James S. Potter

Conditional Deletion of c-myc Does Not Impair Liver Regeneration

The oncogene c-myc encodes a transcription factor that has long been considered essential to liver regeneration, the process by which fully differentiated hepatocytes proliferate in an attempt to maintain a normal functional mass in response to hepatic injury. Experimental liver regeneration can be induced upon 70% partial hepatectomy and is accompanied by an increase in c-myc expression accompanying the synchronous entry of remaining hepatocytes into the cell cycle. Because liver regeneration is an essential process for achieving liver homeostasis, therapies directed at reducing MYC expression in hepatocellular carcinoma are fraught with the theoretical possibility of injuring adjacent noncancerous liver cells, thereby restricting the livers normal regenerative response to injury. To determine if intact c-myc is required for liver regeneration, we reduced hepatic c-myc in c-myc(fl/fl) mice using an adenoviral vector that expresses Cre recombinase. Despite a 90% decrease in hepatic expression of c-myc, restoration of liver mass 7 days later was not compromised. Reconstituted liver retained the same decrease in hepatic c-myc, indicating that hepatocytes deficient in c-myc were able to proliferate in response to partial hepatectomy. Although c-myc is required for embryonic development, our findings indicate that it is not required for the maintenance of the adult liver.

Transfer of Acquired Resistance to Transplantable Leukemia in Mice.

It has been demonstrated that graduated doses (cell-immunization) of leukemic cells 1 and certain normal tissues 2 , 3 can induce resistance to transplantable leukemia in normally susceptible mice. Following cell-immunization injected leukemic cells immediately develop small lesions which soon become necrotic. 4 Lesions do not form immediately following leukemic inoculation of strain C58 mice made resistant by Sto-Li foetal tissues. Once resistance to line I leukemia has been established by means of graduated cell doses it is permanent, while resistance caused by normal tissue implantation may be only temporary with a peak of effectiveness in C58 mice 3 days after implantation of Sto-Li tissue (unpublished data). Of further interest in this problem is the question: Can resistance induced by these 2 methods be transferred to normally susceptible mice? Accordingly, spleen and liver were removed from cell-immunized mice, minced and injected into susceptible C58 mice. Another group of C58 mice was given injections of Sto-Li foetal tissues and 3 days later the spleens were removed from these animals, minced and injected into susceptible C58 mice. Three days after receiving transplants from resistant animals both groups of mice were injected with a normally lethal dose of line I transplantable leukemia. C58 mice treated with normal C58 spleen were also given leukemic inoculations to verify the observed lack of resistance to line I leukemia following implantation of C58 tissue in C58 mice. 2 Control inoculations of normal C58 mice were made to check the potency of the dose of leukemic cells. A summary of the results of these experiments is given in Table I. These preliminary results demonstrate that resistance to a transplantable leukemia may be transferred from cell-immunized mice to normal mice by implantation of tissue from actively immunized mice. Under the conditions of this experiment resistance induced by normal tissues was not transferable. Quantitative effects, duration of the transferred resistance and use of cell-free material are questions for further experiments.

Immunization of Mice Naturally Susceptible to a Transplantable Leukemia

The transplantable leukemia designated as line I 1 was started from a spontaneous case of lymphatic leukemia in April, 1929. The line has passed through 441 transfer generations and, by routine technique, has been inoculated in massive doses into 3625 mice of the highly inbred strain C 58; all but one of these died with the leukemic indications characteristic of this particular line of cells. The single survivor, which at no time showed clinical effects of the inoculation, was inoculated in the 77th transfer generation, in December, 1930. In the 3 ¾ years since that time, the 2925 mice from strain C 58 that have been inoculated with the massive standard dose of cells of line I have all developed leukemia. In the light of this record the natural susceptibility of strain C 58 to leukemic cells of line I appears to be fully established. The present experiments, started in April, 1934, are based on 100% susceptibility to the standard dose. It has been shown previously 2 that between certain limits, reduction of the dosage lengthens the interval before death, and in the early transfers of line I that were used for these experiments (transfers 27-34, Jan.-Mar., 1930) the 18 mice given doses of 6000-9000 cells did not die with leukemia. However, these mice were not tested for an immunizing effect of surviving the small doses. In returning to the study of small doses of the leukemic cells of line I, 346 transfer generations later, the virulence of the cells had become considerably enhanced and it now is found that the minimum dose that will kill is reduced to the order of magnitude of 200 cells. In successive dilutions of the massive standard dose the interval before death is progressively lengthened, as previously reported, 2 but a dilution is reached (1/1024th of standard) that permits a few of the mice to survive and as the dose is further reduced the proportion of survivors increases until every mouse survives (1/524,000th of standard).

Histological observations on resistance to transplantable leukemia in immunized mice

Recently it has been demonstrated that immunization to transplantable lymphatic leukemia in mice can be actively induced by suitable injection of dilute doses of leukemic cells, 1 by injection of normal lymphoid cells, 2 and of embryo skin. 3 Naturally susceptible mice so treated are resistant to injections of normally lethal doses of leukemic cells at subsequent inoculations. This paper records the results of preliminary observations on the fate of malignant lymphoid cells inoculated into immunized mice. A group of 19 mice of strain C58, immunized to line I transplantable lymphatic leukemia by dilute doses of line I cells as already described, 1 were killed at intervals following inoculation of a massive dose; 1 at 13 1/4 hours, 1 at 1 day, 3 at 2 days, 7 at 3 days, 2 at 4 days, 1 at 5 days, 2 at 6 days, and 2 at 7 days. Unimmunized mice inoculated with this line die at 3 1/2 to 4 days following inoculation. The development of lesions of this line of leukemic cells in hosts of strain C58 as previously described 4 forms a basis for comparison with the behavior of these leukemic cells in immunized hosts. Examination of the tissues from the immunized animals shows that at first the inoculated cells continue to proliferate. Lesions may appear in the same areas as in unimmunized mice but never become so large. The percent of cells in division is low (0.3%–1.0%) as compared with active lesions at all stages in unimmunized mice (4%–9%) inoculated with line I. Degeneration of the infiltrating cells usually takes place within the first 4 days. In only 2 animals out of the 14 killed after the second day were active lesions found without necrotic cells, but in these 2 animals necrotic lesions were also found, indicating that recovery was in progress.

Metabolic differences between two transmission lines of mouse leukemia

The experiments of Warburg 1 on the metabolic differences between tumors and normal tissues have stimulated similar investigations in leukemia. Although considerable data have accumulated relative to the metabolism of the cells in leukemia, many of the conclusions have been contradictory. The following investigation has been undertaken as part of the program conducted in these laboratories in which the genetics, pathology and cytology of transmissible leukemia of mice have been reported by MacDowell, Richter and Potter. 2 The genetically controlled material developed in their studies is particularly suitable for metabolic experiments. The oxygen consumption and both aerobic and anaerobic glycolysis of normal lymph nodes and leukemic lymph nodes of 2 distinct transmission lines, designated A and I, have been studied by one of us. All animals were from strain C-58 in which 100% were susceptible to the transmitted disease. Line I killed the inoculated animals in 3 to 4 days, Line A in 6 to 8 days. Infiltration of the lymph nodes in Line I has been shown to be complete in 72 hours, while in Line A, 4 days. Method. The animals were 6 to 8 weeks old. The experimental animals were of the same sex and almost invariably litter mates. Congested, hemorrhagic and necrotic glands were discarded. Four normal animals were sacrificed for each determination of the QQ2, QO2 CO2 and QN2 CO2 of the lymph nodes. In Line I, one or 2 animals were used; in Line A the lymph nodes of 3 mice were combined to obtain sufficient material. The quantity of tissue in each respirometer varied from 30 to 70 mg. moist weight. The nodes of Line I were used 3 days after inoculation, of Line A, 5 to 6 days after inoculation.