Recombinant

DNA

and Surgery

JAMES M. BROWN, M.D.,* ALDEN H. HARKEN, M.D.,* and JOHN B. SHAREFKIN, M.D.t

We have the ability to isolate DNA from tissue, determine its base-pair sequence, and ask if a gene of interest is present. DNA strands can be isolated from one type of cell or organism, cleaved, and inserted (recombined) with DNA from another cell or organism. Recombinant DNA techniques have already improved health care by providing clinically useful quantities of pure human protein hormones such as erythropoietin, insulin, and growth hormone. Furthermore these techniques may increase our understanding of cellular growth control mechanisms to a level that was previously unattainable. They will also increase our knowledge of the development of major diseases and provide a means of specific nontoxic therapies for these diseases. Surgeons will need to understand basic DNA research terminology to keep up with the revolution in medical therapies that these techniques will cause. Our purpose is to begin the process of linking surgery to DNA.

The Significance of DNA to the Surgeon DNA and the control of its expression determines the function of the cell. DNA is the root of tissue repair and regeneration. It is also the root of cancer and other common surgical diseases. DNA research has been useful in the detection and identification of new molecules. Pure human proteins can be synthesized by DNA techniques. The underlying cause of surgical disease can be diagnosed with DNA research techniques and these techniques will change surgical practice. Therefore the surgeon's knowledge of basic DNA biology must expand. Changes in DNA sequence within certain classes of genes called oncogenes have been associated with tumor formation. We are entering an era in which antitumor therapies will not be designed from a combination of Supported in part by the National Institute of Health and the American Heart Association. The opinions contained in this review are exclusively those of the authors and do not necessarily reflect those of the Department of Defense. Address reprint requests to James M. Brown, M.D., Department of Surgery, Box C-305, University of Colorado Health Science Center, 4200 East 9th Ave., Denver, Colorado, 80262. Accepted for publication October 11, 1989.

From the Department of Surgery, * University of Colorado Health Science Center, Denver, Colorado; and the Uniformed Services University of the Health Sciences,t Bethesda, Maryland

judgment and statistics about each anticancer drug's effect on disease free survival. Therapies will include manipulation of gene expression. Oncogene research can identify specific base-pair substitutions in DNA sequences that make cells tumorigenic. Therapeutic agents are being designed to produce DNA sequence-specific suppression of the phenotypic expression of oncogenes. This highly specific therapy contrasts sharply with the present comparatively indiscriminant chemotherapy in which rapidly dividing cells (both neoplastic and normal) are killed. Surgeons will continue to play an active role in the care of patients with cancer and other diseases, but they will play an enhanced role if they understand recombinant DNA language. They will have an important role in the acquisition of tissue for DNA sequence analysis, which will be needed to allow DNA sequence-specific treatment. Thorough understanding of DNA and oncogene research, however, requires a basic understanding of DNA and the techniques of DNA analysis. The purposes of this review are to discuss: (1) the theory behind recombinant DNA techniques, (2) oncogenes and their isolation, (3) oncogenes in non-neoplastic disease, and (4) the potential impact of recombinant DNA technology on clinical medicine and surgery. The Study of DNA DNA Fragment Isolation

The enormity of information contained within the human genome can be difficult to comprehend. To date approximately 600 of the estimated 100,000 human genes have been sequenced.' The human genome consists of

178

VOl. 212.- NO. 2

DNA AND SURGERY

three billion nucleotide base pairs. Assuming one letter per nucleotide, it would take 13 volumes of the Encyclopedia Britannica to list the sequence of genetic nucleotide pairs for a single human cell.2 Furthermore, because there are four nucleic acid bases (cytosine, thymidine, guanine, and adenine), DNA sequences only 20 base pairs in length could exist in 420, or about 1.1 X 1012, different configurations. This means that the study of DNA must be based on exact sequence specificity and on techniques of isolation that preserve the integrity of the DNA molecule. DNA is double stranded (Fig. 1). It can temporarily

FIG. 1. The DNA double helix. The DNA strands can separate to allow replication.

179

unwind its two strands to allow one strand to serve as a template for the transcription of a base-pair-specific sequence of RNA. RNA is transported from the nucleus to the cytoplasm of the cell where translation of the RNA sequence into proteins occurs. The study of DNA can be directed at the DNA in the nucleus or its messenger product, RNA. We can study genes (DNA) or their expression (RNA).34 To study DNA it must first be extracted from a cell culture or tumor tissue sample. Extraction of nucleic acids is usually accomplished by lysis of cells with detergents and digestion of proteins with nonspecific proteases. Second chloroform and phenol are used to remove remaining lipids and proteins. Ethanol is added in the presence of an elevated salt concentration (100 to -500 mmol/L [millimolar]) to precipitate the DNA. Although these procedures result in chromosomal breaks, long enough pieces of DNA remain (about 70,000 base pairs) such that the entire lengths of most functioning gene sequences remain intact. 7-10 After extraction DNA is treated with a restriction enzyme. Restriction enzymes, which were discovered in bacteria, cleave DNA in a highly specific fashion. Each restriction enzyme (of the more than 100 known) recognizes a unique sequence of DNA base pairs and cleaves the DNA only at sites containing this sequence. DNA treated with a restriction enzyme is cut into many fragments corresponding to the number of times the restriction enzyme's recognition sequence occurs (Fig. 2). The crucial point in this process is that restriction enzymes recognize specific short (4 to 8) nucleotide base-pair sequences. Therefore the DNA from many cells treated with a restriction enzyme will be cut into many fragments, all of identical size and sequence. For example, suppose we are interested in studying a gene that exists between point A and point B on a chromosome. A restriction enzyme can be selected that will specifically cut DNA at or near points A and B. If 100,000 cells are in the original sample, then nearly 100,000 copies of the gene or gene fragment will result from restriction enzyme cleavage. Because the restriction enzyme cuts specifically at points A and B, all 100,000 gene copies should have the same sequence and same length.7'0 After restriction enzyme digestion, gel electrophoresis is used to separate DNA fragments (Fig. 3). Agarose (usually at 0.5% to 1.0% solution) is mixed with ethidium bromide. Ethidium bromide fluoresces under ultraviolet light. Because it stereospecifically intercalates with the spiral groove of the DNA helix, ethidium bromide can be used as an indicator for the presence of DNA on a gel. The heated ethidium bromide and agarose solution is cast into a mini (50 mL), midi (150 mL), or large (500 mL) gel tray and is hardened at 4 C. A gel comb is used to create uniformly spaced wells across the width of the gel.

180

BROWN, HARKEN, AND SHAREFKIN

Ann. Surg. * August 1990

l7a bp

llSS~~~~~~~~~~~~~~~bp

R

CXutwNh Md ar

;

S1M

1101 b

IOUb@r

-

-.

X s@*^;iX-

i~ ~ ~ ~ ~ ~b47 p ;;S bp

A2

1

.

.?s

--5-

FIG. 2. Treatment of the virus SV40 DNA with the restriction enzyme HindIlI results in six DNA fragments. Gel electrophoresis of uncut SV40 DNA results in one large, slowly migrating brand (1), whereas treatment with HindlIl results in six fragments (2) that migrate in the gel in inverse proportion to their length. From Darnell J, Lodish H, Baltimore D. Examining the sequences of nucleic acids and proteins. In Molecular Cell Biology. New York: WH Freeman and Co., 1986, p. 247 (Permission granted from the publisher).

j

The sample of DNA is placed into the wells along with appropriate standards (DNA of known length). A charge (5 volts per 1 cm of gel length) is applied across the gel and the charged DNA molecules will migrate. Because of the negative charge on the phosphate backbone of DNA, the DNA fragments will move in a straight line toward the positive electrode, creating a tract called a lane. Fragments of all sizes have the same charge-to-mass ratio, so the only factor determining the rate of migration of a DNA fragment through the gel is its length. Suppose there are many gene fragments (500 base pairs long) after DNA extraction and restriction enzyme cutting. If the gel is illuminated under ultraviolet light there will be a discrete fluorescing band of DNA detected at a distance from its original start point corresponding to a DNA standard size 500 base pairs long. Other bands representing other DNA fragment lengths will also be visible.5'7'0 For example, if the DNA is extracted from a sample of pure SV40 virus particles and cut with a restriction enzyme called HIND III, the DNA will be cut into six fragments because the SV40 virus DNA contains six sites with the sequence recognized by this restriction enzyme (Fig. 2). If a gel is run with the uncut SV40 DNA one large, slowly migrating band will be seen under ultraviolet light. If the DNA is cut with HIND III, however, six faster

migrating (smaller) fragments of DNA will be seen under ultraviolet light as six discrete bands.6 Southern and Northern Blotting Southern blotting (Fig. 4), derived from the name of its inventor, E. M. Southern, is the name given to the process of transferring the product of gel electrophoresis onto a piece of nylon or nitrocellulose filter paper.5 7-'0 Before blotting the DNA in the gel is rendered single stranded or is denatured by soaking the gel in sodium hydroxide. Therefore the DNA is transferred to the filter in a single-stranded form. Nylon is less brittle than nitrocellulose and so has gained favor. The transfer is accomplished by sandwiching the gel next to a piece of nylon filter. By either suction or capillary action, a buffer solution is pulled uniformly through the gel at right angles to the plane of the gel using absorbent paper. The flow of buffer from the gel toward the filter paper transfers the DNA (or RNA in which case the procedure is called Northern blotting) to the filter. The DNA is bound to the nylon by covalently crosslinking the phosphate backbone of the DNA to the nylon using ultraviolet light and baking in a vacuum oven. Because the DNA backbone had been crosslinked to the filter, single-stranded DNA will remain

0H~ ~ ?T9'; DNA AND SURGERY

Vol. 212. No. 2

.0

b

)

&~ C' -"N' )

D

fragments separate according side of the gel (a) the fragments migrate. Shorter fragments move farther than larger fr-agments in an electric field (b). Standard DNA fragments of known length, such as HindIII cut SV40 DNA, can be used as a measuring stick for fragment

FIGs. 3A and B. Gel

electrophoresis.

size. After DNA is placed into

size.

the filter in the same pattern of sizes as on the gel slab. The nucleotide bases of the single-stranded DNA on the filter are in this way exposed so that a so-called DNA probe can be used to determine whether a specific sequence of DNA is on the filter.7'0 on

a

to

181

a

DNA

well

on one

DNA Probes

After extraction, restriction enzyme cutting, gel electrophoresis, and blotting, the fragments of DNA derived from an entire genome are arranged by size on the nylon filter. The desired specific gene sequence can be found by using a segment of DNA called a probe. Use of probes takes advantage of single-stranded DNA's tendency to bind with or hybridize to a complementary single-stranded DNA segment. Probe use also depends on two physical properties of complementary DNA binding called cooperativity and stringency. DNA strand binding is said to be cooperative because the longer and more precisely matched two segments of DNA, the greater the thermal energy needed to separate them. Higher temperatures are required to separate longer segments of complementary DNA compared to shorter, less perfectly matched segments. A probe of 100 base pairs tends to remain bound to complementary DNA (cDNA) at temperatures that would cause denaturing of shorter segments or less perfect matches. If the probe used is an exact base-pair match to the sought for gene, then binding will occur at higher temperatures only to that gene's DNA on the filter paper.7'10 Oligonucleotide (relative short DNA segments) probes can be obtained commercially and are designed to be a perfect base-pair match to a segment ofthe gene of interest. The probe is labeled with P32. The nylon sheet is placed

,1

.- 1,.i-0~: ,~'- ;~i_.~ ~ ~ ~ ~ ~ ..~ ~...~ .

FIG. 4. Southern blotting. DNA cleaved into various lengths by a restriction enzyme is gel electrophoresed. The gel is treated by alkaline washing to render the DNA single stranded. A buffer is pulled through the gel so that the DNA strands migrate to the nylon filter. Radiolabeled shorter DNA fragments called probes are used to search for a segment of DNA on the filter paper. A positive result occurs when the labeled DNA, bound to complementary DNA on the filter, appears on an autoradiograph. From Darnell J, Lodish H, Baltimore D. Examining the sequences of nucleic acids and proteins. In Molecular Cell Biology. New York: WH Freeman and Co., 1986, p. 249 (Permission granted from the publisher).

BROWN, HARKEN, AND SHAREFKIN

182

in a solution with the probe at a temperature resulting in maximum stringency; that is, the conditions that maximize specific DNA binding and minimize nonspecific binding of shorter or imperfectly matched DNA segments. Hybridizing at 60 C, for example, is a more stringent condition than 37 C. If the probe is appropriately designed and the hybridizing reaction is performed at the optimal level of stringency, then the probe will bind only to the gene of interest with an exactly complementary DNA sequence on the filter. The filter paper now containing the probe hybridized to the gene under study is placed into a film cassette at -70 C. At this temperature, silver bromide crystals on the x-ray film are activated. When the film is developed, areas of the filter where specific probe to DNA binding occurred will show up as a dark band. This film exposure process is called autoradiography.7 The specificity of the result can be determined by the specificity of the probe and the conditions (temperature) at which the hybridization is performed. Using these methods, one gene within thousands of other genes can be located. So gels characterize DNA fragments by size. Hybridized blots characterize DNA fragments by exact DNA base-pair sequence. After one probe is used on the electrophoretic blot, that probe can be removed from the nylon with hot alkaline washes, leaving the single-stranded DNA behind. Then another probe, to another gene of interest, can be hybridized to the filter.7-'0 DNA or RNA probes can be used to address simple questions. For example given a piece of tissue, suppose we want to know if the myc oncogene is present. Because the sequence of the myc gene is known, a probe for myc can be designed, purchased, and labeled with P32. After DNA extraction, gel electrophoresis, and blotting, specific probe hybridization appearing on an autoradiograph as a dark spot would indicate that the gene or DNA segment is present in the tissue sample. Extracting the cell's mRNA and using, instead, an RNA probe not only can determine the presence of a gene but an estimate of its activity or expression into mRNA product can be made. That is we can take advantage of a probe's specificity to the DNA or RNA, to which it is complementary. Probes give answers to these questions: Is a gene present? How much of the gene is present? Is the gene's expression enhanced?

Ann. Surg. * August 1990

transforming cancer gene (oncogene) is introduced into the 3T3 cells in culture, the cells lose their contact inhibition and pile on each other to form a visible focus (called focus formation) (Fig. 5). Introducing human tumor tissue DNA into mouse cell culture is done by a process called transfection. DNA from a sample of human tumor tissue is isolated and added directly to a culture flask of mouse 3T3 cells as part of a calcium phosphate precipitate. Surprisingly the mouse 3T3 cells will take up, incorporate, and express the human DNA. 3T3 cells that have lost their growth regulation by this DNA transfection process are said to be transformed. The oncogene in the DNA

DNA from tumor cells or tumor tissue

..

.*

Calcium phosphate-DNA coprecipitate

Apply to NIH-3T3 cells

Clinical Applications

Finding An Oncogene (Focus Formation, Transfection, and Transformation)

How can these basic techniques be used to identify one cancer-causing gene within a sample of tissue? The first step is to develop an assay system for tumorigenicity. This is provided by cultured cells, typically the National Institutes ofHealth mouse 3T3 cell line. 3T3 cells in culture in uniform monolayers because their growth is regulated by a property called contact inhibition. When a

grow

Transformed cells: loss of contact inhibition and focus formation FIG. 5. Transfection of mouse NIH/3T3 cell culture as part of a calcium/ phosphate precipitate. This process, called transfection, results in the uptake, incorporation, and expression of the tumor cell DNA. This will, in some cultures, result in the cell culture equivalent to a tumor called focus formation. By taking cells from the focus of transformed cells and repeating the transfection process, many cells that contain an oncogene can

be grown in cell culture.

Vol. 212 * No. 2

DNA AND SURGERY

that has been spread over thousands of cells in a culture will betray itself by causing cell transformation. This will result in visible focus formation, analogous to a miniature tumor. By taking a sample of cells from the focus of transformed cells and repeating the transfection procedure, many transformed cells can be grown in cell culture. These transformed cells can then be injected into nude mice. Hard tumor formation is the evidence of the presence and expression of a cancer-causing gene.5'7,0 Growing the transformed cells in culture provides a quantity of DNA sufficient for isolation and study. The DNA can be isolated from these cells and cut into fragments with restriction enzymes. Somewhere among these fragments from the transformed cells there may be a sequence of human DNA that can cause cancer. The task then remains to find it and identify its sequence. Locating the gene that transformed the cells can be accomplished by using phage lambda virus as a Trojan horse. This virus consists of a virus coat and single piece of DNA. The two ends of the viral DNA (called the arms) are the only DNA necessary for replication. The middle phage DNA portion, called the 'stuffer' region, can be replaced with another DNA sequence by cutting the arms with restriction enzymes and inserting the DNA from tumor cells. This is accomplished by mixing phage DNA with the stuffer region cut out with DNA isolated from tumor cells. In some phages the cancer-causing segment of DNA will be incorporated into the stuffer region with the help of ligase enzymes. These viruses can be spread over a lawn of bacteria (Fig. 6). In 6 hours many virus colonies will have caused visible areas of lysis in the bacterial lawn. Several of the viral colonies may be derived from viruses that have the human cancer gene incorporated into the stuffer region. The final step takes advantage of a unique feature of human DNA. It contains a specific sequence of 300 base pairs, called the ALU sequence, which is repeated many thousands of times in the human genome. i7'10 A template of the viral colonies on the bacterial lawn is created by touching a nylon filter to the phage-infected lawn (Fig. 6). The nylon filter adsorbs the intact virus particles. The protein of the phage coats can be lysed with detergents, and the DNA is then rendered single stranded by alkaline washing. A probe specific for the ALU sequence labeled with P32 can be hybridized to the nylon sheet and an autoradiograph is made. The one or several phage colonies that contain the human cancer gene detected by its contained ALU sequences will be identified as one or several dark spots on the x-ray film. Because the phage-infected bacterial lawn was originally transferred directly to the filter in mirror image, the phage colony containing the gene can be identified and grown in abundance by repeat culturing. Then by isolating the DNA fragments from the phage DNA and treating the phage DNA with a restriction enzyme to cut out the stuffer region, many copies of the cancer gene can be isolated. The

183 Phage virus colonies growing on bacterial lawn One colony contains oncogene with ALU sequence | Place nitrocellulose filter on plate to pick up phages from each plaque

Nitrocellulose fitter

Alkaline wash

Single-stranded phage DNA bound to filter |

Hybridize with ALU probe and autoradiograph

Autoradiograph signal indicates colony with oncogene

FIG. 6. The phage virus is used to isolate a human oncogene. After mouse NIH/3T3 cells transformed by human oncogene DNA are grown in quantity, their isolated DNA can be incorporated into a phage virus. However only one or several of hundreds or thousands of viral colonies will contain the intact oncogene in addition to the ALU sequence, the repeating DNA base-pair sequence unique to human DNA. After the viral DNA is transferred to the nylon filter, a radiolabeled probe complementary to the ALU sequence can be used to locate the one viral colony that contains a human gene.

ability of this DNA to transform culture cells and cause hard tumor formation can be reconfirmed after repeat transfection of 3T3 cells. Finally the exact base-pair sequence of the gene can be determined." This process, which starts with only a piece of human tumor, can thus exploit recombinant DNA techniques to allow identification, isolation, and exact sequencing of the oncogene that actually converted a normal phenotype to a cancerous phenotype. Polymerase Chain Reaction It is impractical to apply these techniques to clinical medicine now because they might take a dozen workers many months to perform. Advancing DNA research technology, however, may change this situation. For ex-

184

BROWN, HARKEN, AND SHAREFKIN

ample the polymerase chain reaction (PCR) allows rapid amplification of the quantity of a specific DNA sequence from a tissue sample in a matter of hours. 2 The sensitivity of the procedure is so great that the DNA from one bacterium in 1 mL of water can be detected and a patient's tissue can be analyzed with only milligram-sized biopsy samples. The PCR is based on: (1) a bacterial DNA polymerase enzyme from the obligate thermophile bacterium Thermus aquaticus, which remains stable at 90 C, and (2) newly developed thermal cycling machines that can be programmed to repeat cycles of multiple temperatures between 0 C and 100 C. Suppose, for example, that only two copies of a specific gene with a known DNA sequence are present in a tissue sample. By designing appropriate short segments of DNA with sequences complementary to the ends of a segment of this known sequence, primers can be furnished that will allow a DNA polymerase to make copies of each strand of this known segment. By then adding enough free nucleotides as building blocks, and the T. aquaticus DNA polymerase, a reaction sequence can be started to amplify exponentially the number of copies of the desired gene. Using a thermal cycler, the reaction mixture is first heated to 90 C to separate all DNA into single strands. Second, the cycler cools the reaction to 46 C to allow the primers (one complementary to each end of the gene sequence being studied) to combine in sequence-specific fashion with the two gene copies. Third, the reaction mixture is heated to 70 C, the optimal temperature for the T. aquaticus DNA polymerase. The polymerase uses the DNA primers and the free nucleotides to make two segments of DNA, each complementary to the original two DNA segments. When the machine heats the reaction to 90 C again, all the strands separate. After the machine cycles 30 times, which only takes a few hours, the number of copies of the original two genes will be 230! The PCR technology allows a specific gene to be amplified from the DNA of an extremely small tissue sample. The amplified DNA can be run on a gel, blotted, and hybridized to the probe specific for the DNA segment of interest. The PCR also can be used to study expression of genes by detecting the presence of messenger RNA. The viral enzyme reverse transcriptase (RT) makes DNA from an RNA template. Using RT before the PCR reaction with the presence of messenger RNA can be inferred from detection of its complementary DNA sequence. That is, the presence of an oncogene alone may not be as significant as the fact that it is expressed or is transcribed to generate an excessive amount of mRNA and protein. Recent work'3 has demonstrated that the PCR can identify the presence of the human immunodeficiency virus in human blood samples up to 3 years before a positive test for the antibody. The polymerase chain reaction had detected specific retroviral DNA sequences in patients with multiple sclerosis, 14 and has amplified DNA segments from 13,000-year-old prehistoric fossils and 5000-yearold mummies.'5 That is, because the polymerase chain

Ann. Surg. * August t1990

reaction dramatically amplifies a specific DNA (or RNA) segment it has been and will be used to achieve levels of accuracy and specificity heretofore unachievable in clinical medicine.

Proto-oncogenes and Anti-oncogenes

The definition of an oncogene is that of a gene which transforms cells, but a normally functioning oncogene does not cause cancer. This definition derived from studies of tumor causing viruses which identified viral tumor genes."," Surprisingly these genes (or closely related genes) exist in normal vertebrate cells and are called protooncogenes. 18 Closely related cancer associated genes have been grouped into gene families based on function, for example, the ras or myc oncogene families. 16"17 However, oncogene appears in some ways to be a misnomer because oncogenes code for classes of regulatory proteins which are centrally involved in cellular growth regulation. These oncogene protein products have been classified into growth factors, growth factor receptors, and the so called G-proteins which are guanine nucleotide binding proteins located in the plasma membrane and involved in cyclic AMP messenger cascades. Oncogene products may affect the growth of cells by their actions in the nucleus or at the cell membrane. The platelet derived growth factor (PDGF) B chain, for example, is almost identical to the transforming protein product ofthe simian sarcoma virus (SSV). Antibodies to PDGF revert SSV transformed cells to normal phenotype. The SSV protein product has PDGF activity and acts via PDGF receptors.'9'20 Based only on the limited number of observations so far, it seems that oncogenes are important for more than cancer. They seem capable of controlling normal cellular function and wound healing. A classic example of the potential of oncogene research is the ras oncogene.'6 As little as a single point mutation in this gene has accounted for a change in the structure of its coded regulatory protein. This results in unregulated cell growth in vitro. The myc oncogene has been studied in human neuroblastoma tumors (neuroblastoma myc gene). The degree of this gene's amplification (number of extra gene copies) in one study has proven to be a better predictor of disease free survival and tumor behavior than clinical stage of disease. For example, study of the N. myc oncogene in stage II tumors has predicted tumor growth and disease progression.2'22 In contrast to tumor inducing, the retinoblastoma (RB) gene exemplifies the concept of a tumor-suppressing gene or anti-oncogene.2325 In retinoblastomas, both copies of the RB gene are missing. In cells that do not have the RB gene protein product, growth regulation is lost. That is, the term anti-oncogene is meant to imply that the gene is normally present and it or its protein product plays a central role in the regulation of cell growth. Finding a gene which causes neoplasm by its presence is easier than

Vol. 212 * No. 2

DNA AND SURGERY

identifying a gene which causes a tumor by its absence. The RB anti-oncogene illustrates an important point: cell growth is under both positive and negative control. Oncogenes can provide both types of control. Oncogenes or gene deletions have also been detected in tissue from common human tumors including small cell lung,26 breast,27 and colon carcinomas.2830 Single oncogene alterations, however, are often not sufficient to make cells cancerous. Often in these tumors, changes in more than one gene and more than one chromosome appear necessary for tumor growth.

Oncogenes and Non-neoplastic Disease Although their discovery occurred because of cancer research, oncogenes and DNA research will be important in multiple diseases. Oncogenes and their expressed products (proteins) have been measured in human atherosclerotic lesions. In particular, human coronary artery atherosclerotic plaque DNA has been used to transform NIH 3T3 cells in culture.3' The transformed cells have induced solid tumors in nude mice. In contrast, these changes were not induced by transfection with DNA derived from normal coronary artery. These findings suggest that atherosclerotic plaque expresses an oncogene. The same genetic alterations which cause cancer may also account for the smooth muscle proliferation of atherosclerotic plaques! Elevations in the mRNA for platelet derived growth factor have also been found in human atherosclerotic lesions.32 In rat models of myocardial hypertrophy, there is increased expression of the ras, fos, and myc oncogenes.3313 The pattern of oncogene expression is specific to the etiology of the cardiac hypertrophy. For instance, left ventricular pressure overload produces a different pattern of oncogene expression than thyroid induced cardiac hypertrophy.34 These preliminary findings suggest that oncogenes are central regulators of cell function and may show altered expression in many categories of disease. Is Synthetic DNA Therapy Possible?

The technology which allows rapid and automated determination of the presence of a base-pair sequence and level ofexpression of oncogenes is developing. The future of DNA research will involve designed DNA therapies based on knowledge of the DNA sequence alterations responsible for the cancer or disease. For example, recently36 an oncogene c-raf- relating to the tumorigenicity and radiation resistance of the squamous carcinoma cell line SQ-20B has been identified. Transfection of these tumor cells in cell culture using c-raf- 1 DNA in reverse (antisense) orientation contained within a DNA plasmid reduced both the tumor cells tumorigenicity and radiation resistance. That is, based on the knowledge of the DNA sequence which, in part, accounts for the cancerous phenotype, DNA of backwards direction was used to prevent the expression of that DNA sequence. The cells were not

185

killed. Their phenotype was altered toward normal. As another example consider the implications of DNA sequence specific inhibition of human immunodeficiency virus (HIV) expression and replication. Treatment of T cells infected with HIV in culture with an antisense segment of DNA made with phosphothioate oligodeoxynucleotides (altered DNA) prevented the expression of the viral gene rev protein product, which is essential for viral replication.37 The RB gene has been introduced into osteosarcoma cells lacking the RB gene using a nonvirulent viral vector.38 The addition of the RB gene to neoplastic cells suppresses their neoplastic expression in culture. It also suppresses the tumorigenic potential of these cells when injected into nude mice.38 The extraordinary fact about these DNA engineering experiments is that they are designed with complete DNA sequence specificity. That is, the attack on the tumor cell is designed to make it a normal cell again rather than to kill it. The normal bystander cells remain unharmed.

Cloned Genes Can Produce Human Hormones Genetic engineering laboratories have used recombinant DNA techniques to clone genes for the production of human hormones and proteins. Once the sequence of the gene to be cloned is determined, the gene's DNA can be spliced into a bacterial plasmid adjacent to a chosen promoter region. Promoter regions enhance the downstream transcription of sequences. Inserting the DNA next to a promotor region may enhance its expression into the desired protein product. Thus human DNA can be recombined (hence recombinant DNA) with the bacterial chromosomes. In this way bacteria can be 'trained' to make a human protein in therapeutically useful quantities.5 ,7-' Human 'recombinant' erythropoietin promises to lower transfusion requirements in chronically anemic patients.39 40 Recombinant human cytokines (interferons, tumor necrosis factor, and the interleukins) have been used successfully in the treatment of some neoplasms, such as hairy cell leukemia.4 t3 Recombinant human insulin has reduced the development of immunologic resistance to beef and pork insulin." Recombinant human growth hormone has improved the treatment of dwarfism.45 Human tissue plasminogen activator is used to promote reperfusion of occluded coronary arteries in the setting of myocardial ischemia." The astounding fact is that all these treatments occur using pure human protein. They are exact replicas of the naturally occurring human proteins. The basic science of DNA research has allowed these extraordinary improvements in the quality of health care.

Recombinant DNA Techniques Will Change Medical Therapy DNA research techniques have increased our understanding of the cause of cancer and other diseases resulting

186

BROWN, HARKEN, AND SHAREFKIN

from altered cell growth and regulation. These techniques will broadly change medical therapies. The treatment of bowel obstruction may not change, but the treatment of cancer and atherosclerotic disease will. Understanding the recombinant DNA language will enhance our role in surgical treatment and permit us to contribute to the use of DNA sequence-specific therapies as they develop in the future. References 1. Paris Conference (1987). Ninth international workshop on human gene mapping. Human gene mapping 9. Cytogenet Cell Genet 1987; 46:1-762. 2. McKusick VA. Mapping and sequencing the human genome. N EngI J Med 1989; 320:910-915. 3. Watson JD, Crick FHC. Molecular structure of nucleic acids: a structure for deoxyribose nucleic acid. Nature 1953; 171:737738. 4. Jacob F, Monod J. Genetic regulatory mechanisms in the synthesis of proteins. J Mol Biol 1961; 3:318-356. 5. Watson JD, Tooze J, Kurtz DT. Recombinant DNA, A Short Course. New York: WH Freeman, 1983. 6. Darnell J, Lodish H, Baltimore D. Molecular Cell Biology. New York: Scientific American Inc; Freeman Press, 1986. 7. Ausobel FM, Brent R, Kingston RE, et al., eds. Current Protocols in Molecular Biology. New York: J. Wiley and Sons, 1989. 8. Davis LG, Dibner MD, Battey JF. Basic Methods in Molecular Biology. New York: Elsevier, 1986. 9. Old RW, Primrose SB: Studies in Microbiology, Volume 2: Principles of Gene Manipulation. An Introduction to Genetic Engineering. Oxford: Blackwell Scientific, 1985. 10. Maniatis T, Fritsch EF, Sambrook J. Molecular Cloning: A Laboratory Manual. New York: Cold Spring Harbor Laboratory, 1982. 11. Church GM, Gilbert W. Genomic Sequencing. Proc Natl Acad Sci 1984; 81:1991-1995. 12. Saik RK, Gelfand DH, StoffelS, et al. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerane. Science 1988; 239:487-491. 13. Imagawa DT, Lee MH, Wolinsky SM, et al. Human immunodeficiency virus type I infection in homosexual men who remain seronegative for prolonged periods. N Engl J Med 1989; 320: 1458-1462. 14. Poiesz BJ. Detection of sequences homologous to human retroviral DNA in multiple sclerosis by gene amplification. Proc Natl Acad Sci1989; 2878-2882. 15. Paabo S. Ancient DNA: Extraction, characterization, molecular cloning, and enzymatic amplification. Proc Natl Acad Sci 1989; 86:1939-1943. 16. Skinner MA, Iglehart JD. The emerging genetics of cancer. Surg Gynecol Obstet 1989; 168:371-379. 17. Slamon DJ. Proto-oncogenes and human cancers. N Engl J Med 1987; 317:955-957. 18. Oppermann H, Levinson AD, Varmus HE, et al. Uninfected vertebrate cells contain a protein that is closely related to the product of the avian sarcoma virus transforming gene (src). Proc Natl Acad Sci 1979; 76:1804-1808. 19. Johnsson A, Betsholtz L, Heldin CH, Westermark B. The phenotypic characteristics of simian sarcoma virus-transformed human fibroblasts suggest that the v-sis gene product acts solely as a PDGF receptor agonist in cell transformation. EMBO J 1986; 5:15351541. 20. Westermark B, Heldin CH. Platelet-derived growth factor as a mediator of normal and neoplastic cell proliferation. Med Oncol Tumor Pharmacother 1986; 3:177-183. 21. Brodeur GM, Seeger RC, Sather H, et al. Clinical implications of oncogene activation in human neuroblastomas. Cancer 1986; 58:541-545.

Ann. Surg. . August 1990

22. Seeger RC, Brodeur GM, Sather H, et al. Association of multiple copies of the N-myc oncogene with rapid progression of neuroblastomas. N Engl J Med 1985; 313:1111-1116. 23. Weinberg RA. Finding the anti-oncogene. Sci Am 1988; 259:4451. 24. Klein G. The approaching era of the tumor suppressor genes. Science 1987; 238:1539-1545. 25. Ponder B. Gene losses in human tumors. Nature 1988; 335:400402. 26. Lee EY, To H, Shew J, et al. Inactivation of the retinoblastoma susceptibility gene in human breast cancers. Science 1988; 241: 218-221. 27. Law DJ, Olschwang S, Monpezat JP, et al. Concerted nonsyntenic allele loss in human colorectal carcinoma. Science 1988; 241: 961-965. 28. Solomon E, Voss R, Hall V, et al. Chromosome 5 allele loss in human colorectal carcinoma. Nature 1987; 328:616-619. 29. Vogelstein B, Fearon ER, Hamilton SR, et al. Genetic alterations during colorectal-tumor development. N Engl J Med 1988; 319: 525-532. 30. Harbour JW, Lai S, Peng J, et al. Abnormalities in structure and expression of the human retinoblastoma gene in SCLC. Science 1988; 241:353-356. 31. Penn A, Garte SJ, Warren L, et al. Transforming gene in human atherosclerotic plaque DNA. Proc Natl Acad Sci 1986; 83:79517955. 32. Barrett TB, Benditt EP. sis (platelet-derived growth factor B chain) gene transcript levels are elevated in human atherosclerotic lesions compared to normal artery. Proc Natl Acad Sci 1987; 84:10991103. 33. IzumoS, Nadal-Ginard B, Mahdavi V. Proto-oncogene induction and reprogramming of cardiac gene expression produced by pressure overload. Proc NatI Acad Sci 1988; 85:339-343. 34. Simpson PC. Role of proto-oncogenes in myocardial hypertrophy. Am J Cardiol 1988; 62:136-106. 35. Komuro I, Kurabayashi M, Takaku F, Yazaki Y. Expression of cellular oncogenes in the myocardium during the developmental stage and pressure-overloaded hypertrophy of the rat heart. Circ Res 1988; 62:1075-1079. 36. Kasid V, Pfeifer A, Brennan T, et al. Effect of antisense c-raf-l on tumorigenicity of human squamous carcinoma. Science 1989; 243:1354-1356. 37. Matsukura M, Zon G, Shinozuka K, et al. Regulation of viral expression of human immunodeficiency virus in vitro by an antisense phosphorothioate oligodeoxynucleotide against rev (artl trs) in chronically infected cells. Proc Natl Acad Sci 1989; 86: 4244-4248. 38. Huang HS, Yee J, ShewJ, et al. Suppression of the neoplastic phenotype by replacement of the RB gene in human cancer cells. Science 1988; 242:1563-1566. 39. Lin FK, SuggsS, Lin CH, et al. Cloning and expression of the human erythropoietin gene. Proc Natl Acad Sci 1985; 82:7580-7584. 40. EschbachJW, Egrie JC, Downing MR, et al. Correction of the anemia of end-stage renal disease with recombinant human erythropoietin. N Engl J Med 1987; 316:73-78. 41. Dinarello CA, Mier JW. Lymphokines. N EnglJ Med 1987; 317: 940-945. 42. Goldstein D, LaszloJ. Interferon therapy in cancer from imagination to interferon. Cancer Res 1986; 46:4315-4329. 43. Manda T, Shimonura K, MukumotoS, et al. Recombinant human tumor necrosis factor-x: evidence of an indirect mode of antitumor activity. Cancer Res 1987; 47:3707-3711. 44. Goeddel DV, Kleid DG, Bolivard F, et al. Expression in Escherichia coli of chemically synthesized genes for human insulin. Proc Natl Acad Sci 1979; 76:106-110. 45. Goeddel D, Hejneke H, Hozumi T, et al. Direct expression in Escherichia coli of a DNA sequence coding for human growth hormone. Nature 1979; 281:544-548. 46. LoscalzoJ, Braunwald E. Tissue plasminogen activator. N EnglJ Med 1988; 319:925-931.