Transformation/neodifferentiation of human skin fibroblasts by acute oncornaviruses and dexamethasone

We demonstrated that the Kirsten murine sarcoma virus (KiMSV) and the Harvey murine sarcoma virus (HaMSV) converted human skin fibroblasts (HSF) into adipocytes. Adipocytic conversion of HSF by KiMSV and HaMSV was dependent on the presence of glucocorticosteroids. The Kirsten murine leukemia virus, the Harvey murine sarcoma [corrected] virus and the amphotropic helper virus (AP292) were ineffective by themselves. Balb murine sarcoma virus and Moloney murine sarcoma virus were, to a lesser degree, able to effect adipocytic conversion of HSF. In contrast, the feline sarcoma virus and the simian sarcoma virus did not cause this conversion. Together, the results suggest a role for certain oncogenes and glucocorticosteroids in the transformation/neodifferentiation of human cells.     

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.     

Histiocytic conversion of human adult skin fibroblasts by the Snyder-Theilen feline sarcoma virus

Oncogenes are apparently involved in the transformation/neodifferentiation of human cells. We now report a novel example of transformation/neodifferentiation: the specific conversion of cultured human adult skin fibroblasts (HSF) to histiocytes (tissue macrophages (TM)) by the Snyder-Theilen feline sarcoma virus (ST: (FeSV)). The de novo conversion of cultured HSF was demonstrated in a large fraction of ST:FeSV (FeLV)-transformed foci in the presence of dexamethasone (DX). Identification of tissue macrophages in ST:FeSV(FeLV)-infected HSF cultures was established by light-and transmission electron microscopy, histochemistry, immunohistochemistry, adherence-reattachment, latex particle uptake, and secretion of bioactive interleukin-1. The ST:FeSV gene and glucocorticosteroids, or other naturally occurring hormones, may play a role in morphogenetic processes within cells from a variety of normal and diseased tissues in situ, including induction of non-bone marrow, mesenchyme-derived, tissue macrophages.     

Integration and loss of a single v-Ki-ras gene affects tumorigenic potential of human osteosarcoma cells

The human osteosarcoma cell line Te85 clone F-5 is not tumorigenic in vivo. Its transformation with Kirsten murine sarcoma virus (KiMSV) (KHOS) confers full malignant properties and stable non-tumorigenic revertants of this KHOS cell line have been obtained. Here we show that integration and expression of a single copy of the KiMSV proviral DNA, which is totally lost in the HOS 240S revertant, is responsible for the acquisition of tumorigenicity. Cytogenetic analysis and the absence of a residual LTR copy in the revertant cellular genome suggest that the loss of KiMSV provirus is caused either by chromosomal segregation or by recombination not involving the LTR. In addition analysis of the expression of ras proteins revealed no changes in the pattern of c-ras products and the expression of v-ras only in the KHOS cells. All these data suggest that Te85 and HOS 240S cell lines could represent a human alternative recipient system to rodent cells in studies with oncogenes.     

Kirsten murine sarcoma virus transformed cell lines and a spontaneously transformed rat cell-line produce transforming factors

We have examined culture fluids from a variety of Kirsten murine sarcoma virus (KiMSV) transformed rat and mouse cells for the presence of factors which induce normal Rat-1 cells to assume the transformed phenotype. All KiMSV transformants produced transforming factor (TF). Revertants of KiMSV transformed rat or mouse cells failed to relase TF as did normal rat or mouse cells. Cells transformed by a temperature sensitive mutant of KiMSV produced TF at the permissive temperature but not at the nonpermissive temperature. Further, cells from a spontaneous transformant of Rat-1 cells also produced TF. TF is a small polypeptide which competes for the epidermal growth factor receptor. Its effect upon normal cells is reversible and requires de novo RNA and protein synthesis. Cells treated with TF lose the actin fibers observed in normal fibroblasts, assume a transformed cell morphology, become anchorage independent for growth, grow in low concentrations of serum, grow to a high cell density, and have an increased rate of hexose uptake.     

Biological and physical modifications of a murine oncornavirus by 2-deoxy-D-glucose.

2-Deoxy-D-glucose (2-DG) inhibited the release of transforming Kirsten murine sarcoma-leukemia virus [KiMSV(KiMuLV)] from transformed rat kidney (NRK-K) cells. At a concentration of 30 mM 2-DG, RNA synthesis in NRK-K cells was inhibited by approximately 30 percent and protein synthesis was inhibited by as much as 80 percent of control levels. RNA synthesis was not inhibited in nontransformed normal rat kidney (NRK) cells, although protein synthesis was equally suppressed in NRK and NRK-K cells. After treatment with 2-DG, the release of physical particles of KiMSV(KiMuLV) from NRK-K cels was not reduced as determined by equilibrium density gradient centrifugation and assays for RNA-dependent DNA polymerase of culture fluids. The ability to detect virion-associated radioactivity in equilibrium density gradients was dependent on the conditions of labeling. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of KiMSV(KiMulLV) proteins revealed marked structural alterations after propagation of the virus in 30 mM 2-DG. These alterations may account for the observed loss of transforming ability of KiMSV(KiMuLV).     

Changes in phenotypic expression in embryonic and adult cells treated with 5-azacytidine

We have previously shown that 5-azacytidine (5-Aza-CR) induced the formation of biochemically differentiated myotubes, adipocytes, and chondrocytes in the mouse embryo cell line, C3H/10T1/2CL8 (10T1/2), and that the induction of the muscle phenotype was cell cycle specific. Here we show that the adipocyte phenotype is also induced maximally in cells treated during early S phase. During this period, the minimum treatment time required for the subsequent formation of myotubes was 5 min and the number of myotubes formed was dependent on treatment time. The incorporation of ¹⁴C-5-Aza-CR into DNA during the cell cycle, however, was not enhanced during early S phase, suggesting that incorporation of 5-Aza-CR into specific DNA sequences synthesized during early S phase may be required for the expression of the new phenotypes. Single cells, obtained by plating cell suspensions into 16 mm wells at limiting dilution, were treated with 5-Aza-CR during S phase. The resulting clones showed a high frequency of phenotypic conversion, indicating that 5-Aza-CR did not act via a selective mechanism, and several of the clones were capable of expressing more than one phenotype. The cells required more than 2 division cycles after treatment with the analog for the expression of the muscle phenotype and the capacity to differentiate was retained for long periods of time in the absence of cell division. The adult mouse line, CVP3SC6, differentiated into functional striated muscle cells following treatment with 5-Aza-CR. The analog also caused oncogenic transformation in the adult line at the same concentration that was effective at inducing myogenic expression.     

Mouse and rat cells undergo rapidly inducible changes in polypeptide secretion following infection with Kirsten murine sarcoma virus

Characteristic changes in the secreted polypeptides of Kirsten murine sarcoma virus (KiMSV) transformed mouse and rat cell lines could be detected 48 hours after infection of phenotypically normal cells with this virus and correlated with detection of the KiMSV encoded polypeptide p21.     

Prevention of transplantable tumors by adoptive transfer of spleen cells from immunized rats.

Fischer 344 rats were specifically hyperimmunized with allogeneic, nonvirus-producing [Kirsten murine sarcoma virus (KiMSV)] or syngeneic, virus-producing [KiMSV (Rasheed)] rat tumors. Spleen cells taken from these rats adoptively transferred protection against a 100 to 1,000 X rat tumor dose50 cell challenge with several different transplantable rat tumors. Protection was obtained with spleen cells after removal of adherent cells and macrophages but not peritoneal cells. The spleen cells were not directly cytotoxic but required more than 3 days residence in the recipient before protecting the recipient against challenge. No protection against tumor cell challenge was observed when spleen cells were lethally x-ray irradiated before injection into nontreated rats. Spleen cells taken from rats immunized with normal histocompatibility antigens did not protect in this test system.     

Differentiation potential of conditionally immortalized mesenchymal progenitor cells from adult marrow of a H-2Kb-tsA58 transgenic mouse

Primary cultures were initiated from marrow, spleen, and bone explants of an adult H-2K^b-tsA58 transgenic mouse (immortomouse). All cultures were initiated in immortalizing conditions, and an additional marrow culture was first incubated for 1 week in standard conditions and then switched to immortalizing conditions. Marrow cells immediately immortalized were designated the marrow immediate population (MIP); those immortalized after 1 week were termed the marrow delayed population (MDP). MIP and MDP cells both contained a mixture of fibroblastic or flattened cells, and the MIP cells contained an additional subpopulation of adipocytic (Oil Red-O positive) cells. Alkaline phosphatase expression was induced by dexamethasone (10⁻⁷ M) in MDP cells while MIP, spleen, and bone explant cells had only a low level of expression. MDP and MIP cells differentiated into bone when combined with porous calcium phosphate ceramics and implanted subcutaneously into nude mice while bone- and spleen-derived cells did not. Clones were isolated from the MDP and MIP cell populations and tested for differentiated phenotypes. Some MIP-derived clones exhibited adipocytic characteristics while MDP-derived subclones were negative. Histologic examination of porous ceramic implanted clones showed that all of the clones had osteogenic potential. Clones exposed to either dexamethasone, human recombinant bone morphogenetic protein-2, or horse serum plus hydrocortisone showed differences in expression of adipocytic or osteogenic markers. These immortalized cultures have retained both adipocytic and osteogenic potential even after 1 year of continuous culture, and provide a model system for clonal analysis of the developmental potential of marrow-derived mesenchymal precursor cells. © 1996 Wiley-Liss, Inc.     

Proteoglycans from the substratum adhesion sites of MSV-transformed Balb/c 3T3 cells

Kirsten murine sarcoma virus-transformed Balb/c 3T3 cells (KiMSV) are highly tumorigenic and metastatic in the appropriate murine host, are loosely adherent to the tissue culture substratum, and can be readily detached from the substratum by ethylene glycol bis(β-aminoethyl ether) N,N′-tetraacetic acid treatment leaving their adhesion sites as substratum-attached material. Both long-term culture-generated adhesion sites (L-SAM) of KiMSV cells and newly formed adhesion sites of reattaching cells (R-SAM) contain high levels of hyaluronate (HA) and chondroitin sulfate (CS) whereas the R-SAM of parental Balb/c 3T3 cells is enriched in heparan sulfate (HS). A sizable fraction of KiMSV L-SAM proteoglycans (PG) and a smaller fraction of R-SAM PG's aggregate into two size classes of supramolecular complexes, after extraction off the substratum with 4 m guanidine hydrochloride, as determined by chromatography on columns of Sepharose CL2B in several buffer systems. Isopycnic density gradient analyses under associative conditions of KiMSV L-SAM generated three classes of material—high-density G_A1 which contained some HA but principally CS and HS; intermediate-density G_A2 which contained only HA; and low-density G_A3 which contained some HA and principally glycoprotein. R-SAM gradients contained no G_A2 but a sizable amount of “low-density” HA in G_A3. When centrifuged under dissociative conditions, most of G_A1 and all of G_A2 from L-SAM shifted to the top of the gradient, whereas most of the HS-PG in R-SAM remained at the bottom of dissociative gradients. Comparison of these analyses with previous analyses of Balb/c 3T3 extracts demonstrates that (a) KiMSV cells generate adhesion sites with different PG contents than 3T3 sites; (b) the PG's of KiMSV sites have a reduced potential to aggregate into high-molecular-weight complexes but do form intermediate-size complexes not apparent in material from 3T3 sites; (c) these data support the hypothesis that HA is important in detachment of cells from extracellular matrices; and (d) HS-PG's in newly formed adhesion sites of KiMSV cells are considerably different from sites which have “matured”, indicating that there is metabolic activity in these sites during prolonged adherence and movement of transformed cells.     

V-K-ras transformation induces reversion to an earlier developmental form in adult rat adrenal cells

The basis for specific changes in differentiation that accompany ras-oncogene-mediated transformation are not understood. When short-term cultures of fibroblast-like cells from adult rat adrenal glands were transformed with Kirsten murine sarcoma virus (KiMSV), the original stromal characteristics of the target cells (metachromatic extracellular matrix, high collagen production, collagen incorporation into pericellular matrix and a fibroblastic morphology and growth pattern) diminished. In contrast, parenchymal differentiation markers (neutral lipid, delta 5, 3 beta-hydroxysteroid dehydrogenase, 21-hydroxylase and epithelial morphology) were enhanced. These changes in differentiation were initiated concurrently with the over-expression of the transforming protein v-p21, but were unrelated to the levels of v-p21 expression. They were independent of the immortalizing component of transformation mediated by v-K-ras, because they did not take place in spontaneously immortalized lines derived from the same target cells, unless the lines were also transformed with KiMSV. In normal embryonic development, stromal and parenchymal adrenocortical cells arise by divergent differentiation pathways from a common, multipotential mesenchymal precursor. The transformation-induced modulation from a predominantly stromal to a more-parenchymal phenotype is thus reminiscent of reversion to a more primitive, bipotential developmental stage.     

Diltiazem does not increase the in vivo sensitivity of murine hemopoietic progenitor cells to doxorubicin

The effect of doxorubicin and the calcium antagonist, diltiazem, on murine hemopoietic progenitor cells was studied in vivo. Dose-survival curves of murine bone marrow colony forming units (CFU)--spleen and granulocyte macrophage--were determined by in vivo and in vitro methods in DBA/2NCr/BR mice treated with doxorubicin alone or by simultaneous administration of doxorubicin (DX) and diltiazem (DTZ). Time response of bone marrow hematopoietic progenitor cells (HPC) was followed in mice similarly treated. Combination of DTZ with DX did not change the toxic effect of the latter on hemopoietic cells, either in the dose-survival model or in the time-related experiment.     

Viral susceptibility of skin fibroblasts from patients with Huntington disease.

Cultured skin fibroblasts from patients with Huntington disease (HD) and age-matched controls were tested for susceptibility to vesicular stomatitis virus (VSV) and transformation by Kirsten mouse sarcoma virus (KiMSV). The HD and control cells could not be distinguished on the basis of viral replication, plaque morphology, virus yield, or susceptibility to transformation by KiMSV. These findings suggest that the HD gene product, if expressed within peripheral tissue, does not selectively alter or interfere with viral replication.     

LDHk, a uniquely regulated cryptic lactate dehydrogenase associated with transformation by the Kirsten sarcoma virus

A novel isozyme of lactate dehydrogenase is detected in various cells transformed by the Kirsten murine sarcoma virus (KiMSV). This isozyme, designated LDHk, is strongly inhibited by physiological concentrations of oxygen, in an apparently cooperative fashion. LDHk is inhibited by guanosine triphosphate and related compounds, in a noncompetitive fashion. LDHk is found with both 35,000- and 22,000-dalton subunits, although these probably cleave from a 57,000-dalton precursor. In studies utilizing a temperature-sensitive transforming gene mutant of the Kirsten sarcoma virus, we find in vivo expression of LDHk is also temperature-sensitive. In studies using either crude cell-free extracts or purified LDHk, we find the enzyme from cells infected with a temperature-sensitive transforming gene mutant of KiMSV is thermolabile relative to that from wild type KiMSV-infected cells.     

Role of transmembrane 4 superfamily (TM4SF) proteins CD9 and CD81 in muscle cell fusion and myotube maintenance.

The role of transmembrane 4 superfamily (TM4SF) proteins during muscle cell fusion has not been investigated previously. Here we show that the appearance of TM4SF protein, CD9, and the formation of CD9-beta1 integrin complexes were both regulated in coordination with murine C2C12 myoblast cell differentiation. Also, anti-CD9 and anti-CD81 monoclonal antibodies substantially inhibited and delayed conversion of C2C12 cells to elongated myotubes, without affecting muscle-specific protein expression. Studies of the human myoblast-derived RD sarcoma cell line further demonstrated that TM4SF proteins have a role during muscle cell fusion. Ectopic expression of CD9 caused a four- to eightfold increase in RD cell syncytia formation, whereas anti-CD9 and anti-CD81 antibodies markedly delayed RD syncytia formation. Finally, anti-CD9 and anti-CD81 monoclonal antibodies triggered apoptotic degeneration of C2C12 cell myotubes after they were formed. In summary, TM4SF proteins such as CD9 and CD81 appear to promote muscle cell fusion and support myotube maintenance.     

Cytokine production and killer activity of NK/T-NK cells derived with IL-2, IL-15, or the combination of IL-12 and IL-18

NK cell populations were derived from murine splenocytes stimulated by IL-2, IL-15, or the combination of IL-12 and IL-18. Whereas NK cells derived with the latter cytokines consisted of an homogeneous population of NK cells (DX5+CD3-), those derived with IL-2 or IL-15 belonged to two different populations, namely NK cells (DX5+CD3-) and T-NK cells (DX5+CD3+). Among NK cells, only those derived with IL-12/IL-18 produced detectable levels of cytokines, namely IFN-gamma, IL-10, and IL-13 (with the exception of IL-13 production by NK cells derived with IL-2). As for T-NK cells, IL-2-stimulated cells produced a wide range of cytokines, including IL-4, IL-5, IL-9, IL-10, and IL-13, but no IFN-gamma, whereas IL-15-derived T-NK cells failed to produce any cytokine. Switch-culture experiments indicated that T-NK cells derived in IL-2 and further stimulated with IL-12/IL-18 produced IFN-gamma and higher IL-13 levels. Next, we observed that NK/T-NK cell populations exerted distinct effects on Ig production by autologous splenocytes according to the cytokines with which they were derived. Thus, addition of NK cells derived in IL-12/IL-18 inhibited Ig production and induced strong cytotoxicity against splenocytes, whereas addition of NK or T-NK cells grown in IL-2 or IL-15 did not. Experiments performed in IFN-gammaR knockout mice demonstrated that IFN-gamma was not involved in the killer activity of IL-12/IL-18-derived NK cells. The hypothesis that their cytotoxic activity was related to the induction of target apoptosis was confirmed on murine A20 lymphoma cells. Experiments performed in MRL/lpr mice indicated that IL-12/IL-18-derived NK cells displayed their distinct killer activity through a Fas-independent pathway. Finally, perforin was much more expressed in IL-12/IL-18-derived NK cells as compared with IL-2- or IL-15-derived NK cells, an observation that might explain their unique cytotoxicity.     

Antigen-reactive cloned helper T cells. II. Exposure of murine cloned helper T cells to IL 2-containing supernatant induces unresponsiveness to antigenic restimulation and inhibits lymphokine production after antigenic stimulation

Cloned murine helper T lymphocytes (HTL) reactive to alloantigen or to ovalbumin (OVA) become unresponsive to antigenic restimulation after exposure to antigen or to culture supernatant fluids (SF) containing multiple lymphokine activities. Unresponsiveness is manifest by a failure of antigen-stimulated cells to incorporate thymidine or to produce lymphokines after antigenic challenge. Antigen-unresponsive HTL, however, will incorporate thymidine when exposed to an exogenous source of interleukin 2 (IL 2). The duration of unresponsiveness to antigen is correlated with the concentration of IL 2 in SF to which the cloned HTL had been exposed. Chromatographic fractionation of IL 2-containing supernatant from EL-4 thymoma cells (EL-4 SF) yielded a pool of SF that was enriched for IL 2 activity. Exposure of HTL to lymphokines contained in this pool induced unresponsiveness to antigen that was comparable to that observed when HTL were exposed to unfractionated EL-4 SF. Unresponsiveness to antigen also developed after cloned HTL were stimulated with concanavalin A (Con A) or with OVA and syngeneic splenic filler cells. We have used monoclonal antibody (mAb) GK1.5 (anti-L3T4) to investigate the role of lymphokine production in the induction of unresponsiveness. This antibody did not inhibit IL 2-induced thymidine incorporation by cloned HTL, and did not inhibit the induction of unresponsiveness after exposure of cloned HTL to EL-4 SF. In the presence of mAb GK1.5, however, HTL that were stimulated with Con A or OVA did not become unresponsive to antigenic restimulation, an effect that was overcome by the addition of EL-4 SF. These results suggest that HTL become unresponsive to antigen after exposure to IL 2-containing SF, and that stimulation by antigen or Con A can induce the unresponsive state by virtue of stimulating lymphokine production.     

Transforming growth factors released from Kirsten sarcoma virus transformed cells do not compete for epidermal growth factor membrane receptors

Kirsten murine sarcoma virus (KiMSV)-transformed rat kidney cells (KNRK) release small polypeptides (Mr 12,500-15,300) into the culture medium that are capable of stimulating normal rat kidney cells (NRK) to form colonies in soft agar. The transforming growth factors (TGFs) did not compete with epidermal growth factor (EGF) for its receptor and did not induce specific phosphorylation of EGF receptor on NRK cell membranes. These properties differ from the TGFs isolated by other investigators. Our data further establish the heterogeneity of the materials produced by transformed cells that induce transformation-specific changes in normal cells.