Expression and function of protein phosphatase PP2A in malignant testicular germ cell tumours.
Testicular germ cell tumours (TGCT) represent the most common malignancy in young males. We reported previously that two prototype members of the mitogen-activated protein kinase (MAPK) family, the MAPK ERK kinase (MEK) and extracellular signal-regulated kinase (ERK), are inactive in malignant testicular germ cells and become active after drug stimulation, leading to apoptosis of tumour cells. In this study, we asked whether the protein phosphatase PP2A, a known inhibitor of the MEK-ERK pathway, participates in the proliferation and/or apoptosis of primary TGCT (n = 48) as well as two TGCT cell lines (NTERA and NCCIT). Quantitative RT-PCR, immunohistochemistry, western blot analyses and phosphatase assay indicate that primary TGCT as well as TGCT cell lines express PP2A and that PP2A is active in TGCT cell lines. The inhibition of PP2A by application of two PP2A inhibitors, cantharidic acid (CA) and okadaic acid (OA), results in a significant increase in caspase-3-mediated apoptosis of TGCT cell lines. Thereby, PP2A inhibition was accompanied by phosphorylation and activation of MEK and ERK. Functional assays using the MEK inhibitor PD98059 demonstrated that the phosphorylation of MEK and ERK was required for the induction of caspase-3-mediated apoptosis of malignant germ cells. Thus, our data suggest that inhibition of PP2A mediates its apoptosis-inducing effect on TGCT through activation of the MEK-ERK signalling pathway that leads to caspase-3-mediated apoptosis of tumour cells. In addition our results support previous observations that PP2A exerts an anti-apoptotic effect on malignant tumour cells.
Schweyer
S
S
Department of Pathology, University of Göttingen, Göttingen, Germany. sswyer@med.uni-goettingen.de
Bachem
A
A
Bremmer
F
F
Steinfelder
H J
HJ
Soruri
A
A
Wagner
W
W
Pottek
T
T
Thelen
P
P
Hopker
W W
WW
Radzun
H J
HJ
Fayyazi
A
A
D000328:Adult; D000368:Aged; D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western / Q000379:methods; D002193:Cantharidin / Q000494:pharmacology; D053148:Caspase 3 / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D004789:Enzyme Activation; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005419:Flavonoids / Q000494:pharmacology; D006801:Humans; D007150:Immunohistochemistry; D008297:Male; D008875:Middle Aged; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D009373:Neoplasms, Germ Cell and Embryonal / Q000201:enzymology*; D019319:Okadaic Acid / Q000494:pharmacology; D010766:Phosphorylation; D054648:Protein Phosphatase 2 / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013194:Staining and Labeling; D013736:Testicular Neoplasms / Q000201:enzymology*
D005419:Flavonoids; D019319:Okadaic Acid; D048049:Extracellular Signal-Regulated MAP Kinases; D020928:Mitogen-Activated Protein Kinases; D020929:Mitogen-Activated Protein Kinase Kinases; D054648:Protein Phosphatase 2; D053148:Caspase 3; D002193:Cantharidin; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one