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L A Laimins

Publications and source records attributed to L A Laimins.

At least 19 recordsLinked to original sources
PubMed2006

Histone deacetylase 3 localizes to the plasma membrane and is a substrate of Src.

Histone deacetylases (HDACs) negatively regulate gene expression by removing acetyl groups from lysine residues present in histones and other proteins. Histone deacetylase 3 is unique among the Class I family of HDACs, as it is able to shuttle between the nucleus and the cytoplasm, whereas the other family members remain in the nucleus. Histone deacetylase 3 often forms complexes with corepressor proteins that do not associate with the other Class I HDACs, and its phosphorylation correlates with increased enzymatic activity. Here we show that HDAC3 also localizes to the plasma membrane in multiple cell types. Furthermore, c-Src is shown to form a complex with HDAC3 at the plasma membrane and to use HDAC3 as a substrate for phosphorylation. Our results describe a novel localization and binding partner for the HDAC3 protein, as well as implicate c-Src in HDAC3 regulation.

CSK Tyrosine-Protein Kinase↗
PubMed2001

Telomerase activation by human papillomavirus type 16 E6 protein: induction of human telomerase reverse transcriptase expression through Myc and GC-rich Sp1 binding sites.

High-risk human papillomaviruses (HPVs) immortalize keratinocytes by disrupting the retinoblastoma protein (Rb)/p16 pathway and activating telomerase. The E7 oncoprotein targets Rb, while the E6 oncoprotein induces telomerase activity in human keratinocytes. This study has examined the mechanism by which E6 activates telomerase. Expression of human telomerase reverse transcriptase (hTERT), the catalytic subunit of telomerase, was found to be increased in keratinocytes stably expressing HPV type 16 E6, suggesting that E6 acts to increase hTERT transcription. hTERT expression and telomerase activity were activated to significantly higher levels in cells expressing both E6 and E7 than in cells expressing E6 alone. This indicates that E7 may augment E6-mediated activation of hTERT transcription. In transient-transfection assays using hTERT reporters, the induction of hTERT expression by E6 was found to be mediated by a 258-bp fragment of the hTERT promoter, proximal to the ATG initiation codon. Previous studies have demonstrated that overexpression of Myc can activate hTERT expression, suggesting that Myc may be a mediator of E6-mediated hTERT induction. However, in cells stably expressing E6, no strict correlation between the level of Myc and the activation of hTERT was found. Consistent with this observation, mutation of the two Myc binding sites in the hTERT promoter only modestly reduced responsiveness to E6 in transient reporter assays. This indicates that activation of Myc-dependent transcription is not essential for E6-mediated upregulation of hTERT expression. The hTERT promoter also contains five GC-rich elements that can bind Sp1. Mutation of these sites within the 258-bp fragment partially reduced hTERT induction by E6. However, when mutations in the Sp1 sites were combined with the mutated Myc binding sites, all activation by E6 was lost. This indicates that it is the combinatorial binding of factors to Myc and Sp1 cis elements that is responsible for hTERT induction by E6.

Binding Sites↗
PubMed2001

Cellular changes induced by low-risk human papillomavirus type 11 in keratinocytes that stably maintain viral episomes.

Infections by low-risk papillomavirus types, such as human papillomavirus (HPV) type 6 (HPV-6) and HPV-11, induce benign genital warts that rarely progress to malignancy. In contrast, lesions induced by high-risk HPV types have the potential to progress to cancer. Considerable information is available concerning the pathogenesis of high-risk HPV types, but little is known about the life cycle of low-risk HPV types. Although functionally distinct, both high- and low-risk virus types infect keratinocytes and induce virion production upon differentiation. This information suggests that they may share common mechanisms for regulating their productive life cycles. Using tissue culture methods developed to study high-risk HPV types, we examined the ability of HPV-11 to be stably maintained as episomes following transfection of normal human keratinocytes with cloned viral DNA. HPV-11 genomes were found to be maintained in keratinocytes for extended passages in cultures in 14 independent experiments involving transfection of cloned HPV-11 DNA. Interestingly, the HPV-11-positive cells exhibited an extended life span that averaged approximately twofold longer than that of control neomycin-transfected cells. In organotypic cultures, HPV-11-positive cells exhibited altered differentiation patterns, but the extent of disruption was less severe than that seen with high-risk HPV types. In addition, the amplification of HPV-11 DNA, as well as the induction of several viral messages, was observed following differentiation of transfected cells in semisolid media. To determine whether global changes in cellular gene expression induced by HPV-11 were similar to those observed with high-risk HPV-31 (Y. E. Chang and L. A. Laimins, J. Virol. 74:4174-4182, 2000), microarray analysis of 7,075 expressed sequences was performed. A spectrum of cellular genes different from that previously reported for HPV-31 was found to be activated or repressed by HPV-11. The expression of only a small set of genes was similarly altered by both high- and low-risk HPV types. This result suggests that different classes of HPVs have distinct effects on global cellular transcription patterns during infection. The methods described allow for a genetic analysis of HPV-11 in the context of its differentiation-dependent life cycle.

Cell Differentiation↗
PubMed2001

Early polyadenylation signals of human papillomavirus type 31 negatively regulate capsid gene expression.

The L1 and L2 capsid genes of human papillomavirus type 31 (HPV-31) are expressed upon keratinocyte differentiation from a promoter located in the E7 open reading frame (ORF) of the early region. Late transcripts must therefore pass through and ignore the early polyadenylation sequences to use the downstream late AAUAAA element located at the end of the L1 ORF. To identify sequences which modulate downstream capsid gene expression, a variety of substitution mutations were introduced into the early polyadenylation signal and studied first in the context of polycistronic luciferase reporter constructs. Removal of the G/U-rich cleavage stimulation factor (CstF) binding sites and the degenerate cleavage and polyadenylation specificity factor binding sites, UAUAUA, had minimal effect on downstream expression as defined by luciferase activities. This is in contrast to the deletion of the HPV-31 early AAUAAA element, which resulted in a dramatic increase in downstream expression. Additional sequences within the first 800 bp of the L2 ORF were also found to negatively regulate capsid expression in luciferase assays. To determine how these mutations influence gene expression in the context of the complete HPV-31 genome, recombinant genomes were constructed that contained a substitution in the AAUAAA sequence, an inserted strong CstF binding site, an inserted simian virus 40 (SV40) late poly(A) signal, or a substitution of the 5'-most 800 nucleotides of the L2 ORF. Reductions in both transient and stable replication were observed with the recombinant genomes containing the strong CstF site or the late SV40 signal, suggesting that alterations in the strength of the upstream poly(A) signal influence expression of viral replication factors. Similarly, disruption of the L2 ORF resulted in a significant reduction in genome replication and an inability to be maintained stably. In contrast, genomes containing a substitution of the AAUAAA sequence had increased levels of transient and stable replication. Quantitation of late transcripts following keratinocyte differentiation in methylcellulose also showed a reduction in downstream capsid gene expression in lines containing genomes with the strong CstF site or the late SV40 signal mutations, while a significant increase in expression was detected in the lines with genomes lacking the AAUAAA sequence. These studies demonstrate that capsid gene expression in HPV-31 requires an inefficient early poly(A) signal which is defined primarily by the AAUAAA element as well as a major negative regulatory element located within the L2 ORF.

Capsid↗
PubMed2001

Differentiation-dependent chromatin rearrangement coincides with activation of human papillomavirus type 31 late gene expression.

The life cycle of human papillomaviruses (HPVs) is tightly linked to the differentiation status of the host cell. While early genes are expressed during the initial stages of viral infection, late gene expression occurs in the suprabasal layers of the cervical epithelium. Late genes encode E1-E4, a cytosolic protein, and capsid proteins L1 and L2. We have mapped over 30 initiation sites for late transcripts and show that the transcripts initiate in a 200-nucleotide region within the E7 open reading frame. The mechanisms regulating the activation of late gene expression, however, are not yet understood. DNase I hypersensitivity analysis of HPV-31 chromatin in cell lines that maintain viral genomes extrachromosomally indicates that a major shift in nuclease digestion occurs upon differentiation. In undifferentiated cells, hypersensitive regions exist in the upstream regulatory region proximal to the E6 open reading frame. Upon differentiation, a region between nucleotides 659 and 811 in the E7 open reading frame becomes accessible to DNase I. These results indicate that the late transcript initiation region becomes accessible to transcription factor binding upon differentiation. Several complexes mediate chromatin rearrangement, and we tested whether histone acetylation was sufficient for late transcript activation. Treatment with the histone deacetylase inhibitor trichostatin A was found to be insufficient to activate late gene expression in undifferentiated cells. However, it did activate expression of early transcripts. These results suggest that chromatin remodeling around the late promoter occurs upon epithelial differentiation and that mechanisms in addition to histone deacetylation contribute to activation of late gene expression.

Acetylation↗
PubMed2000

The E8E2C protein, a negative regulator of viral transcription and replication, is required for extrachromosomal maintenance of human papillomavirus type 31 in keratinocytes.

The viral E2 protein is a major regulator of papillomavirus DNA replication. An important way to influence viral replication is through modulation of the activity of the E2 protein. This could occur through the action of truncated E2 proteins, called E2 repressors, whose role in the replication cycle of human papillomaviruses (HPVs) has not been determined. In this study, using cell lines that contain episomal copies of the "high-risk" HPV type 31 (HPV31), we have identified viral transcripts with a splice from nucleotide (nt) 1296 to 3295. These transcripts are similar to RNAs from other animal and human papillomaviruses and have the potential to fuse a small open reading frame (E8) to the C terminus of E2, resulting in an E8E2C fusion protein. E8E2C transcripts were present throughout the complete replication cycle of HPV31. A genetic analysis of E8E2C in the context of the HPV31 genome revealed that mutation of the single ATG of the E8 gene, introduction of a stop codon downstream of the ATG, or disruption of the splice donor site at nt 1296 led to a dramatic 30- to 40-fold increase in the transient DNA replication levels in both normal and immortalized human keratinocytes. High-level expression of E8E2C from heterologous vectors was found to inhibit E1-E2-dependent DNA replication of an HPV31 origin of replication construct as well as to interfere with E2's ability to transactivate reporter gene constructs. In addition, HPV31 E8E2C strongly repressed the basal activity of the major viral early promoter P97 independent of E2. E8E2C may therefore exert its negative effect on viral DNA replication through modulating E2's ability to enhance E1-dependent DNA replication as well as by regulating viral gene expression. Surprisingly, HPV31 genomes that were unable to express E8E2C could not be maintained extrachromosomally in human keratinocytes in long-term assays despite high transient DNA replication levels. This suggests that the E8E2C protein may play a role in copy number control as well as in the stable maintenance of HPV episomes.

Base Sequence↗
PubMed2000

Microarray analysis identifies interferon-inducible genes and Stat-1 as major transcriptional targets of human papillomavirus type 31.

Human papillomaviruses (HPVs) infect keratinocytes and induce proliferative lesions. In infected cells, viral gene products alter the activities of cellular proteins, such as Rb and p53, resulting in altered cell cycle response. It is likely that HPV gene products also alter expression of cellular genes. In this study we used microarray analysis to examine the global changes in gene expression induced by high-risk HPV type 31 (HPV31). Among 7,075 known genes and ESTs (expressed sequence tags) tested, we found that 178 were upregulated and 150 were downregulated twofold or more in HPV31 cells compared to normal human keratinocytes. While no specific pattern could be deduced from the list of genes that were upregulated, downregulated genes could be classified to three groups: genes that are involved in the regulation of cell growth, genes that are specifically expressed in keratinocytes, and genes whose expression is increased in response to interferon stimulation. The basal level of expression of several interferon-responsive genes was found to be downregulated in HPV31 cells by both microarray analysis and Northern blot analysis in different HPV31 cell lines. When cells were treated with alpha or gamma interferon, expression of interferon-inducible genes was impaired. At high doses of interferon, the effects were less pronounced. Among the genes repressed by HPV31 was the signal transducer and activator of transcription (Stat-1), which plays a major role in mediating the interferon response. Suppression of Stat-1 expression may contribute to a suppressed response to interferon as well as immune evasion.

3T3 Cells↗
PubMed1999

Human papillomavirus type 31 oncoproteins E6 and E7 are required for the maintenance of episomes during the viral life cycle in normal human keratinocytes.

The E6 and E7 oncoproteins of the high-risk human papillomavirus (HPV) types are able to immortalize human keratinocytes in vitro and likely contribute to the development of anogenital malignancies in vivo. The role of these oncoproteins in the productive viral life cycle, however, is not known. To begin to examine these possible roles, mutations in E6 were introduced in the context of the complete HPV 31 genome. Although transfected wild-type HPV 31 genomes, as well as genomes containing an E6 translation termination linker, an E6 frameshift mutation, and a point mutation in the p53 interacting domain were able to replicate in transient assays, only the wild-type genome was stably maintained as an episome. Interestingly, mutant genomes in either the E6 splice-donor site or splice-acceptor site were reduced in replication ability in transient assays; however, cotransfection of E1 and E2 expression vectors restored this function. In a similar fashion, genomes containing mutant HPV 31 E7 genes, including a translation termination mutant, two Rb-binding site mutants, a casein kinase II phosphorylation site mutant, and a transformation deficient mutant, were constructed. Although transient replication was similar to wild type in all of the E7 mutants, only the casein kinase II mutant had the ability to maintain high copies of episomal genomes. These findings suggest a role for E6 and E7 in the viral life cycle beyond their ability to extend the life span of infected cells.

Casein Kinase II↗
PubMed1999

Differentiation-induced changes in promoter usage for transcripts encoding the human papillomavirus type 31 replication protein E1.

The life cycle of human papillomaviruses (HPVs) is tied to keratinocyte differentiation. One key event in the viral life cycle is the differentiation-dependent increase in viral replication. This increase in replication activity results in an amplification of the HPV genome from approximately 50 copies per cell in basal keratinocytes to thousands of copies of the viral genome per cell in suprabasal keratinocytes. To characterize the events associated with this differentiation-dependent increase in HPV replication, we have initiated studies of mRNAs encoding the HPV replication protein E1 during the differentiation of cell lines that stably maintain episomal HPV DNA. Differentiation induced the expression of several transcripts that hybridized to an E1-specific probe. One of these messages, a 3.7-kb transcript, did not hybridize to a probe specific for the early promoter upstream of the E6 open reading frame. RNase protection analysis confirmed an induction of unspliced messages derived from the differentiation-dependent promoter at nucleotide 742 in the middle of the E7 open reading frame. These observations demonstrate a differentiation-induced increase in E1 mRNAs derived from the viral late promoter and suggest a role for increased E1 expression during amplification of the HPV genome.

Cell Differentiation↗
PubMed1999

Human papillomavirus life cycle: active and latent phases.

Productive infection by human papillomaviruses (HPV) is dependent upon the differentiation of the host cell. Following entry into basal epithelial cells, HPV genomes are established as autonomous replicating extrachromosomal elements and a low level of HPV expression occurs. Upon differentiation of infected cells, productive replication and expression of capsid genes is induced resulting in the synthesis of progeny virions. Evidence from immunosuppressed patients as well as individuals with recurring laryngeal papillomatosis suggest that certain HPV types can exist in a latent state. In latently infected cells, HPV DNA may be present but no differentiation-dependent synthesis of virions occurs. The presence of a latent state for HPVs can be a determining factor in the effectiveness of therapeutic methods for treatment of infections.

Humans↗
PubMed1999

DNA replication of human papillomavirus type 31 is modulated by elements of the upstream regulatory region that lie 5' of the minimal origin.

The viral replication factors E1 and E2 of papillomaviruses are necessary and sufficient to replicate plasmids containing the minimal origin of DNA replication in transient assays. Under physiological conditions, the upstream regulatory region (URR) governs expression of the early viral genes. To determine the effect of URR elements on E1 and E2 expression specifically, and on the regulation of DNA replication during the various phases of the viral life cycle, we carried out a systematic replication study with entire genomes of human papillomavirus type 31 (HPV31), a high-risk oncogenic type. We constructed a series of URR deletions, spacer replacements, and point mutations to analyze the role of the keratinocyte enhancer (KE) element, the auxiliary enhancer (AE) domain, and the L1-proximal end of the URR (5'-URR domain) in DNA replication during establishment, maintenance, and vegetative viral DNA amplification. Using transient and stable replication assays, we demonstrate that the KE and AE are necessary for efficient E1 and E2 gene expression and that the KE can also directly modulate viral replication. KE-mediated activation of replication is dependent on the position and orientation of the element. Mutation of either one of the four Ap1 sites, the single Sp1 site, or the binding site for the uncharacterized footprint factor 1 reduced replication efficiency through decreased expression of E1 and E2. Furthermore, the 5'-URR domain and the Oct1 DNA binding site are dispensable for viral replication, since such HPV31 mutants are able to replicate efficiently in a transient assay, maintain a stable copy number over several cell generations, and amplify viral DNA under vegetative conditions. Interestingly, deletion of the 5'-URR domain leads to increased transient and stable replication levels. These findings suggest that elements in the HPV31 URR outside the minimal origin modulate viral replication through both direct and indirect mechanisms.

Binding Sites↗
PubMed1999

Regulation of human papillomavirus type 31 polyadenylation during the differentiation-dependent life cycle.

The L1 and L2 capsid genes of human papillomavirus type 31 (HPV-31) are expressed late in the differentiation-dependent life cycle from a promoter located in the E7 open reading frame (ORF) of the early region. These late HPV genes are transcribed by RNA polymerase II which reads through the region containing early polyadenylation signals and proceeds to a poly(A) site downstream of L1. In this study, we have investigated the mechanisms regulating differentiation-dependent polyadenylation and read-through in HPV-31. HPV-31 early transcripts were found to utilize a heterogeneous series of polyadenylation sites in undifferentiated cells. The sites for polyadenylation extended over a range of 100 nucleotides from within the E5 ORF to upstream of L2. Upon differentiation, the transcription of early genes increased, but no change in the heterogeneous distribution of 3' ends was detected. The early polyadenylation region was found to contain a single consensus hexanucleotide sequence, AAUAAA, as well as three weak binding sites for the cleavage stimulatory factor, CstF. In contrast to the heterogeneity at the early site, the 3' ends of late transcripts encoding L1 and L2 were localized to a narrow region downstream of the late AAUAAA element. The late polyadenylation signal was found to contain a single high-affinity site for CstF, as well as one consensus hexanucleotide sequence. By using a reporter assay, it was determined that the HPV-31 early polyadenylation sequences allowed significant levels of read-through into the late region in undifferentiated cells. Upon differentiation, this read-through was increased by approximately 50%, indicating that use of the early site decreased. Differentiation was also found to induce a 40% reduction in the levels of CstF subunits, which may contribute to the increased read-through of the early sequence. The insertion of the late high-affinity binding site for CstF into the early polyadenylation region significantly reduced the level of read-through, suggesting that these factors modulate read-through activity. Our studies demonstrate that HPV-31 late gene expression is regulated in a large part by posttranscriptional mechanisms, including the polyadenylation of early transcripts.

Binding Sites↗
PubMed1998

Human papillomavirus oncoproteins alter differentiation-dependent cell cycle exit on suspension in semisolid medium.

The suspension of keratinocytes containing episomal forms of the human papillomavirus (HPV)-31 genome in semisolid medium results in the induction of viral late functions. In this study, the suspension in semisolid medium was used to analyze how HPV deregulates the process of cell cycle exit during differentiation. In cells that contain the entire HPV-31 genome, induction of late protein synthesis was found to be linked with the expression of cyclin A. Consistent with analyses in organotypic rafts, the expression of the high-risk E7 oncoprotein alone was sufficient to retain cyclin A expression during suspension-induced differentiation. The cyclin-dependent kinase inhibitors (CKIs) p27 and p57 were found to be up-regulated in normal keratinocytes, as well as in the lines that express the HPV oncoproteins. The up-regulation of these CKIs is coincident with the inhibition of cyclin/cdk activity in normal keratinocytes. Cells expressing E7 were found to retain significant cdk2-associated kinase activity, although it was partially inhibited, coincident with CKI induction. When the phosphorylation state of Rb was examined during differentiation, cells expressing E7 retained phosphorylated forms of Rb, whereas Rb in normal keratinocytes was hypophosphorylated. As previously reported, E7-expressing cells were found to contain less Rb protein than normal keratinocytes. Interestingly, the Rb levels decreased during normal keratinocyte differentiation, and this differentiation-dependent reduction in Rb levels was enhanced by EG and E7 expression. This study identified proteins that may be critical for cell cycle regulation during normal epithelial differentiation and demonstrated that HPV oncoproteins alter their activities.

CDC2-CDC28 Kinases↗
PubMed1998

Transactivation by the E2 protein of oncogenic human papillomavirus type 31 is not essential for early and late viral functions.

The activation of transcription and of DNA replication are, in some cases, mediated by the same proteins. A prime example is the E2 protein of human papillomaviruses (HPVs), which binds ACCN6GGT sequences and activates heterologous promoters from multimerized binding sites. The E2 protein also has functions in replication, where it complexes with the virally encoded origin recognition protein, E1. Much of the information on these activities is based on transient-transfection assays as well as biochemical analyses; however, their importance in the productive life cycle of oncogenic HPVs remains unclear. To determine the contributions of these E2 functions to the HPV life cycle, a genetic analysis was performed by using an organotypic tissue culture model. HPV type 31 (HPV31) genomes that contained mutations in the N terminus of E2 (amino acid 73) were constructed; these mutants retained replication activities but were transactivation defective. Following transfection of normal human keratinocytes, these mutant genomes were established as stable episomes and expressed early viral transcripts at levels similar to those of wild-type HPV31. Upon differentiation in organotypic raft cultures, the induction of late gene expression and amplification of viral DNA were detected in cell lines harboring mutant genomes. Interestingly, only a modest reduction in late gene expression was observed in the mutant lines. We conclude that the transactivation function of E2 is not essential for the viral life cycle of oncogenic HPVs, although it may act to moderately augment late expression. Our studies suggest that the primary positive role of E2 in the viral life cycle is as a replication factor.

Amino Acid Substitution↗
PubMed1998

Differential requirements for conserved E2 binding sites in the life cycle of oncogenic human papillomavirus type 31.

Human papillomavirus (HPV) E2 proteins regulate viral replication by binding to sites in the upstream regulatory region (URR) and by complex formation with the E1 origin recognition protein. In the genital HPV types, the distribution and location of four E2 binding sites (BS1 to BS4) which flank a single E1 binding site are highly conserved. We have examined the roles of these four E2 sites in the viral life cycle of HPV type 31 (HPV31) by using recently developed methods for the biosynthesis of papillomaviruses from transfected DNA templates (M. G. Frattini et al., Proc. Natl. Acad. Sci. USA 93:3062-3067, 1996). In transient assays, no single site was found to be necessary for replication, and mutation of the early promoter-proximal site (BS4) led to a fourfold increase in replication. Cotransfection of the HPV31 wild-type (HPV-wt) and mutant genomes with expression vectors revealed that E1 stimulated replication of HPV31-wt as well as the HPV31-BS1, -BS2, and -BS3 mutants. In contrast, increased expression of E2 decreased replication of these genomes. Replication of the HPV31-BS4 mutant genome was not further increased by cotransfection of E1 expression vectors but was stimulated by E2 coexpression. In stably transfected normal human keratinocytes, mutation of either BS1, BS3, or BS4 resulted in integration of viral genomes into host chromosomes. In contrast, mutation of BS2 had no effect on stable maintenance of episomes or copy number. Following growth of stably transfected lines in organotypic raft cultures, the differentiation-dependent induction of late gene expression and amplification of viral DNA of the BS2 mutant was found to be similar to that of HPV31-wt. We were unable to find a role for BS2 in our assays for viral functions. We conclude that at least three of the four E2 binding sites in the URRs of HPVs are essential for the productive viral life cycle. The specific arrangement of E2 binding sites within the URR appears to be more important for viral replication than merely the number of sites.

Adenovirus E2 Proteins↗
PubMed1998

Human papillomavirus oncoproteins E6 and E7 independently abrogate the mitotic spindle checkpoint.

The E6 and E7 genes of the high-risk human papillomavirus (HPV) types encode oncoproteins, and both act by interfering with the activity of cellular tumor suppressor proteins. E7 proteins act by associating with members of the retinoblastoma family, while E6 increases the turnover of p53. p53 has been implicated as a regulator of both the G1/S cell cycle checkpoint and the mitotic spindle checkpoint. When fibroblasts from p53 knockout mice are treated with the spindle inhibitor nocodazole, a rereplication of DNA occurs without transit through mitosis. We investigated whether E6 or E7 could induce a similar loss of mitotic checkpoint activity in human keratinocytes. Recombinant retroviruses expressing high-risk E6 alone, E7 alone, and E6 in combination with E7 were used to infect normal human foreskin keratinocytes (HFKs). Established cell lines were treated with nocodazole, stained with propidium iodide, and analyzed for DNA content by flow cytometry. Cells infected with high-risk E6 were found to continue to replicate DNA and accumulated an octaploid (8N) population. Surprisingly, expression of E7 alone was also able to bypass this checkpoint. Cells expressing E7 alone exhibited increased levels of p53, while those expressing E6 had significantly reduced levels. The p53 present in the E7 cells was active, as increased levels of p21 were observed. This suggested that E7 bypassed the mitotic checkpoint by a p53-independent mechanism. The levels of MDM2, a cellular oncoprotein also implicated in control of the mitotic checkpoint, were significantly elevated in the E7 cells compared to the normal HFKs. In E6-expressing cells, the levels of MDM2 were undetectable. It is possible that abrogation of Rb function by E7 or increased expression of MDM2 contributes to the loss of mitotic spindle checkpoint control in the E7 cells. These findings suggest mechanisms by which both HPV oncoproteins contribute to genomic instability at the mitotic checkpoint.

Animals↗
PubMed1998

Activation of papillomavirus late gene transcription and genome amplification upon differentiation in semisolid medium is coincident with expression of involucrin and transglutaminase but not keratin-10.

The life cycle of the papillomaviruses is closely linked to host cell differentiation, as demonstrated by the fact that amplification of viral DNA and transcription of late genes occur only in the suprabasal cells of a differentiated epithelium. Previous studies examining the pathogenesis of papillomavirus infections have relied on the use of organotypic raft cultures or lesions from patients to examine these differentiation-dependent viral activities. In this study, we used a simple system for epithelial differentiation to study human papillomavirus (HPV) late functions. We demonstrate that the suspension of HPV-infected keratinocytes in semisolid medium containing 1.6% methylcellulose for 24 h was sufficient for the activation of the late promoter, transcription of late genes, and amplification of viral DNA. These activities were shown to be linked to and coincide with cellular differentiation. Expression of the late protein E1(wedge)E4 and amplification of viral DNA were detected in the identical set of cells after suspension in methylcellulose. This technique was also used to analyze the differentiation properties of the cells which expressed the late protein E1(wedge)E4. While induction of the spinous layer markers involucrin and transglutaminase was compatible with late promoter induction, expression of the differentiation-specific keratin-10 was shown not to be required for HPV late functions. Interestingly, while the majority of normal human keratinocytes induced filaggrin expression by 24 h, this marker of the granular layer was induced in a smaller subset of HPV type 31 (HPV-31)-positive cells at this time point. The HPV-31-positive cells which expressed filaggrin did not induce the late protein E1(wedge)E4. Use of the methylcellulose system to induce epithelial differentiation coupled with the ability to perform a genetic analysis of HPV functions by using transfection of cloned viral DNA will facilitate the study of the regulation of the papillomavirus life cycle.

Cells, Cultured↗