Toshitsugu Fujita

Human parvovirus B19 (B19V) primarily targets erythroid progenitor cells and is associated with various hematological disorders. However, the detailed mechanisms underlying its life cycle, particularly the viral assembly process, remain largely unknown. In this study, we used in vitro engineered DNA-binding molecule-mediated chromatin immunoprecipitation (in vitro enChIP) to identify host proteins associated with the B19V genome in infected UT7/Epo-S1 cells. Using guide RNAs targeting the viral terminal hairpin region, we successfully enriched viral genomic DNA. Mass spectrometry analysis of the precipitated fractions revealed specific enrichment of desmosomal proteins, including desmoplakin (DSP), desmoglein-1, desmocollin-1, and junction plakoglobin, suggesting that the components of the entire desmosome complex may be associated with the B19V genome. Immunofluorescence microscopy showed that the viral VP2 protein was strongly localized to the extranuclear foci, where it colocalized with DSP. Pull-down assays further demonstrated that VP2, but not VP1, interacted with DSP, indicating that the VP1-unique N-terminal region (VP1u) may inhibit this interaction. Notably, B19V-infected cells displayed reduced cell–cell adhesion and diminished cellular aggregation, implying that these interactions may be involved in the disruption of cell adhesion during infection. These findings revealed a novel mechanism by which B19V exploits the host desmosomal machinery to facilitate viral propagation in infected cells.

Background Cervical cancer (CC), the fourth most common cancer in women, is caused predominantly by human papillomavirus (HPV). Although measuring serum squamous cell carcinoma antigen (SCC Ag) can be useful for detecting recurrence of SCC, a major type of CC, its prognostic value remains unclear. This study focuses on the utility of circulating cell-free HPV (ccfHPV) DNA in plasma as a biomarker, with particular emphasis on its relevance to high-risk HPV subtypes prevalent in Japan. Methods A prospective study of 26 CC patients and 23 females diagnosed with pre-cancerous lesions was conducted. Patients were selected carefully to include only those with single high-risk HPV subtypes (i.e., HPV16, 18, 52, or 58), reflecting regional HPV epidemiology. ccfHPV DNA was isolated from plasma and analyzed by droplet digital PCR targeting the HPV E7 genes. Results The detection rate of ccfHPV DNA in CC patients before clinical treatment was 57.7%. The detection rate correlated significantly with tumor size (r = 0.624, P < 0.01) and clinical stage (r = 0.844, P < 0.01). No ccfHPV DNA was detected in the females with pre-cancerous lesions. By contrast, of the 13 concurrent chemoradiotherapy cases, two relapsed within 6 months post-treatment. In those cases, ccfHPV DNA levels rose earlier than SCC Ag levels. The 11 CC cases in which no ccfHPV DNA was detected within 1 month post-treatment did not relapse. Conclusion ccfHPV DNA is a useful biomarker for advanced-stage CC and for predicting prognosis, particularly in the Japanese clinical setting.

Pancreatic cancer is an aggressive malignancy with a poor prognosis. Single-nucleotide mutations in the KRAS gene are detected in the majority of patients with pancreatic ductal adenocarcinoma (PDAC), the most common type of pancreatic cancer. Identifying KRAS mutations by liquid biopsy could be effective for detecting de novo and recurrent PDAC; however, sensitive and accurate detection remains challenging. We examined the utility of oligoribonucleotide interference-PCR (ORNi-PCR) followed by real-time PCR or droplet digital PCR (ddPCR) for detecting KRAS single-nucleotide mutations by liquid biopsy. A model of cell-free DNA was used to demonstrate that the ORNi-PCR-based methods are more sensitive and accurate for detecting KRAS mutant DNA than conventional real-time PCR or ddPCR. ORNi-PCR-based methods could be useful for early detection of de novo and recurrent PDAC by liquid biopsy for cancer diagnosis.

Blocking PCR is a method that inhibits amplification of DNA possessing a nucleotide sequence complementary to that of a blocker; the method can be used to suppress amplification of target wild-type DNA while amplifying mutated DNA. Previously, we demonstrated that an oligoribonucleotide (ORN) functions as a cost-effective and sequence-specific blocker. This blocking PCR system, named ORN interference-PCR (ORNi-PCR), is compatible with DNA polymerases lacking 5 '-3 ' exonuclease activity but not with those possessing the activity (e.g., Taq DNA polymerase), which can remove a hybridized ORN during DNA extension. Here, we demonstrate that under specific experimental conditions, an intact or phosphorothioated ORN strongly suppresses extension of target DNA by Taq DNA polymerases. This method was applied successfully to real-time ORNi-PCR and one-step real-time reverse transcriptionORNi-PCR using a dual-labeled fluorescent probe to detect a single-nucleotide mutation in DNA and RNA in a sequence-specific manner. The results reaffirm the utility of blocking PCR and provide technical hints for its improvement.

Background Carbonyl reductase 1 (CBR1) is a nicotinamide adenine dinucleotide phosphate (NADPH)-dependent reductase with broad substrate specificity. CBR1 catalyzes the reduction of numerous carbonyl compounds, including quinones, prostaglandins, menadione, and multiple xenobiotics, while also participating in various cellular processes, such as carcinogenesis, apoptosis, signal transduction, and drug resistance. In this study, we aimed to generate transgenic mice overexpressing mouse Cbr1 (mCbr1), characterize the mCbr1 expression in different organs, and identify changes in protein expression patterns. Methods and Results To facilitate a deeper understanding of the functions of CBR1, we generated transgenic mice overexpressing CBR1 throughout the body. These transgenic mice overexpress 3xFLAG-tagged mCbr1 (3xFLAG-mCbr1) under the CAG promoter. Two lines of transgenic mice were generated, one with 3xFLAG-mCbr1 expression in multiple tissues, and the other, with specific expression of 3xFLAG-mCbr1 in the heart. Pathway and network analysis using transgenic mouse hearts identified 73 proteins with levels of expression correlating with mCbr1 overexpression. The expression of voltage-gated anion channels, which may be directly related to calcium ion-related myocardial contraction, was also upregulated. Conclusion mCbr1 transgenic mice may be useful for further in vivo analyses of the molecular mechanisms regulated by Cbr1; such analyses will provide a better understanding of its effects on carcinogenesis and cardiotoxicity of certain cancer drugs.

Hepatocellular carcinoma (HCC) is the most predominant type of liver cancer and is frequently fatal. Alpha‐fetoprotein, alpha‐fetoprotein‐L3, and protein induced by vitamin K absence or antagonist‐II are used as biomarkers to diagnose HCC. However, these biomarkers are not highly specific, especially for early‐stage HCC diagnosis; therefore, more specific biomarkers are needed. Recently, circular RNA (circRNA) biomarkers have been used to diagnose several intractable diseases. In this study, we sought to identify circRNA biomarkers for the specific diagnosis of HCC. To this end, we compared the expression levels of circRNAs in primary HCC and normal tissues using publicly available RNA‐seq data. Our analysis revealed that the expression levels of eight circRNAs were altered in primary HCC tissues compared with normal tissues. To confirm our findings, we examined the expression levels of selected circRNAs in HCC cell lines and normal hepatocytes. The expression level of hsa_circ_0001438, a circRNA that was downregulated in primary HCC, was lower in poorly and well‐differentiated HCC cell lines than in normal hepatocytes. By contrast, the expression level of hsa_circ_0000417, which was increased in primary HCC, was strongly upregulated in a well‐differentiated HCC cell line compared with normal hepatocytes. Thus, hsa_circ_0001438 and hsa_circ_0000417 might be potential biomarkers for the specific diagnosis of HCC. The experimental strategy described here, using publicly available RNA‐seq data, is a useful and cost‐effective method of identifying circRNA biomarkers.

Epigenetic silencing of tumor suppressor genes plays critical roles in oncogenesis. The CDKN2A/p16INK4a gene is one of the most frequently silenced tumor suppressor genes. To elucidate molecular mechanisms of maintenance of the silenced status of the p16INK4a gene, we applied the insertional chromatin immunoprecipitation (iChIP) method. The promoter region of the transcriptionally active and silenced alleles of the p16INK4a gene were tagged with a bacterial DNA-binding protein LexA and its binding elements. Subsequently, the promoter regions were affinity purified with antibody against a tag fused with LexA. Proteins more or less abundantly associated with the silenced promoter were identified by stable isotope labeling using amino acids in cell culture (SILAC), one of quantitative mass spectrometry methods. Knock-down experiments of the identified enzymes revealed essential components of maintenance of epigenetic silencing. The essential enzymes can be good targets of development of drugs reactivating expression of the silenced tumor suppressor genes in cancers. We are currently performing drug discovery research to identify small molecules that bind and inhibit those candidate drug target enzymes. Our locus-specific ChIP technology consisting of iChIP and the engineered DNA-binding molecule-mediated ChIP (enChIP) method is a powerful tool to identify novel drug targets for development of drugs regulating gene expression in intractable diseases. Citation Format: Toshitsugu Fujita, Hodaka Fujii. Identification of regulators of epigenetic silencing of the p16INK4a gene [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3767.

Interleukin (IL)-3 is a pleiotropic cytokine that regulates the survival, proliferation, and differentiation of hematopoietic cells. The binding of IL-3 to its receptor activates intracellular signaling, inducing transcription of immediate early genes (IEGs) such as c-fos, c-jun, and c-myc; however, transcriptional regulation under IL-3 signaling is not fully understood. This study assessed the role of the inhibitor of nuclear factor-κB kinases (IKKs) in inducing IL-3-mediated expression of IEGs. We show that IKK1 and IKK2 are required for the IL-3-induced immediate expression of c-fos and c-jun in murine hematopoietic Ba/F3 cells. Although IKK2 is well-known for its pivotal role as a regulator of the canonical nuclear factor-κB (NF-κB) pathway, activation of IKKs did not induce the nuclear translocation of the NF-κB transcription factor. We further revealed the important role of IKK2 in the activation of c-Jun N-terminal kinase (JNK), which mediates the IL-3-induced expression of c-fos and c-jun. These findings indicate that the IKK2-JNK axis modulates the IL-3-induced expression of IEGs in a canonical NF-κB-independent manner.