analysis of H2AX transcript in breast cancer cells

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Abstract

Cancer is the alteration of the genetic material such that its integrity is lost leading to infinite cell division and proliferation, as well as abnormal physiological functioning of the affected organ or tissue. DNA is efficiently packaged into the cell nucleus by the help of histone proteins. One such histone is H2A, which has a variant H2AX that is involved in DNA repair and determination of cell date after DNA damage. Since cancer is initiated by spontaneous or induced DNA damage, we hypothesised that H2AX would be expressed in higher copy number in normal breast cell lines compared to cancer cells. The aim of the study was to determine the H2AX short+long and long-only transcript expression difference between the cancerous cell lines and the normal cell line. Two cancerous cell lines MCF7 and MDA-MB-231, and one non-tumorigenic cell line (MCF10A) were established in culture. They were harvested and RNA extracted from each of the cell lines. cDNA was synthesised from each of the isolated RNA expect for MDA-MB-231 RNA that was not used since it had been isolated elsewhere and the stored RNA was used instead. Quantitative PCR was used to analyse the cDNA for the H2AX transcript copy numbers. The one cancerous cell lines showed higher expression for both the short + long and long-only transcripts, while the other showed reduced expression compared to normal cell lines. In all cell lines, the short + long transcript copy number was significantly higher compared to long-only transcripts. It was concluded that MDA-MB-231 breast cancerous cells express H2AX histone variants significantly more than the normal cells, while MCF7 cancer cells have down-regulated expression of the variant. These findings necessitate further studies as to the molecular signalling associated with the histone variant and its specific roles in initiation, development and progression of breast cancer or lack thereof.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Table of Contents

Abstract 1

1.0. INTRODUCTION.. 4

1.1. Background. 4

1.2. Hypothesis and statement of the problem.. 8

1.3. Objectives. 9

2.0. LITERATURE REVIEW… 10

2.1. Chromatin and DNA packaging. 10

2.2. H2AX and its phosphorylation. 12

2.3. DNA damage and DNA repair. 15

2.4. Breast cancer and breast cancer cell lines. 18

3.0. MATERIALS AND METHODS. 22

3.1. Establishing breast cell lines in culture. 22

3.2. Cell Counting. 24

3.3. RNA extraction. 25

3.4. Synthesis of cDNA from the extracted RNA.. 26

4.0. RESULTS. 30

4.1. Cell Lines used in this project 30

4.2. RNA extraction. 31

4.3. Synthesis of cDNA.. 31

4.4. H2AX expression analysis using quantitative PCR.. 32

5.0. DISCUSSION.. 36

6.0. Conclusion and Recommendations. 47

Reference List 48

 

 

 

 

 

 

 

 

 

 

 

 

 

1.0. INTRODUCTION

1.1. Background

The genetic material in eukaryotes is organized into DNA and proteins forming a complex structure localized in the nucleus. The complex structure is chromatin that is highly compacted for assembly of the rather long DNA into the small nucleus. However, the chromatin has the property of allowing rapid DNA accessibility for protein synthesis and other processes such as DNA repair and recombination (Lukas et al., 2011). According to Bönisch & Hake (2012), phenotypic plasticity of chromatin is achieved through various mechanisms such as ATP-dependent chromatin remodelling, post-translational modifications of the histone and replacement of canonical histones by variants. Nucleosomes are the building blocks of chromatins and each of these monomers are made up of DNA wrapped around 2 of each H4, H2B, H2A and H3 core histones (Luger et al., 1997). The DNA, which is approximately 160-241 base pairs, is wrapped in 1.7 left-handed super-helical turns forming the nucleosome that has the core particle and the linker region that joins the core particles.

The four core histones on which DNA is wound to form a nucleosome are basic and small proteins that have minimal variation through evolution. However, according to Bönisch & Hake(2012), there are regions of the histones that are more conserved than others. The histone fold domain is the most conserved region, which has 3 α-helices with loops between them. The N-terminal tail is less conserved and structured, and more basic due to an abundance of arginine and lysine (Bönisch & Hake, 2012). The variability of this region allows post-translational modifications that facilitate DNA accessibility for protein synthesis and other processes.  Unlike core histones, linker histones are variable and associate with regions of DNA between nucleosomes. They have a globular domain, central domain and C- and N- terminal tails with similar properties to the N-terminal of a core histone. Their roles include further compaction and spacing of nucleosomes within the complex structure (Luger et al., 2012). Variations may occur in these particles hence changing how chromatin is assembled and ultimately interfering with the cellular process driven by DNA regulations and transcription. Some of these variations could involve how DNA is methylated or levels of histones’ post-translational modifications or incorporation of a variant histone.

Various histone variants have significant roles in specific regions of the genome. H2AZ have been found to play a role in chromatin modification for transcription regulation, while H2AX is involved in DNA damage response (Rogakou et al., 1999). H2AX is a variant of H2A histone and is more common than other variants in all eukaryotes, but differs among them in expression patterns and amino acid sequences (Pinto & Flaus, 2010).  The difference between H2A and H2AX is a single substitution each at the L1 loop and docking domain and two substitutions in the N-terminal tail as shown in figure 1 below (Polo & Jackson, 2011).

Figure 1:Amino acid sequence difference between H2A and H2AX (Li, 2012)

L1 loop substitution effect is of significant importance in chromatin assembly as it’s where the H2A-H2B dimers interact. There are two forms of H2AX mRNA that have been found in proliferating cell cultures; a long form made up of 1600 bases and contains a poly A tail and a short form that is 575 bases long and does not have the poly A (Mannironi et al., 1989). Histone H2AX was identified in 1980, where it was found to preserve genome integrity after DNA damage through phosphorylation of serine at the C-terminal tail (Mannironi et al., 1989). The phosphorylation of serine at the extreme C-terminus of H2AX by Phosphoinositide-3-Kinase-related protein Kinases (PIKKs) is a property it exhibits for its role in DNA repair. The phosphorylation generates the γH2AX mark (Rogakou et al., 1998). As reviewed by Podhorecka et al. (2010), H2AX forms a foci around of double strand breaks within the DNA region and the foci contains DNA repair factors. Phosphorylated H2AX has also been found to induce meiotic sex chromosome inactivation and has significant influence on cell fate after DNA damage (Turner et al., 2004).

DNA damage response (DDR) is an evolutionally mechanism that life has developed over time to identify DNA damage and respond to the signal to enable repair of the genetic material (Jackson & Bartek, 2009). This mechanism involves a complex network that includes cell cycle checkpoints, DNA damage tolerance and repair. Various cellular processes and components affects and are affected by DDR. They include DNA replication, cell cycle, chromatin structure and cell growth. Some events associated with DDR or ineffective response results in various human disorders and the most significant one being cancer. There are a variety of DDR pathways one of which includes phosphorylation of histone H2AX. Incorporation of H2AX variant in the primary stage of chromatin assembly is one among the many mechanisms that ensure access of the packaged DNA for repair and other cellular processes. DDR mechanism involving the histone H2AX is a combination of other factors that initiate the accumulation of DDR proteins, phosphorylation of the histone molecules, spread of the γH2AX and eventual repair of the double stranded DNA. By targeting H2AX in mice, Celeste et al. (2002) produced a knock out mouse H2AX-/-. Compared to positive mice, the mutants were radiation sensitive, immune deficient and growth retarded. Moreover, the males were infertile indicating the significant role of H2AX in maintenance of DNA integrity and hence cancer development as a tumor suppressor protein.

Precision is crucial to avoid mutations that are associated with human disorders such as cancer. To this regard, systemic events efficiently take place within the nucleus during DNA repair. According to Lukas et al. (2012), the foci containing the DDR factors localise at the region of double-strand break site. These factors then spread depending on the length of the DNA and packaging. The spread is regulated through a positive feedback loop that utilizes a mediator protein (MDC1). This protein binds to the γH2AX as well as to ATM kinase and MRN complex resulting in further H2AX phosphorylation at a distance from the DNA break region (Lukas et al., 2012). Proteins for DNA damage recognition are the first to be recruited at the site. These are followed by recruitment of repair proteins to the DNA lesions and the MDC1 acts as the bind for repair proteins and DNA damage checkpoints. The role of H2AX is the mobilization of DNA repair proteins at the site of the breakage. Any mutations of this tumor suppressor protein would means that damage to the DNA would not be repaired, which is a precursor to cancer development. According to a study by Liu et al. (2007), H2AX sensitized tumorigenic cells to induced apoptosis through transcriptional block and chromatin aggregation, while its depletion attenuated apoptosis response of the cancer cells.

In breast cancer cells, it has been found that H2AX mutations are rare (Monteiro et al., 2003). However, there is a significant variation in the H2AX gene copy number in cancer cells (Srivastava et al., 2008). By screening sporadic breast cancer tissues for mutation and gene copy number, Srivastava et al found that there were no mutation for the H2AX, but there was a two-fold reduction in gene copy number. The reduction in copy number significantly reduces the expression of H2AX. There is little evidence of the H2AX transcripts expression in breast cancer tissue compared to a normal one, which was identified as the research gap that this study sought to bridge.

            Real-time quantitative PCR is technique that detects and quantifies DNA. It is a much better technique compared to conventional northern blotting since its sensitive to smaller samples. It uses two steps process, where the first stage involves converting of the mRNA into cDNA using the reverse transcriptase enzyme. Normal PCR process then is carried out using appropriate primers that target the specific genes whose expression is under investigation. According to Guo et al. (2015) real-time qRT-PCR has made analysis of expressions rather simple. This process has been enabled through the utilization of various chemistries such as SYBR Green and TaqMan (Lee & Saunders, 2013). TaqMan probes have fluorescent reporter dye and a quencher in the 5’ and 3’ ends respectively. The probes depend on the activity of DNA polymerase, which has a 5’-nuclease activity. When the probe is not cleaved, the proximity of reporter dye and the quencher prevents detection, but when cleaved and hence template amplified, the process is disrupted and signal is given off. The amount of the fluorescent signal increases exponentially giving the amount of a specific target in a sample (Lee & Saunders, 2013). Another simpler and more economical detection and quantitating method is use of SYBR Green. In this procedure the dye binds to dsDNA and emits light when excited. Accumulation of PCR products, therefore, increases the fluorescent signal for detection and quantification. Although the process is straight forward and sensitive, binding of SYBR Green to any dsDNA reduces its specificity thus requiring extensive optimizations unlike the TaqMan probe (Guo et al., 2015).

1.2. Hypothesis and statement of the problem

In this study, we hypothesised that the mRNA expression of the histone variant H2AX may differ in breast cancer compared to normal breast tissue. This hypothesis has come out due to evidence from peer reviewed articles indicating the role of the variant in the DNA response and its presence at a lower copy number in cancerous compared to normal tissue (Srivastava et al., 2008). The selection of breast cancer cell lines as opposed to other cancers was guided by the association of BRCA1 and H2AX, where they are both abundantly found at the foci of double strand DNA breaks. Mutations in BRCA1 are associated with breast cancer and the same protein is recruited to the site of DNA break by phosphorylation of H2AX. It therefore necessitates determination of the role that H2AX plays in breast cancer considering that the gene copy number is reduced in such cells. This formed the rationale of this study, where the understanding of the relationship between breast cancer and the H2AX histone variant needed to be determined. The expression of the histone variant in breast cancer cells compared to normal cells would add to the evidence base especially because very few studies have been conducted focusing on the role of this histone variant on the carcinogenesis of breast cancer. Determination of differences in the transcript expression would lead to further investigation into the active role of H2AX either during the DNA damage response or DNA repair in breast cancer development and progression.

1.3. Objectives

The main objective of the study was to determine the expression of two transcripts of H2AX in cell lines derived from normal breast tissue and breast cancer tissue. Specifically, the study focused on the following procedural steps to help answer the research questions:

  1. Culture of one normal and two breast cancer-derived cell lines
  2. Extraction of RNA and converting this to cDNA
  3. Determination of H2AX short and long transcript levels by reverse-transcription quantitative PCR (RT-qPCR) in each of the cell lines
  4. Analysis and quantification of the difference between the transcript expression in cancerous cell line compared to normal cells

 

 

 

 

 

 

2.0. LITERATURE REVIEW

2.1. Chromatin and DNA packaging

Packaging of DNA in eukaryotic cells as chromatin evolved through accommodation of conflicting demands of protecting, organizing and storing genetic information while at the same time allowing needed access to the information. The chromatin is a complex structure in which eukaryotic DNA is organized after being bound by histone proteins. It is a substrate for important in vivo genome integrity preservation process that includes replication, transcription and DNA repair (Leung et al., 2014).  Histones are considered to be the most conserved proteins in eukaryotic cells and histone post-translational modifications (PTMs) highly regulate the function and structure of chromatin. According to Mueller-Planitz et al. (2013), nucleosomes are the primary organizational structures of a chromatin that regulate and package eukaryotic genomes. In a chromatin, there is a DNA molecule known as nucleosome fiber (10nm) involved in DNA packaging that regularly interacts with protein globules referred to as nucleosome cores, figure 2 (Golov et al., 2014).

Figure 2: DNA packaging (Golov et al., 2014)

Each globule is wrapped by a region of DNA 145-147bp in length which forms 1.65 left handed super-helical turns and the globule is made up of 8 core histones. The modular organization of a globule is a complex histone octamer made of an (H3-H4)2 tetramer and two H2A-H2B dimers. Along the DNA molecule, histone  is arranged such that the (H3-H4)2 tetramer contacts DNA central part region which is wrapped around the nucleosomal globule while H2A-H2B dimmers makes contact to DNA at entry and exit of the nucleosomal particle (Varas et al, 2015). The sequence-independent nucleotide interaction between nucleosome core and DNA is aided by hydrophobic, ionic and hydrogen bonding of proteins with the sugar-phosphate backbone of DNA.  The core histones has two functional and structural domains known as histone tail and histone fold consisting of three α-helical regions joined together by small loops. The interaction between the core histone nucleosomal DNA, and other histones is facilitated by the histone fold. Between two neighboring nucleosomes, there is a DNA region known as the linker varying in length between 10-90bp among different genome regions, different cells, and different organisms (Varas et al, 2015). The linker can bind with histone H1 which is different both in structure and size from core histones at nucleosome entry-exit sites thereby joining two full superherical turns. The supranucleosomal packaging levels are thought to be maintained by histone H1. Therefore the nucleosome fiber is the basic chromatin structure in eukaryotic cells with exceptions of male gamete chromatin and dinoflagellate chromatin. To date many modified nucleosome forms have been identified occurring along canonical nucleosomes in chromatin. These forms are known to be produced through incorporation of variant histones in nucleosomes and changes made to the nucleosomal globule known as posttranslational modifications (Jianfeng et al., 2012). The best known PTMs which have been observed in various studies include ubiquitination, acetylation, poly-ADP-ribosylation, methylation, SOMUylation, and phosphorylation. PTMs mainly occurs in the histone nonstructured N-terminal tail domain with exceptions of some in globular region of the histone. The variant histones that has been comprehensively characterized include H3.3, H2ABbd, CENP-A (centomeric H3), H5 (variant H1), H2AZ, macroH2A, and H2AX (Jianfeng et al., 2012).

2.2. H2AX and its phosphorylation

H2AX is a chromatin-associated histone and a basic regulator of how cells respond to genotoxic stress and DNA double-strand breaks (DSBs) (Mannironi et al., 1989). The difference between H2A and H2AX is that the latter has a unique C-terminal tail containing a highly conserved SQE motif and a serine residue at position 139 (figure 3). The serine 139 usually responds to DSBs by being rapidly phosphorylated by protein kinases of phosphatidylinositol 3-OH-kinase-related kinase (P13KK) family that includes DNA dependent protein kinase subunit (DNA-PK), ataxia telangiectasia mutated (ATM) or  ATM  and Rad3 related (ATR) (Liu et al., 2008).

Figure 3: H2AX histone showing the structure of the protein (Liu et al., 2008)

Phosphorylation of H2AX core histone variant occurs in the chromatin around the DSBs and suppresses antigen receptor loci translocations during V (D) J recombination (Celeste et al., 2003). In H2A histone pool found in mammalian cells, H2AX variant makes up to 2-25% and it is incorporated into the chromatin in a non-uniform manner. After DSBs are induced the DNA-PKs, ATR and ATM protein kinases starts phosphorylation of H2AX on serine 139 conserved carboxyl terminal forming γ-H2AX foci around the DNA breakage sites (Rogakou et al., 1998). Formation of γ-H2AX foci leads to checkpoint and repair protein binding sites with some proteins catalyzing further covalent modifications ofγ-H2AX to create more binding sites for checkpoint and repair proteins. All these proteins are then assembled into complexes inside chromatin and in the surrounding DSBs. According to Yin et al (2009), H2AX-/- cells display high sensitivity to DSB causing agents, show defective repair of chromosomal DNA double strand breaks, and exhibit high level of DSB-induced and spontaneous genomic stability. They note that although these cells show normal activation of cell cycle checkpoints that depend on p53 and also display apoptotic responses, the cells become defective in G2/Mitosis checkpoints after only a few DSBs are induced. Therefore, the phenotypic characteristics displayed by H2ax-/-  cells indicates  γ-H2AX ability to retain checkpoint and repair proteins around DSBs  may stabilize disrupted DNA strands,  amplify checkpoint signals, and/or promote DNA ends accessibility (Kubota et al., 2014).

In animals, plants and fungi, SQ motif involved in phosphorylation of core histone H2AX is highly conserved. This evolutionary conservation suggest that the mechanism of response to DSB damage is an important process in DNA repair that developed before evolutionary divergence of plants, animals and fungi (Sharma et al., 2015). Evidence based studies has shown that when antibodies specific to SQ  are raised against the mammalian γ-H2AX sequence, they recognize DSBs in bread/wine yeast Saccharamyces cerevisiae, frog Xenopus laevis, and vinegar fly Drosophola melanogaster, after they are exposed to genotoxic agents (Siddiqui et al., 2013, Sharma et al., 2015).  To originate the γ-H2AX foci phosphorylation occurs to about 2000 H2AX molecules that are around each DSB and therefore different studies has concluded that the number of DSB formed is directly proportional to the number of γ-H2AX foci. This makes γ-H2AX the basic marker of early DNA damage and has potential clinical applications. Several studies are already focused on applying the γ-H2AX focus assay to the investigation of carcinogenic/ genotoxic agents or the process of cell aging or turning cancerous, and also evaluate presence of DSB and how they relate diagnostic or environmental exposures and pathological conditions (Scarpato et al., 2013, Laszlo & Fleischer, 2009). After formation of γ-H2AX, dephosphorylation takes place rapidly signifying subsequent effecter proteins recruitment to the site. Therefore, the induction and disappearance of γ-H2AX can be used to monitor functioning of DNA damage repair in a particular cell line with assumptions that the coordinated process in which it operates is required to avoid DSBs being fixed in chromosomal mutations (Scarpato et al.,

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