Differential regulation of E2F transcription factors by p53 tumor suppressor protein.

The cell cycle is under the control of various positive and negative regulators. Two such regulators are the E2F family of transcription factors and the p53 tumor suppressor protein. While E2F proteins are implicated in promoting the S phase of the cell cycle, p53 has the potential to arrest cells in G1 phase and thereby prevent entry into S phase. Because they perform seemingly opposite functions in the control of cell growth, a possibility of functional interactions between E2F and p53 was investigated. It was found that p53 specifically inhibited activated transcription by E2F-5 but not by E2F-1. Investigation into the mechanism of action established that heterodimer formation and the DNA-binding steps were not significantly inhibited by p53. However, the transcriptional activation step of E2F-5 activity, as examined by using a Gal4 DNA-binding domain chimera, was specifically inhibited by p53. Interestingly, p53 could also inhibit transcriptional activation by E2F-4 but not by E2F-2 or E2F-3. The results indicate that p53 differentially regulates the activities of two subclasses (E2F-1/-2/-3 vs. E2F-4/-5) of E2F transcription factors. Detailed analysis using a two-hybrid approach in mammalian cells indicated lack of physical interaction between p53 and E2F-5, DP-1, or E2F-1. Reciprocal analysis revealed that whereas E2F-1 dramatically inhibited p53-activated transcription, E2F-5 or DP-1 did not. Thus, nonreciprocal functional interactions exist between various members of the E2F family of transcription factors and p53 tumor suppressor protein. The complex interplay between various positive and negative regulators of cell growth, such as E2F and p53 proteins, may be crucial in determining the ultimate outcome in terms of cell cycle arrest, cell growth, or apoptosis.

The molecular and functional characterization of E2F-5 transcription factor.

The E2F activity plays a critical role in the control of cell cycle and action of tumor suppressor proteins and is also a target of the transforming proteins of small DNA tumor viruses. We describe here molecular cloning and functional characterization of a fifth member of the E2F family of transcription factors. E2F-5 protein is more homologous to E2F-4 (72% amino acid identity) than to E2F-1, E2F-2, and E2F-3 (35% amino acid identity). Based on structural and functional criteria, the E2F family appears to comprise two distinct sub-families, one composed of E2F-1, E2F-2, and E2F-3 and the other composed of E2F-4 and E2F-5, E2F-5 mRNA is expressed in a wide variety of human tissues. The protein is expressed as multiple species ranging in size from 46 to 54 kDa as a result of differential phosphorylation. The expression of a reporter gene containing E2F binding sites in the promoter is transcriptionally activated by E2F-5 in a cooperative manner with the DP-1 protein. The interaction between E2F-5 and DP-1 is demonstrated using a two-hybrid system in mammalian cells. We have also demonstrated the presence of a strong transactivation domain at the carboxy terminus (273-346 amino acid residues) of E2F-5 protein.

Single-strand breaks in oligodeoxyribonucleotides induced by fission neutrons and gamma radiation and measured by gel electrophoresis: protective effects of aminothiols.

The technique of high-resolution gel electrophoresis using oligodeoxyribonucleotides of known composition as model systems, offers a simple quantitative estimate of DNA damage in aqueous solution induced by ionizing radiation. The fraction of damaged DNA can be quantitatively defined in terms of the increased electrophoretic mobilities of the damaged oligonucleotides, relative to the mobility of the unirradiated and intact oligonucleotides. The usual direct strand breaks can be observed at gamma-ray dosages of 200 Gy. However, at a gamma-ray dosage of 400 Gy, only a broad background, attributed to heterogeneously and multiply damaged oligonucleotide fragments with overlapping and varying electrophoretic mobilities, can be distinguished. On the other hand, individual bands due to resolvable DNA fragments are evident even at dosages as high as 400 Gy for fission neutrons. When double-stranded oligonucleotides are exposed to gamma-ray dosages of 200 Gy, the fraction of damaged DNA approaches 30-40%. This damage can be almost completely suppressed (> 99%) if the irradiations are conducted in aqueous solutions in the presence of 0.5-1.0 mM concentrations of the thiols cysteamine or 3-(3-methylaminopropylamino)propanethiol (WR-151326). The rate constant of reaction of OH radicals with small double stranded oligonucleotides 16 base pairs long, KDNA, is found to be closer to the diffusion-controlled value (> 3 x 10(9) M-1 s-1) than the magnitudes of KDNA for the higher molecular weight, native DNA reported in the literature. These observations suggest that oligonucleotides represent more simple model systems than native DNA in solutions for studying the mechanisms of radioprotection exerted by thiols of different structures.

Mechlorethamine-induced enhancement of radiation sensitivity of guanine.

This study describes and characterizes the interactions of nitrogen mustard mechlorethamine (HN2) with guanine and the radiation sensitivity of guanine in the presence of HN2. Briefly, in an equimolar solution (0.5 mmol dm-3) the pH-dependence (pH 3.0-12.0) and time-dependence (0-36 h) of alkylation of guanine at room temperature were determined using a reverse-phase high-performance liquid chromatography (hplc) column. Based on the hplc peak areas of the product and intact guanine, the optimal pH for alkylation was determined to be 8.0. Similarly, the optimal time required for alkylation was 10 h. Two products, i.e. alkylated guanines, were detected (10:1, peak areas measured at 260 nm) and purified. Structural studies of the products were performed by direct insertion probe-electron impact mass spectrometry. These products were identified as N-(2-chloroethyl)-N-[2-(7-guanyl)ethyl]-methylamine (product 2). At optimal conditions, samples of either guanine or an equimolar solution of guanine and HN2 were 60Co irradiated (gamma-ray) at 25 Gy min-1 at doses up to 400 Gy. Both sets of samples were analysed by hplc. In each case, the sole radiation product observed and characterized was 8-hydroxy-guanine. Dose-yield plots were linear and showed that HN2 enhanced the radiation sensitivity of guanine. This increase in radiation sensitivity is attributed to the differences in electrophilic properties between nitrogen mustard and guanine.

Chromatographic and mass spectral analysis of the radioprotector and chemoprotector S-3-(3-methylaminopropylamino)propanethiol (WR-151326) and its symmetrical disulfide (WR-25595501).

Metabolically active forms of the radioprotective and chemoprotective drug S-3-(3-methylaminopropylamino)propylphosphorothioic acid (WR-151327) are S-3-(3-methylaminopropylamino)propanethiol (WR-151326) and its symmetrical disulfide (WR-25595501). This paper describes applications of sensitive and specific procedures such as capillary column gas chromatography with flame ionization detection, electron impact mass spectrometry and liquid chromatography with electrochemical detection for structural characterization and analysis of the active forms of WR-151327. These chromatographic procedures provide reproducible linear calibration graphs for a relatively wide range of concentrations of the active forms of WR-151327. The described procedures will further facilitate in vivo and in vitro investigations of chemoprotective and radioprotective properties of WR-151327 and its active metabolites.

Radiolysis in aqueous solution of dinucleoside monophosphates by high-energy electrons and fission neutrons.

The radiation chemistry in aqueous solution of the dinucleoside monophosphate d-[CpT] and its sequence isomer d-[TpC] in air or nitrogen was examined using different qualities and quantities of radiations. High-performance liquid chromatography and gas chromatography-mass spectrometry were used to analyze the high-energy electron (13.2 MeV) exposure products or fission-neutron exposure products of d-[CpT] and d-[TpC]. A comparison of product profiles obtained from irradiated d-[CpT] and d-[TpC] suggests that, at relatively low radiation doses (50-250 Gy), products are formed by N-glycosidic or phosphodiester bond-cleavage, while at higher doses (500-1000 Gy) additional products were detected as a consequence of ring-modification mechanisms. The plots of radiation dose-yield and corresponding calculated G values of the released undamaged bases and nucleosides from d-[CpT] and d-[TpC] suggest a base-sequence dependence and a quality- and quantity-dependent response to ionizing radiation. Although the product quantities formed from sequence isomers were slightly different, we found no qualitative differences in the product formed at the lowest doses examined.

Identification and characterization of a nuclear factor that specifically binds to the Rev response element (RRE) of human immunodeficiency virus type 1 (HIV-1).

The Rev protein of human immunodeficiency virus type 1 (HIV-1) differentially transactivates the expression of viral structural proteins by allowing the accumulation of unspliced and singly spliced viral mRNA in the cytoplasm. The cis-acting RNA target sequence for the Rev protein, termed the Rev response element (RRE), is present in the env gene and is predicted to form a highly ordered RNA secondary structure. Recent data indicate that Rev directly binds to RRE and, further, that this binding can be mapped to a 90-nucleotide subfragment at the 5' end of RRE. We now report that RRE also binds specifically and predominantly to a nuclear factor of approximately 56 kD. Mapping of the binding site reveals that the same subfragment that binds Rev also binds this nuclear factor. We designate this protein as NFRRE for nuclear factor, RRE binding. Rev and NFRRE appear to bind simultaneously to RRE. NFRRE is widely distributed in various mammalian cells. We speculate that this factor plays an important role in Rev-mediated transactivation and is likely to be involved in the processing or transport of cellular mRNA.

Inhibition of in vitro transcription by adenosine antibodies.

Antibodies against adenosine markedly inhibited in vitro transcription in isolated BHK 21 nuclei in a dose-dependent manner. The inhibition was specific as it could be completely reversed by the addition of homologous hapten. Addition of RNA at low concentration reversed the inhibition, whereas excess DNA did not have any effect. Adenosine antibodies also inhibited in vitro transcription with calf thymus DNA and E. coli RNA polymerase. Antibodies that react with DNA but not with RNA such as anti-dpA, anti-dpC and anti-DNA failed to inhibit in vitro transcription in isolated nuclei as well as with calf thymus DNA and E. coli RNA polymerase. The results strongly indicate that the binding of adenosine antibodies to RNA is responsible for the inhibition of transcription.

Base-specific binding of adenosine antibodies to double-stranded DNA.

Antibodies raised against adenosine have been reported to react with single-stranded DNA but not with double-stranded DNA. Using a highly sensitive avidin-biotin microELISA we report that these antibodies also react with double-stranded DNA. The binding was specific as it was completely inhibited by the homologous hapten. The results indicate that the antibody populations binding to ssDNA and dsDNA are not cross-reactive. The antibodies were shown to react with the topoisomers of plasmid DNA as assessed by gel retardation assay. The antibodies showed differential binding to restriction fragments of DNA indicating that some of the A residues in dsDNA are accessible to the antibodies.

Antibodies raised against denatured DNA bind to double-stranded DNA.

Antibodies raised against denatured DNA complexed with methylated bovine serum albumin have been reported to react with ssDNA but not with dsDNA. Using a highly sensitive avidin-biotin microELISA, we report that such antibodies also bind to dsDNA. Antibodies which reacted with ssDNA and dsDNA were found to be of IgG type. The antibodies did not react with tRNA and rRNA. The binding of antibodies to dsDNA was partially inhibited by individual deoxyribonucleotides. ssDNA as well as dsDNA inhibited the binding of antibodies to dsDNA. The binding of these antibodies to supercoiled and relaxed forms of pBR322 DNA was demonstrated by gel retardation assay. The cross-reaction with ssDNA was observed even after affinity purification on native DNA-cellulose. The antibodies were also shown to bind to poly(dA-dT).poly(dA-dT).

Radiation-induced volatile hydrocarbon production in platelets.

Generation of volatile hydrocarbons (ethane, pentane) as a measure of lipid peroxidation was followed in preparations from platelet-rich plasma irradiated in vitro. The hydrocarbons in the headspace of sealed vials containing irradiated and nonirradiated washed platelets, platelet-rich plasma, or platelet-poor plasma increased with time. The major hydrocarbon, pentane, increased linearly and significantly with increasing log radiation dose, suggesting that reactive oxygen species induced by ionizing radiation result in lipid peroxidation. Measurements of lipid peroxidation products may give an indication of suboptimal quality of stored and/or irradiated platelets.

Inhibition of in vitro amino acylation and translation by adenosine antibodies.

Adenosine antibodies markedly inhibited in vitro amino acylation of tRNA in a dose-dependent manner. The inhibition was specific as it was reversed by the homologous hapten. Addition of excess tRNA reversed the inhibition indicating that binding of antibodies to tRNA is responsible for inhibition. Adenosine antibodies also inhibited in vitro translation of endogenous mRNAs in rabbit reticulocyte lysate in a dose-dependent manner. The homologous hapten reversed the inhibition showing thereby the immunospecificity of inhibition.

Binding of deoxyadenylate and deoxycytidylate antibodies to double-stranded DNA.

Antibodies raised against deoxyadenylate and deoxycytidylate were found to react with double stranded DNA as assessed by highly sensitive avidin-biotin microELISA. The binding was specific as it was completely inhibited by the homologous hapten. The antibodies did not react with tRNA and rRNA. These antibodies were also shown to react with supercoiled and relaxed forms of pBR322 DNA as demonstrated by gel retardation assay.

Breathing 100% oxygen after global brain ischemia in Mongolian Gerbils results in increased lipid peroxidation and increased mortality.

Exposure of Mongolian gerbils to a 100% oxygen atmosphere after 15 minutes of global brain ischemia resulted in a marked increase in the production of pentane, an in vivo product of lipid peroxidation. Much less pentane production occurred in animals subjected to global brain ischemia then exposed to an air atmosphere and in animals exposed to a 100% oxygen atmosphere without ischemia. Gerbils placed in 100% oxygen for 3-6 hours after 15 minutes of ischemia also had a threefold increase in 14-day mortality compared with gerbils subjected to ischemia and then placed in an air atmosphere. These findings raise a serious question about the use of oxygen-enriched atmospheres during reperfusion following ischemia.