Finished Research Proposal

Module Code: 100C9 Candidate Number: 145244

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Title: Investigating the Role of XRN1 in Ewing Sarcoma

Contents Page Number

1.0 Summary 2

2.0 Introduction 2

2.1 Complete Project Background 2

2.2 Background to Osteosarcoma 3

2.3 Ewing Sarcoma - Comparisons to Osteosarcoma 4

2.4 XRN1 – A Potential Therapeutic Target 6

2.5 Preliminary work 7

2.6 Brief Overview of the Project 8

3.0 Aims and Objectives 9

4.0 Programme of Work 9

4.1 Determine the Rate of Growth of EWS and Control Cell Lines Using Cell Counting at

Defined Time Points 9

4.2 Extract RNA From EWS Cell Lines to Determine the Expression Levels of XRN1 mRNA

Using qRT-PCR 10

4.3 Determine the Expression Levels of XRN1 Protein in EWS Cell Lines Using Western

Blots 11

4.4 Determining the Localisation of XRN1 in EWS Cell Lines Using Immunocytochemistry

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5.0 Gantt Chart 11

6.0 Outcomes – A Summary of What Will be Achieved 12

6.1 Growth Curves 12

6.2 qRT-PCR 12

6.3 Western Blots 12

6.4 Immunocytochemistry 12

7.0 References 13


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1.0 Summary

Osteosarcoma is the most common form of bone cancer, predominantly occurring in

adolescents. Treatment options for Osteosarcoma have not improved for approximately 30

years making a new form of therapy particularly desirable. A potential therapeutic target is the

5’ exoribonuclease XRN1, which is shown to have decreased expression levels in osteosarcoma

cells. Ewing sarcoma (EWS) tumour cells derive from the same stem cell lineage (Mesenchymal

stem cells/MSCs) as osteosarcoma and are the second most common form of bone cancer. This

malignancy has also not seen improvements in therapy for some time. Both the mRNA and

protein expression levels of XRN1 are to be assessed in two different EWS cell lines using qRT-

PCR and Western blots, respectively, to see if the same observation can be made in this

malignancy. The results would provide an insight into where in the MSC differentiation pathway

the defect causing a decrease in XRN1 expression levels occurs since EWS cells do not derive

from the same stage of MSC differentiation as osteosarcoma cells. This information has potential

therapeutic applications as XRN1 could be utilised as a therapeutic target for one or both of the

disorders depending on the outcome of the project.

2.0 Introduction

2.1 Complete Project Background

The most common form of bone cancer is osteosarcoma, which is a primary bone malignancy

particularly prevalent in adolescents (Mirabello et al., 2009). Since the arrival of neoadjuvant

chemotherapy there have been no improvements on the 5 year survival rate, which has

stagnated at approximately 60% for 30 years (Friebele et al., 2015). This reflects the importance

of developing a new therapeutic target to tackle this disease. Very few genetic alterations have

been identified that are consistent in different osteosarcoma patients; meaning it is very

desirable to find a valid molecular therapeutic target (Martin et al., 2012). An exciting potential

target is XRN1, which is a 5’-3’ exoribonuclease that is seen to be downregulated in

osteosarcoma cells (Zhang et al., 2002) – referred to in the paper as hSEP1. The reason why

XRN1 is particularly appealing is because the Newbury lab has demonstrated that apoptosis and

compensatory cell proliferation are increased in response to XRN1 downregulation in the model

organism Drosophila melanogaster. Since a hallmark of cancer cells is the evasion of apoptosis

(Hanahan and Weinberg, 2000) it is hypothesised that this downregulation of XRN1 in

osteosarcoma cells contributes to cellular proliferation, with a secondary mutation being a likely

reason why apoptosis is prevented. It is currently unknown when the event that causes this

downregulation occurs with respect to the differentiation pathway of mesenchymal stem cells

(MSCs) into mature osteoblasts. The word ‘event’ is used because the molecular reasoning for

the decrease in XRN1 is not currently known. Downregulation is thought to the result of a

different defect in the MSC differentiation pathway because the Newbury lab have shown that

there are no mutations in the downregulated XRN1 (Ingham-Clarke., Unpublished)(Ling Ng,

Unpublished).

MSCs are multipotent stem cells that can differentiate into many mesenchymal cells such as

adipose and muscle cells but the most relevant to osteosarcoma is bone (Fig 1). It is possible that

osteosarcoma could potentially be from a developmental defect in MSCs or the result of a

spontaneous defect occurring in differentiated osteoblasts. To test whether XRN1 expression is

related to a developmental defect in MSCs it is logical to study the expression of XRN1 in Ewing


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sarcoma (EWS) as the tumour cells derive from the same stem cell lineage. It should also be

noted that EWS is the second most common form of bone cancer, also having a high incidence

rate in adolescents (Balamuth and Womer, 2010). Because they both derive from MSCs, if XRN1

is downregulated in both cancers it could potentially be the result of a developmental defect in

MSCs, which opens a doorway to a potential new therapeutic target. Specifically, this project

sets out to identify XRN1 expression levels in two EWS cell lines (SK-ES-1 and RD-ES) to allow

comparisons with previous data from the Newbury lab on osteosarcoma cell lines.

Figure 1. Differentiation Pathway of Mesenchymal Stem Cells. This project focuses on the

differentiation pathway that leads to the formation of bone cells (far left). However, it is clear

from this diagram (Dimarino et al., 2013) that it is highly multipotent and can go on to form

many mesenchymal cells.

2.2 Background to Osteosarcoma

Osteosarcoma most commonly affects children and adolescents because it is the stage of life

where rapid growth and development occurs - in particular the elongation of long bones. The

primary tumour cells of osteosarcoma originate from the epiphyseal plate (growth plate) of long

bones such as the femur, pelvis and arms where cells are dividing at the highest rate within the

bone (Fig 2). Patients with osteosarcoma are taller than the age and sex matched general

population reflecting the excessive growth of cells at this location (Cotterill et al., 2004). In

addition to this, the average age of onset for females is younger than males, which correlates

with the fact that females generally undergo a growth spurt sooner than males (Marina et al.,

2004). This is an obstacle for diagnosis because a lot of patients put the joint pain caused by the

tumours down to growth pain (McCarville, 2009). This gives an opportunity for the tumour to

grow and metastasise. A further problem to the diagnosis of osteosarcoma is that laboratory

tests cannot always identify the presence of a tumour. Determining whether a lesion is benign or


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malignant by imaging is also challenging so unfortunately there are multiple factors that

contribute to tumours not being identified as early as possible (McCarville, 2009).

Osteosarcoma cells are osteoblast-like as they have the ability to produce malignant osteoid.

The presence of malignant osteoid results in calcification of immature bone in undesirable

locations, mimicking the normal development of bone in an ectopic location (Fig 2d-h). As for

their cellular origin there is evidence to suggest that they can originate from MSCs, committed

osteoblasts or both (Mutsaers and Walkley, 2014). From these sites cancerous cells most

commonly metastasise to the lungs as well other sites in the body (Saha et al., 2013). Lung

metastases prove fatal in 30-50% of cases, highlighting the importance of identifying the tumour

prior to metastasis (Huang et al., 2009). This form of cancer is also relatively prevalent in large

dogs, whereby the survival rate is less than 20% after the point of diagnosis despite amputation

taking place in many cases (Selvarajah and Kirpensteijn, 2010).

Figure 2. Schematic of Endochondral Ossification (Bone Development) (Gilbert, 2000). The cells

in the mesenchyme all derive from MSCs. (A-B) Mesenchymal cells differentiate into cartilage.

(C) Central chondrocytes undergo apoptosis or hypertrophy to achieve mineralisation of the

extracellular matrix. (D-E) Apoptosis enables blood vessels to invade the bone shaft, which

transfers osteoblasts to the developing bone. (F-H) Hypertrophy, proliferation and

mineralisation by chondrocytes solidify the bone’s structure. Secondary ossification sites occur

at areas that new blood vessels invade.

2.3 Ewing Sarcoma - Comparisons to Osteosarcoma

EWS cells also derive from MSCs and make up the second most common primary sarcoma in

children. Again, it commonly occurs in long bones as well as the ribs, spine and pelvis, however

there are many features that distinguish EWS from osteosarcoma. A key difference lies in the

cellular origin whereby EWS tumours can also occur at a primary site outside of bone and within

soft tissue. When this is the case the disorder is termed extraosseous EWS (Rud et al., 1989).

Furthermore, unlike osteosarcoma that arises solely in the growth plate, EWS tumours occur at

an equal frequency anywhere along the length of the bone (Balamuth and Womer, 2010). Whilst

osteosarcoma cells somewhat resemble osteoblasts, EWS cells are harder to identify as they are


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more poorly differentiated (Longtin, 2003). EWS patients are not significantly taller than the

general population at the average age of onset, however, patients presenting at an age below 15

do show an increase in average height (Cotterill et al., 2004). A likely explanation for

osteosarcoma patients being generally taller is that osteosarcoma cells selectively reside in the

growth plate. Despite these differences being present, EWS encounters similar problems at the

level of diagnosis with growth pains often being put down as the reason for joint pains

(McCarville, 2009).

There are further similarities and differences at the level of cellular morphology, function and

genetics. Osteosarcoma cells are characterised by the secretion of malignant osteoid whereas

EWS cells are identified by their small blue round appearance after haematoxylin and eosin

staining (Iwamoto, 2007), however, small cell osteosarcoma can mimic this appearance (Lee et

al., 2011). With regards to genetic alterations, osteosarcoma frequently exhibits deletions of

Wnt or other Wnt signalling pathway genes (Du et al., 2014). Inherited mutations in the

retinoblastoma gene RB1 are also associated with this disorder as well as mutations in the

tumour suppressor gene p53 (Ottaviani and Jaffe, 2009). These are of course relatively common

mutations exhibited in multiple cancers and not particularly specific to osteosarcoma. On the

other hand, EWS has more unique genetic associations. A fusion of the 5’ portion of the EWSR1

gene from chromosome 22 to an ETS transcription factor family member is a genetic event that

occurs in the majority of EWS cases (Delattre et al., 1992). EWSR1 fuses to the 3’ portion of the

FLI1 gene on chromosome 11 85% of the time with ERG fusions making up 10% of EWSR1-ETS

fusions. The result of this translocation is an oncogenic fusion whose gene product (Termed

EWS-FLI1 – Fig 3) is an aberrant transcription factor that promotes tumorigenicity by both

activating and repressing different genes. Interestingly, the severity of prognosis of a particular

EWS tumour can be related to the breakpoint position of genes within this fusion (Ross et al.,

2013). The EWSR1 portion of the fusion has two main regions that contribute to its function, one

being an RNA binding domain and the other being a transcriptional activation domain. FLI1

encodes a transcription factor with an ETS DNA-binding domain. Together, the oncogenic fusion

product targets multiple genes involved in tumour promoting functions such as angiogenesis and

apoptosis (Sharrocks, 2001). Overall, this non-random chromosomal translocation is used to

classify a broader range of tumours. The EWS family of tumours (EWSFT) groups together

peripheral primitive neuroectodermal tumours (PNET), Askin tumours and EWS based on the

fact that they all possess a EWSR1-ETS family of transcription factor translocation. This is in spite

of the conditions being morphologically heterogeneous (Khoury, 2005).


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Figure 3. Diagram of the Chimeric EWSR1-FLI1 Protein Fusion. The 5’ portion of the EWSR1 gene

on chromosome 22 fuses to the 3’ portion of the FLI1 gene on chromosome 11. This oncogenic

fusion results in the translation of the chimeric protein called EWS-FLI1 diagrammatically

represented above. The N terminal domain of the protein derives from EWSR1 and the C

terminal domain derives from FLI1 (de Alava and Gerald, 2000).

2.4 XRN1 – A Potential Therapeutic Target

As previously mentioned, treatment for these cancers has not improved for far too long.

Osteosarcoma is preferentially treated by surgery (complete amputation or limb salvage) with

chemotherapy often being employed either prior to the surgery to shrink the tumour

(neoadjuvant) or afterwards to help fully eradicate cancer cells (Ferguson and Goorin, 2001). On

the other hand, EWS is responsive to radiotherapy as well as the other aforementioned

treatment options, where appropriate (Donaldson, 2004). With the lack of improvements a new

therapeutic target is essential and XRN1 is a possibility. XRN1 is a 5’ exoribonuclease that

functions to degrade mRNAs and aid the biogenesis of miRNA, which in turn exert their own

post-transcriptional control of gene expression. It is the only cytoplasmic exoribonuclease that

can degrade in a 5’-3’ direction (Jones et al., 2016) and is found in discrete, prominent foci

(Bashkirov et al., 1997) that have subsequently been termed as P or Processing bodies.

Since XRN1 is a well conserved enzyme the Newbury lab were able to study its expression in the

model system Drosophila, where the homologue of XRN1 is called Pacman (Waldron et al.,

2015). It was in this model system that the link between XRN1/Pacman expression levels and

apoptosis/cellular proliferation were made. It was seen that the number of cells undergoing

apoptosis increased in XRN1 null mutant wing imaginal discs. A further observation was that

proliferation, presumably in a compensatory manner also increased, but not to an extent that it

balanced out apoptosing cells meaning a net loss of cells was exhibited. Since a hallmark of

cancer is to evade apoptosis (Hanahan and Weinberg, 2000) it is logical to suggest that the

decreased expression in osteosarcoma cells contributes only to increased cellular proliferation

as opposed to the apoptosis exhibited with Pacman null mutants in Drosophila. This opposing

effect is likely to be a driven by a secondary mutation that blocks apoptosis such as the

frequently mutated p53 (Amaral et al., 2010). However, it needs to be noted that at this time

this is purely speculation. Pulling together the fact that osteosarcoma and EWS derive from the

same stem cell lineage and the observations pertaining to XRN1, it can be summarised that

knowledge on XRN1 expression in EWS could provide information on when XRN1 expression

becomes decreased in the development of MSCs. The null hypothesis is that there is no change

to XRN1 levels. This would still be informative because it would indicate that the defect leading


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to decreased XRN1 expression in osteosarcoma occurs in the late stages of MSC differentiation

where they are already committed to becoming osteoblast-like. On the other hand, if there is a

decreased expression of XRN1 in EWS then it is likely that faulty MSC development (with respect

to XRN1) at an early stage could have a role to play in the pathogenesis of both EWS and

osteosarcoma.

2.5 Preliminary work

The project to be undertaken builds upon recent unpublished work specific to XRN1 expression

levels in osteosarcoma, which was performed in the Newbury lab. The original paper reporting a

link between XRN1 and osteosarcoma was published in 2002 and whilst the methods they used

to quantify XRN1 levels were satisfactory (Measuring absorbance with UV-spectrometry and

semi-quantitative reverse transcription PCR) (Zhang et al., 2002), they have subsequently been

repeated using the more modern technique of TaqMan quantitative reverse transcription (qRT)

PCR to confirm the findings. Western blots were also utilised to measure XRN1 protein levels

because mRNA concentration does not necessarily correlate to protein concentration due to the

possibility of control at the level of protein translation. Incidentally, XRN1 plays a big role in

regulating RNA stability so a nice picture of the interplay of molecules within the cell can be built

from this example. (Fig 4A) Zhang et al revealed decreased expression in three of four

osteogenic sarcoma (OGS) cell lines (TE85/HOS, SAOS – no decrease, U2/U2OS and MG63). (Fig

4B) Furthermore there was obvious decreased expression in six of nine OGS biopsy specimens,

with two of the other three samples showing decreased expression to a lesser extent. Overall,

mRNA expression levels were reduced in most OGS biopsy specimens.

Figure 4. Gels Showing the Decreased XRN1 Expression in OGS Cell Lines/Osteosarcoma cell

line growth curves. (4A) This gel demonstrates the relative expression of XRN1 (Referred to as

hSEP1) in four OGS cell lines compared to one control cell line (Foetal osteoblasts/FOB) (Zhang

et al., 2002). XRN1 and GAP (Housekeeper) were co-amplified in the presence of a radioactive

nucleotide (alpha-32P-dCTP) using RT-PCR before being subjected to electrophoresis on a 2%

agarose gel and processed to view bands based on radioactivity. The M lane has 100bp

molecular weight standards and N has no cDNA to show that the bands are specific to DNA. (4B)

The same procedure except for the method of image development (Ethidium bromide staining

replaced radioactive nucleotide incorporation) was performed for nine OGS specimen, with

decreased expression shown in the majority of samples (Zhang et al., 2002). (4C) When noting

that the control cell line is HOb it can be clearly seen that the three cell lines exhibiting

decreased XRN1 expression (4A) exhibit enhanced growth (Pashler, unpublished).

A C

B


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A B C

The unpublished work from the Newbury lab explored the same four cell lines. Human foetal

osteoblasts/HOb acted as a control throughout since osteosarcoma cells are osteoblast-like. The

first piece of information gathered was how fast/aggressively the cell lines grew (Fig 4C) relative

to the control and each another. Cells were pelleted for subsequent PCR and Western

experiments when they were in their log phase of growth because it is expected that the most

expression occurs at this point. Furthermore, cells are considered to be their healthiest at this

stage because once they plateau (stationary phase) they will begin to die soon after. The growth

curve (4C) only begins to show death on day 6 of HOS cells but the trend would follow with other

cell lines if more time points were incorporated into the graph. The growth curve also provides a

point of reference to look at when studying XRN1 expression levels; with a logical hypothesis

being the cell lines that grew the quickest/most aggressively would have the lowest XRN1

expression levels. With this in mind, qPCR experiments were carried out to determine whether

this was the case. It turned out that the results showed this correlation (Fig 5A). XRN1 levels

were inversely correlated to the speed in which the cell line proliferated (Fig 5C, based on

information from Fig 4C). Furthermore, when looking into XRN1 protein expression levels in the

most aggressive cell line (HOS) it can be seen that levels were greatly decreased (Fig 5B). It is

expected that the Western blot data for the rest of the cell lines would show that decreased

XRN1 protein expression correlates with an increased rate of growth.

Figure 5. Analysing the expression levels of XRN1 mRNA and protein in osteosarcoma cell lines.

(5A) Results of qPCR analysis of XRN1 mRNA levels with 7 biological repeats being incorporated

into the data. (5C) The extent of XRN1 downregulation correlates with the aggressiveness of the

cell line. (5B) Results of a western blot that looked to analyse XRN1 protein expression levels in

the most aggressive osteosarcoma cell line – HOS.

2.6 Brief Overview of the Project

Building upon this work, this project sets out to replicate the above experiments for Ewing

sarcoma cell lines. The two that have been selected are SK-ES-1 and RD-ES. SK-ES-1 has an

epithelial morphology, a modal chromosome number of 49 (making it hyperdiploid) and derives

from an 18 year old Caucasian male (ATCC® HTB86™). Similarly, RD-ES also has an epithelial

morphology and is derived from a 19 year old Caucasian male (ATCC® HTB166™). In contrast to

the previous work in the Newbury lab, MSCs are chosen as a feasible appropriate control

because EWS cells do not necessarily derive from human osteoblasts. It provides a comparison

between a healthy precursor cell and immortalised Ewing sarcoma cells. The ideal control would


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be a healthy version of the tissue each cell line derived from, however this was not possible to

obtain. If the work was on biopsy specimens the same logic could be applied, whereby the ideal

control would be nearby healthy tissue that the tumour cells derived from. However, it also

needs to be considered that gaining biopsies is difficult to do, especially from children. To start

the project, the first step will be to establish the growth curves (4.1) before moving onto RNA

extraction and qRT-PCR selective for XRN1 (4.2). mRNA is a marker for gene expression levels

since it is only present once a gene has been transcribed. Furthermore, these results will be

complemented with data on protein expression levels from Western blots (4.3). The overall

objective is to be able to determine whether XRN1 is downregulated in EWS cell lines or not.

3.0 Aims and Objectives

1 Determine the rate of growth of EWS and control cell lines using cell counting at defined time

points

2 Extract RNA from EWS cell lines to determine the expression levels of XRN1 mRNA using qRT-

PCR

3 Determine the expression levels of XRN1 protein in EWS cell lines using Western blots

4 Determine the localisation of XRN1 in EWS cell lines using immunocytochemistry

4.0 Programme of Work

4.1 Determine the Rate of Growth of EWS and Control Cell Lines Using Cell Counting at Defined

Time Points

To determine the rate of growth of each cell line (SK-ES-1 and RD-ES) the same number of cells

(75,000) will be harvested from each cell line when splitting cells and plated on 6-well plates in

the same volume of media (3ml). The cells in each well will then be incubated and allowed to

grow in parallel. For approximately two weeks the entire contents of one well will be sampled

and counted every 24 hours using a haemocytometer. Daily readings will then be plotted onto a

graph with days on the x axis and cell number on the y axis. This growth curve is expected to

initially take a sigmoidal shape before dropping off once cells die making it more bell shaped.

The lag phase corresponds to the cells adjusting to the plate by adhering to the plastic surface.

The log phase is when the cells are growing exponentially, they are at their healthiest and this is

the fastest rate of mitosis. The stationary phase occurs when cells decrease their rate of mitosis

due to a limited supply of nutrients, which subsequently leads to the death phase as cells starve

and die. The relevant phase for subsequent experiments is the log phase as this is when the cells

are deemed to be at their healthiest and expressing the most mRNA/protein. At this stage cell

pellets will be obtained for subsequent RNA extraction. A total of fourteen pellets will be

obtained with seven being used for PCR analysis and seven for Western Blots. This is a sufficient

number of repeats for statistical analysis. Pictures of cells will also be obtained to compare their

morphology with osteosarcoma cell lines. It is important to determine the rate of growth of the

EWS cell lines relative to the MSC controls, themselves and osteosarcoma cell lines (Using

unpublished data from the Newbury lab). This information can be compared with XRN1

expression to determine whether there is a correlation between XRN1 expression and

aggressiveness of growth like there is in the osteosarcoma cell lines. Furthermore, the log phase

resembles the best time to harvest cells for qRT-PCR and Western blots because cells are at their


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healthiest and expressing the most amount of mRNA/protein.

4.2 Extract RNA From EWS Cell Lines to Determine the Expression Levels of XRN1 mRNA Using

qRT-PCR

To determine mRNA expression levels the total RNA in each cell pellet will be extracted using an

miRNeasy mini kit (Qiagen). The concentration of extracted RNA will then be quantified by a

nanodrop machine before the RNA is frozen at -70oc ready for PCR. This low temperature is used

to prevent any RNase activity. A miRNeasy kit will be used because it extracts miRNA as well as

mRNA, which could potentially be useful in latter experiments. The miRNA may not be needed at

all but it is useful to have the option. To perform a qRT-PCR experiment the RNA will be

amplified and reverse transcribed to cDNA by reverse transcription (RT) PCR. Using the same

amount of RNA from each cell line (100ng/μl) quantitative (Q) PCR will then be performed on the

reverse transcription PCR products using primers that will selectively amplify XRN1 as well as

two different housekeeper proteins (HPRT1 and GAPDH), which act as normalisation controls.

These control assays function to prove the PCR is working and also make XRN1 expression

readings relative to the sizes of particular cells. If cells are bigger then more XRN1 mRNA will be

present; however there will also be more housekeeper mRNA. The usage of seven different

pellets will act as biological repeats, with technical replicates also being incorporated within

both the PCR procedures (Fig 6). A control with no reverse transcriptase will be incorporated in

the RT-PCR procedure to ensure it is only DNA that is being amplified. A control with no cDNA

(Replaced with H2O) will be incorporated in the quantitative PCR reaction to ensure it is only the

cDNA in the samples that is being amplified. TaqMan® reagents will be used throughout the qRT-

PCR procedures. mRNA is a marker of gene expression, therefore this procedure will determine

whether or not XRN1 is downregulated in the EWS cell lines in comparison to the control cell line

(MSCs) and osteosarcoma cell lines (Using previous unpublished data from the Newbury lab) (Fig

5).

Figure 6. Flow chart for

qRT-PCR. The x7 next to

cell pellet refers to the

amount of times this

process is repeated to

generate the XRN1

mRNA expression data.


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Aim Technique Jan Feb Mar Apr May Jun Jul Aug

3.1 Cell Counting to Establish Growth Curves

3.2 RT/Q-PCR to Determine XRN1 mRNA Expression Levels

3.3 Western Blotting to Determine XRN1 Protein Expression Levels

3.4 Immunohistochemistry to Determine XRN1's Cellular Localisation

3.5 Focus on Presentation and Dissertation

4.3 Determine the Expression Levels of XRN1 Protein in EWS Cell Lines Using Western Blots

Western blotting will be employed to assess protein expression levels. Whenever cell pellets are

obtained during the splitting of cells, an equal number of pellets will be set aside for both

western blotting and PCR. This means that comparisons between mRNA and protein expression

levels can be made between cells sampled at the same time. XRN1 protein expression in EWS

cell lines will be compared to levels in the MSCs. The normalisation control will be GAPDH

because previous work in the Newbury lab has shown that this is the protei n with the most

consistent level of expression throughout this procedure. As with qRT-PCR the normalisation

control ensures cell size does not influence expression data. Despite the Western blots having to

be performed at a different time to the PCR it is not an issue because pellets are amenable to

snap-freezing in liquid nitrogen and long term freezing at -70oc. Determination of protein

expression levels is necessary because mRNA expression does not always correlate with

translated protein levels due to various processes pertaining to post-transcriptional control of

gene expression, however it is expected that there will be a positive correlation. These results

will give an indication of how much functional XRN1 is present within the cell. This data will

complement the data from the mRNA experiments as well as those previously performed on

osteosarcoma cell lines in the Newbury lab.

4.4 Determining the Localisation of XRN1 in EWS Cell Lines Using Immunocytochemistry

Immunocytochemistry will be utilised to determine where XRN1 is localised in both the EWS cell

lines as well as the MSC control. XRN1 has previously been exclusively found in P bodies co-

localised with the P body marker DCP2 (decapping enzyme) (Orban and Izaurralde, 2005). The

relevant antibodies for this process are already present in the Newbury lab. The primary

antibodies for XRN1 and DCP2 will be added before the fluorescent secondary antibodies that

bind to these primary antibodies. This stains the specific molecules making them visible under a

fluorescence microscope, which will be followed up by confocal microscopy. This semi-

quantitative technique will determine whether there are more or less P bodies in the EWS ce ll

lines compared to control (MSC) and osteosarcoma cell lines (Using unpublished data on

osteosarcoma cells from the Newbury lab).

5.0 Gantt Chart

The green colour denotes that it will be performed during that month. Although establishing

growth curves only takes a relatively short amount of time (approximately two weeks), there

needs to be an allowance for resuscitating the cells from frozen stocks and growing them up to

confluence. Furthermore, the acquisition of multiple cell pellets for qPCR/Western blotting takes

longer for the slower growing control cell lines (MSCs), which is why the PCR and Westerns take

a relatively long period of time.


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6.0 Outcomes – A Summary of What Will be Achieved

6.1 Growth Curves: The growth curve information will determine the best time to harvest cells

for qPCR and Western experiments. It will also reveal whether the two cell lines grow at the

same rate, and how this compares to the growth rate of the control cell line. Furthermore,

subsequent data on XRN1 expression levels can be compared to the rate at which a particular

cell line grows.

6.2 qRT-PCR: If XRN1 mRNA is expressed at lower levels in EWS cell lines compared to controls

then it will suggest that its downregulation is a result of a dysfunction early in the differentiation

pathway of MSCs. Furthermore it will provide further evidence that the XRN1 expression profile

contributes to EWS and osteosarcoma tumour progression.

6.3 Western Blots: If XRN1 protein levels are also downregulated it will suggest that XRN1 mRNA

levels correspond to protein levels in EWS cell lines. If there are low levels then it provides

further evidence that the dysfunction leading to decreased XRN1 expression occurs early in the

differentiation pathway of MSCs. Furthermore, it will suggest that the XRN1 protein expression

profile contributes to EWS and osteosarcoma tumour progression.

6.4 Immunocytochemistry: If there is a change in localisation of XRN1 in EWS cell lines relative

to control cell lines it would imply that expression levels have an impact on localisation.


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6.0 References Amaral, J.D., Xavier, J.M., Steer, C.J., Rodrigues, C.M., 2010. The Role of p53 in Apoptosis. Discov. Med. 9, 145–152. Balamuth, N.J., Womer, R.B., 2010. Ewing’s sarcoma. Lancet Oncol. 11, 184–192. doi:10.1016/S1470-

2045(09)70286-4 Bashkirov, V.I., Scherthan, H., Solinger, J.A., Buerstedde, J.-M., Heyer, W.-D., 1997. A Mouse

Cytoplasmic Exoribonuclease (mXRN1p) with Preference for G4 Tetraplex Substrates. J. Cell Biol. 136, 761–773.

Cotterill, S.J., Wright, C.M., Pearce, M.S., Craft, A.W., 2004. Stature of young people with malignant bone tumors. Pediatr. Blood Cancer 42, 59–63. doi:10.1002/pbc.10437

de Alava, E., Gerald, W.L., 2000. Molecular biology of the Ewing’s sarcoma/primitive neuroectodermal tumor family. J. Clin. Oncol. Off. J. Am. Soc. Clin. Oncol. 18, 204–213.

Delattre, O., Zucman, J., Plougastel, B., Desmaze, C., Melot, T., Peter, M., Kovar, H., Joubert, I., de Jong, P., Rouleau, G., 1992. Gene fusion with an ETS DNA-binding domain caused by chromosome translocation in human tumours. Nature 359, 162–165. doi:10.1038/359162a0

Dimarino, A.M., Caplan, A.I., Bonfield, T.L., 2013. Mesenchymal stem cells in tissue repair. Front. Immunol. 4, 201. doi:10.3389/fimmu.2013.00201

Donaldson, S.S., 2004. Ewing sarcoma: radiation dose and target volume. Pediatr. Blood Cancer 42, 471–476. doi:10.1002/pbc.10472

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Unpublished Data from the Newbury lab: Pashler, A.L., Ingham-Clarke, O., Ng, Y.

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