Expression Levels of Nuf2 in Cervical Dysplasias and Cancers
K Astbury, L McEvoy, M Ring, CM Martin, JJ O'Leary. Coombe Women's Hospital, Dublin, Ireland; Trinity College Dublin, Dublin, Ireland; Dublin Institute of Technology, Dublin, Ireland
Background: Aneuploidy is a common feature of cervical cancer. Inhibition of p53 and pRb by high risk HPV types has been shown to induce centrosome duplication errors and resultant aneuploidy. More recently abnormal kinetochore function has also been suggested as a potential cause of aneuploidy. Recent publications have demonstrated over-expression of Nuf2 and KNTC2/HEC1 (2 components of the kinetochore) in lung cancer and have also indicated that nuf2 expression levels are affected by HPV E6 and E7.
Design: CaSki, C33A, HeLa and SiHa cervical cancer cell lines were cultured and RNA extracted. Normal cervical RNA was purchased from Biochain Inc. Gene expression profiling of all cell lines was performed using Applied Biosystems human genome survey microarrays and gene expression profiles were compared with expression profiles of normal cervix. Taqman PCR was then performed to validate a number of over-expressed genes, incuding p16IN4A and nuf2. Finally, immunohistochemical analyisis of nuf2 expression was performed in CIN and cervical cancer.
Results: Nuf2 was found to be 39 fold up-regulated in the cervical cancer cell lines compared to normal cervical tissue on gene expression profiling, while HEC1 was 35 fold up-regulated. Further evalation of Nuf2 by TaqMan PCR confirmed its over-expression. Immunohistochemical analysis of 90 normal cervix, CIN and cervical cancer specimens was performed. Nuf2 was not detected in normal cervical tissue or in the squamous cell cervical cancers specimens. CIN 1, 2 and 3 and cGIN cases showed varying intensities of cytoplasmic staining, while all 8 villoglandular cancers tested stained positive for nuf2.
Conclusions: As part of the kinetochore, nuf2 plays an important role in ensuring accurate chromosomal segregation during mitosis. In this study we demonstrate nuf2 gene over-expression in cervical cancer cell lines. The expression of nuf2 at protein level was found to be more variable. While the HeLa cervical cell line, villoglandular adenocarcinomas and pre-invasive cervical lesions stained positively with nuf2 antibody, the squamous cell cervical cancer specimens did not display any staining. We postulate that this may be due either to a structural change in nuf2 protein not detected by the antibody used or to the effect of a naturally ocurring siRNA that interferes with nuf2 protein production.
Monday, March 9, 2009 1:00 PM
Poster Session II # 142, Monday Afternoon