Monensin-resistant mouse Balb/3T3 cell mutant with aberrant penetration of vesicular stomatitis virus.

A mutant (MO-5) resistant to monensin (an ionophoric antibiotic) derived from the mouse Balb/3T3 cell line, was a poor host for vesicular stomatitis virus (VSV) or semliki forest virus (SFV) multiplication. The yield of VSV particles in MO-5 is one 100-fold reduced as is VSV-dependent RNA synthesis. In contrast to a pH-remedial mutant, the abortive production of infectious VSV particles in MO-5 cells was not restored by low pH treatment. The pH values in the endosome and the lysosome of MO-5 cells were 5.2 and 5.4, respectively, values that were comparable to the pH value in Balb/3T3 cells. Assays with [3H]uridine-labeled VSV indicated similar binding of VSV in MO-5: percoll gradient centrifugation analysis of [35S]methionine-labeled VSV-infected Balb/3T3 showed accumulation of VSV in the lysosome fraction 20 min after VSV infection, whereas VSV can be found mainly in endosome/Golgi fraction of MO-5 cells after 40 to 60 min on the percoll gradients. Degradation of [35S]methionine-labeled VSV was observed at a significant rate in Balb/3T3 cells, but not in MO-5 cells. The monensin-resistant somatic cell may thus provide a genetic route to study the mechanism of endocytosis or transport of enveloped viruses.

The mechanism for the activation of latent TGF-beta during co-culture of endothelial cells and smooth muscle cells: cell-type specific targeting of latent TGF-beta to smooth muscle cells.

Transforming growth factor-beta (TGF-beta) is secreted in a latent form and activated during co-culture of endothelial cells and smooth muscle cells. Plasmin located on the surface of endothelial cells is required for the activation of latent TGF-beta (LTGF-beta) during co-culture, and the targeting of LTGF-beta to the cellular surface is requisite for its activation. In the present study, the cellular targeting of LTGF-beta was examined. We detected the specific binding of 125I-large LTGF-beta 1 isolated from human platelets to smooth muscle cells but not to endothelial cells. A mAb against the latency-associated peptide (LAP) of large LTGF-beta 1 complex, which blocked the binding of 125I-large LTGF-beta 1 to smooth muscle cells, inhibited the activation of LTGF-beta during co-culture. The binding of 125I-large LTGF-beta 1 could not be competed either by mannose-6-phosphate (300 microM) or by the synthetic peptide Arg-Gly-Asp-Ser (300 micrograms/ml). These results indicate that the targeting of LTGF-beta to smooth muscle cells is required for the activation of LTGF-beta during co-culture of endothelial cells and smooth muscle cells. The targeting of LTGF-beta to smooth muscle cells is mediated by LAP, and the domain of LAP responsible for the targeting to smooth muscle cells may not be related to mannose-6-phosphate or an Arg-Gly-Asp sequence, both of which have been previously proposed as candidates for the cellular binding domains within LAP.

Ribonuclease V of Escherichia coli: susceptibility of heated ribosomal RNA and stability of R17 phage RNA.

Native ribosomal RNA anid inttact pliage R17 RNA are insensitive to attack by ribonuclease V, an exonucleolytic activity associated with ribosome movement on the substrate RNA. However, ribosomal RNA becomes a substrate when it is heated under conditions that make it a template for protein synthesis, and R17 RNA is attacked if it is either heated or fragmnented. Accessibility of the 5' terminus of an RNA molecule is probably inzcreased by heating or fragmnentation and may determine its susceptibility to ribonuclease V.

Akt-dependent nuclear localization of Y-box-binding protein 1 in acquisition of malignant characteristics by human ovarian cancer cells.

Y-box-binding protein 1 (YB-1), which is a member of the DNA-binding protein family containing a cold-shock domain, has pleiotropic functions in response to various environmental stimuli. As we previously showed that YB-1 is a global marker of multidrug resistance in ovarian cancer and other tumor types. To identify YB-1-regulated genes in ovarian cancers, we investigated the expression profile of YB-1 small-interfering RNA (siRNA)-transfected ovarian cancer cells using a high-density oligonucleotide array. YB-1 knockdown by siRNA upregulated 344 genes, including MDR1, thymidylate synthetase, S100 calcium binding protein and cyclin B, and downregulated 534 genes, including CXCR4, N-myc downstream regulated gene 1, E-cadherin and phospholipase C. Exogenous serum addition stimulated YB-1 translocation from the cytoplasm to the nucleus, and treatment with Akt inhibitors as well as Akt siRNA and integrin-linked kinase (ILK) siRNA specifically blocked YB-1 nuclear localization. Inhibition of Akt activation downregulated CXCR4 and upregulated MDR1 (ABCB1) gene expression. Administration of Akt inhibitor resulted in decrease in nuclear YB-1-positive cancer cells in a xenograft animal model. Akt activation thus regulates the nuclear translocation of YB-1, affecting the expression of drug-resistance genes and other genes associated with the malignant characteristics in ovarian cancer cells. Therefore, the Akt pathway could be a novel target of disrupting the nuclear translocation of YB-1 that has important implications for further development of therapeutic strategy against ovarian cancers.

Induction of vascular endothelial tubular morphogenesis by human glioma cells. A model system for tumor angiogenesis.

We have developed two different models of tumor angiogenesis by human brain tumors: one being tube formation by bovine aortic endothelial (BAE) cells cocultured with tumor cells in vitro, and other being in vivo angiogenesis in mice when tumor cells are transplanted into the dorsal sac. We investigated whether tube formation could be induced in BAE cells in type I collagen gel when these cells were cocultured with seven human glioma cell lines. Four of the seven glioma cell lines, which had high levels of basic fibroblast growth factor (bFGF) mRNA, induced tube formation by BAE cells. The tube formation was blocked by coadministration of anti-bFGF antibody. In in vivo model system of tumor angiogenesis in mice, these four cell lines were highly angiogenic. In contrast, with the other three glioma cell lines, which had poor expression of bFGF, BAE cells showed no apparent tube formation. These three cell lines did not efficiently develop capillary networks in mice. The results demonstrated a correlative relationship in the tubulogenesis of BAE cells, bFGF mRNA levels and angiogenesis in mice. The present study with two model systems of tumor angiogenesis suggests that the angiogenesis of some human glioma cell lines is mediated by bFGF, possibly via paracrine control.

Chinese hamster cell mutant resistant to ML236B (Compactin) is defective in endocytosis of low-density lipo-protein.

A fungal metabolite, ML236B (Compactin), isolated from Penicillium citrinum, is a specific inhibitor of 3-hydroxy-3-methylglutaryl (HMG)-coenzyme A reductase (EC 1.1.1.34). Three ML236B-resistant (ML236Br) mutants, MF-1, MF-2, and MF-3, were isolated from V79 after N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis. The fluctuation test showed 2.2 X 10(-6) mutants per cell per generation of a spontaneous mutation frequency of ML236Br clones. These ML236Br clones showed a four- to fivefold-higher resistance to the drug than did their parental V79. Radioactive acetate, but not mevalonate, incorporation into the sterol fraction increased about 10-fold in ML236Br clones in comparison with that in V79. The cellular level of HMG-coenzyme A reductase in three ML236Br mutants was found to be a few-fold higher than that of V79 when cultured in the presence of lipoproteins. The 125I-labeled low-density lipoprotein-binding assay showed binding activity in three ML236Br clones comparable to that of the parental V79 cells. By contrast, an internalization assay of 125I-labeled low-density lipoprotein into the cells showed significantly reduced activity in three ML236Br clones in comparison with V79.

Transformation by viral and cellular oncogenes of a mouse BALB/3T3 cell mutant resistant to transformation by chemical carcinogens.

The mouse cell line MO-5 is resistant to transformation by various chemical carcinogens and also by UV irradiation (C. Yasutake, Y. Kuratomi, M. Ono, S. Masumi, and M. Kuwano, Cancer Res. 47:4894-4899, 1987). Northern (RNA) blot analysis showed active expression of ras and myc genes in MO-5 and BALB/3T3 cells. The effect of transfection of various oncogenes on transformation was compared in MO-5 cells and parental BALB/3T3 cells. Activated c-H-ras, c-N-ras, and v-mos gene induced transformation foci of MO-5 and BALB/3T3. Introduction of the polyomavirus middle T-antigen (mTag) or the Rous sarcoma virus-related oncogene v-src, however, efficiently transformed BALB/3T3 but not MO-5 cells. Expression and phosphorylation of mTag and the associated c-src proteins were observed in mTag-transfected clones of MO-5 as in BALB/3T3 and phosphorylation of the src protein was observed in v-src-transfected BALB/3T3 and MO-5 clones. Hybrids between mTag- or v-src-induced transformants of BALB/3T3 and untransformed MO-5 maintained the transformation phenotype, suggesting that no dominant suppressor of transformation exists in MO-5. A hybrid clone between BALB/3T3 and MO-5 induced efficient transformation foci after transfection with the mTag gene, suggesting that the deficient transformation phenotype of MO-5 was recessive. Instead, some other alteration of MO-5, plausibly membrane function, might lead to abortive transformation by chemical carcinogens and also by mTag and the v-src gene product.

Chinese hamster cell variants resistant to the A chain of ricin carry altered ribosome function.

Ricin, a toxic lectin from Ricinus communis, is composed of two different polypeptide chains, A and B, and the ricin A chain (RA) blocks protein synthesis. We studied cell lines resistant to cytotoxic action of RA. One low-RA-resistant cell line, AR10, isolated from Chinese hamster ovary (CHO) cells, was resistant to a low dose of RA (1 microgram/ml) and showed a 10-fold-higher resistance to RA and ricin than that of CHO. We further mutagenized AR10 to isolate high-RA-resistant cell lines AR100-6, AR100-9, and AR100-13, which were resistant to higher doses of RA and ricin (100- to 1,000-fold) than CHO was. The binding of [125I]ricin to AR10, AR100-6, AR100-9, and AR100-13 cells was decreased to about 30% of that of CHO. The internalization of [125I]ricin in AR10 cells and in the high-RA-resistant clones was the same. Polyuridylate-dependent polyphenylalanine synthesis, using S-30 extracts from either AR100-9 or AR100-13, was about 100-fold more resistant to the inhibitory action of RA than when CHO, AR10, and AR100-6 cells extracts were used. The protein synthesis with ribosomes (80S) from AR100-9 or AR100-13 was 10- to 100-fold more resistant to RA than it was with parental ribosomes when combined with the S-100 fraction of CHO cells. The polyphenylalanine synthesis assay using the ribosomes constituted from the 60S subunit of AR100-9 and the 40S subunit of CHO indicated that the resistant phenotype of AR100-9 cells is due to an alteration of the 60S ribosomal subunit.

Involvement of interleukin-8, vascular endothelial growth factor, and basic fibroblast growth factor in tumor necrosis factor alpha-dependent angiogenesis.

Tumor necrosis factor alpha (TNF-alpha) is a macrophage/monocyte-derived polypeptide which modulates the expression of various genes in vascular endothelial cells and induces angiogenesis. However, the underlying mechanism by which TNF-alpha mediates angiogenesis is not completely understood. In this study, we assessed whether TNF-alpha-induced angiogenesis is mediated through TNF-alpha itself or indirectly through other TNF-alpha-induced angiogenesis-promoting factors. Cellular mRNA levels of interleukin-8 (IL-8), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and their receptors were increased after the treatment of human microvascular endothelial cells with TNF-alpha (100 U/ml). TNF-alpha-dependent tubular morphogenesis in vascular endothelial cells was inhibited by the administration of anti-IL-8, anti-VEGF, and anti-bFGF antibodies, and coadministration of all three antibodies almost completely abrogated tubular formation. Moreover, treatment with Sp1, NF-kappaB, and c-Jun antisense oligonucleotides inhibited TNF-alpha-dependent tubular morphogenesis by microvascular endothelial cells. Administration of a NF-kappaB antisense oligonucleotide almost completely inhibited TNF-alpha-dependent IL-8 production and partially abrogated TNF-alpha-dependent VEGF production, and an Sp1 antisense sequence partially inhibited TNF-alpha-dependent production of VEGF. A c-Jun antisense oligonucleotide significantly inhibited TNF-alpha-dependent bFGF production but did not affect the production of IL-8 and VEGF. Administration of an anti-IL-8 or anti-VEGF antibody also blocked TNF-alpha-induced neovascularization in the rabbit cornea in vivo. Thus, angiogenesis by TNF-alpha appears to be modulated through various angiogenic factors, both in vitro and in vivo, and this pathway is controlled through paracrine and/or autocrine mechanisms.

Involvement of the transcription factor NF-kappaB in tubular morphogenesis of human microvascular endothelial cells by oxidative stress.

Oxygen radicals are induced under various pathologic conditions associated with neovascularization. Oxygen radicals modulate angiogenesis in cultured human microvascular endothelial cells by an unknown mechanism. Treatment of human microvascular endothelial cells for 15 min with 0.1 to 0.5 mM hydrogen peroxide (H2O2) or 100 U of tumor necrosis factor alpha per ml induced tubular morphogenesis in type I collagen gels. Gel shift assays with nuclear extracts demonstrated that H2O2 increases the binding activities of two transcription factors, NF-kappaB and AP-1, but not of Spl. Tumor necrosis factor alpha increased the binding activities of all three factors. A supershift assay with specific antibodies against JunB, JunD, and c-Jun (Jun family) showed that the antibody against c-Jun supershifted the AP-1 complex after H2O2 treatment. Coadministration of the antisense sequence of NF-kappaB inhibited H2O2-dependent tubular morphogenesis, and the antisense c-Jun oligonucleotide caused partial inhibition. The angiogenic factor responsible for H2O2-induced tubular morphogenesis was examined. Cellular mRNA levels of vascular endothelial growth factor and interleukin-8 (IL-8), but not those of transforming growth factor alpha, were increased after treatment with 0.5 mM H2O2. Coadministration of anti-IL-8 antibody inhibited tubular morphogenesis enhanced by H2O2, and IL-8 itself also enhanced the formation of tube-like structures. Treatment with antisense NF-kappaB oligonucleotide completely blocked H2O2-dependent IL-8 production by endothelial cells. The tubular morphogenesis of vascular endothelial cells after treatment with oxidative stimuli and its possible association with NF-kappaB and IL-8, is examined.

Detection of an isoform of alpha(1)-antitrypsin in serum samples from foals with gastric ulcers.

The objective of this study was to find serum indicators of gastric ulcers in foals. By using two-dimensional electrophoresis of serum proteins, three distinct spots were detected in samples from foals with gastric ulcers detected endoscopically. One of them appeared with high frequency and was identified by partial digestion with trypsin and subsequent nano-electrospray ionisation-tandem mass spectrometry (nanoesi-ms/ms) analysis as an alpha(1)-antitrypsin. Western blot analysis, using an antibody against human alpha(1)-antitrypsin, revealed at least two bands, of molecular weight 58 kDa and 55 kDa, in the sera. The 55 kDa band was detected in 44 of 47 serum samples from foals with gastric ulcers, but in only three of 22 serum samples from healthy foals.

Antimyeloma effects of a novel synthetic retinoid Am80 (Tamibarotene) through inhibition of angiogenesis.

In multiple myeloma (MM), the interaction between myeloma cells and bone marrow microenvironment has an important role in the pathogenesis of MM. We first examined the inducing effect of myeloma cells on migration of human umbilical vein vascular endothelial cells (HUVECs). Five myeloma cell lines produced varying amounts of VEGF, and migration of HUVECs was induced by coculture with myeloma cells. We next examined the inhibitory effect of a novel synthetic retinoid Am80 (Tamibarotene) on both myeloma cells and HUVECs. Am80 is specific for the retinoic-acid receptor-alpha/beta, and has therapeutic effects in all-trans retinoic acid resistant acute promyelocytic leukemia. Am80 slightly inhibited the growth of both myeloma cells and HUVECs, and remarkably inhibited the growth of HUVECs stimulated by VEGF. Am80 showed little growth inhibition of bone marrow stromal cells (BMSCs), but it markedly inhibited migration of HUVECs by cocultured myeloma cells. Am80 inhibited VEGF-induced phosphorylation of VEGF receptor. In addition, VEGF-induced formation of tube-like structures in vitro and neovascularization in mouse corneas were significantly inhibited by Am80. These findings clearly demonstrate that Am80 is a potential inhibitor of angiogenesis caused by the interaction between vascular endothelial cells and myeloma cells, and might be a useful therapeutic agent against MM.

Y box-binding protein-1 binds preferentially to single-stranded nucleic acids and exhibits 3'-->5' exonuclease activity.

We have previously shown that Y box-binding protein-1 (YB-1) binds preferentially to cisplatin-modified Y box sequences. Based on structural and biochemical data, we predicted that this protein binds single-stranded nucleic acids. In the present study we confirmed the prediction and also discovered some unexpected functional features of YB-1. We found that the cold shock domain of the protein is necessary but not sufficient for double-stranded DNA binding while the C-tail domain interacts with both single-stranded DNA and RNA independently of the cold shock domain. In an in vitro translation system the C-tail domain of the protein inhibited translation but the cold shock domain did not. Both in vitro pull-down and in vivo co-immunoprecipitation assays revealed that YB-1 can form a homodimer. Deletion analysis mapped the C-tail domain of the protein as the region of homodimerization. We also characterized an intrinsic 3'-->5' DNA exonuclease activity of the protein. The region between residues 51 and 205 of its 324-amino acid extent is required for full exonuclease activity. Our findings suggest that YB-1 functions in regulating DNA/RNA transactions and that these actions involve different domains.

Circumvention of multiple-drug resistance in human cancer cells by thioridazine, trifluoperazine, and chlorpromazine.

Human KB carcinoma cells were selected in four sequential steps for increasing resistance to colchicine and were found to be cross-resistant to multiple drugs. Thioridazine, a phenothiazine calmodulin inhibitor, almost completely reversed the resistance of the multiple-drug-resistant cells to doxorubicin, vinblastine, dactinomycin, and daunorubicin, and partially reversed the resistance to colchicine and vincristine. Other phenothiazine calmodulin inhibitors, trifluoperazine and chlorpromazine, were also found to show similar but somewhat weaker effects on drug resistance. However, a well-known naphthalenesulfonamide derivative calmodulin inhibitor, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), could not reverse the drug resistance. Very low accumulation of vincristine or daunorubicin was observed in the multiple-drug-resistant KB cells in comparison with accumulation in the parental KB cells. Increased rate of accumulation of the drugs by thioridazine, trifluoperazine, and chlorpromazine was most prominent in the resistant KB-ChR-24 cells than in KB cells. We have observed enhanced efflux of the drugs from the resistant cells, and thioridazine inhibited this efflux. These studies suggest a role for increased drug efflux in the development of the multiple-drug resistance phenotype in human carcinoma cells.

Overcoming drug resistance in cancer cells with synthetic isoprenoids.

A cultured subline (P388/ADM) of mouse P388 leukemia resistant to doxorubicin, vinblastine, vincristine, dactinomycin, and daunorubicin became sensitive again when treated with noncytotoxic doses of either of two synthetic isoprenoids: N-solanesyl-N,N'-bis(3,4-dimethoxybenzyl)ethylenediamine (SDB-ethylenediamine) and N-(p-methylbenzyl)decaprenylamine X HCI (PMB-decaprenylamine). The isoprenoids also reversed resistance to doxorubicin and vincristine in a cultured vincristine-resistant P388 leukemia subline (P388/VCR). Median lethal doses (LD50) for PMB-decaprenylamine and SDB-ethylenediamine administered ip were 123 and 350 mg/kg against mice, whereas the LD50 for verapamil, another modifier of cellular drug resistance, was about 7.6 mg/kg. In vivo experiments with P388/VCR-bearing mice showed that both SDB-ethylenediamine and verapamil overcame vincristine resistance, but PMB-decaprenylamine showed only slight activity. SDB-ethylenediamine was especially effective, overcoming the vincristine resistance at 1 mg drug/kg. Since the structure of SDB-ethylenediamine resembles that of verapamil, a calcium-blocking agent that overcomes drug resistance, it was checked for calcium-blocking activity. However, calcium channel-blocking activity was not observed with 20 micrograms isoprenoid/ml, whereas calcium channel activity was completely blocked by 1 microgram verapamil/ml.

Potentiation of anticancer agents by new synthetic isoprenoids. II. Inhibition of the growth of transplantable murine tumors.

The antitumor effects of combinations of synthetic isoprenoids-decaprenylamine.HCl, N-(p-methylbenzyl)decaprenylamine.HCl [N-(PMB)-decaprenylamine.HCl], and decaprenoic acid-with anticancer agents were studied in male ICR mice with ascites sarcoma 180 (S180) and in male BALB/c mice with fibrosarcoma Meth A. Decaprenylamine.HCl, N-(PMB)-decaprenylamine.HCl, and decaprenoic acid were tested in combination with bleomycin A2 (BLM) on S180. Decaprenoic acid was also examined in combination with BLM or 5-fluorouracil (FUra) for effects on fibrosarcoma Meth A. The dosages of synthetic isoprenoids used in the combination therapy were much below the median lethal dose. In the low-dosage schedule, decaprenylamine.HCl, N-(PMB)-decaprenylamine.HCl or decaprenoic acid considerably enhanced the antitumor effect of BLM on S180; e.g., the T/C value (mean survival time of treated mice/mean survival time of controls) for decaprenylamineHCl plus BLM, N-(PMB)-decaprenylamine.HCl plus BLM, or decaprenoic acid plus BLM was 294, 357, and 279% respectively, and the combinations increased life-span 2.4-fold, 2.3-fold, or 1.8-fold, respectively, as compared to the effects of BLM alone. The combination of BLM or FUra with decaprenoic acid, when administered by iv injection to mice with fibrosarcoma Meth A (solid tumor system), did not produce synergism. However, a preventive effect of decaprenoic acid monotherapy was observed: Decaprenoic acid at 3 mg/kg resulted in 38.9% suppression of tumor growth 21 days after inoculation.

Control of permeation of bleomycin A2 by polyene antibiotics in cultured Chinese hamster cells.

Control of permeation of bleomycin A2, a well-known antitumor antibiotic, in combination with various polyene macrolide antibiotics was analyzed in cultured Chinese hamster cells in vitro. Three polyene antibiotics, filipin, pentamycin, and pimaricin, were found to enhance the action of bleomycin A2 remarkably, while amphotericin B or nystatin could not. Although DNA synthesis and colony-forming activity of polyene-sensitive Chinese hamster V79 cells were synergistically inhibited by the combination of filipin and bleomycin A2, in a polyene-resistant subline (AMBR-1) derived from V79, they were only slightly affected in the presence of both drugs. The cellular uptake of [14C]bleomycin A2 by V79 was enhanced 2- to 4-fold in the presence of increasing doses of filipin or pentamycin, but not in the presence of amphotericin B. The treatment of V79 cells with filipin for 20 to 30 min was enought to block DNA synthesis almost completely when combined with 20 microgram belomycin A2 per ml. The pretreatment of the hamster cells with 6 microgram filipin per ml for 60 min continued to enhance the inhibitory action by bleomycin A2 of DNA synthesis up to 5 hr after the removal of filipin from the cultured medium.

Effect of l-asparaginase and asparagine deprivation on RNA metabolism in mouse leukemia L 5178Y cells in suspension culture.

We analyzed the effect of asparagine starvation and L-asparaginase on RNA metabolism of mouse leukemia cell lines L5178Y, whose growth is dependent on the presence of asparagine, and L5178Y-R, whose growth is independent of the presence of asparagine. The deprivation of asparagine from the medium inhibited cellular protein synthesis by 30 to 40% of the control value in L5178Y cells, but not in L5178Y-R cells, whereas L-asparaginase inhibited synthesis by more than 80% in both L5178Y and L5178Y-R cells. The decrease in protein synthesis caused by asparagine starvation in L5178Y cells was accompanied by a decrease in ribosomal RNA synthesis. The synthesis of rRNA was also markedly blocked when L5178Y and L5178Y-R cells were exposed to L-asparaginase. The rate of synthesis of pulse-labeled RNA decreased significantly in the cells treated with L-asparaginase, and smaller pieces of polyadenylate containing pulse-labeled RNA (presumptive messenger RNA) appeared among monosomes and polysomes. However, the rate of messenger RNA synthesis was constant during asparagine starvation, and a marked accumulation of monosome was observed.

Isolation of retinoic acid-resistant clones from human breast cancer cell line MCF-7 with altered activity of cellular retinoic acid-binding protein.

After ethyl methane sulfonate mutagenesis of the mammary carcinoma cell line, MCF-7, we have isolated three clones, U-2, U-3, and U-9, resistant to retinoic acid. These three clones showed more than a 1000-fold higher level of resistance to retinoic acid than the parental MCF-7 cells when assayed by colony formation in monolayer culture system or by growth curves. The three resistant clones showed a 200-fold higher resistance to 13-cis-retinoic acid, about 10-fold higher resistance to retinol, and about 2-fold higher resistance to retinyl acetate, respectively, than MCF-7. Binding of [3H]retinoic acid or [3H]-retinol to a cellular fraction in situ showed apparent decrease of the specific binding of retinoic acid in U-2, but there was no such specific fraction bound to retinol in U-2 and MCF-7. Sucrose gradient analysis with cytoplasmic fraction showed little, if any, cellular retinoic acid-binding protein in U-2, but a significant amount of the cellular retinoic acid-binding protein could be found in MCF-7. By contrast, there was no activity of cellular retinol-binding protein in both MCF-7 and U-2. The sensitivity or resistance of mammalian cells in culture to retinoic acid is discussed in relation with cellular binding activity for vitamin A.

Interferon gamma-dependent induction of thymidine phosphorylase/platelet-derived endothelial growth factor through gamma-activated sequence-like element in human macrophages.

Thymidine phosphorylase (TP), an enzyme involved in the reversible conversion of thymidine to thymine, is identical to platelet-derived endothelial cell growth factor. TP expression in cancer cells and/or infiltrated macrophages is associated with microvessel density and poor clinical prognosis in patients with various tumor types. However, how TP expression is up-regulated in human tumors is unclear. Of various inflammatory cytokines, such as tumor necrosis factor alpha (TNF-alpha), interleukin 1 alpha (IL-1alpha), and interferon gamma (IFN-gamma), we observed that IFN-gamma most effectively increased the expression of TP in cultured human monocytic U937 cells. Transient transfection of the various deletion constructs of the TP promoter showed that the presence of the -474 to -355 sequence containing gamma-activated sequence-like element was essential for IFN-gamma-dependent activation of the TP gene. Furthermore, the IFN-gamma-dependent transcriptional activity of the promoter construct containing mutations in the gamma-activated sequence-like element was significantly decreased. An electrophoretic mobility shift assay showed that IFN-gamma increased signal transducers and activators of transcription 1 binding to gamma-activated sequence-like element in the TP promoter. IFN-gamma could be a mediator of TP expression in infiltrated monocyte/macrophages, and those monocyte/macrophages expressing TP might play an important role in malignancy and angiogenesis in various human tumors.