Cancer Stem Cells And Apoptosis Biology Essay

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INTRODUCTION

The human body is made up of billions of cells with the DNA as the control centre of every cell. These cells which are specialized have their DNA shortened after every division and eventually die out (12).

In normal cell cycle, a cell duplicates all its components and produces two daughter cells. For successful progression through the cell cycle, sequential activation of a variety of catalytic subunits called cyclin-dependent kinases (Cdks) that are dependent on the periodic synthesis of cell-cycle specific regulatory subunits known as cyclins is required (6). The deregulation of cell-cycle controls is one of the hallmarks of cancer (7, 8).

Unlike normal cells that divide in an orderly way, cancer cells continue to divide without control (5). This leads to the formation of a mass of unused tissue which is called tumour (14). Cancer cells can destroy adjacent tissue; they have high invasive power and can spread to other locations in the body through lymph or blood.

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In addition, cancerous cells also differ from normal cells in cell-cell interaction, organization of the cytoskeleton and interactions with the extracellular matrix (10, 11). Many researchers have established differences in mechanical properties between normal and cancerous cells. (8, 9). By considering the brush layer on the cell surface, Iyer et al (4) reported the quantitative difference between normal and cancerous human cervical cells. They found that normal cells have brushes of one length while cancerous cells have mostly two brush lengths of significantly different densities.

Recent research has shown that mutations leading to tumorigenesis are more numerous and heterogeneous than previously thought (1, 2).

Scientist have divided the causes of cancer into two groups: environment which is thought to be the primary cause and genetics (13).

According to Zosia (3), cancer survival in UK (excluding Scotland) was low especially in the first year after diagnosis and in people aged over 65.

CELL GROWTH

The inherent ability of cells to develop and divide is a defining feature of life. Cell growth can be referred to the ability of a cell to develop (increase in mass) and divide. Cell division results in an increase in cell population and involves one cell called the parent cell giving rise to two calls called the daughter cells. It also allows for continual construction and repair of the organism (15). Cell growth (the increase in cell mass through macromolecular synthesis) requires the synthesis of cellular components in precise, stoichiometric quantities, and must be subject to tight coordinate control (16, 17).

The complex regulatory program through which a cell achieves division is called cell cycle. The cell cycle primarily maintains the genomic constituent and it is a common denominator in all organisms. In addition, the process of cell cycle which leads to the exponential proliferation of cells can give scientist an idea of how cancerous cells develop (18). The cell cycle is divided into mitosis and interphase. Interphase consists of the G1 phase, S phase and the G2 phase. If the process of one phase is not completed the onset of the other phase will not be activated. Cells which have temporally stopped dividing are said to have entered the quiescent cycle (G0 phase) which is a process in which cells maintain a stable, non proliferating state and then, under specific environmental conditions, return to the cell division cycle (19). Most fully differentiated cells like nerve cells may remain in quiescent indefinitely.

The main regulation of initiation of a new cell division is imposed during the G1 phase of the cell cycle (20). G1 phase has been reported to be a period during which an accumulation process begun in the previous S and G2 phases are completed (23).This makes it a highly sensitive phase in the cell growth as there is a great amount of protein synthesis and a high metabolic rate which results in the formation of new organelles (21).Although, the variability of the G1 phase has been published (24), Lodish et al., (22) found that a rapidly dividing human cell which divides every 24 hours spends 9 hours in G1 phase.

In eukaryotic cells, the genetic material in G1 phase exists as chromatin and the DNA is 2n (21). The DNA also has series of proteins called Cyclin-dependent kinases-S-phase promoting factor (SPF) which are "restriction point" that help safeguard it and ensure the proper functioning of the cell (21).

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The G1 CDK proteins activate the transcription factors for a variety of genes including genes which are responsible for DNA synthesis proteins and S phase CDK proteins (22). Activation of a group of cell cycle-dependent transcripts in G1 initiates exit from G1 and entry into S-phase, thereby committing cells to a division cycle (25), in addition, histone deacetylases 1 and 2 act in concert to promote the G1 to S progression (26). Proteins that control exit from G1-phase, including those involved in regulating G1-S transcription, are often found mutated in human tumour cells, suggesting that inactivation of this pathway may be necessary for tumour development(20).

The S-phase is characterized by DNA replication. It is the phase between G1 phase and G2 phase and involves important regulatory pathways. Successful progression through S phase requires that replication be properly regulated and monitored to ensure that the entire genome is duplicated exactly once, without errors, in a timely fashion (30).Prior to S Phase, multiprotein complex called the prereplicative complex (pre-RC) orderly assemble on DNA at origins in a regulated fashion (30). DNA synthesis can occur as fast as 100 nucleotides per second with an error rate of one nucleotide in a billion (32). UbcH7 has been reported to be a regulator of the cell cycle S-phase. Entry into the cell cycle is delayed if UbcH7 is over-expressed whereas; its depletion increases the length of S phase and decreases cell proliferation (27). After replication, Cdt1 which bind to DNA in G1 phase is targeted for degradation, this is to ensure that there is only one round of replication per cycle and that the genome integrity is preserved (28,29).

The eukaryotic genome possesses multiple sites in which DNA replication is initiated (34). During the S-phase, thousands of replication origins are activated at distinct and reproducible times according to a tightly controlled temporal program (35, 36, 37). Early in the S-phase, euchromatin that are transcriptionally active replicates and are localized to the nuclear interior while heterochromatin that are transcriptionally inactive replicates late and are localized towards the nuclear periphery (34).In addition, chromatin formed from DNA replicating early in S-phase is characterized by the deposition of actively acelated histones H3 and H4 while DNA replicating late in S phase is packaged with deacetylated histones (38,39) . Individual chromosome domains that replicate coincidentally are localized in specific regions of the nucleus (35, 36, and 37). With the exception of histone production, the rate of RNA transcription and protein synthesis is very low during the S phase (31). As a result of the outstanding activities occurring in the s-phase of the cell cycle there are checkpoints that monitor cell cycle progression to decrease DNA synthesis following DNA damage. If DNA is damaged, it is repaired by the activation of ATR which is a protein kinase that initiates several complex downstream pathways that cause a halt in the initiation of new replication origins. The pathways also prevent mitosis and replication fork stabilization in order to keep the replication bubble open and DNA polymerase complex attached while the DNA is being fixed (40).Failure to repair DNA lesions may result in blockages of transcription and replication, mutagenesis, and/or cellular cytotoxicity (41) which could lead to aging (42), and carcinogenesis (43,44).

The G2 phase is prior to the start of mitosis after DNA synthesis where the cells synthesizes protein and grow rapidly. It ends when a threshold level of active cyclin B1/CDK1 complex, also known as maturation promoting factor (MPF) has been reached (46). In some cancers, cells proceed directly from DNA replication to mitosis (45). Prior to chromosome segregation, G2 cell cycle checkpoint allows suspension of the cell cycle in response to genotoxic stress through a p53-dependent and p53-independent manner (47). The transcription factor P53 is activated as a result of signals from DNA damage which results in the direct inhibition of CDK1 by three transcriptional targets of p53: p21, Gadd45, and 14-3-3σ.Inactive Cyclin B1/CDk1 is sequestered in the nucleus by p21 (48), while active Cyclin B1/CDK1 complexes are sequestered in the cytoplasm by 14-3-3σ and CDK1 is transcriptionally repressed by p53 (47).

There is a major re-organization of the genome at the end of mitosis as chromosomes decondense and coalesce to form the interphase nucleus (33)

APOPTOSIS

To ensure correct development and function of the multicellular organism, an elaborate cellular homeostasis mechanism is required. This mechanism is called apoptosis (50) otherwise referred to program cell death (51,61). Apoptosis is a form of cell death in which a programmed sequence of events leads to the elimination of cells without releasing harmful substances into the surrounding area (52).It produces cell fragments called apoptotic bodies which surrounding cells are able to engulf and quickly remove so that its contents does not cause damage to surrounding cells(53). Typical cell features that characterize apoptosis include membrane blebbing, DNA fragmentation and chromatin condensation (57). Apoptosis play an important role in the process of gamete maturation as well as in embryo development contributing to the appropriate formation of various organs and structures (55). Distortion of normal pattern of apoptosis induced by different developmental toxicants could result in the formation of inborn anomalies or intrauterine death (56).In addition, upregulated apoptosis is associated with various degenerative and autoimmune diseases such as Parkinson's diseases and systemic lupus erythematosus (SLE) (58) while a very low rate of apoptosis can cause accumulation of malignant cells and give rise tumour formation(59).

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Cells undergoing apoptosis display distinct morphology as seen in figure 1 (63). In the first stage (A), cells begin to shrink as a result of cleavage of lamins and actin filaments in the cytoskeleton. This is followed by chromatin breakdown in the nucleus which leads to nuclear condensation (B) and thereafter, the cells continue to shrink and package themselves in a form that allows for their removal by macrophages which are responsible for clearing the apoptotic cells from tissues in a clean and tidy fashion that avoids many of the problems associated with necrotic cell death (63).The end stages of apoptosis are often characterized by the appearance of membrane blebs (D) or blisters process. Small vesicles called apoptotic bodies are also sometimes observed (D, arrow).

Fig 1: Morphology of an apoptosic trophoblast cell as captured by time-lapse microscopy (images taken over a 6 hour period). Figure copied from (63).

Central in the mediator of apoptosis is a family of cysteinyl aspartate-specific proteases called caspases(60). Caspases are classified by two systems which distinguish them based on their major function and according to the length of their prodomain. There are two groups of caspases based on their major functions; they are pro-apoptotic (caspase-2,-3,-6,-7,-8,-9,-10) and pro-Inflamatory capases (caspse-1,-4,-5,-11,-12). In addition, there are two classes of caspases according to the length of their prodomain. These include initiator caspases (caspase-1,-2,-4,-5,-8,-9,-10,-11,-12) and effector caspases (caspase-3,-6,-7).Initiator caspases are often involved in the interaction with upstream activator molecules such as receptors,they posses long prodomains and their activation mainly take place in large protein complexes.In contrast, effector caspases have short prodomain and they perform the execution steps of apoptosis by cleaving multiple cellular substrates(60). The death inducing signaling complex (DISC) activates caspase-8 and-10 while caspase-9 is activated the apoptosome and caspase-1 and 5 at the inflammasome. Through proteolytic cleavage, the active initiator caspases activate the effector caspases( 62).

INTRINSIC PATHWAY OF APOPTOSIS

This pathway initiates apoptosis through the involvement of the mitochondria following damage of DNA. It could also be triggered in response to defective cell cycle, cellular stress such as UV irradiation or chemicals or to viral infection and in response to growth factor withdrawal (63).This pathway is regulated by the pro- (such as Bad, Bax or Bid) and anti-apoptotic (such as bcl-2 and bcl-XL) Bcl-2 family members, which control mitochondrial release of apoptogenic factors such as cytochrome c and Smac/DIABLO (64).The pro-apoptotic bcl-2 proteins are often found in the cytosol where they act as sensors of cellular damage or stress they relocate to the surface of the mitochondria where the anti-apoptotic proteins are located. It is this reaction between pro- and anti-apoptotic proteins that disrupts the normal function of the anti-apoptotic bcl-2 proteins and can lead to the formation of pores in the mitochondria and the release of cytochrome C and other pro-apoptotic molecules from the intermembrane space (63). After the release of cytochrome c, it forms a complex called apoptosome in the cytosol by interacting with a protein called APAF1 (fig 2). Apoptosome then activates caspase-9 which is the initiator caspase responsible for the effector caspase activation in the intrinsic pathway (65). In addition, the intrinsic pathway requires the activity of a transcription factor, P53, which induces a variety of apoptotic target genes (66).

EXTRINSIC PATHWAY OF APOPTOSIS

Fas receptor and TNF receptor (Tumour necrosis factor) system are the two major death receptors that induce apoptosis in the extrinsic pathway. The Fas receptor which is also known as CD 95 /APO-1 is a transmembrane of lycoprotein death receptor. Its activation is as a result of Fas ligand (Fas-L) binding to cell membranes. As a result of this, FADD (Fas associated death domain) is produced and apoptosis is induced. In the TNF receptor system pathway, the TNF-related apoptosis inducing ligand (TRAIL) binds to TNF-receptor system and produces TRADD (TNF-receptor associated death domain).

Fig 2: General model of the intrinsic and extrinsic apoptosis pathway.

The extrinsic pathway is activated by death receptors and requires FADD and active caspase-8 to cause effector caspase activation. The cell-intrinsic pathway is triggered e.g. by various cytotoxic agents and targets the Bcl-2 family proteins to induce mitochondrial pathway activation. Cytochrome c assembles in the cytosol with APAF1 to form the apoptosome which activates caspase-9 and thereby leads to effector caspase activation. Figure adapted from (64).

Amongst therapeutic compound that mediate apoptosis are Immunotoxins (54), Death-inducing Ligands (67) and antibodies (68).

CANCER STEM CELLS

Stem cells are defined as cells that have the ability to perpetuate themselves through self renewal and to generate mature cells of a particular tissue through differentiation (69). They occur in very small numbers in adult tissues and in higher numbers in fetus and its annexes and safeguard tissue homeostasis in organism through a fine balance of self-renewal and differentiation (81). Stem cells could be totipotent, pluripotent, multipotent,oligopotent or unipotent (74). These properties can be illustrated in vitro using methods like clonogenic assays, where single cells are characterized by their ability to differentiate and self-renew (75, 76). The totipotent ability of the embryonic stem cell has been shown in mouse (82, 83) and human (84) embryos respectively. Furthermore, it has been conclusively demonstrated in mouse that embryonic stem cells can give rise to all tissue types (85). Embryonic cell lines and autologous embryonic stem cells generated through therapeutic cloning have been proposed as promising candidates for future therapies (73).

The public opinion about the use of embryonic stem cell for therapeutic purpose has been divided because of ethical issues that arise from the fact that embryonic stem cells require the deployment of human embryos which has been derived from the potential of coming into existence (81). In another development, adult stem cells have been shown to have therapeutic ability as bone marrow derived stem cells and mesoangioblasts have been shown to regenerate skeletal muscle fibers when transplanted into dystrophic mice (86, 87). The recent identification of different types of multi-potent stem cells, some of which are suitable for protocols of clinical cell therapy, has disclosed new perspectives in the treatment of genetic diseases, including muscular dystrophy(81). Although, stem cells have been shown to have therapeutic effect, a group of stem cells in the body called cancer stem cells have be shown to be of pathogenic interest amongst researchers because they tend to be the cells sustaining the growth of tumors (81). Through progressive genetic alterations, normal cells can be transformed into highly malignant derivates (71). Current evidence indicates that most cancers arise from a single cell that has undergone malignant transformation driven by frequent genetic mutations (72).These cells have the ability to invade and destroy normal tissues (73)

Research has shown that these cancer stem cells are chemoresistant in comparism to their more differentiated progeny and that targeting them in therapy could lead to the cure of human cancers (88, 89). After Dick and colleagues published a seminal report showing that a hierarchy exists among leukemic cells, cancer stem cell research has been a field of interest for many researchers. Cancer stem cells (CSCs) represent malignant subpopulations that initiate and maintain tumorigenic growth in hierarchically organized tumors via their considerable capacity for self-renewal and differentiation (49).

The cancer stem cell (CSC) model of tumor development suggests that the clinical behavior of a tumor will be largely determined by a subpopulation of cells that are characterized by their ability to initiate new tumors (77). CSCs have reportedly been identified in several cancer types, including brain tumors, in which they have been isolated through sphere-formation assays (78). Current evidence indicates that most cancers arise from a single cell that has undergone malignant transformation driven by frequent genetic mutations (72).These cells have the ability to invade and destroy normal tissues (73) by avoiding cell labeling through efflux of the marking dye (79), and with their cell sorting methods (80). Their true measures are in their capacity for self renewal and recapitulation of original tumour (90). CSCs have been found to express several markers of stemness, such as the surface antigen CD133, while the expression of pluripotency transcription factors in CSCs still remains as questionable as it has always been (91). Sheila et al (92) reported a xenograft assay that identified human brain tumor initiating cells that initiated tumours in vivo. They found that CD 133+ brain tumour fraction contains cells that are capable of tumour initiation in NON-SCID (non-obese diabetic, severe combined immunodeficient) mouse brains. Their findings also revealed that Injection of as few as 100 CD133+ cells produced a tumour that could be serially transplanted and was a phenocopy of the patient's original tumour, whereas injection of 105 CD133- cells engrafted but did not cause a tumour. Normal and cancer stem cells often express ABC transporters that confer drug efflux capacity, easily measured by the flow-cytometric detection of the so-called 'side population' in assays evaluating the efflux of fluorescent dyes such as Hoechst 33342 (81).The concept of cancer stem cells revolves on the fact that the cells initiating tumor are organized in hierarchy. In this way, they give rise to mature cells that are more committed with a limited proliferative potential. The cancer stem cells reside at the top in the hierarchy and the size of their niche may be the factor that determines the disease course (93). Report from immunohistochemistry (IHC) study stipulates that ≥1% proportion of CD133+ cells and the presence of clusters with CD133+ cells predisposed glioma patients (grade II to IV) to a poorer survival outcome(94). Cancer stem cells have also been implicated in clinical settings like treatment failure and tumor recurrences, because of their resistance toward chemo- and radiotherapy. According to this view, relapsing tumors evolve from expansion of surviving CSC clones (93).In this regard, CD133 expression after radio- and chemotherapy was increased in patient biopsies from GBM recurrences, compared with biopsies taken prior to treatment (95).

Angiogenesis

Angiogenesis is the formation of new vessels from existing ones. It involves the vascular endothelial growth factor (VEGF) pathway and it's a fundamental event in the process of tumour growth and metastatic dissemination (102). The dependence of tumour growth on the development of neovasculature cannot be over emphasized (103). This assertion was made over 100 years ago (112,113) and the hypothesis was put forward in 1968 (114).

Angiogenesis is useful in the supply of oxygen, nutrients, growth factors hormones and proteolytic enzymes (104,105). It also influences homeostatic factors that control the coagulation and fibrinolytic system, and the dissemination of tumour cells to distal sites. (104,105). An imbalance in the process of angiogenesis contributes to inflammatory, ischaemic, infectious and immune disorders (111). It is now known that there is an 'angiogenic switch' which when 'off', balances the effect of pro-angiogenic molecules with that of anti-angiogenic molecules and when 'on' favours the process of angiogenesis (115).The sources of the Pro- and anti angiogenic molecules include cancer cells, endothelial cells, stromal cells, blood and the extracellular matrix(118). The signals that trigger this switch include mechanical stress, immune/inflammatory response, and genetic mutations (116,117)

Cancer cells in tumours require constant access to blood vessels for growth and metastasis. If these accesses are limited, there will be reduction in mortality and morbidity from these tumors (97). A current model of tumor angiogenesis suggests that it involves recruitment of sprouting vessels from existing blood vessels and incorporation of endothelial progenitors into the growing vascular bed (108).Tumour angiogenesis can also be supported by circulating endothelial precursors (either shed from the vessel wall or mobilized from the bone marrow) (119,120).

The well-established role of VEGF in promoting tumor angiogenesis and the pathogenesis of human cancers has led to the rational design and development of agents that selectively target this pathway (102). In 1996, Teicher (101), postulated that combined administration of antiangiogenic and cytotoxic (chemo- and radiation) therapies would yield maximal benefit because such combinations would destroy two separate compartments of tumors-cancer cells and endothelial cells. Furthermore, cancer cells may express receptors for angiogenic growth factors (e.g., VEGFR1 or VEGFR2), and thus antiangiogenic drugs (e.g., antibody to VEGF) could lead to the direct killing of cancer cells by interfering with survival pathways and/or enhancing sensitivity to other treatments (102). In addition, Antiangiogenetic drugs have produced modest objective responses in clinical trials when administered as a single drug (98, 99). It has been shown that a combination of bevacizumab which is an antibody targeted against the potent angiogenic molecule vascular endothelial growth factor (VEGF), and chemotheraphy produced an unprecedented increase in survival (5 months) in colorectal cancer patients (100). Normalization of the tumour vasculature is an emerging concept in antiangiogenic therapy (96) because increased tumour vascularization and tumour expression of proangiogenic factors has been associated with advanced tumour stage and poor prognosis in a variety of human cancers (106,107). Cancer cells need nutrients for their growth and metastasis which they obtain by co-opting host vessels, sprouting new vessels (angiogenesis), and/or recruiting endothelial cells from the bone marrow (postnatal vasculogenesis) (109).The resulting vessels is structurally and functionally abnormal (110).

METASTASIS

Metastsis is the movement of tumour cells from their primary site to a distal organ (121). It has been regarded as the most fearsome aspect of cancer (125). The process of metastasis involves a series of well regulate events that could be brought to a halt if not properly guarded (127,128).

Malignant tumours have the ability to metastasize(122)

Common locations for metastasized tumours are bones, lungs, liver, and brain (no ref 4 na!).

Santos et al. (123), reported vertebral metastasis of glioblastoma multiforme(GBM) eleven months after surgery .

In 1889, Stephen Paget reported that tumours have affinity for specific organs. He then postulated the 'seed and soil' theory which states that cancer cells (the 'seed') metastasizes to a new environment (the 'soil') with similar characteristics (124). This theory was challenged by James Ewing(126) in 1929 who stated that metastasis occurs by purely mechanical factors that are a result of the anatomical structure of the vascular system. After series of research, it is now known that the process of metastasis is selective (129).