The papillomaviruses are small DNA viruses that encode approximately eight genes, and require the host cell DNA replication machinery for their viral DNA replication. Thus papillomaviruses have evolved strategies to induce host cell DNA synthesis balanced with strategies to protect the cell from unscheduled replication. While the papillomavirus E1 and E2 genes are directly involved in viral replication by binding to and unwinding the origin of replication, the E6 and E7 proteins have auxillary functions that promote proliferation. As a consequence of disrupting the normal checkpoints that regulate cell cycle entry and progression, the E6 and E7 proteins play a key role in the oncogenic properties of human papillomaviruses with a high risk of causing anogenital cancers (HR HPVs). As a consequence, E6 and E7 of HR HPVs are invariably expressed in cervical cancers. This article will focus on the E6 protein and its numerous activities including inactivating p53, blocking apoptosis, activating telomerase, disrupting cell adhesion, polarity and epithelial differentiation, altering transcription and reducing immune recognition.

Overcoming senescence signals in somatic cells is critical to cellular immortalization and carcinogenesis. High-risk human papillomavirus (HPV) can immortalize epithelial cells in culture through degradation of the retinoblastoma protein by HPV E7 and activation of hTERT transcription, the catalytic subunit of telomerase, by the heterodimer HPV E6/E6-associated protein (E6AP). Recent work in our laboratory identified a novel repressor of hTERT transcription, NFX1-91, which is targeted for ubiquitinmediated degradation by HPV type 16 (HPV16) E6/E6AP. In contrast, NFX1-123, a splice variant NFX1, increased expression from an hTERT promoter that was activated by HPV16 E6/E6AP. Here, we show that HPV16 E6 bound both NFX1-91 and NFX1-123 through the common central domain of NFX1 in the absence of E6AP. NFX1-123 positively regulated hTERT expression, as its knockdown decreased hTERT mRNA levels and telomerase activity and its overexpression increased telomerase activity. We identified new protein partners of NFX1-123, including several cytoplasmic poly(A) binding proteins (PABPCs) that interacted with NFX1-123 through its N-terminal PAM2 motif, a protein domain characteristic of other PABPC protein partners. Furthermore, NFX1-123 and PABPCs together had a synergistic stimulatory effect on hTERT-regulated reporter assays. The data suggest that NFX1-123 is integral to hTERT regulation in HPV16 E6-expressing epithelial cells and that the interaction between NFX1-123 and PABPCs is critical to hTERT activity.Normally, somatic cells undergo a finite series of population doublings before entering cellular senescence (24,25). A critical marker of a cell's age is the length of its telomeric DNA (1); with each cellular division, up to 200 nucleotides of DNA are lost at the ends of chromosomes (23,41). Cells that require infinite replicative potential, such as stem cells, protect their telomeric DNA from erosion by constitutively expressing telomerase, a ribonucleoprotein complex that extends telomeric DNA, and thus, these cells avoid senescence. Tumors also overcome cellular senescence in order to continue their growth (22), and many activate telomerase through up-regulation of hTERT, the catalytic subunit of telomerase (63). Thus, hTERT expression and telomerase activity are critical in cellular immortalization and carcinogenesis.Various proteins have been shown to be important regulators of hTERT. They include those that act as transcriptional repressors, including p53, p73, AP-1, and Menin (45, 59, 62, 67), as well as transcriptional activators, such as N-terminally truncated p73, c-Myc, and Sp1 (5,56,57,68,70,74). c-Myc and Sp1 have been shown to bind to the core hTERT promoter and increase hTERT mRNA levels (56,57,70,74), although Sp1 and Sp3 can also recruit histone deactylase to the hTERT promoter to repress expression (73). c-Myc and Sp1 have been found to affect hTERT, but their relative protein levels do not always correlate with the downstream hTERT mRNA and protein expression levels (17,57,69). Other important facto...

The high-risk human papillomavirus (HR HPV) E6 oncoprotein binds host cell proteins to dysregulate multiple regulatory pathways, including apoptosis and senescence. HR HPV16 E6 (16E6) interacts with the cellular protein NFX1-123, and together they posttranscriptionally increase hTERT expression, the catalytic subunit of telomerase. NFX1-123 interacts with hTERT mRNA and stabilizes it, leading to greater telomerase activity and the avoidance of cellular senescence. Little is known regarding what other transcripts are dependent on or augmented by the association of NFX1-123 with 16E6. Microarray analysis revealed enhanced expression of Notch1 mRNA in 16E6-expressing keratinocytes when NFX1-123 was overexpressed. A moderate increase in Notch1 mRNA was seen with overexpression of NFX1-123 alone, but with 16E6 coexpression the increase in Notch1 was enhanced. The PAM2 motif and R3H protein domains in NFX1-123, which were important for increased hTERT expression, were also important in the augmentation of Notch1 expression by 16E6. These findings identify a second gene coregulated by 16E6 and NFX1-123 and the protein motifs in NFX1-123 that are important for this effect. Human papillomaviruses (HPVs) are nonenveloped doublestranded DNA viruses. Of the more than 150 HPVs that have been identified to date, over 30 types are capable of infecting the genital and oral mucosa. HPVs that infect mucosa are divided into low-risk (LR) and high-risk (HR) categories based on their associated risk for anogenital and, increasingly, head and neck cancers (1-11). As these HR HPV infections are linked to malignancies, much focus has been placed on studying the cellular changes these viruses trigger that lead to cancer and on studying the functions of two oncogenes expressed by HR HPV, E6 and E7.HR E6 induces oncogenic changes primarily through its interactions with host cell proteins, and those interactions block apoptotic signaling and drive cellular immortalization (for a review, see reference 12). E6-associated protein (E6AP) is an E3 ubiquitin ligase that is the most well-studied protein partner of HR E6 (13-19). However, HR E6 interacts with other cellular proteins, such as NFX1 splice variants, and these are important in oncogenesis as well (20,21).In human epithelial cells, NFX1 is a gene expressed as two splice variants, . NFX1-91 has been shown to transcriptionally repress telomerase expression in epithelial cells, and HR HPV 16E6 with E6AP targets NFX1-91 for degradation (22,23). Our work has focused on NFX1-123, the longer splice variant of NFX1, and on its role with 16E6 in oncogenesis. Previously we had shown that 16E6 bound NFX1-123 in its central domain, but unlike NFX1-91 it was not degraded; together, 16E6 and NFX1-123 increased hTERT expression and telomerase activity in human foreskin keratinocytes (HFKs) through a posttranscriptional mechanism (20). 16E6 and NFX1-123 increased hTERT by utilizing both the PAM2 motif and R3H domain of the NFX1-123 protein. The N-terminal PAM2 motif is required to interact with cytopl...

High-risk human papillomavirus (HPV) E6 protein induces telomerase activity through transcriptional activation of hTERT, the catalytic subunit of telomerase. HPV type 16 (HPV16) E6 interacts with two splice variants of NFX1 to increase hTERT expression. NFX1-91 is a transcriptional repressor of hTERT that is polyubiquitinated and targeted for degradation by HPV16 E6 in concert with E6-associated protein. We previously showed that NFX1-123 augments hTERT expression through binding to cytoplasmic poly(A) binding proteins (PABPCs). In this study, we determined that unlike NFX1-91, NFX1-123 is a cytoplasmic protein that colocalized with PABPCs but does not shuttle with PABPCs between the nucleus and cytoplasm. NFX1-123 requires both its PAM2 motif, with which it binds PABPCs, and its R3H domain, which has putative nucleic acid binding capabilities, to increase hTERT mRNA levels and telomerase activity in keratinocytes expressing HPV16 E6. In keratinocytes expressing HPV16 E6 and overexpressing NFX1-123, there was increased protein expression from in vitro-transcribed RNA fused with the 5 untranslated region (5 UTR) of hTERT. This posttranscriptional increase in expression required the PAM2 motif and R3H domain of NFX1-123 as well as the coexpression of HPV16 E6. NFX1-123 bound endogenous hTERT mRNA and increased its stability in HPV16 E6-expressing human foreskin keratinocytes, and NFX1-123 increased the stability of in vitro-transcribed RNA fused with the 5 UTR of hTERT. Together, these studies describe the first evidence of posttranscriptional regulation of hTERT, through the direct interaction of the cytoplasmic protein NFX1-123 with hTERT mRNA, in HPV16 E6-expressing keratinocytes.Human papillomavirus (HPV) is a double-stranded DNA tumor virus, and persistent high-risk HPV (HR HPV) infections are associated with cervical and other anogenital cancers (3, 8-10, 19, 41). Dividing epithelial cells are needed for HPV DNA replication and amplification, but differentiated cells are needed for full HPV genome expression. Therefore, HPV drives the upper layers of stratified squamous epithelium to continue to divide as they differentiate, and the HR E6 and E7 proteins are critical to the programmatic disruption of normal epithelium, cellular immortalization in culture, and cervical cancer in mouse models (48, 52). The HR HPV E7 protein drives cells to continue through S phase by targeting pocket proteins for degradation (22, 66), thus allowing the E2F transcriptional factor to activate S-phase genes for DNA replication. The HR HPV E6 protein has several targets in epithelial cells. HR HPV E6 targets p53 for degradation (50), blocking apoptotic and cellular senescence signals due to DNA damage. HR HPV E6 also affects PDZ domain-containing proteins, such as MUPP-1, Scribble, MAGI-1, -2, and -3, PTPN3, and hDlg (17,24,30,36,44,56). Finally, HR HPV E6 blocks cellular senescence signals by activating telomerase (31, 58). The degradation of retinoblastoma protein by E7 and the activation of telomerase by E6 are two critical ste...

The hypothalamic hormone gonadotropinreleasing hormone (GnRH) is released in a pulsatile fashion, with its frequency varying throughout the reproductive cycle. Varying pulse frequencies and amplitudes differentially regulate the biosynthesis and secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) The hypothalamic decapeptide gonadotropin-releasing hormone (GnRH) plays a critical role in reproductive development and function by regulating the biosynthesis and secretion of the pituitary gonadotropins luteinizing hormone (LH) and follicle-stimulating hormone (FSH). GnRH is released into the hypophysial portal circulation and transported to the anterior pituitary, where it binds to specific, high-affinity cell surface receptors on gonadotropes, the pituitary cell type that produces gonadotropins. LH and FSH are heterodimers, each composed of a common a subunit associated noncovalently with a unique 1B subunit (1).The stimulation of gonadotropin secretion by GnRH is dependent on the pulsatile nature of GnRH delivery to the anterior pituitary. Administration of exogenous GnRH in a continuous fashion results in the down-regulation of LH and FSH secretion, whereas pulsatile GnRH stimulates LH and FSH secretion, in several species, including primates and rat (2-4). Furthermore, the frequency and amplitude of GnRH pulses secreted by the hypothalamus, which vary during different phases of the menstrual or estrous cycle, regulate differentially . Similarly, the levels of gonadotropin subunit gene expression in the rat pituitary vary by 2-to 4-fold, depending on the GnRH pulse frequency and amplitude (8)(9)(10)(11) GnRHR activation include inositol phospholipid turnover, which leads to calcium mobilization and protein kinase C activation (19,20).The mechanism by which GnRH is able to regulate differentially LH and FSH biosynthesis and secretion is unknown. Previous studies have been limited by the lack of available cell lines that express the LH and FSH subunit genes and respond to GnRH. We have taken advantage of the availability of the GnRHR cDNA to facilitate studies of the mechanisms of action of GnRH. GH3 cells are a well-characterized pituitary cell line generated from a rat pituitary adenoma that express prolactin (PRL) and growth hormone in a regulated fashion (21). These cells express thyrotropin-releasing hormone (TRH) receptors endogenously and respond to TRH with an increase in PRL gene expression, biosynthesis, and secretion (22). Like the GnRHR, the TRH receptor is a member of the family of G protein-coupled receptors and is coupled to pertussis toxin-insensitive G proteins of the Gq/11 family (23).Furthermore, like GnRH, TRH activates its receptor to stimulate inositol phospholipid turnover, leading to calcium mobilization and protein kinase C activation (24). Thus, the effects of TRH and GnRH appear to be mediated through similar intracellular signal transduction pathways.We hypothesized that GH3 cells may present a useful model for the study of the mechanisms of GnRH action...

Death-associated protein kinase (DAP-Kinase) is a novel serine/threonine kinase whose expression is required for γ interferon-induced apoptosis. A previous study suggested that DAP-Kinase expression may be lost epigenetically in cancer cell lines, because treatment of several nonexpressing cell lines with 5-aza-2′-deoxycytidine resulted in the expression of DAP-Kinase. Using methylation-specific polymerase chain reaction (MSP), we examined the DAP-Kinase CpG island for hypermethylation in cancer. Normal lymphocytes and lymphoblastoid cell lines are unmethylated in the 5′ CpG island of DAP-Kinase. However, in primary tumor samples, all Burkitt’s lymphomas and 84% of the B-cell non-Hodgkin’s lymphomas were hypermethylated in the DAP-Kinase CpG island. In contrast, none of the T-cell non-Hodgkin’s lymphoma samples and 15% or less of leukemia samples examined had hypermethylated DAP-Kinase alleles. U937, an unmethylated, DAP-Kinase–expressing leukemia cell line, was treated with γ interferon and underwent apoptosis; however, Raji, a fully methylated, DAP-Kinase nonexpressing Burkitt’s lymphoma cell line, only did so when treated with 5-aza-2′-deoxycytidine followed by γ interferon. Our findings in cell lines and primary tumors suggest that hypermethylation of the DAP-Kinase gene and loss of γ interferon-mediated apoptosis may be important in the development of B-cell malignancies and may provide a promising biomarker for B-cell–lineage lymphomas.

High-risk human papillomavirus (HR HPV) oncoproteins bind host cell proteins to dysregulate and uncouple apoptosis, senescence, differentiation, and growth. These pathways are important for both the viral life cycle and cancer development. HR HPV16 E6 (16E6) interacts with the cellular protein NFX1-123, and they collaboratively increase the growth and differentiation master regulator, Notch1. In 16E6 expressing keratinocytes (16E6 HFKs), the Notch canonical pathway genes Hes1 and Hes5 were increased with overexpression of NFX1-123, and their expression was directly linked to the activation or blockade of the Notch1 receptor. Keratinocyte differentiation genes Keratin 1 and Keratin 10 were also increased, but in contrast their upregulation was only indirectly associated with Notch1 receptor stimulation and was fully unlinked to growth arrest, increased p21Waf1/CIP1, or decreased proliferative factor Ki67. This leads to a model of 16E6, NFX1-123, and Notch1 differently regulating canonical and differentiation pathways and entirely uncoupling cellular arrest from increased differentiation.

A significant contributor to women's cancer mortality worldwide is cervical cancer, which is caused by high-risk human papillomavirus (HR HPV). The two viral oncoproteins of HR HPV, E6 and E7, partner with host cell proteins to target oncogenic proteins and pathways. Previously, we have shown HR HPV type 16 E6 (16E6) interacts with the host protein NFX1-123 to target telomerase and cellular immortalization, requiring NFX1-123 to fully upregulate telomerase activity. We now report that NFX1-123 is highly expressed in primary cervical cancers. In vitro, cells expressing 16E6 and overexpressing NFX1-123 have extended active growth, decreased senescence marker staining, and more rapid cell cycling compared to 16E6 expressing cells with endogenous amounts of NFX1-123. These findings were associated with increased telomerase activity and augmented expression of its catalytic subunit, hTERT. In complement, HPV 16 positive cervical cancer cell lines with knocked down NFX1-123 had slowed growth and reduced hTERT over time. In cells that express HR HPV E6, greater expression of NFX1-123 can modify active cellular growth and augment hTERT expression and telomerase activity over time, potentially supporting the initiation and progression of HPV-associated cancers.