Our lab studies regulatory aspects of cell growth. We use normal human fibroblasts grown in culture. Our primary interest involves the G 1/S boundary of the cell cycle, when cells make the important commitment to begin DNA replication. More specifically, we concentrate on calcium/calmodulin-dependent pathways involving calmodulin-dependent kinase II or calmodulin-dependent phosphatase (PP2B or calcineurin).
Normal human fibroblasts, such as WS-1 cells, show highly regulated growth, unlike many transformed or cancerous cell lines. We use WS-1 cells to study the normal control mechanisms that may become deranged in cancer cells. Normal human cells are very difficult to transform. A recent report showed the ectopic expression of 3 genes – the mutant ras gene, the largeT antigen gene from the tumor virus SV-40, and the gene encoding the catalytic subunit of telomerase - –ere required for transformation of human cells.
The telomerase gene is required for cells to escape senescence and continue to grow. Telomerase is important in maintaining and capping the ends of chromosomes. Normal human fibroblasts lack telomerase and sensce after about 50 population doublings. We are expressing a telomerase construct (obtained under license from the Geron Corporation) in WS-1 fibroblasts. We expect to extend their usable lifetime in culture, but otherwise maintain normal growth control. We will continue our studies on the control of growth in normal cells, without the previous problems associated with cell cultures that stop growing after 50 population doublings.
We use a variety of approaches, including:
- Labeled thymidine incorporation into DNA as a measure of S-phase.
- Flow analysis of popidium iodide-stained nuclei to determine relative DNA content and calculate the percentage of cells in each phase of the cell cycle.
- Western immunoblot analysis to study expression of proteins involved in the cell cycle.
- Transfection of WS-1 cells using lipofectamine-or retrovirus-mediated gene transfer. The Green Fluorescent Protein (GFP) is used to identify transfected cells, and tag proteins like NFAT that translocate to the nucleus when activated by calcineurin.
- The TRAP assay (Trapeze Assay Kit from Intergen) measures the amount of telomerase activity present in cells.
Sejas,D., K. Haumschild, Y.Wang, and M.D. Mamrack. 1999. Contrasting Effects of Calmodulin-dependent Kinase and Phosphatase Inhibitors in Normal Human Fibroblasts. Mol. Biol. Cell 10s:247
Wang,Y., K. Haumschild, and M.D. Mamrack. 1999. Effect of the Phorbol Ester PMA on Cyclin D Expression in Normal Human Fibroblasts. Mol. Biol. Cell 10s:248.
Hesabi, B., J. D. Manzon, Y.-F., K. D. Haumschild, and M.D. Mamrack. 1997. Growth factor effects on chick cardimyocytes. Mol. Bio. Cell, 2070.
Bi, N. and M.D. Mamrack. 1994. PMA inhibits the growth of human fibroblasts after the induction of immediate-early genes. Exp. Cell Res. 212: 105-112.