Europ. J. Cancer Vol. 3, pp. 481-490. Pergamon Press 1968. Printed in Great Britain

Lysosomes in Relation to Cancer Induction and Treatment*

ANTHONY ALLISON National Institute for Medical Research, Mill Hill, London

THE general theme of this meeting is the biochemistry of cancer, and my allotted task is to discuss lysosomes. All the cancer cells so far studied have been found to contain lyso- somes, but remarkably little is known about their properties and in what respects, if any, they differ from the lysosomes of normal cells. On the other hand, there has been interest in the possibility that lysosomes are involved in oncogenesis, and I shall summarize evidence bearing on this problem. Moreover, marked changes have been reported in the lysosomes of tumour cells subjected to certain therapeutic procedures, and these will also be described.

Classes of carcinogenic agents Agents known to induce cancer fall into

three main classes--physical, chemical and viral. The important physical agents are ultraviolet and ionizing radiations. Chemical carcinogens are numerous and structurally diverse, including polybenzenoid hydrocarbons, heterocyclic nitrogen compounds, aromatic amines, steroids, stilbenes, metals, asbestos, silica and alkylating agents. The oncogenic viruses also vary widely in properties: some multiply in the nucleus, others in the cytoplasm; some contain DNA, others RNA; some contain lipid and are assembled in relation to host cell membranes, while others do not contain lipid and appear to be assembled independently of host cell membranes. Many different cell types can undergo malignant changes when exposed to the right inducing agent.

*Presented at the Second International Symposium on the Biological Characterization of Human Tumors, Rome, April 24-26, 1967.

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Theories of carcinogenesis A central problem in cancer research is to

find a common mechanism in ceils that can be set in motion by such a bewildering variety of oncogenic agents. Broadly, two approaches can be taken. One is to regard viruses as the proximate carcinogenic agents in all cases. Radiations or chemicals are then supposed to activate viruses latent in the tissues and facilitate oncogenesis by the viruses. It seems that the only way in which this hypothesis can be tested is by comparing the effects of chemicals or radiations in animals with and without viruses. However, mice reared under "germ-free" conditions still have viruses [1]. Moreover, although the presence of a virus can be recognized, it is never formally possible to prove the absence of viruses. Hence there is at present no way of critically testing this hypothesis, and only hypotheses that can be tested are useful.

The second approach is to suppose that the same reaction in cells can be initiated indepen- dently by different classes of carcinogenic agents, although two or more can act syner- gically to bring about the common reaction. There may of course be several mechanisms, but it is worth attempting to explain the observations on the basis of one mechanism. In considering what biochemical events might be involved, it is necessary to recall that cancer cells originate by somatic mutation, in the sense that progeny cells inherit the abnormal growth potential and other properties of malignant cells. It is therefore likely that some change in DNA occurs. Pitot and Heidelberger [2] have argued that protein deletions might be perpetuated, but their argument, based on

482 Anthony Allison

analogy with control systems which are im- perfectly understood even in bacteria, is unconvincing in the present state of knowledge.

Mutations are usually produced in either of two ways: point mutations involve substitutions of one or a few base pairs in DNA, and often lead to the formation of a single abnormal protein; [3] whereas chromosomal mutations involve chromosome breakage and re-union, with deletion, inversion or duplication of pieces of chromosomes much larger than are affected by point mutations, although they may be small and difficult to detect at the level of ordinary chromosome cytology. There are reasons for believing that changes of the latter type are more relevant to carcinogenesis than the former.

Chromosomes and malignancy It has been known since the pioneer work of

Boveri in 1914 and of Winge in 1930 that the chromosome constitution of malignant cells is often abnormal. Heteroploidy and other chro- mosomal changes help to explain the properties of fully established tumour cell strains, such as lack of differentiation and invasiveness, but they are secondary and cannot be invoked to explain the original transformation to malig- nancy. However, other evidence suggests that chromosome anomalies precede and lead to malignant changes. Thus, the Ph 1 chromosome, with a partial deletion of chromosome 21, seems to be present before chronic myeloid leukemia develops [4], and may have been produced in some cases by ionizing radiation [5]. Children with Bloom's syndrome and Faconi's anaemia have a marked predisposition to develop leukemia or squamous epithelial tumours [see review by de Grouchy (6)]. When the blood cells or epithelial cells of children with these syndromes are cultured in vitro, numerous chromosome breaks and other aberrations are seen. Radiations, chemicals and viruses causing malignancies are known to increase also the rate of production of chromosome rearrange- ments. I f chromosome anomalies are considered an epiphenomenon to malignancy, it must be postulated that all the above-mentioned con- ditions and treatments independently produce malignant processes and chromosome aber- rations. No definite argument can dispose of this concept, but it is simpler to postulate that chromosomal rearrangements come first and are themselves responsible for the malignant process.

Much attention has been given in recent years to direct interactions, e.g. of alkylating agents or hydrocarbon carcinogens with DNA

[7] and integration of oncogenic virus genomes in DNA, by analogy with lysogenic prophages. However, the fact that agents can react with DNA in vitro does not mean that such reactions occur in living cells. Intercalation of carcinogens between DNA base-pairs would be expected to produce point mutations, rather than chromo- somal effects, and, if this mechanism were important, agents such as proflavine (which certainly intercalate) would be expected to be carcinogenic, which they are not. The corre- lation between carcinogenicity and the capacity of compounds to produce mutations of classical type is poor (see Pitot and Hedelberger [2]). And the variety of oncogenic viruses makes it unlikely that specific interaction with or inter- gration into host-cell DNA is essential for malignant transformation.

The alternative view is that effects on genetic material are indirect and secondary to some other reaction that can be set in motion in many different types of cell by many different stimuli. In looking for such a mechanism our attention has been concentrated on lysosomes, for theoretical reasons and because of a number of experimental observations. The theoretical reasoning is as follows. The most likely indirect mechanism for producing a deletion of genetic material in somatic cells is release of an enzyme capable of attacking DNA. All cells so far investigated (except erythrocytes) have substantial amounts of DNAase in 1ysosomes. Although this enzyme has an acid pH optimum, it functions efficiently at physiological pH provided that the ionic strength is not too high. Lysomal DNAase is capable of breaking both strands of a double helix of DNA with a single hit, and 3'-phosphate is released; [8] breaks produced in this way are much less likely to be repaired by the efficient repair mechanisms present in most cells than breaks of single strands releasing 5'-phosphate, such as are produced by alkaline DNAases. Indeed in most cells the alkaline enzyme is present in rather small amounts and there is a natural inhibitor; several of the enzymes formerly thought to be of this type are phosphodiesterases rather than endonucleases proper.

We have observed that isolated interphase chromosomes (polytene chromsomes of Chiro- nomus) are readily broken by lysosomal DNAase. When selective damage to lysosomes was produced in human diploid cells, numerous chromosome breaks and other aberrations were observed [9]. We have concluded that DNAase entering the nucleus is responsible for these breaks, but do not yet have direct proof that this is so. However, DNAase rapidly enters

Lysosomes in Relation to Cancer Induction and Treatment 483

solated nuclei and inhibits DNA-dependent RNA synthesis. [10] Our recent experiments have shown that addition of highly purified lysosomal DNAase to cultured cells under conditions that allow it to enter the ceils without being degraded is associated with chromosome breakage, which increases the plausibility of the interpretation.

The hypothesis, then, is simple enough. If lysosomal enzymes can be released and gain access to genetic material without at the same time impairing the capacity of cells to divide, they might produce genetic changes (probably deletions) which result in the particular variety of somatic mutations leading to cancerous growth. It can be objected that this mechanism would be wholly nonspecific. However, speci- ficity could result from the fact that only certain parts of the genetic material would be exposed to enzymic attack, and then only at certain stages in the cycle of cell division, e.g. DNA when it is being replicated. Testing the hypothesis proceeds in several steps. The first requirement is that lysosomes be affected by carcinogenic agents of various types; the second prediction is that if selective release of lysosomal enzymes can be achieved, this procedure will under appropriate conditions lead to malignant transformation.

Chemical agents We soon obtained evidence that lysosomes

could be affected by chemical carcinogens. When we followed the uptake of polybenzenoid hydrocarbons and tricycloquinazoline in living cells by fluorescence microscopy, we found that they were concentrated in lysosomes [11]. No nuclear fluorescence was seen. Experiments of this sort on living cells are valuable because during the course of killing and homogenizing cells marked redistribution of carcinogen could take place. Uptake of hydrocarbons in cyto- plasmic particles was described by Graffi [12], who thought that mitochondria were involved, but the disposition of fluorescent organelles in several different cell types corresponds exactly with that of lysosomes, as shown by acid phosphatase staining. It is of interest that, using the fluorescent antibody technique, Tanigaki et al. [13] have recently found 2-acetyl-aminofluorene in the cytoplasm of liver cells; no nuclear fluorescence was detected. In dead cells nonspecific cytoplasmic uptake of hydrocarbons into phospholipid-rich regions, and also nuclear fluorescence, are seen.

Noncarcinogenic hydrocarbons are also taken up into lysosomes, so if this process is relevant to carcinogenesis there must be a difference in

the metabolism and later effects of active and inactive compounds. Information about this is still scanty, but we have been able to inves- tigate one example in some detail. For many years it has been known that hydrocarbon carcinogens have cytotoxic as well as oncogenic effects. The most remarkable instance of their toxity in vivo is the necrosis of rat adrenal cortical cells following administration of 7,12- dimethylbenz(a)anthracene--DMBA [14]. It was shown by Boyland et al. [15] that the adrenal damage is produced by a metabolite of the carcinogen, namely 7-hydroxymethyl- 12-hy- methylbenz (a) anthracene (7-OH- 12-MBA) ; but an isomeric metabolite, 12-hydroxymethyl- 7 - methylbenz (a) anthracene (12- OH-7 - MBA), does not produce adrenal necrosis. We found that in vitro incubation with 7-OH-12-MBA caused a rapid release of enzymes from adrenal lysosomes, but not from kidney or liver lysosomes; 12-OH-7-MBA had no detectable effect on adrenal lysosomes [16]. Thus both the compound specificity and the organ specificity are the same for in vivo necrosis and in vitro release of lysosomal enzymes, and it seems probable that massive release of lysosomal enzymes accounts for adrenal necrosis in vivo.

A marked change in the livers of rats after administration of dimethylaminoazobenzene is accompanied by an increase in lysosomal enzymes in the supernatant fraction [17, 18]. Carbon tetrachloride in doses comparable to those found in vivo releases enzymes from isolated lysosomes [19]. The only steroids that are effective carcinogens are female sex hor- mones; low concentrations of these hormones and their stilbene analogue, diethylstilboestrol, increases permeability of lysosomal membranes much more than do other steroids, many of which have the opposite effect [20].

Particulate carcinogens and metals Particularly interesting with respect to lyso-

somes are particulate carcinogens such as asbestos, silica and metal powders. Exposure of human subjects or experimental animals to asbestos gives rise to tumours, especially to distinctive mesotheliomas of the pleura or peritoneium. The three main types of asbestos (crocidolite, amosite and chrysotile) are all carcinogenic, and the fibres are fully carcino- genic after extraction with organic solvents, so the effect is not due to the small amounts of hydrocarbons associated with the fibres (see Wagner [21]). The effects of different types of asbestos on cells are quite complex, but there is no doubt that the initial uptake is lysosomal. A detailed comparison has also been made of

484 Anthony Allison

the effects of toxic particles (such as silica) and non-toxic particles (such as diamond dust) on cultured cells [22]. Both types of particles were taken up into lysosomes, where the non-toxic particles remained for several days, whereas the silica particles and lysosomal enzymes rapidly escaped into the surrounding cytoplasm, pro- ducing severe damage to the cells. It was concluded that the capacity of silica particles to react with and make permeable lysosomal membranes accounts for their toxicity. There is a close parallelism between the capacity of different particles, including different crystal- line forms of silica, to react with red cell membranes and their fibrogenicity in vivo [23]. We have given reasons for believing that these interactions are due to hydrogen bonding of silicic acid hydroxyl groups with membrane phospholipids [24]. Polyvinylpyridine N-oxide, which like other polymers is taken up into lysosomes, but also readily forms hydrogen bonds with silicic acid, protects against silica toxicity and fibrogenicity.

Thus there is little doubt that the initial effect of silica on cells is lysosomal damage. It is therefore of interest that rats receiving intrathoracic injections of silica developed tumours of the thymus (21), chiefly reticulum- cell sarcomas. These resemble the tumours seen in rats injected with trypan blue [25], which itself becomes localized in lysosomes. The same is true of iron dextran [26], which pro- duces malignant tumours at the site of injection [27], and also carageenan [28, 22]. Powdered nickel oxide or sulphide [29], cobalt [30] and cadmium [31] all produce tumours at the sites of injection. Particles of this type are taken up into lysosomes, as are a number of soluble metal salts [32], some of which are carcinogenic [lead, cadmium].

Co-carcinogenic compounds Co-carcinegens are materials which them-

selves have little oncogenic capacity, but which greatly magnify the oncogenic effect of small doses of hydrocarbons. The best characterised co-carcinogens are croton oil, or its highly purified derivative, phorbol A, non-ionic deter- gents of the Tween and Span group [33] and hyperoxia [34]. Non-ionic detergents of various classes are concentrated in lysosomes and tend to increase the permeability of their membranes [11]. There is good evidence that lysosomes are particularly liable to damage when cells are exposed to high oxygen concentrations [35, 36]. Dilutions of croton oil make lysosomes of cultured cells permeable [11]. G. Weissmann (private communication) has recently found

that phorbol A is highly efficient in releasing enzymes from lysosomes in vitro. Effects on erythrocyte membranes and those of other cytoplasmic organelles were much less than those on lysosomes. There is a close parallelism between effectiveness of various croton oil de- rivatives as co-carcinogens and in unstabilizing lysosomal membranes. All these co-carcino- genic agents would therefore be expected to potentiate the effects of low concentrations of hydrocarbons or their derivatives on lysosomal membranes.

Radiation Several authors have reported effects of

radiation on the activities and intracellular distributions of different lysosomal enzymes in vivo [37-39] see Brandes [40]). Histochemically detectable changes in organelles containing acid phosphatase have been described in several organs after X-radiation; these are detectable in the neighbourhood 1 to 1-5 k rad- - tha t is, not greatly above the lethal dose for the animals in question [40-43]. Release of enzymes from isolated liver lysosomes by ultraviolet or ionizing radiation in vitro has been reported by several workers [44-46]. Again, doses of the order of 1 krad produced detectable release of enzymes.

Since my colleagues and I had found that several of the compounds known to induce photosensitization in human skin (e.g. porphy- rins or anthracene) are concentrated in lyso- somes, we examined the role of lysosomes in photosensitization in vivo and in vitro [47]. This has provided a method for selectively releasing lysosomal enzymes. I f neutral red or some other substance is added to living cells, it is taken up by lysosomes. I f the cells are then illuminated by light of the wavelength absorbed by the photosensitizing substance, photo- oxidative damage to lysosomal membranes is produced. Light of these relatively long wavelengths is not injurious to cells except in the presence of the photosensitizer. The extreme sensitivity of lysosomes to photo- sensitization damage has also been noted by Slater and Riley [48].

ViFUSSS Effects on lysosomes of virus infection have

been reviewed elsewhere [49]. There is now good evidence that on first being taken up into host cells by endocytosis, viruses have their protein coats partially digested by lysosomal enzymes before their nucleic acid can replicate. Later in the replication cycle lysosomes can be seriously damaged, which contributes to cyto-

Lysosomes in Relation to Cancer

pathic effects and leads to classical white-plaque formation (failure of uptake of neutral red into the lysosomes of damaged cells). With lesser changes in the permeability of lysosomes there is red-plaque formation where viruses have multiplied (increased uptake of neutral red into lysosomes). With cell-virus interactions leading to malignant transformation, the latter is usual.

Three other phenomena have attracted much attention in recent years. One is the presence of virus-specific antigens in tumour cells, even when no infectious virus can be recovered. This implies persistence of at least part of the virus genome, a finding which can be accom- modated into the lysosome hypothesis in at least two ways [49]. Chromosome breakage secondary to lysosomal enzyme realease could facilitate incorporation of viral genome by a process of repair: mechanisms of this type are known to be involved in the integration of transforming DNA in bacteria. Alternatively, persistent release of a viral product that becomes associated with membranes could give lysosomal instability over many generations.

The second phenomenon is increased syn- thesis of DNA in host ceils infected with tumour viruses. There is some doubt whether this is an effect specific for tumour viruses. In any case the increased DNA synthesis is rather like that observed in transformation of lymphocytes or in ova which are activated by fertilization or agents that induce parthenogenetic cleavage. There are strong indications that lysosomes are involved in both these phenomena, as suggested by Allison and Mallucci [50] and Hirschhorn and Hirschhorn [51]. Ribosomes from unfer- tilized sea urchin eggs do not synthesize protein until they are treated with a protease [52]. Presumably this protease, which in eggs would be of lysosomal type, removes a protein or peptide inhibitor from ribosomes that already contain messenger RNA. Through such mechanisms lytic enzymes can, para- doxically, activate synthesis; probably de- repression of RNA and DNA synthesis follows.

The third effect of oncogenic viruses on cells that is now well established is that chromo- some aberrations are regularly observed (see Allison [49]. There has been some doubt about the significance of this finding, since chromo- some abnormalities have also been observed in cells infected with non-oncogenic viruses such as herpes simplex or measles. The latter is interesting because the virus can produce severe damage to chromosomes even though it multipliesin the cytoplasm. Hence some cyto- plasmic event (possibly sosomal enzyme acti- vation) leads to chromosome aberrations. All

Induction and Treatment 485

the non-oncogenic viruses producing chromo- somal abnormalities also have cytopathic effects, and this may help to explain why they do not lead to tumour formation. For cells that have undergone the appropriate somatic mutations to produce tumours, the cells must obviously be able to survive and multiply: any virus or other agent that is too damaging cannot be oncogenic. However, the balance between cell damage and oncogenesis must be delicate in many cases, e.g. with polyoma virus in the mouse or physical or chemical carci- nogens which damage numerous ceils at the same time that they induce malignant changes in a few cells.

Observations that cell transformation by viruses is potentiated by radiation (e.g. Stoker [53]) suggests that the two may work by the same mechanism. Recently it has been reported that cells from patients with Fanconi's anaemia - -which develop numerous chromosome aber- rations spontaneously--are more readily trans- formed by simian virus 40 than ceils of control human subjects [54]. This result emphasizes the importance of chromosome changes in the early stages of virus oncogenesis.

Effects of lysosomal enzyme release From the observations that have been

summarized there is enough evidence to make lysosomal enzyme activation at least a possible candidate for a common mechanism in cells set in motion by diverse carcinogenic agents. The second stage of testing the hypothesis has therefore been initiated, examining the effects of lysosomal enzyme release on cells. Selective damage to lysosomes was produced by the photosensitization technique mentioned above in a variety of cell types. These experiments were carried out in collaboration with Dr. W. Russell, Dr. P. Black and Dr. K. Sanford.

The first experiments were carried out with BHK21]C13 hamster cells. After lysosomal damage, more of these cells were able to grow as colonies in agar suspension than with control cells. Some of the colonies so obtained showed marked alterations in morphology, with piled up growth of cells of epithelial type. Other colonies showed parallel growth of cells much like that seen in colonies of untrans- formed cells. However, two clones of both cell types after photosensitization gave tumours in most adult hamsters inoculated subcutaneously with 104 cells. The untransformed BHK21]C13 cells showed tumours after inoculation of 10 v cells. Similar results were obtained in two further experiments of this type. The findings are a little difficult to interpret, in view of the

486 Anthony Allison

production of tumours in controls, but it seems clear that there is an increased malignant potential after lysosomal damage, although this is not always associated with morphological changes in cell colonies of the type often thought characteristic of malignant transformation. However, Sanford [55] in a careful study of many different long-term cell cultures, has also found a very imperfect correlation between morphology and the ability of the cells to grow in vivo. This was our experience also with one of Sanford's rat embryo cell strains. After photosensitization damage to lysosomes, col- onies of cells with the piled-up random growth pattern were obtained, but these failed to produce tumours on inoculation into the anterior chamber of the eye.

With Dr. P. Black weanling hamster kidney cells have been subjected to photosensitization. In extensive experience over several years using these cells for transformation with simian virus 40, Dr. Black has never seen transformation in controls. In one experiment after photo- sensitization a line of transformed cells was obtained with which tumours were produced in adult hamsters. I have obtained two transformed cell lines from hamster embryo cell subjected to photosensitization; these produced tumours in adult hamsters, which were not seen in control experiments. Other studies with rat embryo cells are in progress.

These investigations will be reported fully elsewhere. They suggest quite strongly that lysosomal damage can produce malignant transformation in cells, or facilitate trans- formation occurring spontaneously. The rates of transformation are lower than those obtained with oncogenic viruses in some of the systems but this is perhaps to be expected. The photosensitization technique produces its effects at one moment in time, whereas that of viruses continues and has a greater chance of affecting cells at a vulnerable stage in the mitotic cycle.

Also relevant are experiments of Buzzatti- Traverso and Visconti di Modrone [56], who found after t reatment of Drosophila eggs with neutral red and light somatic mutations with mosaicism. Changes of this type are associated with chromosomal abnormalities, and the mechanism of their production may be the same as that we postulate. The production of cancer is, after all, somatic mutation with mosaicism--the malignant cells having a dif- ferent genetic constitution from the rest.

Malignancy in the Chediak-Higashi syndrome This is a recessively inherited disease charac-

terized by the presence of large lysosomes and melanosomes in several different cell types. I f children with the syndrome do not die of infection early in life, they often develop progressive hyperplasia of the lymphoreticular system which has been defined as malignant [57]. Typically they die in childhood with hepatosplenomegoly and lymphadenopathy, showing at post mortem lymphoid infiltration of most organs.

Thus children with congenitally abnormal lysosomes show a greatly increased suscepti- bility to malignancy. Whether this is associated with persistent infection by a particular micro- organism, or is due to spontaneous appearance of chromosome aberrations (as in Bloom's syndrome or Fanconi's anaemia), or is due to some other mechanism, is at present unknown.

Lysosomes and therapy of cancer cells There is much evidence that lysosomes play

an important role in physiological and patho- logical regressive changes in tissues, e.g. those associated with re-modelling during growth, or following ischaemic injury (see de Duve and Wattiaux [58]). The marked morphological changes in the lysosomes of normal cells following radiation have been mentioned above. Brandes and his colleagues [59, 40] have recently studied lysosomes in mouse mammary gland carcinoma cells submitted to treatment with the alkylating agent Cytoxan or X-radiation. Following both these treat- ments the concentration and size of lysosomes in treated tissues increased, and the reaction products of the histochemical test for acid phosphatase were detected in the cytoplasm and in intercellular spaces. It was concluded that the Cytoxan and ionizing radiation provoked the release and activation of lyso- somal hydrolases and this preceded degradative changes in the tumour cells. These changes were potentiated by concurrent treatment with vitamin A, which labilizes lysosomes.

These observations are of interest in relation to radiation effects and to the possibility that they might be augmented by other more stable agents selectively concentrated in lyso- somes. There is evidence that lysosomes may be involved in various chemotherapeutic effects [60] and this field lies open to investigation.

488 Anthony Allison

lysosomaler Enzyme zumindest einen der mO'glichen Mechanismen bei der Entstehung biisartiger Neoplasien darstellen. 3. Deutliche Veriinderungen der Lysosome, einschliesslich das Freiwerden ihrer Enzyme, wurden in KrebszeUen nach Behandlung mit ionisierenden Strahlen und gewissen Medika- menten beschrieben. Die Rolle der Lysosomen in diesen chemotherapeutischen Wirkungen bedarf weiterer Abklgrung.

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