- Email: [email protected]
Action of Myc in vivo — proliferation and apoptosis
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Action of Myc in vivo — proliferation and apoptosis Stella Pelengaris*, Bettina Rudolph† and Trevor Littlewood‡ The protein products of many dominant oncogenes are capable of inducing both cell proliferation and apoptosis. Recent experiments employing transgenic mice that express an ectopically regulatable myc gene or protein have begun to elucidate the role of the balance between proliferation and apoptosis in Myc-induced carcinogenesis. An outstanding feature of these experiments is the demonstration that the balance between oncogene-induced proliferation and apoptosis in a given tissue can be a critical determinant in the initiation and maintenance of the tumour. Addresses *Biological Sciences, University of Warwick, Coventry CV4 7AL, UK; e-mail: [email protected] † DeveloGen AG, Rudolf-Wissell-Str. 28, D – 37079 Göttingen, Germany ‡ Imperial Cancer Research Fund, 44 Lincoln’s Inn Fields, London WC2A 3PX, UK; e-mail: [email protected] Current Opinion in Genetics & Development 2000, 10:100–105 0959-437X/00/$ — see front matter © 2000 Elsevier Science Ltd. All rights reserved. Abbreviation 4OHT 4-hydroxytamoxifen
factors or the acquisition of additional anti-apoptotic mutations. Analogous to the situation in vitro, it is likely that the net balance between Myc-induced proliferation and apoptosis determines the development of neoplasia (and ultimately the malignancy) potential of the transformed cell in vivo. Addressing the role of oncogenes in vivo, however, is somewhat limited given that the organism develops with the lesion already present, a situation that does not occur in sporadic tumour development in the adult. Ideally, one wishes to determine the immediate and short-term consequences of activating single oncogenes in adult tissues — a situation that most closely resembles the majority of tumours — rather than the combined effects of sustained oncogene activation during development and into the adult. Thus, an animal model in which one could regulate the activity of Myc was required. Moreover, the ability to turn off the oncogenic lesion at various stages of tumour development is essential in determining whether sustained Myc activation is required for tumour maintenance. This has recently been achieved and forms the basis of our review. Is deregulated Myc responsible for tumour initiation, progression, or both?
Introduction The dual potential of Myc
Myc is a potent inducer of both cell proliferation and apoptosis in vitro [1–7,8•,9•,10]. It is capable of preventing cells from exiting the cell cycle and of driving quiescent fibroblasts into continuous cycle [11,12]. Consistent with this, deregulated expression of Myc inhibits both differentiation and the concomitant growth arrest of some cell types. Recent evidence suggests that Myc sensitises cells to a variety of apoptotic triggers rather than directly inducing apoptosis by itself [9•,13,14••]. Experiments in vitro suggest that the relative rates of proliferation and apoptosis induced by deregulated Myc and the presence of survival factors dictates the net outcome of the culture [13,15]. Sensitisaton to apoptotic stimuli is an intrinsic activity of Myc which, under normal cell culture conditions, is suppressed by a milieu of survival factors present in the calf serum used for in vitro culture [15]. Thus, Myc-induced apoptosis is usually only observed when such survival factors are withdrawn or cells express very high levels of Myc. Although some of the survival signalling pathways have been characterised in vitro, little is known about their role in vivo. It has been suggested that the ability of Myc to concomitantly induce proliferation and sensitise cells to apoptosis acts as a ‘fail-safe’ mechanism, guarding against a single proliferative lesion leading to unrestained cell growth [5,8•]. Thus, a cell that acquires deregulated Myc expression also exhibits an enhanced sensitivity to undergo apoptosis. In this scenario, survival of these cells depends on an adequate and continuous supply of survival
The c-myc proto-oncogene is intimately implicated in the control of cell proliferation and its deregulated expression is detected in many tumour cell types [16,17]. It has been assumed that such deregulated expression of Myc is instrumental in the initiation of the neoplastic phenotype. Most data, however, are derived from established tumours or cell lines bearing multiple oncogenic lesions. Given the ability of Myc to drive cell proliferation independently of the requirement for growth factors, it is possible that deregulated Myc expression is selected for during tumourigenesis and is, therefore, a rather late event. What, then, is the evidence that Myc is directly involved in neoplasia? Several conventional transgenic studies employing tissuespecific expression of Myc proteins have attempted to address this question [18]. In these mouse models, the transgene is constitutively expressed in a particular tissue throughout the lifespan of the organism. Although much valuable information has emerged from such studies, interpretation of the resulting phenotypes is especially difficult with oncogenes such as myc that have concomitant and contradictory potentials to induce both cell proliferation and apoptosis. Tumours arising from sustained oncogenic activation often arise after relatively long latent periods and are usually clonal in origin suggesting that additional genetic lesions have contributed to the phenotype. Moreover, there is some evidence that deregulated Myc expression may itself contribute to increased genetic instability [19–21]. Thus, the net observed phenotype seen in conventional Myc transgenics may be as a result predominantly of its
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Table 1 Comparison of proliferation and apoptosis induced by c-Myc in vivo. Location
Promoter
Regulatable by
Observations
Epidermis [27••]
Involucrin
4-hydroxytamoxifen (topical)
Reversible benign papillomatosis and angiogenesis after 21 days. No apoptosis.
β cells of pancreas*
Insulin
4-hydroxytamoxifen (intraperitoneal)
Proliferation and apoptosis. Apoptosis eventually predominates leading to islet cell ablation and diabetes by 7 days
B lymphocytes [26••]
Tetracycline-responsive promoter (tet-o) and immunoglobulin heavy chain enhancer, Eµa expression)†
Doxycycline (oral) (represses Myc)
Reversible T lymphomas and acute myeloid leukaemias
T lymphocytes‡
Lck
4-hydroxytamoxifen (intraperitoneal)
Proliferation but little apoptosis (only seen in an H-Y-TCR background)
T lymphocytes [25]
CD2
Tamoxifen (oral)
Proliferation and apoptosis both observed (but high background)
*S Pelengaris, unpublished data. † The Eµ promoter was used to target the expression of the doxycycline-dependent tetracycline-transactivating protein (tTA) to B lymphocytes. ‡ B Rudolph, unpublished data.
mitogenic activity and Myc’s concomitant potential to induce apoptosis may well have been suppressed by mechanisms unknown during tumourigenesis. This is consistent with the observed oncogenic synergy between Myc and the anti-apoptotic protein Bcl-2 in lymphomagenesis [22,23]. Thus, the notion that Myc-induced apoptosis may act as a ‘fail-safe’ mechanism following activation of the oncogene cannot be addressed using constitutive expression of a myc transgene. These limitations have led to the development of transgenic mouse models in which the expression or activity of Myc is regulated ectopically [24,25,26••,27••]. Such studies have indicated that Myc is sufficient to initiate a benign neoplasia and to maintain a tumour in situ but the precise role of Myc in tumourigenesis is dependent on the tissue type and its environment (Table 1).
Ectopic Myc activation in skin causes proliferation with no detectable apoptosis The epidermis is an ideal tissue in which to investigate the effects of Myc activation as the processes of proliferation, differentiation and cell death are tightly regulated and well understood; moreover, it is a major target of human neoplasia. Expression of the regulatable c-Myc protein, c-MycERTM (human c-Myc fused to the ligand-binding domain of a modified murine oestrogen receptor [12]), was targeted to suprabasal keratinocytes of the epidermis using the involucrin promoter [27••]. Sustained activation of c-MycERTM by topical administration of the specific ligand 4-hydroxytamoxifen (4OHT) results in premalignant papillomatous skin lesions accompanied by angiogenesis (Figure 1a) — a phenotype closely resembling hyperplastic actinic keratosis, a human epithelial precancerous lesion. This complex phenotype completely regresses within 25 days of stopping administration of 4OHT, indicating that it is induced by, and dependent on, the continued activity of a single oncogene product, c-Myc. Although it is likely that not all of the phenotypic changes are the direct result of Myc activation, it is clear that Myc
is able to drive post-mitotic suprabasal cells back into the cell cycle. Concomitant with this is the widespread disruption of normal differentiation. Thus, the primary effect of Myc activation in the suprabasal epidermis appears to be cellular proliferation. But what of apoptosis? c-Myc appears to induce little apoptosis in the interfollicular epidermis of papillomatous lesions (Figure 1a) and although some apoptosis is observable in proliferating keratinocytes of the hair follicles, it is clearly insufficient to offset c-Myc-induced hyperplasia. Nonetheless, Myc is very potent at inducing apoptosis in isolated serumdeprived keratinocytes from the same transgenic animals. This suggests that c-Myc-induced apoptosis in intact skin is most likely suppressed by the presence of excess survival signals such as those provided by soluble trophic cytokines or attachment to extracellular matrix or neighbouring cells. Despite the lack of apoptosis in the papillomatous lesions, the proliferating, dysplastic keratinocytes, rapidly migrate outward and are shed predominantly as nucleated cells that have not completed terminal differentiation, thus forming thickened cornified layers which subsequently lift or flake from the skin surface. The dominant loss of c-Myc-activated keratinocytes through shedding, preventing their accumulation, may account for the rarity of malignant progression of actinic keratosis and the high frequency with which such benign lesions spontaneously regress in human skin. How, then, can deregulation of Myc expression in keratinocytes of the epidermis lead to invasive skin malignancies? Although c-Myc-induced papillomatosis is a completely benign lesion, the accumulation of such premalignant cells within the epidermis could constitute a serious neoplastic risk — further mutations may promote invasiveness leading to basal or squamous cell carcinomas. Secondary mutations that promote cell survival may thus allow occasional rare dermal migrating Myc-activated cells
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Figure 1 Skin
(a) Proliferation (Ki-67)
Apoptosis (TUNEL)
su ba Myc 'off'
pa su Myc 'on'
Pancreas
(b) Proliferation (Ki-67)
Apoptosis (TUNEL)
Myc 'off'
Myc 'on'
Comparison of Myc-induced proliferation and apoptosis in the epidermis and endocrine pancreas. Immunohistochemical staining of tissue sections was performed using Ki-67 antibody, specific for a nuclear protein expressed in proliferating cells, or with TUNEL (TdT-mediated, dUTP nick end labelling) for detection of apoptosis (both brown-staining). (a) Untreated Involucrin–mycERTM transgenic murine skin (‘Myc off’) is identical to that of wild-type animals, with proliferation confined to basal keratinocytes (upper left panel) and TUNEL staining only evident in flattened terminally differentiated keratinocytes prior to enucleation and transformation into dead squames (upper right panel, see arrow). Activation of MycERTM (‘Myc on’) in suprabasal keratinocytes of adult transgenic murine skin is sufficient to generate premalignant papillomatous lesions within 21 days of tamoxifen treatment. Myc induces dramatic proliferation (bottom left panel) but no observable apoptosis within interfollicular epidermis (bottom right panel). The only apoptotic (TUNEL-positive) cells evident are keratinocytes that are about to enter the nonviable cornified layers as nucleated cells (parakeratosis) and which will ultimately be shed (see arrow). ba, basal keratinocytes; su, suprabasal keratinocytes; pa, parakeratosis. Size bar, 50 µm. (b) The pancreatic islets of untreated Insulin–mycERTM mice (‘Myc off’) are identical to those of wild-type animals, with proliferation or apoptosis of pancreatic β cells very rarely detected (upper left and upper right panels, respectively). Activation of MycERTM (‘Myc on’) in β cells of adult transgenic mice induces proliferation (bottom left panel) and extensive apoptosis (bottom right panel) leading to involution of islets after 7 days of tamoxifen treatment. Size bar, 100 µm
Current Opinion in Genetics & Development
to survive despite having left their normal tropic environment (Figure 2a). Indeed, our preliminary observations indicate that loss of p53 may allow invading Myc-expressing keratinocytes to survive and form an invasive carcinoma within the dermis.
Myc activation causes pancreatic β cell apoptosis and diabetes In striking contrast to skin, we find that the predominant effect of Myc activation in the β cells of the adult murine pancreas is apoptosis (S Pelengaris, unpublished data). Although Myc drives post-mitotic β cells into the cell cycle, sustained Myc activation leads to extensive β cell apoptosis resulting in involution of the pancreatic islets and diabetes within 7 days (Figure 1b). This suggests that, unlike Myc activation in the epidermis where apoptosis is suppressed, the early inhibition of cell death is an
absolute requirement for the survival of potentially malignant β cells (Figure 2b). Consistent with this, coexpression of the anti-apoptotic protein Bcl-xL inhibits apoptosis and the islets become significantly enlarged as a result of an increase in proliferation in the absence of cell death (S Pelengaris, unpublished data). We conclude that, in the endocrine pancreas, c-Myc would be highly unlikely to initiate a β cell tumour in the absence of an anti-apoptotic lesion.
The role of Myc-induced proliferation and apoptosis in lymphomas Conventional transgenic animals have, in the past, been employed to demonstrate the involvement of Myc in lymphomagenesis. These models have been updated recently by the generation of a myc gene under the control of a tetracycline-responsive minimal promoter. Expression of this
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Figure 2 A model for Myc-induced in tumourigenesis in different tissues. The outcome of Myc deregulation is dictated by the origin of the tissue in which the initial lesion occurs. (a) In the epidermis, suprabasal keratinocytes bearing deregulated Myc are protected from apoptosis by potent survival signals but are shed from the skin surface. Rare dermal invading keratinocytes bearing activated Myc undergo apoptosis but if these dermal migrants acquire a second anti-apoptotic lesion such as the loss of p53, they may proliferate to form an invasive tumour. (b) In the pancreatic β cells, activation of Myc results in extensive apoptosis culminating in islet involution and diabetes. Clearly, in this tissue, the early suppression of apoptosis is essential for the survival of the Mycderegulated cells and tumourigenesis.
(a)
Shedding Benign papillomatosis
Epidermis
Deregulated keratinocyte Apoptosis Survival signals
Invasive malignancy
Dermis Rare dermal migrant Apoptosis Suppressor of apoptosis e.g. loss of p53 β -cell tumour
(b)
Diabetes Pancreas
Deregulated islet β cell
Islet involution
Suppressor of apoptosis
Apoptosis
Current Opinion in Genetics & Development
gene is regulated by the tetracycline-transactivating protein expressed under the control of the immunoglobulin heavy chain enhancer, Eµ [26••]. Lymphocytes from such transgenic animals express Myc in the absence but not the presence of doxycycline. Whereas no tumours were observed in mice treated with doxycycline, in the absence of doxycycline 100% of the transgenic animals developed tumours and died within 5 months. Despite the use of the Eµ promoter, the majority of the tumours (14/16) were CD4+ CD8+ T cell lymphomas with a small number (2/16) of acute myeloid leukaemias. These tumours appear to derive from the combined effects of Myc on promoting proliferation whilst inhibiting differentiation. No evidence of apoptosis was seen. Consistent with the prolonged latent period before appearance of the tumours, and the clonality of the tumours the authors suggest that additional genetic lesions have contributed to the malignancy [26••]. It should be noted that the expression of Myc in these animals occurred during development and, therefore, mimics the conventional transgenic animals. Nonetheless, it is striking that, in these experiments, repression of Myc expression (by doxycycline treatment) was sufficient to induce tumour regression in 90% of the animals. Tumour elimination in these animals is primarily the result of the restoration of normal haematopoiesis with differentiation of blast-like tumour cells into more mature lymphocytes. Marked apoptosis was also observed in the
regressing tumours. As Myc expression is repressed following doxycycline treatment, it is not clear whether Myc provides the apoptotic stimulus to these cells. Perhaps more likely, the loss of vasculature in the regressing tumour induces apoptosis, perhaps by limiting the supply of soluble survival factors. It is also possible that there is sufficient residual Myc expression in the newly differentiated haematopoietic cells, which may require different or more survival factors compared to their immature predecessors, to induce apoptosis. These data emphasise the central role that Myc may play in lymphomagenesis — suppression of this single lesion leads to tumour regression. Regulatable myc transgenes have also been expressed in the T-lymphocyte compartment. During T cell development, thymocytes differentiate and mature within the thymus through an ordered series of proliferation and maturation events. During this process, thymocytes bearing T cell receptors with appropriate specificities are positively selected and differentiate fully to become mature thymocytes [28]. The remaining thymocytes bearing inappropriate or useless T cell receptors undergo negative selection and undergo apoptosis [29,30]. Activation of a lck-targeted MycERTM protein results in no obvious perubations in thymic ontogeny unless positive selection is enhanced by the use of a H-YTCR genetic background (B Rudolph, unpublished data). Here, compared to H-Y-TCR control litternates, more cells
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are positively selected in lck–c-MycERTM/H-Y-TCR double transgenic mice primarily because of an increase in proliferation without a concomitant effect on apoptosis. c-Myc is very potent in inducing apoptosis in vitro in thymocytes isolated from the lck-c-mycERTM mice, however, suggesting that thymocytes in vivo receive potent survival signals. In contrast to lck–mycER™ mice, Myc activation in CD2–mycER™ mice results in both proliferation and apoptosis [25]. As with conventional T-lymphocyte Myc transgenics (CD2–c-myc, Thy1–c-myc, Eµ–c-myc) [31–33], long-term activation of CD2–MycER™ results in thymic lymphomas, with the majority of tumours being CD4+ CD8+ [25]; however, a high background of spontaneous lymphomagenesis is observed in the absence of 4OHT, suggesting that high levels of transgene expression in these animals allows ligand-independent activation of MycER™ as has been noted previously [27••]. In addition, it is not possible to determine the role of Myc in tumour maintenance in these animals. Given the long latent period before the emergence of these tumours, it must be assumed that additional mutations have been acquired. Perhaps surprisingly, therefore, tumours derived from these thymic lymphomas remain highly sensitive to cMyc-induced apoptosis following explantation in vitro. The authors conclude [27••] that Myc-induced lymphomas do not necessarily have to acquire a secondary mutation that abrogates Myc’s apoptotic activity. The nature of such cooperating mutations is not immediately obvious and their identification would be very revealing. The differences in phenotype between the lck–c-mycERTM and CD2–c-mycERTM transgenic mice may reflect the use of different promoters and/or significant differences in the level of transgene expression.
Conclusions A major step towards the elucidation of the critical balance between Myc-induced proliferation and apoptosis in tumourigenesis has been facilitated by the development of ectopically regulatable oncogenes in transgenic mice. One, perhaps surprising, finding is that Myc alone is sufficient (at least in skin) to initiate and sustain the proliferative premalignant state. The reversibility of the T cell lymphomas observed by Felsher and Bishop [26••] indicates that Myc alone is also sufficient to maintain malignancy in this tumour type. Nonetheless, the formation of a tumour in a given tissue requires the activation of a discrete set of genes — the nature of which is, in a sense, a reflection of the tissue itself. For example, even in the presence of a benign proliferative lesion in the suprabasal epidermis, malignant progression is probably limited by the continued shedding of Myc-activated keratinocytes and the death of rare dermal invading cells by ‘anoikis’. Thus, for skin cancers to arise, the loss of such cells has to be inhibited. In the skin, little apoptosis is detected and would not, therefore, seem to contribute to limiting neoplasia in this tissue. Indeed, widespread apoptosis in a tissue where integrity is important may not be desirable. Similarly, the predominant
effect of activation of lck-driven MycER™ in thymocytes is proliferation rather than apoptosis despite the fact that activation of the same molecule can induce death in isolated thymocytes in vitro ([25]; B Rudolph, unpublished data). By contrast, Myc induces extensive apoptosis in pancreatic β cells. So severe is the apoptosis that the islets involute and diabetes develops. It is clear, therefore, that the early suppression of apoptosis is a prerequisite for Myc-induced tumourigenesis in these cells. We have begun to explain why particular oncogenic lesions are common in tumours originating in a particular tissue. It is clear that activation of Myc is sufficient to initiate neoplasia (at least in the skin) and is required for tumour maintenance in both skin and T lymphomas [25,26••,27••]. In the few instances where the lymphomas failed to regress following suppression of Myc expression [26••], it will be important to determine the nature of any additional lesions that may account for this failure. Nonetheless, the repression of Myc activity alone could potentially realise therapeutic potential.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest •• of outstanding interest 1.
Amati B, Land H: Myc-Max-Mad: a transcription factor network controlling cell cycle progression, differentiation and death. Curr Opin Genet Dev 1994, 4:102-108.
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Bouchard C, Staller P, Eilers M: Control of cell proliferation by Myc. Trends Cell Biol 1998, 8:202-206.
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Desbarats L, Schneider A, Muller D, Burgin A, Eilers M: Myc: a single gene controls both proliferation and apoptosis in mammalian cells. Experientia 1996, 52:1123-1129.
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Eilers M: Control of cell proliferation by Myc family genes. Mol Cell 1999, 9:1-6.
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Evan GI, Littlewood TD: The role of c-myc in cell growth. Curr Opin Genet Dev 1993, 3:44-49.
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Evan G, Harrington E, Fanidi A, Land H, Amati B, Bennett M: Integrated control of cell proliferation and cell death by the c-myc oncogene. Philos Trans R Soc Lond B Biol Sci 1994, 345:269-275.
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Evan GI, Brown L, Whyte M, Harrington E: Apoptosis and the cell cycle. Curr Opin Cell Biol 1995, 7:825-834.
8. Evan G, Littlewood T: A matter of life and cell death. Science 1998, • 281:1317-1322. A very well written review discussing the propensity for growth-deregulating oncoproteins such as Myc, E1A and E2F, to induce apoptosis and propose the model that c-Myc acts to sensitise cells to a variety of apoptotic triggers rather than acting solely as a death effector. This potent mechanism to limit expansion of neoplastic cells and the cooperation between various oncoproteins are discussed. 9. Prendergast GC: Mechanisms of apoptosis by c-Myc. Oncogene • 1999, 18:2967-2987. A very well written review summarising molecular advances in apoptosis with specific attention made to the mechanisms of apoptosis induced by c-Myc. 10. Steiner P, Rudolph B, Muller D, Eilers M: The functions of Myc in cell cycle progression and apoptosis. Prog Cell Cycle Res 1996, 2:73-82. 11. Eilers M, Schirm S, Bishop JM: The MYC protein activates transcription of the alpha-prothymosin gene. EMBO J 1991, 10:133-141. 12. Littlewood TD, Hancock DC, Danielian PS, Parker MG, Evan GI: A modified oestrogen receptor ligand-binding domain as an improved switch for the regulation of heterologous proteins. Nucleic Acids Res 1995, 23:1686-1690.
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13. Evan GI, Wyllie AH, Gilbert CS, Littlewood TD, Land H, Brooks M, Waters CM, Penn LZ, Hancock DC: Induction of apoptosis in fibroblasts by c-myc protein. Cell 1992, 69:119-128. 14. Juin P, Hueber AO, Littlewood T, Evan G: c-Myc-induced •• sensitization to apoptosis is mediated through cytochrome c release. Genes Dev 1999, 13:1367-1381. This elegant in vitro study shows that c-Myc promotes apoptosis of fibroblasts by causing the release of cytochrome c from mitochondria into the cytosol. Cytochrome c release, in itself, however, is not efficient at inducing apoptosis but can cooperate with other triggers of apoptosis such as CD95/Fas and p53. These data provide a mechanism for c-Myc-induced sensitisation to apoptosis. 15. Harrington EA, Bennett MR, Fanidi A, Evan GI: c-Myc-induced apoptosis in fibroblasts is inhibited by specific cytokines. EMBO J 1994, 13:3286-3295. 16. Marcu KB, Bossone SA, Patel AJ: myc function and regulation. Annu Rev Biochem 1992, 61:809-860. 17.
Spencer CA, Groudine M: Control of c-myc regulation in normal and neoplastic cells. Adv Cancer Res 1991, 56:1-48.
18. Morgenbesser SD, DePinho RA: Use of transgenic mice to study myc family gene function in normal mammalian development and in cancer. Semin Cancer Biol 1994, 5:21-36. 19. Mushinski JF, Hanley-Hyde J, Rainey GJ, Kuschak TI, Taylor C, Fluri M, Stevens LM, Henderson DW, Mai S: Myc-induced cyclin D2 genomic instability in murine B cell neoplasms. Curr Top Microbiol Immunol 1999, 246:183-189. [Discussion 190-192]. 20. Felsher DW, Bishop JM: Transient excess of MYC activity can elicit genomic instability and tumorigenesis. Proc Natl Acad Sci USA 1999, 96:3940-3944. 21. Mai S, Hanley-Hyde J, Rainey GJ, Kuschak TI, Paul JT, Littlewood TD, Mischak H, Stevens LM, Henderson DW, Mushinski JK: Chromosomal and extrachromosomal instability of the cyclin D2 gene in induced by Myc overexpression. Neoplasia 1999, 1:241-252. 22. Strasser A, Elefanty AG, Harris AW, Cory S: Progenitor tumours from Emu-bcl-2-myc transgenic mice have lymphomyeloid differentiation potential and reveal developmental differences in cell survival. EMBO J 1996, 15:3823-3834. 23. Strasser A, Harris AW, Bath ML, Cory S: Novel primitive lymphoid tumours induced in transgenic mice by cooperation between myc and bcl-2. Nature 1990, 348:331-333. 24. Berns A: Turning on tumors to study cancer progression [news]. Nat Med 1999, 5:989-990.
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25. Blyth K, Stewart M, Bell M, James C, Evan G, Neil JC, Cameron ER: Sensitivity to myc-induced apoptosis is retained in spontaneous and transplanted lymphomas of CD2-mycER™ mice. Oncogene 2000, in press. 26. Felsher DW, Bishop JM: Reversible tumorigenesis by MYC in •• hematopoietic lineages. Mol Cell 1999, 4:199-207. This paper describes the use of the tetracycline regulatory system to conditionally express myc in haematopoietic cells of transgenic mice. Although sustained myc expression during development leads to malignant T cell lymphomas and acute myeloid leukaemias, inactivation of the transgene causes regression of the majority of established tumours. These results show for the first time that, despite the multistep nature of tumourigenesis in haematopoietic lineages, inactivation of a single genetic lesion, myc, is sufficient to reverse malignancy. 27. ••
Pelengaris S, Littlewood T, Khan M, Elia G, Evan G: Reversible activation of c-Myc in skin: induction of a complex neoplastic phenotype by a single oncogenic lesion. Mol Cell 1999, 3:565-577. This study was the first to demonstrate the importance of a regulatable c-myc transgenic mouse system over conventional transgenics. Expression of a switchable form of the c-Myc oncoprotein, c-MycERTM, was targeted to suprabasal keratinocytes of murine epidermis and activation of the chimaeric protein achieved with administration of tamoxifen. The authors describe the phenotype observed following immediate and short-term activation of c-Myc in the epidermis and, perhaps surprisingly, shows that c-Myc alone is sufficient to induce pre-malignant skin lesions (papillomatosis) with profound angiogenesis. 28. Marrack P, Winslow GM, Choi Y, Scherer M, Pullen A, White J, Kappler JW: The bacterial and mouse mammary tumor virus superantigens; two different families of proteins with the same functions. Immunol Rev 1993, 131:79-92. 29. Tough D, Sprent J: Turnover of naïve- and memory-phenotype T cells. J Exp Med 1994, 179:1127-1135. 30. Shortman K, Egerton M, Spangrude GJ, Scollary R: The generation and fate of thymocytes. Semin Immunol 1990, 2:3-12. 31. Stewart M, Cameron E, Campbell M, Mcfarlane R, Toth S, Lang K, Onions D, Neil JC: Conditional expression and oncogenity of c-myc linked to a CD2 gene dominant control region. Int J Cancer 1993, 53:1023-1030. 32. Spanopoulou E, Early A, Elliot J, Crispe N, Ladyman H, Ritter M, Watt S, Grosveld F, Kioussis D: Complex lymphoid and epithelial thymic tumours in Thy1-myc transgenic mice. Nature 1989, 342:185-189. 33. Adams JM, Harris AW, Pinkert CA, Corcoran LM, Alexander WS, Cory S, Palmiter RD, Brinster RL: The c-myc oncogene driven by immunoglobulin enhancers induces lymphoid malignancy in transgenic mice. Nature 1985, 318:533-538.
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Action of Myc in vivo — proliferation and apoptosis Stella Pelengaris*, Bettina Rudolph† and Trevor Littlewood‡ The protein products of many dominant oncogenes are capable of inducing both cell proliferation and apoptosis. Recent experiments employing transgenic mice that express an ectopically regulatable myc gene or protein have begun to elucidate the role of the balance between proliferation and apoptosis in Myc-induced carcinogenesis. An outstanding feature of these experiments is the demonstration that the balance between oncogene-induced proliferation and apoptosis in a given tissue can be a critical determinant in the initiation and maintenance of the tumour. Addresses *Biological Sciences, University of Warwick, Coventry CV4 7AL, UK; e-mail: [email protected] † DeveloGen AG, Rudolf-Wissell-Str. 28, D – 37079 Göttingen, Germany ‡ Imperial Cancer Research Fund, 44 Lincoln’s Inn Fields, London WC2A 3PX, UK; e-mail: [email protected] Current Opinion in Genetics & Development 2000, 10:100–105 0959-437X/00/$ — see front matter © 2000 Elsevier Science Ltd. All rights reserved. Abbreviation 4OHT 4-hydroxytamoxifen
factors or the acquisition of additional anti-apoptotic mutations. Analogous to the situation in vitro, it is likely that the net balance between Myc-induced proliferation and apoptosis determines the development of neoplasia (and ultimately the malignancy) potential of the transformed cell in vivo. Addressing the role of oncogenes in vivo, however, is somewhat limited given that the organism develops with the lesion already present, a situation that does not occur in sporadic tumour development in the adult. Ideally, one wishes to determine the immediate and short-term consequences of activating single oncogenes in adult tissues — a situation that most closely resembles the majority of tumours — rather than the combined effects of sustained oncogene activation during development and into the adult. Thus, an animal model in which one could regulate the activity of Myc was required. Moreover, the ability to turn off the oncogenic lesion at various stages of tumour development is essential in determining whether sustained Myc activation is required for tumour maintenance. This has recently been achieved and forms the basis of our review. Is deregulated Myc responsible for tumour initiation, progression, or both?
Introduction The dual potential of Myc
Myc is a potent inducer of both cell proliferation and apoptosis in vitro [1–7,8•,9•,10]. It is capable of preventing cells from exiting the cell cycle and of driving quiescent fibroblasts into continuous cycle [11,12]. Consistent with this, deregulated expression of Myc inhibits both differentiation and the concomitant growth arrest of some cell types. Recent evidence suggests that Myc sensitises cells to a variety of apoptotic triggers rather than directly inducing apoptosis by itself [9•,13,14••]. Experiments in vitro suggest that the relative rates of proliferation and apoptosis induced by deregulated Myc and the presence of survival factors dictates the net outcome of the culture [13,15]. Sensitisaton to apoptotic stimuli is an intrinsic activity of Myc which, under normal cell culture conditions, is suppressed by a milieu of survival factors present in the calf serum used for in vitro culture [15]. Thus, Myc-induced apoptosis is usually only observed when such survival factors are withdrawn or cells express very high levels of Myc. Although some of the survival signalling pathways have been characterised in vitro, little is known about their role in vivo. It has been suggested that the ability of Myc to concomitantly induce proliferation and sensitise cells to apoptosis acts as a ‘fail-safe’ mechanism, guarding against a single proliferative lesion leading to unrestained cell growth [5,8•]. Thus, a cell that acquires deregulated Myc expression also exhibits an enhanced sensitivity to undergo apoptosis. In this scenario, survival of these cells depends on an adequate and continuous supply of survival
The c-myc proto-oncogene is intimately implicated in the control of cell proliferation and its deregulated expression is detected in many tumour cell types [16,17]. It has been assumed that such deregulated expression of Myc is instrumental in the initiation of the neoplastic phenotype. Most data, however, are derived from established tumours or cell lines bearing multiple oncogenic lesions. Given the ability of Myc to drive cell proliferation independently of the requirement for growth factors, it is possible that deregulated Myc expression is selected for during tumourigenesis and is, therefore, a rather late event. What, then, is the evidence that Myc is directly involved in neoplasia? Several conventional transgenic studies employing tissuespecific expression of Myc proteins have attempted to address this question [18]. In these mouse models, the transgene is constitutively expressed in a particular tissue throughout the lifespan of the organism. Although much valuable information has emerged from such studies, interpretation of the resulting phenotypes is especially difficult with oncogenes such as myc that have concomitant and contradictory potentials to induce both cell proliferation and apoptosis. Tumours arising from sustained oncogenic activation often arise after relatively long latent periods and are usually clonal in origin suggesting that additional genetic lesions have contributed to the phenotype. Moreover, there is some evidence that deregulated Myc expression may itself contribute to increased genetic instability [19–21]. Thus, the net observed phenotype seen in conventional Myc transgenics may be as a result predominantly of its
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Table 1 Comparison of proliferation and apoptosis induced by c-Myc in vivo. Location
Promoter
Regulatable by
Observations
Epidermis [27••]
Involucrin
4-hydroxytamoxifen (topical)
Reversible benign papillomatosis and angiogenesis after 21 days. No apoptosis.
β cells of pancreas*
Insulin
4-hydroxytamoxifen (intraperitoneal)
Proliferation and apoptosis. Apoptosis eventually predominates leading to islet cell ablation and diabetes by 7 days
B lymphocytes [26••]
Tetracycline-responsive promoter (tet-o) and immunoglobulin heavy chain enhancer, Eµa expression)†
Doxycycline (oral) (represses Myc)
Reversible T lymphomas and acute myeloid leukaemias
T lymphocytes‡
Lck
4-hydroxytamoxifen (intraperitoneal)
Proliferation but little apoptosis (only seen in an H-Y-TCR background)
T lymphocytes [25]
CD2
Tamoxifen (oral)
Proliferation and apoptosis both observed (but high background)
*S Pelengaris, unpublished data. † The Eµ promoter was used to target the expression of the doxycycline-dependent tetracycline-transactivating protein (tTA) to B lymphocytes. ‡ B Rudolph, unpublished data.
mitogenic activity and Myc’s concomitant potential to induce apoptosis may well have been suppressed by mechanisms unknown during tumourigenesis. This is consistent with the observed oncogenic synergy between Myc and the anti-apoptotic protein Bcl-2 in lymphomagenesis [22,23]. Thus, the notion that Myc-induced apoptosis may act as a ‘fail-safe’ mechanism following activation of the oncogene cannot be addressed using constitutive expression of a myc transgene. These limitations have led to the development of transgenic mouse models in which the expression or activity of Myc is regulated ectopically [24,25,26••,27••]. Such studies have indicated that Myc is sufficient to initiate a benign neoplasia and to maintain a tumour in situ but the precise role of Myc in tumourigenesis is dependent on the tissue type and its environment (Table 1).
Ectopic Myc activation in skin causes proliferation with no detectable apoptosis The epidermis is an ideal tissue in which to investigate the effects of Myc activation as the processes of proliferation, differentiation and cell death are tightly regulated and well understood; moreover, it is a major target of human neoplasia. Expression of the regulatable c-Myc protein, c-MycERTM (human c-Myc fused to the ligand-binding domain of a modified murine oestrogen receptor [12]), was targeted to suprabasal keratinocytes of the epidermis using the involucrin promoter [27••]. Sustained activation of c-MycERTM by topical administration of the specific ligand 4-hydroxytamoxifen (4OHT) results in premalignant papillomatous skin lesions accompanied by angiogenesis (Figure 1a) — a phenotype closely resembling hyperplastic actinic keratosis, a human epithelial precancerous lesion. This complex phenotype completely regresses within 25 days of stopping administration of 4OHT, indicating that it is induced by, and dependent on, the continued activity of a single oncogene product, c-Myc. Although it is likely that not all of the phenotypic changes are the direct result of Myc activation, it is clear that Myc
is able to drive post-mitotic suprabasal cells back into the cell cycle. Concomitant with this is the widespread disruption of normal differentiation. Thus, the primary effect of Myc activation in the suprabasal epidermis appears to be cellular proliferation. But what of apoptosis? c-Myc appears to induce little apoptosis in the interfollicular epidermis of papillomatous lesions (Figure 1a) and although some apoptosis is observable in proliferating keratinocytes of the hair follicles, it is clearly insufficient to offset c-Myc-induced hyperplasia. Nonetheless, Myc is very potent at inducing apoptosis in isolated serumdeprived keratinocytes from the same transgenic animals. This suggests that c-Myc-induced apoptosis in intact skin is most likely suppressed by the presence of excess survival signals such as those provided by soluble trophic cytokines or attachment to extracellular matrix or neighbouring cells. Despite the lack of apoptosis in the papillomatous lesions, the proliferating, dysplastic keratinocytes, rapidly migrate outward and are shed predominantly as nucleated cells that have not completed terminal differentiation, thus forming thickened cornified layers which subsequently lift or flake from the skin surface. The dominant loss of c-Myc-activated keratinocytes through shedding, preventing their accumulation, may account for the rarity of malignant progression of actinic keratosis and the high frequency with which such benign lesions spontaneously regress in human skin. How, then, can deregulation of Myc expression in keratinocytes of the epidermis lead to invasive skin malignancies? Although c-Myc-induced papillomatosis is a completely benign lesion, the accumulation of such premalignant cells within the epidermis could constitute a serious neoplastic risk — further mutations may promote invasiveness leading to basal or squamous cell carcinomas. Secondary mutations that promote cell survival may thus allow occasional rare dermal migrating Myc-activated cells
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Figure 1 Skin
(a) Proliferation (Ki-67)
Apoptosis (TUNEL)
su ba Myc 'off'
pa su Myc 'on'
Pancreas
(b) Proliferation (Ki-67)
Apoptosis (TUNEL)
Myc 'off'
Myc 'on'
Comparison of Myc-induced proliferation and apoptosis in the epidermis and endocrine pancreas. Immunohistochemical staining of tissue sections was performed using Ki-67 antibody, specific for a nuclear protein expressed in proliferating cells, or with TUNEL (TdT-mediated, dUTP nick end labelling) for detection of apoptosis (both brown-staining). (a) Untreated Involucrin–mycERTM transgenic murine skin (‘Myc off’) is identical to that of wild-type animals, with proliferation confined to basal keratinocytes (upper left panel) and TUNEL staining only evident in flattened terminally differentiated keratinocytes prior to enucleation and transformation into dead squames (upper right panel, see arrow). Activation of MycERTM (‘Myc on’) in suprabasal keratinocytes of adult transgenic murine skin is sufficient to generate premalignant papillomatous lesions within 21 days of tamoxifen treatment. Myc induces dramatic proliferation (bottom left panel) but no observable apoptosis within interfollicular epidermis (bottom right panel). The only apoptotic (TUNEL-positive) cells evident are keratinocytes that are about to enter the nonviable cornified layers as nucleated cells (parakeratosis) and which will ultimately be shed (see arrow). ba, basal keratinocytes; su, suprabasal keratinocytes; pa, parakeratosis. Size bar, 50 µm. (b) The pancreatic islets of untreated Insulin–mycERTM mice (‘Myc off’) are identical to those of wild-type animals, with proliferation or apoptosis of pancreatic β cells very rarely detected (upper left and upper right panels, respectively). Activation of MycERTM (‘Myc on’) in β cells of adult transgenic mice induces proliferation (bottom left panel) and extensive apoptosis (bottom right panel) leading to involution of islets after 7 days of tamoxifen treatment. Size bar, 100 µm
Current Opinion in Genetics & Development
to survive despite having left their normal tropic environment (Figure 2a). Indeed, our preliminary observations indicate that loss of p53 may allow invading Myc-expressing keratinocytes to survive and form an invasive carcinoma within the dermis.
Myc activation causes pancreatic β cell apoptosis and diabetes In striking contrast to skin, we find that the predominant effect of Myc activation in the β cells of the adult murine pancreas is apoptosis (S Pelengaris, unpublished data). Although Myc drives post-mitotic β cells into the cell cycle, sustained Myc activation leads to extensive β cell apoptosis resulting in involution of the pancreatic islets and diabetes within 7 days (Figure 1b). This suggests that, unlike Myc activation in the epidermis where apoptosis is suppressed, the early inhibition of cell death is an
absolute requirement for the survival of potentially malignant β cells (Figure 2b). Consistent with this, coexpression of the anti-apoptotic protein Bcl-xL inhibits apoptosis and the islets become significantly enlarged as a result of an increase in proliferation in the absence of cell death (S Pelengaris, unpublished data). We conclude that, in the endocrine pancreas, c-Myc would be highly unlikely to initiate a β cell tumour in the absence of an anti-apoptotic lesion.
The role of Myc-induced proliferation and apoptosis in lymphomas Conventional transgenic animals have, in the past, been employed to demonstrate the involvement of Myc in lymphomagenesis. These models have been updated recently by the generation of a myc gene under the control of a tetracycline-responsive minimal promoter. Expression of this
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Figure 2 A model for Myc-induced in tumourigenesis in different tissues. The outcome of Myc deregulation is dictated by the origin of the tissue in which the initial lesion occurs. (a) In the epidermis, suprabasal keratinocytes bearing deregulated Myc are protected from apoptosis by potent survival signals but are shed from the skin surface. Rare dermal invading keratinocytes bearing activated Myc undergo apoptosis but if these dermal migrants acquire a second anti-apoptotic lesion such as the loss of p53, they may proliferate to form an invasive tumour. (b) In the pancreatic β cells, activation of Myc results in extensive apoptosis culminating in islet involution and diabetes. Clearly, in this tissue, the early suppression of apoptosis is essential for the survival of the Mycderegulated cells and tumourigenesis.
(a)
Shedding Benign papillomatosis
Epidermis
Deregulated keratinocyte Apoptosis Survival signals
Invasive malignancy
Dermis Rare dermal migrant Apoptosis Suppressor of apoptosis e.g. loss of p53 β -cell tumour
(b)
Diabetes Pancreas
Deregulated islet β cell
Islet involution
Suppressor of apoptosis
Apoptosis
Current Opinion in Genetics & Development
gene is regulated by the tetracycline-transactivating protein expressed under the control of the immunoglobulin heavy chain enhancer, Eµ [26••]. Lymphocytes from such transgenic animals express Myc in the absence but not the presence of doxycycline. Whereas no tumours were observed in mice treated with doxycycline, in the absence of doxycycline 100% of the transgenic animals developed tumours and died within 5 months. Despite the use of the Eµ promoter, the majority of the tumours (14/16) were CD4+ CD8+ T cell lymphomas with a small number (2/16) of acute myeloid leukaemias. These tumours appear to derive from the combined effects of Myc on promoting proliferation whilst inhibiting differentiation. No evidence of apoptosis was seen. Consistent with the prolonged latent period before appearance of the tumours, and the clonality of the tumours the authors suggest that additional genetic lesions have contributed to the malignancy [26••]. It should be noted that the expression of Myc in these animals occurred during development and, therefore, mimics the conventional transgenic animals. Nonetheless, it is striking that, in these experiments, repression of Myc expression (by doxycycline treatment) was sufficient to induce tumour regression in 90% of the animals. Tumour elimination in these animals is primarily the result of the restoration of normal haematopoiesis with differentiation of blast-like tumour cells into more mature lymphocytes. Marked apoptosis was also observed in the
regressing tumours. As Myc expression is repressed following doxycycline treatment, it is not clear whether Myc provides the apoptotic stimulus to these cells. Perhaps more likely, the loss of vasculature in the regressing tumour induces apoptosis, perhaps by limiting the supply of soluble survival factors. It is also possible that there is sufficient residual Myc expression in the newly differentiated haematopoietic cells, which may require different or more survival factors compared to their immature predecessors, to induce apoptosis. These data emphasise the central role that Myc may play in lymphomagenesis — suppression of this single lesion leads to tumour regression. Regulatable myc transgenes have also been expressed in the T-lymphocyte compartment. During T cell development, thymocytes differentiate and mature within the thymus through an ordered series of proliferation and maturation events. During this process, thymocytes bearing T cell receptors with appropriate specificities are positively selected and differentiate fully to become mature thymocytes [28]. The remaining thymocytes bearing inappropriate or useless T cell receptors undergo negative selection and undergo apoptosis [29,30]. Activation of a lck-targeted MycERTM protein results in no obvious perubations in thymic ontogeny unless positive selection is enhanced by the use of a H-YTCR genetic background (B Rudolph, unpublished data). Here, compared to H-Y-TCR control litternates, more cells
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are positively selected in lck–c-MycERTM/H-Y-TCR double transgenic mice primarily because of an increase in proliferation without a concomitant effect on apoptosis. c-Myc is very potent in inducing apoptosis in vitro in thymocytes isolated from the lck-c-mycERTM mice, however, suggesting that thymocytes in vivo receive potent survival signals. In contrast to lck–mycER™ mice, Myc activation in CD2–mycER™ mice results in both proliferation and apoptosis [25]. As with conventional T-lymphocyte Myc transgenics (CD2–c-myc, Thy1–c-myc, Eµ–c-myc) [31–33], long-term activation of CD2–MycER™ results in thymic lymphomas, with the majority of tumours being CD4+ CD8+ [25]; however, a high background of spontaneous lymphomagenesis is observed in the absence of 4OHT, suggesting that high levels of transgene expression in these animals allows ligand-independent activation of MycER™ as has been noted previously [27••]. In addition, it is not possible to determine the role of Myc in tumour maintenance in these animals. Given the long latent period before the emergence of these tumours, it must be assumed that additional mutations have been acquired. Perhaps surprisingly, therefore, tumours derived from these thymic lymphomas remain highly sensitive to cMyc-induced apoptosis following explantation in vitro. The authors conclude [27••] that Myc-induced lymphomas do not necessarily have to acquire a secondary mutation that abrogates Myc’s apoptotic activity. The nature of such cooperating mutations is not immediately obvious and their identification would be very revealing. The differences in phenotype between the lck–c-mycERTM and CD2–c-mycERTM transgenic mice may reflect the use of different promoters and/or significant differences in the level of transgene expression.
Conclusions A major step towards the elucidation of the critical balance between Myc-induced proliferation and apoptosis in tumourigenesis has been facilitated by the development of ectopically regulatable oncogenes in transgenic mice. One, perhaps surprising, finding is that Myc alone is sufficient (at least in skin) to initiate and sustain the proliferative premalignant state. The reversibility of the T cell lymphomas observed by Felsher and Bishop [26••] indicates that Myc alone is also sufficient to maintain malignancy in this tumour type. Nonetheless, the formation of a tumour in a given tissue requires the activation of a discrete set of genes — the nature of which is, in a sense, a reflection of the tissue itself. For example, even in the presence of a benign proliferative lesion in the suprabasal epidermis, malignant progression is probably limited by the continued shedding of Myc-activated keratinocytes and the death of rare dermal invading cells by ‘anoikis’. Thus, for skin cancers to arise, the loss of such cells has to be inhibited. In the skin, little apoptosis is detected and would not, therefore, seem to contribute to limiting neoplasia in this tissue. Indeed, widespread apoptosis in a tissue where integrity is important may not be desirable. Similarly, the predominant
effect of activation of lck-driven MycER™ in thymocytes is proliferation rather than apoptosis despite the fact that activation of the same molecule can induce death in isolated thymocytes in vitro ([25]; B Rudolph, unpublished data). By contrast, Myc induces extensive apoptosis in pancreatic β cells. So severe is the apoptosis that the islets involute and diabetes develops. It is clear, therefore, that the early suppression of apoptosis is a prerequisite for Myc-induced tumourigenesis in these cells. We have begun to explain why particular oncogenic lesions are common in tumours originating in a particular tissue. It is clear that activation of Myc is sufficient to initiate neoplasia (at least in the skin) and is required for tumour maintenance in both skin and T lymphomas [25,26••,27••]. In the few instances where the lymphomas failed to regress following suppression of Myc expression [26••], it will be important to determine the nature of any additional lesions that may account for this failure. Nonetheless, the repression of Myc activity alone could potentially realise therapeutic potential.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:
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25. Blyth K, Stewart M, Bell M, James C, Evan G, Neil JC, Cameron ER: Sensitivity to myc-induced apoptosis is retained in spontaneous and transplanted lymphomas of CD2-mycER™ mice. Oncogene 2000, in press. 26. Felsher DW, Bishop JM: Reversible tumorigenesis by MYC in •• hematopoietic lineages. Mol Cell 1999, 4:199-207. This paper describes the use of the tetracycline regulatory system to conditionally express myc in haematopoietic cells of transgenic mice. Although sustained myc expression during development leads to malignant T cell lymphomas and acute myeloid leukaemias, inactivation of the transgene causes regression of the majority of established tumours. These results show for the first time that, despite the multistep nature of tumourigenesis in haematopoietic lineages, inactivation of a single genetic lesion, myc, is sufficient to reverse malignancy. 27. ••
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