RESEARCH

New enzyme found in leukemia patients Fatal disease may be

caused by altered DNA

produced by polymerase

that uses RNA from

RNA-containing viruses

Dr. Robert C. Gallo and his coworkers find that a derivative of the drug rifampicin inhibits the DNA polymerase

An enzyme that may be a major link in the chain of events leading to hu­man leukemia has been found in the blood of three leukemia patients. The discovery is particularly important if this fatal disease is caused—in at least some of its forms—by a virus.

In the past five months, Dr. Robert C. Gallo of National Cancer Institute and Dr. Stringner ("Sue") S. Yang and Dr. Robert C. Ting of Bionetics Research Laboratories, Bethesda, Md., have jointly studied the lymphocytes in the peripheral blood of three pa­tients with acute lymphocytic leuke­mia. They found that, in all three patients, these cells contained an RNA-dependent DNA polymerase. On the other hand, they found no detectable amounts of this enzyme in lymphocytes of 48 normal people. Recognizing, of course, that additional data are needed, Dr. Gallo and his group are planning to look for this enzyme in other patients with leukemia or other forms of cancer believed to be caused by RNA viruses.

Dr. Gallo and his associates are the first scientists to report the presence of RNA-dependent DNA polymerase in the cells of cancer patients—in fact, in the cells of any humans. In recent

months, several U.S. scientists have re­ported such an enzyme in RNA viruses and in cancerous tissue-culture cells of mice. Discovery of this enzyme in humans, which in the past few weeks has touched off intense excitement in the cancer research field, was disclosed by Dr. Gallo early last month at a closed meeting of the Pasteur Institute in Paris, France. Dr. Gallo and co­workers made their first published re­port of their findings in the Dec. 5 issue of Nature [228, 927 (1970)].

The enzyme they found is a type of DNA polymerase. That is, it is an enzyme involved in forming the DNA polymer, the basic hereditary material of the cell. DNA polymerase was first isolated back in 1956 by Dr. Ar­thur Kornberg, then at Washington University in St. Louis.

Normally, a cell makes DNA by us­ing another DNA molecule as the tem­plate for assembling the molecule's building blocks, the purine and pyrim-idine nucleotides. Since DNA itself is the template for DNA synthesis, the enzyme required to do this is a DNA-dependent DNA polymerase. The en­zyme that Dr. Gallo and associates found in humans serves a different pur­pose. It forms DNA by using RNA as its template.

The idea that DNA can be made from an RNA template was until re­cently considered by many scientists to be impossible. In fact, the term "DNA-dependent" DNA polymerase was never even used. Since scientists believed that DNA could be formed only off a DNA template, the term would have been regarded as absurdly redundant, like speaking of a canine dog. However, now that either a DNA or an RNA template can be used in synthesizing DNA, the terminology has had to be refined.

Leukemia mechanism. As Dr. Gallo and associates visualize the pro­cess of lymphocytic leukemia, on the basis of the suggestive evidence pres­ently available, the disease may be­gin when an RNA virus enters a nor­mal lymphocyte precursor cell (a so-called "stem cell"). The virus carries with it the RNA-dependent DNA poly­merase. Because of the presence of this enzyme the RNA virus can act as a template for the formation of

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o DMF

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CH3

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\ " 56% Yield

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DNA. The resulting DNA may then become part of the DNA of the host cell's chromosomes. This, in turn, would make the chromosomes abnor­mal and cause the cell to reproduce itself as a cancer cell.

Discussing the possible causes of leukemia, Dr. Gallo speculates that the disease results from the failure of the white cells to mature normally (a proc­ess known as cell differentiation). A white cell when young, he explains, tends to multiply rapidly. As it grows older, however, it completely loses its ability to multiply. If some interven­ing event, such as the altering of the host cell's DNA by an RNA virus, can retard or prevent the cell's process of maturing, the cell will retain much longer than normally its ability to mul­tiply. This, in turn, can cause the great accumulation of immature white blood cells that characterizes leukemia.

The latest research by Dr. Gallo and his group is a direct outgrowth of the dramatic discovery made earlier this year by Dr. Howard M. Temin and Dr. Satoshi Mizutani of University of Wisconsin and by Dr. David Baltimore of MIT. As they reported separately in two papers in the June 27 issue of Nature [226, 1211, 1209 (1970)], they had discovered an RNA-depend-ent DNA polymerase in several RNA viruses that cause cancer in laboratory animals. This finding was soon con­firmed by Dr. Sol Spiegelman of Co­lumbia University and by others. Dr. Spiegelman identified this enzyme in eight cancer-causing RNA viruses, but not in four other RNA viruses that do not cause cancer. In addition, Dr. Maurice Green of St. Louis University has announced that he and coworkers have found a comparable enzyme in mouse cells transformed into cancer­ous cells by an RNA cancer virus. All of these enzymes are able to synthe­size DNA off an RNA template.

Asked whether the enzyme he has found is the same as that originally discovered by Dr. Temin, Dr. Gallo says only that it is "analogous." Thus far, Dr. Gallo has not isolated his en­zyme in completely pure form. The material has, however, been purified by a factor of 200 to 300. Work is continuing in an effort to purify it still further and obtain a clearer under­standing of its properties.

Iconoclast. All of the work on RNA-dependent DNA polymerase runs counter to what Dr. Francis H. C. Crick of Cambridge in the mid-1950's called "the central dogma of molecular biology." According to this belief, ge­netic information flows only in one di-rection-from DNA to RNA. The RNA, in turn, makes possible the for­mation of proteins.

In 1964, the iconoclastic biochemist Howard Temin proposed a theory to

explain what for years had seemed totally incomprehensible—namely, how some viruses containing only RNA can produce cancer in laboratory animals. A cancerous cell, of course, is one that is capable of multiplying. Only if a cancer-causing component is present in the genetically stable DNA mole­cule—not in the transient RNA mole­cule—can a duplicate of this cancer-causing substance, which is part of a gene, be passed from one cell to its daughter cell and then to succeeding generations, as is necessary for the con­tinuing propagation of a tumor.

Dr. Temin proposed that the RNA virus causes cancer by acting as a tem­plate for DNA, which is subsequently incorporated into the host cell's ge­netic material. Since this concept so blatantly contradicted "the central dogma," it was almost universally re­jected. In recent months, however, Dr. Temin's work has been confirmed in many laboratories in the U.S. and elsewhere and is now widely accepted. In fact, every RNA cancer virus tested to date has been found to contain an enzyme capable of forming DNA off the virus's RNA template.

Polymerase inhibition. Part of the significance of the latest NCI-Bionet-ics Research findings is that, if the RNA-dependent DNA polymerase that Dr. Gallo and his group have dis­covered in human leukemic cells is re­quired for the cancer-producing effect of the RNA virus, a substance that in­activates this enzyme might prevent leukemia. Preliminary research in this area has led to encouraging results.

At the suggestion of Dr. Maurice Green of St. Louis University, Dr. Gallo and his group have been testing an antiviral antibiotic, rifampicin, and a number of its derivatives as possible inhibitors of this enzyme. Rifampicin and some of its analogs are made by Gruppo Lepetit, a Dow Chemical sub­sidiary in Milan, Italy, and have been studied by Dr. Green as possible drugs for blocking the action of viral en­zymes in animal cells.

In test-tube experiments, Dr. Gallo has found that rifampicin itself inhibits only very slightly the RNA-dependent DNA polymerase he has been study­ing. On the other hand, a related compound, IV-demethylrifampicin, completely inhibits this enzyme.

Dr. Gallo and coworkers plan in the near future to test JV-demethylrifam-picin and related compounds for treat­ing leukemia and other forms of can­cer in rats and mice. If the compound is found to be effective and safe in animals, it will be tested clinically in patients with leukemia or possibly other malignant diseases. Thus, con­ceivably, a major forward step may be taken in the treatment of virus-caused cancers.

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DEC. 7, 1970 C&EN 49