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S Kitada

Publications and source records attributed to S Kitada.

At least 55 records · Page 3Linked to original sources
PubMed1996

BCL-2 family proteins: regulators of cell death involved in the pathogenesis of cancer and resistance to therapy.

The BCL-2 gene was first discovered because of its involvement in the t(14;18) chromosomal translocations commonly found in lymphomas, which result in deregulation of BCL-2 gene expression and cause inappropriately high levels of Bcl-2 protein production. Expression of the BCL-2 gene can also become altered in human cancers through other mechanisms, including loss of the p53 tumor suppressor which normally functions as a repressor of BCL-2 gene expression in some tissues. Bcl-2 is a blocker of programmed cell death and apoptosis that contributes to neoplastic cell expansion by preventing cell turnover caused by physiological cell death mechanisms, as opposed to accelerating rates of cell division. Overproduction of the Bcl-2 protein also prevents cell death induced by nearly all cytotoxic anticancer drugs and radiation, thus contributing to treatment failures in patients with some types of cancer. Several homologs of Bcl-2 have recently been discovered, some of which function as inhibitors of cell death and others as promoters of apoptosis that oppose the actions of the Bcl-2 protein. Many of these Bcl-2 family proteins can interact through formation of homo- and heterotypic dimers. In addition, several nonhomologous proteins have been identified that bind to Bcl-2 and that can modulate apoptosis. These protein-protein interactions may eventual serve as targets for pharmacologically manipulating the physiological cell death pathway for treatment of cancer and several other diseases.

Apoptosis↗
PubMed1996

Role of basic amino acids in the cleavage of synthetic peptide substrates by mitochondrial processing peptidase.

Our recent experiments using model peptides of rat malate dehydrogenase (MDH) indicated that a proximal arginine and a distal basic amino acid are important for processing by mitochondrial processing peptidase (MPP). [Niidome, T., Kitada, S., Shimokata, K., Ogishima, T., and Ito, A. (1994) J. Biol. Chem. 269, 24719-24722]. To elucidate if the recognition elements apply to other precursor proteins, we analyzed cleavage of model peptides of human ornithine aminotransferase (OAT). Purified peptidase cleaved peptides that corresponded to N-terminal 1-25 and 3-25 at the correct site (Gly17-Val18) at nearly equal rates. Replacement of Arg16 (-2 position) with lysine or alanine reduced the processing efficiency by 95- and 380-fold, respectively. Either deletion from Met1 to Arg10 or replacement of the basic amino acids between them decreased the processing efficiency considerably. A peptide containing Arg7 in addition to Lys4 and Arg10 was more effective than the control peptide. However, a peptide with one and two consecutive basic amino acids in the distal region had a processing efficiency close to the control peptide. These results indicated that processing of OAT was enhanced by an increase in the number of basic amino acids with a suitable distance between them. In other respects, the processing signal of OAT was essentially the same as that of MDH.

Amino Acid Sequence↗
PubMed1996

Immunohistochemical analysis of bcl-2, bax, bcl-X, and mcl-1 expression in prostate cancers.

Proteins encoded by bcl-2 family genes are important regulators of programmed cell death and apoptosis. Alterations in the expression of these apoptosis-regulating genes can contribute to the origins of cancer, as well as adversely influence tumor responses to chemo- and radiotherapy. Using antibodies specific for the Bcl-2, Bax, Bcl-X, and Mcl-1 proteins in combination with immunohistochemical methods, we examined for the first time the expression of these bcl-2 family genes in 64 cases of adenocarcinoma of the prostate, including 10 Gleason grade 2 to 4 tumors, 21 grade 5 to 7 tumors, 17 grade 8 to 10 tumors, 8 lymph node metastases, and 8 bone metastases. In addition, 24 cases of prostatic intraepithelial neoplasia (PIN) or PIN coexisting with carcinoma were also evaluated. All immunostaining results were scored with regard to approximate percentage of positive tumor cells and relative immunostaining intensity. Expression of the anti-apoptotic protein Bcl-2 was present in 16 of 64 (25%) adenocarcinomas and tended to be more frequent in high grade tumors (Gleason grade 8 to 10; 41%) and nodal metastases (38%) than in lower grade (Gleason 2 to 7) primary tumors (16%; P < 0.05). Bcl-X was expressed in all 64 (100%) tumors evaluated. Bcl-X immunointensity was generally stronger in high grade primary tumors (grade 8 to 10) and metastases compared with PIN and low grade neoplasms (P < 0.0001). In addition, the proportion of specimens with > 50% Bcl-X-immunopositive tumor cells also was higher in advanced grade primary tumors (Gleason 8 to 10) and metastases than in PIN and low grade tumors (Gleason 2 to 7; P < 0.005). The anti-apoptotic protein Mcl-1 was expressed in 52 of 64 (81%) tumors, compared with only 9 of 24 (38%) cases of PIN (P < 0.001). In addition, the percentage of Mcl-1-positive cells was typically higher in Gleason grade 8 to 10 tumors and metastases than in PIN or lower grade tumors (P = 0.025). In contrast, the pro-apoptotic protein Bax was expressed in all prostate cancers evaluated, with high percentages of immunopositive cells and strong immunointensity typically occurring regardless of tumor grade. The findings suggest that expression of several anti-apoptotic members of the bcl-2 gene family, including bcl-2, bcl-X, and mcl-1 increases during progression of prostate cancers, a finding that may be relevant to the hormone-insensitive, metastatic phenotype of most advanced adenocarcinomas of the prostate.

Adenocarcinoma↗
PubMed1996

[Primary Non-Hodgkin's lymphoma in the chest wall without preceding disease].

An 80-year-old man was admitted to our hospital with complaints of left-sided chest pain and swelling. A chest roentgenogram revealed a left chest-wall mass. Examination of a biopsy specimen of the mass led to the diagnosis of Non-Hodgkin's lymphoma of diffuse mixed-cell type. Immunohistological examination revealed that it was B-cell type. The chest-wall mass was markedly smaller after radiation therapy, and the patient had a complete remission. About 1 year and 10 months later, Non-Hodgkin's lymphoma developed in the right femur. Combination chemotherapy (cyclophosphamide, adriamycin, vincristine, and prednisolone) and radiation therapy were given. The patient went into a complete remission, and is alive at the time of this writing, 2 years and 6 months after he was first treated. Almost all malignant lymphomas of the chest wall arise in cases of chronic tuberculous pyothorax or tuberculous pleuritis, but in this case there was no preceding disease. When the chest roentgenogram shows a chest-wall mass, we must consider the possibility of malignant lymphoma, even if there is no preceding disease.

Aged↗
PubMed1995

Analysis of elements in the substrate required for processing by mitochondrial processing peptidase.

We have recently demonstrated that synthetic peptides modeled on the extension peptide of malate dehydrogenase can be a good substrate of mitochondrial processing peptidase and that arginine residues present at positions -2 or -3 and distant from the cleavage point were important for recognition by the enzyme (Niidome, T., Kitada, S., Shimokata, K., Ogishima, T., and Ito, A. (1994) J. Biol. Chem. 269, 24719-24722). We further investigated the elements required for substrates of the protease. To analyze the reaction by a more rapid yet quantitative method, we have developed intramolecularly quenched fluorescent substrates. Using the fluorogenic substrates we demonstrated that at least one of the proline and glycine between the distal and proximal arginine residues was also important while other connecting sequences were dispensable. In addition, the protease showed considerable preference for aromatic and, to a lesser extent, hydrophobic amino acids in the P1'-position. These results together with the previous data suggest that the proximal and distal arginine residues, proline and/or glycine between them, and P1' amino acid could be critical determinants for the specific cleavage of the substrates by the protease.

Amino Acid Sequence↗
PubMed1995

Overexpression of the Bcl-2 protein increases the half-life of p21Bax.

Bcl-2 and Bax are homologous proteins which can heterodimerize with each other. These proteins have opposing effects on cell survival when overexpressed in cells, with Bcl-2 blocking and Bax promoting apoptosis. Here we demonstrate that gene transfer-mediated elevations in Bcl-2 protein levels result in a marked increase in the steady-state levels of endogenous p21Bax protein as determined by immunoblotting in the Jurkat T-cell and 697 pre-B-cell leukemia cell lines, but not in several other cell lines including CEM T-cell leukemia, 32D.3 myeloid progenitor, PC12 pheochromocytoma, and NIH-3T3 fibroblasts. Steady-state levels of p21Bax protein were also elevated in the lymph nodes of Bcl-2 transgenic mice in which a BCL-2 transgene is expressed at high levels in B-cells. Northern blot analysis of BCL-2-transfected and control-transfected Jurkat and 697 leukemia cells revealed no Bcl-2-induced alterations in the steady-state levels of BAX mRNAs. In contrast, L-[35S]methionine pulse-chase analysis indicated a marked increase in the half-life (t1/2) of the p21Bax protein in BCL-2-transfected 697 cells compared to control-transfected cells (t1/2 > 24 h versus approximately 4 h), whereas the rate of Bax degradation was unaltered in Bcl-2-transfected CEM cells. The results demonstrate that levels of the proapoptotic p21Bax protein can be post-translationally regulated by Bcl-2, probably in a tissue-specific fashion, and suggest the existence of a feedback mechanism that may help to maintain the ratio of Bcl-2 to Bax protein in physiologically appropriate ranges.

3T3 Cells↗
PubMed1995

FAP-1: a protein tyrosine phosphatase that associates with Fas.

Fas is a cell surface receptor that controls a poorly understood signal transduction pathway that leads to cell death by means of apoptosis. A protein tyrosine phosphatase, FAP-1, capable of interacting with the cytosolic domain of Fas, was identified. The carboxyl terminal 15 amino acids of Fas are necessary and sufficient for interaction with FAP-1. FAP-1 expression is highest in tissues and cell lines that are relatively resistant to Fas-mediated cytotoxicity. Gene transfer-mediated elevations in FAP-1 partially abolished Fas-induced apoptosis in a T cell line. These findings are consistent with an inhibitory effect of FAP-1 on Fas signal transduction.

Amino Acid Sequence↗
PubMed1995

A putative metal-binding site in the beta subunit of rat mitochondrial processing peptidase is essential for its catalytic activity.

Mitochondrial processing peptidase (MPP) consists of alpha- and beta-subunits (alpha-MPP and beta-MPP). beta-MPP has a putative metal-binding sequence (HXXEH). To determine whether the sequence of beta-MPP is essential for the enzymatic activity, we individually mutated the histidines and glutamic acid to arginines and glutamine, respectively. The wild-type and mutated beta-MPPs were co-expressed with alpha-MPP in Escherichia coli. All three mutants had completely lost the activity, whereas the lost activity was recovered on the addition of wild-type beta-MPP. The activity of the wild-type enzyme was reduced by the mutant beta-MPPs. We conclude from these observations that the HXXEH region is involved in the formation of the active site and that beta-MPP is the catalytic subunit of MPP.

Adrenodoxin↗
PubMed1994

Structural requirement for recognition of the precursor proteins by the mitochondrial processing peptidase.

We examined the structural characteristics of the extension peptides responsible for the recognition by the mitochondrial processing peptidase by using preadrenodoxin, which has a long extension peptide of 58 amino acid residues, as the substrate. The deletion of various parts of the extension peptide of pre-adrenodoxin indicated that more than 40 amino acid residues and the presence of basic amino acid residues in the distal portion (20-40 amino acid residues upstream of the cleavage site) were necessary for the recognition of the precursor by the peptidase. The processing of preadrenodoxin was strongly inhibited by the synthetic peptide corresponding to the middle portion of the extension peptide, whereas the peptide corresponding to the amino-terminal portion exhibited weak inhibition of the processing. The replacement of arginine residues in the middle portion of the extension peptide with neutral amino acids resulted in a great decrease in the processing. We conclude that basic amino acids at a position distal to the cleavage site are necessary for the recognition of the precursor proteins by the processing peptidase and that basic amino acids required for the mitochondrial targeting and those for the recognition by the peptidase are separately located in the extension peptide of pre-adrenodoxin.

Adrenodoxin↗
PubMed1994

Arginine residues in the extension peptide are required for cleavage of a precursor by mitochondrial processing peptidase. Demonstration using synthetic peptide as a substrate.

Mitochondrial processing peptidase (MPP) specifically recognizes a large variety of mitochondrial precursor proteins and correctly cleaves off the extension peptides. To determine the structure common to all the extension peptides that is required for specific recognition by MPP, we synthesized various oligopeptides of different chain lengths and amino acid sequences, based on the amino acid sequence of the extension peptide of pre-malate dehydrogenase, and determined kinetic parameters of the cleavage reactions. The minimal length of peptides for effective cleavage was 16 amino acid residues consisting of 11 and 5 residues from the cleavage site to the amino- and carboxyl-terminal sides, respectively. Two sets of basic amino acids in the peptide, the distal arginine residue at position -10 and the proximal ones at positions -3 and -2 relative to the cleavage site, were necessary for effective hydrolysis. Of these two, the residue at position -2 was more important for effective cleavage than the one at position -3 and could not be replaced by a lysine residue. The replacement of the distal arginine by lysine had no effect on the cleavage. Our study demonstrates that use of peptides with the proper length is essential for performing kinetic analyses on the cleavage reaction by MPP and that an arginine residue at position -2 to the cleavage site is necessary for the recognition and cleavage of the extension peptide.

Amino Acid Sequence↗
PubMed1994

Growth of rabbit pigmented and nonpigmented ciliary body epithelium.

Pigmented (PE) and nonpigmented epithelial (NPE) cells were carefully dissected from the ciliary processes of rabbit eyes and maintained in vitro. Both epithelial cultured cells showed hexagonal morphology by light microscopy; abundant granules containing pigment could be seen in PE cells while no pigment was seen in the NPE cells. S-100 protein exists in both cultured cells and was used as a marker for the in vitro confirmation of these cells. Collagen type III was used as a negative marker to rule out stromal cell contamination in tissue cultures. PE cells attached and proliferated well in 20% fetal bovine serum (FBS) in minimum essential media (MEM). Greater proliferation was attained when PE cells were exposed to 20% vitreous extract in addition to 20% FBS-MEM. Media conditioned by PE cells did not significantly stimulate PE cells. NPE cells showed greatest proliferation in 20% FBS-MEM with either 20% concentration of media conditioned by PE cells or when exposed to 20% concentration of vitreous extract. Rabbit vitreous may contain factor(s) which stimulate the proliferation of both cultured cells, and conditioned media from the PE cells may contain factor(s) which stimulate NPE cell proliferation.

Animals↗
PubMed1994

Significance of invasion to the muscularis mucosae on the progression of superficial bladder cancer.

OBJECTIVE: We tried to determine the significance of invasion to the muscularis mucosae on the progression of superficial bladder cancer. METHODS: We subclassified superficial bladder cancer showing lamina propria invasion into two groups: with (pT1b) or without cancer invasion to or near the muscularis mucosae (pT1a). RESULTS: Of 164 bladder cancers, 76 showed no lamina propria invasion and 88 did. In the tumors with lamina propria invasion, 60 were diagnosed as pT1a and 28 as pT1b. The progression rate of pT1b cancer (53.5%) was significantly higher than that of pT1a cancer (6.7%) (P < 0.01). Regardless of size, number, or grade of tumor, the progression rate of pT1b cancer was significantly higher than that of pT1a cancer (P < 0.01). CONCLUSIONS: We stress that patients with superficial cancer invading to or near the muscularis mucosae should be followed up carefully after transurethral resection.

Adult↗
PubMed1994

The content of urokinase-type plasminogen activator and tumor recurrence in superficial bladder cancer.

The contents of urokinase-type plasminogen activator were estimated in superficial bladder cancer from 42 patients undergoing transurethral resection for the first time. Tumor recurred in 11 of 16 patients (68.8%) with high urokinase-type plasminogen activator content (8 ng. urokinase-type plasminogen activator per mg. protein and more) and in 12 of 26 (46.2%) with low urokinase-type plasminogen activator content. In the high content group 8 patients (50%) had disease progression compared to 2 (7.7%) in the low content group (p < 0.01). The progression rates in the high urokinase-type plasminogen activator group with stage pTa disease or tumors more than 1 cm. large were significantly higher than those in the low urokinase-type plasminogen activator group (p < 0.05). The recurrence and progression rates in the high content group with stage pT1, grades 2 and 3, single and multiple tumors, and tumors less than 1 cm. large were higher than those in the low content group. The progression-free survival rate in the high content group was significantly lower than that in the low content group (p < 0.005). In conclusion, urokinase-type plasminogen activator content may be a useful marker to predict recurrence and progression of superficial bladder cancer.

Aged↗
PubMed1994

Reversal of chemoresistance of lymphoma cells by antisense-mediated reduction of bcl-2 gene expression.

The bcl-2 gene is expressed in many types of human tumours and becomes transcriptionally deregulated in the majority of non-Hodgkin's lymphomas as the result of t(14;18) chromosomal translocations. The 26-kDa Bcl-2 protein has been shown to block programmed cell death (apoptosis) induced by many types of stimuli, including a wide variety of chemotherapeutic drugs and radiation. The presence of bcl-2 in tumor cells has been correlated with poor responses to therapy in patients with some types of cancer. To explore further the relevance of bcl-2 to drug resistance, we used antisense (As) approaches to achieve reductions in the levels of steady state Bcl-2 protein levels in t(14;18)-containing human lymphoma cell lines. Both synthetic bcl-2-As oligonucleotides and inducible expression plasmids that produce bcl-2-As transcripts induced reductions in bcl-2 expression, resulting in a marked enhancement in the sensitivity of neoplastic cells to conventional chemotherapeutic drugs such as cytosine arabinoside (ara-C) and methotrexate (MTX). These results suggest that novel therapeutics targeted against bcl-2 could provide the means for improved treatment of cancer by affecting physiological pathways distal to the targets of cytotoxic drugs.

Animals↗
PubMed1994

Evolutionary conservation of function among mammalian, avian, and viral homologs of the Bcl-2 oncoprotein.

The bcl-2 gene was originally cloned because of its involvement in B-cell lymphomas and encodes a 25-kD integral membrane protein that has been shown to inhibit programmed cell death (also termed apoptosis) in a wide variety of circumstances. The Epstein-Barr Virus (EBV) also has been implicated in B-cell malignancies and interestingly contains an open reading frame (BHRF-1) predicting a 19-kD protein with 22% homology to Bcl-2. To compare the functions of p26-Bcl-2 and p19-BHRF-1, we stably introduced expression plasmids encoding these proteins into a murine interleukin-3 (IL-3)-dependent hemopoietic cell line, 32D. Removal of IL-3 from cultures of control-transfected 32D cells resulted in internucleosomal DNA cleavage (a hallmark of programmed cell death) and loss of cell survival. In contrast, 32D cells containing high levels of p26-Bcl-2 or p19-BHRF-2 proteins exhibited prolonged survival and markedly delayed DNA degradation under the same conditions of IL-3 deprivation. As a first attempt to determine the functional importance of amino acid sequences that are conserved between the Bcl-2 and BHRF-1 proteins, we used site-specific mutagenesis to replace two conserved cysteine residues with alanines (positions 158 and 219) in the human Bcl-2 protein. Comparisons of the wild-type and cysteine-minus human Bcl-2 proteins in S49 lymphoma cells revealed equivalent ability to block glucocorticoid-induced cell death and DNA fragmentation, indicating that these two conserved cysteines are not critical for Bcl-2 oncoprotein function. Investigations in 32D cells of an avian homolog of Bcl-2 cloned from the chicken also revealed conservation of function with the human Bcl-2 protein, despite the presence of a 48-amino-acid region of divergent sequence. Taken together, these data demonstrate that despite marked differences in their predicted amino-acid sequences, the human, chicken, and EBV versions of Bcl-2 have retained the structural characteristics necessary to interface with pathways involved in the regulation of programmed cell death in murine cells. The findings thus contribute to the mapping of functional domains in Bcl-2 proteins, and raise the possibility that the EBV-encoded p19-BHRF-1 protein may be able to substitute for p26-Bcl-2 in the development of some types of cancer.

Amino Acid Sequence↗
PubMed1994

Regulation of chemoresistance by the bcl-2 oncoprotein in non-Hodgkin's lymphoma and lymphocytic leukemia cell lines.

BACKGROUND: The bcl-2 gene becomes activated by 14;18 chromosomal translocations in the majority of low-grade non-Hodgkin's lymphomas (NHLs) and is expressed at high levels in the absence of gene rearrangements in a high proportion of B-cell chronic lymphocytic leukemias (B-CLLs). The protein encoded by bcl-2 contributes to neoplastic cell expansion by prolonging cell survival through its ability to block programmed cell death (apoptosis). Because many chemotherapeutic drugs have been shown ultimately to kill tumor cells through mechanisms consistent with programmed cell death, we tested whether the relative levels of bcl-2 oncoprotein influence the sensitivity of lymphoma and leukemia cell lines to killing by conventional cytotoxic drugs commonly used in the treatment of cancer. METHODS: Leukemia cell lines with low levels of bcl-2 expression were stably infected with recombinant bcl-2 retroviruses to achieve elevations in bcl-2 protein levels. Lymphoma cell lines with high levels of bcl-2 expression as the result of 14;18 translocations were either stably transfected with inducible bcl-2 antisense expression plasmids or treated with bcl-2 antisense oligonucleotides to achieve reductions in bcl-2 protein levels. The sensitivity of these genetically modified cells to killing by various antineoplastic drugs was then determined. RESULTS: Gene transfer-mediated elevations in bcl-2 protein levels in lymphocytic leukemia cell lines was correlated with markedly elevated resistance to killing by all cytotoxic drugs tested. Conversely, antisense-mediated reductions in bcl-2 protein levels in t(14;18)-containing NHL cell lines resulted in enhanced sensitivity to all anticancer drugs. CONCLUSIONS: The relative levels of bcl-2 oncoprotein represent one of the key determinants of the sensitivity of lymphocytic cells to killing by essentially all drugs currently available for the treatment of cancer.

Animals↗
PubMed1993

Molecular cloning of the smaller subunit(P52) of rat liver mitochondrial processing protease.

A cDNA encoding the smaller subunit (P52) of mitochondrial processing protease was isolated from a rat liver cDNA library, using cDNA fragment for yeast MAS1 as the probe. The deduced amino acid sequence is highly homologous to those of PEP from Neurospora crassa and MAS1 from Saccharomyces cerevisiae. After in vitro transcription and translation, the precursor peptide was imported into isolated rat liver mitochondria and processed to its mature form.

Amino Acid Sequence↗