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In the midst of life some cells are obliged to die for the good of the
organism of which they are a part. In a final display of biochemical virtuosity,
they manufacture an arsenal of deadly proteins and then turn it upon themselves.
In effect, they commit suicide after first procuring the means of their
own destruction. This extraordinary process of programmed, or physiological,
cell death – long regarded merely as a curiosity – is now the subject of
heightened interest in several disciplines, with implications for the study
of the immune system, cancer and AIDS.

Programmed cell death underlies many familiar phenomena. It remodels
the tadpole, removing the tail and creating the blunt posterior of the mature
frog. It dissolves the juvenile tissues of the caterpillar when the time
comes for metamorphosis. In the embryos of vertebrates it deletes cells
from the developing limb and leaves behind the five separated digits. A
reduced death toll gives ducks their webbed feet.

Equally remarkable findings have emerged from studies of the nematode
worm Caenorhabditis elegans. To make a worm, 1090 cells must be generated
and 131 must die in preordained places and at preordained times. In many
cases the life span of a condemned cell, from genesis to death, is less
than an hour. Two genes, ced-3 and ced-4, are necessary for the process
of cell death to take place, according to Ronald Ellis, Junying Yuan and
Robert Horvitz at the Howard Hughes Medical Institute, Massachusetts Institute
of Technology; if either gene is abnormal, cells that should die are spared
and development goes awry.

Programmed cell death is not confined to development. Adult animals
may control the size of their organs by balancing cell death and cell division.
Some hormones exert their effects by prompting cells to commit suicide.
Glucocorticoid hormones from the adrenal gland, for example, cause death
of cells in the thymus, a gland in the chest with an important role in the
immune system. Other parts of the immune system rely on programmed cell
death to perform many of their most important tasks.

In many cases of programmed cell death, cells die by a recognisably
similar process, which has been named ‘apoptosis’ by Alastair Currie, John
Kerr and Andrew Wyllie at the University of Edinburgh. From start to finish
apoptosis takes just a few hours. The doomed cell separates from its neighbours
and shrinks, while its nucleus changes shape and begins to fragment. Eventually
the cell breaks up into a number of small pieces, which are quickly engulfed
either by neighbouring cells or by ‘refuse-devouring’ cells called macrophages.
Biochemical demolition proceeds in parallel, with the cell’s DNA being shredded
by a powerful enzyme. When the resulting DNA fragments are separated by
size using the technique known as gel electrophoresis, the result is an
unmistakable pattern called a DNA ladder.

Wyllie and his colleague Mark Arends believe that apoptosis is the result
of two switching events: priming and triggering. During the priming phase,
the cell makes the proteins that it will need for its own destruction. This
arsenal includes two important enzymes: a nuclease (whose job is to lacerate
DNA and create the rungs of the DNA ladder) and a transglutaminase, which
forges cross-links between various structures in the cell and causes some
of the changes in shape that accompany death. Because cells often have to
construct these enzymes themselves, chemicals that disrupt the construction
process can sometimes block apoptosis. Cells must, it seems, be healthy
and active before they can die.

With priming complete, the cell waits for the signal that will prompt
it to unleash its self-directed chemical weapons. Triggering is a complex
process, but research on cells in the thymus implicates calcium ions as
a part of the mechanism, according to David McConkey, Sten Orrenius and
Mikael Jondal at the Karolinska Institute in Stockholm. Calcium does not
invariably trigger apoptosis; if additional signals, carried by other molecules,
accompany the calcium signal, then the cell survives. If, on the other hand,
no other signal is forthcoming, the cell switches on its suicide programme
and activates its nuclease. The cell is rather like Owl’s house in Winnie
the Pooh with its bell pull and knocker; the cell interprets incoming signals
according to whether they ring the biochemical bell, knock the door or both.

Once apoptosis is under way and the cell has fragmented, the priority
is for other cells to clear the debris as quickly as possible. Research
by Wyllie, Edward Duvall, and Robert Morris at Edinburgh suggests that dying
cells bear chemical markers that identify them as debris – possibly unusual
sugar molecules. Macrophages and neighbouring cells recognise these markers
and start to devour the remains. This kind of mechanism would have a major
role in the body’s control of inflammation, according to John Savill and
colleagues at the Royal Postgraduate Medical School, Hammersmith Hospital,
and the Imperial Cancer Research Fund in London. The team has found a molecule
called the vitronectin receptor on the surface of macrophages. This molecule
recognises dying white blood cells, an event which leads to their ingestion.
These white blood cells are neutrophils, cells that accumulate at inflamed
sites and wage war on bacteria with the help of chemicals so powerful that
they can injure the body’s own tissues. The combination of apoptosis and
removal by macrophages tidies away potentially harmful neutrophils and helps
to quell the inflammation.

Apoptosis also plays several important roles in the great battle against
infection. It is no exaggeration to say that the complex machinery of the
immune system could not function without it. Take the case of cells that
pose a threat to the body because they have become infected with a virus.
The body’s reaction to such an invasion is to marshal so-called cytotoxic
T cells, which recognise the infected cells and eliminate them. There is
much controversy about whether the actual mechanism involves murder or suicide.
The T cells install perforated proteins into the membranes of their victims,
a practice that could prove fatal on its own account. Yet the evidence shows
that the victims actually die by apoptosis, complete with a DNA ladder,
almost as though they have been persuaded to commit suicide by their encounter
with a cytotoxic T cell.

One idea is that cytotoxic cells transfer the suicide machinery into
their targets, which are then left to do the decent thing. Even so, the
target cell is no passive victim; its own genetic machinery must be working
properly for it to be killed. This conclusion emerged a few years ago from
research by David Ucker, then at the Harvard Medical School. Ucker worked
with a strain of cultured cells which resist being killed by cytotoxic T
cells. This resistance apparently stemmed from a genetic mutation – one
that stopped the cells committing suicide to order. The target of a cytotoxic
T cell must, it seems, acquiesce in its own death.

As the activities of cytotoxic cells make clear, the immune system is
a powerful weapon, which needs to be aimed very carefully. When something
goes awry and the system turns on an improper target – tissues within the
body instead of invaders – the result is autoimmune disease, such as multiple
sclerosis or myasthenia gravis. The immune system has to distinguish between
self and nonself, but it has to be taught how. T cells of various kinds
‘learn’ this lesson early in life – and apoptosis is their teacher. (T
cells must not only learn to avoid attacking the body’s tissues, they must
also learn to detect their targets in association with glycoproteins of
the major histocompatibility complex .

The surface of a T cell bristles with receptor molecules whose job is
to recognise alien substances and trigger an immune reaction. Each T cell
carries receptors of just one type, but the body as a whole makes 100 million
or so different types in an attempt to anticipate as many invaders as possible.
This process inevitably creates many T cells bearing receptors that will
react against the chemical ingredients of the body itself. These self-reactive
T cells are potentially dangerous and so the body must eliminate or inactivate
them early in life. Various mechanisms are likely to be involved in this
exercise; one is cell suicide. Support for this idea has come from researchers
in several laboratories, such as Christopher Smith and colleagues at the
University of Birmingham, Yufang Shi, Beni Sahai and Douglas Green at the
University of Alberta in Canada and Robson MacDonald and Rosemary Lees at
the Ludwig Institute for Cancer Research in Epalinges, Switzerland.

The emerging picture is of a mechanism that is both economical and ingenious.
If the receptors on an immature T cell (one that is developing in the thymus
gland) bind too tightly to any chemical substance that they encounter, the
cell responds by undergoing apoptosis and so destroying itself. At this
stage of development, T cells have not been exposed to the outside world
and so the substances they bind are chemicals native to the body – ‘self
molecules’. T cells which survive this self-inflicted carnage go on to reach
maturity. When their receptors make contact with foreign substances in later
life, their response will be to multiply, instead of destroying themselves.
In short, the same molecular apparatus serves two distinct processes: it
deletes self-reactive T cells and makes sure useful ones multiply.

Apoptosis in immature T cells helps the body to protect itself from
its own immune system. In other immune cells, apoptosis has another, more
positive role: it helps to ensure that the immune response becomes more
finely tuned with time. The cells in question are B cells. These cells mature
in the bone marrow and produce antibodies – proteins that bind to foreign
substances and inactivate them or help destroy them.

One of the most remarkable properties of these cells is their ability
to improve the quality of the antibodies they make as time goes on. The
genes that produce the antibodies mutate, followed by a selection process
in which B cells carrying the best antibodies on their surfaces – those
which bind avidly to their targets – are stimulated to proliferate at the
expense of those bearing poorer antibodies. Research by Yong-Jun Liu and
his colleagues at the University of Birmingham suggests that these poorly
adapted cells commit suicide by apoptosis, leaving the field open to their
better adjusted neighbours.

Work on B cells has also shown how apoptosis could be relevant to the
study of cancer. In a condition called follicular B cell lymphoma, B cells
start to accumulate in an abnormal manner; later on, a rapidly growing malignancy
may develop. Follicular B cell lymphoma is often associated with a translocation
– an accident in which genetic material gets shifted from one chromosome
to another. In this case, the translocation joins a gene called bcl-2 to
a gene that makes parts for antibodies. The antibody genes of B cells are
highly active. The inevitable consequence is that they start to make the
product of the bcl-2 gene in quantity.

Evidence from a number of laboratories now suggests that bcl-2 is a gene
that can suppress apoptosis. When the gene works too rapidly, as it does
after the translocation, cells do not die as they should but accumulate
in a resting state. Although a second genetic mishap is needed before the
cells will proliferate in an uncontrolled manner, it seems likely that bcl-2
is partly responsible for the malignancy. The way in which bcl-2 blocks
apoptosis is still unknown, but David Hockenbery and his co-workers at the
Howard Hughes Medical Institute in St Louis, Missouri, have shown that the
product of the gene resides inside mitochondria, the cellular organelles
which are responsible for energy production.

This influential gene may also be involved in the activities of Epstein-Barr
virus, the agent behind many cases of Burkitt’s lymphoma, a tumour of the
B cells. When Epstein-Barr virus infects B cells it prolongs their normally
short lives. According to work by Gwyn Williams, Sheila Henderson and their
colleagues at the University of Birmingham, the virus does this by preventing
the self-destruction of its host cells. It may either block apoptosis directly,
or indirectly by making use of the bcl-2 gene.

Death by antibody

As Williams points out, cell suicide is a ‘critical control point’ in
cell biology; it is a process that is regulated by built-in mechanisms and
can be encouraged or suppressed. One day it may be possible to manipulate
these built-in mechanisms to develop new forms of cancer therapy. One idea
is simply to encourage tumour cells to destroy themselves. A hint of how
this might be achieved came a couple of years ago from work done by Bernhard
Trauth at the German Cancer Research Centre in Heidelberg and colleagues
from other laboratories in the city. Trauth and his team developed a monoclonal
antibody that could bind to cultured human blood cells and make them undergo
apoptosis. A number of different varieties of B cells and T cells turned
out to be susceptible to the antibodies: when treated, they destroyed themselves.
Mice with artificially induced B cell tumours improved when injected with
the antibodies.

Cancer researchers are becoming increasingly interested in apoptosis.
The growth of a tumour is regulated both by the rate at which its cells
divide and the rate at which they die – an event which can take place by
apoptosis. The importance of this equation was underlined a few years ago
in research by Wyllie and his colleagues at Edinburgh. The researchers
induced tumours in mice using cells containing certain oncogenes (genes
that cause cancer). The most aggressive tumours combined a high rate of
cell division with a low rate of apoptosis. Although cells in other tumours
also divided rapidly, apoptosis slowed their growth.

Cancer is not the only condition which could benefit from renewed interest
in apoptosis. According to a recent proposal by Jean Claude Ameisen and
Andre Capron of the Pasteur Institute in Lille, apoptosis could be involved
in some of the events associated with infection by HIV. One such event is
the body’s gradual loss of helper T cells. Immature cells of this kind sometimes
undergo apoptosis (if they are self reactive) but mature T cells normally
proliferate when stimulated. The researchers say the virus could somehow
prime them to destroy themselves on stimulation. Some other effects associated
with AIDS, such as loss of cells and atrophy in tissues such as bone marrow
and brain, could also be explained if the virus has a tendency to promote
apoptosis.

These ideas are speculative, but they illustrate the importance that
apoptosis has now acquired in the physiological scheme of things. The ability
of cells to die is central to life – a lesson that we can learn from development,
metamorphosis, or the subtle workings of our immune systems. An ability
to manipulate this curious process could one day be central to medical science.

Stephen Young is a freelance science writer based in Wales.

* * *

1: WHEN ORDER GIVES WAY TO CHAOS IN THE CELL

Physiologists distinguish two mechanisms by which cells die: necrosis
and apoptosis. Death by necrosis is untimely death; it follows accidents,
such as encounters with poisons, overheating or lack of oxygen. The dying
cells swell, as do the mitochondria inside them. Enzymes called phospholipases
set to work, attacking the molecules of which membranes are made and causing
widespread damage. Once membranes begin to leak, the cell can no longer
keep chemicals in their proper compartments and dies.

Apoptosis is a much tidier way to die. The doomed cells shrink and repackage
themselves into digestible fragments so that neighbouring cells can consume
them. The cell membrane does not suffer the kind of damage seen in necrosis.
An internally produced enzyme, a nuclease, cuts DNA into conveniently sized
pieces. DNA inside living cells is wound around bead-shaped structures made
up of proteins called histones. Each bead holds a piece of DNA about 150
base pairs long. The beads are separated by stretches of ‘linker’ DNA about
50 base pairs long. During apoptosis, the nuclease cuts the linker DNA but
leaves the beads alone. The result is a set of fragments, some of which
are one bead long, some two beads long, some three beads long and so on,
depending on the exact places where the linker DNA has been cut. When the
fragments are separated by size and visualised by gel electrophoresis, the
result resembles the regularly spaced rungs of a ladder. This pattern does
not accompany necrosis and is one of the clearest signs that a cell has
died by its own hand.

* * *

2: THE IMMUNE SYSTEM AT A GLANCE

The immune system defends the body against invasion and disease. If
its constituent cells, the lymphocytes, were collected together instead
of being dispersed throughout the body the resulting organ would be the
size of the liver. This huge army of cells attacks and destroys invading
microorganisms and commits their details to memory. Should the perpetrators
attack a second time, the response is fast and overwhelming.

Materials that provoke an immune response are called antigens. An antigen
might be a chemical substance on the surface of a virus or bacterium, or
any other large molecule that has invaded the body. Antigens trigger reactions
from two varieties of lymphocyte: B cells, which develop in bone marrow,
and T cells, which develop in the thymus gland. B cells secrete proteins
called antibodies which lock onto antigens and help to destroy them. Each
B cell makes just one variety of antibody, but collectively the cells manufacture
an extraordinarily diverse selection – perhaps as many as 100 million different
types. This diversity comes about while the B cells develop, when their
antibody genes are assembled from a range of possible subsections.

Immature B cells wear antibodies on their surfaces. If these antibodies
engage an antigen the cells are stimulated to proliferate and to secrete
antibodies in quantity. At the same time, other B cells specific to the
same antigen become long-lived memory cells, ready to attack the invader
should it enter the body a second time. Once antibodies attack a bacterium,
the complement system – a kind of chemical kit for drilling holes in membranes
– is activated and the bacterium is destroyed.

T cells come in three main varieties. Cytotoxic T cells kill other cells
that have become infected with viruses. Helper T cells recognise antigens
and release chemical stimulants (interleukins) that spur other cells (notably
the B cells) into action, while suppressor T cells have a dampening effect
on immune reactions.

T cells bear molecules on their surfaces which control their interactions
with other cells. One, the T cell receptor, has the job of recognising antigens
and setting up a suitable response. T cell receptors detect their quarry
only when it is displayed to them by special molecules that sit on the surface
of other cells (glycoproteins made by the genes of the major histocompatibility
complex). This means that T cells confine their attentions to their appointed
targets, namely other cells of various kinds.