Overexpression of Kpnβ1 and Kpnα2 Importin Proteins in Cancer Derives from Deregulated E2F Activity

The Karyopherin superfamily comprises nuclear transport proteins, involved in the shuttling of certain cargo proteins into and out of the nucleus. Karyopherin β1 (Kpnβ1) and Karyopherin α2 (Kpnα2) are importin proteins, which work in concert to transport their cargo into the nucleus. We previously identified increased expression of Kpnβ1 and Kpnα2 in cervical tumours compared to normal epithelium and in transformed cells compared to their normal counterparts. This study therefore aimed to identify the transcription regulatory mechanisms associated with high Kpnβ1 and Kpnα2 levels in cancer cells. Kpnβ1 (−2013 to +100) and Kpnα2 (−1900 to +69) promoter fragments were separately cloned into the reporter vector, pGL3-basic, and luciferase assays revealed both as significantly more active in cancer and transformed cells compared to normal. A series of deletion constructs identified the −637 to −271 Kpnβ1 and −180 to −24 Kpnα2 promoter regions as responsible for the differential promoter activity, and a number of highly conserved E2F binding sites were identified within these regions. Mutation analysis confirmed the requirement of E2F sites for promoter activity, and ChIP analysis confirmed E2F2/Dp1 binding to the Kpnβ1 and Kpnα2 promoters in vivo. Dp1 inhibition resulted in decreased levels of the respective proteins, confirming the role of E2F in the overexpression of Kpnβ1 and Kpnα2 proteins in cancer. E2F activity is known to be deregulated in cervical cancer cells due to the inhibition of its repressor, Rb, by HPV E7. The inhibition of E7 using siRNA resulted in decreased Kpnβ1 and Kpnα2 promoter activities, as did the overexpression of Rb. In conclusion, this study is a first to show that elevated Kpnβ1 and Kpnα2 expression in cancer cells correlates with altered transcriptional regulation associated with deregulated E2F/Rb activitie

Research Communication Deregulated LAP2a Expression in Cervical Cancer Associates with Aberrant E2F and p53 Activities

Summary Lamina-associated polypeptide 2 alpha (LAP2a) plays a role in maintaining nuclear structure, in nuclear assembly/disassembly, and in transcriptional regulation. Elevated LAP2a mRNA expression has been previously reported to associate with certain cancer types. The aim of this study was to investigate LAP2a expression in cervical cancer and transformed cells and to identify factors that associate with its differential expression. LAP2a expression was found to be elevated in cervical cancer tissue by microarray, qRT-PCR, and immunofluorescence analyses. LAP2a also showed elevated expression in cervical cancer cell lines and in transformed fibroblasts compared with normal cells. To determine factors associated with elevated LAP2a in cervical cancer, the effect of inhibiting HPV E7 and E6 oncoproteins was investigated. E7 inhibition resulted in a decrease in phosphorylated Rb and an associated decrease in LAP2a, suggesting a role for E2F in regulating LAP2a expression. This finding was confirmed by inhibiting DP1, a coactivator of E2F, which resulted in decreased LAP2a levels. Inhibition of E6 resulted in elevated p53 and an associated decrease in LAP2a, suggesting that p53 associates with the negative regulation of LAP2a expression. This hypothesis was tested by inhibiting p53 in normal cells, and a resultant increase in LAP2a expression was observed. In conclusion, this study provides evidence for elevated LAP2a expression in cervical cancer and suggests that E2F and p53 activities associate with the positive and negative regulation of LAP2a expression, respectively. 2011 IUBMB IUBMB Life, 00: 000-000, 201

Inhibition of Kpnβ1 mediated nuclear import enhances cisplatin chemosensitivity in cervical cancer

Background Inhibition of nuclear import via Karyopherin beta 1 (Kpnβ1) shows potential as an anti-cancer approach. This study investigated the use of nuclear import inhibitor, INI-43, in combination with cisplatin. Methods Cervical cancer cells were pre-treated with INI-43 before treatment with cisplatin, and MTT cell viability and apoptosis assays performed. Activity and localisation of p53 and NFκB was determined after co-treatment of cells. Results Pre-treatment of cervical cancer cells with INI-43 at sublethal concentrations enhanced cisplatin sensitivity, evident through decreased cell viability and enhanced apoptosis. Kpnβ1 knock-down cells similarly displayed increased sensitivity to cisplatin. Combination index determination using the Chou-Talalay method revealed that INI-43 and cisplatin engaged in synergistic interactions. p53 was found to be involved in the cell death response to combination treatment as its inhibition abolished the enhanced cell death observed. INI-43 pre-treatment resulted in moderately stabilized p53 and induced p53 reporter activity, which translated to increased p21 and decreased Mcl-1 upon cisplatin combination treatment. Furthermore, cisplatin treatment led to nuclear import of NFκB, which was diminished upon pre-treatment with INI-43. NFκB reporter activity and expression of NFκB transcriptional targets, cyclin D1, c-Myc and XIAP, showed decreased levels after combination treatment compared to single cisplatin treatment and this associated with enhanced DNA damage. Conclusions Taken together, this study shows that INI-43 pre-treatment significantly enhances cisplatin sensitivity in cervical cancer cells, mediated through stabilization of p53 and decreased nuclear import of NFκB. Hence this study suggests the possible synergistic use of nuclear import inhibition and cisplatin to treat cervical cancer

IQuaD dental trial; improving the quality of dentistry : a multicentre randomised controlled trial comparing oral hygiene advice and periodontal instrumentation for the prevention and management of periodontal disease in dentate adults attending dental primary care

Peer reviewedPublisher PD

Overexpression of Kpnb1 and Kpna2 Importin Proteins in Cancer Derives from Deregulated E2F Activity

Abstract The Karyopherin superfamily comprises nuclear transport proteins, involved in the shuttling of certain cargo proteins into and out of the nucleus. Karyopherin b1 (Kpnb1) and Karyopherin a2 (Kpna2) are importin proteins, which work in concert to transport their cargo into the nucleus. We previously identified increased expression of Kpnb1 and Kpna2 in cervical tumours compared to normal epithelium and in transformed cells compared to their normal counterparts. This study therefore aimed to identify the transcription regulatory mechanisms associated with high Kpnb1 and Kpna2 levels in cancer cells. Kpnb1 (22013 to +100) and Kpna2 (21900 to +69) promoter fragments were separately cloned into the reporter vector, pGL3-basic, and luciferase assays revealed both as significantly more active in cancer and transformed cells compared to normal. A series of deletion constructs identified the 2637 to 2271 Kpnb1 and 2180 to 224 Kpna2 promoter regions as responsible for the differential promoter activity, and a number of highly conserved E2F binding sites were identified within these regions. Mutation analysis confirmed the requirement of E2F sites for promoter activity, and ChIP analysis confirmed E2F2/Dp1 binding to the Kpnb1 and Kpna2 promoters in vivo. Dp1 inhibition resulted in decreased levels of the respective proteins, confirming the role of E2F in the overexpression of Kpnb1 and Kpna2 proteins in cancer. E2F activity is known to be deregulated in cervical cancer cells due to the inhibition of its repressor, Rb, by HPV E7. The inhibition of E7 using siRNA resulted in decreased Kpnb1 and Kpna2 promoter activities, as did the overexpression of Rb. In conclusion, this study is a first to show that elevated Kpnb1 and Kpna2 expression in cancer cells correlates with altered transcriptional regulation associated with deregulated E2F/Rb activities Citation: van der Watt PJ, Ngarande E, Leaner VD (2011) Overexpression of Kpnb1 and Kpna2 Importin Proteins in Cancer Derives from Deregulated E2F Activity. PLoS ONE 6(11): e27723

HPV E7 inhibition and Rb overexpression affects the activity of the Kpnβ1 and Kpnα2 promoters.

<p>A, B. HPV16 E7 siRNA was used to inhibit E7 expression in CaSki cells and Western blot analysis performed to analyse HPV E7 (A) and Rb (B) levels after HPV E7 knock-down. C, D. Kpnβ1 (C) and Kpnα2 (D) promoter activities were measured after E7 siRNA transfection, and were found to be significantly inhibited after HPV E7 inhibition. E, F. Rb was overexpressed in CaSki cells and Kpnβ1 (E) and Kpnα2 (F) promoter activities were found to be significantly inhibited. Results shown are the mean ± SE of experiments in quadruplicate performed at least two times (* p<0.05).</p

Kpnβ1 and Kpnα2 protein levels in normal, transformed and cancer cells correlate with respective promoter activities.

<p>A. Western Blot analysis of Kpnβ1 and Kpnα2 protein levels in normal WI38, transformed SVWI38, and cervical cancer CaSki cells reveals increased expression in the cancer and transformed cells. B, C. −2013 to +100 Kpnβ1 promoter activity (B) and −1900 to +69 Kpnα2 promoter activity (C) was measured in cell lysates prepared from transfected WI38, SVWI38 and CaSki cells, and was observed to be significantly higher in the transformed and cancer cells compared to the normal cells (*p<0.05). TK-Renilla-Luc was used as a control for transfection efficiency and luciferase activity is expressed relative to Renilla luciferase. Results shown are the mean ± SD of experiments performed in triplicate and repeated at least two times.</p