E2A-ZNF384 and NOL1-E2A fusion created by a cryptic t(12;19)(p13.3; p13.3) in acute leukemia.

A 5-year-old boy who initially presented with ALL and relapsed 4 months later with AML was found to have an add(19) in the leukemia cells. FISH revealed that the add(19) was really a cryptic t(l2;l9)(p13.3;p13.3) interrupting E2A (TCF3). Nucleotide sequences of cloned genomic fragments with the E2A rearrangements revealed that the der(12) contained E2A joined to an intron of the NOLI (p120) gene. Reverse transcriptase (RT)-PCR of patient lymphoblast RNA showed expression of in-frame fusion cDNAs consisting of most of NOL1 fused to the 3' portion of E2A that encoded part of the second transcriptional activation domain and the DNA binding and protein dimerization motifs. The reciprocal der(19) E2A genomic rearrangements included 5' regions of E2A joined to an intron of the ZNF384 (NMP4, CIZ) gene, located approximately 450 kb centromeric to NOL1 on chromosome 12. RT-PCR showed expression of in-frame E2A-ZNF384 fusion cDNAs. To our knowledge, this is the second report of a chromosome translocation in leukemia resulting in two different gene fusions. This is the first report of expression of E2A fusion protein that includes the DNA binding and protein dimerization domains due to a more proximal break in E2A compared to those described previously.

Light-induced giant softening of network glasses observed near the mean-field rigidity transition.

The longitudinal acoustic (LA) mode of bulk GexSe1-x glasses is examined in Brillouin scattering (BS) over the 0.15

Detailed computational study of p53 and p16: using evolutionary sequence analysis and disease-associated mutations to predict the functional consequences of allelic variants.

Deciding whether a missense allelic variant affects protein function is important in many contexts. We previously demonstrated that a detailed analysis of p53 intragenic conservation correlates with somatic mutation hotspots. Here we refine these evolutionary studies and expand them to the p16/Ink4a gene. We calculated that in order for 'absolute conservation' of a codon across multiple species to achieve P<0.05, the evolutionary substitution database must contain at least 3(M) variants, where M equals the number of codons in the gene. Codons in p53 were divided into high (73% of codons), intermediate (29% of codons), and low (0 codons) likelihood of being mutation hotspots. From a database of 263 somatic missense p16 mutations, we identified only four codons that are mutational hotspots at P<0.05 (8 mutations). However, data on function, structure, and disease association support the conclusion that 11 other codons with > or =5 somatic mutations also likely indicate functionally critical residues, even though P0.05. We calculated p16 evolution using amino acid substitution matrices and nucleotide substitution distances. We looked for evolutionary parameters at each codon that would predict whether missense mutations were disease associated or disrupted function. The current p16 evolutionary substitution database is too small to determine whether observations of 'absolute conservation' are statistically significant. Increasing the number of sequences from three to seven significantly improved the predictive value of evolutionary computations. The sensitivity and specificity for conservation scores in predicting disease association of p16 codons is 70-80%. Despite the small p16 sequence database, our calculations of high conservation correctly predicted loss of cell cycle arrest function in 75% of tested codons, and low conservation correctly predicted wild-type function in 80-90% of codons. These data validate our hypothesis that detailed evolutionary analyses help predict the consequences of missense amino-acid variants.

Unusual childhood extramedullary hematologic malignancy with natural killer cell properties that contains tropomyosin 4--anaplastic lymphoma kinase gene fusion.

This report describes an unusual extramedullary hematologic malignancy in an 18-month-old child who presented with a capillary leak syndrome that evolved into hyperleukocytosis with malignant cells. The circulating tumor cells did not express an antigen profile typical of any subtype of leukemia commonly observed in children. Tumor cells were CD3(-)/CD56(+); had germline TCR genes; and strongly expressed CD30, epithelial membrane antigen, and anaplastic lymphoma kinase (ALK) consistent with a null cell anaplastic large cell lymphoma (ALCL). The malignant cells contained a t(2;19)(p23;p13.1) that interrupted ALK and translocated it to the der(19). Reverse transcriptase-polymerase chain reaction and nucleotide sequence analysis revealed fusion of ALK to tropomyosin 4, an ALK fusion partner not described previously in hematologic malignancies. The clinical presentation and phenotypic features of this malignancy were not typical for ALCL because tumor cells expressed both myeloid (CD13, CD33, HLA-DR) and natural killer (NK) cell antigens. The neoplastic cells most resembled NK cells because in addition to being CD3(-)/CD56(+) with germline TCR genes, these cells were CD25(+)/CD122(+)/granzyme B(+) and possessed the functional properties of immature NK cells. The unusual clinical presentation, immunophenotype, and functional properties of these neoplastic cells suggest that this malignancy may be derived from the putative myeloid-NK precursor cell. Furthermore co-expression of NK and ALCL features supports the concept that a minority of null-ALCL may be derived from NK cells and expands the spectrum of phenotypes that can be seen in tumors produced by ALK fusion proteins. (Blood. 2001;98:1209-1216)

An antibody to p16INK4A recognizes a modified form of galectin-3.

Galectin-3 is a carbohydrate binding protein involved in multiple processes including cell-cycle regulation and apoptosis. The ability of galectin-3 to protect cells from apoptosis is dependent upon a region of the protein known as a BH-1 domain for its homology to the anti-apoptotic protein Bcl-2. Here, we show that a monoclonal antibody (MAb) to the human tumor suppressor protein p16INK4A recognizes a post-translationally modified form of human galectin-3. The modified form is detectable in only a subset of cell types expressing galectin-3, indicating that the modification is cell-type-specific. Although there is little amino acid sequence homology between p16INK4a and galectin-3, we show by epitope mapping that the modification directly affects the structure of galectin-3's BH-1 domain. Elucidation of the nature of this modification might provide further insight into galectin-3 function.

The COOH terminus of p18INK4C distinguishes function from p16INK4A.

The INK4 family of proteins consists of four members which can block progression from the G(1)-to-S phase of the cell cycle by inhibiting the activity of cyclin dependent kinases (cdks) 4 and 6. Although the gene encoding p16(INK4a) is commonly inactivated in human tumors, p18(INK4c) is rarely altered. We show here that overexpression of p18(INK4c) does not block cell cycle progression in a T-cell acute lymphocytic leukemia cell line (CEM) sensitive to p16(INK4a)-mediated G(1) arrest. A chimera consisting of the kinase-binding region of p16(INK4a) fused to the COOH terminus of p18(INK4c) is active in all known biochemical assays for INK4 function, but it does not arrest CEM cells. These data imply a novel level of p18(INK4c) regulation mediated through the COOH terminus and suggest that functional differences might underlie the distinct mutational profiles observed for p16(INK4a) and p18(INK4c) in tumors.

Analysis of TP53 mutations in relapsed childhood acute lymphoblastic leukemia.

TP53 is the most commonly mutated gene in human cancer, but TP53 mutations are present in less than 5% of children with acute lymphoblastic leukemia (ALL) at initial presentation. Mutations are detected more frequently in children with relapsed T-cell ALL, but the potential role of TP53 mutations in relapsed B-lineage childhood ALL is not understood as well. The authors determined the nucleotide sequence of amplified DNA from exons 5 to 8 of the TP53 gene in leukemic cells obtained from 17 children with ALL at the time of first bone marrow relapse. All 17 contained only germline TP53 sequences. Review of the published literature disclosed that TP53 mutations have been found in 22% of cases of relapsed ALL. To understand the role of p53 abnormalities in this clinical setting, it will be important for future studies to analyze cases of relapsed ALL with assays capable of interrogating the functional integrity of the p53 pathway.

Histone deacetylase interacts directly with DNA topoisomerase II.

Histone deacetylases (HDACs) modify nucleosomal histones, have a key role in the regulation of gene transcription, and may be involved in cell-cycle regulation, differentiation and human cancer. Purified recombinant human HDAC1 protein was used to screen a cDNA expression library, and one of the clones identified encoded DNA topoisomerase II (Topo II), an enzyme known to have a role in transcriptional regulation and chromatin organization. Coimmunoprecipitation experiments indicate that HDAC1 and HDAC2 are associated with Topo II in vivo under normal physiological conditions. Complexes containing Topo II possess HDAC activities, and complexes containing HDAC1 or HDAC2 possess Topo II activities. HDAC and Topo II modify each other's activity in vitro and in vivo. Our results indicate the existence of a functionally coupled complex between these two enzymes and offer insights into the potential mechanisms of action of both enzymes.

Cell density-dependent VP-16 sensitivity of leukaemic cells is accompanied by the translocation of topoisomerase IIalpha from the nucleus to the cytoplasm.

The resistance of several leukaemic and myeloma cell lines (CCRF, L1210, HL-60, KG-1a and RPMI 8226) to VP-16 was found to increase with cell density and to be maximal (3.5- to 39-fold) in plateau phase cell cultures, as measured by clonogenic and MTT assays. Non-transformed confluent Flow 2000 human fibroblasts and Chinese hamster ovary (CHO) cells were also five- and 15-fold resistant to VP-16 respectively. The transition from log to plateau phase was accompanied by a drastic decrease in topoisomerase (topo) IIalpha content in CHO cells and human fibroblasts, while the leukaemic cells maintained constant cellular levels of topo IIalpha and topo IIbeta. However, the nuclear topo IIalpha content was found to decrease as a result of translocation of the enzyme to the cytoplasmic compartment in the leukaemic cells. This was confirmed by subcellular fractionation experiments, Western blotting analyses and immunocytochemistry studies. The quantity of topo IIalpha in plateau phase cytoplasmic fractions ranged from 18% in L1210 cells to 50% in HL-60 and 8226 cells, as measured by both immunoblotting and quantification of the label in immunofluorescent images. The cytoplasmic fraction from plateau phase cells retained topo II catalytic activity, as measured by the decatenation of kinetoplast DNA. The nuclear-cytoplasmic ratio of topo IIalpha may be critical in determining the sensitivity of leukaemic cells to topo II inhibitors. Cytoplasmic trafficking of topo IIalpha was observed in plasma cells obtained from patients with multiple myeloma, and perhaps contributes to drug resistance in this disease.

Effects of wild-type p53 expression on the quantity and activity of topoisomerase IIalpha and beta in various human cancer cell lines.

The p53 null HL-60 cell line was transfected with plasmids coding for either the wild-type p53 or mutant p53 gene. The stable expression of wild-type p53 resulted in a significant increase in sensitivity to the topoisomerase II poisons etoposide and doxorubicin, but not to the topoisomerase II inhibitors razoxane and ADR-529. HL-60 cells expressing wild-type p53 demonstrated 8- to 10-fold more VP-16 induced DNA breaks by the alkaline elution assay. The effect of inducible expression of wild-type p53 was also studied in the p53 null erythroblastoid cell line K562 and in the human squamous carcinoma cell line SqCC. The inducible expression of wild-type p53 in the K562 cell line resulted in a 3-fold increase in sensitivity to VP-16. The quantity of topoisomerase IIalpha was not altered by the transfection as determined by immunoblotting, while the amount of the beta isoform was increased 2.5-fold in HL-60 cells. The topo II catalytic activity present in nuclear extracts was measured as the decatenation of kinetoplast DNA, and found to be unaltered by p53 expression. Immunostaining for topoisomerase IIalpha was substantially diminished in both stable and inducible wild-type p53 expressing cells when three different antibodies were used (two polyclonal and one monoclonal). However, the addition of VP-16 resulted in a rapid appearance of nuclear fluorescence for topoisomerase IIalpha. No changes in topoisomerase IIbeta immunostaining were observed. These results suggest that an epitope for topoisomerase IIalpha is concealed in cells expressing wild-type p53 and that a complex between topoisomerase IIalpha and p53 may be disrupted by the addition of antitumor drugs.

Quantitative immunofluorescence and immunoelectron microscopy of the topoisomerase II alpha associated with nuclear matrices from wild-type and drug-resistant chinese hamster ovary cell lines.

Topo II alpha is considered an important constituent of the nuclear matrix, serving as a fastener of DNA loops to the underlying filamentous scaffolding network. To further define a mechanism of drug resistance to topo II poisons, we studied the quantity of topo II alpha associated with the nuclear matrix in drug-resistant SMR16 and parental cells in the presence and absence of VP-16. Nuclear matrices were prepared from nuclei isolated in EDTA buffer, followed by nuclease digestion with DNase II in the absence of RNase treatment and extraction with 2 M NaCl. Whole-mount spreading of residual structures permits, by means of isoform-specific antibody and colloidal-gold secondary antibodies, an estimate of the amount of topo II alpha in individual nuclear matrices. There are significant variations in topo II alpha amounts between individual nuclear matrices due to the cell cycle distribution. The parental cell line contained eight to ten times more nuclear matrix-associated topo II alpha than the resistant cell line matrices. Nuclear matrix-associated topo II alpha from wild-type and resistant cell lines correlated well with the immunofluorescent staining of the enzyme in nuclei of intact cells. The amount of DNA associated with residual nuclear structures was five times greater in the resistant cell line. This quantity of DNA was not proportional to the quantity of topo II alpha in the same matrix; in fact they were inversely related. In situ whole-mount nuclear matrix preparations were obtained from cells grown on grids and confirmed the results from labeling of isolated residual structures.

Reconstruction of mandibular defects in irradiated patients.

In this prospective study, mandibular reconstruction using titanium plates was evaluated in 31 patients treated between July 1988 and January 1990. Sixteen patients had prior surgery; 13 had prior radiotherapy. In 11 patients, prior radiation and surgery had failed. Sixteen patients received postoperative radiotherapy either in standard or accelerated fractions. Twelve patients had complications of either intraoral (8), extraoral (5), or combined (1) plate exposure or fistula formation (2). Factors significantly related to complications were poor nutrition, accelerated radiation, and recurrence. Sixty-one percent of all patients healed uneventfully. When patients with complications secondary to recurrence who subsequently died were excluded, the success rate was 73%. Only one patient had an unacceptable result that produced a cosmetic and functional deformity despite secondary repair.