In vitro malignant conversion of low-grade rat urinary bladder carcinoma cells by exposure to N-methyl-N-nitrosourea.

The cause of deeply invasive human bladder carcinoma is unknown. Animal studies suggest that a malignant (invasive) conversion is inducible in low-grade noninvasive tumors by further exposure to a chemical carcinogen. To elucidate what molecular mechanism(s) is involved in the conversion, an in vitro system has been established in which conversion from low- to high-grade carcinoma can be induced. A rat bladder carcinoma cell line D44, derived from an N-methyl-N-nitrosourea (MNU)-induced low-grade noninvasive rat bladder carcinoma was used in the present investigation. Cloned D44 cells (D44c) were exposed to MNU, 50 to 400 micrograms/ml, for 1 h at 37 degrees C once a week for up to 6 weeks. After exposure to MNU, cells with altered morphology were cloned. The yield of altered clones was highest after a total dose of 150 to 200 micrograms of MNU used in 1 to 3 doses. Of 21 clones with altered morphology, 4 clones were further treated with MNU at the initial dose once a week for up to 3 weeks and then subcloned. Thirty-three of these subclones were examined for tumorigenicity in athymic nude mice. Twenty-seven formed highly invasive carcinomas, mostly squamous type, whereas the parental D44c cells failed to develop tumors upon inoculation. Pulmonary metastases were observed in 17 of the 27 clones. Plasminogen activator activity was elevated 4- to 9-fold as compared to parent D44c cells. ras p21 mutations at codon 12 were detected in 5 of 30 clones. These results indicate that the in vitro system described here may provide a useful model to study the molecular mechanisms involved in the conversion of noninvasive bladder carcinomas to metastasizing ones.

Marked enhancement of rat urinary bladder carcinogenesis by heat-killed Escherichia coli.

Chronic urinary tract infection is an important risk factor for the development of carcinoma in the human urinary bladder. To test the effect of chronic persistent inflammation on bladder carcinogenesis, we instilled heat-killed Escherichia coli (1 x 10(8) cells suspended in 0.5 ml of phosphate-buffered 2.1% NaCl solution) twice a week into the heterotopically transplanted rat urinary bladders in which carcinogenesis was initiated by a single dose (0.25 mg) of N-methyl-N-nitrosourea. When compared with the control animals, the rats treated with killed E. coli showed significantly enhanced bladder tumorigenesis, as reflected by an increase in the incidence of tumor (P = 0.05) and a 6- to 40-fold increase in the number of tumors per bladder (P less than 0.0001). The tumors were characterized by intraepithelial clusterings of neutrophils and by chronic inflammation and marked capillary proliferation in the tumor stroma. All of these features were rare in tumors in the control groups. The accelerated cell proliferation induced by killed E. coli treatment appears to play a significant role in the enhancement of tumorigenesis.

Tumor-promoting effect of urinary epidermal growth factor in rat urinary bladder carcinogenesis.

We previously demonstrated that the specific component of rat urine designated as Fraction I (Fr.I), which has been known to enhance carcinogenesis in the rat urinary bladder, contains epidermal growth factor (EGF) and transferrin (TF). The present study was designed to determine whether EGF or TF is responsible for the tumor-enhancing effect of Fr.I. The heterotopically transplanted rat urinary bladder (HTB), which has been developed in our laboratory, was used for the study. Fr.I was prepared from normal rat urine by a method published previously. Fr.I deficient in EGF or TF was prepared by passing this fraction through an Affi-Gel Hz column coupled with anti-rat EGF or TF antibodies, respectively. EGF and TF eluted from the column (designated as eluted EGF and eluted TF) were also tested for tumor-enhancing activity. Fr.I passed through the column coupled with nonimmune rabbit IgG served as control (Fr.I column control). After initiation of carcinogenesis in HTBs by instillation of a single dose of 0.25 mg of N-methyl-N-nitrosourea, test materials were administered into these HTBs once a week for 30 weeks. The results showed that removal of EGF significantly reduced the tumor-enhancing effect of Fr.I (P less than 0.001 as compared to that of the Fr.I column control) and that eluted EGF by itself significantly enhanced the carcinogenesis as compared to that of the vehicle control (P less than 0.006). Removal of TF from Fr.I also reduced the tumor-enhancing effect of Fr.I (P less than 0.01). However, removal of both EGF and TF from Fr.I did not enhance the inhibitory effect demonstrated by the Fr.I which was deficient in EGF. Likewise, combined use of TF and EGF did not exceed the tumor-promoting effect of EGF. The results indicate that EGF in Fr.I may play a significant role in the promotion of bladder carcinogenesis by urine.

Primary structure and inhibitory properties of a proteinase inhibitor produced by Streptomyces cacaoi.

Protein proteinase inhibitors showing sequence homology with Streptomyces subtilisin inhibitor (SSI) have been found to be distributed widely in Streptomyces species, and accordingly have been named SSI-like (SIL) proteins. SIL1 from S. cacaoi was the first of these proteins to be isolated and to be given a serial number. To study the structure-function relationship of SIL proteins, we determined the primary structure of SIL1 and measured its inhibitory activities. It was found to be composed of 110 amino acids and to exist in dimer form. The amino-acid sequence of SIL1 was unique among other characterized SIL proteins in having a one-residue deletion in two regions and a three-residue insertion in the flexible loop region. Sequence comparison indicated that SIL1 was distantly related to other members of the SSI family, and that amino-acid replacements had occurred not only on the surface of the SIL1 molecule but also in the beta-sheet region. The reactive site of SIL1 was considered to be Arg70-Glu71 from sequence alignment with other SSI-family inhibitors. SIL1 inhibited subtilisin BPN' strongly with an inhibitor constant (Ki) of 2.8 x 10(-11) M, like other members of the SSI family possessing an Arg residue at the P1 site. In contrast, SIL1 exhibited weak inhibition toward trypsin with a Ki value of 5.5 x 10(-8) M, possibly as a consequence of insertion of the three residues in the flexible loop region near the reactive site. This contrast seems to be due to the difference in the subsite structure of the two proteinases.

New subtilisin-trypsin inhibitors produced by Streptomyces: primary structures and their relationship to other proteinase inhibitors from Streptomyces.

Three new proteinaceous inhibitors of trypsin and subtilisin of the Streptomyces subtilisin inhibitor (SSI)-like (SIL) protein family were isolated and purified from culture media of Streptomyces strains; SIL5 from S. fradiae, SIL7 from S. ambofaciens and SIL12 from S. hygroscopicus. Their complete amino-acid sequences were determined by sequence analysis of the intact SIL proteins and peptides obtained by enzymatic digestion of S-pyridylethylated proteins. SIL7 showed high sequence similarity to other Arg-possessing SSI-family inhibitors at the P1 site. SIL12 is unique in having a two-residue insertion in the flexible loop region. Based on the amino-acid sequences of these inhibitors and other SSI-family inhibitors whose sequences have already been determined, the phylogenetic relationship of SSI-family inhibitors and Streptomyces strains was considered. Among about 110 amino-acid residues possessed by SSI-family inhibitors, 28 are completely conserved. The contribution of these conserved residues to the function and stability of the inhibitor molecules is discussed on the basis of the results obtained from mutational analysis of SSI and its crystal structure.

Microbial secretion of biologically active human transforming growth factor alpha fused to the Streptomyces protease inhibitor.

A secretory production system for the active form of transforming growth factor alpha (TGF alpha) was established in Streptomyces lividans using a gene encoding the secretory protease inhibitor, Streptomyces subtilisin inhibitor (SSI). It was demonstrated that deletion of one of the putative dual ssi terminators is effective to extracellularly produce a heterologous polypeptide in a fused form. The recombinant fusion protein, SSI::TGF alpha, was purified to homogeneity by a combination of hydrophobic chromatography and reverse-phase high-performance liquid chromatography (RP-HPLC). It was noteworthy that the SSI::TGF alpha hybrid protein exhibited bifunctional activity: the TGF alpha activity for cell growth promotion and the inhibitory activity of SSI. Taken together with the results of analytical gel filtration, these findings strongly indicate that each moiety in the fusion protein correctly folds and the whole hybrid molecule exists in a dimeric form, which results in its bifunctional activity.

Novel host-vector system for selection and maintenance of plasmid-bearing, streptomycin-dependent Escherichia coli cells in antibiotic-free media.

A novel system for selection and maintenance of cells carrying a recombinant plasmid has been developed, using the streptomycin-dependent (Smd) Escherichia coli 4D host and a plasmid vector carrying an rpsL gene from an Sm-resistant (Smr) mutant of E. coli which masks the Smd phenotype. Strain 4D carrying the Smr pBR322 plasmid can grow without Sm. Using this host-vector system, we can select for cells carrying an Smr recombinant plasmid and maintain them in antibiotic-free media.

Chronic lymphocytic leukemia/small lymphocytic lymphoma with Reed-Sternberg-like cells and possible transformation to Hodgkin's disease. Mediation by Epstein-Barr virus.

The pathogenesis of Reed-Sternberg cells and variants (RS-H cells) found in rare cases of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) is unknown. We studied 13 such cases by immunohistochemistry and in situ hybridization for identification of Epstein-Barr virus (EBV) RNA. The RS-H cells in five cases expressed the B-lineage marker CD20 and were negative for CD15. In two cases, the RS-H cells showed expression of both CD20 and CD15, whereas in another six cases, the cells were positive for CD15 but negative for CD20. Three of the cases expressing CD15 showed subsequent evidence of disseminated Hodgkin's disease. Regardless of the phenotype or clinical behavior, the RS-H cells in 12 of 13 cases were found to contain EBV RNA by in situ hybridization, but the surrounding neoplastic lymphocytes were invariably negative for EBV RNA. It is suggested that EBV has an important role in the pathogenesis of the RS-H cells in these rare cases.

Involvement of urine in epithelial-stromal interactions in urinary bladder carcinogenesis.

The role of urine in epithelial-stromal interactions in urinary bladder carcinogenesis was investigated using the 'Stroma' bladder model established in our laboratory. Rats treated with 0.05% N-butyl-N-(4-hydroxybutyl) nitrosamine (BHBN) in drinking water for 4 weeks or age-matched untreated rats served as donors of bladders with denuded epithelium ('stroma' bladders), which were resurfaced 4 days later with urothelial cells from rats treated with 0.05% BHBN for 4 weeks. Subsequently, the transplants received weekly injections of normal rat urine or saline. In the urine-free environment, cell implants developed hyperplastic changes but few tumor formations with no significant difference between the two types of 'stroma' bladder. In contrast, urine instillation stimulated neoplastic growth, and tumor-enhancing effect was significantly accelerated in the BHBN-treated 'stroma' bladder group as compared to the control group. These results suggest that epithelial-stromal interactions are altered in such a way that bladder carcinogenesis is enhanced by prior exposure of the bladder stroma to carcinogen and subsequent urine contact.

Nodular lymphocyte-predominant Hodgkin's disease: study of immunoglobulin light chain protein and mRNA expression.

The techniques of immunohistochemistry and in situ hybridization were applied to 10 formalin- or B5-fixed, paraffin-embedded cases of nodular lymphocyte-predominant Hodgkin's disease to determine whether lymphohistiocytic (L&H) cells contain any detectable amount of immunoglobulin light chain protein or messenger RNA. None of the cases studied demonstrated any detectable amount of either kappa or lambda light chain mRNA within L&H cells or Reed-Sternberg cells despite positive labeling of plasma cells, immunoblasts, and germinal center cells. Polyclonal kappa light chain antibody studies showed positive staining of L&H cells in seven cases, including three costaining with polyclonal lambda light chain antibody. Monoclonal kappa and lambda light chain antibody studies, however, showed no staining of L&H cells despite positive staining of immunoblasts and plasma cells. It is suggested that L&H cells do not synthesize appreciable amounts of light chain immunoglobulin protein and are not closely related to reactive immunoblasts or germinal center cells.

An endogenous target protease, SAM-P26, of Streptomyces protease inhibitor (SSI): primary structure, enzymatic characterization, and its interaction with SSI.

We have been focusing on the potent involvement of the molecular interaction between a protease and a protease inhibitor in the physiological or morphological regulation of Streptomyces cells producing them [Taguchi et al. (1995) J. Bacteriol. 177, 6638-6643; Suzuki et al. (1997) J. Bacteriol. 179, 430-438]. In this study, an extracellular protease, termed SAM-P26, was isolated as a target of endogenous protease inhibitor (SSI) from the culture medium of an SSI non-producing mutant strain derived from Streptomyces albogriseolus S-3253. Complete amino acid sequence determination revealed that SAM-P26 is identical to a protein encoded by the SAM-P20D gene, which was previously found to be located downstream of the gene for SAM-P20, another target protease of SSI. Based on the sequence homology, SAM-P26 was categorized as a member of the chymotrypsin family like SAM-P20. Sequence similarity between SAM-P26 and SAM-P20 was immunologically demonstrated by Western blot analysis using anti-SAM-P20 antiserum. The molecular mass (26 kDa) of SAM-P26 estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was much higher than that calculated from the amino acid sequence of SAM-P26 (18,376.8 Da) and that of the S-pyridylethylated form (18,808.4 Da) of SAM-P26 determined by Matrix-assisted Laser Desorption/Ionization-Time of Flight/Mass Spectrometry. Analytical gel-filtration analysis revealed that SAM-P26 exists as a monomer (18.8 kDa) in the native state. The results as to substrate specificity and inhibitor sensitivity indicated SAM-P26 exhibits chymotrypsin-like activity. For the proteolytic activity, the optimal pH was 10.5 and the optimal temperature was 60 degreesC. The complex formation of SAM-P26 with SSI was confirmed by native-PAGE analysis.

A subtilisin inhibitor produced by Streptomyces bikiniensis possesses a glutamine residue at reactive site P1.

We determined the complete amino acid sequence of a novel subtilisin inhibitor, SIL15, which had been isolated from the culture supernatant of Streptomyces bikiniensis and shown to be a member of the Streptomyces subtilisin inhibitor (SSI)-like (SIL) protein family, and then identified its reactive site. SIL15 is composed of 113 amino acids and exists as a dimer. Compared with other SSI-family inhibitors, SIL15 was found to be unique in that it possesses a Gln residue at the P1 site of the reactive site and has two-residue insertions in two regions, one in the alpha 1-helix and the other in the flexible loop region near the reactive site. Inhibition of subtilisin BPN' by SIL15 (inhibitor constant, 2.7 x 10(-11) M) was due to the presence of a Gln residue at the P1 site, which was well consistent with the results obtained for P1-site mutants of SSI and turkey ovomucoid domain 3.

Three novel subtilisin-trypsin inhibitors from Streptomyces: primary structures and inhibitory properties.

Three novel proteinaceous inhibitors, which had been identified as "Streptomyces subtilisin inhibitor-like (SIL) proteins" and exhibited trypsin inhibition in addition to strong inhibition toward subtilisin BPN', were purified from the culture broth of three Streptomyces strains: SIL10 from S. thermotolerans, SIL13 from S. galbus, and SIL14 from S. azureus. Their primary structures were determined by sequence analysis of intact SIL inhibitors and peptides obtained by enzymatic digestions of S-pyridylethylated SIL inhibitors. These inhibitors were composed of about 110 amino acids and existed as dimer proteins. The reactive site was identified as Lys-Gln for all three inhibitors by sequence analysis of their modified forms in which the reactive-site peptide bond was specifically cleaved by subtilisin BPN' under acidic conditions. Thus, their inhibition toward trypsin and subtilisin BPN' was due to the presence of a Lys residue at the P1 site. Inhibitor constants toward subtilisin BPN' and trypsin were also determined. These inhibitors showed relatively high sequence homology to other SSI-family inhibitors possessing a Lys residue at the P1 site, with amino acid replacements on their molecular surface.

A cold-adapted protease engineered by experimental evolution system.

A new cold-adapted protease subtilisin BPN' mutant, termed m-51, was successfully isolated by use of an evolutionary program consisting of two-step in vitro random mutagenesis, which we developed for the screening of mutant subtilisins with increased activity at low temperature. The m-51 mutant showed 70% higher catalytic efficiency, expressed by the k(cat)/K(m) value, than the wild-type at 10 degrees C against N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide as a synthetic substrate. This cold-adaptation was achieved mainly by the increase in the k(cat) value in a temperature-dependent manner. Genetic analysis revealed that m-51 had three mutations, Ala-->Thr at position -31 (A-31T) in the prodomain, Ala-->Val at position 88 (A88V), and Ala-->Thr at position 98 (A98T). From kinetic parameters of the purified mutant enzymes, it was found that the A98T mutation led to 30% activity increase, which was enhanced up to 70% by the accompanying neutral mutation A88V. The A-31T mutation severely constrained the autoprocessing-mediated maturation of the pro-subtilisin in the Escherichia coli expression system, thus probably causing an activity-non-detectable mutation in the first step of mutagenesis. No distinct change was observed in the thermal stability of any mutant or in the substrate specificity for m-51. In the molecular models of the two single mutants (A88V and A98T), relatively large displacements of alpha carbon atoms were found around the mutation points. In the model of the double mutant (A88V/A98T), on the other hand, the structural changes around the mutation point counterbalanced each other, and thus no crucial displacements occurred. This mutual effect may be related to the enhanced activity of the double mutant.

Functional mapping against Escherichia coli for the broad-spectrum antimicrobial peptide, thanatin, based on an in vivo monitoring assay system.

Previously, we established for the first time an in vivo monitoring assay system conjugated with random mutagenesis in order to study the structure-function relationship of the antimicrobial peptide, apidaecin [Taguchi et al. (1996) Appl. Environ. Microbiol. 62, 4652-4655]. In the present study, this methodology was used to carry out the functional mapping of a second target, thanatin, a 21-residue peptide that exhibits the broadest antimicrobial spectrum so far observed among insect defense peptides [Fehlbaum et al. (1996) Proc. Natl. Acad. Sci. USA 93, 1221-1225]. First, a synthetic gene encoding thanatin was expressed in a fused form with Streptomyces protease inhibitor protein, SSI, under the control of tac promoter in Escherichia coli JM109. Expression of the thanatin-fused protein was found to depend on the concentration of the transcriptional inducer, isopropyl-beta-D-thio-galactopyranoside (IPTG), and to parallel the degree of growth inhibition of the transformant cells. When a PCR random mutation was introduced into the structural gene for thanatin, diminished growth inhibition of the IPTG-induced transformed cells was mostly observed in variants as measured by colony size (plate assay) or optical density (liquid assay) in comparison with the wild-type peptide, possibly depending on the decreased antimicrobial activity of each variant. Next, wild-type thanatin and three variants screened by the in vivo assay, two singly mutated proteins (C11Y and M21R) and one doubly mutated protein (K17R/R20G), were stably overproduced with a fusion partner protein resulting in the efficient formation of inclusion bodies in E. coli BL21(DE3). The products were isolated in large amounts (yield 30%) from the fused protein by successive chemical and enzymatic digestions at the protein fusion linker site. Anti-E. coli JM109 activities, judged by minimum inhibitory concentration, of the purified peptides were in good agreement with those estimated semi-quantitatively by the in vivo assay. Based on the NMR solution structure and molecular dynamics, the structure-function relationship of thanatin is discussed by comparing the functional mapping data obtained here with the previous biochemical data. The functional mapping newly suggests the importance of a hydrogen bonding network formed within the C-terminal loop joining the beta-strands arranged antiparallel to one another that are supposed to be crutial for exhibiting anti-E. coli activity.

Substrate specificity analysis of microbial transglutaminase using proteinaceous protease inhibitors as natural model substrates.

The substrate specificity of microbial transglutaminase (MTG) from Streptomyces mobaraensis (formerly categorized Streptoverticillium) was studied using a Streptomyces proteinaceous protease inhibitor, STI2, as a model amine-donor substrate. Chemical modification and mutational analysis to address the substrate requirements for MTG were carried out around the putative reactive site region of STI2 on the basis of the highly refined tertiary structure and the solvent accessibility index of Streptomyces subtilisin inhibitor, SSI, a homolog of STI2. The results suggest that the P1 reactive center site (position 70 of STI2) for protease subtilisin BPN' or trypsin may be the prime Lys residue that can be recognized by MTG, when succinylated beta-casein was used as a partner Gln-substrate. It is characteristic in that the same primary enzyme contact region of STI2 is shared by both enzymes, MTG and proteases. For quantitative analysis of the TG reaction, we established an ELISA-based monitoring assay system using an anti-SSI polyclonal antibody highly cross-reactive with STI2. Site-specific STI2 mutants were prepared by an Escherichia coli expression-secretion vector system and subjected to the assay system. We reached several conclusions concerning the nature of the flanking amino acid residues affecting the MTG reactivity of the substrate Lys residue: (i) site-specific mutations from Asn to Lys or Arg at position 69 preceding the amine-donor 70Lys, led to enhanced substrate reactivity; (ii) amino acid replacement at 67Ile with Ser led to higher substrate reactivity, (iii) additive effects were obtained by a combination of the positive mutations at positions 67 and 69 as described above, and (iv) Gly at position 65 might be essential for MTG reaction. Moreover, the substrate specificity of guinea pig liver tissue transglutaminase (GTG) was compared with that of MTG using STI2 and its mutants. In contrast to MTG, replacement of Gly by Asp at position 65 was the most favorable for substrate reactivity. Also, 70Lys appeared not to be a prime amine-donor site for GTG-mediated cross-linking, suggesting a difference in substrate recognition between MTG and GTG.

Taxonomic characterization of closely related Streptomyces spp. based on the amino acid sequence analysis of protease inhibitor proteins.

Amino acid sequences of protease inhibitors (Streptomyces subtilisin inhibitor-like proteins) widely distributed in Streptomyces were compared to clarify the taxonomic status of three strains of Streptomyces spp., S. coelicolor A3(2), S. lividans 66 and S. coelicolor Müller, which are closely related by conventional taxonomical procedures. The sequence comparison indicated that S. coelicolor A3(2) is distinct from the type strain S. coelicolor Müller, but belongs to the same taxon as S. lividans 66.

The location and deletion of the genes which code for SSI-like protease inhibitors in Streptomyces species.

The genes coding for the protease inhibitors, SSI and API-2c', have been analyzed by comparing DNA macrorestriction patterns of Streptomyces albogriseolus S-3253 and S. griseoincarnatus KTo-250 with those of inhibitor-deficient mutants. The mutants were found to suffer from chromosomal deletions rather than plasmid loss which resulted in the loss of the relevant genes. Hybridization experiments indicated that the ssi homologs in S. lividans and S. coelicolor A3(2) are located near the end of the linear chromosome.

Effect of downstream message secondary structure on the secretory expression of the Streptomyces subtilisin inhibitor.

The contribution of downstream message secondary structure to the production of Streptomyces subtilisin inhibitor (SSI) was investigated in Streptomyces lividans 66 using a cloned gene. By deletion analysis, two inverted repeats located downstream from the stop codon in the SSI gene were found to exhibit a marked effect on the amount and homogeneity of SSI gene transcript and, consequently, the efficiency of SSI productivity.

Isolation and partial characterization of SSI-like protease inhibitors from Streptomyces.

We attempted to screen a series of Streptomyces subtilisin inhibitor-like (SIL) proteins among several Streptomyces strains by using a highly sensitive assay system established by us. Of six randomly tested strains, four were found to produce SIL inhibitors as their major secreted proteins, suggesting that they might be distributed in a high frequency among this genus. Three inhibitors exhibited inhibition of both subtilisin BPN' and trypsin. Comparison of the amino terminal sequences of these isolated proteins with those of other reported SIL inhibitors revealed that the beta 1- and beta 2-sheets in SSI were highly conserved.