t(11;22)(q23;q11.2) in acute myeloid leukemia of infant twins fuses MLL with hCDCrel, a cell division cycle gene in the genomic region of deletion in DiGeorge and velocardiofacial syndromes

We examined the MLL genomic translocation breakpoint in acute myeloid leukemia of infant twins. Southern blot analysis in both cases showed two identical MLL gene rearrangements indicating chromosomal translocation. The rearrangements were detectable in the second twin before signs of clinical disease and the intensity relative to the normal fragment indicated that the translocation was not constitutional. Fluorescence in situ hybridization with an MLL-specific probe and karyotype analyses suggested t(11;22)(q23;q11.2) disrupting MLL. Known 5′ sequence from MLL but unknown 3′ sequence from chromosome band 22q11.2 formed the breakpoint junction on the der(11) chromosome. We used panhandle variant PCR to clone the translocation breakpoint. By ligating a single-stranded oligonucleotide that was homologous to known 5′ MLL genomic sequence to the 5′ ends of BamHI-digested DNA through a bridging oligonucleotide, we formed the stem–loop template for panhandle variant PCR which yielded products of 3.9 kb. The MLL genomic breakpoint was in intron 7. The sequence of the partner DNA from band 22q11.2 was identical to the hCDCrel (human cell division cycle related) gene that maps to the region commonly deleted in DiGeorge and velocardiofacial syndromes. Both MLL and hCDCrel contained homologous CT, TTTGTG, and GAA sequences within a few base pairs of their respective breakpoints, which may have been important in uniting these two genes by translocation. Reverse transcriptase-PCR amplified an in-frame fusion of MLL exon 7 to hCDCrel exon 3, indicating that an MLL-hCDCrel chimeric mRNA had been transcribed. Panhandle variant PCR is a powerful strategy for cloning translocation breakpoints where the partner gene is undetermined. This application of the method identified a region of chromosome band 22q11.2 involved in both leukemia and a constitutional disorder.

Embryonic expression of the tumor-associated PAX3-FKHR fusion protein interferes with the developmental functions of Pax3

A unique chromosomal translocation involving the genes PAX3 and FKHR is characteristic of most human alveolar rhabdomyosarcomas. The resultant chimeric protein fuses the PAX3 DNA-binding domains to the transactivation domain of FKHR, suggesting that PAX3-FKHR exerts its role in alveolar rhabdomyosarcomas through dysregulation of PAX3-specific target genes. Here, we have produced transgenic mice in which PAX3-FKHR expression was driven by mouse Pax3 promoter/enhancer sequences. Five independent lines expressed PAX3-FKHR in the dorsal neural tube and lateral dermomyotome. Each line exhibited phenotypes that correlated with PAX3-FKHR expression levels and predominantly involved pigmentary disturbances of the abdomen, hindpaws, and tail, with additional neurological related alterations. Phenotypic severity could be increased by reducing Pax3 levels through matings with Pax3-defective Splotch mice, and interference between PAX3 and PAX3-FKHR was apparent in transcription reporter assays. These data suggest that the tumor-associated PAX3-FKHR fusion protein interferes with normal Pax3 developmental functions as a prelude to transformation.

Analysis of transforming activity of human synovial sarcoma-associated chimeric protein SYT-SSX1 bound to chromatin remodeling factor hBRM/hSNF2α

Human synovial sarcoma has been shown to exclusively harbor the chromosomal translocation t(X;18) that produces the chimeric gene SYT-SSX. However, the role of SYT-SSX in cellular transformation remains unclear. In this study, we have established 3Y1 rat fibroblast cell lines that constitutively express SYT, SSX1, and SYT-SSX1 and found that SYT-SSX1 promoted growth rate in culture, anchorage-independent growth in soft agar, and tumor formation in nude mice. Deletion of the N-terminal 181 amino acids of SYT-SSX1 caused loss of its transforming activity. Furthermore, association of SYT-SSX1 with the chromatin remodeling factor hBRM/hSNF2α, which regulates transcription, was demonstrated in both SYT-SSX1-expressing 3Y1 cells and in the human synovial sarcoma cell line HS-SY-II. The binding region between the two molecules was shown to reside within the N-terminal 181 amino acids stretch (aa 1–181) of SYT-SSX1 and 50 amino acids (aa 156–205) of hBRM/hSNF2α and we found that the overexpression of this binding region of hBRM/hSNF2α significantly suppressed the anchorage-independent growth of SYT-SSX1-expressing 3Y1 cells. To analyze the transcriptional regulation by SYT-SSX1, we established conditional expression system of SYT-SSX1 and examined the gene expression profiles. The down-regulation of potential tumor suppressor DCC was observed among 1,176 genes analyzed by microarray analysis, and semi-quantitative reverse transcription–PCR confirmed this finding. These data clearly demonstrate transforming activity of human oncogene SYT-SSX1 and also involvement of chromatin remodeling factor hBRM/hSNF2α in human cancer.

c-myc activation renders proliferation of Epstein-Barr virus (EBV)-transformed cells independent of EBV nuclear antigen 2 and latent membrane protein 1.

Two genetic events contribute to the development of endemic Burkitt lymphoma (BL) infection of B lymphocytes with Epstein-Barr virus (EBV) and the activation of the protooncogene c-myc through chromosomal translocation. The viral genes EBV nuclear antigen 2 (EBNA2) and latent membrane protein 1 (LMP1) are essential for transformation of primary human B cells by EBV in vitro; however, these genes are not expressed in BL cells in vivo. To address the question whether c-myc activation might abrogate the requirement of the EBNA2 and LMP1 function, we have introduced an activated c-myc gene into an EBV-transformed cell line in which EBNA2 was rendered estrogen-dependent through fusion with the hormone binding domain of the estrogen receptor. The c-myc gene was placed under the control of regulatory elements of the immunoglobulin kappa locus composed a matrix attachment region, the intron enhancer, and the 3' enhancer. We show here that transfection of a c-myc expression plasmid followed by selection for high MYC expression is capable of inducing continuous proliferation of these cells in the absence of functional EBNA2 and LMP1. c-myc-induced hormone-independent proliferation was associated with a dramatic change in the growth behavior as well as cell surface marker expression of these cells. The typical lymphoblastoid morphology and phenotype of EBV-transformed cells completely changed into that of BL cells in vivo. We conclude that the phenotype of BL cells reflects the expression pattern of viral and cellular genes rather than its germinal center origin.

The hybrid PAX3-FKHR fusion protein of alveolar rhabdomyosarcoma transforms fibroblasts in culture.

Pediatric alveolar rhabdomyosarcoma is characterized by a chromosomal translocation that fuses parts of the PAX3 and FKHR genes. PAX3 codes for a transcriptional regulator that controls developmental programs, and FKHR codes for a forkhead-winged helix protein, also a likely transcription factor. The PAX3-FKHR fusion product retains the DNA binding domains of the PAX3 protein and the putative activator domain of the FKHR protein. The PAX3-FKHR protein has been shown to function as a transcriptional activator. Using the RCAS retroviral vector, we have introduced the PAX3-FKHR gene into chicken embryo fibroblasts. Expression of the PAX3-FKHR protein in these cells leads to transformation: the cells become enlarged, grow tightly packed and in multiple layers, and acquire the ability for anchorage-independent growth. This cellular transformation in vitro will facilitate studies on the mechanism of PAX3-FKHR-induced oncogenesis.

Pax3 modulates expression of the c-Met receptor during limb muscle development.

Pax3 is a transcription factor whose expression has been used as a marker of myogenic precursor cells arising in the lateral somite destined to migrate to and populate the limb musculature. Accruing evidence indicates that the embryologic origins of axial and appendicular muscles are distinct, and limb muscle abnormalities in both mice and humans harboring Pax3 mutations support this distinction. The mechanisms by which Pax3 affects limb muscle development are unknown. The tyrosine kinase receptor for hepatocyte growth factor/scatter factor encoded by the c-met protooncogene is also expressed in limb muscle progenitors and, like Pax-3, is required in the mouse for limb muscle development. Here, we show that c-met expression is markedly reduced in the lateral dermomyotome of Splotch embryos lacking Pax3. We show that Pax3 can stimulate c-met expression in cultured cells, and we identify a potential Pax3 binding site in the human c-MET promoter that may contribute to direct transcriptional regulation. In addition, we have found that several cell lines derived from patients with rhabdomyosarcomas caused by a t(2;13) chromosomal translocation activating PAX3 express c-MET, whereas those rhabdomyosarcoma cell lines examined without the translocation do not. These results are consistent with a model in which Pax3 modulates c-met expression in the lateral dermomyotome, a function that is required for the appropriate migration of these myogenic precursors to the limb where the ligand for c-met (hepatocyte growth factor/scatter factor) is expressed at high levels.

Amino-terminal protein-protein interaction motif (POZ-domain) is responsible for activities of the promyelocytic leukemia zinc finger-retinoic acid receptor-alpha fusion protein.

Promyelocytic leukemia zinc finger-retinoic acid receptor a (PLZF-RARalpha), a fusion receptor generated as a result of a variant t(11;17) chromosomal translocation that occurs in a small subset of acute promyelocytic leukemia (APL) patients, has been shown to display a dominant-negative effect against the wild-type RARalpha/retinoid X receptor alpha (RXRalpha). We now show that its N-terminal region (called the POZ-domain), which mediates protein-protein interaction as well as specific nuclear localization of the wild-type PLZF and chimeric PLZF-RARalpha proteins, is primarily responsible for this activity. To further investigate the mechanisms of PLZF-RARalpha action, we have also studied its ligand-receptor, protein-protein, and protein-DNA interaction properties and compared them with those of the promyelocytic leukemia gene (PML)-RARalpha, which is expressed in the majority of APLs as a result of t(15;17) translocation. PLZF-RARalpha and PML-RARalpha have essentially the same ligand-binding affinities and can bind in vitro to retinoic acid response elements (RAREs) as homodimers or heterodimers with RXRalpha. PLZF-RARalpha homodimerization and heterodimerization with RXRalpha were primarily mediated by the POZ-domain and RARalpha sequence, respectively. Despite having identical RARalpha sequences, PLZF-RARalpha and PML-RARalpha homodimers recognized with different affinities distinct RAREs. Furthermore, PLZF-RARalpha could heterodimerize in vitro with the wild-type PLZF, suggesting that it may play a role in leukemogenesis by antagonizing actions of not only the retinoid receptors but also the wild-type PLZF and possibly other POZ-domain-containing regulators. These different protein-protein interactions and the target gene specificities of PLZF-RARalpha and PML-RARalpha may underlie, at least in part, the apparent resistance of APL with t(11;17) to differentiation effects of all-trans-retinoic acid.

In vivo and in vitro characterization of the B1 and B2 zinc-binding domains from the acute promyelocytic leukemia protooncoprotein PML.

Acute promyelocytic leukemia (APL) has been ascribed to a chromosomal translocation event which results in a fusion protein comprising the PML protein and retinoic acid receptor alpha. PML is normally a component of a nuclear multiprotein complex which is disrupted in the APL disease state. Here, two newly defined cysteine/histidine-rich protein motifs called the B-box (B1 and B2) from PML have been characterized in terms of their effect on PML nuclear body formation, their dimerization, and their biophysical properties. We have shown that both peptides bind Zn2+, which induces changes in the peptides' structures. We demonstrate that mutants in both B1 and B2 do not form PML nuclear bodies in vivo and have a phenotype that is different from that observed in the APL disease state. Interestingly, these mutations do not affect the ability of wild-type PML to dimerize with mutant proteins in vitro, suggesting that the B1 and B2 domains are involved in an additional interaction central to PML nuclear body formation. This report in conjunction with our previous work demonstrates that the PML RING-Bl/B2 motif plays a fundamental role in formation of a large multiprotein complex, a function that may be common to those unrelated proteins which contain the motif.

Intergenic splicing of MDS1 and EVI1 occurs in normal tissues as well as in myeloid leukemia and produces a new member of the PR domain family.

The EVI1 gene, located at chromosome band 3q26, is overexpressed in some myeloid leukemia patients with breakpoints either 5' of the gene in the t(3;3)(q21;q26) or 3' of the gene in the inv(3)(q21q26). EVI1 is also expressed as part of a fusion transcript with the transcription factor AML1 in the t(3;21)(q26;q22), associated with myeloid leukemia. In cells with t(3;21), additional fusion transcripts are AML1-MDS1 and AML1-MDS1-EVI1. MDS1 is located at 3q26 170-400 kb upstream (telomeric) of EVI1 in the chromosomal region in which some of the breakpoints 5' of EVI1 have been mapped. MDS1 has been identified as a single gene as well as a previously unreported exon(s) of EVI1 We have analyzed the relationship between MDS1 and EVI1 to determine whether they are two separate genes. In this report, we present evidence indicating that MDS1 exists in normal tissues both as a unique transcript and as a normal fusion transcript with EVI1, with an additional 188 codons at the 5' end of the previously reported EVI1 open reading frame. This additional region has about 40% homology at the amino acid level with the PR domain of the retinoblastoma-interacting zinc-finger protein RIZ. These results are important in view of the fact that EVI1 and MDS1 are involved in leukemia associated with chromosomal translocation breakpoints in the region between these genes.

Selective repression of transcriptional activators by Pbx1 does not require the homeodomain.

PBX1 is a homeobox-containing gene identified as the chromosome 1 participant of the t(1;19) chromosomal translocation of childhood pre-B-cell acute lymphoblastic leukemia. This translocation produces a fusion gene encoding the chimeric oncoprotein E2A-Pbx1, which can induce both acute myeloid and T-lymphoid leukemia in mice. The binding of Pbx1 to DNA is weak; however, both Pbx1 and E2A-Pbx1 exhibit tight binding to specific DNA motifs in conjunction with certain other homeodomain proteins, and E2A-Pbx1 activates transcription through these motifs, whereas Pbx1 does not. In this report, we investigate potential transcriptional functions of Pbx1, using transient expression assays. While no segments of Pbx1 activated transcription, an internal domain of Pbx1 repressed transcription induced by the activation domain of Sp1, but not by the activation domains of VP16 or p53. This Pbx1 domain, which lies upstream of the homeodomain and is highly conserved among Pbx proteins, is thus predicted to bind a specific transcription factor. Surprisingly, the repression activity of Pbx1 did not require homeodomain-dependent DNA binding. Thus, Pbx1 may be able to alter gene transcription by both DNA-binding-dependent and DNA-binding-independent mechanisms.

Multiple genetic loci within 11p15 defined by Beckwith-Wiedemann syndrome rearrangement breakpoints and subchromosomal transferable fragments.

Beckwith-Wiedemann syndrome (BWS) involves fetal overgrowth and predisposition to a wide variety of embryonal tumors of childhood. We have previously found that BWS is genetically linked to 11p15 and that this same band shows loss of heterozygosity in the types of tumors to which children with BWS are susceptible. However, 11p15 contains > 20 megabases, and therefore, the BWS and tumor suppressor genes could be distinct. To determine the precise physical relationship between these loci, we isolated yeast artificial chromosomes, and cosmid libraries from them, within the region of loss of heterozygosity in embryonal tumors. Five germ-line balanced chromosomal rearrangement breakpoint sites from BWS patients, as well as a balanced chromosomal translocation breakpoint from a rhabdoid tumor, were isolated within a 295- to 320-kb cluster defined by a complete cosmid contig crossing these breakpoints. This breakpoint cluster terminated approximately 100 kb centromeric to the imprinted gene IGF2 and 100 kb telomeric to p57KIP2, an inhibitor of cyclin-dependent kinases, and was located within subchromosomal transferable fragments that suppressed the growth of embryonal tumor cells in genetic complementation experiments. We have identified 11 transcribed sequences in this BWS/tumor suppressor coincident region, one of which corresponded to p57KIP2. However, three additional BWS breakpoints were > 4 megabases centromeric to the other five breakpoints and were excluded from the tumor suppressor region defined by subchromosomal transferable fragments. Thus, multiple genetic loci define BWS and tumor suppression on 11p15.

Multimeric complexes of the PML-retinoic acid receptor alpha fusion protein in acute promyelocytic leukemia cells and interference with retinoid and peroxisome-proliferator signaling pathways.

The t(15;17) chromosomal translocation, specific for acute promyelocytic leukemia (APL), fuses the PML gene to the retinoic acid receptor alpha (RAR alpha) gene, resulting in expression of a PML-RAR alpha hybrid protein. In this report, we analyzed the nature of PML-RAR alpha-containing complexes in nuclear protein extracts of t(15;17)-positive cells. We show that endogenous PML-RAR alpha can bind to DNA as a homodimer, in contrast to RAR alpha that requires the retinoid X receptor (RXR) dimerization partner. In addition, these cells contain oligomeric complexes of PML-RAR alpha and endogenous RXR. Treatment with retinoic acid results in a decrease of PML-RAR alpha protein levels and, as a consequence, of DNA binding by the different complexes. Using responsive elements from various hormone signaling pathways, we show that PML-RAR alpha homodimers have altered DNA-binding characteristics when compared to RAR alpha-RXR alpha heterodimers. In transfected Drosophila SL-3 cells that are devoid of endogenous retinoid receptors PML-RAR alpha inhibits transactivation by RAR alpha-RXR alpha heterodimers in a dominant fashion. In addition, we show that both normal retinoid receptors and the PML-RAR alpha hybrid bind and activate the peroxisome proliferator-activated receptor responsive element from the Acyl-CoA oxidase gene, indicating that retinoids and peroxisome proliferator receptors may share common target genes. These properties of PML-RAR alpha may contribute to the transformed phenotype of APL cells.

MMSET deregulation affects cell cycle progression and adhesion regulons in t(4;14) myeloma plasma cells

BackgroundThe recurrent immunoglobulin translocation, t(4;14)(p16;q32) occurs in 15% of multiple myeloma patients and is associated with poor prognosis, through an unknown mechanism. The t(4;14) up-regulates fibroblast growth factor receptor 3 (FGFR3) and multiple myeloma SET domain (MMSET) genes. The involvement of MMSET in the pathogenesis of t(4;14) multiple myeloma and the mechanism or genes deregulated by MMSET upregulation are still unclear.Design and MethodsThe expression of MMSET was analyzed using a novel antibody. The involvement of MMSET in t(4;14) myelomagenesis was assessed by small interfering RNA mediated knockdown combined with several biological assays. In addition, the differential gene expression of MMSET-induced knockdown was analyzed with expression microarrays. MMSET gene targets in primary patient material was analyzed by expression microarrays.ResultsWe found that MMSET isoforms are expressed in multiple myeloma cell lines, being exclusively up-regulated in t(4;14)-positive cells. Suppression of MMSET expression affected cell proliferation by both decreasing cell viability and cell cycle progression of cells with the t(4;14) translocation. These findings were associated with reduced expression of genes involved in the regulation of cell cycle progression (e.g. CCND2, CCNG1, BRCA1, AURKA and CHEK1), apoptosis (CASP1, CASP4 and FOXO3A) and cell adhesion (ADAM9 and DSG2). Furthermore, we identified genes involved in the latter processes that were differentially expressed in t(4;14) multiple myeloma patient samples.ConclusionsIn conclusion, dysregulation of MMSET affects the expression of several genes involved in the regulation of cell cycle progression, cell adhesion and survival.

Common chromosomal fragile site FRA16D sequence: identification of the FOR gene spanning FRA16D and homozygous deletions and translocation breakpoints in cancer cells

Fluorescence in situ hybridization of a tile path of DNA subclones has previously enabled the cytogenetic definition of the minimal DNA sequence which spans the FRA16D common chromosomal fragile site, located at 16q23.2. Homozygous deletion of the FRA16D locus has been reported in adenocarcinomas of stomach, colon, lung and ovary. We have sequenced the 270 kb containing the FRA16D fragile site and the minimal homozygously deleted region in tumour cells. This sequence enabled localization of some of the tumour cell breakpoints to regions which contain AT-rich secondary structures similar to those associated with the FRA10B and FRA16B rare fragile sites. The FRA16D DNA sequence also led to the identification of an alternatively spliced gene, named FOR (fragile site FRA16D oxidoreductase), exons of which span both the fragile site and the minimal region of homozygous deletion. In addition, the complete DNA sequence of the FRA16D-containing FOR intron reveals no evidence of additional authentic transcripts. Alternatively spliced FOR transcripts (FOR I, FOR II and FOR III) encode proteins which share N-terminal WW domains and differ at their C-terminus, with FOR III having a truncated oxidoreductase domain. FRA16D-associated deletions selectively affect the FOR gene transcripts. Three out of five previously mapped translocation breakpoints in multiple myeloma are also located within the FOR gene. FOR is therefore the principle genetic target for DNA instability at 16q23.2 and perturbation of FOR function is likely to contribute to the biological consequences of DNA instability at FRA16D in cancer cells.

Cervical nerve root synovial sarcoma in a child with chromosomal (X;18) translocation. Case report and review of the literature.

We report on an 11-year-old female with a history of cervicobrachialgia and progressive weakness of the right arm. Cervical spine MRI showed an enhancing heterogeneous intradural mass occupying the right C6-C7 foramen. She underwent a right C6-C7 foraminotomy with a complete macroscopic removal of the lesion. Pathological examination revealed a synovial sarcoma. Treatment was completed by chemotherapy and proton radiotherapy, and the girl remained free of symptoms for 3 years. After appearance of new symptoms, a local recurrence was confirmed, and despite aggressive treatment with salvage chemotherapy and radiotherapy, the disease progressed beyond medical control, and the child died, 6 years after diagnosis. Early recognition of this rare entity compared to its more benign differential diagnosis is crucial, as an aggressive management is needed.