Masaaki Terada

NEW DELIVERY SYSTEM FOR PLASMID DNA IN VIVO USING ATELOCOLLAGEN AS A CARRIER MATERIAL : THE MINIPELLET

New delivery system for plasmid DNA in vivo using atelocollagen as a carrier material: the Minipellet

MST/MLK2, a Member of the Mixed Lineage Kinase Family, Directly Phosphorylates and Activates SEK1, an Activator of c-Jun N-terminal Kinase/Stress-activated Protein Kinase

c-Jun N-terminal kinases/stress-activated protein kinases (JNKs/SAPKs) are mitogen-activated protein kinase (MAPK)-related protein kinases that are involved in several cellular events, including growth, differentiation, and apoptosis. Mixed lineage kinases (MLKs) form a family of protein kinases sharing two leucine zipper-like motifs and a kinase domain whose primary structure is similar to both the tyrosine-specific and the serine/threonine-specific kinase classes. We have reported that a member of the MLK family, MUK/DLK/ZPK, can activate JNK/SAPK in vivo, and here we show that another member of the MLK family, MST/MLK2, activates JNK/SAPK. Both MUK/DLK/ZPK and MST/MLK2 cause a slight activation of p38/Mpk2 when overexpressed in COS-1 cells, whereas MST/MLK2, but not MUK/DLK/ZPK, activates extracellular response kinase (ERK) to a certain degree. The activity of SEK1/MKK4/JNKK, a MAPK kinase class protein kinase designated as a direct activator of JNK/SAPK, is also induced by MUK/DLK/ZPK or MST/MLK2 overexpression. Furthermore, recombinant MST/MLK2 produced in bacteria directly phosphorylates and activates SEK1/MKK4/JNKK in vitro, showing that MST/MLK2 acts like a MAPK kinase kinase. Taken together, these results suggest that MLK family members are MAPK kinase kinases preferentially acting on the JNK/SAPK pathway.

Gastric cancers of the microsatellite mutator phenotype display characteristic genetic and clinical features

BACKGROUND & AIMS Colon cancer of the microsatellite mutator phenotype (MMP) exhibits significant genotype differences from cancer without the MMP. Twenty-nine MMP-positive gastric cancers were analyzed to clarify if these genotype differences were also associated with distinctive clinicopathologic features. METHODS Alterations of p53, beta2-microglobulin (beta2M ), hMLH1, and hMSH2 genes were analyzed by using polymerase chain reaction, single-strand conformational polymorphism, sequencing, microallelotyping, hypermethylation assays, and immunostaining. The results were contrasted with mutations in BAX, hMSH3, and hMSH6, genes target for the MMP. RESULTS Tumors with the MMP had a significantly lower incidence of p53 gene mutations than the other tumors and often contained beta2M gene somatic mutations. Many tumors contained concomitant genetic and epigenetic alterations in DNA mismatch repair genes, hMLH1, hMSH2, hMSH3, and hMSH6. Gastric cancer of the MMP was associated with well/moderate differentiation, distal location, and better survival. CONCLUSIONS Analysis of somatic alterations in microsatellite sequences and in cancer genes target for the MMP is useful for the classification of groups of gastric cancers with different prognosis. The results further support the concept that (gastric) cancer of the MMP represents a distinctive oncogenic pathway because the mutated cancer genes are usually different from those found in tumors without the MMP.

Helicobacter pylori Infection, Serum Pepsinogen Level and Gastric Cancer: A Case‐Control Study in Japan

We conducted a case‐control study to evaluate the effect of Helicobacter pylori (HP) infection on the risk of gastric cancer in Tokyo, Japan. The sera at the time of diagnosis from 282 gastric cancer cases and 767 sex‐ and age‐matched cancer‐free controls were tested for the presence of anti‐HP IgG antibody (HM‐CAP ELISA kit) and serum pepsinogen (PG) level (PG I and PG II Riabead). No significant association was observed in all sets [matched odds ratio (OR)=1.04, 95% confidence interval: 0.73–1.49]. In subgroup analyses, however, an association was suggested in females [OR=1.57], a younger population (<50 years) [OR=1.86], early cancer [OR=1.53] and small cancer (<40 mm) [OR=1.55]. Furthermore, we observed a tendency for odds ratios to decrease with an increase in age or cancer growth (depth of tumor invasion and tumor size). Considering that the spontaneous disappearance of HP due to extended mucosal atrophy may lead to these decreasing odds ratios, we applied the conditional logistic model adjusted for the PG I/II ratio as a measure of atrophic gastritis. This analysis showed a positive association with HP infection in all sets [OR=1.69; 1.01–2.81], distal cancer [OR=1.88; 1.07–3.31] and intestinal‐type cancer [OR=3.76; 1.39–10.18]. We concluded that the risk of cancer associated with HP infection may be underestimated in studies with cross‐sectional exposure because of spontaneous disappearance of HP due to extended mucosal atrophy.

A faithful method for PCR-mediated global mRNA amplification and its integration into microarray analysis on laser-captured cells.

Quantitative and qualitative analyses of mRNAs from a small number of cells are extremely important for studies on gene expression in various physiological and pathological conditions in multicellular organisms. We present here an effective method for high-fidelity global mRNA amplification for in vivo gene expression profiling of as few as 100 cells obtained by laser-captured microdissection (LCM). This method, called TALPAT, is based on T7 RNA polymerase-mediated transcription, adaptor ligation, and PCR amplification followed by T7-transcription. More than 80% of genes were commonly identified as a more than 3-fold changed gene among three gastric cancer cell lines using cRNA amplified by both TALPAT and the ordinary in vitro T7-transcription. The reproducibility of TALPAT was validated by microarray analysis on 100 breast cancer cells obtained by LCM. For the application of the LCM-TALPAT method, we successfully obtained expression profiles of gastric cancer cells and the mesenchymal cells, enabling us to understand in vivo cell-to-cell cross-talk in the microenvironment.

Elevated serum level of thioredoxin in patients with hepatocellular carcinoma.

Thioredoxin (TRX) is known to contain an active site with aredox-active disulfide and has various biological activities. The objectiveof the present study was to investigate whether circulating TRX levels areelevated in patients with chronic hepatitis (CH) or liver cirrhosis (LC) andhepatocellular carcinoma (HCC). An anti-TRX monoclonal antibody andpolyclonal antibodies that specifically recognize TRX, were generated andused for the development of an ELlSA system to measure TRX levels in humanserum. The geometric mean and its 95% confidence interval of serumlevel of TRX in healthy volunteers was 81.75 ng/ml (74.60-89.59 ng/ml). Theserum level of TRX in LC/CH patients without HCC was 80.87 ng/ml(69.66-93.88 ng/ml). The value was not statistically different from that inserum from normal volunteers (p=0.69). In contrast, the serum level of TRXin patients with HCC was 147.35 ng/ml (125.53-1 72.96 ng/ml), which wassignificantly higher when compared with the level in serum of normalvolunteers (p<0.001) and in serum of LC/CH patients without HCC(p<0.001). In four patients with HCC, the initially high level of serum TRX(>150 ng/ml) decreased below 150 ng/ml after surgical removal of the tumor.The data reported herein revealed that patients with HCC had a significantlyelevated serum level of TRX, suggesting that measurement of serum of TRXmight be a useful clinical parameter when HCC is suspected.

Suppression of Ki-ras p21 levels leading to growth inhibition of pancreatic cancer cell lines with Ki-ras mutation but not those without Ki-ras mutation.

Ki‐ras point mutation characteristically occurs frequently in human pancreatic cancer. To clarify the effect of antisense Ki‐ras RNA on various pancreatic cancer cell lines, a plasmid expressing an antisense Ki‐ras gene fragment (AS‐K‐ras‐LNSX) was transduced into seven human pancreatic cancer cell lines (AsPC‐1, MIA PaCa‐2, PANC‐1, PSN‐1, BxPC‐3, Hs 700T, and Hs 766T) by liposome‐mediated transfection. Western blot analysis showed that transfection of AS‐K‐ras‐LNSX led to a significant reduction in the amounts of Ki‐ras p21 protein in all the pancreatic cancer cell lines except BxPC‐3. The growth of pancreatic cancer cell lines having Ki‐ras point mutations (AsPC‐1, MIA PaCa‐2, PANC‐1, and PSN‐1) was suppressed after transduction of AS‐K‐ras‐LNSX, whereas the effect of the antisense construct on the growth was not significant in cell lines with a wild‐type Ki‐ras gene (BxPC‐3, Hs 700T, and Hs 766T). These results suggest that the pancreatic cancer cells with activated Ki‐ras depend heavily on a Ki‐ras p21–mediated growth signal pathway for their growth because they were far more susceptible to the suppression of the Ki‐ras p21 protein than the cells with wild‐type Ki‐ras. The remarkably increased dependence of the cancer‐cell growth circuitry on one or a few crucial regulatory molecules may thus be a common feature of the cancer cells and implies a novel rationale for the targeting of cancer therapy. Mol. Carcinog. 20:251–258, 1997.

Gene amplification profiling of esophageal squamous cell carcinomas by DNA array CGH

Gene amplification is one of the basic mechanisms that lead to overexpression of oncogenes. DNA array comparative genomic hybridization (CGH) has great potential for comprehensive analysis of both a relative gene-copy number and altered chromosomal regions in cancers, which enables us to identify new amplified genes and unstable chromosomal loci. We examined the amplification status in 32 esophageal squamous cell carcinomas (ESCCs) and 13 ESCC cell lines on 51 frequently amplified loci in a variety of cancers by both DNA array CGH and Southern blot analyses. The 1p34 locus containing MYCL1, 2p24 (MYCN), 7p12 (EGFR), and 12q14 (MDM2) were amplified in one of the 32 cases (3%), and the 17q12 locus (ERBB2) and 8p11 (FGFR1) in two of the 32 cases (6%), while only the 11q13 locus (Cyclin D1, FGF4, and EMS1) was frequently amplified (28%, 9/32), demonstrating this locus to be a major target in ESCCs. One locus, 8q24 (c-MYC) was found to be amplified only in the cell lines. Eight out of 51 loci (15.7%) were found to be amplified in at least one of the 32 primary ESCCs or the 13 ESCC cell lines, suggesting that chromosomal loci frequently amplified in a type of human cancer may also be amplified in other types of cancers. This paper is the first report of an application of DNA array CGH to ESCCs.

Tumor‐infiltrating endothelial cells and endothelial precursor cells in inflammatory breast cancer

Inflammatory breast cancer (IBC) is a specific type of breast tumor that generally has a poor prognosis, in spite of recent advances in treatment. In the present study, semiquantitative reverse transcriptase polymerase chain reaction examination of resected specimens showed that angiogenic factors, not lymphangiogenic factors, are overexpressed in IBC tumors, compared with non‐IBC tumors. Immunohistochemical analysis of the specimens revealed a significantly higher population of tumor‐infiltrating (TI) endothelial cells (ECs) or endothelial precursor cells (EPCs) in tumor‐associated stroma of IBC specimens than in non‐IBC specimens. In a previous study, we examined the phenotype of host cells in response to transplanted IBC cells, using an established human IBC xenograft model (WIBC‐9) (Shirakawa et al., Cancer Res 2001;61:445–51). The data obtained in that study are consistent with the findings of the present study. To explore the therapeutic potential of blocking vascular endothelial growth factor (VEGF) and angiopoietin (Ang) pathways in IBC, established vectors encoding soluble Flt‐1 (sFlt‐1) and soluble Tie2 (sTie2) were injected directly into WIBC‐9. Both vectors produced growth inhibition ratios of WIBC‐9 that were significantly higher than those of a non‐IBC xenograft (MC‐5). Also, both vectors suppressed WIBC‐9 lung metastases. The efficacy correlated with the number of TI ECs/EPCs, which was determined by fluorescence‐activated cell sorting. These ECs/EPCs incorporated acetylated lipoprotein and were integrated within a HUVEC monolayer in vitro culture on day 5.

Cloning of human bone morphogenetic protein type IB receptor (BMPR- IB) and its expression in prostate cancer in comparison with other BMPRs

Bone metastasis is a common event in prostate cancer, and it is known that some of the bone morphogenetic proteins (BMPs) are expressed in prostate cancer cells, while no study on the expression of their receptors, BMPRs, has been reported. Here we report cloning and sequence analysis of the human BMPR-IB cDNA. We also analysed the expression of transcripts of three types of the BMPR genes in human tissues and prostate cancer cell lines. The BMPR-IB mRNA was present in various organs, but the highest level was found in the prostate. Moreover, the amount of BMPR-IB mRNA was significantly low in prostate cancer tissues after androgen withdrawal and was also low in prostate cancer cell lines. RT - PCR analysis showed that the BMPR-IB message was upregulated by androgen stimulation in the LNCaP cell line which expresses the androgen receptor. By contrast, the mRNA levels of BMPR-IA and BMPR-II were not significantly different among non-cancerous and cancerous prostate tissues. It was also suggested that human BMPR-IA and BMPR-IB might have different biological functions in the prostate, although their sequences were 85.3% identical in the serine-threonine kinase domain.