Charlotte Friend

Antibiotic Effect of Tylosin on a Mycoplasma Contaminant in A Tissue Culture Leukemia Cell Line.

Summary A tissue culture line of murine virus-induced leukemia cells was contaminated by a strain of mycoplasma (PPLO) which may be or is related to M. granularum. This organism was resistant to treatment with tetracyclin, kanamycin, erythromydn, chloromycetin, polymixin, stovarsol and iodine, but sensitive to Tylosin. Two consecutive treatments with Tylosin (Purina tylan) at a concentration of 50 μg/ml were non-toxic to the cells and were effective in ridding the cultures of PPLO.

Leukemia of adult mice caused by a transmissible agent.

The attempts to transmit leukemia to adult mice with cell-free material have been numerous, but only a few have met with some measure of success. Two of these reports of successful transmission deserve mention, particularly in connection with Gross’s work, included elsewhere in this monograph. Engelbreth-Holm and Frederiksen2 have reported that 36 of 179 young Ak mice that had been inoculated with cell-free preparations of lymph nodes from leukemic mice of the same strain developed leukemia a t an age when the spontaneous disease was rarely seen. Similar findings in the same strain of mice have recently been described by Schwartz et aL3 These workers, using filtrates of the brain of leukemic animals, obtained a higher percentage of “takes.” Both papers pointed out the possibility that the leukemias may have been of spontaneous origin, but that they were accelerated by the treatment. The present report concerns a disease that has a marked resemblance to leukemia and that has been found to be serially transmissible to adult mice by means of cell-free filtrates. The report stems from earlier studies on the Ehrlich ascites tumor, initiated after the electron microscopic examination of this tumor had shown that some of the cells contained cytoplasmic particles that were suggestive of virus infection.* In the light of Morgan’s findings, elsewhere in this monograph, it is easy to see how misleading such an interpretation may be. In any case, an attempt was made then to explore the possibility of the viral etiology of this tumor. Extracts of the ascites cells were made. After being centrifuged a t high speed, the supernatant fluid was inoculated into infant Swiss mice. The filterable agent under discussion was recovered from a mouse in one of these experiments.

Effect of 2,6-diaminopurine on Virus of Russian Spring Summer Encephalitis in Tissue Culture.

Summary and Conclusions (1) The multiplication of the virus of Russian Spring Summer Encephalitis in tissue culture is inhibited by 2,6-diaminopurine. (2) The action can be blocked as well as reversed by the addition of equimolar concentration of adenine. Guanine is not effective.

Stimulation by dimethyl sulfoxide of erythroid differentiation and hemoglobin synthesis in murine virus-induced leukemic cells.

A fruitful avenue of approach to the complex problems involved in leukemogenesis has been provided by a system which utilizes cell lines that were established in our laboratory from a murine virus-induced erythroleukemia. The details of the establishment and properties of these cell lines, which were derived from the leukemic tissues of Friend virus (FLV)-infected mice, have been described in a recent review.2 Briefly, when fragments of the leukemic spleen and liver are implanted subcutaneously into syngeneic mice, tumors develop at the inoculation site.3 The tumors are transplantable; they do not contain recognizable erythroid cells, but when inoculated into lethally X-irradiated animals, they give rise to spleen colonies that contain both leukemic cells and immature erythroid c e k 4 The tumor cells can also be propagated in tissue culture, where differentiation along the erythroid pathway is observed in the cells of cloned lines as well as of mass-cultured lines. Cells are present a t various levels of maturation, from blast forms to rare orthochromatophilic normoblasts. Tissue culture lines of FLV-induced erythroleukemia cells that have similar characteristics have now also been established by other investigator^.^^^ When the cultured leukemic cells are bioassayed in the syngeneic DBA/2J mice, they produce malignant tumors indistinguishable from the tumors from which the cell lines were derived. The cells of these lines synthesize hemoglobin of the same type as that produced by DBA/2J m i ~ e . ~ . ~ Both the mass-cultured and cloned lines of these cells (FLC) exhibit the following properties:

Hemoglobin biosynthesis in murine virus-induced leukemia cells in vitro.

The potential for exploring the mechanisms controlling differentiation and malignancy of a murine virus-induced erythroleukemia has been demonstrated in our previous studies., 2 3 This leukemia resembles the early stages of di Guglielmos disease in man 4 in that, shortly after the inoculation of the virus into susceptible mice, an acute erythremia develops. Primitive cells and erythroid precursors in the blood, bone marrow, spleen and liver are characteristic of the When fragments of the leukemic spleen or liver are implanted subcutaneously into syngeneic mice, tumors that are transplantable appear at the site of ino~ulation.~ The cells of these tumors, which resemble reticulum cell sarcomas and are without any recognizable erythroid activity,R differentiate along the erythroid pathway when propagated in tissue culture. The origin and characteristics of this system have been described in a recent ~ e v i e w . ~ These leukemic cells, which continue to synthesize virus and maintain their malignancy, are uniquely suited for the study of the regulation of hemoglobin synthesis, since they provide established lines of erythroid precursor cells that can be stimulated to differentiate in vitro. When the cells are grown in medium supplemented with dimethyl sulfoxide (DMSO) a or N,N-dimethylformamide (DMF),l0 there is a decrease in the malignancy of the cells and erythroid differentiation is markedly enhanced. The present communication describes the sequence of events leading to the synthesis of hemoglobin in DMSO-stimulated Friend leukemia cells (FLC).

Effect of 2,6-diaminopurine on the Course of Russian Spring-Summer Encephalitis Infection in the Mouse.∗

Summary The compound 2,6-diamino-purine when administered in doses of 70 mg/kg caused an increase in the survival rates of mice infected with Russian Spring-Summer Encephalitis by the intraperitoneal route. It was effective when treatment was started 24 hours after the inoculation of the virus.

Lymphomas in Mice: Failure of Induction after a Graft-versus-Host Reaction

A mild graft-versus-host reaction induced in (BALB/cJ x DBA/2J) F1 mice by the administration of parental spleen cells that differ at several weak histocompatibility loci did not influence the development of lymphomas in these animals. Rous sarcoma virus also failed to induce tumors in the runt and control animals. Breast carcinomas, presumably due to contamination of the inoculums with mammary tumor virus, occurred in those experimental groups given parental cells, whether or not they were viable or immunologically competent. We found no evidence that the immunologic process—as represented by the graft-versus-host reaction—is causally related to the induction of neoplasia.

Carbonic Anhydrase Isozymes in Cultured Friend Leukemic Cells

Summary Carbonic anhydrase isozymes I and II (CA I and CA II) were assayed in cultured Friend leukemic cells by radial im-munodiffusion. In dimethyl sulfoxide-treated cultures, CA I levels remain constant or decrease somewhat, while CA II levels increase. However, in untreated control cultures both CA I and CA II levels increase. We thank Ms. Ya-Shiou L. Yu for technical assistance.

Variations in the response of cloned murine friend erythroleukemia cells to different inducers

SummaryCells of the line 3BM-78 derived from murine bone marrow cells infected in vitro with polycythemic Friend leukemia virus (FLV-P) produce virus with spleen focus-forming activity (SFFV) and can be induced to synthesize hemoglobin. Fifteen clones, isolated from this line, have been analyzed in detail for the effect of different inducing agents (dimethylsulfoxide, DMSO; hexamethylene bisacetamide, HMBA; and sodium butyrate, SB) on the synthesis of hemoglobin and virus at the clonal level. All the clones proved to be inducible with one or more of the agents, but the degree of the response depended on the type and concentration of the agent used. In general, the effectiveness of the agent—within the usual range of concentration for induction—both for hemoglobin and for virus synthesis, was in the order HMBA>DMSO>SB. Reverse transcriptase activity was, however, more easily induced than hemoglobin synthesis in that stimulation was seen at lower concentrations of the same inducing agent. This clonal analysis confirmed that virus and hemoglobin production are regulated independently in these erythroleukemic cells chronically infected with FLV-P.

Requirement of live Friend leukemia virus for enhanced expression of Hybrid histocompatibility genes.

Summary Bone marrow cells of DBA/2 mice became histoincompatible to irradiated (BALB/c X DBA/2)F1 and (C57BL/6 × DBA/2)F1 hybrids a few hours after infection with Friend leukemia virus (FLV). This alteration was attributed to enhanced expression of Hybrid histocompatibility (Hh) gene(s), i.e., to more effective immunogenicity of the parental-specific alloantigens specified by Hh gene(s). The viral effect required live virions since it was abolished by inactivation of FLV with beta-propiolactone.