Overexpression of manganese superoxide dismutase protects against mitochondrial-initiated poly(ADP-ribose) polymerase-mediated cell death.

Mitochondria have recently been shown to serve a central role in programmed cell death. In addition, reactive oxygen species (ROS) have been implicated in cell death pathways upon treatment with a variety of agents; however, the specific cellular source of the ROS generation is unknown. We hypothesize that mitochondria-derived free radicals play a critical role in apoptotic cell death. To directly test this hypothesis, we treated murine fibrosarcoma cell lines, which expressed a range of mitochondrial manganese superoxide dismutase (MnSOD) activities, with respiratory chain inhibitors. Apoptosis was confirmed by DNA fragmentation analysis and electron microscopy. MnSOD overexpression specifically protected against cell death upon treatment with rotenone or antimycin. We examined bcl-x(L), p53 and poly(ADP-ribose) polymerase (PARP) to identify specific cellular pathways that might contribute to the mitochondrial-initiated ROS-mediated cell death. Cells overexpressing MnSOD contained less bcl-x(L) within the mitochondria compared to control (NEO) cells, therefore excluding the role of bcl-x(L). p53 was undetectable by Western analysis and examination of the proapoptotic protein bax, a p53 target gene, did not increase with treatment. Activation of caspase-3 (CPP-32) occurred in the NEO cells independent of cytochrome c release from the mitochondria. PARP, a target protein of CPP-32 activity, was cleaved to a 64 kDa fragment in the NEO cells prior to generation of nucleosomal fragments. Taken together, these findings suggest that mitochondrial-mediated ROS generation is a key event by which inhibition of respiration causes cell death, and identifies CPP-32 and the PARP-linked pathway as targets of mitochondrial-derived ROS-induced cell death.

Increased tumorigenicity in the human pancreatic cell line MIA PaCa-2 is associated with an aberrant regulation of an IGF-1 autocrine loop and lack of expression of the TGF-beta type RII receptor.

The growth characteristics associated with tumorigenicity were determined in clones of MIA PaCa-2 and PANC-1 pancreatic carcinoma cells. MIA PaCa-2 cells differed from PANC-1 cells in that they rapidly formed tumors in nude mice, formed colonies more rapidly and formed larger colonies in soft agar, and were cloned more efficiently when seeded at low density. MIA PaCa-2 cells but not PANC-1 cells were stimulated to escape quiescence and undergo DNA synthesis with nutrient media lacking growth factors. Both cell lines were stimulated to proliferate with serum-free media containing EGF, transferrin, and insulin. Antibody neutralization assays indicated that an IGF-1 autocrine loop was required for the nutrient stimulation of growth in MIA PaCa-2 cells and for the growth-factor stimulation in both MIA PaCa-2 and PANC-1 cells. Both cell lines were stimulated to proliferate with exogenous IGF-1 in basal media; this stimulation was specifically blocked by antibodies to IGF-1 or its receptor. MIA PaCa-2 and PANC-1 cells expressed similar levels of IGF-1 receptor mRNA and showed similar binding kinetics in receptor binding assays. In contrast to PANC-1 cells, MIA PaCa-2 cells were insensitive to TGF-beta 1 and did not express TGF-beta receptor type II. The results suggest that the growth-factor independence is representative of a more tumorigenic phenotype. We hypothesize that growth-factor independence of MIA PaCa-2 cells is mediated by an aberrant regulation of an IGF-1 autocrine loop. A decreased regulation of this IGF-1 loop may be potentiated by loss of response to TGF-beta.

Prospective study correlating P120 antigen expression with established prognostic factors in breast cancer.

P120 is a nucleolar proliferation antigen found in rapidly dividing cells and a variety of malignancies. Previous retrospective studies have demonstrated that, when detected in human breast cancer, P120 is associated with a poorer prognosis. To determine whether P120 expression correlates with other prognostic factors in breast cancer, we prospectively analysed pathologic and clinical data from 61 patients. P120 was detected in 40 of the 61 specimens (66%). No significant correlation existed between P120 expression and either tumour size or hormone receptors. A significant correlation was found between P120 expression and histological grade, degree of aneuploidy, S-phase fraction, degree of nodal involvement, and stage of disease. P120 is a biological marker indicative of tumour aggressiveness and may play an important role in determining which patients would most benefit from adjuvant therapy.