Down-regulation of ras and myc expression associated with mdr-1 overexpression in adriamycin-resistant tumor cells.

The murine melanoma tumor cells, B16-BL6, are a recognized model for experimental and spontaneous metastasis. B16-BL6 cells express a lower metastatic phenotype upon acquisition of resistance to adriamycin. Using this novel system, the role of ras, c-myc, and multidrug-resistant gene (mdr1) expression in the metastatic and drug-resistant phenotype was examined. The metastatic cells expressed a high level of c-Ki-ras and c-myc, whereas down-regulation of both proto-oncogenes was observed in the adriamycin-resistant cells. The mdr1 gene, which encodes P-glycoprotein of the drug-resistant superfamily gene, was overexpressed in drug-resistant melanoma cells. These results suggest that altered expression of genes that regulate cellular proliferation and growth may be a determinant of metastasis and drug sensitivity of tumor cells.     

Rhythmic patterns in the expression of the ras oncogene in proliferating and differentiating erythroleukaemia cells

Temporal variations in the expression of the ras oncogene, and its protein product, were investigated during hexamethylene bisacetamide (HMBA)-induced differentiation of murine erythroleukaemic (MEL) cells. We highlight the fact that when comparisons were made between untreated, proliferating cells and HMBA-treated, differentiating cells using only one time-point, differences, both for the expression of the gene and the protein, were in most cases insignificant; standard deviations were high and the interpretation could be made that HMBA had little effect. Such interpretation fails to take account of the dynamic nature of the system, with single time-point studies giving incomplete information, which can be misleading. Multiple time analyses showed clearly that rhythmic patterns of expression were modulated by the differentiating agent. Time-dependent changes in the expression of mRNA specific to H- ras and N- ras, as well as in the expression of the Ras protein, when measured over periods of minutes or hours, were apparent. HMBA affected frequency and phasing of the rhythms. Regulation of the dynamics in this way may be crucial to the control of cell function and transformation.     

Activity of topoisomerase I and endonucleases in cells transfected by a ras oncogene

Activities of nuclear endonucleases and topoisomerase I were measured in rat fibroblasts which were at the stages of tumor transformation: control embryonal fibroblasts--CEF; cells immortalised by transfection of S1A segment of SA7 adenovirus--REF-1; intermedius cells transfected once by EJras oncogene--REF-1EJ; and cells transformed after the second transfection by the same oncogene--REF-2EJ. The topoisomerase I and Ca2+, Mg2+-dependent endonuclease was most decreased at the stage of immortalised cells, and the intermedius stage (REF-1EJ) was characterized by the lower activity of Ca2+, Mg2+-dependent endonuclease. The highest activity of Mn2+-dependent endonuclease is seen in REF-2EJ cells. In model experiments the ability of Ca2+, Mg2+-dependent endonuclease to split non-stochastically the EJras oncogene inserted into pBR322 plasmid was shown. The role of the investigated enzymes in the restriction of plasmid integration, cellular immortalisation and recombination of plasmids with chromosomes during cell transformation is discussed.     

Immunohistochemical evaluation of ras oncogene expression in pulmonary and pleural neoplasms

We undertook an immunohistochemical analysis of human bronchopulmonary epithelial neoplasms and pleural mesotheliomas using a monoclonal antibody which recognizes ras oncogene products (p21ras). The monoclonal antibody, RAP-5, recognizes both unaltered and certain mutated p21ras. Formalin fixed and paraffin embedded tissue samples of 187 lung epithelial tumors and 27 pleural mesotheliomas were investigated; normal and bronchiectatic lungs were similarly studied. Normal lung and pleural tissue did not immunostain except for occasional type II pneumocytes. Reactive type II pneumocytes adjacent to carcinomas and bronchiectasis immunostained consistently. Twenty four/34 (71%) squamous carcinomas immunostained. Only 8/50 (16%) adenocarcinomas immunostained focally and weakly whereas 19/24 (79%) bronchioloalveolar carcinomas immunostained. Eleven/18 (61%) large cell carcinomas immunostained with variable intensity. Eleven/13 (85%) carcinoids, 6/7 (85%) well differentiated neuroendocrine carcinomas, and 18/21 (86%) intermediate cell neuroendocrine carcinomas immunostained while none of 20 small cell neuroendocrine carcinomas immunostained. Only a few mesotheliomas were immunostained focally. Two/14 (14%) epithelial type and 1/9 (11%) biphasic type mesotheliomas immunostained weakly; none of 4 spindle cell mesotheliomas immunostained. We conclude that while at least occasional cases of most types of pulmonary epithelial neoplasms express p21ras, the frequency and intensity of the expression are distinctly greater in certain tumor types such as squamous, bronchioloalveolar, and neuroendocrine neoplasm except for the small cell type. Contrary to these lung epithelial neoplasms, most mesotheliomas did not immunostain for p21ras. Whether the enhanced p21ras expression may point to a different mechanism of transformation or may merely reflect differentiation features remains undetermined.     

Adenovirus E1A oncogene expression in tumor cells enhances killing by TNF-related apoptosis-inducing ligand (TRAIL)

Expression of the adenovirus serotype 5 (Ad5) E1A oncogene sensitizes cells to apoptosis by TNF-alpha and Fas-ligand. Because TNF-related apoptosis-inducing ligand (TRAIL) kills cells in a similar manner as TNF-alpha and Fas ligand, we asked whether E1A expression might sensitize cells to lysis by TRAIL. To test this hypothesis, we examined TRAIL-induced killing of human melanoma (A2058) or fibrosarcoma (H4) cells that expressed E1A following either infection with Ad5 or stable transfection with Ad5-E1A. E1A-transfected A2058 (A2058-E1A) or H4 (H4-E1A) cells were highly sensitive to TRAIL-induced killing, but Ad5-infected cells expressing equally high levels of E1A protein remained resistant to TRAIL. Infection of A2058-E1A cells with Ad5 reduced their sensitivity to TRAIL-dependent killing. Therefore, viral gene products expressed following infection with Ad5 inhibited the sensitivity to TRAIL-induced killing conferred by transfection with E1A. E1B and E3 gene products have been shown to inhibit TNF-alpha- and Fas-dependent killing. The effect of these gene products on TRAIL-dependent killing was examined by using Ad5-mutants that did not express either the E3 (H5dl327) or E1B-19K (H5dl250) coding regions. A2058 cells infected with H5dl327 were susceptible to TRAIL-dependent killing. Furthermore, TRAIL-dependent killing of A2058-E1A cells was not inhibited by infection with H5dl327. Infection with H5dl250 sensitized A2058 cells to TRAIL-induced killing, but considerably less than H5dl327-infection. In summary, expression of Ad5-E1A gene products sensitizes cells to TRAIL-dependent killing, whereas E3 gene products, and to a lesser extent E1B-19K, inhibit this effect.     

Metabolism of n-6 fatty acids by NIH-3T3 cells transfected with the ras oncogene

N-6 fatty acid metabolism was compared in NIH-3T3 cells and DT cells, which differ only in the presence of the v-Ki-ras oncogene. Non-dividing cells were incubated with [1-¹⁴C]-labelled fatty acids (18:2n-6, 18:3n-6, 20:3n-6 and 20:4n-6) at different time intervals (2–24 h) and concentration (0–120 M). In both cells lines, the uptake of different fatty acids from the medium was similar and reached a maximum at 6–8 h. All fatty acids reached the same maximum level in DT cells, whereas, the relative uptake of added fatty acids by NIH-3T3 cells was different: 20:4n-6>20:2n-6>18:2n-6=18:3n-6. Throughout the incubation (2–24 h), desaturation and elongation of n-6 fatty acids was more active in DT cells than in NIH-3T3 cells. However, in both cell lines, incubated with different n-6 fatty acid precursors, the levels of radiolabelled 20:4n-6 were relatively constant. In DT cells, phosphatidylcholine was found to be the major fraction labelled with n-6 fatty acids precursors and those of endogenous synthesis, whereas, in NIH-3T3 cells the neutral lipid fraction, particularly triglycerides, was also strongly labelled. In concentration dependent studies, phospholipid labelling by fatty acids was saturable. At lower concentrations, especially in DT cells, phospholipids were labelled predominantly. As the concentration increased there was an overflow into the triglyceride fraction. Since the differences in fatty acid metabolism between the two cell lines cannot be related to the growth rate, it is suggested that they were a consequence of the expression of the v-Ki-ras oncogene.Abbreviations BSA bovine serum albumin - CE cholesterol ester - DG diglyceride - DMEM Dulbecco's modification of Eagle's medium - EL ether lipids (glyceryl ether diesters) - FAME fatty acid methyl ester - FCS fetal calf serum - FFA free fatty acids - HEPES N-2-(hydroxyethyl)piperazine-N-2-ethanesulphonic acid - MG monoglyceride - NL neutral lipid - PC phosphatidylcholine - PE phosphatidylethanolamine - PI phosphatidylinositol - PL phospholipid - s.a specific activity - TG triglyceride - TLC thin layer chromatography     

Model systems of carcinogenesis in vitro: the interaction of the ras oncogene with immortalized cells

Gene transfer experiments have shown that ras effector functions are sufficient to transform cells from a variety of established lines (e. g., mouse NIH3T3 cells). In contrast, primary cells and early passage rodent cells can be transformed by ras oncogenes only at low frequencies, unless cotransfected with collaborating genes such as adenovirus early region IA (EIA) or myc retroviral oncogene homologue. Primary rat embryo fibroblasts (REF) were chosen as a model for the analysis of multistep cellular transformation. Transfection of REF, immortalized by early region of simian adenovirus SA7 with c-Ha-ras oncogene cannot induce their morphological transformation. This phenomenon is observed only after second transfection with the same oncogene. These different cell lines can be used for further analysis of the mechanisms of carcinogenesis.     

Presence of a Kirsten murine sarcoma virus ras oncogene in cells transformed by 3-methylcholanthrene.

Oncogenes have previously been reported in the DNAs of mouse fibroblast lines which had become transformed after in vitro exposure to the carcinogen 3-methylcholanthrene. These oncogenes are now shown to be versions of the cellular Kirsten ras gene and are therefore homologous to oncogenes detected in a variety of human tumor DNAs.     

Massive programmed cell death in intestinal epithelial cells induced by three-dimensional growth conditions: suppression by mutant c-H-ras oncogene expression

Deregulation of molecular pathways controlling cell survival and death, including programmed cell death, are thought to be important factors in tumor formation, disease progression, and response to therapy. Studies devoted to analyzing the role of programmed cell death in cancer have been carried out primarily using conventional monolayer cell culture systems. However the majority of cancers grow as three-dimensional solid tumors. Because gene expression, and possibly function, can be significantly altered under such conditions, we decided to analyze the control and characteristics of cell death using a compatible three- dimensional tissue culture system (multicellular spheroids) and compare the results obtained to those using two-dimensional monolayer cell culture. To do so we selected for study an immortalized, but nontumorigenic line of rat intestinal epithelial cells, called IEC-18, and several tumorigenic variants of IEC-18 obtained by transfection with a mutant (activated) c-H-ras oncogene. The rationale for choosing these cell lines was based in part on the fact that intestinal epithelial cells grow in vivo in a monolayer-like manner and form solid tumors only after sustaining certain genetic mutations, including those involving the ras gene family. We found that the IEC-18 cells, which grow readily and survive in monolayer cell culture, undergo massive cell death within 48-72 h when cultured as multicellular spheroids on a nonadhesive surface. This process was accompanied by a number of features associated with programmed cell death including chromatin condensation (Hoechst 33258 staining) apoptotic morphology, DNA degradation, and a virtual complete loss of colony forming (clonogenic) ability in the absence of apparent membrane damage as well as accumulation of lipid containing vacuoles in the cytoplasm. Moreover, enforced over-expression of a transfected bcl-2 gene could prevent this cell death process from taking place. In marked contrast, three different stably transfected ras clones of IEC-18 survived when grown as multicellular spheroids. In addition, an IEC cell line (called clone 25) carrying its mutant transfected ras under a glucocorticoid inducible promoter survived in three-dimensional culture only when the cells were exposed to dexamethasone. If exposure to dexamethasone was delayed for as long as 48 h the cells nevertheless survived, whereas the cells became irreversibly committed to programmed cell death (PCD) if exposed to dexamethasone after 72 h. These results suggest that intestinal epithelial cells may be programmed to activate a PCD pathway upon detachment from a physiologic two-dimensional monolayer configuration, and that this process of adhesion regulated programmed cell death (ARPCD) can be substantially suppressed by expression of a mutant ras oncogene.(ABSTRACT TRUNCATED AT 400 WORDS)     

Constitutive expression of thrombospondin 1 in MC3T3-E1 osteoblastic cells inhibits mineralization

Thrombospondin 1 (TSP1) is a multifunctional extracellular glycoprotein present mainly in the fetal and adult skeleton. Although an inhibitory effect of TSP1 against pathological mineralization in cultured vascular pericytes has been shown, its involvement in physiological mineralization by osteoblasts is still unknown. To determine the role of TSP1 in biomineralization, mouse osteoblastic MC3T3-E1 cells were cultured in the presence of antisense phosphorothioate oligodeoxynucleotides complementary to the TSP1 sequence. The 18- and 24-mer antisense oligonucleotides caused concentration-dependent increases in the number of mineralized nodules, acid-soluble calcium deposition in the cell/matrix layer, and alkaline phosphatase activity within 9 days, without affecting cell proliferation. The corresponding sense or scrambled oligonucleotides did not affect these parameters. In the antisense oligonucleotide-treated MC3T3-E1 cells, thickened extracellular matrix, well-developed cell processes, increased intracellular organelles, and collagen fibril bundles were observed. On the other hand, the addition of TSP1 to the culture decreased the production of a mineralized matrix by MC3T3-E1 cells. Furthermore, MC3T3-E1 clones overexpressing mouse TSP1 were established and assayed for TSP1 protein and their capacity to mineralize. TSP1 dose-dependently inhibited mineralization by these cells both in vitro and in vivo. These results indicate that TSP1 functions as an inhibitory regulator of bone mineralization and matrix production by osteoblasts to sustain bone homeostasis.     

Transformation by a ras oncogene causes increased expression of protein kinase C-alpha and decreased expression of protein kinase C-epsilon

Rat embryo fibroblasts and liver epithelial cell lines normally express two isoforms of protein kinase C (PKC), PKC alpha and PKC epsilon. Derivatives of these cells transformed by an activated human c-H-ras oncogene display a several-fold increase in expression of PKC alpha and a concomitant decrease in PKC epsilon, at both the protein and mRNA levels. Similar changes are seen when the transformed phenotype is induced by Zn2+ in cells carrying the activated ras oncogene under the control of a metallothionein promoter. Studies using cell lines that express very high levels of PKC beta 1, studies using a specific inhibitor of PKC (CGP 41251), and studies in which PKC activity is down-regulated by treatment with a phorbol ester tumor promoter provide evidence that the effects of the ras oncogene on the expression of PKC alpha and PKC epsilon are mediated mainly through a PKC-independent pathway. The present results provide the first evidence that transformation of cells by an oncogene can alter the relative expression of specific isoforms of PKC. It is possible that these changes contribute to the malignant phenotype of these cells.     

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate and the ras oncogene modulate expression and phosphorylation of gap junction proteins.

Gap junctional intercellular communication is inhibited in response to tumor promoters and oncogene transformation, suggesting that loss of this function is an important step in tumor formation. To elucidate the molecular mechanisms responsible for this inhibition, we examined the expression of gap junction proteins and mRNA in mouse primary keratinocytes after treatment with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) and/or ras transformation. During normal cell growth, keratinocytes expression the alpha 1 (connexin 43) and beta 2 (connexin 26) proteins. Within 5 min of TPA treatment, the alpha 1 protein became rapidly phosphorylated on serine residues and its expression was dramatically reduced by 24 h. The beta 2 protein, after an initial increase in expression, was also significantly reduced 24 h after treatment with TPA. ras transformation caused changes similar to those induced by TPA. The alpha 1 protein underwent an increase in serine phosphorylation, although its expression declined only slightly, while beta 2 expression was greatly reduced. The effects of TPA and ras on alpha 1 expression were additive; treatment of ras-transformed cells with TPA resulted in increased alpha 1 phosphorylation, with greatly decreased protein levels, much lower than those generated by either agent alone. These data provide a likely explanation for the similar and synergistic inhibition of gap junctional intercellular communication by phorbol esters and ras.     

Restoration of cell surface CD4 expression in human immunodeficiency virus type 1-infected cells by treatment with a Tat antagonist.

Productive infection of T lymphocytes with human immunodeficiency virus type 1 (HIV-1) is accompanied by a diminution of surface CD4 receptors. Treatment of chronically HIV-1-infected CD4-negative T cells in vitro with the Tat antagonist Ro 5-3335 resulted in a drug dose-dependent decrease in virus protein production and a reciprocal increase in surface CD4 display. The drug-treated cells remained viable, showed significantly reduced levels of the full-length and spliced HIV-1 mRNAs as detected by Northern (RNA) blot hybridization, and maintained integrated HIV-1 DNA. In immunoprecipitation studies with drug-treated cells, the levels of free 55-kDa CD4 protein increased and gp160 complexed with CD4 decreased in amount. These results show for the first time that certain cytopathogenic effects of chronic HIV-1 infection can be reversed by suppressing virus expression.     

Effects of heat shock on the expression of thrombospondin by endothelial cells in culture

Heat-shock proteins from confluent primary cultures of bovine aortic endothelial cells were analyzed by SDS-polyacrylamide gels. In addition to the increased synthesis of the classical heat-shock proteins, there is an increase of a 180,000-mol wt polypeptide in the growth media of heat-shocked cells. Immunoprecipitation with specific antiserum indicates that the 180,000-mol wt polypeptide is thrombospondin. Assay of mRNA levels coding for thrombospondin after brief hyperthermic treatment (45 degrees C, 10 min), followed by a recovery of 2 h at 37 degrees C, results in a twofold increase in mRNA abundance. In contrast, the activation level of the 71,000-mol wt heat-shock protein mRNA occurs at an earlier time than for thrombospondin mRNA. Immunofluorescence microscopy was used to study the intracellular and extracellular distribution of thrombospondin. Thrombospondin is localized to a prominent pattern of granules of intracellular fluorescence in a perinuclear distribution in cells not exposed to heat. Upon heat treatment, the pattern of granules of intracellular fluorescence appears more pronounced, and the fluorescence appears to be clustered more about the nucleus. There are at least three pools of extracellular forms of thrombospondin: (a) the fine fibrillar extracellular matrix thrombospondin; (b) the punctate granular thrombospondin; and (c) the thrombospondin found in the conditioned medium not associated with the extracellular matrix. When bovine aortic endothelial cells are exposed to heat, the extracellular matrix staining of a fibrillar nature is noticeably decreased, with an increase in the number and degree of fluorescence of focal areas where the punctate granule thrombospondin structures are highly localized. No gross morphological changes in extracellular matrix staining of fibronectin was noted. However, the intermediate filament network was very sensitive and collapsed around the nucleus after heat shock. We conclude that the expression of thrombospondin is heat-shock stimulated.     

Neuropilin-1 expression by tumor cells promotes tumor angiogenesis and progression.

Neuropilin-1 (NRP1) is a VEGF(165) and semaphorin receptor expressed by vascular endothelial cells (EC) and tumor cells. The function of NRP1 in tumor cells is unknown. NRP1 was overexpressed in Dunning rat prostate carcinoma AT2.1 cells using a tetracycline-inducible promoter. Concomitant with increased NRP1 expression in response to a tetracycline homologue, doxycycline (Dox), basal cell motility, and VEGF(165) binding were increased three- to fourfold in vitro. However, induction of NRP1 did not affect tumor cell proliferation. When rats injected with AT2.1/NRP1 tumor cells were fed Dox, NRP1 synthesis was induced in vivo and AT2.1 cell tumor size was increased 2.5- to 7-fold in a 3-4 wk period compared to controls. The larger tumors with induced NRP1 expression were characterized by markedly increased microvessel density, increased proliferating EC, dilated blood vessels, and notably less tumor cell apoptosis compared to noninduced controls. It was concluded that NRP1 expression results in enlarged tumors associated with substantially enhanced tumor angiogenesis.