Oncogene activation and tumor progression. George Klein and Eva KJdn. Department of Tumor Biology, Karolinska Institutet, S-104 01 Stockholm,. Sweden.
Cardnogeiwsfa Vol.5 no.4 pp.429-435, 1984
Commentary
Oncogene activation and tumor progression
George Klein and Eva KJdn Department of Tumor Biology, Karolinska Institutet, S-104 01 Stockholm, Sweden
Introduction 25 years ago (1) Leslie Foulds formulated a number of 'rules' for tumor progression, the process whereby 'tumors go from bad to worse', in Peyton Rous' original description (2). These rules are equally pertinent today and accessible to analysis by the powerful tools of modem biology. Foulds dissected the malignant phenotype into 'unit characteristics', such as growth rate, invasiveness, metastasizability, hormone dependence, etc. In his own words (3): 'the behavior of tumors is determined by numerous characters that, within wide limits, are independently variable, capable of different combinations and assortments and liable to independent progression'. This explains the biological individuality of tumors. No two tumors are exactly alike, even if induced by the same agent and in the same type of target cells of the same inbred host genotype. Foulds pointed out the need for 'particularization or factorial analysis' (4). Foulds' definition of the 'unit characteristics' reflects his working area, experimental pathology. While it was mainly based on the studies of murine tumors, his examples also included human tumors. He emphasized the independent change of metastasizability and hormone responsiveness in human prostatic and thyroid carcinomas and the stepwise changes in the invasiveness of cervical, gastrointestinal and bladder carcinomas. In the mouse system, the possibility of maintaining the tumor beyond the lifespan of the host revealed that progression does not reach an endpoint in the primary host. With the passage of tumor cell populations, new opportunities are created for further microevolutionary changes. They lead to increased independence from local, systemic or drug induced growth restrictions. Foulds' rules are also applicable to changes in the expression of defined cellular markers like histocompatibility antigens (5,6) or differentiation related enzymes (7). In tumors of epithelial tissues the evolution of progressional changes could often be seen directly. Increased invasiveness was detected in sharply outlined focal areas (3), suggesting a clonal event. Experimental analysis of progressional changes in the characteristics of transplanted tumors has first shown that they are due to a Darwinian process of variation and selection within the cell population (5,6). Before the arrival of modern molecular biology, analytical approaches were hampered by the Janus-faced double nature of •Abbreviations: c-onc, cellular oncogenes; v-onc, viral oncogens; LTR, Longterminal repeat; MPC, murine plasmacytoma; BL, human Burkitt lymphoma; LAP, intracisternal A partide; LCL, lymphoblastoid cell lines. © IRL Press Ltd., Oxford, England.
tumor cells. While capable of evolving by mutation and selection, like asexually reproducing microorganisms, their phenotype can also change by differentiation steps, as in normal somatic cells of higher organisms. Molecular distinctions between structural changes at the DNA level and epigenetic changes in regulation have become possible only very recently. Oncogene activation - qualitative or quantitative? Recent developments in cancer research have shown that the increased and/or changed activity of certain single genes, collectively termed as cellular oncogenes (c-onc)* or protooncogenes, can lead to neoplastic development, alone or together with other factors. The oncogenes were originally identified as the cellular homologues of the transforming genes carried by the acute, directly transforming, or class I RNA tumor viruses (for recent reviews, see reference 8). The number of known oncogenes is limited to about 20. They are all highly conserved in evolution, suggesting that they may play an important biological role. It has been debated whether tumorigenic behavior results from the constitutive switch-on of unmodified cellular oncogenes (the 'quantitative' or dosage hypothesis), or from modifications in their DNA sequence, due to point mutations or more extensive DNA-rearrangements (the 'qualitative' hypothesis) (9,10). Probably, both events can occur, and even in the same cell (11). The 'reassortment of unit characteristics' during tumor progression may reflect sequential quantitative and/or qualitative changes in oncogene expression. As a rule, the virally carried oncogenes (\-onc) differ from the corresponding c-onc sequences to various degrees (reviewed by Duesberg, reference 9). Some of these modifications occur because the genes follow the retroviral lifestyle, involving gene processing and the removal of introns. Others may arise from genetic drift. It is unknown whether any of them are essential for the transforming function. In contrast, the point mutations identified in the ras family of oncogenes (12,13,14) that affect one of two specific amino acid positions, are clearly responsible for the acquisition of the transforming potential. The replacement of a single amino acid in a strategically important position is believed to impose major structural changes on the protein. While this speaks for the qualitative model, it is also clear that the constitutive switch-on of a normal c-onc can have a transforming effect as well. This was proven by the transforming capacity of molecular constructs carrying c-mos (15) or c-ras (16) coupled to retroviral longterminal repeats (LTR) regions, containing both promoter and enhancer sequences. Thus for the ras gene, the switch-on of the normal product was shown to have the same effect as a mutational change in the structural gene, without any apparent change in regulatory functions. It has to be noted that not all the viral LTR/c-onc con-
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structs had transforming capacity. This was shown by recombinational analysis using the human and murine c-mos gene. The \-mos carried by the murine sarcoma virus (MSV) retained transforming activity when retroviral LTR sequences were attached to either its 5' or 3 ' end. In contrast, the murine c-mos was only activated if the LTR was attached to the 5' end. The lack of transformation by the 3-LTR construct could be explained by the existence of a regulatory sequence (designated UMF), flanking the 5' end of c-mos. Removal of this sequence resulted in an active product. This illustrates the difficulty of sharp distinctions between the quantitative and the qualitative model. Removal of a flanking regulatory sequence is a structural modification at the DNA level and is thus a qualitative event which determines the transforming capacity. Since the activated c-mos makes a normal product, however, it is also in line with the quantitative model. A sharper distinction between the 'qualitative' and 'quantitative' models could be made if the definitions of qualitative changes on the DNA level were further specified as (0 changes in the structural one gene leading to an altered protein product; and (ii) changes in the regulatory sequences of the gene or position effects leading to the expression of a normally non-expressed protein product or to quantitative changes in normal expression.. LTR/human c-mos constucts had no transforming effect. This was explained by the existence of a small gap in an internal domain of the gene that counteracted the activating influence of attached LTR sequences. It is tempting to speculate that the presence of this additional regulation in the human oncogene may relate to the low transformability of human cells in vitro and the longer lifespan of the species, compared to rodents. C-mos is not expressed in normal cells. Expression at a low level, corresponding to 10 protein molecules/cell, may already lead to transformation. It can therefore be assumed that c-mos is a particularly dangerous oncogene that requires several safeguards against accidental turn-on. The difference between the c-mos in men and mice shows that subtle structural differences acquired during evolution can change the activability of an oncogene. Oncogene activation by chromosomal translocation Two B lymphocyte derived tumors, murine plasmacytoma ( M P Q and human Burkitt lymphoma (BL), carry principally similar chromosomal translocations (for review, see reference 17). The same oncogene, c-myc, and the immunoglobulin genes are involved in both species. In the majority of MPCs, the distal segment of chromosome 15 enters into a reciprocal exchange with part of the IgH-region of chromosome 12 (typical translocation), or the kappa-locus carrying chromosome 6 (variant translocation). In BL, the typical translocation arises from the reciprocal exchange between the distal part of chromosome 8 and the IgHcarrying region of chromosome 14. Less frequently, the exchange occurs with the kappa locus carrying region of the short arm of chromosome 2 or the lambda region of chromosome 22. Since one of the three translocations is regularly present in all BL biopsies and derived lines, but not in other lymphomas or leukemias (except the L2 form of B-cell derived ALL that is believed to originate from the same cell as BL), we have suggested (18) that they act by bringing an oncogene — and presumably the same oncogene - under the influence of a chromosome region that is highly active in the Ig-producing 430
target cell. We surmised that the resulting constitutive switch -on of the oncogene may be an essential event in the neoplastic transformation. Subsequent studies provided molecular evidence (for reviews, see 17, 19, 20) for the basic correctness of this picture. Murine chromosome 15 and human chromosome 8 were found to carry the same oncogene, c-myc, localized at the place where the translocation breakpoint occurs. The typical break of the IgH carrying murine 12 and human 14 chromosome was seen most frequently within the IgH complex itself, although its exact site varied. In all cases, the c-myc gene was found to be transposed to face the IgH sequences head to head (21-31); therefore transcription of the two genes proceeds in the opposite direction and from the two opposite DNA strands. The majority of MPCs produce IgA. In these tumors, c-myc is usually transposed to the Sa-Ca region. Most BLs produce IgM and c-myc is most frequently rearranged to the S/t-Qt area. However, this is only an approximate rule with many exceptions (for review, see 32). There are considerable variations in the location of the breakpoint in and around the c-myc gene. In the typical translocation where c-myc is transposed to the IgH-carrying chromosome, the break can affect the non-coding exon 1, but not the coding exons 2 and 3. It can be upstream of the 5' end of the gene either within the EcoRl fragment or outside it. In the latter case, the Southern blot shows no rearrangement of c-myc, in spite of the translocation. For MPC, this means that the breakpoint must be > 10 kb upstream of exon 1 of c-myc, or > 7 kb downstream of the gene. In BL, it means that the breakpoints are > 17.5 kb in the 5' direction or > 8 kb in the 3' direction (24). How can the resumed activating effect of the IgH region act over such long distances? Perry (32) has emphasized that large chromatin areas may be activated, as reflected by hypomethylation and/or the appearance of DNase I hypersensitive sites. These variations suggest that the molecular mechanism of c-myc activation caused by translocation cannot be explained on a single model. Several alternative mechanisms of activation affecting larger regions of the IgH gene and their flanking sequences have to be assumed to exist. In the variant translocations of BL which involve the light chain genes — kappa from chromosome 2 or lambda from chromosome 22 — the cytogenetic events are different from the typical (IgH) translocation (34,35). The c-myc gene stays in its original position on chromosome 8. The constant genes and part of the variable genes of the light chain locus are transposed to the 3' end of the myc gene giving a tail to head rnyc-light chain orientation. The oncogene is probably activated from the light chain region in the downstream position. It is thus clear that both the typical and variant translocations involve the c-myc gene in both BL and MPC. The variable breakpoints upstream and downstream of the gene 'focus' on the coding exons 2 and 3 that remain invariably intact. This suggests that the c-myc protein plays a crucial role in transformation. The considerable variation in the distance between c-myc and the immunoglobulin genes speaks against the existence of a single enhancer or promoter that would be responsible for the activation of the transposed oncogene. It may be surmised that the activated state of the chromatin in the Ig-gene regions, established at the time when the cell becomes committed to B-cell differentiation may be responsible for the constitutive activation of the inserted myc gene, by mechanisms that remain to be defined.
Oncogene activation and tumor prognaslon
Do the translocations play a decisive role in generating BL andMPC? The translocations are regularly found in primary BL and MPC (36,37). They do not emerge during serial passage in vivo or in vitro. Tetraploid primary MPCs have two translocated and two normal chromosomes. This means that the translocation event must have occurred prior to tetraploidization, i.e., during the microscopical development of the tumor or already at its very inception. Trying to distinguish between a causative and a secondary role in the development of malignancy, great importance must be attached to the regularity of the association between a given tumor-associated trait and the tumor type (38). For BL, the association with the cytogenetic change is absolute at present. Among all BL biopsies and/or derived lines that have been examined in a technically satisfactory way, only one exception can be found, the translocation negative BJAB line (39,40). All other tumors and lines were found to carry one of the three translocations (8; 14, 8;22 or 8;2). BJAB has been derived from an African patient.J.A. It does not have the typical characteristics of BL. While 97% of the African BLs are EBV-DNA and EBNA positive (41), both the J.A. biopsy and the derived BJAB line were EBV-negative. Alone among BL derived lines tested, BJAB has a low agarose clonability, does not grow when injected subcutaneously into nude mice (42) and does not contain the BL-associated gp 69/71 membrane glycoprotein marker (43). If BJAB is eliminated, there is no known exception to the rule that BLs contain one of the three specific translocations. In MPC the association is not equally regular. About 15% of the plasmacytomas do not carry either the typical or the variant translocation. Recently, high resolution banding analysis of three translocation negative tumors (44) revealed an interstitial deletion in the D2/3 band region of chromosome 15, corresponding to the location of the typical translocation breakpoint. This deletion was seen in one of the two homologous chromosomes in diploid tumors, and in two of four chromosomes in tetraploids. In these three tumors c-myc was transcribed at a high level. In one of them, c-myc was rearranged, according to the EcdSl restriction — Southern blot analysis. This suggests that in MPC, c-myc can be regulated by events that are cytogenetically different from but functionally analogous to the usual translocations. The interstitial deletion may have displaced the gene to a functionally active region within the same or another chromosome. The importance of the translocations in plasmacytomagenesis is also illustrated by their occurrence under cytogenetically exceptional circumstances. The AKR 6; 15 mouse strain carries a centromerically fused (Robertsonian) 6; 15 chromosome. Recently we have induced plasmacytomas (PC) in AKR 6; 15 x Balb/c Fj mice (45). Seven of ten PCs had a typical 12; 15 translocation. In the three remaining tumors, the distal segments of the Robertsonian 6; 15 chromosome have been exchanged reciprocally, due to pericentric inversion. The breakpoints were the same as in the usual 6; 15 variant translocation between two different chromosomes. It is unlikely that this extraordinary cytogenetic event would have occurred in three independent tumors, had it not played a crucial role in the determination of the malignant phenotype. The regularity and precision of the translocations seen in MPC and BL and their absence from other types of murine
and human B-cell tumors suggests that this event is necessary for the tumorigenic process. The identical features of the translocations in the two tumors are probably highly meaningful. The breakpoints affect the c-myc oncogene in almost the same fashion (with only minor variations) in the two types of tumors, in spite of their disparate natural history and maturation stage of the cell of origin. Moreover, no other oncogenes than c-myc are known to be shared by the human chromosome 8 and murine 15. Human 8 but not mouse 15 carries c-mos, murine 15 but not human 8 carries c-sis. A possible common denominator in the natural history of MPC and BL may be sought in their relatively long preneoplastic latency period, involving chronic proliferation of the pre-neoplastic target cell. In the EBV-carrying African form of BL, the target cells are presumably first immortalized by EBV and then stimulated by chronic malaria (38). Some of the EBV-carrying B cells cannot follow the normal pathway of maturation. The stimulation by the chronic infection may increase the risk of genetic errors, including illegitimate recombinations between chromosomes. In MPC, the oil (or other foreign body) induced granulomatous reaction within the peritoneal cavity may provide the proliferation predisposition for the chromosomal changes. A similar translocation has also been found in another B-cell derived tumor, the spontaneous immunocytoma of the Louvain strain of rats (46). It arose from a reciprocal exchange between chromosomes 6 and 7 and showed striking banding homologies with the typical 12; 15 translocation in the murine PC. Recently, our laboratory has localized c-myc to rat chromosome 7 (47). The rat immunoglobulin genes have not yet been localized on the chromosome map. In conclusion, it is most reasonable to assure that the translocations play an essential role in the genesis of MPC and BL. This does not mean that they represent the only important change. Secondary changes For the understanding of tumor progression, secondary cytogenetic and molecular events are also of greater interest. Van Ness et al. (48) described an aberrant rearrangement of the kappa locus in one MPC line that also carried the typical 12; 15 translocation. The additional event involved chromosome 6. It was a tripartite translocation, involving chromosomes 6, 12 and 15. This recombinant was quite different from the usual 12; 15 that was also present. The chromosome 15-derived fragment that entered the tripartite rearrangement did not carry c-myc sequences. Perlmutter et al. (49) found a 6; 10 recombinant in the NS-1 murine plasmacytoma line that also contained the typical 12; 15 translocation. This recombination, detected exclusively at the molecular level, has led to the juxtaposition of Q and a single copy element derived from chromosome 10. A corresponding 6; 10 translocation was also found in a second MPC. The chromosome 10-derived sequence was identical in both tumors, it was not homologous to any known oncogene, it was transcribed at a high level, and homologous sequences could be detected in three different species: mouse, rabbit and human. It is possible that the 6; 10 translocation reflects a secondary oncogene activation event that occurs in the course of tumor progression. Rearrangement of the c-mos oncogene found in some mouse plasmacytomas may be another case in point. It may be particularly significant in view of the fact that c-mos is not transcribed in normal tissues at all (50) and 431
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only very rarely in tumors. In the XRPC24 plasmacytoma that carries a typical 12; 15 translocation and has an already activated c-myc, the c-mos gene was highly transcribed as well. The rearranged, cloned sequence, designated as rc-mas, could transform NIH 3T3 cells in transformed assays (51,52). The 5' end of the gene contained an intracisternal A particle (LAP) gene in a head to head orientation. LAP sequences are known to behave as movable genetic elements. It was suggested that the insertion of the LAP sequences may be responsible for the activation of c-mos in analogy with the Hayward model of oncogene activation by retroviral promoter insertion (35). LAP gene integration into the c-mos occurred also in the NS-1 plasmacytoma, another 12; 15 translocation carrier (52). The molecular details were different in the two tumors, however, both with regard to orientation and distance between the LAP and c-mos. In NS-1 c-mos was not detectably transcribed. Chromosome 11 trisomy is another frequent secondary change in murine plasmacytomas (Ohno.E., Migita.S., Wiener,F., Babonits.M., Klein.G., Mushinski.J.F. and Potter.M., in preparation). The occurrence of characteristic secondary changes in a group of tumors that have the same primary change will doubtlessly be encountered to an increasing extent as the submolecular analysis of experimental and human tumors progress. It is already known that Phi chromosome carrying CML is subject to characteristic secondary cytogenetic changes (54). Also, Steel has found (55) that the development of aneuploidy during the prolonged propagation of lymphoblastoid cell lines (LCL) follows non-random pathways, characterized by certain trisomies, but without the appearance of the BL-type translocations. Long-propagated LCL become tumorigenic in nude mice, in parallel with their aneuploidisation (42), illustrating the rule that tumorigenicity can be acquired through several independent pathways. Double or multiple oncogene activation detected by transfection assays In several systems where oncogene activation contributed to the emergence of tumors by one of the three mechanisms so far discussed, namely viral transduction, viral promoter insertion and chromosome translocation, activated oncogenes have also been detected by the focus-forming assay on NIH 3T3 fibroblasts after DNA transfection (for reviews see 56, 57). One of the earliest experiments of this type was carried out with the ALV-induced chicken B-cell lymphoma where c-myc became activated through the insertion of a retroviral promoter (53). Surprisingly, the transforming DNA sequence was not c-myc but another, previously unknown oncogene (58). It was designated as ChBlym-1 or Blym for short (59). The same oncogene was also detected by transfecting NIH 3T3 with the DNA of six Burkitt lymphomas (60), while in another BL line (61) the transfecting sequences were N-ras. ALV-induced chicken B-lymphoma and human BL originates from B-cells at a similar intermediate stage of differentiation. The involvement of the same two oncogenes, c-myc and B-lym in the genesis of these tumors is therefore particularly intriguing. Transfection experiments with DNA of mouse plasmacytoma have also identified an active oncogene but this was different from both c-myc and Blym (62,63). ALV-induced chicken lymphoma develops by a multistep process, including both pre-neoplastic and neoplastic stages 432
(61). Activation of c-myc is probably an early, pre-neoplastic event (62). In the EBV-carrying African form of BL, the situation is probably quite different. EBV is a highly potent transforming (immortalizing) agent in itself. Immortalized LCL can be established from the blood of EBV-seropositive persons at any time, suggesting that they harbor virus carrying B-lymphocytes continuously (64). Such lines are diploid, polyclonal, have a low agarose clonability and do not grow in nude mice. Translocation earring BL lines are monoclonal, have a high agarose clonabilty and grow progressively in nude mice as subcutaneous tumors (42). The rare, c-myc activating chromosomal translocation is therefore probably a late event, perhaps the last step that permits the escape of the cell from some immunological or non-immunological host control that restricts its immediate predecessor. The postulated difference in the role of c-myc in the avian and the human B-lymphoma is entirely conceivable. Already Foulds' comparative analysis of the natural history of different tumors has shown that the same progressional step can occur at different stages of neoplastic development (4). Similar situations have been described in some other systems. Lane et al. (63) found that Abelson virus-induced murine pre-B-cell lymphomas contained transforming sequences that were not present in the viral genome and had no homology with the abl oncogene. The \-abl oncogene itself could be lost during the serial passage of Abelson virusinduced lymphomas, without the loss of the tumorigenic phenotype (65). Transforming DNA sequences derived from MuMTV induced mouse mammary carcinomas differed from any viral sequences (66,67). The DMBA + TPA induced carcinogenic process in the mouse skin was recently studied by Balmain and Pragnell by the transfection technique (68). DNA isolated from carcinogen and/or promoter painted mouse skin had no transforming activity for NIH 3T3 cells, in contrast to papilloma and carcinoma DNA. Transforming sequences isolated from the latter could be assigned to the H-ras family. The lack of any detectable difference in the transforming ability of DNA from the benign papillomas and the invasive carcinomas was puzzling. Balmain suggests that the major chromosomal differences between the purely diploid papillomas and the highly aneuploid carcinomas may be responsible for the difference in tumorigenic behavior. Informative as the NIH 3T3 transfection experiments have been, the relatively easy transformability of this highly aneuploid, immortal cell line, and its notorious predilection for the ras oncogene family has been a source of continuing concern. It is therefore of great interest that Land, Parada and Weinberg (69) have recently succeeded in transforming primary rat embryonic fibroblasts (REF), by transfecting them with two different oncogenes, ras and myc. A molecular clone isolated from the human EJ bladder carcinoma line served as the source of transforming ras. Transfection wim ras alone gave no foci, but certain morphological signs of transformation were present, provided that the transfectants were separated from surrounding untransfected cells or were cloned in soft agar. After they have grown to colonies of 500-5000 cells, they lost the ability to divide, however. In contrast, an established rat fibroblast line could be permanently transformed by ras, and became also tumorigenic in vivo. It was concluded that the inability of 4he activated ras gene to expand transfected REFs in dense monolayer cultures, or to render them tumorigenic in vivo could be com-
Oncogene activation and tumor progression
pensated by cellular functions that existed in immortal cell lines prior to transfection. A second transfection step was then introduced to test whether the myc gene could provide this function. Proviral \-myc clones had no transforming effect on either NIH-3T3 or on rat-1 cells. When introduced into REFs together with ras, under conditions where neither of the two oncogenes had any obvious effect, transformed foci were readily obtained. They were tumorigenic in nude mice or in syngeneic rats, although with certain limitations. In contrast to the ras transformed continuous rat fibroblast line that grew progressively, they only grew to a size of about 2 cm and remained stationary thereafter. The mechanism responsible for the control of the progressive growth of the tumors is not known. This system can be regarded as a model in which the malignant phenotype imposed by the ras gene varies depending on the combination with other cellular events. This 'double transfection' system has led to the definition of two complementation groups. H-ras could be substituted by N-ras or by polyoma middle T, whereas \-myc could be replaced by polyoma large T or by the Ela gene of adenovirus (70). Polyoma middle T is known to induce morphological changes and anchorage independence, whereas large T is required for immortalization and reduced serum dependence (71). This contrasting behavior stimulated speculations concerning the possible modes of action of the products of the two complementation groups. According to this model the genes belonging to the 'myc complementation group' code for nuclear proteins, whereas the genes of the 'ras group' products are membrane associated and are responsible for morphological and functional changes related to the social behavior of cells. Some of these, like anchorage independence may form an important part of tumorigenic behavior in vivo. Active ras oncogenes were found in bladder, colon, lung, pancreas and skin carcinomas, neuroblastomas, sarcomas and different types of hemopoietic malignancies. Mycactivation is associated with myelocytomatosis, myeloid leukemia, bursal lymphomas, Burkitt lymphoma and plasmacytomas. V-/nyc-carrying viruses can induce kidney, pancreas and liver carcinomas and mesotheliomas. Amplified copies of c-myc have been found in neuroendocrine and colonic tumors (72). Thus, neither of the two oncogenes ras and myc are tissue specific. This does not mean that the same oncogenic product can contribute to the transformed phenotypes of any cell. The target cell range is restricted, but the boundaries are wider for some oncogene products than for others. This can be exemplified by the fact that the src protein is expressed in fibroblasts and in certain cells of the hemopoietic system, but only the fibroblasts are transformed. The myb product is also expressed in both types of cells, but it transforms only hemopoietic cells. These and other findings show that the impact of a given oncogene product differs depending on the cell type in which it is expressed. The work of Bishop (73) on the myb oncogene has clearly shown that the oncogene product and the differentiation program of the target cell can interact in different ways. A cell may ignore or suppress the oncogene in a certain phase of differentiation. The amplified myc gene in the HL60 myeloid leukemia line is switched off when the cells are induced to differentiate (74). The stepwise changes in 'unit characteristics' during tumor progression will have to be redefined, not only in terms of what may correspond to the sequential activation of different
oncogenes, but also with regard to the interactions between each oncogene product and the phenotype of the cell where it is expressed. It is probable that there are more than two complementation groups among the known oncogenes. The widely diversified and finely graded phenotypic variation that is manifested as tumor progression is brought about by complex events. Accordingly, the work of Land et al. (69) has demonstrated the existence of minor phenotypic differences between different members of the same complementation group. The present subdivision is heuristically useful, but will be replaced by more complex schemes as analysis proceeds. In addition to the double transfection system with two different oncogenes, the pick-up of two different oncogenes - unlinked and unrelated but complementary in their interaction - by the same transforming virus provides a fascinating tool for this analysis (75). Perspectives In the 1950s, Levan (76), Makino (77) and others developed the 'stemline concept' of tumor growth. It implies that one main stemline is responsible for the progressive growth of each tumor cell population. However, chromosomal and other genetic variation generates new variants that branch off from the stemline. The vast majority of these variants is less adapted for proliferation than the original type. Occasional rare variants may have a selective advantage and can give rise to a new stemline. The natural history of most tumors can be therefore described as a continuous series of stepwise changes. Tumor progression is the expression of these changes at the population level. This microevolutionary process does not put any a priori restriction on the cellular mechanisms that may contribute to this variation, nor is there any obvious reason why only a very limited set of genes should be involved. Nevertheless, the limited evidence surveyed already shows that sequential activation of the presently known oncogenes can be at least a part of this process. DNA-rearrangement by chromosomal transposition represents a particularly intriguing mechanism, since it can bring an oncogene under the cis control of a highly active chromosome region. In the most extensively studied systems, Burkitt lymphoma and murine plasmacytoma, the activated state of the immunoglobulin gene region in B-cells may be more important in switching on the transposed c-myc oncogene, than any one of the presently definable promoter or enhancer sequences. 'Open' regions of the chromatin as possibly reflected by DNA hypomethylation, DNAse I hypersensitive sites, or Z-DNA may be particularly important, as already indicated by their role in the activation of rearranged variable regions within the Ig-gene system itself (78,79). As a result of the translocations, a variety of alternative promoters and/or enhancers may emerge from previously cryptic positions. Molecular comparisons between different tumors that carry the same chromosomal translocation is particularly rewarding, since they reveal the range of permissible variation with regard to breakpoints, orientation and sequence combinations with maintained transforming activity. The invariable conservation of certain regions — like exon 2 and 3 of c-myc — permits, on the other hand, a closer focusing on the products that play an essential role for the neoplastic transformation.
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G.KWn and E.KMn
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