Immunohistochemical analysis of S6K1 and S6K2 expression in human breast tumors.
OBJECTIVE
To express recombinant S6K2 in baculovirus expression system; to purify large quantities of recombinant S6K2 for biochemical studies; to generate and characterise specific MABs against recombinant S6K2; to study the patterns S6K1 and S6K2 expression and subcellular localization in normal, benign and malignant breast tissues.


METHODS
Recombinant baculovirus, expressing wild type S6K2 was generated using Bac-to-Bac system (Invitrogen); recombinant S6K was purified from infected Sf9 cells using affinity purification approach; monoclonal antibodies against recombinant S6K2 were generated; the specificity of generated MABs towards recombinant and endogenous S6K2 were examined by ELISA, Western blotting, immunoprecipitation and immuhohistochemical staining; immunohistochemical detection of S6K1 and S6K2 in human breast tissues was performed using specific monoclonal antibodies towards S6K1 and S6K2.


RESULTS
Large amounts of enzymatically active S6K2 were purified using baculovirus expression system; highly purified preparations of S6K2 were used to generate and characterize anti-S6K2 MABs; elevated levels of S6K1 and S6K2 were found in breast tumors when compared to normal breast tissues; S6K2 is frequently localized in the nuclei of adenocarcinoma tissues, but rarely in fibroadenoma or "normal" breast tissues.


CONCLUSIONS
Production of recombinant S6K2 in large amount and generation of specific monoclonal antibodies towards S6K2 has provided us with excellent tools to study the function and regulation of this important signalling molecule in normal and cancer cells. Immunnohistochemical analysis of S6K1 and S6K2 expression in normal and malignant breast clearly indicates that both kinases are overexpressed in breast tumors, when compared to "normal" tissues. The retention of S6K2 in the nuclei of malignant cells may be caused by disregulation of nucleocytoplasmic shuttling and could subsequently affect cell growth and proliferation.
Savinska
Liliya O
LO
Institute of Molecular Biology and Genetics, NAS of Ukraine, Kyiv, Ukraine.
Lyzogubov
Valeriy V
VV
Usenko
Vasyliy S
VS
Ovcharenko
Galina V
GV
Gorbenko
Olena N
ON
Rodnin
Mykola V
MV
Vudmaska
Mariya I
MI
Pogribniy
Petro V
PV
Kyyamova
Ramziya G
RG
Panasyuk
Ganna G
GG
Nemazanyy
Ivan O
IO
Malets
Milan S
MS
Palchevskyy
Sergey S
SS
Gout
Ivan T
IT
Filonenko
Valeriy V
VV
D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000201:enzymology* / Q000473:pathology; D006801:Humans; D007150:Immunohistochemistry; D011233:Precipitin Tests; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000276:immunology; D038744:Ribosomal Protein S6 Kinases, 90-kDa / Q000096:biosynthesis* / Q000276:immunology; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D011994:Recombinant Proteins; C069329:RPS6KA1 protein, human; D038744:Ribosomal Protein S6 Kinases, 90-kDa; C069330:ribosomal protein S6 kinase, 90kDa, polypeptide 3