Apoptosis causes characteristic morphological changes in cells, including membrane blebbing, cell detachment from the extracellular matrix, and loss of cell-cell contacts. We investigated the changes in focal adhesion proteins during etoposide-induced apoptosis in Rat-1 cells and found that during apoptosis, p130cas (Crk-associated substrate [Cas]) is cleaved by caspase-3. Sequence analysis showed that Cas contains 10 DXXD consensus sites preferred by caspase-3. We identified two of these sites (DVPD 416 G and DSPD 748 G) in vitro, and point mutations substituting the Asp of DVPD 416 G and DSPD 748 G with Glu blocked caspase-3-mediated cleavage. Cleavage at DVPD 416 G generated a 74-kDa fragment, which was in turn cleaved at DSPD 748 G, yielding 47-and 31-kDa fragments. Immunofluorescence microscopy revealed well-developed focal adhesion sites in control cells that dramatically declined in number in etoposide-treated cells. Cas cleavage correlated temporally with the onset of apoptosis and coincided with the loss of p125FAK (focal adhesion kinase [FAK]) from focal adhesion sites and the attenuation of Cas-paxillin interactions. Considering that Cas associates with FAK, paxillin, and other molecules involved in the integrin signaling pathway, these results suggest that caspase-mediated cleavage of Cas contributes to the disassembly of focal adhesion complexes and interrupts survival signals from the extracellular matrix. INTRODUCTIONApoptotic cell death is a fundamental biological process crucial for proper organism development and for maintenance of tissue homeostasis (Raff, 1992). Apoptosis causes characteristic morphological changes that include membrane blebbing, decreased adhesion and intercellular contacts, chromatin condensation, nuclear fragmentation, and the packing of the nuclear fragments into membrane-enclosed apoptotic bodies (Wyllie et al., 1980;Darzynkiewicz et al., 1994). Caspases are a family of highly conserved aspartate-specific cysteine-proteases related to mammalian interleukin-1␤-converting enzyme, which are involved in the final execution phase of apoptosis (Nicholson, 1996;Nicholson and Thornberry, 1997). The substrates for caspases are now known to include a growing number of molecules involved in cytoskeletal regulation and signaling, including ␣-fodrin, gelsolin, growth arrest-specific gene (Gas 2), MEKK-1, and PKC (Brancolini et al., 1995;Emoto et al., 1995;Martin et al., 1995;Ghayur et al., 1996;Cardone et al., 1997;Kothakota et al., 1997;Janicke et al., 1998). Recent studies have shown that attachment to the extracellular matrix (ECM) actively regulates cell survival, and that cells undergoing apoptosis lose their connections to neighboring cells and to the ECM. For example, epithelial and endothelial cells undergo apoptosis ("anoikis") when displaced from the ECM (Frisch and Francis, 1994;Re et al., 1994), whereas cell adhesion to ECM suppresses apoptosis in mammary epithelial cells (Frisch and Francis, 1994;Boudreau et al., 1995;Aharoni et al., 1996).Cell attachment to the ECM i...

No abstract

No abstract

Vascular endothelial growth factor (VEGF) and its receptors are considered the primary cause of tumor-induced angiogenesis. Specifically, VEGFR-2/kinase insert domain receptor (KDR) is part of the major signaling pathway that plays a significant role in tumor angiogenesis, which is associated with the development of various types of tumor and metastasis. In particular, KDR is involved in tumor angiogenesis as well as cancer cell growth and survival. In this study, we evaluated the therapeutic potential of TTAC-0001, a fully human antibody against VEGFR-2/KDR. To assess the efficacy of the antibody and pharmacokinetic (PK) relationship in vivo, we tested the potency of TTAC-0001 in glioblastoma and colorectal cancer xenograft models. Antitumor activity of TTAC-0001 in preclinical models correlated with tumor growth arrest, induction of tumor cell apoptosis, and inhibition of angiogenesis. We also evaluated the combination effect of TTAC-0001 with a chemotherapeutic agent in xenograft models. We were able to determine the relationship between PK and the efficacy of TTAC-0001 through in vivo single-dose PK study. Taken together, our data suggest that targeting VEGFR-2 with TTAC-0001 could be a promising approach for cancer treatment.

Nocodazole, a microtubule-disrupting agent, induced apoptosis in Rat-1 cells, as indicated by changes in cell morphology, DNA fragmentation, and eventual cell death. During nocodazole-induced apoptosis, normally flat cells became rounded in shape and detached from the extracellular matrix. These morphological changes appeared to be closely associated with degradation of focal adhesion proteins, including p130cas, p125(FAK) and paxillin. p130cas was also degraded in cells treated with staurosporine or etoposide, suggesting that degradation of focal adhesion proteins is a characteristic feature of apoptosis. Nocodazole-induced apoptosis was antagonized by Bcl-2: degradation of focal adhesion proteins was suppressed and cell viability was enhanced in bcl-2 over-expressing cells, even in the presence of nocodazole. Further study of the molecular mechanism of Bcl-2 activation should provide an understanding of the apoptosis induced by disruption of the microtubule network.