Ehrlich Mouse Carcinoma Research Articles

Influence of polyhydroxysteroids on [Ca(2+)](i).

Recently, we have shown that two biologically active, disulfated polyhydroxysteroids from the Pacific brittle star Ophiopholis aculeata stimulate Ca(2+) influx into different cell types. In the present study, 45Ca(2+) and two fluorescent calcium probes, quin-2/AM and fura-2/AM, were employed to investigate the course and amplitude of calcium signals induced in different mouse cells using an radio-isotope, spectrofluorimetry, and microcytofluorimetry techniques. The cytotoxic and hemolytic effects were not observed for both steroids at the wide range of concentrations. Steroids did not influence [3H]-uridine incorporation in a variety of cells. The investigated steroids stimulated a rapid increase in cytosolic Ca(2+) content in Ehrlich mouse carcinoma cells, mouse spleen lymphocytes, and mouse peritoneal macrophages in the concentration range 1-100 microg/ml on a dose-dependent basis. Blockers of L-type calcium channels, such as verapamil, diltiazem, nifedipine (1 x 10(-7)M), and 1mM EGTA, inhibited this process and reduced the response of cells to steroid application. The stimulatory effect of steroids on human fibroblast proliferation and mouse macrophage lysosome activity was observed also. It is suggested that the investigated compounds may act as Ca(2+)-agonists and increase Ca(2+)-transport across cell membranes.

Mechanism of action of aclacinomycin A. I. The effect on macromolecular syntheses.

The mechanism of action of aclacinomycin A was investigated with mouse tumor cells and Escherichia coli. The antibiotic inhibited growth of mouse lymphoblastoma L5178Y cells completely at a concentration of 0.0125 μg/ml, and partially at 0.0063-0.0031 μg/ml. A greater inhibition of RNA than DNA synthesis was found in the intact cells of L5178Y: i.e. , approximately 50% inhibition of the former occurred at an antibiotic concentration of 0.07 μg/ml, and that of the latter at 1.0 μg/ml. The degree of preferential inhibition by aclacinomycin A was more pronounced than that by adriamycin. Protein synthesis was not significantly affected. RNA synthesis by RNA polymerase II, from EHRLICH mouse carcinoma cells, was sensitive to aclacinomycin A. By the method employed, approximately 50% inhibition was observed at an antibiotic concentration of 1 μg/ml, with calf thymus DNA or poly(dAdT) as a template. In contrast, RNA polymerase reaction with poly (dIdC) was resistant to the antibiotic. The inhibition of RNA polymerase reaction with poly(dAdT) as template was apparently competitively reversed by increasing concentrations of the template but not of the enzyme, suggesting a direct interaction of the antibiotic with the template. Growth and macromolecular biosyntheses of E. coli were rather resistant to aclacinomycin A. RNA and DNA polymerase reactions with toluenized cells of E. coli P3478 (polA-) were prevented by the antibiotic. RNA synthesis was inhibited by ca. 60% and DNA synthesis by ca. 25% at an antibiotic concentration of 10 μg/ml. RNA polymerase reaction, using E. coli enzyme and calf thymus DNA as a template, was blocked by aclacinomycin A: approximately 50% inhibition was observed at an antibiotic oncentration of 10 μg/ml; whereas DNA polymerase I reaction was not significantly affected by the antibiotic.

In vitro cytocidal effect of L-glutaminase on leukaemic lymphocytes.

MOUSE lymphomas, such as 6C3HED, undergo complete regression after one or more injections of L-asparaginase1, and remissions have been reported in some patients with acute leukaemia after treatment with L-asparaginase2–4. Roberts et al.5 found that purified bacterial L-glutaminase inhibited the growth of Ehrlich mouse carcinoma. Before considering this enzyme for therapeutic studies in man, it is necessary to study in vitro effects of the enzyme on human blood cells. Previous work6 demonstrated that the cells of 6C3HED lymphoma do not survive incubation with very low concentrations of L-asparaginase (1.7 mIU/ml.), while the cells of the in vivo resistant variant of 6C3HED were not killed by 170 mIU of enzyme/ml. The blood lymphocytes from patients with chronic lymphocytic leukaemia were found to be more sensitive than normal lymphocytes to incubation with 170 mIU of L-asparaginase/ml. but not as sensitive as 6C3HED mouse lymphoma which responds in vivo to the enzyme. In this study, the slide chamber method7 was used to compare the sensitivity of normal and leukaemic lymphocytes to glutaminase.

Detection of the antigenic properties of Ehrlich's mouse carcinoma cells after treatment with lonin-3

Detection of the antigenic properties of Ehrlich's mouse carcinoma cells after treatment with lonin-3

Influence of LDH viruses on growth of Ehrlich ascites tumor in mice.

Summary1. Ehrlich Mouse Ascites Carcinoma (EMC) was freed from an endogenous LDH virus by passage in tissue culture, and subsequently carried virus-free for 27 generations in mice. 2. Plasma LDH was from 10- to 40-fold elevated in mice carrying the stock infected tumor. Over 90% of mice carrying the virus-free tumor, however, had normal plasma LDH levels. 3. Growth rate of the virus-free tumor was accelerated by the LDH virus from Ehrlich Mouse Carcinoma itself and also by LDH agents isolated from DBA/59 mammary carcinoma and CD/5 lymphoma. It is suggested that the virus may depress host resistance to the tumor.

A CYTOLYTIC PARASITE IN THE CELLS OF TRANSPLANTABLE, MALIGNANT TUMOURS.

AT this institute two transplantable ascites tumours, the Yoshida rat sarcoma and the Ehrlich mouse carcinoma, have for a number of years been kept alive by intraperitoneal passages and used in immunological research. Through these years each tumour after injection in rats and mice of the same strain has performed very consistently with a very low survival rate—about 5 per cent—and with uniform survival times before death from malignant ascites.

The production of specific sera to Ehrlich's mouse carcinoma on the basis of the phenomena of acquired immunological tolerance

The author attempted to obtain specific antitumor sera on the basis of the phenomenon of an artificially created immunological tolerance. It was shown that only an insignificant degree of immunological approximation can be attained in rabbits with respect to heterologous mice antigens by administering the latter to the embryos, especially when this is supplemented by a subsequent inoculation at an early period of extra-uterine life. This immunological approximation is incomplete and is manifested only by an insignificant reduction in the formation of normal antibodies, inadequate for obtaining “pure” monospecific and antitumor sera to Ehrlich's mouse carcinoma. The antisera to the Ehrlich's carcinoma obtained on the basis of acquired immunological tolerance were more specific than those derived by the usual method of immunization in control rabbits.

The prolonged heterogeneous implantation of mouse tumors in rats

The antigenic content of Ehrlich's mouse carcinoma transplanted for more than a year on young rats was studied with the aid of cancer cell agglutination reaction (in M. M. Kapichnikov's modification). Rat species-specific antigens appear on the surface of cancer cells begining from the first generation of rats, evidently, at the expense of the rat protein adsorption. In addition to this, after 50 generations, certain mouse species-specific antigens disappear from the surface of cancer cells. Identical results are obtained in complement fixation reaction. The loss of mouse antigens is accompanied by a simultaneous decline in the growth of the tumor heterotransplants on mice.

Investigations on a New Heterologous Tumor, "Budapest 1938," Successfully Propagated in Hungarian White Rats

The first report of the successful propagation of a mammalian tumor in a foreign species was that of Putnoky (1). In 1929 in this laboratory he succeeded in transplanting an Ehrlich mouse carcinoma, kindly supplied by Professor Silberstein of Vienna, into rats previously treated with lactic acid. This tumor, which became known as the Ehrlich-Putnoky rat carcinoma, was grown continuously in rats for eight years and ten months, during which more than 300 passages were made, involving over 5000 animals. For a fuller account, see Putnoky's paper (2) in this journal. Unfortunately in August 1938 an outbreak of disease among our animals caused the loss of both the Silberstein strain and the rat-grown Ehrlich-Putnoky tumor. In October 1938 a new stain of the Ehrlich mouse carcinoma was obtained from the Veterinary School of Budapest and designated by us as Bp.V.S. (Fig. 1). This tumor was successfully transplanted into young rats, previously untreated, 11 takes being obtained among 12 animals in the first passage. Since the histologic findings in the 11 tumors were identical, the possibility of an accidental or spontaneous origin is excluded. Fragments of tumor free from necrosis and weighing 0.3 to 0.4 gm. were implanted subcutaneously every eighth day—two separate series, A and B, being maintained. The inoculated animals were kept on a bread and water diet from the time of transplantation.1

Early Stages of Growth in White Rats of the Ehrlich-Putnoky Rat Carcinoma

In 1930 the author (1) recorded for the first time the successful transplantation of the Ehrlich mouse carcinoma into rats. Subsequently Selbie (2) reported the results of histologic observations on the tumour, under the title “Early Stages of the Transplantation of the Ehrlich-Putnoky Tumour into Mice and Rats.” Selbie's work, however, is open to a number of criticisms. In the first place, in a review of the author's earlier studies the following statement is made: “After growing progressively in the rat for about ten days the tumour regressed or became completely necrotic.” On the contrary, as was stated in the original report, though necrotic centers appear in increased number starting from the tenth day, “the tumours in most cases grow progressively until the death of the animal.” Statistical data included in later reports showed that in 250 passages the tumour caused the death of 59 to 82 per cent of the animals (5). These figures apply to Hungarian rats inoculated by a technic developed for the purpose, with the Ehrlich-Putnoky tumour resulting from the heteroplastic transplantation of the Ehrlich mouse carcinoma. Describing his own materials and methods, Selbie writes: “A series of 12 mice and 12 young rats were inoculated subcutaneously with two small pieces of the Ehrlich-Putnoky tumour—on either side of the ventral mid-line—by means of a fine trocar. The tumour material used was taken from a mouse which had been inoculated from one of the tumours sent by Professor Balogh.” In these experiments, then, Selbie was working not with the Ehrlich-Putnoky tumour, which is a tumour that has been maintained in rats for eight years without reinoculation into mice, but, on the contrary, with tumours obtained from mice inoculated with the Ehrlich-Putnoky tumour. In the second place, his transplantations were made with the aid of a trocar, though we have emphasized the importance of employing the technic developed by us, by which large implants are inserted subcutaneously. Finally, in view of the fluctuations in tumour takes and growth, a single series of 12 mice and 12 rats inoculated at the same time would seem an insufficient basis for conclusions.

Dormant Life of Tumour Cells in the Animal Body

WORKING with transplantations of Ehrlich mouse carcinoma (belonging to a strain cultivated in vitro in this Institute for nearly twelve years), we observed that tumours developed in general after two to four weeks. These experiments were carried out on ordinary commercial white mice, all of male sex. The body-weight was 13–20 gm. In several cases we found, however, that tumours developed 6 weeks and even 8, 10, 12 and 16 weeks after the inoculation of the carcinoma cells.

A Bacteriological Study of Mouse Tumors: II. Bacterial Immunity and Tumor Growth; Effect of Bacterial Products on Tumors

The effect of B. danysz on the Ehrlich mouse carcinoma was described in a previous report (1936). Hyperemia and other changes, which were interpreted as being of a regressive nature, were observed in the tumors following intratumoral injection of this highly pathogenic microorganism. The experiments to be described in this paper deal with the attempt to immunize mice against infection with B. danysz by inoculation of a vaccine of that organism, the study of tumor transplants in these animals, and the reaction of the animals as well as the tumors to the injection of virulent B. danysz as compared to that of immunized but non-tumor-bearing animals. The effect of intraperitoneal injections of toxin and heat-killed cultures of B. danysz on the Ehrlich mouse carcinoma was also studied. Torrey and Kahn (1927), during their studies of the effect of proteolytic anaerobes on tumors, attempted to immunize rats actively and passively against B. histolyticus . They then noted the effect of this microorganism on the Flexner-Jobling carcinoma which had been transplanted in these animals. The immunization had no effect on tumor growth, but subsequent injection of cultures of B. histolyticus directly into the tumor apparently caused regression in a number of instances. However, this procedure was not more effective than the injection of B. histolyticus into the tumors of non-treated animals. Lewin (1928) reported that non-specific protein therapy increased the resistance of animals to tumors, for transplants grew poorly in rats and mice that had been subjected to such treatment previous to transplantation. Uhlenhuth (1937), however, found that non-specific protein therapy had no effect on the course of transplanted tumors in mice or rats.

On the Immunity Reactions of a Heterotransplantable Mouse Carcinoma Propagated in Rats for Seven Years

The following investigation was carried out with a tumour strain which can be propagated continuously in mice and in rats. The strain was sent, in 1929, to Professor de Balogh of the Pathological and Cancer Research Institute of the University in Budapest by Professor Silverstein of the Pathological Institute of the University of Vienna. It was then a transplantable mouse tumour, and was described as “Ehrlich's mouse carcinoma.” It is one of the most rapidly, if not the most rapidly, growing of transplantable tumours, and on inoculation into mice takes in 100 per cent. Histologically, it is a very cellular tumour, without any definite structure, and composed of undifferentiated round cells. After several unsuccessful attempts, I was able, in 1929, to transplant this tumour into rats, and it has been propagated continuously in rats since. Detailed accounts of these experiments have been published (Putnoky, 1930, 1933), but a brief account of the technique of transplantation and of the behaviour of the tumour in rats is necessary. The success of the propagation in rats depends mainly on the use of large doses. In my experiments, I use from 300 to 500 mg. of tumour. The material is inserted subcutaneously through an incision in the skin of the back and pushed into a pocket of subcutaneous tissue as distant from the incision as possible. Dr. W. Cramer, to whom I sent several rats with this tumour in 1935, informs me that he has been able to propagate this tumour continuously in rats through 16 generations, using smaller amounts of tumour tissue, from 50 to 100 mg. The tumour has to be transplanted every ten days, but it is not necessary to transplant into very young rats. As a rule, rats weighing 100 to 120 grams were used. The strain of rat appears to be of some importance. In the Budapest rats the tumour grows so rapidly that in ten to fourteen days it attains a weight varying from 30 to 40 grams, and in that period kills a high percentage of the tumour-bearing animals as shown by the following table.

Tumor Lipids: The Comparative Lipid Content of the Periphery and Center

Introduction Bullock and Cramer (1914) analyzed transplantable tumors of mice and rats in an effort to obtain information concerning the presence of lipid substances in rapidly growing cells. In addition to finding that phospholipids were more abundant in rapidly growing tumors than in slowly growing tumors, they found that healthy, non-necrotic portions of a mouse carcinoma contained a lower amount of phospholipid calculated as percentage of dry residue than did necrotic portions of the same tumor. Cholesterol was found only in traces and cholesterol esters were absent from both kinds of tissue. Analyses of peripheral and central portions of Flexner-Jobling rat carcinoma by Uramoto (1932) showed higher free and bound cholesterol and water contents in the center of the tumor, while the values for lipid phosphorus were very nearly the same for both tissues. Bierich and Lang (1933) analyzed intact peripheral and necrotic central portions of a Jensen sarcoma, finding a great increase of cholesterol ester and a great decrease of phospholipid in the necrotic central portion. Brikker and Lasaris (1932) reported opposite results on the Ehrlich mouse carcinoma, since they found a higher proportion of cholesterol to lecithin in the periphery than in the center. The analyses of peripheral and central portions of tumor tissue reported in this paper were undertaken to determine, first, the portions of tumor to be used for future lipid analyses; second, the changes in the lipid partition of a tissue due to necrosis. The blood supply to the center of the transplantable rat tumor used is so poor that, for the most part, the lipids formed from those of the outer or growing tissue should remain within the tumor. Experimental Tumor: The tumor used was rat carcinosarcoma 256 obtained from the Institute of Cancer Research, Columbia University. Young male rats weighing from 70 to 100 gm. were inoculated subcutaneously in the groin by the trocar method. The tumors were removed when they had attained a size such that sufficient tissue could be obtained from the center for analysis. Their age varied from twenty-seven to fifty-two days depending on their growth rate.

A Bacteriological Study of Mouse Tumors

There have been numerous descriptions of the finding of microorganisms in tumors of human, animal, and vegetable origin. A certain divergence exists in the types of bacteria which have been found, and in the interpretation of the significance of their presence. Many have sustained the idea of an etiological relationship between bacteria or microparasites and tumor proliferation. More often the organisms have been regarded as coincidental, or secondary to changes in the tumor or in its overlying surface, or as casual invaders, which, in the case of transplantable tumors, were most probably introduced during one or more of the operations of transplantation, and, as the case might be, were individual to one group of animals or propagated from one generation of tumors to another. As such they might be pathogenic for the animals or carried as harmless saprophytes possibly pathogenic for other species. The older literature has been amply reviewed by Lewin. Kauffmann, in a series of reports, has described his success in isolating from the Ehrlich mouse carcinoma various strains of a T-bacterium, which he believes to be related to the B. tumefaciens of Smith. This organism he discovered in 50 per cent of the tumors; 30 per cent of the tumors showed a banal flora, and 20 per cent were sterile. Kauffmann has studied the biological characteristics of the various T-bacteria with which he was able to produce growths, considered by him to be infectious granulomas, in sunflower and sugar-beet plants, but which in general were non-pathogenic for animals. Other studies have demonstrated that B. tumefaciens apparently has no etiological relationship to malignant growth in animals (Borghi and Luzzatto).

Early Stages of the Transplantation of the Ehrlich-Putnoky Tumour into Mice and Rats

In 1930 Putnoky, from the laboratory of Professor von Balogh, reported the successful propagation of a mouse tumour in rats. The tumour used was obtained from the Institute of General Pathology, Vienna University, and was described as a strain of the Ehrlich mouse carcinoma. Transplantation was effected by introducing pieces of tumour of the size of a lentil into the subcutaneous tissues of the back through an incision. The rats weighed at least 80 gm. After growing progressively in the rat for about ten days, the tumour regressed or became completely necrotic. It was transplanted from rat to rat every eight to ten days and at the time of publication was in its 28th passage in rats. It had been growing in rats continuously for four years and eight months when Professor von Balogh kindly sent two of these animals bearing the tumour to the Imperial Cancer Research Fund. As this is the first reported instance of the continued propagation of a mammalian tumour in a foreign species, it was considered worth while to study the early stages of the tumour9s growth in the rat and mouse. Furthermore, as histological preparations of the Ehrlich-Putnoky tumour differ widely from preparations of two strains of the Ehrlich mouse carcinoma which were kindly supplied by Dr. Santesson of the Radiumhemmet, Stockholm, it was thought that a study of the early stages of the former tumour might help to explain its anomalous appearance.

The Reaction of Mice and of Various Mouse Tumors to the Injection of Bacterial Products

Bacterial products have been used in the treatment of malignant growths since 1882. The literature pertaining to this subject has been reviewed recently by Shear (1). Interest in the problem appears to have been revived by the work of Gratia and Linz (2), who observed that intravenous injection of the filtrate of a broth culture of B. coli into guinea-pigs bearing a transplanted liposarcoma elicited hemorrhage and necrosis within the tumor. Shwartzman and Michailovsky (3) used “agar washings filtrate of meningococcus 44 D group I” and produced the reaction in mouse sarcoma 180. Their results were confirmed in this laboratory (4). Apitz (5) produced hemorrhagic reactions in the Ehrlich mouse carcinoma by injecting B. coli filtrates. Duran-Reynals (6) also employed filtrates of broth cultures of B. coli and produced hemorrhage in 4 transplantable tumors of mice and in a transplantable rat tumor. His filtrate failed to elicit the reaction in animals with certain other types of growth, among which were “19 mice bearing spontaneous malignant mammary gland adenocarcinoma.” Similar negative results with spontaneous mouse tumors were obtained in this laboratory after injection of meningococcus filtrates (7). In a later paper Duran-Reynals (8) describes positive reactions as well as some recessions in spontaneous mouse tumors following treatment with a mixture of filtrates of human typhoid bacillus and 3 strains of mouse typhoid bacilli. Shwartzman (9) has reported recently that a mixture of B. typhosus culture filtrate with homologous antisera produced hemorrhage followed by the complete regression of sarcoma 180 in 21 of 27 treated mice. Fogg (10) has shown that the hemorrhagic reaction and, in many instances, complete retrogression of mouse carcinoma 63 and mouse sarcomas 180 and 37 occurred following the injection of heat-killed cultures of a Gram-negative bacillus belonging to the proteus group. Spontaneous mouse tumors were affected but not destroyed. Furthermore, the alcohol-insoluble fractions of B. proteus, B. typhosus, B. coli, B. pyocyaneus and B. prodigiosus invoked hemorrhage and recession of mouse sarcoma 180. The present paper is a report of further investigations into the problem that have been carried out in this laboratory. It deals with the results of studies pertaining to (1) the toxicity of the bacterial filtrates for mice; (2) the use of cutaneous tumors as test objects for activity of the filtrates; (3) results attending the use of pure strain and stock mice; (4) the effect of filtrates upon different kinds of malignant growths.

The Effect of Prolan on Transplantable Mouse Sarcoma

In 1932 Zondek, Zondek and Hartoch (1) reported an inhibition of growth of the Ehrlich mouse carcinoma following the administration of female sex hormone. An extract of the latter, including both the follicle-ripening component (Prolan A) and the luteinizing component (Prolan B), was injected daily intravenously into adult mice during the three-week period of observation, beginning the day after inoculation with the tumor graft. A daily dose of 200 rat units proved to be the upper limit of toxicity. Using over 400 mice, these authors found that the average tumor growth in animals treated with Prolan over a three-week period was 0.2 grams, contrasted with the average of 1.65 grams in the control tumors. Furthermore it was found that this restriction of growth persisted during later transplants. Inoculation of the inhibited tumors into fresh stock gave the usual percentage of takes, although the weight at three weeks still averaged only 0.24 grams. A third generation failed to grow normally, averaging only 0.14 grams and in some cases failing to take. Cannavo (2) in 1933 reported retardation in the growth of transplanted Ehrlich carcinoma in mice injected with Prolan, 800 rat units before and 1500 units after grafting. Gruhzit (3) in the same year found that Prolan B caused slight retardation in growth of the Flexner-Jobling carcinoma, but no more so than an injection of phenolated water, and had no effect on the percentage of successful inoculationa. Wiesner and Haddow (4) observed neither acceleration nor inhibition of the Jensen rat sarcoma when the animals were coincidentally treated with the gonadotropic hormone of pregnancy urine.

The Effect of the Anterior Pituitary Hormones on the Growth of Mouse Sarcoma

It has been suggested by H. Zondek, B. Zondek, and Hartoch (1) that the so-called ovarian follicle ripening component of the anterior pituitary hormone (Prolan A) may act to hinder tissue growth in general, and particularly the growth of tumors. To test this hypothesis, these authors carried out a series of experiments in which anterior pituitary hormone containing a mixture of both the follicle ripening component (Prolan A) and the luteinizing component (Prolan B) was administered to mice inoculated with transplantable carcinoma. The tumor used was the Frankfurt strain of Ehrlich mouse carcinoma, which had never regressed spontaneously in 1019 inoculations. The plan used by the authors was to give their mice frequent and exceedingly large doses of the hormone. The injections were begun on the day following inoculation with the tumor and were continued for twenty-two days. The tail vein was used for all but the last 4 to 6 injections, which were given subcutaneously. From 0.1 to 0.2 c.c. of the solution of the hormone was injected daily. Each cubic centimeter contained from 500 to 1000 rat units. The maximum daily dose was therefore 200 rat units. On the 23d day the animals were killed and the tumor removed. The results can be summarized as follows: