bcl-x is a member of the bcl-2 gene family, which may regulate programmed cell death. Mice were generated that lacked Bcl-x. The Bcl-x-deficient mice died around embryonic day 13. Extensive apoptotic cell death was evident in postmitotic immature neurons of the developing brain, spinal cord, and dorsal root ganglia. Hematopoietic cells in the liver were also apoptotic. Analyses of bcl-x double-knockout chimeric mice showed that the maturation of Bcl-x-deficient lymphocytes was diminished. The life-span of immature lymphocytes, but not mature lymphocytes, was shortened. Thus, Bcl-x functions to support the viability of immature cells during the development of the nervous and hematopoietic systems.

Aurora kinase A is a member of a new family of serine/threonine kinases that includes Drosophila melanogaster Aurora and Saccharomyces cerevisiae Ipl1 kinase, both of which are essential for controlling normal chromosome segregation and centrosome functions [1][2][3] . Aurora kinase A has been implicated in regulating centrosome function, spindle assembly, spindle maintenance and mitotic commitment in cells [4][5][6][7] . AURKA, encoding aurora kinase A, is a putative oncogene that is amplified and overexpressed in many human cancers [8][9][10][11][12][13] . The molecular targets of aurora kinase A have not been well characterized. We previously reported that phosphorylationmediated feedback between aurora kinase A and protein phosphatase 1 operates through mitosis and that disruption of this interaction results in defects in chromosome segregation 14 .Overexpression of aurora kinase A 8 and loss of wild-type p53 function induce similar phenotypes of centrosome amplification and aneuploidy in cells 15,16 . These observations suggest that gain of aurora kinase A function and loss of wild-type p53 function may be interdependent in common pathways. The finding that human tumors with elevated expression of aurora kinase A have wild-type TRP53 (encoding p53) also suggests that gain of aurora kinase A function may cause loss of wild-type p53 function, contributing to malignant transformation. p53 induces growth arrest or apoptosis in cells exposed to stress and is frequently mutated or deleted in human cancers. Expression of p53 is controlled by Mdm2, which promotes ubiquitination by E3 ubiquitin ligase activity and degradation of p53 by the cytoplasmic 26S proteasome 17 . Stability and activity of p53 are also regulated by post-translational modifications 18-23 including phosphorylation, acetylation, glycosylation and attachment of a small ubiquitin-related modifier protein. Phosphorylation at multiple sites is the predominant mechanism known to stabilize and abrogate Mdm2-mediated ubiquitination and activates p53. In contrast, phosphorylation of the core domain at Thr155 by the COP9 signalosome has been reported to target p53 for degradation 24 . The present study investigated whether phosphorylation by aurora kinase A also regulates p53 activity. RESULTS Aurora kinase A phosphorylates and interacts with p53We first investigated the ability of aurora kinase A to phosphorylate p53 in an in vitro kinase assay. We incubated bacterially expressed glutathione S-transferase (GST) and a GST-p53 fusion protein with aurora kinase A immunoprecipitated from mitotic HeLa cells and γ 32 P ATP. The aurora kinase A immunocomplex clearly phosphorylated GST-p53 (Fig. 1a). To confirm the specificity of aurora kinase A in phosphorylating p53, we used immunoprecipitated wild-type and kinase-inactive aurora kinase A (K162R) in an in vitro kinase assay with GST-p53. Wild-type aurora kinase A phosphorylated p53 but the kinase-inactive mutant did not (Fig. 1b), confirming that aurora A R T I C L E S

Several types of cancer cells, including colorectal cancerderived cell lines, show austerity, the resistance to nutrient starvation, but exactly how cancer cells obtain energy sources under conditions in which their external nutrient supply is extremely limited remains to be clarified. Because autophagy is a catabolic process by which cells supply amino acids from self-digested organelles, cancer cells are likely to use autophagy to obtain amino acids as alternative energy sources. Amino acid deprivation-induced autophagy was assessed in DLD-1 and other colorectal cancer-derived cell lines. The autophagosome-incorporated LC3-II protein level increased after treatment with a combination of autolysosome inhibitors, which interferes with the consumption of autophagosomes. Autophagosome formation was also morphologically confirmed using ectopically expressed green fluorescent protein-LC3 fusion proteins in DLD-1 and SW480 cells. These data suggest that autophagosomes were actively produced and promptly consumed in colorectal cancer cells under nutrient starvation. Autolysosome inhibitors and 3-methyl adenine, which suppresses autophagosome formation, remarkably enhanced apoptosis under amino acid-deprived and glucosedeprived condition. Similar results were obtained in the cells with decreased ATG7 level by the RNA interference. These data suggest that autophagy is pivotal for the survival of colorectal cancer cells that have acquired austerity. Furthermore, autophagosome formation was seen only in the tumor cells but not in the adjacent noncancerous epithelial cells of colorectal cancer specimens. Taken together, autophagy is activated in colorectal cancers in vitro and in vivo, and autophagy may contribute to the survival of the cancer cells in their microenvironment. [Cancer Res 2007;67(20):9677-84]

The function of Aurora-C kinase, a member of the Aurora kinase family identified in mammals, is currently unknown. We present evidence that Aurora-C, like Aurora-B kinase, is a chromosomal passenger protein localizing first to centromeres and then to the midzone of mitotic cells. Aurora-C transcript is expressed at a moderate level albeit about an order of magnitude lower than Aurora-B transcript in diploid human fibroblasts. The level of Aurora-C transcript is elevated in several human cancer cell types. Aurora-C and Aurora-B mRNA and protein expressions are maximally elevated during the G2/M phase but their expression profiles in synchronized cells reveal differential temporal regulation through the cell cycle with Aurora-C level peaking after that of Aurora-B during the later part of the M phase. Aurora-C, like Aurora-B, interacts with the inner centromere protein (INCENP) at the carboxyl terminal end spanning the conserved IN box domain. Competition binding assays and transfection experiments revealed that, compared with Aurora-C, Aurora-B has preferential binding affinity to INCENP and co-expression of the two in vivo interferes with INCENP binding, localization, and stability of these proteins. A kinase-dead mutant of Aurora-C had a dominant negative effect inducing multinucleation in a dose-dependent manner. siRNA mediated silencing of Aurora-C and Aurora-B also gave rise to multinucleated cells with the two kinases silenced at the same time displaying an additive effect. Finally, Aurora-C could rescue the Aurora-B silenced multinucleation phenotype, demonstrating that Aurora-C kinase function overlaps with and complements Aurora-B kinase function in mitosis.

Because autonomous proliferating cancer cells are often exposed to hypoxic conditions, there must be an alternative metabolic pathway, such as autophagy, that allows them to obtain energy when both oxygen and glucose are depleted. We previously reported finding that autophagy actually contributes to cancer cell survival in colorectal cancers both in vitro and in vivo. Pancreatic cancer remains a devastating and poorly understood malignancy, and hypoxia in pancreatic cancers is known to increase their malignant potential. In the present study archival pancreatic cancer tissue was retrieved from 71 cases treated by curative pancreaticoduodenectomy. Autophagy was evaluated by immunohistochemical staining with anti-LC3 antibody, as LC3 is a key component of autophagy and has been used as a marker of autophagy. The results showed that strong LC3 expression in the peripheral area of pancreatic cancer tissue was correlated with a poor outcome (P = 0.0170) and short disease-free period (P = 0.0118). Two of the most significant correlations among the clinicopathological factors tested were found between the peripheral intensity level of LC3 expression and tumor size (P = 0.0098) or tumor necrosis (P = 0.0127). Activated autophagy is associated with pancreatic cancer cells, and autophagy is thought to be a response to factors in the cancer microenvironment, such as hypoxia and poor nutrient supply. This is the first study to report the clinicopathological significance of autophagy in pancreatic cancer. (Cancer Sci 2008; 99: 1813-1819) C ancers are abnormal tissue masses whose growth exceeds and is uncoordinated with that of adjacent normal tissues, and which persist in the same excessive manner after cessation of the stimulus that evoked them.(1) All cancers ultimately depend on the host for their nutrition and blood supply, but as the preexisting vasculature is obviously insufficient to support the cancers' unlimited requirements for energy and nutrition as a result of their unregulated growth, angiogenesis has been considered pivotal to providing proliferating cancer cells with an adequate source of oxygen, energy, and nutrients. However, recent studies have revealed that even after new blood vessels have formed, both the oxygen and glucose supply is insufficient for the aggressively proliferating cancer cells in locally advanced cancers.(2-4) Tumor hypoxia has been used as a marker of poor prognosis; (5,6) however, how cancer cells become more malignant or survive with an extremely poor blood supply, as for example in pancreatic cancer, is poorly understood.(7) When cancer cells are exposed to hypoxia, anaerobic glycolysis increases and provides energy for cell survival, but as the glucose supply is also insufficient because of the poor blood supply, there must be an alternative metabolic pathway that provides energy when both oxygen and glucose are depleted. (8,9) We have reported that several cancer cell lines, including pancreatic cancer-and colorectal cancer-derived cell lines, are resistant to nutrient-deprived co...