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1.
Clin Dev Immunol ; 2012: 785825, 2012.
Article in English | MEDLINE | ID: mdl-22013487

ABSTRACT

The human papillomavirus (HPV) directly infects cervical keratinocytes and interferes with TLR signalling. To shed light on the effect of HPV on upstream receptors, we evaluated TLRs 1-9 gene expression in HPV-negative normal and HPV-positive pre-malignant and malignant ex vivo cervical tissue. Quantitative real-time polymerase chain reaction was performed separately for epithelial and stromal tissue compartments. Differences in gene expression were analyzed by the Jonckheere-Terpstra trend test or the Student's t-test for pairwise comparison. Laser capture microdissection revealed an increase in TLR3 and a decrease in TLR1 mRNA levels in dysplastic and carcinoma epithelium, respectively. In the stroma, a trend of increasing TLR 1, 2, 5, 6, and 9 mRNA levels with disease severity was found. These findings implicate the involvement of TLR3 and TLR1 in early and late cervical carcinogenesis, respectively, suggesting that stromal upregulation of TLRs may play a role in cervical disease progression.


Subject(s)
Carcinoma/immunology , Human papillomavirus 16/immunology , Papillomavirus Infections/immunology , Precancerous Conditions/immunology , Toll-Like Receptors/metabolism , Uterine Cervical Neoplasms/immunology , Carcinoma/pathology , Carcinoma/physiopathology , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/immunology , Cell Transformation, Viral/immunology , Cells, Cultured , Cervix Uteri/pathology , Disease Progression , Epithelial Cells/immunology , Epithelial Cells/metabolism , Epithelial Cells/pathology , Female , Gene Expression Profiling , Gene Expression Regulation, Neoplastic/immunology , Human papillomavirus 16/pathogenicity , Humans , Hyperplasia , Immunity, Innate , Papillomavirus Infections/pathology , Papillomavirus Infections/physiopathology , Precancerous Conditions/pathology , Precancerous Conditions/physiopathology , Stromal Cells/immunology , Stromal Cells/metabolism , Stromal Cells/pathology , Toll-Like Receptors/genetics , Toll-Like Receptors/immunology , Tumor Microenvironment , Uterine Cervical Neoplasms/pathology , Uterine Cervical Neoplasms/physiopathology
2.
Lab Invest ; 90(10): 1482-91, 2010 Oct.
Article in English | MEDLINE | ID: mdl-20479716

ABSTRACT

Interferons (IFNs) are expressed by many cell types and play a pivotal role in the generation of immune responses against viral infections. IFN-κ, a novel type I IFN, displays a tight tropism for keratinocytes and specific lymphoid populations and exhibits functional similarities with other type I IFNs. The human papillomavirus (HPV), the etiological agent for cervical cancer, infects keratinocytes of the uterine cervix and has been shown to directly inhibit the IFN pathway. We evaluated IFN-κ, -ß, and -γ gene expression in HPV-negative normal and HPV-positive pre-malignant and malignant ex vivo cervical tissue covering the entire spectrum of cervical disease. Quantitative real-time polymerase chain reaction and methods previously optimized for detecting low-expressing genes in cervical tissue were used. In contrast to IFN-ß and -γ, IFN-κ mRNA prevalence and levels were unexpectedly higher in diseased compared with normal whole cervical tissue with highest levels observed in invasive carcinoma tissue. Strikingly, laser capture microdissection revealed an absence of IFN-κ mRNA in diseased epithelium, whereas stromal IFN-κ was found exclusively in diseased tissue. IFN-γ and IFN-ß were likewise found to be upregulated in diseased cervical stroma. Immunofluorescence supports the involvement of monocytes and dendritic cells in the stromal induction of IFNs in diseased tissue. Further, using three-dimensional raft cultures in which the viral life cycle can be mimicked, human keratinocytes transfected with full-length HPV16 displayed a significant decrease in IFN-κ mRNA compared with non-transfected human keratinocytes. Altogether, these findings show that IFN-κ is down-regulated in cervical keratinocytes harboring HPV, which may be a contributing factor in the progression of a cervical lesion.


Subject(s)
Cervix Uteri/metabolism , Interferon Type I/metabolism , Keratinocytes/metabolism , Papillomaviridae , Papillomavirus Infections/metabolism , Uterine Cervical Neoplasms/metabolism , Cervix Uteri/pathology , Cervix Uteri/virology , Down-Regulation , Female , Humans , Interferon-beta/metabolism , Interferon-gamma/metabolism , Keratinocytes/pathology , Keratinocytes/virology , Papillomavirus Infections/immunology , Papillomavirus Infections/pathology , Papillomavirus Infections/virology , RNA, Messenger/analysis , Reverse Transcriptase Polymerase Chain Reaction , Uterine Cervical Neoplasms/immunology , Uterine Cervical Neoplasms/pathology , Uterine Cervical Neoplasms/virology
3.
Oncol Rep ; 20(4): 897-903, 2008 Oct.
Article in English | MEDLINE | ID: mdl-18813833

ABSTRACT

Patients with advanced prostate cancer frequently have a poor prognosis as a result of metastasis. The serum prostate-specific antigen (PSA) test is widely used for the diagnosis of prostate cancer. The enzymatic activity of PSA may be involved in the invasion of prostate cancer. We set out to determine the prevalent form of PSA in human prostate adenocarcinoma samples by ELISA and Western blot analysis and its enzymatic activity using a synthetic substrate S-2586 and fibronectin. Our results show that in serum from prostate cancer patients and in tumour homogenates, the prevalent form was PSA bound to alpha1-antichymotrypsin. All homogenates showed enzymatic activity towards a synthetic PSA substrate, whereas only five samples showed activity at 28 kDa towards fibronectin as determined by enzymography, which is most likely due to active PSA. Human prostate cancer LNCaP cells produced largely inactive PSA. In comparison, 22Rv1 cells produced 29-fold less PSA, but with high specific activity. Similarly, our results from the human prostate cancer tissue samples also show that free PSA appears to exist in diverse forms of very different specific activity. Since PSA, as a serine protease, may be involved in the invasion of prostate cancer, our results suggest that prostate cancers have potentially diverse invasive capacity due to differences in specific enzymatic activity of PSA.


Subject(s)
Fibronectins/metabolism , Prostate-Specific Antigen/analysis , Prostatic Neoplasms/chemistry , Adult , Aged , Aged, 80 and over , Cell Line, Tumor , Enzyme-Linked Immunosorbent Assay , Humans , Male , Middle Aged , Prostate-Specific Antigen/physiology
4.
Anal Biochem ; 381(1): 59-66, 2008 Oct 01.
Article in English | MEDLINE | ID: mdl-18601893

ABSTRACT

Optimal sample handling techniques for tissue preparation and storage, RNA extraction and quantification, and target gene detection are crucial for reliable gene expression analysis. Methods for measuring low-expressing genes, such as interferons, in human cervical samples are not described in the scientific literature. To detect interferon mRNA in human cervical samples we obtained normal and dysplastic frozen and formalin-fixed cervical biopsies from colposcopy. Histopathological diagnosis was performed by one pathologist. Cervical keratinocytes were isolated using laser capture microdissection. Immortalized keratinocytes transduced with or devoid of an HPV oncogene were used for initial method development. RNA from samples was extracted and integrity tested to compare tissue storage and extraction methods. The expression of five housekeeping genes was analyzed in cell lines and patient tissue to permit normalization between samples using quantitative real-time polymerase chain reaction. The usefulness of cDNA amplification was assessed for the detection of low-expressing interferon kappa in cervical tissue. Here we report optimal tissue storage conditions, RNA extraction, sample normalization, and transcript amplification, as well as the sensitivity of quantitative real-time polymerase chain reaction and laser capture microdissection, for interferon kappa detection in cervical tissue. Without these optimized techniques, interferon kappa detection would be unattainable in cervical samples.


Subject(s)
Cervix Uteri/metabolism , Epithelium/metabolism , Gene Expression Profiling , Gene Expression Regulation , Interferon Type I/genetics , Lasers , Microdissection/methods , Cell Line , DNA, Complementary/genetics , Female , Humans , Interferon Type I/metabolism , RNA/isolation & purification , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction
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