3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) induces caspase-dependent apoptosis in mononuclear cells.

3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), one of the tryptophan pyrolysates, is a dietary carcinogen and is formed in cooked meat and fish in our daily diet. Trp-P-1 will affect the cells in the blood circulation system before it causes carcinogenicity in target organs such as the liver. In this study, the cytotoxicity of Trp-P-1 was investigated in mononuclear cells (MNCs) from blood. Trp-P-1 (10-15 microM) decreased cell viability and induced apoptosis characterized both by morphological changes and by DNA fragmentation 4 h after treatment. DNA fragmentation was also observed following treatment at 1 nM after 24 h in culture. This result suggested that apoptosis would occur in the body following unexpected intake of foods containing Trp-P-1. To determine the mechanism of apoptosis, we investigated the activation of the caspase cascade in MNCs. Trp-P-1 (10-15 microM) activated the caspase cascade, i.e. the activity of caspase-3, -6, -7, -8 and -9 increased dose-dependently using peptide substrates, the active forms of caspase-3, -8 and -9 were detected by immunoblotting, and cleavage of poly(ADP-ribose) polymerase and protein kinase C-delta as the intracellular substrates for caspases was observed. A peptide inhibitor of caspase-8 completely suppressed activation of all other caspases, while an inhibitor of caspase-9 did not. These results indicated that caspase-8 may act as an apical caspase in the Trp-P-1-activated cascade.

West Nile virus-induced bax-dependent apoptosis.

The mechanism of cell death induced by West Nile virus (WNV), a causative agent of human febrile syndrome and encephalitis, was investigated. WNV-infected K562 and Neuro-2a cells manifested the typical features of apoptosis, including cell shrinkage, chromatin condensation and subdiploid DNA content by flow cytometry. DNA fragmentation into nucleosomal size and changes in outer cell membrane phospholipid composition were also observed in K562 cells. UV-inactivated virus failed to induce the above-mentioned characteristics, suggesting that viral replication may be required for the induction of apoptosis by WNV. Additionally, signals involved in WNV-induced apoptosis are associated with the up-regulation of bax gene expression.

cDNA cloning and sequencing of component C9 of proteasomes from rat hepatoma cells.

The nucleotide sequence of component C9 of rat proteasomes (multicatalytic proteinase complexes) has been determined from a recombinant cDNA clone isolated by screening a Reuber H4TG hepatoma cell cDNA library using synthetic oligodeoxynucleotide probes corresponding to partial amino acid sequences of the protein. The predicted sequence of C9 consists of 261 amino acid residues with a calculated molecular weight of 29,496. The C9 component is a novel protein, differing from known proteins, but its primary structure resembles those of other proteasome components, including C2, C3 and C5, although its similarity to C5 is relatively low, suggesting that proteasomes consist of a family of proteins that have evolved from a common ancestor.

Possible mechanism of nuclear translocation of proteasomes.

Proteasomes (multicatalytic proteinase complexes), which are identical to the ubiquitous eukaryotic 20S particles, are localized in both the cytoplasm and the nucleus, but the mechanism of their co-localization in the two compartments is unknown. On examination of the primary structures of subunits of proteasomes, a consensus sequence for nuclear translocation of proteins, X-X-K-K(R)-X-K(R) (where X is any residue), was found to be present in some subunits and to be highly conserved in the subunits of a wide range of eukaryotes. In addition, proteasomal subunits were found to bear a cluster of acidic amino acid residues and also a potential tyrosine phosphorylation site that was located in the same polypeptide chain as the nuclear location signal. These structural properties suggest that two sets of clusters with positive and negative charges serve to regulate the translocation of proteasomes from the cytoplasm to the nucleus, and that phosphorylation of tyrosine in certain subunits may play an additional role in transfer of proteasomes into the nucleus.

GATA-3 represses gp91phox gene expression in eosinophil-committed HL-60-C15 cells.

To study the regulatory mechanism of gp91phox gene expression in eosinophils, we transiently transfected eosinophil-committed HL-60-C15 cells with gp91phox promoter constructs, and identified a negative element from bp -267 to -246 of the gp91phox gene, the deletion of which caused an 83% increase in promoter activity. Electrophoresis mobility shift assays demonstrated GATA-3 binds to the GATA consensus site from bp -256 to -250. An 81% increment in promoter activity was obtained when a mutation was introduced in the GATA-3 binding site of the bp -267 to +12 construct, which is comparable to that of the bp -245 to +12 construct. We therefore conclude that GATA-3 specifically binding to the GATA site negatively regulates the expression of the gene in HL-60-C15 cells.

cDNA cloning and sequencing of component C5 of proteasomes from rat hepatoma cells.

Proteasomes are multicatalytic proteinase complexes consisting of a set of non-identical polypeptide subunits. A cDNA for component C5 of rat proteasomes was isolated by screening a Reuber H4TG hepatoma cell cDNA library using synthetic oligodeoxynucleotide probes corresponding to partial amino acid sequences of the protein. The polypeptide deduced from the open reading frame consisted of 240 amino acid residues with a calculated molecular weight of 26,479. Computer analysis revealed little similarity of C5 to other proteins reported so far. The primary structure of C5 showed partial sequence homology to that of another component C3, but no regions of homology with the sequence of component C2. Thus C5 is concluded to be a new type of subunit of the proteasome complex.

Expression of chemokine receptors on CD4+ T cells in peripheral blood from HIV-infected individuals in Uganda.

CXCR4, a coreceptor for T cell (T)-tropic HIV-1, is preferentially expressed on naive T cells, whereas CCR5, a coreceptor for macrophage (M)-tropic HIV-1, is preferentially expressed on previously activated memory T cells and the Th1 subset of CD4+ T cells. CCR4 is preferentially expressed on the Th2 subset of CD4+ T cells. A cross-sectional flow cytometry study was conducted to evaluate the expression of CXCR4, CCR5, and CCR4 on the peripheral blood CD4+ T cells from African HIV-1-infected and uninfected Ugandan adults. The plasma viral load in HIV-1-infected individuals was also examined. Upregulation of CCR4 and CCR5 expression but no decrease in CXCR4 expression on CD4+ T cells were obtained in peripheral blood from African adults with progression of the disease. Plasma HIV-1 viremia significantly and inversely correlated with the peripheral CD4+ T cell count but did not correlate with the degree of CCR4 and CCR5 expression on the peripheral CD4+ T cells in HIV-1-infected individuals. Our present data suggest an increase in percentage of activated memory CD4+ T cells in the advanced stage of HIV-1 infection among African adults. There was no evidence of a Th1 to Th2 shift in terms of chemokine receptor expression profile with advancing disease in the peripheral blood of these subjects.

Cell-density dependent expression of the c-myc gene in primary cultured rat hepatocytes.

During culture of mature rat hepatocytes as monolayers, c-myc mRNA was found to be expressed transiently within 2 h, decreasing rapidly to the basal level at 10 h. Then its level increased again to over 10-fold the basal level at 24 h, and remained at this high level during culture. The increase of c-myc mRNA in the second phase was shown by nuclear run-off experiments to be due to an increase of its transcription. The second, but not the first, increase in c-myc expression was inversely proportional to the cell density in culture. The expression of c-myc mRNA was not affected by various hormones including growth factors. These results indicate that hepatocytes in culture at lower cell density tend to move from the Go phase to the G1 phase, but remain in the Go phase when cultured at high cell density.

Coexpression of collagens and collagen-binding heat shock protein 47 in human diabetic nephropathy and IgA nephropathy.

The mechanism of structural changes of the kidney in human diabetic nephropathy (DN) and IgA nephropathy (IgAN) is not yet completely known, but excessive deposition of extracellular matrix (ECM), including various collagens, may be crucial to this process. Heat shock protein (HSP) 47 has been identified as collagen-binding stress protein, shown to have a specific role in the intracellular processing of procollagen molecules during collagen assembly. To determine whether increased deposition of collagens in human DN and IgAN is related to HSP47, we investigated the expression of HSP47 in renal biopsy and autopsy sections obtained from 22 DN and 45 IgAN patients. Five renal biopsy specimens, diagnosed as minor glomerular abnormalities, were simultaneously studied as controls. Monoclonal antibodies specific for HSP47, type III collagen and type IV collagen were used to assess the relative expression of their proteins in paraffin-embedded renal sections by immunohistochemistry. Increased deposition of collagens was closely related to the sclerotic activity of the disease process in DN and IgAN; increased deposition of collagens was often present in relation to a strong expression of HSP47, a stress protein known to regulate collagen synthesis/assembly. By double immunostaining, we found colocalization of collagens and their molecular chaperone HSP47 in the sclerotic glomeruli and tubulointerstitium in DN and IgAN. Our results strongly support a pathologic role for HSP47 in both these diseases and that increased levels of HSP47 may play an important role in the excessive assembly of collagens resulting in glomerulosclerosis and interstitial fibrosis found in DN and IgAN patients.

Direct evidence for nuclear and cytoplasmic colocalization of proteasomes (multiprotease complexes) in liver.

Subcellular localization of the large multicatalytic protease complexes called proteasomes, which have been found in soluble fractions of various cells, was examined by biochemical, immunological, and immunohistological methods. Rat liver nuclei, purified by two different procedures, showed high activities for degrading [3H]methylcasein and various fluorogenic oligopeptides with neutral and weakly alkaline pH optima. On gel filtration, all of these peptidase activities were recovered in a single peak with the unusually large molecular weight of about 600,000. Properties of the proteolytic activity in crude extracts of the nucleus and the cytoplasm were very similar. Immunoelectrophoretic blot analysis showed the presence of appreciable concentrations of proteasomes with similar immunoreactivity in isolated nuclear and cytosolic fractions. Moreover, immunohistochemical staining of human liver showed that proteasomes were predominantly localized in the nuclear matrix but also were present diffusely in the cytoplasm of hepatocytes. These findings indicate the nuclear and cytoplasmic colocalization of proteasomes.

Cisplatin-induced apoptosis in human proximal tubular epithelial cells is associated with the activation of the Fas/Fas ligand system.

The role of Fas and Fas ligand (Fas-L) in the apoptotic cell death process in cisplatin (CP)-treated human proximal tubular epithelial cells (PTECs) was examined. The human PTECs were treated with various concentrations (20-80 microM) of CP for 24 h, and the incidence of apoptosis in CP-treated cells was assessed by trypan blue staining, propidium iodide staining, in situ end labeling, and electron microscopy. The expression of Fas and Fas-L was detected by immunofluorescence microscopy. The results showed that: (1) CP-treatment resulted in a decreased number of live human PTECs and an increased number of dead cells, (2) CP-treated human PTECs showed an increased rate of apoptosis with its typical morphological features, and (3) expression of both Fas and Fas-L was upregulated in CP-treated human PTECs. These results indicate that CP treatment induces apoptosis in human PTECs and the activation of the Fas/Fas-L system may play an active role in the induction of the apoptotic cell death process.

Glucocorticoid-dependent expression of the albumin gene in adult rat hepatocytes.

In primary cultures of adult rat hepatocytes, transcription of the albumin gene, measured as incorporation of [alpha-32P]UTP into mRNA in isolated nuclei, decreased dramatically during culture without addition of serum and hormone, becoming almost negligible 10 h after plating. Of the hormones tested, dexamethasone (0.1 microM) prevented this decrease and restored the transcription within 2 h to the same level as that before culture. The half-maximum dose of dexamethasone for induction of transcription of the albumin gene was about 30 nM. The in vitro finding that expression of the albumin gene is strictly regulated by glucocorticoid was confirmed by an in vivo experiment in adrenalectomized rats showing that the transcription decreased markedly 14 days after adrenalectomy, but was restored rapidly by administration of hydrocortisone. This finding was also supported by identification of a glucocorticoid regulatory sequence from -50 to -62 base pairs between the TATA box and CAT box upstream of the 5'-end of the albumin gene. Cycloheximide inhibited the induction of transcription of the albumin gene by dexamethasone, suggesting that a rapidly induced mediator protein, which is also regulated by glucocorticoid, is involved in the induction of albumin gene expression by glucocorticoid. The albumin gene was also regulated by various other hormones besides glucocorticoid. Glucagon markedly enhanced the transcription induced by dexamethasone, although glucagon alone had no effect. Conversely, epinephrine suppressed stimulation of expression of the albumin gene by dexamethasone. Insulin and triiodothyronine had no effect on transcription of the albumin gene. From these findings we conclude that expression of the albumin gene depends strictly on glucocorticoid, and this dependence is modulated by other hormones.

Separation of yeast proteasome subunits. Immunoreactivity with antibodies against ATP-dependent protease Ti from Escherichia coli.

On two-dimensional gel electrophoresis, proteasomes (multicatalytic proteinase complexes) from the yeast Saccharomyces cerevisiae were separated into a characteristic set of approximately 20 components with molecular weights of 21,000 to 31,000 and isoelectric points of 3.5 to 7.5. The main components were isolated by reverse-phase high performance liquid chromatography on a TSK gel phenyl-5PW RP column and named YC1 to YC11, in order of their elution. Immuno-blot analysis showed that two components (YC1-alpha and YC1-beta) with molecular weights of 30,800 and 28,300 strongly cross-reacted with antibody against the P-component of ATP-dependent protease Ti from Escherichia coli, but no components were found to react with antibodies against the A-component of protease Ti or another ATP-dependent protease La (the Ion gene product) of Escherichia coli. These results indicate a structural relationship between eukaryotic proteasomes and bacterial ATP-dependent protease Ti.

Nonhomologous recombination between the cytochrome b558 heavy chain gene (CYBB) and LINE-1 causes an X-linked chronic granulomatous disease.

We cloned and characterized a genomic DNA fragment including the deletion junction of a chronic granulomatous disease patient with a 25-kb deletion extending to the 5' two-thirds of CYBB. The 3' breakpoint of the deletion exists in exon 7 of CYBB. A LINE-1 element lies at 5 kb upstream of CYBB in normal persons, and the 5' breakpoint of the deletion in the patient is in the LINE-1 element. There are no significant homologies between corresponding normal 5' and 3' regions flanking the breakpoint of the patient, so a nonhomologous recombination is the most possible mechanism for this 25-kb deletion. The analysis also reveals that the patient has a novel 30-bp duplication in the 5' flanking region of the deletion point, which was transmitted by his mother with the deletion. Furthermore we suggest that the deletion occurred in his grandfather.

Human peripheral eosinophils have a specific mechanism to express gp91-phox, the large subunit of cytochrome b558.

Eosinophils as well as neutrophils, monocytes and B lymphocytes are noted for lacking normal cytochrome b558 in patients with X-linked chronic granulomatous disease. The eosinophils of an X-linked male patient, however, fully expressed surface cytochrome b558, generated superoxide anion to a normal extent and definitely expressed the large subunit of cytochrome b558 (gp91-phox). His mononuclear leukocytes contained a diminished amount of gp91-phox mRNA with normal coding sequences. All the coding sequences and a putative poly (A) signal of his gp91-phox gene were normal. These results indicate that eosinophils have a specific mechanism to express gp91-phox and suggest that the mechanism lies at the transcriptional step of the gene.

St. Louis encephalitis virus induced pathology in cultured cells.

Apoptosis is a highly regulated process of cellular self-destruction with diverse functions in multicellular organisms. It is known to be one of the mechanisms of viral pathogenesis. St. Louis encephalitis virus (SLEV), an arthropod-borne flavivirus, causes encephalitis disease of varying severity mostly in North America and in some regions of South America. This virus induces cytopathic effects in vertebrate cell lines, however, the mechanism by which this occurs is yet to be elucidated. SLEV induced cytopathic effects in K562 cells, a human mononuclear cell line, and in Neuro 2a cells, a mouse neuroblastoma cell line. SLEV-infected K562 and Neuro 2a cells underwent apoptotic cell death, whereas neither the cells inoculated with UV-inactivated virus nor the mock-infected cells developed cytopathic effects. The gene expression of regulators of apoptosis was investigated in K562 cells. A rise in the expression of the pro-apoptotic bax gene was detected specifically in the SLEV-infected K562 cells. These findings suggest that up-regulation of bax mRNA is correlated with cytopathic effects in SLEV-infected K562 cells.

Proteasomes (multi-protease complexes) as 20 S ring-shaped particles in a variety of eukaryotic cells.

Latent multicatalytic protease complexes, named proteasomes, were purified to apparent homogeneity from various eukaryotic sources, such as human, rat, and chicken liver, Xenopus laevis ovary, and yeast (Saccharomyces cerevisiae), and their functional and structural properties were compared. They showed latency in breakdown of [methyl-3H]casein, but were greatly activated in various ways, such as by addition of polylysine. They all degraded three types of fluorogenic oligopeptides at the carboxyl side of basic, neutral, and acidic amino acids, and the three cleavage reactions showed different spectra for inhibition, suggesting that they had three distinct active sites. The proteasomes all seemed to be seryl endopeptidases with similar pH optima in the weakly alkaline region. Their physiochemical properties, such as their sedimentation coefficients (19 S to 22 S), diffusion coefficients (2.0-2.6 X 10(-7) cm2 s-1), molecular masses (700-900 kDa), and circular dichroic spectra, were similar. Their amino acid compositions were also very similar. Electron microscopy showed that they had similar well-defined symmetrical morphology, appearing to be ring-shaped particles with a small hole in the center. All the proteasomes seemed to be multisubunit complexes consisting of 15-20 polypeptides with molecular masses of 22-33 kDa and isoelectric points of pH 3-10, but they showed species-specific differences in subunit multiplicity. Moreover, they differed immunologically, as shown by Ouchterlony tests and immunoblotting analyses, although cross-immunoreactivities of some subunits or domains were observed. These results indicate that the sizes and shapes of these proteasomes have been highly conserved during evolution, but that they show species-specific differences in immunoreactivities and subunit structures. Thus proteasomes with similar structure and function seem to be ubiquitously distributed in eukaryotic organisms ranging from man to yeast. This distribution implies the general importance of these proteasomes for proteolysis.

Abnormally high expression of proteasomes in human leukemic cells.

Proteasomes are eukaryotic ring-shaped or cylindrical particles with multicatalytic protease activities. To clarify the involvement of proteasomes in tumorigenesis of human blood cells, we compared their expression in human hematopoietic malignant tumor cells with that in normal peripheral blood mononuclear cells. Immunohistochemical staining showed considerably increased concentrations of proteasomes in leukemic cells from the bone marrow of patients with various types of leukemia and the predominant localization of these proteasomes in the nuclei. Moreover, enzyme immunoassay and Northern blot analysis indicated that the concentrations of proteasomes and their mRNA levels were consistently much higher in a variety of malignant human hematopoietic cell lines than in resting peripheral lymphocytes and monocytes from healthy adults. Proteasome expression was also greatly increased in normal blood mononuclear cells during blastogenic transformation induced by phytohemagglutinin; their expression increased in parallel with induction of DNA synthesis and returned to the basal level with progress of the cell cycle. Thus, abnormally high expression of proteasomes may play an important role in transformation and proliferation of blood cells and in specific functions of hematopoietic tumor cells.

A 25-kb deletion in the 5' region of the cytochrome b558 heavy chain gene (CYBB) in a patient with X-linked chronic granulomatous disease.

We performed molecular genetic analyses of the family of a boy suffering from chronic granulomatous disease (CGD) after immunocytochemically confirming him and his mother to be an X-linked CGD patient and a mosaic carrier, respectively. Southern blot hybridization using cDNA for the cytochrome b558 heavy chain gene (CYBB) as a probe showed that the patient had a deletion in the 5' region of the CYBB and his phenotypically normal mother was heterozygous for this deletion. Polymerase chain reaction analyses of all 13 exons of the patient's CYBB gene demonstrated that the deletion extends from exon 7 or neighboring introns to 5' upstream. The length of the deletion was determined by pulsed-field gel electrophoresis and Southern blotting of genomic DNA using CYBB cDNA and the genetic marker pERT55-5, centromeric to CYBB, as probes. Both probes recognized common SfiI-NotI fragments of 120 kb and 95 kb in normal individuals and the patient, respectively. These results revealed that the patient has a 25-kb deletion spanning from the middle of CYBB to 5' upstream. This is the only report of a large 5' deletion in CYBB and also the first observation that CYBB and pERT55-5 are within 120 kb in Xp21.

Human T-cell leukemia virus type I oncoprotein Tax represses Smad-dependent transforming growth factor beta signaling through interaction with CREB-binding protein/p300.

Human T-cell leukemia virus type I (HTLV-I) Tax is a potent transcriptional regulator that can activate or repress specific cellular genes and that has been proposed to contribute to leukemogenesis in adult T-cell leukemia. Previously, HTLV-I- infected T-cell clones were found to be resistant to growth inhibition by transforming growth factor (TGF)-beta. Here it is shown that Tax can perturb Smad-dependent TGF-beta signaling even though no direct interaction of Tax and Smad proteins could be detected. Importantly, a mutant Tax of CREB-binding protein (CBP)/p300 binding site, could not repress the Smad transactivation function, suggesting that the CBP/p300 binding domain of Tax is essential for the suppression of Smad function. Because both Tax and Smad are known to interact with CBP/p300 for the potentiation of their transcriptional activities, the effect of CBP/p300 on suppression of Smad-mediated transactivation by Tax was examined. Overexpression of CBP/p300 reversed Tax-mediated inhibition of Smad transactivation. Furthermore, Smad could repress Tax transcriptional activation, indicating reciprocal repression between Tax and Smad. These results suggest that Tax interferes with the recruitment of CBP/p300 into transcription initiation complexes on TGF-beta-responsive elements through its binding to CBP/p300. The novel function of Tax as a repressor of TGF-beta signaling may contribute to HTLV-I leukemogenesis. (Blood. 2001;97:2137-2144)