The human double minute-2 gene (hdm2) encodes an oncoprotein that negatively regulates the activity of the tumor suppressor p53. Antagonist peptides and small molecules of the hdm2 oncogenic domain have been shown to activate the p53 response in cells expressing wild-type p53. These results suggest that the p53 hdm2 protein:protein interaction is an interesting target for anti-cancer therapy. Recently, activation of the p53 pathway and anti-tumoral effect using small molecule hdm2 antagonists were described (Vassilev L., 2004). So far, these activities have only been demonstrated in wild-type p53-expressing cells/tumors. However, more than half of the human tumors present aberrations in the p53 protein, meaning that this strategy might only be applicable in half of the human malignancies. It is well known that, apart from p53, cell cycle regulatory proteins like E2F1, p14ARF and pRb also interact directly with hdm2. In this study we have evaluated the p53-independent effects of an hdm2 antagonistic peptide on tumor growth. We have used, the p53-deficient human H1299 lung carcinoma cells as an in vitro model for tumor cell proliferation. A stable tTA-expressing H1299 single cell clone was infected by means of a retroviral vector with a construct encoding a fusion of the antagonist hdm2 peptide (WT peptide) and EGFP. As a control constructs, EGFP alone and an EGFP/MT peptide were used. In the pure population, EGFP expression was found to be doxycyclin regulatable. Interestingly, when inducing the expression of the EGFP fusion proteins, on days 4 and 8, high expressors appeared in the EGFP- and the EGFP/MT-infected population whereas only low and medium expression of the EGFP/wild-type peptide was found. This suggests that high expression of the WT antagonist, affects the viability of p53-deficient cells. Further analysis showed no difference in cell cycle profiles of cells expressing the WT peptide 2 days after induction. On days 4 and 8 the expression of the WT peptide induced a G0/G1 cell cycle arrest, and a reduction of the percentage of cells in S phase. The reduction in S phase observed was confirmed in a clonogenic assay performed over 8 days. Although we found no difference in the number of clones formed in WT peptide-expressing cells the percentage of clusters with a large clone area was lower in the induced as compared to the suppressed population. The mechanism through which the hdm2 antagonist peptide decreases S-phase progression is currently under active investigation using micro-array analysis. In summary, our data for the first time show a growth limiting effect of an hdm2 antagonistic peptide inhibitor in p53-deficient cells. This suggests that therapeutics based on an hdm2 antagonistic effect might be an interesting anti-cancer therapy with a broad applicability towards tumors with different p53 backgrounds.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]