Effect of Oxidative Stress on HAGE Expression

Effect of Oxidative Stress on HAGE Expression Cancer is a group of diseases which involves uncontrolled cell growth, metastasis and invasion of cells to other tissues via lymph node or blood. Cancer is caused by many factors such as (i) Chemicals such 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) Hereditary factors such as mutations in BRCA 1 and BRCA 2, p53 mutations, APC gene mutations, Retinoblastoma gene mutation etc., (v) Hormones which promote growth proliferation such as growth hormones, estrogen, progesterone etc. Cancer is classified into malignant and benign tumours. Malignant tumours are mostly liquid tumours which metastasize to other tissues via lymph node and blood. Benign tumours are mostly solid tumours which stay at one particular tissue and seldom migrate. Malignant and Benign tumours are re classified on the type of cell and tissue from which they originate. They are classified as follows: i) Carcinomas: These are the malignancies of the epithelial cells, which line the internal organs of the body and also present 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) Furthermore the tumours are classified according to (i) site of origin as in lung cancer, breast cancer etc., (ii) type of cell such as rhabdomyosarcoma and acute lymphocytic leukaemia. (Ruddon 2007) The progression from a normal cell to malignant one involves the dysregulation of genes implicated in the control of normal proliferation / death over many years. These are normal functions regulated by proto-oncogenes and tumour suppressor genes. Proto-Oncogene And Tumour Suppressor Gene Protooncogene is found to be normally expressed in all vertebrates including humans. It is also found to be expressed in some insects and yeasts. Protooncogenes play an important role regulating normal cell growth and differentiation. They also perform functions such as signal 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 active oncogene leads to abnormal cell proliferation and thus contributes to the formation of tumour (Alitalo and Schwab 1986). A protooncogene can be converted into an oncogene in a variety of ways such as (i)Transduction induced by retroviruses which cause the integration viral DNA with the host. This DNA on translation give rise to viral proteins which cause the activation of protooncogene, thereby leading to cancer. (ii) Mutation within the protooncogene leading to an increased activity of the pr otein. (iii) chromosomal translocation where the gene for proto oncogene is translocated to other loci, which cause its abnormal expression. For eg in the translocation of abl in chromosome 9 to bcr region 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 inhibit cell growth in variety of ways such as (i) repressing the genes important for cell cycle progression. For eg., pRb (retinoblastoma ) controls the G1 stage by being hypophosphoryated and not allowing the release of transcription factor E2F which promotes G1-S transition. (ii) Stopping cell cycle on detecting DNA damage. DNA damage induces p53 which now leaves from its interacting partner Mdm2 and increases the activity of p21. This p21 protein then inactivates cyclin dependent kinases which are essential for cell cycle progression, (iii) Apoptosis may also be promoted by p53 when damage is irreversible. (iv) inducing DNA repair proteins to repair DNA damage and prevent abnormal cell proliferation. (Yoshida, et al. 2000) There are a number of mechanisms by which a cancer cell survives in a body. This is illustrated in the diagram below.(Weinberg and Hanahan, 2000) Treatment The choice of therapy depends upon the location and stage / grade of the tumour, as well 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 altered proteins which can be used as biomarker which can provide help for the diagnosis and / prognosis of the treatment but can also be used as potential target for immunotherapy. Cancer antigens and their classification Tumour antigen is an antigenic 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 testis antigen, Differentiation antigens, Tumour specific unique antigens, Overexpressed Self Antigens, Viral antigens, Post-translationally, Oncofoetal antigens, Idiotypic Antigens as shown in the table below Copied from (Li, et al. 2005) Amongst all these categories, cancer testis antigen represent the most promising group of antigen to be used in future immunotherapeutic interventions due to their restricted expression to mainly to tumour cells with the exception of placenta and testis which are immune-privileged sites and therefore would not pose any real issue for the risk of autoimmunity.(Simpson, et al. 2005) HAGE HAGE also known as DDX43 and CT13 antigen belongs to this category of antigens and was first identified by Martelange et al using cDNA subtraction approach 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 hypermethylation HAGE is also over expressed in tissues such as testis, placenta, and ovaries. HAGE is usually expressed at the m-RNA level though recent studies in melanoma cells suggest that HAGE could also be expressed at the protein level.(Mathieu, et al. 2010) The name DDX43 comes from the fact that HAGE also belongs to another group of protein known as Dead box proteins and referred to as DDX43. (Abdelhaleem 2004) Dead box proteins These proteins were discovered in the late 1980s. (Gorbalenya, et al. 1989)Dead box proteins belong to the family of RNA helicases and play an important role in the transcription, post transcriptional modifications such as splicing, transport, translation, decay and biogenesis of ribosomes. NTP hydrolysis provides energy to these enzymes and they unwind dsRNA or disrupt RNA-Protein interaction. DDX and DHX are widely studied Dead Box Proteins. There are many classes of DDX and HAGE belongs to DDX43. They are so named because they contain the amino 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 ATP binding and hydrolysis, RNA interaction, remodelling activity etc to these proteins. The consensus sequence of DEAD box family is shown below Copied from (Linder 2006) Regulation and Function of HAGE Both 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. However in cancer, there could be many mechanisms of upregulation of HAGE. The most commonly thought mechanism is the demethylation of DNA which leads to HAGE over-expression. (Roman-Gomez, et al. 2007) HAGE could also be induced due to transformation 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-mRNA splicing, ribosome biogenesis, transcription and initiation of translation. (Rocak and Linder 2004) This project will focus on Head and Neck Cancer. Head and Neck Cancer Head and Neck cancer are the cancers that arise from the upper aerodigestive tract such as nasal 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 anatomy of the head and neck is illustrated below Copied 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 leaf 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, invasive 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 from (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 observed 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 project The observation that HAGE was over-expressed in 40% of HNSCC led to hypothesis that cells under stress 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 must be something in common between these 2 very different forms of cancer. CML and solid tumours are known to have a high expression of reactive oxygen species (ROS), therefore it might be possible that ROS induces HAGE expression. Thus this project will focus on the effect of oxidative stress has on HAGE expression. The effect of other stress such as temperature may also be investigated. METHODS Cell Culture The PCI 13 and PCI 30 cell lines would be supplied by Prof E Tatour. These cell lines are specific 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-RNA The culture media would be removed and washed with DPBS(Dulbeccos phosphate buffer saline). Then DPBS will be removed followed by the addition of RNA-STAT 60. RNA pellets would be then retrieved and re-suspended in ddH2O. RNA extracted is then quantified using NANODROP 8000 UV spectrophotometer. RNA would be then added in measurement wells of the spectrophotometer. The spectrophotometer would be read at 260 and 280nm and the amount of RNA will be calculated. The RNA concentration is calculated in  µg/ µl before being adjusted to 1 µg/ µl by ddH2O. RNA samples 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 will be synthesized. 2  µg of RNA sample will be mixed with 1  µl of oligo-dT primers and diluted to 15  µl of ddH20 in an Eppendorf tube. All RNA samples would follow the same process. UNO Thermoblock will be used to heat the tubes to 70 °C for 5 min which allows the primer-RNA annealing. 10  µl of reverse transcriptase mix, which will be prepared by mixing Muloney Murine Leukemia Virus 5-X buffer, dNTPs, RNasin RNAse inhibitor, M-MLV-reverse transcriptase and ddH20. The tubes will be then pre-heated in a water bath at 39.2 °C for 80 min which allows cDNA synthesis. Tubes will be removed and kept again in the UNO-Thermoblock and heated to 95 °C for five minutes which stops the reaction. These tubes would be stored at -20 °C for future use. Real Time qPCR cDNA generated from RT PCR will be used as a template and the reagents used will be (i) 6.25 µl iQ Sybr Green, (ii) 0.5 µl gene specific sense primer (iii) 0.5 µl gene specific anti-sense primer, (iv) 4.75  µl ddH20 and 0.5 µl cDNA template. These reagent mixtures will be added to all tubes for a specific gene. Samples will be usually carried out in duplicate with a negative control that contains the reaction mixture without cDNA. Rotogene 6000 real-time qPCR analyser will be used to carry out RT qPCR. Relative gene expression using 2ΔCT method will be calculated with the help 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 stock concentration of 100pmol/ µl and will be diluted to a working concentration of 10pmol/ µl. From this working 0.5 µl (5pM) of primers will be used. Total Protein extraction Cells 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 discarded 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 4 °C 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 4 °C. Aliquot of the supernatant will be prepared to be stored at -20 °C for future use. Total Protein Assay Total 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/ml. Protein extracts and standards would be tested in triplicates and duplicates respectively. Assays will be performed in 96-well round bottom plates. 25 µl and 200  µl of reagent A and B will be added to all the samples and the samples will be incubated at room temperature for an hour. Proteins will be then read at 750nm and their concentrations would be calculated based on the standard values. Western Blotting 30  µl 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 weight 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 the proteins run through the gel, they will be transferred to the PVDF membrane with the help of liquid transfer. This membrane will be prepared by washing with 10% methanol for 5 seconds, ddH2O for 5 min and transfer buffer for 10 min. Liquid transfer will be carried out using the following steps. 1. Cold transfer buffer will be used to immerse a gel frame. To this gel frame a sponge pre soaked in transfer buffer will be placed Then 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 milk 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 agitated overnight on a plate rocker at 4 °C. The next 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 except 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 Test Stock Solution Preparation The stock solution of Hanks Buffered Salt Solution (HBSS)will be prepared according to the manufacturers protocol. DCFDA(2,7-dchlorofluoroscein -diacetate) Assay To test ROS levels DCFDA test will be performed. Before conducting the DCFDA assay, the standardization of optimum 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 37 °C. 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 cold HBSS will be added. After this step the cells will be stressed with increasing concentration of H2O2 (see diagram below). The cells will then 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 twice 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 an d 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 procedure will be carried for other sets of wells. DCFDA fluorescence would be measured using Gallios flow cytometer and results would be analysed in Kaluza program. Time Line For The Project During 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 graph below. This will overlap with the time duration, from May to the end of June, during which 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. SUMMARY Cancer 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 research 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.