© 1999 Robert A. Freitas Jr. All Rights Reserved.

Robert A. Freitas Jr., Nanomedicine, Volume I: Basic Capabilities, Landes Bioscience, Georgetown, TX, 1999

10.4.1.1 Apoptosis

It is believed that all nucleated (eukaryotic) cells of all multicellular life on Earth incorporate an evolutionarily conserved self-destruct mechanism called programmed cell death, cell suicide, or simply apoptosis. Scattered reports on cell death have appeared in the literature (and were initially disbelieved) for more than a century, but advances in cytochemistry in the 1980s and early 1990s stimulated an explosion of interest in apoptosis, with ~2500 papers published during 1989-1994²⁰⁶² and ~20,000 publications during 1994-1998.²⁰⁶⁵

Exactly what is apoptosis? Programmed cell death is the outcome of a programmed intracellular cascade of genetically determined steps. During apoptosis, the eukaryotic cell disassembles its DNA and breaks up its contents into membrane-wrapped packets which can then be cleared away without causing inflammation. Animals use apoptosis to eliminate extraneous, virus-infected, or otherwise dangerous cells.²⁰⁷⁵ Apoptosis plays a central role both in development and in homeostasis of metazoans. Cells die by apoptosis in the developing embryo during morphogenesis or synaptogenesis, and in the adult animal during tissue turnover in the skin or gut or at the end of an immune response. Tight coupling of cell death and cell multiplication ensures in some tissues a constant, controlled flux of fresh cells which are crucial to the preservation and optimal functioning of the adult organism.²⁰⁶⁵ Such tissues include those which are environmentally exposed, inside or outside, primarily epithelial tissue and severely insulted liver tissue, plus cells involved in reproductive function.

Apoptosis requires specialized machinery. The central component of this machinery is an irreversible proteolytic system involving a family of protein-chopping enzymes now called "caspases" from "cysteine-containing aspartate-specific proteases" (aka. ICE or Interleukin-1b-Converting Enzyme). Caspases are expressed as 30-50 kD proenzymes with an NH₂-terminal domain, a large ~20 kD subunit, and a small ~10 kD subunit.²⁰⁶⁸ These enzymes participate in a cascade that is triggered in response to proapoptotic signals and culminates in cleavage of a set of proteins, ultimately resulting in an orderly disassembly of the cell.²⁰⁶⁸

Such complex proteolytic systems involve a combination of regulatory proteases, cofactors, feedbacks, and thresholds that converge to control the activity of an effector protease, which in turn carries out the function of the whole process.²⁰⁷¹ These systems keep the effector protease inactive but are able to rapidly activate large amounts of it in response to minute quantities of an appropriate inducer.²⁰⁶⁸ Survival signals from the cell's environment and internal cellular integrity sensors normally hold a cell's apoptotic machinery in check.

Four kinds of events can trigger a suicidal cascade in a eukaryote.²⁰⁶⁹ First, if a cell loses its normal contact with its surroundings or sustains irreparable internal damage, then that cell initiates apoptosis. Second, a cell that simultaneously receives conflicting signals driving or attenuating its division cycle also undertakes apoptosis.²⁰⁷⁴ Third, the immune system can actively direct individual cells to self-destruct, an event called "instructive" apoptosis.^2076-2078 Fourth, perhaps a majority of all apoptosis occurs during gestation and development, probably in response to external signal molecules or to a lack of sufficient levels of molecular signal gradients involved in biostructural development regulation.

Death receptors²⁰⁶⁹ -- cell surface receptors that transmit apoptosis signals initiated by specific death ligands -- play a central role in instructive apoptosis. Death receptors can activate death caspases within seconds of ligand binding, causing the apoptotic demise of the cell within hours.²⁰⁶⁹ Death receptors belong to the tumor necrosis factor (TNF) receptor gene superfamily. (TNF is produced mainly by activated macrophages and T cells in response to an infection.²⁰⁷⁹) The death receptors contain a homologous cytoplasmic sequence termed the "death domain."^2080,2081 Death domains enable death receptors to engage the cell's apoptotic machinery, and mediate functions that are distinct from or even counteract apoptosis.²⁰⁶⁹ In 1998, the best characterized death receptors were CD95 (aka. Fas²⁰⁶⁶ or Apo1) and CD120a (aka. TNFR1, Tumor Necrosis Factor Receptor 1, or p55), but others were known including avian CAR1, Death Receptor 3 (DR3; aka. Apo3, WSL-1, TRAMP, LARD), DR4, and DR5 (aka. Apo2, TRAIL-R2, TRICK-2, KILLER).²⁰⁶⁹

When a caspase cascade is triggered, the first task is to inactivate proteins that protect living cells from apoptosis.²⁰⁶⁸ This accomplished, caspases next begin the direct disassembly of cell structure. One example is the destruction of the nuclear lamina (Section 8.5.4.3), the rigid structure underlying the nuclear membrane that is involved in chromatin organization. Lamina is formed by head-to-tail polymers of intermediate filament proteins called lamins (Section 8.5.3.11). During apoptosis, lamins are cleaved at a single site by caspases, causing the lamina to collapse, contributing to chromatin condensation.^2082,2083

Caspases also reorganize cell structures indirectly by cleaving several proteins involved in cytoskeletal regulation, including gelsolin,²⁰⁸⁴ focal adhesion kinase (FAK),²⁰⁸⁵ and p21-activated kinase 2 (PAK2).²⁰⁸⁶ Cleavage of these proteins results in deregulation of their activity.²⁰⁶⁸ Dissociation of regulatory and effector domains is another hallmark of caspase function. For example, they inactivate or deregulate proteins involved in DNA repair (such as DNA-PK_cs), mRNA splicing (such as U1-70K), and DNA replication (such as replication factor C).^2087,2088

In 1998, 13 mammalian caspases -- named caspase-1 to caspase-13 -- were known.^2067,2068 Caspases cut their substrate proteins at tetrapeptide sites with high specificity and with characteristic motifs.²⁰⁸⁹ For example, caspase-3 recognizes DEVD sequences and, during apoptosis, cleaves and inactivates several significant cellular proteins in the cytosol, nucleus and cytoskeleton.^2090-2092 Caspases participate in apoptosis "in a manner reminiscent of a well-planned and executed military operation;"²⁰⁶⁸ they:

1. cut off contacts with surrounding cells, so the cell balls up;

2. import calcium,²⁰⁷⁵ strongly complexing phosphates;

3. shut down DNA replication and repair, interrupt splicing, disrupt the nuclear structure and condense the chromatin;

4. induce the loss of microvilli;²⁰⁹³

5. destroy cellular DNA by mobilizing apoptotic-unique nucleases^2091,2092 to cleave the double helix at regular intervals in an orderly fashion, first into large fragments of 300-750 kilobases, followed by fragments of ~50 kilobases, then finally oligonucleosomal length DNA fragments of ~200 base pairs^2062-2064 (giving a characteristic pattern by gel electrophoresis called "DNA laddering"²⁰⁹⁴);

6. reorganize the cytoskeleton;

7. induce the cell to display signals in the outer membrane that mark the cell for phagocytosis by neighboring cells or macrophages;²⁰⁹⁵

8. induce downstream blocking of mitochondrial respiratory chain components, resulting in mitochondrial malfunction (via transcriptional induction of redox related genes, the formation of reactive oxygen species, and finally the oxidative degradation of mitochondrial components^2072,2073 including dissipation of mitochondrial inner transmembrane potential and the release of cytochrome c through the outer mitochondrial membrane²¹⁶²), with lysosomal involvement;²²⁹³

9. initiate membrane blebbing (surface blisters) and cell shrinkage; and finally

10. disassemble the cell into multiple membrane-enclosed vesicles called apoptotic bodies without destroying organelle membranes.

In vivo, this process culminates with the engulfment and phagocytization of apoptotic bodies by other cells, preventing inflammation and other complications that would result from a release of intracellular contents. These changes occur in a predictable, reproducible sequence and can be completed within 30-60 minutes.²⁰⁶⁸

Many different ways of activating the apoptotic process are known or suspected.²⁰⁹⁶ Simple detachment of tissue cells from all contacts with the ECM¹⁵⁵³ or manipulation of cell shape⁷¹⁸ have been shown to induce apoptosis experimentally. Apoptosis can also be indirectly initiated by a variety of cellular insults that can damage DNA, including ultraviolet and X-irradiation, hypoxia, and chemotherapeutic neoplastic drugs (e.g., alkylating agents such as nitrogen mustard derivatives, antimetabolites and plant alkaloids) which inflict cell damage that is then translated via the Bcl-2 protein family²⁰⁹⁷ through several poorly understood steps into activation of caspase-9.²⁰⁶⁸ Proteins that sense DNA damage and help trigger apoptosis also affect the cell cycle -- stopping cell division so the damage can be repaired or making the decision (with the activation of tumor suppressor p53) that the damage has gone too far and the cell must die.²⁰⁷⁴ B and T lymphocytes undergo apoptosis in response to anti-IgM antibodies and dexamethasone (a glucocorticoid), respectively.²⁰⁹⁸ The cell death program can also be activated by inhibition of proteasome function,²⁰⁹⁹ or by infection with a wide variety of viruses.²¹¹⁸

Caspases can be directly activated by two distinct intracellular mechanisms that interact with the death receptor complexes. First, because all caspases have similar cleavage specificity, the simplest way to activate a procaspase is to expose it to a previously activated caspase molecule. This caspase cascade is used extensively by cells for the activation of the downstream effector caspases: caspase-3, caspase-6, and caspase-7.²⁰⁶⁸

A second method for activating caspases is the use of apoptotic chaperones, which herd together inactive proenzymes to increase their local concentration and ease them into conformations that promote their activation. This "induced proximity" was first observed in caspase-8 (aka. FLICE, MACH), an initiator caspase that acts downstream of the CD95 death receptor.²⁰⁶⁹ Upon trimeric ligand binding, CD95 receptor molecules are aggregated into a membrane-bound complex. This signaling complex recruits, via the receptor-bound adaptor protein FADD (Fas-Associated protein with Death Domain; aka. Mort-1²¹⁰⁰), several procaspase-8 molecules, resulting in a high local concentration of procaspase-8. Under these conditions, the low protease activity inherent to procaspases is sufficient to drive intermolecular proteolytic activation of the receptor-associated procaspase-8 molecules.²⁰⁶⁸ Proximity-induced activation may also be used to activate caspase-9.²¹⁰¹ Procaspase-9 activation involves a complex with the cofactor Apaf-1 through the CARD (Caspase Recruitment Domain), but activation of caspase-9 also requires cytochrome c (released into the cytosol by mitochondria²⁰⁷³) and deoxyadenosine triphosphate, indicating that caspase activation may require multiple cofactors.

Death receptors can be expressed on both normal and cancerous cells in the human body, so the challenge for conventional drug-based therapy is to find some way to activate death receptors selectively on cancer cells only.^2068,2069 For medical nanorobots, such selectivity should be simple and routine using multiple chemosensors (Section 4.2), a benefit that is characteristic of most nanorobot-based therapeutics. If caspase cascade amplification is sufficient to permit single-site activation of the cascade, then in theory an extracellular nanorobot intending cytocide could press onto the outer surface of a target cell an appropriate ligand display tool. This tool might contain suitably exposed trimeric CD95L (aka. FasL²⁰⁶⁶) ligand (binds to the extracellular domains of three CD95 death receptors), TNF or lymphotoxin a (binds to CD120a), Apo3L ligand aka. TWEAK (binds to DR3), or Apo2L ligand aka. TRAIL (binds to DR4 and DR5).^2069,2070 The binding event would then activate a single death receptor complex, potentially triggering the entire irreversible cytocidal cascade. If necessary, multiple display tools could be employed. This technique avoids the storage requirement for bulky consumables onboard the medical nanorobot.

As another approach, molecular sorting rotors could be used to selectively extract from the cytoplasm specific crucial molecular species of IAPs (Inhibitors of Apoptosis²¹⁰²) that can hold the apoptotic process in check. Examples include survivin, commonly found in human cancer cells,²⁰⁵⁸ the transcription factor NF-kB,²¹⁴² and Akt, which delivers a survival signal that inhibits the apoptosis induced by growth factor withdrawal in neurons, fibroblasts, and lymphoid cells.²¹⁰³ Conversely, decoy receptors (DcRs) that compete with DR4 and DR5 for binding to Apo2L^2104-2106 could be saturated with intrinsically harmless but precisely engineered intracellular "chaff" ligands. With IAPs removed or DcRs blockaded, apoptosis may be free to proceed.

Multicellular plants and animals,^2074,2075 human fibroblasts,²¹⁰⁷ and even slime molds show various forms of apoptotic cell death. Other processes of programmed cell death that may be distinct from either apoptosis or necrosis have been reported in lung fibroblasts,²¹⁰⁸ the caspase-free yeast Saccharomyces cerevisiae,²⁰⁹⁷ and other cells.^2109-2112 In 1998, apoptosis had not yet been reported in any bacterial species, nor was it expected because conventional evolutionary logic does not favor the emergence of cell suicide in the case of obligate single-celled organisms.^2073,2074 Autolysis has been reported in bacteria in specialized circumstances,^{2113-2115,3704} including addiction modules^3318-3320 and phage exclusion,^3320-3322 though these cases, while sometimes termed "programmed cell death,"^{2113,3705-3709} all involve a (messy) necrotic cytocidal outcome or self-damage short of cell death, rather than a (clean) apoptotic cytocidal outcome. Apoptosis also is not found in viruses.

It is theoretically possible that an artificial plasmid could be designed that embodies an engineered self-contained apoptotic system capable of unleashing a non-necrotic cell death process inside a prokaryote. A single such proapoptotic artificial plasmid (similar to artificial chromosomes,²⁴³² already in use by 1998) could then be injected into the target bacterium, giving the desired clean cytocidal outcome. R. Bradbury suggests the following useful components:

1. a restriction enzyme to fragment the bacterial DNA, that is effective against that specific bacteria;

2. DNase to digest the DNA entirely, if not already present;

3. RNase to digest the RNA entirely; if not already present;

4. one or more proteases such as trypsin, to digest internal proteins; and

5. one or more lipases to digest any lipids.

A lambda-phage container holding an engineered vector with only the first two capabilities could still effectively "neuter" the bacterium. Such a biorobot could be relatively harmless to eukaryotic cells, assuming its enzymes lacked nuclear localization targeting sequences; cleanup could be effectively handled by the immune system. Bradbury also suggests another approach -- using a DNA replication blocker such as antisense DNA polymerase, in combination with a mitotic promoter, to induce the cell to attempt to divide forever while lacking any DNA in the progeny. Essential biomolecules would eventually be diluted to such a low level that growth would radically slow and perhaps even death would result. This second approach might yield a faster decline in bacterial activity than simple neutering, due to the diversion of bacterial resources in an attempt to regain intracellular molecular balances. In either approach (as with mechanical approaches; Section 10.4.2), it is necessary to avoid fragmenting the bacterium in a way which allows release of superantigen molecules such as LPS which could cause an immune system overreaction, leading to toxic shock.

Last updated on 24 February 2003