Yohei Hamaguchi

Delineating developmental and metabolic pathways in vivo by expression profiling using the RIKEN set of 18,816 full-length enriched mouse cDNA arrays

We have systematically characterized gene expression patterns in 49 adult and embryonic mouse tissues by using cDNA microarrays with 18,816 mouse cDNAs. Cluster analysis defined sets of genes that were expressed ubiquitously or in similar groups of tissues such as digestive organs and muscle. Clustering of expression profiles was observed in embryonic brain, postnatal cerebellum, and adult olfactory bulb, reflecting similarities in neurogenesis and remodeling. Finally, clustering genes coding for known enzymes into 78 metabolic pathways revealed a surprising coordination of expression within each pathway among different tissues. On the other hand, a more detailed examination of glycolysis revealed tissue-specific differences in profiles of key regulatory enzymes. Thus, by surveying global gene expression by using microarrays with a large number of elements, we provide insights into the commonality and diversity of pathways responsible for the development and maintenance of the mammalian body plan.

MMP-7 (matrilysin) accelerated growth of human umbilical vein endothelial cells

Matrix metalloproteinases (MMP) are considered to play important roles in angiogenesis. In angiogenic processes, endothelial cells secrete MMP-2 or MMP-1 to dissolve the basement membrane or connective tissue around the vessels. MMP-7 (matrilysin) is secreted from the neovasculars induced by cancer and is a metastatic factor of colorectal cancer. The effect of matrilysin on angiogenesis is still unclear, however. We therefore examined the effect of MMP-7 on the proliferation of human umbilical vein endothelial cells (HUVECs) in vitro. Our results showed that recombinant MMP-7 (rMMP-7) accelerated the proliferation of endothelial cells dose-dependently, and did so for endothelial cells cultured not only on type IV collagen, but also on type I collagen. MMP-7 also upregulated MMP-1, -2 secretion, but did not stimulate vascular endothelial growth factor (VEGF) secretion. From this study, we conclude that MMP-7 directly induces angiogenesis, and that therefore MMP-7 would be a good target of cancer therapy.

Analysis of Gene Expression Involved in Brain Metastasis from Breast Cancer Using cDNA Microarray

BackgroundBrain metastases occur in 15% to 30% of breast cancer patients, usually as a late event. The patterns of metastases to different organs are determined by the tumor cell phenotype and interactions between the tumor cells and the organ environment.MethodsWe investigated the gene expression profile occurring in brain metastases from a breast cancer cell line. We used cDNA microarrays to compare patterns of gene expression between the mouse breast cancer cell line Jyg MC (A) and a subline that often metastasis to brain, (B).ResultsBy Microarray analysis about 350 of 21,000 genes were significantly up-regulated in Jyg MC (B). Many candidate genes that may be associated with the establishment of brain metastasis from breast cancer were included. Interestingly, we found that the expression of astrocyte derived cytokine receptors (IL-6 receptor, TGF-beta receptor and IGF receptor) were significantly increased in Jyg MC (B) cells. These results were confirmed by RT-PCR.ConclusionThese results suggest that cytokines produced by glial cellsin vivo may contribute, in a paracrine manner, to the development of brain metastases from breast cancer cells.

Inhibition of osteoclast differentiation and bone resorption by cathepsin K antisense oligonucleotides

We confirmed the expression of cathepsin K, the most abundant and specific cysteine protease found in osteoclasts, at the mRNA level in most of our cases of breast cancer, and even at the protein level in bone metastatic lesions. Therefore, we investigated the functions of cathepsin K in osteoclasts with special attention to bone metastasis from breast cancer. Mouse osteoclast‐like cells (OCLs) were established by coculture of mouse bone marrow cells and osteoblastic cells. Rodent cathepsin K antisense (AS) or random control (CL) oligonucleotides were added on day 0, 3, or 6 of culture. Tartrate‐resistant acid phosphatase staining confirmed the formation of OCLs after 9 d of incubation. AS treatment significantly reduced both the number of TRAP‐positive cells and the percentage of multinuclear cells. For the pit‐forming assay, after 9 d of incubation, mature OCLs were collected and incubated on ivory slices with AS or CL for 48 h. The antisense oligonucleotides also inhibited the bone‐resorbing activity of OCLs. CL treatment did not affect either the number of TRAP‐positive cells or pit formation. Cathepsin K may play important roles in bone resorption as well as in differentiation of osteoclasts. These findings indicate that the inhibition of this enzyme may prevent the development of bone metastasis from breast cancer.

VEGF receptor antisense therapy inhibits angiogenesis and peritoneal dissemination of human gastric cancer in nude mice.

The efficacy of a phosphorothioate antisense oligonucleotide (ASO) for KDR/Flk-1 (KDR/Flk-1-ASO), an endothelial cell–specific vascular endothelial growth factor (VEGF) receptor, was investigated on the peritoneal dissemination and angiogenesis of a human gastric cancer cell line in nude mice. Green fluorescent protein (GFP)–transduced NUGC-4 (NUGC-4-GFP) human gastric cancer cells were implanted into the peritoneal cavity of nude mice. KDR/Flk-1-ASO, -SO, or phosphate-buffered saline was administrated from days 7 to 14, 200 μg/mouse, once a day. The mice were sacrificed on day 28. Disseminated peritoneal tumor nodules expressing GFP were visualized by fluorescence microscopy. KDR/Flk-1-ASO significantly decreased the extent of peritoneal dissemination of the tumors. The number of cells undergoing apoptosis was significantly increased in the KDR/Flk-1-ASO–treated tumors. Microvessel density was significantly reduced in the KDR/Flk-1-ASO–treated tumor nodules. The KDR/Flk-1 antisense strategy, therefore, decreases tumor dissemination apparently by inhibiting angiogenesis.

Telomerase enzyme activity and RNA expression in adriamycin-resistant human breast carcinoma MCF-7 cells

Telomerase activity has been reported in cancer cells after treatment with antineoplastic agents. Assessment of telomerase activity could be a valuable tool to measure the reduction of aggression caused by chemotherapy. This study was designed to investigate the significance of telomerase for chemotherapy with respect to Adriamycin (ADM)-resistance. MCF-7 and its ADM-resistant line (AdrR) were treated with ADM, 5-fluorouracil (5FU) or taxotere (TAXO). Telomerase activity and human telomerase RNA component (hTR) were quantitatively measured by the telomeric repeat amplification protocol assay and RT-PCR, respectively. Cell counting and MTT assay were also performed. In MCF-7, enzyme activity was significantly reduced by ADM and 5FU treatments. In AdrR, 5FU and TAXO reduced enzyme activity, while ADM significantly increased the activity. No significant changes in hTR were seen in these two cell lines after treatment with any of these drugs. When Bcl-2 expression was examined after drug treatments, ADM increased Bcl-2 expression in AdrR cells, while not changing it in MCF-7 cells. We conclude that an unusual reaction of telomerase activity in AdrR may explain, at least in part, one of the mechanisms of the malignant biological behavior related with the drug-resistance to ADM.

Evaluation of dynamic studies of MR mammography for the diagnosis of intraductal lesions with nipple discharge.

ObjectivesWe assessed the utility of dynamic magnetic resonance imaging (MRI) in differentiating benign from malignant lesions of the breast and then applied MRI to diagnose intraductal breast tumors with nipple discharge.MethodsGadolinium (Gd)-enhanced MR mammography was performed on 74 patients with breast tumors and 8 patients with nipple discharge.ResultsThe steepest slopes of the contrast medium uptake (S slope) s from time-intensity curves were significantly different between malignant and benign lesions. At S slope threshold of 0.95%/second, malignancy was predicted with a sensitivity and specificity of 75%. Six of 8 cases with nipple discharge were successfully identified by MR ductography by injecting Gd-DTPA into discharging ducts. Among them, 2 non-invasive ductal carcinomas were differentiated from benign lesions by the S slope value.ConclusionsDynamic MR mammography is an useful modality for differentiating breast lesions and has potential for evaluating intraductal lesions with nipple discharge.

A Multicenter Phase II Study of Docetaxel 60 mg/m2 as First-Line Chemotherapy in Patients with Advanced or Recurrent Breast Cancer

PurposeDocetaxel is an active agent as first-line chemotherapy in patients with advanced breast cancer at a dosage of 100 mg/m2. However, the efficacy of this agent as a first-line drug when used at a lower dosage is unclear. This study was performed to evaluate the clinical efficacy and safety of 60 mg/m2 docetaxel for the treatment of breast cancer.Patients and MethodsThis study enrolled 23 patients with advanced and/or metastatic breast cancer, who had not been treated with an anthracycline or taxane previously. Treatment with docetaxel was continued in patients showing a response until there was evidence of disease progression or unacceptable toxicity.ResultsAmong 20 fully evaluated patients, the overall response rate was 50.0% and the median time to progression was 31 weeks. The most commonly observed adverse events were neutropenia (78.2%) and fatigue (60.9%). Fluid retention occurred in only 8.7% of the patients. Adverse events did not cause discontinuation of the treatment.ConclusionDocetaxel achieved good disease control with mild adverse events in first-line treatment at a dosage of 60 mg/m2.