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.     

A system for deriving revertants of oncogene-transformed human cells

We have previously shown that some transformed derivatives of the human osteosarcoma-derived cell line HOS are killed by treatment with 1 microM ouabain at pH 8.2, whereas their nontransformed counterparts are relatively unharmed by the same conditions. HOS cells transformed by v-Ki-ras and RAS, v-fms, or MET are susceptible to 1 microM ouabain while those transformed by v-fes are not. Here we describe the adaptation of this differentially cytotoxic effect as a method to enrich for cells which revert to a nontransformed phenotype. We have optimized parameters which increase the differential cytotoxicity, including pH and potassium concentration during and subsequent to ouabain treatment. The efficiency of this procedure was tested in mixed cell experiments where model populations were constructed consisting of HOS cells mixed with an excess of v-Ki-ras-transformed HOS cells. Two successive OAK treatments (ouabain/alkaline/K+-free) were sufficient to recover nontransformed cells free of ras-transformants as indicated by genetic markers and morphology. This HOS/ouabain system is currently being used to derive revertants of ras-transformed human cells and could facilitate the isolation of genes interacting in the pathways by which these cells are transformed.     

In vitro differentiation of the human osteosarcoma cell lines, HOS and KHOS.

The osteogenic potential of the two human osteosarcoma cell lines HOS and KHOS; a cell line produced by the transformation of the HOS cells by the Kirsten murine sarcoma virus, was studied in vitro. HOS cells cultured more than 2 weeks formed nodules composed of two morphologically distinct layers, an epithelial-like surface cell layer and a collagen-rich inner cell layer. Alkaline phosphatase (ALPase) activity occurred in the plasma membrane of the surface cell layer, and calcified substances developing along collagen fibers were detected in the collagen-rich inner cell layer. The calcified substances were further examined by analytical electron microscopy and were shown to be hydroxyapatite crystals. In contrast, there was neither ALPase nor the deposition of a calcified substance in the KHOS cells.     

Human smooth muscle myosin light chain-2 gene expression is repressed in ras transformed fibroblast cells.

We have previously characterized human smooth muscle myosin light chain (MLC)-2 isoform by complementary DNA cloning and have shown that this isoform is expressed in a number of nonmuscle cells such as fibroblast cells. In this report, we show that when human osteosarcoma derived clonal cells (TE 85 clone F-5) (HOS), which are immortalized and nontumorigenic, undergo transformation following infection by Kirsten murine sarcoma virus (K-HOS) or by a chemical carcinogen [N-methyl-N-nitro-N-nitrosoguanidine (MNNG-HOS)], the smooth muscle MLC-2 mRNA is repressed. Revertants of transformed K-HOS cells (K-HOS312H) show normal levels of smooth muscle MLC-2 mRNA. Transformation of HOS cells by Ha-ras oncogene sequences, either by retroviral infection or by transfection followed by selection for tumorigenic cells in nude mice, results in complete repression of smooth muscle MLC-2 mRNA level. Treatment of HOS cells with tumor promoting phorbol ester, 12-O-tetradecanoylphorbol-13-acetate, results in repression of smooth muscle MLC-2 mRNA. Smooth muscle MLC-2 mRNA level is repressed in many, but not all, transformed cell lines, suggesting that it is not an indirect consequence of transformation but is specific to the agent that brings about transformation. HOS cells synthesize three MLC-2 protein species resolved by the two-dimensional gel electrophoretic system. The identity of the smooth muscle MLC-2 isoform was established by coelectrophoresis of the in vitro synthesized MLC-2 protein corresponding to the cloned complementary DNA in the two-dimensional gel system along with total [35S]methionine labeled HOS cell proteins. Quantitative analysis of MLC-2 isoforms in different HOS cells indicates that the synthesis of smooth muscle MLC-2 isoform is specifically repressed to an undetectable level in ras transformed and MNNG transformed cells and also following treatment with 12-O-tetradecanoylphorbol-13-acetate.     

In vitro studies of deformation and adhesion properties of transformed cells.

The micropipet aspiration technique and the parallel-plate flow chamber were used to investigate the deformation and detachment properties, respectively, of normal and transformed rat fibroblasts. The normal Cloned Rat Embryo Fibroblasts (CREF) cell line was transfected with the T24 ras oncogene to produce the transformed cell line CREF T24. The CREF T24 cell line was transfected with a Kirsten ras revertant gene (K-rev 1a suppressor) to produce the CT24HKB1 cells, which have the same morphological characteristics as the cells in the CREF line. The cells utilized in this investigation were derived from the parent cell line CREF, the only differences being the presence or absence of the T24 ras oncogene and the Kirsten ras revertant gene. The detachment and deformation properties, therefore, could be related to the metastatic phenotype of the cell rather than inherent differences between disparate cell lines. Results indicated that transfecting the CREF cell line with the ras oncogene greatly modified the detachment and deformation properties. The CREF T24 cells were more easily detached from normal cells and were 50% more deformable. Both CREF and CT24HKB1 showed similar detachment properties. Based on these results, it is speculated that K-rev 1a reversed ras-induced membrane alterations in these cells. Preliminary investigations have demonstrated that both CREF and CREF T24 cells in different phases of the cell cycle differed in morphological characteristics. However, the majority of the cells within a given cell line showed similar deformation characteristics. Current investigations are focusing on characterization of both detachment and deformation properties of these cells as a function of the cell cycle using synchronization techniques.     

Transformation by the k-ras oncogene correlates with increases in phospholipase A2 activity, glycerophosphoinositol production and phosphoinositide synthesis in thyroid cells.

Two cell lines transformed by the k-ras oncogene (KiKi and KiMol cells) and a temperature sensitive clone (Ts), all originated from a normal rat thyroid line (FRTL5 cells), have been employed to analyse the intracellular mechanisms affected by the ras p21. In k-ras transformed cells two phosphoinositide derivatives, glycerophosphoinositol and inositol monophosphate, were markedly increased, whereas inositol bisphosphate and trisphosphate maintained the same level as in normal cells. Cytosolic Ca2+ was also unaffected. This indicates that in epithelial cells the phospholipase C activity is not altered upon ras transformation. The formation of glycerophosphoinositol involved the activation of a phosphoinositide specific phospholipase A2. The higher phospholipase A2 activity in ras transformed cells could be further demonstrated by the increase in total arachidonic acid release. In the Ts clone the increase in glycerophosphoinositol and inositol monophosphate was evident only at the permissive temperature (33 degrees C), whereas it disappeared at 39 degrees C. At 33 degrees C the cells were also characterized by an enriched membrane pool of phosphoinositides. All these changes occurred in parallel with morphological transformation. We propose that cell transformation by the k-ras oncogene affects different steps of the membrane lipid metabolism, among which the most prominent one is the activation of a phosphoinositide specific phospholipase A2. These effects could originate mitogenic metabolites. Moreover, they correlate well with the induction of the malignant phenotype.     

Apoptosis and catastrophic cell death in benzo[a]pyrene-transformed human breast epithelial cells

Apoptosis and mitotic death, bi- and multinucleation, giant cells and micronucleation were investigated in human breast epithelial cell lines transformed by benzo[a]pyrene (BP) (BP1, BP1-E and BP1-E1 cells) and in BP1 cells transfected with the c-Ha-ras oncogene (BP1-Tras cells). Since BP induces apoptosis and the abnormal expression of ras genes elicits catastrophic mitosis, both cell death phenomena were expected to occur in this system, especially in BP1-Tras cells. Regardless of the cell line considered, single-nucleate cells were found to be eliminated preferentially through apoptosis, while bi- and multinucleate cells were eliminated through catastrophic mitosis. Apoptosis and catastrophic mitosis were observed in all cell lines but were significantly more frequent in BP1-Tras cells. The abnormal expression of Ha-ras in the latter cells may enhance in this system the effects of the BP apoptosis path reported for BP-transformed Hepa 1c1c7 hepatoma cells. Transfection with the ras oncogene also enhanced the mitotic disturbances, which produced multi- and micronucleation and mitotic death, possibly because of the genomic instability promoted by this oncogene in the BP-transformed cell line.     

Evidence for two mechanisms by which tumor necrosis factor kills cells

Tumor necrosis factor (TNF) can inhibit the differentiation of preadipocytes to adipocytes and will revert differentiated adipocytes to the preadipocyte state. TNF is not toxic to either adipocytes or preadipocytes when used alone but is highly toxic to these cells when used in conjunction with cycloheximide, yielding virtually 100% killing within 4-6 h of treatment. A cell line (TA1 R-6) was isolated which is resistant to the combined toxic effects of TNF and cycloheximide. This cell line is stable and, unlike the parental cell line, does not morphologically differentiate to adipocytes or express adipocyte-specific mRNAs. It has a more transformed appearance and growth pattern and, while resistant to the toxic effects of TNF and cycloheximide in a 6-h assay, has become sensitive to cytotoxicity induced by TNF used alone in a 3-day assay. The adipocyte differentiation-inducing agents, dexamethasone and indomethacin, block the cytotoxicity induced by TNF alone in the TA1 R-6 line but do not block the rapid cytotoxicity of TNF and cycloheximide in the parental line. These results provide both genetic and pharmacologic evidence that there are at least two distinct or overlapping pathways by which TNF mediates its effects.     

Altered iron homeostasis involvement in arsenite-mediated cell transformation

Chronic exposure to low doses of arsenite causes transformation of human osteogenic sarcoma (HOS) cells. Although oxidative stress is considered important in arsenite-induced cell transformation, the molecular and cellular mechanisms by which arsenite transforms human cells are still unknown. In the present study, we investigated whether altered iron homeostasis, known to affect cellular oxidative stress, can contribute to the arsenite-mediated cell transformation. Using arsenite-induced HOS cell transformation as a model, it was found that total iron levels are significantly higher in transformed HOS cells in comparison to parental control HOS cells. Under normal iron metabolism conditions, iron homeostasis is tightly controlled by inverse regulation of ferritin and transferrin receptor (TfR) through iron regulatory proteins (IRP). Increased iron levels in arsenite transformed cells should theoretically lead to higher ferritin and lower TfR in these cells than in controls. However, the results showed that both ferritin and TfR are decreased, apparently through two different mechanisms. A lower ferritin level in cytoplasm was due to the decreased mRNA in the arsenite-transformed HOS cells, while the decline in TfR was due to a lowered IRP-binding activity. By challenging cells with iron, it was further established that arsenite-transformed HOS cells are less responsive to iron treatment than control HOS cells, which allows accumulation of iron in the transformed cells, as exemplified by significantly lower ferritin induction. On the other hand, caffeic acid phenethyl ester (CAPE), an antioxidant previously shown to suppress As-mediated cell transformation, prevents As-mediated ferritin depletion. In conclusion, our results suggest that altered iron homeostasis contributes to arsenite-induced oxidative stress and, thus, may be involved in arsenite-mediated cell transformation.     

Transfer of potassium. A new measure of cell-cell coupling

Mammalian cells of different species differ in sensitivity to ouabain. This sensitivity is expressed as reduced intracellular K+ content, reduced rates of protein synthesis, and cessation of cell multiplication. Using 86Rb+ as a measure of intracellular K+, we found higher levels of radioactivity in mixtures of ouabain-sensitive and -resistant cells cultured in the presence of ouabain than predicted from pure cultures of the two component cell types. The simplest explanation is that K+ and 86Rb+ are being transferred from ouabain-resistant to ouabain-sensitive cells, enhancing the total intracellular 86Rb+ in the culture. A function, "index of cooperation," expresses this enhancement as a number ranging from 0 to 1, and permits comparisons to be made under various culture conditions and using various cell types. An index of cooperation greater than 0 requires cell contact, since no enhancement occurs when contact between two cell types in the same culture is prevented. The index of cooperation for a number of different cell combinations agrees with other measures of cell-cell interaction associated with gap junctions, such as electrical coupling and metabolic cooperation. Coculture of ouabain-sensitive and ouabain-resistant cells in the presence of ouabain also leads to restoration of the capacity for protein synthesis. Autoradiography shows that this restoration occurs in the sensitive cell type and is dependent upon contact with ouabain-resistant cells. Furthermore, sensitive cells are able to multiply in the presence of ouabain when cocultured with resistant cells. Thus K+, presumably transferred to sensitive cells through gap junctions, is able to counteract the toxic effects of ouabain on intracellular K+ levels and protein synthesis, and to restore growth.     

A new recipient cell line for transfection of biologically active oncogenes]

The properties of the new immortalized rat cell line (REF-1) were analyzed. These cells can be used as recipient ones in transfection assays. REF-1 cells never convert spontaneously to transformed phenotype during long-term passages in vitro unlike NIH3T3 cells. This peculiarity allowed to use REF-1 cells for identification of oncogenes, which induce slow-growing tumors. The following oncogenes were used in gene transfection experiments in order to test their effects: activated human EJ-ras; N-ras; v-myc; v-mos; activated tpr-met and c-hu-met. REF-1 cells, transfected with the members of ras family; v-mos and tpr-met were found to be transformed in vitro and induce tumors in nude mice, on the contrary of c-hu-met- and v-myc-transfected cells, which are non-tumorogenic. A number of clonal cell lines carrying different oncogenes were obtained. The detailed analyses of integration and expression of exogenous sequences of different oncogenes has been presented in 18 clonal cell lines.     

Flavonoids purified from Rhus verniciflua Stokes actively inhibit cell growth and induce apoptosis in human osteosarcoma cells

Many studies have suggested that dietary flavonoids are anticancer agents that induce the apoptosis of cancer cells. However, the effects of flavonoids on the induction of apoptosis in osteosarcoma cells are unclear. Previously, a flavonoid fraction, consisting mainly of protocatechuic acid, fustin, fisetin, sulfuretin, and butein, herein named RCMF (the RVS chloroform-methanol fraction), was prepared from a crude acetone extract of Rhus verniciflua Stokes (RVS). This study evaluated the effects of RCMF on the proliferation and apoptosis using human osteosarcoma (HOS) cells. The mechanism of growth inhibition of the HOS cells by the flavonoid fraction, RCMF, was also assessed. The results demonstrated that RCMF exhibited sensitive growth inhibition and induced apoptosis in HOS cells. PARP cleavage was closely associated with the RCMF-induced apoptosis of the HOS cells. Furthermore, the activation of caspase 8 and Bax, the inhibition of Bcl-2 expression, and the release of cytochrome c are believed to be involved in the RCMF-mediated apoptosis. Collectively, these findings suggest that RCMF is an agent which may be capable of inducing sensitive growth inhibition and apoptosis in HOS cells.     

Ras induces anchorage-independent growth by subverting multiple adhesion-regulated cell cycle events.

Anchorage-independent growth is a hallmark of transformed cells, but little is known of the molecular mechanisms that underlie this phenomenon. We describe here studies of cell cycle control of anchorage-independent growth induced by the ras oncogene, with the use of a somatic cell mutant fibroblast line (ER-1-2) that is specifically defective in oncogene-mediated, anchorage-independent growth. Control, nontransformed PKC3-F4 cells and ER-1-2 cells cannot proliferate in semisolid medium. Three important cell cycle events are dependent on adhesion of these cells to a substratum: phosphorylation of the retinoblastoma protein, pRB; cyclin E-dependent kinase activity; and cyclin A expression. PKC3-F4 cells that express ras (PKC3-F4/ras cells) proliferate in nonadherent cultures, and each of these three events occurs in the absence of adhesion in PKC3-F4/ras cells. Thus, ras can override the adhesion requirement of cellular functions that are necessary for cell cycle progression. ER-1-2 cells that express ras (ER-1-2/ras cells) possess hyperphosphorylated forms of pRB and cyclin E-dependent kinase activity in the absence of adhesion but remain adhesion dependent for expression of cyclin A. The adhesion dependence of pRB phosphorylation and cyclin E-dependent kinase activity is therefore dissociable from the adhesion dependence of cyclin A expression. Furthermore, ectopic expression of cyclin A is sufficient to rescue anchorage-independent growth of ER-1-2/ras cells but does not induce anchorage-independent growth of PKC3-F4 or ER-1-2 cells. However, like pRB phosphorylation and cyclin E-dependent kinase activity, the kinase activity associated with ectopically expressed cyclin A is dependent on cell adhesion, and this dependence is overcome by ras. Thus, the induction of anchorage-independent growth by ras may involve multiple signals that lead to both expression of cyclin A and activation of G1 cyclin-dependent kinase activities in the absence of cell adhesion.     

Establishment of hybridoma secreting human monoclonal antibody against hepatitis B virus surface antigen

The HAT (hypoxanthine, aminopterin, thymidine) sensitive and ouabain resistant human B lymphoblastoid cell line TAW-925 was obtained from 6-thioguanine resistant B lymphoblastoid cell line WI-L2. Hybridomas were obtained at a high frequency (10(-4)-10(-5) when TAW-925 was hybridized with cells transformed with Epstein-Barr virus. Using TAW-925 as a parental cell line, we have obtained a hybridoma which stably secretes human monoclonal antibody against hepatitis B virus surface antigen.     

Codominance of cisplatin resistance in somatic cell hybrids

Intrinsic or acquired resistance to cisplatin in cancer cells remains a major obstacle to successful chemotherapy. The clinically relevant genetic and molecular mechanisms of resistance have not yet been identified. Cisplatin-resistant (CP-r) human KB epidermoid carcinoma cell lines (HeLa) resistant to varying levels of cisplatin after single and multiple selection steps are cross-resistant to other platinum compounds and to methotrexate. Intraspecies hybrids of the sensitive and KB CP-r cells were fused with HeLa D98(OR) CP-s, hypoxanthine-aminopterin-thymidine (HAT) sensitive, ouabain resistant, to determine whether cisplatin resistance is dominant or recessive. Cell-cell hybridization between the sensitive cells and single-step or two-step KB CP-r cells both indicated codominance of cisplatin resistance compared to hybrids between sensitive cell lines (D98(OR)xKB). The hybrids between sensitive cell lines (D98xKB) and a single-step CP-r KB cell line (D98xKB-CP.5) also were cross-resistant to carboplatin and methotrexate. In addition, the relatively slower growth rate of CP-r cells appears to be dominant. In the two-step CP-r KB cell line, KB-CP1, resistance is no more dominant than in the single-step CP-r KB cell line, KB-CP.5, suggesting that one of the two steps of resistance in KB-CP1 may not be dominant. These dominance data suggest that it might be possible to identify one or more genes responsible for cisplatin resistance by gene transfer from a resistant cell line to a sensitive cell line.     

Coordinate regulation of the levels of type III and type I collagen mRNA in most but not all mouse fibroblasts

A mouse genomic clone was isolated by cross-hybridization with a DNA fragment which codes for the NH2-propeptide of chick alpha1(III) collagen. The region of cross-hybridization within the mouse clone was localized, its sequence determined, and an exon coding for the NH2-propeptide of mouse alpha1(III) collagen was identified. This DNA fragment hybridizes to an RNA species of approximately 5300 nucleotides, slightly larger than the major alpha2(I) collagen RNA species. The mouse type III collagen probe was used to examine the effect of transformation on alpha1(III) collagen RNA levels in mouse fibroblasts. The levels of type III and type I collagen mRNA levels were compared in control and sarcoma virus-transformed murine cell lines, as well as in NIH 3T3 cells transformed by members of the human ras oncogenes. The levels of type III RNA decreased about 10-15-fold in Moloney sarcoma virus-transformed cells and in a cell line transformed with a v-mos-containing plasmid, but showed only a 50% decrease in a Kirsten murine sarcoma virus-transformed BALB 3T3 cell line, and increased 4-fold in a Rous sarcoma virus (RSV)-transformed BALB 3T3 cell line. In contrast, the levels of alpha2(I) collagen mRNA are 8- to 10-fold lower in all these cell lines when compared to untransformed cells. NIH 3T3 cells transformed with two human ras oncogenes showed decreased levels of alpha2(I) and alpha1(III) mRNAs. In contrast to the RSV-transformed mouse cell line, RSV-transformed chick embryo fibroblasts contained much smaller amounts of type III RNA than control chick embryo fibroblasts. We conclude that the levels of alpha1(III) and alpha2(I) collagen mRNA are often but not necessarily coordinately regulated by transformation in mouse cells.     

Overexpression of the Polycystin-1 C-Tail Enhances Sensitivity of M-1 Cells to Ouabain

Cells derived from renal cysts of patients with autosomal dominant polycystic kidney disease (ADPKD) are abnormally sensitive to ouabain, responding to physiological ouabain concentrations with enhanced proliferation and increased forskolin-induced transepithelial fluid secretion. This requires activation of the epidermal growth factor receptor (EGFR), Src kinase and the extracellular signal-regulated kinases MEK and ERK. Here, we have determined if the ADPKD phenotype obtained in mouse cortical collecting duct cells by stable overexpression of the C-terminal domain of polycystin-1 (PC-1 C-tail) also elicits the ADPKD-like response to ouabain in the cells. M-1 C20 cells expressing the PC-1 C-tail and M-1 C17 cells lacking expression of this construct were treated with physiological concentrations of ouabain, and cell proliferation, activation of the EGFR-Src-MEK-ERK pathway, forskolin-induced transepithelial Cl^? secretion and the sensitivity of Na,K-ATPase to ouabain were explored. M-1 C20 cells responded to ouabain with increased cell proliferation and ERK phosphorylation. Ouabain also augmented forskolin-induced and cystic fibrosis transmembrane conductance regulator-mediated apical secretion of Cl^? in M-1 C20 cells. These effects required activation of EGFR, Src and MEK. In contrast, ouabain had no significant effects on M-1 C17 cells. Interestingly, approximately 20 % of the Na,K-ATPase from M-1 C20 cells presented an abnormally increased sensitivity to ouabain. Overexpression of PC-1 C-tail in M-1 C20 cells is associated with an ouabain-sensitive phenotype and an increased ability of the cells to proliferate and secrete anions upon ouabain stimulation. This phenotype mimics the ouabain sensitivity of ADPKD cells and may help promote their cystogenic potential.