Apoptosis is a morphologically distinct form of programmed cell death that plays a major role in cancer treatments. This cellular suicide program is known to be regulated by many di erent signals from both intracellular and extracellular stimuli. Here we report that p53 suppressed expression of the cellular FLICE-inhibitory protein (FLIP) that potentially blocks apoptotic signaling in human colon cancer cell lines expressing mutated and wild-type p53. In contrast, the expression of the death receptor KILLER/DR5 (TRAIL-R2) had no e ect on FLIP expression, although exogenous p53 is known to induce KILLER/DR5 expression. In line with these observations, FLIP-negative cancer cells were sensitive to both p53-and KILLER/DR5-mediated apoptosis, whereas cells containing high levels of FLIP underwent apoptotic cell death when triggered by ectopic p53 expression but not by KILLER/DR5 expression. Treating the cells with a speci®c inhibitor of the proteasome inhibited the decrease of FLIP by p53, suggesting that p53 enhances the degradation of FLIP via a ubiquitinproteasome pathway. Thus, the data indicate that p53-mediated downregulation of FLIP may explain the potent sensitization of human cancer cells to the apoptotic suicide program induced by wild-type p53 gene transfer. Oncogene (2001) 20, 5225 ± 5231.

The tumor suppressor gene p53 is a potent transcriptional regulator of genes which are involved in many cellular activities including cell cycle arrest, apoptosis, and angiogenesis. Recent studies have demonstrated that the activation of the transcriptional factor nuclear factor kB (NF-kB) plays an essential role in preventing apoptotic cell death. In this study, to better understand the mechanism reponsible for the p53-mediated apoptosis, the eect of wild-type p53 (wt-p53) gene transfer on nuclear expression of NF-kB was determined in human colon cancer cell lines. A Western blot analysis of nuclear extracts demonstrated that NF-kB protein levels in the nuclei were suppressed by the transient expression of the wt-p53 in a dose-dependent manner. Transduced wt-p53 expression increased the cytoplasmic expression of IkBa as well as its binding ability to NF-kB, thus markedly reducing the amount of NF-kB that translocated to the nucleus. The decrease in nuclear NF-kB protein correlated with the decreased NF-kB constitutive activity measured by electrophoretic mobility shift assay. Furthermore, parental cells transfected with NF-kB were better protected from cell death induced by the wt-p53 gene transfer. We also found that the wtp53 gene transfer was synergistic with aspirin (acetylsalicylic acid) in inhibiting NF-kB constitutive activity, resulting in enhanced apoptotic cell death. These results suggest that the inhibition of NF-kB activity is a plausible mechanism for apoptosis induced by the wt-p53 gene transfer in human colon cancer cells and that anti-NF-kB reagent aspirin could make these cells more susceptible to apoptosis. Oncogene (2000) 19, 726 ± 736.

The CD95 (Fas/APO-1) system regulates a number of physiological and pathological processes of cell death. The ligand for CD95 induces apoptosis in sensitive target cells by interacting with a transmembrane cell surface CD95 receptor. We previously reported that the recombinant adenovirus-mediated transfer of the wildtype p53 gene caused apoptotic cell death in a variety of human cancer cells. To better understand the mechanism responsible for this cell death signaling, we have investigated the potential involvement of the CD95 receptor/ligand system in p53-mediated apoptosis. The transient expression of the wild-type p53 gene upregulated the CD95 ligand mRNA as well as protein expression in H1299 human lung cancer cells de®cient for p53 and in DLD-1 and SW620 human colon cancer cells with mutated p53, all of which constitutively expressed CD95 receptor as shown by a¯ow cytometric analysis, and induced rapid apoptotic cell death as early as 24 h after gene transfer. However, the sensitivity to the cytolytic e ect of agonistic anti-CD95 antibody (CH11) varied among these cell lines: CH11 induced apoptosis in H1299 cells, but not in DLD-1 and SW620 cells despite their abundant CD95 receptor expression, suggesting that the CD95 receptors on DLD-1 and SW620 cells might be inactivated. In addition, an antagonistic anti-CD95 ligand antibody (4H9) that interfered with the CD95-receptor-ligand interaction partially reduced the apoptosis induced by the wild-type p53 gene transfer in H1299 cells, whereas apoptosis of DLD-1 and SW620 cells occurred in the presence of 4H9. Taken together, these ®ndings led us to conclude that the CD95 receptor/ligand system is di erentially involved in p53-mediated apoptosis, suggesting that the restoration of the wild-type p53 function may mediate apoptosis through CD95 receptor/ligand interactions as well as an alternative pathway.

Normal cells in a culture enter a nondividing state after a finite number of population doubling, which is termed replicative senescence, whereas cancer cells have unlimited proliferative potential and are thought to exhibit an immmortal phenotype by escaping from senescence. The p21 gene (also known as sdi1), which encodes the cyclin-dependent kinase inhibitor, is expressed at high levels in senescent cells and contributes to the growth arrest. To examine if the p21 sdi1 gene transfer could induce senescence in human cancer cells, we utilized an adenoviral vector-based expression system and four human cancer cell lines differing in their p53 status. Transient overexpression of p21 sdi1 on cancer cells induced quiescence by arresting the cell cycle at the G 1 phase and exhibited morphological changes, such as enlarged nuclei as well as a flattened cellular shape, specific to the senescence phenotype. We also showed that p21 sdi1 -transduced cancer cells expressed b-galactosidase activity at pH 6.0, which is known to be a marker of senescence. Moreover, the polymerase chain reaction-based assay demonstrated that levels of telomerase activity were significantly lower in p21 sdi1 -expressing cells compared to parental cancer cells. These observations provide the evidence that p21 sdi1 overexpression could induce a senescence-like state and reduce telomerase activity in human cancer cells, suggesting that these novel p21 sdi1 functions may have important implications for anticancer therapy.

Objective:This study aimed to determine whether retrocolic alimentary tract reconstruction is noninferior to antecolic reconstruction in terms of DGE incidence after pancreatoduodenectomy (PD) and investigated patients’ postoperative nutritional status.Summary of Background Data:The influence of the route of alimentary tract reconstruction on DGE after PD is controversial.Methods:Patients from 9 participating institutions scheduled for PD were randomly allocated to the retrocolic or antecolic reconstruction groups. The primary outcome was incidence of DGE, defined according to the 2007 version of the International Study Group for Pancreatic Surgery definition. Noninferiority would be indicated if the incidence of DGE in the retrocolic group did not exceed that in the antecolic group by a margin of 10%. Patients’ postoperative nutrition data were compared as secondary outcomes.Results:Total, 109 and 103 patients were allocated to the retrocolic and antecolic reconstruction group, respectively (n = 212). Baseline characteristics were similar between both groups. DGE occurred in 17 (15.6%) and 13 (12.6%) patients in the retrocolic and antecolic group, respectively (risk difference; 2.97%, 95% confidence interval; −6.3% to 12.6%, which exceeded the specified margin of 10%). There were no differences in the incidence of other postoperative complications and in the duration of hospitalization. Postoperative nutritional indices were similar between both groups.Conclusions:This trial could not demonstrate the noninferiority of retrocolic to antecolic alimentary tract reconstruction in terms of DGE incidence. The alimentary tract should not be reconstructed via the retrocolic route after PD, to prevent DGE.