The hypothesis behind this research proposal is that with tumor clonal progression there is decreasing epidermal growth factor and other polypeptide hormone responsiveness, genetic and cytogenetic change, increasing anchorage-independent growth in agar, increasing metastatic potential, and less sensitivity to chemotherapy and radiation. To test this hypothesis, cloned populations of epidermal growth factor-responsive and epidermal growth factor-nonresponsive cell lines derived from both the primary tumor and synchronous metastatic lesion in the same patient will need to be established. Melanoma will be used as a model. The specific objectives are 1) to establish cell lines from the primary tumor and the synchronous metastasis in the same patient in an adhesive tumor cell culture system under defined reproducible conditions of serum-free hormone-supplemented medium with and without epidermal growth factor, 2) to establish several clones from these cell lines which are homogeneous in their response to epidermal growth factor and have observed in vitro stability, 3) to compare a number of in vitro characteristics of these clones (morphology, anchorage-dependent cloning efficiency, anchorage-independent cloning efficiency in agar, cytogenetics, and drug and radiation sensitivity), 4) to determine if epidermal growth factor independence is associated with autocrine secretion of growth factor by examining the conditioned media of these clonal cell lines, 5) to analyze if either of these epidermal growth factor-dependent and epidermal growth factor-independent clones interact positively or negatively on the in vitro culture behavior of each other by co-cultivation experiments, and 6) to determine the ability of epidermal growth factor-dependent and epidermal growth factor-independent clones derived from different origins (primary or metastatic) to induce subcutaneous tumors and metastatic pulmonary colonies in nude pathogen-free mice. These experiments could answer important questions such as 1) predicting in advance the biological behavior of primary tumors by eliciting if primary tumors already contain clones identical to those present in metastatic lesions (such identity being determined by morphology, anchorage-independence, cytogenetics, drug and radiation sensitivity) or whether this clonal change to more virulent behavior (growth factor independence, chemotherapy and radiation resistance) occurs subsequently after the metastasis and 2) determining if the control of chemotherapy and radiation resistant components of metastatic tumors could be approached by regulating the tumor's response to growth factors.
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