PKR-NF-κB Pathway Upstream of IFN-ß Induction Is Dysregulated in Oncolytic Sindbis Virus-infected HeLa Cells.

BACKGROUND/AIM: Sindbis virus (SINV) is a naturally occurring oncolytic virus that kills cancer cells and is less harmful to normal cells. In this study, a recombinant SINV, which expressed green and blue fluorescent proteins, was used to precisely analyze SINV infection and replication. MATERIALS AND METHODS: Antiviral responses, including IFN-ß mRNA, protein kinase R (PKR), NF-B, and caspase 3/7, were analyzed in SINV-infected cancerous HeLa cells and normal human fibroblast TIG-1-20 cells. RESULTS: SINV could infect, replicate, and proliferate both in HeLa and TIG-1-20 cells, causing lytic infection only in HeLa cells. SINV grew preferentially in HeLa cells causing remarkable apoptosis. IFN-ß mRNA expression was suppressed in SINV-infected HeLa cells compared to that in TIG-1-20 cells. Further analyses of PKR and NF-B upstream of IFN-ß induction revealed that the compromised response in the PKR-NF-B pathway during early infection coincided with IFN induction suppression in HeLa cells. CONCLUSION: Dysregulation of PKR in HeLa cells is the determinant of SINV oncolysis.

Enhancing the Antitumor Immunity of T Cells by Engineering the Lipid-Regulatory Site of the TCR/CD3 Complex.

The engagement of the T-cell receptor (TCR) by a specific peptide-MHC ligand initiates transmembrane signaling to induce T-cell activation, a key step in most adaptive immune responses. Previous studies have indicated that TCR signaling is tightly regulated by cholesterol and its sulfate metabolite, cholesterol sulfate (CS), on the membrane. Here, we report a novel mechanism by which CS modulates TCR signaling through a conformational change of CD3 subunits. We found that the negatively charged CS interacted with the positively charged cytoplasmic domain of CD3ε (CD3εCD) to enhance its binding to the cell membrane and induce a stable secondary structure. This secondary structure suppressed the release of CD3εCD from the membrane in the presence of Ca2+, which in turn inhibited TCR phosphorylation and signaling. When a point mutation (I/A) was introduced to the intracellular immunoreceptor tyrosine-based activation motifs (YxxI-x6-8-YxxL) of CD3ε subunit, it reduced the stability of the secondary structure and regained sensitivity to Ca2+, which abolished CS-mediated inhibition and enhanced the signaling of the TCR complex. Notably, the I/A mutation could be applied to both murine and human TCR-T cell therapy to improve the antitumor efficacy. Our study reveals insights into the regulatory mechanism of TCR signaling and provides a strategy to functionally engineer the TCR/CD3 complex for T cell-based cancer immunotherapy.

Establishment of an epithelioid sarcoma PDCs and PDX to evaluate drug sensitivity.

Epithelioid sarcoma (ES) is a very rare mesenchymal malignancy. Its oncogenesis remains unknown, and few therapies are available for advanced patients. Improved therapies, such as combined therapies, are urgently needed for the treatment of advanced ES. To identify precision drugs for advanced ES patients, the sensitivity of the drugs must be screened and evaluated before they are used for treatment. In the present study, tumour tissue from an ES patient was subjected to NGS sequencing, cell culture and the construction of a PDX model. Using the early generations of tumour-derived cells, we performed a screen and found that the combined drugs ADM and trametinib exhibited coefficiency in tumour inhibition. This conclusion was further confirmed in experiments with later generations of cells and PDX models. Therefore, we suggest that early generations of tumour-derived cells be used to test the sensitivity of ES tumours to candidate drugs. Moreover, we speculate that trametinib may be combined with chemotherapy in ES patients to prolong the duration of the chemotherapeutic response.

Ontogeny of RORγt+ cells in the intestine of newborns and its role in the development of experimental necrotizing enterocolitis.

BACKGROUND: Neonates possess an immature and plastic immune system, which is a major cause of some diseases in newborns. Necrotizing enterocolitis (NEC) is a severe and devastating intestinal disease that typically affects premature infants. However, the development of intestinal immune cells in neonates and their roles in the pathological process of NEC have not been elucidated. RESULTS: We examined the ontogeny of intestinal lamina propria lymphocytes in the early life of mice and found a high percentage of RORγt+ cells (containing inflammatory Th17 and ILC3 populations) during the first week of life. Importantly, the proportion of RORγt+ cells of intestinal lamina propria further increased in both NEC mice and patients tissue than the control. Furthermore, the application of GSK805, a specific antagonist of RORγt, inhibited IL-17A release and ameliorated NEC severity. CONCLUSIONS: Our data reveal the high proportion of RORγt+ cells in newborn mice may directly contribute to the development of NEC.

CCL299, a Benzimidazole Derivative Induces G1 Phase Arrest and Apoptosis in Cancer Cells.

BACKGROUND/AIM: Benzimidazoles are considered potential anticancer candidates. We herein studied the anticancer activity of CCL299, 4-(1H-1,3-benzodiazol-1-yl) benzonitrile. MATERIALS AND METHODS: In this in vitro study, we used ATP assays, flow cytometry, western blotting, and caspase-3/7 assays to evaluate the effects of CCL299 on cell proliferation, cell-cycle progression and apoptosis. RESULTS: ATP assays showed that CCL299 inhibited cell growth in the hepatoblastoma cell line HepG2 and the cervical cancer cell line HEp-2, without exhibiting cytotoxic effects on non-cancer cells and TIG-1-20 fibroblasts. Flow cytometry, western blotting, and caspase-3/7 assays revealed that CCL299 induced G1-phase cell-cycle arrest followed by apoptosis that was associated with up-regulation of p-p53 (Ser15) and p21 expression and the down-regulation of p-CDK2 (Thr160) expression. CONCLUSION: CCL299 exhibits cytotoxic activity via apoptosis in a subset of cancer cells, and should be considered as a promising anticancer candidate agent.

CCL113, a novel sulfonamide, induces selective mitotic arrest and apoptosis in HeLa and HepG2 cells.

Targeting cell­cycle regulation to hinder cancer cell proliferation is a promising anticancer strategy. The present study investigated the effects of a novel sulfonamide, CCL113, on cell cycle progression in cancer cell lines (HeLa and HepG2), a noncancerous cell line (Vero) and a normal human fibroblast cell line (TIG­1­20). The present results showed that treatment with CCL113 significantly decreased the viability of the cancer cells. FACS analyses showed that CCL113 treatment increased the proportion of cancerous and noncancerous cells in the G2/M phase. Analyses of cell cycle regulatory proteins showed that CCL113 treatment inhibited the activity of CDK1 in HeLa cells, possibly due to the decrease in the level of Cdc25B/C proteins and arrest in the M phase. Using time­lapse imaging­assisted analyses of HeLa and Vero cells expressing fluorescent ubiquitination­based cell cycle indicator (FUCCI), it was observed that CCL113 treatment led to a prolonged G2 phase at the G2/M checkpoint and arrest in the M phase in both cell lines. This possibly activated the DNA damage response in noncancerous cells, while inducing mitotic arrest leading to apoptosis in the cancer cells. The results of molecular docking studies suggested that CCL113 might have the potential to bind to the taxol­binding site on ß­tubulin. In conclusion, CCL113 holds potential as a reliable anticancer drug due to its ability to induce mitotic arrest followed by apoptosis of cancer cells and to activate the DNA damage response in noncancerous cells, thereby facilitating exit from the cell cycle.

Alteration of cell cycle progression by Sindbis virus infection.

We examined the impact of Sindbis virus (SINV) infection on cell cycle progression in a cancer cell line, HeLa, and a non-cancerous cell line, Vero. Cell cycle analyses showed that SINV infection is able to alter the cell cycle progression in both HeLa and Vero cells, but differently, especially during the early stage of infection. SINV infection affected the expression of several cell cycle regulators (CDK4, CDK6, cyclin E, p21, cyclin A and cyclin B) in HeLa cells and caused HeLa cells to accumulate in S phase during the early stage of infection. Monitoring SINV replication in HeLa and Vero cells expressing cell cycle indicators revealed that SINV which infected HeLa cells during G1 phase preferred to proliferate during S/G2 phase, and the average time interval for viral replication was significantly shorter in both HeLa and Vero cells infected during G1 phase than in cells infected during S/G2 phase.

A-mating-type gene expression can drive clamp formation in the bipolar mushroom Pholiota microspora (Pholiota nameko).

In the bipolar basidiomycete Pholiota microspora, a pair of homeodomain protein genes located at the A-mating-type locus regulates mating compatibility. In the present study, we used a DNA-mediated transformation system in P. microspora to investigate the homeodomain proteins that control the clamp formation. When a single homeodomain protein gene (A3-hox1 or A3-hox2) from the A3 monokaryon strain was transformed into the A4 monokaryon strain, the transformants produced many pseudoclamps but very few clamps. When two homeodomain protein genes (A3-hox1 and A3-hox2) were transformed either separately or together into the A4 monokaryon, the ratio of clamps to the clamplike cells in the transformants was significantly increased to ca. 50%. We therefore concluded that the gene dosage of homeodomain protein genes is important for clamp formation. When the sip promoter was connected to the coding region of A3-hox1 and A3-hox2 and the fused fragments were introduced into NGW19-6 (A4), the transformants achieved more than 85% clamp formation and exhibited two nuclei per cell, similar to the dikaryon (NGW12-163 x NGW19-6). The results of real-time reverse transcription-PCR confirmed that sip promoter activity is greater than that of the native promoter of homeodomain protein genes in P. microspora. Thus, we concluded that nearly 100% clamp formation requires high expression levels of homeodomain protein genes and that altered expression of the A-mating-type genes alone is sufficient to drive true clamp formation.

Genomic structure of the A mating-type locus in a bipolar basidiomycete, Pholiota nameko.

In the bipolar basidiomycete, Pholiota nameko, the homeodomain protein, A4-hox1, located at the A mating-type locus, is known to regulate mating compatibility. In the present study, we investigated the genomic structure of the P. nameko A mating-type locus and its flanking region. A second homeodomain gene (A4-hox2) was discovered upstream of A4-hox1; this together with the conserved gene order around the A mating-type locus and their similar transcription direction were found in P. nameko, another bipolar mushroom, Coprinellus disseminatus, and two tetrapolar mushrooms, Coprinopsis cinerea and Laccaria bicolor. Analysis of the deduced protein sequences of the homeodomain protein genes from two strains of P. nameko show that the putative functional domains differ from those of the homeodomain proteins of the tetrapolar mushrooms, C. cinerea and L. bicolor.