Joyce L. Hamlin, PhD
Chromosome replication, gene amplification, S-phase damage-sensing checkpoints
The focus of the Hamlin laboratory in the cancer arena relates to the nature of the gross chromosomal instability that is evident in advanced tumors, in particular gene amplification. Next to the loss of p53 activity, amplification of oncogenes is the most frequent large-scale genetic lesion in advanced cancers. Amplified oncogenes are arrayed end to end, either in homogenously-staining regions (HSRs) on the ends of chromosomes or, more often, on small, extrachromosomal double minutes (DMs). The lab was the first to provide evidence that amplificationof the model dihydrofolate reductase (DHFR) gene via either stable HSRs or DMs is initiated by chromosome breaks. These lead to loss of a telomeric fragment, followed by classic bridge-breakage-fusion cycles. This finding was unexpected and extremely important because it unified all of the overt karyotypic rearrangements in tumor cells (translocations, deletions, insertions, inversions, amplification) under one mechanistic umbrella (namely, chromosome breaks). They have recently embarked on a project in which they can engineer a chromosome break specifically at a site downstream from the DHFR gene, in order to test the bridge-breakage-fusion model directly. Fluorescence in situ hybridization and analysis of the repair of the broken ends shows that: 1) indeed, breakage leads to sister chromatid fusion, and 2) fusion is mediated by a repair process that leads to very long-patch erosion and subsequent homologous end-joining, and 3) the process only occurs in cells lacking a functional p53 pathway. By dissecting out the molecular mechanisms responsible for this important genetic rearrangement, the long-range goal is to develop rational strategies for intervention to prevent or reverse the amplification process.
In a second long-standing project, they have used their expertise in mammalian chromosomal replication to study the S-phase damage-sensing checkpoint in cultured cells. This checkpoint is thought to interfere with the maximum kill afforded by radiation and/or DNA damaging agents such as cisplatin. They have had a productive collaboration over the years with James Larner, who is a radiation oncologist. They have shown that the checkpoint inhibits replication solely at the initiation step as opposed to chain elongation. Recently, they have succeeded in cloning and eliciting antibodies to virtually every known protein that is involved in initiating replication in mammalian cells. They intend to use these reagents to determine via chromatin immunoprecipitation experiments which proteins are specifically targeted by the S-phase checkpoint. They will compare the response to ionizing radiation versus cisplatin, with the goal of developing drugs that would negate the checkpoint specifically in tumor cells and increase cell killing by cisplatin. They have begun to focus on a program project or multiinvestigator grant with several other investigators (Dutta, Li, Hecht, Kupfer, Larner, Grant, and Auble) to develop a unified approach to understanding the response to cisplatin in terms of damage-sensing, checkpoint arrest, DNA repair, and recovery.
The lab also has a collaborative grant with Zhifeng Shao to localize, identify, and characterize origins of replication. They have not published together so far, but expect to do so next year. Additionally, they collaborate with Dr. William Pearson on a grant to clone all of the origins of replication from the Chinese hamster and human genomes. Since origin activity, local transcriptional activity, and chromatin architecture have been shown to be co-regulated, and modulated during tumorigenesis, the project is very relevant to understanding molecular mechanisms of cancer development.