Ludwik Gross

“Spontaneous” Leukemia Developing in G3H Mice Following Inoculation, In Infancy, with AK-Emkemic.∗

Summary 1. Seven of 14 C3H mice inoculated when less than 12 hours old with Ak leukemic extracts developed leukemia at 8 to 11 months of age. 2. Four of 6 C3H mice inoculated. when less than 12 hours old, with cell suspensions prepared from Ak embryos. developed leukemia at 8 1/2 months of age. 3. No leukemia resulted when mice more than 12 hours old were used for inoculation. 1. These experiments suggest that spontaneous mouse leukemia may be caused by an agent, which is transmitted from one generation to another, like that of chicken lymphomatosis.

A filterable agent, recovered from Ak leukemic extracts, causing salivary gland carcinomas in C3H mice.

Summary 1. Extracts prepared from livers, spleens, and lymphatic tumors of leukemic Ak mice, were centrifugated at 1400 × g (15 minutes), then 7000 × g (5 to 10 minutes), and finally passed through Selas porcelain niters. The filtered extracts were then inoculated into newborn (less than 16 hours old) C3H or C3H(f) suckling mice. 2. Of the 84 mice inoculated with fresh, filtered extracts, 9 died from leukemia, and 15 others developed bilateral epitheliomas possibly arising in the salivary glands.‡ Of the 54 control litter-mates inoculated with heated (68°C for 30 minutes) extracts, only one developed a cervical tumor. 3. Extracts prepared from Ak leukemic cells may therefore contain 2 different agents, pathogenic for newborn C3H or C3H(f) mice: one, causing leukemia, and another salivary gland epitheliomas. These agents may differ in size. 4. It is assumed that the leukemic agent may interfere with the tumor agent preventing it from exerting its pathogenic properties on the cells of its usual Ak, carrier host, or that mice of the Ak line may be naturally resistant to the tumor agent. 5. Salivary gland tumors have not been observed in untreated mice of either the Ak, the C3H, or C3H(f) inbred lines.

Pathogenic properties, and "vertical" transmission of the mouse leukemia agent.

Summary 1. When centrifugated extracts of Ak mouse leukemia were inoculated into 16 suckling C3H(f) infant mice less than 12 hours old, “spontaneous” leukemia developed in 8 of them at 81/2 to 11 months, and in 5 mice at 14 to 18 months of age. 2. When the centrifugated leukemic extracts were inoculated into 20 C3H(f) infant mice 3 to 6 days old, “spontaneous” leukemia developed in 10 of them, but not before they were 18 months of age. 3. Filtered (Seitz) leukemic extracts were inoculated, in 2 experiments, into 7 C3H(f) infant mice 8 days old, and 2 of them developed leukemia at 23 and 27 months respectively. In 5 additional experiments, leukemic filtrates were inoculated into 25 C3H(f) infant mice less than 12 hours old, and 7 of them developed “spontaneous” leukemia at 61/2 to 9 months of age. These results suggest that the Ak mouse leukemia agent is filterable. 4. In 5 experiments, suckling infant C3H or C3H(f) mice were inoculated with either centrifugated leukemic extracts, leukemic cell suspensions, or with normal Ak-embryo-cell suspensions. After the inoculated infant mice have grown up and reached sexual maturity, they were mated, Of the 10 inoculated parents, 7 eventually developed “spontaneous” leukemia at 9 to 19 months of age. Of the 27 untreated offspring, 13, or 48%, have thus far developed “spontaneous” leukemia at 14 to 20 months of age. The results of these experiments suggest that the mouse leukemia agent is transmitted from parents to their offspring. The transmission of the agent occurs most probably directly through the embryos(2,4). 5. The incidence of spontaneous leukemia among the untreated males and females of the C3H or C3H(f) colonies of mice in our laboratory has been, during the past several years, less than 0.07%.

Development and Serial Cell-Free Passage of a Highly Potent Strain of Mouse Leukemia Virus.

Summary 1. A strain of a filterable mouse leukemia agent has been developed from a spontaneous Ak leukemia through several successive cell-free inoculations of newborn C3H mice. 2. This agent induced acute, generalized leukemia within 2 1/2 to 3 1/2 months in 40 to 100% of the inoculated newborn C3H mice (Bittner substrain). 3. Newborn mice had to be inoculated; adult C3H mice were with few exceptions resistant to inoculation of the cell-free agent. 4. The leukemic agent could be preserved at −70°C from 46 days to 5 1/2 months, with no apparent loss of infectivity.

Effect of thymectomy on development of leukemia in C3H mice inoculated with leukemic passage virus.

Summary 1. Of 39 young and adult C3H mice, thymectomized, and a few days later inoculated intraperitoneally with passage A leukemic filtrate, none developed leukemia. Of 41 normal controls inoculated simultaneously with the same filtrate 18 (44%), developed leukemia at 7 months average age. 2. Of 55 suckling C3H mice inoculated with passage A leukemic filtrate when less than 9 days old, then 1 month later thymectomized, only 1 thus far developed a palpable spleen, and definite leukemia. Of 78 control litter mates inoculated simultaneously with the same filtrates, but not thymectomized, 68 (87%), developed leukemia at 2.7 months average age. 3. Thymectomy, either preceding, or following inoculation of leukemic passage A filtrate, inhibits development of leukemia in otherwise susceptible C3H mice.

Development of myeloid (chloro)-leukemia in thymectomized c3h mice following inoculation of lymphatic leukemia virus.

Summary 1. Thirty-nine C3H mice were thymectomized at 4 to 6 weeks of age and inoculated a few days later with passage A leukemic filtrates. None developed leukemia, although observed until they were 15 months old. Of 41 non-thymectomized adult control mice injected simultaneously with the same filtrates, 63% developed leukemia at 8.7 months average age. 2. Of 60 C3H mice inoculated with passage A leukemic virus at average age of 3 days and thymectomized a month later, 26 (43%) developed leukemia at 10 months average age. Among leukemic mice in this group. 8 developed myelogenous (chloro) leukemia. Of 85 litter-mate controls injected simultaneously with the same filtrates. but not thymectomized, 93% developed lymphatic leukemia at 3.6 months average age. 3. Myelogenous leukemia developing in some virus-injected and subsequently thymectomized mice could be readily transplanted to mice of the same strain, retaining its myelogenous form or changing to lymphoid. 4. Susceptibility of thymectomized mice to the leukemogenic action of passage A virus could be restored by subcutaneous implantation of thymuses from young normal C3H mice. Of 20 C3H mice injected with the leukemic virus, then thymectomized, and which subsequently received subcutaneous implants of normal C3H thymuses, 18 developed leukemia (2 of these were myelogenous), as compared with 35 of 36 non-thymectomized controls that had received the same filtrates.

Induction of leukemia in rats with mouse leukemia (passage A) virus.

Summary 1. A mouse leukemia virus, originally isolated from spontaneous Ak leukemia, then passed serially through newborn mice (passage A), and consistently leukemogenic for suckling mice of susceptible strains, was found to be leukemogenic also for newborn rats. 2. Following intraperitoneal inoculation of passage A filtrates into newborn Sprague-Dawley rats, leukemia developed in from 57% to 73% of the inoculated animals, after a latency varying from 2 1/2 to 4 1/2 months. 3. Either localized thymic lymphosarcomas, or generalized lymphatic leukemia, involving also spleen, liver, and mesenteric lymph nodes, developed in the inoculated animals. A few rats developed acute stem-cell leukemia.

Induction of Parotid Carcinomas and/or Subcutaneous Sarcomas in C3H Mice with Normal C3H Organ Extracts.∗

Summary 1. Parotid tumors or leukemia developed following inoculation of Ak extracts into newborn C3H mice. When extracts from induced C3H leukemias were inoculated into 105 newborn C3H mice (Bittner substrain), 25 (24%) developed leukemia, 12 (11%) parotid tumors, and 7 (7%) subcutaneous sarcomas. Of 296 newborn C3H mice inoculated with parotid tumor extracts, 24 (8%) developed leukemia, 21 (7%) parotid tumors, 15 (5%) subcutaneous sarcomas, 2 submaxillary gland tumors, and 2 medullary adrenal tumors. 2. Centrifuged extracts from normal C3H embryos were inoculated into 103 newborn C3H mice, and 3 developed parotid tumors. Of 9 newborn C3H mice inoculated with C57 Brown/cd embryo extracts, 1 developed parotid tumor. When centrifuged extracts prepared from normal C3H mammary glands were inoculated into 60 newborn C3H mice, 9 developed parotid tumors, and 5 subcutaneous sarcomas. Centrifuged pooled normal C3H(f) and C3H organ (liver, spleen, heart, kidney) extracts fresh, or preserved either by dry-freezing or in 50% glycerine, were inoculated into 74 newborn C3H mice, and 10 (14%) developed parotid tumors. 3. Physiological saline solution or sterile tryptophane broth inoculated into 62 newborn C3H mice, did not induce tumors or leukemia. 4. Ak mice may carry not only a leukemic, but also another oncogenic agent, the latter not pathogenic for its natural carrier. C3H mice may also carry such a masked agent. A blind transfer into a newborn susceptible (C3H) host may sufficiently increase its pathogenic potency to cause parotid tumors. The sarcomas may be caused by another, individually distinct, though biologically similar agent.

High susceptibility of 1 to 14 days old C3H mice to passage A leukemic filtrates.

Summary 1. After 7 consecutive passages through newborn hosts, passage A leukemic filtrates could be inoculated successfully into suckling C3H mice 1 to 14 days old, inducing leukemia, but not parotid tumors, in 62% to 100% of the inoculated mice at 3.3 to 3.7 months average age. 2. The incidence of leukemia was higher in mice inoculated intraperitoneally (89%) than in those injected subcutaneously (69%). 3. Of 122 young, adult C3H mice inoculated intraperitoneally. or intracranially with leukemic filtrates. 37% developed leukemia (none parotid tumors) at 8.8 months average age. 4. Of 310 newborn C3H mice inoculated when less than 16 hours old with the same leukemic filtrates. 64% developed either leukemia (56%), or parotid tumors (8%), at 3.8 months average age. Two mice in this group developed both leukemia and parotid tumors.

Susceptibility of Newborn Mice of an Otherwise Apparently “Resistant” Strain to Inoculation with Leukemia.

Summary 1. A strain of transplantable lymphatic leukemia which originated in a mouse of the Ak line was found to be highly pathogenic for suckling infants of the C3H inbred line. Cell suspensions (20%) prepared from liver, spleen and lymph glands of leukemic mice of the Ak line, reproduced acute leukemia within 12 days when inoculated under the skin (0.05 cc) of newly born suckling mice of the C3H line. 2. This striking susceptibility of suckling C3H infants to inoculation with Ak leukemia appears to be limited to their first few days of life. Practically all infants of the C3H line (82 out of 85) developed leukemia when inoculated within the first 7 days of life; only 27 out of 57, however, were found to be susceptible when inoculated at ages varying from 8 to 15 days. Of 60 infants, 16 to 30 days old, only 15 reacted to inoculation. 3. Forty-nine adult mice of the C3H line were inoculated subcutaneously and 9 intraperitoneally with large doses (0.2 to 1 cc) of Ak leukemia and none reacted. When, however, overwhelming doses (3 cc each) of the Ak leukemic cell suspension were injected into adult mice of the C3H line, 1 out of 5 inoculated subcutaneously, and all 6 inoculated intraperitoneally died from leukemia.