Cloning Of Whole CDNA Sequence And Function Analysis Of The Novel Leukemia Associated Gene LRP15

Many molecular biological aberrantion can be detected in hematopoiesis, including gene mutation, gene deletion, activation of oncogene and inactivation of tumor suppressor gene. Cloning novel leukemia associated gene not only facilitate to explore the mechanism of hematopoiesis, but also to find new target sites for therapy. In order to clone the full length cDNA of a novel leukemia associated candidate gene(LRP15), we assembled the human ESTs(Expression Sequence Tags) fragments obtained from electronic hybridization by a 1.8kb DNA probe, which was only methylated in relapsed leukemia, then designed the primers for rapid amplification of cDNA end(RACE). Bioinformatic data of High Throughout Genomic Sequences(HTGS) and Serial Analysis of Gene Expression(SAGE) were used for chromosome localization and tissue expression analysis. As a result, the full-length cDNA of the novel gene was obtained. Sequence of the novel gene revealed that it has an open reading frame of 780bp encoding a 259 amino acid protein of unknown functions. LRP15 gene is expressed in many different tissues. It was localized in chromosome 3p24. This work is the base of the further study for the novel gene function.Bioinformatic data show that LRP15 may be a leukemia associated gene, which may play an important role in hematopoiesis. In order to define the intracellular localization of the protein coded by the novel gene LRP15, and to predict its function, we analyse the sequence of the promoter of LRP15 gene with bioinformatic data of human genome resources(HGR). The structure and function ofthe LRP15 protein were predicted through RPS-BLAST software. The results of bioinformatic analysis were verified by experiments with enhanced green fluorescence protein(EGFP) and laser confocal microscopy. We show that LRP15 protein has one cAMP-dependent protein kinase phosphorylation site, two casein kinase II phosphorylation sites and one leucine-rich repeat in the N-terminal domain. The protein is localized in cellular nucleus. It is demonstrated that the LRP15 protein is a nucleoprotein and may play an important role in cell development, differentiation and adhesion, and may be associated with DNA repair. The relationship between the novel gene expression and hematopoiesis should be verified by experiments. In this study, expression of LRP15 gene was examined in normal peripheral blood(NPB), normal bone marrow(NBM), normal peripheral blood stem cell(NPBSC), spacimens of leukemia patients and leukemia cell line K562 by RT-PCR. The methylation of LRP15 gene was investigated in K562 cell line by MS-PCR. Then we expose K562 to 5-aza-2'-deoxycytidine and trichostatin(TSA), to determine whether the silencing of LRP15gene by de novo methylation could be reversed. As a result, LRP15 has not been detected in NPB. While about 31.3% of NBM and NPBSC express LRP15(p=0.005), which contain rich hematopoietic progenitor cell. LRP15 expression has no significant difference between AML and ALL. But at the myeloblast (M1,M2) and promyelocyte(M3) stages of development, the positive rate of LRP15 expression is higher than others(p=0.002). Our observations suggest that LRP15 may have a role in hematopoiesis. High levels of LRP15 expression may be especially associated with certain leukemias. It may be important in classification of acute leukemia. Further more, in Ml, M2 and M3, LRP15 has been detected in all relapsed patients, but only 57.1% in de novo patients. And in ALL, there is 60% relapsed patients express LRP15, but only 20% de novo patients do. It indicates that LRP15 expression maypredict the poor prognosis of the acute leukemia. By MS-PCR, we show that the promoter of LRP15 was hypermathylated in K562 cell line, and lose its transcription. After 5-aza-2'-deoxycytidine treatment, with or without TSA, the silencing of LRP15 gene by de novo methylation can be reversed. Our observations demonstrate that the expression of LRP15 was controlled by methylation in its promoter in K562. It suggests methyltransferase inhibitor and deacetylase inhibitor may be eff...