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Physical labeling of papillomavirus-infected, immortal, and cancerous cervical epithelial cells reveal surface changes at immortal stage.

ABSTRACT: A significant change of surface features of malignant cervical epithelial cells compared to normal cells has been previously reported. Here, we are studying the question at which progressive stage leading to cervical cancer the surface alteration happens. A non-traditional method to identify malignant cervical epithelial cells in vitro, which is based on physical (in contrast to specific biochemical) labelling of cells with fluorescent silica micron-size beads, is used here to examine cells at progressive stages leading to cervical cancer which include normal epithelial cells, cells infected with human papillomavirus type-16 (HPV-16), cells immortalized by HPV-16, and carcinoma cells. The study shows a statistically significant (at p < 0.01) difference between both immortal and cancer cells and a group consisting of normal and infected. There is no significant difference between normal and infected cells. Immortal cells demonstrate the signal which is closer to cancer cells than to either normal or infected cells. This implies that the cell surface, surface cellular brush changes substantially when cells become immortal. Physical labeling of the cell surface represents a substantial departure from the traditional biochemical labeling methods. The results presented show the potential significance of physical properties of the cell surface for development of clinical methods for early detection of cervical cancer, even at the stage of immortalized, premalignant cells.

SUBMITTER: Swaminathan Iyer K

PROVIDER: S-EPMC3746186 | biostudies-literature | 2012 Jun

REPOSITORIES: biostudies-literature

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Physical labeling of papillomavirus-infected, immortal, and cancerous cervical epithelial cells reveal surface changes at immortal stage.

Swaminathan Iyer K K Gaikwad R M RM Woodworth C D CD Volkov D O DO Sokolov Igor I

Cell biochemistry and biophysics 20120601 2

A significant change of surface features of malignant cervical epithelial cells compared to normal cells has been previously reported. Here, we are studying the question at which progressive stage leading to cervical cancer the surface alteration happens. A non-traditional method to identify malignant cervical epithelial cells in vitro, which is based on physical (in contrast to specific biochemical) labelling of cells with fluorescent silica micron-size beads, is used here to examine cells at p ...[more]

PMID: 22351422

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Project description:Our previous study implied a correlation of inhibitors of differentiation-1 (Id-1) to cervical cancer development. However, how Id-1 contributes to cervical carcinogenesis is unknown. In the present study, we investigated the role of Id-1 in transforming cervical cells with an in vitro transformation model. The human papillomavirus (HPV) immortalized cervical epithelial cells (H8) were successfully transformed by exposure to the carcinogen N-nitrosopyrrolidine (NPYR). The results showed that both Id-1 RNA and protein expression were significantly increased in transformed H8 cells, suggesting a possible role of Id-1 in cervical cell transformation. Ectopic expression of Id-1 in H8 cells potentiated NPYR-induced cell transformation. In contrast, silencing of Id-1 suppressed NPYR-induced H8 cell transformation. A cDNA microarray assay was performed, which identified suggested potential cell signaling pathways for NPYR-induced H8 cell transformation. The results suggest that Id-1 plays an oncogenic role in the cervix, which sheds light on cervical cancer development and implies potential target for cervical cancer prevention and therapy. Id-1-shRNA H8 and the control pGIPZ-H8 cells, with or without NPYR treatment, were used for cDNA microarray assay. Total RNA was extracted by using TRIZOL Reagent (Life Technologies, Carlsbad, CA, US) following the manufacturer's instructions. Total RNA was further purified by RNeasy micro kit (QIAGEN, GmBH, Germany) and RNase-free DNase Set (QIAGEN, GmBH, Germany). The RNAs were amplified, labeled and purified by using GeneChip 3' IVT Express Kit (Affymetrix, Santa Clara, CA, US) following the manufacturer's instructions. Array hybridization and wash were performed using GeneChipÂ® Hybridization, Wash and Stain Kit (Affymetrix, Santa Clara, CA, US) in a Hybridization Oven 645 (Affymetrix) and Fluidics Station 450 (Affymetrix) following the manufacturer's instructions. The slides were scanned with GeneChipÂ® Scanner 3000 (Affymetrix) using the Command Console Software 3.1 (Affymetrix) with default settings. Raw data were normalized by using the MAS 5.0 algorithm of GeneSpring Software 11.0 (Agilent Technologies, Santa Clara, CA, US).

Project description:IntroductionRelatively little is known about cellular subpopulations in the mature nucleus pulposus (NP). Detailed understanding of the ontogenetic, cellular and molecular characteristics of functional intervertebral disc (IVD) cell populations is pivotal to the successful development of cell replacement therapies and IVD regeneration. In this study, we aimed to investigate whether phenotypically distinct clonal cell lines representing different subpopulations in the human NP could be generated using immortalization strategies.MethodsNondegenerate healthy disc material (age range, 8 to 15 years) was obtained as surplus surgical material. Early passage NP monolayer cell cultures were initially characterized using a recently established NP marker set. NP cells were immortalized by simian virus 40 large T antigen (SV40LTag) and human telomerase reverse transcriptase expression. Immortalized cells were clonally expanded and characterized based on collagen type I, collagen type II, ?1 (COL2A1), and SRY-box 9 (SOX9) protein expression profiles, as well as on expression of a subset of established in vivo NP cell lineage markers.ResultsA total of 54 immortal clones were generated. Profiling of a set of novel NP markers (CD24, CA12, PAX1, PTN, FOXF1 and KRT19 mRNA) in a representative set of subclones substantiated successful immortalization of multiple cellular subpopulations from primary isolates and confirmed their NP origin and/or phenotype. We were able to identify two predominant clonal NP subtypes based on their morphological characteristics and their ability to induce SOX9 and COL2A1 under conventional differentiation conditions. In addition, cluster of differentiation 24 (CD24)-negative NP responder clones formed spheroid structures in various culture systems, suggesting the preservation of a more immature phenotype compared to CD24-positive nonresponder clones.ConclusionsHere we report the generation of clonal NP cell lines from nondegenerate human IVD tissue and present a detailed characterization of NP cellular subpopulations. Differential cell surface marker expression and divergent responses to differentiation conditions suggest that the NP subtypes may correspond to distinct maturation stages and represent distinct NP cell subpopulations. Hence, we provide evidence that the immortalization strategy that we applied is capable of detecting cell heterogeneity in the NP. Our cell lines yield novel insights into NP biology and provide promising new tools for studies of IVD development, cell function and disease.

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Physical labeling of papillomavirus-infected, immortal, and cancerous cervical epithelial cells reveal surface changes at immortal stage.

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Physical labeling of papillomavirus-infected, immortal, and cancerous cervical epithelial cells reveal surface changes at immortal stage.

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