1: Cancer Genet Cytogenet. 2005 Mar;157(2):118-26. Role of multiplex FISH in identifying chromosome involvement in myelodysplastic syndromes and acute myeloid leukemias with complex karyotypes: a report on 28 cases. Barouk-Simonet E, Soenen-Cornu V, Roumier C, Cosson A, Lai JL, Fenaux P, Preudhomme C. Institut de Recherches sur le Cancer de Lille, Unite Inserm 524, 1 place de Verdun, 59045 Lille, France. Chromosomal abnormalities are found by conventional cytogenetic (CC) analysis in about 50% of myelodysplastic syndromes (MDS) and 70% of acute myeloid leukemias (AML). When cytogenetic abnormalities are complex, multiplex fluorescence in situ hybridization (M-FISH) can help clarify complex chromosomal abnormalities and identify rearrangements with prognostic value or cryptic translocations, which could be preliminary steps in identifying new genes. We studied by M-FISH 28 cases of MDS and AML with complex chromosomal abnormalities, 10 of them were therapy-related. M-FISH allowed the characterization of unidentified chromosomal material in 26 cases (93%). One or several unbalanced rearrangements were observed in 27 cases (96%), generally interpreted as deletions or additional material by CC. Among those translocations, 4 involved 3 chromosomes. Eighteen cryptic translocations undetected by CC were found in 13 cases. By FISH analysis using locus specific probes, TP53 deletion, additional copies of MLL, and additional copies or deletions of RUNX1/AML1 were observed in 16, 4, and 3 cases, respectively. Thus, M-FISH is an important tool to characterize complex chromosomal abnormalities which identified unbalanced and cryptic translocations in 96% and 46% of the cases studied, respectively. Complementary FISH helped us identify involvement of TP53, MLL, and RUNX1/AML1 genes in 82% of cases, confirming their probable role in leukemogenesis. Publication Types: Evaluation Studies PMID: 15721632 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 2: Leukemia. 2005 Feb;19(2):197-200. Amplification or duplication of chromosome band 21q22 with multiple copies of the AML1 gene and mutation of the TP53 gene in therapy-related MDS and AML. Andersen MK, Christiansen DH, Pedersen-Bjergaard J. Department of Clinical Genetics, Section of Hematology/Oncology, The Juliane Marie Center, Rigshospitalet, Copenhagen, Denmark. mka@rh.dk Amplification or duplication of the AML1 gene at chromosome band 21q22 was detected by FISH using a locus-specific probe in three out of 171 unselected patients with therapy-related myelodysplasia (t-MDS) or t-AML (1.7%). In two patients AML1 signals were located tandemly on derivative chromosomes, in one patient on a dic(9;21) and in the the other patient on a derivative chromosome 18 made up of interchanging layers of material from chromosomes 9, 14, 18, and 21. In the third patient three single supernumerary copies of AML1 were located on derivatives of chromosomes 19 and 21. All three patients were older, had previously received therapy with alkylating agents without topoisomerase II inhibitors, had complex karyotypes including abnormalities of chromosomes 5 or 7, and presented acquired point mutations of the TP53 gene. No point mutations of the AML1 gene were observed. The results support a pivotal role of impaired TP53 function in the development of gene amplification or duplication in t-MDS and t-AML. PMID: 15618958 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 3: Br J Haematol. 2004 Jun;125(6):709-19. Novel loss-of-function mutations of the haematopoiesis-related transcription factor, acute myeloid leukaemia 1/runt-related transcription factor 1, detected in acute myeloblastic leukaemia and myelodysplastic syndrome. Nakao M, Horiike S, Fukushima-Nakase Y, Nishimura M, Fujita Y, Taniwaki M, Okuda T. Molecular-Targeting Cancer Prevention, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kawaramach-Hirokoji, Kamigyo-ku, Kyoto, Japan. AML1/RUNX1, which encodes a transcription factor essential for definitive haematopoiesis, is a frequent target of leukaemia-associated chromosome translocations. Point mutations of this gene have also recently been associated with leukaemia and myelodysplastic syndrome (MDS). To further define the frequency and biological characteristics of AML1 mutations, we have examined 170 cases of such diseases. Mutations within the runt-domain were identified in five cases: one of de novo acute myeloid leukaemia (AML) and four of MDS. Where multiple time point samples were available, mutations were detected in the earliest samples, which persisted throughout the disease course. Of the five mutations, one was a silent mutation, two were apparent loss-of-function mutations caused by N-terminal truncation, and two were insertions, I150ins and K168ins, which preserved most of the AML1 DNA-binding domain. Both AML1 molecules with insertion mutations were non-functional in that they were unable to rescue haematological defects in AML1-deficient mouse embryonic stem cells. In addition, activating mutations of N-ras, deletion of chromosome 12p, or inactivation of TP53 accompanied some of the AML1 mutations. Together, these observations strongly suggest that one-allele inactivation of AML1 serves as an initial or early event that plays an important role in the eventual development of overt diseases with additional genetic alterations. PMID: 15180860 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 4: Oncogene. 2004 Jul 15;23(32):5476-86. RUNX1 transformation of primary embryonic fibroblasts is revealed in the absence of p53. Wotton SF, Blyth K, Kilbey A, Jenkins A, Terry A, Bernardin-Fried F, Friedman AD, Baxter EW, Neil JC, Cameron ER. Molecular Oncology Laboratory, Institute of Comparative Medicine, Faculty of Veterinary Medicine, University of Glasgow, Bearsden Road, Glasgow G61 1QH, Scotland, UK. S.Wotton@vet.gla.ac.uk The mammalian Runx gene family (Runx1-3) are transcription factors that play essential, lineage-specific roles in development. A growing body of evidence implicates these genes as mutational targets in cancer where, in different contexts, individual family members have been reported to act as tumour suppressors, dominant oncogenes or mediators of metastasis. We are exploring these paradoxical observations by ectopic expression of RUNX genes in primary murine embryonic fibroblasts where, in common with a number of other dominant oncogenes, RUNX1 induces senescence-like growth arrest in the presence of an intact p19(ARF)-p53 pathway. We now report that, in MEFs lacking functional p53, RUNX1 has apparently pro-oncogenic effects on cell growth that include cytoskeletal reorganization, reduced contact inhibition at confluence and accelerated tumour expansion in vivo. On the other hand, RUNX1 conferred no obvious growth advantage at low cell density and actually delayed entry of primary MEFs into S phase. We also found that ectopic RUNX1 interferes with the morphological and growth responses of p53-null MEFs to TGFbeta indicating that these effects are mediated by overlapping pathways. These observations help to elucidate the context-dependent consequences of loss and gain of Runx activity. PMID: 15133495 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 5: Ann Hematol. 2004 Jun;83(6):329-30. Epub 2004 Mar 30. Epigenetic regulation of tumor suppressors in t(8:21)-containing AML. Yang G, Khalaf W, van de Locht L, Jansen JH, van der Reijden BA, Muller-Tidow C, Delwel HR, Serve H, Clapp DW, Hiebert SW. Department of Biochemistry, Vanderbilt University School of Medicine, PRB 512, 23rd and Pierce, Nashville, Tennessee 37232, USA. The t(8;21) is perhaps the most frequent chromosomal translocation associated with acute myeloid leukemia. The translocation creates a fusion protein that consists of the DNA binding domain of the RUNX1 transcription factor fused to the MTG8 transcriptional co-repressor to create a potent transcriptional repressor. Here, we discuss the possibility that the t(8;21) fusion protein represses tumor suppressors that regulate the RAS signaling pathway and the p53 oncogenic checkpoint. Publication Types: Review Review, Tutorial PMID: 15052372 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 6: Exp Hematol. 2004 Jan;32(1):122-30. Granulocyte colony-stimulating factor generates epigenetic and genetic alterations in lymphocytes of normal volunteer donors of stem cells. Nagler A, Korenstein-Ilan A, Amiel A, Avivi L. Bone Marrow Transplantation Department, Institute of Hematology, Chaim Sheba Medical Center, Tel-Hashomer, Israel. OBJECTIVE: Because the effect of granulocyte colony-stimulating factor (G-CSF), which is widely used for allogeneic stem cell transplantation, on DNA function and stability has not yet been unequivocally elucidated, the aim of this study was to determine whether G-CSF leads to epigenetic and/or genetic modifications. MATERIALS AND METHODS: Molecular cytogenetic techniques based on fluorescence in situ hybridization technology were used. RESULTS: Lymphocytes of G-CSF mobilized donors displayed epigenetic (altered replication timing of alleles) and genetic (aneuploidy) alterations similar to those observed in lymphocytes of cancer patients. Specifically, in the donors' lymphocytes, biallelically expressed genes (TP53 and AML1) and a repetitive noncoding DNA sequence associated with chromosome segregation (CEN17) showed loss of synchrony in allelic replication timing (allele-specific replication). Each displayed a highly asynchronous pattern of allelic replication similar to that characterizing monoallelic expressed genes. This non-locus-specific epigenetic phenomenon, which also affects DNA sequences associated with chromosome segregation, was accompanied by aneuploidy. Although the loss of replication synchrony in the lymphocytes of G-CSF mobilized donors was a transient epigenetic modification, aneuploidy remained unchanged. The G-CSF effect also was observed after G-CSF administration in vitro. 5-Azacytidine, a DNA methylation blocking agent, inhibited G-CSF in vitro induction of allele-specific replication. CONCLUSION: G-CSF, probably via changes in DNA methylation capacity, leads to cancer-characteristic DNA modifications in lymphocytes of normal mobilized donors. PMID: 14725909 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 7: Cancer Genet Cytogenet. 2002 Dec;139(2):97-103. Allele-specific replication associated with aneuploidy in blood cells of patients with hematologic malignancies. Korenstein-Ilan A, Amiel A, Lalezari S, Lishner M, Avivi L. Department of Human Genetics and Molecular Medicine, Sackler School of Medicine, Tel-Aviv University, Tel-Aviv, Israel. We hypothesize that coordination between the two DNA parental sets in somatic cells is essential for the stability of the diploid genome, and that its disruption is associated with the many alterations observed in the various cancerous phenotypes. As coordination between two allelic counterparts is well exemplified by synchrony in replication timing, we examined, in blood cells of patients suffering from various hematologic malignancies, replication patterns of five loci. These loci were three cancer-implicated genes (TP53, AML1, and RB1) and two nontranscribed sequences engaged in chromosome segregation. All five loci normally display synchrony in allelic replication timing. In addition, in order to exemplify an asynchronous mode of allelic replication, we followed the replication of allelic counterparts of an imprinted gene (SNRPN), which is distinguished by its asynchronous mode of allelic replication (allele-specific replication). Allelic replication patterns were studied by fluorescence in situ hybridization (FISH), which has been shown to distinguish between nonreplicated and replicated regions of the genome in interphase cells, based on the structure of the specific hybridization signals that are being detected. Using the FISH replication assay we observed, for all loci which normally exhibit synchrony in allelic replication, loss of synchrony when present in blood cells of patients with hematologic malignancies. The loss of synchrony in allelic replication in patients' cells was accompanied by aneuploidy (chromosome losses and gains), the hallmark of cancer. We were able to reinstate the normal pattern of replication in the patients' cells by introducing an inhibitor of DNA methylation. It thus appears loss of allelic coordination is an epigenetic alteration characterizing cancer, which is easily identified by simple cytogenetic means and has a potential use in both cancer investigation and detection. PMID: 12550768 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 8: Gan To Kagaku Ryoho. 2002 Jul;29(7):1107-12. [Molecular mechanisms in leukemogenesis] [Article in Japanese] Mitani K. Dept. of Hematology, Dokkyo University School of Medicine. Mutations in signal transduction molecules, which regulate cell differentiation and proliferation, are involved in the development of leukemia. Aberrations of receptor type tyrosine kinases are known to arise from FLT3 mutations in acute myeloid leukemia (AML) and myelodysplastic syndrome, and c-Kit mutations in mast cell tumors. BCR/ABL found in chronic myelogenous leukemia (CML) is a hallmark of the constitutively active forms of cytoplasmic tyrosine kinases. Downstream of the tyrosine kinase is the RAS GTP-binding protein, and genetic mutations related to this protein have been found in a wide variety of malignant tumors including hematopoietic tumors. In the nucleus, transcription factor-encoding genes are frequently detected as the targets of chromosomal translocations found in specific types of leukemias. For instance, the AML1 gene generates AML1/MTG8 chimera by t (8;21) translocation in AML (M2), AML1/EVI-1 chimera by t (3;21) translocation in blastic crisis of CML, and TEL/AML1 chimera in t (12;21) translocation (pre-B cell type acute lymphoblastic leukemia). Another example of abnormal transcription factors is PML/RAR alpha generated by t (15;17) translocation found in acute promyelocytic leukemia. Mutations or deletions of tumor suppressor genes are frequently found in cell cycle regulators such as p53, RB and p16 genes. Therefore, mutations of any molecules involved in the signal transduction pathways from growth factor receptors to inside the nucleus are thought to contribute to neoplastic transformation of hematopoietic cells. Publication Types: Review Review, Tutorial PMID: 12145988 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 9: Oncogene. 2000 Jul 13;19(30):3434-8. Accommodating haploinsufficient tumor suppressor genes in Knudson's model. Cook WD, McCaw BJ. Bone Marrow Research Laboratories, Royal Melbourne Hospital, Melbourne, Victoria, Australia. Publication Types: Review Review, Tutorial PMID: 10918600 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 10: Blood. 2000 Feb 1;95(3):745-55. CREB-binding protein and p300: molecular integrators of hematopoietic transcription. Blobel GA. Division of Hematology, Children's Hospital of Philadelphia, and the University of Pennsylvania School of Medicine, Philadelphia, PA, USA. Publication Types: Review PMID: 10648382 [PubMed - indexed for MEDLINE] ---------------------------------------------------------------