro, but inactivation of p21 allows for the continued growth of Hus1-deficient cells. Hus1(-/-)p21(-/-) cells display a unique profile of significantly heightened sensitivity to hydroxyurea, a DNA replication inhibitor, and ultraviolet light, but only slightly increased sensitivity to ionizing radiation. Taken together, these results indicate that mouse Hus1 functions in the maintenance of genomic stability and additionally identify an evolutionarily-conserved role for Hus1 in mediating cellular responses to genotoxins.

Radiation-induced GADD45 expression correlates with clinical response to radiotherapy of cervical carcinoma

Background: Recent work has identified a category of genes devoted to the control of genomic stability and prevention of cellular evolution. They encode components of cell cycle checkpoint, i.e., regulatory pathways committed to ordered cell cycle transition and fidelity of replicated DNA under adverse environmental conditions, such as those following exposure to genotoxic agents. Gadd45 belongs to the class II family of DNA damage-inducible (DDI) gene, and its role in DNA repair has been proved in many experimental models. The aim of our study was to correlate gadd45 radio-induction with the responsiveness to radiotherapy of cervical carcinomas, a type of cancer most commonly treated with radiotherapy alone. Methods: By means of a competitive polymerase chain reaction strategy, we compared in 14 patients the gene expression levels before and during external beam radiotherapy, when a dose ranging from 18 to 25 Gy was delivered to the target. Results: We found a correlation between the lack of gadd45 induction and a good clinical response to radiotherapy, in terms of both local control and disease-free survival. Conclusion: Our results support the measure of the induction of gadd45, and possibly of other genes required for regulated G 1-S checkpoint, as a method useful for prognostic evaluation of cervical carcinoma patients.

The product of the DNA damage-inducible gene of Saccharomyces cerevisiae, DIN7, specifically functions in mitochondria.

We reported previously that the product of the DNA damage-inducible gene of Saccharomyces cerevisiae, DIN7, belongs to a family of proteins that are involved in DNA repair and replication. The family includes S. cerevisiae proteins Rad2p and its human homolog XPGC, Rad27p and its mammalian homolog FEN-1, and Exonuclease I (Exo I). Here, we report that Din7p specifically affects metabolism of mitochondrial DNA (mtDNA). We have found that dun1 strains, defective in the transcriptional activation of the DNA damage-inducible genes RNR1, RNR2, and RNR3, exhibit an increased frequency in the formation of the mitochondrial petite (rho(-)) mutants. This high frequency of petites arising in the dun1 strains is significantly reduced by the din7::URA3 allele. On the other hand, overproduction of Din7p from the DIN7 gene placed under control of the GAL1 promoter dramatically increases the frequency of petite formation and the frequency of mitochondrial mutations conferring resistance to erythromycin (E(r)). The frequencies of chromosomal mutations conferring resistance to canavanine (Can(r)) or adenine prototrophy (Ade(+)) are not affected by enhanced synthesis of Din7p. Experiments using Din7p fused to the green fluorescent protein (GFP) and cell fractionation experiments indicate that the protein is located in mitochondria. A possible mechanism that may be responsible for the decreased stability of the mitochondrial genome in S. cerevisiae cells with elevated levels of Din7p is discussed.

Glutamine Deprivation Induces the Expression ofGADD45 and GADD153 Primarily by mRNA Stabilization

The expression of the growth arrest- and DNA damage-inducible genes, GADD45 and GADD153/CHOP (C/EBP-homologous protein), as well as GRP78 (glucose-regulated protein of 78 kDa) was examined in several human breast cell lines subjected to acute glutamine (GLN) deprivation. GLN deprivation caused rapid elevation of GADD45 and GADD153/CHOP mRNA levels in cells that were highly dependent upon GLN for growth and viability. Both GADD mRNAs were rapidly elevated up to several hundred-fold. In contrast, GRP78 expression was elevated by no more than 4-fold by GLN deprivation. The magnitude of GADD up-regulation roughly correlated with the extent of GLN dependence of each cell line. The levels of all three mRNAs were responsive to alterations of ambient GLN content in a physiologically relevant concentration range that corresponded to the affinities of cellular GLN transporters. Provision of GLN-derived metabolites partially inhibited the induction of GADD expression in GLN-deprived cells. Nuclear run-on assays and mRNA decay studies suggested that the primary mechanism leading to increased GADD mRNA levels was not transcriptional, but rather that GADD45 and GADD153/CHOP expression were up-regulated in response to GLN deprivation via marked stabilization of these mRNAs. These results suggest that the expression of GADD genes contributes to growth arrest and/or protection from metabolic damage during GLN-poor conditions.

Relationship between seizure-induced transcription of the DNA damage-inducible gene GADD45, DNA fragmentation, and neuronal death in focally evoked limbic epilepsy.

We investigated the temporal and spatial profile of mRNA transcription for the growth arrest and DNA damage-inducible gene GADD45, DNA fragmentation, and neuronal death in rat brain following focally evoked limbic seizures. GADD45 mRNA was detected by in situ hybridization, whereas fragmented DNA was detected using in situ nick end-labeling by the large (Klenow) fragment of DNA polymerase I. Kainic acid (0.1 microg) was injected into the right amygdala of rats to induce seizures for 45 min, after which diazepam (30 mg/kg) was administered. GADD45 mRNA, DNA fragmentation, and cell death were quantified bilaterally within six limbic brain regions 0-96 h following seizure cessation. All animals underwent seizures of equivalent severity and duration as determined electrographically. In situ hybridization detected bilateral up-regulation of GADD45 mRNA throughout the CA1, CA3, and dentate gyrus of the hippocampus, the piriform and retrosplenial cortices, and the thalamus within 1 h of seizure termination. GADD45 mRNA levels remained elevated for up to 6 h, declining to baseline within all structures by 16 h. Klenow-positive cells were only found within the CA3 pyramidal layer of the ipsilateral hippocampus and appeared 16-72 h following seizure cessation. Morphologic cell death was also restricted to the CA3 subfield. These data demonstrate that focally evoked limbic seizures trigger early bihemispheric GADD45 mRNA transcription within connected limbic structures, whereas subsequent DNA fragmentation and cell death are restricted to selectively vulnerable brain regions.

Identification of genetic factors altering the SOS induction of DNA damage-inducible yebG gene in Escherichia coli

The yebG gene of Escherichia coli is a novel SOS regulon gene, but details of its regulation mechanism and biological function are not yet known. To characterize the regulation of yebG gene as a SOS gene, we identified the genetic factors affecting the SOS induction of yebG gene using yebG- lacZ operon fusion plasmid. We found that the SOS induction of yebG occurs as the cells enter into the stationary growth phase, but its induction is not observed in LB medium in the presence of 1% glucose. A stationary phase SOS induction of the yebG gene does not require the global regulator of stationary phase-specific genes, rpoS, or gyrA functions, but requires cya encoding the adenylate cyclase and hns encoding the histone-like protein H-NS functions. Our results demonstrated that the induction of a DNA damage-inducible yebG gene of E. coli is dependent on cyclic AMP and H-NS.

Identification of genetic factors altering the SOS induction of DNA damage-inducible yebG gene in Escherichia coli.

The yebG gene of Escherichia coli is a novel SOS regulon gene, but details of its regulation mechanism and biological function are not yet known. To characterize the regulation of yebG gene as a SOS gene, we identified the genetic factors affecting the SOS induction of yebG gene using yebG-lacZ operon fusion plasmid. We found that the SOS induction of yebG occurs as the cells enter into the stationary growth phase, but its induction is not observed in LB medium in the presence of 1% glucose. A stationary phase SOS induction of the yebG gene does not require the global regulator of stationary phase-specific genes, rpoS, or gyrA functions, but requires cya encoding the adenylate cyclase and hns encoding the histone-like protein H-NS functions. Our results demonstrated that the induction of a DNA damage-inducible yebG gene of E. coli is dependent on cyclic AMP and H-NS.

Post-transcriptional regulation of the DNA damage-inducible gadd45 gene in human breast carcinoma cells exposed to a novel retinoid CD437.

The biologically active synthetic retinoid CD437 (6-[3-adamantyl-4-hydroxyphenyl]-2-naphthalene, AHPN) and different human breast carcinoma (HBC) cell lines were used to examine the possible mechanism(s) of gadd45 induction. Northern blot analysis of mRNA isolated from MCF-7, MDA-MB-468 and MDA-MB-231 HBC cell lines demonstrated a progressive increase in the 1.4 kb gadd45 transcript after exposure to 1 microM CD437. Western blot analysis showed increased gadd45 protein levels in MDA-MB-468 HBC cells following exposure to CD437. CD437 increased gadd45 mRNA levels by approximately 20-fold in MDA-MB-468 cells, however, the transcriptional activity was increased approximately 2-3-fold as demonstrated by the human gadd45 promoter-luciferase reporter construct and nuclear run-off assays. Sublines of MDA-MB-468 HBC cells expressing stably integrated GADD45 cDNA fragments were obtained and CD437-dependent induction of GADD45 analyzed. We report that approximately 300 nt located in the 5"-untranslated region (5"-UTR) of gadd45 mRNA are involved in the CD437-dependent 4-fold enhanced stability of gadd45 transcripts. MDA-MB-468 cells were stably transfected with either a plasmid having a CMV promoter-driven rabbit beta-globin gene or plasmids having a CMV promoter-driven chimeric gadd45 5"-UTR-rabbit beta-globin gene, where the entire gadd45 5"-UTR (from +1 to +298) or a 45 bp subfragment of the gadd45 5"-UTR (from +10 to +55) was positioned at the 5"-end of the rabbit beta-globin gene. CD437 was found to up-regulate expression of both the chimeric gadd45 -rabbit beta-globin transcripts, suggesting that cis element(s) involved in the CD437-dependent enhanced stability of gadd45 mRNA are contained in the 45 nt of the 5"-UTR of the gadd45 mRNA.

Identification of beta-amyloid-responsive genes by RNA differential display: early induction of a DNA damage-inducible gene, gadd45.

Alzheimer's disease is a neurodegenerative disorder characterized by the extracellular deposition in the brain of amyloid beta-peptide (A beta), presumed to play a pathogenic role. However, the precise molecular mechanisms of its neurotoxicity are not fully understood. Recent studies have suggested that it may exert its toxic effect via activation of transcription factors. We investigated A beta-responsive genes in human preneuron NT2 cells, at early stages of A beta (25-35) exposure, by RNA differential display. A beta induced the expression of (i) the growth arrest and DNA damage-inducible gene (gadd45) implicated in the DNA excision-repair process; (ii) a stress-signaling kinase gene encoding the mitogen-activated protein kinase/Erk kinase kinase-1 (MEKK1); (iii) a new growth factor-inducible immediate-early gene, CYR61, the product of which functions as an extracellular matrix signaling molecule; (iv) other immediate-early genes, such as c-jun and c-fos; (v) the gene encoding the basic fibroblast growth factor (bFGF); (vi) a gene encoding a constituent of the mitochondrial pyruvate dehydrogenase complex, the dihydrolipoamide dehydrogenase-binding protein (E3-BP); and (vii) an unidentified human gene (KIAA0099). A beta not only activates but also respresses genes: (i) the gene encoding "hinge" protein, a subunit of the mitochondrial cytochrome-c reductase and (ii) the SRp55 gene encoding a splicing factor involved in constitutive pre-mRNA splicing and alternative splice site selection. Our results underscored A beta-responsive genes that play key roles in the response (damage/recovery) of neuron cells to A beta exposure. In particular, the strong upregulation of gadd45, indicating DNA damage, was detected early in A beta cytotoxicity. This suggests that DNA strand breaks occurred rapidly in cells exposed to A beta, which may be a critical event in A beta neurotoxicity.

Induction of gadd153 mRNA by nutrient deprivation is overcome by glutamine

The growth arrest and DNA damage-inducible (gadd) genes are co-ordinately activated by a variety of genotoxic agents and/or growth-cessation signals. The regulation of gadd153 mRNA was investigated in renal proximal tubular epithelial cells (LLC-PK1) cultured in a nutrient- and serum-deprived medium. The addition of glutamine alone to LLC-PK1 cells cultured in Earl's balanced salt solution (EBSS) is sufficient to suppress gadd153 mRNA expression, and the removal of only glutamine from Dulbecco's modified Eagle's medium (DMEM) is also sufficient to induce gadd153 mRNA expression. Consistent with these findings, the inhibition of glutamine utilization with acivicin and 6-diazo-5-oxo-L-norleucine (DON) in cells grown in a glutamine-containing medium effectively induces gadd153 expression. Glutamine can be used as an energy source in cultured mammalian cells. However, it is unlikely that deficits in cellular energy stores (ATP) are coupled to gadd153 mRNA expression, because concentrations of ATP, UTP and GTP are all elevated in EBSS-exposed cells, and the addition of α-oxoglutarate to cells grown in EBSS has no effect on gadd153 mRNA expression. In contrast, concentrations of CTP decline substantially in EBSS and glutamine-deprived DMEM-cultured cells. Glutamine also serves as a precursor for the synthesis of protein and DNA. The addition of glutamine to cells grown in EBSS partly restores CTP concentrations. The addition of pyrimidine ribonucleosides (cytidine and uridine) to LLC-PK1 cells also restores CTP concentrations, in a manner commensurate with their relative abilities to overcome gadd153 expression. Finally, glutamine does not completely suppress DNA damage-induced gadd153 expression, suggesting that multiple signalling pathways lead to the expression of gadd153 mRNA under conditions of nutrient deprivation and DNA damage.

Radiosensitive and mitotic recombination phenotypes of the Saccharomyces cerevisiae dun1 mutant defective in DNA damage-inducible gene expression.

The biological significance of DNA damage-induced gene expression in conferring resistance to DNA-damaging agents is unclear. We investigated the role of DUN1-mediated, DNA damage-inducible gene expression in conferring radiation resistance in Saccharomyces cerevisiae. The DUN1 gene was assigned to the RAD3 epistasis group by quantitating the radiation sensitivities of dun1, rad52, rad1, rad9, rad18 single and double mutants, and of the dun1 rad9 rad52 triple mutant. The dun1 and rad52 single mutants were similar in terms of UV sensitivities; however, the dun1 rad52 double mutant exhibited a synergistic decrease in UV resistance. Both spontaneous intrachromosomal and heteroallelic gene conversion events between two ade2 alleles were enhanced in dun1 mutants, compared to DUN1 strains, and elevated recombination was dependent on RAD52 but not RAD1 gene function. Spontaneous sister chromatid exchange (SCE), as monitored between truncated his3 fragments, was not enhanced in dun1 mutants, but UV-induced SCE and heteroallelic recombination were enhanced. Ionizing radiation and methyl methanesulfonate (MMS)-induced DNA damage did not exhibit greater recombinogenicity in the dun1 mutant compared to the DUN1 strain. We suggest that one function of DUN1-mediated DNA damage-induced gene expression is to channel the repair of UV damage into a nonrecombinogenic repair pathway.

Induction of gadd153 mRNA by nutrient deprivation is overcome by glutamine

The growth arrest and DNA damage-inducible (gadd) genes are co-ordinately activated by a variety of genotoxic agents and/or growth-cessation signals. The regulation of gadd153 mRNA was investigated in renal proximal tubular epithelial cells (LLC-PK1) cultured in a nutrient- and serum-deprived medium. The addition of glutamine alone to LLC-PK1 cells cultured in Earl's balanced salt solution (EBSS) is sufficient to suppress gadd153 mRNA expression, and the removal of only glutamine from Dulbecco's modified Eagle's medium (DMEM) is also sufficient to induce gadd153 mRNA expression. Consistent with these findings, the inhibition of glutamine utilization with acivicin and 6-diazo-5-oxo-l-norleucine (DON) in cells grown in a glutamine-containing medium effectively induces gadd153 expression. Glutamine can be used as an energy source in cultured mammalian cells. However, it is unlikely that deficits in cellular energy stores (ATP) are coupled to gadd153 mRNA expression, because concentrations of ATP, UTP and GTP are all elevated in EBSS-exposed cells, and the addition of alpha-oxoglutarate to cells grown in EBSS has no effect on gadd153 mRNA expression. In contrast, concentrations of CTP decline substantially in EBSS and glutamine-deprived DMEM-cultured cells. Glutamine also serves as a precursor for the synthesis of protein and DNA. The addition of glutamine to cells grown in EBSS partly restores CTP concentrations. The addition of pyrimidine ribonucleosides (cytidine and uridine) to LLC-PK1 cells also restores CTP concentrations, in a manner commensurate with their relative abilities to overcome gadd153 expression. Finally, glutamine does not completely suppress DNA damage-induced gadd153 expression, suggesting that multiple signalling pathways lead to the expression of gadd153 mRNA under conditions of nutrient deprivation and DNA damage.

Procedure for the quantitation of gadd45 expression levels in clonal hematopoietic progenitor cells by competitive polymerase chain reaction

Objective: The growth arrest and DNA damage-inducible (gadd) genes represent a family of stress-inducible genes that are coordinately regulated at transcriptional level. Gadd45, in particular, has been linked to a p53-dependent inducible network required for regulated transition from G 1 to S phase of cell cycle following genotoxic insult and growth arrest treatments and has seemingly a pivotal role in DNA repair. Design and methods: Here we show that competitive polymerase chain reaction (PCR) is an adequate method to quantitate gadd45 expression levels in hematopoietic progenitor cell line 32D, whose constitutive gene expression is very low. Results: The sensitivity and reproducibility of our strategy support its usefulness for clinical purposes, to assess the DNA repair capacity of highly purified early myeloid progenitors, whose failure may be responsible for either short-term chemotherapy side effects (bone marrow hypoplasia and peripheral blood cytopenia) or long-term consequences of antiblastic drugs (leukemia and myelodysplasia).

PA26, a novel target of the p53 tumor suppressor and member of the GADD family of DNA damage and growth arrest inducible genes.

Exposure of mammalian cells to hypoxia, radiation and certain chemotherapeutic agents promotes cell cycle arrest and/or apoptosis. Activation of p53 responsive genes is believed to play an important role in mediating such responses. In this study we identified a novel gene, PA26, which maps to chromosome 6q21 and encodes at least three transcript isoforms, of which two are differentially induced by genotoxic stress (UV, gamma-irradiation and cytotoxic drugs) in a p53-dependent manner. A functional p53-responsive element was identified in the second intron of the PA26 gene, in consistance with a mechanism of transcriptional induction of the PA26 gene by p53. No clues to its functions were revealed by sequence analysis, although pronounced negative regulation by serum factors argues for a potential role of PA26 in growth regulation. Immunological analysis suggests that PA26 protein(s) is localized to the cell nucleus. Our results suggest that the PA26 gene is a novel p53 target gene with properties common to the GADD family of growth arrest and DNA damage-inducible stress-response genes, and, thus, a potential novel regulator of cellular growth.

Activation of MYD116 (gadd34) expression following transient forebrain ischemia of rat: implications for a role of disturbances of endoplasmic reticulum calcium homeostasis

MyD116 is the murine homologue of growth arrest- and DNA damage-inducible genes (gadd34), a gene family implicated in growth arrest and apoptosis induced by endoplasmic reticulum dysfunction. The present study investigated changes in MyD116 mRNA levels induced by transient forebrain ischemia. MyD116 mRNA levels were measured by quantitative PCR. After 2 h of recovery following 30 min forebrain ischemia, MyD116 mRNA levels rose to about 550% of control both in the cortex and hippocampus. In the cortex, MyD116 mRNA levels gradually declined to 290% of control 24 h after ischemia, whereas in the hippocampus they remained high (538% of control after 24 h of recovery). To elucidate the possible mechanism underlying this activation process, MyD116 mRNA levels were also quantified in primary neuronal cell cultures under two different experimental conditions, both leading to a depletion of endoplasmic reticulum (ER) calcium pools. Changes in cytoplasmic calcium activity were assessed by fluorescence microscopy of fura-2-loaded cells, and protein synthesis (PS) was evaluated by measuring the incorporation of l-[4,5- 3 H ]leucine into proteins. The first procedure, exposure to thapsigargin (Tg), an irreversible inhibitor of ER Ca 2+-ATPase, produced a parallel increase in cytoplasmic calcium activity and a long-lasting suppression of PS, while the second, immersion in a calcium-free medium supplemented with the calcium chelator EGTA, caused a parallel decrease in cytoplasmic calcium levels and a short-lasting suppression of PS. Exposure of neurons to Tg induced a permanent increase in MyD116 mRNA levels. Exposure of cells to calcium-free medium supplemented with EGTA produced only a transient rise in MyD116 mRNA levels peaking after 6 h of recovery. The results demonstrate that depletion of ER calcium stores without any increase in cytoplasmic calcium activity is sufficient to activate MyD116 expression. A similar mechanism may be responsible for the increase in MyD116 mRNA levels observed after transient forebrain ischemia. It is concluded that those pathological disturbances triggering the activation of MyD116 expression after transient forebrain ischemia are only transient in the cerebral cortex but permanent in the hippocampus.