pre-B cells from normal control, preleukemic, fully leukemic and fully leukemic, nilotinib-treated P190 BCR/ABL transgenic mice

Precursor B-lineage acute lymphoblastic leukemia (pre-B ALL) can be subdivided into different categories based on genetic abnormalities. One type of pre-B ALL is characterized by the presence of the Philadelphia (Ph) chromosome, the derivative chromosome 22 that is one product of a reciprocal translocation between chromosomes 22 and 9. The 22/9 translocation fuses the 5’ part of the BCR gene to the 3’ end of the c-ABL gene. The resulting BCR/ABL fusion encodes a Bcr/Abl protein with deregulated Abl kinase activity. Two major fusion proteins are found in Ph-positive leukemias which differ in molecular weight and the size of the Bcr moiety. The P190 Bcr/Abl protein is common in Ph-positive ALL. Targeted tyrosine kinase inhibitors such as nilotinib are used therapeutically to treat this type of leukemia. The 22/9 translocation fuses the 5’ part of the BCR gene to the 3’ end of the c-ABL gene. The resulting BCR/ABL fusion encodes a Bcr/Abl protein with deregulated Abl kinase activity. Two major fusion proteins are found in Ph-positive leukemias which differ in molecular weight and the size of the Bcr moiety. The P190 Bcr/Abl protein is common in Ph-positive ALL. Targeted tyrosine kinase inhibitors such as nilotinib are used therapeutically to treat this type of leukemia. Mice transgenic for the human P190 form of Bcr/Abl (Jackson Labs strain 017833) develop precursor B-lineage (pre-B) acute lymphoblastic leukemia, on average within 3 months of birth, when on a C57Bl/6J background. Bone marrows were isolated from control C57BLl/6J mice and from transgenic Bcr/Abl mice when they had not yet developed full-blown leukemia (<60 days of age), from Bcr/Abl transgenic mice with overt leukemia and packed bone marrows (>90 days of age), and from fully leukemic mice that had received a seven-day treatment with 75 mg/kg AMN107 (nilotinib). Three mice were used per condition, and cells from each mouse were processed separately. Pre-B cells from these twelve bone marrows were flow-sorted using CD19 and AA4.1 as markers. Total RNA from CD19+ AA4.1+cells used for microarray analysis was isolated by RNeasy (QIAGEN) purification. Double-strand complementary DNA was generated from 5 µg of total RNA using a poly(dT) oligonucleotide that contains a T7 RNA polymerase initiation site and the SuperScript III reverse transcription (Invitrogen). Biotinylated cRNA was generated and fragmented according to the Affymetrix protocol and hybridized to 430 mouse microarrays (Affymetrix).