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 blood cells.

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 neutrophils.

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 cell colonies.

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 quantities.

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 in culture.

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 invading micro-organisms.

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 host defense.

I suspect that the medicine of the 21st Century will concern itself primarily with the means to prevent and treat disease by enhancing host defense mechanisms.

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 the University.

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."

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