MDA-MB-231 human breast cancer cells belong to a highly invasive metastatic cell line that depends on phospholipase D (PLD) activity for survival when deprived of serum growth factors. In response to the stress of serum withdrawal, there is a rapid and dramatic increase in PLD activity. Concomitant with increased PLD activity, there was an increase in the ability of MDA-MB-231 cells to both migrate and invade Matrigel TM . The ability of MDA-MB-231 cells to both migrate and invade Matrigel TM was dependent on both PLD and mTOR, a downstream target of PLD signals. Serum withdrawal also led to a PLD-dependent increase in the expression of the stress factor, hypoxia-inducible factor-1␣. These data reveal that PLD survival signals not only prevent apoptosis but also stimulate cell migration and invasion, linking the ability to suppress apoptosis with the ability to metastasize.The conversion of a normal cell to a malignant cancer cell involves multiple genetic alterations that overcome the many protections built into cells that prevent unwanted proliferation (1). Perhaps the most crucial step in progression to malignancy is gaining the ability to migrate or metastasize to distant sites where the growth of multiple tumors ultimately causes the lethal consequences of the cancer. Although there are several cellular properties that correlate with increased metastatic potential, such as increased protease secretion (2), there has never been a clear genetic event that confers metastatic capability. However, it has been suggested that mutations occurring at early stages of tumorigenesis that confer a proliferative advantage may also contribute to the ability to metastasize at later stages of tumor progression (3).Among the obstacles to be overcome in a developing tumor are default apoptotic programs that cause cells with faulty division signals to undergo apoptosis (1). A cell must generate "survival signals" to suppress these apoptotic programs (4 -6). Interestingly, signals that have been shown to suppress apoptosis have also been linked to cell migration, a hallmark of the metastatic phenotype. Both phosphatidylinositol 3-kinase and phospholipase D (PLD), 2 which provide survival signals in human cancer cells (7-9), have also been linked with cellular processes that contribute to cell migration (8,10). This correlation between survival and cell migration suggests that generating a survival signal early in tumorigenesis could also endow the cell with the ability to migrate. This raises the question as to how the migration would be triggered. One possibility is that although a primary tumor mass is forming, survival signals are selected for in cells deprived of blood serum to suppress the apoptosis that would occur in an unvascularized tumor mass. If the survival response of cells also includes increased cell migration, then in addition to suppression of apoptosis, the response would also include migration to sites where growth factors and nutrition could be obtained.We recently described a survival signal in the highly m...

Phospholipase D (PLD) is elevated in rodent fibroblasts expressing activated H-Ras mutants. We therefore examined the PLD activity in human cancer cells with activating Ras mutations. T24 bladder carcinoma cells express an activated H-Ras gene and Calu-1 lung carcinoma cells express an activated K-Ras gene. We report here that both of these cancer cell lines express highly elevated levels of PLD activity and that the PLD activity is dependent upon Ras. We also show that the PLD activity is dependent upon the Ras effector molecules RalA and phosphatidylinositol-3-kinase (PI3K). PLD activity has been shown to provide a survival signal in breast cancer cell lines that suppressed stress-induced apoptosis. Suppression of PLD activity in the T24 and Calu-1 cells resulted in apoptotic cell death in the absence of serum, indicating that the elevated PLD activity provided a survival signal in these cancer cell lines. Suppression of Ras, RalA, or PI3K also led to apoptosis in the absence of serum. These data indicate that a critical component of Ras signaling in human cancer cells is the activation of PLD and that targeting PLD survival signals in cancer cells could be an effective strategy to induce apoptosis in human cancers with activating Ras mutations.

Our study was undertaken to investigate whether the inflammatory mediator high-mobility group box 1 (HMGB1) can enter the renal tissue and urine and what is the functional change of renal tubular epithelial cells (TECs) interacting with HMGB1 during sepsis. We found that the transcription levels of interleukin 1 (IL-1) and interleukin 6 (IL-6) mRNA in TECs increased significantly during sepsis and these processes can be blocked by splenectomy. We also found out HMGB1 accumulated in the renal tissue and entered urine during sepsis and toll-like receptor 4 (TLR4) was expressed by TECs. In vitro, we demonstrated that HMGB1 induced MAPK and NF-κB activation and G1 cell cycle arrest in TECs. We also found that the mRNA transcription levels of IL-1, IL-6, and tissue inhibitor of metalloproteinases 2 (TIMP2) increased significantly and the IL-1, IL-6, and TIMP2 can be secreted by TECs stimulated by HMGB1. In contrast, LPS RS can block all of the processes above in vitro. In vivo, the increase of the mRNA transcription level of TIMP2 was also observed. These data indicate that HMGB1 accumulates in renal tissue and enters the urine and the interaction between HMGB1 and TLR4 turns TECs into inflammatory promoters during sepsis.