Upon apoptosis induction, BAX undergoes conformational change. This exposes its transmembrane domain, leading to the insertion of BAX into the outer mitochondrial membrane.
Apoptosis is reflected in significant cell morphological changes Table 1. In the earlier phases, a cell undergoing apoptosis loses cell contacts and changes shape. Chromatin condenses in the nucleus and moves toward the nuclear envelope. Condensation of the nucleus pyknosis initiates DNA degradation. Loss of water results in significant cell shrinkage and blebbing of the plasma membrane with little or no morphological changes to the other cellular organelles.
Phosphatidylserine, a lipid present only in the inner layer of the plasma membrane, is now also visible in the outer layer. Nucleus and cytoplasm fragment into apoptotic bodies. Released cellular proteases lead to disintegration of the cellular skeleton, membranes, and proteins. Neighboring macrophages recognize, engulf, and digest apoptotic bodies, completing the process. What is necrosis?
Necrosis is a form of cell injury defined as unregulated cell death resulting from internal or external stresses such as mechanistic injuries, chemical agents, or pathogens. The process is usually rapid and leads to cell swelling oncosis and bursting due to loss of osmotic pressure Table 1. During necrosis, the loss of plasma membrane integrity induces cellular contents to escape to the extracellular space, causing inflammatory responses.
Cell disintegration is preceded by a series of morphological changes, including disruption of cell organelles, such as swelling of the ER and mitochondria, or decay of the Golgi apparatus. An influx of calcium ions from the extracellular matrix activates intracellular nucleases that fragment DNA. Freed lysosomal hydrolases contribute to the degradation of nucleic acids and proteins.
Decay products activate leukocytes, lymphocytes, and macrophages that phagocytose the remnants of dead cells. Necroptosis is a form of regulated cell death that produces necrotic phenotype. It arises in response to stress stimuli PMID: ; 6 , ; 7 , and ; 8 such as interferons, death ligands, or Toll-like receptors.
Figure 4. Figure 5. Autophagy is a natural degradation process of cellular contents during nutrient stress. In the case of macro-autophagy, it involves the formation of double membrane vesicles called autophagosomes that fuse with lysosomes to form autolysosomes.
This process is initiated by the mechanistic target protein of rapamycin mTOR and autophagy-related genes Atgs proteins Figures Autophagy promotes cell survival, providing starved cells with nutrients obtained through the digestion of non-essential cellular components.
It was discovered that macro-autophagy can also be one of the routes for programmed cell death PMID: ; 2. Although significantly less common than apoptosis, it plays a role in regulating some developmental processes. The most well known is the removal of certain larval organs — the salivary glands and midgut — in Drosophila melanogaster during larval—pupal transition PMID; 3 and ; 4.
Autophagic cell death was also observed in in vitro cultures of adult hippocampal neural cells in response to insulin removal PMID: ; 5. The main characteristic of autophagy-dependent cell death is extensive autophagic vacuolization of the cytoplasm, with no changes in chromatin organization as seen in apoptosis Table 1.
Also, cell remnants are not cleared by macrophagic phagocytosis as observed in apoptosis. Species used for generating blot are also shown in Figure S2A. Bottom: diagram of the rpr -HRE deletion constructs tested. The black arrowheads indicate the specific DNA-protein complexes. Loading of equal amounts of labeled wild-type and mutated oligonucleotides is illustrated by formation of comparable amounts of unspecific DNA-protein complexes black arrow.
L—O Reporter gene expression in the PS of stage 15 embryos driven by the fragments described above. A—E Cuticle preparations of the different genotypes with focus on the PS of 1 st instar Drosophila larvae. Dashed, light blue circle in I highlights the absence of GFP-positive cells, whereas closed, light blue circle in J mark the presence of these cells. Note that some cells expressing GFP under the control of the ems -GAL4 driver invaginate deeper than the Ct expressing cells, thus they are still present in ct db7 mutant embryos, indicated by closed, green arrowheads in K and L.
Open, yellow arrowhead in L highlights the absence of these cells in ct mutant embryos. To this end, we analyzed ct deficient cells, which were kept alive by expressing the caspase inhibitor p35 [12] in ct mutant embryos using the PS-specific driver ems -GAL4 [13]. In order to follow the cells normally under the control of Ct, these cells were GFP-labeled using the same driver, which is active only in a subset of Ct-expressing cells Figure 2G, 2K.
Ct is expressed in many different cell and tissue types [14] , thus we tested the Ct switch function in diverse developmental contexts. Co-expression of either the apoptosis inhibitor p35, which rescues the eye size Figure 4C , or of a rpr RNAi construct along with the ct RNAi transgene resulted in a survival of ct deficient cells, as evidenced by the expression of the bristle shaft progenitor maker DE-Cadherin Figure 3J.
Similar results were obtained in other cell types specified by Ct Figure S6 , suggesting that the Ct-dependent switch between cell-type specification and programmed cell death is of general relevance. A, E, I Scanning electron micrographs of individual ommatidia of adult Drosophila fly eyes with indicated genotypes are shown. The closed, red arrowheads in A mark interommatidial bristles, the open, red arrowheads in E mark the absence of these structures.
The closed, light red arrowheads in I indicate the presence of tissue that would normally develop into interommatidial bristles. B, F, J Projections of consecutive confocal sections of one ommatidium of 50 h pupal retinas labeled with DE-Cadherin. Interommatidial bristles are marked by red, closed arrowheads in B. A—M Adult compound eyes of the respective genotypes are shown. L eyeful::ct RNAi ; p35 flies show high frequency of long range metastasis marked by yellow arrowhead , a close-up of which is shown in M.
Eyes of such eyeful::ct RNAi ; p35 flies show undifferentiated and overproliferated eye tissue marked by light blue arrowhead. N Quantification of primary and secondary tumor formation in different genetic backgrounds. O Relative transcript levels of selected genes involved in cell cycle control, DNA damage response, growth control and epigenetic regulation in eye-antennal discs of 3 rd instar larvae of pre-oncogenic control animals ey::Dl and animals with reduced Ct activity ey::Dl;2xct RNAi.
An increase in Casp-3 and PH3 positive cells is seen in the area below the dashed, yellow line highlighting the morphogenetic furrow. By analyzing the Ct- rpr interaction in two well-established in vivo Drosophila cancer models, we asked whether the combined transcriptional regulation of differentiation and apoptosis repression by Ct could represent a cancer prevention mechanism.
In contrast, pre-oncogenic ey :: Dl flies over-expressing Dl exclusively in eye tissue [16] never displayed any eye tumors or invasive tumors but only mildly overgrown eyes Figure 4D, 4N.
Eye-specific inhibition of Ct activity alone only caused a small increase in primary and secondary tumor incidences in both sensitized backgrounds Figure 4E, 4I, 4N , however, these numbers were dramatically increased when Ct function and the ability to activate apoptosis were simultaneously inhibited Figure 4F, 4G, 4K, 4L, 4M, 4N. Consistently, increased numbers of apoptotic cells were found in tumorous tissue with reduced Ct levels ey :: Dl ; 2xct RNAi Figure 4P, 4Q , demonstrating that the coupled regulation of differentiation and apoptosis by a single transcription factor is an important mechanism to suppress cancer.
However, despite increased apoptosis activation in ey :: Dl ; 2xct RNAi eye imaginal discs Figure 4P , which should result in a reduction of tumor growth, tumor formation in these animals was increased Figure 4N, 4Q.
Using the proliferation marker Phosphorylated histone H3 PH3 , we could demonstrate that the tumor growth induced by differentiation loss is due to excessive cell proliferation Figure 4P , which is in line with previous results [4]. What is the molecular basis for this phenotype? Thus we tested its contribution to tumor formation in Ct-induced oncogenic eyes by reducing its level in ey :: Dl ; 2xct RNAi animals. Taken together, these results show that the Ct-dependent tumor growth is in part mediated by the up-regulation of the PI3K signaling pathway and that this pro-tumorigenic effect counteracts the anti-tumorigenic apoptosis effect of Ct.
We found cell clusters expressing the eye differentiation marker ELAV at abnormal, ectopic positions in undifferentiated tissue of 3 rd instar eye-antennal discs Figure S7 , and it had been shown before that changes in the adhesive properties of cells are critical in inducing migratory behavior [19] , [20].
Consistently, transcriptome profiling experiments revealed a reduction in the expression of cell adhesion genes in eye-imaginal discs of Ct depleted animals exhibiting primary and secondary tumor formation ey :: Dl ; 2xct RNAi in comparison to control animals ey :: Dl Figure 5A. Since decreasing the activity of another Ct responsive cell adhesion gene, namely Tissue inhibitor of metalloproteases Timp , also induced an increase in secondary tumors Figure 5B , we asked if restoration of cell adhesion would be able to rescue this phenotype in the Ct loss-of-function setting.
These results demonstrate that regulation of cell adhesiveness is one of the essential Ct-dependent mechanisms to suppress tumor spread. In vertebrates, invasive tumor growth requires the detachment of abnormal cells from tumor tissue and their circulation in the bloodstream [22].
In sum, these results demonstrate that transcriptional coupling of differentiation and apoptosis is a cell-intrinsic mechanism to ensure normal development and to prevent tumor initiation, progression and invasion, which is at least in part achieved by fine-tuning the adhesive properties of cells required for tissue integrity. A Changes in expression of cell adhesion genes in 3 rd instar eye-antennal imaginal discs of ey :: Dl ; 2xct RNAi versus ey :: Dl animals identified by expression profiling experiments.
Red arrows indicate reduced expression, green arrow induced expression of the respective genes in ey :: Dl ; 2xct RNAi animals.
Green arrowhead marks secondary tumor growth in the abdomen. D Quantification of secondary tumor growth rates in different genetic backgrounds. Locations of GFP-labeled eye-imaginal discs and the insect circulatory fluid, the hemolymph, are indicated by arrows. For analysis of the hemolymph, the insect circulatory fluid is extracted by bleeding out the larvae after cutting at the posterior end indicated by dashed, blue line.
We next explored whether the effective regulation of programmed cell death by Ct has been conserved during evolution. The vertebrate homologue of Cut, Cux1, has a well-documented function in cell differentiation during normal development as well as in tumor initiation and progression in specific cancer types [23]. In addition, several studies show that Cux1 represses apoptosis during normal vertebrate development [24] , [25] , [26] , and just recently it has been demonstrated that Cux1 knock-down leads to activated apoptosis and to reduced growth of xenograft tumors in vivo [25] , [27].
To further investigate the mechanistic basis of Cux1 function in mediating apoptosis repression in vertebrates, we suppressed Cux1 in Panc1 pancreatic cancer cells Figure 6A and determined the transcriptional response of human apoptosis genes. Strikingly, mRNA levels of the pro-apoptotic gene puma were consistently elevated, whereas the anti-apoptotic gene Bcl-2 was down-regulated upon Cux1 depletion Figure 6A.
A Relative mRNA expression of eight apoptosis genes after lenti-virus transduced stable Cux1 p knock-down in human Panc1 cancer cells. Stronger effects of KDa reduced versus KDb almost complete p Cux1 knock-down on target gene expression is very likely due to the processed p Cux1 isoform, which can have opposite transcriptional effects to the p full-length form [62].
E—H Reporter gene expression driven by the fragments described above. Closed, yellow arrowheads in E and F mark presence of reporter gene expression, whereas open, yellow arrowheads in G and H mark absence of GFP expression. K Quantification of primary and secondary tumor formation in different genetic backgrounds. Only when Vvl function is reduced and apoptosis is simultaneously inhibited, tumors and metastasis develop.
Does this regulatory layout represent a general mechanism employed by other differentiation factors? This would require a whole suite of cell-type specifying transcription factors to repress cell death genes by interacting with distinct enhancer modules located in their regulatory regions. In addition, these modules should follow a similar functional logic to the rpr -HRE enhancer, in that cell-type specific gene activation is counteracted by strong repressing inputs from linked cis-elements Figure 1L—1O.
In line with this, we found that a different conserved enhancer module on the Drosophila rpr regulatory region rpr -HRE drove expression in CNS midline cells of stage 14 embryos Figure 6F , which never express rpr at this and subsequent developmental stages Figure 6L [29]. Using the JASPAR database [30] , we found consensus binding sequences for POU-domain containing transcription factors on the extended enhancer module, and one of these factors, Ventral veins lacking Vvl , is known to function in midline glial cells and to repress apoptosis [31] , [32].
Our analysis revealed a partial overlap of Vvl and reporter gene expression in rpr -HRE embryos Figure 6J , and consistently ectopic rpr transcripts were detected in several midline cells of vvl mutants Figure 6L, 6M. Revisiting the rpr -HRE enhancer module revealed that extension of the enhancer also led to a complete loss of reporter activity compare Figure 1L , Figure 6E, 6G. Importantly, the functional analogy of Vvl and Ct also extended to the tumor suppression activity, since, like in the case of Ct Figure 4N , primary and secondary tumor frequencies were increased when the ability to activate apoptosis and Vvl function was impaired at the same time Figure 6K.
Furthermore, we identified two unrelated cell-type specifying transcription factors in addition to Ct and Vvl, which showed similar behavior with regards to tumor suppression Figure S8. Together with the fact that the regulatory sequences flanking the Drosophila rpr coding region show significantly less sequence divergence than expected and a high occurrence of conserved transcription factor binding motifs Figure 6B, 6C , these findings lead us to propose that coupling of differentiation and cell death repression via a single transcription factor represents a general cancer prevention mechanism Figure 7 , which could be employed by a large number of developmental regulators in diverse organisms.
A During normal development, cell-type specification factors like Cut ensure the survival of cells by repressing apoptosis while at the same time these factors also induce a specific differentiation program, which generates cells with a specific terminal cell fate.
B In the case of a mutation in a cell-type specification factor those cells unable to differentiate, which are potentially harmful to the organism, are removed by releasing apoptosis repression conferred by the same cell-type specification factor. Thus, the transcriptional coupling of differentiation and apoptosis regulation represents a very fast and efficient cancer prevention mechanism. C Together with other mutations creating a sensitized background, like the over-activation of the Notch N signaling pathway, cells that acquire the inability to differentiate and a resistance to apoptosis activation, two important hallmarks of cancer [1] , [2] , very easily develop into cancer cells.
Programmed cell death is an integral aspect of animal development [33]. Genetic studies in C. In this context, apoptosis is usually regulated by cell signaling pathways [33] , [35] , [36]. In addition to its role in tissue morphogenesis, apoptosis is also required to eliminate potentially deleterious cells, which in most cases involves complex multi-step control mechanisms [33] , [37].
One such situation generating harmful cells is the inability to differentiate or adopt the appropriate cell fate, which very often results in uncontrolled cell proliferation and cancer development, and thus requires the immediate killing of these cells.
However, even though it is established that apoptosis is a protective mechanism against tumorigenesis in cases of aberrant cell differentiation [1] , [34] , [38] , the interplay of the two processes at the mechanistic level has remained unclear.
In our study, we show that the simultaneous and antagonistic regulation of differentiation and apoptosis is a hard-wired developmental program and carried out by individual transcription factors, such as Cut. Our results demonstrate that impairment of differentiation in the cell lineage specified by Cut instantaneously triggers locally restricted apoptosis by releasing transcriptional repression of the pro-apoptotic gene rpr in these cells. Due to its immediate effect, the coupling of differentiation and apoptosis on the transcriptional level represents one of the fastest and most direct mechanisms to eliminate abnormal cells in status nascendi and thereby immediately interferes with their potential to develop into harmful cells.
Interestingly, apoptosis induction as a consequence of aberrant cell-type specification is not only mediated by the cell death promoting gene rpr but also by hid [8]. However, despite the same trigger, which is the inability to properly differentiate, the transcriptional basis for inducing the expression of one of these two apoptosis genes seems to be quite different: in Drosophila early developmental mutants only the expression of the pro-apoptotic gene hid is up-regulated [8] , whereas our study shows that exclusively the transcription of rpr is induced when a factor specifying a distinct cell type is lost.
Although it is currently unknown how hid expression is regulated at the transcriptional level, this raises the possibility that the apoptosis gene hid acts a safeguard when broad positional information at the onset of embryogenesis is absent, whereas rpr might take over this function later in development when individual and specific cell types are defined by transcription factors restricting cell fate choices.
Given the well-known role of the vertebrate homologue of Cut, Cux1, in tumor initiation and progression in specific cancer types [23] , we addressed whether the switch function of the cell specification factor Cut is also relevant in a pathological context.
We found that simultaneous inhibition of Cut function and apoptosis within a sensitized background increases tumor formation and metastasis to secondary sites in the animal. In contrast, down-regulation of Cut and inhibition of apoptosis in a normal developmental context, such as in the Drosophila PS or the developing eye, only results in the survival of the Cut deprived cells, but not in tumor development.
These results demonstrate that cells, which are unable to undergo the cell lineage-specific differentiation program, have to be eliminated, since they have the potential to develop into cancerous cells when other genetic or micro-environmental changes accumulate [19] , [27] , [39]. But why do differentiation-deprived cells form tumors in a cancer-prone tissue environment despite the ability to activate the apoptotic rescue pathway?
This is due to the fact that the transcription factor Cut, as part of its selector gene function, coordinately regulates multiple cellular processes, including differentiation, apoptosis, cell adhesion, but also proliferation, which are all required for proper cell fate specification and the maintenance of a differentiated state thereby preventing tumor formation. If, however, Cut activity is abolished, all its downstream functions are affected, leading not only to the activation of apoptosis, but also to reduced differentiation and adhesion properties and the activation of cell proliferation, which is, in the case of Cut, mediated at least in part by the PI3K signaling pathway.
Thus, loss of Cut function stimulates tumor growth in a sensitized background, since the pro-tumorigenic effects of deregulated proliferation and cell adhesiveness out-compete the anti-tumorigenic apoptosis effects at work. However, when the anti-tumorigenic effect is eliminated in the differentiation-compromised cancer tissue, tumorigenesis is strongly enhanced, which resembles a prevalent situation in aggressive human cancers characterized by the loss of differentiation, the resistance to apoptosis activation and the mis-regulation of adhesion properties [1] , [40] , [41].
Several lines of evidence suggest that the dual role of Cut in differentiation and apoptosis for cancer prevention is conserved in evolution.
First of all, the two vertebrate homologues of Cut, Cux1 and Cux2, code for homeobox-containing transcription factors, which are crucially involved in cell-type specific terminal differentiation [14] , [23] , [42]. Both, Cux1 and Cux2, have similar binding specificities to Drosophila Cut [43] , they also operate as transcriptional repressors and activators of genes in multi-lineage differentiation pathways [26] and, like Drosophila Cut, they act as downstream effectors of the Notch signaling pathway [44] , [45].
In addition to their well-established role in development and differentiation, there are also several examples linking the vertebrate Cut homologue Cux1 to apoptosis and cancer.
First, inhibition or partial disruption of Cux1 function in mice leads to increased apoptosis rates in vivo [24] , [26]. Second, Cux1 regulates normal hematopoiesis, in part by modulating the levels of survival and apoptosis factors [26].
Third, Cux1 plays a prominent role in cancer progression [23]. And fourth, induced down-regulation of Cux1 in subcutaneous xenograft tumors leads to activation of apoptosis and to reduced tumor growth [25]. These processes - cell division and differentiation - are tightly regulated by many signals and signal pathways. Cell differentiation is incompletely understood, but it involves the activation or inactivation of certain genes in response to the cell's interactions with its neighboring cells and with its extracellular matrix ECM.
For example, receptors on the cell will bind to specific molecular elements in the ECM, and this binding activates intracellular signal transduction pathways that turn certain genes on or off. As a result of these interactions, some genes can be expressed in a given cell, but others cannot. For example, in a muscle cell, the genes that encode the contractile proteins actin and myosin are activated, but the gene encoding for insulin synthesis is inactivated.
Some cells, e. Terminally differentiated cells like these i. Cell-cell and cell-ECM interactions are important not only for the induction of differentiation, but also for maintenance of differentiation in some cell types.
One of the hallmarks of tumor cells is that they lose their ability to sense the ECM or neighboring cells. Many cells in an adult are not actively in the process of replicating; this is depicted in the diagram as "cells that cease division," also known as the G 0 phase or the "resting phase.
If conditions require additional cells, the cell will receive signals that promote cell division. These signals will push the cell to complete the G 1 phase cell enlargement and proceed to the S-phase, during which DNA is replicated. In the G 2 phase the cell prepares for division by increasing in size and replicating intracellular organelles. It then divides through mitosis the M-phase. In a sense, the critical juncture is the transition from G 1 to the S-phase.
This transition is carefully regulated by multiple factors, some of which promote the transition. Genes known as proto-oncogenes can be switched on to produce proteins that protein the transition to the S-phase. Counteracting this push to reproduce are genes known as anti-oncogenes also called tumor suppressor genes that inhibit transition to the S-phase.
The video below provides a short visual summary of these events, known as the cell cycle. The two videos below summarize the signaling events that regulate the cell cycle and events occurring during the cell cycle.
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