Synergistic action between viral and environmental factors in the process of malignant transformation Shih-Chao LinAdvisor: Dr. Krzysztof Reiss

Human Polyomavirus JC (JCV)One of Polyomaviridae including BK virus and SV40.dsDNA and neurotropic virus.Upper respiratory tract and digestive tract.Murray et al. Major et. al. 1992Berger et. al. 1998 More than 80% of people exhibit anti- JCV antibodies. Causes progressive multifocal leukoencephalopathy (PML) in immunocompromised patients Transforming antigen of JCV contributes to tumorigenesis.

T-antigen of the human polyomavirus JCOncogenically transforming proteins.

T antigens include large T and small T antigen. Large T antigen regulates viral DNA replication.

T antigen has strong effects in signaling pathways of normal cells. Small T antigen is necessary to complete productive infection cycle.USC.edu

T-Ag and Cell CycleS phaseG2QuiescenceG1 phaseM phaseG0DNA replication preparationDNA replicationMitosis preparationMitosis

Polycyclic Aromatic Hydrocarbons (PAHs)Environmental compounds of various structures and toxicity.Sources: cigarette smoke, deep-fried food, and in natural crude oil.Are carcinogenic and mutagenic and are potent immunosuppressants.Toxipedia.comLaupeze et al. 2002

Carcinogenicity of PAHs Modified from Shenet al. 2006Pradhan et al. 2001 Carcinogenicity due to covalent DNA adducts and oxidative DNA damage .Stowers et al. 1985DNA adductROS production

HypothesisDNA adductsOxidative DNA damageAccumulation of mutationsCellular transformationDevelopment of brain tumorsPAHs JC virusDNA replication (p53, pRb inactivation)Abnormal cell proliferationCompromised DNA repair fidelity

I. Short-term effectiveness of PAHsCell proliferationCell cycle distributionROS accumulationDNA damagesII. Long-term effectiveness of PAHs

Experimental DesignCrude oil from BP-MC252 Deep water horizon (5/20/2010)10% DMSO aromatic extraction and 0.45mm filter sterilizationHPLC

What kinds of PAHs are present in crude oil extracts?

High Pressure Liquid Chromatography (HPLC)Solvent: water,methanol&acetonitrile Pump: provide high pressure to move liquidSamples and injector Inject sample into a columnCoolerColumn: slow down stationary phase of mixtureDAD: measure UV absorbanceFLD: measure fluorescence1234567

PAHs in crude oilFluoranthene (33.4 g/ml)Pyrene (15.4 g/ml) Benzo--Pyrene (10.1 g/ml)Phenanthanene (5.6 g/ml) Chrysene (4.9 g/ml)

With help from Dr.Zeas Lab

What are the effects of PAHs in vitro?

Experimental DesignCrude oil from BP-MC252 Deep water horizon (5/20/2010)10% DMSO aromatic extraction and 0.45mm filter sterilizationSeed R508 into 6-well platesIncubate with PAHs in dilution of 1:50, 1:100, 1:500 and 1:1000 for 24-72 hoursCollect cells for flow cytometry, cell viability count or MTS testPAHs extracts

R503 & R508R503 and R508 are mouse embryo fibroblast (MEF) cells.Cells transfected with pcDNA3.1/Zeo/JCVT expression vector are R503T and R508T.Invitrogen.comReiss et al. 1998

Stable Expression of JCV T-Ag in MEFsT AgNeucleiNeuclei + T Ag10X40XR503TR508TNeuclei + T AgR508R503T

Confirmation of T-Ag in MEFsa-tubulinBs1aBs1a/TR508R508TR503TR503Small T-AgLarge T-AgBs1a: mouse medulloblastoma

cisplatinDMSO1/501/1001/5001/100072h24hSubculture at 48hPAHs extractsCell number and morphology? (R508T)

cisplatinDMSO1/501/1001/5001/1000cisplatinDMSO1/501/1001/5001/100072h24hSubculture at 48hPAHs extractsCell number and morphology? (R508)

Total cell number after treating with PAHsCisplatin: 1mg/mLHigher concentration of PAHs reduce the cell number.Possibilities:PAHs are toxic to cellsPAHs inhibit the cell growth.

Cell viability after treating with PAHsR508T R508High concentration of PAHs cause low amount of cell death in MEFs.

MTS testDetermining cell growth rate.Yellow MTS (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazole) is reduced to purple formazan in the mitochondria of living cells Mitichondria dehydrogenase

Cell Proliferation of MEFs after incubating with PAHsR508TR508High concentration of PAHs inhibits the cell growth in MEFs.Inhibition of cell growth rate is due to the changes in cell cycle progression?

Cell cycle distribution assayCollect cells Fix cells Stain cells with PI flow cytometrysubGG0/G1SG2/M

Cell cycle distribution after treating with PAHs Decrease in cell number could be due to reduced cell growth;Reduced cell growth could result from G0/G1 arrest.Higher but not lower concentration of PAHs caused G0/G1 arrest.

Will PAH extracts induce ROS accumulation in our system?

Reactive oxygen species (ROS) ROS induced by PAHs can be responsible for DNA damage.

Dutta et al. 2010 H2DCFDA is a dye for quantifying ROS by OD photometer

ROS accumulation in MEFs after treating with PAHsR508T-72h R508-72h ***High concentration of PAHs induced ROS production.

Histone H2AXH2AX is required for the structure of chromatin. DNA damage followed by double-stranded DNA breaks leads to phosphorylation of H2AX by kinases at the sites of DNA damage for further DNA repair.abcam.com

R508-gH2A.X (72h)R508T-gH2A.X (72h)cisplatinDMSO1/50 PAHsDAPI gH2A.X MergeDAPI gH2A.X MergecisplatinDMSO1/50 PAHs

II. Long-term effectiveness of PAHsI. Short-term effectiveness of PAHs

Experimental designIncubate cells (R503T, R503, R508T, R508) with PAHs (1/500) Subculture for 6 generationsSplit cells into 6-well plate Clonogenic assay for 7 daysFix cells and stain with crystal violet (clonogenic assay)

Clonogenic assay (aka. colony formation assay)An in vitro survival assay based on the ability of a single cell to grow into a colony.Being used to determine the effectiveness of cytotoxic or carcinogenic agents.1st step to become cancer cells.

Colony formation activities of MEFs after treatment with PAHs Treated with PAHs for 6 generationsR503T R503 R508T R508PAHs+-

SummaryFive kinds of PAHs are present in crude oil. Fluoranthene, Pyrene, Benzo--Pyrene, Phenanthanene, and Chrysene. Higher PAHs Cell number Cell growth rateCell growth rate a. G0/G1 arrest b. ROS accumulation DNA damageLow concentration of PAHs could work synergistically interact to improve the abilities of cells to form colonies.

Future directionsTo confirm the clonogenic ability by using soft agar assay. To investigate intracellular signal transductions in MEFs after incubation with PAHs.To investigate the role of ROS in tumor transformation by administrating ROS scavenger.To administer PAHs to nude mice.

AcknowledgementCommittee members:Krzysztof Reiss Ph.D. Francesca Peruzzi Ph.D. Luis Del Valle M.D.Joy Sturtevant Ph.D.Leonard Meggs M.D.Members of NCR Lab:Anna Wilk Ph.D.Piotr Waligrski Ph.D. Marco PacificiAgnieszka Waligska Ph.D.Duane Jeansonne Ph.D.Collaboration:Om Pakasha Ph.D.Shahriar Koochekpour Ph.D.Arnold Zea Ph.D.David Tate Ph.D.Douglas Johnston Ph.D.

Thank you for your attention.

R508cisplatinDMSOPAHs 1/50PAHs 1/100PAHs 1/500PAHs 1/100020.74%38.28%59.02%48.89%27.39%26.55%35.73%13.22%72.06%61.78%4.01%8.29%R508TROS accumulation in MEFs after incubating with PAHs

* Today Im going to talk about my work and the topic of my work is Synergistic action between viral and environment factors in the process of malignant transformation.In specific, we are trying to figure out the effects of crude oil extracts and transforming antigen in vitro.* First of all, Let me briefly introduce JCV. JCV belongs to polyomavirus family which includes human polyomavirus BK virus and JCV as well as SV40 virus which infected monkey. JCV is a dsDNA virus and JCV is a neurotropic virus. The transmission route of JCV in human population is via upper respiratory tract or digestive tract. Most people have the history of been infected by JCV and they are positive for anti-JCV antibody but most of them have no symptoms. However, JCV still can cause a severe neural disease called PML which is a demyelinating disease especially in immunocompromised patients. Also, Transforming ag of JCV is suspected to contribute to the tumorigenesis.*T ag is one of viral proteins from HCV which is oncogenic. There are two kinds of Tag. One is Large Tag. The other is small TagLarge Tag regulates viral DNA replication. And Small Tag is required for completing productive infectious cycle. In addition, the Tag has strong effects in signal pathway of normal cells.* since were talkn about oncogenisis, we need to know more details about cell cycle. There are four phases in cell cycle progression, G1, S, G2/M phases. when cells are preparing for DNA replication, these cells are in G1 phase; S phase is DNA replication phase; when cells are preparing for mitosis, these cells are in G2 phase; and Mitosis is M phase. The cell cycle is controlled by several regulatory proteins, such as cyclin and cyclin-dependent kinases.For example, the increasing of cyclin E and binding to CDK2 promotes cell cycle from G1 to S phase. Also, these regulatory proteins are controlled by p21. p21 inhibits the complex activities of cyclin D/CDK4 as well as cyclin E/CDK2 and stop cell cycle progression. P21 is controlled by tumor suppressor protein p53. When DNA is damaged, p53 will increase and promote the expression of p21 and ultimately, stopping cell proliferation. another well-known tumor suppressor protein called retinoblastoma protein canl bind to transcriptional factor E2F and prevent cell proliferation. And Tag have been shown that they can bind two these two tumor suppressor proteins, p53 and retinoblastoma protein and promote cell proliferation which may contribute to tumorgenesis.

* The other important factor in this study is polycyclic aromatic hydrocarbons, also called PAHs. PAHs are a group of environmental compounds of various structures and toxicity, as you can see the diagram here. This diagram shows four major types of PAHs.PAHs exist in a lot of sources such as cigarette smoke, deep-dried or over-barbecued food or in natural crude oil. some of PAHs are consider to be carcinogenic and mutagenic. Some reports mentioned that PAHs could suppress immune function which may contribute to malignant transformation. * The mechanism of carcinogenicity of PAHs is due to two pathway. One is PAHs can form covalent bonds to DNA and insert into dsDNA and become a structure called DNA adducts as the picture shows here; and the other way is PAHs can induce the production of reactive oxygen species, ROS, and cause oxidative DNA damages.Both could contribute to the oncogenesis.* So since most people have the history of been infected by JCV but very rarely, they will develop to brain tumors. So other environment factors like PAHs could be involved in the process of tumorgenisis. Therefore, our hypothesis has two factors. This picture is describing these two factors. One is viral factor which is represented by Tag of JCV. The viral factor is responsible for abnormal cell proliferation and unfaithful DNA repair. The other factor is environmental which is presented by PAHs. PAHs could be responsible for formation of DNA adducts and oxidative DMA damages. Combine these two factors could lead to accumulation of DNA mutation and cellular transformation and ultimately malignant transformation could develop to brain tumors. So in part I, we will focus on some experiments to evaluate the short-term effectiveness of PAHs. In part II, we will focus on long-term effects.* The overall experimental design is that we got PAHs from Dr. Koochekpours Lab and they have already extracted the PAHs from crude oil by using DMSO. We will examine what kind of PAHs exist in our samples by using HPLC and some in vitro experiments and we will talk about this part later.So the first question we are going to ask is what kinds of PAHs are present in the crude oil extracts from deep water horizon?*The method we used to examine is by HPLC, High pressure liquid chromatograph.The principle of HPLC, in briefly, is to prepare different gradient of solvents. And the pump of HPLC will provide high pressure to drive liquid through a chromatography column. meanwhile, samples will be injected into the column and the interaction between solvents and column can extract and separate different fractions of mixture based on their mobility. In the end, the detectors, like DAD and FLD can measure the UV absorbance or fluorescent and turn into the concentration of compounds.* With the help of Dr. Zeas Lab, we found there are at least five kinds of PAHs within our crude oil samples. As you can see here the highlights here.

Also, these PAHs have been considered to be carcinogenic.

PAHs DMSO extracts were analyzed by HPLC (Dionex Ultimate 3000) equipped with absorbance detector and the reverse phase C18 column (Waters Spherisorb, 5m ODS2, 4.0x 250 mm) with the injection volume 50l and the detector set to 254 nm.

Next, we want to know what will happen when PAHs treat cells? * The overall experimental design is we will used these PAHs extracts which includes all PAHs that we have identified previously. Incubate the cells with different dilution of PAHs, we will dilute PAHs to 1 to 50 which is the highest conc in out system and 1 to 100, 1 to 500 or 1 to 1000 and incubate cells for different time points. And we will collect these cells for following experiments.* Before showing the in vitro results, Id like to talk a little bit about the cells we used. We used 2 similar kinds of cells, R508 and R503. They are mouse embryo fibroblastoma cells and they both have been transfected with vectors which can express Tag of JCV so called R503T and R508T The picture down here shows how the fibroblast look like.So first, we need to make sure the Tag is expressing in our cells. * We performed immunostaining for Tags by using anti-Tag antibodies. In the fluorescent images presented here, we found the Tag express in both R503T and R508T cells but not those cells without transfection.And we would like to confirm this result by western blotting.

* in western blotting result, We chose, BS1a and Bs1a/T which are mouse medulloblastoma cells as controls for Tag expression. As you can see here, a-tubulin is the internal control and there are the two size of Tag, both Tags are only expressing in cells after transfection but not untransfected cells. * We first treated PAHs on the 508T cells and check the cellular morphology. We used cisplatin which is a chemotherapy drug as a positive control because of its cytotoxicity, and DMSO is a vehicle negative control.When we treated the cells for the first 24hrs with 1 to 500 or 1 to 1000 dilution of PAHs, the cell numbers and cellular morphology is similar to DMSO control. But when we treated cells with higher con of PAHs, like 1 to 50 dilution, the cell number was much lower than DMSO control.This reduction of cell number was getting worse when we incubated for 72hr. And we want to know how about cells without Tag, so we applied PAHs on 508 cells.*So we saw the similar result when we treated 508 cells with PAHs. The cell number was lower in higher conc of PAHs comparing to DMSO control. So we quantified the results by counting total cell number. *This is the quantitative result.For cisplatin control, after 3 days incubation, the cell number was static and much lower than DMSO control. The cell number of DMSO control was increasing but for 1 to 50 of PAHs, the cell number was static and similar to cisplatin-treated group in both 508 and 508T cells. but if we diluted the conc of PAHs, the cell number also increased as in DMSO control.So we think higher conc but not lower conc of PAHs may down lower the cell number in either 508T or 508.We assume there could be two possibilities to cause this result.One is PAHs could kill the cells; the other is PAHs can inhibit the cell growth. So first we performed cell viability count for the 1st possibility.*In this cell viability result, we counted the percentage of viable cells. In cisplatin control, it caused almost up to 30% of cell death in both 508T and 508 cells after 3 day incubation. But 1 to 50 dilution of PAHs group, PAHs caused almost no cell death in the first 2 days and less than 20% of cell death after 3 days, lower than cisplatin control. So we think the PAHs dont have high cytotoxicity and the cytotoxicity of PAHs could not be the major reason to lower down the cell number. Therefore, we performed MTS test to evaluate the cell growth rate.*MTS test is similar to MTT test and has been widely used for determining cell growth rate. The yellow MTS compound can be reduced to purple formazan by mito deh in living cells. By measuring the conc of formazan, we can quantify the cell growth rate.

*We found that the cell growth rate of DMSO control increased after 3 day incubation but cell growth rate in 1 to 50 dilution of PAHs group seem to be inhibited. Also, when we diluted the conc of PAHs to 1 to 500 or 1 to 1000, there is no inhibition in cell growth rate in both 508T and 508 cells.

So we obserbed the cell growth rate is inhibited by higher conc PAHs, and we wondered if the reduction of cell growth rate is due to any changes in cell cycle distribution. So we performed CCD assay by flow cytometry. The diagram here is an example of CCD assay result. In cell cycle distribution assay, we determined the conc of DNA in cells according to PI staining. And assort cells into different cell cycle phases. And the cells in subG phase which I didnt mention earlier are usually treated as dying cells which include apoptotic any necrotic cells.*As you can see the result here, comparing to DMSO control, we found that in both R508T and R508 cells, cell population in G0/G1 increased when treated with higher conc. of PAHs. We didnt see any significant cell cycle distribution changes in lower conc of PAHs gpsThis result suggests that higher but not lower conc of PAHs cause G0/G1 arrest in ccd.And This result also suggests that the decreased total cell number in PAHs treated cells is due to the reduced cell growth. And the reduced cell growth could be resulted from G0/G1 arrest.Next question we are going to ask is will PAH extracts induce ROS accumulation in our system?*As I mentioned earlier, PAHs have been shown that it could lead to ROS production and cause oxidative DNA damages so we are going to evaluate the ROS accumulation in our system. Here we used the dye called H2DCFDA to quantify the ROS. When this dye is taken by cells and go into cytoplasm, it will be activated by intracellular easterase and oxidized by ROS in cytoplasm. And oxidative DCFDA will generate fluoresence. So by measuring the fluorescence, we can quantify the ROS accumulation in cells.*We used ELISA reader to measure the fluorescence of DCFDA. After 72hr of incubation with PAHs, in both R508 and R508T cells, we found that high conc of PAHs really induced ROS accumulation comparing to DMSO control. However, when we diluted the conc of PAHs to 1 to 500 and 1 to 1000, the ROS production failed. *Since we know higher conc of PAHs can induce ROS accumulation in our system so next we want to know if this ROS accumulation cause any DNA damage.Histone H2AX is one of major proteins for the structure of chromatin. When DNA is damaged and generate dsDNA breaks, the H2AX will be phosphorylated by ATM and ATR kinases at DNA damage locations and H2AX can recruit DNA repair proteins for further DNA repair.*We performed the immunostaing for H2AX proteins. In these images, in both 508 and 508T cells, we found that pH2AX expression after treating with 1 to 50 dilution of PAHs for 72hrs but not in DMSO control. So we know after treatment with higher conc of PAHs, there are some DNA damage and DAN repair are ongoing in these cells.In part 2, we will focus on evaluating the long-term effectiveness of PAHs.*So far, we have done some experiments about short-term effects of PAHs in mouse embryo fibroblast cells. Next we would like to know what is the long-term effects of PAHs. So we incubate cells with a moderate conc of PAHs which is 1 to 500. Because 1 to 500 of PAHs wont lead to cell death or inhibit cell growth or make changes in cell cycle distribution.Here is the overall experimental design, we incubated cells with 1 to 500 conc of PAHs and subculture these cells for 6 generations and then split cells to 6-well plate for another 7days incubation so called clonogenic assay. Before showing the result, Id like to introduce clonogenic assay.*The clonogenic assay as known as colony formation assay is an in vitro assay.It is based on the ability of a single cell to grow and form a colony and staining these colonies with crystal violet. Clonogenic assay is widely used for determine the effectiveness of cytotoxic or carcinogenic agents, like PAHs.The ability to grow and form a single colony is the first step to become cancer cell.And the photo here shows how clonogenic assay result looks like. *In this clonogenic assay result, we counted the number of colonies and found that after long-term and lower concentration treatment of PAHs which are black bars, cells seem to have better ability to form colonies than groups without PAHs treatment, the white bars. We dont know yet whether this is a synergistic interaction between Tag and PAHs. So in the future we hope to investigate the detailed mechanism.**In the future directions, we want to evaluate the clonogenic ability by using soft agar assay. And we want to investigate the intracellular protein expression after treating with PAHs.Also, we will use ROS scavenger to further confirm the role of ROS in tumor transformation.Finally, we hope to apply PAHs to animal models.***By using flow cytometry, we found the shift of the fluorescence to the right in both 508T and 508 cells. the diagram down here is a quantitative result showing that higher conc but not lower conc of PAHs can induce ROS accumulation after 72hr incubation.