Myc down-regulation affects cyclin D1/cdk4 activity and induces apoptosis via Smac/Diablo pathway in an astrocytoma cell line.
OBJECTIVE
We investigated the antiproliferative effect of Myc down-regulation via cell proliferation inhibition, cell cycle perturbation and apoptosis in two human astrocytoma models (T98G and ADF) steadily expressing an inducible c-myc Anti-sense RNA.


METHODS
Cell growth experiments were performed using the trypan blue dye exclusion test and cell cycle analysis was evaluated by flow cytometry. Cell cycle molecules were detected by Western blot analysis, co-immunoprecipitation and reverse transcription-polymerase chain reaction assays.


RESULTS
We showed that Myc down-regulation in astrocytoma cells led to G1 accumulation and an inhibition of cell proliferation characterized by S phase delay. Co-immunoprecipitation experiments detected formation of inactive cyclin D1/cdk4 complexes as evaluated by presence of an active unphosphorylated form of retinoblastoma protein, the best characterized target substrate for cyclin D1/cdk4 complex, in ADF pINDc-myc anti-sense 7 cells. We also found that either p57Kip2 "apice" or p27Kip1 "apice" inhibitors bound to cyclin D1/cdk4 complex, thus, suggesting that they cooperated to inhibit the activity of cyclin D1/cdk4. Moreover, c-Myc down-regulation led to activation of the apoptotic mitochondrial pathway, characterized by release of cytochrome c and Smac/Diablo proteins and by reduction of c-IAP levels through activation of proteasome-mediated protein degradation system.


CONCLUSIONS
Our results suggest that c-Myc could be considered as a good target for the study of new approaches in anticancer astrocytoma treatment.
Amendola
D
D
Centro Ricerca S. Pietro, Fatebenefratelli Hospital, Rome, Italy.
De Salvo
M
M
Marchese
R
R
Verga Falzacappa
C
C
Stigliano
A
A
Carico
E
E
Brunetti
E
E
Moscarini
M
M
Bucci
B
B
D017209:Apoptosis / Q000235:genetics*; D051017:Apoptosis Regulatory Proteins; D001254:Astrocytoma / Q000201:enzymology / Q000378:metabolism* / Q000473:pathology; D001483:Base Sequence; D015153:Blotting, Western; D002453:Cell Cycle; D045744:Cell Line, Tumor; D019938:Cyclin D1 / Q000378:metabolism*; D051358:Cyclin-Dependent Kinase 4 / Q000378:metabolism*; D017931:DNA Primers; D015536:Down-Regulation; D020868:Gene Silencing; D016259:Genes, myc; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000502:physiology*; D024101:Mitochondrial Proteins / Q000502:physiology*; D034741:RNA, Small Interfering / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction
D051017:Apoptosis Regulatory Proteins; C412403:DIABLO protein, human; D017931:DNA Primers; D047908:Intracellular Signaling Peptides and Proteins; D024101:Mitochondrial Proteins; D034741:RNA, Small Interfering; D019938:Cyclin D1; D051358:Cyclin-Dependent Kinase 4