The retinoic acid receptor alpha (RAR alpha) and the myl gene are involved in the translocation breakpoint t(15;17)(q22;q21) in acute promyelocytic leukemia (APL). The majority of the breakpoint sites have been mapped within the second intron of the RAR alpha gene; however, the breakpoint sites on the myl gene are variable. Using primer sets derived from exon 2 or exon 3 of the RAR alpha gene and a primer derived from the myl cDNA, we were able to amplify the breakpoint sites of the fusion transcripts of all six APL RNA samples by the reverse transcriptase-polymerase chain reaction (RT-PCR). A DNA fragment of 290 bp (breakpoint A) was amplified using RNA samples from three patients, whereas two DNA fragments of 630 and 774 bp (breakpoint B) were amplified using RNA samples from the other three APL patients. DNA sequence analysis of the amplified fragments suggests that the APL breakpoints clustered within two different introns of the myl gene. Northern blot analysis demonstrated that fusion transcripts RAR alpha/myl and myl/RAR alpha of varying sizes were detected in patients with different breakpoint sites on the myl gene. In addition, we analyzed five APL samples in complete remission and detected t(15;17)- positive cells. We conclude that the t(15;17) breakpoints in APL can be amplified by PCR using a single primer set and that minimal residual disease can be demonstrated in APL using RT-PCR.
Since the translocation breakpoint t(15;17) (q22;q21) in acute promyelocytic leukemia (APL) occurs within the retinoic acid receptor- alpha (RARA) gene, the expression of many genes normally regulated by RARA may be affected by this translocation. To identify genes that may be aberrantly expressed in APL, a subtraction cDNA library of an APL patient with t(15;17) was constructed. A cDNA, pRD1, specifically expressed in APL was identified. DNA sequence analysis of pRD1 showed that this gene is similar to the DNA sequence of annexin VIII, a gene which encodes a vascular anticoagulant. The annexin VIII gene was assigned to chromosome 10, which indicates that specific expression of this gene in APL is not directly involved in the t(15;17) breakpoint region. We have analyzed the expression of annexin VIII gene in nine t(15;17)-positive APL patients and one APL patient with a chromosome 17q-abnormality. We found that all APL samples expressed high levels of the annexin VIII gene. Expression of the annexin VIII gene in all other leukemias, including acute myelogenous leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, and acute lymphoblastic leukemia, was undetectable, except in one patient with acute myelogenous leukemia in which a very low level of expression was detected. Annexin VIII is highly expressed in the APL cell line, NB4. Its expression was significantly reduced after 8 hours of all-trans retinoic acid (ATRA) treatment, whereas the expression of RARA increased several-fold within 4 hours postinduction. Thus, increased expression of RARA preceded the downregulation of annexin VIII after ATRA induction, suggesting an inverse relationship between RARA and annexin VIII expression. Since increased expression of the fusion transcript was seen after ATRA induction and an APL without a t(15;17) translocation expressed high levels of annexin VIII, it appears that increased expression of annexin VIII in APL is not related to the fusion transcript. Therefore, dysregulation of the RARA gene may be related to the overexpression of annexin VIII in APL.
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