Recent studies have revealed that platelet-derived growth factor (PDGF) plays a role in promoting progressive tumor growth in several organs; however, whether PDGF plays such a role in gastric carcinoma is undetermined. We examined whether inhibition of PDGF receptor (PDGF-R) tyrosine kinase signaling by imatinib affects tumor growth and metastasis in an orthotopic nude mouse model of human gastric carcinoma. TMK-1 human gastric carcinoma cells were injected into the gastric wall of nude mice. Groups of mice (n 5 10 each) received sterile water (control), low-dose imatinib (50 mg/kg/day), high-dose imatinib (200 mg/kg/day), cancer chemotherapeutic agent irinotecan (5 mg/kg/week), or imatinib (50 mg/kg/day or 200 mg/kg/day) and irinotecan (5 mg/kg/week) in combination for 28 days. Tumor growth and metastasis were assessed. Resected tumors were analyzed immunohistochemically. Carcinoma-associated fibroblasts, pericytes and lymphatic endothelial cells in stroma expressed high levels of PDGF-R; carcinoma cells did not. Treatment with imatinib alone did not inhibit tumor growth and metastasis; however, treatment with irinotecan alone or combined with imatinib significantly inhibited tumor growth. Only treatment with high-dose imatinib and irinotecan in combination inhibited lymph node and peritoneal metastases. Immunohistochemically, only imatinib alone or in combination with irinotecan was shown to significantly decrease the stromal reaction, microvessel area and pericyte coverage of tumor microvessels. These effects were marked with high-dose imatinib. In conclusion, administration of PDGF-R tyrosine kinase inhibitor in combination with irinotecan appears to impair the progressive growth of gastric carcinoma by blockade of PDGF-R signaling pathways in stromal cells.Recent studies in tumor biology have shown that tumor growth and metastasis are determined not only by cancer cells, but also by a variety of stromal cells. The stroma constitutes a large part of most solid tumors, and the cancer-stromal cell interaction contributes functionally to tumor growth and metastasis.

Bone marrow-derived mesenchymal stem cells (MSCs) are reported to contribute to formation of tumor-promoting stromal cells. We reported recently that, in an orthotopic nude mice model of colon cancer, MSCs traveled to tumor stroma, where they differentiated into carcinoma-associated fibroblast (CAF)-like cells. We also found that CAFs express platelet-derived growth factor receptor (PDGFR) at a high level and that imatinib therapy targeting PDGFR in CAFs inhibits growth and metastasis of human colon cancer. These findings led us to examine whether the tumor-promoting effect of MSCs is impaired by blockade of PDGFR signaling achieved with imatinib. Orthotopic transplantation and splenic injection of human MSCs along with KM12SM human colon cancer cells, in comparison with transplantation of KM12SM cells alone, resulted in significantly greater promotion of tumor growth and liver metastasis. The KM12SM 1 MSC xenograft enhanced cell proliferation and angiogenesis and inhibited tumor cell apoptosis. When tumor-bearing animals were treated with imatinib, there was no significant increase in primary tumor volume or total volume of liver metastases, despite the KM12SM1MSC xenograft, and survival in the mixed-cell group was prolonged by imatinib treatment. Moreover, the ability of MSCs to migrate to tumor stroma was impaired, and the number of MSCs surviving in the tumor microenvironment was significantly decreased. In in vitro experiments, treatment with imatinib inhibited migration of MSCs. Our data suggest that blockade of PDGF signaling pathways influences the interaction between bone marrow-derived MSCs and tumor cells in the tumor microenvironment and, hence, inhibits the progressive growth of colon cancer.

Recent study of murine fibrosarcoma has revealed that plateletderived growth factor (PDGF) plays a direct role in promoting lymphangiogenesis and metastatic spread to lymph nodes. Thus, we investigated the relation between PDGF and PDGF receptor (PDGF-R) expression and lymphatic metastasis in human gastric carcinoma. We examined PDGF-B and PDGF-Rb expression in four human gastric carcinoma cell lines (TMK-1, MKN-1, MKN-45, and KKLS) and in 38 surgical specimens of gastric carcinoma. PDGF-B and PDGF-Rb expression was examined by immunofluorescence in surgical specimens and in human gastric carcinoma cells (TMK-1) implanted orthotopically in nude mice. Groups of mice (n = 10, each) received saline (control) or PDGF-R tyrosine kinase inhibitor imatinib. PDGF-B and PDGF-Rb mRNA expression was significantly higher in patients with lymph node metastasis than in those without and was also significantly higher in diffuse-type carcinoma than in intestinal-type carcinoma. In surgical specimens, tumor cells expressed PDGF-B, but PDGF-Rb was expressed predominantly by stromal cells. Under culture conditions, expression of PDGF-B mRNA was found in all of the gastric cell lines, albeit at different levels. In orthotopic TMK-1 tumors, cancer cells expressed PDGF-B but not PDGF-Rb. PDGF-Rb was expressed by stromal cells, including lymphatic endothelial cells. Four weeks of treatment with imatinib significantly decreased the area of lymphatic vessels. Our data indicate that secretion of PDGF-B by gastric carcinoma cells and expression of PDGF-Rb by tumor-associated stromal cells are associated with lymphatic metastasis. Blockade of PDGF-R signaling pathways may inhibit lymph node metastasis of gastric

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When analyzing the elongation mechanisms in T7 RNA polymerase (T7 RNAP)by using site-directed mutagenesis and a protein expression system, we identified the recognition sites of the rNTP 3-OH group in T7 RNAP. On the basis of three-dimensional crystal structure analysis, we selected and analyzed six candidate sites interacting with the 3-OH group of rNTP in T7 RNAP. We found that the Phe-644 and Phe-667 sites are responsible for the high selectivity of T7 RNAP for rNTPs. Also, we constructed the protein mutations of these residues, F644Y and F667Y, which display a >200-fold higher affinity than the wild type for 3-dNTPs. These findings indicate that the phenylalanine residues of 644 and 667 specifically interact with the 3-OH group. Thus, these mutants, F644Y and F667Y, with incorporation of 3-dNTP terminators, which is similar to native rNTPs, can offer low backgrounds and equal intensities of the sequencing ladders in our method, called "transcriptional sequencing." T7 RNAP1 is a single 99-kDa polypeptide containing in its catalytic domain a highly specific promoter recognition site and a nascent RNA binding domain. T7 RNAP displays a stringent specificity for T7 promoter to initiate transcription. In T7 phage infection and the lytic cycle, T7 RNAP transcribes class II and III of the T7 phage genome (1). Based on three-dimensional structure analysis and mutagenesis of the Klenow fragment, the protein critical regions and structural motifs for polymerase activity have been suggested. Additionally, from sequence homologies of the Klenow fragment, Taq DNA polymerase, and T7 DNA polymerase, structural similarities have also been suggested in motifs A (532-555 aa), B (625-652 aa), and C (805-818 aa) (2). From mutagenesis analysis, the critical sites of primer recognition, metal ion binding, rNTP binding, and polymerase activity have been determined; however these do not necessarily coincide with the homologous motifs. Phenylalanine and tyrosine have been reported to play important roles in discriminating 2Ј-and 3Ј-OH groups in Escherichia coli DNA polymerase I (3, 4) and Taq DNA polymerase (5). These findings indicate that the aromatic ring of phenylalanine or tyrosine is needed to discriminate the 2Ј-and 3Ј-OH groups. Using mutant characterization and three-dimensional structure analysis (6), the catalytic domain of T7 RNAP was separated into the T7 promoter binding region (674 -752 aa), Mg 2ϩ binding sites (Asp-537, Asp-812), and essential regions of polymerase activity (motif A, motif B, motif C). To analyze the mechanism of the polymerase reaction, the interaction sites for the 2Ј-and 3Ј-OH group have been extensively examined (3-5).Recently, Tyr-639 in the active site has been reported to be a discrimination site for 2Ј-OH groups in T7 RNAP (7). The Y639F mutant retains DNA and RNA polymerase activities but cannot discriminate rNTP from 2Ј-dNTPs. To further determine the mechanisms of the polymerase reaction, the 3Ј-OH discrimination site in the elongation step needed to be identified. However, details of the ...

A 53-year-old man was referred to our hospital with bloody stool. Barium enema study and colonoscopy revealed multiple small nodules on the anterior wall of the lower rectum. Biopsy specimens showed proliferation of atypical lymphoid cells forming the nodules. Mucosa-associated lymphoid tissue lymphoma was diagnosed on the basis of histologic and immunohistochemical examinations. No metastasis was detected in lymph nodes or distant organs, indicative of clinical stage I disease. Although the test results were negative for Helicobacter pylori, eradication therapy was performed. The lesion disappeared completely within 9 months after the triple antibiotic therapy. H. pylori eradication therapy may be a useful treatment option regardless of H. pylori status.