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54 Seiten, Note: 9.12
1) Aim of the study
3) Materials and Methods
Estrogen, a steroidal hormone that can act as a potential mitogen and is well implicated to breast cancer and the hormone sensitive cancers often emerge through the hormone information being executed through complementary estrogen receptors (α/β) present in the cell. ER can transduce the hormonal signal through two different mechanisms namely classical genomic pathway of signaling quite common among the steroidal hormones in which the hormone bound receptor readily diffuses across the membrane barriers and bound to the hormone responsive elements (HRE) in the presence of co-activators in the nucleus and affect the expression pattern of genes to produce a response to hormone stimulus and the novel non genomic mechanism of signaling in which the cytosol bound ER exerts its signal by protein-protein interactions with a variety of cytoplasmic signalling molecules to modulate its oncogenic potential (Manavathi & Kumar, 2006) . This pathway appears to be highly complex and co-ordinated by a complicated network of biomolecules affecting the gene expression to cause the biological effects of tumorigenesis and metastasis.
It is important to understand the functions of cellular components that regulate ER signals for breast cancer and one of the most important of all is the microtubules which are basically polymeric cytoskeletal fibres comprised of α/β tubulin heterodimers as their monomeric constituents. Microtubules are often involved in the processes of cell division, cell motility and cell signaling etc and express their functions through a dynamic behaviour characterized by growth and catastrophe under the control of microtubule associated proteins (MAPs). It is reported recently that microtubule network tends to possess an intrinsic tight link with non genomic mode of ER signaling often accomplished through MAPs and one such component that has been discovered is HPIP.
HPIP (Hematopoietic PBX Interacting Protein) protein was initially discovered as a repressor of Hox proteins and mediated transcription process but reports have shown that it has a potential to modulate ER signal by acting as a scaffold protein in the recruitment and activation of cell survival signaling cascades namely Src and phosphatidylinositol-3 kinases (PI3K) for cellular oncogenesis in the presence of an intact microtubule network but its function on microtubule dynamics remains unclear.
Therefore the aim of this project work was to characterize the functional activity of HPIP protein on microtubule dynamics under in vitro conditions and to address this question a defined work plan was adopted.
Estrogen, a steroidal hormone synthesized by developing follicles in the ovaries (Doisy.E.A & Allen.E., 1923) , the corpus lutum, the placenta, breasts etc in the body controls diversified biological activities in the cell including the development of female secondary sex traits, acceleration of fat catabolism, stimulation of endometrial and uterine growth, reducing bone resorption, regulation of cortisol and cholesterol levels etc. Estrogen is also well implicated to numerous human pathophysiological conditions especially estrogen mediated cancers including breast, ovarian, uterine, prostrate, endometrium, osteoporosis, cardiovascular disease, endometriosis and obesity etc (B.J Derro et al., 2006).
Estrogen and Estrogen Receptors (α/β)
Estrogen hormone exhibits its biological effects by readily diffusing across the plasma membrane barrier and directly interacts with cytosolic receptor protein namely estrogen receptor-ER (Jense.EV & Jacobsen.HI., 1962). It is reported that ER exists in two different isoforms as ERα (Chamness.GC & Mcguire.WL., 1972) and ERβ (Gustaffson.J-A˚ et al., 1996) and encoded by two different genes ESR1 and ESR2 located on the sixth and fourteenth human somatic chromosomes respectively. The expression pattern of ERα and ERβ differs significantly in various tissue types with ERα predominantly occurs in endometrium, breast cancer cells, ovarian stroma cells and in the hypothalamus while ERβ is seen at an elevated level in kidney, bone, brain, intestinal mucosa and in prostrate gland etc.
Estrogen action by classical genomic signaling pathways
In this conventional signaling pathway, estrogen readily binds to its complementary receptors ERα/β and an enhanced phosphorylation of the receptor occurs at the physiological doses of E2 (Metzger.D et.al., 1993) and it is crucial for ligand binding, ER dimerization, interaction with cofactors and DNA binding functions. The hormone receptor ER then recruits any of the cofactors of SRC family including SRC1, SRC2 and SRC3 through the interactive binding between LXXLL motifs of SRC coactivators and the AF2 domain in ER. The SRC activation in turn leads onto the recruitment of histone modifying enzymes like CBP/p300 (histone acetyl tranferases), CARM1 (Arginine methyl transferases1), PCAF/GCN5 etc to regulate the expression of hormone targeted genes (Xu.L et al., 1999). However it is reported that the ligand activated ER is also interacting with another large coactivator complex, referred to as TRAP/SMCC/DRIP/ARC complex (TRAP220 complex) to connect ERs with the basal transcription machinery of the cell . It is not known whether the two complexes act sequentially or independently on ER nuclear receptors (Ito.M et al., 1999).
Reports also have shown that ER complex is also shown to be interacting with other novel, unique coactivators namely NSD1, TIF1, PPARγ, ASC2/RAP250 etc prior to its interaction with DNA. Besides the ER and coactivators interaction, reports have shown that the ERs are also interacting with conventional corepressors like N- CoR-nuclear receptor corepressor, SMRT-silencing mediator of retinoid and thyroid receptors, (Mc Kenna NJ et al., 1999) and also RIP 140, SHP etc (Treuter E et al., 1998) to inhibit the transcriptional activation of ER dependant genes and proved as promising targets in anticancer therapies. So far a detailed molecular investigation study has been made in the estrogen mediated classical genomic signal transduction for addressing the complex biological responses that the stimulus hormone elicits in human system. (Fig 1)
ESTROGEN RECEPTOR-DNA INTERACTIONS (Fig 1)
illustration not visible in this excerpt
Source: Stefan Nilsson et al., Physiological Reviews, 2001.
ER-DNA interactions. (W) ER acts as a transcription factor and directly binds to the ERE elements of DNA. (X&Y) ER activates the other transcription factors of the cell Sp1 & AP1 resulting in activation of Sp1, AP1 dependant genes. (Z)ER is functioning as a transcription repressor by inhibiting the NF-κB mediated transcription.
Estrogen non genomic pathway signaling:
A potentially novel mechanism by which estrogen hormone exerts its biological effects is the non-genomic pathway of signaling in cells. It is mediated by interaction of estrogen with membrane bound ER and the subsequent activation of downstream signaling cascade which occurs by protein-protein interactions and DNA binding activities of the transcription factors. This pathway appears to be complex in terms of the biomolecular components involved and the resultant regulation of expression of genes by ligand activated estrogen receptor than the range of functions controlled by ER alone acting as a transcription factor in classical genomic signaling pathway (Linda.B & Maria.S, 2005). It is reported that nearly one-third of the genes in humans that are regulated by ERs do not contain ERE-like sequences (O’Lone.R et al., 2004). The membrane bound ERs include ERα and β only to mediate the signaling cascade. ERs inspite of the absence of transmembrane domain tends to remain and interacts with β-estradiol owing to the post translational modification of palmitoylation in the receptor (Acconcia.F et al., 2004).
The nongenomic actions of β-estradiol that have been reported include the mobilization of intracellular calcium (Improta Brears.T et al., 1999), and the stimulation of adenylate cyclase activity and cAMP production (Aronica SM et al., 1994). Activation of the MAPK signaling pathway by β-estradiol has been extensively studied in several cell types, including breast cancer (Migliaccio.A et al., 1996) endothelial, bone and neuroblastoma cells. The hormone also activates the phosphatidylinositol 3-kinase (PI3K) signaling pathway in endothelial, breast cancer (Castoria et al., 2001) and liver cells.
ERα accomplishes this signal transduction through interactions with well characterized scaffold proteins like HPIP (B.Manavathi et al., 2006) and MNAR (Wong CW et al., 1999) resulting in the activation of Src/PI3K and Src kinases respectively and other proximal signaling molecules including G proteins (Razandi.M et al., 2000), ras (Migliaccio.A et al., 1998). The functions of transcription factors are regulated through protein kinase mediated phosphorylations and so they become the ultimate targets for the non genomic actions of estrogen. The transcription factors ELK-1, C/EBPβ, and CREB [cAMP response element (CRE) binding protein] are all targets for phosphorylation by the MAPK signaling pathway.
The NF-κB transcription factor complex is a target for phosphorylation by the Akt kinase (protein kinase B) and a maximal transcriptional activity of STAT family proteins requires the phosphorylation by β-estradiol activated MAPK, PI3K and Src kinases (Bjornstrom L et al., 2002).
It is reported that Cyclin D1 is a well-defined target for estrogens in breast cancer cells, and it is important for the progression of cells through the G1 phase of the cell cycle (Prall OM et al., 1997).The complex promoter of this gene contains binding sites for multiple transcription factors but does not have a ERE (Estrogen responsive element) sequence. Hence it is suggested that the activation of Cyclin D1 is made through ERs activated CREB (Sabbah.M et al., 2006) and cAMP/Protein kinase A pathways (Castro Rivera.E et al., 2001).
In addition, the cyclin D1 gene is activated by the association of ER that has been activated by β-estradiol with Src kinase and the p85 regulatory subunit of PI3- kinase in breast cancer cells (Castoria.G et al., 2001).The activated MAPK and PI3K signaling pathways target the transcription factors ELK1 and SRF transcription factors which bind to the c-fos promoter of the immediate early c-fos gene and affect its expression in the estrogen non-genomic signaling pathways (Shaywitz AJ et al., 1999).
The best characterized in vivo rapid, membrane-initiated steroid effects are listed Neuronal injury protection (D’Astous.M et al., 2006).
Vasodilatation (Guo X et al., 2005).
Arterial injury protection (Simoncini T et al., 2000).
Cardiomyocyte injury protection (Patten RD et al., 2004).
Breast cancer proliferation and survival (B.Manavathi et al., 2006) & (Marquez DC et al., 2001).
Mechanisms of action of Estrogen Hormone (Fig 2)
illustration not visible in this excerpt
ER overall modes of action in the cell by functioning through a range of diversified pathways to produce its response in the target cell.
1) Classical mechanism of estrogen action.
2) ERE independent genomic action.
3) Ligand independent genomic action.
4) Non-genomic action.
Source: Linda Bjornstrom and Maria Sjoberg, Molecular Endocrinology, 2005.
Microtubule polymers are dynamic, super macromolecular cytoskeletal biological structures of the cell comprised of α and β tubulin heterodimeric proteins as their fundamental constituents (Borisy G.G & E.W. Taylor, 1967). They polymerize in a head to tail fashion leading to the formation of protofilaments. These protofilaments are then laterally arranged to form hollow cylindrical structure microtubules with a diameter of 25nm. An interesting property associated with microtubules is the presence of polarity due to the protofilaments configuration in which the β-tubulin is at the plus (+) or the fast growing end owing to the rapid addition of tubulin heterodimers and α-tubulin occupies the slow growing minus (-) end. The microtubules organize themselves from the centrosome or the microtubule organizing center (MTOC) where the minus (-) is associated and radiate outwards to the periphery of the cell. γ-Tubulin, a protein highly homologous to the α/β-tubulins, is localized at the MTOC, and plays an important role in microtubule nucleation by interacting with α-tubulin (Oakley, 2000).
Microtubules exist in a phase of non-equilibrium characterized by a dynamic growth and shrinkage of the polymer often referred to as dynamic instability of microtubules. It involves a rapid, reversible addition of tubulin heterodimers to the plus (+) end of microtubules by non-covalent interactions between chain and newly added dimer. The dynamics is primarily attributed to the GTP hydrolysis and exchange processes and both the subunits have an inherent site to bind GTP. However β-tubulin has an intrinsic functional activity of hydrolyzing GTP and harbours it at a site that is suitable for subsequent exchange of GTP at a later stage. So the growing chain appears as (GTP: α-tubulin/GDP:β-tubulin)n with the addition of β-tubulin bound GTP caps at the growing(-) end for an enhanced stability which would otherwise be prone for depolymerization, called as microtubule catastrophe. Since β- tubulin has an exchangeable site for GDP-GTP, a second cycle of polymerization proceeds called as microtubule rescue if depolymerization occurs and the process continues to proceed in a cyclic manner in the cell (Georga.A Orr et al, 2003).
Regulation of microtubule dynamics:
Microtubule dynamics are regulated spatially and temporally under in vivo and this phenomenon is well regulated by numerous protein factors often called as microtubule associated proteins-MAPs (Nogales, 2000). The MAPs are so diversified in their mode of action over microtubules and so regulation as such occurs at multiple levels with some proteins regulating tubulin folding, some prteins involved in stabilization of existing MTs such as structural MAPs, and some proteins are interacting with MT ends to influence their dynamics, location and lifespan.
Regulation of activity of MAPs by kinases and phosphatases:
It is reported that the MAPs are subjected for post translational modification mainly phosphorylation by microtubule affinity regulating kinase (MARK) at their N- terminal ends and affect their binding ability to microtubules. As a result the microtubule stability is altered. The most important kinases that regulate MT dynamics are those that control the cell cycle, such as the Cdc2 kinase family (Belmont L et al., 1990, Verde F et al., 1990) , Cdk 5 in neurons (Smith D, 2003), aurora kinases which control centromere localization (Gorbsky, 2004), PAKs that control cell migration (Rayala.S et al., 2005), mitogen activated protein kinases (ERK & JNK) and GSK 3β (Yoshida H et al., 2004 & Goold R.G et al., 2005) which are targets of extracellular signals involved in cell proliferation are contributing for the
dynamics of microtubules. Besides kinases, protein phosphatases also play a key role in the affecting the MT dynamics. Phosphatases of type PP1 which is required to control MT dynamics in and out of mitosis (Tournebize.R et al., 1997) and PP2A which is a regulator of Stathmin for the short steady state maintenance of microtubules and its activation also leads to dephosphorylation of tau MAP (Merrick S.E et al., 1996).
Functions of Microtubules (MT):
(A) MT controlled cell proliferation
The dynamic polymers microtubules perform a diversified range of complex biological a function inside the cell. It includes cell proliferation and mitosis as the most important function. MT dynamics is so crucial for the proper alignment and segregation of chromosomes. Since they have an intrinsic polarity, MTs organize themselves in such a way that the plus end protrudes away from the center while the minus end is at the centrosome poles. Three distinct subsets of mitotic MTs can be characterized: kinetochore MTs (kMTs), whose plus ends attach to sister chromatids at the kinetochore, interpolar or pole-to-pole MTs, which overlap MTs from the opposite pole at the spindle midzone, and astral MTs, extending away from the spindle.
MT turnover is much higher in mitotic cells than in interphase cells (Saxton.W et al., 1984) . In the early prophase stage MT nucleation is increased by 4 fold (Piehl.M et al., 2003) and mostly contributed by the astral tubules and interpolar tubules. During the prometaphase the MT dynamics increases greatly with respect to the kMTs where the polymerized mitotic spindle assembly scan for the kinetochores of the mitotic chromosomes on nuclear envelope breakdown through their plus ends and attain a stability probably by their interactions with the kinetochore associated proteins (Wollmann R et al., 2005)
On the other hand the dynamics of the remaining microtubules tend to show a higher instability because of the availability of free plus ends that are often the targets of cytoplasmic MT depolymerizers even then a subset of astral MTs will remain stable for an effective spindle positioning (Rusan.N et al., 2001). Reports also have shown that the MTs at the minus end are also stabilized by a variety of protein factors including NuMA and TPX2,γ-tubulin, MT assembly promoters by small GTPase Ran, CENP-E etc during the prometaphase. Chromosome associated proteins like chromokinesins play a significant role in the proper alignment and orientation of chromosomes. During anaphase, two mechanisms of K fiber depolymerization exist to drive chromosome segregation. In somatic cells, kMT plus end depolymerization dominates, while treadmilling appears to play a minor role in force production for chromosome segregation leading on cytokinesis. Altogether the MT dependant cell division process seems to be a complex function where a tight coordination among the cellular biomolecules occurs and a detailed analytical study is required to untie the knot.
(B) MT dependant Cell Migration:
MTs also play a key role in the cell migration during the metastatic stage of tumours. This occurs in the presence of an extracellular stimulus and thereby the migrating cell attains a polarity which can be characterized by the localization of signaling molecules, cytoskeleton, centrosome, golgi apparatus and directed membrane trafficking in an asymmetrical way. Studies have shown that MTs are important components for cell migration (Vasiliev.J et.al., 1970). An asymmetrical MT array distribution coupled to abnormally stable subset of MTs at the leading edge of migrating cell are important to attain an appropriate cell geometry for migration. The stability is primarily attributed to FAK signalings (Palazzo.A et al., 2004), increased half life of tubulin at plus ends by cappings (Infante A et al., 2000) and post translational modifications like detyrosination of α-tubulin at the C terminal end (Westermann.S et al., 2003).
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