Breast cancer is uncontrolled growth of breast cells. Mostly it initiates at the cells of lobules of breast which are milk -producing glands. Rarely it can start in the stromal tissues which includes the fatty and fibrous connective tissue of the breast. Millions of women around the world have breast cancer. Thousands of them die annually due to this cancer. The incidence of breast cancer has been found more prevalent in Caucasian ladies than other races. (Breast cancer.org, 2010)
The incidence and death rates of breast cancer increases with age. Current statististics says that the risk of a women to develop breast cancer in her life time is 12.3% (breast cancer.org, 2010). Hormonal exposures like early menarchy, late menopause and hormonal replacement therapy are considered to be the risk factors for breast cancer (Hulka and Moorman 2001). Similarly, obesity especially in post menopausal women is another risk factor for breast cancer (Eliassen et al. 2006). Hamajima et al (2002) says that alcohol consumption is strongly associated with increased risk of breast cancer. Singletary and Gapstur (2001) have suggested that increased level of estrogen and androgen through conversion of alcohol must be the possible mechanism of alcohol to cause breast cancer. Interestingly, breast feeding and multiple pregnancies have been found to be the factors that decrease a woman's risk of breast cancer. (Shantakumar et al. 2007). Regarding genetic predisposition, familial breast cancer is closely associated with mutation in BRCA1 and BRCA2 genes. It has been estimated that 5 to 10 % of breast cancers are due to mutations in these two genes. ( Ford et al. 1998)
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In order to prevent and cure the devastations caused by breast cancer, plethora of researches have been being conducted worldwide to characterize an effective therapeutic agent against this disease. For this purpose, a deep insight on the molecular mechanism of breast cancer and its key players is mandatory before conducting a research on this area.
Cell cycle and Cancer: A General Overview
Cell is the structural and functional unit of life. The cell cycle is a repeated rounds of cell duplication and division where a cell travels through an ordered series of events giving rise to growth and duplication of its contents and ultimately divides into two cells. The process of cell cycle in higher organism is highly ordered and scrupulously regulated. The basic steps of a eukaryotic cell cycles are Gap 1(G1) phase, Synthesis (S) phase, Gap 2 (G2) phase and Mitotic (M) phases. There occurs replication of DNA during the S phase and the equal delivery of the identical chromosomes to daughter cells during the M phase. (Sandal 2002).
Fig 1. General Overview of cell cycle (Collins et al. 1997)
The gap period after the completion of mitosis and before the synthesis of DNA is called G1, which is the period when necessary proteins for the DNA replication are synthesized. In addition, in this phase, the cell monitors both internal and external environments to ensure that all preparations for DNA synthesis have been completed and all the conditions are in favour of cell division. After G1 phase, synthesis of DNA occurs in S phase. During this phase, duplication of cellular content of DNA takes place. This is the longest phase of cell cycle and takes 10-12 hours in a typical 24 hour eukaryotic cell cycle. Once S-phase is over, initiation of G2 phase takes place. This is the second gap phase of cell cycle during which synthesis of protein takes place. The protein synthesised in this phase is required during the separation of duplicated chromosomes and ultimately division of parental cell into two daughter cells in M phase.
M phase is a combination of ordered series of events that leads to the alignment and separation of duplicated chromosomes. This phase consists of four distinct steps viz. Prophase, metaphase, anaphase and telophase, followed by cytokinesis during which parental cell is physically separated into two new daughter cells. ( Maiato H , Deluca J., salmon E., Earnshaw W, 2004) The duration of entire M-phase in a typical eukaryotic cell is 1-2 hours which is much less than S-phase. All the cells of an organism do not continue dividing throughout the life span. After certain intervals of cell cycle, many cells undergo a process of terminal differentiation and become quiescent. This phase is termed as G0 phase of the cell. These cells normally retain their dormant stage unless stimulated by external signal. If triggered by certain stimulus, these cells re- enters the cell cycle and start dividing.
Always on Time
Marked to Standard
In normal cells, an equilibrium exists between cell proliferation and cell death or apoptosis An excessive proliferation of cells leads to a pathological condition called cancer. Thus cancer arises as a result of defects in the cell cycle regulation. As a result of the malfunctions in cell cycle, the mutated cells are allowed to go through the cell cycle accumulating the mutations that leads to tumerogenesis (Foster 2008).
1.2 Regulators of Cell Cycle
In order to ensure well regulation of cell cycle and viability of the daughter cells, there are highly organized controlling centres called checkpoints,in a eukaryotic cell. These checkpoints control the transit through the cell cycle, including the timing and mechanism of the event. There are many checkpoints in a cell cycle; among them two are most critical. One of them exists at the transit point of G1 phase to S-phase called G1/S checkpoint. Another occurs at the borderline of G2 phase and M phase, called G2/M checkpoint.
A family of protein kinases , called cyclin dependent kinases (CDKs) are the key enzymes that regulate the timing control of the cell cycle by rise and fall in their level during the progression of cell cycle. Different CDKs are responsible for the regulation of different points of cell cycle through the process of phosphorylation. Phosphorylation is the process of addition of phosphate residues to a molecule that alters the activity of a protein during cell cycle.
Fig. 2. An overview of double -sword signalling pRB-p53 pathway after DNA damage leading to either Senescence or apoptosis (Irminger-Finger 2010)
The CDKs in cell cycle are controlled by a series of specialized protein called cyclins, hence the name cyclin dependent kinase was coined. The CDKs, unless bound to a particular cyclin, can not manifest their kinase activity. Generally the levels of CDKs remain fairly constant in a cell cycle and their activities alter with reference to the altered level of corresponding cyclin.
It is noteworthy that CDK1 in mammalian cell cycle is same as CDC2 in fission yeast and CDC28 in budding yeast. In mammals cyclin D exists in three sub types viz Cyclin D1, D2 and D3. All the cyclins in cell cycle bind to their cognate CDKs and promote the cell to pass through the corresponding restriction point. Apart from binding of Cyclin with corresponding CDK, there is another enzyme called CDK-activating kinase (CAK) that activates this complex to function. When a cyclin binds to the corresponding CDK, a conformational change on CDK occurs that results into exposure of a domain called phosphorylation site where CAK binds and makes the cyclin-CDK complex fully activated.
In addition to the mechanism of activation, CDK activity is controlled by inhibitory phosphorylation by some inhibitory kinases. The first group of inhibitors are called CIPs (CDK Inhibitory Proteins) which are also designated as cyclin -kinase inhibitors(CKI). p21Cip1/WAF1Â (gene symbol=CDKN1A), p27KIP1Â (gene symbol=CDKN1B), and p57KIP2Â (gene symbol=CDKN1C) are the examples of CIPs. Another group of inhibitors are called INK-4 (Inhibotors of Kinase-4 ) which consists of p15 INK4B, p16 INK4A, p18 INK4C and p19 INK4. All INK4 proteins contain 4 tandem repeats of sequence of aminoacids which were initially identified in ankyrin and hence are referred as ankyrin repeats.
The external receptor-mediated growth promoting signals cause phosphorylation and activation of transcription factors through the induction of certain genes. These genes might be early or delayed response genes. Many of these early response genes are themselves the transcription factors that in turn activate the expression of delayed response genes. For instance, MYC, which is a proto oncogene , is an early response gene that in turn causes the activation of cyclin D and E2F.
RB Pathway and Breast cancer
RB is a tumour suppressor protein that controls the progression of cell through the cell cycle and inhibits the ability of cancer to develop. RB is encoded by retinoblastoma susceptibility gene (pRB) which is located at chromosome 13q14, and inhibits the passage of cell through G1/S checkpoint.
RB is actually a member of a family of pocket proteins consisting of RB, p 107 and p130. These three proteins interact with a large number of proteins. However their action is mediated through their direct biding with E2F family of transcription factors. Thus the inhibitory action of RB protein is achieved through their binding with E2F family. When pRB binds with E2F, it loses its function as a transcription factor and gets sequestered in the cytosol. But when pRB gets phosphorylated by Cyclin D- CDK4/6 complex, E2F gets released and regains it transcriptional activity. Consequently, it binds to the regulatory regions of "E2F-responsive genes" leading to progression of S phase of cell cycle. When this E2F activates the expression of Cyclin E-CDK2 or cyclin A-CDK2 , these complexes target pRB for phosphorylation , and thus maintain the tendency of cell cycle progression.
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Regulation of transcription factor E2F by pRB
Fig 4: Regulation of E2F by pRB
Breast cancer is directly and indirectly correlated with RB pathway. Studies have shown that primary breast tumour negative for RB are commonly more proliferative having poor prognosis. (Crittenden, S.L., Bernstein, D.S., Bachorik, J.L. 2002. A It suggests that breast cancer due to RB deficiency is highly aggressive. Tumour samples lacking RB show higher level of E2F downstream targets that includes several genes whose product is independently correlated with poor outcome of breast cancer. It includes cyclin E, cyclin A , DNA replication factors and chromatin remodelling enzymes .The functional status of RB is directly or indeirectly related with therapeutic response of breast cancer. One of the major clinical problems of in breast cancer treatment is the resistance to hormonal therapy. Anti-estrogen therapies antagonize the proliferative function of estrogen receptor (ER) through a series of reactions that ultimately dephosphorylate RB and arrest cell cycle. RB knockdown or deficient cells do not undergo cell cycle inhibition even after hormonal therapy. They actively proliferate and become hormone independent. Generally pRB exists in hyperphosphorylated condition in the initial stage of S phase and in hypophosphorylated condition while passing to G1 phase from M phase. In addition to the inhibition of cell cycle through E2F, RB palys vital role on control of apoptosis and differentiation of cell and histone modification. (Harbour and Dean 2000).
Loss of heterozygosity (LOH) is one of characteristic features of RB mutation that have been demonstrated in many somatic cancers. Studies have shown that INK4a locus encodes two proteins viz p16 and p14ARF (p19 ARF in mouse) through use of alternative reading frame. This p14 has been found to suppress HDM2 modulating p53-HDM2 negative feedback pathway. Similarly, an experiment on p14-null cell lines have shown that over expression of E2F leads to S phase entry and deregulation of E2F induces the expression of p14. (Komori et al 2005)
Fig. 5. Transcriptional regulation of p14 ARF gene by E2F (Solid arrows indicate normal growth signals mediated by Cyclin /CDK activity. Thick broken arrows indicate abnormal growth due to loss of pRB function) ( Komori et al. 2005)
Key Regulators of G1/S Transition
Studies have demonstrated that over expression of cyclin D1 is the key driving force for human breast carcinoma. Cyclin D1 is encoded by CCND1 gene which is located at 11q13 region of genome, and remains amplified in wide range of human cancers, including 15% of breast cancer (Ormandy et al. 2003). Similarly, cyclin D1 has been found to be over expressed at mRNA and protein level in upto 50% of primary breast cancers(Alle et al. 1998). It indicates that cyclin D1-mediated breast carcinoma arises from transcriptional and post transcriptional level as well. Nevertheless, in vivo study on transgenic mouse mammary gland has shown that H-RAS, c-Myc and c-Neu are even stronger oncogens than cyclin D1 (Muller et al. 1988). It means, cyclin D1 alone is not sufficient to exert its oncogenic potential. Additionally, it has been seen that cyclin D1 is involved in oncogenic actions of c-Neu , Her-2 and Ras but not necessarily in Myc and Wnt-1 induced breast carcinoma (Fu et al. 2004).
Unlike cyclin D1, Cyclin D2 and D3 are not required for normal mammary gland formation , rather just act as downstream targets of mammary oncogens like c-Myc. Thus all 3 types of cylin D are more or less commonly involved in breast carcinoma. Studies have shown that inhibition of cyclin D1 blocks the entry of cells to S phase (Musgrove et al. 1994). Thus role of cyclin D1 for cell proliferation in collaboration with CDK4 and CDK6 has been widely accepted. Another function of cyclin D is to sequester inhibitor proteins like p21 and p27 from Cyclin E-CDK2 complex , thus mediating indirect mechanism of cell proliferation. Cyclin D1 plays vital role on estrogen- induced breast cancer resulting in subsequent enhancement on cyclin E-CDK2 activity and increased G1/S transition. (Said et al 1997)
Cyclin E , in association of cyclin D1 is another main activator of G1/S transition. The levels of cyclin E come to the peak during mid to late G1 phase, thus enhancing the formation of cyclin E- CDK2 complex during that period. The accumulation of cyclin E and formation of active cyclin E-CDK2 complex is the rate limiting event for G1/S transition (Hwang and Clurman 2005).
The oncogenic role of cyclin E has been suggested by studies on cyclin E deficient cells, which have been found to be reluctant to transformation induced by myc alone or combined effort of myc and ras , indicating that cyclin E is another key component of oncogenic signalling (Geng et al 2003). Cells unable to be arrested in G1 allow the damaged cells to proceed to S phase. Although amplification of cyclin E1 locus is rare in breast cancer, the protein product is overexpressed in nearly 40% of breast cancer as a series of isoform ranging from 35 to 50 KDa (Keyomarsi et al. 1995)
A study done on transgenic mouse model says that over expression cyclin E in the mammary gland of mouse results in adenocarcinoma formation only in about 10% of female mice even after 8-13 months , indicating that cyclin E is a weak proto oncogen in breast epithelium. (Bortner and Rosenberg 1997).Rather constitutive over expression of cyclin E , but not of cyclin D1 or cyclin A, in both immortalized human breast epithelium , resulted in chromosome instability, indicating that cyclin E/CDK2 activity in the G1/S -phase transition may be necessary for maintenance of karyotypic stability. (Spruck et al 1997)
Fig. 6. Acting points of cyclin A and cyclin E in cell cycle. (Pines and Hunter 1995)
Cyclin A is a regulatory protein of cell cycle that acts in association with its partner CDK2. The abundance and activity of these complexes during S phase and G2 phase suggests their role in preparation for mitosis. However consensus among scientists does not exists regarding the role of cyclin A.
Controversial Role of cyclin A in M phase:
In the context of unknown activator of CDK1 for mitotic entry, Mitra and Enders (2004) pointed out that cyclin A/CDK2 is a key regulator of CDk1 activation in human cells through the effect of Cdc25B and Cdc25C activity. In contrast, another study by T.K. Fung et.al. (2007) underscored the role of cyclin A as a M-phase promoting factor (MPF). They used RNA interference (RNAi) to investigate that whether cyclin A itself is a comoponent of M-phase promoting factor (MPF) or just a part of machinery that activates MPF. For this purpose, cyclin A and Cyclin B were downregulated, and their requirement for mitosis was studied. The results of this study showed that delayed G2 phase induced by absence of cyclin A was caused by inactivation of cyclin B-Cdc2 through inhibitory phosphorylation. They further suggested that this phenotype can be reversed by expressing RNAi-resistant cyclin A. Their experiment showed that ectopically expressed cyclin A could not replace cyclin B in driving mitosis. Finally, they proposed that among the regulators of Cdc2 phosphorylation , deregulation of a tumour suppressor protein called WEE 1 allowed the activation of cyclin B-Cdc2 even in the absence of cyclin A , thus underscoring the role of cyclin A as a M-phase promoting factor (MPF).
CDK2 and its Partners
Cyclin Dependent Kinase-2 (CDK 2) plays key role in cell growth regulation through sequential phosphorylation events by forming holo enzyme complex with different cyclins and their catalytic partners. It has been seen that at least two phosphorylation events are necessary to release pRB from E2F, first by cyclin D1/CDK4/6 and second by cyclin E/CDK2 (Ludberg and Weinberg 1998).The first step involves inducible holoenzyme complex and second one is auto regulatory feedback loop associated with irreversible step towards entry to S phase. Thus, CDk2 induced phosphorylation of pRB is the ultimate step before release of transcription factor, E2F, and entry into S phase. CDK2, in association with different cyclins, acts at G1/S transition, S phase and G2/M transition, thus regulating critical steps of cell growth and division. Hence pharmacological inhibition of CDK2 might be a potentially effective therapeutic strategy against breast cancer or other cancers.
Studies show that the level of cyclin E and Cyclin E-CDK 2 complex is maximum at G1 phase. It is followed by formation of cyclin A-CDK2 complex. Analysis of physical structure of human cyclin A/CDK2 /ATP complex at 2.3 Angstrom resolution has revealed that cyclin A binds at one side of CDK2 catalytic cleft, inducing large conformational changes on it. As a result alterations on the active site residues of CDK2 relieves the steric blockage at the entrance of the catalytic cleft (Jeffrey et al. 1995).
Interactions between CDK2, pRB and E2F are highly complex. Cyclin A/CDK2 and cyclin E/CDK2 have different functions in regulation of pRB -mediated repression of E2F (Dynlacht et al. 1994). In vitro, E2F1 forms stable complex with cyclin A/CDK2 but not with either sub unit alone. Result shows that 124 amino acids of N-terminal of E2F1 are required for binding of cyclin A/CDK2 complex. Moreover, this binding has been seen to be a direct binding of Cyclin A/CDK2 to E2F which is independent of DP-1 and RB family members. (Xu et al , 1994). Thus, unlike cyclin E/CDK2, cyclin A/CDK2 binds directly to E2F1 and inhibits the DNA -binding activity of E2F1/DP1 via phosphorylation (Xu et al. 1994)
Interstingly, study of Lukas et al (1997) says over expression of E2F alone can initiate S phase even in absence of CDK2 and over expression of CDK2 can replace E2F activity to drive the cell into S phase. Thus it suggests that there is more complex signalling mechanism in this pathway. Recent studies have predicted that there are at least 2 distinct signalling pathways downstream of E2F to initiate S phase(57). Gray-Bablin et al (1996) says that over expression of cyclin E in tumour cells, which also over express p16, can bypass the cyclin D1/CDK4/6/p16/pRB feedback loop; and provides an alternative mechanism by which tumours grow. Thus, this study has declared cyclin E as a redundant cyclin in breast cancer.
B-Myb is an oncoprotein which acts as a target of cyclin A/CDK2, and whose activity is regulated by CDK2 mediated phosphorylation (Ziebold et al. 1997). It acts as an in vitro substrate for cyclin A/CDK2 and not for cyclin D1/CDK4 or cyclin E/CDK2(Saville and Watson 1998) . Six sites in B-Myb fulfil the requirements for recognition by CDK2.(Bartsch et al. 1999). Despite its role in cell proliferation, rapid and dramatic up regulation of cyclin A /CDK2 complex causes apoptosis of human endothelial cells after growth factor deprivation (Levkau et al . 1998).
1.4.3. E2F Family Proteins
E2F is a family of regulatory protein of several genes and proto oncogens like c-myb, DHFR, pRB and cdc2, which control cell growth and proliferation. (Xu et al, 1994). Till now 10 E2F proteins have been identified. They are basically of 3 types. First group consists of activators of transcription which includes E2F1, E2F2, and E2F3a. Similarly, second group of E2F proteins are regarded as repressor of transcription. They involve E2F3b, E2F4, E2F5 and E2F6. These seven members of E2F family form dimerization with their partners DP1or DP2 to become active. However, other three E2F family proteins viz E2F7a, E2F7b and E2F8 form homodimers and play a role of repressor of transcription. (Calzone et al, 2008). Nevertheless, these E2F family members share extensive homology in their structure. They contain DNA binding domain in their N-terminal, and sites for transcriptional activation function at C terminal region. Growth suppressors like RB, p107 have tendency to inhibit E2F dependent transcription. (Corney et al. 2008).
Aim of the Project and Conceptual Framework
This research is a part of characterization of inhibitors of the regulatory protein/complex of RB pathway that lead to breast carcinoma in human beings. Current study will focus on the inhibitor of cyclin A/CDK2 complex . The stability and cytotoxicity of the inhibitor will be measured in vitro using high performance liquid chromatography (HPLC) and Alamr blue assay respectively.
Apart from above quantification, following will be the null hypothesis of this experiment:
"There is no role of cyclin A/CDK2 complex in E2F-1 mediated G1/S transition". OR "Cyclin A is a redundant cyclin during G1/S transition".
To test this hypothesis, cyclin A/CDK2 complex will be inhibited by its specific inhibitor and IC 50 value for the inhibitor will be calculated. The internalization of drug at this phase will be assured by immune fluroscence assay. The level of phosphorylation of E2F-1 will be measured by Western blot assay before and after treatment with inhibitor. Similarly percentage of cells on G1 phase and S phase will be counted by fluorescence activated cell sorting (FACS) assay before and after treatment of cells with inhibitor.(Is it feasible to do??? I am not sure) All the data collected before and after treatment with the inhibitor will be used for comparative study and statistical analysis. If we observe statistically significant decline on the number of cells entering S phase and decreased level of phosphorylation of E2F-1 after the treatment with inhibitor, the null hypothesis will be rejected and alternative hypothesis will be accepted.
Materials and Methods
3.1.1 Cell Lines
MCF - 7 and HacaT cell lines were used in this research where MCF-7 are cytoplasmic estrogen receptor (ER) positive cell lines used in vitro as a good model for human breast adenocarcinoma to study the role of estrogen in breast cancer. These cells are positive for cytokeratin , and negative for desmin, endothelin, neurofilament and vimentin (abcam 2010)
3.1.2 Culture Medium
DMEM was the medium used for the routine sub culture assay. The MCF-7 cell line were maintained in DMEM (Dulbecco's Modified Eagles Medium) which contains all amino acids, growth factors, 10% FBS (Fetal Bovine Serum) and 1% solution of penicillin and streptomycin.
3.2.1. Culture of cells
Culturing and splitting the cells in more than one flask was carried out when they reached nearly 90% or more confluence. The confluence of the cells was checked under a microscope and confirmed whether the cells have occupied 90% or more of the total surface area of the flask.
Whole of the sub culturing and splitting procedure were carried out inside the laminar flow hood sterilized by swabbing with70% ethanol and sometimes by using UV rays as well. For the actual procedure, the old culture suspension was discarded and ,then added with 2mls of phosphate buffered saline (PBS) and rinsed well. After discarding the PBS, trypsin was added to the flask ,mixed well and then incubated at 370c in 5% co2 for 5 minutes. During the incubation new flasks were labelled and pre warmed fresh DMEM was added. 8mls of fresh DMEM was added to large flask. After the incubation, the cells were confirmed to be completely detached from the surface of the flask by observing on the microscope. 4mls of fresh DMEM was added to the flasks for deactivating the trypsin. The cells were resuspended by pipetting up and down. 2mls of this suspended culture was transferred to the newly labelled flask containing fresh DMEM. These flasks were kept at 370c in 5% co2 for incubation.