Oncogenic Interactions Research Articles

P16 methylation does not affect protein expression in cervical carcinogenesis

Previous studies have reported a frequency range of 19–61% for p16 methylation in cervical cancers. However, p16 is strongly expressed in over 90% of cervical cancers and pre-cancers, due to interactions of HPV oncogenes with p53 and pRb. In order to clarify these controversial findings, we developed a new bisulphite sequencing protocol to determine the methylation status of p16. DNA extracted from 17 cell lines and 94 microdissected clinical samples was subjected to methylation analysis. p16 expression was confirmed in Western blot and immunohistochemistry. Complete methylation of p16 was found in none of the dysplastic lesions, but in 26% of the cervical carcinomas. However, immunohistochemistry showed strong p16 expression in all cancers. These findings indicate that p16 methylation does not implicate loss of p16 expression in HPV-induced tumours. In cervical cancer, methylation of p16 does not seem to be an underlying pathogenic mechanism, but may be a result of increasing genetic and epigenetic instability.

On the path to understanding the nature of cancer

In this essay crucial problems of the origin of cancer and the development of malignancy are discussed. The problem of precancer and three ways leading to malignancy are considered: induction of tumor precursors, accumulation of genetic traits common for tumor growth, and the role of inflammation in tumor induction. The nature of viral oncogenes and modes of their action are described in the context of their origin as a component of the viral genome. Oncogenes of RNA-containing viruses and DNA-containing tumorigenic viruses are described together with cellular protooncogenes, which are progenitors of RNA-containing viral oncogenes. Hematological malignancies are described as an intermediate form between simple tumors induced by a single oncogene and more complicated epithelial tumors. The roles of tumor suppressor genes and the interaction of several oncogenes in the formation of carcinomas and also the role of progression in tumor evolution are discussed.

The Challenge of Drugging Undruggable Targets in Cancer: Lessons Learned from Targeting BCL-2 Family Members

The genomic and proteomic revolutions have provided us with an ever-increasing number of mechanistic insights into cancer pathogenesis. Mutated genes and pathologic protein products have emerged as the basis for modern anticancer drug development. With the increasing realization of the importance of disrupting oncogenic protein-protein interaction, new challenges have emerged for classical small molecule and protein-based drug modalities, i.e., the critical need to target flat and extended protein surfaces. Here, we highlight two distinct technologies that are being used to bridge the pharmacologic gap between small molecules and protein therapeutics. With the BCL-2 family of survival proteins as their substrate for intracellular targeting, we conclude that peptide stapling and fragment-based drug discovery show promise to traverse the critical surface features of proteins that drive human cancer.

Prediction and preliminary validation of oncogene regulation by miRNAs

BackgroundMicroRNAs (miRNAs) are one of the most abundant groups of regulatory genes in multicellular organisms, playing important roles in many fundamental cellular processes. More than four hundred miRNAs have been identified in humans and the deregulation of miRNA expression has been also shown in many cancers. Despite the postulated involvement of miRNAs in tumourigenesis, there are only a few examples where an oncogene or a tumour suppressor has been identified as a miRNA target.ResultsHere, we present an in silico analysis of potential miRNA- oncogene interactions. Moreover, we have tested the validity of two possible interactions of miRNAs with genes related to cancer. We present evidence for the down-regulation of c-MYC, one of the most potent and frequently deregulated oncogenes, by let-7 miRNA, via the predicted binding site in the 3'UTR, and verify the suppression of BCL-2 by miR16.ConclusionIn this work both bioinformatic and experimental approaches for the prediction and validation of possible targets for miRNAs have been used. A list of putative targets for different oncomirs, validation of which would be of special interest, is proposed and two such interactions have been experimentally validated.

Human polyomavirus BKV transcriptionally activates DNA methyltransferase 1 through the pRb/E2F pathway

Many DNA tumor virus oncogenes are capable of activating and highjacking the host cell's DNA replication machinery for its own reproduction purposes through targeting and inactivation of the retinoblastoma pocket protein family. Pocket proteins function to regulate cell cycle progression and DNA synthesis through inhibitory interactions with the E2F transcription factors. The interaction of viral oncogenes with the pocket proteins is crucial for their transforming activity. We recently demonstrated that the DNA methyltransferase 1 (DNMT1) gene is an E2F target gene that is transcriptionally activated in cells lacking the retinoblastoma gene (Rb-/-). Overexpression of DNMT1 is implicated in tumor suppressor gene hypermethylation which is associated with tumorigenesis. Given that viral oncogenes potently stimulate E2F activity, we hypothesized that viral infection might activate DNMT1 and thereby promote transformation. Herein, we demonstrate that DNMT1 is strongly activated by the human polyomavirus BKV large T antigen (TAg) and adenovirus E1a. Viral oncogene mutants incapable of binding the pocket proteins are ineffective at activating DNMT1 compared to their wild-type counterparts. Additionally, mutation of the E2F sites within the DNMT1 promoters dramatically abrogates transcriptional activation. These data suggest that viral induction of DNMT1 through modulation of the pRB/E2F pathway may be involved in viral transformation.

Scribble mutants cooperate with oncogenic Ras or Notch to cause neoplastic overgrowth in Drosophila

Cancer is a multistep process involving cooperation between oncogenic or tumor suppressor mutations and interactions between the tumor and surrounding normal tissue. Here we present the first description of cooperative tumorigenesis in Drosophila, by using a system that mimics the development of tumors in mammals. We have used the MARCM system to generate mutant clones of the apical-basal cell polarity tumor suppressor gene, scribble, in the context of normal tissue. We show that scribble mutant clones in the eye disc exhibit ectopic expression of cyclin E and ectopic cell cycles, but do not overgrow due to increased cell death mediated by the JNK pathway and the surrounding wild-type tissue. In contrast, when oncogenic Ras or Notch is expressed within the scribble mutant clones, cell death is prevented and neoplastic tumors develop. This demonstrates, for the first time in Drosophila, that activated alleles of Ras and Notch can act as cooperating oncogenes in the development of epithelial tumors, and highlights the importance of epithelial polarity regulators in restraining oncogenes and preventing tumor formation.

Current perspectives on cytokines for anti-retroviral therapy in AIDS related B-cell lymphomas.

The development of high-grade B-cell lymphoma in Acquired Immunodeficiency Syndrome (AIDS) patients is a relatively late manifestation induced by Human Immunodeficiency Virus-1 (HIV) infection and is considered to be an AIDS-defining condition. Multiple, ongoing molecular and cytogenetic aberrations appear necessary for the development of AIDS-related lymphoma. Studying a panel of human B-cell lines derived from patients with Burkitt's lymphoma (BL) and AIDS-associated Burkitt's lymphoma (AIDS-BL) we had described constitutive expression and secretion of large amounts of Interleukin-16 (IL-16), Macrophage Inflammatory Protein-1alpha (MIP-1alpha), Macrophage Inflammatory Protein-1beta (MIP-1beta), Interleukin-12 (IL-12), Interleukin-10 (IL-10), and Interleukin-7 (IL-7). Some of these cytokines like IL-16, MIP-1beta, MIP-1alpha and Regulated upon activation normal T expressed and secreted (RANTES) are shown to have inhibitory effect on HIV replication. Interestingly, we identified a novel transcription factor family, Macrophage Inflammatory Protein-1alpha Nuclear Protein (MNP), which is suggested as a potential target for anti-retroviral therapy based on the implication of its role and involvement as a key regulator of MIP-1alpha. It is apparent, that HIV induces the production of a cascade of cytokines and cytokine receptors. Some of these molecules serve to increase the infection and replication of HIV per se, and some others serve to induce a state of B-cell growth, activation, and differentiation. This review attempts to delineate the complex mechanisms of viral, B-cell, oncogene, cytokine/cytokine receptor and transcription factor interactions that are involved in AIDS associated lymphomagenesis. Unfolding the relationship between cytokines and the underlying mechanisms of the disease will not only help in understanding the pathophysiology but also will facilitate focusing on the development of new diagnostic and therapeutic strategies.

Oncogenic interaction between BCR-ABL andNUP98-HOXA9 demonstrated by the use of an in vitro purging culture system

Chronic myelogenous leukemia (CML) is a clonal stem cell disease caused by the BCR-ABL oncoprotein and is characterized, in its early phase, by excessive accumulation of mature myeloid cells, which eventually leads to acute leukemia. The genetic events involved in CML's progression to acute leukemia remain largely unknown. Recent studies have detected the presence of the NUP98-HOXA9 fusion oncogene in acute leukemia derived from CML patients, which suggests that these 2 oncoproteins may interact and influence CML disease progression. Using in vitro purging of BCR-ABL-transduced mouse bone marrow cells, we can now report that recipients of bone marrow cells engineered to coexpress BCR-ABL with NUP98-HOXA9 develop acute leukemia within 7 to 10 days after transplantation. However, no disease is detected for more than 2 months in mice receiving bone marrow cells expressing either BCR-ABL or NUP98-HOXA9. We also provide evidence of high levels of HOXA9 expressed in leukemic blasts from acute-phase CML patients and that it interacts significantly on a genetic level with BCR-ABL in our in vivo CML model. Together, these studies support a causative, as opposed to a consequential, role for NUP98-HOXA9 (and possibly HOXA9) in CML disease progression.

Cell-specific transgene expression from a widely transcribed promoter using Cre/lox in mice.

Mice carrying two or more transgenes are used frequently to evaluate oncogene interactions during carcinogenesis. However, neoplastic transformation typically results in reduced expression both of differentiation-specific genes and of transgenes that use their promoters. In contrast, the more widely expressed metallothionein (MT) gene remains expressed at a high level in certain neoplasms, including those developing in pancreas. We have developed a system to maintain high-level, tissue-specific transgene expression during pancreatic carcinogenesis that uses Cre recombinase and a lox site-containing target transgene. Cre was expressed in pancreatic acinar cells under control of the elastase promoter (EL). Cre-mediated target transgene recombination placed a previously silent open-reading frame, encoding rat transforming growth factor alpha (TGFalpha), under control of the MT gene promoter. As long as DNA rearrangement does not occur in other cell types that express MT, TGFalpha expression will be restricted to acinar cells. Development of an effective target transgenic mouse required evaluation of multiple lineages to identify one with sufficient TGFalpha expression to induce pancreatic lesions after transgene rearrangement.

Transgenic mouse models in carcinogenesis research and testing

Double transgenic mice bearing fusion genes consisting of mouse albumin enhancer/promoter-mouse c- myc cDNA and mouse metallothionein 1 promoter-human TGF-α cDNA were generated to investigate the interaction of these genes in hepatic oncogenesis and to provide a general paradigm for characterizing both the interaction of nuclear oncogenes and growth factors in tumorigenesis. In addition, these mice provide an experimental model to test how environmental chemicals might interact with the c- myc and TGF-α transgenes during the neoplastic process. We show experimental evidence that co-expression of TGF-α and c- myc transgenes in mouse liver promotes overproduction of ROS and thus creates an oxidative stress environment. This phenomenon may account for the massive DNA damage and acceleration of hepatocarcinogenesis observed in the TGF-α/c- myc mouse model. Also, the role of mutagenesis in hepatocarcinogenesis induced by 2-amino-3,8-dimethylimidazo(4,5- f)-quinoxaline (MeIQx) was demonstrated in C57BL/lacZ (Muta™ Mice) and double transgenic c- myc/lacZ mice that carry the lacZ mutation reporter gene. The MeLQx hepatocarcinogenicity was associated with an increase in in vivo mutagenicity as scored by mutations in the lacZ reporter gene. These results suggest that transgenic mouse models may provide important tools for testing both the carcinogenic potential of environmental chemicals and the interaction/cooperation of these compounds with specific genes during the neoplastic process.

Carcinogenicity and mutagenicity of heterocyclic amines in transgenic mouse models

Double transgenic mice bearing fusion genes consisting of mouse albumin enhancer/promoter-mouse c– myc cDNA and mouse metallothionein1 promoter-human TGF α cDNA were generated to investigate the interaction of these genes in hepatic oncogenesis and to provide a general paradigm for characterizing both the interaction of nuclear oncogenes and growth factors in tumorigenesis. In addition, these mice provide an experimental model to test how environmental chemicals might interact with the c– myc and TGF α transgenes during the neoplastic process. Treatment of the double transgenic mice with both genotoxic agents such as diethylnitrosamine and 2-amino-3-methylimidazo-[4,5- f]quinoline (IQ) as well as the tumor promoter phenobarbital greatly accelerated the neoplastic process. To investigate the role of mutagenesis in the carcinogenic process, 2-amino-3,8-dimethyl-imidazo[4,5- f]quinoxaline (MeIQx) induced mutagenesis and hepatocarcinogenicity was examined in C57BL/lacZ (Muta TMMice) and double transgenic c– myc/lacZ mice that carry the lacZ mutation reporter gene. The MelQx hepatocarcinogenicity was associated with an increase in in vivo mutagenicity as scored by mutations in the lacZ reporter gene. These results suggest that transgenic mouse models may provide important tools for testing both the carcinogenic potential of environmental chemicals and the interaction/cooperation of these compounds with specific genes during the neoplastic process.

Interaction of c- myc with transforming growth factor α and hepatocyte growth factor in hepatocarcinogenesis

Double transgenic mice bearing fusion genes consisting of mouse albumin enhancer/promoter-mouse c- myc cDNA and mouse metallothionein 1 promoter-human TGF- α cDNA were generated to investigate the interaction of these genes in hepatic oncogenesis and to provide a general paradigm for characterizing the interaction of nuclear oncogenes and growth factors in tumorigenesis. Coexpression of c- myc and TGF- α as transgenes in the mouse liver resulted in a tremendous acceleration of neoplastic development in this organ as compared to expression of either of these transgenes alone. The two distinct cellular reactions that occurred in the liver of the double transgenic mice prior to the appearance of liver tumors were dysplastic and apoptotic changes in the existing hepatocytes followed by emergence of multiple focal lesions composed of both hyperplastic and dysplastic cell populations. These observations suggest that the interaction of c- myc and TGF- α, during development of hepatic neoplasia contributes to the selection and expansion of the preneoplastic cell populations which consequently increases the probability of malignant conversion. These studies have now been extended to examine the interaction of hepatocyte growth factor (HGF) with c- myc during hepatocarcinogenesis in the transgenic mouse model. While sustained overexpression of c- myc in the liver leads to cancer, coexpression of HGF and c- myc in the liver delayed the appearance of preneoplastic lesions and prevented malignant conversion. Similarly, tumor promotion by phenobarbital was completely inhibited in the c- myc/HGF double transgenic mice whereas phenobarbital was an effective tumor promoter in the c- myc single transgenic mice. The results indicate that HGF may function as a tumor suppressor during early stages of liver carcinogenesis, and suggest the possibility of therapeutic application for this cytokine. Furthermore, we show for the first time that interaction of c- myc with HGF or TGF- α results in profoundly different outcomes of the neoplastic process in the liver.

Transgenic mouse models in carcinogenesis: interaction of c-myc with transforming growth factor alpha and hepatocyte growth factor in hepatocarcinogenesis.

1. Overexpression of the c-myc oncogene is associated with a variety of both human and experimental tumours, and cooperation of other oncogenes and growth factors with the myc family are critical in the evolution of the malignant phenotype. 2. Double transgenic mice bearing fusion genes consisting of mouse albumin enhancer/promoter-mouse c-myc cDNA and mouse metallothionein 1 promoter-human transforming growth factor (TGF-alpha) cDNA were generated to investigate the interaction of these genes in hepatic oncogenesis and to provide a general paradigm for characterizing the interaction of nuclear oncogenes and growth factors in tumourigenesis. 3. Coexpression of c-myc and TGF-alpha as transgenes in the mouse liver resulted in a tremendous acceleration of neoplastic development in this organ as compared to expression of either of these transgenes alone. The two distinct cellular reactions that occurred in the liver of the double transgenic mice prior to the appearance of liver tumours were dysplastic and apoptotic changes in the existing hepatocytes followed by emergence of multiple focal lesions composed of both hyperplastic and dysplastic cell populations. 4. These observations suggest that the interaction of c-myc and TGF-alpha, during development of hepatic neoplasia contributes to the selection and expansion of the preneoplastic cell populations which consequently increases the probability of malignant conversion. 5. We have now extended these studied and examined the interaction of hepatocyte growth factor (HGF) with c-myc during hepatocarcinogenesis in the transgenic mouse model. While sustained overexpression of c-myc in the liver leads to cancer, coexpression of HGF and c-myc in the liver delayed the appearance of preneoplastic lesions and prevented malignant conversion. Similarly, tumour promotion by phenobarbitone was completely inhibited in the c-myc/HGF double transgenic mice whereas phenobarbitone was an effective tumour promoter in the c-myc single transgenic mice. 6. The results indicate that HGF may function as a tumour suppressor during early stages of liver carcinogenesis, and suggest the possibility of therapeutic application for this cytokine. Furthermore, we show for the first time that interaction of c-myc with HGF or TGF-alpha results in profoundly different outcomes of the neoplastic process in the liver.

Anticlastogenic and antioxidative activities of plant flavonoids in mice

Double transgenic mice bearing fusion genes consisting of mouse albumin enhancer/promoter-mouse c–myc cDNA and mouse metallothionein1 promoter-human TGFα cDNA were generated to investigate the interaction of these genes in hepatic oncogenesis and to provide a general paradigm for characterizing both the interaction of nuclear oncogenes and growth factors in tumorigenesis. In addition, these mice provide an experimental model to test how environmental chemicals might interact with the c–myc and TGFα transgenes during the neoplastic process. Treatment of the double transgenic mice with both genotoxic agents such as diethylnitrosamine and 2-amino-3-methylimidazo-[4,5-f]quinoline (IQ) as well as the tumor promoter phenobarbital greatly accelerated the neoplastic process. To investigate the role of mutagenesis in the carcinogenic process, 2-amino-3,8-dimethyl-imidazo[4,5-f]quinoxaline (MeIQx) induced mutagenesis and hepatocarcinogenicity was examined in C57BL/lacZ (MutaTMMice) and double transgenic c–myc/lacZ mice that carry the lacZ mutation reporter gene. The MelQx hepatocarcinogenicity was associated with an increase in in vivo mutagenicity as scored by mutations in the lacZ reporter gene. These results suggest that transgenic mouse models may provide important tools for testing both the carcinogenic potential of environmental chemicals and the interaction/cooperation of these compounds with specific genes during the neoplastic process.

ONCOGENES IN RHEUMATOID ARTHRITIS

The evolving knowledge of the actions and interactions of (proto)oncogenes in cancer has deeply influenced the understanding of other nonmalignant diseases. In RA, the longstanding pathohistologic evidence of transformed-appearing synovial cells at the site of bone and cartilage attachment and joint destruction can now be explained in terms of alterations of cell regulation, cell cycle, and apoptotically triggered cell death. The detection of upregulated oncogenes and their gene products at these sites supported the hypothesis of an aberrant synovial cell type invading the joint. Interestingly, there are hints that this transformation of synovial cells may require more than one activated oncogene. A model was introduced by Carson and Ribero in 1993. In this model, a primary stimulus affects the cell and leads to the enhanced transcription of an oncogene (i.e., c-myc). A second stimulus activates other oncogenes and determines if this cell (i.e., a synovial fibroblast) proliferates (marked by the presence of bcl-2 mRNA) or undergoes apoptosis (marked by fas mRNA and Fas expression at the cell surface). This co-upregulation might explain why some investigators could not detect a significant upregulation of oncogenes in cultured synovial fibroblasts devoid of their normal milieu. Based on the results of the specific activity of Fas and perforin and recent data from our laboratory, we have modified Carson's model to include these data. As there exists an established retroviral model in which the tax sequence of the HTLV retrovirus initiates central oncogene transcription similar to those activated in RA, the retroviral particles, which do not resemble any other known retrovirus but are detectable in the synovial fluid, might well be an important stimulus in the pathogenesis of RA. To simplify the puzzling events of oncogene interactions in RA, we have summarized the data and propose that an oncogene network acts as a pathogenic mechanism in the synoviocytes of the rheumatoid joint. Similar to the "cytokine network" regulating the T-cell-dependent pathway, the "oncogene network" is presumably the major T-cell-independent pathway in RA (Fig. 4).

Gastric cancer

Gastric cancer remains one of the most common causes of cancer mortality worldwide, despite a decline in incidence in Western countries. An important recent finding has been the recognition of an association between Helicobacter pylori and gastric cancer. The mechanisms, however, by which H. pylori infection and other environmental factors influence gastric tumorigenesis remain to be elucidated. The recent identification of growth factors and their receptors has provided insights into the regulation of cell growth, proliferation, and differentiation. Furthermore, studies of the effects and interactions of growth factors, oncogenes, and tumor suppressor genes on biological behavior of human gastric cancers are in progress and may aid in therapeutic decision-making. Fortuitously, molecular analysis requires small amounts of tumor tissue, which can be obtained from endoscopic biopsies. Regarding the treatment of gastric cancer, promising data on the use of neoadjuvant chemotherapy to increase surgical resectability have been obtained. However, locoregional failure continues to be the major obstacle and the addition of adjuvant intraperitoneal chemotherapy is specifically aimed at this problem. Novel therapeutic approaches are clearly needed in the management of advanced disease in view of the relatively poor results thus far with available compounds. Current Opinion in Gastroenterology 1993, 9:930-937

Altered RB expression is a prognostic clinical marker involved in human bladder tumorigenesis.

We are now beginning to understand the development of bladder cancer at the molecular level. Tumor evolution involves the interaction of both oncogenes and tumor suppressor genes. One of the key tumor suppressor genes in this process is the retinoblastoma (RB) gene. Much has been learned recently about the role of this gene in the tumor progression and prognosis of bladder cancer, although several questions are still unanswered. The progress made on this subject in our laboratory as well as others will be the focus of this report.

Molecular determinants of metastatic transformation.

In recent years, experimental systems have developed to analyze genetic and epigenetic regulation of the metastatic phenotype. Numerous studies have uncovered a potent role for transforming oncogenes in metastatic conversion. In addition, it has been shown that oncoprotein products operate in a dose-dependent fashion. The continued expression of oncoproteins is required to induce and regulate metastatic dissemination of tumor cells and, consequently, many of the signal transduction pathways that are controlled by the oncogene products can regulate metastasis. Exogenous growth factors that act through these same pathways also alter metastatic potential. Some primary and immortalized cells can be transformed by oncogenes but remain completely benign and nonmetastatic. Malignant transformation can be achieved in these cells through the cooperative interaction of specific oncogenes or loss of active suppression regulated by recessive genetic determinants. Therefore, it is likely that tumor cells acquire the metastatic phenotype through the cooperative interaction of dominant and recessive genetic alterations. This model is consistent with the correlative data accumulating in studies of human tumor specimens where more malignant carcinomas often contain both activating mutations in oncogenes and either inactivating mutations or loss of tumor-suppressor genes.

Genetic regulation of mammary carcinogenesis in the rat by susceptibility and suppressor genes.

Rat strains differ in their susceptibilities to mammary carcinogenesis. Strains such as Wistar-Furth are very susceptible to chemically induced carcinogenesis. This phenotype is controlled by autosomally dominant susceptibility genes. Strains such as the Copenhagen are resistant to spontaneous and induced mammary carcinogenesis. This phenotype is controlled by an autosomal dominant gene termed the mammary carcinoma suppressor (MCS). Strains with intermediate susceptibility such as the F344 carry neither the MCS nor susceptibility genes. Both the MCS and susceptibility genes are chiefly active within the mammary parenchyma. Both genes act at carcinogenesis stages beyond carcinogen metabolism and DNA adduction. The MCS gene completely inhibits both palpable and microcarcinomas. It does not inhibit focal alveolar hyperplasias. Its gene product acts solely within the mammary epithelial cell in which it is produced. We are currently investigating the interactions of various oncogenes and the MCS gene. In addition, efforts are underway to identify and clone this gene.

Functional analysis of a complex oncogene arrangement in biotype III Agrobacterium tumefaciens strains

The ubiquitous grapevine-associated octopine/cucumopine Ti plasmids of biotype III Agrobacterium tumefaciens strains carry two T regions, TA and TB, with a complex oncogene arrangement. Within the octopine/cucumopine group, two main strain types were identified: 'large TA' strains with a TA region resembling the TL region of the biotype I octopine strain Ach5 and 'small TA' strains with a similar T region organization as the 'large TA' strains but with a large internal TA deletion. Structural and functional studies of the representative 'large TA' strain Tm4 revealed six oncogenes. Each oncogene was inserted in a disarmed vector and tested for biological activity using the corresponding oncogenes of Ach5 as standards. Five Tm4 oncogenes, TA-iaaM, T-ipt, T-6b, TB-iaaH and TB-iaaM, were shown to be active, the IS-interrupted TA-iaaH gene was inactive. To study the role of each gene in the pTiTm4 context, several single and multiple pTiTm4 mutations were constructed. It was shown that whereas TA-iaaM and TB-iaaH are essential for tumour formation on grapevine, T-ipt, T-6b and TB-iaaM are not. The avirulence of the TA-iaaM- mutant was shown to be due to an inhibitory effect of the T-ipt gene, since a TA-iaaM-/T-ipt- double mutant was fully virulent. We conclude that the TA-iaaM gene of 'large TA' strains is specifically required to counteract the tumour growth inhibiting activity of the T-ipt gene. Both TA-iaaM and T-ipt are absent from the 'small TA' strains. A model on the roles and interactions of the different oncogenes in 'large TA' and 'small TA' strains is presented.