Convenient clustering method of cancer cell lines by their one-dimensional protein modification profiles

It is important to know physiological properties of cancer cells for effective radiation therapy of patients. We decided to develop a convenient proteomic approach to investigate molecules corresponding to the physiological properties of individual cancer cell lines. The protein molecules we focused for this purpose were those with modification of side chain amino acid, since in many cases the modification is reversible and is utilized for regulation of the function of protein. The overall profile of modified proteins should have substantial potential in reflecting cellular physiological properties.The proteomic approach we are currently developing contains 1D SDS-PAGE of total cell lysate followed by quantitative Western blotting using anti-modified amino acid antibody. Thus, the profile contains one-dimensional list of the molecular weight of modified protein and its fluorescence signal intensity detected after ECL reaction. To perform the Western blotting analysis quantitatively, which is the key point of this work, we have made several improvements. The most important one is triplicated standard and sample data acquisition on single membrane, which is difficult and laborious to perform by 2D PAGE analysis, for the collection of accurate and reliable data. The profiling data of different cell lines will be further processed by band matching then statistical analysis to create hierarchical trees among them.As an initial analysis we are planning to investigate tyrosine-phosphorylated protein profile of 35 individual human cell lines (32 cancer cell lines and 3 normal fibroblasts) whose X-ray radiation sensitivity have been known.第76回日本生化学会大

Global profiling of human tyrosine kinase and phosphatase gene expression and of protein tyrosine phosphorylation

Both protein kinases and phosphatases represent distinct large protein families, whose members are similar to each other in sequence, structure and biochemical properties, in the human genome. The human genome has been estimated to contain between 500 and 1000 protein kinases. The expression status of overall kinase and phosphatase genes and the phosphorylation status of their substrates in individual cells seems to represent the detailed character of the cells since these protein families play key roles in variety of intercellular and intracellular signaling pathways by transmitting, amplifying or integrating upstream signals. In the 22,000 spots on the Agilent Custom OligoDNA Microarray, there are spots corresponding to 68 tyrosine kinase genes and 74 tyrosine phosphatase genes. Thirty three human cancer cell lines and two normal human fibroblast cells were analyzed by the above microarray using a mixture of 11 human tissue RNAs (Clontech) as a reference for each sample. The RNA expression data of twenty one kinase genes and fourteen phosphatase genes were selected after elimination of spots with p-value of more than 0.05 in at least 12 cell lines. For the profiling of tyrosine phosphorylation substrates in individual cell lines, we have developed a qualitative and quantitative anti-phosphotyrosine antibody Western blotting (qqPYW) method. By this method, molecular weight and signal intensity of each band detected was normalized by two steps, using internal molecular weight markers constructed by chemically tyrosine-phosphorylated proteins, to make precise matching to the bands in different samples to be practical. By this study, both gene expression data and protein phosphorylation data have become to be obtained at the quality that they can be compared to see any possible correlation. It will be necessary to establish further systematic methodology, such as identification of phosphorylated substrate proteins and in vivo interaction assay, to confirm the correlation observed.第２６回日本分子生物学会年

Prediction of radiation morbidity from polymorphisms in candidate genes among prostate cancer patients treated with carbon ion therapy.

Heavy ion beams possess high linear energy transfer components and a prominent Bragg peak in the human body. These properties promote higher relative biological effectiveness and a favorable dose distribution. Some patients, however, develop adverse effects in the rectum and /or bladder/urethra. Using DNA samples collected from 95 Japanese prostate cancer patients who underwent radiotherapy with the Heavy Ion Medical Accelerator in Chiba, 905 SNPs were genotyped from 127 candidate genes for radiation susceptibility. These genes were selected from our previous gene expression analyses of cultured human cell lines and mouse strains that had exhibited variable radiosensitivities. The SNPs in the candidate genes were selected from jSNP and dbSNP databases and their allele frequencies were examined using 133 healthy controls. The acute and late radiation morbidities were scored using Late Effects of Normal Tissues-subjective, objective, management, and analytic criteria. Significant differences were observed of genotype frequencies for SNPs in candidate gene loci. Area under the curve of the Receiver Operating Characteristic Curve (ROC) was estimated for each SNP marker for radiation morbidity to construct a predictive score. The top 7 markers were used in constructing predictive scores for dysuria within 3 months, and the top 5 markers at 6 months after starting radiotherapy. The top 10 markers were used in constructing predictive scores for polyuria within 3 months, and the top 11 markers at 6 months after starting radiotherapy. The distribution of ten predictive scores according to response values showed the potential discriminating power of these analyses.ASHG 55th Annual Meetin