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Induction of p16 during immortalization by HPV 16 and 18 and not during malignant transformation
1Division of Basic Medical Sciences, Faculty of Medicine, Memorial University of Newfoundland, St John's, Canada.
Abstract:
The p16 (MTS1) tumour-suppressor gene is a cyclin-dependent kinase (cdk) inhibitor that decelerates the cell cycle by inactivating the cdks that phosphorylate the retinoblastoma tumour-suppressor gene (Rb) protein (pRb). In cervical cancers, pRb is inactivated by the HPV E7 oncoprotein or by mutations. The hypothesis of earlier reports was that the disruption of the p16/cdk-cyclin/Rb cascade is essential for malignant cervical transformation/carcinogenesis. We previously established in vitro model systems of cervical cancer representing four steps of oncogenic progression initiated by the two most common oncogenic HPVs in ectocervical and endocervical epithelial cells. This report used these systems to investigate the role of p16 in cervical cancers. A dramatic enhancement of the p16 RNA level was observed after immortalization by HPV 16 or 18. Furthermore, the p16 protein was newly observed following immortalization. However, no further changes were found for RNA or protein levels after serum selection or malignant transformation. For three cervical carcinoma cell lines, similar high levels of p16 expression were seen. Point mutations or homozygous deletions of p16 were not observed in the in vitro systems or in clinical specimens. These results suggest that the inactivation of the p16/cdk-cyclin/Rb cascade does not occur during malignant transformation but occurs during the immortalization by HPV in HPV-harbouring premalignant lesions, the in situ equivalent of immortalized cells. Also suggested is that p16 has no role in the specific malignant transformation step from immortal premalignant lesions during the carcinogenesis of HPV-initiated cervical cancers.
Insights
The p16 tumor suppressor gene
Area of Science:
- Oncology
- Molecular Biology
- Virology
Background:
- The p16 (MTS1) gene acts as a tumor suppressor by inhibiting cyclin-dependent kinases (cdks) and decelerating the cell cycle.
- In cervical cancers, the retinoblastoma protein (pRb) is inactivated by HPV E7 or mutations, leading to the hypothesis that p16/cdk-cyclin/Rb cascade disruption is crucial for malignant transformation.
- Previous research suggested that p16's inactivation is essential for cervical cancer development.
Purpose of the Study:
- To investigate the role of the p16 gene in cervical cancer development using established in vitro models.
- To determine if p16 inactivation occurs during malignant transformation or earlier stages of HPV-induced cervical carcinogenesis.
- To analyze p16 RNA and protein expression levels throughout the oncogenic progression of cervical cells.
Main Methods:
- Utilized in vitro model systems of cervical cancer representing four stages of oncogenic progression initiated by HPV 16 or 18.
- Quantified p16 RNA and protein levels after immortalization, serum selection, and malignant transformation.
- Screened for p16 point mutations and homozygous deletions in in vitro models and clinical cervical cancer specimens.
Main Results:
- A significant increase in p16 RNA and the emergence of p16 protein were observed after immortalization by HPV 16 or 18.
- No further changes in p16 RNA or protein levels were detected after serum selection or malignant transformation.
- High p16 expression was consistent across three cervical carcinoma cell lines, with no observed p16 mutations or deletions in vitro or in clinical samples.
Conclusions:
- The inactivation of the p16/cdk-cyclin/Rb cascade occurs during HPV-mediated immortalization of premalignant cervical lesions, not during malignant transformation.
- p16 plays no role in the specific malignant transformation step from immortalized premalignant lesions in HPV-initiated cervical cancers.
- These findings suggest that HPV-induced immortalization, rather than malignant transformation, is the critical stage involving p16 pathway alterations in cervical carcinogenesis.