Nutlin-3a, an MDM2 Antagonist, Induces Cellular Senescence in Adult T-Cell Leukemia/Lymphoma Cells.

Adult T cell Leukemia/Lymphoma (ATLL) is a neoplasm of T-lymphocyte origin etiologically associated with HTLV-I and is known to be resistant to standard anti-cancer therapies. Recently, a small-molecule antagonist of MDM2, Nutlin-3a, has been developed, which inhibits the p53-MDM2 interaction and activates p53 signaling. The p53 tumor suppressor retains its wild-type conformation and transcriptional activity in half of all human tumors. Since anti-proliferative effects are imparted through a variety of mechanisms that include cell cycle arrest, apoptosis, and cellular senescence, and hence its activation may offer a therapeutic benefit in malignancies.

Considering the future clinical use of Nutlin-3a, we characterized the sensitivity of leukemia cell lines to Nutlin-3a in the present study. Nutlin-3a requires not only wild-type p53 but also functional signaling in the p53 pathway. Thus, we first performed p53 mutation-analysis in all cell lines used, then evaluated the efficacy of Nutlin-3a. Here, we report for the first time that Nutlin-3a induces cellular senescence in hematological cell lines.

Material and methods : We used 19 hematological cell lines including 8 HTLV-I related cell lines: 5 ATLL cell lines and 3 HTLV-I infected T-cell lines. The p53 mutations were examined using cDNA of each cell line, amplified by PCR targeting ORF, and sequenced using an ABI-PRISM model 310 Genetic Analyzer. Cell proliferation was assessed by MTS assay after 5 days incubation with 10μM Nutlin-3a. Apoptosis was evaluated by Annexin V binding and propidium iodide (PI) staining. Sodium butyrate (SB), known to induce cellular senescence in many types of cells, was used and senescence was assessed by SA-β-galactosidase (SA-β-gal) staining. We also analyzed expressions of p53, MDM2, p21, caspases, and pro- or anti-apoptotic proteins by Western blot.

Results and Discussion : In mutation-analysis, 10 cell lines had the wild-type and 9 cell lines had mutated p53. Nine of 10 cell lines with wild-type p53 showed more than 60% inhibition of cells growth following Nutlin-3a treatment. In contrast, 8 of 9 cell lines with mutated p53 were not affected by Nutlin-3a. In ATLL cell lines with wild-type p53, Western blot analysis after 12-48h exposure to Nutlin-3a showed an increased level of p53, the p53 targets MDM2 and p21 especially in wild type cells. Of note, some of these cell lines were stained by SA-β-gal, but showed faint signals of apoptotic cell death on Annexin V and PI staining. Although it has been reported that p53 dependent proteins puma, noxa, and Bax play key roles in p53 dependent apoptosis, we did not find any change in these factors. Although 7 days of incubation with 1mM SB led senescence even in p53-mutated cell lines and these cells were stained by SA-β-gal, these cells were not stained by SA-β-gal when treated with Nutlin-3a.

Moreover, mutated cell lines did not show any apoptotic change following Nutilin-3a treatment. These findings suggest that cellular senescence is more important than p53 dependent apoptosis in Nutlin-3a treatment in ATLL cells.