Quantitative analysis using ELISA of vascular endothelial growth factor and basic fibroblast growth factor in human colorectal cancer,liver metastasis of colorectal cancer and hepatocellular carcinoma

Angiogenesis consists of the sprouting of capillaries from pre-existing vessels. It is well-known that tumor growth is angiogenesis-dependent. Vascular endothelial growth factor （VEGF） and basic fibroblast growth factor （bFGF） stimulated vascular endothelial cell proliferation and are involved in the neoplastic angiogenesis of several types of tumors including those of the intestinal tract. Authors usually investigated VEGF and using immunohistochemistry bFGF protein expressions or Western blotting and VEGF and bFGF transcripts using reverse transcriptase Dolymerase chain reaction （RT-PCR）.

Platelet-activating factor in cirrhotic liver and hepatocellular carcinoma

AIM： Platelet-activating factor （PAF） is a pro-inflammatory and angiogenic lipid mediator. Here we aimed to investigate levels of PAF, lyso-PAF （the PAF precursor）, phospholipase A2 （PLA2, the enzymatic activity generating lyso-PAF）, acetylhydrolase activity （AHA, the PAF degrading enzyme） and PAF receptor （PAF-R） transcripts in cirrhotic liver and hepatocellular carcinoma （HCC）. METHODS： Twenty-nine patients with HCC were enrolled in this study. Cirrhosis was present in fourteen patients and seven had no liver disease. Tissue PAF levels were investigated by a platelet-aggregation assay. Lyso- PAF was assessed after its chemical acetylation into PAR AHA was determined by degradation of [^3H]-PAE PLA2 levels were assessed by EIA. PAF-R transcripts were investigated using RT-PCR. RESULTS： Elevated amounts of PAF and PAF-R transcripts 1 （leukocyte-type） were found in cirrhotic tissues as compared with non-cirrhotic ones. Higher amounts of PAF and PAF-R transcripts 1 and 2 （tissue-type） were found in HCC tissues as compared with non-tumor tissues. PLA2, lyso-PAF and AHA levels were not changed in cirrhotic tissues and HCC. CONCLUSION： While the role of PAF is currently unknown in liver physiology, this study suggests its potential involvement in the inflammatory network found in the cirrhotic liver and in the angiogenic response during HCC.

Hallmarks in colorectal cancer:Angiogenesis and cancer stem-like cells

Carcinogenesis is a multistep process that requires the accumulation of various genetic and epigenetic aberrations to drive the progressive malignant transformation of normal human cells.Two major hallmarks of carcinogenesis that have been described are angiogenesis and the stem cell characteristic of limitless replicative potential.These properties have been targeted over the past decade in the development of therapeutic treatments for colorectal cancer(CRC),one of the most commonly diagnosed and lethal cancers worldwide.The treatment of solid tumor cancers such as CRC has been challenging due to the heterogeneity of the tumor itself and the chemoresistance of the malignant cells.Furthermore,the same microenvironment that maintains the pool of intestinal stem cells that contribute to the continuous renewal of the intestinal epithelia also provides the necessary conditions for proliferative growth of cancer stem-like cells.These cancer stem-like cells are responsible for the resistance to therapy and cancer recurrence,though they represent less than 2.5%of the tumor mass.The stromal environment surrounding the tumor cells,referred to as the tumor niche,also supports angiogenesis,which supplies the oxygen and nutrients needed for tumor development.Anti-angiogenic therapy,such as with bevacizumab,a monoclonal antibody against vascular-endothelial growth factor,significantly prolongs the survival of metastatic CRC patients.However,such treatments are not completely curative,and a large proportion of patient tumors retain chemoresistance or show recurrence.This article reviews the current knowledge regarding the molecular phenotype of CRC cancer cells,as well as discusses the mechanisms contributing to their maintenance.Future personalized therapeutic approaches that are based on the interaction of the carcinogenic hallmarks,namely angiogenic and proliferative attributes,could improve survival and decrease adverse effects induced by unnecessary chemotherapy.

VEGF in hepatocellular carcinoma and surrounding cirrhotic liver tissues

We read with a great interest the recent work of Deli and colleagues. in the World Journal of Gastroenterology reporting vascular endothelial growth factor （VEGF） expression in hepatocellular carcinoma （HCC） and cirrhotic liver tissues. This well-documented work shows that VEGF was significantly higher in surrounding cirrhotic liver tissues than in HCC. Authors assessed VEGF expression using immunohistochemistry. The immunohistochemical staining is an efficient tool to assess the percentage of cells stained positively for VEGF but is not really efficient to estimate their true VEGF content. Evaluation of the VEGF protein by an enzyme-linked immunosorbent assay 0ELISA） has been reported, by us and others, to be an efficient tool in order to assess tissue VEGF expression. We have, thus, tested whether the ELISA method might be an efficient tool in order to confirm data reporting higher amounts of VEGF in surrounding cirrhotic liver tissues than in HCC. Deli and colleagues. also correctly pointed out that basic fibroblast growth factor （bFGF） has been reported to act cooperatively on VEGF expression. We have, thus, also assessed bFGF tissue levels in order to search for a putative link between VEGF and bFGF levels in cirrhotic tissues.

IgD class switch recombination is not controlled through the immunoglobulin heavy chain 3′ regulatory region super-enhancer

In secondary lymphoid organs,mature B cells express membrane immunoglobulin(Ig)of M and D isotypes(IgM and IgD,respectively)of the same specificity through alternative splicing of a pre-mRNA encompassing the VDJ variable region and Cμand Cδheavy chain constant exons.1 After encountering antigen,B cells undergo class switch recombination(CSR)by which the Cμgene is substituted with Cγ,Cεor Cα,thereby generating IgG,IgE and IgA antibodies of the same antigenic specificity but with new effector functions.CSR requires the DNA-editing enzyme activation-induced deaminase(AID).

The IgH 3′ regulatory region super-enhancer does not control IgA class switch recombination in the B1 lineage

The bone marrow-derived B2 population represents the vast majority of bone marrow,blood,lymph node and splenic B-cells.Mouse B1 B-cells mostly originate during embryonic life in the liver and represent the main B-cell population in the pleural and peritoneal cavities.1,2,3,4,5 B1 and B2 B-cells differ in their origin,antigen specificity,cell surface markers,tissue distribution and capacity for class switch recombination(CSR).Schematically,B1 B-cells appear earlier than B2 B-cells during fetal development and maintain their self-renewal ability throughout their life.

Uracil-DNA glycosylase is not implicated in the choice of the DNA repair pathway during B-cell class switch recombination

Mature B-cells express membrane IgM and IgD(of same specificity)through alternative splicing of a pre-mRNA encompassing constant(C)μand Cδgenes.After encountering antigen,Bcells undergo class switch recombination(CSR)that substitutes the Cμgene with Cγ,Cε,or Cα,thereby generating IgG,IgE,and IgA antibodies with the same antigenic specificity but new effector functions.DNA-editing enzyme activation-induced deaminase(AID)is essential for CSR by targeting switch(S)regions preceding Cμ(namely,the Sμdonor region)and the Cγ,Cε,and Cαgenes(namely,the Sγ,ε,αacceptor regions).1,2 CSR is controlled in cis by IgH locus super-enhancers3 and in trans by a wide spectrum of enzymes and proteins.1,2 Among them,the role of the uracil DNA glycosylase(UNG)remains controversial.UNG is a key enzyme of base excision repair,which carries out faithful repair.Some authors estimate that during CSR,the UNG enzymatic activity removes the AID-induced dC to dU converted base of singlestrand DNA,generating abasic sites and leading to DNA strand breaks.1 For other authors,the role of UNG is to stabilize the S–S synapse and to recruit DNA repair factors that facilitate the endjoining process.4,5 Thus,the classical non-homogenous end joining pathway would be increased over the alternative end joining(A-EJ)pathway in UNG-deficient mice,4 suggesting an intriguing role of UNG in promoting the A-EJ pathway.

High-throughput sequencing reveals similar molecular signatures for class switch recombination junctions for theγandαisotypes

INTRODUCTION After encountering an antigen,B cells undergo class switch recombination(CSR),which substitutes the Cμgene with Cγ,Cε,or Cαto generate IgG,IgE,and IgA antibodies with the same antigenic specificity but new effector functions.1 The DNAediting enzyme activation-induced deaminase(AID)is essential for CSR by targeting switch(S)regions preceding Cμ(namely,the Sμdonor region)and the Cγ,Cε,and Cαgenes(namely,the Sγ,ε,αacceptor regions).1 Cis-and trans-controlled DNA double strand beaks are generated during this process.2–6 Recruitment of DNA repair factors that facilitate the end-joining process is a crucial step of class switch recombination.Two pathways are implicated in this end joining.The classical non-homogenous end joining(c-NHEJ)pathway ligates DNA ends with no or little homology.By contrast,the alternative end joining(A-EJ)pathways is used to ligate DNA ends that have microhomology.

Class switch recombination junctions are not affected by the absence of the immunoglobulin heavy chain E_(μ) enhancer

After encountering antigen,B-cells undergo class switch recombination(CSR)that substitutes the constant(C)μgene with Cγ,Cε,or Cα,thereby generating IgG,IgE,and IgA antibodies with new effector functions but the same antigenic specificity.1 The DNA-editing enzyme activation-induced deaminase(AID)is required for CSR by targeting specific DNA switch(S)regions preceding the C region,except Cδ.2 Sμis the donor region,while Sγ,ε,αare the acceptor regions.CSR is controlled in cis by the immunoglobulin heavy chain(IgH)3’regulatory region(3’RR).3 The 3’RR is essential to poise AID on the S acceptor region.During CSR IgH,intrachromosomal interactions(schematized in Fig.1a)are found between the 3’RR and the intronic Eμenhancer.1,4 Looping allows transcriptional binding activators to enhancers to facilitate CSR.However,CSR is only modestly influenced by Eμdeletion.5–7 During CSR,two different DNA repair pathways take place:the classical nonhomologous end joining(c-NHEJ)and the alternative end joining(A-EJ)pathways.

Retraction Note:3′RR and 5′Eμimmunoglobulin heavy chain enhancers are independent engines of locus remodeling

The authors have retracted this Correspondence.After the publication of this correspondence,it came to the authors’attention that the control RAG2^(−/−)mouse was a RAG2^(−/−)γc^(−/−)mouse.This point resulted in the conclusions being considered invalid.The authors apologize to the journal and its readers for any inconvenience caused.

Retraction Note:Trans-silencing effect of the 3′RR immunoglobulin heavy chain enhancer on Igκtranscription at the pro-B cell stage

The authors have retracted this Correspondence.After the publication of this correspondence,it came to the authors’attention that the control RAG2^(−/−)mouse was a RAG2^(−/−)γc^(−/−)mouse.This point resulted in the conclusions being considered invalid.The authors apologize to the journal and its readers for any inconvenience caused.

Molecular analysis of γ1, γ3, and α class switch recombination junctions in APOBEC3-deficient mice using high-throughput sequencing

Activation-induced deaminase(AID)is required for immunoglobulin(Ig)class switch recombination(CSR),in which the constant(C)μgene of IgM is substituted with C_(γ),C_(ε),or C_(α),thereby generating IgG,IgE,and IgA antibodies,respectively,with new effector functions but the same antigenic specificity.1 AID targets specific DNA switch(S)regions preceding C regions except for Cδ.2 Sμis usually the donor region,while Sγ,ε,αare the acceptor regions.AID deaminates C into U on single-stranded DNA by targeting the WRCY(W=A/T,R=A/G,and Y=C/T)hot motif and,to a lesser extent,the SYC(S=G/C,Y=C/T)cold motif.3,4 AID is a member of the apolipoprotein B editing complex(APOBEC)family.Among APOBEC genes,a family of evolutionarily conserved cytidine deaminases,APOBEC3 is implicated in diverse cell functions including innate immunity against retroviruses.4 The DNA-editing APOBEC3 enzymes have recently attracted attention due to their involvement in cancer and potential applications in gene editing.5–7 While a single copy of each APOBEC3 gene is present in rodents,seven copies of each APOBEC3 gene are found in humans.

Trans-silencing effect of the 3′RR immunoglobulin heavy chain enhancer on Igκtranscription at the pro-B cell stage

Due to their impact on nuclear organization,enhancers are master regulators of cell fate.1,2 The immunoglobulin heavy chain(IgH)locus undergoes numerous changes(such as transcription,accessibility,DNA breaks,and mutations)throughout B-cell differentiation.Several of these events are controlled by the IgH 3′regulatory region(3′RR).The 3′RR is the master control element of mature B-cell IgH transcription,3 somatic hypermutation(SHM),4,5 conventional class switch recombination(CSR),4,6–10 and locus suicide recombination(LSR).11 In contrast,the 3′RR is expected to be dispensable for V(D)J recombination.12,13 During Bcell development,the heavy and light chain loci are poised for their VDJ and VJ rearrangements,respectively.The IgH locus rearranges first,with D-J joining at the pro-B-cell stages,followed by V-DJ joining at the pre-B-cell stage.The Igk locus is poised for VJ rearrangements at the pre-B cell stage.