Expression of tissue factor and vascular endothelial growth factor is associated with angiogenesis in colorectal cancer

We examined the expression of tissue factor (TF) and vascular endothelial growth factor (VEGF) and the microvessel density (MVD) in 100 patients with colorectal cancer, and we investigated the relationship of the expression of TF or VEGF with angiogenesis. TF antigen was positive in 57.0% of all specimens. Incidence of TF expression was 41.2%, 45.5%, 52.6%, 84.6%, and 81.3% in tumors from patients in clinical stages I, II, IIIA, IIIB, and IV, respectively. TF expression was correlated with the Dukes' classification (P = 0.01) and the clinical stage of colorectal cancer (P = 0.02). VEGF antigen was positive in 64.0% of all specimens. Incidence of VEGF expression was 41.2%, 57.6%, 73.7%, 84.6%, and 75.0% in tumors from patients in clinical stages I, II, IIIA, IIIB, and IV, respectively. VEGF expression was correlated with the Dukes' classification (P = 0.01) but showed a weak association with the clinical stage (P = 0.08). MVD was significantly associated with the depth of invasion (P = 0.01), lymph node metastasis (P = 0.001), and liver metastasis (P = 0.02). The mean values of MVD were 7.5 +/- 2.8, 10.1 +/- 5.7, 14.6 +/- 5.8, 13.5 +/- 3.9, and 15.9 +/- 4.2 in tumors from patients in clinical stages I, II, IIIA, IIIB, and IV, respectively. A close relationship between VEGF and MVD (P < 0.001) and a significant correlation between TF expression and MVD were observed (P = 0.02). TF-positive carcinomas presented high MVD and VEGF expression (P < 0.001) more frequently than did TF-negative tumors. These results suggest that involvement of TF in the process of metastasis and progression of colorectal cancer may depend on increased angiogenesis.     

DNA vaccine

The study of DNA vaccine has been started after the initial report showing that plasmids encoding a reporter gene are able to induce in vivo expression of the encoded protein. While some of DNA vaccines have already been tested in clinical trials, a lot of new methods and technologies to augment the immunogenicity of DNA vaccines are under evaluation in preclinical studies. We examined two approaches incorporating helper antigens or co-stimulatory molecules in CTL antigen and showed the enhancement of tumor antigen-specific CD8+ T cell induction and in vivo tumor rejection capacity. These data and the recent approaches may provide us an important hint for establishing new therapeutic vaccines.     

IL-3 can not replace GM-CSF in inducing human monocytes to differentiate into Langerhans cells

Receptors for granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3) share a common beta subunit. We recently reported that GM-CSF acts in concert with transforming growth factor-beta1 (TGF-beta1) and Notch ligand Delta-1 (Delta-1) to promote the differentiation of human blood monocytes into Langerhans cells. In the present study, we examined whether IL-3, in place of GM-CSF, can induce the development of Langerhans cells from blood monocytes in the presence of TGF-beta1 and Delta-1, because the IL-3 receptor alpha chain was substantially expressed on monocytes. However, the generation of Langerhans cells was not obtained by the combination of IL-3, TGF-beta1 and Delta-1, even though GM-CSF and IL-3 exhibited a similar effect with respect to the differentiation of monocytes into macrophages and dendritic cells. The addition of GM-CSF to the culture supplemented with IL-3, TGF-beta1 and Delta-1 restored the differentiation of monocytes toward Langerhans cells. A microarray analysis revealed that a number of genes including Langerhans cell markers, E-cadherin and Langerin, were specifically expressed in cells from GM-CSF-containing cultures but not in those from IL-3-containing cultures. These data suggest that IL-3 can not replace GM-CSF to induce the differentiation of human monocytes into Langerhans cells in culture.     

Three‐dimensional co‐culture of C2C12/PC12 cells improves skeletal muscle tissue formation and function

Engineered muscle tissues demonstrate properties far from native muscle tissue. Therefore, fabrication of muscle tissues with enhanced functionalities is required to enable their use in various applications. To improve the formation of mature muscle tissues with higher functionalities, we co‐cultured C2C12 myoblasts and PC12 neural cells. While alignment of the myoblasts was obtained by culturing the cells in micropatterned methacrylated gelatin (GelMA) hydrogels, we studied the effects of the neural cells (PC12) on the formation and maturation of muscle tissues. Myoblasts cultured in the presence of neural cells showed improved differentiation, with enhanced myotube formation. Myotube alignment, length and coverage area were increased. In addition, the mRNA expression of muscle differentiation markers (Myf‐5, myogenin, Mefc2, MLP), muscle maturation markers (MHC‐IId/x, MHC‐IIa, MHC‐IIb, MHC‐pn, α‐actinin, sarcomeric actinin) and the neuromuscular markers (AChE, AChR‐ε) were also upregulated. All these observations were amplified after further muscle tissue maturation under electrical stimulation. Our data suggest a synergistic effect on the C2C12 differentiation induced by PC12 cells, which could be useful for creating improved muscle tissue. Copyright © 2014 John Wiley & Sons, Ltd.     

Expression of hepatocyte growth factor(hgf) and C-met gene in human gastric-cancer cell-lines

Tissue P-glycoprotein immunostaining was performed in seventy one patients with follicular large, mixed, or small cell non-Hodgkin's lymphomas. Only six patients (8.5%) demonstrated positive immunostaining for P-glycoprotein. All patients had complete and durable responses to initial therapy. P-glycoprotein (MDR) fails to predict for primary drug resistance in follicular lymphomas and is constitutionally expressed in only a few follicular lymphomas at present.     

A stomach oncofetal antigen recognized by monoclonal antibody GC302

A monoclonal antibody, GC302, was established by fusing murine myeloma NS/1 cells with the splenocytes of a BALB/c mouse immunized with a human gastric cancer cell line, NU-GC-3. The serological specificity of GC302 was analyzed by an anti-mouse Ig mixed-hemadsorption (MHA) test on a panel of human cell lines, and an immunoperoxidase method using the frozen sections of tumors and normal tissues of adult and fetus. GC302 reacted with cancers of the stomach and colorectum but did not react with hepatocellular carcinomas, melanomas, or astrocytomas in the MHA tests. By the immunoperoxidase method, GC302 was found not to react with normal adult gastric mucosa, but to react with the mucosa in the fetal stomach, intestinal metaplasia, and almost all of the cancer of the stomach. GC302 also reacted with the normal mucosa of the intestine, colon, and rectum as well as with cancers of these origins. In normal liver sections, the antibody reacted with the bile ducts, but not with the hepatic cells. These results indicate that the antigen detected by GC302 is characterized as an oncofetal antigen in the stomach, and also as a differentiation antigen whose localization discriminates between the gastrointestinal tracts of the forgut origin and those of the midgut and hindgut origin. The molecular weight of the GC302 antigen was estimated to beca. 40,000 by the Western blot analysis. Periodic acid treatment on the antigen suggested that the antigenic determinant is a carbohydrate.     

mRNA expression of leukemia-associated antigens in patients with acute myeloid leukemia for the development of specific immunotherapies

Specific immunotherapies for patients with acute myeloid leukemia (AML) using leukemia-associated antigens (LAA) as target structures might be a therapeutic option to enhance the graft-vs.-leukemia effect observed after allogeneic stem cell transplantation or to prolong a complete remission (CR) achieved by chemotherapy. Significant mRNA expression of LAA is a prerequisite for such immunotherapies. Here, previously characterized antigens associated with solid tumors (TAA) and newly characterized LAA were investigated for their expression in up to 60 AML patients and in leukemia cell lines. To investigate their specificity for leukemic blasts, the mRNA expression was also characterized in PBMN and CD34 positive cells of healthy volunteers and in a panel of normal tissues. The following antigens showed high mRNA expression in AML patients: MPP11 was detected in 43/50 (86%), RHAMM in 35/50 (70%), WT1 in 40/60 (67%), PRAME in 32/50 (64%), G250 in 18/35 (51%), hTERT in 7/25 (28%) and BAGE in 8/30 (27%) of AML patients. Real-time RT-PCR showed a tumor-specific expression of the antigens BAGE, G250 and hTERT, as well as highly tumor-restricted expression for RHAMM, PRAME and WT1. The antigen MPP11 was overexpressed. These antigens might be candidates for immunotherapies of leukemia patients and, because of their simultaneous expression, also for polyvalent vaccines.     

Bax Induction Activates Apoptotic Cascade via Mitochondrial Cytochrome c Release and Bax Overexpression Enhances Apoptosis Induced by Chemotherapeutic Agents in DLD-1 Colon Cancer Cells

Cancer cells express different levels of apoptosis-promoting Bax protein. The present study evaluated whether induction of Bax initiates apoptosis and whether Bax overexpression enhances apoptosis induced by several chemotherapeutic agents in DLD-1 colon cancer cells, which originally express a high level of endogenous Bax protein and a low level of Bcl-2 protein. To investigate these two points, parental DLD-1 cells were transfected with the Tet-On Bax induction system ( pTet-On and pTRE-Bax plasmids), and stable transduced cells were obtained. Induction of Bax by the Tet-On system initiated cytochrome c release from mitochondria, caspase-3 activation, and apoptosis to some extent in DLD-1 cells. Apoptosis induced by a chemotherapeutic agent, 5-fluorouracil, mitomycin C, paclitaxel, doxorubicin, or cisplatin, was enhanced by Bax overexpression. These findings suggest that Bax -overexpression-based gene therapy combined with chemotherapy would be effective in the treatment of colon cancer.     

Bcl-2 in cell-cycle regulation of hematopoietic cells by transforming growth factor-beta1

We reported that several growth factors regulate the doubling time of hematopoietic progenitor cells by modulating the time required to pass through the G1 phase. As recent studies revealed the link between cell death and cell-cycle progression, we asked if cell death regulators such as Bcl-2 play a role in regulating the cell-cycle of hematopoietic cells by growth factors. Among growth factors, transforming growth factor-beta1 (TGF-beta1), a negative regulator of hematopoiesis, was chosen. When a large number of cells was required for analysis, we used IL-3-dependent Ba/F3 cells instead of primary hematopoietic progenitor cells because the response of Ba/F3 cells to TGF-beta1 was similar to that of primary hematopoietic progenitor cells. TGF-beta1 decelerated the cell-cycling of hematopoietic cells by inducing a delay in G1 to S phase transition, an event associated with increase in the level of Bcl-2 as well as p27, a cyclin/cyclin-dependent kinase inhibitor. In experiments using Ba/F3 cells with the potential to produce Bcl-2 in an inducible manner, Bcl-2 apparently functions upstream of p27. The effects of TGF-beta1 on Bcl-2 and p27 expression as well as cell growth were abrogated by c-kit ligand. These findings suggest that Bcl-2 plays a crucial role in regulating the cell-cycle of hematopoietic progenitor cells.