Normal Growth and Differentiation in a Spontaneously Immortalized Near-Diploid Human Keratinocyte Cell Line, NIKS

Allen-Hoffmann, B. Lynn; Schlosser, Sandy J.; Ivarie, C. A. R.; Sattler, Carol A.; Meisner, Lorraine F.; O’Connor, Seán

doi:10.1046/j.1523-1747.2000.00869.x

Cited by 218 publications

(212 citation statements)

References 51 publications

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“…LCM analysis revealed the focal nature of IFN-k expression (data not shown) along with the importance of cervical stromal cells in IFN-k induction. The focal and low-expressing nature of IFN-k, 28 along with the potential differences in the occurrence of stromal cells among specimens, may explain why IFN-k was not detected in some HPV-positive cervical specimens. However, when IFN-k was detected, IFN-g, IFN-b, and IL-10 were always present (data not shown).…”

Section: Discussionmentioning

confidence: 99%

IFN-κ, a novel type I IFN, is undetectable in HPV-positive human cervical keratinocytes

DeCarlo

Severini

Edler

et al. 2010

Laboratory Investigation

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Interferons (IFNs) are expressed by many cell types and play a pivotal role in the generation of immune responses against viral infections. IFN-k, 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-k, -b, and -g 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-b and -g, IFN-k 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-k mRNA in diseased epithelium, whereas stromal IFN-k was found exclusively in diseased tissue. IFN-g and IFN-b 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-k mRNA compared with non-transfected human keratinocytes. Altogether, these findings show that IFN-k is down-regulated in cervical keratinocytes harboring HPV, which may be a contributing factor in the progression of a cervical lesion. Interferons (IFNs) have a critical role in the generation of an innate immune response against invading pathogens and, as a consequence, are an approved therapy for many diseases 1,2 including human papillomavirus (HPV)-associated diseases. 3,4 On infection, IFNs are synthesized and secreted to evoke anti-viral, 5 anti-tumor, 6 and immunoregulatory (reviewed in Stetson and Medzhitov 7 ) activity conferring protection to neighboring cells. Two main classes of IFNs exist. The type I IFNs, consisting of IFN-a, -b, -k, -d, -e, -o, and -t, share a common receptor and downstream signaling pathways (reviewed in Pestka et al. 8 ) and are chiefly involved in generating anti-viral activity in response to pathogens. The sole member of the type II IFN subclass, IFN-g, uses a discrete type II IFN receptor, 9 distinct signaling pathways and is primarily involved in the regulation of immune and inflammatory responses. IFN-k, a newly identified type I IFN, has been shown to signal through the type I IFN pathway 10 and displays a tight tropism for keratinocytes, monocytes (MCs), and dendritic cells (DCs). 10 IFN-k has been shown to display similar anti-viral activity to other type I IFNs. 11 However, it is distinct from other type I IFNs, as it...

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Section: Discussionmentioning

confidence: 99%

IFN-κ, a novel type I IFN, is undetectable in HPV-positive human cervical keratinocytes

DeCarlo

Severini

Edler

et al. 2010

Laboratory Investigation

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“…37 MEFs were maintained in high glucose DMEM (http://www.lifetechnologies.com/order/catalog/product/11965118) containing 10% FBS, 2 mM L-glutamine (http://cellgro.com/ products/cell-culture-reagents/amino-acids-vitamins/L-glutamineliquid-2.html), 100 mM MEM non-essential amino acids (https:// www.lifetechnologies.com/order/catalog/product/11140050), 0.055 mM b-mercaptoethanol (Midwest Scientific-0482-1) and 10 mg/ml gentamycin (http://www.lifetechnologies.com/order/catalog/product/15710064). Spontaneously immortalized near-diploid human keratinocyte cell line (NIKS) 45 were maintained in Fmedia, which is 3 parts Dulbecco's modified Eagle's medium to 1 part Ham's F12 media (https://www.lifetechnologies.com/order/ catalog/product/11765070) supplemented with the following components: 5% fetal bovine serum, 24 ug/ml adenine, 8.4 ng/ml cholera toxin (http://www.labome.com/product/EMD-Millipore/ 227036-1MG.html), 10 ng/ml epidermal growth factor (http://www.sigmaaldrich.com/catalog/product/sigma/ e4127), 2.4 mg/ml hydrocortisone (http://www.sigmaaldrich.com/catalog/ product/sigma/h0888), 5 mg/ml insulin, 1% penicillin-streptomycin (https:// www.lifetechnologies.com/order/catalog/ product/15140122), and 0.2% fungizone (http://www.omegascientific.com/ shop/fungizone-amphotericin-b/). CCHMC-SCC1 head and neck cancer cells were cultured from a tumor obtained with IRB approval at the time of surgical resection.…”

Section: Methodsmentioning

confidence: 99%

“…To determine the regulation of DEK during mitosis in non-transformed cells, which harbor relatively low DEK expression levels, we utilized a near-diploid immortalized keratinocyte cell line (NIKS) 45 and murine embryonic fibroblasts (MEFs). We found that endogenous DEK is largely absent from mitotic chromosomes from prophase through anaphase, but reassociates in telophase.…”

Section: Introductionmentioning

confidence: 99%

DEK over-expression promotes mitotic defects and micronucleus formation

Matrka

Hennigan

Kappes³

et al. 2015

Cell Cycle

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The DEK gene encodes a nuclear protein that binds chromatin and is involved in various fundamental nuclear processes including transcription, RNA splicing, DNA replication and DNA repair. Several cancer types characteristically over-express DEK at the earliest stages of transformation. In order to explore relevant mechanisms whereby DEK supports oncogenicity, we utilized cancer databases to identify gene transcripts whose expression patterns are tightly correlated with that of DEK. We identified an enrichment of genes involved in mitosis and thus investigated the regulation and possible function of DEK in cell division. Immunofluorescence analyses revealed that DEK dissociates from DNA in early prophase and re-associates with DNA during telophase in human keratinocytes. Mitotic cell populations displayed a sharp reduction in DEK protein levels compared to the corresponding interphase population, suggesting DEK may be degraded or otherwise removed from the cell prior to mitosis. Interestingly, DEK overexpression stimulated its own aberrant association with chromatin throughout mitosis. Furthermore, DEK colocalized with anaphase bridges, chromosome fragments, and micronuclei, suggesting a specific association with mitotically defective chromosomes. We found that DEK over-expression in both non-transformed and transformed cells is sufficient to stimulate micronucleus formation. These data support a model wherein normal chromosomal clearance of DEK is required for maintenance of high fidelity cell division and chromosomal integrity. Therefore, the overexpression of DEK and its incomplete removal from mitotic chromosomes promotes genomic instability through the generation of genetically abnormal daughter cells. Consequently, DEK over-expression may be involved in the initial steps of developing oncogenic mutations in cells leading to cancer initiation

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“…Cell lines-Near-diploid immortalized foreskin keratinocytes (NIKS) [22] in the presence or absence of the human papillomavirus type 16 (HPV16) E6 oncogene [23] were grown in culture to 70% confluency. These cells were harvested and used to mimic normal and diseased cervical tissue in initial experiments to assess HKG suitability and cDNA amplification uniformity.…”

Section: Sample Preparationmentioning

confidence: 99%

Gene expression analysis of interferon κ in laser capture microdissected cervical epithelium

DeCarlo

Escott

Werner

et al. 2008

Analytical Biochemistry

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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 κ 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 κ detection in cervical tissue. Without these optimized techniques, interferon κ detection would be unattainable in cervical samples. KeywordsCervical keratinocytes; Frozen and formalin-fixed cervical tissue; Housekeeping genes; Interferons; Interferon κ; Laser capture microdissection; Quantitative real-time polymerase chain reaction Quantitative real-time polymerase chain reaction (qRT-PCR) 1 for the molecular analysis of disease is a powerful and widely used tool. Extraction of high-quality RNA for use in gene expression analysis techniques such as reverse transcription (RT), qRT-PCR, and cDNA microarrays is of great importance. Although obtaining high-quality RNA from cell lines is relatively straightforward, the complexity and heterogeneity of human tissue pose considerable challenges for linking gene expression patterns with disease state. Nonetheless,

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Normal Growth and Differentiation in a Spontaneously Immortalized Near-Diploid Human Keratinocyte Cell Line, NIKS

Cited by 218 publications

References 51 publications

IFN-κ, a novel type I IFN, is undetectable in HPV-positive human cervical keratinocytes

IFN-κ, a novel type I IFN, is undetectable in HPV-positive human cervical keratinocytes

DEK over-expression promotes mitotic defects and micronucleus formation

Gene expression analysis of interferon κ in laser capture microdissected cervical epithelium

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