Increased expression of cellular RNA-binding proteins in HPV-induced neoplasia and cervical cancer.

The expression profile of a panel of RNA-binding proteins (heterogeneous ribonucleoprotein (hnRNP) A1, hnRNP C1/C2, hnRNP H, hnRNP I, ASF/SF2, SR proteins, HuR and U2AF(65)) and markers of differentiation, proliferation and neoplasia (cytokeratin (CK) 13, CK-14, proliferating cell nuclear antigen (PCNA), Syndecan-1 and p16INK4a) were analyzed in 50 formalin fixed paraffin embedded cervical tissues using immunohistochemistry. The samples included histologically normal cervical epithelium, human papillomavirus (HPV) induced low-grade and high-grade pre-malignant lesions and cervical cancers. All samples were tested for HPV DNA using nested PCR. Forty-nine of the 50 tissue samples tested positive for HPV, 27 tissue samples (54%) were HPV-16 positive and 4 samples (8%) were HPV-18 positive. The immunohistochemistry results detected different expression levels of the various proteins in basal epithelial cells in histologically normal epithelium followed by an increase in expression in the intermediate layers, whereas the superficial layers remained negative for all tested RNA-binding proteins. Expression of all RNA-binding proteins increased in neoplastic lesions and highest expression was detected in cervical cancers. p16INK4a had a stronger association with high-grade lesions when compared with the RNA-binding proteins. The expression profile of the RNA-binding proteins is similar to PCNA expression in histologically normal epithelium as well as in lesions (low-grade and high-grade) and cervical cancers. As PCNA expression has been suggested to mimic HPV E6/E7 expression in cervical epithelium, the results suggest the RNA-binding protein analyzed here regulate HPV early gene expression directly and late gene expression indirectly.

CD10-/ALDH- cells are the sole cisplatin-resistant component of a novel ovarian cancer stem cell hierarchy.

It is long established that tumour-initiating cancer stem cells (CSCs) possess chemoresistant properties. However, little is known of the mechanisms involved, particularly with respect to the organisation of CSCs as stem-progenitor-differentiated cell hierarchies. Here we aimed to elucidate the relationship between CSC hierarchies and chemoresistance in an ovarian cancer model. Using a single cell-based approach to CSC discovery and validation, we report a novel, four-component CSC hierarchy based around the markers cluster of differentiation 10 (CD10) and aldehyde dehydrogenase (ALDH). In a change to our understanding of CSC biology, resistance to chemotherapy drug cisplatin was found to be the sole property of CD10-/ALDH- CSCs, while all four CSC types were sensitive to chemotherapy drug paclitaxel. Cisplatin treatment quickly altered the hierarchy, resulting in a three-component hierarchy dominated by the cisplatin-resistant CD10-/ALDH- CSC. This organisation was found to be hard-wired in a long-term cisplatin-adapted model, where again CD10-/ALDH- CSCs were the sole cisplatin-resistant component, and all CSC types remained paclitaxel-sensitive. Molecular analysis indicated that cisplatin resistance is associated with inherent- and adaptive-specific drug efflux and DNA-damage repair mechanisms. Clinically, low CD10 expression was consistent with a specific set of ovarian cancer patient samples. Collectively, these data advance our understanding of the relationship between CSC hierarchies and chemoresistance, which was shown to be CSC- and drug-type specific, and facilitated by specific and synergistic inherent and adaptive mechanisms. Furthermore, our data indicate that primary stage targeting of CD10-/ALDH- CSCs in specific ovarian cancer patients in future may facilitate targeting of recurrent disease, before it ever develops.

Raman spectroscopic evaluation of efficacy of current paraffin wax section dewaxing agents.

During a spectroscopic study to identify biochemical changes in cervical tissue with the onset of carcinogenesis, residual paraffin wax contributions were observed on almost all dewaxed formalin-fixed paraffin-processed (FFPP) tissue sections examined. Subsequently, the present study was formulated to evaluate the efficacy of current dewaxing agents using Raman spectroscopy. Three cervical FFPP sections were subjected to each of the protocols. Sections were dewaxed using four common dewaxing protocols, namely, xylene, Histoclear, heat-mediated antigen retrieval (HMAR) using xylene and citrate buffer, and Trilogy (combined deparaffinization and unmasking of antigens). The potential for hexane as a dewaxing agent was also evaluated. Sections were dewaxed in multiple dewaxing cycles using xylene, Histoclear, and hexane. Residual paraffin wax contributions remained at 1062 cm(-1), 1296 cm(-1), and 1441 cm(-1). HMAR using xylene and citrate buffer, and HMAR using Trilogy, showed a similar efficacy, resulting in incomplete removal of wax. Hexane was shown to be the most effective dewaxing agent, resulting in almost complete removal of wax. Immunohistochemistry was carried out on dewaxed slides, and those dewaxed with hexane displayed a stronger positivity (approximately 28%). Implications for histopathology and immunohistochemistry are considered, as well as problems that residual wax poses for spectroscopic evaluation of dewaxed FFPP sections with a view to disease diagnosis.

The role of claudin-1 and claudin-7 in cervical tumorigenesis.

BACKGROUND/AIM: The claudin family of proteins are key constituents of tight junctions and the aberrant expression of these proteins can contribute to de-stabilisation of tight junctions and thus to loss of cell polarity and cohesion. Increased expression of claudin-1 and claudin-7 has been observed in pre-invasive cervical lesions and cervical carcinomas. The present study attempted to assess the effect of claudin-1 and claudin-7 overexpression on the HeLa cervical carcinoma cell line, in terms of cell proliferation/viability, permeability, invasion and migration. MATERIALS AND METHODS: HeLa cells were stably transfected with expression vectors containing the claudin-1 and claudin-7 genes to produce two separate stable cell lines expressing claudin-1 and claudin-7, respectively. The stable cell lines were examined with regard to their invasion and migration abilities, cell permeability and cell proliferation/viability and compared to non-claudin-1 or -7 transfected HeLa. RESULTS: The present study found that claudin-1 and claudin-7 affected the migratory ability of HeLa cells, reducing their ability to migrate in a gap closure assay compared to non-claudin-transfected HeLa cells. Monolayers of claudin-1 and claudin-7 transfected cells also displayed an increased transepithelial electrical resistance indicating decreased permeability compared to non-claudin-transfected HeLa. The study found that claudin-1 or claudin-7 expression had no effect on the proliferation or viability of HeLa cells. Claudin-1 or -7 expression also did not affect the invasive ability of HeLa cells with both stable cells lines and non-claudin-transfected HeLa cells all showing low invasive ability. CONCLUSION: The results of the present study indicate that claudin-1 and claudin-7 overexpression alone does not contribute to increased tumorigenesis in cervical carcinoma, instead claudin-1 and - 7 expression in HeLa cells contribute to reducing the migratory ability of cells and decrease their permeability.

Raman spectroscopic analysis of human skin tissue sections ex-vivo: evaluation of the effects of tissue processing and dewaxing.

Raman spectroscopy coupled with K-means clustering analysis (KMCA) is employed to elucidate the biochemical structure of human skin tissue sections and the effects of tissue processing. Both hand and thigh sections of human cadavers were analyzed in their unprocessed and formalin-fixed, paraffin-processed (FFPP), and subsequently dewaxed forms. In unprocessed sections, KMCA reveals clear differentiation of the stratum corneum (SC), intermediate underlying epithelium, and dermal layers for sections from both anatomical sites. The SC is seen to be relatively rich in lipidic content; the spectrum of the subjacent layers is strongly influenced by the presence of melanin, while that of the dermis is dominated by the characteristics of collagen. For a given anatomical site, little difference in layer structure and biochemistry is observed between samples from different cadavers. However, the hand and thigh sections are consistently differentiated for all cadavers, largely based on lipidic profiles. In dewaxed FFPP samples, while the SC, intermediate, and dermal layers are clearly differentiated by KMCA of Raman maps of tissue sections, the lipidic contributions to the spectra are significantly reduced, with the result that respective skin layers from different anatomical sites become indistinguishable. While efficient at removing the fixing wax, the tissue processing also efficiently removes the structurally similar lipidic components of the skin layers. In studies of dermatological processes in which lipids play an important role, such as wound healing, dewaxed samples are therefore not appropriate. Removal of the lipids does however accentuate the spectral features of the cellular and protein components, which may be more appropriate for retrospective analysis of disease progression and biochemical analysis using tissue banks.

Adenovirus E4orf4 induces HPV-16 late L1 mRNA production.

The adenovirus E4orf4 protein regulates the switch from early to late gene expression during the adenoviral replication cycle. Here we report that overexpression of adenovirus E4orf4 induces human papillomavirus type 16 (HPV-16) late gene expression from subgenomic expression plasmids. E4orf4 specifically overcomes the negative effects of two splicing silencers at the two late HPV-16 splice sites SD3632 and SA5639. This results in the production of HPV-16 spliced L1 mRNAs. We show that the interaction of E4orf4 with protein phosphatase 2A (PP2A) is necessary for induction of HPV-16 late gene expression. Also an E4orf4 mutant that fails to bind the cellular splicing factor ASF/SF2 fails to induce L1 mRNA production. Collectively, these results suggest that dephosphorylation of SR proteins by E4orf4 activates HPV-16 late gene expression. Indeed, a mutant ASF/SF2 protein in which the RS-domain had been deleted could itself induce HPV-16 late gene expression, whereas wild type ASF/SF2 could not.

Identification of a 17-nucleotide splicing enhancer in HPV-16 L1 that counteracts the effect of multiple hnRNP A1-binding splicing silencers.

Human papillomavirus type 16 (HPV-16) infections can in rare cases persist and cause lesions that may progress to cervical cancer. Cells in the lesions are not permissive for virus production, nor are cervical cancer cells. The intracellular environment is such that it prevents production of the highly immunogenic, viral structural proteins L1 and L2. One may speculate that inhibition of L1 and L2 expression is a prerequisite for persistence and cancer progression. We have therefore investigated how expression of HPV-16 L1 is regulated. We found that the only splice site in the HPV-16 late region, which is used to produce L1 mRNAs, is under control of a splicing enhancer located in the 17 nucleotides immediately downstream of the splice site. However, the function of this enhancer in cervical cancer cells is largely overshadowed by multiple splicing silencers in the late region which bind to hnRNP A1. High levels of hnRNP A1 therefore inhibit HPV-16 L1 expression. Immunohistological analysis of cervical epithelia revealed that hnRNP A1 is expressed primarily in the lower layers of the epithelium. hnRNP A1 is undetectable in terminally differentiated cells that can express HPV-16 late genes, which supports the conclusion that high levels of hnRNP A1 inhibit HPV-16 L1 expression.

A downstream polyadenylation element in human papillomavirus type 16 L2 encodes multiple GGG motifs and interacts with hnRNP H.

Production of human papillomavirus type 16 (HPV-16) virus particles is totally dependent on the differentiation-dependent induction of viral L1 and L2 late gene expression. The early polyadenylation signal in HPV-16 plays a major role in the switch from the early to the late, productive stage of the viral life cycle. Here, we show that the L2 coding region of HPV-16 contains RNA elements that are necessary for polyadenylation at the early polyadenylation signal. Consecutive mutations in six GGG motifs located 174 nucleotides downstream of the polyadenylation signal resulted in a gradual decrease in polyadenylation at the early polyadenylation signal. This caused read-through into the late region, followed by production of the late mRNAs encoding L1 and L2. Binding of hnRNP H to the various triple-G mutants correlated with functional activity of the HPV-16 early polyadenylation signal. In addition, the polyadenylation factor CStF-64 was also found to interact specifically with the region in L2 located 174 nucleotides downstream of the early polyadenylation signal. Staining of cervix epithelium with anti-hnRNP H-specific antiserum revealed high expression levels of hnRNP H in the lower layers of cervical epithelium and a loss of hnRNP H production in the superficial layers, supporting a model in which a differentiation-dependent down regulation of hnRNP H causes a decrease in HPV-16 early polyadenylation and an induction of late gene expression.

A 57-nucleotide upstream early polyadenylation element in human papillomavirus type 16 interacts with hFip1, CstF-64, hnRNP C1/C2, and polypyrimidine tract binding protein.

We have investigated the role of the human papillomavirus type 16 (HPV-16) early untranslated region (3' UTR) in HPV-16 gene expression. We found that deletion of the early 3' UTR reduced the utilization of the early polyadenylation signal and, as a consequence, resulted in read-through into the late region and production of late L1 and L2 mRNAs. Deletion of the U-rich 3' half of the early 3' UTR had a similar effect, demonstrating that the 57-nucleotide U-rich region acted as an enhancing upstream element on the early polyadenylation signal. In accordance with this, the newly identified hFip1 protein, which has been shown to enhance polyadenylation through U-rich upstream elements, interacted specifically with the HPV-16 upstream element. This upstream element also interacted specifically with CstF-64, hnRNP C1/C2, and polypyrimidine tract binding protein, suggesting that these factors were either enhancing or regulating polyadenylation at the HPV-16 early polyadenylation signal. Mutational inactivation of the early polyadenylation signal also resulted in increased late mRNA production. However, the effect was reduced by the activation of upstream cryptic polyadenylation signals, demonstrating the presence of additional strong RNA elements downstream of the early polyadenylation signal that direct cleavage and polyadenylation to this region of the HPV-16 genome. In addition, we identified a 3' splice site at genomic position 742 in the early region with the potential to produce E1 and E4 mRNAs on which the E1 and E4 open reading frames are preceded only by the suboptimal E6 AUG. These mRNAs would therefore be more efficiently translated into E1 and E4 than previously described HPV-16 E1 and E4 mRNAs on which E1 and E4 are preceded by both E6 and E7 AUGs.

Cross-reactivity of some antibodies to human epitopes with shrimp Pandalus borealis proteins: a possible aid in validation and characterization of crustacean cells in vitro.

Cell characterization of primary cultures in vertebrates is well established but not in marine invertebrates. This fact is hampering advances in the development of tissue cultures from this species. In the present study, a panel of antibodies to structural proteins, stress proteins, oncogenes and proliferation antigens, developed against mammalian antigens, were tested in paraffin sections of the crustacean Pandalus borealis tissues. Several tissues were analysed: hepatopancreas, gills, ovaries, epithelium under the cuticle and abdominal muscle. Specific antibodies to crustacean proteins are not commercially available. The immunocytochemical results show that antibodies to human epitopes cross-react with antigens in the crustacean Pandalus borealis indicating that some cellular proteins are highly conserved in evolution. Cytokeratin, proliferating cell nuclear antigen, ras and p-glycoprotein were detected by immunocytochemistry in Pandalus borealis. No immunoreactivity for Ki-67 and metallothionein was observed. This system can help in validation and characterization of invertebrate cultures.