Radiographic evidence of treatment with bisphosphonates.

Bony disease is typically evident with radiographic examination. Loss of bone mass consistent with osteoporosis is evident on plain dental radiographs, and it is reasonable to expect that anti-resorptive treatment of osteoporosis would lead to changes in radiodensity of structures visible on dental radiographs. Review of a number of radiographs of patients receiving anti-resorptive (bisphosphonate) treatment appears to confirm increased radiodensity of the structures, which may have implications in risk assessment of complications following dental procedures.

Targeting survivin and p53 in pediatric acute lymphoblastic leukemia.

Despite advances in treatment and outcomes for patients with pediatric acute lymphoblastic leukemia (ALL), there continue to be subsets of patients who are refractory to standard chemotherapy and hematopoietic stem cell transplant. Therefore, novel gene targets for therapy are needed to further advance treatment for this disease. RNA interference technology has identified survivin as a potential therapeutic target. Survivin, a member of the inhibitor of apoptosis (IAP) proteins and chromosome passenger complex, is expressed in hematologic malignancies and overexpressed in relapsed pediatric ALL. Our studies show that survivin is uniformly expressed at high levels in multiple pediatric ALL cell lines. Furthermore, silencing of survivin expression in pediatric ALL cell lines as well as primary leukemic blasts reduces viability of these cells. This includes cell lines derived from patients with relapsed disease featuring cytogenetic anomalies such as t(12;21), Philadelphia chromosome t(9;22), t(1;19) as well as a cell line carrying t(17;19) from a patient with de novo ALL. Furthermore, inhibition of survivin increases p53-dependent apoptosis that can be rescued by inhibition of p53. Finally, a screen of randomly selected primary patient samples confirms that survivin-specific small interfering RNA and survivin-targeted drug, YM155, effectively reduce viability of leukemic blasts.

Chromosomes with delayed replication timing lead to checkpoint activation, delayed recruitment of Aurora B and chromosome instability.

Certain chromosome rearrangements display a significant delay in chromosome replication timing (DRT) that is associated with a subsequent delay in mitotic chromosome condensation (DMC). DRT/DMC chromosomes are common in tumor cells in vitro and in vivo and occur frequently in cells exposed to ionizing radiation. A hallmark for these chromosomes is the delayed phosphorylation of serine 10 of histone H3 during mitosis. The chromosome passenger complex, consisting of multiple proteins including Aurora B kinase and INCENP is thought to be responsible for H3 phosphorylation, chromosome condensation and the subsequent segregation of chromosomes. In this report, we show that chromosomes with DRT/DMC contain phosphorylated Chk1, consistent with activation of the S-M phase checkpoint. Furthermore, we show that INCENP is recruited to the DRT/DMC chromosomes during all phases of mitosis. In contrast, Aurora B kinase is absent on DRT/DMC chromosomes when these chromosomes lack serine 10 phosphorylation of H3. We also show that mitotic arrest deficient 2 (Mad2), a member of the spindle assembly checkpoint, is present on DRT/DMC chromosomes at a time when the normally condensed chromosomes show no Mad2 staining, indicating that DRT/DMC activates the spindle assembly checkpoint. Finally, cells with DRT/DMC chromosomes have centrosome amplification, abnormal spindle assembly, endoreduplication and significant chromosome instability.

Large scale mapping of methylcytosines in CTCF-binding sites in the human H19 promoter and aberrant hypomethylation in human bladder cancer.

The methylation status of binding sites of the insulator protein, CTCF, in the H19 promoter has been suggested as being critical to the regulation of imprinting of the H19/IGF2 locus located in chromosome 11p15. In this study, we have analyzed the methylation of all of seven potential CTCF-binding sites in the human H19 promoter since the methylation status of these sites has not been reported. We found that all the binding sites except the sixth were hypermethylated whereas only the sixth binding site showed allele-specific methylation in normal human embryonic ureteral tissue. We also analyzed the methylation status of these sites in human-mouse somatic-cell-hybrid clones containing a single copy of human chromosome 11 and which were treated with 5-aza-2'-deoxycytidine (5-aza-CdR) to yield clones which expressed human IGF2 and H19 mutually exclusively of each other. In most of the clones, a correlation between methylation of the sixth CTCF-binding site and expression of IGF2 was observed. Therefore, we analyzed the methylation status of this site in human bladder cancer and found hypomethylation of the paternal allele in two of six informative cases. These results demonstrate that only the sixth CTCF-binding site acts as a key regulatory domain for switching between H19 or IGF2 expression, whereas the other sites are not subject to allele-specific methylation. Loss of methylation imprinting of H19 is linked to hypomethylation of the paternal allele in human bladder cancer, unlike the situation in Wilms' tumor and colon cancer where the maternal allele becomes hypermethylated.

Delayed replication timing leads to delayed mitotic chromosome condensation and chromosomal instability of chromosome translocations.

Chromosomal rearrangements are found in virtually all types of human cancers. We show that certain chromosome translocations display a delay in mitotic chromosome condensation that is associated with a delay in the mitosis-specific phosphorylation of histone H3. This delay in mitotic condensation is preceded by a delay in both the initiation as well as the completion of chromosome replication. In addition, chromosomes with this phenotype participate in numerous secondary translocations and rearrangements. Chromosomes with this phenotype were detected in five of seven tumor-derived cell lines and in five of thirteen primary tumor samples. These data suggest that certain chromosomal rearrangements found in tumor cells cause a significant delay in replication timing of the entire chromosome that subsequently results in delayed mitotic chromosome condensation and ultimately in chromosomal instability.

Spontaneously immortalized cell lines obtained from adult Atm null mice retain sensitivity to ionizing radiation and exhibit a mutational pattern suggestive of oxidative stress.

The study of Ataxia-telangiectasia (A-T) has benefited significantly from mouse models with knockout mutations for the Atm (A-T mutation) locus. While these models have proven useful for in vivo studies, cell cultures from Atm null embryos have been reported to grow poorly and then senesce. In this study, we initiated primary cultures from adult ears and kidneys of Atm homozygous mice and found that these cultures immortalized readily without loss of sensitivity to ionizing radiation and other Atm related cell cycle defects. A mutational analysis for loss of expression of an autosomal locus showed that ionizing radiation had a mutagenic effect. Interestingly, some spontaneous mutants exhibited a mutational pattern that is characteristic of oxidative mutagenesis. This result is consistent with chronic oxidative stress in Atm null cells. In total, the results demonstrate that permanent cell lines can be established from the tissues of adult mice homozygous for Atm and that these cell lines will exhibit expected and novel consequences of this deficiency.

Positional cloning of a novel Fanconi anemia gene, FANCD2.

Fanconi anemia (FA) is a genetic disease with birth defects, bone marrow failure, and cancer susceptibility. To date, genes for five of the seven known complementation groups have been cloned. Complementation group D is heterogeneous, consisting of two distinct genes, FANCD1 and FANCD2. Here we report the positional cloning of FANCD2. The gene consists of 44 exons, encodes a novel 1451 amino acid nuclear protein, and has two protein isoforms. Similar to other FA proteins, the FANCD2 protein has no known functional domains, but unlike other known FA genes, FANCD2 is highly conserved in A. thaliana, C. elegans, and Drosophila. Retroviral transduction of the cloned FANCD2 cDNA into FA-D2 cells resulted in functional complementation of MMC sensitivity.

pRB induces Sp1 activity by relieving inhibition mediated by MDM2.

pRB activates transcription by a poorly understood mechanism that involves relieving negative regulation of the promoter specificity factor Sp1. We show here that MDM2 inhibits Sp1-mediated transcription, that MDM2 binds directly to Sp1 in vitro as well as in vivo, and that MDM2 inhibits the DNA-binding activity of Sp1. Forced expression of pRB relieves MDM2-mediated repression, and interaction of pRB with the MDM2-Sp1 complex releases Sp1 and restores DNA binding. These results suggest a model in which the opposing activities of MDM2 and pRB regulate Sp1 DNA-binding and transcriptional activity.

P53 mutation and MDM2 amplification frequency in pediatric rhabdomyosarcoma tumors and cell lines.

BACKGROUND: The p53 tumor suppressor gene is the most commonly mutated gene in human cancer, and mutations arise in a wide variety of tumor types. Wild-type p53 functions as a regulator of apoptosis, so mutations in the p53 gene are generally associated with aggressive tumors and a poor prognosis. PROCEDURE: We have investigated the p53 mutation and MDM2 amplification frequencies in biopsies from pediatric rhabdomyosarcoma (RMS) tumors and cell lines by SSCP and Southern analyses. RESULTS: A mutation was detected in only 1 of 20 tumor specimens (5%), whereas the frequency in established RMS cell lines was significantly higher (6/10, 60%). p53 Mutations were more common in cell lines derived from tumors previously exposed to chemotherapy compared to those derived from tumors at di-agnosis, and it is likely that these mutations enhanced the probability of successful long-term culture. The frequency of MDM2 gene amplification in patient biopsies was also low (2/20, 10%). Interestingly, complete responses to treatment were obtained in the two patients with tumors that demonstrated amplification of MDM2. The response to treatment of patients with tumors wild-type for p53 and without MDM2 amplification was quite varied, indicating that expression of a wild-type p53 gene at diagnosis cannot always facilitate a favorable outcome. CONCLUSIONS: p53 mutation and MDM2 gene amplification frequencies are extremely low in RMS tumors, but a wild-type p53 genotype is not always associated with a favorable prognosis.

Localization of the Fanconi anemia complementation group D gene to a 200-kb region on chromosome 3p25.3.

Fanconi anemia (FA) is a rare autosomal recessive disease manifested by bone-marrow failure and an elevated incidence of cancer. Cells taken from patients exhibit spontaneous chromosomal breaks and rearrangements. These breaks and rearrangements are greatly elevated by treatment of FA cells with the use of DNA cross-linking agents. The FA complementation group D gene (FANCD) has previously been localized to chromosome 3p22-26, by use of microcell-mediated chromosome transfer. Here we describe the use of noncomplemented microcell hybrids to identify small overlapping deletions that narrow the FANCD critical region. A 1.2-Mb bacterial-artificial-chromosome (BAC)/P1 contig was constructed, bounded by the marker D3S3691 distally and by the gene ATP2B2 proximally. The contig contains at least 36 genes, including the oxytocin receptor (OXTR), hOGG1, the von Hippel-Lindau tumor-suppressor gene (VHL), and IRAK-2. Both hOGG1 and IRAK-2 were excluded as candidates for FANCD. BACs were then used as probes for FISH analyses, to map the extent of the deletions in four of the noncomplemented microcell hybrid cell lines. A narrow region of common overlapping deletions limits the FANCD critical region to approximately 200 kb. The three candidate genes in this region are TIGR-A004X28, SGC34603, and AA609512.

Structural basis for amide hydrolysis catalyzed by the 43C9 antibody.

Among catalytic antibodies, the well-characterized antibody 43C9 is unique in its ability to catalyze the difficult, but desirable, reaction of selective amide hydrolysis. The crystallographic structures that we present here for the single-chain variable fragment of the 43C9 antibody, both with and without the bound product p -nitrophenol, strongly support and extend the structural and mechanistic information previously provided by a three-dimensional computational model, together with extensive biochemical, kinetics, and mutagenesis results. The structures reveal an unexpected extended beta-sheet conformation of the third complementarity determining region of the heavy chain, which may be coupled to the novel indole ring orientation of the adjacent Trp H103. This unusual conformation creates an antigen-binding site that is significantly deeper than predicted in the computational model, with a hydrophobic pocket that encloses the p -nitrophenol product. Despite these differences, the previously proposed roles for Arg L96 in transition-state stabilization and for His L91 as the nucleophile that forms a covalent acyl-antibody intermediate are fully supported by the crystallographic structures. His L91 is now centered at the bottom of the antigen-binding site with the imidazole ring poised for nucleophilic attack. His L91, Arg L96, and the bound p -nitrophenol are linked into a hydrogen-bonding network by two well-ordered water molecules. These water molecules may mimic the positions of the phosphonamidate oxygen atoms of the antigen, which in turn mimic the transition state of the reaction. This network also contains His H35, suggesting that this residue may also stabilize the transition-states. A possible proton-transfer pathway from His L91 through two tyrosine residues may assist nucleophilic attack. Although transition-state stabilization is commonly observed in esterolytic antibodies, nucleophilic attack appears to be unique to 43C9 and accounts for the unusually high catalytic activity of this antibody.

Skeletal muscle and small-conductance calcium-activated potassium channels.

Skeletal muscle becomes hyperexcitable following denervation and when cultured in the absence of nerve cells. In these circumstances, the bee venom peptide toxin apamin, a blocker of small-conductance calcium-activated potassium (SK) channels, dramatically reduces the hyperexcitability. In this report, we show that SK3 channels are expressed in denervated skeletal muscle and in L6 cells. Action potentials evoked from normal innervated rat skeletal muscle did not exhibit an afterhyperpolarization, indicating a lack of SK channel activity; very low levels of apamin binding sites, SK3 protein, or SK3 mRNA were present. However, denervation resulted in apamin-sensitive afterhyperpolarizations and increased apamin binding sites, SK3 protein, and SK3 mRNA. Cultured rat L6 myoblasts and differentiated L6 myotubes contained similar levels of SK3 mRNA, although apamin-sensitive SK currents and apamin binding sites were detected only following myotube differentiation. Therefore, different molecular mechanisms govern SK3 expression levels in denervated muscle compared with muscle cells differentiated in culture.

Embryonic stem cells can be used to construct hybrid cell lines containing a single, selectable murine chromosome.

Microcell-mediated chromosome transfer is a useful technique for the study of gene function, gene regulation, gene mapping, and functional cloning in mammalian cells. Complete panels of donor cell lines, each containing a different human chromosome, have been developed. These donor cell lines contain a single human chromosome marked with a dominant selectable gene in a rodent cell background. However, a similar panel does not exist for murine chromosomes. To produce mouse monochromosomal donor hybrids, we have utilized embryonic stem (ES) cells with targeted gene disruptions of known chromosomal location as starting material. ES cells with mutations in aprt, fyn, and myc were utilized to generate monochromosomal hybrids with neomycin phosphotransferase-marked murine Chr 8, 10, or 15 respectively in a hamster or rat background. This same methodology can be used to generate a complete panel of marked mouse chromosomes for somatic cell genetic experimentaion.

Duplication of ATR inhibits MyoD, induces aneuploidy and eliminates radiation-induced G1 arrest.

Chromosome 3q alterations occur frequently in many types of tumours. In a genetic screen for loci present in rhabdomyosarcomas, we identified an isochromosome 3q [i(3q)], which inhibits muscle differentiation when transferred into myoblasts. The i(3q) inhibits MyoD function, resulting in a non-differentiating phenotype. Furthermore, the i(3q) induces a 'cut' phenotype, abnormal centrosome amplification, aneuploidy and loss of G1 arrest following gamma-irradiation. Testing candidate genes within this region reveals that forced expression of ataxia-telangiectasia and rad3-related (ATR) results in a phenocopy of the i(3q). Thus, genetic alteration of ATR leads to loss of differentiation as well as cell-cycle abnormalities.

Data sets, partitions, and characters: philosophies and procedures for analyzing multiple data sets.

We compared four approaches for analyzing three data sets derived from staphylinoid beetles, a superfamily whose known species diversity is roughly comparable to that of vertebrates. One data set is derived from adult morphology and the two molecular data sets are from 12S ribosomal RNA and cytochrome b mitochondrial DNA. We found that taxonomic congruence following conditional data combination, herein called compatible evidence (CE), resolved more nodes compatible with an initial conservative hypothesis than did total evidence (TE), conditional data combination (CDC), or taxonomic congruence (TC). CE sets a base of nodes obtained by CDC analysis and then investigates what further agreement may arise in a universe where these nodes are accepted as given. We suggest that CE75-75 may be appropriate for future studies that aim to both generate a well-corroborated tree and investigate conflicts between data sets, partitions, and characters. CE75-75 is a 75% bootstrap consensus CDC tree followed by combinable-component consensus of a 75% bootstrap consensus of each homogeneous set of partitions having hierarchical structure.

Amplification of MDM2 inhibits MyoD-mediated myogenesis.

One obvious phenotype of tumor cells is the lack of terminal differentiation. We previously classified rhabdomyosarcoma cell lines as having either a recessive or a dominant nondifferentiating phenotype. To study the genetic basis of the dominant nondifferentiating phenotype, we utilized microcell fusion to transfer chromosomes from rhabdomyosarcoma cells into C2C12 myoblasts. Transfer of a derivative chromosome 14 inhibits differentiation. The derivative chromosome 14 contains a DNA amplification. MDM2 is amplified and overexpressed in these nondifferentiating hybrids and in the parental rhabdomyosarcoma. Forced expression of MDM2 inhibits MyoD-dependent transcription. Expression of antisense MDM2 restores MyoD-dependent transcriptional activity. We conclude that amplification and overexpression of MDM2 inhibit MyoD function, resulting in a dominant nondifferentiating phenotype.

Mapping of the ARIX homeodomain gene to mouse chromosome 7 and human chromosome 11q13.

The recently described homeodomain protein ARIX is expressed specifically in noradrenergic cell types of the sympathetic nervous system, brain, and adrenal medulla. ARIX interacts with regulatory elements of the genes encoding the noradrenergic biosynthetic enzymes tyrosine hydroxylase and dopamine beta-hydroxylase, suggesting a role for ARIX in expression of the noradrenergic phenotype. In the study described here, the mouse and human ARIX genes are mapped. Using segregation analysis of two panels of mouse backcross DNA, mouse Arix was positioned approximately 50 cM distal to the centromere of chromosome 7, near Hbb. Human ARIX was positioned through analysis of somatic cell hybrids and fluorescence in situ hybridization of human metaphase chromosomes to chromosome 11q13.3-q13.4. These map locations extend and further define regions of conserved synteny between mouse and human genomes and identify a new candidate gene for inherited developmental disorders linked to human 11q13.

Localization of two potassium channel beta subunit genes, KCNA1B and KCNA2B.

The gating properties and current amplitudes of mammalian voltage-activated Shaker potassium channels are modulated by at least two associated beta subunits (Kv beta 1.1 and Kv beta 1.2). The human Kv beta 1.1 gene (KCNA1B) resides on chromosome 3, as indicated by somatic cell hybrid mapping. More precise localization of KCNA1B to 3q26.1 was obtained with fluorescence in situ hybridization (FISH) and was corroborated by PCR screening of the CEPH YAC library. The human Kv beta 1.2 gene (KCNA2B) resides on chromosome 1, as indicated by somatic cell hybrid mapping, and has been localized by FISH to 1p36.3.

Microcell mediated chromosome transfer maps the Fanconi anaemia group D gene to chromosome 3p.

Fanconi anaemia (FA) is an autosomal recessive disorder characterized by progressive pancytopenia, short stature, radial ray defects, skin hyperpigmentation and a predisposition to cancer. Cells from FA patients are hypersensitive to cell killing and chromosome breakage induced by DNA cross-linking agents such as mitomycin C (MMC) and diepoxybutane (DEB). Consequently, the defect in FA is thought to be in DNA crosslink repair. Additional cellular phenotypes of FA include oxygen sensitivity, poor cell growth and a G2 cell cycle delay. At least 5 complementation groups for Fanconi anaemia exist, termed A through E. One of the five FA genes, FA(C), has been identified by cDNA complementation, but no other FA genes have been mapped or cloned until now. The strategy of cDNA complementation, which was successful for identifying the FA(C) gene has not yet been successful for cloning additional FA genes. The alternative approach of linkage analysis, followed by positional cloning, is hindered in FA by genetic heterogeneity and the lack of a simple assay for determining complementation groups. In contrast to genetic linkage studies, microcell mediated chromosome transfer utilizes functional complementation to identify the disease bearing chromosome. Here we report the successful use of this technique to map the gene for the rare FA complementation group D (FA(D)).

Novel DNA binding motifs in the DNA repair enzyme endonuclease III crystal structure.

The 1.85 A crystal structure of endonuclease III, combined with mutational analysis, suggests the structural basis for the DNA binding and catalytic activity of the enzyme. Helix-hairpin-helix (HhH) and [4Fe-4S] cluster loop (FCL) motifs, which we have named for their secondary structure, bracket the cleft separating the two alpha-helical domains of the enzyme. These two novel DNA binding motifs and the solvent-filled pocket in the cleft between them all lie within a positively charged and sequence-conserved surface region. Lys120 and Asp138, both shown by mutagenesis to be catalytically important, lie at the mouth of this pocket, suggesting that this pocket is part of the active site. The positions of the HhH motif and protruding FCL motif, which contains the DNA binding residue Lys191, can accommodate B-form DNA, with a flipped-out base bound within the active site pocket. The identification of HhH and FCL sequence patterns in other DNA binding proteins suggests that these motifs may be a recurrent structural theme for DNA binding proteins.