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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 is administered to cure, control, or ease cancer symptoms.
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) [5-8]. 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. 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 of substantial interest from a therapeutic point of view is the cell membrane which contains surface proteins as well as intrinsic membrane proteins . Surface proteins that are restricted in their expression to specific cancer(s) could be utilised for antibody-based therapy; signalling pathways regulated by these surface proteins could also be targeted for drug based therapy.
Current technologies have the ability to profile these cell surface proteome by tagging proteins of intact cells and could therefore provide a better understanding into the regulation of the cell surface proteome in response to a range of intracellular and extracellular signals.
Our preliminary work has successfully identified protein disulphide isomerase (PDI) as protein with altered expression in certain types of cancer including breast, bladder, ovarian and prostate cancer clinical samples . PDI is primarily located within the endoplasmic reticulum in most cells where it is associated with the correct folding of nascent proteins . However, what makes it an attractive target is that it also has high expression on the surface of some cancer cells (REF) therefore making the targeting of csPDI more accessible than the intracellular version. This project aims to culture a range of established tumour cells lines including those used in preliminary work and to characterise the expression of PDI, with special attention into its localisation on the cell surface. This will be primarily achieved by using cell culture and immunocytochemical techniques. The secondary objective of this investigation is based upon the understanding that csPDI has been shown to play an important role in invasion in aggressive tumour types via its role in mediating integrin-dependent cell adhesion and thus if we could therapeutically target csPDI we could prevent the invasive process.
1.4 Protein Disulphide Bonds
Protein Disulphide Isomerase is a major form of intracellular proteins found in many species and corresponds to approximately 0.4% to 0.7% of the total cellular protein content [12-13]. Cellular functioning relies on the ability of proteins to adopt correct folds by catalysing disulphide bond formation. PDI is a soluble protein involved in catalysing these disulphide interchanges which result in the formation, reduction and the rearrangement of disulphide bonds [12, 14].
In eukaryotic cells, disulphide bonds are generally formed in the lumen of the rough endoplasmic reticulum (ER) [15-16]. The unique quality control of the ER means that proteins which fail to fold or assemble properly cannot proceed any further i.e. to the Golgi compartment. PDI thus helps to stabilise bond formation. PDI contains a reactive cysteine molecule that functions to attack any exposed disulphides of a misfolded protein; the resulting mixed disulphide is reshuffled forming a different disulphide bond (Figure 1). Native disulphide bonds are less likely to be attacked by PDI, since they are often hidden in the stable tertiary structure and, thus, not as easily exposed for attack .
The formation of disulphide bonds function to stabilize folded forms of a protein in several ways: by bonding two segments of the protein chain together ; by forming the nucleus of a hydrophobic core of the folded protein whereby local residue condense around the disulphide bond onto each other. And also by acting to destabilize the unfolded form of the protein by lowering its entropy . Once the folded proteins have left the ER folding assistance is no longer able to reverse unfolding events.
1.4 Protein Disulphide Isomerase
Two groups independently published the first identification of PDI-like activity in 1963. The group of Brunó Straub (315, 316), later president of Hungary, found that extracts from both pigeon and chicken pancreas were able to stimulate the reoxidation of reduced ribonuclease. In parallel, Anfinsen and co-workers , as part of the work on ribonuclease that earned Anfinsen the 1972 Nobel Prize in Chemistry with Moore and Stein, detailed studies showing acceleration of reactivation of ribonuclease by a microsomal system from rat liver. Anfinsen's group subsequently partially purified the enzyme responsible [21-23]. Soon after in the 1970s PDI was purified to homogeneity, followed by its 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) .
The major breakthroughs in the study of PDI came in the 1980s with the publication of the first highly cited review specifically on the enzyme (87), which raised awareness of the enzyme, and the publication of the sequence of rat PDI (70). This sequence predicted two regions with a high degree of homology to thioredoxin, a small cytoplasmic enzyme involved in thiol-dependent redox reactions (
The PDI protein is 508 amino acids in length with a C-terminal ER sequence (KDEL). The KDEL sequence is thought to mediate interactions between PDI and the KDEL receptor on the membranes of the Golgi and intermediate compartments. The PDI-KDEL complex is then recycled back to the ER. Secreted PDI retains the KDEL anchor . Studies on cultured rat hepatocytes (12) and pancreatic cells (13) support this work and showed secreted PDI to associates with the cell surface (14).
However there has also been research which claims that PDI could be transported from the cell despite the C-terminal KDEL anchor. Theories as to how PDI escapes recycling include a defect in the retention system, saturation of the KDEL receptor and escape from a salvage compartment. These ideas imply an unregulated leakage of PDI. …………et al found that an overexpression of PDI in the Chinese hamster ovary cells was seen to cause an enhanced secretion of PDI but not other resident ER proteins containing the KDEL sequence, thewse findings were supported by HT1080 cells . These observations imply that a lot more research is required into the structure and function of PDI in terms of the role of KDEL in PDI secretion. PDI in actual fact have multiple functions depending on the family member which is bound to KDEL?
No published structure exists for full-length mammalian PDI, despite decades of trials from multiple groups. NMR studies have shown PDI to have a modular structure with five domains: a, b, b′, a′ and c (FIG 2). The a and a′ domains contain a thioredoxin-like sites  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 . The c domain at the C end is rich carboxy terminal has a KDEL sequence for ER protein retention  The two catalytic a domains have a conserved CXXC motif, which is the redox-active site. When PDI functions as an oxidase, the two cysteine residues form an unstable disulfide bond and, via a mixed disulfide, this bond is transferred to the client protein . Apart from oxidizing substrates, PDI also has the ability to reduce and isomerize disulfide bonds, the latter by direct rearrangement of intramolecular disulfide bonds  or by cycles of substrate reduction and subsequent oxidation . The active sites of most PDI family members consist of a CGHC motif. The central and immediately surrounding residues are important in determining the pKa values of the active site cysteines, and therefore the preference for oxidation or reduction of disulfide bonds [87,88]. The crystal structure of yeast PDI (PDIp) revealed that the four thioredoxin domains are arranged in the shape of a 'twisted U', with the two active sites facing each other, suggesting cooperativity between the active sites .
Several hydrophobic patches were identified on the surface of PDIp, forming a continuous hydrophobic surface which may be crucial for interaction with partly folded substrates . The b½¢ domain contains the principal peptide binding site , and PDI has chaperone activity as well as oxidoreductase activity . Interaction with unfolded substrates does not depend on PDI's oxidoreductase activity , as PDI can also act as a chaperone for proteins without cysteines . Therefore, chaperone activity and oxidoreductase activity are not necessarily coupled. PDI is not the only oxidoreductase in the ER. In humans, 19 other ER-resident proteins with at least one thioredoxin-like domain have been identified, and the list is still growing (Table 1) .
Figure 2. Domain architecture of PDI. The boundaries for the domains a and b are those defined by NMR (Kemmink et al. 1996, 1999), while those for the a' domain are defined by homology to domain a.
PDI has two active sites, each with a WCGHCK motif that is similar to the active site of thioredoxin. This conserved motif is characterised by a pair of vicinyl cysteine residues that shuttle between the disulphide and dithiol form. 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 an insight has been seen into understanding the mechanisms underlying these processes.
1.5 PDI Family
The multitude of PDI family members reflects both importance and difficulty of introducing correct disulphide bonds into client proteins. 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. Among various tissues the liver contains the largest amount of PDI protein, followed by the kidneys and fat tissues .
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 disulphide bond [REF].
1.6 PDI function
PDI has been found to be secreted from a variety of cell types, among which hepatocytes  pancreatic exocrine cells , endothelial cells, activated platelets . Mutagenesis studies are now helping to unravel the catalytic mechanism of PDI and work in yeast and other systems is clarifying the physiological roles of the multiple PDI-related proteins. In 1963, Goldberger et al identified PDI as a catalyst of native disulphide bond formation in the refolding of RNase A (Goldberger et al. 1963), but other functions have since been found for this ubiquitous protein. In human thyroid cells, surface PDI is responsible for the shedding of the ectodomain of thyrotropin [36-39]. PDI also has redox-independent foldase activity, it assists folding of proteins with no disulphides [40-41] A variety of In vitro studies have also been successful in demonstrating the catalysis and kinetics of PDI activities against a range of substrates [42-45]. 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
Recently, a number of reports indicated that PDI is, in effect, a marker for the release of intracellular contents from damaged cells (for example, activating tissue factor via catalysis of thiol-disulphide exchange and thus initiating the blood-clotting cascade at the site of wound damage) (253).
A number of chaperone and anti-chaperone activities have also been attributed to PDI [46-47] with a potential involvement in a quality control system that targets misfolded proteins for degradation . PDI is known to facilitate the secretion of human lysozyme from Saccharomyces cerevisiae cells, a reflection of its chaperone activity . However in 1994 Puig et al suggested that under certain conditions PDI could facilitate misfolding and aggregation of substrates; this has been termed anti-chaperone activity. Denatured lysozyme aggregates were found in the presence of low concentrations of PDI. This hypothesis still remains questionable as to whether this activity has any 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 . Nonetheless 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.
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.
1.7 Cell surface characterisation
Why cell surface, and why not the intracellular version, talk about the environment
The presence of PDI in the endoplasmic reticulum has been well established. However, the interest now lies in the expression of PDI on the external surface of mammalian cells. Akagi et al showed immunoreactive PDI on the plasma membrane of rat exocrine pancreatic cells and Krishna Rao and Houseman isolated from chicken-embryo retina cell membranes a protein with 99% homology to chicken PDI. PDI has also been identified on the surface of B cells  and platelets . 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  Burgess et al 2000 showed that platelets activation is accompanied by an increased amount of the reduced form of the PDI active sites . More recently a link between thiols on platelet surface has been found to be involved in integrin-mediated platelet adhesion [REF]
Recent studies have found two distinct activities of csPDI: The first activity involves thiol-disulphide-exchange activity. Lahav et al showed that csPDI are involved in catalysing disulphide bond formation between integrin's and their ligands, thus promoting covalently linked adhesion of platelets to other platelets and to other vascular cells [REF]. The second activity of csPDI termed denitrosation where csPDI functions to catalyse the release of NO (nitric oxide) from RSNOs (s-nitrosothiols) . It has been suggested that after secretion, PDI binds to the cell surface through electrostatic interactions . In any case, interaction with other surface proteins is plausible, since PDI, like other members of its family, is a soluble protein, not likely to be inserted into the membrane.
A variety of functions have been suggested for PDI located on the cell surface, thus targeting csPDI may be of potential therapeutic benefit. The basis of which is believed to involve the thioredoxin sites of PDI reducing activity of the cell exterior (Mandel et al., 1993), allowing disulphide links between macromolecules present on the cell surface, to be reshuffled or reduced. Both Lawereance et al 1996 and Jiang et al 1999 supported this view by illustrating the level of cell surface thiols in lymphocytes and in fibrosarcoma cells to be positively correlated to the amount of cell surface PDI (Lawrence et al., 1996; Jiang et al., 1999). However there has been suggestion of an inverse link by Tager et al 1997 who found an increase in the number of thiols on membrane proteins was seen upon the inhibition of surface-bound PDI with anti-PDI antibodies in B cell chronic lymphocytic leukemia (Tager et al., 1997).
1.8 PDI and cancer
What is of interest in that PDI was shown by immunohistochemistry and Western blots to be overexpressed in the invasive low-generation tumours? Although some PDI staining was also observed in the less invasive, high-generation tumours, the expression was mainly confined to the tumour periphery. In addition, a direct link of PDI in cellular adhesion has been shown in retina cells from chicken embryo, where a cell surface adhesion protein was identified to be PDI . Furthermore... showed that incubation of glioblastoma cells with PDI antibody inhibits adhesion of U373 cells to the plastic surface, indicating a role of PDI in glioma cell adhesion.
The functionality of heat-shock proteins at the cell surface has begun to be elucidated, with recent work focusing on heat-shock protein-receptor interactions.
With the aid of our project and the confirmation of over-expressed PDI on the surface of a panel of human cell line, the findings could hold strong therapeutic and prognostic potential given PDIs status as sensor of cell pathophysiology
In this project the aim is to determine if csPDI could be used 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 primarily using cell culture and immunocytochemical techniques.
More on preliminary work required
To carry out cell passaging on a range of tumour cell line (breast MCF-7, breast MDA-MB 231, Bladder RT-112, bladder Ej128, Ovarian a2780, Ovarian IGROV 1, Prostate PC-3 and prostate)
To obtain growth curves by manually counting adherent cells
To carry out immunocytochemical techniques in order to characterise the presence of PDI, in particular on the plasma membrane
To verify the presence of PDI on the cell surface using confocal microscopy with the aid of multiple staining.
3.0 Materials and Methods
3.1 Cell lines
Established cancer cell lines (SW-620 (colon), PC-3 (prostate), DU145 (prostate), MDAMB-231 (breast), MCF-7 (breast), RT112 (bladder), EJ138 (bladder), A2780 (ovarian) and IGROV1 (ovarian)) were cultured as adherent monolayers. These cells were obtained from the ICT cell culture bank (University of Bradford, UK) and maintained in Roswell Park Memorial Institute medium (RPMI-1640) supplemented with 10% fetal bovine serum (FBS), 1 mM Sodium Pyruvate and 2 mM L-glutamine: All purchased from Sigma Aldrich Poole UK. Cells were grown in monolayers and propagated at 37oC in a 95% air and 5% CO2 humidified incubator. Upon reaching 60-80% confluence, cells were passaged at a 1/10 split ratio.
3.2 Routine maintenance of cell culture
For routine subculture, cells in T-25 corning flasks (Corning ltd, Surrey UK) were rinsed twice with Hanks Balanced Salt Solution (HBSS) (Sigma chemical co). Cells were lifted using 3ml of 0.25% v/v trypsin /ethylenediaminetetracetic acid EDTA solution (Sigma Aldrich Poole UK) for 5-10 minutes at 37oC. Briefly, detached cells were then resuspended into 10ml of fresh medium to stop any enzymatic action and centrifuged for 5 mins (1000 g). The supernatant containing trypsin was disposed off and the cell pellet was then further resuspended into 10ml of fresh medium. To determine the cell count a haemocytometer grid (Naubeur 0.0025mm) was used. The number of cells in suspension was determined by pipetting 10µl of cell suspension into the haemocytometer and cells counted using a light microscope x100 magnification. A mean count from five grids of both chambers was taken (4 corners and the centre). Cell count was expressed as mean cell count x104ml.
3.3 Growth curves
To determine the growth rate of the cell lines, growth curves were conducted as follows: cells were trypsinised and seeded into eight T-25 flasks to makeup exactly 10ml of complete medium. On days 2, 4 and 7 after setting up the flasks on day 0; cells from 2 flasks were resuspended and the cell count was determined in exactly the same process mentioned above (3.2). The growth curve of time versus cell number (fig 3) allowed determining how long it took the cells to reach 60-80% confluence
In order to characterise the expression of Protein Disulphide Isomerise on the surface of cancer cells immunocyotochemical techniques were used. The following materials for staining: 4% paraformaldehyde store at 4°C (Cambridge laboratories), phosphate buffered saline (PBS) powder pH 7.4 (Sigma Aldrich), Normal goat serum (NGS) (Vector laboratories), a-PDI mouse monoclonal antibody (Abcam #ab2792), Alexa 488 Goat secondary antibody (), Vectashield Hard set medium with DAPI (vectorlabs), and Wheat germ Agglutin (WGA) ()
To set up coverglass 2mls of 3x105 cells/ml were seeded overnight onto sterilised coverglass (22mm x 22mm) in 6 well multiwell plates (costar coming incorporated). For immunoflourescence, cells were washed twice in PBS to remove the medium, transferred to a fresh 6-well plate before they were fixed in 4% paraformaldehyde for 20 minutes. The cells were then washed thrice in PBS each washing step lasting 5 minutes. A 100μl of blocking buffer (PBS containing 2% NGS) was added to prevent non-specific binding and the coverglass incubated for 30 minutes. The primary antibody was added directly (1:200 dilute with NGS) without washing and the cells were incubated for a further 30 minutes. All incubation steps were carried out at room temperature. Immediately after primary antibody incubation, cells were washed thrice, counterstained with secondary antibody (Alexa Fluor-488 - labeled goat anti mouse IgG antibody used at 1:50) and incubated for a final 30 mins in the dark. Subsequently the covergrass washed three times. The coverglass were mounted onto slided in a droplet of Vectashield Hard set Medium with DAPI 4' 6- diamidino-2-phenylindole to stain the nuclei and viewed with a confocal microscope (COMPANY) at x63 magnification.
3.4.1 Multiple staining
WGA is a red dye staining the membrane. This method was a modification of (1984) and allowed to characterise the presence of PDI on the cell membrane. Cells were seeded onto sterilised coverglass and incubated overnight (37°C). After two washes in PBS, the coverglass were transferred to a clean six multiwell plate and fixed with 4% PFA for 15 minutes at 37°C. The cells were then washed 3 times in PBS and 100µl of WGA diluted in PBS (2.5ug/ml) was added and incubated at room temperature for a further 15mins. Labelling with primary and secondary antibody was carried out it to the procedure as described in 3.4.1.
The study was designed to characterise the expression of protein disulphide isomerase (PDI) on the surface of a range of cancer cell lines by utilising cell culture and immunocytochemistry techniques. Preliminary work involved attempts to optimise the antibody for PDI staining, optimise WGA for membrane staining as well identifying the effects of Tritonx100 on PDI characterisation. Further investigation involved multiple staining to clarify and confirm the surface expression of PDI using immunocytochemistry.
Cell culture growth curve
Growth curves were conducted to identify a standard pattern of growth for MCF-7 and RT112 cells. Briefly cells were resuspended in eight x T25 flasks at a concentration of 3 x104 cells/ml and left to grow for 7 days. On days 2, 3, and 7 cells were trypsinised and the total cell count averaged from 2 flasks (3.2) Figure 3 displays the results. During an initial lag phase the rate of growth for both cell line was very slow. Cell division then starts to accelerate into the exponential phase. RT112 cells had a tendency to grow at a faster rate then MCF7; where by on day 7 cell number reached 76.5 x104cell/ml. MCF-7 had a slower tendency to grown and only reached a maximum of 54.9x104cells/ml by day 7 which is nearly two thirds of that of RT112 cells. The exponential phase of growth was 2 days for RT112 cells and 5 days for MCF7. Cells for all further experiments were selected from the exponential phase of their respective growth curves. Therefore, in order to obtain optimum conditions RT112 cells were resuspended every 3 or 4 days and MCF-7 cells every 7 days.