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At least 19 recordsLinked to original sources

PubMed1980

Antipain inhibits N-methyl-N'-nitro-N-nitrosoguanidine-induced transformation and increases chromosomal aberrations.

The morphologic transformation induced in Syrian hamster embryo cells by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) (0.25 microgram/ml of medium) is inhibited by posttreatment with antipain (6-600 microgram/ml), a protease inhibitor, but is unaffected by pretreatment. DNA replication relative to untreated controls is not affected by MNNG, antipain, or the combination of the two; no synergistic lethality of antipain and MNNG occurred as reflected in the cloning efficiency. Antipain was ineffective in influencing MNNG-induced sister chromatid exchanges, but it increased frequencies of chromosomal aberration (per metaphase) at 10, 26, and 40 hr when cells were treated with MNNG at 0.25 microgram/ml of medium followed by antipain 10 min later, the procedure used in the transformation studies. Antipain also increased the average number of aberrations at the second mitosis (34 hr) when the MNNG concentration was doubled. Chromatid exchanges increased 26 hr posttreatment with the combination of MNNG and antipain used for transformation. No difference in MNNG-induced aberrations was observed when antipain preceded MNNG by 24 hr. Although the mode of actin of antipain is unknown, antipain does not inhibit transformation by suppressing chromosomal rearrangements that could convert recessive mutations to the homozygous state.

Animals↗

PubMed1980

Differential antimutagenic effects of caffeine and the protease inhibitor antipain on mutagenesis by various mutagens in Escherichia coli.

The effects of caffeine (2 mg/ml) and the protease inhibitor antipain (1.75 mg/ml) in the plating agar medium on the yields of prototrophic revertants induced by 10 mutagens in E. coli uvrA- strains were tested. Mutagenesis by 4-nitroquinoline 1-oxide was greatly diminished by both caffeine and antipain. UV mutagenesis was decreased moderately by caffeine, and greatly by antipain. X-Ray mutagenesis was decreased very slightly by both caffeine and antipain. Mutagenesis by N-hydroxyurethan was inhibited moderately by caffeine, and greatly by antipain; that by methyl methanesulfonate was inhibited moderately by both caffeine and antipain, and that by N-methyl-N'-nitro-N-nitrosoguanidine was not suppressed by caffeine but was inhibited moderately by antipain. Mutagenesis by ethyl methanesulfonate was inhibited greatly by caffeine, but only slightly by antipain. The antimutagenic effect of caffeine was strong on furylfuramide (AF-2) mutagenesis, moderate on that of mitomycin C (tested with B2r type strain) and negligible on that of N-methyl-N-nitrosourea. These diverse antimutagenesis patterns are briefly discussed in relation to the current idea that antipain antimutagenesis is due to inhibition of inducible error-prone repair.

Antipain↗

PubMed1989

Antipain-mediated suppression of X-ray-induced chromosomal aberrations in human lymphocytes.

The protease inhibitor antipain is known to modulate the number of chromosomal aberrations induced by the S-phase-dependent alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine. Experiments have now been carried out to see if antipain might also effect the yield of aberrations induced by X-rays, which are S-independent and thus produce chromosomal aberrations by a different mechanism. The results show that human lymphocytes exposed to 0.4 or 1.5 Gy of X-rays at 48 h of culture and fixed at 3, 6, 8, 10 or 12 h thereafter contain 27-52% fewer chromatid breaks if the cells are also treated with antipain before irradiation. Because previous studies postulated that antipain could affect the induction of chromosomal aberrations by suppressing free radical reactions within cells, we also tested whether antipain affects X-ray-induced aberrations when present only during the time of irradiation, as is the case for free radical scavengers, such as L-cysteine. The results indicate that, in contrast to L-cysteine, antipain can suppress the induction of X-ray-induced aberrations even when administered as late as 2 h after irradiation, suggesting that the effects of antipain on aberrations are not attributable to its interference with short-lived radicals within the cells. Although the exact mechanism whereby antipain decreases the yield of chromosome aberrations induced by the S-independent agent X-rays is unknown, these data indicate that the formation of chromosome aberrations by S-independent agents too can involve an antipain-sensitive process.

Antipain↗

PubMed1985

Antipain-mediated suppression of sister chromatid exchanges induced by an inhibitor of poly (ADP-ribose) polymerase.

Exposure of mammalian cells to inhibitors of poly(ADP-ribose) polymerase, such as 3-aminobenzamide (3AB) results in the induction of sister chromatid exchanges (SCEs). The mechanism for the induction of SCEs by 3AB is unknown but is thought to be related to the incorporated halogenated pyrimidine used in SCE analysis. In this characteristic, 3AB-mediated SCE induction is similar to the elevated SCE frequency found in Bloom's syndrome (BS) cells. Recently, it has been reported that certain protease inhibitors, such as antipain, will inhibit SCE induction in BS cells. We now report that antipain will also suppress 3AB-induced SCE frequency. As has been reported for BS cells, the effects of antipain on SCE induction are partial, reducing SCE frequency by 0.15 to 0.40 SCE/chromosome (5-25% of the total induced frequency), and 30 microM concentrations of antipain are saturating. Antipain has no effect on baseline SCE frequency. These effects appear to involve free-radical production because dimethylsulfoxide (DMSO), a free-radical scavenger, will mimic the effects of antipain on 3AB-induced SCEs. Both antipain and DMSO will also reduce the elevated SCE frequency found in cells exposed to high (100 microM or more) levels of bromodeoxyuridine (BrdUrd). High exogenous levels of BrdUrd produce some of the same biological effects as 3AB exposure. Thus, a minor fraction of the elevated SCE frequency seen in cells exposed to 3AB or to high levels of BrdUrd appears to be similar to that found in cultured BS cells and is probably due to some free-radical-producing process.

Animals↗

PubMed1978

Prevention of normal mouse uterine maturation by antipain and elastatinal: suppression of peroxidase activity.

We had shown previously that the serine protease inhibitors, antipain and leupeptin, restrict uterine DNA snythesis and function in adult mice. The present study is an extension, focusing on the perimaturation period. The protease inhibitors, antipain and elastatinal were tested and the end points were uterine development and peroxidase activity in the uterus. Four week old mice were treated with eigher antipain (3 mg), elastatinal (5 mg) or vehicle (control) twice daily for 3 weeks. The uteri were excised, weighed and homogenized. Subcellular fractions were prepared by differential centrifugation. Each fraction was analyzed for peroxidase activity. The weights and protein content of uteri from control mice averaged greater than twice those in either antipain or elastatinal treated mice (p less than 0.001). Peroxidase activity was sharply curtailed by both inhibitors, antipain being more active than elastatinal. Mechanistically, antipain and elastatinal may act directly on the uterus either by inhibiting key enzymes or by preventing the formation of macromolecules crucial to normal function. Alternatively, the protease inhibitors may interfere with ovarian function, thereby restricting normal uterine development.

Animals↗

PubMed1988

Suppression of the radiation-induced expression of a tumor-associated antigen in human cell hybrids by the protease inhibitor antipain.

The effect of the protease inhibitor antipain (1.25 micrograms/ml) on the radiation-induced expression of a tumor-associated antigen in human cell hybrids has been investigated. A variety of treatment protocols have been studied where antipain was present before, during and at various times post-irradiation. It was found that antipain suppressed the radiation-induced expression of the tumor-associated antigen in all treatment protocols. The most effective suppression was obtained in those protocols where the protease inhibitor was present for the first 4 h post-irradiation. A possible explanation for this observation is that antipain may inhibit an error-prone DNA repair process. However, it is clear that this is not the only mechanism whereby the inhibitor can exert its effect since suppression was obtained even when antipain was added 10 days post-irradiation, a time when any DNA repair processes would be expected to be over.

Antigens, Neoplasm↗

PubMed1977

A protease inhibitor blocks SOS functions in Escherichia coli: antipain prevents lambda repressor inactivation, ultraviolet mutagenesis, and filamentous growth.

Inhibition of DNA synthesis in E. coli by treatment with carcinogenic and mutagenic agents results in the coordinate expression of a group of diverse functions (SOS functions) including lambda prophage induction, filamentous growth, and an error-prone DNA repair activity (SOS repair) believed to be responsible for ultraviolet mutagenesis. It has been proposed that this SOS induction proceeds via irreversible proteolytic inactivation of repressor(s) for SOS functions. To test this hypothesis, we investigated the effect of a protease inhibitor, antipain [(1-carboxy-2-phenylethyl)carbamoyl-L-arginyl-L-valylargininal], on SOS induction. We found that 0.5 mM antipain (which has no effect on cell growth, overall RNA and protein synthesis, or induction of beta-galactosidase) drastically decreases mutagenesis. Antipain also blocks expression of thermally induced mutator activity (another manifestation of SOS repair) and filamentous growth in a tif-1 mutant that expresses SOS functions at 42 degrees without inhibition of DNA synthesis or detectable DNA damage. Furthermore, antipain inhibits thermal induction of lambda prophage in the tif-1 mutant without affecting the kinetics of thermal induction of lambdacI857 prophage. This lambda mutant codes a temperature-sensitive repressor that is directly destroyed by heat and does not require the SOS induction pathway for inactivation at 42 degrees. From our results we conclude that antipain inhibits lambda prophage induction by blocking proteolytic inactivation of lambda repressor and that it inhibits the induction or expression of SOS repair and filamentous growth. Our results suggest a role for proteolytic cleavage in the regulation of SOS functions.

Carbamates↗

PubMed1978

Antipain inhibits thyroxine-induced synthesis of carbamyl phosphate synthetase I in tadpole liver.

The increased activity of carbamyl phosphate synthetase I [carbamoyl-phosphate synthase (ammonia); ATP: carbamate phosphotransferase (diphosphorylating), EC 2.7.2.5] in tadpole liver observed during thyroxine-induced metamorphosis was markedly inhibited by intraperitoneal injection of the microbial protease inhibitor antipain (0.1 micrometermol/g of body weight, twice daily). A somewhat less than maximal inhibition was seen when antipain was given only during the first 2 days of thyroxine treatment. On the other hand, little inhibition was observed when the inhibitor was given after the third or fourth day of thyroxine treatment. Antipain also inhibited thyroxine-induced increases of ornithine transcarbamylase (EC 2.1.3.3), arginase (EC 3.5.3.1), and succinate-cytochrome c reductase (EC 1.3.99.1) activities. Among other microbial protease inhibitors tested, chymostatin was nearly as effective as antipain, leupeptin was less effective, and pepstatin was ineffective. Analysis of the total liver protein and of the immunoprecipitate by sodium dodecyl sulfate/polyacrylamide gel electrophoresis showed that the inhibition was due to decreased amount of the enzyme protein. Antipain had no significant effect on leucine incorporation into total protein of tadpole liver. These results indicate the involvement of a proteolytic step in the pretranscriptional events in thyroxine-stimulated enzyme induction.

Animals↗

PubMed1979

Conditions for inhibiting and enhancing effects of the protease inhibitor antipain on x-ray-induced neoplastic transformation in hamster and mouse cells.

Using cultured normal hamster embryo cells and the heterploid mouse C3H cell line 10T1/2, clone 8, we have studied the effect of the protease inhibitor antipain on x-ray-induced neoplastic transformation. We found in both cell systems that, while there was no effect on cell survival as compared to irradiated controls, the addition of antipain at a concentration of 6 microgram/ml to the cultures 24 hr prior to irradiation resulted in enhanced transformation as compared to the frequency in cultures exposed to radiation alone. Yet the addition of antipain to cultures 10 min after irradiation resulted in a decreased transformation rate. This decrease was not found when antipain was added to the mouse cells 24 hr after irradiation or to the hamster cells 48 hr after irradiation. These results suggest that the protease inhibitor antipain has more than one mechanism of action in modulating the fixation and expression of transformation by x-irradiation, possibly by the modification of DNA repair.

Animals↗

PubMed1996

Involvement of antipain-sensitive protease activity in the interferon-beta-induced UV-refractoriness of Cockayne syndrome fibroblasts.

Fibroblast cells obtained from two siblings and a female patient with Cockayne syndrome (CS), when pretreated with human interferon (HuIFN)-beta prior to irradiation with UV light (254 nm wavelength), exhibited transiently induced fibrinolytic protease activity immediately after the irradiation in association with increased refractoriness to UV cell-killing. A protease inhibitor, antipain, inhibited the induction of protease activity in lysates of the CS fibroblasts from these 3 cases after the combination of HuIFN-beta pretreatment and UV irradiation, whereas elastatinal and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) inhibited the activity less than antipain did. Antipain also suppressed the increase in UV-refractoriness of HuIFN-beta-pretreated CS fibroblasts, as revealed by culturing cells for 24 h in medium containing the inhibitor immediately after UV exposure and thereafter evaluating the ability of colony formation by the cells. Thus, an antipain-sensitive protease may be involved in the UV-refractoriness induced by HuIFN-beta in CS fibroblast strains.

Antipain↗

PubMed1994

Involvement of antipain-sensitive protease activity in suppression of UV-mutagenicity by human interferon-alpha.

To study the relationship between the transient elevation of protease activity and hypomutability observed in hypermutable human RSa cells pretreated with human interferon (HuIFN)-alpha and then irradiated with far-ultraviolet light (UV), protease inhibitors capable of specifically inhibiting the activity were investigated. Of ten inhibitors tested, antipain showed the greatest inhibitory effect. Antipain also prevented the suppression of UV-mutagenicity by HuIFN-alpha in RSa and xeroderma pigmentosum-derived fibroblast cells, as shown by culturing cells in medium containing antipain immediately after UV exposure and evaluating the generation of clones resistant to ouabain- or 6-thioguanine-mediated cytotoxicity. Thus, an antipain-sensitive protease may be involved in the hypomutability induced by HuIFN-alpha.

Antipain↗

PubMed1989

Inhibition by leupeptin and antipain of the intracellular proteolysis of Ii.

Intracellular cleavage of Ii was evaluated in immunoprecipitates of radiolabeled Raji cells treated with protease inhibitors (leupeptin, antipain, chymostatin, and pepstatin) or blockers of endosomal function (chloroquine and monensin). Immunoprecipitates with anti-class II and anti-Ii(12-28) sera and VIC-Y1 MoAb revealed Ii cleavage products of 21,000 and 10,000 daltons (p21 and p10) only in leupeptin- and antipain-treated cells. Both p21 and p10 were judged to be N-terminal products because they were recognized with anti-Ii(12-28) and not with anti-Ii(183-193) or anti-Ii(192-211) sera. p10 might be derived from p21 because its intensity was increased in inverse proportion to p21 as a function of leupeptin or antipain concentration. p21, but not p10, was recognized by anti-class II antibody and thus might originate from class II-associated Ii. In pulse-chase studies, p21 and p10 appeared at 2 hr and later after Ii synthesis. p25, an Ii C-terminal fragment, was about 60% reduced by leupeptin or antipain. Intracellular proteolytic cleavage of class II-associated Ii appeared to follow two pathways leading either to N-terminal p21 and p10 or to C-terminal p25. Such cleavages might regulate or catalyze foreign antigen binding to class II.

Antibodies, Monoclonal↗

PubMed1998

Protease activation following UV irradiation is linked to hypomutability in human cells selected for resistance to combination of UV and antipain.

In order to examine the relationship between activation of an antipain-sensitive protease and suppression of mutability in UV (UVC)-irradiated human cells, a human cell variant with the high protease activity induced by UV was established and characterized for its susceptibility to UV-induced mutagenicity. Cells of a hypermutable cell strain, RSa, were mutagenized with ethyl methanesulfonate and irradiated with 10 J/m2 UV, followed by exposure to 20 mM antipain for 34 h. Whereas the combined treatment was totally lethal to RSa cells not treated with ethyl methanesulfonate, one surviving clone was isolated from the mutagenized cells and designated UVAP-1. When fibrinolytic protease activity was measured from extracts of the cell, it was found that the protease activity was elevated promptly after UV irradiation, reaching the maximum at 10 min post-irradiation. This protease activity was inhibited by antipain. After UV irradiation the phenotypic mutation frequencies of UVAP-1 cells were much lower than those of the parent RSa cells, as evaluated by the generation of clones resistant to ouabain-killing. Furthermore, mutation at the K-ras codon 12 in genomic DNA was detected in RSa cells but not in UVAP-1 cells. Thus, the protease activation was correlated with the decreased levels of UV-mutagenicity in UVAP-1 cells, supporting the possible involvement of the antipain-sensitive protease activity in the regulation of cellular mutability following UV irradiation.

Antipain↗

PubMed1983

Role of cellular proteinases in acute myocardial infarction. I. Proteolysis in nonischemic and ischemic rat myocardium and the effects of antipain, leupeptin, pepstatin and chymostatin administered in vivo.

To test the hypothesis that cellular proteinases contribute to ischemic myocellular death, measurements were made of tyrosine release (an index of overall proteolysis) from incubated slices of nonischemic and ischemic myocardium obtained at various times after coronary artery occlusion in rats. Proteolysis failed to increase in ischemic myocardium throughout the first 24 hours of occlusion, when irreversible damage develops, indicating that cellular proteinases do not undergo generalized activation in this phase. These data represent the first assessment of myocardial proteolysis throughout the development of ischemic death, and suggest that cellular proteinases do not play a causal role in this process. However, the possibility remains that ischemia selectively accelerates the breakdown of vital proteins, a phenomenon that may not be detected by measuring overall proteolysis. To determine whether future studies on the effects of proteolytic inhibition on infarct size are feasible, the ability of the proteinase inhibitors antipain, leupeptin, pepstatin and chymostatin, given in vivo, to interfere with proteolysis in ischemic myocardium was also evaluated. Leupeptin (10 or 40 mg/kg) inhibited proteolysis in a dose-related fashion (-49 and -72%, respectively, p less than 0.001). Antipain (20 mg/kg) decreased protein breakdown by 60% (p less than 0.001). The combination of antipain (20 mg/kg), leupeptin (40 mg/kg) and pepstatin (5 mg/kg) suppressed proteolysis almost completely at both 15 minutes (-88%, p less than 0.001) and at 6 hours (-72%, p less than 0.05) of ischemia, that is, throughout the development of irreversible injury. These results demonstrate that whatever proteolysis is occurring during acute myocardial infarction is largely mediated by cathepsins A, B, D, L and H and by calcium-activated neutral protease (that is, the enzymes sensitive to the inhibitors used). Because antipain, leupeptin and pepstatin significantly suppress such proteolysis, these agents might be useful in further assessing any potential contribution of cellular proteinases to the production of ischemic myocellular death. In addition, this study provides a new experimental model that affords serial assessments of regional myocardial proteolysis during the evolution of myocardial infarction.

Animals↗

PubMed1981

Protease inhibitor antipain suppresses 12-O-tetradecanoyl-phorbol-13-acetate induction of plasminogen activator in transformable mouse embryo fibroblasts.

Plasminogen activator (PA) activity was analyzed in normal and transformed 10T1/2 mouse fibroblasts treated with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) and the protease inhibitors antipain, leupeptin, and soybean trypsin inhibitor (SBTI). TPA induced PA activity in normal 10T1/2 cells was inhibited by antipain. Transformed 10T1/2 cells maintained high levels of PA activity which were not further stimulated by the addition of TPA. Similarly, antipain inhibited the PA activity of the transformed cultures. Leupeptin and SBTI had no effect. These findings, in light of the fact that antipain has been shown to suppress the promotional effect of TPA in X-ray induced malignant transformation, may suggest a definite role for proteases in the transformational event or maintenance of the transformed state.

Animals↗

PubMed1996

Suppression of UV- and interferon-alpha-refractoriness by antipain in human IFr cells established from RSa cells sensitive to both stimuli.

The human cell line IFr is a variant with an increased resistance to cell proliferation inhibition (CPI) by human interferon (HuIFN)-alpha, established from RSa cells with unusually high-sensitivity to CPI. IFr cells were later found to have increased resistance to the cell-killing effects of far-ultraviolet (UV) irradiation. Here, in cell lysates extracted from UV-irradiated IFr cells but not in those from irradiated RSa cells, fibrinolytic protease activity was found to be elevated promptly and transiently after irradiation. Treatment of IFr cells with HuIFN-alpha alone also resulted in the elevation of protease activity, but not that of RSa cells. Both the activity elevated after UV irradiation and after HuIFN-alpha treatment was inhibited to the greatest extent by antipain in vitro. Moreover, the refractoriness of IFr cells to UV cell-killing and to HuIFN-alpha CPI was suppressed by culturing with medium containing antipain immediately after UV irradiation or during HuIFN-alpha exposure. In similarly treated RSa cells, there was no modulation of UV- or HuIFN-alpha-susceptibility. These comparative characteristics between the two cell lines suggested that antipain-sensitive proteases and/or cellular functions may be involved in increased resistance to UV and HuIFN-alpha of IFr cells.

Antipain↗

PubMed1985

C-myc expression is reduced in antipain-treated proliferating C3H 10T1/2 cells.

In this report, we demonstrate that treatment of proliferating irradiated and nonirradiated C3H 10T1/2 cells with the protease inhibitor antipain is associated with a reduction in c-myc expression. Under conditions in which antipain treatment results in reduced c-myc transcripts, there is no effect on total RNA synthesis, growth rate, or saturation density. Antipain may be a useful inhibitor in which to further study the role of c-myc in cellular physiology.

Animals↗

PubMed1980

Cytotoxic effects of protease inhibitors on human cells. 1. High sensitivity of xeroderma pigmentosum cells to antipain.

Antipain had little effect on UV-survival and UV-induced sister chromatid exchanges in normal and xeroderma pigmentosum (XP) cells, suggesting that it may not affect DNA repair. Antipain itself produced a small, but significant, amount of sister chromatid exchanges. XP cells showed very high sensitivity to the cytotoxic effect of antipain, but an SV40-transformed XP cell strain did not demonstrate high sensitivity.

Adolescent↗