Restoration of p53 pathway by nutlin-3 induces cell cycle arrest and apoptosis in human rhabdomyosarcoma cells.
OBJECTIVE
Seventy to eighty percent of rhabdomyosarcoma (RMS) tumors retain wild-type p53. The tumor suppressor p53 plays a central role in inducing cell cycle arrest or apoptosis in response to various stresses. p53 protein levels are regulated by MDM2 through ubiquitin-dependent degradation. In this study, we evaluated whether nutlin-3, a recently developed small-molecule antagonist of MDM2, has an effect on p53-dependent cell cycle arrest and apoptosis in cultured human RMS cell lines.


METHODS
Five RMS cell lines with different p53 statuses and MDM2 expression levels were treated with nutlin-3. Gene expression patterns, cell viability, cell cycle, and apoptosis after nutlin-3 treatment, and antitumor activity of combination treatment with vincristine or actinomycin D were assessed.


RESULTS
Significant p53 activation was observed in wild-type p53 cell lines after nutlin-3 treatment. p53 activation led to cell cycle arrest in parallel with increased p21 expression. Furthermore, these cell lines underwent p53-dependent apoptosis, concomitant with elevation of proapoptotic genes and activation of caspase-3. The effect of nutlin-3 was almost the same in terms of half maximal inhibitory concentration and apoptosis whether or not MDM2 was overexpressed. Nutlin-3 did not induce either cell cycle arrest or apoptosis in p53 mutant cell lines. A combination of vincristine or actinomycin D with nutlin-3 enhanced the antitumor activity in RMS cell lines with wild-type p53.


CONCLUSIONS
Nutlin-3 effectively restored p53 function in both normal MDM2 expression and MDM2 overexpression RMS cell lines with wild-type p53. p53 restoration therapy is a potential therapeutic strategy for refractory RMS with wild-type p53.
Miyachi
Mitsuru
M
Department of Pediatrics, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto, Japan.
Kakazu
Naoki
N
Yagyu
Shigeki
S
Katsumi
Yoshiki
Y
Tsubai-Shimizu
Satoko
S
Kikuchi
Ken
K
Tsuchiya
Kunihiko
K
Iehara
Tomoko
T
Hosoi
Hajime
H
D000903:Antibiotics, Antineoplastic / Q000494:pharmacology*; D017209:Apoptosis; D002453:Cell Cycle / Q000187:drug effects*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D003609:Dactinomycin / Q000494:pharmacology; D004357:Drug Synergism; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology*; D020125:Mutation, Missense / Q000235:genetics; D009379:Neoplasms, Muscle Tissue / Q000378:metabolism*; D010879:Piperazines / Q000494:pharmacology*; D051736:Proto-Oncogene Proteins c-mdm2 / Q000037:antagonists & inhibitors*; D012208:Rhabdomyosarcoma / Q000378:metabolism*; D016159:Tumor Suppressor Protein p53 / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D014750:Vincristine / Q000494:pharmacology
D000903:Antibiotics, Antineoplastic; D007093:Imidazoles; D010879:Piperazines; D016159:Tumor Suppressor Protein p53; D003609:Dactinomycin; C482205:nutlin 3; D014750:Vincristine; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2