CCND1 a Cell Cycle Regulator and Proto-Oncogene

Nikolic, Margareta (1993) CCND1 a Cell Cycle Regulator and Proto-Oncogene. PhD thesis, University of Glasgow.

Abstract

The CCND1 gene codes for a recently discovered member of the cyclin D family of proteins and is thought to function during the G1 phase of the cell cycle. Amplification and transcript over-expression of CCND1 have in the past two years been reported in several types of human tumours, providing the initial evidence for the contribution of its product in the development of cancer. This investigation was aimed at clarifying the involvement of the CCND1 gene in transformation. The CCND1 gene dosage, transcript and protein levels were characterised in a panel of newly derived SCC cell lines, revealing the same frequency of amplification as reported in tumour biopsies. The frequency differed from the previous analyses of established cell lines and may reflect the methods used to derive the cell lines. In all cases amplification resulted in upregulated levels of the CCND1 mRNA and protein (p34cycD1), although amplification was not necessary for over-expression of CCND1. These findings further emphasise the status of this gene as the target of 11q13 amplification. Interestingly, the cell lines that exhibited CCND1 amplifications had also increased expression of the tumour suppressor protein pRb, a potential regulator or substrate of p34cycD1 SCC derived cell lines, original tumour biopsies and xenografts revealed an exclusively nuclear localisation of p34cycD1, as previously determined for fibroblast cell lines. Serum stimulation of GO arrested fibroblasts induced an increase of p34cycD1 levels four hours from exposure, which reached a maximum between 8-12 hours and declined thereafter. The same oscillation pattern was observed in EOF stimulated keratinocytes where cell cycle delay was achievable through serum deprivation. To analyse the stage during transformation at which a cell might find it beneficial to over-express p34cycD1, two types of transfection analyses were carried out. An established, poorly tumourigenic SCC cell line (SCC12) that originated from a skin SCC, was lipofected with sense or antisense Cyl-1 cDNA expression vectors and the derived clones examined for their ability to form tumours in immunodeficient mice. No alterations in growth patterns or tumourigenicity of the transfected cells were observed. Alternatively, human foreskin primary keratinocytes transfected with Cyl-1 cDNA expression vectors alone or in combination with HPV-16 E6 or E7expression constructs exhibited the ability to overcome the Ml restrictive stage and therefore achieve an extended life span. It remains speculative if in vivo, overexpression of CCND1 may contribute to immortalisation of epithelial cells. Examination of rodent fibroblast cell lines and their fos over-expressing clones revealed the lack of correlation between p34cycD1 levels and fos induced transformation. In conclusion, these studies may suggest a significant contribution of CCND1 over-expression in the initial stages of epithelial transformation. Increased levels of p34cycD1 could permit escape of epithelial cells from growth control and therefore differentiation, resulting in their indefinite lifespan which allows the occurrence and fixation of other genetic alterations. However, these results do not contradict the potential involvement of CCND1 in later stages of tumour development.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Adviser: Brad Ozanne
• Keywords: Genetics
• Date of Award: 1993
• Depositing User: Enlighten Team
• Unique ID: glathesis:1993-74761
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 27 Sep 2019 16:36
• Last Modified: 27 Sep 2019 16:36
• URI: https://theses.gla.ac.uk/id/eprint/74761