Objective: It has been reported that acute leukemia with the fusion product of MLL-related molecule is determined to be the poor prognosis. To make clear one of its biological characters, we cultured AML blasts with t (11; 19) (q23; p13.1), which produced fusion protein of MLL and ELL, and the morphological changes as well as their DNA, and RNA were examined.
Method: Bone marrow cells were obtained from two patients with MLL-ELL fusion gene after obtaining informed consent. There were over 90% blastic cells in bone marrow. Mononuclear cells were obtained with Ficoll (SG. 1077), and were cultured in DMEM with 10% FCS in CO2 incubator. The morphology of the cells was observed for 3 months. Also, DNA was extracted, and MLL-ELL translocation was analyzed with genomic Southern blotting with probe x which contains BamHI-digested 0.9 kb fragment from MLL cDNA. RNA was extracted with GTC method, and 1st strand cDNA was synthesized with oligo-dT primer, and PCR was performed with MLL common primer and ELL antisense primer. The reaction of DNA sequencing was performed with BigDye Terminator Cycle Sequencing kit (Applied Biosystems), and analyzed with ABI Prism 3700 DNA analyzer.
Result: After one month of the cultures, fibroblastoid cells were expanded without any blastic cells onto them. Southern analysis, RT-PCR study, and DNA sequence revealed that the fusion cDNA product with MLL and ELL was observed in fibroblastoid cells.
Discussion: AML blasts with MLL-ELL fusion gene can convert their morphology into the fibroblastoid cells. The fibroblasts have been reported to be resistant to the chemotherapeutic agents. This morphological conversion may contribute one of the causes of poor prognosis in AML patients with MLL-ELL fusion gene. We now characterize precisely these fibroblastoid cells in point of the expression of the specific proteins, and the functions including the binding and proliferating capability to the non-adherent blastic cells.