HPV16 E6 Oncoprotein Inhibits Apoptosis Induced during Serum–Calcium Differentiation of Foreskin Human Keratinocytes

Alfandari, Jacklin; Magal, Sharon Shnitman; Jackman, Anna; Schlegel, Richard; Gonen, Pinhas; Sherman, Levana

doi:10.1006/viro.1999.9675

Cited by 38 publications

(34 citation statements)

References 52 publications

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“…TM -293 cells were infected with the recombinant retrovirus HPV16 E6, kindly provided by L. Sherman (46). The cells were grown to 75% confluence and infected at a multiplicity of infection of 2-5.…”

Section: Retroviral Infection Of T-rex Tm -293 Cells-for Constitutivementioning

confidence: 99%

Cell Cycle Regulation and p53 Activation by Protein Phosphatase 2Cα

Ofek¹,

Ben-Meir²,

Kariv-Inbal³

et al. 2003

Journal of Biological Chemistry

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Protein phosphatase 2C (PP2C) dephosphorylates a broad range of substrates, regulating stress response and growth-related pathways in both prokaryotes and eukaryotes. We now demonstrate that PP2C␣, a major mammalian isoform, inhibits cell growth and activates the p53 pathway. In 293 cell clones, in which PP2C␣ expression is regulated by a tetracycline-inducible promoter, PP2C␣ overexpression led to G 2 /M cell cycle arrest and apoptosis. Furthermore, PP2C␣ induced the expression of endogenous p53 and the p53-responsive gene p21. Activation of the p53 pathway by PP2C␣ took place both in cells harboring endogenous p53, as well as in p53-null cells transfected with exogenous p53. Induction of PP2C␣ resulted in an increase in the overall levels of p53 protein as well as an augmentation of p53 transcription activity. The dephosphorylation activity of PP2C␣ is essential to the described phenomena, as none of these effects was detected when an enzymatically inactive PP2C␣ mutant was overexpressed. p53 plays an important role in PP2C␣-directed cell cycle arrest and apoptosis because perturbation of p53 expression in human 293 cells by human papillomavirus E6 led to a significant increase in cell survival. The role of PP2C␣ in p53 activation is discussed.

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Section: Retroviral Infection Of T-rex Tm -293 Cells-for Constitutivementioning

confidence: 99%

Cell Cycle Regulation and p53 Activation by Protein Phosphatase 2Cα

Ofek¹,

Ben-Meir²,

Kariv-Inbal³

et al. 2003

Journal of Biological Chemistry

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“…The opposing effects of BPV-1 E6 and E7 may be implicated in the life cycle, replication, and maintenance of BPV-1. Recent studies have shown that the high-risk HPV-16 E6 and E7 oncogenes either increase or decrease apoptosis depending on the cell lines and apoptotic stimuli employed (Puthenveettil et al, 1996;Jones et al, 1997;Iglesias et al, 1998;Magal et al, 1998;Rapp and Chen, 1998;Stoppler et al, 1998;Alfandari et al, 1999;Liu et al, 2000b;Mannhardt et al, 2000;Finzer et al, 2002). Interestingly, while normal human diploid fibroblasts that express HPV-16 E7 are highly predisposed to undergo apoptosis in response to growth factor deprivation, they have decreased propensity to undergo apoptosis in response to TNF-and Fas treatment (Jones et al, 1997;Thompson et al, 2001).…”

Section: Discussionmentioning

confidence: 99%

Opposing effects of bovine papillomavirus type 1 E6 and E7 genes on Fas-mediated apoptosis

Liu

Gao

et al. 2005

Oncogene

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Programmed cell death (PCD), best exemplified by apoptosis, is a genetically programmed process of cellular destruction that is indispensable for normal development and homeostasis of multicellular organisms. Tumor necrosis factor a (TNF) and related cytokines are employed by host defenses to eliminate virally infected cells through induction of apoptosis. Many viruses have evolved specific gene products to modulate this process. We have recently shown that the bovine papillomavirus type 1 (BPV-1) E6 and E7 genes independently sensitize mouse cells to TNF-induced apoptosis. In this report, we investigated the effect of E6 and E7 expression on Fasmediated apoptosis. In contrast to TNF-mediated apoptosis, E6 and E7 demonstrated opposite effects: while E7 potentiated apoptosis triggered by an agonistic Fas antibody, E6 attenuated the effect. The mitochondrial pathway leading to the activation of caspases appears to be involved in Fas-mediated apoptosis in C127 cells. To further explore the mechanisms by which E6 and E7 modulate Fas-mediated apoptosis, we examined the surface expression of Fas in cells expressing E6 and E7. Significantly, levels of surface Fas expression correlated with the opposing effects of E6 and E7 on Fas-mediated apoptosis. Specifically, while E7 increased the surface expression of Fas, E6 reduced surface Fas expression. Mutational analysis demonstrated a correlation of E6's ability to downregulate surface Fas expression and apoptosis. Since the tumor suppressor p53 can be targeted for degradation by human papillomavirus and has been shown to induce apoptosis by upregulating surface Fas expression, we investigated the role of p53 in BPV-1 E6 and E7 modulation of Fas-mediated apoptosis. Our results demonstrated that the modulatory effects by E6 and E7 could occur in the absence of p53. Interestingly, the reduced Fas protein level on the cell surface is not accompanied by a decrease in total Fas levels in E6-expressing cells. Instead, considerably more Fas protein is found in the cytoplasm of cells expressing E6. These results highlight a novel activity of E6 and E7 that may be involved in viral pathogenesis.

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“…In vitro, keratinocyte differentiation has been successfully induced by several factors, such as osmotic stress, growth factors, high calcium concentrations, or serum supplementation [16,22,23,27,46]. To investigate whether the keratinocytes isolated from piglets retain the ability to differentiate in vitro after the isolation procedure, normal keratinocyte growth medium (EKGM) was replaced with a differentiation medium.…”

Section: Effect Of Calcium and Serum On Cell Differentiation Of Keratmentioning

confidence: 99%

“…By adjusting the plane of focus of the microscope, we were able to classify enlarged cells with a flattened morphology and a cornified envelope as differentiated cells. To induce differentiation, the medium was supplemented either with serum (SeDM) [23] or calcium (CaDM) [16]. Keratinocytes growing as a monolayer in low calcium (60 M) can be induced to terminally differentiate by adding calcium to the culture medium (> 1 mM).…”

Section: Porcine Keratinocytes Retain Their Ability To Differentiatementioning

confidence: 99%

“…In these systems overgrowth was often observed between keratinocytes and neurons and the simultaneous use of different media for the individual cell types is hampered as they provide only one coherent medium room. However, knowing that keratinocytes and neurons require different media compositions and that serum and calcium are important differentiation factors for keratinocytes [2,16,22,23,27,56], the establishment of a compartmented chamber provides a tool for co-culture studies.…”

Section: Compartmented Co-culture Of Keratinocytes and Drg Neuronsmentioning

confidence: 99%

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Isolation and cultivation of primary keratinocytes from piglet skin for compartmentalized co-culture with dorsal root ganglion neurons

Ponce

Heintz

Schäfer

et al. 2017

JCB

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Abstract. Keratinocytes are the main cell population in the epidermis, where they coexist with a variety of other cell types. Their successful isolation and cultivation have afforded opportunities to study epidermal functions. Human keratinocytes have been studied most extensively, but their source is limited by the skin supply. In a previous work, we developed an in vitro co-culture model of porcine keratinocytes with porcine sensory neurites to investigate functional interaction. However, a detailed description of the isolation of porcine keratinocytes and their culture conditions has not been given in detail. Here, we present the isolation procedure and a characterization of keratinocytes derived from new-born piglets, using simple assays based on conventional and fluorescence microscopy. Media, coating substrates and plating densities were tested with respect to cell viability, proliferation and morphology. Growing keratinocytes in EpiLife keratinocyte growth medium (EKGM) on human collagen type I substrate was best to support proliferation. The minimum plated density was 500 viable cells/cm 2 for primary and 1000 viable cells/cm 2 for subcultured cells. Population doubling (PD) and generation time (tg) depended on the plating densities. Keratinocytes seeded at a density of 5000 viable cells/cm 2 had a PD of 4.36 ± 0.60 per passage and tg of 1.69 ± 0.24 days. Our results show that the optimal isolation and culture conditions for keratinocytes from piglets differ from those for keratinocytes from adult pigs and humans. Thus, the information obtained from the characterization allowed the performance and optimization of a co-culture and contributes to further investigations in epidermal homeostasis and cutaneous sensation.

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HPV16 E6 Oncoprotein Inhibits Apoptosis Induced during Serum–Calcium Differentiation of Foreskin Human Keratinocytes

Cited by 38 publications

References 52 publications

Cell Cycle Regulation and p53 Activation by Protein Phosphatase 2Cα

Cell Cycle Regulation and p53 Activation by Protein Phosphatase 2Cα

Opposing effects of bovine papillomavirus type 1 E6 and E7 genes on Fas-mediated apoptosis

Isolation and cultivation of primary keratinocytes from piglet skin for compartmentalized co-culture with dorsal root ganglion neurons

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