Springer protocols feed by Cancer Research 
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This is an RSS file. You can use it to subscribe to this data in your favourite RSS reader or to display this data on your own website or blog.Gene Gun Her2/neu DNA Vaccination: Evaluation of Vaccine Efficacy in a Syngeneic Her2/neu Mouse Tumor Model
Genetic vaccination using naked plasmid DNA is an immunization strategy both against infectious diseases and cancer. In order to improve the efficacy of DNA vaccines, particularly in large animals and humans, different strategies have been pursued. These vaccination strategies are based on different application routes, schedules, and coexpression of immunomodulatory molecules as adjuvants. Our mouse tumor model offers the possibility to investigate Her2/neu DNA vaccines in different settings, i.e., intramuscular or intradermal application with or without coexpression of adjuvants. Protection from tumor growth in tumor chal...
Source: Springer protocols feed by Cancer Research - June 26, 2015 Category: Cancer & Oncology Source Type: news
Aptamer Targeting the ERBB2 Receptor Tyrosine Kinase for Applications in Tumor Therapy
Aptamers are an emerging class of molecules in cancer therapy. They can be easily synthesized and are considered cost-effective drug candidates. In this book chapter we describe the selection and characterization of DNA aptamers specific to the human epidermal growth factor receptor 2 (ERBB2/HER2), an oncogenic tyrosine kinase. First, a DNA aptamer library is applied and ERBB2-specific aptamers are selected using SELEX. Binders are subcloned into a pGEM-T vector, sequenced, and characterized using biochemical and cell biological techniques. By multimerizing the selected ERBB2 aptamers, it might be possible to significantly...
Source: Springer protocols feed by Cancer Research - June 26, 2015 Category: Cancer & Oncology Source Type: news
Molecular Analysis of Human Papillomavirus Virus-Like Particle Activated Langerhans Cells In Vitro
Langerhans cells (LC) are the resident antigen-presenting cells in human epithelium, and are therefore responsible for initiating immune responses against human papillomaviruses (HPV) entering the epithelial and mucosal layers in vivo. Upon proper pathogenic stimulation, LC become activated causing an internal signaling cascade that results in the up-regulation of co-stimulatory molecules and the release of inflammatory cytokines. Activated LC then migrate to lymph nodes where they interact with antigen-specific T cells and initiate an adaptive T-cell response. However, HPV manipulates LC in a suppressive manner that alter...
Source: Springer protocols feed by Cancer Research - November 12, 2014 Category: Cancer & Oncology Source Type: news
Retroviral Expression of Human Cystatin Genes in HeLa Cells
Retroviral gene transfer is a highly efficient and effective method of stably introducing genetic material into the genome of specific cell types. The process involves the transfection of retroviral expression vectors into a packaging cell line, the isolation of viral particles, and the infection of target cell lines. Compared to traditional gene transfer methods such as liposome-mediated transfection, retroviral gene transfer allows for stable gene expression in cell populations without the need for lengthy selection and cloning procedures. This is particularly helpful when studying gene products that have negative effect...
Source: Springer protocols feed by Cancer Research - November 12, 2014 Category: Cancer & Oncology Source Type: news
A High-Throughput Cellular Assay to Quantify the p53-Degradation Activity of E6 from Different Human Papillomavirus Types
A subset of human papillomaviruses (HPVs), known as the high-risk types, are the causative agents of cervical cancer and other malignancies of the anogenital region and oral mucosa. The capacity of these viruses to induce cancer and to immortalize cells in culture relies in part on a critical function of their E6 oncoprotein, that of promoting the poly-ubiquitination of the cellular tumor suppressor protein p53 and its subsequent degradation by the proteasome. Here, we describe a cellular assay to measure the p53-degradation activity of E6 from different HPV types. This assay is based on a translational fusion of p53 to Re...
Source: Springer protocols feed by Cancer Research - November 12, 2014 Category: Cancer & Oncology Source Type: news
Robust HPV-18 Production in Organotypic Cultures of Primary Human Keratinocytes
The productive program of the human papillomaviruses takes place in terminally differentiating squamous epithelia. In this chapter, we provide the protocols for robust production of HPV-18 in organotypic cultures of early passages of primary human keratinocytes. A critical step is the generation of genomic HPV plasmids in vivo by using Cre-loxP-mediated excisional recombination from a vector plasmid. We discuss the rationale for this approach. This system produces high yields of infectious virus and facilitates genetic analyses of HPV protein functions and their regulation in the context of recapitulated host tissue enviro...
Source: Springer protocols feed by Cancer Research - November 12, 2014 Category: Cancer & Oncology Source Type: news
Genetic Methods for Studying the Role of Viral Oncogenes in the HPV Life Cycle
Human papillomaviruses are the causative agents of several cancers, but only a minority of HPV infections progress to malignancy. In order to better understand HPV biology during the normal, differentiation-dependent life cycle, a cell culture model that maintains the complete episomal genome and permits host cell differentiation is critical. Furthermore, the use of cloned DNA as a starting material is important to facilitate genetic analyses. In this chapter, procedures for isolating human keratinocytes, establishing cell lines maintaining HPV16 genomes, and inducing cellular differentiation, which permits analysis of bot...
Source: Springer protocols feed by Cancer Research - November 12, 2014 Category: Cancer & Oncology Source Type: news
Methods to Assess the Nucleocytoplasmic Shuttling of the HPV E1 Helicase and Its Effects on Cellular Proliferation and Induction of a DNA Damage Response
Replication of the human papillomavirus (HPV) double-stranded DNA genome in the nucleus of infected cells relies on the viral proteins E1 and E2 in conjunction with the host DNA replication machinery. This process is tightly linked to the replication of cellular DNA, in part through the cyclin-dependent phosphorylation of E1, which inhibits its export out of the nucleus to promote its accumulation in this compartment during S-phase. It has been recently shown that accumulation of E1 in the nucleus, while a prerequisite for viral DNA replication, leads to the inhibition of cellular proliferation and the activation of a DNA ...
Source: Springer protocols feed by Cancer Research - November 12, 2014 Category: Cancer & Oncology Source Type: news
HPV Binding Assay to Laminin-332/Integrin α6β4 on Human Keratinocytes
Human papillomaviruses (HPVs) have been shown to bind to Laminin-332 (Ln-332) on the extracellular matrix (ECM) secreted by human keratinocytes. The assay described here is an important tool to study HPV receptor binding to the ECM. The assay can also be modified to study the receptors required for HPV infection and for binding to tissues. We previously showed that Ln-332 is essential for the binding of HPV11 to human keratinocytes and that infectious entry of HPV11 requires α6β4 integrin for the transfer of HPV11 from ECM to host cells (Culp et al., J Virol 80:8940–8950, 2006). We also demonstrated that s...
Source: Springer protocols feed by Cancer Research - November 12, 2014 Category: Cancer & Oncology Source Type: news
Replication of Human Papillomavirus in Culture
Human papillomaviruses (HPV) are the major factor in causing cervical cancer as well as being implicated in causing oral and anal cancers. The life cycle of HPV is tied to the epithelial differentiation system, as only native virus can be produced in stratified human skin. Initially, HPV research was only possible utilizing recombinant systems in monolayer culture. With new cell culture technology, systems using differentiated skin have allowed HPV to be studied in its native environment. Here, we describe current research studying native virions in differentiated skin including viral assembly, maturation, capsid protein i...
Source: Springer protocols feed by Cancer Research - November 12, 2014 Category: Cancer & Oncology Source Type: news
A Real-Time PCR Approach Based on SPF1 Primers and the INNO-LiPA HPV Genotyping Extra Assay for the Detection and Typing of Human Papillomavirus
A highly sensitive SPF10 real-time PCR was developed to achieve simultaneous amplification and detection of the human papillomavirus (HPV) target. That way, LiPA analysis of the HPV-negative samples can be avoided, reducing workload and cost. Here, we describe in detail a SYBR Green I-based real-time PCR assay based on SPF10 primers using the LightCycler® 480 system to generate and detect HPV amplicons, which are compatible with the LiPA assay. (Source: Springer protocols feed by Cancer Research)
Source: Springer protocols feed by Cancer Research - November 12, 2014 Category: Cancer & Oncology Source Type: news
Evolution and Classification of Oncogenic Human Papillomavirus Types and Variants Associated with Cervical Cancer
The nomenclature of human papillomavirus (HPV) is established by the International Committee on Taxonomy of Virus (ICTV). However, the ICTV does not set standards for HPV below species levels. This chapter describes detailed genotyping methods for determining and classifying HPV variants. (Source: Springer protocols feed by Cancer Research)
Source: Springer protocols feed by Cancer Research - November 12, 2014 Category: Cancer & Oncology Source Type: news
Homogeneous, Bioluminescent Proteasome Assays
Protein degradation is mediated predominantly through the ubiquitin–proteasome pathway. The importance of the proteasome in regulating degradation of proteins involved in cell-cycle control, apoptosis, and angiogenesis led to the recognition of the proteasome as a therapeutic target for cancer [1–6]. The proteasome is also essential for degrading misfolded and aberrant proteins, and impaired proteasome function has been implicated in neurodegerative and cardiovascular diseases [7, 8]. Robust, sensitive assays are essential for monitoring proteasome activity and for developing inhibitors of the proteasome. Pepti...
Source: Springer protocols feed by Cancer Research - October 15, 2014 Category: Cancer & Oncology Source Type: news
Detection of p53 Protein Transcriptional Activity by Chromatin Immunoprecipitation
p53 is a key transcriptional mediator that controls the expression of hundreds of target genes necessary to maintain cellular homeostasis and genome integrity. An important cellular function that is dependent on p53 transcriptional activity is apoptosis or programmed cell death. Indeed, inhibition of p53 transcriptional activity is often observed in cancers as a result of mutations within its DNA-binding domain. In this chapter, we describe the use of chromatin immunoprecipitation and real-time quantitative polymerase chain reaction to detect p53 transcriptional activity in cancer cells and tumor tissues. This technique en...
Source: Springer protocols feed by Cancer Research - October 15, 2014 Category: Cancer & Oncology Source Type: news
Detection of p53 Protein Aggregation in Cancer Cell Lines and Tumor Samples
The p53 protein plays a central role in regulating apoptosis. The loss of functional p53 is common in many cancers. In cancer cells, the dysfunctional p53 protein often maintains a misfolded, inactive conformation due to genetic mutations or posttranslational deregulation. The misfolded p53 protein can aggregate and form amyloid-like oligomers and fibrils, which abrogate the pro-apoptotic functions of p53. Therefore, the aggregation of p53 may be a crucial factor in carcinogenesis, tumor progression, and the response of cancer cells to apoptotic signals. In this chapter, we provide details on various methods for detecting ...
Source: Springer protocols feed by Cancer Research - October 15, 2014 Category: Cancer & Oncology Source Type: news
