Familial adenomatous polyposis coli (FAP) is a disease characterized by the development of multiple colorectal adenomas, and affected individuals carry germline mutations in the APC gene. With the use of a conditional gene targeting system, a mouse model of FAP was created that circumvents the embryonic lethality of Apc deficiency and directs Apc inactivation specifically to the colorectal epithelium. loxP sites were inserted into the introns around Apc exon 14, and the resultant mutant allele (Apc580S) was introduced into the mouse germline. Mice homozygous for Apc580S were normal; however, upon infection of the colorectal region with an adenovirus encoding the Cre recombinase, the mice developed adenomas within 4 weeks. The adenomas showed deletion of Apc exon 14, indicating that the loss of Apc function was caused by Cre-loxP-mediated recombination.

The protein p73 is a structural and functional homologue of the p53 tumour-suppressor protein but, unlike p53, it is not induced in response to DNA damage. The tyrosine kinase c-Abl is activated by certain DNA-damaging agents and contributes to the induction of programmed cell death (apoptosis) by p53-dependent and p53-independent mechanisms. Here we show that c-Abl binds to p73 in cells, interacting through its SH3 domain with the carboxy-terminal homo-oligomerization domain of p73. c-Abl phosphorylates p73 on a tyrosine residue at position 99 both in vitro and in cells that have been exposed to ionizing radiation. Our results show that c-Abl stimulates p73-mediated transactivation and apoptosis. This regulation of p73 by c-Abl in response to DNA damage is also demonstrated by a failure of ionizing-radiation-induced apoptosis after disruption of the c-Abl-p73 interaction. These findings show that p73 is regulated by a c-Abl-dependent mechanism and that p73 participates in the apoptotic response to DNA damage.

The ubiquitously expressed c-Abl protein tyrosine kinase localizes to both the nucleus and cytoplasm. The nuclear form of c-Abl is activated in the cellular response to genotoxic stress. Here we show that cytoplasmic c-Abl is activated by oxidative stress. The results also demonstrate that mitochondrial cytochrome c is released in the cellular response to H 2 O 2 and that this effect is mediated by a c-Abl-dependent mechanism. In concert with these results, we show that H 2 O 2 -induced apoptosis is attenuated in c-Abl-deficient cells. These findings demonstrate that cytoplasmic c-Abl is involved in the apoptotic response of cells to oxidative stress.Normal cellular metabolism is associated with the production of reactive oxygen species (ROS) 1 and, as a consequence, damage to DNA and proteins (1, 2). The generation of ROS is also known to induce apoptosis; however, the molecular mechanisms responsible for ROS-induced apoptosis are unclear. Studies have indicated that ROS induce activation of topoisomerase II-mediated cleavage of chromosomal DNA and thereby apoptosis (3). Other work has suggested that ROSinduced apoptosis is p53-dependent (4, 5) and that p53-induced apoptosis is mediated by . In addition, the p66 shc adaptor protein (5) and the p85 subunit of phosphatidylinositol 3-kinase (PI3K) (4) have been implicated in the apoptotic response to oxidative stress.The nuclear form of the c-Abl tyrosine kinase is activated in the cellular response to genotoxic stress (9). Nuclear c-Abl has been implicated in the apoptotic response to DNA damage by mechanisms in part dependent on p53 and its homolog, p73 (10 -14). c-Abl also functions as an upstream effector of the proapoptotic SAPK/JNK and p38 mitogen activated protein kinase (MAPK) pathways in the genotoxic stress response (9,15,16). Other studies have demonstrated that c-Abl phosphorylates p85 and thereby inhibits PI3K activity in the apoptotic response to DNA damage (17). Additional evidence supporting a role for c-Abl in apoptosis has been provided by the findings that cells deficient in c-Abl or expressing a dominant-negative c-Abl mutant exhibit an attenuated apoptotic response to genotoxic agents (18,19).Recent work has shown that c-Abl phosphorylates protein kinase C (PKC) ␦ in cells treated with H 2 O 2 (20). The present results demonstrate that the cytoplasmic, and not the nuclear, form of c-Abl is activated in the cellular response to H 2 O 2 . We also show that H 2 O 2 induces mitochondrial cytochrome c release and apoptosis by a c-Abl-dependent mechanism. MATERIALS AND METHODSCell Culture-COS7 cells and MEFs derived from wild-type and c-Abl Ϫ/Ϫ mice (21) were cultured in Dulbecco's modified Eagle's medium containing 10% heat-inactivated fetal calf serum, 2 mM L-glutamine, 100 units/ml penicillin, and 100 g/ml streptomycin. DLD1 cells were grown as described (7). Cells were treated with H 2 O 2 (Sigma), 30 mM N-acetyl-L-cysteine (NAC; Sigma), or 10 M cis-platinum (Sigma).Analysis of Kinase Activity-Cell lysates were prepared in lysis buffer (10 mM Tr...

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Protein kinase C (PKC) ␦ is cleaved by caspase-3 to a kinase-active catalytic fragment (PKC␦CF) in the apoptotic response of cells to DNA damage. Expression of PKC␦CF contributes to the induction of apoptosis by mechanisms that are presently unknown. Here we demonstrate that PKC␦CF associates with p73␤, a structural and functional homologue of the p53 tumor suppressor. The results show that PKC␦CF phosphorylates the p73␤ transactivation and DNA-binding domains. One PKC␦CF-phosphorylation site has been mapped to Ser-289 in the p73␤ DNA-binding domain. PKC␦CF-mediated phosphorylation of p73␤ is associated with accumulation of p73␤ and induction of p73␤-mediated transactivation. By contrast, PKC␦CF-induced activation of p73␤ is attenuated by mutating Ser-289 to Ala (S289A). The results also demonstrate that PKC␦CF stimulates p73␤-mediated apoptosis and that this response is attenuated with the p73␤(S289A) mutant. These findings demonstrate that cleavage of PKC␦ to PKC␦CF induces apoptosis by a mechanism in part dependent on PKC␦CF-mediated phosphorylation of the p73␤ Ser-289 site.

The nuclear p300/CBP proteins function as coactivators of gene transcription. Here, using cells deficient in p300 or CBP, we show that p300, and not CBP, is essential for ionizing radiation-induced accumulation of the p53 tumor suppressor and thereby p53-mediated growth arrest. The results demonstrate that deficiency of p300 results in increased degradation of p53. Our findings suggest that p300 contributes to the stabilization and transactivation function of p53 in the cellular response to DNA damage.In the exposure of cells to ionizing radiation (IR), 1 the formation of DNA double-strand breaks is associated with increases in p53 levels and the transactivation function of p53 (1-3). Activation of p53 in the response to IR induces transcription of the p21 (WAF1, Cip-1) gene (4). Thus, the growth arrest function of p53 is regulated at least in part by p21-mediated inhibition of cyclin-Cdk complexes and the proliferating cell nuclear antigen (PCNA) (1). In addition, p53-dependent induction of the bax gene contributes to the apoptotic response to DNA damage (5). Other genes implicated in p53-induced growth arrest and apoptosis include GADD45 (3), mdm2 (6, 7), cyclin G (8), and IGF-BP3 (9).Recent work has demonstrated that the DNA-dependent protein kinase (DNA-PK) is necessary but not sufficient for activation of p53 sequence-specific DNA binding (10). Phosphorylation of the p53 N-terminal region by DNA-PK may contribute to the transactivation function and stability of p53 (11,12). Other studies have shown that the ataxia telangiectasia-mutated (ATM) protein phosphorylates p53 on serine 15 in vitro (13,14). The findings that the p53 serine 15 site is phosphorylated in IR-treated cells (15,16) and that this effect is diminished in AT cells (16) have supported a role for ATM in the regulation of p53. The p300/CBP proteins (17-20) have also been implicated as coactivators of the p53 transactivation function (21,22). The N-terminal domain of p53 interacts with the C-terminal region of p300/CBP. Acetylation of the p53 C-terminal domain by p300/CBP stimulates the DNA binding activity of p53 (23). A dominant negative form of p300/CBP has also been found to inhibit p53-mediated transactivation and the G 1 arrest and apoptotic responses (24).Cells derived from p300-deficient embryos exhibit severe defects in proliferation (25). Consequently, in the present work, we have established cells expressing ribozymes specific for p300 or CBP such that the transfectants are selectively deficient in either protein. Our results demonstrate that p300, and not CBP, is essential for IR-induced increases in both p53 levels and the p53 transactivation function. MATERIALS AND METHODSCell Culture-MCF-7 cells were maintained in Dulbecco's modified Eagle's medium containing 10% heat-inactivated bovine serum, 2 mM L-glutamine, 10 units/ml penicillin, and 10 g/ml streptomycin. The active p300 (p300-R), inactive p300 (p300-RI), active CBP (CBP-R), or inactive CBP (CBP-RI) ribozymes (26) were stably introduced into cells by LipofectAMINE (Life Tech...

The cellular response to ionizing radiation (IR) includes the induction of apoptosis. The p300/CBP proteins possess histone acetyltransferase activity and function as transcriptional coactivators of p53. We have prepared cells de®cient in p300 or CBP to de®ne the roles of these proteins in the cellular response to DNA damage. The present results demonstrate that p300, but not CBP, contributes to IR sensitivity of cells. The results also demonstrate that IR-induced apoptosis is impaired in the p300-, but not CBP-, de®cient cells. These ®ndings indicate that p300 functions in the apoptotic response to DNA damage.

The formation of the bipolar spindle is responsible for accurate chromosomal segregation during mitosis. The dynamic instability of microtubules has an important role in this process, and has been shown to be an effective target for cancer chemotherapy. Several agents that target non-microtubule mitotic proteins, including the motor protein Eg5, Aurora kinases and Polo-like kinases, are currently being developed as chemotherapeutic drugs. However, because the efficacies of these drugs remain elusive, new molecular targets that have essential roles in tumor cells are desired. Here, we provide in vivo evidence that transforming acidic coiled-coil-3 (Tacc3) is a potential target for cancer chemotherapy. Using MRI, we showed that Tacc3 loss led to the regression of mouse thymic lymphoma in vivo, which was accompanied by massive apoptosis. By contrast, normal tissues, including the thymus, showed no overt abnormalities, despite high Tacc3 expression. in vitro analysis indicated that Tacc3 depletion induced multi-polar spindle formation, which led to mitotic arrest, followed by apoptosis. Similar responses have been observed in Burkitt's lymphoma and T-ALL. These results show that Tacc3 is a vulnerable component of the spindle assembly in lymphoma cells and is a promising cancer chemotherapy target.