Alterations in intracellular Ca2؉ homeostasis and cytochrome c release from mitochondria have been implicated in the regulation of apoptosis, but the relationship between these events remains unclear. Here we report that enforced expression of either Bax or Bak via adenoviral gene delivery results in the accumulation of the proteins in the endoplasmic reticulum (ER) and mitochondria, resulting in early caspase-independent BCL-2-sensitive release of the ER Ca 2؉ pool and subsequent Ca 2؉ accumulation in mitochondria. The inhibition of ER-to-mitochondrial Ca 2؉ transport with a specific inhibitor of mitochondrial Ca 2؉ uptake attenuates cytochrome c release and downstream biochemical events associated with apoptosis. Bax and Bak also directly sensitize mitochondria to cytochrome c release induced by immediate emptying of ER Ca 2؉ pool. Our results demonstrate that the effects of the "multidomain" proapoptotic BCL-2 family members Bak and Bax involve direct effects on the endoplasmic reticular Ca 2؉ pool with subsequent sensitization of mitochondria to calcium-mediated fluxes and cytochrome c release. These effects modulate the kinetics of cytochrome c release and apoptosis.

Previous studies have demonstrated that Ca2؉ is released from the endoplasmic reticulum (ER) in some models of apoptosis, but the mechanisms involved and the functional significance remain obscure. We confirmed that apoptosis induced by some (but not all) proapoptotic stimuli was associated with caspase-independent, BCL-2-sensitive emptying of the ER Ca 2؉ pool in human PC-3 prostate cancer cells.

Purpose: Krüppel-like factor 4 (KLF4) is a zinc-finger protein that plays important roles in stem cells and the development of gastric cancers. However, the role of KLF4 in primary lung cancer is unknown. The purpose of this study is to determine possible roles of KLF4 in lung cancer. Experimental Design: The KLF4 expression in primary lung cancer tissues and casematched normal lung tissues were determined by protein and mRNA analyses. The effects of KLF4 on cell proliferation, clonogenic formation, and cell cycle progression were determined in cultured lung cancer cells or bronchial epithelial cells after enforced KLF4 overexpression or small interfering RNA knockdown. The in vivo antitumor activity of KLF4 was evaluated by using stably transfected lung cancer cells and by adenovector-mediated gene delivery. The effect of KLF4 in regulating p21 and cyclin D1 was also evaluated. Results: KLF4 protein and mRNA levels were dramatically decreased in most primary lung tumors compared with in case-matched normal lung tissues. Enforced expression of KLF4 resulted in marked inhibition of cell growth and clonogenic formation. The tumor-suppressive effect of KLF4 was associated with its role in up-regulating p21 and down-regulating cyclin D1, leading to cell cycle arrest at the G 1 -S checkpoint. Knockdown of KLF4 promoted cell growth in immortalized human bronchial epithelial cells. The enforced expression of KLF4 gene to lung cancer cells by ex vivo transfection or adenovector-mediated gene transfer suppressed tumor growth in vivo. Conclusions: Our results suggest that KLF4 plays an important role in suppressing the growth of lung carcinoma. (Clin Cancer Res 2009;15(18):5688-95)