In many types of cancer, including cervical cancer, the nuclear retinoic receptor ß2 (RARß2) gene is epigenetically modified and unable to be significantly induced. RARß2 can act as a tumor suppressor, since loss of its expression is associated with human tumor progression. One mechanism of RARß2-mediated growth inhibition is based on its ability to constitutively repress the AP-1 transcription factor, but this mechanism has never been demonstrated in cervical cancer cells. In this thesis the biological consequences of reconstitute RARß2 receptor expression in cervical cancer cells was investigated. For this purpose, human papillomavirus HPV-18 positive HeLa cells were stably transfected with RARß2 cDNA under the control of the ß-actin promoter. The characterization of the RARß2 tranfectants revealed a strongly reduced AP-1 binding to the corresponding specific oligonucleotides, even in the absence of atRA treatment. In HeLa cells, the AP-1 reduction correlates with diminished HPV-18 oncogene transcription and slower cellular growth. Western blot analysis demonstrated that the only member of the AP-1 family consistently reduced in HeLa RARß2 clones was c-Jun, despite ongoing gene expression. In order to understand by which mechanism c-Jun is reduced, and since the phosphorylation of c-Jun is important for protein stabilization, HeLa RARß2 clones were treated with different c-Jun-N-terminal kinase (JNK) stimulators. The treatments resulted in a c-Jun increase at RNA and protein levels, and led to a reconstitution of AP-1 binding similar to non-transfected HeLa controls. However, the reconstitution of AP-1 binding levels did not have an inductor effect on the HPV-18 oncogenes, the expression of which has been postulated to be AP-1 dependent. Other classical AP-1 regulated genes such as metalloproteinases were up regulated as expected. In conclusion, the data of this thesis indicate that RARß2 induced a destabilization and accelerated degradation of c-Jun at the protein level responsible for the AP-1 reduction. The mechanism of AP-1 trans-repression in HeLa cells is different from that postulated for other cancer cell models. All changes produced in HeLa cells after RARß2 constitutive expression lead to reduced cell proliferation, which can be associated with the RARß2 tumor suppressor function.
|Supervisor:||Clayton, Prof. Dr. Christine|
|Date of thesis defense:||28 October 2003|
|Faculties / Institutes:||Service facilities > German Cancer Research Center (DKFZ)|
|Subjects:||570 Life sciences|
|Controlled Keywords:||RAR beta, AP-1, c-Jun|
|Uncontrolled Keywords:||RAR beta , AP-1 , c-JunRAR beta , AP-1 , c-Jun|