We introduced a functional p16 cDNA into non-small cell lung cancer (NSCLC) cell lines expressing dierent combinations of normal and mutated p16, p53, and Rb genes via a recombinant adenovirus to determine the eect of exogenous p16 expression on cell growth. Analysis of p16-de®cient cells infected with Adv/p16 identi®ed growth arrest of the cells in the G 0 ± G 1 phase early on. Apoptosis was identi®ed to occur by the 5th day after infection which corresponded with increased p16 expression, reduced Rb expression, and increased Rb hypophosphorylation, but only occurred in cells expressing functional p53. Further analysis indicated that the expression of the anti-apoptotic protein bcl-2 was greatly reduced in the NSCLC cell lines H460 and A549 (both 7p16, +p53, +Rb), again only by the 5th day after Adv/p16 infection, but no aect on Bax expression was observed. H1299 cells (7p16, 7p53, +Rb) infected with Adv/p16 only exhibited apoptosis by an additional infection with Adv/p53 which also corresponded with a down-regulation of bcl-2. In addition, the infection of A549 cells with Adv/p16 followed by a subsequent infection with Adv/Rb lead to a signi®cant decrease in apoptosis which correlated with an increase in bcl-2 expression. These studies suggest that p16 is capable of mediating apoptosis in NSCLC cell lines expressing wild-type p53, through a direct down-regulation of Rb and an indirect down-regulation of the anti-apoptotic protein bcl-2. Oncogene (2000) 19, 1589 ± 1595.
The p16 tumor suppressor gene is frequently inactivated in human cancer tissues and cell lines. We previously reported that wild-type p16 expression from an adenovirus vector (Adv/p16) induced p53-dependent apoptotic cell death in non-small cell lung cancer (NSCLC) cell lines. Here we show the potential mechanism of apoptosis induced by Adv/p16 infection. Infection of human NSCLC cell line A549, which carries the wild-type p53 gene, with Adv/p16 resulted in activation of caspase-3, accompanied by the cleavage of its substrate poly (ADP-ribose) polymerase (PARP), on day 3 of infection. The retinoblastoma (Rb) cell cycle regulator protein was also cleaved after activation of caspase-3; when the levels of Rb signi®cantly diminished, apoptosis began. When A549 cells were pretreated with the caspase-inhibitory peptide N-acetyl-asp-Glu-Val-Asp-CHO (aldehyde) (Ac-DEVD-CHO), Adv/p16-mediated apoptosis and Rb cleavage were greatly inhibited. Furthermore, MDM2, a negative regulator of p53 expression was upregulated 3 days after Adv/p16 infection, and MDM2 was subsequently cleaved by caspase-3; MDM2 cleavage was inhibited by Ac-DEVD-CHO treatment. These data implied that cleavage of Rb, in addition to activation of caspase-3, represented a mechanism by which Adv/p16 induced apoptotic cell death in human NSCLC cells. Our results support the clinical relevance of Adv/p16 as a treatment for p16-null human NSCLC that express wild-type p53.
. Basaloid squamous carcinoma of the esophagus is very rare. We report two cases of basaloid squamous carcinoma of the esophagus. Both tumors histologically consisted of solid cell nests with intervening fibromyxoid stroma. In some tumor nests were comprised of pseudoglandular structures containing myxoid matrix, and displayed focal immunoreactivity for laminin. Thoracic esophagectomy with lymph node dissection was followed by intrathoracic esophagogastrostomy in both patients. The patients had uneventful postoperative courses. Regular periodic follow-up showed no evidence of recurrence or metastasis in the 22-month postoperative period.
Clinical trials of adenoviral p53 gene therapy provide the evidence that the bystander effect induced by the wild-type p53 gene transfer on adjacent tumor cells contributes to tumor progression; its mechanism, however, remains uncharacterized. We report in this work that injection of adenovirus expressing the human wild-type p53 gene (Ad5CMVp53) into established human colorectal tumors in nu/nu mice resulted in CD95 ligand (CD95L) overexpression, followed by a massive neutrophil infiltration. Culture supernatants of human colorectal cancer cells infected with Ad5CMVp53 exhibited a potent chemotactic activity against murine polymorphonuclear neutrophils, which could be abolished by the anti-CD95L mAb (NOK-1). In vivo cell depletion experiments indicated that neutrophils were in part responsible for the antitumor effect of the Ad5CMVp53 infection. Our data directly suggest that overexpression of CD95L by the wild-type p53 gene transfer induces neutrophil infiltration into human colorectal tumors, which may play a critical role in the bystander effect of p53 gene therapy.
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