Synthesis of the cyclic and acyclic acetal derivatives of 1-(3-C-ethynyl-beta-D-ribo-pentofuranosyl)cytosine,a potent antitumor nucleoside. Design of prodrugs to be selectively activated in tumor tissues via the bio-reduction-hydrolysis mechanism

We have designed and synthesized the acetal derivatives of 1-(3-C-ethynyl-beta-D-ribo-pentofuranosyl)cytosine (ECyd, 1), the 2',3'-O-nitrobenzylidene derivatives 2 and 3 and the 5'-O-(alkoxy)(nitrophenyl)methyl derivatives 6-10 as potential prodrugs of ECyd. These prodrugs can be selectively activated in tumor tissues via a bio-reduction-hydrolysis mechanism owing to the characteristic properties of tumor tissues, such as hypoxia and lower pH. Although the 2',3'-O-(4-nitrobenzylidene) derivatives 2 and 3 were converted bio-reductively into the corresponding 4-aminobenzylidene derivatives by rat S-9 mix, the reduction products, that is, the corresponding amino congeners 4 and 5, proved to be rather stable in an aqueous solution at pH 6.5 used as a pH model for acidic tumor tissues. In contrast, the 5'-O-(alkoxy)(4-nitropheny)methyl derivatives 6-8 were also reduced by rat S-9 mix to the corresponding amino congeners 11-13, which were hydrolyzed to release ECyd more effectively at pH 6.5 than at pH 7.4. Accordingly, the acyclic acetals 6-8 may be efficient prodrugs of ECyd, that are effectively reduced under physiological conditions releasing ECyd in acidic tumor tissues.     

Allogeneic tumor-specific cytotoxic T lymphocytes

Summary We report the development of cytotoxic T lymphocytes specific for an allogeneic brain tumor in a rat model. DA strain cytotoxic T cell precursors stimulated by an allogeneic tumor (9L gliosarcoma) from the Fischer rat could generate a population of cytotoxic T lymphocytes that lysed the allogeneic 9L tumor but failed to lyse other targets, including Fischer concanavalin-A(ConA)-stimulated lymphoid blast targets. DA T cells depleted of reactivity to the Fischer haplotype (DA-f) retained reactivity to the 9L tumor, demonstrating that T cell precursors with specificity for normal Fischer alloantigens were not required for the generation of a response to the 9L Fischer tumor. The preferential lysis of the tumor target did not simply reflect a higher density of Fischer target antigens on the tumor than that found on normal Fischer ConA blast targets. First, the relative densities of class I antigen on the 9L tumor and normal Fischer ConA blasts were comparable. Second, cytotoxic T cells could not be generated from DA-f precursors when Fischer ConA blasts were used as stimulators. If DA-f T cells were simply responding to the higher density of Fischer antigen found on 9L tumor, it would have been expected that the ConA blasts expressing comparable levels of antigen to that found on the tumor would have generated cytotoxicity for both the 9L and ConA targets. We conclude that the cytotoxic T cells are specific for a determinant expressed only by the tumor. Such tumor-specific cytotoxic T cells could be useful in vivo for adoptive immunotherapy of brain tumors.     

RNK granule extract cytolysis: increased tumor susceptibility and release of proteochondroitin sulphate inhibitor in high NaCl

The insensitivity of the natural killer (NK)-resistant L5178Y-F9 murine T-cell lymphoma to granule extracts from a rat NK leukemia could be preferentially reversed in increased NaCl (0.25 M) compared with the NK- and granule extract-sensitive SL2-5. The high salt effect predominated in the binding rather than the lytic phase of the extract reaction similar to the activity of extract inhibitory supernates preferentially produced from L5178Y-F9 cells. Exposure of the L5178Y-F9 to 0.25 M NaCl was associated with an increased production of inhibitory supernate and an increased sensitivity of the cell as an extract target. Pretreatment of inhibitor-containing supernatant or inhibitor-producing L5178Y-F9 cells with pronase or chondroitinase AC reduced the inhibitory activity of the resultant supernates, and L5178Y-F9 supernates treated with anti-chondroitin sulphate AC antibodies exhibited reduced inhibitory activity. These observations and the previously reported molecular weight heterogeneity and protease sensitivity of the inhibitor argue that chondroitin sulphate AC-containing proteoglycans released from the tumor cell surface may inhibit cytolysin activity, contributing to the preferential resistance of the L5178Y-F9 to rat NK granule extract cytolysis.     

The influence of hypoxia on bioluminescence in luciferase-transfected gliosarcoma tumor cells in vitro

Firefly luciferase catalyzes the emission of light from luciferin in the presence of oxygen and adenosine triphosphate. This bioluminescence is commonly employed in imaging mode to monitor tumor growth and treatment responses in vivo. A potential concern is that, since solid tumors are often hypoxic, either constitutively and/or as a result of treatment, the oxygen available for the bioluminescence reaction could be reduced to limiting levels, leading to underestimation of the actual number of luciferase-labeled cells during in vivo experiments. We present studies of the oxygen dependence of bioluminescence in vitro in rat 9 L gliosarcoma cells tagged with the firefly luciferase gene (9L(luc)). We demonstrate that the bioluminescence signal decreases at pO(2) 相似文献   

Treatment of murine lupus with F(ab')2 fragments of monoclonal antibody to L3T4. Suppression of autoimmunity does not depend on T helper cell depletion

Treatment with mAb to the L3T4 Ag on Th cells can inhibit autoimmunity in mice. However, the mechanism by which anti-L3T4 inhibits autoimmunity is not known. In these studies, lupus-prone NZB/NZW F1 (B/W) mice were treated with F(ab')2 fragments of mAb to L3T4 to determine whether Th cell depletion is required for the beneficial effects of anti-L3T4. We first showed that treatment of female B/W mice with F(ab')2 anti-L3T4 from age 5 to 9 mo significantly reduced autoantibody production without depleting L3T4+ cells. However, treatment was complicated by the development of a host immune response to the rat mAb fragments. To circumvent this problem, female B/W mice were treated with a single high-dose of intact rat mAb to L3T4 (GK1.5) at age two mo. to induce immune tolerance to the mAb. Then, after recovery of L3T4+ cells, the mice were treated from age four to 14 mo with either F(ab')2 anti-L3T4 (0.5 mg 3 times per wk), intact anti-L3T4, or saline. In mice tolerized by this regimen, neither the F(ab')2 rat mAb nor the intact rat mAb elicited a host response. The mAb fragments bound target Ag but did not deplete the Th cells, whereas intact mAb to L3T4 profoundly depleted the L3T4+ cells. Despite this difference, both therapies had the same substantial beneficial effects on autoimmunity. They significantly decreased anti-DNA Ab production, improved renal function and prolonged survival. The initial tolerizing dose, by itself, did not inhibit autoimmunity. These findings show that anti-L3T4 suppresses autoimmunity by directly altering Th cell function through the L3T4 Ag, and not solely by depleting Th cells. They also document the detrimental effects of the host immune response to therapy with anti-L3T4 mAb, and they demonstrate a new strategy by which this response may be prevented.     

Chemotactic response of rat mammary adenocarcinoma cell clones to tumor-derived cytokines

A cytokine with an apparent molecular weight of 53,000 daltons was isolated from serum-free medium conditioned by MTLn3 cells or from homogenates of MTLn3 cells, a highly metastatic variant of the rat 13762NF mammary adenocarcinoma. The chemotactic responses of MTLn3 and the low metastatic variant MTLn2 cells to this cytokine were tested in vitro using modified Boyden chambers. Both the chemotactic and chemokinetic movements of MTLn3 cells were stimulated by the MTLn3-derived cytokine. In addition, the MTLn3-derived cytokine stimulated a relatively small, but significant chemotactic migration of MTLn2 tumor cells, while these cells did not respond to medium conditioned by MTLn2 cells. MTLn3 cells themselves did not respond chemotactically to type I collagen or medium conditioned by MTLn2 cells. These results suggest that the chemotactic response may be a function of metastatic potential of the invading tumor cells. The production of tumor cytokines that enhance tumor cell motility may thus represent a phenotypic difference between 13762NF tumor cell subpopulations of high and low metastatic potential.     

Monoclonal antibodies as probes for differentiation and tumor-associated antigens: a Forssman specificity on teratocarcinoma stem cells

A set of monoclonal antibodies derived by fusing P3-NS1/1-Ag4-1 myeloma cells with spleen cells from a rat immunized with mouse spleen were screened for activity against a tumor cell panel. One of these antibodies was found to react only with mouse embryonal carcinoma cells and no other tumor cell type tested, including differentiated derivatives of teratocarcinomas. In the adult mouse, this antigen is expressed by subpopulations of cells in the spleen, bone marrow, lymph node, brain, kidney and testes, although not in liver and thymus. This antigen has a species and tissue distribution consistent with that of Forssman antigen. The molecules which carry this specificity on the embryonal carcinoma cells appear to be glycolipids.     

Administration of monoclonal anti-T cell antibodies retards murine lupus in BXSB mice

Murine lupus in BXSB mice is associated with B cell hyperactivity, monocyte proliferation, and impaired T cell function. However, the significance of these abnormalities, and the relationship among them, has not been clearly established. To examine the role of T cells in the pathogenesis of autoimmune disease in BXSB mice, we depleted specific T cell subsets from BXSB males by using rat IgG2b monoclonal antibodies (MAb) to either Thy-1.2 (on all T cells) or L3T4 (on "helper/inducer" T cells). A single injection of anti-Thy-1.2 (6 mg i.v.) at age 3 mo produced a sustained 40 to 50% reduction in circulating T cells for 6 mo. Treatment prevented monocytosis, reduced anti-DNA antibody concentration, and retarded renal disease, but it did not prolong life. Repeated injections of rat MAb to Thy-1.2 were precluded by the development of a host immune response to rat immunoglobulin (Ig) that can cause anaphylaxis in BXSB mice. In contrast, rat MAb to L3T4 stimulated little or no immune response to rat Ig. We therefore were able to treat BXSB mice weekly with anti-L3T4 (2 mg i.p.) from age 3 to 12 mo. Treatment reduced circulating L3T4+ cells beneath the level of detection by fluorescence analysis. It also significantly reduced monocytosis, anti-DNA antibody production, renal disease, and mortality. These findings establish that monocytosis and autoimmunity in BXSB mice are promoted by T cells. They extend our previous observation that MAb to L3T4 retard autoimmunity in NZB/NZW F1 mice. Our finding that treatment with MAb to L3T4 is effective in two strains of lupus-prone mice suggests that treatment with MAb to Leu-3/T4, the human homologue for L3T4, may be effective in people with systemic lupus erythematosus.     

Calcium influx into neurons can solely account for cell contact- dependent neurite outgrowth stimulated by transfected L1

We have used monolayers of control 3T3 cells and 3T3 cells expressing transfected human L1 as a culture substrate for rat PC12 cells and rat cerebellar neurons. PC12 cells and cerebellar neurons extended longer neurites on human L1 expressing cells. Neurons isolated from the cerebellum at postnatal day 9 responded equally as well as those isolated at postnatal day 1-4, and this contrasts with the failure of these older neurons to respond to the transfected human neural cell adhesion molecule (NCAM). Human L1-dependent neurite outgrowth could be blocked by antibodies that bound to rat L1 and, additionally, the response could be fully inhibited by pertussis toxin and substantially inhibited by antagonists of L- and N-type calcium channels. Calcium influx into neurons induced by K+ depolarization fully mimics the L1 response. Furthermore, we show that L1- and K+(-)dependent neurite outgrowth can be specifically inhibited by a reduction in extracellular calcium to 0.25 microM, and by pretreatment of cerebellar neurons with the intracellular calcium chelator BAPTA/AM. In contrast, the response was not inhibited by heparin or by removal of polysialic acid from neuronal NCAM both of which substantially inhibit NCAM-dependent neurite outgrowth. These data demonstrate that whereas NCAM and L1 promote neurite outgrowth via activation of a common CAM-specific second messenger pathway in neurons, neuronal responsiveness to NCAM and L1 is not coordinately regulated via posttranslational processing of NCAM. The fact that NCAM- and L1-dependent neurite outgrowth, but not adhesion, are calcium dependent provides further evidence that adhesion per se does not directly contribute to neurite outgrowth.     

Filter elution analysis of the effect of alpha-difluoromethylornithine on X-ray-induced DNA damage in 9L cells

We used the filter elution technique to study DNA single- and double-strand scission under denaturing alkaline and nondenaturing conditions in X-irradiated 9L rat brain tumor cells. The amount of DNA damage determined by the alkaline elution assay was similar for different lysis conditions (sodium dodecyl sulfate and sarkosyl) and DNA fluorometric assays (Hoechst 33258 and 3,5-diaminobenzoic acid dyes). Therefore, results of the filter elution assay obtained with the various methods can be compared directly. Using these assays, we found that there was no significant change in the susceptibility to X-ray-induced DNA damage, measured either as single- or double-strand breaks, in 9L cells depleted of polyamines by treatment with alpha-difluoromethylornithine. Results obtained by filter elution are different from results obtained with viscoelastometry, which suggests that the two methods may resolve the effects of changes in DNA structure in different ways.     

Sequential isolation of proteoglycan synthesis mutants by using herpes simplex virus as a selective agent: evidence for a proteoglycan-independent virus entry pathway.

A novel mouse L-cell mutant cell line defective in the biosynthesis of glycosaminoglycans was isolated by selection for cells resistant to herpes simplex virus (HSV) infection. These cells, termed sog9, were derived from mutant parental gro2C cells, which are themselves defective in heparan sulfate biosynthesis and 90% resistant to HSV type 1 (HSV-1) infection compared with control L cells (S. Gruenheid, L. Gatzke, H. Meadows, and F. Tufaro, J. Virol. 67:93-100, 1993). In this report, we show that sog9 cells exhibit a 3-order-of-magnitude reduction in susceptibility to HSV-1 compared with control L cells. In steady-state labeling experiments, sog9 cells accumulated almost no [35S]sulfate-labeled or [6-3H]glucosamine-labeled glycosaminoglycans, suggesting that the initiation of glycosaminoglycan assembly was specifically reduced in these cells. Despite these defects, sog9 cells were fully susceptible to vesicular stomatitis virus (VSV) and permissive for both VSV and HSV replication, assembly, and egress. HSV plaques formed in the sog9 monolayers in proportion to the amount of input virus, suggesting the block to infection was in the virus entry pathway. More importantly, HSV-1 infection of sog9 cells was not significantly reduced by soluble heparan sulfate, indicating that infection was glycosaminoglycan independent. Infection was inhibited by soluble gD-1, however, which suggests that glycoprotein gD plays a role in the infection of this cell line. The block to sog9 cell infection by HSV-1 could be eliminated by adding soluble dextran sulfate to the inoculum, which may act by stabilizing the virus at the sog9 cell surface. Thus, sog9 cells provide direct genetic evidence for a proteoglycan-independent entry pathway for HSV-1, and results with these cells suggest that HSV-1 is a useful reagent for the direct selection of novel animal cell mutants defective in the synthesis of cell surface proteoglycans.     

Effect of nitrogen-containing derivatives of the plant triterpenes betulin and glycyrrhetic acid on the growth of MT-4, MOLT-4, CEM, and Hep G2 tumor cells

Derivatives of the available plant triterpenes glycyrrhetic acid and betulin (betulin succinates and amides of betulonic and 18beta-glycyrrhetic acids containing fragments of long-chain amino acids and a peptide) were synthesized. The inhibitory action of these compounds on the growth of MT-4, MOLT-4, CEM. and Hep G2 tumor cells and their effect on the apoptosis of these cells were studied. It was shown that betulonic acid amides are more effective inhibitors of the tumor cell growth than the corresponding amides of glycyrrhetic acid. It was also found that betulonic acid amides containing fragments of caprylic, pelargonic, and undecanoic acids are more effective inhibitors of tumor cell growth than betulinic acid. The 17-dipeptide derivative of betulonic acid N-{N-[3-oxo-20(29)-lupen-28-oyl]-9-aminononanoyl}-3-amino-3-phenylpropionic acid exhibited the maximum inhibitory activity toward the tumor cells studied. Data on the induction of apoptosis in tumor cells by betulin derivatives at a concentration of 10 microg/ml were obtained by flow cytometry. The amides of betulonic acid proved to be the most effective inducers of apoptosis.     

RNK granule extract cytolysis: differential inhibitor production by an NK-resistant vs an NK-sensitive murine lymphoma

Natural killer (NK) cell-resistant tumors exist despite their ability to bind cells from the effector population. Tumor sensitivity to NK activity was therefore examined at the level of susceptibility to cytolysin-containing NK cell cytotoxic granule extracts. The NK-sensitive SL2-5 murine lymphoma was markedly more susceptible than the NK-resistant L5178Y-F9 to solubilized granule preparations from the rat NK tumor cell line RNK-16, and this corresponded also with tumor sensitivity to hypotonic lysis. However, the resistant L5178Y-F9 was better able to inhibit the extract activity than the SL2-5. Dissociation of the binding and lysis phases of the cytolysin reaction based on their differential temperature requirements, 4 degrees C for binding and 37 degrees C for lysis, permitted an examination of the cytolysin/tumor interaction prior to lysis. The residual cytotoxic activity was lower after extract exposure to the L5178Y-F9 compared with the SL2-5 consistent with possible inhibitor production. Finally, supernatant material collected from the L5178Y-F9 was a better inhibitor of granule extract lysis and acted preferentially in the extract-binding phase. The inhibitor appears to be protein in nature, relatively stable, and exhibits molecular weight heterogeneity ranging from 2000 to greater than 300,000.     

Polyomavirus transforms rat F111 and mouse NIH 3T3 cells by different mechanisms.

Polyomavirus middle tumor antigen (mT) was expressed in a line of mouse NIH 3T3 cells under control of the dexamethasone-regulatable mouse mammary tumor virus promotor. Contrary to rat F111 cells which were rendered anchorage independent by mT expression alone (L. Raptis, H. Lamfrom, and T.L. Benjamin, Mol. Cell. Biol. 5:2476-2487, 1985), mT-producing NIH 3T3 cells were unable to grow in agar even after full mT induction. The mT:pp60c-src-associated phosphatidylinositol kinase was activated in these cells to a degree similar to that in fully transformed cells expressing the small and large T antigens, in addition to mT. We therefore propose that the stimulation of this phosphatidylinositol kinase, although apparently necessary, is not sufficient for transformation of NIH 3T3 cells by polyomavirus.     

Comparative proteomics study reveals that bacterial CpG motifs induce tumor cell autophagy in vitro and in vivo

Unmethylated CpG dinucleotides, present in bacterial DNA, are recognized in vertebrates via the Toll-like receptor 9 (TLR9) and are known to act as an anticancer agent by stimulating immune cells to induce a proinflammatory response. Although the effects of CpG-oligodeoxynucleotides (CpG-ODNs) in immune cells have been widely studied, little is known regarding their molecular effects in TLR9-positive tumor cells. To better understand the role of these bacterial motifs in cancer cells, we analyzed proteome modifications induced in TLR9-positive tumor cells in vitro and in vivo after CpG-ODN treatment in a rat colon carcinoma model. Proteomics analysis of tumor cells by two-dimensional gel electrophoresis followed by mass spectrometry identified several proteins modulated by bacterial CpG motifs. Among them, several are related to autophagy including potential autophagic substrates. In addition, we observed an increased glyceraldehyde-3-phosphate dehydrogenase expression, which has been shown to be sufficient to trigger an autophagic process. Autophagy is a self-digestion pathway whereby cytoplasmic material is sequestered by a structure termed the autophagosome for subsequent degradation and recycling. As bacteria are known to trigger autophagy, we assessed whether bacterial CpG motifs might induce autophagy in TLR9-positive tumor cells. We showed that CpG-ODN can induce autophagy in rodent and human tumor cell lines and was TLR9-dependent. In addition, an increase in the number of autophagosomes can also be observed in vivo after CpG motif intratumoral injection. Our findings bring new insights on the effect of bacterial CpG motifs in tumor cells and may be relevant for cancer treatment and more generally for gene therapy approaches in TLR9-positive tissues.     

The combined effect of lymphokine activated killer cell and radiation therapy on rat brain tumorin vitro

Thein vitro effect of a combined treatment with lymphokine activated killer (LAK) cell and radiation therapy on rat brain tumor was examined using⁵¹Cr release assay. The tumor cell-line used in this experiment was 9L rat brain tumor derived from a Fischer 344 rat. LAK cells were obtained by culturing rat lymphocytes with recombinant human interleukin 2 for at least 3 days. The cytotoxic activity of the LAK cells was examined by⁵¹Cr release assay. Irradiation was done by exposing the microtiter plate in which the¹⁵Cr labeled 9L cells and LAK cells were cultured to a¹³⁷Cs gamma cell unit. Without irradiation, there was 18% cytotoxicity in the 1:100 tumor-to-LAK cell ratio specimen after 24 hrs cocultivation. However, if 5 Gy of irradiation was given, followed by 12 hrs incubation, the cytotoxicity was enhanced significantly at the same cell ratio (30%). This enhancement effect was the most prominent when the cell ratio was 1:100 and the irradiation dose was 5 Gy. To generate the enhancement effect, an incubation time of over 8 hrs both before and after irradiation was required. The supernatant of the LAK cells showed 19.8% and 11.4% cytotoxicity with and without irradiation, respectively. This result indicates the participation of a cytotoxic factor released from LAK cells.This work is supported in part by grant from Univeristy of Tsukuba Project Research.     

A 32P postlabeling assay for determining the incorporation of bromodeoxyuridine into cellular DNA

Randerath's procedure for 32P postlabeling of 3'-monophosphate deoxyribonucleotides from digests of cellular DNA has been modified. 3'-Monophosphate deoxyribonucleotides are converted to 3',5'-bis[32P]phosphate deoxyribonucleotides with polynucleotide kinase and [32P]ATP; these products are enzymatically converted by P1 nuclease and polynucleotide kinase into 5'-[32P]monophosphate deoxyribonucleotides, which are separated from [32P]ATP on an anion-exchange column eluted with 0.1 M NaH2PO4, pH 6.5. Labeled mononucleotides in the effluent are separated by high-performance liquid chromatography. Values for the base composition of calf thymus DNA determined with this modified assay compare very favorably with reported values. The assay was used to measure the level of incorporation of the clinically useful agent bromodeoxyuridine into the DNA of 9L rat brain tumor cells. The modified assay appears to be a very accurate method for the determination of levels of base analogs incorporated into DNA.