Nicholas Coleman

Integration of high-risk human papillomavirus: a key event in cervical carcinogenesis?

An important occurrence in cervical carcinogenesis is deregulated expression of the high‐risk human papillomavirus (HR‐HPV) oncogenes E6 and E7. Several risk factors for cervical neoplastic progression are likely to contribute to viral oncogene deregulation, particularly integration of HR‐HPV into the host genome. Integration represents a by‐product of viral infection that is detected in almost 90% of cervical carcinomas. The mechanism of integration is not fully understood, although there is a clear predilection for chromosomal common fragile sites, most likely due to their accessibility for insertion of foreign DNA. Recent work has suggested that an important intermediate stage in cervical carcinogenesis is characterized by transcriptionally silent HR‐HPV integrants, which co‐exist with viral episomes in infected cells. As episome‐derived E2 protein inhibits integrant transcription, clearance of episomes (eg by host innate immunity) is associated with loss of integrant silencing and integrant selection. The process of integration and subsequent clonal selection of integrants can therefore be considered as two independent and biologically distinct events. Indeed, integrated HPV may be viewed as selectable because it represents a form of the virus that is resistant to host mechanisms of viral clearance, enabling infected cells to maintain viral oncogene expression and avoid cell death. Care should be taken in interpreting studies of HPV integration frequency in clinical samples, as the techniques used have assessed either the presence of integrated viral DNA or evidence of transcriptional activity from integrants, but not both. Copyright

DC-SIGNR, a DC-SIGN homologue expressed in endothelial cells, binds to human and simian immunodeficiency viruses and activates infection in trans

DC-SIGN, a C-type lectin expressed on the surface of dendritic cells (DCs), efficiently binds and transmits HIVs and simian immunodeficiency viruses to susceptible cells in trans. A DC-SIGN homologue, termed DC-SIGNR, has recently been described. Herein we show that DC-SIGNR, like DC-SIGN, can bind to multiple strains of HIV-1, HIV-2, and simian immunodeficiency virus and transmit these viruses to both T cell lines and human peripheral blood mononuclear cells. Binding of virus to DC-SIGNR was dependent on carbohydrate recognition. Immunostaining with a DC-SIGNR-specific antiserum showed that DC-SIGNR was expressed on sinusoidal endothelial cells in the liver and on endothelial cells in lymph node sinuses and placental villi. The presence of this efficient virus attachment factor on multiple endothelial cell types indicates that DC-SIGNR could play a role in the vertical transmission of primate lentiviruses, in the enabling of HIV to traverse the capillary endothelium in some organs, and in the presentation of virus to CD4-positive cells in multiple locations including lymph nodes.

Organization of Human Papillomavirus Productive Cycle during Neoplastic Progression Provides a Basis for Selection of Diagnostic Markers

ABSTRACT The productive cycle of human papillomaviruses (HPVs) can be divided into discrete phases. Cell proliferation and episomal maintenance in the lower epithelial layers are followed by genome amplification and the expression of capsid proteins. These events, which occur in all productive infections, can be distinguished by using antibodies to viral gene products or to surrogate markers of their expression. Here we have compared precancerous lesions caused by HPV type 16 (HPV16) with lesions caused by HPV types that are not generally associated with human cancer. These include HPV2 and HPV11, which are related to HPV16 (supergroup A), as well as HPV1 and HPV65, which are evolutionarily divergent (supergroups E and B). HPV16-induced low-grade squamous intraepithelial lesions (CIN1) are productive infections which resemble those caused by other HPV types. During progression to cancer, however, the activation of late events is delayed, and the thickness of the proliferative compartment is progressively increased. In many HPV16-induced high-grade squamous intraepithelial lesions (CIN3), late events are restricted to small areas close to the epithelial surface. Such heterogeneity in the organization of the productive cycle was seen only in lesions caused by HPV16 and was not apparent when lesions caused by other HPV types were compared. By contrast, the order in which events in the productive cycle were initiated was invariant and did not depend on the infecting HPV type or the severity of disease. The distribution of viral gene products in the infected cervix depends on the extent to which the virus can complete its productive cycle, which in turn reflects the severity of cervical neoplasia. It appears from our work that the presence of such proteins in cells at the epithelial surface allows the severity of the underlying disease to be predicted and that markers of viral gene expression may improve cervical screening.

Compromised lymphocytes infiltrate hepatocellular carcinoma: the role of T-regulatory cells.

Hepatocellular carcinoma (HCC) has a poor prognosis with limited therapeutic options. We propose that local immune responses in patients with HCC are held in check by tumor‐infiltrating CD4+CD25+ T‐regulatory lymphocytes (Treg cells), which suppress the activity and proliferation of effector CD4+ and CD8+ T cells. The phenotype and cell cycle status of tumor‐infiltrating lymphocytes (TILs) in HCC were analyzed via immunohistochemistry of sections from patients undergoing surgery for HCC and via flow cytometry of peripheral blood mononuclear cells and TILs isolated from patients with HCC. Circulating and tumor‐infiltrating T‐cell function and activation status were assessed via proliferation and flow cytometry. More than 96% of TILs were quiescent as measured via Mcm‐2 or Ki‐67 expression, while less than 10% of CD8+ T cells expressed perforin or granzyme B. CD4+CD25+ Treg cells comprised 8.7% (1.4–13.8) of TILs and always exceeded the proportion in distant nontumor tissue (2.4% [1.5–5.6]; P = .014). Treg cells isolated from HCC suppressed proliferation of autologous circulating CD4+CD25− cells and perforin expression and proliferation of autologous CD8+ T cells. The proportion of circulating Treg cells in patients with HCC was similar in healthy controls (7.2% [1.2–23.3] and 9.2% [1.6–30.2], respectively), but the proportion of circulating Treg cells that were also transforming growth factor β1+ was elevated in HCC compared with controls (55.5% [8.2–73.9] and 2.0% [0–4.9], respectively; P = .003). In conclusion, TILs are compromised and contain a subpopulation of suppressive CD4+CD25+Foxp3+ Treg cells. Functional deletion of tumor‐infiltrating Treg cells could enhance tumor‐specific immunotherapy. (HEPATOLOGY 2005;41:722–730.)

cis Expression of DC-SIGN Allows for More Efficient Entry of Human and Simian Immunodeficiency Viruses via CD4 and a Coreceptor

ABSTRACT DC-SIGN is a C-type lectin expressed on dendritic cells and restricted macrophage populations in vivo that binds gp120 and acts intrans to enable efficient infection of T cells by human immunodeficiency virus type 1 (HIV-1). We report here that DC-SIGN, when expressed in cis with CD4 and coreceptors, allowed more efficient infection by both HIV and simian immunodeficiency virus (SIV) strains, although the extent varied from 2- to 40-fold, depending on the virus strain. Expression of DC-SIGN on target cells did not alleviate the requirement for CD4 or coreceptor for viral entry. Stable expression of DC-SIGN on multiple lymphoid lines enabled more efficient entry and replication of R5X4 and X4 viruses. Thus, 10- and 100-fold less 89.6 (R5/X4) and NL4–3 (X4), respectively, were required to achieve productive replication in DC-SIGN-transduced Jurkat cells when compared to the parental cell line. In addition, DC-SIGN expression on T-cell lines that express very low levels of CCR5 enabled entry and replication of R5 viruses in a CCR5-dependent manner, a property not exhibited by the parental cell lines. Therefore, DC-SIGN expression can boost virus infection in cis and can expand viral tropism without affecting coreceptor preference. In addition, coexpression of DC-SIGN enabled some viruses to use alternate coreceptors like STRL33 to infect cells, whereas in its absence, infection was not observed. Immunohistochemical and confocal microscopy data indicated that DC-SIGN was coexpressed and colocalized with CD4 and CCR5 on alveolar macrophages, underscoring the physiological significance of these cis enhancement effects.

Acquisition of High-Level Chromosomal Instability Is Associated with Integration of Human Papillomavirus Type 16 in Cervical Keratinocytes

Whereas two key steps in cervical carcinogenesis are integration of high-risk human papillomavirus (HR-HPV) and acquisition of an unstable host genome, the temporal association between these events is poorly understood. Chromosomal instability is induced when HR-HPV E7 oncoprotein is overexpressed from heterologous promoters in vitro. However, it is not known whether such events occur at the “physiologically” elevated levels of E7 produced by deregulation of the homologous HR-HPV promoter after integration. Indeed, an alternative possibility is that integration in vivo is favored in an already unstable host genome. We have addressed these issues using the unique human papillomavirus (HPV) 16-containing cervical keratinocyte cell line W12, which was derived from a low-grade squamous intraepithelial lesion and thus acquired HPV16 by “natural” infection. Whereas W12 at low passage contains HPV16 episomes only, long-term culture results in the emergence of cells containing integrated HPV16 only. We show that integration of HPV16 in W12 is associated with 3′ deletion of the E2 transcriptional repressor, resulting in deregulation of the homologous promoter of the integrant and an increase in E7 protein levels. We further demonstrate that high-level chromosomal instability develops in W12 only after integration and that the forms of instability observed correlate with the physical state of HPV16 DNA and the level of E7 protein. Whereas intermediate E7 levels are associated with numerical chromosomal abnormalities, maximal levels are associated with both numerical and structural aberrations. HR-HPV integration is likely to be a critical event in cervical carcinogenesis, preceding the development of chromosomal abnormalities that drive malignant progression.

Colorectal cancer screening: prospects for molecular stool analysis

Colorectal cancer is common. As many patients present with advanced disease, an effective screening test would have substantial clinical benefits. Recent progress in understanding the biology of colorectal cancer (and of cancer cells in general) has led to possible new approaches to screening. In particular, there are prospects of developing tests based on analysis of stool, which promise improved accuracy, safety, affordability and patient compliance.

HPV: from infection to cancer

Infection with HPV (human papillomavirus) 16 is the cause of 50% or more of cervical cancers in women. HPV16 infection, however, is very common in young sexually active women, but the majority mount an effective immune response and clear infection. Approx. 10% of individuals develop a persistent infection, and it is this cohort who are at risk of cancer progression, with the development of high-grade precursor lesions and eventually invasive carcinoma. Effective evasion of innate immune recognition seems to be the hallmark of HPV infections, since the infectious cycle is one in which viral replication and release is not associated with inflammation. Furthermore, HPV infections disrupt cytokine expression and signalling with the E6 and E7 oncoproteins particularly targeting the type I IFN (interferon) pathway. High doses of IFN can overcome the HPV-mediated abrogation of signalling, and this may be the basis for the therapeutic effects on HPV infections of immune-response modulators such as the imidazoquinolones that induce high levels of type I IFNs by activation of TLR (Toll-like receptor) 7. Using the unique W12 model of cervical carcinogenesis, some of these IFN-related interactions and their relevance in the selection of cells with integrated viral DNA in cancer progression have been investigated. Our data show that episome loss associated with induction of antiviral response genes is a key event in the spontaneous selection of cervical keratinocytes containing integrated HPV16. Exogenous IFN-beta treatment of W12 keratinocytes in which the majority of the population contain episomes results only in the rapid emergence of IFN-resistant cells, loss of episome-containing cells and a selection of cells containing integrated HPV16 in which the expression of the transcriptional repressor E2 is down-regulated, but in which E6 and E7 are up-regulated.

Global microRNA profiles in cervical squamous cell carcinoma depend on Drosha expression levels

Gain of chromosome 5p is seen in over 50% of advanced cervical squamous cell carcinomas (SCCs), although the genes responsible for the selective advantage provided by this abnormality are poorly understood. In the W12 cervical carcinogenesis model, we observed that 5p gain was rapidly selected over ∼15 population doublings and was associated with the acquisition of a growth advantage and invasiveness. The most significantly upregulated transcript following 5p gain was the microRNA (miRNA) processor Drosha. In clinically progressed cervical SCC, Drosha copy‐number gain was seen in 21/36 clinical samples and 8/10 cell lines and there was a significant association between Drosha transcript levels and copy‐number gain. Other genes in the miRNA processing pathway, DGCR8, XPO5 and Dicer, showed infrequent copy‐number gain and over‐expression. Drosha copy‐number and expression were not elevated in pre‐malignant cervical squamous intraepithelial lesions. Importantly, global miRNA profiling showed that Drosha over‐expression in cervical SCC appears to be of functional significance. Unsupervised principal component analysis of a mixed panel of cervical SCC cell lines and clinical specimens showed clear separation according to Drosha over‐expression. miRNAs most significantly associated with Drosha over‐expression are implicated in carcinogenesis in other tissues, suggesting that they regulate fundamental processes in neoplastic progression. Our evidence suggests that copy‐number driven over‐expression of Drosha and consequent changes in miRNAs are likely to be important contributors to the selective advantage provided by 5p gain in cervical neoplastic progression. Copyright

Placental expression of DC-SIGN may mediate intrauterine vertical transmission of HIV.

Mechanisms of transplacental transmission of human immunodeficiency virus (HIV) are poorly understood. DC‐SIGN is a C‐type lectin able to bind HIV gp120 with high affinity, mediating HIV adsorption to the surface of dendritic cells for up to several days. Via this mechanism, DC‐SIGN significantly enhances the infection of CD4+ co‐receptor (CCR5 or CXCR4)+ T lymphocytes in trans. In this study, DC‐SIGN‐specific serum was developed to investigate the cell type responsible for the high level of DC‐SIGN RNA expression previously observed in the placenta. DC‐SIGN expression was shown on CD68+ HLA‐II+ CD14low S100+/− CD83− CD86− cmrf‐44− villous cells consistent with Hofbauer cells and also on CD68+ HLA‐II+ CD14high S100− CD83− CD86− cmrf‐44− decidual macrophages. The DC‐SIGN+ Hofbauer cells co‐express CD4 and the chemokine receptors, CCR5 and CXCR4, observations which may account for the ability of these cells to become infected with HIV. These fetal DC‐SIGN+ cells are separated by only a layer of trophoblast from both DC‐SIGN+ maternal cells and maternal blood, potential sources of HIV in infected mothers. Previous studies have suggested that this trophoblast layer is frequently breached during pregnancy. It is therefore proposed that DC‐SIGN may facilitate the transplacental transmission of HIV. Copyright