Identification of the 12q15 amplicon within the homogeneously staining regions in the embryonal rhabdomyosarcoma cell line RMS-YM.

Gene amplification represents one of the molecular mechanisms of oncogene overexpression in many types of tumors. Homogeneously staining regions (HSRs) are cytogenetic hallmarks of gene amplification. Rhabdomyosarcoma is the most common malignant soft-tissue tumor in children. RMS-YM is an embryonal rhabdomyosarcoma cell line that possesses 3 HSRs. This cytogenetic finding suggests the presence of gene amplifications associated with tumor development or progression in RMS-YM. Here, using fluorescence in situ hybridization, we detected high amplification of the MDM2 gene in the HSRs of RMS-YM. We also refined the region of the amplicon and identified that the FRS2 gene and others are amplified in RMS-YM. MDM2 and FRS2 play important roles as a regulator of p53 and a mediator of FGF signaling, respectively, and thus are potential molecular targets for therapy in many different tumors. RMS-YM may be useful for studies of the molecular pathways of tumorigenesis and tumor progression in rhabdomyosarcoma and for in vitro evaluation of newly developed therapeutic agents that target MDM2 or FRS2.

PAX3-NCOA2 fusion gene has a dual role in promoting the proliferation and inhibiting the myogenic differentiation of rhabdomyosarcoma cells.

We analyzed a complex chromosomal translocation in a case of embryonal rhabdomyosarcoma (RMS) and showed that it generates the fusion gene PAX3 (paired box 3)-NCOA2 (nuclear receptor coactivator 2). To understand the role of this translocation in RMS tumorigenesis, we established two types of stable mouse myoblast C2C12 cell lines expressing PAX3-NCOA2 and PAX3-FOXO1A (forkhead box O1A), respectively. Compared with control cells, PAX3-NCOA2 cells grew faster, were more motile, were less anchorage dependent, progressed more quickly through the G1/S phase of cell cycle and showed greater transcriptional activation of the PAX3 consensus-binding site. However, PAX3-NCOA2 cells proliferated more slowly and differentiated more weakly than did PAX3-FOXO1A cells. Both PAX3-NCOA2 cells and PAX3-FOXO1A cells formed tumors in nude mice, although the PAX3-NCOA2-induced tumors grew more slowly. Our results may explain why NCOA2 rearrangement is mainly found in embryonal rhabdomyosarcoma, which has a better prognosis than alveolar rhabdomyosarcoma, which expresses the PAX3-FOXO1A fusion gene. These results indicate that the PAX3-NCOA2 fusion gene has a dual role in the tumorigenesis of RMS: promotion of the proliferation and inhibition of the myogenic differentiation of RMS cells.

The chromosomal constitution of postmitotic neurons, assessed by neuronal nuclear transfer into oocytes and in ES cell lines derived from them.

Spectral karyotyping (SKY) was used to assess the chromosomal constitution of embryos generated by nuclear transfer (NT) of neuronal nuclei (N-NT) or cumulus cell nuclei (C-NT) into oocytes and of their embryonic stem cell derivatives (ntES cells). We detected chromosomal changes during the first mitotic cleavage and in the condensed chromatids of NT embryos. We also found clonal translocations in the ntES cells that were derived from NT embryos cloned from neuronal nuclei. The differentiation potentials of the ntES cells showing chromosomal rearrangements were partly restricted. Our findings indicate that balanced or unbalanced chromosomal translocations can occur in early NT embryogenesis, suggesting that a DNA repair system is activated during both NT embryogenesis and ntES cell establishment. We observed a higher incidence of chromosomal changes in N-NT than in C-NT embryos, which may reflect a higher frequency of double-stranded (ds) DNA breaks in the neuronal genome.

Multicolor banding technique, spectral color banding (SCAN): new development and applications.

Conventional banding techniques can characterize chromosomal aberrations associated with tumors and congenital diseases with considerable precision. However, chromosomal aberrations that have been overlooked or are difficult to analyze even by skilled cytogeneticists were also often noted. Following the introduction of multicolor karyotyping such as spectral karyotyping (SKY) and multiplex-fluorescence in situ hybridization (M-FISH), it is possible to identify this kind of cryptic or complex aberration comprehensively by a single analysis. To date, multicolor karyotyping techniques have been established as useful tools for cytogenetic analysis. However, since this technique depends on whole chromosome painting probes, it involves limitations in that the origin of aberrant segments can be identified only in units of chromosomes. To overcome these limitations, we have recently developed spectral color banding (SCAN) as a new multicolor banding technique based on the SKY methodology. This new technique may be deemed as an ideal chromosome banding technique since it allows representation of a multicolor banding pattern matching the corresponding G-banding pattern. We applied this technique to the analysis of chromosomal aberrations in tumors that had not been fully characterized by G-banding or SKY and found it capable of (1) detecting intrachromosomal aberrations; (2) identifying the origin of aberrant segments in units of bands; and (3) precisely determining the breakpoints of complex rearrangements. We also demonstrated that SCAN is expected to allow cytogenetic analysis with a constant adequate resolution close to the 400-band level regardless of the degree of chromosome condensation. As compared to the conventional SKY analysis, SCAN has remarkably higher accuracy for a particular chromosome, allowing analysis in units of bands instead of in units of chromosomes and is hence promising as a means of cytogenetic analysis.

A novel infant acute lymphoblastic leukemia cell line with MLL-AF5q31 fusion transcript.

Infant acute lymphoblastic leukemia (ALL) is characterized by the presence of the proB phenotype (CD10(-)/CD19(+)), poor prognosis and frequent rearrangement of the mixed lineage leukemia (MLL) gene. The most frequent rearrangement is t(4;11)(q21;q23), the role of whose product, the MLL-AF4 fusion transcript, has been extensively studied in leukemogenesis. In a cell line of infant leukemia with MLL rearrangement denoted KP-L-RY, panhandle PCR amplification of cDNA revealed the presence of a fusion transcript, MLL-AF5q31, indicating that AF5q31 is also a partner gene of MLL. In this fusion transcript the MLL exon 6 is fused in frame to the 5' side of the putative transactivation domain of AF5q31. The AF5q31 protein is a member of the AF4/LAF4/FMR2-related family of proteins, which have been suggested to play a role in hematopoietic cell growth and differentiation. The MLL-AF5q31 fusion transcript, although probably rare, appears to be associated with the pathogenesis of infant ALL like MLL-AF4. Co-expression of HoxA9 and Meis1 genes in the KP-L-RY cell line indicated possible functional similarity between MLL-AF4 and MLL-AF5q31. Further understanding of the function of AF5q31 as well as the specific leukemogenic mechanism of MLL-AF5q31 awaits future studies.

Involvement of the NUP98 gene in a chromosomal translocation t(11;20)(p15;q11.2) in a patient with acute monocytic leukemia (FAB-M5b).

We report here a case of acute monocytic leukemia (M5b subtype according to the French-American-British [FAB] classification) with chromosomal translocation t(11;20)(p15;q11.2). Fluorescence in situ hybridization analysis with a probe for the NUP98 gene, which is located at chromosome band 11p15, showed that the probe hybridized to both derivative chromosomes 11 and 20 as well as to the remaining normal chromosome 11, indicating that the NUP98 gene was split and involved in this translocation. This is the first report of t(11;20)(p15;q11.2) involving the NUP98 gene in overt leukemia.

A variant form of myelodysplastic syndrome with Ph- minor-BCR/ABL transcript.

This study concerns a patient with minor (m)-BCR/ABL transcript-positive and Philadelphia (Ph) chromosome-negative myelodysplastic syndrome (MDS). The patient was a 78-year-old man whose condition was diagnosed as refractory anemia with excess of blasts in transformation. Molecular genetic studies, using reverse transcriptase polymerase chain reaction analysis detected m-BCR/ABL messenger RNA. We used spectral karyotyping to analyze metaphase cells but could not detect a Ph chromosome. Fluorescence in situ hybridization, however, revealed fusion signals of BCR and ABL probes on an apparently normal chromosome 22.

Agranular CD4+CD56+ blastic natural killer leukemia/lymphoma.

Blastic natural killer cell leukemia/lymphoma (blastic NKL/L) is characterized by blastic morphology and a distinctive immunophenotype combining blastic features and cytologically resembling acute myeloid or lymphoid leukemia. The clinical, pathologic, and cytogenetic features of blastic NKL/L have not yet been systematically identified. We report herein a case of blastic NKL/L with skin lesion, adenopathy, and systemic lymphoadenopathy. The identified tumor cells were positive for CD4 and CD56, and negative for T-cell, B-cell, and myeloid markers. T-cell receptor beta, gamma, delta, and immunoglobulin heavy chain genes in the bone marrow cells showed germ-line configurations. Southern blot analysis with a terminal probe did not reveal any Epstein-Barr virus infection. Although patients diagnosed as blastic NKL/L have generally shown chemotherapy resistance and poor prognosis, our patient was treated with a combined chemotherapy, which is also used for acute lymphoblastic leukemia, and has maintained complete remission (CR) for more than 13 months. In addition to clinical investigations, we thoroughly analyzed his karyotype by using a combination of G-banding and a new technique, spectral karyotyping. The karyotype was described as 45, XY, der(1)t(1;20)(p32;q11.2), der(6) (1pter-->1p32:: 6p21.1-->6q13:: 7q11.2-->7qter), der(7) t(7;20)(q11.2;q11.2), t(13;14)(q14;q32), der(13)t(6;13) (p21.1; q14), -20.

Isolation and characterization of a human thiamine pyrophosphokinase cDNA.

A human thiamine pyrophosphokinase cDNA clone (hTPK1) was isolated and sequenced. When the intact hTPK1 open reading frame was expressed as a histidine-tag fusion protein in Escherichia coli, marked enzyme activity was detected in the bacterial cells. The hTPK1 mRNA was widely expressed in various human tissues at a very low level, and the mRNA content in cultured fibroblasts was unaffected by the thiamine concentration of the medium. The chromosome localization of the hTPK1 gene was assigned to 7q34.

Cloning and characterization of LUN, a novel ring finger protein that is highly expressed in lung and specifically binds to a palindromic sequence.

We isolated cDNAs encoding a novel RING finger protein (LUN), the mRNAs of which were expressed at high levels in the lung. In situ hybridization revealed that LUN mRNAs were expressed in the alveolar epithelium of the lung. The LUN gene locus was assigned to chromosome 9p21, which contains candidate tumor suppressor genes associated with loss of heterozygosity in more than 86% of small cell lung cancers. We clarified that LUN is localized to the nucleus and reveals Zn(2+)-dependent DNA binding activity. The region from amino acids 51 to 374 of LUN is responsible for DNA binding. Furthermore, we identified a novel palindromic binding consensus (5'-TCCCAGCACTTTGGGA-3') for the LUN binding. Interestingly, this LUN binding palindromic sequence is found in the upstream transcriptional regulatory region of the E-cadherin gene and two intervening regions of the talin gene. Our results suggested that LUN might be an important trans-acting transcriptional regulator for lung cancer-associated genes including E-cadherin and talin genes.

Identification and characterization of SEB, a novel protein that binds to the acute undifferentiated leukemia-associated protein SET.

SET, the translocation breakpoint-encoded protein in acute undifferentiated leukemia (AUL), is a 39-kDa nuclear phosphoprotein and has an inhibitory activity for protein phosphatase 2A (PP2A). SET is fused to a putative oncoprotein, CAN/NUP214, in AUL and is thought to play a key role in leukemogenesis by its nuclear localization, protein-protein interactions and PP2A inhibitory activity. Here, we describe the isolation and characterization of a novel cDNA encoding a protein with 1542 amino-acid residues that specifically interacts in a yeast two-hybrid system as well as in human cells with SET. This new protein, which we name SEB (SET-binding protein), is identified as a 170-kDa protein by immunoprecipitation with a specific antibody and is localized predominantly in the nucleus. SEB1238--1434 is determined as a SET-binding region that specifically binds to SET182--223. SEB also has an oncoprotein Ski homologous region (amino acids 654--858), six PEST sequences and three sequential PPLPPPPP repeats at the C-terminus. SEB mRNA is expressed ubiquitously in all human adult tissues and cells examined. The SEB gene locus is assigned to the chromosome 18q21.1 that contains candidate tumor suppressor genes associated with deletions in cancer and leukemia. Although the function of SEB is not known, we propose that SEB plays a key role in the mechanism of SET-related leukemogenesis and tumorigenesis, perhaps by suppressing SET function or by regulating the transforming activity of Ski in the nucleus.

Development of spectral colour banding in cytogenetic analysis.

We developed a novel chromosome banding technique-spectral colour banding (SCAN). With this technique we displayed a multicolour banding pattern that almost entirely correlated with the corresponding G-banding pattern. With SCAN analysis we could identify the chromosome-band origin of double minute chromosomes in gastric cancer. Our preliminary use of this technique suggests that it has significant clinical applications for cytogenetic analysis.

Fusion of MOZ and p300 histone acetyltransferases in acute monocytic leukemia with a t(8;22)(p11;q13) chromosome translocation.

Histone acetyltransferase p300 functions as a transcriptional co-activator which interacts with a number of transcription factors. Monocytic leukemia zinc finger protein (MOZ) has histone acetyltransferase activity. We report the fusion of the MOZ gene to the p300 gene in acute myeloid leukemia with translocation t(8;22)(p11;q13). FISH and Southern blot analyses showed the rearrangement of the MOZ and p300 genes. We determined the genomic structure of the p300 and the MOZ genes and the breakpoints of the translocation. Analysis of fusion transcripts indicated that the zinc finger and acetyltransferase domains of MOZ are fused to a largely intact p300. These results suggest that MOZ-p300, which has two acetyltransferase domains, could be involved in leukemogenesis through aberrant regulation of histone acetylation.

Characterization of complex chromosomal abnormalities in B-cell lymphoma by a combined spectral karyotyping (SKY) analysis and fluorescence in situ hybridization (FISH) using a 14q telomere probe.

We report a case of non-Hodgkin's lymphoma of unknown origin with invasion into bone marrow and brain. This case showed complex chromosomal abnormalities, including five clonal marker chromosomes (mar) and four additional materials of unknown origin (add) that could not be identified by means of conventional G-banding. Spectral karyotyping (SKY) analysis could not only determine the origin and organization of all thus far unidentified structural chromosomal abnormalities but also detect two cryptic unbalanced translocations, which had been erroneously considered to be normal on the basis of G-banding analysis, and correct one abnormality misidentified by G banding. Among these abnormalities, we identified the new partner site of the 14q32 translocation, 22q13, and the jumping translocations involving 2p23 as a new donor chromosome. Furthermore, by using fluorescence in situ hybridization (FISH) with the probes specific for the 14q telomere, we could identify the unbalanced translocation of t(3;14)(q27;q32), which had been erroneously considered to be normal chromosome 3 on the basis of not only G-banding but also of SKY analysis. This translocation is one of the most frequent chromosomal abnormalities in B-cell lymphoma, especially diffuse large cell lymphoma. After SKY and FISH analysis, the original descriptions in the G-band karyotype were modified for a total of 13 chromosomes. The combination of SKY and FISH using the 14q telomere probe was therefore considered very useful for the characterization of complex cytogenetic cases in B-cell lymphoma.

Aggressive natural killer cell leukemia/lymphoma: a comprehensive cytogenetic study by spectral karyotyping.

A 38-year-old male presented with fever and hepatosplenomegaly. Cells that had infiltrated to the bone marrow were consistent immunophenotypically and genotypically with natural killer (NK) cells. Oligoclonal Epstein-Barr virus infection was detected in the bone marrow cells. The patient was diagnosed as a case of aggressive NK cell leukemia/lymphoma. Combined chemotherapy was not effective and death occurred shortly after presentation. Although the karyotype of this case was too complicated to be accurately identified only by G-banding, spectral karyotyping (SKY) analysis not only identified all chromosomal materials of unknown origin, but also detected the cryptic translocation on the apparently normal chromosome. Moreover, SKY analysis identified der(4)t(4;14)(q12;q11.2). The chromosomal band 14q11.2 is a recurring breakpoint in T-cell non-Hodgkin's lymphoma, and is also the locus of the delta chain of the T-cell receptor. To our knowledge, t(4;14)(q12;q11.2) in T-cell or NK-cell malignancies has not been previously reported.

PEBP2alphaA/CBFA1 mutations in Japanese cleidocranial dysplasia patients.

Cleidocranial dysplasia (CCD) is an autosomal dominant human bone disease whose genetic locus has been located on chromosome 6p21, where the PEBP2alphaA/CBFA1 gene essential for osteogenesis also maps. Previously, several heterozygous mutations in PEBP2alphaA/CBFA1 were found in CCD patients. In this study, we identified six different types of mutations in PEBP2alphaA/CBFA1 in Japanese CCD patients. Four cases were similar to those reported previously: two were nonsense mutations in the Runt domain, one was a hemizygous deletion, and the other was a missense mutation in the Runt domain which abolished the DNA-binding activity of Runx2/PEBP2alphaA/CBFA1. The remaining two mutations were novel: one had a heterozygous gt-to-tt mutation at the splice donor site (gt) between the exon3-intron junction, which resulted in abnormal exon3 skipping, and the other had a mutation in exon7, which led to the introduction of a translational stop codon in the middle of the transactivation domain. Thus, defects in either the DNA-binding domain or transactivation domain of Runx2/PEBP2alphaA/CBFA1 can cause CCD. The results not only provide a strong genetic evidence that mutations involving in PEBP2alphaA/CBFA1 contribute to CCD, but also provide a useful tool to study how Runx2/PEBP2alphaA/CBFA1 plays its pivotal role during osteoblastic differentiation.

Mre11 is essential for the maintenance of chromosomal DNA in vertebrate cells.

Yeast Mre11 functions with Rad50 and Xrs2 in a complex that has pivotal roles in homologous recombination (HR) and non-homologous end-joining (NHEJ) DNA double-strand break (DSB) repair pathways. Vertebrate Mre11 is essential. Conditionally, MRE11 null chicken DT40 cells accumulate chromosome breaks and die upon Mre11 repression, showing frequent centrosome amplification. Mre11 deficiency also causes increased radiosensitivity and strongly reduced targeted integration frequencies. Mre11 is, therefore, crucial for HR and essential in mitosis through its role in chromosome maintenance by recombinational repair. Surprisingly perhaps, given the role of Mre11 in yeast NHEJ, disruption of NHEJ by deletion of KU70 greatly exacerbates the effects of MRE11 deficiency, revealing a significant Mre11-independent component of metazoan NHEJ.

Combined spectral karyotyping and DAPI banding analysis of chromosome abnormalities in myelodysplastic syndrome.

Spectral karyotyping (SKY) is a new molecular cytogenetic technique that allows simultaneous visualization of each chromosome in a different color. We have used SKY for comprehensive analysis of 20 myelodysplastic syndromes (MDSs) (13 primary MDSs, 3 therapy-related MDSs, and 4 acute leukemias developed from MDS, including 1 cell line established from a secondary leukemia), previously analyzed by G-banding. To locate the chromosomal breakpoints, DAPI-counterstained band images from all metaphases were transformed to G-band-like patterns. By using SKY, it was possible to identify the origin and organization of all clonal marker chromosomes (mar), as well as the origin of all abnormalities defined as additional material of unknown origin (add) or homogeneously staining regions (hsr) by G-banding. In total, SKY identified the chromosomal basis of 38 mar, add, and hsr, corrected 8 abnormalities misidentified by G-banding, and revealed 6 cryptic translocations in 5 cases. Total or partial chromosomal loss (mainly of -5/5q- and -7/7q-) is the most frequent cytogenetic abnormality in MDS. In 3 of 11 cases with -5/5q- and in 4 of 8 with -7/7q-, lost material was detected by SKY in unbalanced translocations. A total of 60 chromosomal losses were identified by G-banding in 16 cases with multiple chromosome abnormalities involving at least 3 chromosomes. For 26 of these losses (43%), SKY analysis suggested that the losses were not complete, but had been translocated to a variety of partner chromosomes. Moreover, SKY analysis revealed that a ring chromosome in a case of acute leukemia developed from MDS contained three to six segments that originated from chromosome 21 material. Fluorescence in situ hybridization showed the amplification of the AML1 gene on regions derived from chromosome 21, providing the first evidence of amplification involving this gene in MDS. Genes Chromosomes Cancer 26:336-345, 1999.