Fibrosarcoma Line Research Articles

The DEXD/H-box RNA helicase RHII/Gu is a co-factor for c-Jun-activated transcription.

Tandem affinity purification (TAP) and mass spectrometric peptide sequencing showed that the DEAD-box RNA helicase RHII/Gu is a functional interaction partner of c-Jun in human cells. The N-terminal transcription activation region of, c-Jun interacts with a C-terminal domain of RHII/Gu. This interaction is stimulated by anisomycin treatment in a manner that is concurrent with, but independent of, c-Jun phosphorylation. A possible explanation for this effect is provided by the observation that RHII/Gu translocates from nucleolus to nucleoplasm upon anisomycin or UV treatment or when JNK signaling is activated by overexpression of a constitutively active form of MEKK1 kinase. Several experiments show that the RNA helicase activity of RHII/Gu supports c-Jun-mediated target gene activation: dominant-negative forms of RHII/Gu, as well as a neutralizing antibody against the enzyme, significantly interfered with c-Jun target gene activity but not with transcription in general. These findings clarify the mechanism of c-Jun-mediated transcriptional regulation, and provide evidence for an involvement of RHII/Gu in stress response and in RNA polymerase II-catalyzed transcription in mammalian cells.

Locus Control Region of the Human CD2 Gene in a Lentivirus Vector Confers Position-Independent Transgene Expression

Vectors derived from murine leukemia virus (MLV) have been used in many human gene therapy clinical trials. However, insertion of the locus control regions (LCRs) derived from the beta-globin gene locus or the CD2 gene into MLV vectors frequently led to vector rearrangement. Since the human immunodeficiency virus (HIV) sequence diverges significantly from the MLV sequence, we tested whether the LCR sequence is more stable in the context of an HIV vector. Clones derived from human fibrosarcoma line HT1080 cells transduced with an HIV vector containing the T-cell-specific CD2 LCR exhibit the same wide range of transgene expression as clones lacking the LCR. In contrast, Jurkat and primary T-cell clones derived from the transduction of the LCR-containing vector show, on average, a three- to fourfold increase in transgene expression relative to that of the control vector. This is consistent with previous observations that the CD2 LCR contains a T-cell-specific enhancer. In addition, the clones derived from the LCR-containing vector have a much lower clonal variation in transgene expression than those derived from the control vector. We also demonstrate that the level of transgene expression is proportional to the vector copy number. These results suggest that the human CD2 LCR sequence is compatible with HIV vector sequences and confers enhanced integration site-independent and copy number-dependent expression of the transgene. Thus, HIV vectors may represent the ideal vehicle to deliver genes controlled by various cis-acting elements such as LCRs.

Striking Bimodal Methylation of the Repeat Unit of the Tandem Array Encoding Human U2 snRNA (the RNU2 Locus)

The genes encoding human U2 small nuclear RNA are arrayed in tandem (the RNU2 locus) and have undergone concerted evolution for >35 Myr. Tandem organization of repetitive sequences may facilitate recombination that underlies concerted evolution, but could risk instability. Since DNA methylation plays a crucial role in genome stability, we investigated the methylation status of the RNU2 locus to understand the forces maintaining array stability and homogeneity. We found that a region of ∼1.5 kb spanning the U2 promoter, U2 gene sequence, and CT microsatellite is completely unmethylated, whereas the rest of the repeat is heavily methylated. Since the U2 transcription enhancer DSE and CT microsatellite mark the boundaries between methylated and unmethylated domains, they might function as cis-acting elements for establishing and maintaining proper methylation at the RNU2 locus. Interestingly, the RNU2 locus in human fibrosarcoma line HT1080 is hypomethylated, and de novo methylation did not occur in an artificial U2 tandem array introduced by stable transfection. The observed bimodal methylation pattern may be important for both efficient transcription of U2 gene and maintenance of nearly perfect tandem arrays in somatic cells.

31P magnetic resonance spectroscopy as a predictor of efficacy in photodynamic therapy using differently charged zinc phthalocyanines.

Photodynamic therapy (PDT) is a developing approach to the treatment of solid tumours which requires the combined action of light and a photosensitizing drug in the presence of adequate levels of molecular oxygen. We have developed a novel series of photosensitizers based on zinc phthalocyanine which are water-soluble and contain neutral (TDEPC), positive (PPC) and negative (TCPC) side-chains. The PDT effects of these sensitizers have been studied in a mouse model bearing the RIF-1 murine fibrosarcoma line studying tumour regrowth delay, phosphate metabolism by magnetic resonance spectroscopy (MRS) and blood flow, using D2O uptake and MRS. The two main aims of the study were to determine if MRS measurements made at the time of PDT treatment could potentially be predictive of ultimate PDT efficacy and to assess the effects of sensitizer charge on PDT in this model. It was clearly demonstrated that there is a relationship between MRS measurements during and immediately following PDT and the ultimate effect on the tumour. For all three drugs, tumour regrowth delay was greater with a 1-h time interval between drug and light administration than with a 24-h interval. In both cases, the order of tumour regrowth delay was PPC > TDEPC = TCPC (though the data at 24 h were not statistically significant). Correspondingly, there were greater effects on phosphate metabolism (measured at the time of PDT or soon after) for the 1-h than for the 24-h time interval. Again effects were greatest with the cationic PPC, with the sequence being PPC > TDEPC > TCPC. A parallel sequence was observed for the blood flow effects, demonstrating that reduction in blood flow is an important factor in PDT with these sensitizers. © 1999 Cancer Research Campaign

A comparison in individual murine tumors of techniques for measuring oxygen levels

Purpose: To investigate the relationship between different techniques for measuring oxygen levels in a murine tumor model. Methods and Materials: Using the murine fibrosarcoma line KHT-C, five techniques of measuring oxygen levels—the Eppendorf pO 2 Histograph, EF5 binding, the comet assay, a paired survival assay, and an in vivo growth delay assay—were assessed. In these experiments, three or more techniques were applied in different combinations to measure the oxygen levels in individual tumors. Results: Statistically significant correlations were observed between the hypoxic proportions calculated from the paired survival assay with those from EF5 binding. The comet assay was found to have a statistically significant correlation with the paired survival analysis and the growth delay analysis. No statistically significant correlation was found between the Eppendorf pO 2 Histograph measurements and those from the other techniques, although there were weak correlations with the paired survival assay and EF5 binding. For technical reasons, a comparison was not made between EF5 binding and the growth delay assay. Conclusions: The correlations found between EF5 binding and the comet assay with the radiobiological assays suggest that these techniques have potential for predicting outcome following radiation treatment. The lack of correlation seen between the pO 2 Histograph data and the radiobiological assays is in contrast to results from early clinical trials.

Expression Cloning of Mouse cDNA of CMP-NeuAc:Lactosylceramide α2,3-Sialyltransferase, an Enzyme That Initiates the Synthesis of Gangliosides

Expression cloning of a cDNA for the alpha2,3-sialyltransferase (GM3 synthase) (EC 2.4.99.-) gene was performed using a GM3-lacking mouse fibroblast line L cell and anti-GM3 monoclonal antibody. Plasmids from a cDNA library generated with poly(A)+ RNA of a mouse fibrosarcoma line CMS5j and pdl3027 (polyoma T antigen) were co-transfected into L cells. The isolated cDNA clone pM3T-7 predicted a type II membrane protein with 13 amino acids of cytoplasmic domain, 17 amino acids of transmembrane region, and a large catalytic domain with 329 amino acids. Introduction of the cDNA clone into L cells resulted in the neo-synthesis of GM3 and high activity of alpha2,3-sialyltransferase. Among glycosphingolipids, only lactosylceramide showed significant activity as an acceptor, indicating that this gene product is a sialyltransferase specific for the synthesis of GM3. An amino acid sequence deduced from the cloned cDNA showed the typical sialyl motif with common features among alpha2,3-sialyltransferases. Among various mouse tissues, brain, liver, and testis showed relatively high expression of a 2.3-kilobase mRNA, whereas all tissues, more or less, expressed this gene.

Towards the development of peptide mimotopes of carbohydrate antigens as cancer vaccines.

Tumor-associated carbohydrate antigens are considered important targets in efforts to develop cancer vaccines. To further enhance vaccine efforts, we are developing peptide mimotopes of tumor-associated carbohydrate antigens that can elicit functional immune responses. Mapping peptide epitopes with anticarbohydrate antibodies can lend to defining structural relationships that can go undetected by screening of carbohydrate antigens alone. Here we contrast reactivity patterns for peptides using monoclonal antibodies (MAbs) directed to the neolactoseries related Lewis Y (LeY) and sialyl-Lewis X (sLeX) antigen and the GD3/GD2 ganglioside antigen. We observe that representative MAbs cross-react with a WRY-containing peptide and that this motif type is isolated by the respective monoclonal in peptide phage display screening. Primary immunization with multiple antigen peptide preparations with QS-21 adjuvant efficiently elicited cytotoxic IgM antibodies for a murine Meth A fibrosarcoma line expressing sLeX. The cytotoxicity of IgG polyclonal response was found to be as effective as IgM in mediating complement-dependent cytotoxicity against the Meth A line. These experiments suggest that peptide mimotopes of the LeY and sLeX tumor-associated carbohydrate antigen and QS-21 adjuvant could be considered as an immunogenic therapeutic vaccine in carcinoma and melanoma patients in the minimal residual disease setting.

Lipid characteristics in metastatic cells.

Interest in lipid characteristics of metastatic cells was aroused by the consideration that the various lipid components of cell membranes influence a broad spectrum of cell surface biological functions which are involved in different steps of the metastatic cascade. Correlation between invasive properties and characteristics of cell surface components has been appropriately studied in a limited number of metastatic cell systems isolated by in vivo and in vitro procedures. The major findings of this study have been reported in this review. Among membrane lipid components, glycolipids and phospholipids appeared particularly affected in tumor cells which acquired a metastatic phenotype. In fact, the reduction of complex gangliosides typical of transformed cell lines was even more evident in a highly metastatic variant selected from RSV-transformed murine fibroblasts. The reduction of complex gangliosides, mainly GD1a, particularly affected the adhesion sites of this variant. In a fibrosarcoma line, T3 cells, the metastatic properties appeared to be correlated with the content and cell surface expression of Gb3ose, a glycolipid characteristic of this line. Moreover, a particularly high level of ether-linked lipids was found in high metastatic variants isolated from murine melanoma and fibrosarcoma lines, as well as in human mammary carcinomas. The findings considered in this review are discussed for their possible relevance to the invasive properties of metastatic cells.

Production of retroviral vectors for gene therapy with the human packaging cell line FLYRD18.

The development of gene therapy is hampered by the difficulty of producing large stocks of retroviral vectors at high titer. This study aimed to improve culture conditions and to intensify the production of retroviruses by FLYRD18, a packaging cell line derived from the HT1080 human fibrosarcoma line. Batch virus production proved to be feasible in unsupplemented basal medium and provided significantly higher titers and productivities than medium supplemented with 10% serum. For longer-term production, however, AIM-V complete serum-free medium and basal medium supplemented with 2% serum gave superior results. Serum supplementation should nevertheless be optimized to take into account the presence of inhibitors of viral production. In monolayer cultures with 0.2 mL/cm(2), the cell concentration was increased up to 2 x 10(6) cells/mL without loss of cell productivity. A semicontinuous production process, which enables the collection of larger amounts of viruses from the same culture, has also been successfully used. Suspension culture processes were prevented by the anchorage dependency of the FLYRD18 cell line. Microcarrier cultures were able to produce viruses but will require further investigation and optimization for their performance to become competitive with monolayer cultures. In the course of this study, more than a 10-fold increase of titer has been achieved.

A human fibrosarcoma inhibits systemic angiogenesis and the growth of experimental metastases via thrombospondin-1.

Concomitant tumor resistance refers to the ability of some large primary tumors to hold smaller tumors in check, preventing their progressive growth. Here, we demonstrate this phenomenon with a human tumor growing in a nude mouse and show that it is caused by secretion by the tumor of the inhibitor of angiogenesis, thrombospondin-1. When growing subcutaneously, the human fibrosarcoma line HT1080 induced concomitant tumor resistance, preventing the growth of experimental B16/F10 melanoma metastases in the lung. Resistance was due to the production by the tumor cells themselves of high levels of thrombospondin-1, which was present at inhibitory levels in the plasma of tumor-bearing animals who become unable to mount an angiogenic response in their corneas. Animals carrying tumors formed by antisense-derived subclones of HT1080 that secreted low or no thrombospondin had weak or no ability to control the growth of lung metastases. Although purified human platelet thrombospondin-1 had no effect on the growth of melanoma cells in vitro, when injected into mice it was able to halt the growth of their experimental metastases, providing clear evidence of the efficacy of thrombospondin-1 as an anti-tumor agent.

Peptides derived from a wild-type murine proto-oncogene c-erbB-2/HER2/neu can induce CTL and tumor suppression in syngeneic hosts.

In this analysis, we examined whether peptides derived from a wild-type murine proto-oncogene, c-erbB-2, function as tumor rejection Ags. Expression of murine c-erbB-2 examined by means of reverse transcription-PCR was observed in several normal adult tissues, such as intestine, kidney, and testis. We then transduced human and murine c-erbB-2 cDNA into two mutually noncross-reactive fibrosarcoma lines of BALB/c origin, CMS7 and CMS17. In BALB/c mice immunized with CMS17HE (CMS17 transduced with human c-erbB-2 cDNA), the growth of subsequently challenged CMS7HE (CMS7 transduced with human c-erbB-2 cDNA) was significantly suppressed. CTL against human c-erbB-2-expressing cells were generated from BALB/c spleen cells in vivo and in vitro sensitized by CMS17HE. The CTL activity was also directed against murine c-erbB-2-expressing cells, CMS7ME and CMS17ME, and was blocked by anti-CD8 or anti-Kd mAbs. A series of peptides of human or murine c-erbB-2 compatible with the Kd binding motif was synthesized. The CTL were reactive with P1.HTR (H-2d) pulsed with three of these peptides, p63-71 (human c-erbB-2 derived), p63-71(A) (murine c-erbB-2 derived), and p780-788 (common for human and murine c-erbB-2). Spleen cells immunized in vivo and in vitro with syngeneic spleen cells pulsed with these peptides became cytotoxic for CMS17HE and/or CMS17ME, but not CMS17neo (CMS17 transduced with control vector). The growth of CMS7ME was suppressed in mice immunized with the murine c-erbB-2-derived peptide, p63-71(A) or p780-788. There was no apparent pathologic change in mice that rejected CMS7ME after vaccination with these peptides.

Enhancement of lung-colonizing potential of murine tumor cell lines co-cultivated with activated macrophages.

In order to explore the influence of activated macrophages on tumor cells, we cultured a series of weakly metastatic clones isolated from the murine T3 fibrosarcoma line (T3 clones) and the B16-F10 melanoma cells on feeder layers of C. parvum- or thioglycollate-elicited macrophages, or 'resident' (unstimulated) macrophages. Co-cultivation with C. parvum-elicited macrophages, but not with resident macrophages, induced an increase of the lung-colonizing potential of T3 clones and B16-F10 cells. An enhancement of lung-colonizing potential was also found in tumor cells grown in media conditioned by C. parvum-elicited macrophages. Thioglycollate-elicited macrophages also generated a pro-clonogenic activity which was however effective only on T3 clones but not on B16-F10 cells. The increase in the lung-colonizing potential of tumor cells stimulated by activated macrophages was retained to some degree after subcultivation in tissue culture medium or transplantation into syngeneic animals. In conclusion, our data support the notion that macrophages at different states of activation may enhance lung colonization of tumor cells by inducing a partially stable change of phenotype.

Involvement of oxidants and oxidant-generating enzyme(s) in tumour-necrosis-factor-α-mediated apoptosis: role for lipoxygenase pathway but not mitochondrial respiratory chain

Cellular signalling by the inflammatory cytokine tumour necrosis factor alpha (TNF alpha) has been suggested to involve generation of low levels of reactive oxygen species (ROS). Certain antioxidants and metal chelators can inhibit cytotoxicity and gene expression in response to TNF alpha in numerous cell types. However, neither the source nor function of TNF alpha-induced oxidant generation is known. Using specific inhibitors, we ruled out involvement of several oxidant-generating enzymes [cyclo-oxygenase (indomethacin), cytochrome P-450 (metyrapone), nitric oxide synthase (NG-methyl-L-arginine), NADPH oxidase (iodonium diphenyl), xanthine oxidase (allopurinol), ribonucleotide reductase (hydroxyurea)] in TNF alpha-mediated apoptosis of the murine fibrosarcoma line, L929. We also demonstrated no role for mitochondrial-derived radicals/respiratory chain in the lytic pathway using specific inhibitors/uncouplers (rotenone, KCN, carboxin, fluoroacetate, antimycin, malonate, carbonyl cyanide p-trifluoromethoxyphenylhydrazone) and chloramphenicol-derived respiration-deficient cells. Significant ROS (H2O2, O2-.) generation was not observed in response to TNF alpha in L929 cells using four separate assays. Also, prevention of intracellular H2O2 removal by inhibition of catalase did not potentiate TNF alpha-mediated cell death. These data suggest that neither H2O2 nor O2-. plays a direct role in TNF alpha cytotoxicity. Finally, we suggest a central role for lipoxygenase in TNF alpha-mediated lysis. Three inhibitors of this radical-generating signalling pathway, including an arachidonate analogue (5,8,11,14-eicosatetraynoic acid), could protect cells against TNF alpha. The inhibitor nordihydroguaiaretic acid is also a radical scavenger, but it could not protect cells from ROS toxicity at concentrations that effectively prevented TNF alpha killing. Therefore protection by nordihydroguaiaretic acid cannot be due to scavenging of cytotoxic H2O or O2-.. The lipoxygenase product, (12S)-hydroxyeicosatetraenoic acid, was also significantly protective. As this analogue can act as a substrate for certain lipoxygenases, this effect may be due to prevention of generation of physiological products.

Composition of ether-linked sub-classes of glycerophospholipids in clones with a different metastatic potential isolated from a murine fibrosarcoma line (T3 cells).

An increase of ether-linked sub-classes of choline and ethanolamine glycerophospholipids has been shown in different types of tumor cells, and correlated with some of their specific biological parameters. In the present study, we examined the composition of ether-linked lipids in a series of clones with a different lung-colonizing potential isolated in our laboratory from a highly metastatic fibrosarcoma line (T3 cells). We found good correlation between the metastatic potential of T3 isolates and increased proportions of both alkylacyl and alkenylacyl subclasses in choline glycerophospholipids (CGP). Moreover, propagation of a weakly metastatic T3 clone in tissue culture led to the emergence of a sub-clone which expressed high metastatic potential together with a high level of alkylacyl and alkenylacyl CGP. No differences were found in the alkylacyl and alkenylacyl-ethanolamine glycerophospholipids (EGP) between the strongly and weakly metastatic T3 clones. We discuss the accumulation of alkylacyl and alkenylacyl CGP in metastatic cells for its possible role in metastatic diffusion by generation of platelet-activating factor (PAF).

The distal region of the long arm of human chromosome 1 carries tumor suppressor activity for a human fibrosarcoma line

Loss or inactivation of tumor suppressor genes has been implicated by indirect methods in the etiology of most human cancers. In the functional studies presented here, tumor suppressors on human chromosome 1 were investigated using microcell-mediated chromosome transfer. Translocated chromosomes from normal human cells representing most of 1q, or all of 1p and a small portion of 1q translocated onto the region of the X chromosome encoding HPRT, were transferred into human fibrosarcoma cell line HT1080. Analysis of HT1080 microcell hybrids showed a tumor suppressor activity associated with 1q. All HT1080 cells carrying transferred 1q in a ratio of 1:1 with the HT1080 genome showed a more flattened morphology and a reduced ability to form tumors in nude mice compared to parental HT1080 cells. Diploid HT1080 cells carrying a single extra 1q also had a longer population doubling time and showed a loss of ability to clone in soft agar. Tumors arose from 1q-containing clones with a longer latency period, and a large majority of the cells comprising these tumors had lost the transferred chromosome. These results indicate the presence on chromosome 1q23-qter of a tumor suppressor gene or genes that can act to suppress transformation of a human fibrosarcoma cell line.

Satellite DNA sequences flank amplified DHFR domains in marker chromosomes of mouse fibrosarcoma cells.

This study centers on marker chromosomes carrying expanded chromosomal regions which were observed in two independent derivatives of the AA12 murine fibrosarcoma line, the 10(-3) M MTX-res H2 and the 5 x 10(-7) M MTX-res E. Previous characterization of the marker chromosomes of MTX-res variants showed their common derivation from a marker chromosome (m) of the parental line, endowed with two interstitial C-bands. Cytogenetic evidence pointed to one C-band of m as the site involved in the chromosomal rearrangements leading to the HSR/ASR chromosomes. ISH of a 3H-labeled satellite DNA probe allowed satellite sequences flanking the HSR/ASR in the marker chromosomes, where the C-band was no longer visible, to be detected. FISH experiments using biotinylated DHFR and satellite DNA probes showed that the respective target sequences are contiguous in new marker chromosomes. They also allowed inter- and intrachromosomal rearrangements to be seen at DHFR amplicons and satellite sequences. Double-color FISH using digoxygenated satellite DNA and biotinylated pDHFR7 showed that in a marker chromosome from the H2 cell line the two target sequences are not only adjacent, but closer than 3 Mb, as indicated by overlapping of the different fluorescence signals given by the two probes. Another marker chromosome in the E variant was shown to display a mixed ladder structure consisting of a head-to-head tandem of irregularly-sized satellite DNA blocks, with two symmetrical interspersed DHFR clusters.

Antitumor response independent of functional B or T lymphocytes induced by the local and sustained release of interleukin-2 by the tumor cells.

Transfection of tumor cells with a vector containing the entire coding sequence of human interleukin-2 (hIL-2) was previously shown to convert the tumorigenic murine fibrosarcoma line CMS-5 into a non-tumorigenic line. The failure of the IL-2-secreting tumor to grow in conventional (immunocompetent) mice was attributed to the activation of CD8+ T cells that exhibited tumor specificity and memory. In order to determine whether or not the IL-2 produced by the tumor may be activating tumor cytotoxic effector cells other than B or T cells we have repeated this study using immunodeficient SCID and SCID-beige mice as syngeneic tumor recipients. In contrast to the rapid growth of the wild-type tumor, the hIL-2-transfected cells (N2A/IL2/CMS5) did not grow, or grew more slowly and regressed, in the mice that lack functional B and T cells. The inhibition of tumor growth associated with the local release of IL-2 was reversed in mice treated with antiasialo-GM1 antibodies specific for natural killer (NK) lineage cells. In contrast to the studies with conventional mice, the IL-2-dependent effector cells in the immunodeficient mice exhibited no evidence of memory. In vitro analysis of spleen cells from tumor-bearing mice revealed the presence of effector cells able to lyse YAC-1 target cells as well as the wild-type CMS-5 and the IL-2-transfected variant tumor lines but unable to lyse P815 cells. The pattern of selective target cell killing and the kinetics of killing were indistinguishable from those observed using tumor necrosis factor alpha (TNF alpha) the mediator associated with natural cytotoxicity cell killing of tumor cells. Histopathology of the IL-2-secreting tumors in SCID mice reveals the presence of infiltrating lymphoid cells and macrophages that were not observed in the CMS-5 tumors. Consistent with the notion that the tumor killing in the SCID mice was mediated by TNF alpha, mice bearing IL-2-secreting tumors had elevated levels of serum TNF alpha and little or no effector cell activity, or TNF alpha was found in tumor-bearing mice treated with anti-asialo-GM1 antibody. The results indicate that the cytokine-induced tumor regression observed in the IL-2-transfected tumors is a more complex phenomenon than previously recognized and one that is mediated by effector cells of the NK cell and/or monocyte/macrophage lineages, in addition to CD8+ T cells.

Activation of the nuclear factor κB is not sufficient for regulation of tumor necrosis factor-induced interleukin-6 gene expression

After treatment of L929 cells, a murine fibrosarcoma line, with tumor necrosis factor (TNF), a nuclear κB-like transcription factor is rapidly induced as identified by gel shift mobility assays using the κB-responsive sequence of the immunoglobulin or interleukin-6 (IL-6) genes as a DNA probe. When induction was carried out under conditions of increased or decreased cytotoxicity, which correlates with altered IL-6 gene expression, nuclear factor κB (NF-κB) activation was also demonstrated, but the abundance of the protein/DNA complex observed remained unchanged. Also activation of NF-κB as a function of time following TNF treatment did not reveal a correlation between the abundance of the protein/DNA complex and the TNF-induced IL-6 mRNA levels. Moreover, in L929 cells resistant to TNF cytotoxicity, the κB-like factor still became fully activated by TNF, although the IL-6 gene was only marginally expressed. In conclusion, discrepancies between the abundance of the activated NF-κB-like factor and the IL-6 mRNA production upon treatment with TNF indicate that (an) additional transcription factor(s) and/or (a) regulating mechanism(s) is (are) necessary for fine regulation of the level of IL-6 gene expression in response to cytokine stimulation.

CHEMOSENSITIZATION OF MURINE FIBROSARCOMA CELLS TO DRUGS AFFECTED BY THE MULTIDRUG RESISTANCE PHENOTYPE BY THE ANTIDEPRESSANT TRAZODONE - AN EXPERIMENTAL-MODEL FOR THE REVERSAL OF INTRINSIC DRUG-RESISTANCE

A variety of resistance phenotypes to cytotoxic agents in bacteria, protozoa parasites and mammalian cells are mediated by evolutionarily conserved proteins of the mdr family. The finding that chloroquine resistance in the malarial parasite, Plasmodium falciparum, that is mediated by an mdr-1 gene product can be circumvented by tricyclic antidepressant drugs has stimulated the present study to assess whether this class of agents might also modulate the multidrug resistance (MDR) phenotype(s) in mammalian tumor cells. The possible chemosensitizing effects of nine antidepressant drugs have been tested against the UV-2237M murine fibrosarcoma line and its MDR variant. At nontoxic concentrations all nine antidepressants markedly enhanced the cytotoxicity of ADR against the parental cells but were much less effective against the MDR cells. The most active antidepressant, trazodone, also enhanced the cytotoxicities of vinblastine and vincristine, but not those of actinomycin D, mitomycin C, or 5-fluorouracil. The parental cells treated with trazodone exhibited an increased accumulation of intracellular ADR, but lacked detectable alterations in the expression and drug-binding activity of plasma membrane P-glycoprotein, and trazodone did not affect the activities of isolated protein kinase C and calmodulin. These data suggest that the antidepressant drug trazodone may be useful in the reversal of the intrinsic drug resistance of tumor cells that express low levels of P-glycoprotein.

Different pathways for chromosomal integration of transfected circular pSVneo plasmids in normal and established rodent cells

The chromosomal integration of transfected circular plasmid pSV2neo molecules was investigated in normal and established mammalian fibroblasts, including two secondary cultures of normal mouse fibroblast lines, one established mouse-hamster hybrid cell line, R44, and the human fibrosarcoma line, HT1080. The physical organization of the integrated molecules was studied by restriction analysis. The results showed that whereas the normal fibroblasts predominantly integrated one head-to-tail partial dimeric molecule, the established cells predominantly integrated distinctly different molecular forms including deleted monomeres (HT1080) and various complex concatemeric molecules (R44), and frequently at more than one chromosomal site (R44). We also constructed a head-to-tail dimeric version of the plasmid, which in the case of the normal fibroblasts again integrated as a partial dimeric molecule in at least 50% of these cells. This result excluded the possibility that the normal mouse transfectants were selected for the integration of two functional neo genes. Thus, it is concluded that the distinctly different molecular forms integrated in normal and established cells demonstrate the operation of different integration pathways, the possible nature of which is discussed.