Oncogenes: Their role in neoplasia

ONCOGENES:

THEIR ROLE IN NEOPLASIA

STANLEY A. BROSMAN, BRIAN C.-S. LIU, PH.D.

M.D.

From the Department of Urology, Kaiser Permanente Medical Center, and the Department of Immunology and Microbiology, UCLA Center for the Health Sciences, Los Angeles, California

Both clinicians and scientists in the field of cancer have been stimulated by the recent burst of knowledge in cancer biology. The molecular and genetic analysis of cancer cells has been made possible by rapid technologic progress in molecular biology and the fusion of ideas and perspectives from the fields of virology, chemical carcinogenesis, cytogenetics, and tumor biology. l-8 The focus of much of this interest has been on oncogenes. How oncogene function relates to what is already known about cancer biology is the subject of this article. Oncogenes are involved in the process of cellular transformation, and their role can be understood in terms of what has already been learned about carcinogenesis. By definition, oncogenes are genes that cause cancer. They represent altered versions of a group of normal genes that exist in every cell. These normal genes are known as proto-oncogenes. The proto-oncogenes, of which more than forty have been discovered in the cellular genome and an additional ten or more in the genomes of tumor viruses, encode proteins that involve the cell’s structure, growth, and mitotic activity. When these proto-oncogenes are changed by (1) point mutations, (2) translocation from their usual position on one chromosome to another, or (3) have their transcriptioninitiation sites altered, they may produce a protein with a different configuration, an excess of their protein product; or they may express this product at an inappropriate time of the cell cycle. These changes have been shown to confer transforming properties leading to neoplasia, and these altered genes are referred to as oncogenes.

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Discovery of Oncogenes The history of oncogenes follows two disparate but converging lines of investigation. The first was a study of RNA viruses, or retroviruses, and their ability to cause cancer. In the early 19OOs, a farmer in New York discovered a tumor in one of his hens, which he brought to Dr. Peyton Rous at the Rockefeller Institute. Dr. Rous transplanted this sarcoma, as well as induced its formation into healthy chickens by means of a cell-free infiltrate.’ Unfortunately, study and understanding of virology was in its infancy, and it was not until the 1950s that the Rous sarcoma virus (RSV) was “rediscovered” and used to study neoplasia in cell culture.8 In the early 197Os, scientists found that this virus contained a single gene responsible for oncogenesis among its regular complement of genes.g This gene was named src for sarcoma. The protein that it encodes was designated p60 src because its molecular weight is 60,000 daltons. The src gene is not required for viral growth or function, but its continuous function is required for oncogenesis. If the src gene is removed or its products inactivated, the transformed cells return to normal. In 1975, Bishop and Varmus at the University of California, San Francisco, discovered that src was not really a viral gene.‘JOJ1 They learned that it was almost an exact copy of a normal gene found in all chicken cells. They speculated that the normal gene, a proto-oncogene, was picked up by a retrovirus in the course of a mammalian infection and somehow in the process, became a cancer gene or oncogene. This virus could be injected into some

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types of healthy cells, and cellular transformation would ensue. The second line of investigation centered on the Heubner-Todaro12 hypothesis promulgated in 1969. This virogene-oncogene hypothesis stated in part that covert viral oncogenes might be present in normal cells. The concept was developed by evidence that retroviral genes could be transmitted from parents to offspring of many animal species as components of normal cellular chromosomes. Todaro and Heubne+ predicted that these retroviruses might include oncogenes, as well as the genes necessary for their own replication. The oncogenes might be activated within a cell by a variety of mechanisms that include chemicals or other carcinogens, mutagens such as radiation, or invading viruses. 1.5,11 It has been known that DNA from certain naturally occurring human tumors can induce malignant transformation of the NIH/3T3 cell line. This is a mouse fibroblast cell line that is highly susceptible to DNA transfection and subsequent transformation. Although only 10 per cent of human tumor DNAs are capable of transforming this particular cell line, oncogenes have been detected in tumors representative of each of the major types of human cancer. An oncogene present in the T24 and EJ bladder carcinoma cell lines was the first to be isolated by molecular cloning techniques.14 This oncogene is a member of the ru,s family of murine sarcoma viruses. Using special DNA probes, the bladder cancer oncogene was found to be derived from a sequence of similar structure within the normal human genome.15 Unlike the T24 oncogene, which exhibits a high degree of transforming activity, the normal cellular gene does not have transforming activity. The genetic change responsible for producing transformation capabilities is a point mutation in which the nucleotide thymidine replaced guanine at the twelfth amino acid residue. These important findings provided evidence for a molecular mechanism in carcinogenesis and demonstrated that a small change in the structure of certain proteins could lead to dramatic changes in the biologic activity of these molecules and to the pleotropic changes associated with cellular transformation. This particular TUSgene, and the other oncogenes, has been highly conserved during evolution.1e~17There is only a 1 per cent divergence of the protein sequence in the cellular ras gene between the mouse and human. This is in contrast 2

to an 18 per cent divergence between mouse and human fi-globin sequences. These genes have been identified in every vertebrate species, as well as drosophila and even yeast cells. Clearly, the presence of these genes throughout the biologic kingdom indicates that they serve important biologic functions. Function of Proto-Oncogenes and Oncogene Products Whatever the specific mechanisms may be that convert proto-oncogenes to oncogenes, they exert their effect by means of the proteins they encode. l8 At the present time, there is only a partial understanding of what these protein products are and what they do but these small peptides participate in cellular mitosis. In their natural state, oncogenes are relatively quiescent and control orderly mitotic events. They are probably active during embryogenesis, wound healing, tissue repair, and normal cell replacement. They may be involved in cellular functions other than growth. For example, the cellular src gene is expressed at a much higher level in cells of the nervous system than in other tissues, although their function in this tissue is unknown. l9 For purposes of discussion, oncogenes can be divided into three groups according to their biochemical mechanisms and sites of action and a fourth group containing those whose mechanism has not yet been determined (Table I). 1. Protein phosphorylation This group acts by adding energy-rich phosphate to the amino acids tyrosine, serine, and threonine that are contained in the cell membrane.20 These oncogenes are known as protein kinases and contain the Rous sarcoma virus oncogene, STC, that acts on vinculin, the Abelson leukemia oncogene, abl, that is associated with chronic myelogenous leukemia, and oncogenes that code for growth factors and growth factor receptors.21,22 Protein kinases act at the inner surface of the plasma membrane where they catalyze the addition of a phosphate molecule to other proteins.23 This phosphorylation involves the transfer of the energy-rich terminal phosphate group of adenosine triphosphate to the proteins being modified. While most protein kinases phosphorylate the amino acids serine and threonine, this particular group of oncogenes phosphorylate tyrosine. This function of tyrosine phosphorylation is shared by the viral oncogene and

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TABLE

I.

Oncogene function

Function

and location Chromosomal Location

Oncogene

1. Protein phosphorylation (protein kinases)

src abl fes

1 9 15 1 18 7 22 7 5 17 8 11 X 6 12 1 8 2 6 11 1 16 1 17 2 1 3 4 X 7 1

ark? yes

Protein kinases acting as growth factor and growth factor receptors

2. Guanosine triphosphate

3. DNA binding (replication

binding

and transcription)

Unknown

its normal cellular proto-oncogene. Exactly how this particular function leads to the varied effect of cell transformation is not well under-

stood, but there are several possibilities. The oncogene could link phosphate to a number of target proteins, altering the function of each one. The normal transduction of exogenous signals through the membrane may be affected.24 The diverse or pleotropic nature of the cancer cell might be explained by the presence of a substrate protein which functions in several cellular pathways. Since tyrosine phosphorylation is involved in complex systems affecting transmembrane signal transduction, alterations in these systems could allow the development of the cancerous state. Vinculin is a substrate for the transforming tyrosine protein kinase p60 produced by the WC oncogene. In the transformed cell, vinculin contains twenty times the amount of phosphate as it does in the normal cell. Vinculin is local-

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met sis (PDGF) erb-B (EGF) fms (CSF-1) neu mos H-ras-1 H-ras-2 K-ras- 1 K-ras-2 N-ras n-w N-myc m yb ets ski fos L-myc erb-A rel B-lym raf-1 raf-2 PKS-1 PKS-2 fgr

ized in adhesion plaques, where it is interposed between the cell’s plasma membrane and the ends of the actin bundles. Here it connects the bundles of cytoskeletal figments to an anchoring protein in the plasma membrane. This allows increased cell mobility and is associated with the anchorage independent growth typical of many cancer cells. Excessive phosphorylation of vinculin decreases its ability to act as an anchoring protein .25 However, vinculin phosphorylation alone does not explain the rearrangement of cytoskeletal elements. It is possible that STCalso acts on membrane substrates that affect lipid binding to vinculin.25 Growth

factors

and growth

factor

receptors

There has been a great deal of interest in the role of growth factors in cellular transformation and their relation to oncogenes.2es27Spom and Todaro2s

suggested

that

one way

a tumor

cell

may become autonomous is by the production

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of growth factors for which it possesses functional receptors. Somatic mutational events can predispose to cancer by alterations in growth factors, their receptors, or the intracellular postreceptor pathway. Most oncogenes do not encode growth factors but indirectly influence the expression of genes that do release growth factors. l8 Several oncogenes have functions that are similar or identical to those of growth factors. Epidermal growth factor (EGF) and plateletderived growth factor (PDGF) have been studied extensively. 26,27They deliver their signal by binding to specific protein receptor molecules embedded in the plasma membrane. The receptors have protein kinase activity which appears to be tyrosine-specific. This action is similar to that in the previously described protein kinase group. These growth receptors span the membrane. That part of the molecule which is outside the cell recognizes the growth factor and a catalytic domain inside the cell accomplishes phosphorylation. When a growth factor binds to its receptor, a signal is transmitted across the membrane and protein kinase activity is increased. The protein encoded by the viral and cellular sis oncogene is almost identical to PDGF which is a powerful mitogen. 2g,3oThe ti proto-oncogene is actually the gene coding for the B chain of PDGF. Cells containing an activated ti oncogene proliferate because of the synthesis and secretion of PDGF-like growth factor. Whereas the real PDGF delivers its signal only on appropriate occasions, the h-encoded protein may do so continually, causing unregulated cell division. Other tyrosine kinase encoding genes that act as growth factors include the erb-B protooncogene that codes for a truncated version of epidermal growth factor (EGF) receptor and the product of the fms proto-oncogene that is related to the receptor for macrophage colony stimulating factor (CSF-1).27 Messing31.32 has demonstrated that bladder carcinoma cells actively produce transforming growth factors similar to EGF, although as yet there has been no link to an oncogene. Neal and co-workers33 examined normal and neoplastic human urothelium for the presence of EGF receptors, using an immune peroxidase stain and a monoclonal antibody. They found that whereas the controls did not stain for this receptor, the more invasive tumors stained more frequently (21 of 24) than superficial tumors (7 of 24). The poorly differentiated tumors stained

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more (18 of 21) than moderately tumors (10 of 27). 2. Guankine

triphosphate

differentiated

binding

This group of oncogenes is involved with membrane signal transduction and contains the ras family that has been identified in many human solid tumors. The products are small peptides with molecular weights of 21,000 daltons (~21). The chromosomes containing this gene have been localized; and when a mutation is present, it tends to occur at the twelfth or the sixty-first amino acid. In normal bladder, codon 12 consists of guanine-guanine-cytosine which produces glycine. In the ras oncogene from bladder cancer cells, codon 12 consists of guanine-thymine-cytosine which produces valine. This single change has been shown to produce a transforming peptide. Attempts to reproduce this phenomenon with all of the other nineteen amino acids successfully induced transforming activity with the exception of a single one, proline. Why these two sites appear to have such high susceptibility for mutational changes is unknown, but conformational changes in the tertiary structure of the protein will affect function. Several carcinogens have been shown to produce point mutations in the TU.Sgene. Nitrosomethylurea produces a mutation in which guanine is replaced by adenine in codon 12. Dimethylbenzanthracene causes a mutation in codon 61. Other hydrocarbons and nitrosamines have produced similar mutations. The pus oncogene acts on guanosine triphosphate (GTP) which is found on the inner face of the plasma membrane. The ras gene product may be one of a group of G proteins that play an active role in the cell’s response to external stimuli.34 They interact with membrane receptors and effector molecules inside the cell. The effecters mediate the cellular responses to the original exogenous stimulus. All of the G proteins are made of three subunits, but it is the alpha subunit that binds to guanine and appears to carry out the effector function of GTP hydrolysis. It is this alpha subunit that appears to be encoded by the ra.s gene. Unregulated activity of the ras oncogene, which may or may not result in overexpression, could lead to a disturbance in the orderly transduction of exogenous signals that would disrupt the regulation of mitotic activity. The cellular activities of the ras oncogene have been studied employing microinjection

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techniques.35 Within thirty minutes, rat embryo fibroblasts that have been injected with p2l show changes in cell surface morphology and the induction of pinocytosis. These processes are fundamental in the maintenance of cell homeostasis, and alterations in the rate of pinocytosis exert profound effects on various intracellular processes controlling cell proliferation. The ability and efficiency of the TCISoncogene to induce transforming activity may be related to DNA methylation .36 The methylation pattern of cytosine at critical sites on normal genes serves as a regulatory signal for expression, High levels of DNA methylation are associated with the absence of expression while low levels correlate with active expression3’ The transforming activity of the r’as oncogene has been shown to be abrogated by producing high levels of methylation in this gene.3e This suggests that the transforming activity of this gene is reversible by an epigenetic mechanism. 3. DNA binding All of the oncogenic proteins discussed so far have acted primarily on the cell plasma membrane. There is a group of oncogenes, notably the myc oncogene family, that acts within the cell nucleus as a DNA binding protein and participates in DNA replication.38*39 The myc oncogene was one of the first cellular genes to be linked to neoplasia and is overexpressed in a number of human cancers including neuroblastoma and small cell lung cancer.38,3e Blocking the myc protein with polyclonal or monoclonal antibodies inhibits DNA synthesis and DNA polymerase activity but not RNA transcription.40 The proto-myc gene product appears in growing cells shortly before they begin synthesizing DNA. A large amount of proto-myc protein may cause cells to replicate indefinitely, a condition known as immortalization, The cell line NIH13T3, which was mentioned previously, is an example of an immortalized cell line. While immortalization alone does not convert a normal cell into a cancer cell, the activation of a second oncogene might be enough to confer transforming properties on the cell. It has been shown in tissue culture that a cell line immortalized by the myc oncogene can display features typical of cancer when an active rus oncogene is introduced.41s42 The fos proto-oncogene is another nuclear acting gene that is associated with cellular differentiation through a mechanism of transcrip-

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tional transactivation.43,44 The administration of growth factors induces fos activity that is rapidly followed by the induction of myc messenger RNA, 45 This implies that there is a cascade of cell membrane, cytosol, and nuclear events that precede cellular transformation. Mechanisms of Oncogene Activation There are three mechanisms that explain how genetic changes may cause the malfunction of a proto-oncogene or its product.5,4e These include mutations that might change the method in which a protein acts; overproduction of a gene product or amplification, that may occur by translocation of a gene into the vicinity of a transcriptional enhancer, insertion of retroviral DNA or transduction into a rstroviral genome; and deregulation of a quiescent protooncogene resulting in the expression of the product at an inappropriate time. Point mutation as a mechanism for structural and functional abnormalities has been previously mentioned in relation to the MS oncogene and bladder cancer. Although point mutations have been demonstrated in laboratory experiments, they have been identified in only a few human cancers. Examination of the Harvey TCLSgene identified in bladder cancer cell lines indicates that although the point mutation at codon 12 produces transformation of the NIH/3T3 cell line, there is no overproduction of the gene product or a change in its biochemical product. This mutation has not been found in a survey of more than twenty-nine human cancers, including ten bladder, ten lung, and nine colon carcinomas4’ There have been a few reports of cancers associated with mutations at amino acid 61, and there are likely to be other mutation sites as well.4s Unfortunately, the 3T3 transformation assay seems to detect mutations occurring primarily at codon 12. More common mechanisms associated with abnormal oncogene function are chromosomal translocations and rearrangements.49-51 In Burkitt lymphoma, there is a reciprocal translocation between the distal regions of the long arms of chromosomes 8 and 14. The myc protooncogene is located on the distal segment of chromosome 8. The translocation places the myc proto-oncogene in the promoter region for IgH (heavy chain). In its original site this myc proto-oncogene was repressed, but in the new site it becomes activated and a neoplastic cell

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develops which produces large amounts of IgH. Similar translocations have been described in chronic myelogenous leukemia and other types of lymphocytic leukemias. There are specific sites on chromosomes that are known as fragile sites and are prone to breakage and rearrangement. Large numbers of these fragile sites have been demonstrated in a variety of cancers at places where rearrangements are known to occur.5l Yunis and Soreng52 believe that these may provide a physical basis for somatic recombination. Another mechanism involved with oncogene activity is the loss of a regulating nucleotide sequence that leads to overexpression. The myc oncogene contains three exons, but only the second and third produce the DNA binding protein product. The first exon acts as an “operator” and regulates the transcription activity of the other exons. Sometimes when myc is translocated, it loses the first exon and its activity becomes deregulated resulting in overexpression of its product. An increase in the number of genes producing a product is known as amplification. This is seen in cells containing double minute chromosomes that are small supernumerary chromosomes and in areas of a chromosome known as homogeneously staining regions. Both of these sites contain repetitively duplicated cellular genes that produce excessive amounts of gene product. An increase in the number of copies of a gene has been described in several neoplasms and correlates with aggressive tumor behavior. The chromosomal location of oncogenes may have an influence on other genes in the same area. Several oncogenes are located near the genes that code for some of the cell growth factors and their receptors. This proximity suggests that there may be some type of coordinate control exerted by the proto-oncogene. On the short arm of chromosome 1, one of the T~S proto-oncogenes that is associated with neuroblastoma is near the gene that produces nerve growth factor. In patients with Wilms tumor associated with aniridia, gonadoblastoma, and mental retardation, there is a deletion on the short arm of chromosome 11 that is close to another of the TUS gene loci. Some of the children with Beckwith-Wiedemann syndrome, which is also associated with Wilms tumor, have a duplication of the chromosomal material on the short arm of chromosome 11 that includes the site of a TUSprotooncogene.

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Role of Oncogenes in Carcinogenesis Integration of our current knowledge of oncogene activity with established concepts in cancer biology may help us to understand the role of the oncogene. e,53-55The presence of active oncogenes in human cancers has been sought by many investigators but until recently most have been unsuccessful. Slamon et ~1.~~ have demonstrated cellular oncogene activity in tissue obtained from fresh human tumors. They studied tumors from 54 patients representing twenty different cell types. This included 9 patients with renal carcinoma. They tested for the presence of fifteen different cellular oncogenes. More than one cellular oncogene was active in all of the tumors examined. Four cellular oncogenes (myc, fos, H-ras, K-ras) were expressed in nearly all of the tumors studied; other oncogenes were expressed infrequently, and some were not detected. Oncogene activity was compared between normal and malignant tissues from the same organ in 14 patients. Five oncogenes were expressed at higher levels in some tumors than in normal tissue. However, there was poor correlation between levels of cellular oncogene expression in a given tumor and the ratio of expression in tumor tissue compared with normal tissue. Although these data add evidence to support the multi-gene cancer hypothesis, they do not shed light on the necessity of these transcriptionally active oncogenes in the cancer process. Slamon and co-workers5’ have also reported that amplification of the HER-2heu gene is a significant predictor of overall survival and time to relapse in patients with breast cancer who have nodal me&stases. Their data indicate that the presence of gene amplification has greater prognostic value than estrogen and progesterone receptors and is equivalent to and independent of the number of positive lymph nodes. The greater the number of gene copies, the worse is the prognosis. Similar findings relating amplification to prognosis have been made for neuroblastoma and small cell lung carcinoma by studying the myc family of oncogenes. 58-80 The N-myc and L-myc gene expression is restricted to particular tissues and during certain stages of development. The c-myc gene has a more generalized pattern of expression. Patients with neuroblastoma who have a large number of c-myc copies have a worse prognosis regardless of their tumor stage, although most high-

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stage tumors have many copies of the c-myc gene.58 Stage IV-S which has a better prognosis is associated with no increase in the number of gene copies. Patients with Wilms tumor have enhanced expression of N-myc without the presence of gene amplification. They also have low levels of c-myc expression which suggests that this family of nuclear genes interact with each other in some type of regulatory function.e1 Viola and co-workerse2 examined patients with benign prostatic hyperplasia and with prostatic carcinoma by immunohistology to detect the expression of the T(LSoncogene product ~21. This protein was not detected in 19 patients with benign hyperplasia but was found in some patients with prostate carcinoma. The detection of the antigen related to the grade of the disease. It was identified in 33 per cent of grade 1, 66 per cent of grade 2, and 100 per cent of patients with grade 3 disease. These authors suggest that the TUSoncogene protein may represent a new class of prostate tumor markers. These same investigators using histochemical staining have identified the presence of the p21 antigen in the most superficial cells of the bladder and urethral urothelium.e3 In patients with severe dysplasia and high-grade urothelial carcinoma, there was an intense staining reaction for the p21 antigen that greatly exceeded that seen in the normal epithelium or in patients with low-grade tumors. Piehl and Stamep4 have also detected T(IS oncogene expression in prostatic cancer cells, while Fujita and Aaronson65 have identified point mutations and rus amplifcation in human urethelial tumors. In our laboratory, we have identified a rusrelated oncogene product in the urine of patients with transitional cell carcinoma (TCC).ee-es Instead of identifying an expected p21 protein, we found a protein with a molecular weight of 55 kilodaltons (~55) which was antigenically related to the MS oncogene. The T(ISp55 expression correlates with the stage and grade of the cancer. Slightly elevated expression (2 + ) was found in 2123 noncancer patients, while 26/29 samples from cancer patients had elevated (3 + ) p55 expression. The 3 cancer patients with minimal expression (1 + ) had previously undergone resection of their tumors. This implies that TUS p55 may be a useful marker for patients with TCC. We have also detected rus-related oncogene products within the cytoplasm of exfoliated cells obtained by bladder washings from patients with TCC.ea

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The use of an immunoperoxidase technique with immunocytologic staining of oncogene products within the cell may improve the sensitivity of detecting aggressive tumor cells. These data indicate that oncogenes are active in human neoplasms, and their expression can be detected. A large body of evidence has accumulated that suggests that most human cancers are the result of multiple events that must surely involve multiple genes functioning at various levels of expression over a long period of time.41z42 This correlates with the principles of tumor initiation and promotion that have been widely demonstrated and accepted in human carcinogenesis. The one-gene-one-cancer hypothesis was developed based on work with retroviruses and the NIH/3T3 cell line. These studies indicated that an aberrant genetic event, such as a mutation in a single gene, could account for the pleotropic changes seen in carcinogenesis . An example is the rus oncogene mutation in the T24 bladder cancer cell line. However, there is not likely to be much relevance of these single-gene models to human cancers.41 Although there is general acceptance of the concept that activation of a single cellular proto-oncogene may cause it to function like a viral oncogene and induce cellular transformation, this hypothesis also assumes that activated proto-oncogenes are involved not only in the few natural tumors that are caused by those viruses containing oncogenes, but also in tumors that do not contain viruses. Furthee defined distinct stages in the development and progression of neoplasms by studying hormone-dependent tumors. He found that some tumors could be induced by a prolonged overproduction of a stimulating hormone while other tumors developed when there was a prolonged deficiency of an inhibitory hormone. In his study of thyroid tumors, he found that prolonged exposure to thyroid-stimulating hormone could induce a proliferative lesion in the thyroid, while its absence led to tumor formation in the pituitary. Tumors associated with a hormonal imbalance are usually dependent on the continued overproduction of the stimulating hormone or the persistent lack of the inhibitory hormone, and may stop growing or regress when the abnormality is corrected. As the tumor cell population increases, mutations and chromosomal rearrangements and other aberrations occur in which a subclone can continue to grow in a hormonally normal environment.

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These autonomous clones may remain hormonally responsive in that their growth can be stimulated by the original hormone imbalance, although they are no longer dependent on it. As additional changes occur in the cellular environment and within the cell, the tumor may become totally unresponsive although complete cellular autonomy is never certain. Both breast and prostate cancers are examples of this type of tumor cell behavior. Foulds70 stated that the total growth of a cell population represented the sum of its intrinsic and responsive growth rates. Normal cells have a low intrinsic and a high responsive growth rate. As cells transform, there is a progressive alteration in this ratio. The most malignant cells, i.e., those with the capacity and ability for metastasis, have the highest intrinsic growth rate and are the least responsive to environmental change. Yet highly malignant tumors have reverted to normal functioning cells when their environment was altered. An example is the mouse teratocarcinoma which kills nearly all of its syngeneic hosts. 71 When the tumor is implanted or the tumor-bearing host is attached to a normal embryo host, the tumor differentiates in the strong inductive embryonic environment and participates in the formation of all normal tissues. Similar results have been obtained in cell fusion studies, and we are familiar with the differentiation of neuroblastomas into benign tumors in some patients. The importance of the cellular environment in the maintenance of normal tissue differentiation and behavior and the induction of neoplasia when this environment is altered has been emphasized by Rubin. 72 As normal embryonic development proceeds, the ability of tissues to change from one purpose to another is progressively decreased but never completely lost, Cunha et ~1.~~ demonstrated this concept by transplanting normal adult rat bladder epithelium into the embryonic mesenchyme which induces prostatic epithelium. Within a short time, the bladder mucosa converted to the acinar epithelium of the prostate.74 When a similar experiment was done using transitional cell carcinoma, the cancer changed into an adenocarcinoma characteristic of the prostate. This demonstrates that even malignant carcinomas can remain partly under the control of its associated mesenchyme. Proto-oncogenes that are important regulators of mitotic activity are likely to be expressed during embryonic development and during the

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life of those cells which continue to divide and differentiate. The induction of their activity is probably mediated by environmental and homeostatic processes. Our knowledge of oncogene activity and the multistep development of human cancers can be correlated with the fundamental principles in neoplasia which have been accepted for many years. George and Eva Klein75 have offered the concept of “conditioned tumorigenicity” which states that the transforming effect of any activated oncogene is restricted to a very narrow period of time during the differentiation and maturation of a cell. Thus the expression of an oncogene may be variable in time, duration, intensity, and relate to the particular conditions and stimuli that are present at any given moment. The reversibility of the neoplastic process in some situations suggests that there are periods of time and special conditions during which abnormal gene activity can be abrogated. The likelihood of a feedback or a control mechanism was suggested by Knudson7e based on studies of several hereditary cancers. These unusual neoplasms, as exemplified by retinoblastoma and some instances of nephroblastoma, have revealed a group of genes that are important in cellular transformation. Knudson refers to them as anti-oncogenes because they produce cancer in a recessive mode. Sager77,78refers to these genes as tumor suppressor genes and considers them to be dominant since both normal copies need to be lost for the tumor phenotype to be expressed. One normal allele is adequate to protect the host against a particular cancer. In distinction, the typical oncogene produces cell transformation in the heterozygous state. Both nephroblastoma and retinoblastoma are associated with recessive mutations at a single site.7el77In retinoblastoma, there is a deletion of a particular band on the long arm of chromosome 13 (13 of 14) resulting in a deficiency of the enzyme esterase D. In nonhereditary cases of retinoblastoma, a mutation has been demonstrated in this same gene. Examples of chromosomal deletion have been found in patients with Wilms tumor. The short arm of chromosome 11 (~13) has been found to be the deleted segment. In a few instances, patients with the nonhereditary form of nephroblastoma have shown a deletion of the same band. The absence of a single enzyme or a group of enzymes that ordinarily control cell growth may permit the development of a neoplasm. Likewise, the mutation UROLOGY

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occurring in one of these genes would alter the function of its product. Fearon et ~1.‘~ studied TCC from 12 patients and found 5 tumors in which there was a somatic loss of genes on llp resulting in homozygosity or hemizygosity of the nondeleted alleles in the tumor cells. This change was found in nearly all of the tumor cells and suggests that the llp deletion conferred a selective growth advantage. Anti-oncogenes are probably more numerous and more difficult to find than oncogenes. They show a much narrower tissue specificity and may play an important role in tissue differentiation. By performing cytogenetic analyses of somatic cell hybrids, it has been found the loss of chromosome 11 is associated with an increased expression of neoplasia suggesting that there may be a large group of anti-oncogenes located on this chromosome.80 Additional evidence for the presence of anti-oncogenes has been provided by Noda et al.*’ who discovered a group of genes that could suppress the transformed phenotype and produce a reversion of cellular differentiation. These particular genes could block the expression of activated rus, fes, and src but not the transforming effect of mos or sis. This implies that there may be groups of anti-oncogenes which act on the differentiation of cells at varying times and that they are also regulated by some type of feedback mechanism. Carcinogens such as radiation, chemicals, or viruses may act by altering the tissue environment, disrupt the normal control mechanism for oncogenes and anti-oncogenes, induce higher levels of normal proto-oncogene expression such as seen in some cancers with gene rearrangements, or inducing structurally aberrant gene products. 5 There is evidence that all of these mechanisms may occur at various stages of the neoplastic process. There is an increasing amount of experimental and clinical data which indicates that oncogene activation plays an important role during the development of human and animal cancers and can explain many of the mechanisms involved in neoplastic transformation. The exact stage at which the various oncogenes become activated is unclear. It is unknown if they are involved in the causation of cancer, and it is unlikely that they act as single genes without environmental control or that once activated they are solely responsible for autonomous cell growth. 41~82But the knowledge obtained from studying these genetic aberrations has provided a great insight into the molecular basis for can-

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cer and will yield new opportunities for prevention, diagnosis, and treatment of neoplasia. Department of Urology Kaiser Permanente Medical Center Los Angeles, California 90027 (DR. BROSMAN) References 1. Bishop MJ: Viruses, genes and cancer, Cancer 55: 2329 (1985). 2. Yunis JJ: The chromosomal basis of human neoplasia, Science 221: 227 (1983). 3. Cooper GS, et al: Molecular cloning of a new transforming gene from a chemically transformed human cell line, Nature 311: 29 (1983). 4. Gordon H: Oncogenes, Proc Mayo Clin 60: 697 (1985). 5. Bishop JM: The molecular genetics of cancer, Science 235: 305 (1987). 6. Fidler IJ, and Hart JR: Biologic diversity in metastatic neoplasms: origins and implications, ibid 217: 998 (1982). 7. Rous PA: A sarcoma of the fowl transmissible by an agent seoarable from the tumor cells. I EXD Med 13: 397 (1911). ‘8. Temin H, and Rubin H: Chara&eristics of an assay for Rous sarcoma virus and Rous sarcoma cells in tissue culture, Virology 6: 669 (1958). 9. Stehelin D, Varmus HE, Bishop JM, and Vogt PK: DNA related to the transforming gene of avian sarcoma virus are present in the DNA of uninfected vertebrates, Nature 260: 170 (1976). 10. Bishop MJ: Oncogenes, Sci Am 246: 80 (1982). 11. Varmus HE: I. The discovery of cellular oncogenes and their role in neoplasia, Cancer 55: 2324 (1985). 12. Huebner RJ, and Todaro GJ: Oncogenes of RNA tumor viruses as determinants of cancer. Proc Nat1 Acad Sci USA 64: 1087 (1969). 13. Todaro GJ, and Huebner RJ: The viral oncogene hypothesis: new evidence. ibid 69: 1009 (1972). 14. Reddy EP,‘Reynolds RK, ‘San&s E, and Barbacid M: A point mutation is responsible for the acquisition of transforming properties by the T24 human bladder carcinoma oncogene, Nature 366: 149 (1982). 15. Capon DJ, et al: Complete nucleotide sequences of the T 24 human bladder carcinoma oncogene and its normal homologue, ibid 302: 33 (1983). 16. Persson H, et oE: Antibodies to human c-myc oncogene product. Evidence of an evolutionarily conserved protein induced during cell proliferation, Science 225: 687 (1984). 17. Spector DH, Varmus HE, and Bishop JM: Nucleotide sequences related to the transforming gene of avian sarcoma virus are present in the DNA of uninfected vertebrates, Proc Nat1 Acad Sci USA 74: 4102 (1978). 18. Weinberg RA: The action of oncogenes in the cytoplasm and nucleus, Science 230: 770 (1984). 19. Brugge JS, et aE: Neurones express high levels of a structurally modified, activated form of pp6Oc-src, Nature 316: 554 (1985). 20. Hunter T, and Cooper JA: Protein-tyrosine kinases, Ann Rev Biochem 54: 897 (1985). 21. Collett MS, and Erikson RL: Protein kinase activity associated with the avian sarcoma virus src gene product, Proc Nat1 Acad Sci USA 75: 2021 (1978). 22. Seeburg PH, Colby WW, Capon DJ, and Goeddel DV: Biological properties of human c-Ha-ras 1 genes mutated at codon 12, Nature 312: 71 (1984). 23. Ingebritsen TS, and Cohen P: Protein phosphatases. Properties and role in cellular regulations, Science 221: 331 (1983). 24. Rozengart E: Early signals in the mitogenic response, ibid 234: 161 (1986).

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25. Born P, and Burger MM: The cytoskeletal protein vinculin contains transformation-sensitive, covalently bound lipid, ibid 235: 476 (1987). 26. Stiles CD: The biologic role of oncogenes-insights from platelet-derived growth factors, Cancer Res 46: 5215 (1985). 27. Goustin AS, Leof EB, Shipley GD, and Moses HL: Growth factors and cancer, ibid 46: 1015 (1986). 28. Spom MB, and Todaro GJ: Autocrine secretion and malignant transformation of-cells, N Engl J Med 303: 878 (1980). 29. Owens AK, Pantazis P, and Antoniades HN: Simian sarcoma virus-transformed cells secrete a mitogen identical to platelet-derived growth factor, Science 225: 54 (1984). 30. Doolittle RF, et al: Simian sarcoma virus oncogene, v-sis, is derived from the gene (or genes) encoding a platelet derived growth factor, ibid 221: 275 (1983). 31. Messing E: Growth factors and human bladder tumors, J Urol 131: 111 (1984). 32. IDEM: Transforming growth factors (TFG’s) produced by bladder carcinoma, (Abstract 613) ibid 133: 267 (1985). 33. Neal DE, et oE: Epidermal-growth-factor receptors in human bladder cancer; comparison of invasive and superficial tumours, Lancet 1: 366 (1985). 34. Hurley JB, et al: Homologies between transducing G proteins and ras gene products, Science 226: 860 (1984). 35. Bar-Sagi D, and Feramisco JR: Induction of membrane ruffling and fluid-phase pinocytosis in quiescent fibroblasts by ras proteins, ibid 233: 1061 (1986). 36. Bore110 MG, et al: DNA methylation affecting the transforming activity of the human Ha-ras oncogene, Can Res 47: 62 (1987). 37. Jones PA: DNA methylation and cancer, ibid 46: 461 (1986). 38. Donner P, Greiser-Wilke I, and Moelling K: Nuclear localization and DNA binding of the transforming gene product of avian myelocytomatosis virus, Nature 296: 262 (1982). 39. Persson H, and Leder P: Nuclear localization and DNA binding properties of a protein expressed by human c-myc oncogene, Science 225: 718 (1985). 40. Studzinski GD, Brelui S, Feldman SC, and Watt RZ: Participation of c-myc protein in DNA synthesis of human cells, ibid 234: 467 (1986): 41. Land H. Parada LE and Weinberg RA: Cellular oncogenes and multistep carcinogenesis, ibid 222: 771 (1983). 42. Balmain A: Transforming ras oncogenes and multistage carcinogenesis, Br J Cancer 51: 1 (1985). 43. Sambucetti LC, and Currau T: The fos protein complex is associated with DNA in isolated nuclei and binds to DNA cellulose, Science 234: 1417 (1986). 44. Colletta G, Cirafici AM, and Vecchio G: Induction of the c-fos oncogene by thyrotropic hormone in rat thyroid cells in cultures, ibid 233: 458 (1986). 45. Setoyama C, et al: Transcriptional activation encoded by the v-fus gene, Proc Nat1 Acad Sci USA 83: 3213 (1986). 46. Tabin CJ, et al: Mechanism of activation of a human oncogene, Nature 366: 143 (1982). 47. Feinberg AP, et al: Mutation affecting the 12th amino acid of the c-Ha-ras oncogene product occurs infrequently in human cancer, Science 220: 1175 (1983). 48. Capon DJ, et al: Activation of Kl-ras2 gene in human colon and lung carcinomas by two different point mutations, Nature 304: 507 (1983). 49. Finger LR, et al: A common mechanism of chromosomal translocation in T- and B-cell neoplasia, Science 234: 982 (1986). 50. Leder R et al: Translocations among antibody genes in human cancer, ibid 222: 765 (1983). 51. Croce CM, and Klein G: Chromosome translocations and human cancer, Sci Am 252: 54 (1985). 52. Yunis JJ, and Soreng AL: Constitutive fragile sites and cancer, Science 226: 1199 (1984). 53. Poste G, and Fidler IJ: The pathogenesis of cancer metastasis, Nature 283: 139 (1979). 54. Frost P, and Kerbal RS: On a possible epigenetic mecbani.sm(s) of tumor cell heterogeneity, Can Metast Rev 2: 375 (1983).

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55. Klein G, and Klein E: Evolution of tumours and the impact of molecular oncology, Nature 315: 190 (1985). 56. Slamon DJ, deKernion JB, Verma IM, and Cline MJ: Expression of cellular oncogenes in human malignancies, Science 224: 256 (1984). 57. Slamon DJ, et al: Human breast cancer: correlation of relapse and survival with amplification of the HER-2 neu oncogene, ibid 235: 177 (1987). 58. Seeger RC, et al: Association of multiple copies of the Nmyc oncogene with rapid progression of neuroblastomas, N Engl J Med 313: 1111 (1985). 59. Wong AJ, et al: Gene amplification of c-myc and N-myc in small cell carcinoma of the lung, Science 233: 461 (1986). 60. Santos E, et al: Malignant activation of a k-ras oncogene in lung carcinoma but not in normal tissue of the same patients, ibid 223: 661 (1984). 61. Nissen PD, et al: Enhanced expression of the N-myc gene in Wilms tumor, Cancer Res 46: 6217 (1986). 62. Viola VM, et al: Expression of ras oncogene p21 in prostate cancer, N Engl J Med 314: 133 (1986). 63. Viola MV, et aE: Ras oncogene p21 expression is increased in premalignant lesions and high grade bladder carcinoma, J Exp Med 161: 1213 (1985). 64. Piehl D, and Stamey T: Ras oncogene expression in prostate cancer, J Urol 135: 1027 (1986). 65. Fujita J, and Aaronson SA: Point mutations and gene amplification affecting ras proto-oncogenes in human urinary tract tumors, (Abstract 611), ibid 133: 266A (1985). 66. Liu BC-S, et al: Onto-fetal E7/G4 antigen and ras related oncogene products in urine of patients with transitional cell carcinoma as prognostic indicators, ibid (in press, 1987). 67. Stock LM, Brosman SA, Fahey JL, and Liu BC-S: Ras related oncogene protein as a tumor marker in transitional cell carcinoma of the bladder, ibid 137: 789 (1987). 68. Jacobs MA, et al: Detection of ras related oncogene products within the cytoplasm of exfoliated bladder tumor cells by immunoperoxidase technique, Urology (in press). 69. Furth J: Conditioned and autonomous neoplasms: a review, Cancer Res 13: 477 (1953). 70. Foulds L: The natural history of cancer, J Chronic Dis 8: 2 (1958). 71. Mintz B, and Fleischman RA: Teratocarcinomas and other neoplasms as developmental defects in gene expression, Adv Cancer Res 34: 211 (1981). 72. Rubin H: Cancer as a dynamic developmental disorder, Cancer Res 45: 1935 (1985). 73. Cunha GR, et al: Epithelial-mesenchymal interactions in prostatic development. I. Morphological observations of prostatic induction by urogenital sinus mesenchyme in epithelium of the adult rodent urinary bladder, J Cell Bio196: 1662 (1983). 74. Fujii H, Cunha GR, and Norman JT: The induction of adenocarcinomatous differentiation in neoplastic bladder epithelium by an embryonic prostatic inducer, J Urol 128: 858 (1982). 75. Klein G, and Klein E: Conditioned tumorigenicity of activated oncogenes, Cancer Res 46: 3211 (1986). 76. Knudson AG Jr: Hereditary cancer, oncogenes and antioncogenes, ibid 45: 1437 (1985). 77. Sager R: Genetic suppression of tumor formation: a new frontier in cancer research, ibid 46: 1573 (1986). 78. IDEM: Resistance of human cells to oncogenic transformation, Cancer Cell 2: 487 (1984). 79. Fearon ER, Feinberg Ap, Hamilton SH, and Vogelstine B: Loss of genes on the short arm of chromosome 11 in bladder cancer, Nature 318: 377 (1985). 80. Srivatsan ES, Benedict WF, and Stanbridge EJ: Implication of chromosome 11 in the suppression of neoplastic expression in human cell hybrids, Cancer Res 46: 6174 (1986). 81. Noda M, Selinger Z, S&nick EM, and Bassin RH: Flat revertants isolated from Kirstens sarcoma virus-transformed cells are resistant to the action of specific oncogenes in B cells, Proc Nat1 Acad Sci USA 80: 5602 (1983). 82. Duesberg PH: Activated proto-oncogenes: sufficient or necessary for cancer? Science 228: 669 (1985).

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