Cancer is a set of diseases characterised by unregulated cell growth. It can lead to the invasion of its surrounding tissues and can spread (metastasise) to other parts of the body. The cause of cancer is believed to involve interactions between genetic susceptibility and environmental toxins [1-2]. People of all ages can get affected by cancer with the risk for most types increasing with age [3-6]. According to Cancer Research UK; Breast, Lung, Bowel and Prostate cancers together account for over half of all new cancer cases each year [7-8]. To date there is no magic bullet discovered for the treatment of cancer, thus therapy can vary depending on the tumour type, the cancer type as well as differences between individuals.
In terms of cancer treatment, chemotherapy means treatment with cell killing (cytotoxic) drugs. Chemotherapy is a standard option for most types of cancer: it is often used in combination with other treatment modalities such as surgery and radiation. Chemotherapeutic agents work by entering the bloodstream and reaching all parts of the body. Depending on the type or how advanced the cancer is chemotherapy can be administered to cure, control, or ease cancer symptoms.
Get your grade
or your money back
using our Essay Writing Service!
However, chemotherapy works by damaging dividing cells : which include both normal and cancerous cells explaining why chemotherapy can cause toxic side effects (e.g. bone marrow toxicity, damage to the skin, hair follicle and lining of the digestive system) [9-12]. It is important to acknowledge, not all patients undergoing chemotherapy benefit from treatment however will not be spared of the side-effects; the treatment regimen could also lead to death in a small number of patients and one need to understand these grave consequences before starting the treatment. The ability to differentiate between normal and cancerous cells is critical to be spared of unwanted side effects.
1.3 Why targeted therapy?
Unlike conventional chemotherapy, the aim of targeted therapy is to interact with specific molecules involved in processes and pathways used by cancer cells to grow, divide and spread throughout the body. One area substantial interest from therapeutic point of view is the cell membrane which contains surface proeins aswell as intrinsic membrane proteins. Current technologies have the ability to profile the cell surface proteome by tagging proteins of intact cells. Research has directed an unexpected pattern of expression of specific proteins on the surface plasma membrane especially in cancer. The profiling of the cell membrane proteomes could potentially provide new insights and uncover disease related alterations. One protein with altered protein expression is protein disulfide isomerise (PDI) which preliminary work has suggested in a panel of different tumour cell lines. PDI is mainly located in the endoplasmic reticulum in most cells where it is associated with the correct folding of nacent proteins. However what makes an attractive target is that it has high expression on the cell surface of some cancers which makes csPDI more accessible as a target than the intracellular version. This project aims to culture a range of tumour cells lunes and characterise the expression of PDI with special attention into its expression on the cell surface of tumour cells.
1.4 What is PDI?
Protein disulphide isomerase (PDI) is a multidomain member of the thioredoxin superfamily involved in catalysing thiol disulphide interchanges. PDI can function in two interrelated ways: by working as a molecular chaperone and assisting the folding of the polypeptides or alternatively as an oxidoreductase catalysing the formation, reduction and isomerisation of disulphide bonds. The enzymatic activity of the PDI was first discovered in the early 1960s by Anfinsen's group . In the 1970s PDI was purified to homogeneity, followed by a cDNA clone being isolated in the 1980s. More recently, attention has again focused on PDI following the identification of an oxidase, Ero1, which catalyses the oxidation of PDI in the endoplasmic reticulum (ER) 
1.5 Endoplasmic Reticulum
In eukaryotic cells, disulfide bonds are generally formed in the lumen of the rough endoplasmic reticulum (ER) and very rarely in the cytosol. The unique quality control of the ER means that proteins which fail to fold or assemble cannot proceed any further i.e. to the Golgi compartment. Thus disulfide bonds are located in lysosomal and secretory proteins as well as the exoplasmic domains of membrane proteins. The formation of disulfide bonds can stabilizes folded forms of a protein in several ways: it can bond two segments of the protein chain; form the nucleus of a hydrophobic core of the folded protein whereby local residues may condense around the disulfide bond onto each other, and also the disulfide bond formation can act to destabilize the unfolded form of the protein by lowering its entropy . Most proteins fold in the ER contain disulfide bonds. So why exactly are disulfide bonds so important? During folding disulfide bonds can restrict the flexibility of the polypeptide giving directionality to the folding process and could provide additional stability to the folded protein. Once the folded proteins have left the ER folding assistance is no longer able to reverse unfolding events.
Always on Time
Marked to Standard
The location and role of PDI within the endoplasmic reticulum has been well established . More recently PDI has been reporteed to be observed on the plasma membrane of several cell types [18-19], including megakaryocytes and platelets . Interestinngly surface-associated PDI is reported to be secreted from rat exocrine pancreatic cells transport PDI to the cell membrane and secrete it into the lumen .In support of these findings Turano et al found PDI to be expressed in other cellular components, suggesting a potential functional applications outside the ER . The ER is known to play several pivatol roles in cancer and with the aid of our project, the confirmation of overexpressed PDI in the ER in a panel of human cell line could hold strong therapeutic and prognostic potential given PDIs status as sensor of cell pathophysiology.
To date several functions have been attributed to PDI. In human thyroid cells, surface PDI is responsible for the shedding of the ectodomain of thyrotropin . Specifically, Ryser et al  have shown that activation and translocation of receptor-bound diphtheria toxin, as well as entry of receptor-bound (HIV) human immunodeficiency virus, require the presence of csPDI. The oxidation of cysteine residues into disulfide bonds occur during the folding process and is essential for proteins to reach their native structure. Moreover, the prevention of oxidation eventually leads to apoptosis.
Among various tissues, the liver contains the largest amount of PDI protein, followed by the kidneys and fat tissues, and it has been shown that fasting and refeeding affect the PDI protein and its enzyme activities. PDI is one of the endoplasmic reticulum stress proteins and it plays an essential role in cell survival under stress conditions . To reach their native state PDI require the formation of inter- or intra-molecular disulfide bonds via a mechanism known as oxidative protein folding . This process involves the synthesis of post transcriptionally modified secretory proteins being properly folded. Disulfide isomerase are therefore involved in catalyzing the isomerisation and formation of these disulfide bonds .
A variety of In vitro studies have been successful in demonstrating the catalysis and kinetics of PDI activities against a range of substrates [27-30]. However, these in vitro studies are highly dependent on the conditions and a lack of explanation as to how in vivo functioning of PDI catalyses these reactions. An insight into the meachanism of PDI function was given by the use of mutant forms of PDI and individual or domain combinations in different assays [31-33]
In 1994 Puig et al suggested that certain conditions could cause PDI to facilitate misfolding and aggregation of substrates; this has been termed anti-chaperone activity. Denatured lysozyme aggregates was found in the presence of low concentrations of PDI. The hypothesis remains questionable as to whether this activity has significance in vivo, although it has been reported that the anti-chaperone activity may be due to the binding of partially aggregated substrates to PDI . However, since PDI is present in the lumen of the ER at millimolar concentrations, folding substrates in vivo are unlikely to give a true representation as to PDIs contribution to anti-chaperone activity.
1.6 PDI composition
The PDI protein is 508 amino acids in length, has a typical C-terminal ER retrieval sequence (KDEL). NMR studies have shown that PDI has a modular structure with five domains: a, b, b′, a′ and c′). The a and a′ domains are homologous to thioredoxin, a small protein involved in many cytoplasmic redox reactions (Freedman et al., 1994);. The middle b and b′ domains do not show any significant homology to thioredoxin, but their secondary structure is similar to that of the a and a′ domains (Ferrari and Soling, 1999). The c domain at the C end is rich of acidic residues typical of calcium binding proteins (Lucero and Kaminer, 1999) and has a KDEL sequence for ER protein retention (Denecke et al., 1992)
PDI has two active sites, each with a WCGHCK motif which is similar to the active site of thioredoxin. This conserved motif is characterised by a pair of vicinyl cysteine residues which shuttle between the disulphide and dithiol form. The reactions that this enzyme catalyses require individual active sites to be maintained in either the oxidised. disulphide form, for disulphide bond formation, or the reduced dithiol form, for isomerisation or reduction of disulphide bonds (see Figure 1). The redox state of the active site is determined by its reduction potential and this in turn determines the reaction that PDI is able to catalyse. How the active sites are maintained in either their reduced or oxidised state, and how the ER maintains an oxidising environment, have been the subject of intense speculation over the past 40 years; but it is only recently, with the discovery of Ero1, that we are beginning to understand the mechanisms underlying these processes.
1.7 PDI Family
This Essay is
a Student's Work
This essay has been submitted by a student. This is not an example of the work written by our professional essay writers.Examples of our work
The multitude of PDI family members reflects both importance and difficulty of introducing correct disulfide bonds into client proteins. PDI has been reported to have chaperone and anti-chaperone activities  and may be involved in a quality control system that targets misfolded proteins for degradation . PDI is also known to facilitate the secretion of human lysozyme from Saccharomyces cerevisiae cells, a reflection of its chaperone activity . Recently, many homologues similar in structure to PDI have been identified. These PDI homologues have two or more CXXC motifs (where X is a variable amino acid), each of which is predicted to function as a site for the formation, reduction, or isomerization of a disulfide bond.PDI is a member of a large family of proteins that are resident within the ER and are thought to be involved in oxidative protein folding. In mammalian cells, this family includes ERp57, ERp72, ERp44, PDIR, P5, ERdj5 and PDIp; and in yeast, it includes Eps1, Eug1, Mpd1 and Mpd2. To date 14 human PDI family members have been identified in the ER, which contain one to four Tx domain motif  possibly accounting for the complexity of the ER folding machinery and suggesting specificity amongst the PDI. Although the name of the family implies that all members have a role in disulphide isomerization, only a subset are able to catalyse this reaction efficiently, whereas others are probably not directly involved in native disulphide bond formation.
PDI has been wrongly ascribed a number of different roles, mainly as a result of its ability to bind to hydrophobic affinity probes and its abundant representation in cDNA libraries. These roles include phosphoinositide-specific phospholipase C and glycosylation site binding protein.
1.9 PDI and cancer
Cellular functioning rely on the ability of proteins to adopt correct folds by catalysing disulfide bond formation. Crystal structure analysis of yeast PDI has increased our understanding of the mechanism and function PDI. Bladder RT112 cell line is a carcinoma originated from Caucasian urinary bladder. Breast MCF 7 cell line is also MCF isolated in 1970 from a Caucasian.
Preliminary work has been successful in demonstrating that PDI is over-expressed in the majority of a panel of breast, colon and bladder cancer clinical samples, as well as in tumour xenograft material. PDI is mainly located in the endoplasmic reticulum (ER) in most cells where it is associated with the correct folding of nascent proteins. However what makes PDI attractive as a potential target is that it is has been shown to be highly expressed on the cell surface of some cancer cells, making csPDI easily accessible as a target in comparison to the intracellular version.
In this project the aim is to determine if csPDI as a potential therapeutic target by characterising the expression of csPDI in a broad range of cancer types using established tumour cell lines. This will be achieved primarly using cell culture and immunocytochemical techniques
To carry out cell passaging on a range of tumour cell line (Breast MCF-7, Bladder RT-112)
To obtain growth curves by manually counting adherent cells
To carry out immunocytochemical techniques
We verified the presence of PDI on the cell surface using flow cytometry and confocal microscopy