Biomedical subjects

Yoshio Miki

Publications and source records attributed to Yoshio Miki.

29 records · Page 2Linked to original sources

PubMed2004

Expression profiling to predict postoperative prognosis for estrogen receptor-negative breast cancers by analysis of 25,344 genes on a cDNA microarray.

Estrogen receptor (ER) status is an essential determinant of clinical and biological behavior of human breast cancers. While ER-positive breast cancers respond well to adjuvant hormone therapy, ER-negative tumors are generally resistant. To date, no attempts have succeeded in finding molecular markers for classifying ER-negative breast cancers with respect to postoperative prognosis. To identify a set of prognostic markers for this type of cancer, we used a cDNA microarray consisting of 25,344 human genes to investigate expression profiles of ten primary breast cancers from patients who had died of breast cancer within 5 years after surgery (5y-D) and 10 from patients who had survived disease-free for more than 5 years (5y-S). Sets of genes characterizing each group were identified by Mann-Whitney and random-permutation tests. We documented 71 genes with higher expression in the 5y-D group than in the 5y-S group, and 15 with higher expression in the 5y-S group than in the 5y-D group. Semi-quantitative RT-PCR experiments were carried out to confirm the results of the microarray analysis. We established a scoring system for predicting postoperative prognosis of ER-negative breast cancers on the basis of aberrant gene expression. The list of genes reported here provides valuable information with regard to progression of breast cancer and is a source of possible target molecules for development of novel drugs to treat patients with ER-negative breast cancers.

Adult↗

PubMed2004

Relatively common mutations of the Bloom syndrome gene in the Japanese population.

Bloom syndrome (BS) is a rare autosomal recessive genetic disorder characterized by lupus-like erythematous facial telangiectasia, sun sensitivity, infertility, stunted growth and a high predisposition to various types of cancer. Chromosomal abnormalities are hallmarks of this disorder, and high frequencies of sister chromatid exchanges and quadriradial configurations in lymphocytes and fibroblasts are diagnostic features. BLM is the causative gene for BS. We investigated the mutation in the BLM gene in 4 Japanese BS kindreds. Taken together with previously documented mutations, 2 kindreds were homozygous for 631delCAA and 2 were compound heterozygous for 631delCAA. The silent mutation of A1055C (Thr to Thr) was detected in control Japanese individuals. The 6-bp deletion/7-bp insertion at position 2,281, which most Askenazi Jewish BS patients carry, was not detected in 200 Japanese alleles. These results suggest that 631delCAA is a relatively common mutation among the Japanese BS patients.

Adenosine Triphosphatases↗

PubMed2004

Genome-wide gene-expression profiles of breast-cancer cells purified with laser microbeam microdissection: identification of genes associated with progression and metastasis.

Breast carcinoma is a complex disease characterized by accumulation of multiple genetic alterations, and the understanding of the molecular basis of mammary tumorigenesis is still incomplete. In this study we analyzed gene-expression profiles of 81 surgical specimens of 12 ductal carcinoma in situ (DCIS) and 69 invasive ductal carcinoma (IDC). After applying laser-microbeam micro-dissection to all samples we achieved 98-99% pure populations of breast cancer cells, and of normal breast epithelial cells used as controls. A cDNA-microarray analysis of 23,040 genes in these samples and a subsequent unsupervised hierarchical clustering distinguished two tumor groups, mainly in terms of estrogen-receptor (ER) status. We then undertook a supervised analysis and identified 325 genes that were commonly either up- or down-regulated in both pathologically discrete stages (DCIS and IDC), indicating that these genes might play important roles in malignant transformation of breast ductal cells. In addition, we searched invasion-associated gene candidates whose expression was altered in IDC, but not in DCIS, and identified 24 up-regulated genes and 41 down-regulated genes. Furthermore, we identified 34 genes that were expressed differently in tumors from patients with lymph node metastasis as opposed to no metastasis. On that basis we developed a scoring system that correlated well with the metastatic status. Tumors from all of the 37 test patients with lymph-node metastasis yielded positive scores by our definition, whereas 38 of the 40 tumors (95%) without lymph node metastasis had negative scores. Our data should provide useful information for identifying predictive markers for invasion or metastasis, and suggest potential target molecules for treatment of breast cancers.

Adult↗

PubMed2003

Protein kinase Cdelta is responsible for constitutive and DNA damage-induced phosphorylation of Rad9.

The mammalian homolog of the Schizosaccharomyces pombe Rad9 is involved in checkpoint signaling and the induction of apoptosis. While the mechanisms responsible for the regulation of human Rad9 (hRad9) are not known, hRad9 is subject to hyperphosphorylation in the response of cells to DNA damage. The present results demonstrate that protein kinase Cdelta (PKCdelta) associates with Rad9 and that DNA damage induces this interaction. PKCdelta phosphorylates hRad9 in vitro and in cells exposed to genotoxic agents. The functional significance of the interaction between hRad9 and PKCdelta is supported by the finding that activation of PKCdelta is necessary for formation of the Rad9-Hus1-Rad1 complex. We also show that PKCdelta is required for binding of hRad9 to Bcl-2. In concert with these results, inhibition of PKCdelta attenuates Rad9-mediated apoptosis. These findings demonstrate that PKCdelta is responsible for the regulation of Rad9 in the Hus1-Rad1 complex and in the apoptotic response to DNA damage.

Apoptosis↗

PubMed2002

Activation of SAPK/JNK signaling by protein kinase Cdelta in response to DNA damage.

The cellular response to genotoxic stress includes activation of protein kinase Cdelta (PKCdelta). The functional role of PKCdelta in the DNA damage response is unknown. The present studies demonstrate that PKCdelta is required in part for induction of the stress-activated protein kinase (SAPK/JNK) in cells treated with 1-beta-d-arabinofuranosylcytosine (araC) and other genotoxic agents. DNA damage-induced SAPK activation was attenuated by (i) treatment with rottlerin, (ii) expression of a kinase-inactive PKCdelta(K-R) mutant, and (iii) down-regulation of PKCdelta by small interfering RNA (siRNA). Coexpression studies demonstrate that PKCdelta activates SAPK by an MKK7-dependent, SEK1-independent mechanism. Previous work has shown that the nuclear Lyn tyrosine kinase activates the MEKK1 --> MKK7 --> SAPK pathway but not through a direct interaction with MEKK1. The present results extend those observations by demonstrating that Lyn activates PKCdelta, and in turn, MEKK1 is activated by a PKCdelta-dependent mechanism. These findings indicate that PKCdelta functions in the activation of SAPK through a Lyn --> PKCdelta --> MEKK1 --> MKK7 --> SAPK signaling cascade in response to DNA damage.

Base Sequence↗

PubMed2002

Conserved domains in the chicken homologue of BRCA2.

Germline mutation of the BRCA2 gene causes a high risk of developing breast and ovarian cancer. Although the BRCA2 protein has been implicated in homologous recombination (HR) of DNA and in transcription, it is still unclear how mutation leads to tumorigenesis. We have identified a non-mammalian homologue of BRCA2 from chicken, which encodes a protein with 3397 amino acids (aa) and shows only 40% identity to human BRCA2. However, comparison of the mammalian and chicken sequences revealed remarkably high homology in several segments. These include a N-terminal region (approximately 100 aa), which was previously shown to possess intrinsic transcriptional activity, and a C-terminal region (aa residue approximately 2480-approximately 3180 in human BRCA2), which has not been clearly assigned any function. In contrast, although the eight BRC repeats of mammalian BRCA2 are believed to play an important role in HR by interacting with Rad51, the BRC3, BRC5, and BRC6 repeats exhibit virtually no similarity to their mammalian counterparts. Among 311 missense mutations listed as unclassified variants in the NIH Breast Cancer Information Core database, only 83 of these sites are identical in chicken BRCA2. Thus, chicken BRCA2 may provide a means to identify domains and residues associated with cancer predisposition.

Amino Acid Sequence↗

PubMed2002

Genetic and histological assessment of surgical margins in resected liver metastases from colorectal carcinoma: minimum surgical margins for successful resection.

HYPOTHESIS: There have been few reports on the minimum surgical margins (SMs) required for successful liver resection in patients with colorectal metastases. This minimum requirement may be narrower than the previously recommended margin of 10 mm. OBJECTIVES: To identify the minimum margins by assessing the presence of micrometastases around the tumor using genetic and histological techniques, and to investigate whether SMs are associated with patterns of tumor recurrence or patient survival. DESIGN: Prospective and retrospective studies. SETTING: Tertiary referral cancer center. PATIENTS AND METHODS: Fifty-eight patients who underwent 62 liver resections for hepatic metastasis from colorectal cancer between December 1, 1996, and November 30, 2000, were included in the study. Tissue samples taken from the tumor, surrounding liver parenchyma, and Glisson pedicle near the tumor were tested for K-ras and p53 mutations using the mutant allele-specific amplification method. For the retrospective study on patient outcomes, 194 patients who had undergone liver resections between 1980 and 2000 were analyzed according to their SMs. RESULTS: Of the 62 sets of samples from liver metastases, 39 were positive for K-ras and p53 gene mutations or both. Micrometastases in the liver parenchyma surrounding colorectal metastases were present in 2.0% (4/199) of tested samples and were located within 4 mm of the tumor border. Micrometastases via Glisson pedicle were more common (14.3% [3/21]), but these were also confined to a short distance from the tumor edge (</=5 mm). Of the 5 micrometastases detected by genetic analysis, only 2 were confirmed by histopathological examination. The analysis of patient outcomes demonstrated that the incidence of cut-end recurrence (relapse in the bed of resection) decreased from 20.0% to a range of 5.6% to 7.5% when the SM is 2 mm or more. The incidence of definite cut-end recurrence in patients with SMs less than 2 m, 2 to 4 mm, and 5 mm or wider was 13.3% (6/45), 2.8% (1/36), and 0% (0/102), respectively. The SM was not a significant prognostic factor in patient survival. CONCLUSIONS: Micrometastases around liver tumors are not common, and most are confined to the immediate vicinity of the tumor border. We propose an SM of 2 mm as a clinically acceptable minimum requirement, which carries approximately a 6% risk of margin-related recurrence. Because liver resection provides the only chance of cure, complete removal of the tumor with a minimum margin is justified when technically unavoidable because of the size, location, number of tumors, or successive resections.

Adult↗

PubMed2002

Identification and characterization of the human protein kinase-like gene NTKL: mitosis-specific centrosomal localization of an alternatively spliced isoform.

Although the centrosome has an essential role in mitosis, its molecular components have not been fully elucidated. Here, we describe the molecular cloning and characterization of the human gene NTKL, which encodes an evolutionarily conserved kinase-like protein. NTKL mRNA is found ubiquitously in human tissues. NTKL is located on 11q13 and is mapped around chromosomal breakpoints found in several carcinomas, suggesting that NTKL dysfunction may be involved in carcinogenesis. Alternative splicing generates two variant forms of NTKL mRNA that encode protein isoforms with internal deletions. When fused to green fluorescent protein, the full-length product and one of the variant proteins are found in cytoplasm. The other variant product also exists in the cytoplasm during interphase, but is found in the centrosomes during mitosis. Endogenous NTKL protein is also localized to the centrosomes during mitosis. This cell-cycle-dependent centrosomal localization suggests that NTKL is involved in centrosome-related cellular functions.

Adaptor Proteins, Vesicular Transport↗

PubMed2002

Mutation analysis of the mel-18 gene that shows decreased expression in human breast cancer cell lines.

BACKGROUND: Mammalian mel-18 is a member of the polycomb group, and it acts as a transcriptional repressor with DNA binding activity. Murine mel-18 negatively regulates the cell cycle through the c-myc/cdc25 cascade, and mice haploinsufficient for mel-18 develop mammary gland tumors. In addition, the human homolog of mel-18 is located at 17q, on which candidate tumor suppressor genes for breast cancer have been suggested for a long time. These observations indicate that the mel-18 gene may be a tumor suppressor gene for breast cancer. To investigate this possibility, we examined the expression of mel-18 mRNA in human breast cancer cell lines and searched for mel-18 gene mutations in sporadic and familial breast cancers. METHODS: The expression of mel-18 mRNA was examined in five breast cancer cell lines by RT-PCR, and somatic and germline mutations of the mel-18 gene were analyzed by the PCR-SSCP and sequence methods in 48 sporadic breast cancers, including 16 cases with loss of heterozygosity (LOH) at the mel-18 locus, and in 23 cases from 18 breast cancer families, respectively. RESULTS: We found that most cell lines examined here showed decreased expression of mel-18 mRNA, however, no alteration other than a single nucleotide change that did not lead to amino acid alteration in one patient was identified. CONCLUSION: Our results reveal that mel-18 gene mutations are exceedingly rare in human breast cancers, and a reduction of mel-18 expression in human breast cancer cell lines would support a role for mel-18 haploinsufficiency in breast carcinogenesis.

Adult↗

PubMed2002

C421A polymorphism in the human breast cancer resistance protein gene is associated with low expression of Q141K protein and low-level drug resistance.

Breast cancer resistance protein (BCRP) confers multidrug resistance to cancer cells against agents such as SN-38 (an active metabolite of irinotecan), mitoxantrone, and topotecan. Among 59 human tumor cell lines tested, 6 cell lines, A549, NCI-H460, KM-12, HT-29, OVCAR-5, and RPMI8226, showed high BCRP expression. BCRP cDNA was isolated from 11 cancer cell lines and three variant cDNAs [G34A substituting Met for Val-12 (V12M), C421A substituting Lys for Gln-141 (Q141K), and 944-949 deletion lacking Ala-315 and Thr-316 (delta315-6)] were identified. G34A and C421A variants were polymorphisms, and 944-949 deletion was a splicing variant. C421A BCRP-transfected PA317 cells showed markedly decreased protein expression and low-level drug resistance compared with wild-type BCRP-transfected cells when transfectants expressed similar levels of BCRP mRNA. G34A or 944-949-deleted BCRP-transfected PA317 cells showed similar or somewhat lower protein expression and drug resistance compared with wild-type BCRP-transfected cells. Of 124 healthy Japanese volunteers, 67 were wild-type, 48 were heterozygous, and 9 were homozygous for the C421A allele. These results suggest that some people possess the C421A polymorphic BCRP gene and express low amounts of Q141K BCRP. In addition to that, C376T polymorphism in exon 4 substituting stop codon for Gln-126 was found in 3 of the 124 general Japanese population. This C376T polymorphism may also have high impact because active BCRP protein will not be expressed from the C376T allele. Therefore, people with C376T and/or C421A polymorphisms may express low amounts of BCRP, and this low BCRP expression might result in hypersensitivity of normal cells to such anticancer drugs as irinotecan and mitoxantrone.

ATP Binding Cassette Transporter, Subfamily G, Mem↗

PubMed2001

Human regulatory factor X 4 (RFX4) is a testis-specific dimeric DNA-binding protein that cooperates with other human RFX members.

Regulatory factor X (RFX) members are evolutionarily conserved transcription factors that share a highly conserved winged helix DNA-binding domain. Human RFX4 has been isolated from breast cancer as a partial cDNA encoding a short RFX-type DNA-binding domain fused to the estrogen receptor, but the entire structure of RFX4 has been unknown. Here, we report the molecular cloning and characterization of human RFX4. RFX4 contains evolutionarily conserved regions, including a RFX-type DNA-binding domain, a dimerization domain, and other conserved regions, and is closely related to RFX1, RFX2, and RFX3 in structure. The expression of RFX4 is restricted to testis. In vitro synthesized RFX4 protein bound to typical RFX binding sites in a sequence-dependent manner. Immunoprecipitation analyses showed that RFX4 interacts physically with RFX2, RFX3, and RFX4 itself but not with RFX1. In contrast to other mammalian RFX members that form dimers, RFX4 is revealed to have no distinct transcriptional activation domains. By using a chimeric protein of RFX1 and RFX4, the C-terminal domain of RFX4 was shown to be a possible transcriptional repression domain. Taken together, these results indicate that RFX4 is the first mammalian member of RFX family without transcriptional activation capacity and might function through selective interactions with other RFX members in transcriptional regulation.

Amino Acid Sequence↗