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Shift from apoptotic to necrotic cell death during human papillomavirus-induced transformation of keratinocytes.

ABSTRACT: Oncogenic transformation is a complex, multistep process, which goes through several stages before complete malignant transformation occurs. To identify early processes in carcinogenesis, we used an in vitro model, based on the initiating event in cervical cancer, papillomavirus transformation of keratinocytes. We compared gene expression in primary keratinocytes (K) and papillomavirus-transformed keratinocytes from early (E) and late (L) passages and from benzo[a]pyrene-treated L cells (BP). The transformed cells exhibit similar transcriptional changes to clinical cervical carcinoma. The number of transcripts expressed progressively decreased during the evolution from K to BP cells. Bioinformatic analysis, validated by detailed biochemical analysis, revealed substantial contraction of both pro- and antiapoptotic networks during transformation. Nonetheless, L and BP cells were not resistant to apoptotic stimuli. At doses of cisplatin that led to 30-60% apoptosis of K and E cells, transformed L and BP cells underwent 80% necrotic cell death, which became the default response to genotoxic stress. Moreover, appreciable necrotic fractions were observed in the cervical carcinoma cell line, HeLa, in response to comparable doses of cisplatin. The shrinkage of biochemical networks, including the apoptotic network, may allow a cancer cell to economize on energy usage to facilitate enhanced proliferation but leaves it vulnerable to stress. This study supports the hypothesis that the process of cancer transformation may be accompanied by a shift from apoptosis to necrosis.

SUBMITTER: Kravchenko-Balasha N

PROVIDER: S-EPMC2670175 | biostudies-literature | 2009 Apr

REPOSITORIES: biostudies-literature

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Shift from apoptotic to necrotic cell death during human papillomavirus-induced transformation of keratinocytes.

Kravchenko-Balasha Nataly N Mizrachy-Schwartz Sarit S Klein Shoshana S Levitzki Alexander A

The Journal of biological chemistry 20090216 17

Oncogenic transformation is a complex, multistep process, which goes through several stages before complete malignant transformation occurs. To identify early processes in carcinogenesis, we used an in vitro model, based on the initiating event in cervical cancer, papillomavirus transformation of keratinocytes. We compared gene expression in primary keratinocytes (K) and papillomavirus-transformed keratinocytes from early (E) and late (L) passages and from benzo[a]pyrene-treated L cells (BP). Th ...[more]

PMID: 19221178

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Project description:Human papillomavirus (HPV) infection of the genital tract is common; however, only about 10 to 15% of infections persist, and approximately 10 to 15% of these persistent infections result in cancer. Basal epidermal stem cells are the presumed target cells for HPV infection, providing a reservoir of latently infected cells that persist over time and initiate lesions. However, it is not known whether stem cell density has any influence on transformation of human keratinocytes by HPV. We explored the relationship between stem cell properties of normal human keratinocytes and their susceptibility to transformation by HPV16 DNA. Normal human keratinocyte isolates (NHKc) derived from different donors were cultured in three-dimensional anchorage-free suspension to assess their spheroid-forming ability. NHKc spheroids were then plated back into plastic monolayer culture and transfected with full-length HPV16 DNA, which we have previously shown to integrate into the host cell genome upon transfection. Spheroid-derived NHKc (SD-NHKc) and fluorescence-activated cell sorting-purified populations of basal stem-like keratinocytes, expressing low levels of epidermal growth factor receptor and high levels of integrin alpha 6 (EGFRlo/ITG?6hi), responded to transfection with HPV16 DNA with more vigorous proliferation, greater immortalization efficiency, and faster progression to differentiation resistance than autologous mass-cultured cells. Conversely, cells committed to terminal differentiation (EGFRhi/ITG?6lo) grew slowly after transfection with HPV16 and failed to generate immortalized or DR clones. HPV16 DNA induced stem cell properties in mass-cultured NHKc. We conclude that HPV16 preferentially immortalizes basal keratinocytes with stem cell properties and that these cells readily achieve a differentiation-resistant phenotype upon immortalization by HPV16.IMPORTANCE This paper explores the relationship between the stem cell properties of normal human epidermal cells in culture and these cells' susceptibility to transformation by HPV16 DNA, the HPV type present in about 50% of cervical cancers. We report variable susceptibilities to HPV16-mediated transformation among different keratinocyte isolates derived from neonatal foreskin. Our findings provide strong experimental evidence that HPV16 preferentially transforms basal keratinocytes with stem cell properties. Insights gained from these studies increase our understanding of the host cell-specific factors influencing individual susceptibility to HPV-driven transformation and the contributing factors leading to preneoplastic and neoplastic progression of HPV-positive lesions.

Project description:Human papillomaviruses induce a host of anogenital cancers, as well as oropharyngeal cancer (HPV+OPC); human papillomavirus 16 (HPV16) is causative in around 90% of HPV+OPC cases. Using telomerase reverse transcriptase (TERT) immortalized foreskin keratinocytes (N/Tert-1), we have identified significant host gene reprogramming by HPV16 (N/Tert-1+HPV16) and demonstrated that N/Tert-1+HPV16 support late stages of the viral life cycle. Expression of the cellular dNTPase and homologous recombination factor sterile alpha motif and histidine-aspartic domain HD-containing protein 1 (SAMHD1) is transcriptionally regulated by HPV16 in N/Tert-1. CRISPR/Cas9 removal of SAMHD1 from N/Tert-1 and N/Tert-1+HPV16 demonstrates that SAMHD1 controls cell proliferation of N/Tert-1 only in the presence of HPV16; the deletion of SAMHD1 promotes hyperproliferation of N/Tert-1+HPV16 cells in organotypic raft cultures but has no effect on N/Tert-1. Viral replication is also elevated in the absence of SAMHD1. This new system has allowed us to identify a specific interaction between SAMHD1 and HPV16 that regulates host cell proliferation and viral replication; such studies are problematic in nonimmortalized primary keratinocytes due to their limited life span. To confirm the relevance of our results, we repeated the analysis with human tonsil keratinocytes (HTK) immortalized by HPV16 (HTK+HPV16) and observed the same hyperproliferative phenotype following CRISPR/Cas9 editing of SAMHD1. Identical results were obtained with three independent CRISPR/Cas9 guide RNAs. The isogenic pairing of N/Tert-1 with N/Tert-1+HPV16, combined with HTK+HPV16, presents a unique system to identify host genes whose products functionally interact with HPV16 to regulate host cellular growth in keratinocytes.IMPORTANCE HPVs are causative agents in human cancers and are responsible for around of 5% of all cancers. A better understanding of the viral life cycle in keratinocytes will facilitate the development of novel therapeutics to combat HPV-positive cancers. Here, we present a unique keratinocyte model to identify host proteins that specifically interact with HPV16. Using this system, we report that a cellular gene, SAMHD1, is regulated by HPV16 at the RNA and protein levels in keratinocytes. Elimination of SAMHD1 from these cells using CRISPR/Cas9 editing promotes enhanced cellular proliferation by HPV16 in keratinocytes and elevated viral replication but not in keratinocytes that do not have HPV16. Our study demonstrates a specific intricate interplay between HPV16 and SAMHD1 during the viral life cycle and establishes a unique model system to assist exploring host factors critical for HPV pathogenesis.

Project description:Keratins, formerly known as cytokeratins, are the major epithelial-specific subgroup of intermediate filament proteins. Adult hepatocytes express keratin polypeptides 8 and 18 (K8/K18), whereas cholangiocytes express K8/K18 and keratins 7 and 19 (K7/K19). Keratins function primarily to protect hepatocytes from apoptosis and necrosis, which was revealed using several genetic mouse models. This cytoprotective function was further clarified by the identification of natural human keratin variants that are normally silent, but become pathogenic by predisposing their carriers to apoptosis during acute or chronic liver injury mediated by toxins, virus infection, or metabolic stress. During apoptosis, caspases cleave K18 and K19 at conserved aspartates (human K18/K19: (235) Val-Glu-Val-Asp(?) ) and K18 at a unique aspartate (human K18: (394) Asp-Ala-Leu-Asp(?) ), with the latter exposed epitope becoming recognized by the M30 antibody in blood and tissues. Additional K18-containing protein backbone epitopes are detected using the M6 and M5 (termed M65) antibodies. Intact K18 and its associated fragments, which are released into blood during apoptosis and necrosis in various diseases, have been analyzed by enzyme-linked immunosorbent assay using the M30/M65 antibodies or their signal ratios. Furthermore, M30/M65 levels have been used as diagnostic and prognostic biomarkers in acute and chronic liver diseases, including nonalcoholic steatohepatitis and acute liver failure. Other keratin biomarkers include K8/K18/K19-related tissue polypeptide antigen, K18-related tissue polypeptide-specific antigen, and K19-related CYFRA-21-1, which have been evaluated mostly in patients with epithelial tumors.Keratins and their fragments are released into blood during liver and other epithelial tissue injury. The epithelial specificity of K18/K19, epitope unmasking upon caspase digestion, keratin abundance, and relative keratin stability render them useful biomarkers for hepatocyte and cholangiocyte apoptosis and necrosis. However, the precise biochemical nature and release mechanism of circulating keratins remain unknown. (Hepatology 2016;64:966-976).

Dataset Information

Shift from apoptotic to necrotic cell death during human papillomavirus-induced transformation of keratinocytes.

Publications

Shift from apoptotic to necrotic cell death during human papillomavirus-induced transformation of keratinocytes.

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