Iron depletion results in Src kinase inhibition with associated cell cycle arrest in neuroblastoma cells.

Iron is required for cellular proliferation. Recently, using systematic time studies of neuroblastoma cell growth, we better defined the G1 arrest caused by iron chelation to a point in mid-G1, where cyclin E protein is present, but the cyclin E/CDK2 complex kinase activity is inhibited. In this study, we again used the neuroblastoma SKNSH cells lines to pinpoint the mechanism responsible for this G1 block. Initial studies showed in the presence of DFO, these cells have high levels of p27 and after reversal of iron chelation p27 is degraded allowing for CDK2 kinase activity. The initial activation of CDK2 kinase allows cells to exit G1 and enter S phase. Furthermore, we found that inhibition of p27 degradation by DFO is directly associated with inhibition of Src kinase activity measured by lack of phosphorylation of Src at the 416 residue. Activation of Src kinase occurs very early after reversal from the DFO G1 block and is temporally associated with initiation of cellular proliferation associated with entry into S phase. For the first time therefore we show that iron chelation inhibits Src kinase activity and this activity is a requirement for cellular proliferation.

Two cell cycle blocks caused by iron chelation of neuroblastoma cells: separating cell cycle events associated with each block.

Studies have presented evidence that besides the well described S phase block, treatment of cancer cell lines with the iron chelator deferrioxamine (DFO) also results in an earlier block in G1 phase. In this article, measurements of cell cycle regulatory proteins define this block at a very specific point in G1. DFO treatment results in markedly decreased cyclin A protein levels. Cyclin E levels that accumulate in early to mid-G1 are increased in cells treated with DFO as compared to the resting cells. The DFO S phase block is shown after cells are arrested at G1/S by (aphidicolin) then released into DFO. The same S phase block occurs with DFO treatment of a neuroblastoma cell line relatively resistant to the G1 DFO block. These experiments clearly differentiate the S phase DFO block from the earlier block pinpointed to a point in mid-G1, before G1/S when cyclin E protein increases but before increased cyclin A synthesis. Apoptosis was observed in cells inhibited by DFO at both cell cycle arrest points.

Antagonism of endogenous growth hormone-releasing hormone (GHRH) leads to reduced proliferation and apoptosis in MDA231 breast cancer cells.

GHRH, in addition to stimulating the release of growth hormone (GH) from the pituitary, is a trophic factor for pituitary somatotrophs. Growth hormone-releasing hormone is also expressed in the gonads, gastrointestinal tract, pancreas, thymus, and lymphocytes, as well as in tumors of the pancreas, lung, central nervous system, and breast. Since GHRH has mitogenic effects, we examined the hypothesis that GHRH is an autocrine/paracrine growth factor in neoplastic breast tissue. The effect of disrupting endogenous GHRH on cell growth and apoptosis of MDA231 cells was examined through the use of a competitive GHRH antagonist, [N-acetyl-Tyr1, D-Arg2] fragment 1-29Amide (GHRHa). Cell proliferation was determined by direct cell counting and tritiated thymidine incorporation. Apoptosis was analyzed by examination of DNA laddering and nuclear condensation. GHRHa resulted in a dose-dependent, transient, and reversible decrease in cell number, proliferation rate, and tritiated thymidine uptake. Conversely, GHRHa led to a marked and dose-dependent increase in both DNA laddering and nuclear condensation. These results indicate that disruption of endogenous GHRH action in MDA231 cells results in both decreased cellular proliferation and increased apoptosis. Taken together, the findings suggest that endogenous GHRH acts as an autocrine/paracrine factor in the regulation of growth of at least some breast cancer cell types.