KRISTIN MCNALLY- For a tumor to develop in the body requires that a single cell sequentially acquire a change (mutation) in a series of critical genes. Each change must enable that cell to grow faster than the surrounding cells so that the mutated cell produces a population large enough that the next growth-promoting mutation can occur in the cell that has already acquired the first mutation. If acquisition of the ultimate mutation, i.e., the genetic change that renders the cell that has acquired all the other required changes to now be able to form a malignant tumor, were to be blocked, the chance of developing cancer would be greatly reduced. Recently, it has been discovered that mutations are introduced by specialized polymerases that can replicate past DNA damage, but make mistakes. One of these polymerases is Pol zeta, composed of two parts, hRev3 and hRev7. Members of the Carcinogenesis Laboratory recently showed that by using antisense to prevent expression of the protein coded by hREV3, the catalytic subunit of Pol zeta, can dramatically reduce the frequency of mutations induced by various carcinogens. Kristin is using a similar approach (siRNA) to eliminate expression of the newly-discovered hREV7 gene, coding for the non-catalytic subunit of Pol zeta, to see if this polymerase plays a role in human cell mutagenesis. Cell strains found to have a decreased level of hRev7 protein as a result of siRNA are being tested to see if the frequency of  mutations induced by UV or chemical carcinogens is reduced or eliminated. 

DAN APPLEDORN- MSU-1.1 cells, which are derived from normal human skin fibroblasts, have acquired an unlimited lifespan in culture by turning on telomerase. Unlike normal human fibroblasts, these cells can be malignantly transformed by over-expression of an H-Ras oncogenes, which activates Rho GTPases, Cdc42 and Rac1. These H-Ras oncogene-expressing cells rapidly form tumors in athymic mice, while the parental MSU-1.1 cells do not. To identify down-stream effectors of Cdc42 and Rac1 and their role in carcinogenesis, Affymetrix Gene Chip analysis was used to compare cells that do and do not express these genes. Of particular interest was my finding that the vascular endothelial growth factor (VEGF) gene exhibited reduced expression in cells that have reduced levels of Rac1. These cells also showed a lower frequency of tumor formation. Ongoing studies are examining the mechanism by which Rac1 and CDC42 act as effectors of the H-as oncogene.

PIRO LITO- By use of Affymetrix Gene Chip analysis, we discovered that the gene Sprouty 2 is not expressed in normal human fibroblasts but is expressed at an enhanced level in the MSU-1.1 cell line (a cell line intermediate between normal and malignant).  Sprouty 2 is expressed at very high levels in MSU-1.1 cells malignantly transformed by expression of the H-Ras oncogene. Protein expression follows this same pattern. I have been studying the role of Sprouty 2 in malignant transformation. Sprouty 2 has been found to bind Cby, an E3 ubiquitin ligase, that ubinquinates the epidermal growth factor receptor (EGFR) and targets it for degradation.  I have found that cells expressing high levels of Sprouty 2 express high levels of the of EGFR protein.  When the H-Ras expressing malignant cells were transfected with a Sprouty 2 specific siRNA, such cells were no longer found to form tumors in athymic mice and the EGFR protein level was markedly reduced. From this and other experiments, I conclude that EGFR protein must be expressed at high levels and be activated by EGF or another suitable ligand for the H-Ras oncogene-transformed cells to be malignantly transformed. 

JIE ZHANG- LRP12 is a novel gene that was discovered in our laboratory (Qing et al, 1999) because it was expressed in normal human fibroblasts but not expressed in the malignantly transformed cell strains derived from them—or in the majority of cell lines derived from fibroblastic tumors from patients. LRP12 was found to be a member of the low-density lipoprotein receptor-related protein family of endocytotic receptors, with 73% identity to LRP3. Like all LRP's, LRP12 is a transmembrane protein. Studies using the yeast two-hybrid assay demonstrated that the protein SARA, binds to a section of LRP12 protein. I have found that SARA binds to the protein at a site immediately adjacent to the cytoplasmic side of the cell membrane. I have determined that the LRP12 protein serves to bring SARA in contact with the TGFbeta receptor, causing enhanced signaling by this pathway. Sarcoma cells that lack LRP12 protein exhibit reduced signaling through TGFbeta pathway and this correlates with their ability to form sarcomas in athymic mice.  

JESSICA APOSTOL - A third newly-discovered human polymerase, Pol kappa, is considered to play an essential role in the replication of DNA containing adducts formed by exposure to the polycyclic carcinogen designated benzo(a)pyrene diol epoxide (BPDE). To determine whether this protein is required for allows human cells to be able to replicate past fork-blocking BPDE damage without making mutations, Jessica has used various approaches to reduce expression of hPol kappa in a derivative of human cell strain MSU-1.1. She recently obtained evidence that this protein was successfully eliminated by using siRNA. The cells lacking hPol kappa are currently being examined to determine whether the loss of this protein results in a significant increase in the frequency of mutations induced by BPDE. Jessica will determine the nature of the mutations induced by this carcinogen in the absence hPol kappa and compare them with that induced by BPDE in normal cells. This should give her insight into the polymerases responsible for induction of mutations by this human carcinogen found in cigarettes.

RICK TOBEY - By use of Affymetrix Gene Chip analysis, we discovered that the gene Fibulin 5, which is expressed in normal human fibroblasts, is expressed at an reduced levels in the MSU-1.1 cell line (a cell line intermediate between normal and fully malignant).   Fibulin 5 is not expressed  in MSU-1.1 cells malignantly transformed by expression of the H-Ras oncogene. Protein expression follows this same pattern. We previously have found that the Fibulin 1 gene follows the same pattern of expression and that expression of Fibulin 1 in the malignant cells blocks tumor formation—indicating the Fibulin 1 is a tumor suppressor gene. I am investigating whether Fibulin 5 has a similar role. The Fibulin proteins are produced within cells and are secreted and form part of the extracellular matrix.  I have expressed Fibulin 5 in MSU-1.1 malignantly transformed cells  by expression of the H-Ras oncogenes, and I am testing them to determine whether their tumor forming ability has been impaired.