Chronic myeloid leukemia (CML) is characterized by the reciprocal translocation t(9;22)(q34;q11.2) which fuses the ABL1 oncogene on chromosome 9 with the BCR gene on chromosome 22. It is the BCR/ABL protein that drives the neoplasm and the ABL/BCR is not necessary for the disease. In the majority of CML cases, the BCR/ABL fusion gene is cytogenetically recognizable as a small derivative chromosome 22(der 22), which is known as the Philadelphia (Ph) chromosome. However, approximately 2-10% of patients with CML involve cryptic or complex variant translocations with deletions on the der(9) and/or der(22) occuring in roughly 10-15% of CML cases. Fluorescence in situ hybridization (FISH) analysis can help identify deletions and complex or cryptic rearrangements. Various BCR/ABL FISH probes are available, which include dual color single fusion, dual color extra signal (ES), dual color dual fusion and tri color dual fusion probes. To test the utility of these probes, six patients diagnosed with CML carrying different complex variant Ph translocations were studied by G-banding and FISH analysis using the BCR/ABL ES, BCR/ABL dual color dual fusion, and BCR/ABL tricolor probes. There are differences among the probes in their ability to detect variant rearrangements, with or without accompanying chromoso me 9 and/or 22 deletions, and low level disease.

Past anthrax attacks in the United States have highlighted the need for improved measures against bioweapons. The virulence of anthrax stems from the shielding properties of the Bacillus anthracis poly-γ-d-glutamic acid capsule. In the presence of excess CapD, a B. anthracis γ-glutamyl transpeptidase, the protective capsule is degraded, and the immune system can successfully combat infection. Although CapD shows promise as a next generation protein therapeutic against anthrax, improvements in production, stability, and therapeutic formulation are needed. In this study, we addressed several of these problems through computational protein engineering techniques. We show that circular permutation of CapD improved production properties and dramatically increased kinetic thermostability. At 45 °C, CapD was completely inactive after 5 min, but circularly permuted CapD remained almost entirely active after 30 min. In addition, we identify an amino acid substitution that dramatically decreased transpeptidation activity but not hydrolysis. Subsequently, we show that this mutant had a diminished capsule degradation activity, suggesting that CapD catalyzes capsule degradation through a transpeptidation reaction with endogenous amino acids and peptides in serum rather than hydrolysis.

PURPOSE: This study was designed to analyze the effect of two different ablation modes ("temperature control" and "power control") of a microwave system on procedural outcome in porcine kidneys in vivo. METHODS: A commercially available microwave system (Avecure Microwave Generator; MedWaves, San Diego, CA) was used. The system offers the possibility to ablate with two different ablation modes: temperature control and power control. Thirty-two microwave ablations were performed in 16 kidneys of 8 pigs. In each animal, one kidney was ablated twice by applying temperature control (ablation duration set point at 60 s, ablation temperature set point at 96°C, automatic power set point; group I). The other kidney was ablated twice by applying power control (ablation duration set point at 60 s, ablation temperature set point at 96°C, ablation power set point at 24 W; group II). Procedural outcome was analyzed:(1) technical success (e.g., system failures, duration of the ablation cycle), and (2) ablation geometry (e.g., long axis diameter, short axis diameter, and circularity). RESULTS: System failures occurred in 0% in group I and 13% in group II. Duration of the ablation cycle was 60 ± 0 s in group I and 102 ± 21 s in group II. Long axis diameter was 20.3 ± 4.6 mm in group I and 19.8 ± 3.5 mm in group II (not significant (NS)). Short axis diameter was 10.3 ± 2 mm in group I and 10.5 ± 2.4 mm in group II (NS). Circularity was 0.5 ± 0.1 in group I and 0.5 ± 0.1 in group II (NS). CONCLUSIONS: Microwave ablations performed with temperature control showed fewer system failures and were finished faster. Both ablation modes demonstrated no significant differences with respect to ablation geometry.

The incidence of adenocarcinoma of the lung is increasing in the United States, however, the difficulties in obtaining lung cancer families and representative samples of early to late stages of the disease have lead to the study of mouse models for lung cancer. We used Spectral Karyotyping (SKY), mapping with fluorescently labeled genomic clones (FISH), comparative genomic hybridization (CGH) arrays, gene expression arrays, Western immunoblot and real time polymerase chain reaction (PCR) to analyze nine pairs of high-invasive and low-invasive tumor cell strains derived from early passage mouse lung adenocarcinoma cells to detect molecular changes associated with tumor invasion. The duplication of chromosomes 1 and 15 and deletion of chromosome 8 were significantly associated with a high-invasive phenotype. The duplication of chromosome 1 at band C4 and E1/2-H1 were the most significant chromosomal changes in the high-invasive cell strains. Mapping with FISH and CGH array further narrowed the minimum region of duplication of chromosome 1 to 71-82 centimorgans (cM). Expression array analysis and confirmation by real time PCR demonstrated increased expression of COX-2, Translin (TB-RBP), DYRK3, NUCKS and Tubulin-alpha4 genes in the high-invasive cell strains. Elevated expression and copy number of these genes, which are involved in inflammation, cell movement, proliferation, inhibition of apoptosis and telomere elongation, were associated with an invasive phenotype. Similar linkage groups are altered in invasive human lung adenocarcinoma, implying that the mouse is a valid genetic model for the study of the progression of human lung adenocarcinoma.

Cell migration plays an important role in tumor cell invasion and metastasis. Previously, we reported that caspase-8 contributes to cell migration and adhesion, a novel nonapoptotic function of an established apoptotic factor. Herein, we report that pro-caspase-8 is capable of restoring cell migration/adhesion to caspase-8-null cells, establishing the first biological function of a pro-caspase. The catalytic activity of caspase-8 was not required for cell motility. Stimulation of motility with epidermal growth factor induced the phosphorylation of caspase-8 on tyrosine-380 and the interaction of caspase-8 with the p85 alpha subunit of phosphatidylinositol 3-kinase. Tyrosine-380 was required for the restoration of cell motility and cell adhesion in caspase-8-null cells, demonstrating the importance of the caspase-8-p85 interaction for these nonapoptotic functions. These results suggest that caspase-8 phosphorylation converts it from a proapoptotic factor to a cell motility factor that, through tyrosine-380, interacts with p85, an established cell migration component.

Tumor suppressor gene BRCA1 is frequently mutated in familial breast and ovarian cancer. BRCA1 plays pivotal roles in maintaining genomic stability by interacting with numerous proteins in cell cycle control and DNA repair. Irofulven (6-hydroxymethylacylfulvene, HMAF, MGI 114, NSC#: 683863) is one of a new class of anticancer agents that are analogs of mushroom-derived illudin toxins. Preclinical studies and clinical trials have demonstrated that irofulven is effective against several tumor cell types. The exact nature of irofulven-induced DNA damage is not completely understood. Previously, we have demonstrated that irofulven activates ATM and its targets, NBS1, SMC1, CHK2 and p53. In this study, we hypothesize that irofulven induces DNA double-strand breaks and that BRCA1 may affect chemosensitivity by controlling cell cycle checkpoints, DNA repair and genomic stability in response to irofulven treatment. We observed that irofulven induces the formation of chromosome breaks and radials as well as the activation and foci formation of gamma-H2AX, BRCA1 and RAD51. We also provided evidence that irofulven induces the generation of DNA double-strand breaks. By using BRCA1-deficient or proficient cells, we demonstrated that in response to irofulven, BRCA1 contributes to the control of S and G2/M cell cycle arrest, is critical for repairing DNA double-strand breaks and for RAD51-dependent homologous recombination. Furthermore, we found that BRCA1 deficiency results in increased chromosome damage and chemosensitivity after irofulven treatment.

The Fanconi anemia-BRCA pathway of genes are frequently mutated or epigenetically repressed in human cancer. The proteins of this pathway play pivotal roles in DNA damage signaling and repair. Irofulven is one of a new class of anticancer agents that are analogues of mushroom-derived illudin toxins. Preclinical studies and clinical trials have shown that irofulven is effective against several tumor cell types. The exact nature of irofulven-induced DNA damage is not completely understood. Previously, we have shown that irofulven activates ATM and its targets, NBS1, SMC1, CHK2, and p53. In this study, we hypothesize that irofulven induces DNA double-strand breaks and FANCD2 may play an important role in modulating cellular responses and chemosensitivity in response to irofulven treatment. By using cells that are proficient or deficient for FANCD2, ATR, or ATM, we showed that irofulven induces FANCD2 monoubiquitination and nuclear foci formation. ATR is important in mediating irofulven-induced FANCD2 monoubiquitination. Furthermore, we showed that FANCD2 plays a critical role in maintaining chromosome integrity and modulating chemosensitivity in response to irofulven-induced DNA damage. Therefore, this study suggests that it might be clinically significant to target irofulven therapy to cancers defective for proteins of the Fanconi anemia-BRCA pathway.[Mol Cancer Ther 2006;5(12):3153-61].

CHK2 and p53 are frequently mutated in human cancers. CHK2 is known to phosphorylate and stabilize p53. CHK2 has also been implicated in DNA repair and apoptosis induction. However, whether p53 affects CHK2 activation and whether CHK2 activation modulates chemosensitivity are unclear. In this study, we found that in response to the DNA damage agent, irofulven, CHK2 activation, rather than its expression, is inversely correlated to p53 status. Irofulven inhibits DNA replication and induces chromosome aberrations (breaks and radials) and p53-dependent cell cycle arrest. Pretreatment of cells with the DNA polymerase inhibitor, aphidicolin, resulted in reduction of irofulven-induced CHK2 activation and foci formation, indicating that CHK2 activation by irofulven is replication-dependent. Furthermore, by using ovarian cancer cell lines expressing dominant-negative CHK2 and CHK2-knockout HCT116 cells, we found that CHK2 activation contributes to the control of S and G2/M cell cycle arrests, but not chemosensitivity to irofulven. Overall, this study demonstrates that in response to irofulven-induced DNA damage, the activation of CHK2 is dependent on DNA replication and related to p53 status. By controlling cell cycle arrest and DNA replication, p53 affects CHK2 activation. CHK2 activation contributes to cell cycle arrest, but not chemosensitivity.

Translocations and unique chromosome break points in melanoma will aid in the identification of the genes that are important in the neoplastic process. We have previously shown a unique translocation in malignant melanoma cells der(12)t(12;20). The transcription factor E2F1 maps to 20q11. Increased expression of E2F has been associated with the autonomous growth of melanoma cells, however, the molecular basis has not yet been elucidated. To this end, we investigated E2F1 gene copy number and structure in human melanoma cell lines and metastatic melanoma cases. Fluorescent in situ hybridization (FISH) analysis using a specific E2F1 probe indicated increased E2F1 gene copies in melanoma cell lines compared to normal melanocytes. We also observed increased copies of the E2F1 gene in lymph node metastases of melanoma. In addition, Western blot analysis demonstrated increased E2F1 protein levels in 8 out of 9 melanoma cell lines relative to normal melanocytes. Inhibition of E2F1 expression with RNAi also reduced melanoma cell growth. Our results suggest that the release of E2F activity by elevated E2F1 gene copy numbers may play a functional role in melanoma growth.