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DNA amplification is an important mechanism of tumor progression that allows cancer cells to up-regulate expression of critical genes such as oncogenes. Recent studies using comparative genomic hybridization revealed increased DNA copies on chromosome 20q in seven melanoma cell lines and eight archival metastatic melanoma lesions. We performed FISH analysis of metaphase spreads in 13 melanoma cell lines and nine primary melanoma specimens using a variety of probes specific for chromosome 20. All 13 cell lines (100%) and 8/9 primary tumors (89%) showed extra copies of chromosome 20 relative to tumor ploidy. Additionally, 6/14 cell lines (43%) and 2/8 primary tumors (25%) showed translocated chromosome 20 material. Cytological evidence for gene amplification was found in one of the 13 cell lines with an add(20)(p13). These data suggest that over-representation of a gene(s) important for melanoma pathogenesis occurs on the chromosome 20 long arm.

<p> Cancer cells are characterized by elevated ROS levels, which provide these cells with a distinct survival advantage, promoting proliferation, invasion, and resistance to apoptotic stimuli. In order to maintain ROS below a critical threshold that would otherwise result in death, cancer cells have appropriately adapted their anti-oxidative machinery. Here, I studied superoxide dismutase 1 (SOD1) as a potential contributor to cancer cell survival under conditions of high oxidative stress. I determined that SOD1 is up-regulated in a majority of cancer cells in which the activity of another dismutase, MnSOD, is reduced, and found evidence to suggest that SOD1 is essential for maintaining mitochondrial integrity in these cells. Additionally, I explored a possible mechanism by which mitochondrial SOD1 increases in cancer cells, and found evidence indicating that this is due to decreased MULAN levels. Last, I tested whether blocking SOD1 activity could sensitize cancer cells to chemotherapeutic treatment, and determined that additional mechanisms must exist to compensate for SOD1 activity loss.</p>

Chromosomal translocations involving the mixed-lineage leukemia (MLL) gene lead to acute myeloid or lymphoid leukemias that are often associated with poor prognosis, particularly in infants. The translocations result in in-frame fusion between MLL and one of the over sixty known partner genes that are heterogeneous in nature. AF4 and AF9 are two of the most common MLL fusion partners. Importantly, they also interact specifically with each other in subnuclear foci. An AF4-mimetic peptide which disrupts the AF4-AF9 interaction is able to cause necrotic cell death in leukemic cell lines harboring MLL-AF4 or MLL-AF9 fusion oncogenes, emphasizing the importance of the AF4-AF9 interaction in leukemias caused by these fusions. AF4 and AF9 have been isolated as components of multiple transcription elongation complexes whose other members include the RA polymerase II regulator pTEFb and the chromatin modifying enzyme Dot1. Dot1 and its unique histone H3 lysine 79 methyltransferase activity are implicated in leukemias associated with MLL chimeric ncogenes. Data presented in this dissertation indicate that both AF4 and Dot1 are critical co-factors for MLL-AF9 associated leukemias. Interruption of the AF4---MLL-AF9 interaction, either by mutations or by overexpression of a dominant negative AF4 fragment, abolishes MLL-AF9---mediated hematopoietic cell immortalization as well as MLL target gene activation. In addition, loss of Dot1 function compromises cell viability by triggering apoptosis in hematopoietic cells immortalized by MLL-fusion oncoproteins. These findings poses AF4 and Dot1 as potential therapeutic targets for treatment of acute leukemias associated with MLL fusio oncogenes / acase@tulane.edu