The effect of the cyclin-dependent kinase inhibitor flavopiridol on anaplastic large cell lymphoma cells and relationship with NPM-ALK kinase expression and activity.
BACKGROUND
The loss of cell cycle regulation due to abnormal function of cyclin-dependent kinases (cdk) occurs in tumors and leads to genetic instability of chemotherapy-resistant cells. In this study, we investigated the effect of the cdk inhibitor flavopiridol in anaplastic large cell lymphomas, in which unrestrained proliferation depends on NPM-ALK tyrosine kinase activity.


METHODS
Effects of flavopiridol were examined in ALK-positive and -negative anaplastic large cell lymphoma cells by means of immunoblotting and immunofluorescence analyses to assess cdk expression and activity, quantitative real time reverse transcriptase polymerase chain reaction to measure drug-induced changes in transcription, and FACS analyses to monitor changes in proliferation and survival.


RESULTS
Treatment with flavopiridol resulted in growth inhibition of anaplastic large cell lymphoma cells, along with accumulation of subG(1) cells and disappearance of S phase without cell cycle arrest. Consistent with flavopiridol activity, phosphorylation at cdk2, cdk4, cdk9 sites on RB and RNA polymerase II was inhibited. This correlated with induction of cell death through rapid mitochondrial damage, inhibition of DNA synthesis, and down-regulation of anti-apoptotic proteins and transcripts. Notably, flavopiridol was less active in ALK-positive cells, as apoptosis was observed at higher concentrations and later time points, and resistance to treatment was observed in cells maintaining NPM-ALK signaling. NPM-ALK inhibition affected proliferation but not survival of anaplastic large cell lym-phoma cells, whereas it resulted in a dramatic increase in apoptosis when combined with flavopiridol.


CONCLUSIONS
This work provides the first demonstration that targeting cdk is effective against anaplastic large cell lymphoma cells, and proves the critical role of NPM-ALK in the regulation of responsiveness of tumor cells with cdk dysregulation.
Bonvini
Paolo
P
Clinica di Oncoematologia Pediatrica, Azienda Ospedaliera-Università di Padova, 35128 Padova, Italy. paolo.bonvini@unipd.it
Zorzi
Elisa
E
Mussolin
Lara
L
Monaco
Giovanni
G
Pigazzi
Martina
M
Basso
Giuseppe
G
Rosolen
Angelo
A
D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis; D001973:Bromodeoxyuridine / Q000494:pharmacology; D002453:Cell Cycle; D002469:Cell Separation; D002470:Cell Survival; D004305:Dose-Response Relationship, Drug; D005419:Flavonoids / Q000494:pharmacology*; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D017728:Lymphoma, Large-Cell, Anaplastic / Q000188:drug therapy*; D010880:Piperidines / Q000494:pharmacology*; D011505:Protein-Tyrosine Kinases / Q000096:biosynthesis*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013347:Subcellular Fractions; D013997:Time Factors
D000970:Antineoplastic Agents; D005419:Flavonoids; D010880:Piperidines; C077990:alvocidib; C107023:p80(NPM-ALK) protein; D011505:Protein-Tyrosine Kinases; D001973:Bromodeoxyuridine