Increased High-Risk Human Papillomavirus Viral Load Is Associated With Immunosuppressed Microenvironment and Predicts a Worse Long-Term Survival in Cervical Cancer Patients.

Objectives: To evaluate the correlation between tumor-infiltrating lymphocytes (TILs) and the viral load of high-risk human papillomavirus (HR-HPV) in cervical cancer patients.

Methods: A total of 62 cervical cancer patients were recruited during 1993-1994 and assigned into four groups treated with radiotherapy alone or radiotherapy combined with chemotherapy and/or thermotherapy. Ki67+ tumor cells, CD4+, CD8+, FoxP3+, OX40+ and granzyme B+ TILs were detected by immunohistochemistry.

Prognostic significance of human papillomavirus viral load in correlation with different therapeutic modalities in cervical cancer patients.

Purpose: High-risk human papillomavirus (HR-HPV) infections were the causal factor in the development of cervical cancer, but the significance of HPV viral load in the prediction of the response to current therapeutic approaches had not reached consensus. The present study was performed to assess the high risk HPV viral load of cervical cancer patients who underwent radiotherapy alone or in combination with chemotherapy or hyperthermotherapy or both in correlation to long-term survival.

Methods: 116 cervical cancer patients were recruited and assigned into four groups of different therapeutic modalities.

A cis-acting diversification activator both necessary and sufficient for AID-mediated hypermutation.

Hypermutation of the immunoglobulin (Ig) genes requires Activation Induced cytidine Deaminase (AID) and transcription, but it remains unclear why other transcribed genes of B cells do not mutate. We describe a reporter transgene crippled by hypermutation when inserted into or near the Ig light chain (IgL) locus of the DT40 B cell line yet stably expressed when inserted into other chromosomal positions. Step-wise deletions of the IgL locus revealed that a sequence extending for 9.

Activation-induced cytidine deaminase-mediated hypermutation in the DT40 cell line.

Depending on the species and the developmental stage of B cells, activation-induced cytidine deaminase (AID) triggers immunoglobulin (Ig) gene diversification by gene conversion, hypermutation or switch recombination. The bursal B cell line DT40 usually diversifies its rearranged Ig light chain (IgL) gene by gene conversion, but disruption of the RAD51 gene paralogues or deletion of the psiV conversion donors induces hypermutation. Although not all aspects of somatic hypermutation can be studied in DT40, the compact size of the chicken IgL locus and the ability to modify the genome by targeted integration are powerful experimental advantages.

Gene function analysis using the chicken B-cell line DT40.

Quidquid agis, prudenter agas et respice finem!-Whatever you do, do it wisely and consider the goal. In consideration of that sage advice, the chicken B-cell line DT40 is an excellent model cell system to study the function of vertebrate genes. In addition to being highly amenable to gene manipulations, the recent influx of genome and gene/protein resources allows for the straightforward selection, design, and targeting of candidate genes for knockout analysis.

Protein evolution by hypermutation and selection in the B cell line DT40.

Genome-wide mutations and selection within a population are the basis of natural evolution. A similar process occurs during antibody affinity maturation when immunoglobulin genes are hypermutated and only those B cells which express antibodies of improved antigen-binding specificity are expanded. Protein evolution might be simulated in cell culture, if transgene-specific hypermutation can be combined with the selection of cells carrying beneficial mutations.

Biotechnology and the chicken B cell line DT40.

Protein optimization is a major focus of the biotech and pharmaceutical industry. Various in vitro technologies have been developed to accelerate protein evolution and to achieve protein optimization of functional characteristics such as substrate specificity, enzymatic activity and thermostability. The chicken B cell line DT40 diversifies its immunoglobulin (Ig) gene by gene conversion and somatic hypermutation.

Isolation of nuclear and cytoplasmic proteins from DT40 cells.

Nuclear and cytoplasmic proteins from DT40 B cells are extracted by serial fractionation in this protocol. The protein extracts are suitable for the detection of DNA-protein interaction, protein-protein interaction, DNase I footprinting analysis, and related techniques. This is a general protocol that was adapted as shown here to our use due to the fragile nature of DT40 cells and their nuclei.

Targeted transfection of DT40 cells.

In order to understand the function of genes, transfection can be used as a method in which artificially prepared knockout and expression constructs are being introduced into cell lines. Since many genes are essential for embryonic development and a homozygous deletion results in non-viable embryos, gene disruption in a cell line by using transfected constructs might be an alternative choice. Electroporation is often used to stably transfect knockout and knockin vectors into DT40 cell line.

Subcloning Dt40 by limiting dilution.

Subcloning by limited dilution can be used to derive clonally related cell populations from a heterogeneous DT40 cell culture. For example, if one suspects that a drug resistant population may represent the progeny of more than one transfectant, the protocol can be used to isolate genetically homogeneous mutant clones. Other uses are the excision of floxed DNA sequences after Cre recombinase expression or fluctuation analysis to determine mutation rates (see Protocols 'Excision of floxed-DNA sequences by transient induction of Mer-Cre-Mer' and 'Analysis of sIgM expression by FACS').

Target screening by PCR.

After the stable transfection of the knockout vector, the transfectants successful for targeted integration can be screened by PCR. A primer located upstream of the 5' targeting arm of the construct can be used for this PCR together with a primer from the resistance marker. PCR screening is more advantageous than Southern screening not only for speed but also for scaling up the screening.

Immunoglobulin gene conversion and hypermutation assay by FACs.

Immunoglobulin (Ig) gene conversion and hypermutation rates can be quantified by FACS. The spontaneous DT40 variant, C118, and C118-derived DT40 mutants can be used to analyze gene conversion phenotype. The C118 has a frameshift in its rearranged V segment.

Excision of floxed-DNA sequences by transient induction of Mer-Cre-Mer.

Disruption of multiple genes and complementation of the phenotypes is restricted by the number of available drug-resistance genes. It is therefore highly desirable to recycle the drug-resistance genes using a site-specific recombination system like Cre/loxP. DT40(Cre1) cell line, which is transgenic with an inducible Cre recombinase (MerCreMer) is useful for transient induction of Cre/loxP recombination.

Basic cell culture conditions.

The DT40 cell line is derived from ALV (avian leukosis virus)-transformed bursal B cell of chicken. This cell line is suspension cell, and can be cultured in cell culture bottles and in culture plates (6-, 24- and 96-well etc.).

Genome resources for the DT40 community.

The chicken B cell line DT40 is a tool that is uniquely situated to study the function of genes due to its high rate of homologous recombination. As any tool is only as good as the resources behind it, the recent boon and continuing output of available genomic information has only widened the possibilities for the DT40 community. Besides the release of the chicken genome, the public databases are expanding rapidly with a wealth of experimentally produced data including various chicken cell specific EST's, full-length cDNA's, non-coding RNA's and expressed SAGE tags.

Immunoglobulin gene conversion or hypermutation: that's the question.

Chicken B cells develop their primary immunoglobulin (Ig) gene repertoire by pseudogene templated gene conversion within the bursa of Fabricius. The DT40 cell line is derived from bursal B cells and continues to diversify its rearranged Ig light chain in cell culture. Ig gene conversion of DT40 requires expression of the AID gene which was earlier shown to be needed for Ig hypermutation and switch recombination in mammalian B cells.

Dt40 gene disruptions: a how-to for the design and the construction of targeting vectors.

Genome projects have provided comprehensive gene catalogs and locus maps for many model organisms. Although sequence comparison and protein domain searches may suggest evolutionary conserved gene functions, genetic systems are still needed to determine the role of genes within living cells. Due to high ratios of targeted to random integration of transfected DNA constructs, the chicken B cell line DT40 has been widely used as a model for gene function analysis by gene knockout.

A role for PCNA ubiquitination in immunoglobulin hypermutation.

Proliferating cell nuclear antigen (PCNA) is a DNA polymerase cofactor and regulator of replication-linked functions. Upon DNA damage, yeast and vertebrate PCNA is modified at the conserved lysine K164 by ubiquitin, which mediates error-prone replication across lesions via translesion polymerases. We investigated the role of PCNA ubiquitination in variants of the DT40 B cell line that are mutant in K164 of PCNA or in Rad18, which is involved in PCNA ubiquitination.

Activation-induced cytidine deaminase initiates immunoglobulin gene conversion and hypermutation by a common intermediate.

Depending on the species and the lymphoid organ, activation-induced cytidine deaminase (AID) expression triggers diversification of the rearranged immunoglobulin (Ig) genes by pseudo V (psiV) gene- templated gene conversion or somatic hypermutation. To investigate how AID can alternatively induce recombination or hypermutation, psiV gene deletions were introduced into the rearranged light chain locus of the DT40 B-cell line. We show that the stepwise removal of the psiV donors not only reduces and eventually abolishes Ig gene conversion, but also activates AID-dependent Ig hypermutation.

Immunoglobulin gene conversion: insights from bursal B cells and the DT40 cell line.

Chicken B cells diversify their immunoglobulin (Ig) light and heavy chain genes by pseudogene templated gene conversion within the bursa of Fabricius. Although Ig gene conversion was initially believed to occur only in birds, it is now clear that most farm animals also use this elegant mechanism to develop an immunoglobulin gene repertoire. The best model to study Ig gene conversion remains the chicken Ig light chain locus due to its compact size and the fact that all the pseudogene donors are sequenced.