Quantitation of CDC6 and MCM5 mRNA in cervical intraepithelial neoplasia and invasive squamous cell carcinoma of the cervix

Abstract

CDC6 and MCM5 play essential roles in eukaryotic DNA replication. Several studies have highlighted the potential of these proteins as molecular markers of dysplastic and malignant cells in histopathological diagnosis. The mode of expression of CDC6 and MCM5 mRNA and their significance in normal, dysplastic and malignant cervical cells remains to be elucidated. Using a quantitative real-time RT PCR assay, we compared CDC6 and MCM5 mRNA expression in normal cervical epithelium, cervical intraepithelial neoplasia and invasive squamous cell carcinoma of the cervix. Our study cohort comprised 20 normal cervical biopsies, 20 CIN3 and eight invasive squamous cell carcinomas. All samples were formalin fixed and paraffin embedded. Total RNA was extracted and analysed for expression of GAPDH, CDC6 and MCM5 using real-time quantitative TaqMan RT-PCR. A linear increase in MCM5 and CDC6 mRNA expression is observed in normal cervix, CIN3 and invasive cervical carcinoma. The overall difference in MCM5 mRNA expression in the normal cervix, CIN3 and invasive cohort groups is highly statistically significant (P ¼ 0.001). An increase in CDC6 mRNA expression in CIN3 and invasive cervical squamous cell carcinoma was observed; however, the overall difference between cohort groups was not found to be statistically significant (P ¼ 0.104). Increased transcription of MCM5 and CDC6 occurs as a consequence of cervical neoplastic progression. This pattern of increased mRNA expression in CIN3 and invasive cervical carcinoma directly correlates with findings at the phenotypic protein expression level. This study further confirms the importance of MCM5 and CDC6 in malignant transformation and in the pathogenesis of cervical dysplasia. In all eukaryotes, a conserved mechanism of DNA replication exists, which ensures that DNA replication occurs only once in a single cell cycle. 1 This mechanism is often termed the 'licensing' of DNA replication. 2 DNA replication requires the regulated assembly of pre-replicative complexes (pre-RC) onto DNA during the G1 phase of the cell cycle. Pre-RCs render the chromatin competent or 'licensed' to replicate. Among the proteins known to assemble to form the pre-RC are cell division cycle protein 6 (CDC6) and mini chromosome maintenance (MCM) proteins. 3,4 Biological analysis of CDC6 suggests that it functions as a clamp loader in which ATP binding and hydrolysis induce conformational changes, which result in the recruitment or loading of MCMs onto DNA. 3 Once the MCMs are recruited to the pre-RC, CDC6 protein is then phosphorylated by Cyclin A/CDK2 in S phase of the cell cycle. This results in the translocation of CDC6 from its chromatin sites to the cytoplasm where it is degraded by the anaphase promoting complex (APC)/cyclosome. 5-8 Relocalisation of CDC6 to the cytoplasm prevents reinitiation of replication. In addition to conferring replication competence, studies also suggest that the level/ modification of CDC6 in G2 phase functions as a checkpoint control, which ensures that the S phase nucleus has com...

Evaluation of RNA Markers for Early Detection of Cervical Neoplasia in Exfoliated Cervical Cells

“…Detecting molecular changes indicative of disease in these extracts requires a large fraction of abnormal cells from the lesion, a robust change in expression that can withstand dilution, or a change in expression that extends beyond the morphologically detected lesion (i.e., the existence of a field effect). Although dilution cannot be discounted, it should be noted that the level of discrimination in this study using extracts of exfoliated cervical cells was similar to the immunohistochemistry measures of CDC6 and MCM in tissue samples of lesions shown by Murphy et al (22).…”

“…The minichromosome maintenance proteins MCM5 and MCM7 are normally up-regulated in the G 0 to G 1 -S phase transition and initiate and promote DNA replication. Increased expression at both the protein and RNA level has been shown in cervical tissue (21)(22)(23). MCM7 might also be a direct target for HPV (24,25).…”

“…MCM7 might also be a direct target for HPV (24,25). Several immunohistochemistry studies have implicated CDC6 and MKI67 as proliferation markers often correlated with the grade of neoplasia (22,26,27). SHCBP1 was originally implicated by our own microarray study and had been described as a growth factor stimulated link of signaling pathways governing cell cycle progression (28).…”

Rb family proteins in gastric cancer (Review)

“…pRb2/ p130 is the only pocket protein phosphorylated in G0 (82,86) and in terminally differentiated cells and animal tissues (87). During quiescence state pRb2/p130 binds the E2F4 and E2F5 transcription factors inhibiting transcription of their target genes such as cyclin A2, CDC2 and CDC6, all involved in cell cycle progression and neoplastic transformation (88)(89)(90).…”

p16INK4a and p14ARF mRNA expression in Pap smears is age-related

“…However, despite RNA derived from exfoliated cervical cells being challenging, other mRNA-based biomarkers such as HPV oncogenes, 25 Brn3a, 26 or MCM5, 27 were successfully tested on cervical smears and biopsies.…”