The main focus of this project is to define the potential autocrine/paracrine mechanisms regulating the growth of human tumors and utilize these as rational targets for early detection and intervention of malignant disease. We have developed several investigative strategies to achieve this goal. Our initial effort was accomplished by acclimating tumor cell lines to grow in protein-free/peptide-free medium (R0), thereby forcing the cell to express maximum survival capabilities in a nutrient poor environment. RPMI-1640 without phenol red was used as the base medium supplemented with L-glutamine (2nM) and sodium selenite (30nM). Under these conditions we have been able to establish long-term growth (2-5 yrs) from over 50 epithelial cancer lines including those of: lung (small cell/non-small cell), breast, colon, ovarian, pancreatic, prostate, and neuroblastoma/glioblastoma lineage. During the adaptation process all cells express a varying latency or acclimation period of between 5-14 days before abundant growth is achieved. This delay is thought to reflect the time required for gene activation and the accumulation of endogenous growth factors to critical levels since R0 conditioned media supports the growth of unadapted cells without a proliferative lag phase. Biochemical analysis of the conditioned media from R0 cells revealed the presence of protein/peptides such gastrin releasing peptide (GRP), insulin-like growth factors (IGF-I/IGF-II), transforming growth factor alpha (TGF-a), adrenomedullin (AM) and transferrin (Tf) which are established mitogens of tumor. Since R0 base medium contains no exogenous protein/peptides, the only source of such products was for the R0 adapted cells themselves. These conditions may mirror steps in carcinogenesis which involve clonal expansion of the initiated cell. For proliferation to occur in any cell process (carcinogenesis, wound repair, or embryogenesis) two fundamental requirements are necessary: 1) Activation of a signal transduction pathway (i.e. cAMP, Ca+2 flux, PI turnover, phosphorylation) by an inducer factor (IF) which triggers initial cell growth mechanisms and 2) Intracellular cofactor (metal ions and vitamins) availability mediated by a competency factor (CF) which allows growth to take place. It is the interplay between these two factors (IF/CF) that drives proliferation and underlies the clonal expansion process. Thus by identifying the IF/CF and their respective receptor systems expressed during R0 adaptation of tumor cell lines we have been able to generate appropriate molecular and immunological reagents to evaluate their involvement during normal to malignant conversion in pathological specimens. In addition, since disruption of IF/CF interactions result in growth cessation, these factors make potential targets to intervene in the carcinogenesis pathway by blocking the promotion event. Recent studies using this approach have identified an AM binding protein produced by human tumor cell lines adapted to R0 conditions.

Agency
National Institute of Health (NIH)
Institute
Division of Clinical Sciences - NCI (NCI)
Type
Intramural Research (Z01)
Project #
1Z01SC000192-07
Application #
6433321
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
7
Fiscal Year
2000
Total Cost
Indirect Cost
Name
Clinical Sciences
Department
Type
DUNS #
City
State
Country
United States
Zip Code
Martinez, Alfredo; Zudaire, Enrique; Julian, Miguel et al. (2005) Gastrin-releasing peptide (GRP) induces angiogenesis and the specific GRP blocker 77427 inhibits tumor growth in vitro and in vivo. Oncogene 24:4106-13
Julian, Miguel; Cacho, Monica; Garcia, Mario A et al. (2005) Adrenomedullin: a new target for the design of small molecule modulators with promising pharmacological activities. Eur J Med Chem 40:737-50
Martinez, Alfredo; Zudaire, Enrique; Portal-Nunez, Sergio et al. (2004) Proadrenomedullin NH2-terminal 20 peptide is a potent angiogenic factor, and its inhibition results in reduction of tumor growth. Cancer Res 64:6489-94
Martinez, Alfredo; Oh, Hae-Ryong; Unsworth, Edward J et al. (2004) Matrix metalloproteinase-2 cleavage of adrenomedullin produces a vasoconstrictor out of a vasodilator. Biochem J 383:413-8
Martinez, Alfredo; Julian, Miguel; Bregonzio, Claudia et al. (2004) Identification of vasoactive nonpeptidic positive and negative modulators of adrenomedullin using a neutralizing antibody-based screening strategy. Endocrinology 145:3858-65
Zudaire, Enrique; Martinez, Alfredo; Ozbun, Laurent L et al. (2004) Characterization of adrenomedullin in non-human primates. Biochem Biophys Res Commun 321:859-69
Ajona, Daniel; Castano, Zafira; Garayoa, Mercedes et al. (2004) Expression of complement factor H by lung cancer cells: effects on the activation of the alternative pathway of complement. Cancer Res 64:6310-8
Abasolo, Ibane; Yang, Luping; Haleem, Riffat et al. (2003) Overexpression of adrenomedullin gene markedly inhibits proliferation of PC3 prostate cancer cells in vitro and in vivo. Mol Cell Endocrinol 199:179-87
Martinez, Alfredo; Pio, Ruben; Zipfel, Peter F et al. (2003) Mapping of the adrenomedullin-binding domains in human complement factor H. Hypertens Res 26 Suppl:S55-9
Zudaire, E; Martinez, A; Cuttitta, F (2003) Adrenomedullin and cancer. Regul Pept 112:175-83