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A model for human osteosarcoma in hamsters.

An animal model for human osteosarcoma was established in newborn Syrian golden hamsters by injecting productively infected TE-85 cells (cultured human osteosarcoma cells) adjacent to the femur. Tumors were palpable 10 to 15 days following cell injection and enlarged progresively until the animal died (mean survival time was 36 days). The tumor take was 100% and all animals developed pulmonary metastases. Osteoid and bone were identified in sections of the tumor by light and electron microscopy. Invasion of the marrow spaces and adjacent skeletal muscle by the malignant osteoblasts, anaplastic sarcoma cells and multinucleated giant cells was frequently observed. The tumor was transplantable. TE-85 cell surface antigens were demonstrable by immunofluorescence on the surface of the cells of the induced tumors. The present tumor model had unique immunologic characteristics in that no antibodies were demonstrable in the sera of the tumored animals.

Animals

Purification and biochemical characterization of a virus-specific reverse transcriptase from human osteosarcoma tissue.

A RNA-dependent DNA polymerase (RTase) was purified from human osteosarcoma tissue by successive column chromatography of the microsomal fraction on DEAE-cellulose (DE-23 and DE-52) and phosphocellulose. The purified enzyme has a molecular weight of about 68,000, a pH optimum of 8.1, a Mg2+ optimum of 0.8 mM, Mn2+ optimum of 1.0 mM and a KCl optimum of 60 mM. The enzyme transcribes (rA)n . (dT)12, (rC)n . (dG)12-18 and (2-O-methyl C)n . (dG)18, but is unable to transcribe (dA)n . (dT)10. The enzyme has no catalytic activity in the presence of oligodeoxynucleotide initiators alone, indicating the absence of terminal deoxynucleotidyl transferase. The purified enzyme is able to transcribe the heteropolymeric regions of a 70S RNA from R(Mu)LV. The presented data support the presence of a RNA-dependent DNA polymerase in human osteosarcoma tissue with biochemical properties, resembling those of C-type RNA tumor viruses.

Adolescent

An animal model for human osteosarcoma.

Osteosarcomas formed in antilymphocyte serum (ALS)-treated hamsters when 2x10(6) TE-85 human osteosarcoma cells (maintained in tissue culture) infected with M-MSV (RD-114) virus were injected adjacent to the femur or the scapula; undifferentiated sarcomas formed when 1 x 10(6) cells were injected subcutaneously. Tumors were palpable 10 to 14 days after the cells were injected and grew progressively until the animals died (mean survival time was 30 days). All animals had pulmonary metastases. Neither the subcutaneous sarcomas nor the metastases contained bone or osteoid; however, the osteosarcomas adjacent ot the femur and scapula contained collagen, osteoid and calcified bone when observed by light and electron microscopy. These results indicate that the TE-85-M-MSV cell-ALS hamster system is an animal model for the study of osteosarcomas of human cell origin.

Animals

Transformation of human osteosarcoma cells by a chemical carcinogen.

A human osteosarcoma clonal cell line (TE-85, clone F-5) was treated in vitro with various levels of 7,12-dimethylbenz[a]anthracene or dimethyl sulfoxide (control). Cells treated only with the carcinogen underwent morphologic alteration in vitro, and one of these altered cell lines produced tumors subcutaneously and intracerebrally when injected into NIH nude mice.

9,10-Dimethyl-1,2-benzanthracene

Placenta-like alkaline phosphatases from human osteosarcoma cells.

Hormone-induced alkaline phosphatases in human osteosarcoma cells (LM) were extracted and purified. Characterization of the purified enzyme showed two distinct isoenzymes. One isoenzyme was heat labile, was homoarginine inhibited, and had the electrophoretic migration of alkaline phosphatase of human osseous origin. Immunodiffusion showed that this isoenzyme reacted positively only against anti-bone alkaline phosphatase antibodies. The second isoenzyme was heat stable, was inhibited by phenylalanie, and had the same electrophoretic migration as did alkaline phosphatase extracted from mature normal human placenta. This second isoenzyme had the same antigenicity as did the normal placental enzyme. Like the D-variant placental phenotype, this second isoenzyme was inhibited by L-leucine and ethylenediaminetetraacetic acid.

Alkaline Phosphatase

A virally induced osteosarcoma in rats. A model for immunological studies of human osteosarcoma.

Inoculation of Moloney sarcoma virus into the medullary canal of the tibia in newborn Wistar-Lewis rats resulted in an initially localized osteosarcoma which usually metastasized to the lung and resulted in the death of the animal within four to five weeks. Tumor cells were grown in tissue culture and used as target cells in the assay of lymphocyte-mediated cytotoxicity using a microcytotoxicity and a radioisotope labeling method. Lymphocyte-mediated cytotoxicity was demonstrated throughout the course of the clinical disease as well as in a small number of animals which showed spontaneous regression of their tumors. Serum factors which could "block" or augment the cellular response were also identified. This model resembles the spontaneous osteosarcoma of humans in many respects and may be useful for studies of the human disease.

Animals

Effect of xenogeneic immune RNA on normal human lymphocytes against human osteosarcoma cells in vitro.

New Zealand White rabbits were immunized with whole-cell suspensions of TE-85 cells (from a human osteosarcoma) maintained in tissue culture. RNA was extracted from the lymphoid tissues of the immunized animals. Normal human peripheral blood lymphocytes were pretreated with both the whole-cell immune RNA (IRNA) and the Sephadex column-eluted fractions of the whole-cell IRNA. Significant stimulation of the cytotoxic effect of the lymphocytes was observed following whole-cell IRNA pretreatment and pretreatment with peak III fractions eluted from the column. This increase in inhibition was observed whether the target cells were TE-85 (the immunizing cells), L.M. and M.Mc. (two unrelated osteosarcoma primary cell cultures), or TE-85-M-MSV cells (a cell line capable of producing a human osteosarcoma in immunosuppressed hamsters). No inhibition was observed when cells from other types of human tumors were used as target cells. The results suggested that the transferred immunity was directed against tumor-specific osteosarcoma antigens.

Antigens, Neoplasm

Isolation and partial purification of plasma membrane-associated antigens from human osteosarcoma (TE-85) cells in tissue culture.

The plasma membrane of a cloned line of TE-85 (human osteosarcoma) cells subcultured for the last 4 years was isolated. The isolation was by hypotonic swelling, cell homogenization, and discontinuous sucrose gradient ultracentrifugation for 16 hr. Tumor-specific water-soluble antigens were identified by limited papain digestion of the isolated plasma membrane. Fractionation by diethylamino-ethyl anion-exchange column chromatography yielded antigenic fractions that inhibited the reaction of immune serum (from an unrelated patient with osteosarcoma) with the plasma membrane of TE-85 cells in tissue culture by indirect immunofluorescence test. Microimmunodiffusion confirmed the specificity of the isolated antigen against the sera of other patients with osteosarcoma. The definition of the antigen fraction may permit evaluation of antigen-antibody interaction in tumor immunity.

Antigen-Antibody Complex

A technique for developing established cell lines from human osteosarcomas.

A method is described which has been successfully used to develop two human osteogenic sarcomas into established lines in culture. This method provides a means whereby cells growing from explanted tumor tissue can be immediately cloned and the fibroblastic (nonneoplastic) cells thus selected against. Both lines have been passaged for over 100 population doublings since cloning and have retained the ability to form colonies from single cells plated at low density without the use of feeder layers or conditioned medium. In culture, the osteogenic sarcoma cells are nonfibroblastic, pile up, and appear to retain a morphological similarity to the in vivo tumors from which they were derived. A karyotype of cells derived from one of the tumors containing a marker chromosome is also presented.

Adolescent

Effect of human leukocyte interferon on the growth of human osteosarcoma cells in tissue culture.

Nine osteosarcoma cell lines, originally developed from six osteosarcoma tumours in five patients, and two cell lines of non-tumour origin (glia and fibroblast) were grown in vitro in the presence of human leukocyte interferon (L-IF). L-IF exerted a dose-dependent inhibition of growth in all these lines. The inhibitory activity displayed characteristics typical of interferons. Inhibition of cell growth occurred at a much lower L-IF concentration for the osteosarcoma than for the non-tumour-derived lines. Inhibition of tumour cell growth was observed at concentrations obtained in the serum of osteosarcoma patients treated with interferon.

Cell Count

The ultrastructure of human osteosarcoma: a study of nine cases.

Electron microscopic study of 9 osteosarcomas corroborates the light microscopic observation that cells with osteoblastic, chondroblastic and fibroblastic features occur in osteosarcomas. Two types of multinucleated giant cells were observed, one was osteoclast-like. The other had anaplastic and degenerative changes. The intercellular matrix is composed of collagen fibers, amorphous ground substance with osteoid and tumor bone. Contrary to many previous electron microscopic reports, osteosarcomas contain more than one cell type.

Adolescent

Dialyzable transfer factor in the treatment of human osteosarcoma: an analytic review.

In conclusion, then, we would answer the seven questions raised earlier concerning transfer factor as follows: Certianly, as shown by clinical results, it does exist. It does have a definite immunologic effect in humans, boosting cell-mediated immunity, as shown by a rise in the level of active T cells. Its clinical effects have been demonstrated repeatedly, and it should become useful in still other clinical situations as further research provides more effective therapeutic modalities. Transfer factor from selected donors appears to provide prophylaxis against metastasis when administered to osteosarcoma patients with no clinically evident metastases at the time of surgical removal of the primary tumor; whether this treatment is superior to chemotherapeutic prophylaxis is conjectural and controversial. Its mechanism of action has not been demonstrated as yet, although many theories exist. The best evidence is that the effects are both specific and nonspecific. It appears to be produced by T lymphocytes. The exact nature of the substance we call "transfer factor" remains to be elucidated. Further research should provide more conclusive answers to these questions.

Animals