Constitutive aggregates of intermediate-sized filaments of the vimentin and cytokeratin type in cultured hepatoma cells and their dispersal by butyrate.
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Biomedical subjects
Publications and source records attributed to E Borenfreund.
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The endoskeletal structure of tumor cells with a characteristic cytoplasmic abnormality initiated in rat liver by the in vivo adminisration of the carcinogen diethylnitrosamine was studied in clonal cell lines established and propagated in vitro. The bulk of the cytoplasmic and plasma membrane protein was removed by extraction with Triton X-100, and subsequently the juxtanuclear detergent-insoluble fraction containing filaments of 100-150 A was released into citrate buffer at pH 2.8. Analysis of this fraction by sodium dodecyl sulfate acrylamide gel electrophoresis revealed the pressence of five major proteins that banded with apparent molecular weights of about 66, 57, 52, 48 , and 43 x 10(3), the last of which comigrated with actin. The proteins thus resembled those from intermediate-sized filaments of both the vimentin (57 x 10(3)) and the prekeratin types obtained from various vertebrate cells. They also appeared to be related to the polypeptides of intermediate-sized filaments from Mallory bodies induced by griseofulvin in the livers of mice and to some of the polypeptides seen in isolates of Mallory bodies from human alcoholics. These results indicated that a major component of the carcinogen-induced lesion consisted of intermediate-sized filaments. The possible significance in cell transformation of this stably maintained aggregate of filaments that binds concanavalin A and displaces the nucleus is discussed. The close resemblance of this lesion to that seen in the cells of cirrhotic livers of alcoholics (Mallory's alcoholic hyalin) raises a question regarding the possible oncogenic status of such cells in humans.
Washed, non-viable sperm from AKR and NZB mice contain infectious murine type C viruses (MuLV), whereas similarly treated sperm from Balb/c and probably C57Bl/6 mice do not. Interaction of washed, viable AKR, Balb/c and NZB sperm with tissue culture cells leads to a transfer of infectious MuLV to these cells. The most likely mechanism is infection by virus closely associated with the sperm. Another possible mechanism is the introduction of proviral DNA. These experiments indicate that type C viruses can be found with mouse sperm. They suggest sperm could horizontally transmit these MuLV to animals during copulation and to the germ line of mice during penetration of ova.
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A late-gestation neonatal antigen (gamma foetal antigen; gamma-FA) immunologically and biochemically unrelated to murine alpha-foetoprotein, was identified in several spontaneous and carcinogen-induced sarcomas and hepatic carcinomas of the mouse and rat. An approximate mol. wt of 35,000 for gamma-FA from both foetus and tumour was obtained by molecular-sieve chromatography and sucrose-gradient centrifugation. Radial immunodiffusion analyses of organ extracts indicated that gamma-FA could be found in several neonatal tissues, the highest concentration occurring in the spleen. In the 2-month-old mouse, only splenic tissue contained gamma-FA and at much lower levels than in the organ of the newborn mouse.
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Epithelioid liver cells were established in culture from rats sacrificed 10 to 18 weeks after the administration of the hepatocarcinogen diethylnitrosamine in their drinking water. After approximately 3 months in vitro, more than half the cells propagated from rats that developed hepatocellular carcinomas had bean-shaped, acentrically displaced nuclei with large juxtanuclear homogeneous appearing areas resembling the hyalin or Mallory bodies in the livers of chronic alcoholics. These abnormalities were not seen in the livers of origin, but were retained in the carcinomas that formed after the cultured cells containing such juxtanuclear hyalin inclusions were inoculated into young rats or nude (i.e., thymusless) mice; these features persisted upon reestablishment and continuous passage of the tumor cells in culture. The cells were further characterized by their karyotypes and their growth properties in liquid media and soft agar. By transmission electron microscopy the hyaline bodies in the culture tumor cells were shown to consist of a disorganized meshwork of filaments. Examination by incubating cells with cytochalasin B and by using antiactin and anti-DNase antisera as indirect immunofluorescence probes also revealed a disturbance in the cytoskeleton.
Epithelial liver cells isolated from rats after oral diethylnitrosamine administration were established in culture. When propagated in vitro for 2--3 months, over half the cells contained acentric nuclei and large juxtanuclear inclusions resembling the hyaline deposits (Mallory bodies) in cirrhotic livers of alcoholics. The morphological changes and disarrayed filaments in these bodies were retained on serial passages for many months. Cells inoculated into nude mice or newborn rats produced carcinomas from which cells with these abnormalities were reestablished in continuous culture.
Bacteriophage T7 DNA can penetrate Syrian hamster embryonic cells after a mandatory initial pretreatment with DEAE-dextran. In 3 h an extracellular complex between T7DNA and the cell monolayer is formed which is equivalent to 105 T7 genomes per cell. During the ensuing 24-48 h of cell growth, an average of 102-103 T7 genomes are transported to the nucleus in 90% of the cells of the culture.
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Epithelioid cells from the livers of normal and genetically impaired (Gunn) rats were established in long-term cultures in vitro. These cells grew as flat, epithelioid cobble-stone-type monolayers and showed a diploid karyotype. They secreted rat serum albumin and proteins into their growth media and contained aryl hydrocarbon hydroxylase. Such cells were transformed by treatment with methylazoxymethanol acetate; they then exhibited an irregular, piling growth pattern, acquired the ability to grow in soft agar, and thereafter grew as tumors in hamsters given cortisone and in nude mice. These malignant spindle-cell tumors were reestablished in culture and still secreted serum albumin. The transformed cells became highly multinucleate when exposed to cytochalasin B and thus behaved like tumor cells. This behavior was not shown by the original cells. Cells transformed by benzo[a]pyrene failed to grow in soft agar culture or as tumor in animals. Cells were not affected by diethylnitrosamine.
Penetration of somatic mammalian cells by spermatozoa occurred after simple admixture in culture. With sperm labeled in vivo, autoradiography revealed incorporation of DNA into nuclei of recipient cells, indicating release of DNA after entrance by sperm. This system provides a new approach to study the molecular biology of information transfer and of haploid gene expression.
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