Specific Activity Of DNA Research Articles

DNA microarray for detection and identification of sulfur oxidizing bacteria in Biogas Clean-up System

Knowledge of gene function and coexistence in the sulfur oxidizing bacteria (SOB) communities plays a significant role in driving the sulfide oxidation process in biogas clean-up process, including the stability and balance within the system. Microarray techniques can be responding to achieve as specific-detection, quantitative-identification, and high-throughput tools for microbial characterization in various environments. This research designed the microarray for monitoring SOB bacteria based on the entire genome and functional genes: soxAXBYZ and fccAB, using genome sequencing of non-SOB as a negative control, and then SOB microarray used to detect and identify SOB species-strain in the starch industry. The design of the DNA microarray revealed 61,788 probes which covered 722 strains of SOB and 35 strains of non-SOB. The quality test results demonstrated the DNA concentration, specific activity (28-36 pmol/μg), and yield (6-7 μg) of the genomic DNA had high-quality and could be repeated in the future with little noise signal. The expression level of recirculating sludge has increased in level of expression than the starting sludge. Thiothrix, Syntrophomonas, Paracoccus Magnetospirillum, Arcobacter, Sulfuricurvum, Acinetobacter, and Hydrogenophaga gained the gene expression level that involved with sulfide oxidation. The overexpression could be due to the biotrickling filter have more nutrients and optimal conditions for SOB growth to synthesize the necessary proteins for cell adaptation, such as SoxXA, SoxYZ, SoxB, and FccAB related to bacterial activity that oxidized the H2S in biogas.

Phenylbutyrate exerts adverse effects on liver regeneration and amino acid concentrations in partially hepatectomized rats

Phenylbutyrate is recommended in urea cycle disorders and liver injury to enhance nitrogen disposal by the urine. However, hypothetically there may be adverse responses to the use of phenylbutyrate in the treatment of liver disease because of its role as a histone deacetylase inhibitor and its stimulatory effect on branched-chain alpha-keto acid dehydrogenase, the rate-limiting enzyme in the catabolism of branched-chain amino acids (BCAA; valine, leucine and isoleucine). We report the effects of phenylbutyrate on liver regeneration and amino acid levels in plasma of partially hepatectomized (PH) rats. Phenylbutyrate or saline was administered at 12-h intervals to PH or laparotomized rats. Phenylbutyrate delayed the onset of liver regeneration compared to the saline-treated controls, as indicated by lower hepatic DNA specific activities 18 and 24() h post-PH, decreased hepatic fractional protein synthesis rates 24h post-PH and lowered the increases in liver weights and hepatic protein and DNA contents 48h after PH. Hepatic DNA fragmentation (a hallmark of apoptosis) was higher in the phenylbutyrate-treated animals than in controls. Phenylbutyrate decreased the glutamine and BCAA concentrations and the ratio of the BCAA to aromatic amino acids (phenylalanine and tyrosine) in the blood plasma in both hepatectomized and laparotomized animals. In conclusion, the delayed onset of liver regeneration and the decrease in BCAA/AAA ratio in blood suggest that phenylbutyrate administration may be disastrous in subjects with acute hepatic injury and BCAA supplementation is needed when phenylbutyrate is used therapeutically.

Apoptosis induction is involved in UVA-induced autolysis in sea cucumber Stichopus japonicus

Autolysis easily happens to sea cucumber (Stichopus japonicus, S. japonicus) for external stimulus like UV exposure causing heavy economic losses. Therefore, it is meaningful to reveal the mechanism of S. japonicas autolysis. In the present study, to examine the involvement of apoptosis induction in UVA-induced autolysis of S. japonicas, we investigated the biochemical events including the DNA fragmentation, caspase-3 activation, mitogen-activated protein kinases (MAPKs) phosphorylation and free radical formation. Substantial morphological changes such as intestine vomiting and dermatolysis were observed in S. japonicus during the incubation after 1-h UVA irradiation (10W/m2). The degradation of the structural proteins and enhancement of cathepsin L activity were also detected, suggesting the profound impact of proteolysis caused by the UVA irradiation even for 1h. Furthermore, the DNA fragmentation and specific activity of caspase-3 was increased up to 12h after UVA irradiation. The levels of phosphorylated p38 mitogen activated protein kinase (MAPK) and phosphorylated c-Jun.-N-terminal kinase (JNK) were significantly increased by the UVA irradiation for 1h. An electron spin resonance (ESR) analysis revealed that UVA enhanced the free radical formation in S. japonicas, even through we could not identify the attributed species. These results suggest that UVA-induced autolysis in S. japonicas at least partially involves the oxidative stress-sensitive apoptosis induction pathway. These data present a novel insight into the mechanisms of sea cucumber autolysis induced by external stress.

Physical and functional interaction of Rnf2 with Af9 regulates basal and aldosterone-stimulated transcription of the α-ENaC gene in a renal collecting duct cell line

The physical and functional interaction of Rnf2 (RING finger protein 2), a central component of the PRC (Polycomb repressive complex) 1 and Af9 (ALL1-fused gene from chromosome 9 protein), an aldosterone-sensitive transcription factor, in regulating basal and aldosterone-stimulated transcription of the α-ENaC (epithelial Na+ channel α-subunit) gene was explored in mIMCD3 CD (collecting duct) cells. Since Rnf2 lacks DNA-specific binding activity, other factors must mediate its site-specific chromatin recruitment. Rnf2 and Af9 co-localized in the nucleus and co-immunoprecipitated. A GST (glutathione transferase)–Af9 carboxy-terminal fusion protein directly interacted with in vitro translated Rnf2 in GST pull-down assays. Rnf2 knock down enhanced basal and aldosterone-stimulated α-ENaC mRNA levels and α-ENaC promoter activity. ChIP/QPCR (chromatin immunoprecipitation/quantitative PCR) assays demonstrated enrichment of Rnf2, H2AK119 (mono-ubiquitinated histone H2A lysine 119), and H3K27me3 (histone H3 lysine 27 trimethylated), a PRC2 chromatin mark, at multiple α-ENaC promoter subregions corresponding to regions of known Af9 enrichment, under basal conditions. Sequential ChIP confirmed Rnf2–Af9 co-occupancy of the α-ENaC promoter. Aldosterone provoked early and sustained depletion of Rnf2, ubiquitinated H2AK119, and trimethylated H3K27 associated with the subregions of the α-ENaC promoter. Thus, Af9 mediates site-selective physical and functional recruitment of Rnf2 to the α-ENaC promoter to constrain basal α-ENaC transcription in collecting duct cells, and aldosterone reverses this process.

The potential use of protein kinase D inhibitors for prevention/treatment of epidermal tumors

The serine/threonine kinase protein kinase D (PKD) has been proposed to be a pro-proliferative, anti-differentiative signal in epidermal keratinocytes. Indeed, the phorbol ester tumor promoter, 12-O-tetradecanoylphorbol 13-acetate (TPA) induces biphasic PKD activation, which mirrors the biphasic response of initial differentiation followed by proliferation and tumor promotion seen in TPA-treated keratinocytes in vitro and epidermis in vivo. Our objective was to test the idea that PKD's pro-proliferative and/or anti-differentiative effects in keratinocytes contribute to TPA-induced tumorigenesis. Using western analysis and assays of keratinocyte proliferation and differentiation, we investigated the effect of inhibitors of PKD on keratinocyte function. We found that overexpression of a constitutively active PKD mutant increased, and of a dominant-negative PKD mutant decreased, keratinocyte proliferation. A recently described selective PKD inhibitor showed low potency to inhibit keratinocyte proliferation or PKD activation. Therefore, we tested the ability of known only relatively selective PKD inhibitors on keratinocyte function and protein kinase activation. H89 {N-[2-(p-bromocinnamylamino) ethyl]-5-isoquinoline-sulfonamide}, a reported inhibitor of PKD and cAMP-dependent protein kinase, enhanced the effect of a differentiating agent on a marker of keratinocyte differentiation. Another reported non-selective PKD inhibitor, resveratrol stimulated differentiation and inhibited proliferation. The protein kinase C/PKD inhibitor Gö6976 blocked the increase in proliferation (as measured by DNA specific activity) induced by chronic TPA without affecting the initial TPA-elicited differentiation. Our results support the idea that relatively selective PKD inhibitors, such as Gö6976, H89 and resveratrol, might be useful for preventing/treating epidermal tumorigenesis without affecting keratinocyte differentiation.

Testicular x-ray irradiation in adult mice as a model to study spermatogonial proliferation

Testicular irradiation (300 r) of sexually mature mice provides a good model to study spermatogonial proliferation. DNA specific activity can be used under these conditions as a suitable assay of the proliferation and differentiation of testicular cells.

Effects of neonatal thyroidectomy on thymidine metabolism and deoxyribonucleic acid synthesis in the developing rat cerebellum.

Thyroid deficiency at birth caused a temporal decrease of the DNA content in rat cerebellum during the periods from 7 to 21 days of life as compared with the normal. At 2 h following the subcutaneous injection of [3H]-thymidine, the specific radioactivity of cellular dTTP in thyroid deficient rats was apparently increased on the 14th and 21st days, although the endogenous pool sizes of dTTP did not significantly differ between the normal and thyroidectomized rats throughout the entire ages studied. The ratio of incorporation of thymidine into cerebellar DNA was initially determined by the specific radioactivity and then expressed as the ratio of [3H]-DNA to [3H]-dTTP, the relative specific radioactivity of DNA. The age peak of incorporation occurred at 7 days in the normal; on the other hand, the specific activity of DNA in thyroid deficiency attained a maximum at 14 days and the relative specific activity appeared to be prolonged up to 14 days. The corresponding changes were observed in the formation of dTTP, suggesting that a transient retardation of cerebellar DNA synthesis taking place by neonatal thyroidectomy may be in part attributable to the variance of phosphorylation of thymidine. Indeed, thymidine kinase activity that regulated salvage pathway for DNA synthesis displayed a parallel variation in thyroid deficiency to characteristic age dependence of DNA synthesis.

Evidence for an enterotropic hormone: compensatory hyperplasia in defunctioned bowel.

The relative contributions of luminal and systemic factors to the development of compensatory intestinal hyperplasia were assessed by comparing the adaptive response to jejunal resection in functioning and defunctioned ileum. In Thiry--Vella loops of upper ileum, mucosal contents of RNA and DNA increased by 16--21 per cent 2--7 days after jejunectomy (P = 0.05--0.01), but DNA specific activity was unchanged. In lower ileum remaining in continuity with the nutrient stream, nucleic acids were elevated by 27--86 per cent (P less than 0.001) and specific activity by 34--36 per cent (P = 0.05--0.005). Proximal to the site of resection, duodenal hyperplasia was equally rapid but less intense than in the distal bowel. Full adaptation requires both intraluminal nutrients and enterotropic hormones to be present.

Modulation of DNA Binding Properties of CCAAT/Enhancer Binding Protein Epsilon by Heterodimer Formation and Interactions with NFkappaB Pathway.

Specific regulation of gene expression is achieved through interaction of multiple transcriptional activators and repressors with their cognate DNA sites. This activity is subject to several levels of control, including post-translational modification by phosphorylation, acetylation, selective degradation and interaction with co-activators and co-repressors.Recent studies directed at discovering C/EBP- responsive genes resulted in identification of a diverse and degenerate collection of putative C/EBP binding sites. Identification of binding sites for each individual member of the C/EBP family is complicated by common co-expression of several of these proteins in the same cell and their ability to form hetero-dimeric complexes with each other and structurally-related ATF/CREB proteins. Activity of C/EBP proteins is regulated by phosphorylation mediated through several major kinase pathways, that affect DNA-binding, subcellular localization in the nucleus, and interaction with cell cycle proteins.Our studies of C/EBP-e phosphorylation identified several sites, including one for p38 MAPK within the transactivation domain of C/EBP-e. As a result of phosphorylation at Threonine-75 (T75), C/EBP-e DNA binding and specific transcriptional activity was significantly enhanced.In this study, we identified consensus binding sequences for C/EBP-e and for heterodimers of C/EBP-e with its most common dimerization partner ATF4. Furthermore, we find that C/EBP-e- DNA interaction is highly upregulated by interaction with activated NFkappaB pathway protein p65-RelA. This interaction depends on the T75 phosphorylation status of C/EBP-e and can be enhanced by introduction of phosphomimetic Thr to Asp mutation. By using selective removal of p65-RelA and siRNA mediated p65 knock-down, we demonstrate that activated NFkappaB pathway is required for high level expression of genes with C/EBP-e responsive promoter. This novel interaction may play an important role in regulation of a C/EBP-e - dependent genes in myeloid cells, for example at sites of infection or other inflammations.

1104. Imaging of sec-R Directed and PEG-Stabilized Gene Transfer Nanoparticles in CF Mice

Compacted DNA targeted to the serpin enzyme complex receptor (sec-R) can deliver amounts of human CFTR DNA sufficient for partial correction of the chloride transport defect and NOS-2 downregulation in the noses of CF knock out mice. To examine the distribution of these compacted DNA particles in vivo, we used radioscintigraphy imaging. We modified our DNA compacting agent (a polymer of poly-L-lysine, chemically conjugated to the C105Y sec-R targeting ligand) with radiolabeled I-125. Prior to dosing, 12 S489X/FABP-hCFTR were imaged by X-ray for proper anatomical alignment with radiograph images. Following complex formulation, 9 mice were dosed intranasally (IN) with 25 |[mu]|g of sec-R targeted radiolabeled hCFTR DNA (specific activity 50 |[mu]|Ci each), while 3 control mice received administration of free I-125 (specific activity 50 |[mu]|Ci each). We used a small animal gamma scanner to analyze radio images at 2, 4, 24, and 48 hrs following nasal administration of the dose. At each time 3 animals were sacrificed, frozen and stored for subsequent sectioning. For each mouse, I-125 scintigraphic images, x-ray images, and autoradiographs were aligned and analyzed. Targeted delivery resulted in higher total retained activity than the carrier control. Both test and control groups begin to exhibit thyroid uptake by 2 hrs, while significantly higher activities were observed in the nasal and lower airways for animals that received the targeted complexes vs. controls. The highest observed activity was in thyroid, liver and bladder. Autoradiographic images correlated well with their corresponding in vivo scintigraphic images. For mice that received targeted complexes, strong signals were measured in both abdominal (stomach and liver) and bladder regions indicating possible ingestion during or aspiration following administration, as well as secretion into the bladder. This rapid clearance by 2 hrs was observed for all mice. Regional analysis showed significantly (p<0.05) higher activities for the targeted delivery in nose, lung and liver vs. controls, 2 and 4 hrs post administration. By 24 hrs, activities in the upper airways and lungs were back to the background level, while activity in the thyroid remained significant at 24 and 48 hrs. The data indicate a rapid clearance of sec-R targeted compacted DNA (by 2 hrs), followed by the appearance of at least the labeled component of the complex (the ligand) in the thyroid and bladder by 4 hrs. We also examined expression conferred by second generation PEG-stabilized compacted nanoparticles containing the Firefly luciferase gene using bioluminescent imaging. Expression was detected in lung 2 and 4 days following intra-tracheal (IT) but not IN administration. Lung homogenates from both methods of administration exhibited luciferase activity although levels in the IT group were significantly higher. These data suggest that a threshold level of expression, achieved only with IT administration, is necessary for successful bioluminescent detection . Development of these real-time imaging techniques has allowed us to better assess the localization and site of activity of our gene transfer complex formulations.

832. Radioscintigraphy of sec-R Directed CFTR Gene Transfer in CF Mice Following Intranasal Administration

Previously, we have shown that compacted DNA targeted to the serpin enzyme complex receptor (sec-R) can deliver amounts of a human CFTR vector sufficient for partial correction of the chloride transport defect and NOS-2 downregulation in the CF knock out mouse nose. To examine the distribution of these compacted DNA particles in vivo following administration, we used radioscintigraphy imaging. We modified our DNA compacting agent (a polymer of poly-L-lysine, chemically conjugated to the C105Y sec-R targeting ligand) with radiolabeled I-125. Prior to dosing, 12 S489X/FABP-hCFTR were imaged by X-ray for proper anatomical alignment with radiograph images. Following complex formulation, 9 mice were dosed intranasally (IN) with 25 μg of sec-R targeted radiolabeled hCFTR DNA (specific activity 50 μCi each), while 3 control mice received administration of free I-125 (specific activity 50 μCi each). We used a small animal gamma scanner produced at Thomas Jefferson National Lab to analyze radio images at 2, 4, 24, and 48 hrs following nasal administration of the dose. At each time 3 animals were sacrificed, frozen and stored for subsequent sectioning. For each mouse, I-125 scintigraphic images, x-ray images, and autoradiographs were aligned and analyzed. Targeted delivery resulted in higher total retained activity than the carrier control at the assayed time points. Both test and control groups begin to exhibit thyroid uptake by 2 hrs. By 2 hrs, scintigraphic and autoradiographic data indicated high activities in the nasal and lower airways in animals that received the targeted complexes compared to controls. The highest observed activity was in thyroid, liver and bladder. Autoradiographic images correlated well with their corresponding in vivo scintigraphic images. For mice that received targeted complexes, strong signals were measured in both abdominal (stomach and liver) and bladder regions indicating possible ingestion during or aspiration following administration, as well as secretion into the bladder. This rapid clearance by 2 hrs was observed for all mice. Regional analysis showed significantly (p < 0.05) higher activities for the targeted delivery in nose, lung and liver compared with controls, 2 and 4 hrs post administration. By 24 hrs, activities in the upper airways (nasal region) and lungs were back to the background level, while activity in the thyroid remained significant at 24 and 48 hrs. The data indicate a rapid clearance of sec-R targeted compacted DNA (by 2 hrs), followed by the appearance of at least the labeled component of the complex (the ligand) in the thyroid and bladder by 4 hrs. Intranasal administration may also result in aspiration or ingestion of the targeted complexes. Alternatively, these complexes or their components may have access to and enter the circulation and accumulate in the liver.

High-Porosity Activated Carbon as a Possible Matrix For Native DNA and Dextran-Sulfate Immobilization

In this study, specific and nonspecific activity of calf thymus DNA and Dextran-Sulfate (DS)-containing synthetic carbonic adsorbents (0.7-7mg of each ligands per 1 cm3 of activated carbonic beads, 0.3-0.6mm diameter, bulk density gamma=0.1-0.2g/cm3) have been compared in stir-bath and micro-column in vitro tests. DS coating as well as DNA coating does not demonstrate deep influence on the unspecific adsorptive activity of carbonic matrix toward creatinine, vitamin B12, and unconjugated bilirubin. No essential difference has been found in the specific activity of DNA and DS containing adsorbents toward anti-ds- and anti-ss-DNA-antibodies, as well as antibodies against DNA-protein complexes (anti-DNP-antibodies): in both cases the percentage of decrease of appropriate antibody concentration varied between 35 and 51% for single-pass microcolumn experiments with moderate enhancement of extraction efficacy (up to 60-75%) due to additional recirculation (2 h) or preliminary plasma dilution in 2-5 times. In the micro-column experiments with the proinflammatory cytokines DNA or DS-coating did not diminish TNF-alpha, IL-1beta, and IL-6 adsorption from 3% BSA solution, but even improves to some extent its removal compared with uncoated matrix.

Structural and Functional Characteristics of Homing Endonucleases

Mobile genetic elements constitute a remarkably diverse group of nonessential selfish genes that provide no apparent function to the host. These selfish genes have been implicated in host extinction, speciation and architecture of genetic systems. Homing endonucleases, encoded by the open reading frames embedded in introns or inteins of mobile genetic elements, possess double-stranded DNA-specific endonuclease activity. They inflict sequence-specific double-strand breaks at or near the homing site in intron- or intein-less allele. Subsequently, through nonreciprocal exchange the insertion sequence (intron or intein) is transferred from an intein- or intron-containing allele to an intein- or intron-less allele. The components of host double-strand break repair pathway are thought to finish the “homing” process. Several lines of evidence suggest that homing endonucleases are capable of promoting transposition into ectopic sites within or across genomes for their survival as well as dispersal in natural populations. The occurrence of inteins at high frequencies serves as instructive models for understanding the mechanistic aspects of the process of homing and its evolution. This review focuses on genetic, biochemical, structural, and phylogenetic aspects of homing endonucleases, and their comparison with restriction endonucleases.

Cigarette smoke exposure and hypercholesterolemia increase mitochondrial damage in cardiovascular tissues.

A shared feature among cardiovascular disease risk factors is increased oxidative stress. Because mitochondria are susceptible to damage mediated by oxidative stress, we hypothesized that risk factors (secondhand smoke and hypercholesterolemia) are associated with increased mitochondrial damage in cardiovascular tissues. Atherosclerotic lesion formation, mitochondrial DNA damage, protein nitration, and specific activities of mitochondrial proteins in cardiovascular tissues from age-matched C57 and apoE(-/-) mice exposed to filtered air or secondhand smoke were quantified. Both secondhand smoke and hypercholesterolemia were associated with significantly increased mitochondrial DNA damage and protein nitration. Tobacco smoke exposure also resulted in significantly decreased specific activities of mitochondrial enzymes. The combination of secondhand smoke and hypercholesterolemia resulted in increased atherosclerotic lesion formation and even greater levels of mitochondrial damage. These data are consistent with the hypothesis that cardiovascular disease risk factors cause mitochondrial damage and dysfunction.

Response of MG63 osteoblast-like cells to titanium and titanium alloy is dependent on surface roughness and composition

The success of an implant is determined by its integration into the tissue surrounding the biomaterial. Surface roughness and composition are considered to influence the properties of adherent cells. The aim of this study was to determine the effect of chemical composition and surface roughness of commercially pure titanium (Ti) and Ti–6Al–4V alloy (Ti–A) on MG63 osteoblast-like cells. Unalloyed and alloyed Ti disks were machined and either fine-polished or wet-ground, resulting in smooth (S) and rough (R) finishes, respectively. Standard tissue culture plastic was used as a control. Surface topography and profile were evaluated by cold field emission scanning electron microscopy and profilometry, while chemical composition was determined using Auger electron spectroscopy and Fourier transform infrared spectroscopy. The effect on the cells was evaluated 24 h postconfluence by measuring cell number, [ 3H]-thymidine incorporation into DNA, cell and cell layer alkaline phosphatase specific activity (ALPase), osteocalcin and collagen production, [ 35S]-sulfate incorporation into proteoglycan, and prostaglandin E 2 (PGE 2) and transforming growth factor- β (TGF- β) production. When compared to plastic, the number of cells was reduced on the pure Ti surfaces, while it was equivalent on the Ti-A surfaces; [ 3H]-thymidine incorporation was reduced on all surfaces. The stimulatory effect of surface roughness on ALPase in isolated cells and the cell layer was more pronounced on the rougher surfaces, with enzyme activity on Ti-R being greater than on Ti-A-R. Osteocalcin production was increased only on the Ti-R surface. Collagen production was decreased on Ti surfaces except Ti-R; [ 35S]-sulfate incorporation was reduced on all surfaces. Surface roughness affected local factor production (TGF- β, PGE 2). The stimulatory effect of the rougher surfaces on PGE 2 and TGF- β was greater on Ti than Ti-A. In summary, cell proliferation, differentiation, protein synthesis and local factor production were affected by surface roughness and composition. Enhanced differentiation of cells grown on rough vs. smooth surfaces for both Ti and Ti-A surfaces was indicated by decreased proliferation and increased ALPase and osteocalcin production. Local factor production was also enhanced on rough surfaces, supporting the contention that these cells are more differentiated. Surface composition also played a role in cell differentiation, since cells cultured on Ti-R surfaces produced more ALPase than those cultured on Ti-A-R. While it is still unknown which material properties induce which cellular responses, this study suggests that surface roughness and composition may play a major role and that the best design for an orthopaedic implant is a pure titanium surface with a rough microtopography.

Effects of lithium chloride as a potential radioprotective agent on radiation response of DNA synthesis in mouse germinal cells.

Mouse spermatogonial germ cells are highly sensitive to ionizing radiation. Lithium salts are reported to stimulate the postirradiation recovery of hematopoietic marrow cells. We have, therefore, examined whether administered lithium chloride (LiCl) would also be able to protect the mouse germinal cells against radiation injury. Taking DNA synthesis as an endpoint, our results show that the testicular DNA-specific activity in irradiated mice was higher by 61% on average when they had been pretreated with LiCl both 24 h and 1 h prior to gamma-irradiation (2.0 Gy). It was also observed that the DNA synthetic activity in the germinal cells fully recovered after LiCl pretreatment at doses of 40 mg per kg body weight prior to total body irradiation of 0.05-0.25 Gy, whereas at doses of 0.5-6.0 Gy, following the same procedure of LiCl pretreatment, only an incomplete recovery was observed. The dose reduction factor for LiCl is 1.84. The current findings indicate that pretreatment with LiCl provides considerable protection against radiation damage in mouse spermatogonia.

Resistance to apoptosis induced by alkylating agents in v‐Ha‐ras‐transformed cells due to defect in p53 function

In this study, we examined the susceptibility of various oncogene-transformed NIH/3T3 cells to apoptosis induced by alkylating agents. Only v-Ha-ras-transformed cells showed marked resistance to apoptotic death induced by these drugs. Upon treatment with methylmethane sulfonate (MMS), NIH/3T3 cells exhibited normal G1 checkpoint function accompanied by the accumulation of p53 and p21CIP1/WAF1 protein. However, no such effects were observed in v-Ha-ras-transformed cells. To further examine the functional status of p53 in ras-transformed cells, we determined the DNA sequence, protein half-life, protein-complexing activity, and specific DNA-binding activity of p53. The results showed that ras transformants and parental NIH/3T3 cells had the same p53 protein half-life of 40 min or less, the same normal wild-type p53 cDNA sequence, and the same co-immunoprecipitable cellular proteins complexed with p53. In electrophoretic mobility gel-shift assays, however, nuclear extracts of cells treated with MMS, ras-transformed cells, and normal cells displayed distinct patterns of binding between p53 ad its consensus binding site. Furthermore, western blot analysis showed that the bcl-2 and bax proteins were constitutively elevated in ras-transformed cells but not in parental NIH/3T3 cells. Heat-shock protein 70 (hsp70), which has been found to be negatively regulated by wild-type p53, was also dramatically induced in ras-transformed cells but not in NIH/3T3 cells in response to MMS. Thus, our data suggest that an activated ras oncogene can suppress alkylating agent-induced apoptotic cell death by means of a defect in the signal transduction pathway regulating p53 function and alteration in the expression of apoptotic (bax) or anti-apoptotic proteins (bcl-2 and hsp70). Mol. Carcinog. 18:221–231, 1997. © 1997 Wiley-Liss, Inc.

Resistance to apoptosis induced by alkylating agents in v-Ha-ras-transformed cells due to defect in p53 function

In this study, we examined the susceptibility of various oncogene-transformed NIH/3T3 cells to apoptosis induced by alkylating agents. Only v-Ha-ras-transformed cells showed marked resistance to apoptotic death induced by these drugs. Upon treatment with methylmethane sulfonate (MMS), NIH/3T3 cells exhibited normal G1 checkpoint function accompanied by the accumulation of p53 and p21CIP1/WAF1 protein. However, no such effects were observed in v-Ha-ras-transformed cells. To further examine the functional status of p53 in ras-transformed cells, we determined the DNA sequence, protein half-life, protein-complexing activity, and specific DNA-binding activity of p53. The results showed that ras transformants and parental NIH/3T3 cells had the same p53 protein half-life of 40 min or less, the same normal wild-type p53 cDNA sequence, and the same coimmunoprecipitable cellular proteins complexed with p53. In electrophoretic mobility gel-shift assays, however, nuclear extracts of cells treated with MMS, ras-transformed cells, and normal cells displayed distinct patterns of binding between p53 and its consensus binding site. Furthermore, western blot analysis showed that the bcl-2 and bax proteins were constitutively elevated in ras-transformed cells but not in parental NIH/ 3T3 cells. Heat-shock protein 70 (hsp70), which has been found to be negatively regulated by wild-type p53, was also dramatically induced in ras-transformed cells but not in NIH/3T3 cells in response to MMS. Thus, our data suggest that an activated ras oncogene can suppress alkylating agent-induced apoptotic cell death by means of a defect in the signal transduction pathway regulating p53 function and alteration in the expression of apoptotic (bax) or anti-apoptotic proteins (bcl-2 and hsp70).

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.

DNA Synthesis in Adult Feline Ventricular Myocytes

Adult mammalian ventricular myocytes are terminally differentiated cells, and the prevailing perception has been that DNA synthesis and repair are not active. We tested the hypothesis that there is potential for DNA synthesis and repair by studying the ability of whole-cell extracts from adult myocytes to incorporate [alpha-32P]dCTP into damaged plasmids. Left ventricular myocytes were isolated from adult cat hearts by collagenase dissociation. Cells were maintained in room air (control extract, CE) or made ischemic (IE) with N2 displacement of O2 and extracted for total protein. The nicked form of the plasmid was produced by exposure to an Fe3+/ascorbic acid free radical generating system. Both IE and CE degraded the supercoiled form of the plasmid and incorporated [alpha-32P]dCTP into the nicked (32P/DNA mass; CE = 2.2, IE = 3.0) and linear forms (32P/DNA mass; CE = 28.7, IE = 25.2). Exposure of plasmids to UV light did not inhibit incorporation of label. Inhibition studies with the cell extracts suggested a participation of polymerase delta in myocyte DNA repair/synthesis. Myocyte extract was as active as extract from rapidly growing COS cells at incorporating labeled nucleotides into plasmid DNA. The ability of intact myocytes to incorporate [alpha-32P]dCTP into endogenous DNA was measured in isolated cells made permeable with saponin. Studies were done in room air or N2. Permeable cells incorporated [alpha-32P]dCTP into nuclear DNA, but maximal specific activity of DNA was observed at 15 minutes with ischemia and at 60 minutes with room air control cells (ischemia, 1.34 +/- 0.5, 0.86 +/- 0.33, 0.60 +/- 0.04; air, 1.0, 1.28 +/- 0.20, 1.87 +/- 0.38, at 15, 30, and 60 minutes, respectively). These data indicate that mammalian adult ventricular myocytes can actively repair and/or synthesize both exogenous and endogenous DNA. A DNA synthetic response to cellular damage may have important pathological and clinical implications.