The roles of supernumerical X chromosomes and XIST expression in testicular germ cell tumors.
OBJECTIVE
An overabundance of X chromosomes in testicular germ cell tumors and the identification of the candidate testicular germ cell tumor susceptibility gene TGCT1 on Xq27 highlight the potential involvement of X chromosomes in testicular germ cell tumor pathogenesis. The current study was designed to shed light on the question whether the multiple X chromosomes in testicular germ cell tumor are active or inactive through a complex mechanism of X chromosomal gain and XIST expression.


METHODS
We analyzed 4 testicular germ cell tumor derived cell lines and 20 primary testicular germ cell tumor tissues. The number of X chromosomes was determined by fluorescence in situ hybridization using the X chromosome specific probe. The expression patterns of XIST and the 3 X-linked genes androgen receptor (AR), fragile X mental retardation (FMR1 ) and Glypican 3 (GPC3 ) were studied by reverse transcriptase-polymerase chain reaction. Bisulfite genomic sequencing was used to analyze the methylation patterns of the AR, FMR1 and GPC3 genes. The relative expression levels of the 2 X-linked proto-oncogenes ARAF1 and ELK1 were assayed by quantitative reverse transcriptase-polymerase chain reaction.


RESULTS
XIST expression was common in seminomatous testicular germ cell tumors (2 of 2 or 100% of seminoma derived cell lines and 10 of 12 or 83% of seminomatous testicular germ cell tumor tissues) but not in nonseminomatous testicular germ cell tumors (0 of 2 or 0% nonseminoma derived cell lines and 2 of 8 or 25% of nonseminomatous testicular germ cell tumor tissues). However, X chromosomal gain was consistently observed in the 2 types of tumors. XIST expression in testicular germ cell tumors and normal testicular parenchyma was not associated with methylation of the AR, FMR1 or GPC3 genes. After determining the expression patterns of AR, FMR1 and GPC3 in testicular germ cell tumor samples we concluded that multiple X chromosomes in testicular germ cell tumors were predominantly hypomethylated and active regardless of XIST expression. The biological significance of excess active X chromosomes in testicular germ cell tumors was suggested by enhanced expression of the 2 X-linked oncogenes ARAF1 and ELK1 in the testicular germ cell tumor derived cell lines.


CONCLUSIONS
The current data may suggest the potential oncogenic implications of X chromosomal gain in testicular germ cell tumors.
Kawakami
Takahiro
T
Department of Urology, Shiga University of Medical Science, Otsu, Japan.
Okamoto
Keisei
K
Sugihara
Hiroyuki
H
Hattori
Takanori
T
Reeve
Anthony E
AE
Ogawa
Osamu
O
Okada
Yusaku
Y
D041321:Chromosomes, Human, X; D019175:DNA Methylation; D015972:Gene Expression Regulation, Neoplastic / Q000502:physiology; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D008297:Male; D009367:Neoplasm Staging; D009373:Neoplasms, Germ Cell and Embryonal / Q000235:genetics* / Q000473:pathology; D062085:RNA, Long Noncoding; D022661:RNA, Untranslated / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D018239:Seminoma / Q000235:genetics* / Q000473:pathology; D012729:Sex Chromosome Aberrations; D013736:Testicular Neoplasms / Q000235:genetics* / Q000473:pathology; D013737:Testis / Q000473:pathology; D014157:Transcription Factors / Q000235:genetics*; D014407:Tumor Cells, Cultured
D062085:RNA, Long Noncoding; D022661:RNA, Untranslated; D014157:Transcription Factors; C077885:XIST non-coding RNA