Biomedical subjects

H Saya

Publications and source records attributed to H Saya.

At least 91 records · Page 5Linked to original sources

PubMed1994

Polyclonal uses of T-cell receptor (TCR)alpha and beta genes for cytotoxic T lymphocytes in human metastatic melanoma: possible involvement of TCR alpha in tumor-cell recognition.

Identification of genetic structure and diversity of T-cell receptor (TCR)alpha and beta genes for cytotoxic T lymphocytes (CTLs) infiltrating human cancers is important for the better understanding of molecular mechanisms of host defense at tumor sites. cDNAs of TCR alpha and beta genes of 22 different melanoma-specific CTL clones established from the tumor-infiltrating lymphocytes of 2 patients were sequenced for analysis of their genetic structure and diversity. V alpha 7.2-J alpha 10-C alpha was found in 4 of 22 clones, 2 of which also used the same beta-chain. The other 20 clones showed different combinations of alpha and beta use. At deduced amino-acid levels, 7 of 9 clones from one patient used a threonine residue at the 26th position in the complementarity-determining region (CDR)1 of TCR alpha. Eight of 13 clones used a threonine at the 99th or a serine residue at the 100th position in CDR3 of TCR alpha CTL clones with the same or different TCR alpha showed the same or different patterns of cytotoxicity, respectively. These results suggest that CTLs usually do not demonstrate clonal expansion at tumor sites of metastatic melanoma's but rather that polyclonal T cells capable of binding to multiple melanoma determinants through CDR3 of TCR alpha accumulate in the tumor.

Base Sequence↗

PubMed1994

Germline p53 gene mutations in subsets of glioma patients.

BACKGROUND: Heritable germline mutations of the p53 gene have been described in patients with Li-Fraumeni syndrome, occasionally in nonfamilial malignancies such as multifocal osteosarcoma, in a small subgroup of young patients with two or more primary malignancies, and in patients with sporadic breast carcinoma. We recently reported that multifocal gliomas are frequently associated with other primary malignancies, and we hypothesized that genetic alterations may account for this phenomenon. PURPOSE: We examined the frequency of germline p53 gene mutations in patients with glioma and either multifocality of lesions, history of an additional primary (different) malignancy, or a family history of cancer. METHODS: Lymphocytes from 51 glioma patients were analyzed for germline p53 gene mutations using RNA-polymerase chain reaction analysis, single-strand conformation polymorphism, and gene sequencing techniques. RESULTS: Germline p53 gene mutations were detected in six of 19 patients with multifocal glioma, including two with family history of cancer, one with another primary malignancy, and two with all three risk factors; one of four patients with unifocal glioma, another primary malignancy, and a family history of cancer; and two of 15 patients with unifocal glioma and a family history of cancer but no second malignancies. No mutations were detected in the patient with unifocal glioma and another malignancy or in the 12 control patients with unifocal glioma and no second malignancies or family history of cancer. Patients having mutations were younger than other patients in the same group. CONCLUSIONS: Germline p53 mutations are frequent in patients with multifocal glioma, glioma and another primary malignancy, and glioma associated with a family history of cancer, particularly if these factors are combined. IMPLICATIONS: Relatives at high risk can be identified for genetic counseling, early cancer detection, and possible enrollment in chemoprevention trials.

Adult↗

PubMed1994

Differential expression of two fibroblast growth factor-receptor genes is associated with malignant progression in human astrocytomas.

Malignant astrocytomas, which are highly invasive, vascular neoplasms, compose the majority of nervous system tumors in humans. Elevated expression of fibroblast growth factors (FGFs) in astrocytomas has implicated the FGF family of mitogens in the initiation and progression of astrocyte-derived tumors. In this study, we demonstrated that human astrocytomas undergo parallel changes in FGF-receptor (FGFR) expression during their progression from a benign to a malignant phenotype. FGFR type 2 (BEK) expression was abundant in normal white matter and in all low-grade astrocytomas but was not seen in malignant astrocytomas. Conversely, FGFR type 1 (FLG) expression was absent or barely detectable in normal white matter but was significantly elevated in malignant astrocytomas. Malignant astrocytomas also expressed an alternatively spliced form of FGFR-1 (FGFR-1 beta) containing two immunoglobulin-like disulfide loops, whereas normal human adult and fetal brains expressed a receptor form (FGFR-1 alpha) containing three immunoglobulin-like disulfide loops. Intermediate grades of astrocytic tumors exhibited a gradual loss of FGFR-2 and a shift in expression from FGFR-1 alpha to FGFR-1 beta as they progressed from benign to malignant phenotype. These results suggest that differential expression and alternative splicing of FGFRs may be critical in the malignant progression of astrocytic tumors.

Alternative Splicing↗

PubMed1994

Basic fibroblast growth factor and fibroblast growth factor receptor I are implicated in the growth of human astrocytomas.

Malignant astrocytomas are highly invasive, vascular neoplasms that comprise the majority of nervous system tumors in humans. A strong association has previously been made between malignancy in human astrocytic tumors and increased expression of certain fibroblast growth factor (FGF) family members, including basic and acidic FGF. The influence of endogenous basic FGF on glioblastoma cell growth in vitro was evaluated using basic FGF-specific antisense oligonucleotides. These studies indicated that human glioblastoma cell growth in vitro, can be inhibited by suppressing basic FGF expression. Human astrocytomas also exhibited changes in FGF receptor (FGFR) expression during the course of their progression from a benign to a malignant phenotype. FGFR2 (bek) expression was abundant in normal white matter and in all low grade astrocytomas, but was not observed in glioblastomas. Conversely, FGFR1 (flg) expression was absent or barely detectable in normal white matter, but was significantly elevated in glioblastomas. Glioblastomas also expressed an alternatively spliced form of FGFR1 containing two immunoglobulin-like disulfide loops (FGFR1 beta), whereas normal human adult and fetal brain expressed a form of the receptor containing three immunoglobulin-like disulfide loops (FGFR1 alpha). Intermediate grades of astrocytic tumors exhibited a gradual loss of FGFR2 and a shift in expression from FGFR1 alpha to FGFR1 beta as they progressed from a benign to a malignant phenotype. The underlying cytogenetic changes that contribute to these alterations are not entirely understood, but abnormalities in the p53 tumor suppressor gene may influence expression of bFGF as well as the FGFR. These results suggest that alterations in FGFR signal transduction pathways may play a critical role in the malignant progression of astrocytic tumors.

Astrocytes↗

PubMed1994

The CD44 adhesion molecule and metastasis.

The family of related cell surface adhesion molecules designated CD44 are multifunctional proteins displayed by a wide variety of normal and malignant tissues. CD44 properties in vitro include hyaluronate-mediated adhesion, motility, hyaluronate degradation, self-aggregation, and adhesion to lymphoid tissue. These properties are among several that are required by invasive and metastatic tumor cells. Several in vivo experiments indicate that tumor cells transfected to overexpress specific CD44 isoforms display an enhancement in metastatic potential. Numerous CD44 isoforms exist as a result of alternative splicing, and specific isoforms enhance tumor cell metastasis more efficiently than others. This suggests that regulation of CD44 alternative splicing is an important determinant of metastatic potential. Several human tumors display specific alterations in CD44 isoform expression, and the extent of clinical disease in specific tumors correlates with the CD44 isoform expression pattern. In this review we analyze these data that suggest that CD44 plays an important role in tumor metastasis.

Animals↗

PubMed1994

Increase in the capability of interleukin 2 gene-transduced renal cell carcinoma cells to induce cytotoxic lymphocytes.

The immunogenicity of human cancer cells transfected with interleukin 2 (IL-2) gene, a potent vaccine candidate, has not yet been fully investigated. Human renal cell carcinoma (RCC) cells transduced with human IL-2 gene (RCC-IL-2) were investigated in vitro for the capability to induce lymphokine-activated killer (LAK) or cytotoxic T lymphocyte (CTL) activity in peripheral blood mononuclear cells (PBMC) or tumor-infiltrating lymphocyte (TIL). The RCC-IL-2 cells stimulated PBMC to demonstrate LAK activity, and also stimulated autologous TILs to proliferate and exhibit cytotoxicity relatively restricted to autologous tumor cells. In contrast, both parental RCC and RCC transduced with neomycin gene alone failed to induce these activities. These results indicate that RCC-IL-2 cells are more potent than the other RCC cells with regard to inducing cytotoxic lymphocytes against autologous tumor cells.

Base Sequence↗

PubMed1994

Detection of cellular proteins that interact with the NF2 tumor suppressor gene product.

The neurofibromatosis type 2 (NF2) gene was recently cloned, and the protein it encodes (merlin) was revealed to belong to a family of proteins that link cytoskeletal components with proteins in the cell membrane. To elucidate the biological function of merlin, we produced a bacterial fusion protein consisting of glutathione S-transferase and merlin and used it to detect five merlin-binding cellular proteins, designated p165, p145, p125, p85 and p70, by a protein-binding assay. p165 and merlin were phosphorylated on serine/threonine residues, and immunoprecipitation showed that p85 bound the native form of merlin. Although the entire merlin-ezrin-radixin-moesin (MERM) homology domain of merlin was found to be essential for binding to all five proteins, the MERM homology domains of ezrin and moesin did not bind to any of the five proteins. Since most reported NF2 mutations are in the region we determined was necessary for binding, the mutations probably impair binding. Therefore, the formation of the protein complex is probably crucial for tumor suppression.

Base Sequence↗

PubMed1993

Role and regulation of expression of 92-kDa type-IV collagenase (MMP-9) in 2 invasive squamous-cell-carcinoma cell lines of the oral cavity.

The present study was undertaken to determine the role of the metalloproteinase MMP-9 in the invasive phenotype of squamous-cell carcinoma of the oral cavity and the regulation of its expression. Zymographic analysis of conditioned medium from 2 highly invasive squamous-cell-carcinoma cell lines indicated large amounts of an enzyme which was indistinguishable, in size (92 kDa) from the MMP-9 pro-enzyme. Conversion of the 92-kDa gelatinase into a lower-molecular-weight species (84 kDa), identical in size to the activated gelatinase, was evident when both cell lines, which are avid secretors of urokinase, were cultured in the presence of plasminogen. Penetration of an extracellular-matrix-coated filter was dramatically reduced in the presence of the collagenase inhibitor, tissue inhibitor of metalloproteinase-2, suggesting a critical role for MMP-9 in the invasive process. Immunohistochemical studies demonstrating the presence of MMP-9 in tumor cells of resected squamous-cell cancers suggested that secretion of this collagenase by cells in vitro was reflective of the in vivo setting. Since several phorbol-ester response elements are present in the MMP-9 promoter, we determined the role of protein-kinase-C pathways in the regulation of MMP-9 expression in cultured SCC. Treatment of cells with PMA resulted in a more-than-20-fold increase in the level of protein and mRNA. Conversely, culturing of cells in the presence of the protein-kinase-C inhibitor, calphostin-C, led to a dose-dependent decrease in the amount of MMP-9 mRNA and protein, suggesting that the constitutive expression of this collagenase reflects activation of this signal transduction pathway. In summary, our data suggest that, for a sub-population of squamous-cell carcinomas, secreted MMP-9 is an important determinant of the invasive phenotype, and that the expression of this metalloproteinase is regulated by protein-kinase-C pathways.

Amino Acid Sequence↗

PubMed1993

Bacterial expression of an active tyrosine kinase from a protein A/truncated c-src fusion protein.

The carboxy-terminal half of the c-src protein fused to the protein A moiety was expressed in bacteria. The protein A/truncated c-src fusion protein, which does not have SH2 and SH3 domains, is found in the periplasmic space allowing for a simple one-step purification and demonstrated high efficiency in autophosphorylation and exogeneous substrate phosphorylation. The missense mutation at codon 294 (Ile-->Thr), which is located in the ATP-binding domain of the c-src, resulted in dramatic reduction of tyrosine kinase activity of the fusion protein. Using the fusion protein, we also revealed that staurosporin, a well-known kinase inhibitor, directly affects autophosphorylation of the C-terminal half of the c-src protein. This truncated c-src expression system provides a good source of enzyme for diverse experiments and is an ideal model for understanding the implication of structural alterations in the catalytic activity of the c-src kinase by site-directed mutagenesis experiments.

Alkaloids↗

PubMed1993

Expression of CD44R1 adhesion molecule in colon carcinomas and metastases.

CD44 is a cell surface adhesion molecule postulated to control lymphocyte recirculation by facilitating entry into lymphoid tissue. Tumour cells transfected to overexpress the epithelial variant CD44R1 readily gain access to lymph nodes and distant metastatic sites in animal models, possibly by mimicking circulating lymphocytes. To investigate if human tumours display altered CD44 expression we performed reverse transcriptase a polymerase chain reaction (PCR) analysis of CD44 in 49 specimens from normal colonic mucosa, primary colon and rectal tumours, normal liver, and metastases of 20 patients. The haematopoietic variant CD44H was the principal isoform amplified in all of the specimens, However, 12/14 primary tumours and 16/16 metastatic potential. Moreover, the relative increase with only 2 of 13 normal mucosa specimens. This increase in CD44R1 relative to CD44H expressed by human colon carcinoma cells may increase their metastatic potential. Moreover, the relative increase in PCR amplification of CD44R1 compared with that of CD44H may provide a sensitive method for detecting primary and metastatic colon carcinoma cells in small biopsy specimens.

Base Sequence↗

PubMed1993

Novel variants of CD44 arising from alternative splicing: changes in the CD44 alternative splicing pattern of MCF-7 breast carcinoma cells treated with hyaluronidase.

CD44 is a cell-surface glycoprotein postulated to play a role in a variety of biological processes, including lymphocyte homing and tumor-cell metastasis. Several isoforms of CD44 have been identified in human cells, and the genesis of some of these isoforms has been attributed to alternative splicing. In the study presented here we amplified three novel transcript variants of CD44 from human cell lines using a reverse transcriptase-polymerase chain reaction strategy. Two of the novel isoforms differed from previously described CD44 isoforms as a result of alternative splicing that occurred at previously reported splice junctions. The third novel CD44 isoform was generated from a previously unreported alternative splice junction near the 5' end of the open reading frame. Southern blot analysis of genomic DNA revealed that these novel isoforms and all of the previously described CD44 isoforms arose from alternative splicing. The capability of cells to modify their CD44 alternative splicing pattern was demonstrated in MCF-7 cells, which altered their CD44-isoform expression pattern in response to treatment with hyaluronidase. A better understanding of mechanisms regulating CD44 alternative splicing may provide insights into diverse processes, including tumor-cell metastasis and lymphocyte homing.

Alternative Splicing↗

PubMed1993

Detection of p53 alterations in human astrocytomas using frozen tissue sections for the polymerase chain reaction.

The polymorphism of amino acid residue 72 on the human p53 tumor-suppressor gene is a useful marker for detecting intragenic loss of heterozygosity (LOH). We examined the LOH of the p53 gene in human malignant astrocytomas by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis using DNA extracted from frozen tissue sections under histologic examination. Eleven of 16 informative cases (69%) of the malignant astrocytomas were found to have LOH in the p53 gene. Sequential frozen sections were analyzed by immunohistochemistry using anti-p53 antibody PAb1801 to detect overexpression of the p53 protein, which is presumably altered if it is detectable. Ten of the 11 cases that had LOH of the p53 gene overexpressed the p53 protein. Moreover, 4 of the 11 patients with LOH of the p53 gene developed a second neoplasm in addition to an astrocytoma, possibly indicating genetic instability in these patients. These data suggest that alterations of the p53 gene may play an important role in the genesis of malignant astrocytoma. The combination of the PCR-RFLP method and immunohistochemical analysis using frozen tissue sections is a practical diagnostic tool for examination of human malignancies, including astrocytomas. astrocytomas.

Astrocytoma↗

PubMed1993

Epidemiology, cytogenetics, and molecular biology of brain tumors.

The incidence of primary brain tumors has increased dramatically among elderly North Americans during the past two decades. Numerous chromosomal abnormalities have been associated with these tumors; various subsets of these abnormalities are specific to certain types of brain tumors. Astrocytic gliomas may exhibit losses of genetic information from chromosomes 9p, 10q, 11p, 13q, 17p, or 22. Mutations of the p53 gene are found mostly in the malignant astrocytic forms and have been linked to malignant tumor transformation and progression. Functional and structural abnormalities of the neurofibromatosis 1 (NF1) gene and overexpression of the epidermal growth factor receptor have been associated with expression of the malignant glioma phenotype. Other less clearly defined abnormalities in astrocytomas include mutations of the retinoblastoma (RB) gene and overexpression of platelet-derived growth factor; transforming growth factor-alpha and -beta; the c-erb B-1, c-myc, ras, c-fos, and ros oncogenes; and insulin-like growth factor I and II. In other glioma tumors, p53 mutations are either infrequent, as in oligodendrogliomas, or absent, as in ependymomas. Occasionally, medulloblastomas exhibit p53 mutations and loss of genetic information from chromosomes 6q and 16q or expression of the c-erb B-2 oncogene. Loss of heterozygosity in chromosome 22 is the most frequent event in meningiomas, suggesting the presence of a tumor-suppressor gene in this chromosome.

Aged↗

PubMed1993

Proliferating cell nuclear antigen (PCNA) expression in chronic lymphocytic leukemia (CLL).

Chronic Lymphocytic Leukemia (CLL) is usually an indolent disorder which in some patients assumes an aggressive clinical course. In order to assess at presentation the prognosis of a given patient, several staging systems and prognostic variables have been proposed including the expression of the Proliferating Cell Nuclear Antigen (PCNA). PCNA is a 36 kd nuclear protein, the regulation of which is cell cycle-dependent. In CLL, PCNA levels correlate with cell proliferation, clinical stage and the lymphocyte doubling time (LDT). Furthermore, preliminary data suggests that PCNA expression may also predict response to Fludarabine-based chemotherapy. Since PCNA is a cofactor for Delta DNA polymerase, PCNA overexpression in CLL may also reflect the intrinsic DNA repair activity of the leukemic cells and thus their resistance to chemotherapy. Further studies aiming at modulation of PCNA expression in CLL cells may clarify this issue and may offer a future new therapeutic strategy with which to treat this disorder.

Antigens, Neoplasm↗

PubMed1993

p53 protein immunostaining in routinely processed paraffin-embedded sections.

Mutation of the p53 tumor suppressor gene is the most common genetic alteration in human tumors. The altered protein product of the mutant gene is stabilized in tumor cells and can be detected using monoclonal antibody immunohistochemistry. We report a technique for immunostaining of the altered p53 protein in routinely processed paraffin-embedded tissue sections, comparing this method with a frozen-section immunostain method for concordance. Similarly processed tumor cell lines with known mutations or deletions of the p53 gene served as positive or negative controls. The results in 25 human gliomas and 20 colorectal carcinomas showed a 100% correspondence of positive reactivity in the colorectal carcinomas and an 83% correspondence in the gliomas. In no case did the paraffin-embedded sections react in the absence of frozen section reactivity (that is, there were no false positives). Three of 18 gliomas showed reactivity in frozen sections without reactivity in the companion paraffin-embedded sections. This discrepancy could have been caused by technical factors such as length of fixation time. We propose that this new paraffin-embedded section immunostaining method will be of value as a screening technique for the investigation of p53 mutations in archived human tumors. The data thus obtained may then be correlated with clinical information and perhaps be of value in diagnosis or predicting outcome for various human cancers.

Adenocarcinoma↗

PubMed1992

Prognostic value of proliferating cell nuclear antigen expression in chronic lymphoid leukemia.

Chronic lymphocytic leukemia (CLL) is a usually indolent disease that can assume an aggressive clinical course in some patients. To develop assays that would be predictive of how a particular patient's disease would evolve, we studied the expression of proliferating cell nuclear antigen (PCNA) by Western blotting in 40 patients with CLL. The concentration of PCNA, a cofactor for delta DNA-dependent DNA polymerase, is indicative of the proliferative state of the cell. Significantly lower PCNA levels were observed in earlier stage CLL when compared with more advanced disease. The leukemic cell proliferative rate, assessed by lymphocyte doubling time and flow cytometry, also correlated significantly with the level of PCNA expression. These results suggest that a high level of PCNA in the cells of CLL patients at presentation identifies a subgroup of patients whose CLL cells have a higher proliferative activity and who may, therefore, have a potentially shorter survival.

Actins↗

PubMed1992

Alternative splicing of neurofibromatosis type 1 gene transcript in malignant brain tumors: PCR analysis of frozen-section mRNA.

The neurofibromatosis type 1 (NF1) gene encodes a 360-residue region showing significant homology to the catalytic domains of both mammalian GTPase-activating protein (GAP) and yeast IRA protein. The product of the GAP-related domain of the NF1 gene (NF1-GRD) has been shown to stimulate ras GTPase and consequently to inactivate ras protein. We previously reported that the NF1-GRD has two types of transcripts, type I and type II, which are generated by an alternative splicing mechanism, and that the differential splicing of the NF1-GRD may be related to differentiation of neuroectodermal cells. Here we examined the differential expression of type I and type II transcripts of NF1-GRD in clinical samples of supratentorial malignant brain tumors by the RNA-polymerase chain reaction (PCR) method using frozen tissue sections. Our observations revealed that normal cerebrum predominantly expressed the type II NF1-GRD transcript, whereas primitive neuroectodermal tumors predominantly expressed the type I transcript. Additionally, although the type I/type II ratio in astrocytomas varied widely among tissue samples, all glioblastomas showed higher type I/type II ratios than adjacent brain samples. The RNA-PCR analysis using frozen tissue sections is a useful and sensitive method for detecting genetic markers in clinical tissue samples.

Astrocytoma↗

PubMed1992

Structural alterations of the p53 gene in human leukemias.

We have investigated alterations of the p53 gene in human leukemias by polymerase chain reaction-mediated restriction fragment length polymorphism analysis. This method detects the codon 72 polymorphism of the p53 gene, allowing identification of loss of heterozygosity (LOH) of the p53 gene. In this study, at least two specimens were obtained from each patient to compare the allele status at different points of clinical course. Of 21 cases examined 7 were heterozygous at this polymorphic site and were evaluable for LOH study. Only one patient of Philadelphia chromosome-positive acute lymphoblastic leukemia (ALL) lost the heterozygosity in the specimen of her last relapse. Leukemic cells from her repeated relapses including the last one revealed to have the clonally rearranged immunoglobulin H chain gene of the same size, indicating that alteration of the p53 gene in this patient might account for the lethal relapse as a clonal evolution event. Northern-blot analysis of the p53 gene showed that one case of CD7(+) ALL had altered p53 mRNA. Overall, it is demonstrated that alterations of the p53 gene might be involved in the pathogenesis or progression of at least some human leukemias, although the alterations in leukemias seemed to be not as frequent as in solid tumors.

Base Sequence↗