title: Evidence that active demethylation mechanisms maintain the genome of carcinoma in situ cells hypomethylated in the adult testis.
abstract: BACKGROUND
  Developmental arrest of fetal germ cells may lead to neoplastic transformation and formation of germ cell tumours via carcinoma in situ (CIS) cells. Normal fetal germ cell development requires complete erasure and re-establishment of DNA methylation. In contrast to normal spermatogonia, the genome of CIS cells remains unmethylated in the adult testis. We here investigated the possible active and passive pathways that can sustain the CIS genome hypomethylated in the adult testis.
  
  
  METHODS
  The levels of 5-methyl-cytosine (5mC) and 5-hydroxy-methyl-cytosine (5hmC) in DNA from micro-dissected CIS cells were assessed by quantitative measurements. The expression of TET1, TET2, APOBEC1, MBD4, APEX1, PARP1, DNMT1, DNMT3A, DNMT3B and DNMT3L in adult testis specimens with CIS and in human fetal testis was investigated by immunohistochemistry and immunofluorescence.
  
  
  RESULTS
  DNA from micro-dissected CIS cells contained very low levels of 5hmC produced by ten eleven translocation (TET) enzymes. CIS cells and fetal germ cells expressed the suggested initiator of active demethylation, APOBEC1, and the base excision repair proteins MBD4, APEX1 and PARP1, whereas TETs - the alternative initiators were absent. Both maintenance and de novo methyltransferases were detected in CIS cells.
  
  
  CONCLUSIONS
  The data are consistent with the presence of an active DNA de-methylation pathway in CIS cells. The hypomethylated genome of CIS cells may contribute to phenotypic plasticity and invasive capabilities of this testicular cancer precursor.
authors.affiliation: University Department of Growth and Reproduction GR-5064, Rigshospitalet, Blegdamsvej 9, DK-2100 Copenhagen, Denmark.
authors.forename: D G
authors.initials: DG
authors.lastname: Kristensen
authors.affiliation: University Department of Growth and Reproduction GR-5064, Rigshospitalet, Blegdamsvej 9, DK-2100 Copenhagen, Denmark.
authors.forename: J E
authors.initials: JE
authors.lastname: Nielsen
authors.affiliation: University Department of Growth and Reproduction GR-5064, Rigshospitalet, Blegdamsvej 9, DK-2100 Copenhagen, Denmark.
authors.forename: A
authors.initials: A
authors.lastname: Jørgensen
authors.affiliation: University Department of Growth and Reproduction GR-5064, Rigshospitalet, Blegdamsvej 9, DK-2100 Copenhagen, Denmark.
authors.forename: N E
authors.initials: NE
authors.lastname: Skakkebæk
authors.affiliation: University Department of Growth and Reproduction GR-5064, Rigshospitalet, Blegdamsvej 9, DK-2100 Copenhagen, Denmark.
authors.forename: E
authors.initials: E
authors.lastname: Rajpert-De Meyts
authors.affiliation: University Department of Growth and Reproduction GR-5064, Rigshospitalet, Blegdamsvej 9, DK-2100 Copenhagen, Denmark.
authors.forename: K
authors.initials: K
authors.lastname: Almstrup
mesh_terms: D002278:Carcinoma in Situ / Q000235:genetics* / Q000473:pathology; D002454:Cell Differentiation; D002471:Cell Transformation, Neoplastic / Q000235:genetics; D019175:DNA Methylation / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics; D005333:Fetus / Q000378:metabolism / Q000473:pathology; D015894:Genome, Human; D006801:Humans; D007150:Immunohistochemistry; D008297:Male; D009373:Neoplasms, Germ Cell and Embryonal / Q000235:genetics* / Q000473:pathology; D013736:Testicular Neoplasms / Q000235:genetics* / Q000473:pathology; D013737:Testis / Q000378:metabolism / Q000473:pathology
chemical_list: D004268:DNA-Binding Proteins