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Cell darwinism, apoptosis, free radicals and haematological malignancies
Medical Hypotheses (2001) 56(1), 52–57 © 2001 Harcourt Publishers Ltd doi: 10.1054/mehy.2000.1108, available online at http://www.idealibrary.com on
Cell darwinism, apoptosis, free radicals and haematological malignancies A. Cucuianu Cancer Institute Cluj, Haematology Department, Cluj-Napoca, Romania
Summary Haematopoiesis can be interpreted as an ecosystem composed of billions of cells interacting according to Darwinian rules. Mutation, by promoting cell diversity, ensures versatility in coping with internal and external challenges. Most mutated cells are eliminated through apoptosis. However, if mutation generates relative resistance to apoptosis it may result in growth advantage for the mutated cells. The probability of monoclonality and malignancy is significantly increased if the normal multiclonal environment is damaged by a pathologic proapoptotic process that spares the apoptosis resistant clones. Paroxysmal nocturnal haemoglobinuria, myelodysplastic syndromes, chronic myeloid leukaemia, secondary acute leukaemias and immunosuppression-related non-Hodgkin’s lymphomas can be interpreted as ‘opportunistic’ clonal and malignant diseases. Free radicals (FRs) are closely linked to apoptosis and have been incriminated in oncogenesis. Conditions associated with increased FR formation or impaired FR disposal may provide the enhanced apoptotic background against which an apoptosis-resistant clone may gain growth advantage. © 2001 Harcourt Publishers Ltd
INTRODUCTION
Haematopoiesis as a ‘chaotic’ darwinian system
Darwin’s ‘survival of the fittest’ principle, is, in the words of Daniel Dennet a ‘universal acid’ kind of law, applying to any physical, chemical, biological or social motion. In order to survive in an ever-changing environment, a system has to be adaptable and versatile (1). Versatility implies diversity. Darwinian principles are obvious in large, open biological systems such as populations and species, but may also apply to the cellular units forming individual organisms (2,3). I shall try to outline an interpretation of oncogenesis in haematological malignancies as a particular growth pattern arising from disturbances in the local ecological balance.
Organisms are complex ecosystems composed of millions of clones of ‘self’ cells that interact and compete among themselves and with thousands of ‘non-self’ species of bacteria, fungi, protosoares, mites and worms. They compete for food, ligands, electric stimuli and living space. As living systems are characterized by the capacity to copy and reproduce information, ‘success’ in this competition is represented by cell or signal proliferation. Cell darwinism is more evident in tissues where diversity and adaptability are essential in dealing with a highly diverse spectrum of environmental challenges, such as the haematopoietic tissue. Small, randomly acquired genetic differences account for blood cell diversity (4). Lymphocytes have an enormous versatility conferred by the hypervariable regions of the immunoglobulin and T-cell receptor genes. It can be postulated that in an immunocompetent organism, at any moment, there are millions of B-cell clones with different immunoglobulin specificities, capable of fitting almost any possible antigen (5). If antigen A actually appears, in other words if the environmental conditions change in the A direction, the A algorithmic
Received 9 December 1999 Accepted 28 February 2000 Published online 8 December 2000 Correspondence to: Andrei Cucuianu, Cancer Institute Cluj, Haematology Department, Bvd. 21 Decembrie Nr 73, R-3400, Cluj-Napoca, Romania. Phone: +40 64 192766; Fax: +40 64 198365; E-mail: [email protected]
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Cell darwinism, apoptosis, free radicals and haematological malignancies
pathway, involving the A’ immunoglobulin gene mutation will be ‘chosen’ over the many other pathways at hand at that moment, therefore, the A’ clone will be selected to deal with the A antigen (6). This process is not ‘targeted’ along a linear trajectory, but involves the small, gradual, fractal changes characteristic to chaotic, natural systems (7). Such a process is never ‘perfect’, there will always exist a small risk of failure or aberrant response.
Cell darwinism, cell death and proliferation Apoptosis represents the way most cells die, through an active, energy-consuming chain of reactions involving DNA destruction by specific enzymes, notably the caspase cascade. Apoptosis begins in early embryonic development and is instrumental in normal growth and development (8). Apoptosis can be triggered by various physical, chemical and biological challenges. The products of several oncogenes and tumour-suppressor genes (p53, p73, c-abl, bax, fas, BRCA1 and BRCA2) promote apoptosis (9–12) while other genes like bcl-2, bcl-x, mcl-1, inhibit it (13). As it has already been mentioned, in the haematopoietic environment, the main diversity-inducing mechanism is the capacity of cells to mutate, through DNA breakage and rearrangement. Most mutated cells in which repair mechanisms fail, die in apoptosis (10). Some mutated cells survive, and it is reasonable to think that part of them will represent an adaptation, a gain for the organism. Therefore apoptosis may act as a way to promote the ‘relatively good’ mutations, rather than a way to preserve the status quo. However the possibility that some ‘bad for the organism, good for themselves’ mutated cells may persist will always exist. During the youth of a healthy organism, when the number of clones is at its highest and the apoptotic pressure is relatively mild, the fittest cells relative to the environmental challenges are indeed selected. However, if the apoptotic pressure increases, in the context of proapopotic overexposure (e.g. radiation, chronic infection, chemicals), or simply with aging, as more and more clones disappear and the very number of cells becomes an issue, the qualities needed for cellular selection will have less to do with functionality than with the surviving process itself; therefore cells that have acquired flaws in their suicide program (such as cells with bcl-2 overexpression or p53 alterations) will have an advantage even if they have also acquired other mutations that make them less functional. It has been shown that senescent human fibroblasts and lymphocytes display resistance to apoptosis as well as impaired functionality and that their accumulation may contribute to agerelated pathology (14,15). © 2001 Harcourt Publishers Ltd
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Cell darwinism, apoptosis and cancer In this perspective, one can imagine cancer cells as being very good at survival, most of the time at the expense of functionality. As the capacity to mutate is a given of every living system, we can assume that aberrant mutations occur frequently. Still, aberrant clones rarely gain growth advantage. However if normal clones are destroyed or suppressed, and an aberrant clone happens to be resistant to that particular toxic process, its relative advantage may become decisive. Several malignant cell types are currently considered ‘supersurvivors’ rather than ‘superproliferators’, cells that possess ‘relatively good tricks’ that help them stay alive. Much evidence points to the relative resistance of malignant cells to apoptosis. The antiapoptotic gene bcl-2 is overexpressed in follicular NHL (16). In normal lymphatic germinal centres bcl-2 is underexpressed, and intense mutation and apoptosis occurs; 99% of the cells that enter the germinal centres die in apoptosis (17 ). On the other hand, the germinal centre is the place where most NHL clones are thought to arise: in an intensely apoptotic environment, an apoptosis-resistant clone may gain relative growth advantage. Proapoptotic genes like p53 were found to be underexpressed in several malignant cell lines (18). The bcr-abl gene, specific to chronic myeloid leukaemia also seems to inhibit apoptosis (19). Although many specific single genetic abnormalities have so far been described in malignant haemopathies, most authors agree that other events are needed for oncogenesis. Healthy persons may possess such aberrant clones without displaying monoclonal patterns or progressing to malignancy (20,21). If an event leading to widespread apoptosis occurs (such as radiation, infection, immune aggression or simply the passing of time), clones that are relatively resistant to apoptosis will get selected and thrive. Such clones may appear to regulatory mechanisms as ‘solutions’ to cellular poverty. Growth advantage leads to monoclonal, non-diverse patterns of growth. Most cancers are monoclonal or oligoclonal, however monoclonality does not always spell malignancy (22). The ‘malignant’ status, implying cancerrelated lethality, usually means that dominant clones are undifferentiated (anaplastic) and that normal clones are destroyed or inhibited. The majority of clonal growth patterns never progress to cancer or are not even diagnosed during the limited time of life. However, as mutations occur permanently even within the dominant clone, the chance of any given subclone, anaplastic ones included, to become dominant is greater in an oligoclonal, less competitive environment than in a multiclonal, pluralistic one. It has been demonstrated that in healthy older persons, clonal haematopoiesis is common (23) and indeed, malignant haemopathies are more frequent in the elderly. Several clonal and malignant haematological diseases may fit this ‘opportunistic’ pathogenetic pattern. Medical Hypotheses (2001) 56(1), 52–57
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1. Monoclonal gammopathy of undetermined significance (MGUS), a feature that occurs relatively often in elderly healthy subjects (1–5%) is 1000 times more frequent than multiple myeloma (MM), its malignant counterpart; the chance of MM to occur is however thousands of times higher in persons with MGUS than in the general population (24). 2. Myelodysplastic syndromes (MDS) are clonal haematopoietic stem cell disorders carrying a high risk of transformation to acute myeloid leukaemia (AML); cell proliferation in most MDS forms is sluggish, and increased apoptosis is frequently encountered in haemopoietic progenitors (25). However, against this background of increased apoptosis, the emergence of an anaplastic, apoptosisresistant AML clone is quite likely. 3. Aplastic anaemia (AA), is a condition defined by suppression or destruction of normal haematopoietic stem cells. In AA, the remaining haematopoiesis is sometimes clonal and the risk of clonal and malignant haemopathies to develop is increased (26). Another interesting ‘natural experiment’ is the AA – paroxysmal nocturnal haemoglobinuria (PNH) syndrome. PNH is a non-malignant clonal haematopoietic stem cell disease in which blood cells lack the phospho-inosytol-glycan A gene (PIG-A) gene codifying a glycosil-phosphoinosytol (GPI) anchor for various membrane receptors. The pathogenesis of PNH implies an autoimmune T-cell mediated attack against haematopoietic stem cells, the site of the attack being represented by GPIanchored receptors with the consecutive development of AA; if a PIG-A mutated, GPIdeficient clone is present, its cells are spared the autoimmune attack, restoring clonal haematopoiesis by themselves. However, PNH cells are deficient, being vulnerable to complement mediated lysis. Moreover, the incidence of subsequent clonal diseases such as MDS or overtly malignant ones like AML is much increased (27,28). 4. Non-Hodgkin’s lymphoma (NHL) occurs much more frequently in AIDS and in post-transplant immunocompromised patients and virtually 100% of the cases are Epstein-Barr virus (EBV)-related NHLs (29,30). As most people have gone through infectious mononucleosis during adolescence, EBV-infected and transformed clones persist in many individuals; however, they rarely gain growth advantage unless a debilitating event such as HIV infection or prolonged immunosuppression occurs. 5. The genetic conditions associated with unstable DNA like Werner’s and Bloom’s syndromes and Fanconi’s anaemia also provide interesting clues. Werner’s Medical Hypotheses (2001) 56(1), 52–57
disease, whose cause seems to be a defect in DNA helicases (31), is associated with signs of premature aging and a high incidence of cancer. The high mutation rate appearing as a consequence of the unstable genome, leads to increased apoptosis, therefore to premature ageing. Against this background of mutation/apoptosis, there is a higher chance for cancer clones to appear and progress. Similarly, the main feature in Fanconi’s anaemia is bone marrow aplasia and a high risk of acute leukaemia (32). 6. Chronic myeloid leukaemia (CML), in its chronic phase is not really a malignant disorder, as it only displays clonality with the presence of the bcr-abl gene, increased proliferation, but not anaplastic transformation or impaired functionality (22). However, sooner or later, malignant transformation (the blastic phase) occurs in virtually 100% of the cases. It has been shown that in the early chronic phase, multiclonal progenitors are still present, but their numbers diminish in time, so that in late chronic phase they are undetectable (33). Though it is quite clear that the bcr-abl gene product can offer a growth advantage to the leukaemic clone, nobody has so far explained why in CML, or in acute leukaemias for that matter, normal clones are destroyed or suppressed. This aspect is of prime importance since it is the infectious and haemorrhagic complications related to the lack of normal cells rather than the presence of leukaemic cells that eventually kill the patients. Since it has been found that bcr-abl transcripts exist in blood cells of healthy persons (20,21) a model resembling the pathogenesis of PNH can be proposed: in persons harbouring the bcr-abl gene, widespread apoptosis of the normal stem cells induced by toxic agents (radiation, chemicals, etc) could result in the selection of the bcr-abl, apoptosis-resistant clone. The high probability of blastic transformation may be explained by both a low degree of competition in a clonal environment and an increased likelihood that the ‘really bad’ mutation appears due to the high number of mitoses. One may also speculate that after years of intense proliferation, the initial CML clone will eventually exhaust its proliferative potential; a previously transformed anaplastic subclone may move in to fill the vacuum, leading to the acute blastic phase. It is notable that so far, the only treatment that can cure CML is allogeneic bone marrow transplantation, which, aside from the benefits brought by high-dose cytotoxic therapy and graft-versus-leukaemia effect, also populates the recipient marrow with multiclonal donor stem cells. © 2001 Harcourt Publishers Ltd
Cell darwinism, apoptosis, free radicals and haematological malignancies
Free radicals, apoptosis and cancer Apoptosis is the death-pathway common to several toxic agents (radiation, chemotherapy, infectious and immune aggression), as well as to the most toxic of them all, aging. Although different, all these agents generate free radicals (FR) like oxygen reactive species, nitrogen oxide and alkyl radicals. Mitochondrial respiration itself leads to oxygen reactive species production. Therefore, FR are produced as a consequence of life itself. FR are intimately linked to apoptosis. Oxidative stress can induce apoptotic death, and mitochondria have a central role, since cytochrome c release in the cytoplasm and the opening of the permeability transition pore are important events in the apoptotic cascade (34–37). As it is known that FR have a mutagenic effect on DNA, there seems to exist a connection between the fact that FR promote mutation but also trigger the mechanism that eliminates mutated cells. In fact FR will promote apoptosis and mutation concomitantly: if the mutation happens to alter a gene involved in apoptosis, the cell will become relatively immune to FRmediated injury (Fig. 1). According to the mitochondrial theory, aging is a vicious circle in which the gradual accumulation of somatic mutations of mitochondrial DNA (mtDNA), leads to altered production and disposal of oxygen reactive species, which in their turn accelerate the rate of mutation (38). MtDNA seems to be very unstable and in fact, apart from aging, mtDNA abnormalities have been found in a number of human pathologies, including dysplastic and neoplastic diseases (39). The antiapoptotic effect of the bcl-2 product, which is localized in mitochondria, endoplasmic reticulum and nuclear membranes, sites of increased FR generation, is probably exerted through the
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control of oxidative damage to cellular constituents (40). Evidence exists that the bcr-abl gene product exerts its antiapoptotic effect through blockage of mitochondrial release of cytochrome c and therefore failure to induce an increase in oxygen-reactive species (41). It is also notable that retinoids induce their apoptotic effect by stimulating the intracellular production of FR (42). If we hypothesize that FR are the main hurdles that cells have to overcome, and that cancer cells are ‘supersurvivor’ cells, we can imagine that in a FR-damaged environment, malignant cells should be the ones endowed with better defensive systems. Bcl-2 overexpression can therefore be considered as a ‘good trick’ relative to the production and disposal of free radicals, thus inhibiting apoptosis and leading to a growth advantage for the cancer clone. It has been shown that hepatoma cells are less sensitive to anoxic stress than normal hepatocytes (43). In fact, many malignant cell types derive their energy from anaerobic glycolysis, and are therefore less exposed to oxidative stress (44). Lactate dehydrogenase (LDH), an enzyme which is essential in anaerobiosis is a marker of aggressiveness in many tumours; some evidence exists that elevated LDH is not merely the result of increased cell destruction but also of synthesis upregulation in tumour cells (45).
Cell darwinism and cancer therapy Both radiotherapy and chemotherapy induce apoptosis in cancer as well as in normal cells. However, both chemotherapy and radiotherapy often fail to eradicate malignant clones. In fact, the more chemotherapy is given, the higher the aggressiveness of relapses. In these
Fig. 1 Free radicals (FR), resulting from various physiologic and pathologic processes, induce DNA damage. Most cells in which DNA repair mechanisms fail, die in apoptosis. However some mutations, involving apoptosis-related genes, may confer resistance to FR-induced apoptosis. If high FR exposure persists, FR-resistant clones may become more prominent, while normal clones, having intact suicide mechanisms, will suffer continuous loss, resulting in a monoclonal and subsequently malignant growth pattern. © 2001 Harcourt Publishers Ltd
Medical Hypotheses (2001) 56(1), 52–57
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may give an important growth advantage to an abnormal clone, if this clone happens to possess a way to avoid or better deal with that particular challenge. As malignant clones harbour mutations in genes involved in apoptosis, but the presence of such mutations is not sufficient in itself for oncogenesis, one can speculate that the likelihood of cancer is greatly increased in the context of widespread apoptosis. As free radicals are closely linked to apoptosis, they may be the main factors that destabilize the cellular environment by tilting the balance towards clones that somehow avoid their proapoptotic effects. Prevention and treatment lowers the odds for cancer appearance and progression, by acting both on malignant and normal clones.
cases therapy only accelerates the selection process that has possibly led to malignancy in the first place. There is no established therapy at the moment that aims at restoring or boosting the normal multiclonal environment. However, some established treatments may do just that. In leukaemias, allogeneic stem cell transplantation repopulates the recipient marrow with multiclonal haematopoiesis. Despite the effects of myeloablative chemotherapy, radiotherapy and the graft-versusleukaemia effect, the leukaemic clone may persist and relapse may or may not occur, possibly in relation to the viability of the donor’s multiclonal haematopoiesis in a foreign environment. Chemotherapy itself may lead to remissions and cures not necessarily by killing all the neoplastic cells but by radically changing the apoptotic ‘rules of the game’, somehow eliminating the suppression of the normal clones (for example the elimination of an autoimmune suppressive mechanism). Reports exist on the benefits of aggressive immunosuppressive therapy in clonal diseases such as myelodysplastic syndromes, and to prolonged complete remissions in acute leukaemia after haematopoietic growth factor treatment in the absence of chemotherapy (46,47). The relatively recent finding that certain types of gastric lymphomas are associated with Helicobacter pylori (HP) infection and that HP eradication can be a cure in these cases (48), may also support this hypothesis, as it is known that HP induces apoptosis in lymphocytes (49). The best cancer therapy of the moment is prevention. Recent evidence points out to the role of diet in preventing cancer. Antioxidants in soy beans, garlic, tomatoes, red grapes play a central role in cancer-preventing diets (50). As alcohol and tobacco are both FR inducers and cancer-risk factors, avoiding them would be synergic with antioxidant diets (51). Few, if any targeted, anti-cancer-clone therapies exist at the moment. Much is expected from gene therapy. However, even if malignant cells are indeed targeted and eliminated, the hypothetical underlying defect of the ‘normal’, multiclonal environment may persist, setting the stage for relapses or other malignancies. In the near future, when all human genes will be mapped, geneticists may find that the real difference between cancer patients and healthy subjects lies not in the cancer clone site on the genetic map, but elsewhere, within the multiclonal, non-malignant environment regions.
17.
CONCLUSIONS
18.
Living organisms can be viewed as diverse and apparently chaotic cell systems in which cells and clones interact according to Darwinian rules. The more clones there are, the smaller the chance for any given clone to gain growth advantage. A process that affects normal clones
19.
Medical Hypotheses (2001) 56(1), 52–57
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21. Bose S., Deininger M., Gora-Tybor J., et al. The presence of typical and atypical BCR-ABL fusion genes in leucocytes of normal individuals: biologic significance and implications for the assessment of minimal residual disease. Blood 1998; 92: 3362–3367. 22. Butturini A., Gale R. P. Relationship between clonality and transformation in acute leukemia. Leukemia Research 1991; 1: 1–7. 23. Champion K. M., Gilbert J. G., Asimakoupulos F. A. et al. Clonal hematopoiesis in normal elderly women: implications for the myeloproliferative disorders and myelodysplastic syndromes. Br J Haematol 1997; 97: 920–926. 24. Blade J., Guillermo A. L., Rozman C. et al. Malignant transformation and life expectancy in monoclonal gammopathy of undetermined significance. Br J Haematol 1992; 81: 391–394. 25. Parker J. E., Mufti G. J. Ineffective haemopoiesis and apoptosis in myelodysplastic syndromes. Br J Haematol 1998; 101: 220–230. 26. van Kamp H., Landegent J. E., Jansen R. P., et al. Clonal hematopoiesis in patients with acquired aplastic anemia. Blood 1991, 78: 3209–3214. 27. Rotoli B., Luzzato L. Paroxysmal nocturnal hemoglobinuria. Sem Hematol 1989; 36: 201–205. 28. Takeda J., Myata T., Kawagoe K. et al. Deficiency of the GPI anchor caused by a somatic mutation of the PIG-A gene in paroxysmal nocturnal hemoglobinuria. Cell 1993; 73: 703–711. 29. Sverdlow S. H. Post-transplant lymphoproliferative disorders: a morphologic, phenotypic and genotypic spectrum of disease. Histopathology 1992, 20: 373–385. 30. Aboulafia D. Epidemiology and pathogenesis of AIDS-related lymphomas. Oncology 1998, 12: 1068–1081. 31. Epstein C. J., Motulsky A.G. Werner syndrome: entering the helicase era. Bioessays 1996; 18: 1025–1027. 32. Garcia-Higuera I., Kuang Y., D’Andrea A. D. The molecular and cellular biology of Fanconi anemia. Curr Opin Hematol 1999; 6: 83–88. 33. Frassoni F., Podesta M., Piaggio G., et al. Normal primitive haematopoietic progenitors are more frequent than their leukaemic counterpart in newly diagnosed patients with chronic myeloid leukaemia but rapidly decline with time. Br J Haematol 1998; 104: 538–545. 34. Mathieu J., Chancerelle Y., Herodin F et al. Oxidative stress and apoptosis. Ann Pharm Fr 1996; 54: 193–201. 35. Lenaz G. Role of mitochondria in oxidative stress and ageing. Biochim Biophys Acta 1998; 1366: 53–67.
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36. Richter C. Reactive oxygen and nitrogen species regulate mitochondrial Ca2+ homeostasis and respiration. Biosci Rep 1997; 17: 53–66. 37. Hampton M. B., Fadeel B., Orrenius S. Redox regulation of caspases during apoptosis. Ann NY Acad Sci 1998; 854: 328–335. 38. Papa S., Skulachev V. P. Reactive oxygen species, mitochondria and aging. Mol Cell Biochem 1997; 174: 305–319. 39. Johns D. R. Mitochondrial DNA and disease. N Eng J Med 1993; 333: 638–644. 40. Korsmeyer S. J., Yin X. M., Oltvai Z. N. et al. Reactive oxygen species and the regulation of cell death by the Bcl-2 family. Biochim Biophys Acta 1995; 1271: 63–66. 41. Amarante-Mendes G., Kim C. N., Liu L. et al. Bcr-Abl exerts its antiapoptotic effect against diverse apoptotic stimuli through blockage of mitochondrial release of cytochrome c and activation of caspase-3. Blood 1998; 91: 1700–1705. 42. Zheng A., Mantymaa P., Saily M. et al. An association between mitochondrial function and all-trans retinoic acid-induced apoptosis in acute myeloblastic leukemia cells. Br J Haematol 1999; 105: 215–224. 43. Kobryn C. E., Fiskum G. Differential sensitivity of AS-30D rat hepatoma cells and normal hepatocytes to anoxic cell damage. Am J Physiol 1992; 262: 1384–1387. 44. Hamilton E., Fennell M., Stafford D. M. Modification of tumor glucose metabolism for therapeutic benefit. Acta Oncol 1995; 34: 429–433. 45. Griffini P., Freitas I., Vigorelli E., Van Noorden CJ. Changes in the zonation of lactate dehydrogenase activity in lobules of rat liver after experimentally induced colon carcinoma metastases. Anticancer Res 1994; 14: 2537–2540. 46. Jonasova A., Neuwirtova R., Cermak J., et al. Cyclosporin A therapy in hypoplastic MDS patients and certain refractory anemias without hypoplastic bone marrow. Br J Haematol 1997; 100: 304–309. 47. Bassan R., Rambaldi A., Amaru R. Unexpected remission of acute myeloid leukemia after GM-CSF. Br J Haematol 1994; 87: 835–838. 48. Isaacson P. G. Gastrointestinal lymphomas of T and B-cell type. Mod Pathol 1999; 12: 151–158. 49. Reinacher-Schick A., Petrasch S., Burger A. et al. Helicobacter pylori induces apoptosis in mucosal lymphocytes in patients with gastritis. Z Gastroenterol 1998; 36: 1021–1026. 50. Cummings J. H., Bingham S. A. Diet and the prevention of cancer. BMJ 1998; 317: 1636–1640. 51. Gerber M., Corpet D. Energy balance and cancers. Eur J Canc Prevent 1999; 8: 77–89.
Medical Hypotheses (2001) 56(1), 52–57
Cell darwinism, apoptosis, free radicals and haematological malignancies A. Cucuianu Cancer Institute Cluj, Haematology Department, Cluj-Napoca, Romania
Summary Haematopoiesis can be interpreted as an ecosystem composed of billions of cells interacting according to Darwinian rules. Mutation, by promoting cell diversity, ensures versatility in coping with internal and external challenges. Most mutated cells are eliminated through apoptosis. However, if mutation generates relative resistance to apoptosis it may result in growth advantage for the mutated cells. The probability of monoclonality and malignancy is significantly increased if the normal multiclonal environment is damaged by a pathologic proapoptotic process that spares the apoptosis resistant clones. Paroxysmal nocturnal haemoglobinuria, myelodysplastic syndromes, chronic myeloid leukaemia, secondary acute leukaemias and immunosuppression-related non-Hodgkin’s lymphomas can be interpreted as ‘opportunistic’ clonal and malignant diseases. Free radicals (FRs) are closely linked to apoptosis and have been incriminated in oncogenesis. Conditions associated with increased FR formation or impaired FR disposal may provide the enhanced apoptotic background against which an apoptosis-resistant clone may gain growth advantage. © 2001 Harcourt Publishers Ltd
INTRODUCTION
Haematopoiesis as a ‘chaotic’ darwinian system
Darwin’s ‘survival of the fittest’ principle, is, in the words of Daniel Dennet a ‘universal acid’ kind of law, applying to any physical, chemical, biological or social motion. In order to survive in an ever-changing environment, a system has to be adaptable and versatile (1). Versatility implies diversity. Darwinian principles are obvious in large, open biological systems such as populations and species, but may also apply to the cellular units forming individual organisms (2,3). I shall try to outline an interpretation of oncogenesis in haematological malignancies as a particular growth pattern arising from disturbances in the local ecological balance.
Organisms are complex ecosystems composed of millions of clones of ‘self’ cells that interact and compete among themselves and with thousands of ‘non-self’ species of bacteria, fungi, protosoares, mites and worms. They compete for food, ligands, electric stimuli and living space. As living systems are characterized by the capacity to copy and reproduce information, ‘success’ in this competition is represented by cell or signal proliferation. Cell darwinism is more evident in tissues where diversity and adaptability are essential in dealing with a highly diverse spectrum of environmental challenges, such as the haematopoietic tissue. Small, randomly acquired genetic differences account for blood cell diversity (4). Lymphocytes have an enormous versatility conferred by the hypervariable regions of the immunoglobulin and T-cell receptor genes. It can be postulated that in an immunocompetent organism, at any moment, there are millions of B-cell clones with different immunoglobulin specificities, capable of fitting almost any possible antigen (5). If antigen A actually appears, in other words if the environmental conditions change in the A direction, the A algorithmic
Received 9 December 1999 Accepted 28 February 2000 Published online 8 December 2000 Correspondence to: Andrei Cucuianu, Cancer Institute Cluj, Haematology Department, Bvd. 21 Decembrie Nr 73, R-3400, Cluj-Napoca, Romania. Phone: +40 64 192766; Fax: +40 64 198365; E-mail: [email protected]
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pathway, involving the A’ immunoglobulin gene mutation will be ‘chosen’ over the many other pathways at hand at that moment, therefore, the A’ clone will be selected to deal with the A antigen (6). This process is not ‘targeted’ along a linear trajectory, but involves the small, gradual, fractal changes characteristic to chaotic, natural systems (7). Such a process is never ‘perfect’, there will always exist a small risk of failure or aberrant response.
Cell darwinism, cell death and proliferation Apoptosis represents the way most cells die, through an active, energy-consuming chain of reactions involving DNA destruction by specific enzymes, notably the caspase cascade. Apoptosis begins in early embryonic development and is instrumental in normal growth and development (8). Apoptosis can be triggered by various physical, chemical and biological challenges. The products of several oncogenes and tumour-suppressor genes (p53, p73, c-abl, bax, fas, BRCA1 and BRCA2) promote apoptosis (9–12) while other genes like bcl-2, bcl-x, mcl-1, inhibit it (13). As it has already been mentioned, in the haematopoietic environment, the main diversity-inducing mechanism is the capacity of cells to mutate, through DNA breakage and rearrangement. Most mutated cells in which repair mechanisms fail, die in apoptosis (10). Some mutated cells survive, and it is reasonable to think that part of them will represent an adaptation, a gain for the organism. Therefore apoptosis may act as a way to promote the ‘relatively good’ mutations, rather than a way to preserve the status quo. However the possibility that some ‘bad for the organism, good for themselves’ mutated cells may persist will always exist. During the youth of a healthy organism, when the number of clones is at its highest and the apoptotic pressure is relatively mild, the fittest cells relative to the environmental challenges are indeed selected. However, if the apoptotic pressure increases, in the context of proapopotic overexposure (e.g. radiation, chronic infection, chemicals), or simply with aging, as more and more clones disappear and the very number of cells becomes an issue, the qualities needed for cellular selection will have less to do with functionality than with the surviving process itself; therefore cells that have acquired flaws in their suicide program (such as cells with bcl-2 overexpression or p53 alterations) will have an advantage even if they have also acquired other mutations that make them less functional. It has been shown that senescent human fibroblasts and lymphocytes display resistance to apoptosis as well as impaired functionality and that their accumulation may contribute to agerelated pathology (14,15). © 2001 Harcourt Publishers Ltd
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Cell darwinism, apoptosis and cancer In this perspective, one can imagine cancer cells as being very good at survival, most of the time at the expense of functionality. As the capacity to mutate is a given of every living system, we can assume that aberrant mutations occur frequently. Still, aberrant clones rarely gain growth advantage. However if normal clones are destroyed or suppressed, and an aberrant clone happens to be resistant to that particular toxic process, its relative advantage may become decisive. Several malignant cell types are currently considered ‘supersurvivors’ rather than ‘superproliferators’, cells that possess ‘relatively good tricks’ that help them stay alive. Much evidence points to the relative resistance of malignant cells to apoptosis. The antiapoptotic gene bcl-2 is overexpressed in follicular NHL (16). In normal lymphatic germinal centres bcl-2 is underexpressed, and intense mutation and apoptosis occurs; 99% of the cells that enter the germinal centres die in apoptosis (17 ). On the other hand, the germinal centre is the place where most NHL clones are thought to arise: in an intensely apoptotic environment, an apoptosis-resistant clone may gain relative growth advantage. Proapoptotic genes like p53 were found to be underexpressed in several malignant cell lines (18). The bcr-abl gene, specific to chronic myeloid leukaemia also seems to inhibit apoptosis (19). Although many specific single genetic abnormalities have so far been described in malignant haemopathies, most authors agree that other events are needed for oncogenesis. Healthy persons may possess such aberrant clones without displaying monoclonal patterns or progressing to malignancy (20,21). If an event leading to widespread apoptosis occurs (such as radiation, infection, immune aggression or simply the passing of time), clones that are relatively resistant to apoptosis will get selected and thrive. Such clones may appear to regulatory mechanisms as ‘solutions’ to cellular poverty. Growth advantage leads to monoclonal, non-diverse patterns of growth. Most cancers are monoclonal or oligoclonal, however monoclonality does not always spell malignancy (22). The ‘malignant’ status, implying cancerrelated lethality, usually means that dominant clones are undifferentiated (anaplastic) and that normal clones are destroyed or inhibited. The majority of clonal growth patterns never progress to cancer or are not even diagnosed during the limited time of life. However, as mutations occur permanently even within the dominant clone, the chance of any given subclone, anaplastic ones included, to become dominant is greater in an oligoclonal, less competitive environment than in a multiclonal, pluralistic one. It has been demonstrated that in healthy older persons, clonal haematopoiesis is common (23) and indeed, malignant haemopathies are more frequent in the elderly. Several clonal and malignant haematological diseases may fit this ‘opportunistic’ pathogenetic pattern. Medical Hypotheses (2001) 56(1), 52–57
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1. Monoclonal gammopathy of undetermined significance (MGUS), a feature that occurs relatively often in elderly healthy subjects (1–5%) is 1000 times more frequent than multiple myeloma (MM), its malignant counterpart; the chance of MM to occur is however thousands of times higher in persons with MGUS than in the general population (24). 2. Myelodysplastic syndromes (MDS) are clonal haematopoietic stem cell disorders carrying a high risk of transformation to acute myeloid leukaemia (AML); cell proliferation in most MDS forms is sluggish, and increased apoptosis is frequently encountered in haemopoietic progenitors (25). However, against this background of increased apoptosis, the emergence of an anaplastic, apoptosisresistant AML clone is quite likely. 3. Aplastic anaemia (AA), is a condition defined by suppression or destruction of normal haematopoietic stem cells. In AA, the remaining haematopoiesis is sometimes clonal and the risk of clonal and malignant haemopathies to develop is increased (26). Another interesting ‘natural experiment’ is the AA – paroxysmal nocturnal haemoglobinuria (PNH) syndrome. PNH is a non-malignant clonal haematopoietic stem cell disease in which blood cells lack the phospho-inosytol-glycan A gene (PIG-A) gene codifying a glycosil-phosphoinosytol (GPI) anchor for various membrane receptors. The pathogenesis of PNH implies an autoimmune T-cell mediated attack against haematopoietic stem cells, the site of the attack being represented by GPIanchored receptors with the consecutive development of AA; if a PIG-A mutated, GPIdeficient clone is present, its cells are spared the autoimmune attack, restoring clonal haematopoiesis by themselves. However, PNH cells are deficient, being vulnerable to complement mediated lysis. Moreover, the incidence of subsequent clonal diseases such as MDS or overtly malignant ones like AML is much increased (27,28). 4. Non-Hodgkin’s lymphoma (NHL) occurs much more frequently in AIDS and in post-transplant immunocompromised patients and virtually 100% of the cases are Epstein-Barr virus (EBV)-related NHLs (29,30). As most people have gone through infectious mononucleosis during adolescence, EBV-infected and transformed clones persist in many individuals; however, they rarely gain growth advantage unless a debilitating event such as HIV infection or prolonged immunosuppression occurs. 5. The genetic conditions associated with unstable DNA like Werner’s and Bloom’s syndromes and Fanconi’s anaemia also provide interesting clues. Werner’s Medical Hypotheses (2001) 56(1), 52–57
disease, whose cause seems to be a defect in DNA helicases (31), is associated with signs of premature aging and a high incidence of cancer. The high mutation rate appearing as a consequence of the unstable genome, leads to increased apoptosis, therefore to premature ageing. Against this background of mutation/apoptosis, there is a higher chance for cancer clones to appear and progress. Similarly, the main feature in Fanconi’s anaemia is bone marrow aplasia and a high risk of acute leukaemia (32). 6. Chronic myeloid leukaemia (CML), in its chronic phase is not really a malignant disorder, as it only displays clonality with the presence of the bcr-abl gene, increased proliferation, but not anaplastic transformation or impaired functionality (22). However, sooner or later, malignant transformation (the blastic phase) occurs in virtually 100% of the cases. It has been shown that in the early chronic phase, multiclonal progenitors are still present, but their numbers diminish in time, so that in late chronic phase they are undetectable (33). Though it is quite clear that the bcr-abl gene product can offer a growth advantage to the leukaemic clone, nobody has so far explained why in CML, or in acute leukaemias for that matter, normal clones are destroyed or suppressed. This aspect is of prime importance since it is the infectious and haemorrhagic complications related to the lack of normal cells rather than the presence of leukaemic cells that eventually kill the patients. Since it has been found that bcr-abl transcripts exist in blood cells of healthy persons (20,21) a model resembling the pathogenesis of PNH can be proposed: in persons harbouring the bcr-abl gene, widespread apoptosis of the normal stem cells induced by toxic agents (radiation, chemicals, etc) could result in the selection of the bcr-abl, apoptosis-resistant clone. The high probability of blastic transformation may be explained by both a low degree of competition in a clonal environment and an increased likelihood that the ‘really bad’ mutation appears due to the high number of mitoses. One may also speculate that after years of intense proliferation, the initial CML clone will eventually exhaust its proliferative potential; a previously transformed anaplastic subclone may move in to fill the vacuum, leading to the acute blastic phase. It is notable that so far, the only treatment that can cure CML is allogeneic bone marrow transplantation, which, aside from the benefits brought by high-dose cytotoxic therapy and graft-versus-leukaemia effect, also populates the recipient marrow with multiclonal donor stem cells. © 2001 Harcourt Publishers Ltd
Cell darwinism, apoptosis, free radicals and haematological malignancies
Free radicals, apoptosis and cancer Apoptosis is the death-pathway common to several toxic agents (radiation, chemotherapy, infectious and immune aggression), as well as to the most toxic of them all, aging. Although different, all these agents generate free radicals (FR) like oxygen reactive species, nitrogen oxide and alkyl radicals. Mitochondrial respiration itself leads to oxygen reactive species production. Therefore, FR are produced as a consequence of life itself. FR are intimately linked to apoptosis. Oxidative stress can induce apoptotic death, and mitochondria have a central role, since cytochrome c release in the cytoplasm and the opening of the permeability transition pore are important events in the apoptotic cascade (34–37). As it is known that FR have a mutagenic effect on DNA, there seems to exist a connection between the fact that FR promote mutation but also trigger the mechanism that eliminates mutated cells. In fact FR will promote apoptosis and mutation concomitantly: if the mutation happens to alter a gene involved in apoptosis, the cell will become relatively immune to FRmediated injury (Fig. 1). According to the mitochondrial theory, aging is a vicious circle in which the gradual accumulation of somatic mutations of mitochondrial DNA (mtDNA), leads to altered production and disposal of oxygen reactive species, which in their turn accelerate the rate of mutation (38). MtDNA seems to be very unstable and in fact, apart from aging, mtDNA abnormalities have been found in a number of human pathologies, including dysplastic and neoplastic diseases (39). The antiapoptotic effect of the bcl-2 product, which is localized in mitochondria, endoplasmic reticulum and nuclear membranes, sites of increased FR generation, is probably exerted through the
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control of oxidative damage to cellular constituents (40). Evidence exists that the bcr-abl gene product exerts its antiapoptotic effect through blockage of mitochondrial release of cytochrome c and therefore failure to induce an increase in oxygen-reactive species (41). It is also notable that retinoids induce their apoptotic effect by stimulating the intracellular production of FR (42). If we hypothesize that FR are the main hurdles that cells have to overcome, and that cancer cells are ‘supersurvivor’ cells, we can imagine that in a FR-damaged environment, malignant cells should be the ones endowed with better defensive systems. Bcl-2 overexpression can therefore be considered as a ‘good trick’ relative to the production and disposal of free radicals, thus inhibiting apoptosis and leading to a growth advantage for the cancer clone. It has been shown that hepatoma cells are less sensitive to anoxic stress than normal hepatocytes (43). In fact, many malignant cell types derive their energy from anaerobic glycolysis, and are therefore less exposed to oxidative stress (44). Lactate dehydrogenase (LDH), an enzyme which is essential in anaerobiosis is a marker of aggressiveness in many tumours; some evidence exists that elevated LDH is not merely the result of increased cell destruction but also of synthesis upregulation in tumour cells (45).
Cell darwinism and cancer therapy Both radiotherapy and chemotherapy induce apoptosis in cancer as well as in normal cells. However, both chemotherapy and radiotherapy often fail to eradicate malignant clones. In fact, the more chemotherapy is given, the higher the aggressiveness of relapses. In these
Fig. 1 Free radicals (FR), resulting from various physiologic and pathologic processes, induce DNA damage. Most cells in which DNA repair mechanisms fail, die in apoptosis. However some mutations, involving apoptosis-related genes, may confer resistance to FR-induced apoptosis. If high FR exposure persists, FR-resistant clones may become more prominent, while normal clones, having intact suicide mechanisms, will suffer continuous loss, resulting in a monoclonal and subsequently malignant growth pattern. © 2001 Harcourt Publishers Ltd
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may give an important growth advantage to an abnormal clone, if this clone happens to possess a way to avoid or better deal with that particular challenge. As malignant clones harbour mutations in genes involved in apoptosis, but the presence of such mutations is not sufficient in itself for oncogenesis, one can speculate that the likelihood of cancer is greatly increased in the context of widespread apoptosis. As free radicals are closely linked to apoptosis, they may be the main factors that destabilize the cellular environment by tilting the balance towards clones that somehow avoid their proapoptotic effects. Prevention and treatment lowers the odds for cancer appearance and progression, by acting both on malignant and normal clones.
cases therapy only accelerates the selection process that has possibly led to malignancy in the first place. There is no established therapy at the moment that aims at restoring or boosting the normal multiclonal environment. However, some established treatments may do just that. In leukaemias, allogeneic stem cell transplantation repopulates the recipient marrow with multiclonal haematopoiesis. Despite the effects of myeloablative chemotherapy, radiotherapy and the graft-versusleukaemia effect, the leukaemic clone may persist and relapse may or may not occur, possibly in relation to the viability of the donor’s multiclonal haematopoiesis in a foreign environment. Chemotherapy itself may lead to remissions and cures not necessarily by killing all the neoplastic cells but by radically changing the apoptotic ‘rules of the game’, somehow eliminating the suppression of the normal clones (for example the elimination of an autoimmune suppressive mechanism). Reports exist on the benefits of aggressive immunosuppressive therapy in clonal diseases such as myelodysplastic syndromes, and to prolonged complete remissions in acute leukaemia after haematopoietic growth factor treatment in the absence of chemotherapy (46,47). The relatively recent finding that certain types of gastric lymphomas are associated with Helicobacter pylori (HP) infection and that HP eradication can be a cure in these cases (48), may also support this hypothesis, as it is known that HP induces apoptosis in lymphocytes (49). The best cancer therapy of the moment is prevention. Recent evidence points out to the role of diet in preventing cancer. Antioxidants in soy beans, garlic, tomatoes, red grapes play a central role in cancer-preventing diets (50). As alcohol and tobacco are both FR inducers and cancer-risk factors, avoiding them would be synergic with antioxidant diets (51). Few, if any targeted, anti-cancer-clone therapies exist at the moment. Much is expected from gene therapy. However, even if malignant cells are indeed targeted and eliminated, the hypothetical underlying defect of the ‘normal’, multiclonal environment may persist, setting the stage for relapses or other malignancies. In the near future, when all human genes will be mapped, geneticists may find that the real difference between cancer patients and healthy subjects lies not in the cancer clone site on the genetic map, but elsewhere, within the multiclonal, non-malignant environment regions.
17.
CONCLUSIONS
18.
Living organisms can be viewed as diverse and apparently chaotic cell systems in which cells and clones interact according to Darwinian rules. The more clones there are, the smaller the chance for any given clone to gain growth advantage. A process that affects normal clones
19.
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