Thyroid cell transformation requires the expression of the HMGA1 proteins.

Elevated expression of HMGA1 and HMGA2 proteins is correlated with a highly malignant phenotype in several human tumors. We previously demonstrated that the block of HMGA2 protein synthesis prevented rat thyroid cell transformation by murine retroviruses. Suppression of HMGA2 synthesis was associated with lack of induction of HMGA1 proteins suggesting that both HMGA1 and HMGA2 play a role in the process of neoplastic transformation. To determine the role of the HMGA1 gene in thyroid cell transformation, we blocked HMGA1 protein synthesis by an antisense methodology. Here we report that transfection of an HMGA1 cDNA antisense construct into a normal rat thyroid cell line (FRTL-5 Cl2), followed by infection with Kirsten murine sarcoma virus (KiMSV), generated a transformed cell line that expresses high levels of the v-ras-Ki oncogene and that does not require thyroid-stimulating hormones for growth. However, this cell line does not show the malignant phenotype, i.e., it neither grows in soft agar nor induces tumors after injection in athymic mice. Moreover, the lack of the neoplastic phenotype in the virus-infected thyroid cells carrying the HMGA1 antisense construct correlates with the absence of induction of AP-1 transcriptional activity.

Retrovirus transformation associated secretory phosphoproteins of mouse and mink cells.

Using antisera to major excreted protein (MEP) of Kirsten sarcoma virus transformed NIH 3T3 (KNIH) cells, we have identified phosphoproteins from the media of feline sarcoma virus (FeSV) transformed mink cells. These secretory phosphoproteins from FeSV-transformed mink cells are of 35 kDa M.W. and they do not have autophosphorylation activity. A comparative analysis of MEP from the media of transformed mouse and mink cells was performed on the basis of proteolytic cleavage products and acid hydrolyzed products of the phosphoproteins. While a marked difference was observed in the peptide map, a common 32P-linked molecule was observed following acid hydrolysis of both species of phosphoproteins.

Isolation and characterization of Kirsten murine sarcoma virus-transformed mouse keratinocytes resistant to transforming growth factor beta.

BALB/MK (MK) is a continuous murine keratinocyte line whose cells are strictly dependent on exogenous epidermal growth factor (EGF) for growth in culture. A derivative cell, KC, resulted from Kirsten murine sarcoma virus transformation, and these cells no longer require EGF for their growth. Despite differences in MK and KC growth conditions, both cell lines are growth inhibited by picomolar concentrations of transforming growth factor-beta (TGF-beta). When MK and KC cells were maintained in the presence of TGF-beta, resistant variants eventually proliferated only from the KC population. In an attempt to determine the mechanism of development of TGF-beta resistance, the TGF-beta-resistant cells (KCR cells) were compared with TGF-beta-sensitive KC cells with regard to growth properties, TGF-beta 1 binding characteristics, and gene expression. KCR cells continued to synthesize DNA and proliferated in the presence of TGF-beta 1 concentrations up to 2 nM, which was 500-fold greater than the ED50 for the sensitive cells. Although the KCR cells possess similar receptor numbers and affinity for TGF-beta 1, we observed differences in affinity cross-linking studies. The KCR cells expressed more of the type III, high molecular weight cell surface binding protein and less of the type II than the KC cells. The type I moiety was clearly altered to a smaller size in some, but not all, KCR cells. In gene regulation studies, there was no apparent difference in c-Ki-ras and v-Ki-ras mRNA levels in the KC and KCR cells. Additionally, expression of TGF-alpha and TGF-beta 1 mRNA was similar in MK, KC, and KCR cells. The expression of proliferation-associated genes, such as c-myc and MGSA/c-gro/kc, which were markedly decreased by TGF-beta 1 in the MK and KC cells, was not altered by TGF-beta 1 in the KCR cells. The data suggest that the loss of TGF-beta 1 responsiveness in the KCR cells was due to an alteration in the TGF-beta receptor that did not permit signal transduction, although the existence of postreceptor alterations cannot be excluded.

A rat mutant cell clone showing temperature-dependent transformed phenotypes with functional expression of the src gene product.

The cellular mutant B814 isolated from a Fischer rat cell line shows temperature-sensitivity of focus formation on infection with Moloney murine sarcoma virus (Mo-MSV) and Rous sarcoma virus (RSV). An RSV-transformed clone (S814-2) isolated from B814 cells shows temperature-sensitive transformed phenotypes for morphology, growth in soft agar, and glucose uptake. The expression, phosphorylation, and tyrosine kinase activity of pp60v-src in S814-2 were not affected at the nonpermissive temperature, and virus rescued from this clone had wild-type transforming ability, suggesting that a cellular factor altered in S814-2 is responsible for the cellular steps of transformation after the function of pp60v-src. In addition, the cellular 36K protein, a possible candidate as a target of pp60v-src, was phosphorylated at the nonpermissive temperature in S814-2, indicating that phosphorylation of the 36K protein is not correlated with transformed phenotypes.

Cytoarchitecture of Kirsten sarcoma virus-transformed rat kidney fibroblasts: butyrate-induced reorganization within the actin microfilament network.

Murine sarcoma virus-transformed rat fibroblasts (KNRK cells) undergo marked cytoarchitectural reorganization during in vitro exposure to sodium-n-butyrate (NaB) resulting in restoration of (1) a more typical fibroblastoid morphology, (2) proper cell-to-cell orientation, and (3) substratum adherence. Augmented cell spreading, involving greater than 90% of the population, was a function of culture density and time of exposure to NaB (2 mM final concentration). Induced cell spreading reflected a 2.5- to 3.0-fold increase in both total cellular actin content and deposition of actin into the detergent-resistant cytoskeleton. Cytoskeletal actin deposition in response to NaB was accompanied by the formation of occasionally dense, parallel alignments of F-actin-containing microfilaments and by a dramatic increase in the size and incidence of actin-enriched membrane ruffles. Long-term NaB-treated cells exhibited parallel orientations of microfilaments similar to those found in untransformed fibroblasts. Increased cytoskeletal actin occurred within 24 hr of NaB exposure, correlating with the initial reorganization of actin-containing microfilaments detected microscopically, and reflected concomitant 3-fold increases in cellular alpha-actinin and fibronectin content. In contrast, the amount of vimentin, tropomyosin, and tubulin in NaB-treated cells was significantly decreased. NaB-induced morphologic restructuring of sarcoma virus-transformed fibroblasts, thus, impacts on all three basic cytoskeletal systems. Selective increases, however, were evident in particular cytoskeletal proteins (actin, alpha-actinin, fibronectin) implicated in microfilament networking and cell spreading.

Immortalization of murine connective tissue-type mast cells at multiple stages of their differentiation by coculture of splenocytes with fibroblasts that produce Kirsten sarcoma virus.

Mature connective tissue mast cells (CTMC) have not been previously available as a cell line from any species. Here we describe 15 novel mast cell lines (KiSV-MC) that were derived by coculturing murine splenocytes with fibroblasts that produce a Ki-ras-containing murine sarcoma virus. Some of the KiSV-MC lines are similar to CTMC in that they synthesize predominantly heparin proteoglycans, and contain up to 35 micrograms of histamine and 2.2 units of carboxypeptidase A/10(6) cells in secretory granules which stain red with Safranin. Other cell lines display phenotypic characteristics intermediate to CTMC and mucosal-like mast cells in being predominantly Safranin-, having lower amounts of histamine and carboxypeptidase A, and in synthesizing chondroitin sulfate E proteoglycans in preference to heparin proteoglycans. When the individual KiSV-MC lines were compared, a linear relationship was found between the number of Safranin+ granules, the cellular contents of histamine and carboxypeptidase A, and the biosynthesis of heparin relative to chondroitin sulfate E proteoglycans. Upon sensitization with monoclonal IgE and exposure to hapten-specific antigen, the cells exocytose the contents of their secretory granules. Thus, these immortalized cells provide the first source of CTMC-like lines for chemical and functional analysis and illustrate that murine mast cells can express a continuum of phenotypes.

Kirsten murine sarcoma virus abolishes interferon gamma-induced class II but not class I major histocompatibility antigen expression in a murine fibroblast line.

The effect of infecting fibroblasts with Kirsten murine sarcoma virus/murine leukemia virus (Ki-MSV/MLV) on constitutive and IFN-gamma-induced H-2 antigen expression was investigated. The fibroblasts used were two established cell lines (C3H10T1/2 and BALB/c3T3) and fresh embryo fibroblasts from C3H mice. Class I antigens were expressed constitutively by BALB/c3T3; infection with MLV, MSV or the two together had little effect on this constitutive expression. Class I antigens (H-2K, H-2D) were strongly induced on all three types of fibroblast by rIFN-gamma, and infection had little effect on this. None of the fibroblasts expressed constitutively detectable levels of class II antigen; however, C3H10T1/2 fibroblasts could be induced for both H-2A and H-2E by IFN-gamma. Infection of C3H10T1/2 with helper-free Ki-MSV, or MSV together with MLV, completely abolished this induction of class II antigens, while infection with MLV alone had little effect, implying that the abolition of class II induction was due to genomic regions of Ki-MSV not shared with Ki-MLV, probably the v-Ki-ras gene.

Retrovirus infection alters growth factor responses of T lymphocytes.

A murine helper/inducer T cell clone, D10.G4, has been infected with Kirsten-murine sarcoma virus (KiSV) pseudotyped with an amphotropic murine leukemia virus. The resultant Ki-ras-expressing lines (KiSV-D10) remain dependent on exogenous factors for continued growth but display distinctly different mitotic responses to certain cytokines as compared to the uninfected parent clone. Unlike the parent D10.G4 cells, these KiSV-D10 cells can be maintained in vitro indefinitely in the presence of recombinant interleukin 2 (IL 2), and they all display a maximal proliferative response to purified or recombinant interleukin 1 (IL 1). The IL 1-induced proliferation is shown not to be dependent or secretion of the T cell autocrine growth factors IL 2 or B cell stimulatory factor-1 (BSF-1). The KiSV-D10 lines show certain differences from one another and parent D10.G4 cells in their secretory and proliferative responses to T cell receptor- and BSF-1 mediated signals. These viral oncogene-expressing T cell lines, which remain responsive to and dependent on physiologic growth factors, should prove valuable for analyzing the mechanisms of action of single oncogenes and the intracellular events in T lymphocyte activation.

Induction of endogenous xenotropic retrovirus expression by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate.

The tumor promotor 12-O-tetradecanoylphorbol-13-acetate (TPA) was examined for its ability to induce endogenous retrovirus from a high-passage clone of Kirsten sarcoma virus-transformed Balb/c (K-Balb) mouse cells. TPA activated virus in a concentration-dependent manner (0.0016 to 4.0 microM). Exposure to 1mM actinomycin D inhibited virus induction, suggesting that cellular RNA synthesis is required de novo by this inducer. A broad-spectrum neutralizing antibody to murine type C virus, gp70, was shown to neutralize the infectivity of the induced virus. The activated virus had the host range of the xenotropic Balb virus:2, and after removal of the inducer, the activated state decayed rapidly. TPA stimulated DNA, RNA, and protein synthesis in K-Balb cells, indicating that the mechanism of induction may be different from that of previously identified virus inducers. The effects observed using the well-defined K-Balb system offer an opportunity to study the modulation of retrovirus gene expression by TPA.

Hydrocortisone promotes the neodifferentiation of Kirsten murine sarcoma virus transformed human skin fibroblasts to adipose cells: relevance to oncogenic mechanisms.

Here we have demonstrated that transformation of human skin fibroblasts (SF) by the Kirsten murine sarcoma virus (KiMSV) is associated with their neodifferentiation into preadipose cells. Hydrocortisone (HC) promotes the transformation/neodifferentiation of such preadipocytes into mature fat cells. The effects of HC on the expression of adipocyte-containing foci and on the total number of transformed foci present in KiMSV-treated cultures appeared to be dose-dependent and was optimal at a concentration of about 500 ng/ml, or 1.25 X 10(-6) M. Although increasing serum concentrations (2-15%) increased the total number of transformed foci, it had no effect on the expression of adipocyte-containing foci in the presence of HC. The virus-induced preadipocytes undergoing partial conversion in the presence of HC were capable of clonal expansion and extensive proliferative activity. In contrast, mature adipocytes were terminally differentiated and as such have lost their ability to proliferate. The results suggest a role for a ras oncogene and HC in the transformation/neodifferentiation of human cells that might ultimately lead to cancer in some fraction of such cells.

Activation of intracisternal a particles by 5-azacytidine in mouse Ki-BALB cell line.

5-Azacytidine activates the production of intracisternal A particles (IAPs) in mouse Ki-BALB cell line as ascertained by electron microscopy scanning and numeration. Efficiency of the activation is higher than that obtained with iododeoxyuridine. The increase in particle production is concentration and time dependent. The results obtained in our system correlate the high IAP expression after drug treatment with a demethylation of IAP-related genes sequences.

Stimulation of cell proliferation by vitamin A derivatives on murine sarcoma virus-transformed mouse cells in serum-free culture.

The effect of retinoids (Rds) on cell proliferation was studied in serum-free culture condition, using non-transformed and transformed derivatives of BALB 3T3. Cell proliferation of an SV40-transformed line was inhibited significantly by Rd treatment. However, proliferation of two cell lines that were transformed by a Kirsten and Moloney strain of murine sarcoma virus (MSV) and produced growth factor into culture medium, was remarkably stimulated by Rds. Addition of serum masked both the inhibitory and stimulatory effects of Rds.

Rat mutant cells showing temperature sensitivity for transformation by wild-type Moloney murine sarcoma virus.

To clarify the cellular target(s) of onc gene products of Moloney murine sarcoma virus (Mo-MSV), we isolated seven mutant cells that exhibit temperature sensitivity for transformation by wild-type Mo-MSV from Fischer rat cell line. Five strains of these mutant cells showed normal virus production at the nonpermissive temperature when infected with Mo-MSV, suggesting that viral replication is not affected by these cellular mutations. Four of these mutants were also temperature sensitive (ts) for transformation by Kirsten murine sarcoma virus (Ki-MSV), whereas the other three mutants were not ts, suggesting that our mutants isolated with Mo-MSV can be divided into two classes as regards temperature sensitivity to transformation by Ki-MSV.

BALB and Kirsten murine sarcoma viruses alter growth and differentiation of EGF-dependent balb/c mouse epidermal keratinocyte lines.

Clonal BALB/c mouse epidermal keratinocyte (BALB/MK) cell lines were established in tissue culture. Despite their aneuploid nature, the lines were nontumorigenic, and retained in vitro properties similar to those of primary diploid keratinocytes. These included the constitutive expression of keratin and terminal differentiation in response to a calcium concentration greater than 1.0 mM in the medium. The cells also demonstrated an absolute requirement for nanomolar concentrations of epidermal growth factor (EGF) for their proliferation. BALB or Kirsten murine sarcoma viruses are acute transforming retroviruses, which have been shown to transform fibroblastic and hematopoietic cells. Infection of BALB/MK or its clonal sublines with either virus leads to the rapid acquisition of EGF-independent growth. The cells concomitantly lose their sensitivity to calcium-induced terminal differentiation. Thus these retroviruses can rapidly confer upon epithelial keratinocytes in culture growth properties that resemble those of malignant epidermoid carcinoma cells.

Enhanced viral transformation of skin fibroblasts from neurofibromatosis patients.

Enhanced viral transformation of cultured skin fibroblasts (SF) from patients with neurofibromatosis (NF) was observed, compared with cultures established from normal, age-matched controls. Cultures of skin fibroblasts from persons with and without clinical NF in families in which the disorder had been diagnosed were examined for transformability by Kirsten murine sarcoma virus. The viral transformation results were compared with those obtained with SF cultures initiated from controls in families without history of any disorder with an hereditary component, or cancer. The data show that 63 percent of cultures from patients with clinical NF were transformed, compared with 7 percent of control cultures (P = less than 0.0054). Cultures of skin fibroblasts from persons without the classical features of NF, but in families in which the disorder had been recognized, also exhibited a relatively high transformation rate, since 75 percent were transformed. Neurofibromatosis can be included among other hereditary disorders in which enhanced transformability of cultures of SF by an oncogenic virus may be demonstrated.

The virion RNA species of the Kirsten murine sarcoma-leukemia virus complex released from a clonally related series of mouse cells.

We have characterized the virion RNA species of Kirsten sarcoma (KiSV) and Kirsten leukemia (KiLV) viruses released from a clonally related series of mouse cells (14). We have identified the KiLV and KiSV genome RNAs. In addition to the viral RNA species we find large amounts of a virus-like RNA (VL30 RNA), which is heterogeneous and shows variability in its expression. The amount of VL30 RNA in virions does not correlate with the state of transformation of the cells releasing the virus or the ability of the virus to transform other cells. Characterization of RNA rescued from non-producer cells has revealed a sarcoma virus (KiSV CB3) with an oligonucleotide fingerprint different from that of a standard KiSV RNA, suggesting that it has lost some viral sequences. The oligonucleotide fingerprints of KiLV and VL30 RNAs are distinct from each other and from those reported for other murine leukemia virus RNAs.

Virus RNA species in kirsten murine sarcoma virus-transformed mink cells.

The nuclear and cytoplasmic RNAs from Kirsten murine sarcoma virus (KiMuSV)-transformed non-producer mink cells were studied for the species of virus-specific RNA by fractionation in agarose gels, transfer to diazotized aminophenylthioether paper and hybridization to complementary DNA probe. In both nuclei and cytoplasm, only genome-length KiMuSV-specific RNA was detected. No subgenomic virus RNA species was detected in poly(A+) or poly(A-) RNA fractions. The same observations were made in KiMuSV-transformed mink cells superinfected with feline leukaemia viruses. The significance of these findings is discussed.

The isolation and characterization of a clonally related series of murine retrovirus-infected mouse cells.

Starting with cloned NIH 3T3 mouse cells we have isolated a series of related lines infected with the Kirsten murine sarcoma/leukaemia (MSV/MLV) virus complex. These lines exhibit all three possible infected cell phenotopes: (i) transformed MSV/MLV producers; (ii) non-transformed MLV producers; (iii) transformed non-producers. We have also selected non-transformed revertants from one of the non-producer clones. This series of lines allows the study of the expression of the virus genome against a constant background of cellular gene expression. We have further characterized the lines with regard to anchorage dependence of growth, tumorigenicity and the presence of a rescuable sarcoma genome. The non-producer clones are uniform in their transformed properties. The revertants contain rescuable sarcoma virus, biologically indistinguishable from the original transforming virus, implying that the reversion is due to a change in cellular rather than viurs genetic information.