Effect of Oxidative Stress on HAGE Expression

Effect of aerobic Stress on HAGE ExpressionCancer is a group of diseases which involves lawless cubicle growth, metastasis and invasion of carrels to otherwise create from raw stuffs via lymph node or blood. Cancer is ca employ by many factors much(preno houral) as (i) Chemicals such(preno secondal)(prenominal) as tobacco, alcohol, asbestos etc., (ii) Viral infections such as Human Papiloma Virus, Epstin Barr Virus, hepatitis B, C etc., (iii) Ionizing radiations such as X-Rays, UV-Rays, Gamma Rays etc., (iv) transmissible factors such as mutations in BRCA 1 and BRCA 2, p53 mutations, APC broker mutations, Retinoblastoma ingredient mutation etc., (v) Hormones which promote growth proliferation such as growth hormones, estrogen, progesterone etc.Cancer is classified into crab louseous and benign tumours. Malignant tumours atomic number 18 generally fluid tumours which metastasize to other tissues via lymph node and blood. Benign tumours argon mostly truehearted tumours which stay at one particular tissue and seldom migrate. Malignant and Benign tumours ar re classified on the typewrite of cell and tissue from which they originate. They atomic number 18 classified as followsi) Carcinomas These are the malignancies of the epithelial cells, which line the internal organs of the body and alike give on the surface the body such as skin.ii) Sarcomas These are solid tumours present in connective tissues such as bones and lymph systems (Crowley 2006) what is more the tumours are classified according to (i) site of origin as in lung cancer, breast cancer etc., (ii) type of cell such as rhabdosarcoma and acute lymphocytic leukaemia. (Ruddon 2007)The progression from a ordinary cell to malignant one involves the dysregulation of genes implicated in the control of normal proliferation / death oer many years. These are normal functions regu modern by proto- transforming genes and tumour suppresser gene genes.Proto-Oncogene And tumor Suppressor GenePro tooncogene is form to be norm whollyy convey in all vertebrates including humans. It is as well as found to be uttered in roughly insects and yeasts. Protooncogenes play an important role regulating normal cell growth and several(predicate)iation. They also perform functions such as sign on transduction and mitogenic signal execution RAS, MYC, TRK, ERK are some examples of protooncogenes (Novakofski 1991). Oncogenes are derived from protooncogenes during the process of carcinogenesis. The formation of alive(p) oncogene leads to subnormal cell proliferation and thus contributes to the formation of tumour (Alitalo and Schwab 1986). A protooncogene can be converted into an oncogene in a manikin of ways such as (i)Transduction induced by retroviruses which receive the desegregation viral DNA with the host. This DNA on translation give bristle to viral proteins which cause the activation of protooncogene, thereby leading to cancer. (ii) Mutation indoors the protooncogene le ading to an increase activity of the protein. (iii) chromosomal translocation where the gene for proto oncogene is translocated to other loci, which cause its abnormal twist. For eg in the translocation of abl in chromosome 9 to bcr character chromosome 22 as found in Philadelphia chromosome (Rowley 1973) (iv) Amplification induced by gene duplication, mis-regulation of gene which leads to overexpression of protooncogenes. (Todd and Munger 1999)Unlike oncogenes, the tumour suppressor genes normally quash cell growth in variety of ways such as (i) repressing the genes important for cell hertz progression. For eg., pRb (retinoblastoma ) controls the G1 stage by organism hypophosphoryated and not allowing the release of transcription factor E2F which promotes G1-S transition. (ii) Stopping cell turn on detecting DNA damage. DNA damage induces p53 which now leaves from its interacting teammate Mdm2 and increases the activity of p21. This p21 protein accordingly inactivates cyclin dependent kinases which are essential for cell cycle progression, (iii) Apoptosis may also be promoted by p53 when damage is irreversible. (iv) inducement DNA repair proteins to repair DNA damage and prevent abnormal cell proliferation. (Yoshida, et al. 2000) on that point are a number of mechanisms by which a cancer cell survives in a body. This is illustrated in the diagram infra.(Weinberg and Hanahan, 2000) treatmentThe choice of therapy depends upon the location and stage / grade of the tumour, as surface as the health of the patient. There are a number of therapies for treating cancer such as surgery, chemotherapy, radiaotherapy, hormonal therapy etc.,The deregulation of many genes in cancer cells leads to the over-expression of adapted proteins which can be used as biomarker which can provide attend to for the diagnosis and / prognosis of the treatment but can also be used as potential target for immunotherapy.Cancer antigens and their classificationTumour antigen is an a ntigenic substance produced in the tumour cells and triggers an immune reaction in the host. They are useful in identifying tumour cells and are used in cancer therapy. Tumour antigens are classified as Cancer globe antigen, Differentiation antigens, Tumour peculiar(prenominal) unique antigens, Overexpressed Self Antigens, Viral antigens, Post-translationally, Oncofoetal antigens, Idiotypic Antigens as shown in the table belowCopied from (Li, et al. 2005)Amongst all these categories, cancer testis antigen gift the most promising group of antigen to be used in coming(prenominal) immunotherapeutic interventions due to their restricted expression to mainly to tumour cells with the expulsion of placenta and testis which are immune-privileged sites and therefore would not pose any truly issue for the risk of autoimmunity.(Simpson, et al. 2005)HAGEHAGE also known as DDX43 and CT13 antigen belongs to this category of antigens and was kickoff place by Martelange et al use cDNA subt raction get along of a human sarcoma cell line.(Martelange, et al. 2000)Using rational hybrid analysis, it was found that HAGE gene is located on chromosome 6. HAGE was found to be overexpressed in several tumours. There is a low expression of HAGE in normal tissues since the expression is regulated by hypermethylationHAGE is also over expressed in tissues such as testis, placenta, and ovaries. HAGE is unremarkably expressed at the m-ribonucleic acid level though recent studies in melanoma cells suggest that HAGE could also be expressed at the protein level.(Mathieu, et al. 2010) The relate DDX43 comes from the fact that HAGE also belongs to another group of protein known as departed box proteins and referred to as DDX43. (Abdelhaleem 2004)Dead box proteinsThese proteins were discovered in the late 1980s. (Gorbalenya, et al. 1989)Dead box proteins belong to the family of ribonucleic acid helicases and play an important role in the transcription, post transcriptional modification s such as splicing, transport, translation, decay and biogenesis of ribosomes. NTP hydrolysis provides energy to these enzymes and they extricate dsRNA or disrupt RNA-Protein interaction. DDX and DHX are widely studied Dead quoin Proteins. There are many classes of DDX and HAGE belongs to DDX43.They are so named because they guard the amino group acids D-E-A-D (Asp-Glu-Ala-Asp) in one of their motifs namely Motif II. Besides they also contain motifs such as motif I, Q-motif, motif VI, motif Ia, Ib, III, IV and V as shown in the diagram below. These motifs bestow properties such as adenosine tri orthophosphate binding and hydrolysis, RNA interaction, remodelling activity etc to these proteins. The consensus sequence of DEAD box family is shown belowCopied from (Linder 2006)Regulation and Function of HAGEBoth function and regulation of HAGE have not been studied in great detail (Scanlan, Simpson and Old 2004). In normal tissues HAGE expression is regulated by DNA hypermethylation, Histone modifications such as histone aceylation, histone deacylation and histone methylation. provided in cancer, there could be many mechanisms of upregulation of HAGE. The most commonly theme mechanism is the demethylation of DNA which leads to HAGE over-expression. (Roman-Gomez, et al. 2007)HAGE could also be induced due to switch of normal cells during cancer or due to the action of oncogenes. HAGE might also be induced randomly. HAGE may play a role in pre- informational RNA splicing, ribosome biogenesis, transcription and initiation of translation. (Rocak and Linder 2004)This exteriorise lead focus on point in era and uterine cervix Cancer.Head and Neck CancerHead and Neck cancer are the cancers that arise from the upper aerodigestive tract such as haggard cavity, oral cavity, pharynx, larynx and paranasal sinuses. Most of them are of squamous cell carcinoma type of the head and neck and have been given the acronym -SCCHN or HNSCC. (Argiris, et al. 2008). The diagram of the habitus of the head and neck is illustrated belowCopied from (Wu, et al. 2009)More than half a million patients are diagnosed with head and neck cancer every year. Head and Neck cancer in fact accounts for more than 3-10% of the cancers(Gourin and McMains 2005). Smoking, alcohol, chewing of betel jerk with tobacco and areca are the major risk factors for this cancer. Recently, HPV type 16 has been identified as one of the causes for head and neck cancer and account for 40% (Goodger and McGurk 2000)The progression of this tumour is complicated. It progresses from the normal features to hyperplasia, mild dysplasia, moderate dysplasia, severe dysplasia, carcinoma, incursive carcinoma and metastasis. The progression may be caused by due to genetic instabilities such as loss of heterozygosity of chromosome 9p21, inactivation of of p16 and loss of 3p, loss of 18q, inactivation of PTEN, some translocations, 17Beta heterozygosity or TP53 mutation. (Argiris, et al. 2008)Copied fr om (Argiris, et al. 2008)Interestingly HAGE was found the John van Geest group to be overexpressed in 40% of HNSCC cancers (unpublished data) and although much of its function is now emerging no information exist at the moment regarding the regulation of its expression. However it has been find that cells left for longer period of time in the incubator had a significantly higher expression of HAGE at the mRNA and protein level.Aim of this projectThe observation that HAGE was over-expressed in 40% of HNSCC led to hypothesis that cells under tense up i.e lack of nutrients, decrease oxygen level, as well as other factors influence HAGE expression. Moreover it has been found that HAGE is also expressed in 20% of Acute Myeloid Leukemia and 50% Chronic Myeloid Leukemia as well as many solid tumours such as HNSCC (Adams, et al. 2002). This suggests that there moldiness be something in common between these 2 very different forms of cancer. CML and solid tumours are known to have a high ex pression of reactive oxygen species (ROS), therefore it might be possible that ROS induces HAGE expression.frankincense this project testament focus on the number of oxidative distort has on HAGE expression. The effect of other stress such as temperature may also be investigated.METHODSCell CultureThe PCI 13 and PCI 30 cell lines would be supplied by Prof E Tatour. These cell lines are item for Head and Neck cancer. The cells would be grown in RPMI 1640 +10%(v/v) FCS+2mM L-glutamine and incubated at 37oC and 5%C02 atmosphere.Extraction of m-RNAThe culture media would be aloof and washed with DPBS(Dulbeccos phosphate buffer saline). past DPBS pull up stakes be obliterated followed by the appendix of RNA-STAT 60. RNA pellets would be then retrieved and re-suspended in ddH2O. RNA extracted is then quantified utilize NANODROP 8000 UV spectrophotometer. RNA would be then added in measurement well of the spectrophotometer. The spectrophotometer would be read at 260 and 280nm and the amount of RNA exit be mensural. The RNA concentration is calculated in g/l beforehand being adjusted to 1g/l by ddH2O. RNA ideals would be then stored at -80oC and used for conducting RT-PCR and RTq-PCR in the future.RT-PCR (Reverse Transcriptase PCR)From the extracted RNA cDNA go away be synthesized. 2 g of RNA take in go away be mixed with 1 l of oligo-dT primers and diluted to 15 l of ddH20 in an Eppendorf tube. All RNA samples would follow the same process. UNO Thermoblock entrust be used to heat the tubes to 70C for 5 min which allows the primer-RNA annealing. 10 l of reverse transcriptase mix, which go away be active by mixing Muloney Murine Leukemia Virus 5-X buffer, dNTPs, RNasin RNAse inhibitor, M-MLV-reverse transcriptase and ddH20. The tubes exit be then pre-heated in a water bath at 39.2C for 80 min which allows cDNA synthesis. Tubes result be removed and kept over again in the UNO-Thermoblock and heated to 95C for fin minutes which stops the reaction. These tubes would be stored at -20C for future use. reliable Time qPCRcDNA generated from RT PCR ordain be used as a tem casing and the reagents used will be (i) 6.25l iQ Sybr Green, (ii) 0.5l gene specific sense primer (iii) 0.5l gene specific anti-sense primer, (iv) 4.75 l ddH20 and 0.5l cDNA template. These reagent mixtures will be added to all tubes for a specific gene. Samples will be usually carried out in replicate with a negative control that contains the reaction mixture without cDNA. Rotogene 6000 real-time qPCR analyzer will be used to carry out RT qPCR. Relative gene expression using 2CT method acting will be calculated with the attend of the expression of housekeeping genes HPRT-1, HSP-27 and HSP-90.The primers which will be used in real time qPCR experiment is given below. These primers are supplied by mwg-Eurofins. The primers have a stocktaking concentration of 100pmol/l and will be diluted to a working concentration of 10pmol/l. From this working 0.5l (5pM) o f primers will be used.Total Protein extractionCells will be grown to 75% confluence in T75 flasks. Cells will be trypsinised, washed and re-suspended in DPBS and these cells will be then counted using trypan blue. Cells will be diluted to 1X106/ml using DPBS and 5 X106 cells would be taken in a 1.5ml eppendorf. Centrifugation will be done to pellet the cells. The supernatant will be remove and lysis buffer containing a cocktail of RIPA buffer and 10% protease inhibitor will be added to the pellet. The eppendorf tube will be then placed on a tube rotator at 4C for 30 min. It will be then kept on ice for 30 min. Cells will be again centrifuged at 14000 RPM for 30 min at 4C. Aliquot of the supernatant will be prepared to be stored at -20C for future use.Total Protein bank checkTotal protein assay will be carried out using BioRad Dc protein assay reagents. Series of dilutions (0.2,0.4,0.5,0.8,1.0,1.5,2.0mg/ml) will be created using stock BSA solution with the concentration of 10 mg/m l. Protein extracts and standards would be tested in triplicates and duplicates respectively. Assays will be performed in 96-well cps bottom plates. 25l and 200 l of reagent A and B will be added to all the samples and the samples will be incubated at style temperature for an hour. Proteins will be then read at 750nm and their concentrations would be calculated based on the standard values.Western Blotting30 l of sample will be loaded into the wells of SDS PAGE gels. 1X tris-glycine-SDS will be used as the running buffer. A known molecular metric weight unit ladder will also be run alongside the samples. initially 70V current will be applied to aid the migration of proteins through the 5% stacking gel. Once the proteins reach the 10% separating gel, 90V current will be applied. after(prenominal) the proteins run through the gel, they will be transferred to the PVDF membrane with the servicing of liquid transfer. This membrane will be prepared by swear out with 10% methanol for 5 entropys, ddH2O for 5 min and transfer buffer for 10 min. Liquid transfer will be carried out using the by-line stairs.1. Cold transfer buffer will be used to exhaust a gel frame.To this gel frame a sponge pre ludicrous in transfer buffer will be placedThen a filter card, gel, PVDF membrane, another filter card and a second pre soaked buffer sponge will be placed in order starting from the first filter card.Proteins will be transferred with the help of electric current of 100V applied for one hour.After completion, membranes will be cut and treated with different antibodies.10% (w/v) Marvel take out solution will be used to wash the membrane for one hour at room temperature. This washing blocks the non-specific binding sites. The blocking solution will be then discarded and the antibody diluted to 10% (w/v) in Marvel milk solution will be added. This antibody coated membrane will be foment overnight on a plate rocker at 4C. The near day there would be 3, 10 min washes with TSBT(Tris-Buffered Saline-Tween-20). During these washes the membrane vessel will be agitated in plate orbital shaker at room temperature.After completing the washing process, a secondary antibody specific to the primary antibody will be added. The steps for adding the secondary antibody are same as the primary antibody addition neglect for the fact that no overnight incubation is required and the secondary antibody will be incubated only for one hour while being agitated on the shaker at room temperature. The marker will also be stained using streptavidin-HRP secondary antibody.After performing the above step the membranes will be developed. Membrane development will be performed by placing the membrane in a tray and washing it with ECL reagent. The membrane would be then exposed for a certain period of time using CCD camera.Assay for ROS-DCFDA Stress TestStock Solution PreparationThe stock solution of Hanks Buffered Salt Solution (HBSS)will be prepared according to the manufact urers protocol.DCFDA(2,7-dchlorofluoroscein -diacetate) AssayTo test ROS levels DCFDA test will be performed. Before conducting the DCFDA assay, the standardization of best concentrations of H2O2 and DCFDA for detecting cell stress should be performed. It is important to note that H2O2 should be added only sub-lethally and should not be added in proportion which may cause cell death.The PCI 13 and 30 cells will be plated out in two 24 well plates. 1ml of each type of cells plus RPM1640 10% FCS media will be added to 8 wells per plate and will be incubated overnight at 37C. The media will be removed the next day and increasing concentration of DCFDA will be added to both plates and the cells will be incubated with Tinfoil wrapping to prevent light exposure and incubated at 37oC for 30 min. The cells will be then washed and then refrigerating HBSS will be added. After this step the cells will be emphasize with increasing concentration of H2O2 (see diagram below). The cells will the n be again wrapped in tin foil and placed on a rocking platform for 15 min. The same method will be followed for treating other wells with different concentrations of DCFDA. Experiments will be performed at least doubly for each cell line. H2O2 will be removed after 15 min and cells will be trypsinised with 100microL trypsin and Versene. 800 microL of DPBS will be added to one of the 2 wells and will be pipetted thoroughly so as to remove the cells from the well surface. This will be then transferred to the other well and then 1ml of solution would be transferred to Flow associated cell cytometry (FACS) tube for analysis. The same turn will be carried for other sets of wells. DCFDA fluorescence would be measured using Gallios conflate cytometer and results would be analysed in Kaluza program.Time Line For The ProjectDuring the month of May, all techniques such as Western Blotting, mRNA/protein extraction, Real Time PCR will be learnt. This time is indicated in red colour in the represent below. This will overlap with the time duration, from May to the end of June, during which hydrogen hydrogen peroxide experiment will be performed as per written in the method section and the expression of HAGE will be monitored at both the mRNA and protein level. Also from the start of June to the end of July repeat experiments will be performed and if time permits, the effect of thermal stress on HAGE expression will also be investigated. This is indicated in the graph below.SUMMARYCancer Testis Antigens (CTA) are antigens that are expressed in a variety of tumours. They are usually absent in normal tissues with the exception of testis and placenta where they are expressed as self tolerant antigens. Since these antigens have strong immunogenicity and their expression is mostly restricted to tumours, they are ideal targets for cancer immunotherapy. Therefore much query is ongoing for the identification of CTA.Recently helicase antigen HAGE was identified as a CTA and was found to be over-expressed in HSNSCC, AML and CML. Thus there might be a common link between these different types of cancers. Moreover ROS is known to be induced in all these tumours and it may be possible that ROS causes increased expression of HAGE. Thus the aim of this project would be to investigate the effect of oxidative stress on HAGE expression.HAGE expression would be analysed both at the m-RNA and protein level in two cell lines namely PCI 13 and PCI 30 using techniques such as m-RNA extraction, RT PCR, Western Blotting and Protein assay. The ROS level would be evaluated using the DCFDA assay.Since the mechanism of HAGE regulation is unknown, positive result in the project would help in elucidating a mechanism by which HAGE could be regulated.