**2.1. Morphological and biochemical features**

Morphological features that share both caspase-dependent and independent apoptotic pathways are: a) loss of plasma membrane symmetry and loss of cell-to-cell contact and cell round-up, b) chromatin condensation (picnosis) and fragmentation and nuclear breakdown, c) overall cell shrinkage and cytoskeleton alterations although the majority of the cell organelles remain intact, d) membrane blebbing and formation of the membrane-enclosed particles called apoptotic bodies that contain nuclear or cytoplasmic material and will be engulfed by phagocytes or neighbouring cells [6].

68 Apoptosis and Medicine

et al. 2012)

be prevalent.

**2. Apoptosis** 

**2.1. Morphological and biochemical features** 

accordance with to the NCCD are described:

refers to a suicidal type of death. However, as more mechanisms of programmed cell death are being elucidated, the Nomenclature Committee on Cell Death (NCCD) recommends caution when using the term apoptosis [5]. Historically, the classical methods to define cell death rely on morphological criteria. Thus, apoptosis was termed programmed cell death type I, autophagy named as programmed cell death type II and necrosis as a type of death lacking characteristics of both type I and type II. As more biochemical methods become available over the last decades, a more definite and concise classification of types of cell death has become necessary. Therefore, cell death types can be classified according to morphological appearance, biochemical features, functional criteria or immunological aspects. In the following figure (Figure 1) the classification of different types of cell death, in

**Figure 1.** Classification of different types of cell death according to the NCCD (modified from Galluzzi

It is important to mention that a single stimulus can trigger more than one mechanism of cell death simultaneously within a cell. However, only the most efficacious mechanism will

Morphological features that share both caspase-dependent and independent apoptotic pathways are: a) loss of plasma membrane symmetry and loss of cell-to-cell contact and cell Because apoptotic cells are eaten so quickly, there are few dead cells left on tissue sections. This is the reason why apoptosis was neglected by pathologists for a long time, even though apoptosis is the main mechanism for discarding of harmful or unwanted cells in multicellular organisms.

Along with the morphological transformation described, there are several biochemical alterations that take place. For instance, activation of endonucleases, some of them dependent on Ca2+ and Mg2+ channels, that cleaves genomic DNA. In the apoptotic process, this event gives rise to internucleosomal DNA double-strand breaks with fragments of multiples of 180 bp, resulting in the typical pattern of *DNA ladder* that can be detected by electrophoresis on agarose gels. Other changes include loss of the inner mitochondrial transmembrane potential [7] and exposure of the phospholipid phosphatidylserine to the outer cell membrane, which allows phagocytes to detect and engulf these apoptotic cells [8].

**Figure 2.** Features of Apoptosis, Oncosis and Necrosis (taken from Hail et al., 2006)

The most distinguishable feature of apoptosis is the formation of apoptotic bodies. On the other hand, oncosis is characterized by cytoplasmic swelling, dilation of organelles and vacuolization and plasma membrane blebbing. The cell will finally die by cytolysis, which is a typical hallmark of necrosis [9]. Apoptotic cells can also lose their plasma membrane and eventually undergo a secondary necrosis. However, this phenomenon has only been observed *in vitro*. According to Majno and Joris, necrosis is not a type of cell death, but

rather, it refers to changes subsequent to any type of death. They prefer to use the term oncosis to describe a nonprogrammed or accidental type of cell death characterized by swelling [10]. Moreover, a new modality of cell death has recently emerged, termed **necroptosis** to indicate a regulated form of necrosis [11].

Cell Death and Cancer, Novel Therapeutic Strategies 71

**Substrate Funtion**

DNA damage repair

Cellular structure

**PARP, DSBs, Rad51 and Rad9, DNA-**

**Actin, Gas2, FAK, PAK2, Fodrin and** 

**C1, C2 and U170** mRNA processing

**Phosphatase 2A, Raf1 and Rb** Cell cycle

**Pro-IL-1β** Cytokine

**PKC and Akt** Apoptosis and cell cycle

**Bid, Bcl-2, Bcl-xL and XIAP** Apoptosis regulation

**D4-GDP Inhibitor** Rho GTPases regulator **Laminin A/C and B and NuMA** Nuclear shape maintenance

**ICAD** DNA fragmentation **Huntingtin** Huntington disease

**MDM2** Transcription factor

**4. Extrinsic and intrinsic apoptotic pathways** 

**Table 1.** Proteins substrates of caspases (Blank and Shiloh 2007; Cohen et al 1997)

**ACINUS** Regulation of chromatin condensation

**SREBP-1 and SREBP-2** Sterol regulatory element binding proteins

Caspase activity can be regulated both in a positive and in a negative manner. Negative caspase regulators are called Inhibitors of Apoptosis Proteins (IAPs) that bind to the catalytic site of caspases neutralizing its activity [19], or targeting them to degradation by ubiquinitation [20,21]. Some examples of IAPs are: XIAP, c-IAP1, c-IAP2, NAIP and survivin. On the other hand, Smac/DIABLO and Omi/HtrA2 act as positive regulators by

There are at least two main well characterized apoptotic routes: the death receptor or extrinsic pathway and the mitochondrial or intrinsic pathway. The extrinsic pathway plays a major role in tissue homeostasis and responds to external cues, coming especially from the immune system, whereas the intrinsic pathway is triggered as a response to various internal insults such as DNA damage, cytosolic calcium overload, starvation, oxidative stress,

The death receptor pathway is initiated by extracellular stimuli that are recognized by a subgroup of the tumour necrosis factor receptor (TNF-R) family named dead receptors (Fas/CD95/APO-1, TNFR1, TRAIL R1/DR4 and TRAIL R2/DR5). Upon binding of their ligands (FAS, TNFα and TRAIL) these receptors become activated and interact via their death domain with the protein motif **F**as-**a**ssociated **d**eath **d**omain (FADD) in adapter proteins, forming the **D**eath **i**nducing **s**ignalling **c**omplex (DISC), which binds to the prodomain of the initiator caspase-8 [25]. Thus, caspase-8 is activated by dimerization,

radiation, cytotoxic agents, etc., and involves mitochondrial destabilization [23,24].

**PKCs and ATM** 

inactivating IAPs [22].

**Gelsolin** 

Apoptosis can be divided into three stages: initiation, integration/decision and execution/degradation [12]. The initiation phase mainly depends upon cell type and apoptotic stimulus. The integration/decision phase consists of the activation of proteases, nucleases and other effector molecules. The execution/degradation phase involves morphological and biochemical changes that are common to all apoptotic mechanisms, regardless of the stimulus that initiated the process. The classical apoptosis is defined as a type of programmed cell death characterized by the activation of zymogens known as caspases, which are **c**ystein-dependent **asp**artate-directed proteases. Both caspasedependent and caspase-independent cell death mechanisms share multiple characteristics, such as mitochondrial membrane permeabilization (MMP), DNA fragmentation, etc. Hence, (MMP) can commit cells to die even in the absence of caspase activation, by releasing factors such as apoptosis inducing-factor (AIF) or endonuclease G (endo G).
