Cultured hepatocytes from adult Fischer 344 rats were transformed by virion or cloned simian virus 40 (SV40) DNA using the calcium phosphate method. Transformation by SV40 occurred in either serum-supplemented medium or chemically defined medium (CDM). The frequency was greatest in serum-supplemented medium but transformants did not remain differentiated. In contrast, SV40 transformants developed less frequently in CDM, but retained differentiated functions. The frequency of transformation was enhanced by treatments that stimulated cell proliferation, in particular supplementing CDM with epidermal growth factor. Hepatocytes transformed in CDM were epithelial in morphology, secreted albumin, transferrin, hemopexin, and expressed the enzyme glucose-6-phosphatase, all characteristics of normal liver. Transformants did not produce detectable levels of α-fetoprotein, a marker of fetal or abnormal liver. We conclude that ( a ) hepatocytes can be transformed by transfection with SV40 DNA; ( b ) the frequency of transformation is enhanced by stimulating DNA synthesis; and ( c ) the transformed cells retain specific functions of normal hepatocytes in situ . Using this system it will be possible to study transformation of hepatocytes by viral and cellular oncogenes and to determine their effects on hepatocellular differentiation.

https://cancerres.aacrjournals.org/content/canres/46/8/4018.full.pdf

Transformation of Rat Hepatocytes by Transfection with Simian Virus 40 DNA to Yield Proliferating Differentiated Cells

A method is described for the culture of rat ovarian surface epithelial cells, i.e., the cellular component thought to be the source of most ovarian cancers. These cells in culture have a characteristic epithelial morphology which distinguishes them from other ovarian cell types. Cultured surface epithelial cells are histochemically positive for 17 beta-hydroxysteroid dehydrogenase and negative for delta 5-3 beta-hydroxysteroid dehydrogenase, the same as in cryostat sections of whole rat ovary. Ultrastructurally, cultured surface epithelial cells have basement membranes, microvilli, and apical intercellular junctions. Kirsten murine sarcoma virus was used to produce three transformed cell lines from pure first-passage cultures of these cells. These three lines retained 17 beta-hydroxysteroid dehydrogenase activity and showed slight delta-3 beta-hydroxysteroid dehydrogenase activity. Tumors resulting when these cells were injected s.c. or i.p. into immunosuppressed female rats were highly malignant, resembling histologically human ovarian endometrioid stromal sarcoma. This is the first demonstration of the susceptibility of ovarian surface epithelium to an oncogenic virus.

https://cancerres.aacrjournals.org/content/canres/41/6/2063.full.pdf

Transformation of cultured rat ovarian surface epithelial cells by Kirsten murine sarcoma virus.

Fischer rat thyroid epithelial cells (FRT) growing continuously in culture were infected with the Kirsten murine sarcoma virus KiMSV(KiMuLV) and found to produce this virus constitutively. Although the morphology of the FRT cells did not change appreciably, the cells became malignant after infection with KiMSV(KiMuLV) as shown by the growth of infected cells in semi-solid media (uninfected FRT cells did not grow) and by the tumorigenicity of infected cells when injected into syngeneic animals (uninfected FRT cells or FRT cells infected with non-transforming retro-viruses were not tumorigenic). The induced tumors morphologically resembled moderately differentiated carcinomas. Two markers of thyroid epithelial differentiation were absent in the original FRT clone and remained unexpressed after transformation. Fully differentiated rat thyroid epithelial cells (FRT-L cells) infected with another strain of the Kirsten murine sarcoma virus, the KiMSV(MolMuLV), were also transformed as demonstrated by the ability also of these cells to grow as carcinomas (after in vitro transformation) in syngeneic animals. Our results clearly demonstrate that the Kirsten murine sarcoma virus can transform in vitro cells of epithelial as well as of fibroblastic origin.

Transformation of rat thyroid epithelial cells by Kirsten murine sarcoma virus

Apoptosis caused by inadequate or inappropriate cell-matrix interactions is defined as anoikis. Although transformed cells are known to be anoikis-resistant, the underlying mechanisms have not been well understood. We investigated the mechanisms of anoikis resistance of tumor cells. We observed that cell aggregation in suspension promoted cell survival and proliferation. We demonstrated a correlation between tumor cell aggregation in suspension and cell growth in soft agar. Analysis of tyrosine kinase-mediated cell survival and growth signaling pathways revealed increased levels of tyrosine-phosphorylation of PECAM-1 and Pyk2 in cell aggregates. We also showed that PECAM-1 and Pyk2 physically interact with each other, and that PECAM-1 carrying a deletion of exons 11-16 could no longer bind to Pyk2. Furthermore, RNA interference-mediated reduction of Pyk2 and PECAM-1 protein levels reduced cell aggregation and inhibited the growth of tumor cells in soft agar. The data demonstrated that Pyk2 and PECAM-1 were critical mediators of both anchorage-independent growth and anoikis resistance in tumor cells.

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Cell aggregation induces phosphorylation of PECAM-1 and Pyk2 and promotes tumor cell anchorage-independent growth

Differentiated, cloned rat thyroid epithelial cells (424 cells) were infected with a wild-type and a temperature-sensitive strain of the myeloproliferative variant of the Moloney murine sarcoma virus. The thyroid cells were productively infected and transformed by both virus strains and displayed some of the typical properties of malignant cells, such as morphological changes, growth in soft agar, and in vivo tumorigenicity. The acquisition of the transformed phenotype by the virus-infected cells was accompanied by a loss of the typical differentiated features of the thyroid epithelium, such as thyroglobulin (TG) secretion, iodide uptake, and dependence for growth on six factors including thyrotropin, the physiological thyroid stimulator. TG mRNA could not be demonstrated in cells transformed by both viral strains, suggesting a block at the level of the TG gene transcription. While the transformed state of the cell clones infected with the temperature-sensitive strain could be reverted by shifting the cultures to the temperature nonpermissive for transformation (39 degrees C), no reversion of the differentiated functions took place after such a shift, showing that the v-mos oncogene irreversibly shuts off the differentiation of thyroid epithelial cells in vitro. These results demonstrate, for the first time, an oncogenic potential of the v-mos oncogene family towards differentiated epithelial cells in vitro.

A mos oncogene-containing retrovirus, myeloproliferative sarcoma virus, transforms rat thyroid epithelial cells and irreversibly blocks their differentiation pattern.

We studied the transformation of epithelial, diploid cell lines (RL-33 and RL-34) derived from W rat liver by the Kirsten murine sarcoma virus. On days 4-5 after virus infection, the epithelial cells began to pile up focally, forming small projections and releasing round cells from the foci. The epithelial cells grew in chains or as islets and grew in suspension above the cells attached to the bottom of the flasks when the cultures reached the confluent stage. The virus titration pattern was "one-hit." Three classes of transformed cells were isolated with respect to virus release and antigen expression: 1) virus producer, 2) non-producer, and 3) sarcoma-positive, leukemia-negative cells. When transplanted sc into newborn rats, the transformed cells produced sarcomas. The transformed cells formed within 1-3 days larger aggregates than those of their normal counterpart cells when suspended in liquid growth medium above an agar base. Aggregate properties (size, viability, and proliferation) of transformed cells correlated with growth in soft agar and tumorigenicity. RNA-dependent DNA polymerase and type C virus particles were readily induced in the normal rat liver epithelial cells after exposure to 5-iodo-2'-deoxyuridine.

R. J. Huebner

Papers

Transformation of Rat Liver Epithelial Cells by Kirsten Murine Sarcoma Virus