Recently we reported that cIAP1, an inhibitor of apoptosis, is overexpressed through 11q22 amplification in cell lines derived from esophageal squamous cell carcinomas (ESC) and is associated with resistance of ESC to drug-induced apoptosis. In cervical squamous cell carcinoma (CSCC) cell lines, amplification and overexpression of cIAP1 was also observed. CSCC cell lines with cIAP1 amplification showed significant resistance to radiation-induced cell death as compared with lines showing no cIAP1 amplification. Immunohistochemical analysis of 70 primary CSCCs from patients treated only with radiotherapy demonstrated that both overall survival and local recurrence-free survival was significantly poorer among patients with tumors showing high levels of nuclear cIAP1 staining than among patients whose tumors revealed little or no nuclear cIAP1. Multivariate analysis showed nuclear cIAP1 staining to be an independent predictive factor for local recurrence-free survival after radiotherapy among patients with CSCC. These findings demonstrate that cIAP1 may play an important role in the development/progression of CSCC and that cIAP1 could be a novel predictive marker for resistance to radiotherapy in individual CSCC patients.

Gene silencing is widely used to clarify the gene function. Although RNA interference (RNAi) can expect a handy and large effect for gene silencing, the development of the screening system of a target gene is important. Green fluorescent proteins (GFPs) and Reef Coral fluorescent proteins (RCFPs) are well known that can make stable fluoresce without any co-factors in almost all cells. Both of the function of the target gene and the fluorescent of GFPs/RCFPs are most probably kept, even if a lot of genes are made as a fusion protein with these fluorescent proteins. When GFP or RCFP is connected with C-terminal of a target protein, and if a target protein receives the resolution by RNAi, the fluorescence of GFP or RCFP in the downstream of a target protein cannot be translated to a protein and fluorescence disappears. We developed the high throughput screening system for RNAi. Fluorescence-Activated Cell Sorter (FACS) measurement of the fluorescence of 293 cells after the expression vector of a target gene and RNAi vector were co-transfected to 293 cells, a handy screening of RNAi can be established.

Ex vivo expansion of CD34 positive cells in the umbilical cord blood (UCB) was investigated in liquid culture system with various combination of cytokines (stem cell factor [SCF], Thrombopoietin [TPO], interleukin [IL]-6,soluble IL-6 receptor, Flt-3/Flk-2 Ligand) and FACS analysis of cell surface markers indicated an absolute CD34+ cell numbers increased after 12 days of culture ( 20 -fold) . And these cytokines were significantly effective in increasing non-lineage-committed hematopoietic progenitor cells (CD34+CD38- subpopulation) ,though CD34+CD133+ subpopulations were slightly decreased. The tendency to decrease of CD90 and CD117 after 12days culture were also observed though a big change was not seen for CD123 and HLA-DR after 12days culture.

The polyploidization of tetraploid Meth-A cells by staurosporine (SS), an inhibitor of protein kinases, was examined. Tetraploid Meth-A cells were polyploidized well by SS, but diploid cells were not. When the drug concentration was gradually reduced, polyploidization progressed further. Cell clones having octaploid and decaploid DNA content were obtained from the cell population 20 days after the drug removal. The durations of the G₁, S and G₂/M phases of the polyploid cells were almost the same as those of the parent tetraploid cells. These findings suggest that polyploidization by SS is responsible for the production of polyploid Meth-A cells whose ploidy is not a power of 2.

Asparagine depletion in the serum during L-asparaginase treatment causes the the death of lymphoblasts that lack ability to synthesize asparagine. Cellular levels of asparagine synthetase (AS) inversely correlate with cellular sensitivity to L-asparaginase. Human leukemia cells that do not express AS in detectable quantities are hypersensitive to the effects of L-asparaginase. In the present studies, we have established a flow cytometry for AS protein detection assay with the monoclonal anti-AS antibody, 3G6. We compared AS expression in single-cell suspensions with standard biochemical and Western blot assays to validate the fluorescence-activated cell sorting (FACS) method. FACS yielded a linear relationship between the mean fluorescence intensity and AS activity. Using this standard curve, FACS-analyzed AS activity in leukemia cells ranged from 25.8 to 436 pmol asparagine formed/mg protein /min, similar to those obtained by Western blot analyse and even to AS mRNA levels by RT-PCR. Thus, AS-FACS can rapidly assess the heterogeneity of steady-state AS in single-cell suspensions and may be useful for assay in peripheral blood cells, bone marrow cells and leukemic cells. This quantitative assay system should be developed as a potential application of AS estimation for fresh leukemia cells.

Genomics and the genomics-based applications require technologies for high throughput analyses of biomolecules. Together with various technologies developed to date including DNA microarray and lab-on-achip, micro-bead technology is one of the key technologies for this purpose. The micro-bead technology have been successfully applied to the analysis of DNA and is thought to have great potential to be flexibly applied to the analyses of proteins, handling of which is much more difficult due to their diverse characteristics as compared to DNA. The micro-bead technology utilizes instruments and techniques developed for cytometry. Numbers of micro-beads with different biomolecules assigned by the fluorescence code are reacted in a single vessel. Then, the fluorescence from each micro-bead is analyzed by flow cytometry, image cytometry or so on. The micro-bead based technology allows multiplex analysis of various biomolecules. Further, it also allows automated analysis when paramagnetic beads are used. We have developed the technique for the preparation of fluorescently coded paramagnetic beads together with the instruments for the automatic operation. As the first example, we have applied our platform to the SNP typing. After the allele specific ligation, the products are captured on the fluorescently coded paramagnetic beads through sequence specific hybridization between the complimentary tags, one immobilized on the bead and the other connected to the SNP detection oligonucleotide. The paramagnetic beads are washed and allowed for the cytometry analysis. The recent progress of the micro-bead technology will realize more flexible, high throughput and cost effective analyses in genomics and medical inspection applications.

Comparative genomic hybridization (CGH) has been used for a screening of the genes that are responsible for genomic copy-number changes. For this purpose, chromosomal CGH is being replaced by array CGH. Recently, we have applied CGH to lineage analyses in undifferentiated-type gastric carcinomas. We used the DNA that was taken with laser capture microdissection from multiple portions in individual tumors and amplified through DOP-PCR. Comparing the pattern of chromosomal loss/gain at the arm level between poorly differentiated adenocarcinomas (POR) and signet ring cell carcinomas (SIG) and between the tumors with minor tubular component (TC) and those without TC, we have found that most of the POR without TC derives from SIG and that the genetic pathway may be different between the tumors with TC and those without TC. Using the dendrograms that were based on intratumoral heterogeneity analyses, we have demonstrated that large part of the POR with TC derives from tubular adenocarcinoma and small part of them from SIG. Thus, the CGH-based lineage analysis may be a useful tool for the demonstration of genetic lineage between the lesions of different morphology at different loci, and thereby, for a reappraisal of tumor classification.

Little is known about genetic alterations during malignant progression of meningioma. We have reported our investigation into the genetic pathway underlying the development of intracranial meningioma using comparative genomic hybridization (CGH). Benign meningiomas displayed only a few genetic changes such as monosomy 22. Anaplastic meningiomas manifested more aberrations than typical meningiomas, frequently exhibiting losses of 1p, 2p, 6q, chromosome 10 and 14q, and gain of 20q, in addition to monosomy 22. The average number of alteration sites in each patient with typical meningioma was significantly less than those in each patient with atypical and with anaplastic meningioma. Anaplastic meningiomas showed the chromosomal changes seen in atypical meningiomas together with other aberrations. These CGH findings suggest that losses of 1p, 2p, 6q, chromosome 10 and 14q, and gain of 20q are genetic changes implicated in the malignant progression of meningioma. In this article, we review the studies on CGH in cases of meningioma.