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Biomedical subjects

S Sakiyama

Publications and source records attributed to S Sakiyama.

At least 91 records · Page 5Linked to original sources

Mutations of the p53 gene in human lung cancer from chromate-exposed workers.

We examined p53 mutations in 20 cancer samples from 19 chromate workers with lung cancer by Polymerase chain reaction-Single strand conformation polymorphism analysis and direct sequencing. Six missense mutations were identified in 4 (20%) of the 20 chromate lung cancer samples. Fewer mutations were found in the patients with lung cancers who had been exposed to chromate than in those who had not. However, the pattern of p53 mutations in lung cancer patients exposed to chromate differed from that of common lung cancers in 3 respects. There were no apparent G to T transversions, which are common base changes in lung cancers. Half of the mutational sites (3/ 6) had changes of AT base-pairs, and 2 of 4 mutational tumor samples had double missense mutations. Our results suggested that chromate exposure may induce point mutation of the p53 gene.

Adenocarcinoma↗

Interaction of DA41, a DAN-binding protein, with the epidermal growth factor-like protein, S(1-5).

Recently, we have identified a new protein (DA41) which can associate with candidate tumor-suppressor DAN protein. In the present study, we have searched for DA41-interacting protein(s), using a yeast two-hybrid system. An adult rat lung cDNA library was screened by using a truncated form of DA41 (1-308) which lacks a DAN-binding region as bait. One of the positive clones, T16, contained a cDNA sequence of 1934 nucleotides with a single open reading frame of 493 amino acids. A data base search revealed that T16 exhibited a strong sequence similarity to the human epidermal growth factor (EGF)-like protein, S(1-5). The region encoding amino acids 155-232 of DA41 was identified for the interaction with T16. Since DAN and S(1-5) proteins are known to suppress and stimulate DNA synthesis, respectively, it is possible that functional interaction of DAN with S(1-5) through DA41 might play an important role(s) in the regulation of cell growth.

Adaptor Proteins, Signal Transducing↗

Identification of a receptor-type protein tyrosine phosphatase expressed in postmitotic maturing neurons: its structure and expression in the central nervous system.

We have isolated a rat cDNA encoding a receptor-type protein-tyrosine-phosphatase (RTP) expressed in brain and kidney (RPTP-BK) and characterized its expression in the developing central nervous system. RPTP-BK has seven fibronectin type III-like repeats in the extracellular region and a unique catalytic phosphatase domain in the cytoplasmic region. Bacterial expression of its phosphatase domain showed that the dephosphorylation of phosphotyrosine residues was mediated by the cytoplasmic catalytic domain. Sequence comparison revealed that RPTP-BK is homologous with GLEPP1, a rabbit PTP expressed in renal glomerular epithelia, and has the same phosphatase domain as murine PTPphi expressed in macrophages. RPTP-BK has also significant homology with Drosophila DPTP10D in the phosphatase domain, whose expression is localized exclusively in growth cones of the embryonal brains. The gene for RPTP-BK is well conserved among other species, and the expression in the brain but not in the kidney is developmentally regulated during the neonatal stage. Hybridization in situ showed that RPTP-BK is highly expressed in the postmitotic maturing neurons of the olfactory bulb, developing neocortex, hippocampus and thalamus. Because the expression of RPTP-BK in the developing neocortex is correlated with the stage of axonogenesis in cortical neurons, RPTP-BK might be crucial in neural cell development of the mammalian central nervous system.

Amino Acid Sequence↗

Expression of antisense RNA to S100A4 gene encoding an S100-related calcium-binding protein suppresses metastatic potential of high-metastatic Lewis lung carcinoma cells.

S100A4 (also known as pEL98/mts1/p9Ka/18A2/42A/calvasculin /FSP1/CAPL), a member of S100-related calcium-binding proteins, has been implicated to play a role in metastasis. In the present study, we examined the effect of antisense S100A4 RNA on metastatic potential of Lewis lung carcinoma (LLC) cells. High-metastatic All cells were transfected with the expression vector containing S100A4 cDNA in an inverted (antisense) orientation under the transcriptional control of the mouse metallothionein promoter. Treatment of a stably transfected clone (AS10 cells) with Zn2+ resulted in the suppression of the experimental metastatic ability, which was accompanied with the expression of antisense S100A4 RNA and the suppression of the S100A4 expression at both the mRNA and the protein levels. To further confirm the effect of antisense S100A4 RNA, we established several clones after retroviral transduction with an antisense S100A4 construct. Notably, reduced metastatic potential was also evident in these clones. In the antisense S100A4 RNA-expressing cells, cell motility and in vitro invasiveness were found to be suppressed.

Animals↗

Interleukin-1 beta converting enzyme (ICE) is preferentially expressed in neuroblastomas with favourable prognosis.

To determine whether interleukin-1 beta converting enzyme (ICE) plays a role in the programmed cell death of neuroblastoma, we studied ICE expression in primary tumours. In patients in stages I, II and IVS, ICE mRNA was detected in 22 of 32 (69%) tumours, while only 5 of 26 (19%) tumours expressed ICE in stages III and IV (P < 0.001). ICE mRNA was expressed in 27 of 47 (57%) tumours without MYCN amplification, but it was not detected in any tumours with MYCN amplification (P < 0.01). Immunohistochemically, the cytoplasm was stained in all 15 neuroblastomas examined. The nuclei were stained in 12 neuroblastomas without MYCN amplification, whereas only 1 of 3 tumours with MYCN amplification had positive staining in the nuclei. In ganglioneuromas, high levels of ICE mRNA were expressed, but immunostaining showed that the protease expression was confined to the cytoplasm. These observations suggest that ICE may be associated with the spontaneous regression often seen in favourable neuroblastomas and that localisation of ICE protease in the cell may be important for the cell death pathway. Double staining for ICE and TUNEL showed that they were co-localised in some nuclei, but the distribution of ICE protease expression was not necessarily the same as that of DNA fragmentation, suggesting that the protease expression probably preceded DNA fragmentation during the apoptotic process. ICE may play an important role in regulating the apoptotic process of neuroblastoma.

Blotting, Northern↗

A product of DAN, a novel candidate tumour suppressor gene, is secreted into culture medium and suppresses DNA synthesis.

Our previous studies have shown that the DAN gene product possesses an ability to revert phenotypes of transformed rat fibroblasts and represents a candidate tumour suppressor gene for neuroblastoma. In the present study, characterisation of DAN was carried out using rat fibroblast 3Y1 cells and their DAN-overexpressor counterparts (S-9). The N-terminal region of DAN (amino acids 1-24) was highly hydrophobic and DAN protein was found to be secreted into the culture medium. When DAN was treated with PNGase F, a enzyme that cleaves most N-linked carbohydrate residues, the mobility of both cytoplasmic and secreted DAN was increased in SDS-polyacrylamide gel electrophoresis, suggesting DAN is N-glycosylated, irrespective of its localisation. When partially purified, DAN was able, when added to the culture, to suppress DNA synthesis of Rous sarcoma virus-transformed 3Y1 cells, which lack the expression of DAN.

Animals↗

The genomic analysis of human DAN gene.

DAN gene is shown to be localized at human chromosome 1p36.11-p36.13, which resides within the consensus deletion observed in neuroblastoma. In the present study, we have isolated the genomic DNA containing the entire human DAN gene and determined its nucleotide sequence. Structural analysis revealed that DAN gene is composed of four exons and spans approximately 15 kb. Its overall structure was basically the same as that of rat DAN gene. Two distinct transcription initiation sites (major and minor) were identified by the primer extension experiment. Putative TATA and CAAT-like elements are present 38 and 366 bp upstream from the major transcription start site, respectively, however, no apparent TATA-like sequence was found in the upstream region of the minor transcription start site. The 400-bp region immediately upstream from the major transcription initiation site was strongly GC-rich (79% GC). Genomic Southern experiments demonstrated that the allelic loss of DAN gene might occur in neuroblastoma. Interestingly, there exist two dinucleotide repeats, (CA)7 and (CA)8, in the first intron of DAN gene, raising the possibility to distinguish two alleles of DAN gene in some of the cancer cells.

Base Composition↗

Identification of essential cis-acting regulatory elements for transcription of the rat DAN gene.

The DAN gene was initially isolated as one of the genes whose expression is significantly decreased in a variety of transformed rat fibroblasts 3Y1 cells when compared with the parental 3Y1 cells. In the present study, we have isolated the genomic clone of the DAN gene from a 3Y1 genomic library and characterized the possible regulatory elements responsible for the transcription of the DAN gene. The transcription initiation site was determined by a primer extension experiment. Putative TATA and CAAT-like elements were present 31 and 358 bp upstream from the transcription start site, respectively. Transient transfection of a series of DAN-chloramphenicol acetyltransferase (CAT) gene constructs, which contain different portions of the 5'-flanking region (2,236 bp) of the DAN gene and the CAT gene, was used to localize a regulatory element. These experiments demonstrated the presence of the regions that regulate DAN gene expression positively (-57 to +118) and negatively (-1,232 to -636). The electrophoretic mobility-shift assays revealed that 3Y1 and SR-3Y1 nuclear extracts specifically interact with the positive (-57 to +118) and the negative (-1,226 to -987) regulatory regions, respectively.

Animals↗

Identification of a new cellular protein that can interact specifically with DAN.

Recently, we demonstrated that the DAN gene product contains a growth- and/or a tumor-suppressive activity in vitro. In the present work, using a yeast two-hybrid system, we searched for cellular proteins that can associate with the DAN gene product. A cDNA clone, termed DA41, was initially isolated from an adult rat lung cDNA library. The DA41 gene was expressed in all adult tissues examined, however, the levels of expression varied significantly among the different tissues. Like the DAN gene product, the DA41 protein was similarly restricted to the cytoplasm. Sequence analysis revealed that DA41 cDNA is 2,167 nucleotides in length and contains a single open reading frame (ORF) of 582 amino acids (61,945 daltons). A homology search revealed that the DA41 gene product shares no structural similarity with those filed in the data base. In a synchronous 3Y1 cell culture, DA41 mRNA was expressed at a low level in quiescent cells; however, its level was significantly increased between the G1 and S phases of the cell cycle. On the other hand, the expression level of DAN mRNA did not change throughout the cell cycle progression. These results suggest that the DAN-DA41 complex might play a crucial role in the regulation of the cell cycle progression.

Adaptor Proteins, Signal Transducing↗

A study of cognitive development and behavior problems in mentally retarded children.

Cognitive development in seventy-one mentally retarded children (19 autistic, 52 non-autistic; aged 7-19 years) from a school for handicapped children was studied, using Ohta's scale for evaluating cognitive development level based on language comprehension (Ohta's stage), and other developmental scales. Behavior problems were also examined. The present study reports on the utility of Ohta's stage in non-autistic children, and the relationship between cognitive development level and behavior problems in mentally retarded children. In non-autistic children, there were temporal correlations between Ohta's stage and other development scales (a standard developmental test, speech development, symbolic play development, imitation development), suggesting that in non-autistic children as well, Ohta's stage may serve well as a scale for cognitive development, and reflect symbolic representational functioning. In non-autistic children, most behavior problems in feeding, elimination and sleeping, hyperkinesis, hypokinesis, stereotyped behaviors, self-injurious behavior and licking were closely associated with cognitive development level, and were more often noted in children of lower cognitive development level rather than only in the severely mentally retarded children. Some behavior problems may often occur in the sensorimotor period and hardly occur in the symbolic representational period.

Adolescent↗

[DAN gene].

DAN gene has been isolated by means of differential screening method between rat fibroblast 3Y1 and Rous sarcoma virus-transformed 3Y1 (SR-Y1) cells. DAN expression was suppressed in some of the transformed cells and its re-expression reverted the various transformed phenotypes such as colony formation in soft agar and tumor formation in mice. When DAN was overexpressed in 3Y1 cells, these cell showed the retardation of the entry into the S phase. DAN cDNA encodes a 27-kD protein which consists of 178 amino acids. The deduced amino acid sequence has no homology with known proteins. The amino terminal of DAN is highly hydrophobic and DAN is indeed secreted into the culture medium. This secreted DAN, when added to the culture of SR-3Y1 cells, suppressed DNA synthesis. Human DAN is mapped to 1p36.11-p36.13, a region known to have highly significant linkage with the genesis and/or progression of human neuroblastoma. Aberrant bands on Southern blot were detected in some of the neuroblastoma cases. Rat and human genomic DANs were isolated. CAT and the electrophoretic mobility shift assays revealed the presence of possible positive and negative regulatory elements at -57 approximately +118 and -1,232 approximately -987 of rat genome, respectively.

Animals↗

Inhibition of peritoneal dissemination of colon carcinoma in syngeneic mice immunized with interleukin-2-producing cells.

We have examined the antitumor effect of murine colon carcinoma cells engineered to produce human interleukin-2 (IL-2) in syngeneic mice. Subcutaneous inoculation of retrovirally-transduced cells with IL-2 gene formed small tumors, but they became regressed spontaneously. Consequently, the inoculated mice showed prolonged survival. Histological examination of the tumors derived from IL-2-producers revealed predominant infiltration of macrophages around tumor necrotic masses. Thus, inoculation of IL-2-producing cells could protect the mice from subsequent subcutaneous or intraperitoneal challenges with wild-type cells, suggesting the induction of acquired immunity due to the effect of tumor vaccination.

Animals↗

Evaluation of the bystander effect in experimental brain tumors bearing herpes simplex virus-thymidine kinase gene by serial magnetic resonance imaging.

Antitumor effects of herpes simplex virus-thymidine kinase (HSV-tk) gene transfer followed by ganciclovir (GCV) administration were studied by serial magnetic resonance imaging (MRI) with reference to the bystander effect. Mixed populations of 9L-gliosarcoma cells transduced with the HSV-tk gene (TK cells) and wild-type 9L cells were implanted into the brain of syngeneic Fisher rats at various ratios (total cell number, 10(5) cells; percentage of TK cells, 100%, 25%, 10%, or 0%). Rats were treated with GCV (30 mg/kg per day) or saline for 14 days and tumor masses were visually monitored using MRI. All of the saline-treated rats (regardless of TK cell percentage) and GCV-treated rats inoculated with 0% TK cells died between day 19 and day 31 (mean survival, 22.9 days) due to progressive tumor growth. The GCV-treated rats inoculated with more than 10% of TK cells lived significantly longer than the saline-treated rats (p < 0.01). The mean survivals of GCV-treated groups were 50.7, 70.0, and longer than 100 days for 10%, 25%, and 100% TK cells, respectively. MRI study revealed that reduction in tumor size and disappearance of tumor were observed in the GCV-treated rats inoculated with 10% or 25% TK cells. Complete regression of the tumor was, however, observed only in the rats implanted with 100% TK cells. The present results show that the bystander effect is clearly observed in vivo in a TK percentage-dependent manner, and a population of more than 25% of TK-positive cells is required for complete tumor elimination.

Animals↗

Specific interaction of pRB with a rat genomic DNA fragment, REC11.

The retinoblastoma gene product (pRB) has been known to contain a sequence-nonspecific DNA binding activity. It is unknown whether pRB can recognize and bind to specific DNA sequences. We have recently identified a rat genomic DNA fragment, termed REC11, which can interact with rat Cdc37-related protein (RCdc37). In this study, we have found that pRB could interact with the REC11, DNA in a sequence-specific manner. A series of GST-RB deletion mutants was used in the gel shift assays to define the domain of pRB responsible for this interaction. GST-RB (385-611) and GST-RB (612-928) completely lost the binding activity, while GST-RB (555-682) retained an activity to associate with the REC11 DNA, indicating that the continuous spacer region of pRB might be important for this sequence-specific DNA binding activity.

Animals↗

Reduced messenger RNA expression level of p21 CIP1 in human colorectal carcinoma tissues and its association with p53 gene mutation.

p21 Cip1 was first isolated as one of the cyclin-dependent kinase (Cdk) interacting proteins induced by wild-type p53 gene product, and it appears to play an essential regulatory role in the control of cell proliferation as a potent, tight-binding inhibitor of cyclin-Cdk complex that blocks the G1/S transition of the cell cycle. We have now examined the p21 Cip1 mRNA expression levels in 16 surgically excised human colorectal tumor and non-tumor tissues by Northern-blot analysis with reference to the identification of p53 gene mutations. p53 gene mutations were detected in 6 tumor tissues but not in the other 10 tissues by the polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) method and following direct sequencing. The mean p21 Cip1 mRNA expression level in tumor tissues was significantly suppressed compared to that of non-tumor tissues, irrespective of p53 gene mutations. In p53 gene mutation-detected cases, the mean expression level of p21 Cip1 mRNAs of tumor tissues was about 60% of that of cases without p53 gene mutation. Moreover, the relative mRNA expression levels of p21 Cip1 significantly decreased as the pathohistological stages progressed by Dukes' staging system, while in patients with liver metastasis these levels were significantly suppressed compared to those of patients without organ metastasis. These results indicate that reduced expression of p21 Cip1 mRNA is critical for growth activity and malignant potential of human colorectal carcinoma, and that the decrease in p21 Cip1 mRNA level is due to p53 gene mutation as well as other mechanisms during human colorectal carcinogenesis.

Adult↗

Augmentation of in vivo growth of Lewis lung carcinoma cells transduced with granulocyte macrophage-colony stimulating factor gene.

Cloned high-metastatic Lewis lung carcinoma. A11 cells were retrovirally transduced with either granulocyte macrophage-colony stimulating factor (GM-CSF) or beta-galactosidase gene and examined for their tumorigenicity. GM-CSF-engineered A11 cells produced a much higher amount of GM-CSF than the parental and control cells. Unexpectedly, GM-CSF-engineered A11 cells grew more rapidly than the control cells, while in vitro growth rates of these cells were almost the same. The enhanced tumor growth seemed to be unique to GM-CSF among various cytokines, because interleukin 2 (IL-2), interleukin 4 (IL-4) and interleukin 6 (IL-6) producer cells exhibited suppressed tumor growth.

Animals↗

Association of p53 gene mutation with decreased chemosensitivity in human malignant gliomas.

Loss of p53 function is involved in tumorigenesis of various human cancers, but the relation between mutation of the p53 tumor-suppressor gene and the chemo- and radiosensitivity of tumors remains unclear. Mutated p53 gene in malignant glioma is often associated with progression and recurrence of malignancy, and these events are closely linked with increased resistance to both chemotherapy and radiation. We have examined the status of the p53 gene in malignant gliomas obtained from 34 patients (glioblastoma: 29 cases, anaplastic astrocytomas: 5 cases). The chemosensitivities of these specimens using 28 kinds of anti-cancer agents were determined using an in vitro assay system. Overall, 12 mutated cases of p53 gene were found in malignant glioma samples. The mean numbers of effective agents were 0.58 for the tumor samples with p53 mutations and 5.00 for tumors without mutations. Our data indicate that p53 gene mutation predisposes to decreased cell killing via chemotherapy in malignant gliomas.

Adolescent↗