title: Genetic and epigenetic changes of HPV16 in cervical cancer differentially regulate E6/E7 expression and associate with disease progression.
abstract: OBJECTIVE
  The study was aimed at understanding the complex interactions of genetic and epigenetic events in expression of HPV16 E6/E7 and progression of cervical carcinoma. For this, expression of E6/E7 was done in 36 samples, along with the physical status, methylation and LCR sequence variations. Later, the genetic and epigenetic studies were extended to 239 samples to find out the association of these factors with progression of cervical cancer.
  
  
  METHODS
  E6/E7 expression was quantified by real-time PCR. Physical status of HPV16 was determined by mutiplex-PCR of whole E2 ORF using overlapping primers and E6 ORF and validated by real-time PCR. Methylation status of P97 promoter/enhancer was analyzed by methylation sensitive restriction analysis (MSRA). Viral lineage and variations in LCR was ascertained by sequencing LCR/E6/E7 ORFs.
  
  
  RESULTS
  Samples with episomal unmethylated virus showed comparatively high expression of E6/E7 than episomal methylated, integrated unmethylated and integrated methylated forms of HPV16. Variations in the LCR, particularly in the binding sites of negatively regulating transcription factors, also contribute to high expression of E6/E7. The integrated form significantly increases with decrease of episomal form during tumor progression. Methylation of the promoter/enhancer gradually decreased with tumor progression and is inversely correlated to integration. Two novel variants were observed in E6 gene in European- and North-American-1-lineages. Log-rank test revealed better prognosis of the patients with episomal methylated HPV16 compared to the other forms.
  
  
  CONCLUSIONS
  Our results show higher expression of E6/E7 in samples with episomal unmethylated virus having sequence variations in LCR.
authors.affiliation: Department of Oncogene Regulation, Chittaranjan National Cancer Institute, Kolkata, India.
authors.forename: Dipanjana
authors.initials: D
authors.lastname: Mazumder Indra
authors.forename: Ratnesh Kumar
authors.initials: RK
authors.lastname: Singh
authors.forename: Sraboni
authors.initials: S
authors.lastname: Mitra
authors.forename: Sankhadeep
authors.initials: S
authors.lastname: Dutta
authors.forename: Chandraditya
authors.initials: C
authors.lastname: Chakraborty
authors.forename: Partha Sarathi
authors.initials: PS
authors.lastname: Basu
authors.forename: Ranajit Kumar
authors.initials: RK
authors.lastname: Mondal
authors.forename: Susanta
authors.initials: S
authors.lastname: Roychoudhury
authors.forename: Chinmay Kumar
authors.initials: CK
authors.lastname: Panda
mesh_terms: D045744:Cell Line, Tumor; D019175:DNA Methylation; D018450:Disease Progression; D044127:Epigenesis, Genetic; D005260:Female; D015967:Gene Expression Regulation, Viral; D052162:Human papillomavirus 16 / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D009856:Oncogene Proteins, Viral / Q000096:biosynthesis / Q000235:genetics*; D016366:Open Reading Frames; D050725:Papillomavirus E7 Proteins / Q000096:biosynthesis / Q000235:genetics*; D030361:Papillomavirus Infections / Q000473:pathology / Q000821:virology*; D011401:Promoter Regions, Genetic; D060888:Real-Time Polymerase Chain Reaction; D012097:Repressor Proteins / Q000096:biosynthesis / Q000235:genetics*; D002583:Uterine Cervical Neoplasms / Q000473:pathology / Q000821:virology*; D002578:Uterine Cervical Dysplasia / Q000378:metabolism / Q000821:virology*
chemical_list: C068651:E6 protein, Human papillomavirus type 16; D009856:Oncogene Proteins, Viral; D050725:Papillomavirus E7 Proteins; D012097:Repressor Proteins; C059731:oncogene protein E7, Human papillomavirus type 16