Michelle H. Townsend

Evaluation of various glyphosate concentrations on DNA damage in human Raji cells and its impact on cytotoxicity

Abstract Glyphosate is a highly used active compound in agriculturally based pesticides. The literature regarding the toxicity of glyphosate to human cells has been highly inconsistent. We studied the resulting DNA damage and cytotoxicity of various glyphosate concentrations on human cells to evaluate DNA damaging potential. Utilizing human Raji cells, DNA damage was quantified using the comet assay, while cytotoxicity was further analyzed using MTT viability assays. Several glyphosate concentrations were assessed, ranging from 15 mM to 0.1 &mgr;M. We found that glyphosate treatment is lethal to Raji cells at concentrations above 10 mM, yet has no cytotoxic effects at concentrations at or below 100 &mgr;M. Treatment concentrations of 1 mM and 5 mM induce statistically significant DNA damage to Raji cells following 30–60 min of treatment, however, cells show a slow recovery from initial damage and cell viability is unaffected after 2 h. At these same concentrations, cells treated with additional compound did not recover and maintained high levels of DNA damage. While the cytotoxicity of glyphosate appears to be minimal for physiologically relevant concentrations, the compound has a definitive cytotoxic nature in human cells at high concentrations. Our data also suggests a mammalian metabolic pathway for the degradation of glyphosate may be present. HighlightsSignificant DNA damage and cellular death occurs when Raji cells are exposed to glyphosate concentrations at or above 10 mM.Glyphosate concentrations below 10 &mgr;M do not induce significant DNA damage and cells maintain full long term viability.Intermediate glyphosate concentrations induce initial damage, but undergo repair that leads to eventual viability.Discrepancies in regards to cytotoxicity may be due to inconsistent time points that show alternative damage.

Non-small-cell lung cancer cell lines A549 and NCI-H460 express hypoxanthine guanine phosphoribosyltransferase on the plasma membrane

In both males and females, lung cancer is one of the most lethal cancers worldwide and accounts for >30% of cancer-related deaths. Despite advances in biomarker analysis and tumor characterization, there remains a need to find suitable biomarker antigen targets for treatment in late-stage lung cancer. Previous research on the salvage pathway enzyme TK1 shows a unique relationship with cancer patients as serum levels are raised according to cancer grade. To expand this analysis, the other salvage pathway enzymes were evaluated for possible upregulation within lung cancer. Adenine phosphoribosyltransferase, deoxycytidine kinase, and hypoxanthine guanine phosphoribosyltransferase (HPRT) were assessed for their presentation on two non-small-cell lung cancer cell lines NCI-H460 and A549. In the present study, we show that deoxycytidine kinase and adenine phosphoribosyltransferase have no significant relationship with the membrane of NCI-H460 cells. However, we found significant localization of HPRT to the membrane of NCI-H460 and A549 cells. When treated with anti-HPRT antibodies, the average fluorescence of the cell population increased by 24.3% and 12.9% in NCI-H460 and A549 cells, respectively, in comparison with controls. To ensure that expression was not attributed to cytoplasmic HPRT, confocal microscopy was performed to visualize HPRT binding on the plasma membrane. After staining NCI-H460 cells treated with both fluorescent antibodies and a membrane-specific dye, we observed direct overlap between HPRT and the membrane of the cancer cells. Additionally, gold-conjugated antibodies were used to label and quantify the amount of HPRT on the cell surface using scanning electron microscopy and energy-dispersive analysis X-ray. Further confirming HPRT presence, the gold weight percentage of the sample increased significantly when NCI-H460 cells were exposed to HPRT antibody (P=0.012) in comparison with isotype controls. Our results show that HPRT is localized on the surface of these non-small-cell lung cancer cell lines.

Biomarker analysis and clinical relevance of TK1 on the cell membrane of Burkitt’s lymphoma and acute lymphoblastic leukemia

TK1 is an enzyme involved in DNA synthesis and repair. TK1 is usually found elevated in cancer patients’ serum, which makes it a useful tumor proliferation biomarker that strongly correlates with cancer stage, metastatic capabilities, and recurrence risk. In this study, we show that TK1 is upregulated and localizes on the plasma membrane of Burkitt’s lymphoma, acute promyelocytic leukemia, T cell leukemia, and acute lymphoblastic leukemia (ALL). Using flow cytometry, we confirmed that TK1 localizes on the surface of Raji, HL60, and Jurkat cell lines and on ALL clinical samples. Using fluorescent microscopy, we found a strong association of TK1 with the plasma membrane in Raji, HL60, and Jurkat cell lines. These findings were also confirmed by scanning electron microscopy. Our study also shows that this phenomenon does not occur on normal resting or proliferating lymphocytes. In addition, we show that membrane TK1 is found in all oligomeric forms ranging from monomer to tetramer and exhibits enzymatic activity. These findings suggest TK1 as a possible target for immunotherapy with the potential to be utilized in the treatment of hematological cancers.

Characterization of two related Erwinia myoviruses that are distant relatives of the PhiKZ-like Jumbo phages.

Bacteriophages are a major force in the evolution of bacteria due to their sheer abundance as well as their ability to infect and kill their hosts and to transfer genetic material. Bacteriophages that infect the Enterobacteriaceae family are of particular interest because this bacterial family contains dangerous animal and plant pathogens. Herein we report the isolation and characterization of two jumbo myovirus Erwinia phages, RisingSun and Joad, collected from apple trees. These two genomes are nearly identical with Joad harboring two additional putative gene products. Despite mass spectrometry data that support the putative annotation, 43% of their gene products have no significant BLASTP hit. These phages are also more closely related to Pseudomonas and Vibrio phages than to published Enterobacteriaceae phages. Of the 140 gene products with a BLASTP hit, 81% and 63% of the closest hits correspond to gene products from Pseudomonas and Vibrio phages, respectively. This relatedness may reflect their ecological niche, rather than the evolutionary history of their host. Despite the presence of over 800 Enterobacteriaceae phages on NCBI, the uniqueness of these two phages highlights the diversity of Enterobacteriaceae phages still to be discovered.

A review of HPRT and its emerging role in cancer

Hypoxanthine guanine phosphoribosyltransferase (HPRT) is a common salvage housekeeping gene with a historically important role in cancer as a mutational biomarker. As an established and well-known human reporter gene for the evaluation of mutational frequency corresponding to cancer development, HPRT is most commonly used to evaluate cancer risk within individuals and determine potential carcinogens. In addition to its use as a reporter gene, HPRT also has important functionality in the body in relation to purine regulation as demonstrated by Lesch–Nyhan patients whose lack of functional HPRT leads to significant purine overproduction and further neural complications. This regulatory role, in addition to an established connection between other salvage enzymes and cancer development, points to HPRT as an emerging influence in cancer. Recent work has shown that not only is the enzyme upregulated within malignant tumors, it also has significant surface localization within some cancer cells. With this is mind, HPRT has the potential to become a significant biomarker not only for the characterization of cancer, but also for its potential treatment.

Abstract 2149: Unusual expression of HPRT on the surface of the colorectal cancer cell lines HT29 and SW620

Colorectal cancer is one of the most common cancers in the world, affecting nearly 1.2 million people in the United States alone. The aim of this study is to investigate the salvage pathway enzyme HPRT as a possible biomarker in two colorectal cancer cell lines: HT29 and SW620. HPRT is a transferase in the purine salvage pathway that functions primarily by catalyzing the conversion of hypoxanthine to inosine monophosphate and guanine to guanosine monophosphate. Because of its role in proliferation and cell cycle regulation, we hypothesized an increase in HPRT expression within cancer cells, which could potentially lead to presentation on the surface of the cell. HPRT surface localization was assessed utilizing confocal microscopy, flow cytometry, and scanning electron microscopy. These techniques allowed us to visualize HPRT on the plasma membrane and quantify expression. There was statistically significant expression of HPRT on the surface of both HT29 and SW620 cells with a 28% and 58% fluorescent shift in the population, respectively. Confocal microscopy images revealed overlap between cells stained with a membrane dye and anti-HPRT FITC antibody, which indicates a direct relationship between HPRT and the plasma membrane of SW620 cells. To visualize the location of HPRT on the plasma membrane, anti-HPRT antibodies were labelled with gold and protein presence was quantified using an electron microscope, which measured the gold elemental weight percentage of each sample. When exposed to anti-HPRT antibody, the gold weight percentage of the samples significantly increased with 12.4% gold in SW620 cells and 11.2% gold in HT29 cells, indicating significant HPRT surface presence. The observed localization of HPRT to the plasma membrane was variable between the two different cancer cell lines. SW620 cells, which are faster growing and more aggressive, had significantly higher HPRT presentation that HT29 cells. These data collectively suggest that HPRT may be a possible biomarker target for the identification and possible treatment of colorectal cancer cells. Citation Format: Evita G. Weagel, Michelle H. Townsend, Michael D. Anderson, Edwin J. Velazquez, K Scott Weber, Richard A. Robison, Kim L. O9Neill. Unusual expression of HPRT on the surface of the colorectal cancer cell lines HT29 and SW620 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2149. doi:10.1158/1538-7445.AM2017-2149

Examination of Hypoxanthine Guanine Phosphoribosyltransferase as a biomarker for colorectal cancer patients

ABSTRACT The aim of this study is to investigate these enzymes as possible biomarkers in two colorectal cancer cell lines: HT29, SW480, SW620, and Colo205. With 1,168,929 individuals currently diagnosed with colorectal cancer in the United States, there remains a need to find biomarkers to improve diagnosis and expand treatment options for patients. Due to their role in proliferation and cell cycle regulation, we hypothesized an increase in salvage pathway enzyme (APRT, DCK, and HPRT) expression and possible presentation within colon cancer cells. Enzyme surface localization was assessed utilizing confocal microscopy, flow cytometry, and scanning electron microscopy. General protein expression was evaluated utilizing immunohistochemistry and Western blot analysis. While we found no statistically significant presence of either APRT or DCK on the membranes of SW620, Colo205, and HT29 cells, but found significant expression of HPRT on the surface of HT29, SW480, and SW620 cells. The average population fluorescence increased by 28%, 58%, and 40% in HT29, SW620, and SW480 cells, respectively, when compared to isotype controls. Confocal microscopy images revealed direct overlap between SW620 cells stained with a membrane dye and anti-HPRT antibody, indicating co-localization on the plasma membrane. In addition, cells treated with gold labelled HPRT antibody experienced significant changes in gold weight percentage on both SW620 and HT29 cells when compared to isotype controls. When evaluating expression within normal tissue, there was insignificant levels of HPRT binding. These data collectively suggest that HPRT may be a possible biomarker target for the identification and treatment of colorectal cancer.

The expansion of targetable biomarkers for CAR T cell therapy

Biomarkers are an integral part of cancer management due to their use in risk assessment, screening, differential diagnosis, prognosis, prediction of response to treatment, and monitoring progress of disease. Recently, with the advent of Chimeric Antigen Receptor (CAR) T cell therapy, a new category of targetable biomarkers has emerged. These biomarkers are associated with the surface of malignant cells and serve as targets for directing cytotoxic T cells. The first biomarker target used for CAR T cell therapy was CD19, a B cell marker expressed highly on malignant B cells. With the success of CD19, the last decade has shown an explosion of new targetable biomarkers on a range of human malignancies. These surface targets have made it possible to provide directed, specific therapy that reduces healthy tissue destruction and preserves the patient’s immune system during treatment. As of May 2018, there are over 100 clinical trials underway that target over 25 different surface biomarkers in almost every human tissue. This expansion has led to not only promising results in terms of patient outcome, but has also led to an exponential growth in the investigation of new biomarkers that could potentially be utilized in CAR T cell therapy for treating patients. In this review, we discuss the biomarkers currently under investigation and point out several promising biomarkers in the preclinical stage of development that may be useful as targets.

Membrane expression of thymidine kinase 1 and potential clinical relevance in lung, breast, and colorectal malignancies

BackgroundLung, breast, and colorectal malignancies are the leading cause of cancer-related deaths in the world causing over 2.8 million cancer-related deaths yearly. Despite efforts to improve prevention methods, early detection, and treatments, survival rates for advanced stage lung, breast, and colon cancer remain low, indicating a critical need to identify cancer-specific biomarkers for early detection and treatment. Thymidine kinase 1 (TK1) is a nucleotide salvage pathway enzyme involved in cellular proliferation and considered an important tumor proliferation biomarker in the serum. In this study, we further characterized TK1’s potential as a tumor biomarker and immunotherapeutic target and clinical relevance.MethodsWe assessed TK1 surface localization by flow cytometry and confocal microscopy in lung (NCI-H460, A549), breast (MDA-MB-231, MCF7), and colorectal (HT-29, SW620) cancer cell lines. We also isolated cell surface proteins from HT-29 cells and performed a western blot confirming the presence of TK1 on cell membrane protein fractions. To evaluate TK1’s clinical relevance, we compared TK1 expression levels in normal and malignant tissue through flow cytometry and immunohistochemistry. We also analyzed RNA-Seq data from The Cancer Genome Atlas (TCGA) to assess differential expression of the TK1 gene in lung, breast, and colorectal cancer patients.ResultsWe found significant expression of TK1 on the surface of NCI-H460, A549, MDA-MB-231, MCF7, and HT-29 cell lines and a strong association between TK1’s localization with the membrane through confocal microscopy and Western blot. We found negligible TK1 surface expression in normal healthy tissue and significantly higher TK1 expression in malignant tissues. Patient data from TCGA revealed that the TK1 gene expression is upregulated in cancer patients compared to normal healthy patients.ConclusionsOur results show that TK1 localizes on the surface of lung, breast, and colorectal cell lines and is upregulated in malignant tissues and patients compared to healthy tissues and patients. We conclude that TK1 is a potential clinical biomarker for the treatment of lung, breast, and colorectal cancer.

Metastatic colon adenocarcinoma has a significantly elevated expression of IL-10 compared to primary colon adenocarcinoma tumors

ABSTRACT Classical anti-inflammatory cytokines are known to play a role in both cancer progression as well as cancer elimination. We evaluated the anti-inflammatory cytokines IL-10 and TGF-β in patients with colon adenocarcinoma and metastatic colon adenocarcinoma using immunohistochemical assays to determine the expression of the cytokines between various malignant tissues. We found tissues stained with TGF-β showed no significant upregulation within malignant tumors when compared with normal tissue controls. We observed high levels of TGF-β presence in most tissues similar to GAPDH expression. Within both colon adenocarcinoma and metastatic carcinomas there was a significant variability among patients in the expression of IL-10. While some patients experienced insignificant increases in the cytokine compared with controls, other patients had a clear upregulation of the protein within their tissue. In addition, there was an increase in the number of patients positive for IL-10 upregulation within metastatic tumors when compared with primary tumors. These data indicate that there is substantial variability between patients in regards to IL-10 expression, which may further aid in characterizing tumors and evaluating metastatic potential.