MethodsCells. SW1 and B16F10 mouse melanoma cells were maintained in DMEM supplemented with 10% FBS, L-glutamine, and antibiotics.Constructs. ATF2 peptides were cloned in frame into a hemagglutinpenetratin (HA-penetratin) pcDNA3 vector (6). ATF2 peptide, corresponding to amino acids 50-100 of human ATF2, shares 100% homology with the murine sequence (corresponding amino acids in the mouse ATF2 cDNA are 33-82). Jun2-luciferase (Jun2-luc) and TRE-luc constructs were previously described (8, 10). The ATF2 50-100 peptide was Melanomas are among the aggressive tumor types because of their notorious resistance to treatment and their high capacity to metastasize. ATF2 is among transcription factors implicated in the progression of melanoma and its resistance to treatment. Here we demonstrate that the expression of a peptide spanning amino acids 50-100 of ATF2 (ATF2 50-100 ) reduces ATF2 transcriptional activities while increasing the expression and activity of c-Jun. Altering the balance of Jun/ATF2 transcriptional activities sensitized melanoma cells to apoptosis, an effect that could be attenuated by inhibiting c-Jun. Inhibition of ATF2 via RNA interference likewise increased c-Jun expression and primed melanoma cells to undergo apoptosis. Growth and metastasis of SW1 and B16F10 mouse melanomas were inhibited by ATF2 50-100 to varying degrees up to a complete regression, depending on the mode (inducible, constitutive, or adenoviral delivery) of its expression. Thus, by attenuating ATF2 and inducing c-Jun activity, ATF2 50-100 inhibits melanoma growth and metastasis.This article was published online in advance of the print edition.

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Clinical trials of recombinant human interleukin-12 (rhIL-12) delivered by intravenous administration have shown dose-limiting toxicities with limited tumor responses at the doses tested. We have previously reported that intratumoral injection of an adenovirus vector expressing murine interleukin-12 (Adv.RSV-mIL-12) was effective in inducing antitumor immune responses, tumor regression, and long-term survival in mice with established metastatic cancer in the liver. We now report additional studies in the same murine tumor model to assess the safety of intratumoral Adv.RSV-mIL-12 injection. At vector doses that were previously shown to be therapeutically effective, no inflammation in the liver or lungs, and no significant elevations in serum creatinine and aminotransferases were seen after vector injection. Serum elevations of IL-12 and interferon-gamma (IFN-gamma) were 17- and 19-fold lower than peak levels after intravenous recombinant IL-12 at the maximal tolerated dose in clinical trials. No elevations in serum proinflammatory cytokines (interleukin-6, tumor necrosis factor-alpha) were noted up to 2 weeks after vector injection. No systemic dissemination of the vector was detected on polymerase chain reaction (PCR) assays at therapeutically effective vector doses. At higher supratherapeutic vector doses of Adv.RSV-mIL-12, however, inflammation in the liver and lungs with elevation in serum aminotransferases were seen, but not in controls injected with the equivalent particle number of an empty adenoviral vector. These results support the cautious testing in patients with hepatic metastases of adenovirus mediated IL-12 gene delivery by intratumoral injection.