Analysis of the aryl hydrocarbon receptor and a truncated form (AHR C[upper case symbol for greek Delta]Δ553) in cancer cells

The aryl hydrocarbon receptor (AhR) is a ligand-activated bHLH-PAS protein that binds its partner, the aryl hydrocarbon receptor nuclear translocator (Arnt), in the nucleus to initiate the expression of proteins involved with detoxification. Published work suggests cross-talk between both proteins and cellular pathways involving the transcription factors, HIF -1 , ER, and NFKB, whose activity is typically upregulated in cancer. This thesis focuses on using a truncated form of AhR, AhR CΔ553, which is thought to act as a dominant-negative to sequester Arnt from its other binding partners. To test this hypothesis, we transfected HeLa cells with AhR CΔ553 fused to pEGFP or a vector under a tetracycline-inducible promoter. Stable cell lines expressing pEGFP-AhR CΔ553 have been generated and confirmed to have nuclear localization. We were also interested in confirming endogenous localization patterns of AhR and Arnt to study the role of p23 in the nuclear translocation of AhR. While we were successful in showing AhR translocating to the nucleus in treated MCF-7 cells, we couldn\u27t clearly see nuclear AhR in Hepalclc7 cells, the cell line with knockdown levels of p23. To compare DNA damage generated in Jm·kat and Hepalclc7 cells, we looked for reactive oxygen species (ROS) production and quantified DNA damage after exposure to benzo[a]pyrene (B[a]P) and some of its derivatives. Hepalclc7 cells were prone to a wide variety of DNA damage, but Jurkat cells did not appear to undergo damage specifically through ROS production. Finally, we wanted to confirm apoptosis in HeLa cells after being cocultured with Trichomonas vaginalis. The G3 lab strain was more aggressive than Tl , but Parp, and apoptotic marker, was not observed in HeLa cells, suggesting that experimental conditions need to be further optimized

Analysis of the aryl hydrocarbon receptor and a truncated form (AHR C[upper case symbol for greek Delta]Δ553) in cancer cells

The aryl hydrocarbon receptor (AhR) is a ligand-activated bHLH-PAS protein that binds its partner, the aryl hydrocarbon receptor nuclear translocator (Arnt), in the nucleus to initiate the expression of proteins involved with detoxification. Published work suggests cross-talk between both proteins and cellular pathways involving the transcription factors, HIF -1 , ER, and NFKB, whose activity is typically upregulated in cancer. This thesis focuses on using a truncated form of AhR, AhR CΔ553, which is thought to act as a dominant-negative to sequester Arnt from its other binding partners. To test this hypothesis, we transfected HeLa cells with AhR CΔ553 fused to pEGFP or a vector under a tetracycline-inducible promoter. Stable cell lines expressing pEGFP-AhR CΔ553 have been generated and confirmed to have nuclear localization. We were also interested in confirming endogenous localization patterns of AhR and Arnt to study the role of p23 in the nuclear translocation of AhR. While we were successful in showing AhR translocating to the nucleus in treated MCF-7 cells, we couldn\u27t clearly see nuclear AhR in Hepalclc7 cells, the cell line with knockdown levels of p23. To compare DNA damage generated in Jm·kat and Hepalclc7 cells, we looked for reactive oxygen species (ROS) production and quantified DNA damage after exposure to benzo[a]pyrene (B[a]P) and some of its derivatives. Hepalclc7 cells were prone to a wide variety of DNA damage, but Jurkat cells did not appear to undergo damage specifically through ROS production. Finally, we wanted to confirm apoptosis in HeLa cells after being cocultured with Trichomonas vaginalis. The G3 lab strain was more aggressive than Tl , but Parp, and apoptotic marker, was not observed in HeLa cells, suggesting that experimental conditions need to be further optimized

Characterization of Serine Proteases in Trichomonas vaginalis

Trichomonas vaginalis, an anaerobic protozoan parasite, causes trichomoniasis the most prevalent non-viral STD. While males are usually asymptomatic, females may have symptoms such as inflammation and vaginal secretions. Trichomoniasis is also a cause of many complications during pregnancy. Infection is treated with metronidazole or tinidazole.However, there is a need to develop alternative chemotherapies because of drug resistance. Serine proteases have been shown to be virulence factors in various parasites such as Toxoplasma gondii and Entamoeba histolytica. T. gondii invasion of host cells has been shown to be blocked by serine protease inhibitors. T. vaginalis has been suspected to have serine protease activity, but this enzyme has not been studied in any detail. We have searched the T. vaginalis genome database and found potential serine protease candidate genes. We have cloned 10 of these genes using PCR with the goal of ultimately expressing these genes for recombinant protein expression. Currently we are also studying the effect of serine protease inhibitors on T. vaginalis growth

Characterization of Serine Proteases in Trichomonas vaginalis

Trichomonas vaginalis, an anaerobic protozoan parasite, causes trichomoniasis the most prevalent non-viral STD. While males are usually asymptomatic, females may have symptoms such as inflammation and vaginal secretions. Trichomoniasis is also a cause of many complications during pregnancy. Infection is treated with metronidazole or tinidazole.However, there is a need to develop alternative chemotherapies because of drug resistance. Serine proteases have been shown to be virulence factors in various parasites such as Toxoplasma gondii and Entamoeba histolytica. T. gondii invasion of host cells has been shown to be blocked by serine protease inhibitors. T. vaginalis has been suspected to have serine protease activity, but this enzyme has not been studied in any detail. We have searched the T. vaginalis genome database and found potential serine protease candidate genes. We have cloned 10 of these genes using PCR with the goal of ultimately expressing these genes for recombinant protein expression. Currently we are also studying the effect of serine protease inhibitors on T. vaginalis growth

Super-enhancers promote transcriptional dysregulation in nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) is an invasive cancer with particularly high incidence in Southeast Asia and Southern China. The pathogenic mechanisms of NPC, particularly those involving epigenetic dysregulation, remain largely elusive, hampering clinical management of this malignancy. To identify novel druggable targets, we carried out an unbiased high-throughput chemical screening and observed that NPC cells were highly sensitive to inhibitors of cyclin-dependent kinases (CDK), especially THZ1, a covalent inhibitor of CDK7. THZ1 demonstrated pronounced antineoplastic activities both in vitro and in vivo An integrative analysis using both whole-transcriptome sequencing and chromatin immunoprecipitation sequencing pinpointed oncogenic transcriptional amplification mediated by super-enhancers (SE) as a key mechanism underlying the vulnerability of NPC cells to THZ1 treatment. Further characterization of SE-mediated networks identified many novel SE-associated oncogenic transcripts, such as BCAR1, F3, LDLR, TBC1D2, and the long noncoding RNA TP53TG1. These transcripts were highly and specifically expressed in NPC and functionally promoted NPC malignant phenotypes. Moreover, DNA-binding motif analysis within the SE segments suggest that several transcription factors (including ETS2, MAFK, and TEAD1) may help establish and maintain SE activity across the genome. Taken together, our data establish the landscape of SE-associated oncogenic transcriptional network in NPC, which can be exploited for the development of more effective therapeutic regimens for this disease. Cancer Res; 77(23); 6614-26. ©2017 AACR