Biomedical subjects

Michelle A Ozbun

Publications and source records attributed to Michelle A Ozbun.

8 recordsLinked to original sources

PubMed2006

Sumoylation dynamics during keratinocyte differentiation.

SUMO modification regulates the activity of numerous transcription factors that have a direct role in cell-cycle progression, apoptosis, cellular proliferation, and development, but its role in differentiation processes is less clear. Keratinocyte differentiation requires the coordinated activation of a series of transcription factors, and as several crucial keratinocyte transcription factors are known to be SUMO substrates, we investigated the role of sumoylation in keratinocyte differentiation. In a human keratinocyte cell line model (HaCaT cells), Ca2+-induced differentiation led to the transient and coordinated transcriptional activation of the genes encoding crucial sumoylation system components, including SAE1, SAE2, Ubc9, SENP1, Miz-1 (PIASx beta), SUMO2 and SUMO3. The increased gene expression resulted in higher levels of the respective proteins and changes in the pattern of sumoylated substrate proteins during the differentiation process. Similarly to the HaCaT results, stratified human foreskin keratinocytes showed an upregulation of Ubc9 in the suprabasal layers. Abrogation of sumoylation by Gam1 expression severely disrupted normal HaCaT differentiation, consistent with an important role for sumoylation in the proper progression of this biological process.

Calcium↗

PubMed2005

Human papillomavirus type 31b infection of human keratinocytes does not require heparan sulfate.

Oncogenic human papillomaviruses (HPVs) are difficult to study experimentally as they replicate at low levels in vivo. This has precluded the purification of high-risk HPV virions from in vivo lesions. Virus-like particles (VLPs) and pseudovirions from low- and high-risk HPV types can emulate various aspects of HPV virion attachment and infections. These studies suggest that HPV infection is mediated by alpha6-integrin and/or heparan-sulfonated receptors. However, whether VLPs and pseudovirions accurately reflect the infection process of HPV virions has not been verified. We generated infectious HPV31b virions from organotypic (raft) tissues and performed experimental infections in a variety of cells. Successful infection following viral attachment, internalization, and nuclear transport was assayed by detecting newly synthesized, spliced HPV transcripts using reverse transcription (RT)-PCR or RT-quantitative PCR. Most human epithelial cells were infected with HPV31b at a multiplicity of infection as low as 1 to 10 viral genome equivalents per cell. HPV31b infection was detected in other cell lines, including COS-7 monkey kidney cells, but higher viral multiplicities of infection were required. Heparin preparations of various molecular weights or heparinase I treatment of cells prevented HPV31b infection of COS-7 cells and C-33A human cervical cancer cells in reproducible and dose-dependent manners. However, these reagents were unable to block infection of human keratinocytes, including HaCaT and N/TERT-1 cells and low-passage human foreskin keratinocytes. These data suggest that HPV31b infection of human keratinocytes, the natural host cell for HPV infections in vivo, does not require a heparan-sulfonated receptor, whereas heparan sulfate is important for infection of some other cells.

Animals↗

PubMed2005

The minor capsid protein L2 contributes to two steps in the human papillomavirus type 31 life cycle.

Prior studies, which have relied upon the use of pseudovirions generated in heterologous cell types, have led to sometimes conflicting conclusions regarding the role of the minor capsid protein of papillomaviruses, L2, in the viral life cycle. In this study we carry out analyses with true virus particles assembled in the natural host cell to assess L2's role in the viral infectious life cycle. For these studies we used the organotypic (raft) culture system to recapitulate the full viral life cycle of the high-risk human papillomavirus HPV31, which was either wild type or mutant for L2. After transfection, the L2 mutant HPV31 genome was able to establish itself as a nuclear plasmid in proliferating populations of poorly differentiated (basal-like) human keratinocytes and to amplify its genome to high copy number, support late viral gene expression, and cause formation of virus particles in human keratinocytes that had been induced to undergo terminal differentiation. These results indicate that aspects of both the nonproductive and productive phases of the viral life cycle occur normally in the absence of functional L2. However, upon the analysis of the virus particles generated, we found an approximate 10-fold reduction in the amount of viral DNA encapsidated into L2-deficient virions. Furthermore, there was an over-100-fold reduction in the infectivity of L2-deficient virus. Because the latter deficiency cannot be accounted for solely by the 10-fold decrease in encapsidation, we conclude that L2 contributes to at least two steps in the production of infectious virus.

Capsid Proteins↗

PubMed2005

Using an immortalized cell line to study the HPV life cycle in organotypic "raft" cultures.

The papillomavirus life cycle is tied to the differentiation of the stratified squamous epithelium that this virus infects. The ability to study the papillomavirus life cycle is facilitated by organotypic culturing techniques that allow one to closely recapitulate this terminal differentiation process in the laboratory. Current techniques allow for the establishment of recombinant wild-type or mutant human papillomavirus (HPV) genomes in transfected early-passage human foreskin keratinocytes (HFKs). These cells can then be used in organotypic culture to investigate the role of individual viral genes in different aspects of the viral life cycle. When using early-passage HFKs, there is a need for the transfected HPV genome to extend the life span of the cells in order to have sufficient cell generations in which to carry out organotypic culturing. The recent isolation of a spontaneously immortalized HFK cell line that supports the complete HPV life cycle has further allowed investigators to study wild-type or mutant papillomaviral genomes that do not confer immortalization. In this chapter, we describe the methodologies that permit the study of the HPV life cycle in this HFK cell line.

Animals↗

PubMed2004

Propagation of infectious human papillomavirus type 16 by using an adenovirus and Cre/LoxP mechanism.

Human papillomavirus type 16 (HPV16) infection is a major risk factor for the development of squamous cell cancers of the cervix and of the head and neck. A major barrier to understanding the progression from initial infection to cancer has been the lack of in vitro models that allow infection, replication, and persistence of the viral genome as an episome in differentiated epithelial cells. To overcome this barrier, we designed an adenoviral delivery vector that contained a full HPV16 genome flanked by LoxP homologous recombination sites and a fluorescent reporter that was expressed only after the HPV genome was excised by Cre recombinase. This system delivered circular HPV16 genomes to cervical epithelial cells and well differentiated human airway epithelia. After delivery, the HPV16 genome replicated and persisted as an episome in cervical keratinocytes. These cells developed an immortalized phenotype and a dysplastic epithelial appearance. Moreover, induction of differentiation led to the expression of late genes and production of infectious HPV16 virions. This work provides a means of introducing biologically active HPV genomes into epithelial cells, which are normally difficult to transfect. These methods allow the study of HPV genome replication and gene expression in the earliest stages of HPV genome establishment, and they may provide a means to study nononcogenic HPV viral types.

Adenoviridae↗

PubMed2002

Variable expression of some "housekeeping" genes during human keratinocyte differentiation.

We investigated the expression levels of four cellular "housekeeping" genes during epithelial differentiation. Differentiation is a dynamic process and various cellular RNAs have been targeted for use as internal controls during differentiation of human keratinocytes, but the consistent expression of such standards has not been previously validated. We used the organotypic (raft) culture system to grow stratified and differentiated epithelium in vitro. We compared cellular RNAs from epithelial tissues of both normal human keratinocytes and keratinocytes whose differentiation scheme is altered by the replication of human papillomavirus. Using ribonuclease protection assays to quantify RNA expression levels, we found that beta-actin and glyceraldehyde-3-phosphate dehydrogenase levels fluctuated during epithelial differentiation, whereas cyclophilin RNA and 28S-ribosomal RNA were the most consistently expressed during epithelial differentiation. These stably expressed cellular RNAs can be targeted as controls to permit quantitative expression analyses of cellular and pathogen RNAs during epithelial differentiation under various experimental conditions.

Actins↗

PubMed2002

Infectious human papillomavirus type 31b: purification and infection of an immortalized human keratinocyte cell line.

Human papillomaviruses (HPVs) are aetiological agents of human malignancies, most notably cervical cancers. The life-cycles of HPVs are dependent on epithelial differentiation, and this has impeded many basic studies of HPV biology. The organotypic (raft) culture system supports epithelial differentiation such that infectious virions are synthesized in raft tissues from epithelial cells that replicate extrachromosomal HPV genomes. The CIN-612 9E cell line maintains episomal copies of HPV type 31b (HPV31b), an HPV type associated with cervical cancers. Many previous studies, including our own, have focused on characterizing the later stages of the HPV31b life-cycle in CIN-612 9E raft tissues. In this study, we have used the raft system to generate large numbers of HPV31b viral DNA (vDNA)-containing particles. We found a biologically contained homogenization system to be efficient at virion extraction from raft epithelial tissues. We also determined that vDNA-containing particles could be directly quantified from density-gradient fractions. Using an RT-PCR assay, the presence of newly synthesized, spliced HPV31b transcripts was detected following HPV31b infection of the immortalized HaCaT epithelial cell line. Spliced E6 and E1( wedge )E4 RNAs were detected using a single round of RT-PCR from cells infected with a dose as low as 1.0 vDNA-containing particle per cell. Spliced E1*I,E2 transcripts were found in cells infected with an HPV31b dose as low as 10 vDNA-containing particles per cell. Infectivity was blocked by HPV31 antiserum, but was not affected by DNase I. This work lays a foundation for a detailed analysis of the early events in HPV infection.

3T3 Cells↗

PubMed2002

Human papillomavirus type 31b infection of human keratinocytes and the onset of early transcription.

Human papillomaviruses (HPVs) cause a number of human tumors and malignancies, including cervical cancers. Epithelial differentiation is required for the complete HPV life cycle and can be achieved using the organotypic (raft) culture system. The CIN-612 9E cell line maintains episomal copies of HPV type 31b (HPV31b), an HPV type associated with cervical cancers. When grown in the raft system, CIN-612 9E cells form a differentiated epithelium such that infectious virions can be synthesized. Many aspects of the later stages of the HPV31b life cycle have been investigated in CIN-612 9E raft tissues. We used a biologically contained homogenization system for efficient virion extraction from raft epithelial tissues. Purified HPV31b virions were used to infect low-passage-number human foreskin keratinocytes and a variety of epithelial cell lines. Newly synthesized, spliced HPV31b transcripts were detected by reverse transcription and PCR (RT-PCR) following HPV31b infection. HPV31b infection was most efficient and reproducible in HaCaT cells. The onset of viral transcription following infection was also investigated using RT-PCR techniques. Spliced E1(*)I,E2 RNAs were present as early as 4 h postinfection (p.i.), whereas the other major viral transcripts were detected by 8 to 10 h p.i. Furthermore, we characterized the structures and temporal expression of seven novel spliced early transcripts expressed following infection.

Animals↗