Introduction
The development and progression of breast cancer have been attributed to a series of cellular and molecular events, most of which are not well understood (Veeck & Esteller 2010). However, cell and molecular biologists agree that normal cells become cancerous when they undergo genetic modifications that make them acquire growth and multiplication advantages. If a normal cell becomes cancerous, then it does not perform standard physiological functions in the body. Multiple studies with regard to general cancer conditions have identified two classes of genes in the development of cancers in human beings (Huang, Nayak, Jankowitz, Davidson & Oesterreich 2011). The first category contains the tumour suppressor genes, which have been shown to prevent cells from growing and surviving.
The second category is characterised by oncogenes that facilitate the processes of cell growth and survival (Grønbæk, Hother & Jones 2007). Grønbæk and colleagues (2007) assert that the mechanisms of cell alterations ultimately modify the proteins that encode nucleotides, alter the number of genes and increase the rate of gene transcription. Cellular studies show that epigenetic and genetic changes cause abnormal cell activities through interfering with the normal pathways involved in cell functions. Methylation is an example of epigenetic change that has been widely studied. Bird (2002) asserts that methylation happens after DNA replication and it is characterised by the addition of methyl groups to DNA molecules at the sites with cytosine residues (Huang et al 2011). Methylation of DNA occurs in regions with a high concentration of guanidine residues.
In healthy cells, genes with DNA that is hypermethylated are not expressed. On the contrary, hypomethylation of DNA molecules has been associated with increased gene activities.
DNA methylation
Research has identified three enzymes that are associated with DNA methylation in human beings. First, DNA methyltransferase 1 (DNMT1) is involved in maintaining methylation patterns in cells. Second, DNA methyltransferase 3A (DNMT3A) is concerned with regulating de novo methylation processes in cells. It has also been suggested that DNA methyltransferase 3B (DNMT3B) has the same functions as that of DNMT3A (Chen et al 2007; Chedin 2010). The three enzymes are expressed at different rates, but their modes of expression do not always correlate with hypomethylation and hypermethylation. The variations are caused by the action of miRNAs that regulate the molecular events of DNMT (Blair and Yan 2012).
There are different mechanisms for DNA methylation. One of the best explained mechanisms is through the involvement of TET1-3 proteins, which belong to hydroxylases (Tahiliani et al 2009). The molecules catalyse the conversion of 5mc, 5HMC, 5fC and 5caC (Ito, D’Alessio, Taranova, Hong, Sowers & Zhang 2010; He et al 2011; Pfaffeneder et al 2011).
Point mutations due to DNA methylation
Grønbæk and colleagues (2007) show that point mutations are promoted in regions characterised by 5mc residues through many ways. A cytosine that could be methylated could encounter deamination to form a thymine. If the changes are not corrected by independent molecular events in cells, then they result in disease due to point mutations in regions coding for genes. In most cases, genes that are affected are those that are concerned with the regulation of cell growth and survival. More than 30% of human diseases that are associated with point mutations have been shown to have alterations at the CpG dinucleotides.
Chromatin and histone modification
Chromatin is the physiological template for the human genome. The basic unit of chromatin is the nucleosome that is characterised by about 200bp of DNA. The base pairs are organised in small basic units of proteins that exist as octamers. A collection of octamers is known as histone. Thus, histone is the major molecular material that is involved in organising and maintaining the structural integrity of DNA and genes. The human DNA has been found to lie on the surface while histone materials are found in the inner parts of histone molecules. Nucleosomes are part of euchromatin and heterochromatin. Also, euchromatin and heterochromatin are found in mitotic chromosomes. However, heterochromatin and euchromatin of the human genome differ significantly in terms of structure and function. Heterochromatin is packed tightly to prevent transcription factors from accessing histone. On the other hand, euchromatin is not tightly packed. Thus, factors involved in transcription could easily access major regions of DNA that are involved in the initiation of transcription of DNA (Richards & Elgin 2002)
Research has also shown that cancer could be caused by epigenetic modifications that alter the normal structure of histone. DNA molecules in every cell nucleus wraps around histone materials, which have an extensive altered N-terminal tails that could also be flexible. The rate at which transcription of DNA occurs greatly depends on specific alterations that could either tighten or loosen molecules of DNA from histone (Blair and Yan 2012).
Some alterations of histone correlate with active genes such as trimethylation in histone H3. Some changes could also be connected with genes that could be repressed such as H3K9me3. Acetylation of histone occurs when histone acetyltransferases (HATs) catalyse the addition of acetyl groups to lysine residues. The remove of such residue is catalysed by histone deacetylase (HDACs). Methyl groups are removed through the action of histone demethylases (HDMs). Another important modification of histone is phosphorylation, which ensures that essential residues in histone are phosphorylated. The roles of the enzymes that catalyse the activities of phosphorylation, methylation, and acetylation are not clearly understood (Blair & Yan 2012).
Breast cancer
Research shows that BRAC1 and BRAC2 genes could be responsible for coding proteins that make breast cells grow and multiply abnormally. Disease progression and degrees of severity vary among the different types of breast cancer (Stecklein, Jensen & Pal 2012). If a patient lacks the three receptors that signify the presence of breast cancer, then such a patient is said to be triple negative. It has been shown that a patient presenting with cancer characterised by this state (triple negative) could be difficult to treat. In such patients, there are no readily available hormonal therapy approaches that could be used to treat cancer. Patients who present with cancers characterised by estrogen receptor (ER) tumours are treated using hormonal therapy. Clinical data also show that trastuzumab (Herceptin®) has been used to treat patients with human epidermal growth receptor (HER) positive tumours (Blair & Yan 2012). Epidermal growth factor has been correlated with cancers with high rates of metastasis and invasiveness. Such tumours are quite difficult to treat, especially when they are treated at advanced stages. At such advanced stages, the tumours could have spread to other body organs where they cause physiological damage. However, the effect of EGFR on the development and progression of breast cancers has been reduced through the use of tyrosine kinase inhibitors. An example of such inhibitors is gentinib. The pharmacological product has also been shown to target cells with over-expression of HER2.
Many forms of breast cancer are hypomethylated (Soares, Pinto, Cunha, Andre, Barão, Sousa & Cravo 1999). The observation is similar to findings from other studies (Hinshelwood & Clark 2008). Hypermethylation in noncancerous cells does not occur in the same regions like those in cancer cells (Hinshelwood & Clark 2008). This is a major observation that differentiates molecular events between caner and healthy cells. In order for cancer to progress, tumour suppressor genes need to be suppressed. If they are suppressed, then they lose the ability to prevent the growth of tumours associated with the progression of cancer. DNA hypermethylation results in unstable genes that could lead to cancer. In fact, a significant number of unstable genes results in various forms of cancer because they code for abnormal proteins that facilitate cells to grow abnormally.
DNMTs could be involved in gene-based methylation that is common in breast cancer (Girault, Tozlu, Lidereau & Bièche 2003). However, a weak correlation between DNMTs and breast cancer has been demonstrated. Methylation of the promoter of ER could result in the down-regulation of ER. Breast cancers with over-expressed ER and PR are treated through the use of drugs that imitate the receptors (Blair & Yan 2012). Scientists have concentrated on deciphering the roles of HMTs and HDMs in the development and progression of breast cancer. Such efforts have shown that there are elevated levels of H3K27 methyltransferases, which enhance the activities of zeste homologue 2 (EZH2). Cancer states that are characterised by elevated levels of H3K27 methyltransferases have fast rates of metastasis. Such cancers are difficult to treat and manage. Breast cancer is also associated with over-expression of lysine-specific demethylases that are involved in the removal of methyl groups from lysine residues. Research has demonstrated that there is over-expression of H3K4 demethylase in ER negative tumours. In fact, this is being used as a diagnostic marker for breast cancers that have high chances of metastasising.
The discovery of the genes and molecules involved in the development and progression of breast cancers has been very sequential. The orderly manner in which scientists have been discovering breast cancer-specific genes and molecules has greatly impacted the management of the disease. For example, hormonal therapy has been adopted in the treatment of breast cancers that are characterised by ER. In the future, further research could result in the identification of more genes and molecules with regard to breast cancer. Therefore, more treatment approaches would be designed in the future.
Study aims and objectives
Cell migration processes are important biological events that are regulated by different cells in the body (Girault et al 2003; Francisco et al 2003). One of best studied results of cell migration is metastasis of tumours, which ensures that cancerous cells move from their organ of origin to other organs. It would be expected that cancerous cells are hypermethylated and that they would have higher rates of cell invasion than normal cells. The proposed study aims at accomplishing two objectives. First, it aims at establishing the invasion patterns of normal and cancer cells. Second, it aims at assessing the rate at which normal and breast cancer cells invade cell barriers in order to attack neighbouring organs.
Hypothesis
The study assumes that there will be no differences in cell invasion patterns between the normal and breast cancer cells in vitro. It also hypothesises that the two types of cells have the same rates of cell invasion.
Materials and methods
The study will be conducted in a fully equipped molecular biology laboratory that will have a carbon dioxide incubator, fluorescent plate reader and laminar flow hood for cleaning air, among other equipment.
Cell lines
The study will utilise two types of cells. The first type of cell lines will be the HBT-22, a cell line that is derived from MCF-7 cells that are known to be non-invasive. Second, CCL-121 cell line will be used it is assumed that cells in this category are invasive because they are derived from HT1080 cells.
Plates and reagents
The following plates will be used based on the plating procedures needed in the experiment: 6 well plates, 12 well plates, 24 well plates and 96 well plates. The following reagents would be used in the study: 5x BME coating solution, 10x buffer to be used for coating purposes, 10x cell dissociation buffer, cell harvesting solution, and calcein AM that will be dissolved in DMSO. Other reagents would be DPBS (a wash buffer) that will be enriched with Mg and Ca ions, serum free medium (SFM), and IMDM solution with about 10% FBS.
Protocol
All the procedures in the study will culminate in determining the cells that would pass travel across the membrane and standard curve. The procedures will be carried out in three days.
Day 1 (starving cells and coating with the BME buffer)
Cells will be removed from cell growth cultures and starved for 24 hours. The period of starvation would be characterised by absence of nutrients that would support cell growth and proliferation Cells from cultures will be washed with PBS in order to eliminate all serum. Afterwards, SFM medium will be added to the cells. Based on the desired working concentration, the coating buffer will be diluted and used to coat Transwell inserts. The plates will be maintained in growth medium for a period of 24 hours, with 5 percent carbon dioxide and a temperature of 37 degrees Celsius.
Day 2
Cell dissociation medium will be utilised to harvest cells that were starved for 24 hours. The harvesting medium will be removed through spinning the cell solutions and resuspending in a medium without serum. Cell concentration will be determined and right seeding concentration be used, which will be serum-free. In a 96 well plate, a blank well will be left for background calculations. Some receiver wells will be set, containing serum that would act as a chemoattractant while other wells will not contain serum. The cell plates will be incubated for a period of 18 hours.
Day 3 (preparation of a standard curve)
The study will involve the preparation of independent curves for each category of cell lines and essay. Cells will be harvested and collected in a tube. Calcein AM vials will be warmed in the laboratory and mixed with about 30µg of DMSO. Cells will be pelleted and resuspended in a diluted amount of CDS ( about 1x). A serial dilution of cells will be made and will start with the highest concentration of cells to the lowest concentration (no cells, but only 1x CDS). Three volumes (triplicates) of 50µl of each serial dilution will be added to different wells on a 96 well plate. The thawed Calcein AM solution will be diluted with CDS, specifically, at a ratio of 2.4:1. Fifty mcrolitres of the resulting mixture will be added to every of the prepared plate. The microplate will be incubated under dark conditions for exactly one hour. After the incubation, the plate will be read using fluorescence reader. Excitation will be set at 485nm while emission will be maintained at 520nm.
Analysis
The average relative fluorescence units (RFU) with regard to each serial dilution will be obtained through calculating the mean RFU for concentration and subtracting the reading of wells that will not have cells (background). In order to obtain a standard curve, cell concentration will be plotted on the Y-axis while RFU will be plotted on the X-axis. Based on the equation y= mx + b, the number of cells will be represented by y in the equation while x would represent RFU for each sample. The total number of cells passing through the BME solution be determined through multiplying the number of cells represented by y in the equation by an established multiplication factor. The proportion of invasion with regard to each well will be determined through dividing the amount of cells obtained in the above step by the original number of cells in the plate. It would be expected that there would be minimal or no movement of the HTB-22 cell line (noninvasive cells) either in the presence or absence of serum, which would attract cells to the basement membrane.
Expenditure of main reagents
Figure 1.Table showing the reagents to be used in the study and their approximated prices.
Health and safety
Most of the reagents that will be used the study would be dangerous if they will not be handled well. Personal protection equipment will be utilised to minimise exposure to the chemicals. All procedures will be carried out in air hood to promote safety. In addition, hand gloves will be used to avoid hand contact with the chemicals, which would have the potency of leading to health and safety hazards in the laboratory. In case an accident occurs when handling a certain reagent, then its safety sheet would be read in order to initiate the right first aid procedures.
References
Bird, A, 2002, ‘DNA methylation patterns and epigenetic memory’, Genes & development, vol. 16, no. 1, pp. 6-21.
Blair, LP, & Yan, Q, 2012, ‘Epigenetic mechanisms in commonly occurring cancers’, DNA and cell biology, vol. 31, no. 1, pp. 49-61.
Chedin, F, 2010, ‘The DNMT3 family of mammalian de novo DNA methyltransferases’, Progress in molecular biology and translational science, vol. 101, no.1, pp. 255-285.
Chen, T, Hevi, S, Gay, F, Tsujimoto, N, He, T, Zhang, B.,… & Li, E, 2007, ‘Complete inactivation of DNMT1 leads to mitotic catastrophe in human cancer cells’, Nature genetics, vol. 39, no. 3, pp. 391-396.
Francisco, J. A., Cerveny, C. G., Meyer, D. L., Mixan, B. J., Klussman, K., Chace, D. F.,… & Wahl, A. F. (2003). cAC10-vcMMAE, an anti-CD30–monomethyl auristatin E conjugate with potent and selective antitumor activity. Blood, 102(4), 1458-1465.
Girault, I, Tozlu, S, Lidereau, R, & Bièche, I, 2003, ‘Expression analysis of DNA methyltransferases 1, 3A, and 3B in sporadic breast carcinomas’, Clinical Cancer Research, vol. 9, no. 12, pp. 4415-4422.
Grønbæk, K, Hother, C, & Jones, PA, 2007, ‘Epigenetic changes in cancer. Apmis, vol. 115, no. 10, pp. 1039-1059.
He, YF, Li, BZ, Li, Z, Liu, P, Wang, Y, Tang, Q,… & Xu, GL, 2011, ‘Tet-mediated formation of 5-carboxylcytosine and its excision by TDG in mammalian DNA’, Science, vol. 333, 6047, pp. 1303-1307.
Hinshelwood, R. A., & Clark, S. J. (2008). Breast cancer epigenetics: normal human mammary epithelial cells as a model system. Journal of molecular medicine, 86(12), 1315-1328.
Huang, Y, Nayak, S, Jankowitz, R, Davidson, NE, & Oesterreich, S, 2011, ‘Epigenetics in breast cancer: what’s new?’, Breast Cancer Research, vol. 13, no. 6, pp. 1-11.
Ito, S, D’Alessio, AC, Taranova, OV, Hong, K, Sowers, LC, & Zhang, Y, 2010, ‘Role of Tet proteins in 5mC to 5hmC conversion, ES-cell self-renewal and inner cell mass specification’, Nature, vol. 466, no. 7310, pp. 1129-1133.
Pfaffeneder, T, Hackner, B., Truß, M, Münzel, M, Müller, M, Deiml, CA,… & Carell, T, 2011, ‘The discovery of 5‐formylcytosine in embryonic stem cell DNA’, Angewandte Chemie, vol. 123, no. 31, pp. 7146-7150.
Richards, EJ, & Elgin, SC, 2002, ‘Epigenetic codes for heterochromatin formation and silencing: rounding up the usual suspects’, Cell, vol. 108, no. 4, pp. 489-500.
Soares, J, Pinto, AE., Cunha, CV, Andre, S, Barão, I, Sousa, JM, & Cravo, M, 1999, ‘Global DNA hypomethylation in breast carcinoma’, Cancer, vol. 85, no. 1, pp. 112-118.
Stecklein, SR., Jensen, RA, & Pal, A, 2012, ‘Genetic and epigenetic signatures of breast cancer subtypes’, Front Biosci (Elite Ed), vol. 4, no. 1, pp. 934-949.
Tahiliani, M, Koh, KP, Shen, Y, Pastor, WA, Bandukwala, H, Brudno, Y,… & Rao, A, 2009, ‘Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1’, Science, vol. 324, no. 5929, pp. 930-935.
Veeck, J, & Esteller, M, 2010, ‘Breast cancer epigenetics: from DNA methylation to microRNAs’, Journal of mammary gland biology and neoplasia, vol. 15, no. 1, pp. 5-17.