Transformation of non-tumorigenic osteoblast-like human osteosarcoma cells by hexavalent chromates: alteration of morphology, induction of anchorage-independence and proteolytic function.

Soluble chromium (VI) compounds either alone or in combination with 3-methylcholanthrene (MC) were used to transform non-tumorigenic osteoblast-like human osteosarcoma cells (HOS TE85). The Cr(VI) compounds were highly toxic to these cells with LC50 values in the range of approximately 0.5-1.0 microM. Continuous passaging of the treated cells resulted in sustained increase in anchorage-independent (AI) colony formation. Treatment with Cr(VI) and MC resulted in substantial increase in AI growth. At the XVth passage, a number of individual AI colonies were expanded in culture and used for further studies. The cells are refractory in appearance and grow as 'nests' rather than as monolayers. The cell lines have relatively high plating efficiency (PE) in soft agar and respond to promotional effect of phorbol-12-myristate-13-acetate by an increase in PE and in the size and number of AI colonies. While the isolated cells are not tumorigenic when tested in athymic nude mice, most of the lines possess higher levels of plasminogen activator (PA) activity, considered as one of the markers of transformation. This is also reflected in the increase in the steady state level of urokinase type PA mRNA. These results show that Cr(VI) compounds are capable of promoting human cells to an altered phenotype characteristic of a stage in the carcinogenesis cascade.     

Association of decreased intercellular communication with the immortal but not the tumorigenic phenotype in human mammary epithelial cells

Intercellular communication was compared in early passage cultures of human mammary epithelial cells (HMEC) from normal and malignant breast tissues and immortalized nontumorigenic human breast cell lines (184A1 and 184B5). A clonogenic assay for the cell-mediated transfer of toxic metabolites of 6-thioguanine (TG) between cells was used as a measure of intercellular communication. We examined the effects of wild-type TG-sensitive (TGs) HMEC density on the survival of mutant TG-resistant (TGr) immortalized HMEC in TG-containing medium. Survival rates of TGr HMEC cocultured with TGs normal HMEC, malignant HMEC, or immortalized nontumorigenic HMEC were dependent on the density of TGs cells. For example, the percentage of recovery of TGr cells cocultured with 10(5) TGs immortalized, normal, or carcinoma HMEC was 88 +/- 4, 41 +/- 10, or 2.0 +/- 1.7, respectively. Gap junction-mediated intercellular communication between homologous HMEC types was also studied, as quantitated on the basis of Lucifer yellow dye transfer between cells in culture. Results from the dye transfer studies supported those from the metabolic cooperation studies. These results using nonimmortalized tumor cells differ from previous reports in which immortal tumor cells have been found to communicate less than their normal counterparts. Previous reports suggesting that tumor cell lines communicate less than normal cells may have resulted from the confounding influence of the immortal phenotype on the tumor phenotype.     

Oligonucleotide microarray expression analysis of genes whose expression is correlated with tumorigenic and non-tumorigenic phenotype of HeLa x human fibroblast hybrid cells

In order to understand the differences and similarities between tumorigenic and non-tumorigenic HeLaxhuman fibroblast hybrids, gene expression profiles were examined with synthetic oligonucleotide arrays containing nearly 7000 gene probe sets. We used two pairs of genetically related hybrids, each pair representing individual clones of non-tumorigenic and tumorigenic segregant hybrids, respectively. Analysis of six possible comparisons, utilizing two algorithms, identified 204 genes with differential expression. The greater number of differentially expressed genes was observed when non-tumorigenic hybrids were compared with tumorigenic segregants. Fifteen and 14 genes, respectively, were consistently found to be differentially expressed in non-tumorigenic and tumorigenic cells. Among those 29 differentially expressed genes, three (intestinal alkaline phosphatase, caveolin-1, and solute carrier family2, member3) have been reported previously to be associated with the tumorigenic phenotype, using the same hybrid pairs. In addition, among the genes previously detected by differential display, 78% of them exhibited more than 5-fold change, demonstrating a high consistency between the two methods of differential gene expression. These findings suggest that synthetic oligonucleotide arrays are a powerful and highly reproducible tool to identify those genes whose expression is associated with certain phenotypes.     

Induction of morphological transformation, anchorage-independent growth and plasminogen activators in non-tumorigenic human osteosarcoma cells by lead chromate

The possible carcinogenicity of insoluble chromium (VI) compound, PbCrO4, in human cells has been tested using a nontumorigenic human osteosarcoma cell line (HOS, TE 85). Electron microscopic studies show that PbCrO4 is phagocytosed by HOS cells and accumulates within the vacuoles in the cytoplasm. A number of cell lines have been isolated following multiple treatment of HOS cells with PbCrO4. These cell lines are morphologically different from HOS cells, form anchorage-independent colonies in soft agar and form quickly regressing small tumor nodules in athymic nude mice. The cellular and secreted plasminogen activator (PA) levels of 5 cell lines isolated after PbCrO4 treatment are increased up to 8 fold and up to 10 fold respectively as compared to untreated HOS controls. SDS-PAGE analysis in the presence of copolymerized substrates is consistent with increase in 55 kDa urokinase-type PA (u-PA) and 68 kDa tissue-type PA (t-PA). These results show that PbCrO4 treatment leads to stable phenotypic changes indicative of the transformation of HOS cells.     

Differential sensitivity of tumorigenic and genetically related non-tumorigenic cells to cytotoxic polyunsaturated fatty acids

A series of closely related rat brain cell lines that differ in their ability to form tumors has been used to investigate the selectivity of cytotoxic polyunsaturated fatty acids. The colony-formation ability of tumorigenic F4 cells was markedly reduced when the cells were challenged with GLA and EPA. In contrast, the non-tumorigenic revertants were less affected. All retransformed tumorigenic variants exposed to GLA were as sensitive as their parental tumorigenic cells and more sensitive than the non-tumorigenic clones. However, two out of three retransformed tumorigenic variants exposed to EPA were less sensitive than either the parental tumorigenic or non-tumorigenic clones. The addition of ferrous chloride to the culture medium increased the cytotoxicity of GLA in tumorigenic but not in non-tumorigenic variants. These results suggest that tumorigenicity per se is characterized by a high sensitivity to PUFAs exogenously administered at appropriate concentrations and that the sensitivity is fatty acid specific.     

The free cytoplasmic calcium concentration of tumorigenic and non-tumorigenic human somatic cell hybrids

The fluorescent indicator of Ca2+ concentration, quin-2, has been used to measure the concentration of free Ca2+ in the cytoplasm of tumorigenic and non-tumorigenic human somatic cell hybrids. The cell hybrids were derived from the fusion of a HeLa derivative (D98 AH2) and normal human fibroblasts. The calcium concentration of the tumorigenic cell lines was 180 +/- 7nM and the level in the non-tumorigenic cells was 136 +/- 6nM. This difference was statistically highly significant (P less than 0.001). Control experiments are reported which show that the level of 3a2+ measured is not influenced by cell density or by the concentration of quin-2-tetra-(acetoxymethyl)ester used in these experiments. The possible implications of this elevated level of cytoplasmic calcium in tumorigenic cells are discussed.     

Loss of chromosome 3p arm differentiating tumorigenic from non-tumorigenic cells derived from the same SV40-transformed human mammary epithelial cells

After immortalization of human normal mammary epithelial cells by replication-defective SV40 genome integration, 2 cultures were developed independently. Both had the same integration site, in band 9q21, but rapidly diverged karyotypically. After a few passages, one, designated SC2T2, exhibited near-diploid (a) and the other, designated SL2T2, near-tetraploid (b) karyotypes. The simplest formulas were 44, X, -X, der(3;22) (q10;q10), der(4)t(4;9)(q34;q12), +8, +9, add(13)(pl), der(19) t(8; 19)(q21;p 13.3), add(22)(p 1) for karyotype (a) and 93, XXXX, add(1)(q12), add(11)(q13), +20 for karyotype (b). A number of alterations were further acquired with passages. Both cell cultures were tumorigenic, but their efficiency of grafting in nude mice largely differed: it was low for SL2T2 and high for SC2T2 cultures. All cultures of the xenografted tumors, obtained from either SL2T2 or SC2T2, exhibited the same clonal anomalies as those characterizing karyotype (a). It was concluded that only cells with karyotype (a) were tumorigenic, and that the difference in the tumorigenic potential of cultures SC2T2 and SL2T2 was related to their richness in cells with this karyotype. The comparison of the various karyotypes, together with data obtained in other cell types transformed by SV40, suggests that the acquisition of tumorigenicity in S2T2 mammary epithelial cells may be related to the loss of chromosome 3p arm. © 1995 Wiley-Liss, Inc.     

Neoplastic transformation of human osteoblast cells to the tumorigenic phenotype by heavy metal-tungsten alloy particles: induction of genotoxic effects

Heavy metal-tungsten alloys (HMTAs) are dense heavy metal composite materials used primarily in military applications. HMTAs are composed of a mixture of tungsten (91-93%), nickel (3-5%) and either cobalt (2-4%) or iron (2-4%) particles. Like the heavy metal depleted uranium (DU), the use of HMTAs in military munitions could result in their internalization in humans. Limited data exist, however, regarding the long-term health effects of internalized HMTAs in humans. We used an immortalized, non-tumorigenic, human osteoblast-like cell line (HOS) to study the tumorigenic transforming potential of reconstituted mixtures of tungsten, nickel and cobalt (rWNiCo) and tungsten, nickel and iron (rWNiFe). We report the ability of rWNiCo and rWNiFe to transform immortalized HOS cells to the tumorigenic phenotype. These HMTA transformants are characterized by anchorage-independent growth, tumor formation in nude mice and high level expression of the K-ras oncogene. Cellular exposure to rWNiCo and rWNiFe resulted in 8.90 +/- 0.93- and 9.50 +/- 0.91-fold increases in transformation frequency, respectively, compared with the frequency in untreated cells. In comparison, an equivalent dose of crystalline NiS resulted in a 7.7 +/- 0.73-fold increase in transformation frequency. The inert metal tantalum oxide did not enhance HOS transformation frequency above untreated levels. The mechanism by which rWNiCo and rWNiFe induce cell transformation in vitro appears to involve, at least partially, direct damage to the genetic material, manifested as increased DNA breakage or chromosomal aberrations (i.e. micronuclei). This is the first report showing that HMTA mixtures of W, Ni and Co or Fe cause human cell transformation to the neoplastic phenotype. While additional studies are needed to determine if protracted HMTA exposure produces tumors in vivo, the implication from these in vitro results is that the risk of cancer induction from internalized HMTAs exposure may be comparable with the risk from other biologically reactive and insoluble carcinogenic heavy metal compounds (e.g. nickel subsulfide and nickel oxide).     

Characterization of the glutametergic system in MG-63 osteoblast-like osteosarcoma cells

BACKGROUND: The glutamate system is a fairly complex bioregulation pathway, consisting of several ionotropic Glu receptors (iGlu.Rs), the metabotropic Glu receptors (mGlu.Rs), Glu transporters (EAATs) and glutamine synthetase (GS), which metabolizes glutamate to glutamine. MATERIALS AND METHODS: We characterized the MG-63 human osteoblast-like osteosarcoma cells with regards to mRNA expression of the (a) NMDA.R group of iGlu.Rs, (b) mGlu.Rs, (c) EAAT1 transporter, and (d) GS, using specific primers for their detection by RT-PCR. RESULTS: Our data confirm the mRNA expression of the NR1, NR2A, NR2B and NR2D subunits of NMDA.R and of GS mRNA in MG-63 cells. In addition, we documented, for the first time, the mRNA expression of the NR3A, EAAT1, mGlu.R1, mGlu.R2, mGlu.R3, mGluR.4, mGlu.R5 and mGlu.R8 mRNA. However, we did not detect the mRNA expression of NR2C, NR3B, mGlu.R6 and mGlu.R7 mRNA. CONCLUSION: Our data suggest that MG-63 cells can be used as a model for studying the possible role of Glu beyond CNS.     

Resistance of murine LTA cells to oncogene--mediated progression from tumorigenic to metastatic phenotype

We have previously shown that expression of the H-ras oncogene alone does not induce progression of a tumorigenic but non-metastatic murine fibroblast cell line (LTA) to a metastatic phenotype. Because myc genes, alone or with ras, have been implicated at different stages of progression in other systems, we examined the ability of v-myc, alone and in combination with H-ras, to induce malignant conversion of LTA. We found no increase in either "spontaneous" (assessed after s.c. injection into nude mice) or "experimental" (assessed after i.v. injection into chick embryos) metastatic ability in spite of high levels of v-myc RNA in LTA cells transfected with v-myc alone. Serial in vivo passaging did not consistently select for either myc expression or metastatic ability. Myc transfected cells expressing high levels of myc RNA were subsequently transfected with H-ras. LTA cells expressing both oncogenes at high levels remained non-metastatic. LTA cells thus are resistant to the effects of myc and ras oncogenes (alone and in combination) on a specific stage of tumor progression, that of malignant conversion, and may offer a good model for studying mechanisms of resistance to these oncogenes.     

RasGRP3, a Ras activator, contributes to signaling and the tumorigenic phenotype in human melanoma

RasGRP3, an activator for H-Ras, R-Ras and Ras-associated protein-1/2, has emerged as an important mediator of signaling downstream from receptor coupled phosphoinositide turnover in B and T cells. Here, we report that RasGRP3 showed a high level of expression in multiple human melanoma cell lines as well as in a subset of human melanoma tissue samples. Suppression of endogenous RasGRP3 expression in these melanoma cell lines reduced Ras-GTP formation as well as c-Met expression and Akt phosphorylation downstream from hepatocyte growth factor (HGF) or epidermal growth factor (EGF) stimulation. RasGRP3 suppression also inhibited cell proliferation and reduced both colony formation in soft agar and xenograft tumor growth in immunodeficient mice, demonstrating the importance of RasGRP3 for the transformed phenotype of the melanoma cells. Reciprocally, overexpression of RasGRP3 in human primary melanocytes altered cellular morphology, markedly enhanced cell proliferation and rendered the cells tumorigenic in a mouse xenograft model. Suppression of RasGRP3 expression in these cells inhibited downstream RasGRP3 responses and suppressed cell growth, confirming the functional role of RasGRP3 in the altered behavior of these cells. The identification of the role of RasGRP3 in melanoma highlights its importance, as a Ras activator, in the phosphoinositide signaling pathway in human melanoma and provides a new potential therapeutic target.     

Differentiation and the tumorigenic and metastatic phenotype of murine melanoma cells

Using B16 F10 murine melanoma cells and sublines generated from the JB/MS melanoma which exhibit various degrees of melanogenesis, the relationships among differentiation, tumorigenicity, and metastatic potential were examined. The effect of melanocyte-stimulating hormone (MSH), which specifically stimulates differentiation of melanocytes, was also studied. All melanoma lines tested were capable of growing as experimental pulmonary metastases but, surprisingly, the undifferentiated and amelanotic JB/MS-w cells failed to grow as primary subcutaneous tumors. JB/MS-w cells, which had few surface MSH receptors, did not respond to MSH with an increase in melanin production, unlike the other cell lines. Although in vitro treatment with MSH did not change the rates of growth of primary tumors by these cell lines, such treatment decreased the number of pulmonary metastases from B16 F10, JB/MS cells, JB/MS-b1 cells and JB/MS-w cells. Conversely, MSH treatment significantly increased the rates of pulmonary metastases from JB/MS-p cells. The expression of surface melanoma antigens, urokinase-type plasminogen activity and susceptibility to natural killer cells were examined. MSH did not significantly alter surface melanoma antigen expression, but increased the natural killer cell susceptibility of B16 F10, JB/MS and JB/MS-b1 cells, cells which possess abundant surface MSH receptors. There was an inverse correlation between differentiation (pigmentation) and proliferation in vitro, and the more pigmented melanoma cells (B16 F10, JB/MS and JB/MS-b1) expressed relatively lower levels of class-I MHC, relatively higher levels of class-II MHC and the highest metastatic capacity. These results demonstrate that MSH possesses the capacity to regulate not only melanogenesis, but also other factors critical to the metastatic growth of the cells.     

Camptothecin derivatives induce regression of human ovarian carcinomas grown in nude mice and distinguish between non-tumorigenic and tumorigenic cells in vitro

We have recently shown that the plant alkaloid 20(S)-camptothecin and its derivatives 9-nitro-20(S)-camptothecin(9NC) and 9-amino-2O(S)-camptothecin(9AC) inhibit the growth of a variety of human tumors xenografted in nude mice. In this report, we demonstrate that 9NC and 9AC effectively inhibit growth, and subsequently induce regression, of human ovarian tumors grown in nude mice. Tumor regression is accompanied by degenerative changes in the tumor cells as assessed by microscopic observations of histological sections prepared from the tumors. Parallel experiments in vitro show that 9NC inhibits in a similar manner the growth of human ovarian carcinoma cells, regardless of their ability to induce tumors when xenografted in nude mice, and induces similar morphological changes in both non-tumorigenic and tumorigenic cells, as assessed by microscopic observation. Flow cytometry studies show that 9NC-induced growth inhibition of the non-tumorigenic cells is associated with accumulation of these cells in G₂. In contrast, 9NC-induced growth inhibition of the tumorigenic cells is associated with the generation of cells containing a reduced DNA content, that is, cells programmed to die. In conclusion, camptothecins appear to be cytostatic for non-tumorigenic, but cytotoxic for tumorigenic cells, an important finding from viewpoints of cell biology, pharmacology and cancer chemotherapy.     

Downregulation of Betaig-h3 gene is causally linked to tumorigenic phenotype in asbestos treated immortalized human bronchial epithelial cells

Although Betaig-h3 gene has been suggested to modulate cell adhesion and tumor formation, its physiological functions are not well understood. Using human papillomavirus immortalized human bronchial epithelial (BEP2D) cells, we found that Betaig-h3 expression was markedly decreased in asbestos-induced tumorigenic cells. Fusion of tumorigenic and control BEP2D cells resulted in the recovery of Betaig-h3 gene expression to control level and the loss of tumorigenic phenotype. Furthermore, ectopic expression of Betaig-h3 gene in asbestos-induced tumorigenic cells inhibited cell growth in vitro, anchorage independent phenotype, as well as tumorigenicity in nude mice. Betaig-h3 gene is ubiquitously expressed in various normal human tissues, with the exception of the brain, where there is little or no expression. In contrast, there was a decrease or absence in expression of the Betaig-h3 gene in 14 human tumor cell lines of diverse histological types examined, when compared with normal human cells or tissues. The result strongly suggests that loss of Betaig-h3 expression is a frequent event in human cancer and causally related to acquisition of tumorigenic phenotype in asbestos-treated BEP2D cells.     

Co-inoculation of tumorigenic rat prostate mesenchymal cells with non-tumorigenic epithelial cells results in the development of carcinosarcoma in syngeneic and athymic animals

Co-inoculation of NbF-I and NbE-I s.c. into either adult male syngeneic rats or athymic nude mice induced the development of tumors that resembled carcinosarcoma on histopathologic evaluation. These tumors were composed of a mixture of adenocarcinoma and fibrosarcoma and were induced by the mixtures of NbF-I and NbE-I cells at a ratio ranging from 0.001 to 3.2; inoculation of NbF-I alone resulted in the development of fibrosarcoma. Flow-cytometric analysis showed that the epithelial cells subcloned from the carcinosarcoma had a DNA profile like that of their parental cell line and remained non-tumorigenic. When co-inoculated with the tumorigenic fibroblasts in syngeneic hosts, however, the subcloned epithelial cells again formed carcinosarcomas. Our results indicate that cell fusion between epithelial cells and fibroblasts is an unlikely explanation for tumorigenicity. We propose that prostatic fibroblasts exert a directive influence on their adjacent epithelial cells through a paracrine mechanism that determines epithelial growth and tumorigenicity in vivo.