Naomi Galili

The PAX3-FKHR fusion protein created by the t(2;13) translocation in alveolar rhabdomyosarcomas is a more potent transcriptional activator than PAX3.

Alveolar rhabdomyosarcomas are pediatric solid tumors with a hallmark cytogenetic abnormality: translocation of chromosomes 2 and 13 [t(2;13) (q35;q14)]. The genes on each chromosome involved in this translocation have been identified as the transcription factor-encoding genes PAX3 and FKHR. The NH2-terminal paired box and homeodomain DNA-binding domains of PAX3 are fused in frame to COOH-terminal regions of the chromosome 13-derived FKHR gene, a novel member of the forkhead DNA-binding domain family. To determine the role of the fusion protein in transcriptional regulation and oncogenesis, we identified the PAX3-FKHR fusion protein and characterized its function(s) as a transcription factor relative to wild-type PAX3. Antisera specific to PAX3 and FKHR were developed and used to examine PAX3 and PAX3-FKHR expression in tumor cell lines. Sequential immunoprecipitations with anti-PAX3 and anti-FKHR sera demonstrated expression of a 97-kDa PAX3-FKHR fusion protein in the t(2;13)-positive rhabdomyosarcoma Rh30 cell line and verified that a single polypeptide contains epitopes derived from each protein. The PAX3-FKHR protein was localized to the nucleus in Rh30 cells, as was wild-type PAX3, in t(2;13)-negative A673 cells. In gel shift assays using a canonical PAX binding site (e5 sequence), we found that DNA binding of PAX3-FKHR was significantly impaired relative to that of PAX3 despite the two proteins having identical PAX DNA-binding domains. However, the PAX3-FKHR fusion protein was a much more potent transcriptional activator than PAX3 as determined by transient cotransfection assays using e5-CAT reporter plasmids. The PAX3-FKHR protein may function as an oncogenic transcription factor by enhanced activation of normal PAX3 target genes.

Residual disease at the end of induction therapy as a predictor of relapse during therapy in childhood B-lineage acute lymphoblastic leukemia.

PURPOSE More than 95% of children with B-lineage acute lymphoblastic leukemia (ALL) achieve a clinical remission after the induction phase of chemotherapy (first 28 days) as evaluated by morphologic criteria. However, relapse occurs in approximately 30% of these children. The objective of this study was to determine whether the outcome of patients in clinical remission at the end of induction therapy could be predicted using a highly sensitive method to detect residual disease. PATIENTS AND METHODS All children diagnosed with B-lineage ALL at the Childrens Hospital of Philadelphia during a 2-year period were eligible. The extent of residual leukemia was quantitated in remission marrow samples obtained at the end of induction therapy in 44 children using a phage clonogenic assay in association with complementarity-determining-region 3 (CDR3)-polymerase chain reaction (PCR). RESULTS Residual disease was a significant predictor of outcome independent of WBC count, age, or sex. The estimated relapse-free survival (RFS) during therapy was 50.4% (+/- 12.6%) for patients with high residual disease (> or = 0.6% leukemia cells among total marrow B cells) versus 91.9% (+/- 5.5%) for those with lower levels (P < .002). There were no significant differences in off-treatment RFS between patients with high or low residual disease who completed therapy in continuous remission (P = .82). The overall estimated RFS was 32.3% (+/- 11.6%) for patients with high residual disease versus 62.6% (+/- 10.7%) for patients with lower levels of residual leukemia cells, with a median follow-up of 5.3 years for patients in continuous remission (P < .008). CONCLUSION PCR detection of high residual disease at the end of induction therapy identifies patients at increased risk for relapse during therapy.

Molecular residual disease status at the end of chemotherapy fails to predict subsequent relapse in children with B-lineage acute lymphoblastic leukemia.

PURPOSE We have investigated whether the extent of residual leukemia in the marrows obtained at the completion of chemotherapy can predict subsequent relapse in children with B-lineage acute lymphoblastic leukemia (ALL). PATIENTS AND METHODS Marrow samples of 24 patients were examined for residual disease at the end of treatment using a quantitative method based on the polymerase chain reaction (PCR) amplification of the complementarity determining region-3 of the immunoglobulin heavy chain. RESULTS Of the 15 patients who remain in continuous bone marrow remission (range, 41 to 98 months), 14 had no detectable leukemic cells; one patient had a very low level (one in approximately 335,000 marrow cells) of residual leukemic cells that underwent clonal evolution. Among the nine patients who had a marrow relapse after the completion of treatment, eight patients whose relapses occurred 4 to 54 months from the end of therapy had no detectable leukemic cells, whereas only the one patient who relapsed 2 months after the completion of therapy had detectable residual disease. CONCLUSION These observations indicate that the absence of detectable residual leukemia by PCR at the end of chemotherapy is not sufficient to assure that the patient is cured and suggest that frequent serial monitoring is required for the early prediction of relapse off therapy.

Genomic organization of the human PAX3 gene: DNA sequence analysis of the region disrupted in alveolar rhabdomyosarcoma

Mutations in the human PAX3 gene have previously been associated with two distinct diseases, Waardenburg syndrome and alveolar rhabdomyosarcoma. In this report we establish that the normal human PAX3 gene is encoded by 8 exons. Intron-exon boundary sequences were obtained for PAX3 exons 5, 6, 7, and 8 and together with previous work provide the complete genomic sequence organization for PAX3. Difficulties in obtaining overlapping genomic clone coverage of PAX3 were circumvented in part by RARE cleavage mapping, which showed that the entire PAX3 gene spans 100 kb of chromosome 2. Sequence analysis of the last intron of PAX3, which contains the previously mapped t(2;13)(q35;q14) translocation breakpoints of alveolar rhabdomyosarcoma, revealed the presence of a pair of inverted Alu repeats and a pair of inverted (GT)n-rich microsatellite repeats within a 5-kb region. This work establishes the complete structure of PAX3 and will permit high-resolution analyses of this locus for mutations associated with Waardenburg syndrome, alveolar rhabdomyosarcoma, and other phenotypes for which PAX3 may be a candidate locus.

Rnf4, a RING protein expressed in the developing nervous and reproductive systems, interacts with Gscl, a gene within the DiGeorge critical region

A yeast 2‐hybrid screen was performed to identify possible transcriptional modulators interactive with goosecoid‐like (gscl), a transcription factor with suppressive activity, expressed during early brain and gonad development. The screen resulted in the identification of a RING protein known as rnf4 or snrf. Gscl/rnf4 interactions were confirmed by affinity chromatography and by immunoprecipitation. Northern analysis confirmed earlier reports of ubiquitous rnf4 expression in adult tissues. Immunohistochemical analysis of mouse embryos revealed expression primarily in the developing nervous system, with strong expression in the dorsal root ganglia and developing gonads. In contrast to previous reports, both cytoplasmic and nuclear expression of rnf4 was documented. The results reported here confirm and extend earlier reports of rnf4 expression. They suggest for the first time, that in addition to acting as a modulator of transcriptional activity, rnf4 may function, as do other RING proteins, to promote the formation of intracytoplasmic complexes involved in shuttling information between the cytoplasm and the nucleus. Dev Dyn;218:102–111.

Sequence-ready physical map of the mouse chromosome 16 region with conserved synteny to the human velocardiofacial syndrome region on 22q11.2.

Abstract. Proximal mouse Chromosome (Chr) 16 shows conserved synteny with human Chrs 16, 8, 22, and 3. The mouse Chr 16/human Chr 22 conserved synteny region includes the DiGeorge/Velocardiofacial syndrome region of human Chr 22q11.2. A physical map of the entire mouse Chr 16/human Chr 22 region of conserved synteny has been constructed to provide a substrate for gene discovery, genomic sequencing, and animal model development. A YAC contig was constructed that extends ca. 5.4 Mb from a region of conserved synteny with human Chr 8 at Prkdc through the region conserved with human Chr 3 at DVL3. Sixty-one markers including 37 genes are mapped with average marker spacing of 90 kb. Physical distance was determined across the 2.6-Mb region from D16Mit74 to Hira with YAC fragmentation. The central region from D16Jhu28 to Igl-C1 was converted into BAC and PAC clones, further refining the physical map and providing sequence-ready template. The gene content and borders of three blocks of conserved linkage between human Chr 22q11.2 mouse Chr 16 are refined.

Human T lymphocytes become glucocorticoid-sensitive upon immune activation

Abstract T lymphocytes activated in mixed lymphocyte cultures were found to be sensitive to the lytic effect of glucocorticoids (methylprednisolone and hydrocortisone). Human thymocytes, freshly separated blood lymphocytes, and phytohemagglutinin blasts were resistant. In addition, the activated T lymphocytes isolated from the synovial fluid of arthritic patients were also glucocorticoid sensitive whereas the blood lymphocytes of the same patients were resistant. The glucocorticoid sensitivity was not accompanied by elevation of the cytoplasmatic receptors to steroids. The direct sensitivity of activated T cells to glucocorticoids may explain partly the immunosuppressive activity of these drugs.

Immunohistochemical analysis of the expression of two serine-threonine kinases in the maturing mouse testis.

Previously we identified two intronless serine-threonine kinase genes (Tsk1 and Tsk2) located 3 kb apart on mouse chromosome 16 (Galili, N., Baldwin, H.S., Lund, J., Reeves, R., Gong, W., Wang, Z., Roe, B.A., Emanuel, B.S., Nayak, S., Mickanin, C., Budraf, M.L., Buck, C.A., 1997. A region of mouse chromosome 16 is syntenic to the DiGeorge, velocardiofacial syndrome minimal critical region. Gen. Res. 7, 17-26). Tsk1 was identical to a putative testicular kinase reported by Bielke et al. (Bielke, W., Blaschke, R.J., Miescher, G.C., Zurcher, G., Andres, A.C., Ziemiecki, A., 1994. Characterization of a novel murine testis-specific serine/threonine kinase. Gene 13, 235-239). Here we document the expression patterns of each Tsk throughout spermiogenesis showing an initial association of Tsk1 with cells in meiotic metaphase and a later association of Tsk2 with tail-like structures in the lumen of the seminiferous tubule.

Gscl, a gene within the minimal DiGeorge critical region, is expressed in primordial germ cells and the developing pons.

Gscl, a paired‐type homeobox gene, has been implicated in the pathology of DGS/VCFS by virtue of its genomic location and its structural similarity to the Gscgene family. Immunohistochemical and in situ studies were performed to examine the expression pattern of this gene during embryonic development. A polyclonal antibody, generated to the full‐length protein and shown to be specific for GSCL by both Western blotting and immunofluorescence, was used for immunohistochemical localization. Both in situ and antibody staining localized GSCL expression to a cluster of cells in the pons region of the developing brain. This GSCL expression pattern showed partial overlap with that of Pax6. More detailed immunohistochemistry revealed the GSCL in primordial germ cells during migration from the epithelium of the hindgut and later as they colonize the developing gonads. GSCL was not detected in tissues affected in DGS/VCSF. Dev. Dyn. 1998;212:86–93.