In this revised proposal, we reiterate that our studies have shown that transferrin receptor density is directly related to the state of cellular proliferation and that the highest density of transferrin receptor is found on cells in active phases of the cell cycle. Further studies relate the increased density of transferrin receptor found on the cell surface to increased receptor synthesis which occurs within hours after cells are exposed to stimulus for proliferation. Since it has been hypothesized that this increased density of transferrin receptor is regulated by an iron requirement during cellular proliferation, we have recently completed a number of experiments that lend strong support to this hypothesis. These studies utilize the class IIIA metal, gallium (Ga), which when bound to transferrin as transferrin-Ga, causes dose-related inhibition of cellulary iron uptake and arrest of proliferation of leukemic cells in tissue culture. This effect of transferrin-Ga different from the effects of other transferrin bound metals studied so far, but is similar to the effects seen with a monoclonal antibody (3A7.7), which results in inhibition of transferrin binding and associated iron uptake. In vitro studies proposed will beter elucidate the effect of transferrin-Ga on inhibition of cellular iron uptake and cellular proliferation, and in vivo studies will be performed on patients receiving gallium infusion for small cell lung cancer. We plan to measure iron utilization by proliferating cells under various tissue culture conditions. In added studies, we will further examine the specific effect by which transferrin-Ga appears to inhibit acidification of the endosome. Studies of the iron-containing enzyme ribonucleotide reductase, which may reflect the necessary iron requirement for cellular proliferation, will also be undertaken. Further studies utilizing a Chinese hamster-human hybrid cell system have permitted the specific mapping of the human genes for transferrin receptor and p97 antigen (a membrane iron-binding protein). These studies will be continued, including identification of conditions that affect expression of human receptor as well as p97 antigen on these cells. Further studies of our exciting recent finding of transferrin synthesis by malignant small cell lung cancer cell lines will be continued.
