Absolute quantification of four isoforms of the class I phosphoinositide-3-kinase catalytic subunit by real-time RT-PCR.
Class I phosphoinositide-3-kinase (PI3K) consists of four isoforms of the catalytic subunit, p110alpha, -beta, -delta and -gamma, generated from the genes PIK3CA, -B, -D and -G, respectively. These isoforms show different tissue distribution and some specific and indispensable functions in various biological pathways such as development, inflammation, autoimmunity and malignancy. In human cancers, frequent genomic amplification and gain-of-function mutations of PIK3CA were reported, which suggests an oncogenic potential. However, the role played by the other three isoforms in human cancer remains to be determined. We wanted to investigate the relationship between all the isoforms in human cancers. Here, we have established a system for the simultaneous absolute-quantification of all four isoforms by real-time reverse transcription polymerase chain reaction (RT-PCR). The reliability of this system was confirmed using three main criteria: (i) good correlation of each standard curve, (ii) high specificity of the PCR reactions and (iii) excellent reproducibility. Using this system, we investigated human monocytic leukemia cells (U937) to analyze expression of all four isoforms. The biological implications of the expression level of the four isoforms of class I PI3K catalytic subunit are discussed.
Nakamura
Hiroyuki
H
Dan
Shingo
S
Akashi
Tetsuyuki
T
Unno
Michiaki
M
Yamori
Takao
T
D020134:Catalytic Domain / Q000235:genetics*; D045744:Cell Line, Tumor; D004587:Electrophoresis, Agar Gel; D015870:Gene Expression; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000737:chemistry* / Q000145:classification / Q000235:genetics* / Q000378:metabolism*; D017434:Protein Structure, Tertiary / Q000235:genetics; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D020298:U937 Cells
D007527:Isoenzymes; D019869:Phosphatidylinositol 3-Kinases