Winding Your Way Through DNA Symposium
San Francisco, California
Saturday Morning, September 26, 1992
David W. Golde, M.D.
Head, Division of Hematologic Oncology, Memorial Sloan-Kettering Cancer
Center, New York City
I'm delighted to be back in San Francisco, where my medical career began
as an intern at the University of California in the summer of 1966. San
Francisco was a very special place that year, and UCSF has become a very
special medical center. I'm going to tell you about how recombinant DNA
has led to important treatments for human disease, in the form of a
story, rather than a didactic lecture.
About 22 years ago, I sat in a small laboratory on the hill at UCSF and
pondered what was to become my lifetime work: the study of how blood
cells are produced and how they function. I knew this was an important
topic because the Bible so clearly says so, and because blood, through
the ages, has been known for its central role in the life of complex
multi-cellular animals, including man.
The blood cells function in host defense. The constant vigilance and
battle we wage to protect ourselves from an increasingly hostile
environment. The cells circulating in the blood come in several
varieties, and each is essential to life. There are both red and white
The red blood cells are unique in that they contain no nucleus
and are filled with a red protein known as hemoglobin. Their
only function is to carry oxygen to the tissues. A decrease in their
production leads to anemia.
Among the white blood cells, the neutrophils and
monocytes protect us against bacterial and fungal invasion.
Macrophages, which derive from blood monocytes, are found in all
the tissues where they are critical to the ecology of the body in
cleaning up debris and dead cells as well as toxic particles. The
platelets are essential in host defense against bleeding; they
plug disruptions in blood vessels, and initiate the coagulation
reactions which allow proteins in the plasma to form a clot.
The T-lymphocytes are critical in the recognition of foreign
tissues and micro-organisms and in controlling the functions of other
host cells. Sub-types of T-lymphocytes can kill cancer cells and
virally-infected cells, and are therefore essential in host defense
against tumors and viruses. The B-lymphocytes produce
immunoglobulins, the antibodies that coat and help destroy
invading micro-organisms as well as toxins.
These cells hurtle wildly through the bloodstream. The neutrophil lives
only about eight hours; the platelet, ten days; and the red blood cell
lives for months. Some investigators have estimated that the red blood
cell travels more than 200 miles in its lifetime. Some T-lymphoctyes
exist for the life of the individual.
In order to provide sufficient cells for host defense, red and white
blood cells are produced at a prodigious rate. Ten billion cells are
produced every hour, every day, in our bodies, and this is only at
baseline. In times of need, the production of red cells can increase
tenfold, and there is no theoretical limit to the production of
Surprisingly, all the blood cells are produced from the single mother
cells known as the stem cells. These reside primarily in the
bone marrow but also circulate in the blood. These cells have the
capacity of giving rise to precursors for all the cellular elements of
blood. Through a tightly regulated system, these precursor cells
undergo proliferation and differentiation, that is, they
divide and specialize, resulting in the production of more mature
cells, which are then released into the blood stream.
You can see that if new genetic information were placed into the stem
cell it would subsequently appear in all of the hematogenic cells
in the blood, but would not appear in the cells of, for example, muscle
or other tissues.
How does the process of blood cell production work? How is it
regulated? And what goes wrong in disease states?
In 1966, the same year I was an intern on the wards of UC Medical
Center, investigators in Israel and in Australia developed a culture
system where colonies of white blood cells would grow from bone marrow
cultured in semi-solid media. It was found that these colonies would
only form in the presence of certain factors that were released from
other cells. These factors were named "colony stimulating
factors" although little was known of their nature or importance.
This is a picture (slide) of white blood cells that formed in these
cultures, and each white dot you see here represents the progeny of a
single progenitor cell.
A number of scientists who were here at the University of California
taught me how to do these experiments, Mary Maloney and Harvey Patt, but
perhaps more importantly, scientists such as the late Gordon Tompkins
taught me how to interpret them. I don't know if the bus to Marin from
UC Medical Center still runs, but in that bus, there was a rush to get
to the rear of the bus (not the front), because in the rear sat Gordie
Tompkins and on Fridays, especially, we'd stop and buy beer and we'd be
able to talk to him at least as far as Mill Valley. Those that had to
go to Santa Rosa sometimes arrived Friday night in a shaky state.
During the 1970's I, and other investigators, searched for the cellular
origin of the colony stimulating factors. Where did they come from?
What cells made them? We identified the activated T-lymphocyte and the
macrophages as primary producers of colony stimulating factors doing the
very simple types of experiments that are described in this slide.
Culture supernatants were taken from isolated macrophages and
isolated T-lymphocytes and tested in the culture system I showed you for
the ability to stimulate colonies. So macrophages were grown in these
cultures and then the medium that was conditioned by the macrophages was
tested to see if it would stimulate the production of the white blood
In 1984 we succeeded in purifying a very tiny amount of granulocyte
macrophage colony stimulating factory (GMCSF). The quantity we
isolated was not enough to provide a single dose for a single patient.
These studies, however, led to the molecular cloning of the
complimentary DNA for GMCSF, thanks to a collaboration with
scientists at the Genetics Institute.
This tiny amount of GMCSF took ten years to isolate, and one rule of
protein chemistry is that if you get two proteins on a gel, the one
you're interested in will be the smaller one. This tiny amount of
protein was not enough to actually do experiments with, but it was
enough to get the gene sequence. The cDNA was inserted into the Ferrari
of plasmids. This is a true racehorse of expression vectors, and you
put the sequence in here for GMCSF, take the entire plasmid, which is
armed with very strong promoters, and this particular plasmid can be
expressed in the mammalian cells. You put that into mammalian cells in
culture and you have an incredible factory that can produce huge
amounts, kilograms, of a protein that we only had in microgram
With adequate quantities of recombinant GMCSF in hand, we can then ask
the question: Does this substance that stimulates bone marrow colonies
in culture, also stimulate white cell production in humans? As you can
see, I'm smiling, so you already know the answer. Amazingly, little
more than a year after the molecular cloning of GMCSF, we had the
opportunity to test the activity of this material in patients. We found
that the recombinant material dramatically stimulated the production of
white blood cells. Here you see the results in a single patient: the
white blood cell count rose from a baseline of about 2,000 to as high as
16,000 with stimulation of the eosinophils, monocytes, and a great
stimulation in the production of neutrophils, just as the material did
We could now control the production of host defense cells.
Let me repeat that: We could now control the production of host
defense cells. That is, recombinant technology made it possible to
conceive of positively regulating a host defense, making an individual a
better defender against disease but allowing them to make more host
defense cells that also function better. Other colony stimulating
factors were cloned and tested in the clinic and we now have at our
disposal a whole new class of powerful agents that can control blood
cell production and function. With recombinant erythropoietin,
the red cell hormone, we can stimulate the production of red blood
cells. As soon as we have the tools to regulate the production of all
the cellular elements of blood, we will have achieved a state where we
can control host defense.
How do these blood stimulating factors work? The key to hormone action
as we understand it today is the receptor. There is probably no more
graceful a receiver in the history of football than Lynn Swann. Here he
is shown pulling the hormone, that is the football, and in the process,
stepping on a hapless Dallas Cowboy. The receptor for GMCSF and
many other hematopoietic hormones have been molecularly cloned,
and I show you here their structure in the form of a diagram. The GMCSF
molecule binds to its specific receptor, setting up a signal which
regulates the expression of new genes in the target cell. The details
of the signaling process are not yet defined but it is likely that
phosphorylation and de-phosphorylation of critical proteins are
important intermediary steps. The precise mechanism of how the
hematopoietic hormones function at the cellular level will be worked out
by the turn of century and will provide us with a host of new
therapeutic avenues thanks to recombinant DNA technology.
In addition to receptors fixed to the cell surface, cells can also
produce soluble receptors that float out into the area around the
cells and are capable of binding hormone in the extra-cellular milieu.
Such soluble receptors have been identified for all of the hematopoietic
hormones and many of the growth factors. Last night you learned
about RNA splicing and how from a single gene a different variety
of the protein can be made. This is an example of control of protein
function by alternative splicing. Since the soluble receptor is missing
the portion that would lock it into the cell membrane, it comes flying
out of the cell and is able to bind the hormone outside the cell. About
half of the receptors made are locked into the membrane and these
function in the signaling process. The hematopoietic hormones not only
stimulate the production of cells, but they also increase the
function of individual cells. As can be seen in this scanning
electron micrograph, GMCSF and its neutrophils in the bottom panel
caused a marked ruffling of the membrane and increased intracellular
communications. These changes are associated with heightened killing
capacity of the cells against micro-organisms and increases in function.
These are "angry" neutrophils; these are neutrophils prepared to kill
While the life of the flesh is in the blood, the death also lurks there.
This handsome young woman is suffering from acute leukemia and
the spots that you see on her face and chest are due to bleeding in the
skin caused by inadequate production of platelets.
This is what the blood looks like in leukemia, with a dramatic increase
in the number of abnormal and non-functioning white cells. The white
blood cells grow out of control and if not treated this quickly leads to
the death of the individual from either infection or bleeding.
We are now combating a new blood disease caused by a virus with a
devastating impact throughout the world. The HIV virus, which
likely originated in monkeys, is highly pathogenic to humans. It leads
to inactivation of a class of lymphocytes in the blood that disrupts the
immune system, preventing a normal host defense response to
micro-organisms which otherwise would have low pathogenicity. Cancer,
AIDS, and auto-immune diseases, such as lupus erythmatosis and
rheumatoid arthritis, all progress because of defective host
defense. Thus host defense was the first, and will be the final,
frontier in human therapeutics. The application of molecular biology
has provided us with new tools that will allow us to positively impact
I suspect that the medicine of the 21st Century will concern itself
primarily with the means to prevent and treat disease by enhancing host
With the availability of the colony stimulating factors we found new
weapons to treat disease. I show as an example the course of a professor
at the University of California at Los Angeles, who had a type of
leukemia known as hairy cell leukemia. He was admitted to the
hospital with low blood pressure (known as hypotension) and in a
state of shock. Antibiotics were ineffective and his circulating level
of neutrophils was zero. Experimentally we gave GMCSF and surprisingly
we found that the white blood cellcount began to rise; it rose from a
level of zero up to many thousands, an as soon as the neutrophil count
began to rise, the patient's fever went away and blood pressure returned
to normal. We stopped the treatment with the colony stimulating factor
and surprisingly he was thereafter able to maintain an adequate
neutrophil count of 2,000. We gave him a recombinant GMCSF to stimulate
neutrophil production and to our pleasure and surprise we found that
these cells functioned normally and were able to overcome a lethal
infection. Subsequently, he was treated with another product of
recombinant DNA technology, alpha-interferon, with excellent
control of his leukemia. He is currently alive and well and teaching at
This is the essence of natural host defense therapy. Using the body's
natural regulators, produced by recombinant technology to enhance the
body's resistance to attack from outside by micro-organisms, as well as
from within by cancer.
Clinically useful products of biotechnology now comprise a very long
list, including hormones such as human insulin to treat diabetes,
growth hormone to treat dwarfism, the interferons are used in
cancer therapy and the treatment of viral infections, and I have already
discussed the colony stimulating factors. Replacement of clotting
factors, such as Factor VIII which is deficient in
hemophilia, the production of vaccines such as those for
hepatitis and hopefully, for HIV, and the replacement of enzymes
and other molecules deficient in genetic disease will dramatically
change the practice of medicine as we know it.
I'm now going to show you a shocking picture. It is of a man who
ultimately died of a condition called Graft-versus-Host Disease.
He received a bone marrow transplant from an identical tissue-matched
sibling. The transplanted bone marrow gave rise to immune cells which
recognized the new host as foreign and led to this lethal complication.
If host defense cells are capable of destroying the entire body,
certainly they can destroy a tumor. Why don't we recognize and destroy
our own cancer cells? The answer to this important question is
uncertain. But it appears that we do not recognize our tumor cells as
foreign to a degree sufficient to mount an effective attack. With the
newfound ability to stimulate host defense cells, strategies are being
developed to use molecularly tailored monoclonal antibodies, to direct
white blood cells to the site of the tumor so that malignant blood cells
can be killed by the body's own defense. The little white triangles
here are representative of altered monoclonal antibodies that are
directed at an antigen on the tumor. Then with the stimulation of host
defense cells, these will come and home in on the tumor and hopefully
provide an effective control.
I would like to end my talk with a discussion of the Star Wars-like
technology for treating disease--the actual transfer of genes.
Soon we should be able to treat this important disease--sickle cell
anemia--by transferring into the hematopoietic stem cells that I
showed you a normal beta hemoglobin gene. The defect in sickle
cell anemia is a single nucleotide change in the beta hemoglobin gene.
Shortly we should be able to fix this dramatic abnormality by gene
therapy. While the repair of genetic abnormalities with gene transfers
is feasible with the technology at hand, the treatment of cancer by gene
transfer is somewhat more speculative. Nonetheless, before the end of
this century we will be able to clinically alter tumor cells by
inserting genes that shut off their malignant capability or that make
them easily recognized by the immune system. Similarly, we may be able
to introduce genes which when activated by drugs, leads to the death of
the cancer cell.
Who is the most important worker in the research laboratory? Clearly,
it is the president of the United States, for without a coherent and
far-reaching national biomedical policy and financial support, the
promise of molecular technology will not be fulfilled.
Lastly, it is necessary that we put technology in its proper
perspective: it is a tool and a tool which should be used to improve
the human condition. Technologies may change but the fundamentals of
human accomplishment will not.
In closing, I hearken back to the 60's when I first came to this
beautiful city. I leave you with the spiritual imperative exemplified
by the Beatles which saluted the creative energy of mankind. "People
think the Beatles know what's going on. We don't. We're just doing it."