Appropriate resources and expression technology necessary for human proteomics on a whole-proteome scale are being developed. We prepared a foundation for simple and efficient production of human proteins using the versatile Gateway vector system. We generated 33,275 human Gateway entry clones for protein synthesis, developed mRNA expression protocols for them and improved the wheat germ cell-free protein synthesis system. We applied this protein expression system to the in vitro expression of 13,364 human proteins and assessed their biological activity in two functional categories. Of the 75 tested phosphatases, 58 (77%) showed biological activity. Several cytokines containing disulfide bonds were produced in an active form in a nonreducing wheat germ cell-free expression system. We also manufactured protein microarrays by direct printing of unpurified in vitro-synthesized proteins and demonstrated their utility. Our 'human protein factory' infrastructure includes the resources and expression technology for in vitro proteome research.

Constitutive nuclear factor (NF)-kB activation is thought to be involved in survival, invasion, and metastasis in various types of cancers. However, neither the subtypes of breast cancer cells with constitutive NF-kB activation nor the molecular mechanisms leading to its constitutive activation have been clearly defined. Here, we quantitatively analyzed basal NF-kB activity in 35 human breast cancer cell lines and found that most of the cell lines with high constitutive NF-kB activation were categorized in the estrogen receptor negative, progesterone receptor negative, ERBB2 negative basal-like subtype, which is the most malignant form of breast cancer. B reast cancer is a disease of the mammary epithelium, which is composed of two major types of differentiated cells: luminal epithelial cells and basal or myoepithelial cells.(1) Recent studies have identified self-renewing pluripotent stem cells in mammary epithelium and suggest a model in which these stem cells could differentiate into the luminal-or basal-restricted lineages. Molecular taxonomic analyses of breast cancers by gene expression profiling have identified five breast cancer subtypes: luminal A, luminal B, basal-like, ERBB2-positive, and normal breast-like.(2) This classification is closely associated with the differentiation model of mammary epithelium. Luminal-and basallike breast cancer subtypes express genes characteristic of the two distinct types of epithelial cells. These subtypes show different clinical courses and responses to therapeutic agents. The basallike subtype has been associated with aggressive behavior and poor prognosis and typically does not express estrogen receptor (ER), progesterone receptor (PR), or ERBB2 ("triple-negative" phenotype).(3) Therefore, patients with basal-like subtype are unlikely to benefit from currently available targeted therapeutic strategies, such as hormone therapy and Herceptin (Roche, Basel, Switzerland). It is thus crucial to identify effective molecular targets for this subtype of breast cancer.Nuclear factor (NF)-κB transcription factors are important regulators of the genes necessary for innate and adaptive immune responses and for the survival and proliferation of certain cell types. The NF-κB family is composed of five different proteins, including RelA, RelB, c-Rel, and the precursor and processed products of the NFKB1 (p105/p50) and NFKB2 (p100/p52) genes.These proteins homodimerize and/or heterodimerize to form active transcription factors. Two distinct NF-κB pathways have been proposed: the classical pathway, which activates the RelA-p50 complex, and the alternative pathway, which activates the RelBp52 complex.(4) In normal cells, activation of the classical and alternative pathways is tightly regulated by inhibitor of NF-κB (IκB) family proteins and a p100 protein, respectively. Both NF-κB pathways are aberrantly activated and involved in tumor development in various cancers, including breast cancer.(5,6) Previous studies have revealed that hormone-independent breast cancer cells exhibit cons...

ErbB2-negative breast tumors represent a significant therapeutic hurdle because of a lack of effective molecular targets. Although NOTCH proteins are known to be involved in mammary tumorigenesis, the functional significance of these proteins in ErbB2-negative breast tumors is not clear. In the present study, we examined the expression of activated NOTCH receptors in human breast cancer cell lines, including ErbB2-negative and ErbB2-positive cell lines. Activated NOTCH1 and NOTCH3 proteins generated by ;-secretase were detected in most of the cell lines tested, and both proteins activated CSL-mediated transcription. Down-regulation of NOTCH1 by RNA interference had little or no suppressive effect on the proliferation of either ErbB2-positive or ErbB2-negative cell lines. In contrast, down-regulation of NOTCH3 significantly suppressed proliferation and promoted apoptosis of the ErbB2-negative tumor cell lines. Down-regulation of NOTCH3 did not have a significant effect on the ErbB2-positive tumor cell lines. Down-regulation of CSL also suppressed the proliferation of ErbB2-negative breast tumor cell lines, indicating that the NOTCH-CSL signaling axis is involved in cell proliferation. Finally, NOTCH3 gene amplification was detected in a breast tumor cell line and one breast cancer tissue specimen even though the frequency of NOTCH3 gene amplification was low (<1%). Taken together, these findings indicate that NOTCH3-mediated signaling rather than NOTCH1-mediated signaling plays an important role in the proliferation of ErbB2-negative breast tumor cells and that targeted suppression of this signaling pathway may be a promising strategy for the treatment of ErbB2-negative breast cancers.

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To determine whether methylation of the hMLH1 promoter is related to increasing age and gastric carcinogenesis, we examined hMLH1 methylation and expression in 100 gastric cancers. hMLH1 methylation and aberrant protein expression were observed in 9 and 13 cancers, respectively. Normal and intestinal metaplastic tissues adjacent to cancers with hypermethylation did not exhibit any hMLH1 methylation, indicating that it may be specific to gastric cancers. The frequency of hMLH1 methylation significantly increased with age. These results suggest that hMLH1 methylation plays an important role in gastric carcinogenesis in old people.

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The DNMT3A (DNA methyltransferase 3A) and DNMT3B genes encode putative de novo methyltransferases and show complex transcriptional regulation in the presence of three and two different promoters respectively. All promoters of DNMT3A and DNMT3B lack typical TATA sequences adjacent to their transcription start sites and contain several Sp1-binding sites. The importance of these Sp1-binding sites was demonstrated by using a GC-rich DNA-binding protein inhibitor, mithramycin A, i.e. on the basis of decrease in the promoter activities and mRNA expression levels of DNMT3A and DNMT3B. Overexpression of Sp1 and Sp3 upregulated the promoter activities of these two genes. The physical binding of Sp1 and Sp3 to DNMT3A and DNMT3B promoters was confirmed by a gel shift assay. Interestingly, Sp3 overexpression in HEK-293T cells (human embryonic kidney 293T cells) resulted in 3.3-and 4.0-fold increase in DNMT3A and DNMT3B mRNA expression levels respectively by quantitative reverse transcriptase-PCR, whereas Sp1 overexpression did not. Furthermore, an antisense oligonucleotide to Sp3 significantly decreased the mRNA levels of DNMT3A and DNMT3B. These results indicate the functional importance of Sp proteins, particularly Sp3, in the regulation of DNMT3A and DNMT3B gene expression.

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