Isolation and characterization of new markers for human brain tumors using molecular biological techniques

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In this research project, we first established improved molecular biological procedures for efficient isolation of cDNA clones of differentially expressed mRNAs from small materials : (1) modified differential screening procedure, (2) directional tag PCR subtraction, an efficient subtractive cDNA cloning procedure, and (3) cDNA library DNA-Southern blot procedure which can replace Northern blot analysis.Using these procedures, we successfully isolated 22 distinct rat cDNA clones whose corresponding mRNAs were preferentially expressed in the fetal brain during the brain development. We then isolated their human homologues and analyzed the mRNA expressions in various human tissues. We found that the expression pattern of the human neuronatin was notable. The neuronatin mRNA was selectively expressed in fetal human brain among normal human tissues and in human pituitary adenomas among various human brain tumors, suggesting the utilization as a new marker for the pituitary adenomas. We also analyzed the mRNA patterns of human glioma cell lines exhibiting different biological characters using the procedures described above. We identified 9 cDNA clones whose corresponding mRNAs were expressed more abundantly in the cells which showed hetero-transplantability and higher growth rate than the control cells. These cDNA clones included novel clones and those involved in the protein synthesis, signal transduction, and nucleotide metabolism. These findings showed that our improved procedures are useful to analyze molecular basis of tumor cells and to identify new tumor markers.In addition, we analyzed gene mutations in human brain tumors to understand the molecular backgrounds of different mRNA expressions, and found frequent gene mutations of tumor suppressor genes, such as p53 and p16, in human brain tumors.