An In Vivo and In Vitro Comparison of the Effects of b2-a2 and b3-a2 p210BCR-ABL Splice Variants on Murine 32D Cells

Abstract

The Philadelphia (Ph) chromosome, a characteristic cytogenetic marker of chronic myeloid leukaemia (CMJL), is caused by a reciprocal translocation juxtaposing the 3′ region of the ABL gene onto the 5′ region of the BCR gene. Due to conservation of the reading frame, but depending on the site of the breakpoint in the BCR gene, two alternatively spliced variants of the p210BCR-ABL mRNA (known as b2-a2 and b3-a2) are produced. To investigate whether there are any biological differences between these splice variants we have transfected the b3-a2 or b2-a2 cDNA into a murine myeloid cell line, 32D. We have also included the previously prepared 32Dp210 cell line (which expresses the b3-a2 transcript) in all of our comparisons. RT-PCR analysis indicated that transcription levels were comparable between the variants. Morphological examination of the cells expressing either of the BCR-ABL transcripts indicated that these cells were more mature with increased cytoplasm:nuclear ratios compared to the 32D parental and 32Dneo vector control cells. However, the 32Dp210 cells had a very different appearance from the other panel members and flow karyotyping indicated a clonal ewolution and cytogenetic instability in these cells alone. At 106 and 107 cell doses all 32D cells; expressing BCR-ABL caused ill health and tissue infiltration in SCID mice with such rapidity that statistical analysis was not informative. However, at the 105 and 104 dosage levels there were similar survival rates between mice injected with 32Db2-a2 or 32Db3-a2 while mice injected with 32Dp210 had a significantly shorten-survival time. The study of this 32D cell line panel indicated that there were no overt differences in the biological properties conferned by the b3-a2 or b2-a2 transcripts to the 32D cells although these transcripts were able to confer in vitro and in vivo biological effects. This punel of BCR-ABL expressing 32D cells provides a useful model for CML disease progression studies.