The cell cycle

The actual mechanics of cell division, according to Dick McIntosh at the University of Denver, require significantly more instructions than it takes to build a moon rocket or supercomputer. First of all, the cell needs to duplicate all of its molecules, that is DNA, RNA, proteins, lipids, etc. At the organelle level, several hundred mitochondria, large areas of ER, new Golgi bodies, cytoskeletal structures, and ribosomes by the million all need to be duplicated so that the daughter cells have enough resources to grow and, in turn, divide themselves. All these processes make up the ‘cell cycle’. Some cells will divide on a daily basis, others live for decades without dividing. The cell cycle is divided into phases, starting with interphase, the period between cell divisions (about 23 hours), and mitosis (M phase), the actual process of separating the original into two daughter cells (about 1 hour). Interphase is further split into three distinct periods: gap 1 (G1, 4–6 hours), a synthesis phase (S, 12 hours), and gap 2 (G2, 4–6 hours). Generally, cells continue to grow throughout interphase, but DNA replication is restricted to the S phase. At the end of G1 there is a checkpoint. If nutrient and energy levels are insufficient for DNA synthesis, the cell is diverted into a phase called G0. In 2001 Tim Hunt, Paul Nurse, and Leeland Hartwell received the Nobel Prize for their work in discovering how the cell cycle is controlled.

Tim Hunt found a set of proteins called cyclins, which accumulate during specific stages of the cell cycle. Once the right level is reached, the cell is ‘allowed’ to progress to the next stage and the cyclins are destroyed. Cyclins then start to build up again, keeping a score of the progress at each point of the cycle, and only allowing progression to the next stage if the correct cyclin level has been reached.

Mitosis

Once the cell is ready to divide, it enters the part of the cell cycle called mitosis (M phase) which is broken down into five further phases: prophase, prometaphase, metaphase, anaphase, and telophase. The first is prophase, during which the chromosomes condense to become discrete structures. In prometaphase the nuclear membrane breaks down and the nucleoli become indistinct.

The chromosomes now become further compacted, coiling and supercoiling as they condense into clearly visible paired sausagelike structures (see Figure 8c, d, e), each made of two chromatids, joined together by a structure called the centromere. The centromere provides the site of attachment to the mitotic spindle (the microtubular framework which brings about the separation of chromosome to the daughter cells) at a structure called the kinetochore. The mitotic spindle is formed from cytoplasmic microtubules, organized by a pair of centrioles which have previously replicated and migrated to either end of the cell (as described in Chapter 2). This stage completes prometaphase, which is followed by metaphase, where the mitotic spindle microtubules apply tension to the chromosomes to align them in the centre of the spindle itself, at which point they form the ‘metaphase plate’.

The next stage is anaphase, in which one chromatid from each chromosome is separated and moved to opposite ends of the mitotic spindle ( Figure　9 ). This is brought about by a shortening of the microtubules that run from the kinetochores to the spindle poles, and a lengthening of the microtubules that run from one end of the spindle to the other, brought about by adjacent tubules sliding over one another. The arrival of each group of chromatids at the poles of the spindle completes the last stage of mitosis, called telophase. To complete division of the whole cell into two daughter cells, a contractile ring of actin filaments forms around the middle of the cell, pinching the cytoplasm into two halves rather like a belt pulling tighter and tighter, a process called cytokinesis ( Figure　9b ). Once the daughter cells have separated their chromosomes will begin to relax, decondensing to their interphase confi guration as part of the newly formed nucleus. A new nuclear envelope is formed at the surface of the decondensing chromosomes, which already carry the building blocks for new nuclear pores on their surfaces. Mitosis is now complete, and the cell enters interphase again, either to begin a process of further redoubling for the next division, or to leave the cell cycle (G0) and embark on a journey of differentiation to fulfll a specialized tissue function, as will be described in Chapter 5.

9. Cell division. (a) Section through cell during anaphase with a set of chromatids pulled towards each pole by the mitotic spindle, (b) two daughter cells beginning to separate at the end of division, ‘pinching off’ from each other with a central furrow

Meiosis

Meiosis is a reduction division, used by multicellular organisms to produce special cells (gametes) which have a single copy of DNA (haploid), in readiness for fusion with another gamete to produce a cell (zygote) with the normal two copies of DNA (diploid), from which the embryo will develop into a new organism. Gametes can be sperm and eggs as in most animals, pollen and ovules in plants, and spores in other life forms such as fungi. The creation of a new organism by fusion of gametes from two different organisms is defined as sexual reproduction. Plants, of course, are not as dependent on this process as are the majority of animals, having ways of asexual reproduction open to them. It is beyond the scope of this book to delve too deeply into the genetic consequences of sexual reproduction, but suffice to say that the constant mixing of genes brought about in this manner provides the variety which upon which the pressures of natural selection drive evolution.

The mechanics of meiosis are relatively simple: two rounds of chromosome division are completed without a round of DNA replication in between. By dividing a diploid cell twice, four haploid gametes are produced. Meiosis begins with the two matching (homologous) chromosomes (one maternal, one paternal) in a diploid cell coming together, at which point DNA may be ‘swapped’ in a process called crossing over. The first division separates one of each pair of chromosomes to two new daughter cells, which then divide directly producing four gametes which now have half the original DNA complement (haploid). The molecular mechanics of chromosome separation via the microtubules of the spindle in meiosis are pretty much the same as they are in mitosis. Numerically, sperm production considerably outstrips egg production, as a fertile human male produces 1000 sperm in the same time as a single heartbeat, whereas human females are born with around 500 eggs to last a lifetime.

DNA replication

Before a cell can divide, it must produce two copies of its DNA, one for each daughter cell. The two strands of DNA from the mother cell are separated, and copies are made using the original strands of DNA as a template. Remembering that the nucleotide base A always pairs with T and C with G, if one strand has a sequence ATCG then the new strand will have a sequence TAGC. The opposite old strand is TAGC and its daughter strand is ATCG. In this way, two identical copies of a DNA sequence are made. This is called semi-conservative replication, and normally provides an exact copy. Any mistakes in copying generate mutations, leading to alterations in the genetic message which are passed to the daughter cells. DNA is made in a single uninterrupted burst that takes around a third of the time that a new cell needs before it divides again (cell cycle). In bacteria, the cell cycle may last only a few minutes, and a couple of hours in simple eukaryotes such as yeast. In contrast, most mammalian cell cycles are around 24 hours. Only a small proportion of cells in the human body divide daily. For example, we manufacture a new layer of skin cells every day, and reline the surface of our intestines continuously, but some nerve cells will last a lifetime. When DNA replication is required, it takes place in around 100 ‘replication factories’ distributed throughout the nucleus. DNA is fed in to the replication machinery like film into a projector, and emerges as two films. Exactly what happens at the molecular level could fill a book twice the size of this one, but here is a brief outline of the overall process of the transmission of genetic information between cells.

The first requirement is to separate the two strands of the DNA helix to provide templates for the formation of new strands, one on each half of the original DNA. This is (relatively) simple in prokaryotes where the DNA lies naked in the cell. In eukaryotes, it would take far too long to replicate the DNA starting at one end only, so that the DNA is opened up by an enzyme called helicase at around 1000 different sites along its length. If you consider the topology of this process using pieces of twisted string, it requires one strand of the continuous helix to be cut before a short stretch of double stranded DNA can be unwound. Then the main enzyme of replication (DNA polymerase) locks on to the opened DNA strands at these ‘replication forks’ and adds new nucleotide bases (in the correct order) to the new strands at a rate of 100 bases per second ( Figure　10 ). Bacteria can do this at even greater rates—up to 1000 bases per second. Despite the rapid rate, replication is extremely accurate, with enzymes that proofread and correct any mismatched nucleotide, usually leaving only one error in every billion nucleotides.

Transcription

Transcription is the first step in the process by which genetic information generates new proteins. One strand of the DNA (known as the coding strand) is used as a template to make an RNA sequence (messenger RNA or mRNA;　Figure　11 ). This messenger RNA will then be used as a template for protein production.

10. DNA replication. (a) A replication fork. Helicase unwinds the double stranded DNA so that each strand can be copied. DNA polymerase enzymes, one each strand, assemble the complementary strands working in opposite directions (arrows). In bacteria, the DNA strands are continuous and circular, so replication starts at one point and continues round to complete the entire DNA molecule

In a similar way to replication, RNA is synthesized along the DNA template but with RNA polymerase rather than DNA polymerase. Messenger RNA also uses a different nucleotide base, uracil (U), instead of the thymine (T) in DNA. Before the newly transcribed RNA passes out of the nucleus, the leading end is capped, and a tail is fixed to the trailing end. The parts of the copied DNA sequence that do not code for protein are removed by a process called RNA splicing. The new mRNA is then tagged with proteins that will target it to a nuclear pore prior to passage into the cytoplasm, where it will combine with a ribosome to form the machinery for making a new protein.

(b) In animal cells replication is started simultaneously at many points.At the top (1) is double stranded DNA (grey strand, black strand) showing the places or origins where replication starts (X). DNA polymerases begin to copy both strands in the direction indicated by the arrows (2, 3). A number of DNA polymerase enzymes replicate each strand resulting in fragments of the newly synthesized DNA strand (3). As the replication bubbles grow these fragments are linked together and checked for accuracy to make two exact copies of the starting DNA (4)

11. RNA polymerase is a large complex of proteins, which binds to specific sites at the start of genes. It unwinds the DNA helix and copies one strand to give a complementary RNA strand. The sugar molecules in RNA are different to those found in DNA. Three of the four bases (A, C, G) are the same but T is substituted for U. As the RNA polymerase moves along the DNA, its strands recombine to form a helix

Although the mechanics of transcription are understood, how genes are selected for transcription is less clear. The phenomenon of ‘gene expression’ has occupied thousands of molecular biologists for many decades. Basic genes required to maintain the cell in good working order (housekeeping genes) are always expressed but many other genes will only be required at specific times during the life of the organism. Some genes, such as the two globin proteins that make up haemoglobin, are required in such amounts that red cells switch to over 90% globin production. The cell can also switch genes on and off in a very complex pattern and the production or non-production of proteins and enzymes is the cell’s main way of responding to any change of circumstance. We have around 200 different cell types in our bodies, all with specialized roles. These differentiated cells result from different genes being switched on or off in different tissues. Cells also have to be able to respond rapidly to external changes by switching genes on and off. These switches are controlled by a group of around 3000 different proteins called transcription factors. Some genes require many factors, others only a few. Transcription factors reside in the cytoplasm and must enter the nucleus to access their genes. Transcription factors required for rapid responses shuttle in and out of the nuclear pores, maintaining a constant state of readiness.

How cells move

Much of what we know of the mechanisms of how a cell moves comes from watching fibroblast cells in tissue culture. The cell moves by extending a broad leading edge, a lamellipodium (see Figure 3c) which moves by a cycle of attachment and detachment of the underside membrane to underlying surface, rather like the incoming tide moving up the beach. The upper surface of the lamellipodium has ruffles, active waves of folded membrane, which flow towards the rear of the cell. All this activity is generated under the cell membrane by the addition of subunits to the front ends of a branching network of actin filaments. At the rear of the cell, the actin filaments break down, ready to be recycled to the front of the lamellipodium. Finger-like projections termed filopodia (similar to microvilli) ‘feel out’ gaps between cells, allowing fibroblasts to move through solid tissue. This migration through tissues is entirely normal for both fibroblasts and white blood cells as they go about their normal duties of cell maintenance and immune defence, but at the same time represents the major problem in cancer. Tumours are initiated by a local loss of control of cell division. If the mass of newly divided cells stays put, then the resulting tumour is benign and can usually be successfully removed by surgery or killed by radiation therapy. The biggest problem with cancer is metastasis, where cells dissociate from the primary tumour, penetrate the surrounding tissue, and ultimately access the bloodstream, from which point they can generate secondary tumours virtually anywhere in the body. An understanding of exactly how cells (both normal and tumour) move through tissue barriers is the first step to find treatments which could inhibit metastasis, and new drugs which could stop the spread of cancer cell away from the original (primary) site. In the last few years, some progress has been made in identifying both genes that are active in metastasis (unsurprisingly those that are also involved in cell migration) and inhibitors of their mechanisms. Compounds from such diverse sources as citrus peel (modified citrus pectin) and olive oil (oleamides) have shown anti-metastatic properties.

Movement, an emergent property?

René Dutrochet, one of the pioneers of cell biology, remarked in 1824 that ‘life, as far as physical order is concerned, is nothing more than movement; and death is simply the cessation of this movement’. Nearly 200 years later, just how cells combine the various components of the cytoskeleton to function as a single moving entity is still a bit of a mystery. Cell movement requires biochemical cues (signals), an energy supply, and the reorganization of structural elements. Cytoskeletal elements need to grow and shrink and to arrange themselves for action, but when, with how much force, and when to stop? Acccording to Guenter Albrecht-Buehler from Northwestern University in Chicago, a cell biologist studying the behaviour of cells in culture, ‘the functions of the organism initiate and control the interactions between its molecules’—which is a way of saying the whole is greater than the sum of the parts. Something that happens as a result of the interaction of many complex systems is called an emergent property. In nature, the classic examples come from social insects, such as the massive cathedral-like structures produced by termite colonies, or even the production of a honeycomb. The movement of a cell could be considered an emergent property of the molecules of the cytoskeleton, supported by energy production from mitochondria, and information stored in DNA. Thus it seems feasible that the millions of cells within a tissue produce emergent properties to fulfll the purpose of the tissue, and then add another level as tissues form organs, and yet further levels as an entire organism. This view may go some way to accounting for (if not exactly explaining) the complexities of our own existence.

Is cell movement completely random?

After 30 years examining the behaviour of individual cells in culture, and recording an enormous amount of time lapse footage, the studies of Albrecht-Buehler have produced some fascinating observations which may suggest that cells both require and indeed possess ‘intelligence’ of their surroundings to act as they do. The idea that a cell ‘knows’ where it is going is reinforced by their behaviour when they meet other cells and then, after contact, move off in opposite directions. This shows a ‘choice’ within the cell in reaction to an unexpected event in their migration.Albrecht-Buehler’s published observations of the behaviour of cells in a culture dish appear to indicate directed movement and the apparent appreciation of other cells at a distance of several cell diameters. Albrecht-Buehler terms this ‘cell intelligence’, and points out that this behaviour requires incoming information, which he suggests may involve infrared light. He suggests that cells can signal to each other over a distance of several cell diameters by the emission and reception of infrared light. In the absence of any great body of work in this area of research, it is hard to assess the true significance of these studies, but they are extremely intriguing. In his own words, Albrecht-Buehler suggests that ‘cell behaviour is controlled by very complex data integration systems that are, so far, unknown to biology’.

If what controls cell movement in the relative simplicity of a plastic dish is complex, then so is cell movement within the human body. Blood cells are pumped around the circulation to provide oxygenation of tissues by red cells and to maintain immune surveillance with a variety of white blood cells called leucocytes. To reach sites of injury or infection, leucocytes must leave the circulatory flow. This is achieved by attaching themselves to the wall of small blood vessels, first rolling to a stop using adhesive molecules called lectins, a bit like grappling irons, then stabilizing the attachment using stronger adhesive proteins called integrins. At this point, leucocytes migrate between the endothelial cells that line the blood vessels (rather like elbowing a way through a crowd), ready to confront invading bacteria, and maybe commit suicide by bursting to release their antibacterial contents (this will be described in more detail in Chapter 6). Once the bacteria have succumbed, their corpses are engulfed by macrophages that have also migrated to the site. The speed and specificity of this response is impressive, albeit that there are ‘patrolling’ cells constantly coursing through the whole blood system. There is also a regular directed migration in the production of another class of white blood cells (T cells), which leave the bone marrow as immature precursor cells and complete their development in the thymus using similar homing and adhesion molecules. Although there are general ideas about the mechanisms of migrations in the body, we are far from understanding the whole picture.

How old are our cells?

Benjamin Franklin’s assertion that the only two certainties in life are death and taxes only partially applies to cells. The options for ‘the end’ in cellular terms are to divide or die. A cell ‘avoids’ death by dividing, but nevertheless sacrifices its own unique existence by becoming two daughter cells. Most daughter cells are identical, and will have identical fates, which is to differentiate to perform a particular function, eventually die, and be replaced. This may involve a lifespan of no more than a few hours, as is the case for some white blood cells (neutrophils), or up to 120 days for red blood cells. Most cells throughout our bodies will be replaced over the course of our lifetime, with an average of seven to ten years according to Jonas Frisen, from the Karolinska Institute in Stockholm. There are, however, three types of cell that we carry from the cot to the grave: the neurons of the cerebral cortex of the brain; cells in the inner part of the eye lens; and, perhaps surprisingly, heart muscle cells, which must have contracted a mind boggling three billion times in anyone who reaches 100 years of age. The cells in the inner part of the lens are structurally very similar to keratinized skin cells in that they are largely filled with keratin fibres, but laid down in a highly organized crystalline arrangement allowing the maximum amount of light to travel through them. In time, this crystalline organization may break down and the cells of the eye lens become opaque, resulting in cataracts.

Cell death

As we have seen, the life span of cells within the human body may vary between hours and decades. The actual process of death may come about in a variety of ways from a variety of causes. Direct trauma such as mechanical damage, or extreme heat or cold, produces instant and direct effects such as rupture of the cell membrane, or instant destruction of proteins. This produces a balance of disorder over order that cannot be reversed, so the cell dies, rather like the sudden and usually violent death of an individual, maybe in a road accident or war situation. In cells, this type of death is termed necrosis.

A different and more intriguing cell death is one which is a very necessary process in all multicellular organisms, originally identified in studies of development but subsequently as a ‘suicide pathway’ for all cells that have failed to reproduce themselves in a satisfactory manner, ending any threat posed by the continued replication of ‘rogue’ cells. This is known as programmed cell death, or ‘apoptosis’ (from Greek, ‘the falling of the leaves’), first recognized in the 1970s by Alastair Currie, John Kerr, and Andrew Wylie, all pathologists in Aberdeen University, who observed (mainly by electron microscopy) a characteristic series of changes leading to cell death. It took over a decade before this process was fully accepted as a major biological mechanism for maintaining the　status quo of cells in tissues. Apoptosis does not trigger an immune response (as can occur with necrosis), as the cell contents are effectively ‘recycled’, with the fragmented remnants of the dead cell taken up (phagocytosed) by their healthy neighbours. Apoptosis is routine in developmental processes such as the removal of webbing between fingers in humans, the loss of tadpole tails in amphibians, and insect metamorphosis. Cells that are no longer required during development begin to shrink, and their surfaces generate spherical protuberances called blebs ( Figure 12a ), a very active process when observed by time-lapse microscopy, with the surface of the cell resembling boiling mud pools in volcanic sites. Inside the cell, nuclear contents break down, and the chromatin aggregates into characteristic dense masses that became the benchmark appearance of apoptotic cells ( Figure　12c ), as the DNA is chopped up into small pieces.

12. Apoptosis (programmed cell death). (a) Initially, the surface of a cell becomes blebbed, (b) mitochondria lose their internal structure, (c) the contents of the nucleus aggregate, and (d) pores develop in the surface of mitochondria

Once it was realized that apoptosis was a universal process in the demise of cells, the search began in earnest, to find how this ‘cell suicide’ was initiated. Two pathways emerged, dependent on whether apoptosis was triggered externally (extrinsic) or internally (intrinsic). Extrinsic apoptosis results from signals received at the cell membrane, which bind to ‘death receptors’ on the cell surface that then initiate the process of cell suicide. This type of apoptosis usually occurs as a method of cell killing in immunological reactions. Intrinsic apoptosis occurs during embryonic development where it is programmed for a particular stage or, alternatively, is triggered as a result of extensive damage to DNA, such as that caused by ionizing radiation. Apoptosis is also triggered when DNA replication is compromised and, despite the presence of repair mechanisms, the proofreading quality control systems still find inaccuracies that would lead to production of mutated proteins.

The mechanics of cell suicide

So how does a cell go about ending its own existence? The actual mechanism of ‘cell suicide’ depends upon mitochondria, termed the ‘angels of death’ by Nick Lane in his book, P ower, Sex and Suicide: Mitochondria and the Meaning of Life . The first change occurs in the mitochondrial inner membrane, which becomes damaged by aberrant biochemical activity, leading to the formation of pores in the mitochondrial membrane ( Figure　12b , d). At this point, the mitochondrion becomes committed to trigger apoptosis, and releases cytochrome　c (a protein crucial to its normal function of energy production) which exits through the newly formed pores. This information came to light as a result of some neat experiments in which apoptotic mitochondria were introduced into perfectly healthy cells, resulting in apoptosis. The released cytochrome　c binds to several other proteins in the cytoplasm to form a complex called the apoptosome which, in turn, activates a cascade of ‘executioner enzymes’ which not only kill the cell but cause fragmentation of the nucleus and cytoplasm into bite-size pieces ready to be phagocytosed by neighbouring cells.

Because intrinsic cell death occurs as part of a developmental programme, it seemed likely that there were actually genes for apoptosis. Working with　Caenorhabditis elegans (a much studied nematode worm), where it is known that exactly 131 of the total of 1090 cells die by apoptosis during development, Bob Horovitz from Cambridge in the USA found several programmed cell death genes. These studies led to the award of a Nobel Prize in 2002, shared with John Sulston and Sydney Brenner from Cambridge in the UK.

Several of these ‘cell death’ genes were those genes that were routinely mutated in many mammalian cancer cells, confi rming that apoptosis was a mechanism to delete cells with damaged DNA. Mutations in the cell death genes blocked apoptosis, allowing cells with damaged or mutated DNA to develop abnormally and generate tumours. David Lane, who lost his father to cancer when he was only 19, dedicated his research efforts to explore the changes that occur when normal cells become cancerous. In 1979, he found p53, a protein that is inactivated or deficient in almost all cancer cells. If normal p53 is present at the right levels in dividing cells, and a cell has damaged DNA (or replicates its DNA inaccurately), then the p53 triggers apoptosis, or, failing that, initiates a pathway to senescence, halting all division, hence inspiring its description as a tumour suppressor and the ‘guardian of the genome’. Apoptotic and senescent cells are recognized by cells of the immune system (see next chapter) and removed by phagocytosis. Thus, even at advanced stages of cancer, a restoration of the ‘p53 response’ will bring powerful defence responses into play to shrink tumours and stop further growth. Unfortunately, getting normal p53 into cancer cells is not a simple process, and several molecular biological strategies are currently under investigation. The promise of gene therapy as the answer to multiple diseases has still to be realized, largely because the practicalities of introducing genes and their products into cells in the right amounts and at the right time without unacceptable side effects are still proving extremely difficult. Interestingly, China appears to have made the most progress in p53-based treatments, with a drug called Gendicine having been approved for head and neck cancer in 2003, whereas the US Food and Drink Administration by 2010 had not approved any p53-based treatments.