scholarly journals E6/E7 Functional Differences among Two Natural Human Papillomavirus 18 Variants in Human Keratinocytes

Viruses ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 1114
Author(s):  
Emily Montosa Nunes ◽  
Valéria Talpe-Nunes ◽  
João Simão Sobrinho ◽  
Silvaneide Ferreira ◽  
Vanesca de Souza Lino ◽  
...  
Keyword(s):  
Single Cells ◽  
Human Keratinocytes ◽  
Host Cells ◽  
Migration And Invasion ◽  
Primary Human Keratinocytes ◽  
Epithelial Development ◽  
Cervical Adenocarcinomas ◽  
Human Papillomavirus 18 ◽  
And Migration ◽  
Hpv 18

It is suggested that HPV-18 variants from the A lineage have higher oncogenic potential compared to B variants. Some studies show uneven distribution of HPV-18 variants in cervical adenocarcinomas and squamous cell carcinomas. Regarding HPV-18 variants’ functions, the few studies reported focus on E6, and none were performed using natural host cells. Here, we immortalized primary human keratinocytes (PHKs) with E6/E7 of HPV-18 A1 and B1 sublineages and functionally characterized these cells. PHK18A1 reached immortalization significantly faster than PHK18B1 and formed a higher number of colonies in monolayer and 3D cultures. Moreover, PHK18A1 showed greater invasion ability and higher resistance to apoptosis induced by actinomycin-D. Nevertheless, no differences were observed regarding morphology, proliferation after immortalization, migration, or epithelial development in raft cultures. Noteworthy, our study highlights qualitative differences among HPV-18 A1 and B1 immortalized PHKs: in contrast to PHK18A1, which formed more compact colonies and spheroids of firmly grouped cells and tended to invade and migrate as clustered cells, morphologically, PHK18B1 colonies and spheroids were looser, and migration and invasion of single cells were observed. Although these observations may be relevant for the association of these variants with cervical cancer of different histological subtypes, further studies are warranted to elucidate the mechanisms behind these findings.

scholarly journals Characterization of serum antibodies from women immunized with Gardasil: A study of HPV-18 infection of primary human keratinocytes

Vaccine ◽  
2016 ◽  
Vol 34 (27) ◽  
pp. 3171-3177 ◽  
Author(s):  
Hsu-Kun Wang ◽  
Qing Wei ◽  
Zina Moldoveanu ◽  
Warner K. Huh ◽  
Huong Lan Vu ◽  
...  
Keyword(s):  
Human Keratinocytes ◽  
Serum Antibodies ◽  
Primary Human Keratinocytes ◽  
Hpv 18

Cotransfection of HPV-18 and v-fos DNA Induces Tumorigenicity of Primary Human Keratinocytes

Virology ◽  
1993 ◽  
Vol 196 (2) ◽  
pp. 855-860 ◽  
Author(s):  
Xu Fang Pei ◽  
Jeanne M. Meck ◽  
David Greenhalgh ◽  
Richard Schlegel
Keyword(s):  
Human Keratinocytes ◽  
Primary Human Keratinocytes ◽  
Hpv 18

scholarly journals Alternative Fates of Keratinocytes Transduced by Human Papillomavirus Type 18 E7 during Squamous Differentiation

2002 ◽  
Vol 76 (6) ◽  
pp. 2964-2972 ◽  
Author(s):  
Wei-Ming Chien ◽  
Francisco Noya ◽  
Heather M. Benedict-Hamilton ◽  
Thomas R. Broker ◽  
Louise T. Chow
Keyword(s):  
Human Papillomavirus ◽  
Cyclin E ◽  
Dna Amplification ◽  
Human Keratinocytes ◽  
S Phase ◽  
Primary Human Keratinocytes ◽  
P27kip1 Protein ◽  
Differentiated Cells ◽  
Hpv 18

ABSTRACT The human papillomavirus type 18 (HPV-18) E7 protein promotes S-phase reentry in postmitotic, differentiated keratinocytes in squamous epithelium to facilitate vegetative viral DNA amplification. To examine the nature and fate of the differentiated cells that reenter S phase, organotypic cultures of primary human keratinocytes transduced with HPV-18 E7 were pulse-chase-pulse-labeled with 3H-thymidine (3H-TdR) and bromodeoxyuridine (BrdU). The kinetics of the appearance of doubly labeled suprabasal cells demonstrate that E7 expression did not promote prolonged S phase. Rather, there was a considerable lag before a small percentage of the cells reentered another round of S phase. Fluorescence in situ hybridization analysis, indeed, revealed a small fraction of the cells with more than 4n chromosomes in the differentiated strata. Differentiated cells positive for 3H-TdR, BrdU, or both often had enlarged nuclei or were binucleated. These results suggest that S phase is not followed by cell division, although nuclear division may occur. Interestingly, a significant fraction of differentiated cells that entered S phase subsequently accumulated p27kip1 protein with a kinetics preceding the accumulation of cyclin E. We conclude that E7-transduced, differentiated keratinocytes that enter S phase have two alternative fates: (i) a low percentage of cells undergoes endoreduplication, achieving higher than 4n ploidy, and (ii) a high percentage of cells accumulates the p27kip1, cyclin E, and p21cip1 proteins, resulting in arrest and preventing further S-phase reentry.

scholarly journals Coordinated action of human papillomavirus type 16 E6 and E7 oncoproteins on competitive endogenous RNA (ceRNA) network members in primary human keratinocytes

BMC Cancer ◽  
2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Brigitta László ◽  
László Antal ◽  
Eszter Gyöngyösi ◽  
Anita Szalmás ◽  
Szilárd Póliska ◽  
...  
Keyword(s):  
Regulatory Network ◽  
Human Keratinocytes ◽  
Functional Enrichment ◽  
Host Cells ◽  
Pathway Enrichment Analysis ◽  
Biological Processes ◽  
Primary Human Keratinocytes ◽  
Cerna Network ◽  
E6 And E7 Oncoproteins ◽  
E6 And E7

Abstract Background miRNAs and lncRNAs can regulate cellular biological processes both under physiological and pathological conditions including tumour initiation and progression. Interactions between differentially expressed diverse RNA species, as a part of a complex intracellular regulatory network (ceRNA network), may contribute also to the pathogenesis of HPV-associated cancer. The purpose of this study was to investigate the global expression changes of miRNAs, lncRNAs and mRNAs driven by the E6 and E7 oncoproteins of HPV16, and construct a corresponding ceRNA regulatory network of coding and non-coding genes to suggest a regulatory network associated with high-risk HPV16 infections. Furthermore, additional GO and KEGG analyses were performed to understand the consequences of mRNA expression alterations on biological processes. Methods Small and large RNA deep sequencing were performed to detect expression changes of miRNAs, lncRNAs and mRNAs in primary human keratinocytes expressing HPV16 E6, E7 or both oncoproteins. The relationships between lncRNAs, miRNAs and mRNAs were predicted by using StarBase v2.0, DianaTools-LncBase v.2 and miRTarBase. The lncRNA-miRNA-mRNA regulatory network was visualized with Cytoscape v3.4.0. GO and KEEG pathway enrichment analysis was performed using DAVID v6.8. Results We revealed that 85 miRNAs in 21 genomic clusters and 41 lncRNAs were abnormally expressed in HPV E6/E7 expressing cells compared with controls. We constructed a ceRNA network with members of 15 lncRNAs – 43 miRNAs – 358 mRNAs with significantly altered expressions. GO and KEGG functional enrichment analyses identified numerous cancer related genes, furthermore we recognized common miRNAs as key regulatory elements in biological pathways associated with tumorigenesis driven by HPV16. Conclusions The multiple molecular changes driven by E6 and E7 oncoproteins resulting in the malignant transformation of HPV16 host cells occur, at least in part, due to the abnormal alteration in expression and function of non-coding RNA molecules through their intracellular competing network.

scholarly journals Human Papillomavirus (HPV) Type 18 Induces Extended Growth in Primary Human Cervical, Tonsillar, or Foreskin Keratinocytes More Effectively than Other High-Risk Mucosal HPVs

2009 ◽  
Vol 83 (22) ◽  
pp. 11784-11794 ◽  
Author(s):  
Michael J. Lace ◽  
James R. Anson ◽  
Aloysius J. Klingelhutz ◽  
John H. Lee ◽  
Aaron D. Bossler ◽  
...  
Keyword(s):  
High Risk ◽  
Human Keratinocytes ◽  
Human Papillomaviruses ◽  
Population Doubling ◽  
Primary Human Keratinocytes ◽  
Hpv 16 ◽  
Epithelial Origin ◽  
Hpv Types ◽  
Population Doublings ◽  
Hpv 18

ABSTRACT Mucosal high-risk (HR) human papillomaviruses (HPVs) that cause cervical and other anogenital cancers also are found in ∼25% of head and neck carcinomas (HNCs), especially those arising in the oropharynx and the tonsils. While many HR HPV types are common in anogenital cancer, over 90% of HPV-positive HNCs harbor HPV type 16 (HPV-16). Using a quantitative colony-forming assay, we compared the ability of full-length mucosal HPV genomes, i.e., the low-risk HPV-11 and HR HPV-16, -18, and -31, to persist in and alter the growth of primary human keratinocytes from the foreskin, cervix, and tonsils. The HR HPV types led to the formation of growing keratinocyte colonies in culture independent of the site of epithelial origin. However, HPV-18 induced colony growth in all keratinocytes >4-fold more effectively than HPV-16 or HPV-31 and >20-fold more efficiently than HPV-11 or controls. HPV-11-transfected or control colonies failed to expand beyond 32 to 36 population doublings postexplantation. In contrast, individual HR HPV-transfected clones exhibited no apparent slowdown of growth or “crisis,” and many maintained HPV plasmid persistence beyond 60 population doublings. Keratinocyte clones harboring extrachromosomal HR HPV genomes had shorter population doubling times and formed dysplastic stratified epithelia in organotypic raft cultures, mirroring the pathological features of higher-grade intraepithelial lesions, yet did not exhibit chromosomal instability. We conclude that, in culture, the HR HPV type, rather than the site of epithelial origin of the cells, determines the efficacy of inducing continued growth of individual keratinocytes, with HPV-18 being the most aggressive mucosal HR HPV type tested.

scholarly journals Sp100 Provides Intrinsic Immunity against Human Papillomavirus Infection

mBio ◽  
2013 ◽  
Vol 4 (6) ◽  
Author(s):  
Wesley H. Stepp ◽  
Jordan M. Meyers ◽  
Alison A. McBride
Keyword(s):  
Human Papillomavirus ◽  
Dna Replication ◽  
Human Keratinocytes ◽  
Hpv Infection ◽  
Nuclear Bodies ◽  
Host Cells ◽  
Viral Transcription ◽  
Primary Human Keratinocytes ◽  
Dna Viruses ◽  
Hpv Dna

ABSTRACTMost DNA viruses associate with, and reorganize, nuclear domain 10 (ND10) bodies upon entry into the host nucleus. In this study, we examine the roles of the ND10 components PML, Sp100, and Daxx in the establishment of human papillomavirus type 18 (HPV18) infection of primary human keratinocytes. HPV18 DNA or HPV18 quasivirus was introduced into primary human keratinocytes depleted of each ND10 protein by small interfering RNA technology, and genome establishment was determined by using a quantitative immortalization assay and measurements of viral transcription and DNA replication. Keratinocyte depletion of Sp100 resulted in a substantial increase in the number of HPV18-immortalized colonies and a corresponding increase in viral transcription and DNA replication. However, Sp100 repressed viral transcription and replication only during the initial stages of viral establishment, suggesting that Sp100 acts as a repressor of incoming HPV DNA.IMPORTANCEThe intrinsic immune system provides a first-line defense against invading pathogens. Host cells contain nuclear bodies (ND10) that are important for antiviral defense, yet many DNA viruses localize here upon cell entry. However, viruses also disrupt, reorganize, and modify individual components of the bodies. In this study, we show that one of the ND10 components, Sp100, limits the infection of human skin cells by human papillomavirus (HPV). HPVs are important pathogens that cause many types of infection of the cutaneous and mucosal epithelium and are the causative agents of several human cancers. Understanding how host cells counteract HPV infection could provide insight into antimicrobial therapies that could limit initial infection.

scholarly journals The E6 and E7 Proteins of the Cutaneous Human Papillomavirus Type 38 Display Transforming Properties

2003 ◽  
Vol 77 (3) ◽  
pp. 2195-2206 ◽  
Author(s):  
Sandra Caldeira ◽  
Ingeborg Zehbe ◽  
Rosita Accardi ◽  
Ilaria Malanchi ◽  
Wen Dong ◽  
...  
Keyword(s):  
Human Keratinocytes ◽  
Human Papillomaviruses ◽  
Host Cells ◽  
Primary Human Keratinocytes ◽  
Transition Control ◽  
Skin Cancers ◽  
E6 And E7 ◽  
E7 Proteins ◽  
Nonmelanoma Skin Cancers

ABSTRACT Several studies have suggested the involvement of cutaneous human papillomaviruses (HPVs) in the development of nonmelanoma skin cancers. Here we have characterized the in vitro properties of E7 proteins of three cutaneous HPV types, 10, 20, and 38, which are frequently detected in skin specimens. We show that HPV38 E7 is able to inactivate the tumor suppressor pRb and induces loss of G1/S transition control, a key event in carcinogenesis. In contrast, HPV10 and HPV20 E7 proteins do not display these in vitro transforming activities. We also show that the two early proteins E6 and E7 of HPV38 are sufficient to corrupt the cell cycle and senescence programs in primary cells, inducing active and long-lasting proliferation of primary human keratinocytes, the natural host cells. Our study shows that E6 and E7 of this cutaneous HPV type have transforming activity in primary human cells, suggesting a role for HPV38 infection in skin carcinogenesis. In further support of such a role, we detected HPV38 DNA in approximately 50% of nonmelanoma skin cancers, but only in 10% of healthy skin specimens (P < 0.001).

scholarly journals Global Effects of Human Papillomavirus Type 18 E6/E7 in an Organotypic Keratinocyte Culture System

2004 ◽  
Vol 78 (17) ◽  
pp. 9041-9050 ◽  
Author(s):  
Peggy A. Garner-Hamrick ◽  
J. M. Fostel ◽  
Wei-Ming Chien ◽  
N. Sanjib Banerjee ◽  
Louise T. Chow ◽  
...  
Keyword(s):  
Human Papillomavirus ◽  
Culture System ◽  
Human Keratinocytes ◽  
Protein Translation ◽  
Primary Human Keratinocytes ◽  
Raft Culture ◽  
Dna And Rna ◽  
E6 And E7 ◽  
E7 Proteins ◽  
Hpv 18

ABSTRACT The effects of human papillomavirus type 18 (HPV-18) E6 and E7 proteins on global patterns of host gene expression in primary human keratinocytes grown in organotypic raft culture system were assessed. Primary human keratinocytes were infected with retroviruses that express the wild-type HPV-18 E6 and E7 genes from the native differentiation-dependent HPV enhancer-promoter. Total RNA was isolated from raft cultures and used to generate probes for querying Affymetrix U95A microarrays, which contain >12,500 human gene sequences. Quadruplicate arrays of each E6/E7-transduced and empty vector-transduced samples were analyzed by 16 pairwise comparisons. Transcripts altered in ≥12 comparisons were selected for further analysis. With this approach, HPV-18 E6/E7 expression significantly altered the expression of 1,381 genes. A large increase in transcripts associated with DNA and RNA metabolism was observed, with major increases noted for transcription factors, splicing factors, and DNA replication elements, among others. Multiple genes associated with protein translation were downregulated. In addition, major alterations were found in transcripts associated with the cell cycle and cell differentiation. Our study provides a systematic description of transcript changes brought about by HPV-18 E6/E7 in a physiologically relevant model and should furnish a solid source of information to guide future studies.

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