Abstract
BackgroundThe 21-kDa Vaccinia virus VH1-related (VHR) dual-specific protein phosphatase (encoded by the DUSP3 gene) plays a critical role in cell cycle progression and is itself regulated during the cell cycle. We have previously demonstrated using RNA interference that cells lacking VHR arrest in the G1 and G2 phases of the cell cycle and show signs of beginning of cell senescence.MethodsIn this report, we evaluated successfully the expression levels of VHR protein in 62 hysterectomy or conization specimens showing the various (pre) neoplastic cervical epithelial lesions and 35 additional cases of hysterectomy performed for non-cervical pathologies, from patients under 50 years of age. We used a tissue microarray and IHC technique to evaluate the expression of the VHR phosphatase. Immunofluorescence staining under confocal microscopy, Western blotting and RT-PCR methods were used to investigate the localization and expression levels of VHR.ResultsWe report that VHR is upregulated in (pre) neoplastic lesions (squamous intraepithelial lesions; SILs) of the uterine cervix mainly in high grade SIL (H-SIL) compared to normal exocervix. In the invasive cancer, VHR is also highly expressed with nuclear localization in the majority of cells compared to normal tissue where VHR is always in the cytoplasm. We also report that this phosphatase is highly expressed in several cervix cancer cell lines such as HeLa, SiHa, CaSki, C33 and HT3 compared to primary keratinocytes. The immunofluorescence technique under confocal microscopy shows that VHR has a cytoplasmic localization in primary keratinocytes, while it localizes in both cytoplasm and nucleus of the cancer cell lines investigated. We report that the up-regulation of this phosphatase is mainly due to its post-translational stabilization in the cancer cell lines compared to primary keratinocytes rather than increases in the transcription of DUSP3 locus.ConclusionThese results together suggest that VHR can be considered as a new marker for cancer progression in cervix carcinoma and potential new target for anticancer therapy.
Highlights
The 21-kDa Vaccinia virus VH1-related (VHR) dual-specific protein phosphatase plays a critical role in cell cycle progression and is itself regulated during the cell cycle
VHR expression and localization in cervix biopsies VHR expression at the protein level was studied using a tissue microarray mounted in normal exocervix (n = 16), low-grade squamous intraepithelial lesions (SIL) (n = 11), high-grade SILs (n = 18), invasive squamous cell carcinoma (SCC) (n = 12), normal endocervix (n = 19), adenocarcinoma (n = 12) and adenocarcinoma in situ (n = 9)
The VHR score was statistically higher in high grade SIL (H-SIL) and SCCs (p < 0.0001 and p < 0.05 respectively) compared to normal exocervical epithelium (Fig 1, upper panel)
Summary
The 21-kDa Vaccinia virus VH1-related (VHR) dual-specific protein phosphatase (encoded by the DUSP3 gene) plays a critical role in cell cycle progression and is itself regulated during the cell cycle. The human genome contains 61 genes for Vaccinia virus H1-like, or 'dual-specific' protein phosphatases (DUSPs) [1], most of which have poorly understood functions. Many of these genes encode phosphatase that dephosphorylate the mitogen-activated protein kinases (e.g. MKP1/DUSP1, PAC1/DUSP2, MKP3/DUSP6, etc) or regulate the cell cycle (e.g. CDC14A and CDC14B). The elimination of VHR by RNA interference resulted in cell cycle arrest in G1/S and G2/M [3,4] This effect of VHR knock-down was counteracted by downmodulation of the levels of Erk and Jnk or by modest levels of Mek and Jnk inhibitors. The permissive role of VHR in cell cycle regulation prompted us to ask if VHR levels are perturbed in cancer cells
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