Conversion through homologous recombination of the gene encoding Simian virus 40 115,000-molecular-weight super T antigen to a gene encoding a normal-size large T antigen variant.

We have previously cloned the gene encoding a 115,000-Mr super T antigen (115K super T antigen), an elongated form of the Simian virus 40 large T antigen, originating from the rat cell line V 11 F1 clone 1, subclone 7 (May et al., J. Virol. 45:901-913, 1983). DNA sequence analysis has shown that the 115K super T antigen gene contains notably an in-phase duplication of a sequence located in the region of tsA mutations. We have also shown that the 115K super T antigen gene is able to induce the formation of transformed foci in transfected rat cells. After rat cell cultures were transfected with the cloned gene encoding 115K super T antigen, we obtained a large number of transformants as reported in this paper. In these transformants, we detected a very high frequency of new T antigen variants, as shown by immunoprecipitation of the cell extracts with anti-simian virus 40 tumor serum followed by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. Based on these results and all of the data presently available, it appears likely that the input plasmid or cosmid DNAs containing the cloned gene were first subjected to recombination events that yield new variant T antigen genes before these recombinant genes become integrated. The new variant T antigens observed in the transformants were predominantly those comigrating with normal-size large T antigen. In fact, these latter variants appeared to be indistinguishable from wild-type large T antigen as judged by restriction mapping by Southern blotting of the total genomic DNA of the transformants. Models of intermolecular or intramolecular homologous recombination occurring between or within the input plasmid or input cosmid DNA molecules are proposed to account for the formation of such revertants.

Transcriptional activation plays a role in the induction of apoptosis by transiently transfected wild-type p53.

The p53 tumor suppressor gene has been implicated in the induction of apoptosis in several cell systems. We have recently reported than transiently-transfected wt p53 is capable of inducing apoptosis in certain transformed cell lines. We demonstrated by quantitative analysis using flow cytometry that apoptosis was restricted to the population expressing wt, but not mutant, p53. In the present study we use this model system to analyse the functional domain of p53 in the induction of apoptosis. Several constructs expressing mutations or deletions in the C-terminal oligomerization domain, the N-terminal transactivation domain or the central DNA-binding domain were introduced into HeLa cells, and the ability of the expressed proteins to induce apoptosis was evaluated. All the functional domains were found to be necessary for the induction of apoptosis. In addition, cycloheximide and actinomycin D inhibited wt p53-induced apoptosis. We therefore conclude that p53 acts in this cell system, at least in part, as a transcriptional activator in the induction of apoptosis.

Human transforming growth factor alpha: sequence analysis of the 4.5-kb and 1.6-kb mRNA species.

Two transforming growth factor alpha (TGF alpha) mRNA species with the apparent sizes of 4.5 and 1.6 kb were identified in all human cell lines analysed. The cDNA corresponding to the 4.5-kb species was entirely sequenced, revealing the presence of a 3'-untranslated region (3'-UTR) of 3571 nucleotides, which contained several potential polyadenylation signals. Our results indicate that the 1.6-kb species is derived from the same precursor by alternative polyadenylation. In addition, we present evidence suggesting that TGF alpha-specific mRNAs could be initiated from transcription start points (tsp) located upstream from the tsp previously identified by Jakobovitz et al. [Mol. Cell. Biol. 8 (1988) 5549-5554].

Phenothiazines inhibit copper and endothelial cell-induced peroxidation of low density lipoprotein. A comparative study with probucol, butylated hydroxytoluene and vitamin E.

The effect of two phenothiazines, chlorpromazine (CPZ) and trifluoperazine (TFP) on the copper and endothelial cell-induced peroxidation of low density lipoprotein (LDL) has been studied and compared to that of drugs previously shown to protect LDL against peroxidation: probucol (PBC) and butylated hydroxytoluene (BHT). Incubation with CPZ or TFP inhibited in a dose-dependent manner LDL peroxidation induced either by copper ions or by cultured endothelial cells. Both the electrophoretic mobility and the thiobarbituric reactive substance content of LDL returned to almost normal values in the presence of 50 microM CPZ or TFP. The two studied phenothiazines also strongly inhibited the hydrolysis of LDL phosphatidylcholine which accompanies copper or endothelial cell-induced peroxidation of the particle. CPZ and TFP were as effective as PBC and BHT in inhibiting the LDL peroxidation. Whereas copper or endothelial cell-oxidized LDL were recognized and rapidly catabolized by mouse peritoneal macrophages, CPZ- or TFP-, as well as PBC- or BHT-treated LDL were not. Moreover, it was found that, in contrast to vitamin E, neither CPZ nor PBC reacted with model peroxy radicals formed by gamma irradiation of aerated ethanol. The possible mechanisms underlying this protective effect of phenothiazines against LDL oxidative modification are discussed.

[Non-hypertensive desoxycorticosterone treatment enhances the progression of atherosclerosis in WHHL rabbit]. / La déoxycorticostérone à dose non hypertensive accélère la progression de l'athérosclérose chez le lapin WHHL.

Coexistence of hypertension and lipid disorders enhances the development of atherosclerosis. However it is still unclear whether this promoting effect of hypertension results only from hemodynamic changes or whether part of it is mediated by humoral or neurogenic factors independently of blood pressure alteration. The aim of this study is to determine whether mineralocorticoids, which are known to be involved in the pathogenesis of hypertension, can influence the atherosclerotic process in Watanabe heritable hyperlipidemic rabbits (WHHL) independently of pressure changes. For this purpose, DOCA (200 or 400 mg/kg) or vehicle were implanted subcutaneously for 4 weeks in 3 months old WHHL or New Zealand (NZ) rabbits, without nephrectomy and with a fluid intake solution of 1% NaCl +0.2% KCl. DOCA treatment, independently of hemodynamic changes, significantly increases the size of atherosclerotic lesions in parallel with the aortic cholesterol esters content in the arch and thoracic aorta of WHHL rabbits. Plasmatic and aortic cholesterol and triglyceride content remains unchanged by DOCA treatment. Alteration of endothelial function usually found in WHHL rabbits is accentuated only for the dose of 400 mg/kg. Aortic sensitivity to serotonin is not altered, but the maximal contraction to this agonist is decreased in both strains by mineralocorticoid treatment. These results indicate the importance of non-hemodynamic factors related to hypertension which are implicated also in atherogenesis and support the clinical observations that a reduction of arterial pressure in hypertensive atherosclerotic patients is not sufficient to reduce the progression of this vascular disease.

Immunological evidence for the association between simian virus 40 115-kDa super T antigen and hsp70 proteins in rat, monkey, and human cells.

Immunological evidence was provided that in subclone 7 cell line, which is derived from SV40 transformed cells, 115-kDa super T antigen, a transformation-competent, elongated form of large T antigen was physically complexed with hsp70 proteins. This conclusion was first based on the coimmunoprecipitation from unstressed or heat shocked subclone 7 cells of both super T antigen and hsp70 proteins. This was observed with any one of a set of anti-T monoclonal antibodies reacting to determinants located either in the C-terminal region or in the N terminal region. Reciprocally coimmunoprecipitation of both hsp70 and super T was also observed in the anti-hsp70 peptide serum-immunoprecipitate. The formation of complexes between hsp70 proteins and super T antigen in subclone 7 cells was also confirmed by Western blot experiments. Moreover, when expressed in cell lines originating from human (Hela cells) or monkey (CV1P cells) species following transfection with the relevant plasmid, super T antigen again displayed the ability to associate with hsp70 proteins. Considering that super T antigen was obtained in laboratory experiments as a stable evolutionary variant of SV40 large T antigen, it is suggested that the marked ability of super T antigen to associate with heat shock protein could be selectively advantageous under certain conditions.

In vitro and ex vivo inhibition of the modification of low-density lipoprotein by indapamide.

The effect of an antihypertensive drug, indapamide, on copper- and endothelial cell-induced peroxidation of human low-density lipoprotein (LDL) was studied and compared with that of drugs previously shown to protect LDL against peroxidation: probucol and vitamin E and other thiazidic and nonthiazidic diuretics (clopamide, hydrochlorothiazide, and furosemide). Incubation with indapamide inhibited in a dose-dependent manner LDL peroxidation induced either by copper ions or by cultured endothelial cells. Both electrophoretic mobility and thiobarbituric acid-reactive substances (TBARS) content of LDL returned to almost normal values in the presence of 1 microM indapamide. This drug was at least 10 times more potent than probucol and vitamin E in inhibiting LDL peroxidation. No inhibitory effect has been observed with clopamide, hydrochlorothiazide, and furosemide in the same experimental conditions. Homozygote Watanabe rabbits were treated orally with indapamide (10 mg/kg/d for 3 days) to evaluate the potential protective effect of the compound on LDL peroxidation in vivo. Purified LDL from placebo and treated rabbits were submitted to peroxidation induced by copper ions, and indapamide was effectively able to protect LDL in these experimental conditions. This effect was especially obvious 6 and 8 h after the start of the incubation when LDL of the placebo-treated animals were modified. The mechanism of action of these drugs was examined in vitro using the 1,1-diphenyl-2-picryl-hydrazyl (DPPH) test and in kinetic studies of arachidonic acid photoperoxidation. Indapamide as well as vitamin E and probucol were effective free radical scavengers, but the other diuretic molecules were not.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of p53 as a transcription factor in the induction of apoptosis.

The tumor suppressor gene p53 plays a major role in the protection of cells from DNA damage. Activation of the protein in response to irradiation or genotoxic agents, and possibly by other signals, results in growth arrest at the G1 phase of the cell cycle or in apoptosis. While it has been shown that the ability of p53 to function as a sequence-specific transcriptional activator is necessary for the induction of growth arrest, the mechanism of p53-mediated apoptosis is not clear yet. In the present report we summarize the results obtained by several groups concerning p53-mediated apoptotic pathway. We suggest that p53 may induce apoptosis via a complex network of interacting pathways, which may be transcriptionally dependent or independent, depending on external signals and on the cellular context. Whatever the mechanisms are, the outcome-cell death by apoptosis-is a key function of the tumor suppressor p53.

Non-hypertensive deoxycorticosterone-salt treatment accelerates atherosclerosis progression in Watanabe heritable hyperlipidemic rabbit.

Epidemiological studies have indicated that hypertension enhances the development of atherosclerosis in patients with lipid disorders. However, it was still unclear whether this promoting effect resulted only from hemodynamic changes or whether part of it was mediated by humoral or neurogenic factors independent of blood pressure alteration. The aim of this study was to determine whether mineralocorticoids, which are known to be involved in the pathogenesis of hypertension, could be implicated in the atherosclerotic process independent of pressure changes. For this purpose, the effect of deoxycorticosterone (DOCA, 200 mg/kg s.c.) on aortic atherosclerosis was studied in Watanabe heritable hyperlipidemic (WHHL) rabbits in comparison with New Zealand rabbits. After 4 weeks of treatment, DOCA significantly increased the size of atherosclerotic lesions in the arch and thoracic aorta (+115%) in parallel with the aortic cholesterol ester content (+83%). The vascular free cholesterol and triglyceride content remained unchanged on DOCA treatment, as were arterial pressure and plasma cholesterol levels. None of these effects was observed in New Zealand rabbits. DOCA did not accentuate the alteration of endothelial function usually found in WHHL rabbits. The sensitivity to serotonin was not altered, but the maximal contraction to this agonist was decreased in both strains by mineralocorticoid treatment.

S 12340: a potent inhibitor of the oxidative modification of low-density lipoprotein in vitro and ex vivo in WHHL rabbits.

Oxidative modification of low-density lipoprotein (LDL) is thought to play a key role in the formation of foam cells and in the initiation and progression of the atherosclerotic plaque. After evaluation of a large number of original drugs, S 12340 was found to be the most potent compound in inhibiting in a dose-dependent manner the human LDL oxidative modification induced either by copper ions or by cultured endothelial cells. Both the electrophoretic mobility and the thiobarbituric acid reactive substances returned to almost normal values in the presence of 0.5 microM of S 12340. Watanabe heritable hyperlipidemic rabbits were treated orally for 3 days or for 1 month with S 12340 to evaluate the potential protective effect of the compound on LDL oxidative modification induced ex vivo. Purified LDL from placebo and treated rabbits were submitted to oxidation, and S 12340 was effectively able to protect LDL in a dose-dependent manner and at doses as low as 10 mg/kg/day. Purified LDL from animals sacrificed at various times after oral administration of S 12340 were protected against oxidation for at least 6 h after the last administration of the compound. These findings are in good agreement with the plasma and LDL levels of S 12340 in these WHHL rabbits. S 12340, probucol and vitamin E were all able to decrease the optical density of the 1,1-diphenyl-2-picrylhydrazyl solution, demonstrating their free radical scavenging properties. The pharmacological properties of the compound suggest that S 12340 may be of potential interest for a new therapeutic approach to atherosclerosis.

Hypercholesterolemia increases coronary endothelial dysfunction, lipid content, and accelerated atherosclerosis after heart transplantation.

Hyperlipidemia may increase endothelial damage and promote accelerated atherogenesis in graft coronary vasculopathy. To study the effects of hypercholesterolemia on coronary endothelial dysfunction, intimal hyperplasia, and lipid content, a porcine model of heterotopic heart transplantation, allowing nonacute rejection without immunosuppressive drugs, was used. A high cholesterol diet was fed to donor and recipient swine 1 month before and after transplantation. The endothelial function of coronary arteries of native and transplanted hearts from cholesterol-fed animals was studied in organ chambers 30 days after implantation and compared with endothelial function in arteries from animals fed a normal diet. The total serum cholesterol increased 3-fold in donors and recipients. Endothelium-dependent relaxations to serotonin, to the alpha(2)-adrenergic agonist UK14,304, and to the direct G-protein activator sodium fluoride were decreased significantly in allografted hearts compared with native hearts from both groups. Relaxations to the calcium ionophore A23187 and bradykinin were decreased significantly in allografts from animals fed the high cholesterol diet. The prevalence of intimal hyperplasia was significantly increased in coronary arteries from hypercholesterolemic swine. There was a significant increase in the lipid content of allograft arteries of hypercholesterolemic recipients. Hypercholesterolemia causes a general coronary endothelial dysfunction, increases the prevalence of intimal hyperplasia, and augments the incorporation of lipids in the vascular wall after heart transplantation. Hyperlipidemia accelerates graft coronary atherosclerosis through its effects on the endothelium.

Synthesis and evaluation of 3',5'-di-tert-butyl-4'-hydroxyflavones as potential inhibitors of low density lipoprotein (LDL) oxidation.

The novel flavones 6-28, which display structural analogies with the two well-known lipid peroxidation inhibitors, probucol [1] and butylated hydroxytoluene [2], were synthesized and studied in vitro for their ability to inhibit the copper sulfate or endothelial cell-induced lipid peroxidation of human low-density lipoprotein (LDL). Most of the flavones were active in the range of 0.1-1 microM.

Calcium antagonists prevent monocyte and endothelial cell-induced modification of low density lipoproteins.

Low density lipoprotein (LDL) incubated in the presence of the calcium antagonists verapamil, nifedipine and flunarizine were more resistant than control LDL to human monocyte- or endothelial cell-induced modification, as assessed by electrophoretic mobility in agarose gel, thiobarbituric acid reactive substance content, and degradation by J774 macrophages. The efficiency of the drugs was: flunarizine greater than nifedipine greater than verapamil. Moreover, a 24 h preculture with calcium antagonists significantly impaired the ability of cells to modify LDL in the absence of the drugs. All the studied drugs also inhibited copper-induced autooxidation of LDL. None of the studied calcium antagonists, at concentrations up to 10(-4) M, significantly reacted with free radicals as assessed by the 1,1-diphenyl-2-picrylhydrazyl test. It is suggested that such a protective effect of calcium antagonists against LDL peroxidation could play a role in the previously reported antiatherogenic effect of these drugs.