The Cytogenetics of Human Tumors

The Cytogenetics of Human Tumors‘**]

BY K. D. ZANG AND H. SINGER[*]

Chromosome changes can be observed in many tumors, suggesting that a connection exists between chromosome aberrations and tumor formation. A general introduction to the techniques and objectives ofresearch in cytogenetics is folIowed by a discussion of the modern theories of the etiology of tumors, using human meningioma as an example. Meningiomas are relatively benign, slowly growing tumors of the meninges.

1. Introduction

Depending on the techniques involved and the aims of the particular research project, the field of work of the cytogeneticist lies between that of the chemist and molecular biologist on the one side, and that of the pathologist and anatomist on the other. The cytogeneticist also works with morphological structures, there- by obtaining information not accessible by purely histomorphological techniques about the fine structure of genetic material. This information, however, does not provide any evidence of the function of these structures. The aim of the geneticist to discover cor- relations between functions and structures can be realized only through a comparison of the cytogenetic findings with morphological, physiological, and chemical evidence. In this article we shall give a brief outline of the techniques and aims of the cytogeneticist, using as an example the chromosome pattern observed in human tumors. Special attention is paid to meningiomas, as the chromosome changes in these tumors are clear and distinctive, and are thus particularly suitable for purposes of demonstration.

2. The Preparation of Human Chromosomes

The chromosomes, carriers of the genetic information of the cell, exist in the non-dividing nucleus in an extremely decondensed form whose finer structure cannot be revealed by light microscopy. In the course of cell division in mammals they contract to form 1-10 pm long structures arranged in a three-dimensional system. This produces considerable overlapping in cell preparations (see Figure 1) and until a few years ago it was impossible to identify and count the individual chromosomes. Until 1956 the chromosome number in man was assumed to be “about 48” [I].

[*] Dr. K. D. Zang and Dr. H. Singer Max-Planck-Institut fur Psychiatrie, Neuropathologische Abteilung

Fig. 1 . Division of a brain tumor cell (meningioma) of man. Hematox- ylin-Eosin staining, magnification 1200 X .

8 Miinchen 23, Kraepelinstrasse (Germany) [**I The investigations reported in this paper were carried out with the support of the Deutsche Forschungsgerneinschaft. [l] For references see G . Heberer: Handbuch der Humangenetik. Thieme-Verlag, Stuttgart 1968, Vol. Ijl, pp. 145ff. 121 P . C. Nowell, Cancer Res. 20, 462 (1960).

The present form of routine chromosome analysis was made possible by three fundamental findings. In 1960, Nowell discovered that lymphocytes in the circu- lating blood, which do not normally divide, undergo

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transformation into lymphoblasts and divide after the addition of an extract from Phaseolus vulgaris. (This finding is of no importance in the investigation of tumor cells, since such cells undergo spontaneous division.) An important technical improvement was the pretreatment of the cells with a hypotonic solution. This produces a swelling of the cells, resulting in improved separation of the chromosomes [31. The third fundamental advance is based on the treatment of cells with colchicine derivatives. It has permitted the number of human chromosomes to be definitely established as 46 14.53.

Colchicine has several effects: one is that the spindle appara- tus (see Section 4), to which the chromosomes are fixed, is destroyed, so that the chromosomes have a better chance to spread out. Furthermore, the chromosomes contract more, acquire clearer outlines, and can be more easily identified under the microscope. The cytostatic effect of colchicine is also very advantageous: on blocking of the cell division, more mitotic cells accumulate and are available for analysis. (For further particulars of the preparation of chromosomes see 16-81.)

The modern cytogeneticist works with cell suspensions or monolayer tissue cultures, in contrast to the section preparations formerly used. Air drying of the cells after hypotonic treatment further improves the spreading out of the chromosomes. This is followed by fixation in the usual way (e.g. with acetic acid/alcohol or for- malin) and staining with a stain as specific as possible for chromatin structures (e.g. Feulgen or Orcein). In cells prepared in this way the chromosomes lie mainly in one optical plane, so that photography and further

Fig. 2. Normal human metaphase chromosomes. Orcein staining, magnification 1300 x .

[3] T. C. Hsu, J. Heredity 43, 167 (1952). [41 J. nio and H. Levan, Hereditas 42, 1 (1956). [5] C. E. Ford and J. L. Humerton, Nature (London) 178, 1020 (1956). [6] J. J . Yunis: Human Chromosome Methodology. Academic Press, New York 1965. [7] R. Turpin and J. Lejeune: Les Chromosomes Humains. Gauthier-Villus, Paris 1965. (81 R. A. Pfeiffer: Karyotyp und Phanotyp der autosomden Chromosomenaberrationen des Menschen. G. Fischer-Verlag, Stuttgart 1968.

analysis are substantially facilitated (Figure 2). Most of the methods of preparation employed are modifi- cations of the method developed by Moorhead e f al. I9J.

3. The Preparation and Culture of Tumor Tissues

Preparation of cells is preceded by treatment of the tissue in a manner that varies according to the respective tissue and to the aim of the investigation. Three methods of preparing tissues are used in the cytogenetic study of human tumors: 1) direct preparation; 2) short-time culture; 3) long-time culture of biopsy material. The direct preparation method is the most reliable for ensuring that the chromosome sets are representative of the tumor sampled. This procedure is particularly suitable for malignant tumors which at any moment contain a relatively high percentage of dividing cells. In the sudden change from body temperature to am- bient temperature the cell metabolism and the contin- uation of initiated cell divisions are slowed down considerably; in addition, no more cells start the process of division. For analysis the tissue is com- minuted mechanically and is then subjected to enzyme treatment (e.g. with trypsin) to achieve maximum pos- sibleconversion of the tissue into a cell suspension. The chromosomes are prepared as described in Section 2.

However, the number of dividing cells is often insuffi- cient for evaluation. In such cases the cell suspension is incubated for some hours in a culture medium, consisting of an isotonic salt solution enriched with supplements whose necessity for the growth of the tissue in question is determined empirically. Besides amino acids, vitamins, trace elements, etc., the addition of 10-30% of homologous or heterologous serum is recommended in many cases. By addition of a small amount of Colcemid (deacetylmethylcolchicine, 0.1 to 1.Opg per ml of medium, for 2-16 hours) the cells beginning to divide during this period are collected. The chromosomes are prepared in the same way as described above (Section 2). Prolonged culture of the tumor biopsy material presents both advantages and disadvantages in chromosome analysis. In this method an attempt is made to culture the tumor in vitro for a longer period of time. Either small pieces of the tumor are placed in a glass vessel in a plasma clot (a coagulated mass of plasma and homologous embryo extract, generally chicken plasma and chick embryo extract), which is covered with a layer of nutrient medium, or a cell suspension is allow- ed to establish itself on the bottom of the vessel. In the first case the tumor grows out radially around the original particle, which is both anchored by the clot and nourished by it, and in the second case a uniform sheet of cells is produced. In both techniques the nutrient medium is renewed regularly once or twice a week, and is maintained at a uniform physiological

191 P. S. Moorhead, P . C. Nowell, W . J . Mellman, D . M . Battips, and D. A . Hungerford, Exper. Cell Res. 20, 613 (1960).

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f

Fig. 3. Schematic representation of a normal cell division cycle. Top: mitosis. Bottom: interphase. For details see text.

pH. When the cells have multiplied sufficiently, they are freed from the bottom of the flask by enzyme treatment. One part is used for chromosome analysis, and the other can be cultured again (subculture). The advantage of this method is that chromosome analysis can be repeated as often as desired; the disad- vantage is that on more prolonged culture a selection or evolution of certain cell types can occur, so that the chromosome sets found do not necessarily correspond to those of the original tumor. As the chromosomal aberrations of human tumors will be demonstrated in the following section on brain tumors, some particulars of their culture may be mentioned. We used particle cultures (in accordance with the third method), in which the chromosomes of the primary cultures could be analyzed even after a few days. Culture of the tumor particles was performed on cover slips, which were placed in test-tubes filled with the culture medium. The tubes were slowly rotat- ed to ensure oxygen saturation for the tissue [lo]. The chromosomes were prepared directly on the cover slips, without preliminary removal of the cells. In this way the cell sheet was preserved, and almost all the cell division figures present were available for study [I*].

4. Cell Division and Possible Abnormalities

Under normal conditions the body cells always repro- duce in the same way. Figure 3 gives a schematic representation of the cell cycle. In the prophase the ____ [lo] G. Kersring: Die Gewebezuchtung menschlicher Hirnge- schwiilste. Springer, Berlin 1961. [ I l l J. Lejeune, R. Turpin, and M. Goutier, Rev. franc. Etudes clin. biol. 5, 406 (1960).

thread-like chromatin structures condense to form “chromosomes” with two genetically identical chromatids. During the next stage, the metaphase, the chromosomes contract further and arrange themselves in the “equatorial plate”, a disk-shaped structure in the central plane of the cell. The nuclear membrane has disappeared. Each chromosome is stretched between two spindle fibers which end in the centrioles. In the anaphase the two chromatids of each chromosome separate at the centromere, the point of spindle- fiber attachment, and migrate by the progressive shortening of the spindle fibers t o their respective poles. In the telophase a new nuclear membrane is formed around the halved chromosome sets, and the chromosomes again disappear by despiralization. The following GI phase (from “growth”) is a period of high metabolic activity without observable changes in the cell morphology. Most of the body cells are found in this phase. It can last for days, even months and years, as long as the cell is not preparing to divide. In the latter case the S phase (from “synthesis”) begins, during which the nucleus doubles its DNA content (e.g. the DNA content of each chromatid) as can be measured cytophotometrically. During this phase the basic structure of the chromosome, a DNA strand wound in a double helix, is doubled. This synthesis phase lasts on average 6-8 hours. The following, G2 phase is a short period of high metabolic activity, generally lasting less than an hour, during which the nucleus prepares for its subsequent division. In the following prophase each chromosome again consists of two chromatids instead of one. Mitotic division occurs in this way in all body cells; on average it takes about an hour. In tumor cells the GI phase is greatly shortened, and in certain circum-

Aiigew. Cliem. intermit. Edit. 1 Vol. 7 (1968) J No. 9 71 1

stances amounts to but a few hours. In these cells reproduction has apparently become an end in itself, the interphase being limited to the minimum time neces- sary to provide the structures and energy required for further division. Defects in division can also occur in normal mitosis and lead to numerical and structural changes in the nucleus, but the number of such pathologically altered cells remains relatively small. In tumor cells, on the other hand, they occur in especially high numbers (see Section 6) .

Fig. 5. Tetraploid chromosome set after endoreplication (see text). The homologous chromosomes are arranged in pairs.

densedform and begin a new cellcyclewithout preceding division (endomitosis). Here too doubled chromosome sets are found in the next cell division but the paired arrangement of the chromosomes is generally missing (Figure 6) . Besides these numerical chromosome aberrations, structural anomalies also play an essential role

( b )

Fig. 4. rations.

Left row: arrangement of the chromosomes in the metaphase plate. Right row: separation in the anaphase. disjunction, (c) anaphase lag.

Mechanism of the most common types of chromosome aber-

(a) normal division, (b) non-

Fig. 4 shows the mechanism of numerical chromosome aberrations. As a result of the non-separation of a centromere, both chromatids can migrate to one daughter cell, so that the chromosome concerned is doubled in one daughter cell and absent in the other (non-disjunction). The second possibility is the absence of the spindle fiber on one side of a chromosome, so that one chromatid does not move to its cor- responding pole. It can either move to the other side or remain where it is and finish in either one of the daughter cells according to chance (lag). As a result of the absence of the spindle fiber attachment the affected chromosome is quickly lost.

Another common aberration of the chromosome set is its multiplication (polyploidy). This is one of the fundamental defects of cell division in tumors. Here the doubling process of the chromosomes takes place several times in succession, without any mitosis occurring in the meantime (endo-reduplication). The process is first recognizable in the next cell division, in which the chromosomes mostly appear arranged in double pairs (Figure 5) .

Another possibility is that the chromosomes begin to contract as if in prophase, but owing to the absence of spindle fiber formation they soon again lose the con-

1t b5L S_

Fig. 6 Tetraploid chromosome set after endomitosis, a special form of endoreplication (see text) The chromosomes are arranged randomly.

in tumors. Figure 7 shows the commonest structural chromosome aberrations and their formation (for details see legend). The possible origins of these structural aberrations are discussed in Section 6.

5. The Morphology of Human Chromosomes

In considering human chromosomes prepared under standardized conditions, we shall first mention the various shapes of these chromosomes (see Figure 2).

712 Angew. Chem. internut. Edit. / VoI. 7 (1968) No. 9

Fig. 7. Schematic representation of the most common structural chromosome aberrations. (a) Break. (b) Reciprocal translocation, generally combined with a change in the shape of both partners; a translocation involving fragments of similar size is not recognizable morphologically. (c) Formation of a dicentric chromosome by translocation. The two re- combined fragments ( l /GHJKL) become lost a s they contain n o centromere (-a- or -0-). (d) Central fusion after break of both partners in the immediate neighborhood of the centroinere; the chromosome formed f rom the remainder of the two chromosome arms often becomes lost because of its missing or incomplete centromere. (e) Formation of a ring chromosome after a break a t each end of the chromosome. The two fragments can recombine, but will in any case be lost. (f) Pericentric inversion, i.e. a double break as in (e); the middle piece is set in again, but in a n inverted form (shown white in the figure). (9) Telomeric or tandem fusion. In this one chromosome is broken at the end of the arm and the other near the centromere. During 1-ecornbination the almost complete a r m of the second chromosome becomes attached t o the first. The process takes place in marker chromosomes having one short and one excessively long arm.

Independently of the differences in size, three main types can be distinguished: 1. mediocentric chromosomes (the centromere, the point of spindle fiber attachment, occurs approximately in the middle); 2. submediocentric chromosomes (the centromere lies between the middle and the end); 3. subteIocentric or acrocentric chromosomes (the centromer is in the immediate vicinity of the end of the chromosome). A classification of human chromosomes according to size and shape was suggested in 1960 in the “Denver nomenclature” [12J, and was then revised and improved in London in 1964rl3J and in Chicago in 1966[14J.

I121 Human Chromosome Study Group: Proposed Standard System of Nomenclature of Human Mitotic Chromosomes. Lancet i, 1063 (1960).

Figure 8 gives a schematic representation of the normal human chromosome set. Figure 9 shows the arrangement of a normal female chromosome set ob- tained under standard conditions of preparation (karyogram). The autosome pairs (chromosomes with purely somatic information) are numbered from 1 to 22; the gonosomes (sex chromosomes, which besides containing information for the normal development of sex also carry somatic genes) are not numbered. I n addition to the numbers the letters A-G are frequently employed; this is Piitau’s classification (1960) [L51,

Fig. 8 . Schematic representation of the normal human set of chromosomes, in order of decreasing size and morphological characteristics (Denver classification).

Fig. 9. Normal female chromosome set arranged according to the Denver classification (karyogram).

1131 The London Conference on the Normal Human Karyotype. Cytogenetics 2, 264 (1963). [I41 Chicago Conference: Standardization in Human Cytogene- tics. Birth Defects. Orig. Article Series 11, 2 (1966), issued by the National Foundation -- March of Dimes. 1151 K. Putau, Amer. J. hum. Genetics 12, 250 (1960).

Angew. Chem. itrternat. Edit. Vol. 7 (1968) No. 9 713

which places special emphasis on the chromosome shape. Its use is especially recommended for the chromosome groups C , D, F, and G, in which it is often impossible to determine the order of the chromosome pairs within the group. This identification, as well as the allocation of the respective pairs (whose partners derive from the father’s and the mother’s side), is often greatly facilitated by the use of autoradiography. For this purpose, use is made of the fact that during the S phase the chromosomes follow a certain sequence in their reduplication by labeling them with radio- active thymicline. Figure 10 shows such a labeling pattern of metaphase chromosomes [161.

Fig. I I . Metaphase chromosomes in chronic myeloic leukemia. The arrows show the Phl-chromosome and its homologous partner [l7a].

Fig. 10. Metaphase chromosomes labeled with tritiated thymidine. At the moment of fixationthechromosomesshow different labeling patterns, with, however, a general agreement between homologous partners. Upper figure: Microphotographic representation of the metaphase chromosomes. Lower figure: Demonstration of the uptake of radio- active thymidine by covering the slide with a photoemulsion. The arrows indicate the position of the chromosome pair A,.

6. Chromosome Changes in Human Tumors

The human tumors studied most intensively with regard to their chromosomes are the leukemias, probably because blood or bone marrow are readily accessible without an operation, and because repeated examinations are possible. In one type of leukemia, chronic myeloic leukemia, a definite and reproducible chromosome aberration is demonstrable: the “Phila- delphia” chromosome, a deleted chromosome of the G-groupr171 (see Figure 11).

[I61 W. Schmid, see [6], pp. 91 ff. [17] P. C. Nowell and D. A. Hungerford, J . nat. Cancer Inst. 25, 85 (1960).

_____~

The importance of this finding lies in its regularity and in the fact that the chromosome is found only in pathologically altered cells. From this one can con- clude that the Philadelphia chromosome is not a congenital defect. Such structurally abnormal but typical and recognizable chromosomes are called “marker” chromosomes in cytogenetics. A similar, defined chromosome aberration, in the sense of a marker chromosome, occurs in Walden- stroms macroglobulinemia [1*1. In the mosaic form (hence not in all cells) there appears an additional 47th chromosome, which generally has the size of chromosome No. 2 and the shape of chromosome No. 5. This is also a pathological cell strain, which is demonstrable in the blood, but, just as with leukemia, is not a “tumor” in the strict sense. Such marker chromosomes have also been described in most solid tumors of man, though they are seldom the only pathological finding in the tumor chromosomes. In most cases there are considerable numerical deviations, generally consisting in a moderate to considerable increase in the chromosome numbers between the diploid (46) and tetraploid (92) chromosome sets. Analysis is made more difficult in most cases by the fact that the chromosome changes are not the same in all tumor cells. The number of chromosomes varies from cell to cell so that only a “modal number” can be given which again can differ from case to case. A great number of chromosome breaks and all types of recombinations given in Figure 7 can also occur. As an example, reference will be made to HeLa cells, a per- manent cell line, derived originally from a carcinoma of the cervix and used today throughout the world for genetic and biochemical studies. The chromosome number and the morphology of these cells can vary considerably from laboratory to laboratory. However, the establishment of such a perma- nent cell line, which derives from a tumor and is reproducible in subcultures for as long as required, is an exception. Nor- mally, the capacity of cells for division and growth in vitro is limited. After an average of 50 subcultures (about 1--11/2

years) the reproduction capacity of the cultures usually di- minishes and the cells die. This occurs not only with cultures

[17a] We are grateful to Dr. F. Back, Institut fur Hamatologie GSF, Assoziation Euratom, Miinchen, for this photograph. 1181 C. Bottura, J . Ferrari, and A. A . Veiga, Lancet i, 1170 (1961).

714 Angew. Chem. internat. Edit. Vol. 7 (1968) No. 9

of tumor origin but also with fibroblast cultures from normal human connective tissue. That further cultivation is no longer feasible is indicated in normal and so far euploid cultures (i.e. cultures with a normal diploid set of chromosomes) by in- creasing polyploidization and the beginning of structural chromosome aberrations.

Up to the present the chromosome sets of about 200 malignant human primary tumors have been described [ex. 19-231.The results are unsatisfactory for the above reasons. It is impossible to find a typical combination of chromosomal changes for a particular tumor, e.g. cancer of the stomach. A similar lack of agreement exists between different kinds of tumors. The occasionally expressed presumption that the chromosome El6 increases t o a substantially greater extent in tumor tissue than the other chromosomes should also be treated with reserve. Deletion of the long arm of chromosomes of the C-group are a common finding in tumors and can lead to chromosomes morphologically indistinguishable from chromosome No. 16. As only two chromosomes of this type are normally present, a considerable increase can easily be simulated in this way, without the relative decrease in the numerous chromosomes of the C-group being noticed. A similar finding, i.e. the relative decrease in chromosomes of the G-group, has been discussed by several authors ~ - 2 7 1 on the basis of the material reported in the literature (see Sec- tion 7).

In our studies on brain tumors we looked for chromosome changes in relatively benign and slow growing tumors. In this we paid special attention to meningiomas, tumors that do not originate from the brain but from the meninges, forming a histologically particularly uniform system. As a rule they grow only by displacing the surrounding tissue, without infiltrative and metastasizing growth like malignant tumors.

We have demonstrated the loss of a chromosome of the G-group[ZsJ as the basic change in meningiomas. This can occur as the only finding. Furthermore, this loss is sometimes demonstrable as a mosaic pattern together with cells having a completely normal chromosome complement, and in some tumors only normal cells could be found. (Perhaps in this small group a cell line with a missing G chromosome simply could not be detected.) Some tumors in our series of over 30 cytogenetically investigated meningiomas showed more severe histological abnormalities; in this group, besides the G-chromosome, 1-5 other chromosomes were missing (Figs. 12 and 13). However, each chromosome set was uniform and characteristic for the tumor concerned. No great variations from cell to cell, such as those described for malignant tumors, were found in any of the samples investigated by us.

[191 T. Ishihara, Y. Kikuchi, and A. A . Sandberg, J . nat. Cancer Inst. 30, 1303 (1963).

[201 S. Makino, M. S . Sasaki, and A . Tonomirra, J. nat. Cancer Inst. 32, 741 (1964).

[211 A. A. Sandberg, T. Ishihara, T. Miwa, and T. S. Hauschkn, Cancer Res. 21, 678 (1961).

[221 A. J . Spriggs, Brit. J . Radiol. 37, 210 (1964). [231 H. J. Lubs and J. H. Salmon, J . Neurosurg. 22, 160 (1965). (241 H. van Steenis, Nature (London) 209, 819 (1966). [2Sl J . W. I. M . Simons, Nature (London) 209, 818 (1966). [26] N . S. Kiseleva, Lancet i, 616 (1964). [271 A . Levan, Hereditas 55, 28 (1966). [281 K . D. Zang and H. Singer, Nature (London) 216, 84 (1967).

Fig. 12. the G-group (uniform cell strain in the whole tumor).

Karyogram of a rneningioma cell. Loss of a chromosome of

Fig. 13 . Karyogram of a meningioma cell. Loss of a chromosome of the G-group and three other chromosomes of different groups (uniform cell strain in the whole tumor).

In some tumors, but even here in only a few cells, there was a doubling of the set of chromosomes and small structural changes, such as are shown in Figure 7. These variations are summarized in Figures 14 and 15. However, it should be emphasized once more that the structural aberrations described in the benign meningiomas examined by us are rare and of limited importance compared to the constant numerical aberrations. Two chromosomes of the G-group are also missing in the endoreplicated cell shown in Figure 5 . We assume that the loss of the G-chromosome was the primary event and the doubling of the chromosome set was the secondary event, possibly occurring in vitro. It is rather unlikely that the chromosome aberrations found by us were influenced by the formation of clones during the period of cultivation. It has been possible to show in over a thousand brain tumors cultured at the Neuropathologisches lnstitut of Bonn University and at our Institute, that the medium used by us is especially suitable for the propagation of the primary structure of these tumors, though in a two- dimensional system. Neither an overgrowth of connective tissue cells nor further dedifferentiation of nuclear structures has been observed. Comparison with cultures of the same tumors in a typical nutrient medium for fibroblasts was very informative (McCoy 5 A). The cultures grew more slowly, the morphology of the cell sheets was clearly changed, and chromosome analysis showed almost entirely normal chro-

A q e w . Chem. intertint. Edit. / Vol. 7 (1968) 1 No. 9 715

Fig. 14. Dicentric chromosomes in a meningioma.

Fig. 15. a meningioma.

Ring chromosomes and multiradial recombination figures in

mosome sets [291. This example shows the danger of a selective propagation of the connective tissue of a tumor by using an inappropriate technique, and of subsequent interpretation of the observed normal chromosome sets as evidence that the tumor in question i s benign. On the other hand, we find in the culture of glioblastomas, the true carcinoma of the brain, the same abundance of numerical and structural chromosome changes as described in other malignant tumors 1291.

7. The Relationship between Chromosome Aberrations and Tumor Formation

In his theory of cancer, Boveri in 1914[3ol was the first to propose a relationship between chromosome aberrations and tumor formation. The chromosome aberrations found in human tumors in the last ten years have established his theory. Three possible types of relationship are discussed today: 1. It has been suggested that tumors are caused by chromosome changes, as chromosome changes have been demonstrated in many tumors and in the remainder the morphological changes were of such an order that they could not be detected by light microscopy. 2. The chromosome changes are not the primary cause of tumor formation, but the result of it. They can be present or lacking, and vary in most tumors from sample to sample. 3. Morphological chromosome changes can be one of the possible ways in which tumors arise; conversion of normal into tumor tissue (neoplastic transformation) can, however, also occur in another way, without morphological chromosome changes being observable.

[29] K. D. Zung, unpublished. [30] T. Boveri: Zur Frage der Entwicklung maligner Tumoren. Fischer-Verlag, Jena 1914.

A common feature of these three at first apparently contradictory views is that today it is no longer possible to maintain the argument that tumors occur without changes in the genetic structure of the cells. Conse- quently, chromosome changes in the widest sense must also be present. But these changes need not necessarily be detectable microscopically. It must, however, be left an open question whether the chromosome aberrations found are the (constant) visible expression of these genetic changes, or whether they can be jnter- preted only as an expression of uncontrolled cell multiplication [311. On this point even the appearance of marker chromosomes gives no clear information; for the present it only indicates a unicellular and not a multicellular origin of a particular tumor cell population 1321.

In chronic myeloic leukemia the deletion of the long arm of a G-chromosome occurs, resulting in a loss of about 40% of the chromosomal DNA. In meningiomas a whole chromosome of this group is missing, and in many malignant tumors with a great increase in the number of chromosomes there seems to be a relative decrease in the chromosomes of the G-group. Consequently, it can be postulated that there is a relationship between this stereotype chromosome aberration and the tumor formation. This hypothesis does not reject other possibilities. It cannot be denied that during the development of (malignant) tumors the chromosome set becomes increasingly abnormal, and that there is no specific correlation between this and the malignity. Thus, in the late stages of chronic myeloic leukemia there is found, apart from the Phila- delphia chromosome, an abundance of numerical and structural aberrations. It should rather be supposed that in growing tumors those cells multiply preferentially whose chromosome sets have a genetic constitution promoting their metabolism and reproductive capacity under the respective in vivo or in vitro conditions. It may well be argued that, as a result of several deletions and translocations, cells with morphologically and numerically different chromosome sets are genetically identical. We are merely attempting, on the basis of our findings, to support the hypothesis that the loss of G-chromosome material could trigger off uncontrolled cell multiplication (see Section 7.1.).

7.1. Chromosome Changes and Tumor Formation due to Exogenous Effects

There are essentially three groups of exogenous factors which are known to induce tumors: 1. ionizing radiation; 2. oncogenic viruses; 3. mutagenic chemicals. It is also known that all these factors can cause chromosome aberration. Using human cells, Koprowski et al. [331 established for the first time in 1962 that chromosome aberrations can be caused by the SV40 virus in tissue culture. These aberrations began with the loss

[31] K . Boyreuther, Nature (London) 186, 6 (1960). [32] C. E. Ford, J . C. Hamerton, and R . H . Mole, J. cellular comparat. Physiol. 52, 235 (1958). [33] H. Koprowski, J . A . Poiztdii, F. Jeiuen, R. G . Radvin, P . S . Moorheod, and E . Sokselu, J. cellular comparat. Physiol. 59,281 (1962).

716 Aiigetv. Chem. iiitrrnnt. Edit. / Vol. 7 (1968) / No. 9

of chromosomes of the G-group. Considerable variations in the chromosome number and all the described structural changes, as observed in the chromosome sets of malignant tumors, arose on further culture. These findings have been confirmed repeatedly, and have been found in the meantime also in tissue cultures infected with Rous sarcoma virus, polyoma virus, and adenovirus type 12. All these viruses can cause tumors in vivo in animals. On the other hand, however, non- oncogenic viruses, e.g. the measles virus, also have the capacity to cause chromosome breaks ‘341.

- @i&ifi:

Fig 16 Chromatid breaks and recombination figures In the chromosomes of a peripheral lymphocyte after cyclophosphamide therapy [34al.

Fig. 17. Chromosome breaks and multiradial recombination figures in chromosomes of peripheral lymphocytes after radium and X-ray therapy [34a].

It is also known that ionizing radiation can cause chromosome changes in vivo and in vifvo (see Figure 16) and that tumors can be induced by X-rays. An inter- esting example is the case described by Engel1351 in which a chronic myeloic leukemia with the typical Philadelphia chromosome occured in a patient several years after X-ray treatment for a bronchial carcinoma.

1341 W. W. Nichols, Hereditas 50, 53 (1963). [34a] We are grateful to Dr. M . 5uuc/1i1zger, Strahlenbiologisches Institut der UniversitLt Miinchen, for this photograph. 1351 E. Engel, Lancet ii, 291 (1965).

~ ~

A finding familiar to the chemist is the production of tumors by the application of mutagenic chemicals. With most substances the induced transformation of the cells is associated with a change in the chromosomes. The effects of urethane [361 and mustard gas 1371

in inducing chromosome changes were the first described. Since that time it has been discovered that many alkylating agents have the capacity to cause chromosome aberrations (cf. Fig. 16), and the same occurs with many purine and pyrimidine derivatives [381. Most DNA-base analogs have a mutagenic effect; it is assumed that they act directly on the DNA of the cell nucleus and alter the chromosome structure. Thus, for example, the effect of urethane can be partly counteracted by thymine [391. Other chemicals, such as caffeine and colchicine, as well as some other cytostatics, do not affect the DNA but alter or impede the function of the spindle fibers 1381. Considerable chromosome aberrations are also produced in this way.

There is a great similarity between the visible effects of all the chemical, radiological, and viral agents capable of inducing both tumors and chromosome aberrations; primarily, there are disturbances in chromosome disjunction, and, furthermore, chromosome breaks occur. Apparently these exogenous agents have definite and constant predilection sites for the induction of breaks. The broken ends of chromosomes have a predisposition to recombination, i.e. t o combine with other sites where breaks have taken place, so that numerous recombinations are possible after the simul- taneous occurrence of breaks in several chromosomes in the same cell cycle. Thus cell strains differing in chromosome morphology but genetically identical can be produced.

7.2. Chromosome Changes and Tumor Formation due to Endogenous Factors

There is some evidence for the assumption that endogenous as well as the described exogenous factors can play a part in tumor formation and in the increase in chromosome changes. As a n example, we may recall the existence of “ tumor families”, in which tumors occur over the course of several gener- ations; the tumors a re generally malignant, and can show similar or different localization and morphology [39,401. Such families are said t o have a “predisposing factor”, but no more definite etiological and pathogenic information can be given.

A correlation has also been found between chromosome anomalies and immunological diseases. Many autoimmune diseases and tumors have been associated, e.g. pernicious anemia with carcinoma of the stomach, as well as Hashimoto’s thyroiditis with cancer of the

[36] F. Oelilkers, Z . Vererbungsl. 81, 313 (1943). [37] C. Auerbach, Proc. Roy. SOC. Edinburgh, Sect. B 62, 284 (1947). [38] For references, see E. Grundmnnn: Allgemeine Cytologie. Thieme, Stuttgart 1964. 1391 0. Y. Verscliuer: Handbuch der Humangenetik. Thieme, Stuttgart 1964, Vol. 111/1, pp. 671 ff. [40] P. C. Koller, Europ. J. Cancer 3, 279 (1967).

717 Airgcw. Chcm. internut. Edit. Vol. 7 (1968) i No. 9

thyroid gland [411. On the other hand, mothers having demonstrable thyroid antibodies (Hashimoto’s thyroiditis) have children with chromosome anomalies, especially children with a G-trisomy (Down’s syndrome), more commonly than healthy mothers. In addition, patients with numerical or structural chromosome anomalies are more inclined to develop malignant tumors than the general population [421.

Chromosome aberrations can be caused in vitro in primary normal fibroblast cultures if an extract of genetically foreign lymphocytes is added. It has not yet been made clear whether a classical immune mechanism is involved, or something else1431. Chromosome changes also arise in normal tissue cultures after activation of lysosomal enzymes (441; a tumor-inducing factor could, for example, produce a breakdown of lysosomes, whose released enzymes could cause chromosome breaks to recombine in a way that leads to the formation of tumor cells.

We must assume that the majority of the possible morphological chromosome changes are not consistent with cell life. Many findings indicate that immunological mechanisms can lead to chromosome changes. For this reason it can be argued that such immune reactions have a kind of self-protection function, in which the “abnormal” cells are eliminated through an alteration of the chromosome structure. A possible defect in this self-protection system could be that not all cells undergo lethal chromosomal damage and some become so changed that, on the contrary, they exhibit uncontrolled growth 141,451.

[41] For references see Ph. J . Fialkow, Blood 30, 388 (1967). [42] R. W. Miller, New England J. Med. 87, 275 (1966). [43] Ph. J . Fialkow and S. M . Gartler, Nature (London) 211, 713 (1966). [44] A . C. Allison and G. R . Patron, Nature (London) 207, 1170 (1965). [45] K . H. Bauer: Das Krebsproblem. Springer, Berlin 1963.

8. Outlook

Human chromosomes are very unsuitable for the investigation of many questions in genetics, especially biochemical genetics, mainly because of their small size. On the other hand, some of the difficulties could be eliminated by greater cooperation in morphological, virological, and biochemical research. Thus, there is a possibility of fractionating dividing cells and con- centrating the chromosomes [461. The separation of chromosomes or chromosome groups by physical methods could yield further information on chromosome structure. An “in vitro system” of concentrated chromosomes of defined morphology could reveal the functional differences between individual chromosomes. On the cytogenetic side, experiments are already being undertaken to culture strains with defined chromosome aberrations. Biochemical methods can be used to try to find structural o r functional differences between these cells and normal cells, Already, for example, a series of observations indicate that in chronic myeloic leukemia the activity of the alkaline leukocyte phosphatase is decreased, whereas in patients with Down’s syndrome it is increased. The goal of drawing up a gene map of the human chromosomes is still far in the future, and will probably never be attained o n the same scale as in the case of the giant chromosomes from the salivary gland of Drosophila melanogaster, but it is not completely hopeless; for instance, a large number of genes can already be localized on the human X-chromosome, and their sequence and approximate distance apart can be determined.

Received: July 5, 1968 [A 654 IE] German version: Angew. Chem. 80. 726 (1968)

Translated by Express Translation Service, London

[46] J. Mendelsohn, D . Moore, and N . Salzman, J. molecular. Biol. 32, 101 (1968).

Element and Compound. On the Scientjfic History of Two Fundamental Chemical Concepts[**]

BY E. STRoKERI*l

A wish is often expressed that any purely factual ac- to the past; they still find themselves as a precipitate in count be completed by an historical dimension, where contemporary scientific methods - a sediment in not only a science’s past, like a venerable arsenal of the present-day concepts. factual knowledge possessed in earlier times, but also the thought-structures which led to such knowledge in

In the following article we do not wish to a mere inventory of what was regarded at different

the first place, should be called to memory. These thought-structures, however, never belong exclusively

times as element and as but rather to examine a few, typical, clearly definable thought-

.

(“1 Prof. Dr. Elisabeth Stroker structures, which have become what they now are .. Seminar fur Philosophie der Technischen Universitat 33 Braunschweig, Pockelsstrasse 14 (Germany)

through the changing usage of the concepts of “element” and of “compound” in the course of the history Of chemistry. The alchemistic doctrines of

[**I Based on a paper read at the general meeting of the Cesell- schaft Deutscher Chemiker, in Berlin (September 18-23, 1967).

718 Angew. Chem. internat. Edit. ,’ Vol. 7 (1968) 1 No. 9

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