Identification of glioma-specific RFX4-E and -F isoforms and humoral immune response in patients.
For regulatory factor X4 (RFX4), two alternatively spliced variants, RFX4-A and -B, were reported in the testis. In this study, we identified transcript variants RFX4-C, -D, -E, and -F, and demonstrated by reverse transcription-polymerase chain reaction (RT-PCR) that RFX4-A, -B and -C mRNAs were expressed only in the testis, and RFX4-D mRNA was expressed only in normal brain tissues. In tumors, RFX4-E and -F in addition to RFX4-D mRNA were expressed in gliomas by rapid amplification of cDNA ends and RT-PCR analyses. Expression of RFX4 mRNA was not observed in other tumors, such as lung, esophageal, stomach, colon or liver cancers. Quantitative real-time RT-PCR using common primer pairs detecting all of the variant transcripts showed high expression in normal testis, low expression in the brain (1% compared to the expression in testis), and overexpression in 17 of 61 gliomas (28%). Western blot analysis using DC28 monoclonal antibody (mAb) produced against recombinant RFX4-D C-terminus protein showed expression of RFX4-A and -C proteins, but not RFX4-B protein, in the testis, and expression of RFX4-D protein in the brain. Moreover, expression of RFX4-E and -F proteins, but not RFX4-D protein, was observed in gliomas. Immunohistochemistry analysis using DC28 mAb showed positive staining in the nuclei of spermatocytes in the testis and glioma cells. Antibody against RFX4 was detected in the sera of 3 of 58 (5%) glioma patients by enzyme-linked immunosorbent assay, suggesting the immunogenicity of RFX4-E and -F proteins in glioma patients.
Matsushita
Hirokazu
H
Department of Immunology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Japan.
Uenaka
Akiko
A
Ono
Toshiro
T
Hasegawa
Kosei
K
Sato
Shuichiro
S
Koizumi
Fumihito
F
Nakagawa
Kazuhiko
K
Toda
Masahiro
M
Shingo
Tetsuro
T
Ichikawa
Tomotsugu
T
Noguchi
Yuji
Y
Tamiya
Takashi
T
Furuta
Tomohisa
T
Kawase
Takeshi
T
Date
Isao
I
Nakayama
Eiichi
E
D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D017398:Alternative Splicing; D000917:Antibody Formation; D015153:Blotting, Western; D001921:Brain / Q000502:physiology; D001932:Brain Neoplasms / Q000276:immunology*; D002648:Child; D002675:Child, Preschool; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D020869:Gene Expression Profiling; D005910:Glioma / Q000276:immunology*; D006801:Humans; D007150:Immunohistochemistry; D008297:Male; D008875:Middle Aged; D020033:Protein Isoforms; D000071701:Regulatory Factor X Transcription Factors; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013737:Testis / Q000502:physiology; D014157:Transcription Factors / Q000096:biosynthesis*
D020033:Protein Isoforms; D000071701:Regulatory Factor X Transcription Factors; D014157:Transcription Factors; C506397:RFX4 protein, human