the e. i?. a · hermann, merkle, and bouin’s solutions. the first fixation was found best suited...

MALIGNANCY OF THE CROWN-GALL AND ITS ANALOGY TO ANIMAL CANCER’

ISAAC LEVIN AND MICHAEL LEVINE From the Department of Cancer Research of the Monte3ore Hospital arid lfome,

New York

Rec:eived for publication, December 17, 1919

METHODS EMPLOYED IN THE INVESTIGATIONS

In 1907, Dr. E. I?. Smith showed that the disease of plants called crown-gall-tumor-like in nature-could be reproduced arti- ficially by an inoculation of a normal plant with a pure culture of a bacillus which he called Bacterium tumefaciens. Smith maintains that this or a similar disease can be produced only by this particular organism, that all other tumor-like formations observed in plants due to other parasites, like Plasmodiophora brassicae, are entirely different pathological entities, and that only crown-gall is a true plant tumor. In his estimation, this tumor is practically identical, biologically, with animal and human cancer. Since crown-gall is caused by the action of the bacterium described by him, he concludes in one of his recent articles that to a biologist the conclusion is almost irresistible that human cancer must be due to a parasite and that one parasite may well be the cause of the most diverse forms, as we have seen to be the case in plants.” This deduction is so sweeping in character, so far-reaching in its generalization, and the results of Dr. Smith’s work have become so widely known and quoted by the medical profession that it has seemed to the writers to be desirable to renew his investigations, to analyze the material from the viewpoint of animal pathology, and to attempt to deter- mine the true analogy between the crown-gall and animal cancer.

1 Presented before the American Association for Cancer Research, June 14, 1919.

243

TBE JOURNAL OF CANCER REBBARCR, YOL V, NO. 3

244 IBAAC LEVIN AND MICHAEL LEVINE

The writers have inoculated with the pure culture of Bacte- rium tumefaciens the following plants: Growing tips, stems, petioles, and blades of Rosa (Rose), Ricinus (castor oil), Pelargon- ium (house geranium), Lycopersicum (tomato), Nicotianum (tobacco), Helianthus (sun-flower), Bryophyllum calycinum and Ficus elastica (India-rubber tree). Galls formed were fixed at various stages of development in Carnoy, Flemming strong,

FIG. 1. CROWN-GALL ON A STEM OF A RUBBER PLANT

Hermann, Merkle, and Bouin’s solutions. The first fixation was found best suited for the more woody galls. After embedding in paraffin, the sections were cut from 5 to 30 micra in thickness, and were stained with Flemming’s triple stain, methyl green counterstained with fuchsin, or Delafield’s hematoxylin counterstained with eosin. The most satisfactory preparations were ob- tained by fixing with Carnoy solution and staining with gentian violet and safranin. In these preparations the cell walls stain a

MALIGNANCY O F THE CROWN-GALL 245

beautiful deep violet, while the cytoplasm and chromatin material stain a much fainter purple. The nucleolus takes a bright ruby red color.

FIG. 2. a, CROWN-CALL ON A STEM OF A R O S E ; WART-LIKE BENIGN GALL. h, LONGITUDINAL SECTION OF THE SAME STEM

The gross study of the material shows that in nearly every inoculated plant there developed a distinct crown-gall within one or two months after inoculation. Usually these galls grow slowly and are not very large in comparison with the size

246 ISAAC LEVIN AND MICHAEL LEVINE

FIG. 3. LONGITUDINAL SECTION OF A STEM OF RICINUS PLANT

The’gall is benign, the normal stem tissue is only displaced

MALIGNANCY OF THE CROWbJ-GALL 244

of the plant itself. Figure 1 shows a gall which grew for over six months on a rubber plant; the plant itself is 6 feet high. Most frequently the gall grows out of the surface of the stem and does

FIG. 4. a, RICINUS PLANT INOCULATED AT THE APICAL PORTION OF THE STEM; SHOWS DWARFING OF THE PLANT. 11, CONTROL PLANT

not seem to affect the rest of the plant (fig. 2). In other instances the growth displaces and distorts a part of the cortical area of the stem (fig. 3). Only in a small percentage of the inoculated


plants does the crown-gall affect the tissues of the plant more deeply. Figure 4 shows a photograph of the growing regions of two plants, one inoculated with the microorganism, and the other a normal control plant. While the latter grew up above the tip,

Fra. 5 . FELARGONIUM Shows necrosis of the inoculated part of the plant

the inoculated plant ceased to grow and became dwarfed, and the leaves drooped. At the same time, even in the inoculated plant, the lower part of the stem as well as the root apparently remained normal. Figure 5 presents an instance where the part of the plant above the point of inoculation, and even to a certain

MALIGNANCY OF THE CROWN-CALL 249

degree below the point of inoculation, became necrotic. Cross (fig. 6) or longitudinal (fig. 7) sections of such plants show that these crown galls grow invasively, destroy and replace normal

FIG. 6. LONGITUDINAL SECTIONS O F Two STEMS OF RICINUS PLANTS Show invasive growth of the gall and destruction of the normal stem tissue

plant tissue, and occasionally develop further along the length of the stem in a manner which produces the appearance of a secondary metastatic tumor above or below the primary growth (fig. 8).


In accordance with the findings of Smith, the writers have also observed in many instances the formation of small leafy shoots at the periphery of a crown-gall (figs. 9 and 10).

The microscopical analysis of the material shows that the crown-gall consists of a number of uniformly small, young, un- differentiated cells which contain a greater amount of cytoplasm

than the normal parenchyma cells within which they develop (fig. 11).

The small slowly growing crown-galls which frequently develop when the inoculation is done at the bases of leaves (fig. 12) present a very striking picture when examined microscopically. In the center of thc growth there is an area of characteristic crown- gall cells, and surrounding this area the crown-gall cells appear

The nuclei frequently show mitosis.

MALIGNANCY OF THE CROWN-GALL 251

to.be much larger in size and present all the characteristics of normal parenchyma cells (fig. 13). Apparently in these crown- galls the peripheral gall cells became differentiated into adult

1'10. 8. LONGITUDINAI~ SECTIONS SHOWING AT a AND b CONDITIONS SOMEWHAT SIMILAR TO METASTASIS FORMATION

normal tissue cells. Frequently there were observed new irregular conducting systems formed within the crown-gall tissue (fig. 14), also apparently as a further differentiation of the crown-gall cells.


Microscopical study of a large number of the leafy sheets which developed at the periphery of a crown-gall showed that the start- ing point and the base of each leafy shoot is ordinary crown-gall

l h ~ . !). L O ~ . ( J ~ L D I A A L SLLT~ )h OF A :;XIJ O F A ‘I’OBACCO I’LANT The apical portion of the stcm was removed and the cut surface inoculated.

The crown-gall shows the development of leafy shoots.

tissue with the identical gall cells (fig. 15), indicating that the crown-gall cells themselves gave rise to the formation of the leafy shoot.


DISCUSSION

The analysis of the material shows that crown-gall is undoubtedly a neoplastic disease and that the pathogenesis of the condition consists in an abnormal proliferation of a group of cells.

FIG. 10. TOBACCO LEAF INOCULATED AT TIIE M:r, RIB Shows at a a small gall with a leafy shoot

It is self-evident, therefore, that there must exist many points Since a of analogy between the crown-gall and animal cancer.


plant presents a much less complex organization than the animal, crown-gall tissue undoubtedly offers an ideal material for the study of the phenomena connected with cell proliferation. All this does not imply that crown-gall or any other neoplastic disease in animal or plant must of necessity be a tumor, even less so a malignant tumor-a cancer.

FIG. 11. MICROPHOTOGRAPH Shows crown-gall tissue invading the normal stem tissue

In comparing new growth in animals and plants one must take into consideration the fact that an adult vertebrate is not capable of reproducing complete organs, while the highest plants may and do constantly reproduce with ease all their organs, leaves and branches as well as roots. Reproduction of parts of the organism, and consequently cell proliferation, is a function of an adult plant which may be induced with the greatest of ease.

255 MALIGNANCY O F THE CROWN-GALL

It is self-evident that since an adult plant cell proliferates so as to reproduce an organ, a young crown-gall cell may do likewise. This characteristic of a normal plant cell explains the striking

.

FIG. 12. FOUR LEAVES OF RICINUS INOCULATED AT THE UPPER PORTION OF PETIOLES

Shows small wart-like galls

phenomenon, not encountered in any neoplastic disease in the animal, that a crown-gall may form within its own cells, or rather


as a transformation of its cells, not only adult differentiated tissues (parenchyma) but even rudimentary organs (conducting system) or a whole rudimentary organism (leafy shoot), All these various types of structures found in a crown-gall do not indicate, as appears to be the opinion of Smith, that the crown- gall is analogous to all the types of human cancer, but rather that it is different from any type of animal tumor.

FIG. 13. MICROPHOTOGRAPH O F A G A L L S H O W N IN F I G U R E 12 The characteristic gall tissue is surrounded by newly formed parenchyma

tissue.

In order to formulate clearly the position which the crown-gall occupies among the neoplastic diseases, one must take into consideration the-fact that the crown-gall is usually a benign condition and ionlyip-arely does it act in a manner analogous to a malignant tumor in an animal.

MALIGNANCY O F THE CROWN-GALL 257

The true mechanism of the formation of a benign crown-gall can be conceived only in the light of the difference in the structure of an animal and a plant organism. An animal organism reacts frequently to an attack of any injurious agent with the so-called inflammatory processes, which are accompanied by certain activ- ities of the lymphoid tissues and proliferation and migrations of lymph cells. These cells, and not the special tissues of the injured region, offer protection, envelop and destroy the injurious

FIG. 14. RIIUROPHOTDYRAPH Crown-gall showing newly formcd irregular conducting system

agent, or neutralize it. When the latter is destroyed, the inflammatory process ceases and functions of repair or replacement of the injured tissue take place, This repair, which ends in the formation of a scar, is a type of neoplasia which differs radically from tumor formation. It continues just as long as is necessary to replace the lost tissu.e and then ceases; while tumor formation is a neoplasia which has no reason for its formation in the needs of the organism, and has no finality in its development.


The highest organized plants do not possess any specialized lymphoid tissue to take care of the functions of the protection of the organism against an injury or of the repair of the injured or lost tissue. One of the methods of protection of plants against injury consists in the presence of a cellulose wall. It seems plausible to suppose that as a matter of self-protection a plant may respond to an injury by a proliferation of the cells of the

FIG. 15. MICI~OIJIIOTO(;ILAPII At a crown-yd1 tissue, and at b the tissue of LL leafy shoot

region which was subjected to the injury. The small wart-like galls, which grow very rapidly for a time and then remain sta- tionary, as the writers have observed in anumber of instances, are products of such functions of repair and protection and do not represent true tumors. That the Bacterium tumefaciens may be found even within the crown-gall cells or in the irnmc- diate neighborhood, still further emphasizes the truth of this conception.


Thus a small benign crown-gall is a condition analogous to granulation tissue and a scar in the animal organism. A crown- gall which grew to comparatively large size and still does not affect in any way the general welfare of the plant may be com- pared to a benign tumor in an animal, but is probably more nearly analogous to a large callus, which develops after a fracture of a long bone, or to a cheloid in the human.

Smith ascribes a great deal of importance to the phenomenon of development of leafy shoots in a certain number of crown- galls, and considers this type of gall to be identical with human embryoma. This finding he considers the main proof for his contention that all types of human cancer may be reproduced in plants by the aid of the Bacterium tumefaciens. In the opinion of the writers there is no analogy between a human embryoma and the crown-gall with a leafy shoot sprouting from it. An embryoma is a growth consisting of an irregular combination of various fetal tissues. It is more akin to a malformation than to a true tumor. A malignant tumor may develop subsequently within an embryoma in the same manner as it develops within normal tissue. The leafy shoot on the other hand appears on a fully developed crown-gall and is identical with a shoot which develops in a normal part of a plant. It simply indicates that crown-gall tissue as well as normal plant tissue may reproduce complete organs and thus still more widens the gap between the animal tumors and the crown-gall.

The malignant type of crown-gall is undoubtedly quite analogous to animal cancer. This condition occurs very rarely, as is seen from the results of the field studies on sugar beets of Town- send, who found that the destruction of the beets by the gall is not sufficient to influence the tonnage of the crop.

The most enthusiastic supporters of the parasitic theory of cancer admit that the formation of benign tumors cannot be due to a parasite. In other words, they admit that the mechanism of the formation of benign and malignant tumors must differ from each other. The same is undoubtedly true as regards the formation of the usual benign type of crown-gall and the by far less frequent condition when the gall dwarfs or destroys the plants

THE JOURNAL OF CANCER RESEARCE, VOL. V, NO. 3

260 . ISAAC LEVIN AND MICHAEL LEVINE

and thus acts as a malignant tumor. When two plants of the same size, the same age, growing in the same soil under identical conditions of heat, light, moisture and nutrition, are inoculated in the same regions with the same quantity of an identical culture of Bacterium tumefaciens, and there develops in one plant a small, benign, wart-like structure and in the other a large malignant tumor which may destroy the plant, it is difficult to con- ceive that the same microorganism, and only the microorganism, created the two conditions. The only possible explanation of the phenomenon lies in the fact that in the second plant, for some unknown reason, the cell proliferation which began only as a protection against the bacterial invasion and apparently ceased when the bacteria were rendered harmless, suddenly received an impetus for limitless proliferation. It seems then that the first impetus to the cell proliferation and formation of a benign crown- gall may be caused by the Bacterium tumefaciens. But the transformation of this protective or inflammatory benign cell proliferation into a malignant tumor is due, as in every type of animal and human cancer, to some mechanism within the organism of the host, independent of the microorganisms, the nature of which is unknown.

CONCLUBION

1. Crown-gall is a neoplastic disease, and offers an ideal material for the cytological study of cancer.

2. Crown-gall occurs both as a benign and a malignant condition.

3. The benign crown-gall is analogous to granuloma or cheloid in the human and is caused by Bacterium tumefaciens. 4. The malignant crown-gall is analogous to animal cancer,

and Bacterium tumefaciens is not the direct cause of the malignant transformation.

the e. i?. a · hermann, merkle, and bouin’s solutions. the first fixation was found best suited...

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