Human Cervical Epithelium Research Articles

Human serum albumin-bound paclitaxel nanoparticle inhibits cervical carcinoma cell proliferation and oxidative damage through CYP3A4 and CYP2C8

BackgroundCervical cancer (CC) is currently the most common malignant tumour in the female reproductive tract, and paclitaxel (PTX) is a commonly used chemotherapeutic agent, but tumour cell resistance will seriously affect the therapeutic efficacy of PTX. Nanoparticle human serum albumin-bound paclitaxel (Nano-HSA-PTX) is a novel drug delivery modality that may have superior effects to PTX alone. ObjectiveTo clarify the effect of Nano-HSA-PTX on cervical carcinoma (CC) cells and the underlying mechanisms. MethodsAfter the preparation of Nano-HSA-PTX, its morphology was observed by electron transmission microscope (TEM), and its entrapment efficiency (EE%) and drug loading rate (DL%) were detected. Nano-HSA-PTX was compared with conventional PTX for drug metabolism. Additionally, CC HeLa and SiHa cells were purchased and divided into three groups to treat with Nano-HSA-PTX, PTX and normal saline, respectively. MTT, cell cloning, Transwell and cell scratch assays were carried out to determine cell proliferation, invasion and migration, flow cytometry and Western blotting were performed to detect apoptosis rate and apoptosis-related protein expression, and PCR was conducted to quantify oxidative damage indicators. Further, CYP3A4 and CYP2C8 expression patterns in CC cells (HeLa and SiHa) and human normal cervical epithelia (End1/E6E7) and the changes of their levels under the intervention of Nano-HSA-PTX were measured. Subsequently, C57BL/6mice were purchased for subcutaneous tumorigenesis experiment to observe the impact of Nano-HSA-PTX on tumor growth. ResultsUnder TEM, Nano-HSA-PTX was complete and arranged compactly, with a stable structure and markedly higher EE% and DL% than PTX (P < 0.05). Under Nano-HSA-PTX intervention, the proliferation, invasion, migration and oxidative damage of HeLa and SiHa were significantly decreased compared with the control and PTX groups, while the apoptosis was increased (P < 0.05). Besides, elevated CYP3A4 and CYP2C8 levels were observed in CC cells, which were inhibited by Nano-HSA-PTX and PTX (P < 0.05). Finally, tumorigenesis experiments in nude mice revealed that Nano-HSA-PTX could inhibit tumor growth. ConclusionCompared with PTX, Nano-HSA-PTX has a superior effect of inhibiting CC activity. And this mechanism of action was carried out by inhibiting the expression of CYP3A4 and CYP2C8.

Three-dimensional models of the cervicovaginal epithelia to study host-microbiome interactions and sexually transmitted infections.

2D cell culture systems have historically provided controlled, reproducible means to analyze host–pathogen interactions observed in the human reproductive tract. Although inexpensive, straightforward, and requiring a very short time commitment, these models recapitulate neither the functionality of multilayered cell types nor the associated microbiome that occurs in a human. Animal models have commonly been used to recreate the complexity of human infections. However, extensive modifications of animal models are required to recreate interactions that resemble those in the human reproductive tract. 3D cell culture models have emerged as alternative means of reproducing vital elements of human infections at a fraction of the cost of animal models and on a scale that allows for replicative experiments. Here, we describe a new 3D model that utilizes transwells with epithelial cells seeded apically and a basolateral extracellular matrix (ECM)-like layer. The model produced tissues with morphologic and physiological resemblance to human cervical and vaginal epithelia, including mucus levels produced by cervical cells. Infection by Chlamydia trachomatis and Neisseria gonorrhoeae was demonstrated, as well as the growth of bacterial species observed in the human vaginal microbiota. This enabled controlled mechanistic analyses of the interactions between host cells, the vaginal microbiota, and STI pathogens. Affordable and semi high-throughput 3D models of the cervicovaginal epithelia that are physiologically relevant by sustaining vaginal bacterial colonization, and facilitate studies of chlamydial and gonococcal infections.

Bacterial vaginosis and health-associated bacteria modulate the immunometabolic landscape in 3D model of human cervix

Bacterial vaginosis (BV) is an enigmatic polymicrobial condition characterized by a depletion of health-associated Lactobacillus and an overgrowth of anaerobes. Importantly, BV is linked to adverse gynecologic and obstetric outcomes: an increased risk of sexually transmitted infections, preterm birth, and cancer. We hypothesized that members of the cervicovaginal microbiota distinctly contribute to immunometabolic changes in the human cervix, leading to these sequelae. Our 3D epithelial cell model that recapitulates the human cervical epithelium was infected with clinical isolates of cervicovaginal bacteria, alone or as a polymicrobial community. We used Lactobacillus crispatus as a representative health-associated commensal and four common BV-associated species: Gardnerella vaginalis, Prevotella bivia, Atopobium vaginae, and Sneathia amnii. The immunometabolic profiles of these microenvironments were analyzed using multiplex immunoassays and untargeted global metabolomics. A. vaginae and S. amnii exhibited the highest proinflammatory potential through induction of cytokines, iNOS, and oxidative stress-associated compounds. G. vaginalis, P. bivia, and S. amnii distinctly altered physicochemical barrier-related proteins and metabolites (mucins, sialic acid, polyamines), whereas L. crispatus produced an antimicrobial compound, phenyllactic acid. Alterations to the immunometabolic landscape correlate with symptoms and hallmarks of BV and connected BV with adverse women’s health outcomes. Overall, this study demonstrated that 3D cervical epithelial cell colonized with cervicovaginal microbiota faithfully reproduce the immunometabolic microenvironment previously observed in clinical studies and can successfully be used as a robust tool to evaluate host responses to commensal and pathogenic bacteria in the female reproductive tract.

Transcriptomic profile of VEGF-regulated genes in human cervical epithelia.

Cervical epithelial cells play a central role in cervical remodeling (CR) during pregnancy and cervical events during menstrual cycle, including mounting physical and immunological barriers, proliferation and differentiation, maintenance of fluid balance, and likely in withstanding the mechanical force exerted by the growing fetus prior to term. In the present study, we attempt to decipher the specific roles of VEGF in fetal human cervical epithelial cells by delineating VEGF signature genes using RNA sequencing in order to characterize the specific biological effects of VEGF in these cells.Out of a total of 25,000 genes screened, 162 genes were found to be differentially expressed in human cervical epithelial cells, of which 12 genes were found to be statistically significantly differentially expressed. The differentially expressed genes (162) were categorized by biological function, which included (1) proliferation, (2) immune response, (3) structure/matrix, (4) mitochondrial function, and (5) cell adhesion/communication and others (pseudogenes, non-coding RNA, miscellaneous genes, and uncharacterized genes). We conclude that VEGF plays a key role in CR by altering the expression of genes that regulate proliferation, immune response, energy metabolism and cell structure, and biological processes that are essential to development and likely CR.

Overexpression of SOCS4 inhibits proliferation and migration of cervical cancer cells by regulating JAK1/STAT3 signaling pathway

Purpose: To delve into the related molecular mechanism of suppressor of cytokine signaling 4 (SOCS4) on cervical cancer cell proliferation and migration. Methods: Quantitative real-time polymerase chain reaction and western blot assays were employed to examine SOCS4 mRNA or protein expression in four human cervical cancer cell line (HeLa, SW-732, AV3, and CaSki) and normal cervical epithelium immortalized cell line (H8) MTT cell viability assays were applied to verify the cell proliferation of HeLa after overexpression of SOCS4. Wound scratch healing assays and transwell assays were applied to examine cell migration and invasion of HeLa after overexpression of SOCS4. Flow cytometry and western blot assays were applied to check the role of SOCS4 in the apoptosis of cervical cancer cells. The western blot assays were applied to examine the protein expression of JAK1, p-JAK1, STAT3, and p-STAT3 in HeLa after overexpression of SOCS4. Results: In this study, the results revealed that the mRNA and protein expression level of SOCS4 was lower in four human cervical cancer cell line than normal cervical epithelium immortalized cell line, respectively. Overexpression of SOCS4 inhibited the proliferation migration, and invasion of cervical cancer cells as well as promotes apoptosis of cervical cancer cells. Meanwhile, overexpression of SOCS4 in HeLa would inhibit phosphorylation of JAK1 and STAT3 protein. Conclusion: SOCS4 inhibited the proliferation and migration of cervical cancer cells by regulating the JAK1/STAT3 pathway.

Evolution of the exclusively human pathogen Neisseria gonorrhoeae: Human-specific engagement of immunoregulatory Siglecs.

Neisseria gonorrhoeae causes the sexually transmitted disease gonorrhea exclusively in humans and uses multiple strategies to infect, including acquisition of host sialic acids that cap and mask lipooligosaccharide termini, while restricting complement activation. We hypothesized that gonococci selectively target human anti‐inflammatory sialic acid‐recognizing Siglec receptors on innate immune cells to blunt host responses and that pro‐inflammatory Siglecs and SIGLEC pseudogene polymorphisms represent host evolutionary adaptations to counteract this interaction. N. gonorrhoeae can indeed engage multiple human but not chimpanzee CD33rSiglecs expressed on innate immune cells and in the genitourinary tract––including Siglec‐11 (inhibitory) and Siglec‐16 (activating), which we detected for the first time on human cervical epithelium. Surprisingly, in addition to LOS sialic acid, we found that gonococcal porin (PorB) mediated binding to multiple Siglecs. PorB also bound preferentially to human Siglecs and not chimpanzee orthologs, modulating host immune reactions in a human‐specific manner. Lastly, we studied the distribution of null SIGLEC polymorphisms in a Namibian cohort with a high prevalence of gonorrhea and found that uninfected women preferentially harbor functional SIGLEC16 alleles encoding an activating immune receptor. These results contribute to the understanding of the human specificity of N. gonorrhoeae and how it evolved to evade the human immune defense.

Abstract 3308: Mitophagy induction and interference with cancer-specific apoptosis in SHetA2-treated cells

Abstract Background: SHetA2 is a small molecule flexible heteroarotinoid (Flex-Het) compound that exhibited in-vivo cancer therapeutic and chemoprevention activity, lack of mutagenicity or teratogenicity, and a pharmacologic profile suitable for an oral drug. First-in-human clinical trials of SHetA2 for ovarian cancer prevention and cervical cancer treatment are in development. Autophagy is induced in cancer cells by a wide variety of therapeutic drugs and commonly interferes with the drugs efficacies. Because SHetA2 binds to mortalin and induces mitochondrial damage and apoptosis in cancer, but not in healthy cells, we hypothesized that this drug induces mitochondria-selective autophagy (mitophagy) in cancer, but not healthy cells; and that the mitophagy interferes with SHetA2-induction of apoptosis. Methods: Cultures of human ovarian and cervical cancer cell lines and human ovarian surface epithelium (HOSE) were treated with various concentrations and combinations of SHetA2, 3-methyladenine (3-MA), Beclin-1 siRNA, and caspase inhibitors. Autophagy was evaluated by electron microscopy (EM) and fluorescent imaging of LC3B processing in cells and by Western blot (WB) analysis of p62, Beclin-1 and LC3B processing. Mitophagy was measured by EM of cells and WB analysis of BNIP3 total levels and co-immunoprecipitation with LC3B in protein extracts. Apoptosis was measured by Annexin-V and propidium iodide staining of cells and by WB analysis of caspase 9 and 3 and PARP-1 cleavage. Result: Induction of autophagy by SHetA2 treatment was demonstrated by the development of double-membraned autophagosomes and accumulation of the processed LC3B II in cancer, but not in HOSE cells. Mitophagy was demonstrated by the presence of mitochondria inside the autophagosomes and by reduction of total BNIP3 in association with increased BNIP3/LC3B complexes in SHetA2 treated cells. Autophagic flux was demonstrated by gradual reduction of p62. Conversion of autophagy to apoptosis was associated with reappearance of basal p62 levels and cleavage of Beclin-1, PARP-1, and caspases 9 and 3 about 24 hours after initiation of treatment. Interference of the autophagy with apoptosis was demonstrated by increased levels of apoptosis in cells co-treated with SHetA2 and autophagy inhibitors (3-MA or Beclin-1 siRNA) along with reduced apoptosis in cells co-treated with SHetA2 and an autophagy inducer (rapamycin). This was observed in cell lines harboring mutant or no p53, but not in an ovarian cancer cell line with wild type p53. Conclusion: SHetA2-induction of autophagy may be initiated by mitochondrial damage and reduces, but does prevent, SHetA2-mediated apoptosis in ovarian and cervical cancer cell lines, but not in a cell line with wild type p53 or in HOSE cells. Combination of SHetA2 with an autophagy inhibitor may improve the therapeutic efficacy in clinical trials of patients harboring mutant or loss of p53 protein. Funded by: RO1CA196200 and RO1CA200126 Citation Format: Vishal Chandra, Andrew Long, Chioniso Patience Masamha, Doris Mangiaracina Benbrook. Mitophagy induction and interference with cancer-specific apoptosis in SHetA2-treated cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3308. doi:10.1158/1538-7445.AM2017-3308

Gene promoter methylation and protein expression of BRMS1 in uterine cervix in relation to high-risk human papilloma virus infection and cancer.

Cervical cancer is strongly related to certain high-risk types of human papilloma virus infection. Breast cancer metastasis suppressor 1 (BRMS1) is a tumor suppressor gene, its expression being regulated by DNA promoter methylation in several types of cancers. This study aims to evaluate the methylation status of BRMS1 promoter in relation to high-risk types of human papilloma virus infection and the development of pre-cancerous lesions and describe the pattern of BRMS1 protein expression in normal, high-risk types of human papilloma virus-infected pre-cancerous and malignant cervical epithelium. We compared the methylation status of BRMS1 in cervical smears of 64 women with no infection by high-risk types of human papilloma virus to 70 women with proven high-risk types of human papilloma virus infection, using real-time methylation-specific polymerase chain reaction. The expression of BRMS1 protein was described by immunohistochemistry in biopsies from cervical cancer, pre-cancerous lesions, and normal cervices. Methylation of BRMS1 promoter was detected in 37.5% of women with no high-risk types of human papilloma virus infection and was less frequent in smears with high-risk types of human papilloma virus (11.4%) and in women with pathological histology (cervical intraepithelial neoplasia) (11.9%). Methylation was detected also in HeLa cervical cancer cells. Immunohistochemistry revealed nuclear BRMS1 protein staining in normal high-risk types of human papilloma virus-free cervix, in cervical intraepithelial neoplasias, and in malignant tissues, where staining was occasionally also cytoplasmic. In cancer, expression was stronger in the more differentiated cancer blasts. In conclusion, BRMS1 promoter methylation and aberrant protein expression seem to be related to high-risk types of human papilloma virus-induced carcinogenesis in uterine cervix and is worthy of further investigation.

The Pathogenesis of Human Cervical Epithelium Cells Induced by Interacting with Trichomonas vaginalis

Background Trichomonas vaginalis is a protozoan parasite that occurs in the urogenital-vaginal tract and is the primary causative agent of trichomoniasis, a common sexually transmitted disease in humans. The aggregation of this protozoan tends to destroy epithelial cells and induce pathogenesis.Principal FindingsThis study cultured T. vaginalis and human cervical epithelial cells (Z172) under the same conditions in the experiments. Following co-culturing for ten hours, the protozoans became attached to Z172, such that the cells presented a round shape and underwent shrinkage. Time-lapse recording and flow cytometry on interacted Z172 revealed that 70% had been disrupted, 18% presented a necrosis-like morphology and 8% showed signs of apoptosis. Gene expression profiling revealed in the seven inflammatory Z172 genes as well as in T. vaginalis genes that code for adhesion proteins 65 and 65-1.SignificanceThese results suggest that cytopathogenic effects progress while Z172 is in contact with T. vaginalis, and the resulting morphological changes can be categorized as disruption.

Cancer cell uptake of polymer hydrogel nanotubes.

To gain deeper understanding of how the shape and size of particles influence their interaction with cells, the uptake process of polymer hydrogel nanotubes (PNTs) by cancer cells was characterized. The PNTs were composed of crosslinked poly(ethylene glycol) segments with a diameter of 300 nm and a polydispersed length in the range of 2 to 20 μm. The uptake of PNTs into human cervical carcinoma epithelia (HeLa) cells cultured in vitro was monitored by confocal laser fluorescence microscopy and details of PNT entry into cells were revealed by a series of confocal Z-section images and time-lapsed video microscopy. It was found that PNTs initiating contact with the cell membrane through the tips of the tubes were readily taken up by the cells, whereas the uptake of tubes approaching the cell surface in parallel manner was very difficult. The length of the tubes did not appear to affect cellular uptake. In fact, it was found surprisingly that PNTs as long as > 10 μm were internalized by these non-phagocytic cells. The rate of PNT uptake varied significantly and was probably influenced by the angle of attachment. Better understanding of the interaction between PNTs and cells may provide important insight in the use of PNTs as potential anticancer drug delivery vehicles.

156P - Simultaneous Inhibition of Chaperone Activity and Chaperone Expression in Human Cancer Cells Sensitizes Them to Chemotherapy and Radiotherapy

ABSTRACT Aim: We propose to combine inhibitors of chaperone activity with inhibitors of chaperone expression for better targeting tumors because (i) cancer cells are addicted to the chaperone activity of heat-shock protein 90 (Hsp90), (ii) the Hsp90 chaperone activity inhibitors (e.g. 17AAG) may have a 100-fold higher affinity to Hsp90 in malignant cells than in normal cells, (iii) these Hsp90 inhibitors cause induction of cytoprotective chaperones Hsp70 and Hsp27 that impairs antitumor effects of the Hsp90 inhibition. Methods: MCF-7 and HeLa cell lines derived from human breast and cervical tumors and human normal epithelium or fibroblasts were subjected to 17AAG and inhibitors of the Hsp induction (quercetin, triptolide, NZ28), and conventional drugs (doxorubicin, paclitaxel) or clinically relevant doses of gamma-photon radiation. The cell death/survival was assessed in annexin V-staining and clonogenic assays. The expression of certain cellular proteins was analyzed by western blotting. Results: It was found that 10–100 nM 17AAG significantly retarded proliferation in both the cancer cell lines. As markers of the Hsp90 activity inhibition, the specific depletion of Akt, survivin and HIF-1alpha as well as up-regulation of Hsp70 and Hsp27 were revealed in those cell samples. Co-treatment with 10–30 µM quercetin or 2–4 nM triptolide, or 0.3–1 µM NZ28 prevented the Hsp up-regulation in the 17AAG-treated cancer cells and sensitized them to doxorubicin and paclitaxel, and gamma-radiation. Importantly, no enhanced cytotoxicity was observed in normal human fibroblasts and epithelial cells co-treated by the same way. The enhanced chemo- and radiosensitization of cancer cells appears to be due to simultaneous blockade of both the Hsp90-dependent antiapoptotic pathways and the 17AAG-induced up-regulation of Hsp70 and Hsp27. Conclusions: Combined administration of inhibitors of the Hsp90 chaperone activity and blockers of the concomitant induction of Hsp70 and Hsp27 enables us to selectively sensitize tumors to chemo- and radiotherapy. In perspective, such an approach may be developed into an effectual pharmacological strategy for cancer treatment. Disclosure: All authors have declared no conflicts of interest.

Establishment of a novel method for primary culture of normal human cervical keratinocytes

Background Cervical keratinocytes are recovered at a low numbers and frequently associated with contaminating human fibroblasts which rapidly overgrow the epithelial cells in culture with medium supplemented with 10% fetal bovine serum (FBS). However, it is difficult to initiate keratinocyte cultures with serum-free keratinocyte growth medium alone because cell attachment can be poor. Therefore, the culture of these cells is extremely difficult. In this study, we described a modified culture medium and coated culture plastics for growing normal human cervical epithelial cells in vitro. Methods Normal cervical epithelial tissue pieces were obtained and digested with type I collagenase to dissociate the cells and a single cell suspension produced. The cells were cultured on plastic tissue culture substrate alone or substrate coated with collagen type I from rat tail, with modified keratinocyte serum-free medium (K-SFM) supplemented with 5% FBS. After attachment, the medium were replaced with K-SFM without FBS. The expression of basal keratins of the ectocervical epithelium, K5, K14 and K19 were assayed by immunofluorescence with monoclonal antibodies to identify the cell purity. Results Our results indicate that cells attached to the culture plastic more quickly in K-SFM supplemented with 5% FBS than in K-SFM alone, as well as to tissue culture plastic coated with collagen type I than plastic alone. The modified medium composed of K-SFM and 5% FBS combined with a specific tissue culture plastic coated with collagen type I from rat tail was the best method for culture of normal cervical epithelial cells. K5, K14 and K19 were assayed and keratinocyte purity was nearly 100%. Conclusion A novel, simple and effective method can be used to rapidly obtain highly purified keratinocytes from normal human cervical epithelium.

PL.89 Functional Effects of Oestradiol on Cytokine Responses of Human Ectocervical Epithelial Cells to Toll-Like Receptor 2 and 4 Ligands

We have demonstrated that the pattern recognition toll-like receptors (TLR) 2&4 are upregulated during pregnancy in human cervical epithelium. We sought to determine whether oestradiol (E2) alters the immunological responses...

Viral Load of High-Risk Human Papillomaviruses as Reliable Clinical Predictor for the Presence of Cervical Lesions

Infections with high-risk human papillomaviruses (Hr-HPV) can cause malignant transformation of the human cervical epithelium. HPV DNA tests generally are very sensitive to detect cervical neoplastic lesions but also identify transient HPV infections. As a consequence, the specificity and positive predictive value are low. We analyzed viral load of Hr- and possibly Hr-HPV types more than seven orders of magnitude (on a log10 scale) in 999 consecutive BD-SurePath liquid-based cervical cytology samples from routine cervical screening enriched with atypical squamous cells of undetermined significance (n = 100), low-grade squamous intraepithelial lesions (LSIL; n = 100), and high-grade squamous intraepithelial lesions (HSIL; n = 97) using type-specific multiplex quantitative real-time PCR and the BSGP5+/6+-PCR/MPG assay. In the 36-month follow-up, 79 histologically verified CIN2+ and 797 double-negative cytology cases were identified. Viral loads in LSIL and HSIL were significantly increased compared with no intraepithelial lesion or malignancy in both the quantitative PCR (qPCR) and BSGP5+/6+-PCR/MPG assay (P < 0.0001). The mean viral loads in LSIL and HSIL were not significantly different. Using a newly determined high viral load cut off for 14 Hr-HPV types, the sensitivity for prevalent CIN3+ remained at 100% for both assays compared with the minimal detection threshold. The specificity (corresponding to double-negative cytology at subsequent screening episodes) increased substantially (qPCR, from 91.1% to 95.7%; BSGP5+/6+-PCR/MPG, from 79.8% to 96.2%). Compared with DNA positivity alone, high Hr-HPV viral loads could reduce the amount of false positive results detected by the BSGP5+/6+-PCR/MPG and qPCR by 81.4% and 52.1%, respectively. Quantitative type-specific HPV DNA assays show high flexibility in defining thresholds that allow optimizing clinical accuracy for cervical cancer precursors.

CsrRS and Environmental pH Regulate Group B Streptococcus Adherence to Human Epithelial Cells and Extracellular Matrix

Streptococcus agalactiae (group B Streptococcus or GBS) is a common colonizer of the gastrointestinal and genital tracts and an important cause of invasive infections in newborn infants and in adults with predisposing chronic conditions or advanced age. Attachment to epithelial surfaces at mucosal sites is a critical step in the successful colonization of a human host, and regulation of this process is likely to play an important role in both commensalism and dissemination to cause invasive disease. We found that inactivation of the CsrRS (or CovRS) two-component system increased GBS adherence to epithelial cells derived from human vaginal, cervical, and respiratory epithelium, as well as increasing adherence to extracellular matrix proteins and increasing biofilm formation on polystyrene. Neutral (as opposed to acidic) pH enhanced GBS binding to vaginal epithelial cells and to fibrinogen and fibronectin, effects that were partially dependent on CsrRS. The regulatory effects of CsrRS and environmental pH on bacterial adherence correlated with their effects on the expression of multiple surface adhesins, as assessed by quantitative reverse transcription-PCR. We conclude that GBS adherence to epithelial and abiotic surfaces is regulated by the CsrRS two-component system and by environmental pH through their regulatory effects on the expression of bacterial surface adhesins. Dynamic regulation of GBS adherence enhances the organism's adaptability to survival in multiple niches in the human host.

Hox B4 as potential marker of non-differentiated cells in human cervical cancer cells

Cervical cancer (CC) is a common malignancy in women worldwide. Cervical tumorigenesis involves a multistep process in which accumulations of genetic alterations are present. Homeotic genes, such as HOX gene re-expression, have been reported in a wide variety of tumors. In order to know the role of HOX B4 gene expression in CC, in the present study, two-dimensional polyacrylamide gel electrophoresis, matrix-assisted laser desorption/ionization, and time-of-flight mass spectrometry were used for differential screening of protein expression in CC. Immunohistochemical analysis was performed on the cervical tissue microarray (TMA) to detect the Hox B4 protein. Hox B4 peptide was detected among 15 increased spots differentially observed in CC. Using TMA, Hox B4 protein was also immunodetected in the nuclei of cervical epithelial tumor cells, while in normal cervical epithelium, it was absent. Interestingly, it was possible to detect the Hox B4 protein in the precursor lesions. Hox B4 protein is present in the precursor lesions as CC cells, suggesting that Hox B4 could be a protein related to the neoplastic state (non-differentiated cells) of human cervical epithelium.

Abstract A14: Finding biomarkers in the differential expression patterns between epithelial layers in cervical dysplasia

Abstract Normal human cervical epithelium consists of a differentiating continuum of cell layers. It has been hypothesized that the most basal layer consists of stem cells and that cells migrate towards the surface as they mature and differentiate. Hence, cells in different layers of the epithelium are expected to be at different stages of maturity and express different genes. Carcinogenesis is a multi-step process frequently occurring over many years. Understanding the early genetic changes, most of which manifest as changes in this maturation process, will hopefully lead to better biomarkers for detecting precancers at highest risk to progress. Objective: By studying differences in expression between cervical epithelial layers and across various grades of cervical intraepithelial neoplasia (CIN), we hope to gain a better understanding of the molecular basis of the carcinogenic process, which will hopefully lead to novel biomarkers for the early detection of cervical cancer. Materials and Methods: CIN biopsies representing various histopathological grades of dysplasia were used for this study. These included some from a previous study that had been stored at −80°C in RNAlater, which we embedded in O.C.T., as well as newer biopsies that were fixed immediately in Tissue-Tek Xpress Molecular Fixative and embedded in paraffin (MFPE). Specimens were sectioned on to slides following standard protocols for their embedding medium. Microdissection to separate various layers of epithelium was performed manually with a needle. The epithelium was collected as 2 layers. RNA was extracted using the TRIzol reagent method for frozen specimens and the Qiagen RNeasy FFPE kit for MFPE specimens. mRNA was labeled using the Agilent Low Input Quick Amp Labeling kit and tested on the Agilent Whole Human Genome 4×44K Microarray. Data was normalized against the array median intensity and data from replicate probes was averaged. Genes that are differentially expressed in different layers of the epithelium and/or in different grades will be identified for further study and validation through immunohistochemistry. Results and Conclusions: An earlier attempt to use laser capture microdissection was hampered by slow cutting speed and processing time, leading to low-quality RNA and weak expression array signal. Using manual microdissection, one CIN II case was done from the frozen samples. Duplicate samples of the stroma left over from microdissection were analyzed concurrently with the two epithelial layers. The scatter observed in comparing the duplicate samples was used to define what constituted differential expression between the epithelial layers, resulting in the identification of 114 genes overexpressed in the superficial layer and 2 genes overexpressed in the basal layer. Subsequent attempts to repeat this procedure showed that many of our frozen samples had poor quality RNA. Hence, we sought out newer MFPE specimens. Results from some of these samples will also be presented. A comparison of the results between the multiple dysplastic grades should be revealing as we hope to identify genes whose altered expression may play a role in cancer progression. Our success with manual microdissection suggests that this analysis is feasible. Work is ongoing to expand our sample size and to better comprehend the biochemical implications of our results. Citation Information: Cancer Prev Res 2011;4(10 Suppl):A14.

Abstract C21: Wnt inhibitory factor 1 is silenced by promoter hypermethylation in cervical cancer and its restoration suppresses tumor growth

Abstract Background and Purpose: Cervical cancer is the leading cause of morbidity and mortality among women worldwide. It is also the second most common cause of cancer deaths in women. Recent studies suggest that aberrant activation of the Wingless-type (Wnt) pathway plays an important role in cervical cancer. However, the mechanisms and implications of Wnt activation in human cervical cancer are yet to be determined. We hypothesized that the Wnt inhibitory factor 1 (WIF1), a secreted Wnt antagonist, might be silenced in human cervical cancer. Therefore, we characterized the methylation status of WIF1 gene, its mRNA and protein expression in human cervical cancer samples. We also determined the effects of WIF1 treatment on tumor growth in a xenograft mouse model. Methods: To study WIF1 promoter methylation, genomic DNA was isolated from human normal and cancerous cervical samples, processed for bisulfite modification using EZ DNA methylation kit and WIF1 methylation-specific PCR was performed. WIF1 mRNA and protein expression in human cervical normal epithelium and tumor samples were assessed by real-time RTPCR and immunohistochemistry, respectively. To determine the tumor suppressive effects of WIF1, tumor xenograft studies were performed by injecting HeLa cells subcutaneously into the flanks of nude mice. Palpable tumors were treated for 7 weeks with peritumoral injection of pCI-blast-WIF1 expression vector that expresses the full WIF1 protein. Results: Our study demonstrates that WIF1 promoter hypermethylation is a frequent mechanism leading to WIF1 down-regulation as observed by decreased WIF1 mRNA and protein expression in cervical cancer compared with normal tissue. Furthermore, treatment of human cervical cancer in a xenograft mouse model by WIF1 gene transfer significantly inhibited the tumor growth by decreasing the expression of TCF-4, β-catenin, c-myc, cyclin D1 and CD44. WIF1 elicited its tumor suppressive effects by acting at multiple levels in Wnt/β-catenin pathway, in addition to modulating the expression of antiapoptotic (Bcl-2) and apoptotic proteins (p53, p21 and caspase-3), leading to a significant reduction in tumor cell proliferation and induction of massive apoptosis. Of major clinical relevance, while vector treated tumors were highly infiltrative, WIF1 treated tumors presented mainly with pushing borders, a characteristic feature of benign tumors. In addition, WIF1 treatment decreased the expression of angiogenic factors, VEGF and CD31, and significantly reduced the tumor vascular irrigation. Conclusions: Our findings for the first time demonstrate that WIF1 is silenced by promoter hypermethylation and identify WIF1 down-regulation as an important mechanism of Wnt activation in cervical cancer. Remarkably, our in vivo study emphasizes the anti-invasive, anti-angiogenic and tumor suppressive effects of WIF1 and therefore its potential therapeutic value in the treatment of cervical cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr C21.

The HPV16 transcriptome in cervical lesions of different grades

Infections with high-risk human papillomaviruses (HPV), mainly HPV type 16, can cause malignant transformation of the human cervical epithelium and cervical cancer (CxCa). Very little is known about the quantitative expression of HPV16 transcripts in cervical lesions of different grades. We have analysed the viral transcriptome in 80 HPV16 DNA positive cervical smears including lesions of different cytological grades, using nucleic acid sequence-based amplification (NASBA)–Luminex hybridisation assays quantifying spliced and unspliced HPV16 transcripts. Based on the quantitative analysis of single transcripts, highly significant changes in transcript levels were observed between different grades of cervical lesions. In conclusion, quantitative expression changes of HPV16 transcript markers may be involved in tumour progression. This study provides a basis for selection of candidate RNA markers for diagnostics of HPV16-related disease.

Role and predictive strength of transglutaminase type 2 expression in premalignant lesions of the cervix

The demonstration that type 2 transglutaminase (TG2) can incorporate polyamine into the E7 oncoprotein of human papillomavirus (HPV) type 18 has led to the hypothesis that TG2 can have a role in the host cellular response to HPV infection. The aim of this study was to investigate whether HPV-related pathology, in infected human cervical epithelium, was associated with modulation of TG2 expression. Normal controls and HPV-infected cervical biopsies were analyzed for the expression of TG2, and the findings were compared with lesion grade. The correlation between TG2 expression and p16, a marker for HPV-induced dysplasia, and the retinoblastoma protein (Rb), a target of the E7 protein of HPV, was also investigated. Results obtained showed that TG2 was absent in normal squamous mucosa, whereas it was present in 100% CIN I lesions. Low-grade lesions showed significantly higher TG2 expression than high-grade lesions (P<0.0001). In 94% of CIN I more than 50% of the cells were positive for TG2, with a strong staining intensity (+3), whereas a decreased staining intensity and a low number of positive cells were found in CIN II/III. In CIN I cases, both nuclear and cytoplasmic staining were found in cells exhibiting classical morphological features of HPV infection. In addition, during progression from low-grade squamous intraepithelial lesions to severe dysplasia, TG2 expression was inversely correlated with p16 (Pearson: −0.930), whereas a positive correlation was observed between the expression of TG2 and pRb (Pearson: 0.997). TG2 is expressed in HPV infection as an early phenomenon, not restricted to high-risk genotypes. TG2 upregulation is probably part of host cell reaction against HPV-induced tissue modification. It may act as a cellular antioxidant defense factor, playing an important role in counteracting oxidative damage in neoplastic disease.