SANDRA O'REILLY- Transformation of normal human fibroblasts into malignant cells, able to form fibrosarcomas, is a multi-step process involving selection & clonal expansion of genetically altered cells. To study the mechanisms involved, a human fibroblast lineage, designated MSU-1, consisting of normal, finite life span cells, an immortal diploid derivative strain, MSU-1.0, and its near-diploid derivative strain, MSU-1.1, was developed. MSU-1.1, but not MSU-1.0 cells, can be malignantly transformed by exposure to ionizing or UV radiation, or chemical carcinogens. The trans-formation involves changes in such genes as TELOMERASE, p53, MET, MMP2, & LRP12, or overexpression of an H-, K-, or N-RAS oncogene.

JEANNINE SCOTT- In addition to using translesion synthesis to continue replication of DNA that contains carcinogen-induced damage, human cells use various methods to avoid such error-prone replication. These involve use of an undamaged copy of the blocked DNA, such as the homologous copy provided by the allelic copy of that chromosome or the newly-synthesized daughter strand of the sister duplex of the DNA in which replication has been stopped. Such damage avoidance involves error-free gene conversion mechanisms. How cells signal the replicating fork to undertake such temporary use of an undamaged copy of the blocked sequence as a template is not yet clear. However, the process has recently been shown to involve protein complexes, i.e., hMms2 complexing with hUbc13, and allowing hUbc13-mediated mono- or poly-ubiquitination of PCNA through lysine-63. Jeannine is testing these hypotheses  by eliminating one or other of these proteins by use of siRNA or antisense RNA in human cells and then determining the effect on the frequency of gene conversion (homologous recombination) and also the effect on the frequency of mutations. If an error-free gene conversion pathway is blocked in cells that have acquired fork-blocking lesions in their DNA, the expected effect is an increase in the frequency of mutations.

IGOR ZLATKIN- Because the cells that form human cancers are derived from normal human cells, the human immune system frequently does not recognize and kill the cancer cells. To make the immune system more sensitive to cancer cells, we are developing a type of immunotherapy that could supplement standard cancer therapies.  In proof-of-principle experiments, we have injected human tumor-derived cells that express a highly antigenic, non-human protein referred to as PA19 into athymic mice. When the tumor cells express PA19 protein, we find that in many cases no tumors develop in a 6 month test period.  When the tumor cells do not express PA19 protein, tumors form at each site of injection within a few weeks. Evidence suggests that PA19 stimulates the cellular immune response and that it is responsible for the death of the cancer cells.  We are studying what types of tumors such a therapy may be useful for and how best to administer this agent.

YUN WANG- Determining the role of specialized human cell DNA polymerases in error-free and error-prone replication past DNA damage that blocks replication (fork-blocking lesions) is critical for determining the mechanisms of translesion synthesis and how carcinogens induce mutations that could lead to cells becoming malignant. XP variants patients have a normal ability to carry out repair of DNA lesions induced by sunlight, but nevertheless are highly prone to develop skin cancer. Researchers in the Carcinogenesis Laboratory have shown that skin fibroblasts from these patients are very sensitive to mutations induced by UV radiation, which would explain their susceptibility to skin cancer. More recently, others have shown that cells from XP variants lack polymerase eta, which is able to replicate past UV damage without making errors. Yun is determining what polymerase(s) are responsible for replicating past UV lesions in a such a highly error-prone manner. In particular she is testing whether human Pol iota plays a role.

TERRY MCMANUS - To determine the mechanisms involved in how mutations can be induced in human cells by carcinogenic agents, such as UV or chemicals, Terry is using various approaches, such as antisense, ribozymes, or siRNA to eliminate various mRNAs coding for specific proteins in such cells. Cells lacking specific proteins, e.g., those involved in translesion synthesis or in genetic recombination, are identified using antibodies and then tested for their response to specific carcinogens and compared with the response of normal cells. For such studies, he also prepares synchronized populations of cells and uses these to determine the effects of loss of specific proteins on the ability of carcinogen-treated cells to move through the cell cycle.