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Chronic myelogeneous leukemia (CML) is genetically characterized by fusion of the bcr and abl genes on chromosomes 22 and 9, respectively. In most cases, the fusion involves a reciprocal translocation t(9;22)(q34;q11), which produces the cytogenetically distinctive Philadelphia chromosome (Ph1). Fusion can be detected by Southern (DNA) analysis or by in vitro amplification of the messenger RNA from the fusion gene with polymerase chain reaction (PCR). These techniques are sensitive but cannot be applied to single cells. Two-color fluorescence in situ hybridization (FISH) was used with probes from portions of the bcr and abl genes to detect the bcr-abl fusion in individual blood and bone marrow cells from six patients. The fusion event was detected in all samples analyzed, of which three were cytogenetically Ph1-negative. One of the Ph1-negative samples was also PCR-negative. This approach is fast and sensitive, and provides potential for determining the frequency of the abnormality in different cell lineages.

One of the most common chromosomal abnormalities in acute leukemia is a reciprocal translocation involving the HRX gene (also called MLL, ALL-1, or HTRX) at chromosomal locus 11q23, resulting in the formation of HRX fusion proteins. Using the yeast two-hybrid system and human cell culture coimmunoprecipitation experiments, we show here that HRX proteins interact directly with the GADD34 protein. We have found that transfected cells overexpressing GADD34 display a significant increase in apoptosis after treatment with ionizing radiation, indicating that GADD34 expression not only correlates with apoptosis but also can enhance apoptosis. The amino-terminal third of the GADD34 protein was necessary for this observed increase in apoptosis. Furthermore, coexpression of three different HRX fusion proteins (HRX-ENL, HRX-AF9, and HRX-ELL) had an anti-apoptotic effect, abrogating GADD34-induced apoptosis. In contrast, expression of wild-type HRX gave rise to an increase in apoptosis. The difference observed here between wild-type HRX and the leukemic HRX fusion proteins suggests that inhibition of GADD34-mediated apoptosis may be important to leukemogenesis. We also show here that GADD34 binds the human SNF5/INI1 protein, a member of the SNF/SWI complex that can remodel chromatin and activate transcription. These studies demonstrate, for the first time, a gain of function for leukemic HRX fusion proteins compared to wild-type protein. We propose that the role of HRX fusion proteins as negative regulators of post-DNA-damage-induced apoptosis is important to leukemia progression.The disruption of the human homologue of the Drosophila Trithorax (trx) gene, HRX, by chromosomal translocations resulting in the juxtaposition of genetic elements and formation of HRX fusion genes is one of the most common genetic alterations in human acute leukemia (52). These translocations occur in approximately 10% of acute lymphoid leukemias (ALLs), 5% of acute myeloid leukemias (AMLs), and 85% of topoisomerase II inhibitor-related secondary leukemias in adults. Furthermore, these translocations are present in half of all the de novo leukemias in children younger than 1 year (26).HRX, also referred to as ALL-1, MLL-1, or HTRX (13, 18, 52, 60), is a ubiquitously expressed 3,969-amino-acid nuclear protein (28) with unknown biologic function. HRX shares at least two regions of strong homology with the similarly sized Drosophila Trx: a series of centrally located zinc finger-like domains and a carboxy-terminal stretch of 210 amino acids. In Drosophila, Trx controls body segment patterning as a positive transcriptional regulator of the homeotic selector genes of the Antennapedia and bithorax complexes (7). Studies with transgenic mice have shown that the function of Hrx in mice has features in common with that of Trx in Drosophila. Hrx has been demonstrated to be required for proper segment identity and to function positively as a regulator of Hox gene expression in Hrx heterozygous and homozygous deficient mice (58).To date, more than 26 differen...

Accurate Gleason score, pathologic stage, and surgical margin (SM) information is critical for the planning of post-radical prostatectomy management in patients with prostate cancer. Although interobserver variability for Gleason score among urologic pathologists has been well documented, such data for pathologic stage and SM assessment are limited. We report the first study to address interobserver variability in a group of expert pathologists concerning extraprostatic soft tissue (EPE) and SM interpretation for radical prostatectomy specimens. A panel of 3 urologic pathologists selected 6 groups of 10 slides designated as being positive, negative, or equivocal for either EPE or SM based on unanimous agreement. Twelve expert urologic pathologists, who were blinded to the panel diagnoses, reviewed 40x whole-slide scans and provided diagnoses for EPE and SM on each slide. On the basis of panel diagnoses, as the gold standard, specificity, sensitivity, and accuracy values were high for both EPE (87.5%, 95.0%, and 91.2%) and SM (97.5%, 83.3%, and 90.4%). Overall kappa values for all 60 slides were 0.74 for SM and 0.63 for EPE. The kappa values were higher for slides with definitive gold standard EPE (kappa=0.81) and SM (kappa=0.73) diagnoses when compared with the EPE (kappa=0.29) and SM (kappa=0.62) equivocal slides. This difference was markedly pronounced for EPE. Urologic pathologists show good to excellent agreement when evaluating EPE and SM. Interobserver variability for EPE and SM interpretation was principally related to the lack of a clearly definable prostatic capsule and crush/thermal artifact along the edge of the gland, respectively.

One of the most common chromosomal abnormalities in acute leukemia is a reciprocal translocation involving the HRX gene at chromosome locus 11q23, resulting in HRX fusion proteins. Using the yeast two-hybrid system, in vitro binding studies, and human cell culture coimmunoprecipitation experiments, we show here that a region of the HRX protein that is consistently retained in HRX leukemic fusion proteins interacts directly with SET, another protein implicated in leukemia. We have identified the binding sites on HRX for SET and show that these sequences are clustered near the A⅐T hooks that have been shown to bind DNA. We also show that carboxyl-terminal SET sequences, possibly the acidic tail of SET, bind to HRX. We have also found serine/ threonine-specific protein phosphatase activity in anti-HRX coimmunoprecipitates. Using the phosphatase inhibitor okadaic acid and Western blotting, the phosphatase was identified as protein phosphatase 2A (PP2A). Mutation of a single amino acid in one of the SET binding sites of HRX resulted in lower amounts of both coimmunoprecipitated SET protein and coimmunoprecipitated PP2A. These results suggest that the leukemogenic effects of HRX fusion proteins may be related to interactions with SET and PP2A.