HPV-16 impairs the subcellular distribution and levels of expression of protein phosphatase 1γ in cervical malignancy.

BACKGROUND: The high risk Human Papillomavirus (HPV) E6 oncoproteins play an essential role in the development of cervical malignancy. Important cellular targets of E6 include p53 and the PDZ domain containing substrates such as hScrib and Dlg. We recently showed that hScrib activity was mediated in part through recruitment of protein phosphatase 1γ (PP1γ). METHODS: Expression patterns of hScrib and PP1γ were assessed by immunohistochemistry of HPV-16 positive cervical intraepithelial neoplasm (CIN), classified as CIN1 (n = 4), CIN2 (n = 8), CIN3 (n = 8), cervical carcinoma tissues (n = 11), and HPV-negative cervical tissues (n = 8), as well as by subfractionation assay of the HPV-16 positive cervical cancer cell lines, CaSki and SiHa. To explore the effects of the HPV-16 oncoproteins, we have performed siRNA knockdown of E6/E7 expression, and monitored the effects on the expression patterns of hScrib and PP1γ. RESULTS: We show that PP1γ levels in HPV-16 positive tumour cells are reduced in an E6/E7 dependent manner. Residual PP1γ in these cells is found mostly in the cytoplasm as opposed to the nucleus where it is predominantly found in normal cells. We have found a striking concordance with redistribution in the pattern of expression (9/11; 81.8%) and loss of PP1γ expression in HPV-16 positive cervical tumours (2/11; 18.2%). Furthermore, this loss of PP1γ expression and redistribution in the pattern of expression occurs progressively as the lesions develop (8/8; 100%). CONCLUSION: Together, these results suggest that PP1γ may be a novel target of the HPV-16 oncoproteins and indicate that it might be a potential novel biomarker for HPV-16 induced malignancy.

A novel interaction between hScrib and PP1γ downregulates ERK signaling and suppresses oncogene-induced cell transformation.

Previous studies have shown that the cell polarity regulator hScrib interacts with, and consequently controls, the ERK signaling pathway. This interaction occurs through two well-conserved Kinase Interacting Motifs, which allow hScrib to bind ERK1 directly, resulting in a reduction in the levels of phospho-ERK. This suggests that hScrib might recruit a phosphatase to regulate this signaling pathway. Using a proteomic approach we now show that Protein Phosphatase 1γ (PP1γ) is a major interacting partner of hScrib. This interaction is direct and occurs through a conserved PP1γ interaction motif on the hScrib protein, and this interaction appears to be required for hScrib's ability to downregulate ERK phosphorylation. In addition, hScrib also controls the pattern of PP1γ localization, where loss of hScrib enhances the nuclear translocation of PP1γ. Furthermore, we also show that the ability of hScrib to interact with PP1γ is important for the ability of hScrib to suppress oncogene-induced transformation of primary rodent cells. Taken together, these results demonstrate that hScrib acts as a scaffold to integrate the control of the PP1γ and ERK signaling pathways and explains how disruption of hScrib localisation can contribute towards the development of human malignancy.

Sequential effects of the proteasome inhibitor bortezomib and chemotherapeutic agents in uterine cervical cancer cell lines.

Although the prognosis of uterine cervical cancer has improved due to the advances of treatment modalities, survival of recurrent or metastatic cervical cancer remains poor. Cisplatin is an effective radiosensitizer, but its single agent activity in recurrent cervical cancer is disappointing. Inactivation of tumor suppressors through ubiquitin-mediated degradation by human papillomavirus is known to be a critical step in the carcinogenesis of uterine cervix. Bortezomib, a selective inhibitor of the proteasome, has been shown to inhibit the growth of several solid tumors. To determine the role of bortezomib in cervical cancer as a chemotherapeutic agent, we studied its biological properties. Bortezomib efficiently inhibited the proteasomal activities in cervical cancer cells, and an increased expression of tumor suppressors such as p53, hDlg and hScrib became evident. In addition, sequential or concomitant treatment of bortezomib and cisplatin stimulated the expression of p53, hScrib and p21 and the stimulation was markedly influenced by the order of drugs in HeLa cells. We further confirmed that the concomitant use of bortezomib and cisplatin has synergistic inhibitory effects on the growth of xenograft tumors derived from HeLa cells. Our data establish the possibility that the concomitant use of bortezomib and cisplatin could be an alternative choice in cases resistant to conventional chemotherapy, and sequential effects must be considered for advanced and therapy-resistant cervical cancer patients.

Regulation of SIRT1 determines initial step of endometrial receptivity by controlling E-cadherin expression.

Sirtuin 1 (SIRT1), originally found as a class III histone deacetylase, is a principal modulator of pathways downstream of calorie restriction, and the activation of SIRT1 ameliorates glucose homeostasis and insulin sensitivity. We examined the role of SIRT1 in the regulation of uterine receptivity using Ishikawa and RL95-2 endometrial carcinoma cell lines. Exogenous expression of SIRT1 significantly enhanced E-cadherin expression, while small interfering RNA-mediated depletion of endogenous SIRT1 resulted in a significant reduction of E-cadherin expression. A SIRT1 activator resveratrol elevated E-cadherin expression in a dose dependent manner, while SIRT1 repressors nicotinamide and sirtinol exhibited a dose dependent reduction of E-cadherin expression. We also showed that both forced expression of SIRT1 and activation of SIRT1 promote E-cadherin-driven reporter gene constructs, and SIRT1 is localized at E-cadherin promoter containing E-box elements in Ishikawa cells. Using an in vitro model of embryo implantation, we demonstrate that exogenous expression of SIRT1 and stimulation of SIRT1 activity resulted in the Ishikawa cell line becoming receptive to JAR cell spheroid attachment. Furthermore, resveratrol enhanced E-cadherin and Glycodelin protein expression at sites of intercellular contact, suggesting an additive role of resveratrol in promoting implantation. The initial step of human reproduction depends on the capacity of an embryo to attach and implant into the endometrial wall, and these results revealed the novel mechanism that activation and increased expression of SIRT1 play an important role in uterine receptivity.

Synthesis of functionalized pentacenes from isobenzofurans derived from C-H bond activation.

The synthesis of unsymmetric functionalized pentacenes from 1,4-anthraquinones and functionalized isobenzofurans, which were prepared by transformation via C-H bond activation, was successfully accomplished. Examples of the synthesis of pentacenes with functional groups at the 5-position are still rare. These obtained functionalized pentacenes are highly soluble in hexane, toluene, and THF.

A pilot study of weekly paclitaxel administration for patients with relapsed cervical cancer after heavy medication.

No standard therapy has been established for patients with relapsed cervical cancer after applying radical hysterectomies including lymphadenectomies, radiotherapy, and platinum-based chemotherapy. This study was designed to evaluate the effectiveness and safety of weekly paclitaxel (TXL) therapy in patients who suffered a cervical cancer relapse after heavy treatment. The candidates for the study included patients with cervical cancer that recurred after radical therapy (including lymphadenectomies), postoperative radiotherapy, and platinum-based chemotherapy, the lesions of which could be evaluated by imaging diagnosis. Patients received 80 mg/m2 of TXL by intravenous drip in one hour. Premedications included 10 mg of dexamethasone (iv), 50 mg of cimetidine (iv), and 50 mg of diphenhydramine (po) administered 30 minutes before the TXL treatment. This procedure was repeated weekly on an ongoing basis. The median progression-free survival was 14 months (range: 0 to 24 months), and the median overall survival 19 months (range: 6 to 24 months). Grade-3 or higer hematologic toxicity was observed for leukocyte (total WBC) and neutrophil/granulocyte in one patient (12.5%), but was controllable with GCSF. The weekly TXL therapy was effective against cervical cancer relapse after heavy treatment and its toxicity was tolerable.

[Evaluation of QOL with orally disintegrating antiemetic tablets in outpatient chemotherapy].

An investigation into the effect of orally disintegrating antiemetic tablets (ramosetron OD 0.1 mg) on 16 outpatients undergoing chemotherapy for recurrent gynecologic malignancies was carried out using a questionnaire to evaluate their quality of life (QOL). The patients were divided into 2 groups: a control group in which the patients received only an intravenous drip infusion of 0.3 mg of ramosetron on the day of their chemotherapy and a double antiemetic therapy group in which the patients were treated with orally disintegrating antiemetic tablets for 4 days in addition to the intravenous drip. When outpatient chemotherapy (paclitaxel 80 mg/m2/1 h div + CBDCA AUC 2/1 h div) was performed, a comparison was made between the 2 groups, based on the results of a QOL questionnaire for antiemetic therapy during cancer chemotherapy (Ishihara's QOL survey method). This study found a greater improvement in QOL in the double antiemetic therapy group on days 2 to 4, a period when deterioration in QOL is usually observed. These findings suggest that orally disintegrating antiemetic tablets as a measure to reduce delayed nausea and vomiting are useful for improving QOL.