Alfred B. Jenson

Ancient papillomavirus-host co-speciation in Felidae.

BackgroundEstimating evolutionary rates for slowly evolving viruses such as papillomaviruses (PVs) is not possible using fossil calibrations directly or sequences sampled over a time-scale of decades. An ability to correlate their divergence with a host species, however, can provide a means to estimate evolutionary rates for these viruses accurately. To determine whether such an approach is feasible, we sequenced complete feline PV genomes, previously available only for the domestic cat (Felis domesticus, FdPV1), from four additional, globally distributed feline species: Lynx rufus PV type 1, Puma concolor PV type 1, Panthera leo persica PV type 1, and Uncia uncia PV type 1.ResultsThe feline PVs all belong to the Lambdapapillomavirus genus, and contain an unusual second noncoding region between the early and late protein region, which is only present in members of this genus. Our maximum likelihood and Bayesian phylogenetic analyses demonstrate that the evolutionary relationships between feline PVs perfectly mirror those of their feline hosts, despite a complex and dynamic phylogeographic history. By applying host species divergence times, we provide the first precise estimates for the rate of evolution for each PV gene, with an overall evolutionary rate of 1.95 × 10-8 (95% confidence interval 1.32 × 10-8 to 2.47 × 10-8) nucleotide substitutions per site per year for the viral coding genome.ConclusionOur work provides evidence for long-term virus-host co-speciation of feline PVs, indicating that viral diversity in slowly evolving viruses can be used to investigate host species evolution. These findings, however, should not be extrapolated to other viral lineages without prior confirmation of virus-host co-divergence.

Mouse Fibroblasts Lacking RB1 Function Form Spheres and Undergo Reprogramming to a Cancer Stem Cell Phenotype

Activation of the RB1 pathway triggers the cell-cycle arrest that mediates cell-cell contact inhibition. Accordingly, mutation of all three RB1 family members leads to loss of contact inhibition and outgrowth of fibroblasts into spheres where cell-cell contacts predominate. We present evidence that such outgrowth triggers reprogramming to generate cells with properties of cancer stem cells. Fibroblasts with only a single RB1 mutation remain contact inhibited; however, if this contact inhibition is bypassed by forcing the RB1(-/-) cells to form spheres in suspension, cells with properties of cancer stem cells are also generated. These cells not only form tumors in nude mice but also generate differentiated cells. We propose that contact inhibition imposed by the RB1 pathway performs an unexpected tumor suppressor function by preventing cell outgrowth into structures where cells with properties of cancer stem cells can be generated from differentiated somatic cells in advancing cancers.

Human papillomavirus in metastatic squamous carcinoma from unknown primaries in the head and neck: A retrospective 7 year study

GOAL Human Papillomavirus (HPV) is found to be increasingly implicated in head and neck cancers. The objective of this study was to determine the primary site of origin of HPV positive squamous carcinomas metastatic to lymph nodes of the neck. METHODS Surgical pathology records from January 1, 2000 to July 31, 2007 were used to identify surgically removed neck lymph nodes with the diagnosis of metastatic squamous carcinoma. Specimens in formalin-fixed, paraffin-embedded blocks were examined for HPV (+) by analyzing sequencing data generated by PCR and immunostaining for the expression of the p16INK biomarker, which is overexpressed if Rb is not present. H & E stained slides were also reviewed for histological classification. The available retrospective demographics were extracted from the charts to determine trends of confounding factors. RESULTS Of the 43 patient samples, 41 contained adequate DNA to test for HPV. The mean age of the 41 patients was 62 years. All of the patients smoked and 39/41 patients consumed alcohol. The overall HPV (+) incident rate was 27% (11/41) by PCR with strongly diffuse or strong focal p16 staining. 9 of the 34 males and 2 of the 7 females had HPV (+) carcinomas. The average age of the 2 HPV (+) females was 44, compared to the HPV (-) females who averaged 70. The average age of the HPV (+) males was 56 compared with the average age 55 of the HPV (-) males. HPV (+) carcinomas appeared to arise from multiple sites in the oropharynx, particularly the tonsils and tongues, including unknown primaries. By histological exam, most metastatic HPV(+) squamous carcinomas were poorly differentiated (basaloid) microscopically and grossly cystic. CONCLUSION The 27% HPV (+) squamous cancers metastatic to neck lymph node originated from multiple sites in the oropharynx. The HPV (+) female population, although a total of only 2, tended to be much younger than the HPV (-) ones, whereas the HPV (+) male population was similar in age to the HPV (-) male population.

Detection of Cervical Cancer Biomarker Patterns in Blood Plasma and Urine by Differential Scanning Calorimetry and Mass Spectrometry

Improved methods for the accurate identification of both the presence and severity of cervical intraepithelial neoplasia (CIN) and extent of spread of invasive carcinomas of the cervix (IC) are needed. Differential scanning calorimetry (DSC) has recently been shown to detect specific changes in the thermal behavior of blood plasma proteins in several diseases. This methodology is being explored to provide a complementary approach for screening of cervical disease. The present study evaluated the utility of DSC in differentiating between healthy controls, increasing severity of CIN and early and advanced IC. Significant discrimination was apparent relative to the extent of disease with no clear effect of demographic factors such as age, ethnicity, smoking status and parity. Of most clinical relevance, there was strong differentiation of CIN from healthy controls and IC, and amongst patients with IC between FIGO Stage I and advanced cancer. The observed disease-specific changes in DSC profiles (thermograms) were hypothesized to reflect differential expression of disease biomarkers that subsequently bound to and affected the thermal behavior of the most abundant plasma proteins. The effect of interacting biomarkers can be inferred from the modulation of thermograms but cannot be directly identified by DSC. To investigate the nature of the proposed interactions, mass spectrometry (MS) analyses were employed. Quantitative assessment of the low molecular weight protein fragments of plasma and urine samples revealed a small list of peptides whose abundance was correlated with the extent of cervical disease, with the most striking plasma peptidome data supporting the interactome theory of peptide portioning to abundant plasma proteins. The combined DSC and MS approach in this study was successful in identifying unique biomarker signatures for cervical cancer and demonstrated the utility of DSC plasma profiles as a complementary diagnostic tool to evaluate cervical cancer health.

The ZEB1 Transcription Factor Acts in a Negative Feedback Loop with miR200 Downstream of Ras and Rb1 to Regulate Bmi1 Expression

Background: Oncogenic Ras muations in cancer cells cause tumor-suppressing senescence in primary cells through repression of Bmi1. Results: Rb1 pathway status regulates a ZEB1-miR-200 loop downstream of Ras to control expression of Bmi1. Conclusion: Rb1 and ZEB1-miR200 are key downstream effectors of Ras that act on Bmi1. Significance: Rb1 and ZEB1-miR-200 link Ras to Bmi1 to regulate a cellular choice between oncogene-induced senescence and tumor progression when Ras is mutated. Ras mutations are frequent in cancer cells where they drive proliferation and resistance to apoptosis. However in primary cells, mutant Ras instead can cause oncogene-induced senescence, a tumor suppressor function linked to repression of the polycomb factor Bmi1, which normally regulates cell cycle inhibitory cyclin-dependent kinase inhibitors (cdki). It is unclear how Ras causes repression of Bmi1 in primary cells to suppress tumor formation while inducing the gene in cancer cells to drive tumor progression. Ras also induces the EMT transcription factor ZEB1 to trigger tumor invasion and metastasis. Beyond its well-documented role in EMT, ZEB1 is important for maintaining repression of cdki. Indeed, heterozygous mutation of ZEB1 is sufficient for elevated cdki expression, leading to premature senescence of primary cells. A similar phenotype is evident with Bmi1 mutation. We show that activation of Rb1 in response to mutant Ras causes dominant repression of ZEB1 in primary cells, but loss of the Rb1 pathway is a hallmark of cancer cells and in the absence of such Rb1 repression Ras induces ZEB1 in cancer cells. ZEB1 represses miR-200 in the context of a mutual repression loop. Because miR-200 represses Bmi1, induction of ZEB1 leads to induction of Bmi1. Rb1 pathway status then dictates the opposing effects of mutant Ras on the ZEB1-miR-200 loop in primary versus cancer cells. This loop not only triggers EMT, surprisingly we show it acts downstream of Ras to regulate Bmi1 expression and thus the critical decision between oncogene-induced senescence and tumor initiation.

Human papillomavirus in various lesions of the head and neck.

The association of human papillomavirus with benign and malignant epithelial lesions of the head and neck has been studied by a peroxidase-antiperoxidase technique having immunospecificity against genus-specific structural antigens of the papillomaviruses. More than 360 specimen blocks from 144 patients were evaluated. There was evidence of human papillomavirus antigen in three out of eight patients with childhood-onset laryngeal papillomas (37.5%) and in four out of eight patients with adult-onset papillomas (50%). A patient with an unusual flat, wartlike lesion appearing as an oral cavity leukoplakia had detectable papillomavirus antigen in it. None of the 13 cases of inverting papilloma or any of the malignant lesions studied showed evidence for the presence of papillomavirus antigen. There is currently only suggestive evidence for the oncogenic potential of human papillomavirus in the head and neck.

Different thresholds of ZEB1 are required for Ras-mediated tumour initiation and metastasis

Ras pathway mutation is frequent in carcinomas where it induces expression of the transcriptional repressor ZEB1. Although ZEB1 is classically linked to epithelial-mesenchymal transition and tumour metastasis, it has an emerging second role in generation of cancer-initiating cells. Here we show that Ras induction of ZEB1 is required for tumour initiation in a lung cancer model, and we link this function to repression Pten, whose loss is critical for emergence of cancer-initiating cells. These two roles for ZEB1 in tumour progression can be distinguished by their requirement for different levels of ZEB1. A lower threshold of ZEB1 is sufficient for cancer initiation, whereas further induction is necessary for tumour metastasis.

Sequential Inductions of the ZEB1 Transcription Factor Caused by Mutation of Rb and Then Ras Proteins Are Required for Tumor Initiation and Progression

Background: Ras mutation drives tumor initiation as well as invasion. Results: The ZEB1 transcription factor is sequentially induced with mutation of Rb1 and Ras, and these inductions are required for Ras-mediated tumor initiation and then invasion. Conclusion: ZEB1 plays a critical role in initiation and progression of Ras-mediated tumors. Significance: Induction ZEB1 is important for tumor initiation and invasion in a model of Ras-initiated cancer. Rb1 restricts cell cycle progression, and it imposes cell contact inhibition to suppress tumor outgrowth. It also triggers oncogene-induced senescence to block Ras mutation. Loss of the Rb1 pathway, which is a hallmark of cancer cells, then provides a permissive environment for Ras mutation, and Ras is sufficient for invasive tumor formation in Rb1 family mutant mouse embryo fibroblasts (MEFs). These results demonstrate that sequential mutation of the Rb1 and Ras pathways comprises a tumor initiation axis. Both Rb1 and Ras regulate expression of the transcription factor ZEB1, thereby linking tumor initiation to the subsequent invasion and metastasis, which is induced by ZEB1. ZEB1 acts in a negative feedback loop to block expression of miR-200, which is thought to facilitate tumor invasion and metastasis. However, ZEB1 also represses cyclin-dependent kinase (cdk) inhibitors to control the cell cycle; its mutation in MEFs leads to induction of these inhibitors and premature senescence. Here, we provide evidence for two sequential inductions of ZEB1 during Ras transformation of MEFs. Rb1 constitutively represses cdk inhibitors, and induction of ZEB1 when the Rb1 pathway is lost is required to maintain this repression, allowing for the classic immortalization and loss of cell contact inhibition seen when the Rb1 pathway is lost. In vivo, we show that this induction of ZEB1 is required for Ras-initiated tumor formation. ZEB1 is then further induced by Ras, beyond the level seen with Rb1 mutation, and this Ras superinduction is required to reach a threshold of ZEB1 sufficient for repression of miR-200 and tumor invasion.

Macaca fascicularis papillomavirus type 1: a non-human primate betapapillomavirus causing rapidly progressive hand and foot papillomatosis.

Papillomaviruses (PVs) are a group of small, non-enveloped DNA viruses that cause mucosal or cutaneous neoplasia in a variety of animals. Whilst most papillomas will regress spontaneously, some may persist or undergo malignant transformation. In this study, aggressive, persistent and extensive warts were observed on the hands and feet of a cynomolgus macaque (Macaca fascicularis). The presence of PV in the wart biopsies was identified by immunohistochemistry and PCR amplification of PV DNA. The genomic DNA of this PV was cloned and sequenced, and the PV was designated M. fascicularis papillomavirus type 1 (MfPV-1). Its genome was 7588 bp in length and the organization of its putative open reading frames (E1, E2, E6, E7, L1, L2 and E4) was similar to that of other PVs. MfPV-1 had a short non-coding region (NCR) of 412 bp. Molecular analysis of MfPV-1 genomic DNA classified it into the genus Betapapillomavirus, to which all epidermodysplasia verruciformis (EV)-type PVs belong. Diseases caused by PVs of the genus Betapapillomavirus are usually associated with natural or iatrogenic immunosuppression. The genomic characterization performed in this study showed that MfPV-1 clustered within the genus Betapapillomavirus and also contained EV-type-specific motifs in its NCR. Further characterization of this virus and its host interactions may allow us to develop a non-human primate model for human betapapillomaviruses, a genus populated by human PV types causing EV.

Papillomavirus antibody prevalence in free-ranging and captive bottlenose dolphins (Tursiops truncatus).

Genital epithelial tumors of Atlantic bottlenose dolphins (Tursiops truncatus [Tt]) and Burmeisters porpoises (Phocoena spinipinnis) were formerly shown to be associated with papillomavirus (PV) infection. Papillomaviruses are highly prevalent viruses involved in the development of various tumor types in a wide range of animals, and so-called high-risk PVs contribute to malignant progression. In marine mammals, the incidence and prevalence of PV infection, transmission pathways, and persistence of infection are largely unknown. Using virus-like particles of bottlenose dolphin PV type 1 (TtPV1) as the antigen, enzyme-linked immunosorbent assay (ELISA) studies were conducted to evaluate PV antibody prevalence in bottlenose dolphins. In total, sera obtained from 115 dolphins were examined. Fifty-one percent of captive dolphins (n=18 of 35) and 90% of free-ranging dolphins (n=72 of 80) were antibody positive. Higher ELISA reactivity was observed among males compared with females. Sexually immature dolphins appeared more likely to seroconvert with age. Besides determining their PV antibody prevalence, each animal was also assessed for the presence of orogenital tumors. Interestingly, the mean age of free-ranging dolphins with tumors (n=21) was 11.2 yr compared with 29.9 yr in captive dolphins with tumors (n=9). Results from the current study suggest PV infection in bottlenose dolphins is common, that the main route of PV transmission among them may be horizontal, and that orogenital neoplasia may develop in early life stages of certain free-ranging bottlenose dolphins.