| ObjectiveAcute myeloid leukemia (AL) is comprised of a group of clonal hematological malignances, and the mechanisms of leukemogenesis always involve a lot of molecular events and multiple steps. Recurrent chromosome aberrations can be detected in about 55% AL patients, among which translocation is one of the most frequent. Many genes coding for various transcription factors (TFs) are involved in translocations, leading to the generation of fusion genes composed of both the disrupted TFs and its partners. Such fusion genes can be translated into fusion proteins, which often disturb the normal proliferation, differentiation and apoptosis pathways of hematological cells, and play an important role in leukemogenesis and/or disease progression. Therefore, detection of the recurrent chromosome translocations, molecular cloning and functional study of the involved genes will shed light on deciphering the molecular mechanisms of leukemogenesis, providing molecular markers for making diagnosis and minimal residual disease (MRD) detection, and allow the development of new target therapeutic strategies. RUNX1 (alias: AML1) is located at 21q22, encoding an important TF which is a key regulator of the normal proliferation and differentiation of the hematological cells. AML1 is among one of the most frequent targets of chromosome translocations, and it can translocate with many different partners. At present, more than 30 kinds of translocations involving AML1 have been reported, with about 20 different partners been successfully cloned. The current study will add a new case of translocation involving AML1 and will report the results of molecular cloning and preliminary functional analyses of the fusion genes.Methods1. R-banding and G-banding technique were used to detect the chromosome abnormality of the bone marrow (BM) specimen and PHA-stimulated peripheral blood (PB) sample from an AML-M2 patient. Chromosome painting (CP) and M-FISH were applied to confirm the results of the karyotypic analyses. FISH analyses with the AML1-ETO dual color, dual fusion probe alone and in combination with the chromosome painting probe for chromosome 11 were used to detect the involvement of the AML1 gene. RT-PCR was used to exclude the cryptic expression of the AML1-ETO fusion gene. The clinical characteristics of this patient were investigated for a 10-month follow-up. All the experiments were performed with informed consent.2. 3'-RACE was applied to investigate the partner gene of AML1 in this translocation. RT-PCR was used to detect the fusion transcripts, the expression of the uninvolved AML1 and LPXN allele and to clone the full-length coding sequences (CDS) of the target transcripts which were afterwards inserted to a series of eukaryotic expression vectors to perform the functional analyses. All constructs were verified by sequencing.3. With transient transfections, we detected the subnuclear localization of fusion proteins encoded by full-length CDS of wild-type AML1B, LPXN, two of the representative transcripts of AML1-LPXN named AL, ALs and LPXN-AML1, respectively. Co-transfections of wild-type AML1B, AL and ALs with CBFβwere also performed to detect their co-localization in the nucleus.4. Electrophoretic mobility shift assay (EMSA) was applied to detect the combination of the transcript AL and ALs with a DNA probe which contains a specific AML1 binding site. Luciferase reporter gene assay with the pM-CSF-R-luc reporter plasmid was used to detect the impact of the transcript AL and ALs on the normal transcriptional activities of the wild type AML1B protein.5. We constructed some models with NIH3T3 cells which had stable higher expression of AL, ALs, LPXN-AML1 and LPXN, respectively. Growth rate of the above cells was detected with the cell counting kit 8 (CCK-8) and growth curve was made according to it. Growth ability of such cells in the soft agar and their tumorogenicity in the subcutaneous of the BALB/c nude mice were also investigated to detect the malignant transformation ability of the above genes. Results1. Conventional karyotypic analysis of the BM sample revealed a new form of chromosome abnormality: t(11;21)(q1?3;q22), which was further confirmed with CP and M-FISH. Karyotype of the PHA-stimulated PB sample showed to be normal. FISH analyses with a dual-color, dual fusion AML1-ETO probe alone and together with a CP probe for chromosome 11 detected the disruption of one AML1 allele, while the ETO gene remained intact in this translocation. Expression of the AML1-ETO fusion transcript was not detected with RT-PCR. Clinical investigation showed that the patient had poorer response to chemotherapy and had a short survival.2. The result of 3'-RACE showed that AML1 was fused with LPXN, which is located in 11q12.1 in this translocation. RT-PCR detected four kinds of AML1-LPXN transcripts and one LPXN-AML1 transcript. For AML1-LPXN, we only choose two representative transcripts named AL and ALs, respectively, in the following experiments. The genomic breakpoints in this translocation lied in intron 6 of AML1; between base 835 and 836 in exon 8, and between base 984 and 985 in exon 9 of LPXN, respectively.3. As reported in the literature, wild-type AML1B protein was found to localize in the nucleus; the CBFβprotein was found to localize in the whole cell, predominantly in the cytoplasm; while the LPXN protein was located in the cytoplasm. Both fusion proteins encoded by transcript AL and ALs were located in the nucleus as wild-type AML1B, while LPXN-AML1 fusion protein had the same localization with wild-type LPXN. In addition, co-localization of proteins encoded by transcript AL and ALs with CBFβ, respectively, in the nucleus were also visualized.4. EMSA detected the specific combination of proteins encoded by transcript AL and ALs with the designed DNA probe which contains a specific AML1 binding site, and such combination was enhanced when AL and ALs was co-transfected with CBFβ, respectively. The result of the luciferase reporter gene assay showed that proteins encoded by transcript AL and ALs could both inhibit the transactivation ability of wild-type AML1B on the M-CSFR reporter in a dose-dependent manner.5. NIH3T3 cells which had stable and higher expression of AL, ALs, LPXN-AML1 fusion gene and LPXN gene, respectively, grew more rapidly than cells that were transfected only with pEGFP-N1 vector. Also, such stably-selected cells could grow in colonies in the soft agar, and could form solid tumors when injected in the subcutaneous of the BALB/c nude mice.Conclusions1. We have identified a new acquired chromosome translocation with the disruption of the AML1 gene, i.e. t(11;21)(q1?3;q22).2. Fusion partner of AML1 in this translocation was LPXN, which is located at 11q12, thus the karyotype of the above translocation was refined as t(11;21)(q12;q22). The genomic breakpoints in this translocation lied in intron 6 of AML1; between base 835 and 836 in exon 8, and between base 984 and 985 in exon 9 of LPXN, respectively, resulting in the formation of four kinds of AML1-LPXN variants and one kind of LPXN-AML1 reciprocal transcript.3. Transcript AL and ALs had the same localization with the wild-type AML1B and both could co-localize with CBFβin the nucleus, thus proteins encoded by transcript AL and ALs could compete with wild-type AML1B to combine with CBFβand may inhibit the transcriptional activity of wild-type AML1B protein. LPXN-AML1 was localized in the cytoplasm as wild-type LPXN protein, thus we speculated that LPXN-AML1 may not affect the transcriptional activity of wild-type AML1B protein.4. Proteins encoded by transcript AL and ALs could inhibit the transactivation ability of wild-type AML1B protein on the promoter of the M-CSFR gene by competing with the wild-type AML1B protein to combine with its target core DNA sequences.5. Fusion transcript AL and ALs,LPXN-AML1 and wild-type LPXN gene could confer the NIH3T3 cell with growth advantage and malignant transformation abilities. |
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