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

The human cell line IF^r is a variant with an increased resistance to cell proliferation inhibition (CPI) by human interferon (HuIFN)-α, established from RSa cells with unusually high-sensitivity to CPI. IF^r 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 IF^r cells but not in those from irradiated RSa cells, fibrinolytic protease activity was found to be elevated promptly and transiently after irradiation. Treatment of IF^r cells with HuIFN-α 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-α treatment was inhibited to the greatest extent by antipain in vitro. Moreover, the refractoriness of IF^r cells to UV cell-killing and to HuIFN-α CPI was suppressed by culturing with medium containing antipain immediately after UV irradiation or during HuIFN-α exposure. In similarly treated RSa cells, there was no modulation of UV- or HuIFN-α-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-α of IF^r cells. © 1996 Wiley-Liss, Inc.     

Increased levels of UV-induced protease activity in human UVAP-1 cells exposed to gravity-changing stress: involvement of E-64-sensitive proteases in suppression of UV mutagenicity

Under the 1G condition, the increase in antipain-sensitive protease activity promptly after UV (mainly 254 nm wavelength) irradiation in cultured human cells is detected and found to be one of the intriguing events involved in suppression of cell mutability. It was found that two cell lines, RSa and its variant UVAP-1 cells are applicable; the former is hypermutable and not susceptible to protease activation, while the latter is hypomutable and susceptible. In the present study it was investigated whether the increase in protease activity by UV irradiation is also observed in hypomutable human UVAP-1 cells exposed to gravity-changing stress and whether the increase is involved in suppression of UV mutagenicity. Exposure of human UVAP-1 cells to gravity-changing stress such as free-fall and parabolic flight prior to UV irradiation resulted in a pronounced increase in protease activity, but not to hypergravity conditions (2 and 10G) prior to UV irradiation. To characterize the proteases, components of lysates from the cells exposed to free-fall prior to UV irradiation were fractionated by high performance liquid chromatography, indicating two separate fractions with highly increased levels of E-64-sensitive protease activity. In the cells treated with E-64 during their exposure to free-fall, K-ras codon 12 base substitution mutation was detected after UV irradiation, although the mutation was not detected after UV irradiation alone. Thus, the increase in E-64-sensitive protease activity may be involved in the suppression of UV mutagenicity in UVAP-1 cells exposed to free-fall.     

Involvement of LEU13 in interferon-induced refractoriness of human RSa cells to cell killing by X rays

Culture of human cells with human interferon alpha and beta (IFNA and IFNB) results in increased resistance of the cells to cell killing by X rays. To identify candidate genes responsible for the IFN-induced X-ray resistance, we searched for genes whose expression levels are increased in human RSa cells treated with IFNA, using an mRNA differential display method and Northern blotting analysis. RSa cells, which showed increased survival (assayed by colony formation) after X irradiation when they were treated with IFNA prior to irradiation, showed increased expression levels of LEU13 (IFITM1) mRNA after IFNA treatment alone. In contrast, IF(r) and F-IF(r) cells, both of which are derived from RSa cells, showed increased X-ray resistance and high constitutive LEU13 mRNA expression levels compared to the parental RSa cells. Furthermore, the IFNA-induced resistance of RSa cells to killing by X rays was suppressed by antisense oligonucleotides for LEU13 mRNA. LEU13, a leukocyte surface protein, was previously reported to mediate the actions of IFN such as inhibition of cell proliferation. The present results suggest a novel role of LEU13 different from that in the inhibition of cell proliferation, involved in IFNA-induced refractoriness of RSa cells to X rays.     

Supervision of cellular response to radiation by human interferon

Human interferon (HuIFN) conferred human RSa cells to increased resistance to ultraviolet light, 4-nitroquinoline-1-oxide and X-ray in association with enhancement of DNA-repair capacity. The HuIFN actions were summarized by the supervision of cellular response, possibly via plasminogen activator-like protease induction.     

Search for gravity-responsive genes by PCR-based mRNA differential display in human cells

Mechanisms of molecular responses of human cells to gravity change and/or space radiation are one of the most important physiological problems in space science. We have previously reported that expression levels of several genes are changed in cultured human cells after UVC irradiation, and a few of those genes are responsible for UVC sensitivity. In this study, to find candidates for genes that play roles in susceptibility of human cells to gravity stressors, including those responsible for genetic stability in humans, we analyzed genes expressed differentially after gravity stress in human cells, using a PCR-based mRNA differential display (D.D.) method. Cells used were RSa and its variant cell lines, with discrepant sensitivity to radiation cell-killing and mutagenicity [correction of mutagenecity].     

Hypermutable change of human UV(r)-1 cells by p53 overexpression.

The p53 protein has been reported to regulate cellular responses to genetic stress such as far-ultraviolet light (UV), protecting human cells from mutation. Levels of p53 protein in hypermutable RSa cells were found here to increase soon after UV irradiation, while those in UV(r)-1 cells, a hypomutable variant of RSa cells, showed a delayed increase. Three cell lines overexpressing wild-type p53 in UV(r)-1 cells exhibited higher sensitivity to UV mutagenicity than did control U-V-7 cells transfected with vector alone, assessed using the ouabain-resistance phenotypic mutation test and identification of K-ras codon 12 base substitution mutation. On the other hand, U-V-7 cells showed UV-induced elevation of antipain-sensitive protease activity, but p53 transfectants did not. Moreover, antipain treatment to U-V-7 cells was increased susceptibility to UV mutagenicity. Thus, p53 protein overproduction may sensitize human cells, at least those tested, to UV mutagenicity, in association with inhibition of protease activity.     

The Roles of HSP27 and Annexin II in Resistance to UVC-Induced Cell Death: Comparative Studies of the Human UVC-Sensitive and -Resistant Cell Lines RSa and APr-1

We have reported that heat shock protein 27 (HSP27) and annexin II are involved in the protection of human cells against UVC-induced cell death. In this study we tried to confirm the combined roles of HSP27 and annexin II in cell death after UVC irradiation. In RSa cells with sensitivity to UVC, expression of annexin II decreased after UVC irradiation, but not in AP^r-1 cells with increased resistance to UVC. HSP27 siRNA-transfected AP^r-1 cells were sensitized to UVC lethality and showed decreased annexin II expression after UVC irradiation. In contrast, transfection of RSa cells with HSP27 cDNA increased their resistance to UVC lethality and caused increased annexin II expression. Furthermore, over-production of annexin II in RSa cells resulted in increased resistance to UVC lethality. This study indicates the involvement of cellular HSP27 expression in the UVC susceptibility of human cells, which occurs in association with regulation of annexin II expression.     

Increase in ultraviolet sensitivity by overexpression of calpastatin in ultraviolet-resistant UVr-1 cells derived from ultraviolet-sensitive human RSa cells

Human RSa cells are highly sensitive to apoptotic-like cell death by ultraviolet irradiation (UV) while UVr-1 cells are their variant with an increased resistance to UV. Three days after UV at 10 J/m2, the viability of RSa cells was approximately 17% while that of UVr-1 cells was 65%. This different survival might reflect apoptotic cell death since apoptosis-specific DNA ladder was more clearly observed in RSa cells than in UVr-1 cells after UV. Addition of ALLN/calpain inhibitor I to the culture medium after UV resulted in similar survival (14 - 18%) between RSa and UVr-1 cells. Immunoblot analysis showed down-regulation of protein kinase CTheta, Src, Bax and mu-calpain after UV was more prominent in UVr-1 than in RSa cells. Activated mu-calpain appeared within 1 h post-UV only in UVr-1 cells. The expression of calpastatin, a specific endogenous inhibitor of calpain, was higher in RSa than in UVr-1 cells. To further examine the role of calpain in UV-induced cell death, cDNA of human calpastatin was transfected into UVr-1 cells. The results showed that overexpression of calpastatin suppressed down-regulation of Src, mu-calpain and Bax. Concomitantly, colony survival after UV was reduced in calpastatin-transfected cells as compared to vector control cells. Our results suggest that activation of calpain might account for, at least in part, the lower susceptibility to UV-induced cell death in UVr-1 cells.     

Isolation and characterization of a novel large protease accumulated in mammalian cells in the presence of inhibitors

We have isolated and characterized a novel, large, multicatalytic protease from mammalian cells. This protease was designated PABI (protease accumulated by inhibitors). When baby hamster kidney (BHK) cells were grown in medium containing leupeptin, a potent serine-cysteine protease inhibitor, the trypsin-like protease activity (PABI) in the cells increased its level more than 100-fold over the control. This increase was also observed in other cultured cells such as COS, HepG2, and skin fibroblast cells. The activity was also elevated by treatment with other protease inhibitors including chymostatin or trans-epoxysuccinyl-L-leucylamide-(4-guanidino)butane. Immunoblot analysis, by employing antisera prepared against the purified PABI, also showed a concomitant increase of this protein in BHK, COS, and HepG2 cells on leupeptin treatment. PABI was purified to a homogeneous state from leupeptin-treated BHK cells. PABI is a glycoprotein of molecular weight 700,000. PABI was found to be a multimer of a major subunit of apparent Mr of 84,000, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electron microscopic analysis. PABI dissociates into subunits only under reducing conditions in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. PABI has both trypsin-like and chymotrypsin-like protease activities toward synthetic substrates. Both activities were inhibited by phenylmethanesulfonyl fluoride, aprotinin, bovine pancreas trypsin inhibitor, and chymostatin. Leupeptin inhibited only the trypsin-like activity of PABI. p-Chloromercuribenzoate had no effect on either activity. Furthermore, PABI degraded collagen type I and fibronectin. These results indicate that PABI is a novel protease which differs from any known proteases including cytosolic high molecular weight proteases. The physiological function of PABI is yet to be determined.     

Involvement of aldolase A in X-ray resistance of human HeLa and UV(r)-1 cells

To find novel proteins involved in radio-resistance of human cells, we searched for nuclear proteins, whose expression levels alter after X-ray irradiation in HeLa cells, using agarose fluorescent two-dimensional differential gel electrophoresis following mass spectrometry. We identified 6 proteins, whose levels were increased in nuclei 24 h after irradiation at 5 Gy, including aldolase A. Nuclear aldolase A levels increased twofold after the irradiation, however, total aldolase A levels did not change. When the expression of aldolase A was suppressed by its specific siRNA, sensitization of the suppressed cells to X-ray-induced cell death was observed. In addition, UV^r-1 cells with higher aldolase A expression exhibited lower sensitivity to X-ray-induced cell death than the parental RSa cells with lower aldolase A expression. These results suggest that aldolase A may play a role in the radio-response of human cells, probably in nuclei, in addition to its glycolytic role in the cytosol.     

Role of proteases in the release of porcine epidemic diarrhea virus from infected cells

Porcine epidemic diarrhea virus (PEDV), a causative agent of pig diarrhea, requires a protease(s) for multicycle replication in cultured cells. However, the potential role of proteases in the infection process remains unclear. In order to explore this, we used two different approaches: we infected either Vero cells in the presence of trypsin or Vero cells that constitutively express the membrane-associated protease TMPRSS2 (Vero/TMPRSS2 cells). We found that PEDV infection was enhanced, and viruses were efficiently released into the culture fluid, from Vero cells infected in the presence of protease, while in cells without protease, the virus grew, but its release into the culture fluid was strongly hampered. Cell-to-cell fusion of PEDV-infected cells and cleavage of the spike (S) protein were observed in cells with protease. When infected Vero cells were cultured for 3 days in the absence of trypsin but were then treated transiently with trypsin, infectious viruses were immediately released from infected cells. In addition, treatment of infected Vero/TMPRSS2 cells with the protease inhibitor leupeptin strongly blocked the release of virus into the culture fluid. Under electron microscopy, PEDV-infected Vero cells, as well as PEDV-infected Vero/TMPRSS2 cells treated with leupeptin, retained huge clusters of virions on their surfaces, while such clusters were rarely seen in the presence of trypsin and the absence of leupeptin in Vero and Vero/TMPRSS2 cells, respectively. Thus, the present study indicates that proteases play an important role in the release of PEDV virions clustered on cells after replication occurs. This unique observation in coronavirus infection suggests that the actions of proteases are reminiscent of that of the influenza virus neuraminidase protein.     

Effects of leupeptin and pepstatin on protein turnover in adult rat hepatocytes in primary culture

Addition of pepstatin, an inhibitor of acid protease, to 2-day cultures of rat hepatocytes rapidly inhibited the activity to hydrolyze hemoglobin (Hb), but did not affect the activity to hydrolyze α-N-benzoyl-dl-arginine-β-naphthylamide (BANA). On the other hand, addition of leupeptin, an inhibitor of thiol protease, inhibited the activity of BANA hydrolase and caused a sixfold increase in the activity of Hb hydrolase within 1 day. Neither protease inhibitor affected the rate of protein synthesis. Release of amino acids from hepatocytes into Hanks' salt solution was measured by the ninhydrin method. Pepstatin inhibited the release only 15% within 2 days, but leupeptin inhibited it 65% within 10 h. These two inhibitors had additive inhibitory effects on the release, suggesting that they inhibit the degradations of different groups of proteins. The inhibitory effect of leupeptin gradually decreased after 10 h, which is consistent with the observed induction of a protease activity mentioned above. A preferential involvement of leupeptin-sensitive protease in the degradation of proteins with longer half-lives was suggested from studies on [¹⁴C]leucine release from hepatocytes prelabeled for 30 h. On the other hand, the two inhibitors had similar effects on the release of [¹⁴C]leucine from hepatocytes labeled for only 1 h. Their inhibitory effects were again additive, but there was no reduction in the inhibition by leupeptin on prolonged incubation, suggesting that proteins with short half-lives were not substrates for the induced protease. These results suggest that in hepatocytes, proteins with longer half-lives are degraded more by cathepsin B than by cathepsin D, while those with short half-lives are degraded equally by these two proteases.     

Suppression of UV mutagenicity by human interferon

Effects of human interferon (HuIFN)-alpha on UV mutagenicity were examined in a human cell strain, RSa, and xeroderma pigmentosum (XP)-derived fibroblasts (XP1KY). The frequency of ouabain-resistance mutation in UV-irradiated RSa cells was unusually high (Suzuki et al., 1985), but that in cells pretreated with HuIFN-alpha before irradiation was reduced. 6-Thioguanine-resistance mutation was also depressed in XP1KY cells treated with HuIFN-alpha before irradiation. However, the depression of UV mutagenicity by HuIFN-alpha was lessened by treatment with cycloheximide immediately after UV irradiation. The relationship between HuIFN-depressed UV mutagenicity and HuIFN-affected DNA-repair and repair-related functions is discussed.     

Further evidence for a possible role of trypsin-like activity in the adherence of Porphyromonas gingivalis.

The present study was undertaken to evaluate a possible correlation between the level of trypsin-like activity and the adherence properties of Porphyromonas gingivalis. It was demonstrated that strains with high cell-associated trypsin-like activity attach in higher numbers to human epithelial cells than strains with low levels of trypsin-like activity. To a lesser extent, the same tendency was also noted for the agglutination of human erythrocytes. The ability of P. gingivalis to attach to erythrocytes and epithelial cells was found to be affected by the presence of arginine and thiol protease inhibitors (leupeptin, p-chloromercuriphenylsulfonic acid). The inhibition profile was partially dependent on the age of the bacterial cells used in the adherence assay. It is suggested that adherence of mid-log P. gingivalis cells involves primarily trypsin-like proteases, whereas 2-day-old cells possess additional specific attachment mechanism(s).     

Reduction in cell entry of Eimeria tenella (Coccidia) sporozoites by protease inhibitors, and partial characterization of proteolytic activity associated with intact sporozoites and merozoites

The role of proteases in the invasion of host cells by Eimeria tenella (Wisconsin strain) was studied in vitro. Protease inhibitors were used to treat sporozoites before inoculation or were applied to cultured chicken kidney cells before infection. The inhibitors antipain, leupeptin, aprotinin, L-1-tosylamide-2-phenyl-ethyl chloromethyl ketone (TPCK), or N-alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK) reduced parasite invasion to 16-66% of control after treatment of cultured cells or sporozoites with 5- or 50-micrograms/ml concentrations of inhibitors in the culture medium. Phenylmethylsulfonyl fluoride (PMSF) reduced invasion to 32-57.7% at concentrations of 1-4 mM. The optimum pH for hydrolysis of azocasein by intact sporozoites or merozoites was determined over a range of pH 5.0 to pH 9.0. Sporozoites were highly active over a broad range from pH 5.5 to pH 9.0, with an apparent optimum at pH 8.0. Merozoites had a much lower specific activity with pH optima at 7.0 and 8.5. The protease activity of sporozoites or merozoites could be inhibited completely by the addition of 50 micrograms/ml of leupeptin, TPCK, or TLCK or of 4 mM PMSF. Antipain inhibited proteases of sporozoites but not of merozoites. Pepstatin had little effect on either sporozoites or merozoites. The results suggest that parasite proteases of Eimeria may be necessary for invasion of host cells.     

Alterations in Leishmania donovani 3'-nucleotidase/nuclease activity banding pattern in polyacrylamide gels

1. In the absence of protease inhibitors, partial purification of the 3'-nucleotidase/nuclease (3'-N'ase) from detergent extracts of Leishmania donovani leads to the appearance of bands, at 42,000 and 38,000 Da, of enzyme activity which, in SDS-PAGE, migrate faster than 43,000 Da, the apparent weight of the intact polypeptide. 2. The generation of these faster migrating species is a consequence of detergent extraction and is prevented by the addition of the protease inhibitors PMSF and leupeptin, suggesting that degradation by an endogenous protease is responsible. 3. Treatment of leishmanial membranes with exogenous proteases yields activity at 38,000 Da which is membrane-associated. Protease treatment has no effect on living cells. 4. The observed changes in enzyme migration are discussed in terms of enzyme stability and regulation. 5. The advantages and limitations of the in situ gel activity assay are considered.     

A UV-resistant mutant without an increased repair synthesis activity, established from a UV-sensitive human clonal cell line

When cells of a human clonal cell line, RSa, with high sensitivity to UV lethality, were treated with the mutagen, ethyl methanesulfonate, a variant cell strain, UVr-1, was established as a mutant resistant to 254-nm far-ultraviolet radiation (UV). Cell proliferation studies showed that UVr-1 cells survived and actively proliferated at doses of UV-irradiation that greatly suppressed the proliferation of RSa cells. Colony-formation assays also confirmed the increased resistance of UVr-1 cells to UV. The recovery from a UV-induced inhibition in DNA synthesis, as [methyl-3H]thymidine uptake into cellular DNA, was more pronounced in UVr-1 cells than in RSa cells. Nevertheless, there was no significant difference in the activity of UV-induced DNA repair synthesis in either cell line, as estimated by the extent of unscheduled DNA synthesis and DNA repair replication. UVr-1 cells were also more refractory to 4-nitroquinoline 1-oxide (4NQO), but the activity of DNA repair synthesis induced by 4NQO in UVr-1 cells was much the same as in the RSa cells. Both N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) sensitivity and MNNG-induced DNA repair synthesis activity in UVr-1 cells were similar to that of RSa cells. These characteristics of UVr-1 cells are discussed in the light of a previously reported UV-resistant variant, UVr-10, which had an increased DNA repair synthesis activity.     

Genetic or chemical protease inhibition causes significant changes in the Bacillus subtilis exoproteome

Bacillus subtilis is a prolific producer of enzymes and biopharmaceuticals. However, the susceptibility of heterologous proteins to degradation by (extracellular) proteases is a major limitation for use of B. subtilis as a protein cell factory. An increase in protein production levels has previously been achieved by using either protease-deficient strains or addition of protease inhibitors to B. subtilis cultures. Notably, the effects of genetic and chemical inhibition of proteases have thus far not been compared in a systematic way. In the present studies, we therefore compared the exoproteomes of cells in which extracellular proteases were genetically or chemically inactivated. The results show substantial differences in the relative abundance of various extracellular proteins. Furthermore, a comparison of the effects of genetic and/or chemical protease inhibition on the stress response triggered by (over) production of secreted proteins showed that chemical protease inhibition provoked a genuine secretion stress response. From a physiological point of view, this suggests that the deletion of protease genes is a better way to prevent product degradation than the use of protease inhibitors. Importantly however, studies with human interleukin-3 show that chemical protease inhibition can result in improved production of protease-sensitive secreted proteins even in mutant strains lacking eight extracellular proteases.     

Protease inhibitors suppress the formation of tight junctions in gastrointestinal cell lines.

Tight junctions (TJ) of the fascia occludens type can be induced in the human colon adenocarcinoma cell lines HT29 and Caco-2 by treatment with 320 mM cesium sulfate. This process can be completely inhibited by the protease inhibitors leupeptin and antipain. The concentration for 50% inhibition was 32 microM leupeptin and 270 microM antipain, respectively. In the polarized colon carcinoma cell line Caco-2, the spontaneous formation of histotypical TJ and the development of transepithelial electrical resistance do not occur when the cells are cultured in medium containing 400 microM leupeptin. Following the removal of leupeptin, zonula occludens type TJ and electrical resistance develop synchronously during a period of 4 h. Dihydroleupeptin, the alcohol analog of leupeptin, inhibits neither the spontaneous nor the induced assembly of TJ fibrils. Thus, the aldehyde group of leupeptin is essential for activity. These data suggest that the salt-induced as well as the spontaneous formation of TJ involve cellular proteases which are susceptible to protease inhibitors.     

Engineered Protease-resistant Antibodies with Selectable Cell-killing Functions

Molecularly engineered antibodies with fit-for-purpose properties will differentiate next generation antibody therapeutics from traditional IgG1 scaffolds. One requirement for engineering the most appropriate properties for a particular therapeutic area is an understanding of the intricacies of the target microenvironment in which the antibody is expected to function. Our group and others have demonstrated that proteases secreted by invasive tumors and pathological microorganisms are capable of cleaving human IgG1, the most commonly adopted isotype among monoclonal antibody therapeutics. Specific cleavage in the lower hinge of IgG1 results in a loss of Fc-mediated cell-killing functions without a concomitant loss of antigen binding capability or circulating antibody half-life. Proteolytic cleavage in the hinge region by tumor-associated or microbial proteases is postulated as a means of evading host immune responses, and antibodies engineered with potent cell-killing functions that are also resistant to hinge proteolysis are of interest. Mutation of the lower hinge region of an IgG1 resulted in protease resistance but also resulted in a profound loss of Fc-mediated cell-killing functions. In the present study, we demonstrate that specific mutations of the C_H2 domain in conjunction with lower hinge mutations can restore and sometimes enhance cell-killing functions while still retaining protease resistance. By identifying mutations that can restore either complement- or Fcγ receptor-mediated functions on a protease-resistant scaffold, we were able to generate a novel protease-resistant platform with selective cell-killing functionality.