ÐÓ°ÉÔ­´´

ALL KINDS of cells and organisms, from bacteria to people, respond to
harmful environmental conditions by synthesising stress proteins, also known
as heat-shock proteins. As the severity of the stress increases, so does
the production of the proteins. They appear in response to a host of events
such as high temperature, lack of oxygen, and exposure to heavy metals,
ethanol and, in mammals at least, to physical trauma.

Researchers know of at least 30 stress proteins, identified from the
mass of cellular proteins separated by gel electrophoresis. Cells make them
in varying amounts, depending on the type and severity of stress. The functions
of the proteins are still unclear. Preliminary evidence suggests, however,
that those made by an organism under stress protect it from damage that
further exposure would cause. In this issue, David Miller describes what
we know about the role of stress proteins that are always present in cells,
even in the absence of stress. They often appear to protect other proteins
by binding to them.

Whatever their function after stress, the appearance of extra heat-shock
proteins is a sure sign that the organism has been stressed. Biologists
might be able to use this knowledge in at least three ways: to measure stress
or pollution in the environment; to improve medical diagnosis and therapy;
and in biotechnology. In each case, there is a commercial application that
is about to reach the marketplace.

Current methods of measuring pollution in the environment have limitations.
It is not easy to assess the severity of contamination by sampling and testing
for various chemicals. In water, for example, the heavy metal cadmium may
be chelated in a form that renders it harmless; measuring cadmium levels
alone cannot distinguish between safe and toxic forms of the metal. Alternatively,
measurements may indicate that the level of a heavy metal in water is low,
but it may accumulate to toxic levels in the tissues of organisms, such
as the livers of fish.

Williams Welch, a cell biologist at the University of California at
San Francisco, thinks that stress proteins will provide a better method
for determining environmental contamination. ‘Why not let the organism tell
you that there is a problem?’ says Welch.

The stress proteins synthesised by an organism are a highly sensitive
indicator of environmental stress, according to Welch. The amount of stress
protein it produces indicates the severity of the pollution; a knowledge
of which ones it makes reveals the type of stress. For example, cells make
one particular stress protein, identified by its size as a subunit molecular
weight of 32 kilodaltons, in response to heavy metals, but not in response
to other stress agents. So if researchers identify this protein in an organism,
they know that heavy metals are present.

Some industries could use this knowledge, Welch argues, to monitor the
pollution of waterways near factories, for instance, by placing a specific
test organism at a site where contamination is suspected. Laboratory workers
could then analyse the organism at regular intervals to determine which
stress proteins are present and in what quantities.

Proteins for profit

This application is being pursued by a newly formed Canadian company
called CB Research International Corporation based in British Columbia,
with an American affiliate in Long Beach, California. The company, which
is the first in the field, plans to market diagnostic tools to measure stress
proteins. The diagnostic kits might use antibodies to various stress proteins
to determine their levels, or gene probes that detect the proteins indirectly,
by targeting levels of messenger RNA, the chemical mediator between genes
and proteins.

The company also plans to sell transgenic organisms that could act as
custom-made monitors of specific pollutants. The transgenic organism would
carry a ‘promoter’ from a stress-protein gene attached to a ‘reporter’ gene.
The promoter is a piece of DNA that lies alongside a gene and ensures its
activation in the right circumstances. The idea is that a particular kind
of environmental stress will activate the stress promoter and cause the
cell to produce the reporter protein, which could be anything that is easy
to detect. The level of the reporter protein indicates the severity of the
stress.

Occupational health workers or environmental health officers could monitor
the exposure of humans to pollution in a similar way, by taking regular
blood samples and measuring the stress response of the white blood cells
known as lymphocytes. An increase in the levels of stress proteins in lymphocytes
above normal levels will indicate that a worker may have been exposed to
a toxic compound. Welch, who also acts as a consultant to CB Research International,
says that the monitoring is simple enough to be done as often as once a
month.

Stress proteins will also play an important role in hyperthermia, the
application of heat to tumours. Doctors are increasingly using heat, applied
to tumours once or twice a week, in conjunction with a course of daily radiation
treatment. Surface tumours are most easily heated with microwaves, but radiofrequency
waves or ultrasound can be used to heat deep-seated tumours.

The level of stress proteins increases after the first heat treatment
and, at the same time, the tumour cells develop a temporary resistance to
the damage caused by heat. This resistance is known as thermotolerance.
As the stress proteins decrease again, the resistance also disappears. This
link between the amounts of stress proteins in a cell and its tolerance
to heat is the reason why scientists believe that stress proteins do something
to protect cells from the harmful effects of the environment.

A clinician can decide when to give the second heat treatment, making
sure that the temporary resistance to heat has disappeared first, by measuring
the stress proteins. At Stanford, in the Department of Radiation Oncology,
we are running a trial to test this use of stress proteins.

The proteins could also help doctors to determine the extent of many
kinds of injury. When traumas occur in organs of the body, it may be difficult
to track down the site and the extent of the damage. ÐÓ°ÉÔ­´´s have recently
shown that a stress response occurs in the heart and brain of people following
the transient blockage of blood vessels, ischaemia, during a heart attack
or stroke. According to Welch, it may be possible to diagnose the extent
of damage caused by ischaemia by measuring the stress proteins released
into the blood. Further studies may reveal stress proteins that are specific
to a particular organ, enabling doctors to pinpoint the site of damage.

Stress proteins may also play a role in the development of atherosclerosis,
the disease of the walls of the arteries that often leads to ischaemia,
according to Paul Berberian from Wake Forest University in North Carolina.
Fatty plaques develop on the walls of diseased arteries and increase in
size to the point where they impede blood flow and stimulate clotting. Berberian
has found that the cholesterol-rich cells in the plaque have increased levels
of the most prominent stress protein, the 70 kilodalton protein. No one
yet knows how the stress proteins contribute to the build-up of plaques,
however.

An aid to recovery

Doctors may soon exploit the phenomenon of thermotolerance, or tolerance
to stress, to reduce the effect of trauma during surgery or other therapies.
Mary Barbe from the Medical College of Pennsylvania and her colleagues have
shown that inducing cells to produce stress proteins by heating them mildly
can protect the retina of rats from subsequent damage by light. William
Currie and his colleagues from Dalhousie University, Nova Scotia, have demonstrated
that the hearts of rats exposed to mild hyperthermia recover more quickly
from ischaemia than do hearts that have not been heated. So using hyperthermia
to stimulate cells to produce stress proteins might help to protect the
retina during eye surgery or the heart following ischaemia.

Parasitologists are studying stress proteins as well. Parasites that
cause malaria, schistosomiasis and sleeping sickness, for instance, suffer
a heat shock when they invade a human. Their body temperatures rise from
that in its primary host, such as a snail, to 37 C in humans. Douglas Young
from the Medical Research Council’s Tuberculosis Unit at Hammersmith Hospital,
in London, has shown that the parasites secrete large amounts of the 70
kilodalton stress protein, and that the patient responds by making antibodies
against it. The bacteria that causes leprosy, legionnaires’ disease, syphilis
and Lyme disease also elicit an antibody response, but against a different
stress protein.

Parasites provoke proteins

Because the stress proteins stimulate the body to produce antibodies,
they could be candidates for vaccines against these pathogens. The stress
proteins made by all organisms have much the same structure – they share
‘highly conserved’ regions – so a vaccine against stress protein should
protect against many diseases caused by parasites. The structural similarity
of stress proteins, however, may cause problems for the human host. Repeated
injections of a protein vaccine may break down the host’s tolerance of self.
The antibodies produced may turn around and attack host stress proteins
causing an autoimmune reaction. At the moment, it is unclear who benefits
from the stress proteins during infection, the pathogen or the host. But
either way, manipulating the level of stress proteins during infection may
reduce the severity of the infection.

The booming field of biotechnology also stands to benefit from the application
of stress proteins and their promoters. Industry now produces many proteins
that have important uses in medicine, such as insulin and growth hormone,
from genetically engineered bacteria and yeast cultured in large fermentation
vats. Various sorts of environmental insults, such as crowding, overheating
and nutritional deficits cause a stress response in the yeast or bacteria
that reduces the amount of the desired protein they can manufacture. CB
Research International plans to develop strains of yeast and bacteria that
produce unusually high levels of one or more stress proteins and so are
resistant to, or tolerant of, environmental stress in the vat.

The company also has industrial outlets in mind for its pollution indicator.
This consists of a promoter from a stress protein linked to a reporter protein.
Company scientists will transfer the construct into the yeast or bacteria
along with the gene for the protein being made for commercial purposes.
Any stressful situation in the vat would activate the promoter and cause
the calls to manufacture the reporter protein. By measuring the amount of
the reporter protein, for example by detecting a change in the colour of
the medium, technologists could receive an early warning of stress in the
vat.

Biotechnologists are also exploring the opposite approach: to link the
gene for a protein of commercial interest to the promoter of a stress protein.
Then, as harmful conditions develop in the vat, cells will reduce their
synthesis of many normal proteins but increase the production of the stress
proteins. Cells will make more of the commercially important protein as
well, because the gene for the protein is linked to the stress promoter.
Technicians can then easily harvest the desired protein from a medium containing
few other normal proteins.

Tolerance to stress can be used to advantage in agriculture too. Genetically
engineered plants containing extra copies of the genes for stress proteins
could grow in environments that normally plants cannot tolerate. It may
be possible to produce crop plants resistant to heat, drought or salt. As
the greenhouse effect continues to promote global warming, humanity will
increasingly rely on plants that can grow at higher temperatures. ÐÓ°ÉÔ­´´s
have yet to determine which of the stress proteins can provide this protection

Dr Robin Anderson is senior research associate in radiation oncology
at Stanford University Medical Center.