Heparin (GAG-hed) inhibits LCR activity of human papillomavirus type 18 by decreasing AP1 binding.
BACKGROUND
High risk HPVs are causative agents of anogenital cancers. Viral E6 and E7 genes are continuously expressed and are largely responsible for the oncogenic activity of these viruses. Transcription of the E6 and E7 genes is controlled by the viral Long Control Region (LCR), plus several cellular transcription factors including AP1 and the viral protein E2. Within the LCR, the binding and activity of the transcription factor AP1 represents a key regulatory event in maintaining E6/E7 gene expression and uncontrolled cell proliferation. Glycosaminoglycans (GAGs), such as heparin, can inhibit tumour growth; they have also shown antiviral effects and inhibition of AP1 transcriptional activity. The purpose of this study was to test the heparinoid GAG-hed, as a possible antiviral and antitumoral agent in an HPV18 positive HeLa cell line.


METHODS
Using in vivo and in vitro approaches we tested GAG-hed effects on HeLa tumour cell growth, cell proliferation and on the expression of HPV18 E6/E7 oncogenes. GAG-hed effects on AP1 binding to HPV18-LCR-DNA were tested by EMSA.


RESULTS
We were able to record the antitumoral effect of GAG-hed in vivo by using as a model tumours induced by injection of HeLa cells into athymic female mice. The antiviral effect of GAG-hed resulted in the inhibition of LCR activity and, consequently, the inhibition of E6 and E7 transcription. A specific diminishing of cell proliferation rates was observed in HeLa but not in HPV-free colorectal adenocarcinoma cells. Treated HeLa cells did not undergo apoptosis but the percentage of cells in G2/M phase of the cell cycle was increased. We also detected that GAG-hed prevents the binding of the transcription factor AP1 to the LCR.


CONCLUSIONS
Direct interaction of GAG-hed with the components of the AP1 complex and subsequent interference with its ability to correctly bind specific sites within the viral LCR may contribute to the inhibition of E6/E7 transcription and cell proliferation. Our data suggest that GAG-hed could have antitumoral and antiviral activity mainly by inhibiting AP1 binding to the HPV18-LCR.
Villanueva
Rita
R
Departamento de Genética y Biología Molecular, Centro de Investigación y de Estudios Avanzados, Apartado Postal 14-740, México D.F. 07000, México. rvr1032000@yahoo.com.mx
Morales-Peza
Néstor
N
Castelán-Sánchez
Irma
I
García-Villa
Enrique
E
Tapia
Rocio
R
Cid-Arregui
Angel
A
García-Carrancá
Alejandro
A
López-Bayghen
Esther
E
Gariglio
Patricio
P
D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D002453:Cell Cycle / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D004268:DNA-Binding Proteins / Q000378:metabolism; D015536:Down-Regulation / Q000187:drug effects; D005260:Female; D006367:HeLa Cells; D006493:Heparin / Q000494:pharmacology* / Q000627:therapeutic use; D052161:Human papillomavirus 18 / Q000187:drug effects* / Q000235:genetics*; D006801:Humans; D019901:Locus Control Region / Q000187:drug effects* / Q000235:genetics; D051379:Mice; D008819:Mice, Nude; D008822:Mice, Transgenic; D008954:Models, Biological; D011485:Protein Binding / Q000187:drug effects; D012045:Regulatory Sequences, Nucleic Acid; D018808:Transcription Factor AP-1 / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects; D002583:Uterine Cervical Neoplasms / Q000188:drug therapy / Q000473:pathology; D023041:Xenograft Model Antitumor Assays
D000998:Antiviral Agents; D004268:DNA-Binding Proteins; D018808:Transcription Factor AP-1; D006493:Heparin