S65

Poster Sessions

Acute leukemia / Burkitt lymphoma

P001 Acute myeloid leukemia, myelodysplasticsyndrome, multiple myeloma andnon-Hodgkin’s lymphoma carrying mutatedCCAAT/enhancer-binding protein alpha

O. Fuchs, D. Provaznikova, M. Kocova, A. Kostecka,R. Neuwirtova, P. Kobylka, J. Cermak, J. Brezinova,J. Schwarz, P. Salaj, K. Benesova. Department of CellPhysiology, Institute of Haematology and Blood Transfusion,Prague, Czech Republic

The transcription factor CCAAT/enhancer binding protein(C/EBP)α is a myeloid-specific transcription factor which isrequired for neutrophil differentiation. C/EBPα is encoded byan intronless gene that maps to human chromosome 19q13.1.C/EBPα is a member of the basic region leucine zipper(bZIP) class of DNA-binding proteins. The loss of functionof C/EBPα has leukemogenic potential. Four deletions, oneduplication, one insertion and three point mutations weredetected in the transcription factor CCAAT/enhancer bind-ing protein (C/EBP)α of analysed samples, consisting 10/92(10.9%) of AML patients’ samples, 2/42 (4.8%) of MDSpatients’ samples, 1/3 (33,3%) of non-Hodgkin’s patients’samples and 3/8 (37.5%) of multiple myeloma patients’samples. None C/EBPα mutations were detected in CMLand ALL patients. This work was supported by the InternalGrant Agency of the Ministry of Health of Czech RepublicNR/9045-3.Aim: We studied the presence of mutations in the C/EBPα ofpatients with hematologic malignancies.Patients and methods: Patient-derived bone marrow orperipheral blood samples (92 AML, 42 MDS, 7 CML, 1ALL, 8 multiple myeloma and 3 non-Hodgkin’s lymhomapatients’samples) were Ficoll-Paque PLUS purified and totalRNA and genomic DNA were isolated. Complementary DNAwas synthetised from total RNA using SuperScript II reversetranscriptase. The PCR reaction was carried out using ge-nomic DNA or cDNA, Advantage-GC Genomic PolymeraseMix and two or four overlapping primer pairs, which coverthe entire coding region of human C/EBPα. PCR productswere electrophoresed on agarose gels, purified and sequencedusing BigDye Terminator v1.1 Cycle Sequencing Kit in bothdirections. GenBank Accession No. NM_004364.2 was usedfor evaluation of obtained sequences.Results: 392_393insA was detected in AML patient andcaused truncated protein by introduction of terminationcodon. 722_746del was found in AML M4 patient. This

deletion of six nucleotides changes the whole DNA- bind-ing and homodimerization or heterodimerization regions ofthe C/EBPα. 600_1055del was detected in AML patientand also means the change in bZIP region of the C/EBPα.1038_1213del was detected in one AML patient, two multiplemyeloma patients and one non-Hodgkin’s lymphoma patient.584_589dup was observed in four AML patients, two MDSpatients and one multiple myeloma patient. This duplicationmeans insertion of two amino acids in proline and histidinerich region of C/EBPα which has some role in growth in-hibition properties of C/EBPα. 637delT was found in AMLpatient and causes truncated protein C/EBPα with changes inbZIP region. Three point mutations in bZIP region of oneAML patient changed 3 amino acids in this region of C/EBPα.Conclusion: All detected C/EBPα mutations have not beendescribed till now. C/EBPα mutations play some role not onlyin AML and MDS but probably also in multiple myelomaand non-Hodgkin’s lymphoma.

P002 Microenvironment influence on cyclin A1mRNA expression in AML blasts

M. Golemovic, L. Rnjak, M. Sucic, Z. Mitrovic,M. Pisk-Mikulic, B. Labar, D. Batinic. Department ofLaboratory Medicine, Department of Clinical Pathology andDepartment of Medicine, University Hospital Center Zagreb,Zagreb, Croatia

The cyclins constitute a family of proteins involved in pro-gression through the cell cycle. Among them, cyclin A1(CycA1) is expressed in hematopoietic progenitor cells and inacute myeloid leukemia (AML), and its role in the develop-ment of AML has been previously demonstrated in transgenicmice. The aim of this study was to determine whether bonemarrow stromal support induces changes in CycA1 mRNAexpression in cultured human AML blasts and whether cellcontact or cytokine support was crucial factor in that case.The CycA1 mRNA expression was analyzed in bone marrowmononuclear cells (BMMNC) of 27 AML patients and 7healthy BM donors. With regard to FAB-classification, therewere 6/27 M1, 6/27 M2, 4/27 M3, 6/27 M4 and 5/27 M5AML cases. All patients included in the study were adultswith median age 38.5 years (range, 22-77 years).The RNAwas isolated from cells on day 0 and following 48 hrs ofculture in different in vitro conditions. There were three dif-ferent serum-fee medium culture conditions: (1) co-culture ofblasts on confluent human stromal cell line HS5; (2) culturein serum free-medium alone (BIT9500, StemCell Tech.); and