Transfer of murine intracisternal A particle phenotype in chloramphenicol-resistant cytoplasts

Malech, Harry L.; Wivel, Nelson A.

doi:10.1016/0092-8674(76)90083-0

Cited by 22 publications

(2 citation statements)

References 23 publications

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“…As previously reported (2), these cell fragments die within 48-72 hr after separation, but in the past 2 years, workers in several laboratories have reported reconstitution and subsequent viability of mammalian cells by fusing karyoplasts from a specific cell line to cytoplasts from the same cell line (4-6) (homospecific) or to other cell lines (7,8) (9) that allows them to be distinguished from the 3T3 cells. Cells to be enucleated were plated in Falcon 3013, 25-cm2 flasks (Falcon Plastics, Oxnard, CA) as described (10).…”

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confidence: 86%

Selection of reconstituted cells from karyoplasts fused to chloramphenicol-resistant cytoplasts

Shay¹

1977

Proc. Natl. Acad. Sci. U.S.A.

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Murine Balb/3T3 and murine A-MT-BU-A1 mammary tumor cells were separated in the presence of cytochalasin B into enucleated cytoplasmic components (cytoplasts) and nucleated subcellular components (karyoplasts). Karyoplasts were derived from 3T3 cells, while cytoplasts were derived from A-MT-BU-A1 cells that were both chloramphenicol-resistant (CAPr) and sensitive to hypoxanthine/aminopterin/thymidine (HATS). CAPr has been shown to be cytoplasmically transmitted (possibly a mitochondrial gene mutation), while sensitivity to medium containing HAT has been shown to be transmitted by the nucleus (i.e., nuclear gene mutation). Such CAPr cytoplasts derived from A-MT-BU-A1 cells were then fused, using polyethylene glycol, to HAT-resistant 3T3 karyoplasts. The mononucleated reconstituted cells produced by such procedures were cloned in medium containing both HAT and CAP. Some of the reconstituted cells survived, because they were resistant to both drugs, while the nuclear and cytoplasmic whole cell contaminants were killed by one or the other of the two drugs. The results of these experiments indicate that reconstituted cells that are derived from two different cell lines are viable, as indicated by their ability for long-term proliferation in culture. Most of the clones derived resembled morphologically the 3T3 nuclear donor parent cells, but some of the clones did not resemble either parental cell line. It is anticipated that such selection techniques will permit more complete analysis of interrelationships between nucleus and cytoplasm.Cytochalasin B can be used to separate animal cells growing in monolayer culture into nuclear [karyoplasts (1, 2) and minicells (3)] and cytoplasmic [cytoplasts (1, 2)] components. As previously reported (2), these cell fragments die within 48-72 hr after separation, but in the past 2 years, workers in several laboratories have reported reconstitution and subsequent viability of mammalian cells by fusing karyoplasts from a specific cell line to cytoplasts from the same cell line (4-6) (homospecific) or to other cell lines (7, 8) (heterospecific). These studies indicate that such reconstitution experiments are feasible, but convincing evidence that such reconstituted cells after several mitotic divisions are not whole cell contaminants that detached during enucleation or that failed to enucleate during the centrifugation is still lacking. The main reason such reconstituted cells have not been satisfactorily identified, as opposed to whole cell contaminants, is due primarily to the lack of both nuclear and cytoplasmic markers that would permit the identification of reconstituted cells, at a time when the properties of such cells may have been modified by the influence of a new combination of nucleus and cytoplasm.

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confidence: 86%

Selection of reconstituted cells from karyoplasts fused to chloramphenicol-resistant cytoplasts

Shay¹

1977

Proc. Natl. Acad. Sci. U.S.A.

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“…Microscopy genic properties but also for their ultrastructural properties, by means of transmission electron microscopy. The AMT cells contain intracisternal A virus particles (IAP), which are excellent morphologic markers, These particles are not shed from the cells but are only transmitted vertically (that is, by mitosis)[14, 151 . The techniques for electron microscopy are standard procedures previously described[3, 161 .…”

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confidence: 99%

Nuclear control of tumorigenicity in cells reconstructed by PEG‐induced fusion of cell fragments

Shay¹,

Clark

1979

J Supramol Struct

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The techniques of somatic cell hybridization have provided a valuable means of studying mechanisms of regulation of mammalian cell differentiation and transformation. Most previous studies have indicated that fusions between tumorigenic and nontumorigenic cells result in hybrid cells that are usually tumorigenic. In recent years it has been demonstrated that the phenotypic expression of tumorigenicity is at least partially due t o the extensive chromosome loss that occurs in most interspecific and some intraspecific hybrid cells. In the present study we have utilized enucleation techniques that permit cells to be divided into nuclear (karyoplast) and cytoplasmic (cytoplast) cell fragments. Even though these nuclear and cytoplasmic fragments are metabolically stable for short periods of time, in our hands they ultimately degenerate. Viable cells can be reconstructed by PEG-induced fusion of karyoplasts to cytoplasts. Since reconstructed cells apparently do not segregate chromosomes, they may provide a clearer understanding of the interactions between the nucleus and the cytoplasm in the control of the expression of tumorigenicity. We have reconstructed cells using karyoplasts from the tumorigenic Y-1 cell line and cytoplasts from a nontumorigenic cell line, A-MT-BU-AI. In addition we have reconstructed cells containing Y-1 cytoplasts and A-MT-BU-A1 karyoplasts. The reconstructed cells produced were assayed for tumorigenicity by their ability t o grow in soft agar and in nude mice. The results of these experiments indicate that the reconstructed cells containing a tumorigenic nucleus and a nontumorigenic cytoplasm ultimately are tumorigenic and conversely the reconstructed cells containing a nontumorigenic nucleus and a tumorigenic cytoplasm are nontumorigenic. These experiments support the concept that with these cell lines the nucleus (karyoplast) is sufficient t o control the phenotypic expression of tumorigenicity.Key words: cell enucleation, cell reconstruction, nuclear control of tumorigenicity Hybrid cells produced by fusing tumorigenic and nontumorigenic cells from different species in most instances are tumorigenic. The tumorigenic phenotypes expressed in such hybrids have been partially explained by the extensive chromosome losses that occur in

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Interferon treatment of murine meth‐a sarcoma cells: Effects on the malignant phenotype and expression of tumor‐specific and H‐2 antigens

Wivel

Pitha

1982

Intl Journal of Cancer

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A virus-free methycholanthrene-induced sarcoma (Meth-A) in BALB/c mice was grown in culture and treated with purified mouse interferon (alpha and beta mixture) prior to testing for oncogenicity in the host animal. Use of interferon in vitro caused growth inhibition, but not cytotoxic effects; such effects were fully reversible upon interferon removal from the system. There was a significant decrease in tumor incidence in mice challenged with interferon-treated cells, but this could be overcome by sufficiently increasing the number of cells in the inoculating dose. By transplanting these BALB/c sarcoma cells into Sprague-Dawley nude mice, the effects of interferon could be negated. Since these mice have an unaltered activity of NK cells, the results suggest that NK cells do not play a major role in rejection of the Meth-A tumor, but that the reduction in tumor incidence is dependent on the presence of functional T cells. Interferon caused a detectable reduction in the expression of the tumor-specific transplantation antigen (TSTA) associated with Meth-A cells, but increased the expression of H-2 antigens. It has recently been shown that H-2 antigens play a role in host recognition of the TSTA of methylcholanthrene-induced sarcomas. It is suggested that the increased expression of H-2 antigens on interferon-treated Meth-A cells could lead to an increased frequency of recognition by T-cells.

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Transfer of murine intracisternal A particle phenotype in chloramphenicol-resistant cytoplasts

Cited by 22 publications

References 23 publications

Selection of reconstituted cells from karyoplasts fused to chloramphenicol-resistant cytoplasts

Selection of reconstituted cells from karyoplasts fused to chloramphenicol-resistant cytoplasts

Nuclear control of tumorigenicity in cells reconstructed by PEG‐induced fusion of cell fragments

Interferon treatment of murine meth‐a sarcoma cells: Effects on the malignant phenotype and expression of tumor‐specific and H‐2 antigens

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