- Email: [email protected]
Chromosome patterns in primary neoplasia
86
Experimental
CHROMOSOME
PATTERNS THEODORE
Cell Research, Suppl. 9, 86-98 (1963)
IN PRIMARY
NEOPLASIA
S. HAUSCHKA
New York State Department of Health, Roswell Park Memorial Bu$alo, N.Y., U.S.A.
Institute,
ONCOGENY by chromosomal variation is the somatic mutation hypothesis in its crudest form [3, 401. Experimentalists have shown an instinctive aversion to this seemingly untestable idea [4]. Moreover, the hypothesis is nebulous, regarding the molecular mechanisms of carcinogenesis by the “nearer causes” of cancer-the viruses, chemical mutagens, ionizing radiation and hormonal imbalance. Search for a trend, or lack of trend, in a broad sampling of tumor karyotypes is at best a circumstantial approach to the unanswered question: Are chromosome anomalies capable of initiating cancer? Or do they play a merely secondary role in tumor progression? The vast body of information on nuclear irregularities in transplantable or cultured neoplasms is irrelevant for oncogenic mechanisms. However, the quest for chromosomal pecularities in autochthonous tumors has been so prolific, that today we may attempt to discern cytogenetic patterns, where yesterday there was uncertainty and disagreement among the cytologists themselves (e.g. [l. lo]). Control patterns Karyotypic constancy in many normal somatic tissues is well documented [9, lo]. Diploidy predominates in the mitoses of such tissues and also in hyperplastic repair growth. A few organs, liver, thymus, marrow and gonads, contain secondary classes of balanced polyploids. For example, an exactly countable 16-ploid megakaryocyte metaphase with 360 chromosomes from an X0 female with a basic chromosome number of 45 was photographed by Hauschka [lo]. This approaches the upper limit of polyploidy in nonmalignant human marrows. Aneuploidy as a byproduct of normal cell proliferation occurs in all carefully examined tissues. Up to 12 per cent aneuploid elements in normal human marrows [30, 311 arise by mitotic error during hemopoiesis. Such cells appear to be non-viable or non-competitive. Transient karyotypic anomalies in somatic cell populations may be increased by surprisingly small amounts of radiation. The incidence of aneuExperimental
Cell Research, Suppl. 9
Chromosome pafferns in primary neoplasia
87
ploid metaphases in the marrows of 23 patients with hyperthyroidism was 9 per cent, i.e. within normal limits, prior to therapy [24]. Two days after therapeutic doses of 5-15 millicuries of orally administered radio-iodine 1311, aneuploidy rose to 33 per cent (range 24-53 per cent). However, when these marrows were re-examined one month later, the temporary flare-up of chromosome irregularities had disappeared [ 161. Follow-ups on large groups of such patients do not yet indicate whether their leukemia risk is higher than expected. Bender and Gooch [2] report chromosome aberrations persisting for 24 years in the leucocytes of 8 otherwise normal, healthy men who accidentally received whole-body irradiation ranging from 23 to 365 rads of mixed gammarays and fission neutrons. From these and related clinical findings it seems apparent that most of the random irregularities occurring in somatic nuclei during an individual life-span are physiologically inferior to the balanced diploid genome, hence incapable of initiating neoplastic growth (except perhaps under special localized selective pressures). The recent discoveries [lo, 311 which explain certain human developmental and endocrine disorders in terms of well-defined chromosome abnormalities-X0, XXX, XXXX, XXY, XYY, G-trisomy, D-trisomy, etc.permit two further generalizations: (a) Abnormalities in the zygotic chromosome complement are usually deleterious or lethal for the developing organism. (b) Zygotic heteroploidy as such does not constitute a precondition of neoplasia any more than does fortuitous somatic aneuploidy. Fig. 1 shows the karyotype of a perfectly healthy man with 47 instead of 46 chromosomes. His normal anatomy and mentality are explained by the fact that the extra chromosome is a largely heterochromatic Y, which contains non-essential genes.
Obsolete patterns Some defunct interpretations in the recent literature continue to confuse one of the cardinal questions in tumor cytology: Are there many primary tumors with exactly diploid chromosome sets? In a study of “early” avian and mammalian’ neoplasms, Bayreuther [1] claimed almost universal diploidy. But he inadvertently counted nonmalignant diploid stromal mitoses among the metaphases examined. Precise karyotyping as in Fig. 1, an essential prerequisite for the detection of pseudodiploidy, was precluded by the size of this 10,000 metaphase survey of 7 species. Experimental
Cell Research, Suppl. 9
T. S. Hauschka Even the most exacting technical standards are no guarantee against pitfalls of interpretation. Nowell and Hungtirford’s report [25] of consistent diploidy in cultures of blood from acute human leukemias was verified at our laboratory [28]. However, their interpretation of the dividing leucocytes as “leukemic elements” was invalidated when we examined the marrows of the same patients by the “direct squash” method. The modal karyotypes of these leukemic marrows were all aneuploid or pseudo-diploid, and contained distinct marker chromosomes. These abnormal metaphases represent the in vivo, whereas the diploid cells dividing leukemic stem cells functioning in vitro are apparently normal leucocytes. The otherwise very useful blood culture method is therefore, untrustworthy for chromosome study in acute leukemia [12, 281.
Neoplastic stemline patterns There are primary tumors with stem cell karyotypes morphologically indistinguishable from the characteristic diploid chromosome ‘set of the species. The majority of mammary adenocarcinomas of the mouse, the milkagent tumors, belong in this category (Fig. 2). Another case in point is’ the more than 80 per cent diploid chromosome number distribution in a mouse plasmacytoma [9]. The stemline of the latter was highly stable and remained diploid even after prolonged serial transplantation and exposure to the antimetabolite amethopterin. Assuming for the discussion that this is not a virus tumor, and has a true diploid karyotype, the neoplastic change might have arisen by mutation, in the most modern sense of the word “mutation”. The ultrafine resolution of molecular genetics in T 4 bacteriophage has reduced the mutable site to the level of individual nucleotides [5]. The somatic mutation hypothesis of neoplasia thereby gains an almost impregnable sanctuary in the DNA helix: base changes within the triplet “codons” of somatic nuclei seem, for the time being, quite fool-proof against an experimental attack by cancer research. The chromosomes of the mouse (Fig. 2) are so much alike that it is difficult Fig. l.-Karyotypte of an XYY leucocytes and skin was 47 XYY. and was fertile. x 2100. Fig. 2.-Diploid tumor. x 1400.
metaphase
Fig. 3.-Aneuploid ovarian carcinoma. Experimental
man whose somatic chromosome constitution This 44-year old individual had no anatomical
with 40 chromosomes
endo-reduplication x 900.
Cell Research, Suppl. 9
metaphase
of normal morphology with
in metaphases of or mental defects
from a mouse mammary
47 diplo-chromosomes
from
a human
Chromosome pafterns in primary neoplasia
Experimenfal
89
Cdl Rescnrch,
Sup@.
9
T. S. Hauschka to distinguish between true- and pseudo-diploidy. With the human karyotype (Fig. 1) this is much easier, and numerous pseudo-diploid leukemias [26, 23, 291 and carcinomas [lo] have been recorded. However, the majority of primary human and other mammalian neoplasms is frankly aneuploid [S, 8, 13-15, 17, 20, 22, 32, 34-38, 40). Although very little information on cytological variability is available for TABLE
I. Chromosome patterns in 73 malignant
human ascites exudates and
13 benign effusions.” Number of Cases Malignant 1 2 10 9 34 17 Benign 2 11
Cases with diploid or pseudo-diploid mode
-
Cases with aneuploid mode
2 lo* 6 32 13
Hansen-Melander ef al., 1956 [8] Ising and Levan, 1957 1151 KoIIer, 1956, 1960 [18, 191 Makino et al., 1959 [22] Ishihara et al., 1962 [14] Spriggs et al., 1962 [34]
-
Spriggs et al., 1962 [34] Ishihara et al., 1962 [14]
1
3 2 4 2 11
References
a The malignant exudates include carcinomas of breast, lung, bladder, ovary, colon, stomach, reticulosarcoma, neuroblastoma, melanoma. b Most of these cases had hyper-diploid modes.
precancerous lesions [lo] and early carcinoma in situ, gross chromosomal changes may occur before invasion of surrounding tissues. This difficult material was painstakingly investigated by Spriggs et al. [33] in six patients showing epithelial instability of the cervix uteri, including live with carcinoma in situ. Two of the latter had. sharp aneuploid modes, while balanced tetraploidy was surprisingly frequent in three cases. The karyotypes of 73 human malignant ascitic exudates compiled in Table I ranged from hypodiploid to hypertetraploid giving no indication of a “preferred” chromosome constitution. Most of these neoplasms had distinctively individualized stem cell karyotypes differing from one another as well as from the diploid norm. Only 3 among the 73 effusions showed modal concentrations of diploid cells [34]; they were: (a) an oat cell carcinoma of the lung with 47 per cent probably nonmalignant diploid cells and a secondary mode with 79 chromosomes; Experimental
Cell Research, Suppl. 9
Chromosomepatterns in primary neoplasia
91
(b) a carcinoma of the colon with a 53 per cent diploid peak and a secondary mode at 72 chromosomes; (c) a very heterogeneous carcinoma of the ovary with a 17 per cent diploid component and three minor modes at 75, 76 and 79 chromosomes. All 13 benign serous fluids (Table I) contained close to 100 per cent diploid metaphases, a finding of considerable diagnostic value. TABLE II. Frequency of aneuploid modal karyotypes plasms examined directly in patient material without or colchicine treatment.” Number of cases 6 5 13 1 1 4 35 1 1 1 4 1 1
Type of neoplasia Pulmonary carcinoma Mammary carcinoma Gastric carcinoma Colonic carcinoma Rectal carcinoma Ovarian carcinoma Uterine and cervical carcinoma Urethral carcinoma Skin carcinoma Squamous cell carcinoma Maxillary carcinoma Reticulosarcoma Glioma
among 74 human neorecourse to tissue culture
Aneuploid modal karotype 5 5
“Diploid” modal karyotype 1 -
12
1 1
1 4
-
34 1 1 1 4
-
1
1
1
-
a Summarized from data of Roller, 1947 [17]; Hansen-Melander et al., 1956 [8]; Manna, 1957 1231; Ising and Levan, 1957 [15]; Wakabayashi and Ishihara, 1958 [38]; Makino et aL, 1959 [22]; Ishihara, 1959 1131; Spriggs et al., 1962 1341; and Hauschka [ll].
The 74 human carcinomas and other tumors summarized in Table II represent the neoplastic population profile as it exists in uiuo: All these tumors were fixed immediately upon removal from the patients, and were studied without recourse to the vicissitudes of tissue culture or colchicine pretreatment. The largely aneuploid cytological findings are therefore not confused by artifacts. Since more than half of these tumors were untreated, therapy cannot be held responsible for the impressive 69174 incidence of heteroploidy. Chromosome counting is, of course, limited to well-spread metaphases which constitute a rather small slice of the malignant cell population. Because different components of a neoplasm may differ in their capacity to Experimental
Cell Research, Suppl. 9
92
T. S. Hauschka
divide, it is possible to under- or over-estimate the proportion of stem cell karyotypes. To overcome this difficulty, Stich and Steele [36] have measured the DNA content in the nuclei of interphase, metaphase, and telophase in 8 human carcinomas and sarcomas. Using Patau’s two-wavelength method with an accuracy of + 4 per cent, they obtained a precise index of dividing and non-dividing cells. On the whole, the telophase DNA measurements for individual tumors fell within a narrow range, as compared with the much wider distribution of corresponding metaphase values, and the extreme scatter of hyperploid multiples among the interphase nuclei. It became apparent that only a fraction of the metaphases completes mitosis. Endo-reduplication metaphases (Fig. 3) often revert to interphase without intervening cytokinesis. The higher polypoid DNA interphase classes [36] may reflect repetitive endomitoses. Fig. 4 shows an endo-reduplication metaphase from a mouse lymphoma with about 80 four-stranded diplochromosomes, equivalent to 320 telophase chromosomes, or a 16-ploid DNA value. The DNA was built up to this high level by three successive endo-reduplications. Thus, tumor growth in volume does not proceed entirely by increase in cell number. Nuclear and compensatory cytoplasmic growth of individual cells may contribute substantially to total tumor size, in addition to the mitotic increment of the principal stem-cells.
Tissue und species patterns The modes of chromosome numbers in the tumors of various mammalian species are mainly grouped around diploid and tetraploid. Hypotetraploidy may be so extensive that it approaches a seemingly triploid number. Such tumors are not to be regarded as triploids in the genetic sense. Leukoses, on the whole, exhibit a near-diploid trend, whereas the patterns of neoplasms from other tissues extend over the entire range of viable chromosome complements. It has been suggested by Levan [al] that the frequently hypo-tetraploid constitution of transplanted mouse tumors may indicate preferred karyotypes of high viability, which would, in their degree of aneuploidy, differ from one species to the next. In support of this concept Takayama and Makino [37] refer to the “rigid patterns” found in long-term tissue cultures of human cell strains. This trend of diverse unbalanced genomes toward a rather uniform karyotypic compromise was especially pronounced in venereal tumors of the dog, secured from different Japanese localities about 1800 miles apart [37]. The Experimental
Cell Research, Suppl. 9
Chromosome patterns in primary
Fig. 4.-Endo-reduplication metaphase with about chemically-induced mouse lymphoma. x 2600.
neoplasia
80 four-stranded
93
diplochromosomes
from a
normal diploid chromosome constitution of the dog is 2n = 78. The modal chromosome number of live venereal dog tumors was 59, and these neoplasms exhibited striking karyological similarity also in their abnormal metacentric marker chromosomes. Although selection of a preferred nuclear type of high viability arising spontaneously in each dog seems a reasonable explanation, infectious passage of tumor cells from dog to dog cannot be entirely ruled out. In the opinion of the Japanese authors, “a long distance (1800 miles) may form no serious bar to the transmission of the tumor by copulation because of the considerable moving ability of a dog”. On the other hand, immunogenetic differences between dogs would seem to constitute a serious obstacle to the copulatory homografting of malignant cells. Experimental
Cell Research, Suppl. 9
T. S. Hauschka Patterns of etiology Theoretically the most intriguing feature to emerge from the karyology of tumors would be a correlation of chromosome pattern with causation. While this is rather unlikely, it does appear a remote possibility, if we classify, for example, the mouse leukemias according to the various known types of leukemogenesis (Table III). TABLE
III. Aneuploidy
in 64 normal mouse marrows and in 53 primary leukemias. (Summarized according to etiology.)
Type of leukemia
Total mice
Number with diploid= mode
Per cent aneuploid cells
Total metaphases counted
murine
References
Nonleukemic controls
64
64
7.5-18.9
1269
]6,35, 391
“Spontaneous” leukemia Radiation induced leukemia Chemically* induced leukemia
26 4 16
15 1 0
30.0-96.1 54.4-92.5 85.5
1371 395 551
if5920,391
4 5
4 4
very low very low
776 733
Graffi virus leukemia Gross virus leukemia
16,f-401 I351
111 [ll
a “Diploid” refers only to number (2n = 40) and may include pseudodiploids. * 9, lo-dimethyl-1, P-benzanthracene. References: Bayreuther, 1961 [l]; Ford et aZ., 1958 [6]; Kurita and Yosida, 1961 [18]; Stich, 1960 [32]; Wakonig and Stich, 1960 [36].
By comparison with the diploid control marrows, the chemically induced leukemias are grouped at the opposite extreme, being consistently aneuploid. Moreover, Stich [35] observed the modal number of 41 in most of these leukemias, perhaps indicating a particular oncogenic chromosome constellation; it would seem impossible to verify this morphologically in the acrocentric murine karyotype. Like the frequently diploid breast tumors of mice, which have a viral etiology, most of the virus-induced mouse leukemias exhibit no apparent chromosome changes [l]. Before postulating diploidy as a general attribute of virus tumors we should, however, consider the pronounced chromosomal aberrations found within 24 hr following in vitro infection of Chinese hamster cells with Herpes simplex virus [7]. These observations implicate at least some animal viruses as potential mutagens on the gross chromosomal level. Experimental
Cell Research, Suppl. 9
95
Chromosome patterns in primary neoplasia Patterns related to pathophysiology and therapy Nuclear patterns related to histologic derivation and to are beginning to appear in the tumors most investigated karyology of all malignancies, the human leukemias. Table IV summarizes the “direct” marrow studies done Memorial Institute during the last few years [28-311. Sixty TABLE IV. Chromosome patterns
pathophysiology with respect to at Roswell Park control marrows
in 60 human control and 56 leukemic marrows.
Summarized from 5198 “direct squash” counts of Sandberg el al. [28-311. Modal chromosome number Total counts
Source of marrow 60 control donors 3 chronic lymphocytic
leukemias
27 13 chronic acute myeloblastic myelocytic leukemias leukemias
13 acute lymphoblastic
leukemias
Pseudo 45
46
46
1649
-
60
46
-
3
47-49
50-54
85-97
-
-
-
-
-
-
-
-
”
1637 928
-3
9? 81
14= 2”
938
-
2
-
I 7
1 2
2
’ All with Ph’ Chromosome; the 9 cases tabulated under 2n = 46 were examined by us on wet-mounted impermanent slides, prior to the discovery of the Ph’ marker chromosome. Their classification as true diploids is therefore questionable. * 2 is minimal number of pseudo-diploid cases since the 8 marrows with modes of 46 were not closely examined for structural anomalies.
had sharp diploid modes, containing only 12.2 kO.8 per cent aneuploid metaphases. About half of the leukemic patients were examined before any kind of therapy; however, their chromosome complements were distributed in much the same way as in those receiving chemotherapy. The only observed chemotherapeutic effects on the marrow karyotype were two instances of remission in acute leukemia during which the aneuploid leukemic stem cells temporarily vanished from-the marrow; the malignant karyotypes reappeared when the leukemia became resistant to the treatment. The nuclear characteristics of the four histologic leukemia types (Table IV) fall into a superficial, but perhaps relevant, pattern with regard to departure from diploidy. The acute myeloblastic leukemias were narrowly grouped around diploidy, while the acute lymphoblastic series had higher chromosome numbers, including 2 near-tetraploids. The latter two patients [30] and two similar cases [27] have all survived long beyond the usual life expectancy in Experimental
Cell Research, Suppl. 9
T. S. Hauschka
96
this virulent disease. This holds out some promise of utilizing chromcsome constitution in prognosis. Three chronic lymphocytic leukemias were diploid, but in their crowded metaphases accurate counting was difficult and the mitotic index was unusually low. The majority of the chronic myelocytic cases contained the “Philadelphia minute” Phi chromosome discovered by Nowell and Hungerford [25, 261. This important finding has now been verified in over 100
Fig. 5.-Diagrammatic representation of normal flow of messenger RNA (unbroken arrow) from a diploid 2n chromosome set toward normal cytoplasmic protein synthesis (NCP). Genetic misinformation (broken arrows) from mutated intrinsic DNA (IDNA), extrinsic viral DNA (EDNA) or RNA (ERNA) competes with normal RNA messengers for blank ribosomes.
patients all over the globe [29]. While one robin in Philadelphia does not make a spring, the Ph’ chromosome does make a strong case for the chromosomal etiology of at least one type of neoplasm, chronic myelocytic leukemia. However, the little Phi may be merely the crude outward expression of the loss of a regulator gene for myelopoiesis, linked to a piece of chromosome large enough for microscopic detection. CONCLUSIONS
The genotypic environment of the individual gene, the degree of balance in the karyotype as a whole, is all-important in determining phenotypic manifestation. Orderly flow of messenger RNA from a diploid set of genes to the cytoplasm can be upset as to kind, amount, or timing by three mechanisms, all of which change the nucleic information in the altered cell: Experimental
Cell Research, Suppl. 9
Chromosomepatterns in primary neoplasia
97
(a) mutational change in the narrow sense; (b) chromosomd aberrations large enough to be seen with the light microscope; (c) infection by viral or other extrinsic DNA or RNA. The balance in the integrated diploid cell between coded messages from the genes and their properly timed execution on the surfaces of ribosomes effects normal cytoplasmic protein synthesis (NCP). This is graphically expressed in Fig. 5 by means of a simple equation. The constant K in the denominator signifies the restricted availability of a relatively limited supply of uninformed ribosomes in the cytoplasm. The numerator is subject to misleading information from changes in the intrinsic nuclear DNA (IDNA), or from extrinsic DNA and RNA (EDNA and ERNA). By changing the variety, amount or timing of RNA messengers in the numerator, protein synthesis is disoriented; the cell is in danger of being isolated from growth regulation by the host. This isolation leads to one of three possible consequences; metabolic deficiency, cell death, or cancer. There is, then, no serious conflict between the chromosomal and other integrative theories of ontogeny. Neoplastic transformation-by whatever mechanism-“has to be something which eventually affects the genome” (Heidelberger, quoted in [lo]) and which has protoplasmic continuity through chromosomal replication. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.
BAYREUTIIER, K., Nature 186, 6 (1960). BENDER, M. A. and GOOCH,P. C., Radiation Res. 16, 44 (1962). BOVERI, T. H., Zur Frage der Entstehung maligner Tumoren. Gustav Fischer, Jena, 1914. BURDETTE, W. J., in Radiation Biology and Cancer, p. 349. Univ. of Texas Press, Austin, 1959. CRICK, F. H. C., Sci. Am. 207, 66 (1962). FORD, C. E., HAMERTON,J. L. and MOLE, R. H., J. Cellular Comp. Physiol. 52, Suppl. 1, 235 (1958). HAMPAR, B. and ELLISON, S. A., Nature 192, 145 (1961). HANSEN-MELANDER, E., KULLANDER, S. and MELANDER, Y., J. Nat. Cancer Inst. 16, 1067 (1956). HAUSCHKA, T. S., J. Cellular Comp. Physiol. 52 Suppl. 1, 197 (1958). Cancer Res. 21, 957 (1961). Unpublished observations. HUNGERFORD,D. and NOWELL, P. C., J. Nat. Cancer Inst. 29, 545 (1962). ISHIHARA, T., Gann 50, 403 (1959). ISHIHARA, T., KIKUCHI, Y. and SANDBERG,A. A., Personal communication (1962). ISING, U. and LEVAN, A., Acta Pathol. Microbial. Stand. 40, 13 (1957). KOEPF, G. F. and SANDBERG,A. A., Personal communication (1962). KOLLER, P. C., Bit. J. Cancer 1, 38 (1947). Ann. N.Y. Acad. Sci. 63, 793 (1956). in Cell Physiology of Neoplasia, pp. 9-37. Univ. of Texas Press, Austin, 1960. KURITA, Y. and YOSIDA, T. H., Gann 52, 257 (1961).
7 - 631807
Experimental
Cell Research, Suppl. 9
T. S. Hauschka 21. 22. 23, 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40.
LEVAN, A., Ann. N.Y. Acad. Sei. 63, 774 (1956). Mar&o, S., ISHIHARA, T. and TONOMURA, A., 2. Krebsforsch. 63, 184 (1959). MANNA, G. K., Proc. 2001. Sot., Calcutta, Mookerjee Memorial Volume, p. 95, 1957. MOORE, G. E., KOEPF, G. F. and SANDBERG, A. A., Am. J. Roentgenol. Rad. Therapy and Nucl. Med. In press (1963). NOWELL, P. C. and HUNGERFORD, D. A., J. Natl. Cancer Inst. 25, 85 (1960). ibid. 27, 1013 (1961). SANDBERG, A. A., Unpublished observations. SANDBERG, A. A., ISHIHARA, T., CROSSWHITE, L. H. and HAUSCHKA, T. S., Cancer Res. 22, 748 (1962). ~ Blood 20, 393 (1962). SANDBERO, A. A., ISHIHARA, T., MIWA; T. and HAUSCHKA, T. S., Cancer Res. 21, 678 (1961). SANDBERG, A. A., KOEPF, G. F., CROSSWHITE, L. H. and HAUSCHKA, T. S., Am. J. Human Genet. 12, 231 (1960). SPRIGGS, A. I. and BODDINGTON, M. M., Lancet 11 (July 21), 153 (1962). SPRIGGS, A. I., BODDINGTON, M. M. and CLARKE, C. M., Lancet 1 (June 30), 1383 (1962). Brit. Med. J. In press (1963). STICH, H. F., J. Natl. Cancer Inst. 25, 649 (1960). STICH, H. F. and STEELE, H. D., ibid. 28, 1207 (1962). TAKAYAMA, S. and MAKINO, S., Z. Krebsforsch. 64, 253 (1961). WAKABAYASHI, M. and ISHIHARA, T., Cytologia 23, 341 (1958). WAKONIG, FL, and STICH, H. F., J. Natl. Cancer Inst. 25, 295 (1960). WINGE, o., Z. Zellforsch. mikroskop. Anat. 10, 683 (1930).
Experimental
Cell Research, Suppl. 9
Experimental
CHROMOSOME
PATTERNS THEODORE
Cell Research, Suppl. 9, 86-98 (1963)
IN PRIMARY
NEOPLASIA
S. HAUSCHKA
New York State Department of Health, Roswell Park Memorial Bu$alo, N.Y., U.S.A.
Institute,
ONCOGENY by chromosomal variation is the somatic mutation hypothesis in its crudest form [3, 401. Experimentalists have shown an instinctive aversion to this seemingly untestable idea [4]. Moreover, the hypothesis is nebulous, regarding the molecular mechanisms of carcinogenesis by the “nearer causes” of cancer-the viruses, chemical mutagens, ionizing radiation and hormonal imbalance. Search for a trend, or lack of trend, in a broad sampling of tumor karyotypes is at best a circumstantial approach to the unanswered question: Are chromosome anomalies capable of initiating cancer? Or do they play a merely secondary role in tumor progression? The vast body of information on nuclear irregularities in transplantable or cultured neoplasms is irrelevant for oncogenic mechanisms. However, the quest for chromosomal pecularities in autochthonous tumors has been so prolific, that today we may attempt to discern cytogenetic patterns, where yesterday there was uncertainty and disagreement among the cytologists themselves (e.g. [l. lo]). Control patterns Karyotypic constancy in many normal somatic tissues is well documented [9, lo]. Diploidy predominates in the mitoses of such tissues and also in hyperplastic repair growth. A few organs, liver, thymus, marrow and gonads, contain secondary classes of balanced polyploids. For example, an exactly countable 16-ploid megakaryocyte metaphase with 360 chromosomes from an X0 female with a basic chromosome number of 45 was photographed by Hauschka [lo]. This approaches the upper limit of polyploidy in nonmalignant human marrows. Aneuploidy as a byproduct of normal cell proliferation occurs in all carefully examined tissues. Up to 12 per cent aneuploid elements in normal human marrows [30, 311 arise by mitotic error during hemopoiesis. Such cells appear to be non-viable or non-competitive. Transient karyotypic anomalies in somatic cell populations may be increased by surprisingly small amounts of radiation. The incidence of aneuExperimental
Cell Research, Suppl. 9
Chromosome pafferns in primary neoplasia
87
ploid metaphases in the marrows of 23 patients with hyperthyroidism was 9 per cent, i.e. within normal limits, prior to therapy [24]. Two days after therapeutic doses of 5-15 millicuries of orally administered radio-iodine 1311, aneuploidy rose to 33 per cent (range 24-53 per cent). However, when these marrows were re-examined one month later, the temporary flare-up of chromosome irregularities had disappeared [ 161. Follow-ups on large groups of such patients do not yet indicate whether their leukemia risk is higher than expected. Bender and Gooch [2] report chromosome aberrations persisting for 24 years in the leucocytes of 8 otherwise normal, healthy men who accidentally received whole-body irradiation ranging from 23 to 365 rads of mixed gammarays and fission neutrons. From these and related clinical findings it seems apparent that most of the random irregularities occurring in somatic nuclei during an individual life-span are physiologically inferior to the balanced diploid genome, hence incapable of initiating neoplastic growth (except perhaps under special localized selective pressures). The recent discoveries [lo, 311 which explain certain human developmental and endocrine disorders in terms of well-defined chromosome abnormalities-X0, XXX, XXXX, XXY, XYY, G-trisomy, D-trisomy, etc.permit two further generalizations: (a) Abnormalities in the zygotic chromosome complement are usually deleterious or lethal for the developing organism. (b) Zygotic heteroploidy as such does not constitute a precondition of neoplasia any more than does fortuitous somatic aneuploidy. Fig. 1 shows the karyotype of a perfectly healthy man with 47 instead of 46 chromosomes. His normal anatomy and mentality are explained by the fact that the extra chromosome is a largely heterochromatic Y, which contains non-essential genes.
Obsolete patterns Some defunct interpretations in the recent literature continue to confuse one of the cardinal questions in tumor cytology: Are there many primary tumors with exactly diploid chromosome sets? In a study of “early” avian and mammalian’ neoplasms, Bayreuther [1] claimed almost universal diploidy. But he inadvertently counted nonmalignant diploid stromal mitoses among the metaphases examined. Precise karyotyping as in Fig. 1, an essential prerequisite for the detection of pseudodiploidy, was precluded by the size of this 10,000 metaphase survey of 7 species. Experimental
Cell Research, Suppl. 9
T. S. Hauschka Even the most exacting technical standards are no guarantee against pitfalls of interpretation. Nowell and Hungtirford’s report [25] of consistent diploidy in cultures of blood from acute human leukemias was verified at our laboratory [28]. However, their interpretation of the dividing leucocytes as “leukemic elements” was invalidated when we examined the marrows of the same patients by the “direct squash” method. The modal karyotypes of these leukemic marrows were all aneuploid or pseudo-diploid, and contained distinct marker chromosomes. These abnormal metaphases represent the in vivo, whereas the diploid cells dividing leukemic stem cells functioning in vitro are apparently normal leucocytes. The otherwise very useful blood culture method is therefore, untrustworthy for chromosome study in acute leukemia [12, 281.
Neoplastic stemline patterns There are primary tumors with stem cell karyotypes morphologically indistinguishable from the characteristic diploid chromosome ‘set of the species. The majority of mammary adenocarcinomas of the mouse, the milkagent tumors, belong in this category (Fig. 2). Another case in point is’ the more than 80 per cent diploid chromosome number distribution in a mouse plasmacytoma [9]. The stemline of the latter was highly stable and remained diploid even after prolonged serial transplantation and exposure to the antimetabolite amethopterin. Assuming for the discussion that this is not a virus tumor, and has a true diploid karyotype, the neoplastic change might have arisen by mutation, in the most modern sense of the word “mutation”. The ultrafine resolution of molecular genetics in T 4 bacteriophage has reduced the mutable site to the level of individual nucleotides [5]. The somatic mutation hypothesis of neoplasia thereby gains an almost impregnable sanctuary in the DNA helix: base changes within the triplet “codons” of somatic nuclei seem, for the time being, quite fool-proof against an experimental attack by cancer research. The chromosomes of the mouse (Fig. 2) are so much alike that it is difficult Fig. l.-Karyotypte of an XYY leucocytes and skin was 47 XYY. and was fertile. x 2100. Fig. 2.-Diploid tumor. x 1400.
metaphase
Fig. 3.-Aneuploid ovarian carcinoma. Experimental
man whose somatic chromosome constitution This 44-year old individual had no anatomical
with 40 chromosomes
endo-reduplication x 900.
Cell Research, Suppl. 9
metaphase
of normal morphology with
in metaphases of or mental defects
from a mouse mammary
47 diplo-chromosomes
from
a human
Chromosome pafterns in primary neoplasia
Experimenfal
89
Cdl Rescnrch,
Sup@.
9
T. S. Hauschka to distinguish between true- and pseudo-diploidy. With the human karyotype (Fig. 1) this is much easier, and numerous pseudo-diploid leukemias [26, 23, 291 and carcinomas [lo] have been recorded. However, the majority of primary human and other mammalian neoplasms is frankly aneuploid [S, 8, 13-15, 17, 20, 22, 32, 34-38, 40). Although very little information on cytological variability is available for TABLE
I. Chromosome patterns in 73 malignant
human ascites exudates and
13 benign effusions.” Number of Cases Malignant 1 2 10 9 34 17 Benign 2 11
Cases with diploid or pseudo-diploid mode
-
Cases with aneuploid mode
2 lo* 6 32 13
Hansen-Melander ef al., 1956 [8] Ising and Levan, 1957 1151 KoIIer, 1956, 1960 [18, 191 Makino et al., 1959 [22] Ishihara et al., 1962 [14] Spriggs et al., 1962 [34]
-
Spriggs et al., 1962 [34] Ishihara et al., 1962 [14]
1
3 2 4 2 11
References
a The malignant exudates include carcinomas of breast, lung, bladder, ovary, colon, stomach, reticulosarcoma, neuroblastoma, melanoma. b Most of these cases had hyper-diploid modes.
precancerous lesions [lo] and early carcinoma in situ, gross chromosomal changes may occur before invasion of surrounding tissues. This difficult material was painstakingly investigated by Spriggs et al. [33] in six patients showing epithelial instability of the cervix uteri, including live with carcinoma in situ. Two of the latter had. sharp aneuploid modes, while balanced tetraploidy was surprisingly frequent in three cases. The karyotypes of 73 human malignant ascitic exudates compiled in Table I ranged from hypodiploid to hypertetraploid giving no indication of a “preferred” chromosome constitution. Most of these neoplasms had distinctively individualized stem cell karyotypes differing from one another as well as from the diploid norm. Only 3 among the 73 effusions showed modal concentrations of diploid cells [34]; they were: (a) an oat cell carcinoma of the lung with 47 per cent probably nonmalignant diploid cells and a secondary mode with 79 chromosomes; Experimental
Cell Research, Suppl. 9
Chromosomepatterns in primary neoplasia
91
(b) a carcinoma of the colon with a 53 per cent diploid peak and a secondary mode at 72 chromosomes; (c) a very heterogeneous carcinoma of the ovary with a 17 per cent diploid component and three minor modes at 75, 76 and 79 chromosomes. All 13 benign serous fluids (Table I) contained close to 100 per cent diploid metaphases, a finding of considerable diagnostic value. TABLE II. Frequency of aneuploid modal karyotypes plasms examined directly in patient material without or colchicine treatment.” Number of cases 6 5 13 1 1 4 35 1 1 1 4 1 1
Type of neoplasia Pulmonary carcinoma Mammary carcinoma Gastric carcinoma Colonic carcinoma Rectal carcinoma Ovarian carcinoma Uterine and cervical carcinoma Urethral carcinoma Skin carcinoma Squamous cell carcinoma Maxillary carcinoma Reticulosarcoma Glioma
among 74 human neorecourse to tissue culture
Aneuploid modal karotype 5 5
“Diploid” modal karyotype 1 -
12
1 1
1 4
-
34 1 1 1 4
-
1
1
1
-
a Summarized from data of Roller, 1947 [17]; Hansen-Melander et al., 1956 [8]; Manna, 1957 1231; Ising and Levan, 1957 [15]; Wakabayashi and Ishihara, 1958 [38]; Makino et aL, 1959 [22]; Ishihara, 1959 1131; Spriggs et al., 1962 1341; and Hauschka [ll].
The 74 human carcinomas and other tumors summarized in Table II represent the neoplastic population profile as it exists in uiuo: All these tumors were fixed immediately upon removal from the patients, and were studied without recourse to the vicissitudes of tissue culture or colchicine pretreatment. The largely aneuploid cytological findings are therefore not confused by artifacts. Since more than half of these tumors were untreated, therapy cannot be held responsible for the impressive 69174 incidence of heteroploidy. Chromosome counting is, of course, limited to well-spread metaphases which constitute a rather small slice of the malignant cell population. Because different components of a neoplasm may differ in their capacity to Experimental
Cell Research, Suppl. 9
92
T. S. Hauschka
divide, it is possible to under- or over-estimate the proportion of stem cell karyotypes. To overcome this difficulty, Stich and Steele [36] have measured the DNA content in the nuclei of interphase, metaphase, and telophase in 8 human carcinomas and sarcomas. Using Patau’s two-wavelength method with an accuracy of + 4 per cent, they obtained a precise index of dividing and non-dividing cells. On the whole, the telophase DNA measurements for individual tumors fell within a narrow range, as compared with the much wider distribution of corresponding metaphase values, and the extreme scatter of hyperploid multiples among the interphase nuclei. It became apparent that only a fraction of the metaphases completes mitosis. Endo-reduplication metaphases (Fig. 3) often revert to interphase without intervening cytokinesis. The higher polypoid DNA interphase classes [36] may reflect repetitive endomitoses. Fig. 4 shows an endo-reduplication metaphase from a mouse lymphoma with about 80 four-stranded diplochromosomes, equivalent to 320 telophase chromosomes, or a 16-ploid DNA value. The DNA was built up to this high level by three successive endo-reduplications. Thus, tumor growth in volume does not proceed entirely by increase in cell number. Nuclear and compensatory cytoplasmic growth of individual cells may contribute substantially to total tumor size, in addition to the mitotic increment of the principal stem-cells.
Tissue und species patterns The modes of chromosome numbers in the tumors of various mammalian species are mainly grouped around diploid and tetraploid. Hypotetraploidy may be so extensive that it approaches a seemingly triploid number. Such tumors are not to be regarded as triploids in the genetic sense. Leukoses, on the whole, exhibit a near-diploid trend, whereas the patterns of neoplasms from other tissues extend over the entire range of viable chromosome complements. It has been suggested by Levan [al] that the frequently hypo-tetraploid constitution of transplanted mouse tumors may indicate preferred karyotypes of high viability, which would, in their degree of aneuploidy, differ from one species to the next. In support of this concept Takayama and Makino [37] refer to the “rigid patterns” found in long-term tissue cultures of human cell strains. This trend of diverse unbalanced genomes toward a rather uniform karyotypic compromise was especially pronounced in venereal tumors of the dog, secured from different Japanese localities about 1800 miles apart [37]. The Experimental
Cell Research, Suppl. 9
Chromosome patterns in primary
Fig. 4.-Endo-reduplication metaphase with about chemically-induced mouse lymphoma. x 2600.
neoplasia
80 four-stranded
93
diplochromosomes
from a
normal diploid chromosome constitution of the dog is 2n = 78. The modal chromosome number of live venereal dog tumors was 59, and these neoplasms exhibited striking karyological similarity also in their abnormal metacentric marker chromosomes. Although selection of a preferred nuclear type of high viability arising spontaneously in each dog seems a reasonable explanation, infectious passage of tumor cells from dog to dog cannot be entirely ruled out. In the opinion of the Japanese authors, “a long distance (1800 miles) may form no serious bar to the transmission of the tumor by copulation because of the considerable moving ability of a dog”. On the other hand, immunogenetic differences between dogs would seem to constitute a serious obstacle to the copulatory homografting of malignant cells. Experimental
Cell Research, Suppl. 9
T. S. Hauschka Patterns of etiology Theoretically the most intriguing feature to emerge from the karyology of tumors would be a correlation of chromosome pattern with causation. While this is rather unlikely, it does appear a remote possibility, if we classify, for example, the mouse leukemias according to the various known types of leukemogenesis (Table III). TABLE
III. Aneuploidy
in 64 normal mouse marrows and in 53 primary leukemias. (Summarized according to etiology.)
Type of leukemia
Total mice
Number with diploid= mode
Per cent aneuploid cells
Total metaphases counted
murine
References
Nonleukemic controls
64
64
7.5-18.9
1269
]6,35, 391
“Spontaneous” leukemia Radiation induced leukemia Chemically* induced leukemia
26 4 16
15 1 0
30.0-96.1 54.4-92.5 85.5
1371 395 551
if5920,391
4 5
4 4
very low very low
776 733
Graffi virus leukemia Gross virus leukemia
16,f-401 I351
111 [ll
a “Diploid” refers only to number (2n = 40) and may include pseudodiploids. * 9, lo-dimethyl-1, P-benzanthracene. References: Bayreuther, 1961 [l]; Ford et aZ., 1958 [6]; Kurita and Yosida, 1961 [18]; Stich, 1960 [32]; Wakonig and Stich, 1960 [36].
By comparison with the diploid control marrows, the chemically induced leukemias are grouped at the opposite extreme, being consistently aneuploid. Moreover, Stich [35] observed the modal number of 41 in most of these leukemias, perhaps indicating a particular oncogenic chromosome constellation; it would seem impossible to verify this morphologically in the acrocentric murine karyotype. Like the frequently diploid breast tumors of mice, which have a viral etiology, most of the virus-induced mouse leukemias exhibit no apparent chromosome changes [l]. Before postulating diploidy as a general attribute of virus tumors we should, however, consider the pronounced chromosomal aberrations found within 24 hr following in vitro infection of Chinese hamster cells with Herpes simplex virus [7]. These observations implicate at least some animal viruses as potential mutagens on the gross chromosomal level. Experimental
Cell Research, Suppl. 9
95
Chromosome patterns in primary neoplasia Patterns related to pathophysiology and therapy Nuclear patterns related to histologic derivation and to are beginning to appear in the tumors most investigated karyology of all malignancies, the human leukemias. Table IV summarizes the “direct” marrow studies done Memorial Institute during the last few years [28-311. Sixty TABLE IV. Chromosome patterns
pathophysiology with respect to at Roswell Park control marrows
in 60 human control and 56 leukemic marrows.
Summarized from 5198 “direct squash” counts of Sandberg el al. [28-311. Modal chromosome number Total counts
Source of marrow 60 control donors 3 chronic lymphocytic
leukemias
27 13 chronic acute myeloblastic myelocytic leukemias leukemias
13 acute lymphoblastic
leukemias
Pseudo 45
46
46
1649
-
60
46
-
3
47-49
50-54
85-97
-
-
-
-
-
-
-
-
”
1637 928
-3
9? 81
14= 2”
938
-
2
-
I 7
1 2
2
’ All with Ph’ Chromosome; the 9 cases tabulated under 2n = 46 were examined by us on wet-mounted impermanent slides, prior to the discovery of the Ph’ marker chromosome. Their classification as true diploids is therefore questionable. * 2 is minimal number of pseudo-diploid cases since the 8 marrows with modes of 46 were not closely examined for structural anomalies.
had sharp diploid modes, containing only 12.2 kO.8 per cent aneuploid metaphases. About half of the leukemic patients were examined before any kind of therapy; however, their chromosome complements were distributed in much the same way as in those receiving chemotherapy. The only observed chemotherapeutic effects on the marrow karyotype were two instances of remission in acute leukemia during which the aneuploid leukemic stem cells temporarily vanished from-the marrow; the malignant karyotypes reappeared when the leukemia became resistant to the treatment. The nuclear characteristics of the four histologic leukemia types (Table IV) fall into a superficial, but perhaps relevant, pattern with regard to departure from diploidy. The acute myeloblastic leukemias were narrowly grouped around diploidy, while the acute lymphoblastic series had higher chromosome numbers, including 2 near-tetraploids. The latter two patients [30] and two similar cases [27] have all survived long beyond the usual life expectancy in Experimental
Cell Research, Suppl. 9
T. S. Hauschka
96
this virulent disease. This holds out some promise of utilizing chromcsome constitution in prognosis. Three chronic lymphocytic leukemias were diploid, but in their crowded metaphases accurate counting was difficult and the mitotic index was unusually low. The majority of the chronic myelocytic cases contained the “Philadelphia minute” Phi chromosome discovered by Nowell and Hungerford [25, 261. This important finding has now been verified in over 100
Fig. 5.-Diagrammatic representation of normal flow of messenger RNA (unbroken arrow) from a diploid 2n chromosome set toward normal cytoplasmic protein synthesis (NCP). Genetic misinformation (broken arrows) from mutated intrinsic DNA (IDNA), extrinsic viral DNA (EDNA) or RNA (ERNA) competes with normal RNA messengers for blank ribosomes.
patients all over the globe [29]. While one robin in Philadelphia does not make a spring, the Ph’ chromosome does make a strong case for the chromosomal etiology of at least one type of neoplasm, chronic myelocytic leukemia. However, the little Phi may be merely the crude outward expression of the loss of a regulator gene for myelopoiesis, linked to a piece of chromosome large enough for microscopic detection. CONCLUSIONS
The genotypic environment of the individual gene, the degree of balance in the karyotype as a whole, is all-important in determining phenotypic manifestation. Orderly flow of messenger RNA from a diploid set of genes to the cytoplasm can be upset as to kind, amount, or timing by three mechanisms, all of which change the nucleic information in the altered cell: Experimental
Cell Research, Suppl. 9
Chromosomepatterns in primary neoplasia
97
(a) mutational change in the narrow sense; (b) chromosomd aberrations large enough to be seen with the light microscope; (c) infection by viral or other extrinsic DNA or RNA. The balance in the integrated diploid cell between coded messages from the genes and their properly timed execution on the surfaces of ribosomes effects normal cytoplasmic protein synthesis (NCP). This is graphically expressed in Fig. 5 by means of a simple equation. The constant K in the denominator signifies the restricted availability of a relatively limited supply of uninformed ribosomes in the cytoplasm. The numerator is subject to misleading information from changes in the intrinsic nuclear DNA (IDNA), or from extrinsic DNA and RNA (EDNA and ERNA). By changing the variety, amount or timing of RNA messengers in the numerator, protein synthesis is disoriented; the cell is in danger of being isolated from growth regulation by the host. This isolation leads to one of three possible consequences; metabolic deficiency, cell death, or cancer. There is, then, no serious conflict between the chromosomal and other integrative theories of ontogeny. Neoplastic transformation-by whatever mechanism-“has to be something which eventually affects the genome” (Heidelberger, quoted in [lo]) and which has protoplasmic continuity through chromosomal replication. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.
BAYREUTIIER, K., Nature 186, 6 (1960). BENDER, M. A. and GOOCH,P. C., Radiation Res. 16, 44 (1962). BOVERI, T. H., Zur Frage der Entstehung maligner Tumoren. Gustav Fischer, Jena, 1914. BURDETTE, W. J., in Radiation Biology and Cancer, p. 349. Univ. of Texas Press, Austin, 1959. CRICK, F. H. C., Sci. Am. 207, 66 (1962). FORD, C. E., HAMERTON,J. L. and MOLE, R. H., J. Cellular Comp. Physiol. 52, Suppl. 1, 235 (1958). HAMPAR, B. and ELLISON, S. A., Nature 192, 145 (1961). HANSEN-MELANDER, E., KULLANDER, S. and MELANDER, Y., J. Nat. Cancer Inst. 16, 1067 (1956). HAUSCHKA, T. S., J. Cellular Comp. Physiol. 52 Suppl. 1, 197 (1958). Cancer Res. 21, 957 (1961). Unpublished observations. HUNGERFORD,D. and NOWELL, P. C., J. Nat. Cancer Inst. 29, 545 (1962). ISHIHARA, T., Gann 50, 403 (1959). ISHIHARA, T., KIKUCHI, Y. and SANDBERG,A. A., Personal communication (1962). ISING, U. and LEVAN, A., Acta Pathol. Microbial. Stand. 40, 13 (1957). KOEPF, G. F. and SANDBERG,A. A., Personal communication (1962). KOLLER, P. C., Bit. J. Cancer 1, 38 (1947). Ann. N.Y. Acad. Sci. 63, 793 (1956). in Cell Physiology of Neoplasia, pp. 9-37. Univ. of Texas Press, Austin, 1960. KURITA, Y. and YOSIDA, T. H., Gann 52, 257 (1961).
7 - 631807
Experimental
Cell Research, Suppl. 9
T. S. Hauschka 21. 22. 23, 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40.
LEVAN, A., Ann. N.Y. Acad. Sei. 63, 774 (1956). Mar&o, S., ISHIHARA, T. and TONOMURA, A., 2. Krebsforsch. 63, 184 (1959). MANNA, G. K., Proc. 2001. Sot., Calcutta, Mookerjee Memorial Volume, p. 95, 1957. MOORE, G. E., KOEPF, G. F. and SANDBERG, A. A., Am. J. Roentgenol. Rad. Therapy and Nucl. Med. In press (1963). NOWELL, P. C. and HUNGERFORD, D. A., J. Natl. Cancer Inst. 25, 85 (1960). ibid. 27, 1013 (1961). SANDBERG, A. A., Unpublished observations. SANDBERG, A. A., ISHIHARA, T., CROSSWHITE, L. H. and HAUSCHKA, T. S., Cancer Res. 22, 748 (1962). ~ Blood 20, 393 (1962). SANDBERO, A. A., ISHIHARA, T., MIWA; T. and HAUSCHKA, T. S., Cancer Res. 21, 678 (1961). SANDBERG, A. A., KOEPF, G. F., CROSSWHITE, L. H. and HAUSCHKA, T. S., Am. J. Human Genet. 12, 231 (1960). SPRIGGS, A. I. and BODDINGTON, M. M., Lancet 11 (July 21), 153 (1962). SPRIGGS, A. I., BODDINGTON, M. M. and CLARKE, C. M., Lancet 1 (June 30), 1383 (1962). Brit. Med. J. In press (1963). STICH, H. F., J. Natl. Cancer Inst. 25, 649 (1960). STICH, H. F. and STEELE, H. D., ibid. 28, 1207 (1962). TAKAYAMA, S. and MAKINO, S., Z. Krebsforsch. 64, 253 (1961). WAKABAYASHI, M. and ISHIHARA, T., Cytologia 23, 341 (1958). WAKONIG, FL, and STICH, H. F., J. Natl. Cancer Inst. 25, 295 (1960). WINGE, o., Z. Zellforsch. mikroskop. Anat. 10, 683 (1930).
Experimental
Cell Research, Suppl. 9