Search

Abstract Experiments were conducted to determine the fate of nitrogen (N) remobilized as a result of ozone (O3 )-induced accelerated senescence in hybrid poplar subjected to declining N availability concurrent with O3 stress. Cuttings were grown in sand culture where the supply of N to the plant could be controlled on a daily basis and reduced in half of the plants when desired. Plants all initially received 3·57 mm N daily until approximately the 20 leaf stage after which daily supply of N was reduced to 0·71 mm. Plants were grown in open-top chambers in the field (Rock Springs, PA, USA) and received charcoal-filtered air, half also received supplemental O3 to a level of 0·08 µL L-1 . Allocation of newly acquired N was determined with 15 N. The specific allocation (mg labelled N mg-1 total N) of labelled N to upper, expanding leaf N was not affected by O3 , but was strongly affected by N treatment. However, O3 increased the relative partitioning of labelled N to the expanding leaves and the roots. The balance between partitioning of newly acquired N to the upper leaves and roots was not affected by O3 , but was reduced by N withdrawal. Calculated net N flux was strongly negative in the lower leaves of O3 -exposed, N withdrawal plants. Nitrogen uptake was not reduced by O3 . The allometric relationships between the roots and shoots were not affected by O3 or N availability. The relative contribution of newly acquired versus remobilized N to new growth appears to be determined by N supply. Ozone exposure alters the allocation of newly acquired N via alterations in plant size, whereas N availability exerts a strong effect upon both plant size and N allocation. [ABSTRACT FROM AUTHOR]

Abstract Background In many tree species the perception of short days (SD) can trigger growth cessation, dormancy entrance, and the establishment of a chilling requirement for bud break. The molecular mechanisms connecting photoperiod perception, growth cessation and dormancy entrance in perennials are not clearly understood. The peach [Prunus persica (L.) Batsch] evergrowing (evg) mutant fails to cease growth and therefore cannot enter dormancy under SD. We used the evg mutant to filter gene expression associated with growth cessation after exposure to SD. Wild-type and evg plants were grown under controlled conditions of long days (16 h/8 h) followed by transfer to SD (8 h/16 h) for eight weeks. Apical tissues were sampled at zero, one, two, four, and eight weeks of SD and suppression subtractive hybridization was performed between genotypes at the same time points. Results We identified 23 up-regulated genes in the wild-type with respect to the mutant during SD exposure. We used quantitative real-time PCR to verify the expression of the differentially expressed genes in wild-type tissues following the transition to SD treatment. Three general expression patterns were evident: one group of genes decreased at the time of growth cessation (after 2 weeks in SD), another that increased immediately after the SD exposure and then remained steady, and another that increased throughout SD exposure. Conclusions The use of the dormancy-incapable mutant evg has allowed us to reduce the number of genes typically detected by differential display techniques for SD experiments. These genes are candidates for involvement in the signalling pathway leading from photoperiod perception to growth cessation and dormancy entrance and will be the target of future investigations.

Abstract Background Dormancy associated MADS-box (DAM) genes are candidates for the regulation of growth cessation and terminal bud formation in peach. These genes are not expressed in the peach mutant evergrowing, which fails to cease growth and enter dormancy under dormancy-inducing conditions. We analyzed the phylogenetic relationships among and the rates and patterns of molecular evolution within DAM genes in the phylogenetic context of the MADS-box gene family. Results The peach DAM genes grouped with the SVP/StMADS11 lineage of type II MIKCC MADS-box genes. Phylogenetic analyses suggest that the peach SVP/StMADS11-like gene family, which contains significantly more members than annual model plants, expanded through serial tandem gene duplication. We found evidence of strong purifying selection acting to constrain functional divergence among the peach DAM genes and only a single codon, located in the C-terminal region, under significant positive selection. Conclusion Because all DAM genes are expressed in peach and are subjected to strong purifying selection we suggest that the duplicated genes have been maintained by subfunctionalization and/or neofunctionalization. In addition, this pattern of selection suggests that the DAM genes are important for peach growth and development.

We have synthesized an oxetane derivative of the benzimidazole compound mebendazole (OBD9) with enhanced solubility and strong anticancer activity in multiple types of cancer cells, especially colorectal cancer. In this report, we provide evidence that OBD9 suppresses colorectal cancer growth by interfering with the Wnt signaling pathway, a main driver of cell growth in colorectal cancer. Specifically, we find that OBD9 induces autophagic degradation of TNIK (traf2 and Nck-interacting kinase), which promotes T-cell factor-4 (TCF4)/beta-catenin-mediated gene expression. Thus, OBD9 as a TNIK inhibitor blocks Wnt/beta-catenin signaling at the final step of transcriptional activation. We suggest that OBD9 provides a potential novel autophagy-mediated, Wnt-damping therapeutic strategy for the treatment of colorectal cancer., (©2022 American Association for Cancer Research.)

The malignancy potential is correlated with the mechanical deformability of the cancer cells. However, mechanical tests for clinical applications are limited. We present here a Triangular Correlation (TrC) between cell deformability, phagocytic capacity, and cancer aggressiveness, suggesting that phagocytic measurements can be a mechanical surrogate marker of malignancy. The TrC was proved in human prostate cancer cells with different malignancy potential, and in human bladder cancer and melanoma cells that were sorted into subpopulations based solely on their phagocytic capacity. The more phagocytic subpopulations showed elevated aggressiveness ex vivo and in vivo. The uptake potential was preserved, and differences in gene expression and in epigenetic signature were detected. In all cases, enhanced phagocytic and aggressiveness phenotypes were correlated with greater cell deformability and predicted by a computational model. Our multidisciplinary study provides the proof of concept that phagocytic measurements can be applied for cancer diagnostics and precision medicine., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Rosaceae is the most important fruit-producing clade, and its key commercially relevant genera (Fragaria, Rosa, Rubus and Prunus) show broadly diverse growth habits, fruit types and compact diploid genomes. Peach, a diploid Prunus species, is one of the best genetically characterized deciduous trees. Here we describe the high-quality genome sequence of peach obtained from a completely homozygous genotype. We obtained a complete chromosome-scale assembly using Sanger whole-genome shotgun methods. We predicted 27,852 protein-coding genes, as well as noncoding RNAs. We investigated the path of peach domestication through whole-genome resequencing of 14 Prunus accessions. The analyses suggest major genetic bottlenecks that have substantially shaped peach genome diversity. Furthermore, comparative analyses showed that peach has not undergone recent whole-genome duplication, and even though the ancestral triplicated blocks in peach are fragmentary compared to those in grape, all seven paleosets of paralogs from the putative paleoancestor are detectable.

Unlabelled: Metastatic tumors have been shown to establish permissive microenvironments for metastases via recruitment of bone marrow-derived cells. Here, we show that metastasis-incompetent tumors are also capable of generating such microenvironments. However, in these situations, the otherwise prometastatic Gr1(+) myeloid cells create a metastasis-refractory microenvironment via the induction of thrombospondin-1 (Tsp-1) by tumor-secreted prosaposin. Bone marrow-specific genetic deletion of Tsp-1 abolished the inhibition of metastasis, which was restored by bone marrow transplant from Tsp-1(+) donors. We also developed a 5-amino acid peptide from prosaposin as a pharmacologic inducer of Tsp-1 in Gr1(+) bone marrow cells, which dramatically suppressed metastasis. These results provide mechanistic insights into why certain tumors are deficient in metastatic potential and implicate recruited Gr1(+) myeloid cells as the main source of Tsp-1. The results underscore the plasticity of Gr1(+) cells, which, depending on the context, promote or inhibit metastasis, and suggest that the peptide could be a potential therapeutic agent against metastatic cancer., Significance: The mechanisms of metastasis suppression are poorly understood. Here, we have identified a novel mechanism whereby metastasis-incompetent tumors generate metastasis-suppressive microenvironments in distant organs by inducing Tsp-1 expression in the bone marrow–derived Gr1+myeloid cells. A 5-amino acid peptide with Tsp-1–inducing activity was identified as a therapeutic agent against metastatic cancer.

Increased levels of ADAM12 have been reported in a variety of human cancers. We have previously reported that urinary ADAM12 is predictive of disease status in breast cancer patients and that ADAM12 protein levels in urine increase with progression of disease. On the basis of these findings, the goal of this study was to elucidate the contribution of ADAM12 in breast tumor growth and progression. Overexpression of both the ADAM12-L (transmembrane) and ADAM12-S (secreted) isoforms in human breast tumor cells resulted in a significantly higher rate of tumor take and increased tumor size. Cells expressing the enzymatically inactive form of the secreted isoform, ADAM12-S, had tumor take rates and tumor volumes similar to those of wild-type cells, suggesting that the tumor-promoting activity of ADAM12-S was a function of its proteolytic activity. Of the two isoforms, only the secreted isoform, ADAM12-S, enhanced the ability of tumor cells to migrate and invade in vitro and resulted in a higher incidence of local and distant metastasis in vivo. This stimulatory effect of ADAM12-S on migration and invasion was dependent on its catalytic activity. Expression of both ADAM12 isoforms was found to be significantly elevated in human malignant breast tissue. Taken together, our results suggest that ADAM12 overexpression results in increased tumor take, tumor size, and metastasis in vivo. These findings suggest that ADAM12 may represent a potential therapeutic target in breast cancer.

Breast cancer preferentially metastasizes to bone. We therefore addressed the role of Notch signaling in osteoblast-cancer cell interactions and in bone metastasis. Human bone marrow osteoblasts selectively enhanced the expression of Notch3 and its ligand Jagged1 in human breast cancer cell lines. Osteoblasts also stimulated cancer cell colony formation in soft agar, which was reduced by a chemical inhibitor of Notch signaling and anti-transforming growth factor beta1 (TGFbeta1) antibody. TGFbeta1, a major prometastatic product of osteoblasts, also stimulated cancer cell Notch3 expression. Notch3 knockdown in the cancer cells by stable short hairpin RNA interference decreased the osteoblast- and TGFbeta1-stimulated colony formation as well as TGFbeta1-mediated Smad3/Smad2 phosphorylation; Jagged1 level was coordinately reduced. In addition, expression of snail, a regulator of epithelial-mesenchymal transition, and the mesenchymal markers fibronectin and vimentin was attenuated by reducing Notch3 levels. To study the role of Notch3 signaling in bone metastasis, cancer cells were inoculated into athymic mice, either into femoral bone marrow cavities or into the systemic circulation via the left ventricle. Compared with robust osteolysis in mice receiving control cells, osteolytic lesions were significantly reduced following inoculation of cells with constitutively reduced Notch3 expression. Taken together, our results suggest that enhanced Notch3 expression in breast cancer cells, triggered by osteoblasts and their secretion of TGFbeta1 in the bone marrow niche, may stand as a novel mechanism for promoting bone metastasis.

Inflammation in the tumor bed can either promote or inhibit tumor growth. Peroxisome proliferator-activated receptor (PPAR)alpha is a central transcriptional suppressor of inflammation, and may therefore modulate tumor growth. Here we show that PPARalpha deficiency in the host leads to overt inflammation that suppresses angiogenesis via excess production of the endogenous angiogenesis inhibitor thrombospondin-1 and prevents tumor growth. Bone marrow transplantation and granulocyte depletion show that PPARalpha expressing granulocytes are necessary for tumor growth. Neutralization of thrombospondin-1 restores tumor growth in PPARalpha-deficient mice. These findings suggest that the absence of PPARalpha activity renders inflammatory infiltrates tumor suppressive and, thus, may provide a target for inhibiting tumor growth by modulating stromal processes, such as angiogenesis.

Neuropilin-1 (NRP1) is expressed by endothelial cells and neurons and serves as a receptor for both vascular endothelial growth factor (VEGF), an angiogenesis factor, and semaphorin 3A (Sema3A), a mediator of axonal guidance. We show here that NRP1 is also expressed in keratinocytes in vitro and in vivo. However, nothing has been reported about the regulation or function of keratinocyte NRP1. Using NRP1 promoter constructs in HaCaT cells, a keratinocyte cell line, we could demonstrate that a neuron restrictive silencer element (NRSE) was implicated in transcriptional repression of the NRP1 gene. Electrophoretic mobility shift assays demonstrated that the neuron restrictive silencer factor (NRSF) binds to NRSE. Overexpression of NRSF in HaCaT cells decreased NRP1 RNA and protein, whereas a dominant negative NRSF increased NRP1. Furthermore, the histone deacetylase inhibitor trichostatin A, an inhibitor of NRSF silencing activity, also increased NRP1 levels. NRP2 expression was not affected. Epidermal growth factor (EGF) and heparin-binding EGF-like growth factor (HB-EGF) strongly up-regulated NRP1 expression, concomitant with down-regulation of NRSF. Other keratinocyte mitogens such as keratinocyte growth factor (KGF) had no effect. To address function, HaCaT cells were exposed to two NRP1 ligands, VEGF165 and Sema3A. Neither had an effect on proliferation, whereas Sema3A, but not VEGF165, inhibited cell migration. Down-regulation of NRP1 by NRSF overexpression reduced Sema3A activity. It was concluded that NRSF is a transcription factor that silences NRP1 expression and thereby diminishes the Sema3A mediated inhibition of HaCaT keratinocyte migration.

Neuropilin-1 (NRP-1), a recently identified co-receptor for vascular endothelial growth factor, is expressed by several nongastrointestinal tumor types and enhances prostate cancer angiogenesis and growth in preclinical models. We investigated the expression and regulation of NRP-1 and the effect of NRP-1 overexpression on angiogenesis and growth of human colon adenocarcinoma by immunohistochemistry and in situ hybridization. NRP-1 was expressed in 20 of 20 human colon adenocarcinoma specimens but not in the adjacent nonmalignant colonic mucosa. By reverse transcriptase-polymerase chain reaction analysis, NRP-1 mRNA was expressed in seven of seven colon adenocarcinoma cell lines. Subcutaneous xenografts of stably transfected KM12SM/LM2 human colon cancer cells overexpressing NRP-1 led to increased tumor growth and angiogenesis in nude mice. In in vitro assays, conditioned medium from NRP-1-transfected cell lines led to an increase in endothelial cell migration, but did not affect endothelial cell growth. Epidermal growth factor (EGF) led to induction of NRP-1 in human colon adenocarcinoma cells and selective blockade of the epidermal growth factor receptor (EGFR) decreased constitutive and EGF-induced NRP-1 expression. Blockade of the Erk 1/2 and P38 mitogen-activated protein kinase signaling pathways also led to a decrease in constitutive and EGF-induced NRP-1 expression. These findings demonstrate the ubiquitous expression of NRP-1 in human colon cancer and suggest that NRP-1 may contribute to colon cancer angiogenesis and growth. This study also suggests that EGF and mitogen-activated protein kinase signaling pathways play an important role in NRP-1 regulation in colon cancer cells.

Primary tumor growth and metastasis depend on angiogenesis, which is determined by the balance between proangiogenic and antiangiogenic molecules. Interferon (IFN)-alpha and -beta inhibit angiogenesis through downregulation of interleukin-8, matrix metalloproteinase-9, and basic fibroblast growth factor. To provide evidence for the causal role of IFN-alpha/beta in the induction of neoplasms, their angiogenesis, and hence, progressive growth, we carried out experiments using 129S6 IFN-alpha/beta receptor -/- mice back-crossed to BALB/c nude mice. Subcutaneous angiogenesis was determined following implantation of gelfoam sponges containing 0.4% agarose and several proangiogenic molecules. Tumorigenicity and production of lung metastasis were determined subsequent to subcutaneous and intravenous injections, respectively, of highly metastatic A375SM human melanoma cells. Carcinogenesis was induced by chronic exposure of mice to UVB radiation (5 kJ/m2, 3 times/week). Angiogenesis, tumorigenicity, and production of metastasis, as well as development of autochthonous skin tumors, were all accelerated in IFN-alpha/beta receptor -/- mice as compared to control mice. Collectively, the data show that inability to respond to endogenous IFN-alpha/beta (through a mutation in the IFN-alpha/beta receptor) leads to increased susceptibility to carcinogenesis, enhanced angiogenesis, tumorigenicity, and metastasis.

Neuropilin-1 (NRP1) is a 130-kDa transmembrane receptor for semaphorins, mediators of neuronal guidance, and for vascular endothelial growth factor 165 (VEGF(165)), an angiogenesis factor. A 2.2-kb truncated NRP1 cDNA was cloned that encodes a 644-aa soluble NRP1 (sNRP1) isoform containing just the a/CUB and b/coagulation factor homology extracellular domains of NRP1. sNRP1 is secreted by cells as a 90-kDa protein that binds VEGF(165), but not VEGF(121). It inhibits (125)I-VEGF(165) binding to endothelial and tumor cells and VEGF(165)-induced tyrosine phosphorylation of KDR in endothelial cells. The 3' end of sNRP1 cDNA contains a unique, 28-bp intron-derived sequence that is absent in full-length NRP1 cDNA. Using a probe corresponding to this unique sequence, sNRP1 mRNA could be detected by in situ hybridization differentially from full-length NRP1 mRNA, for example, in cells of liver, kidney, skin, and breast. Analysis of blood vessels in situ showed that NRP1, but not sNRP1, was expressed. sNRP1 was functional in vivo. Unlike control tumors, tumors of rat prostate carcinoma cells expressing recombinant sNRP1 were characterized by extensive hemorrhage, damaged vessels, and apoptotic tumor cells. These results demonstrate the existence of a naturally occurring, soluble NRP1 that is expressed differently from intact NRP1 and that appears to be a VEGF(165) antagonist.

The cytokine interferon-beta is a regulator of cell replication and function, including invasion and induction of angiogenesis. The goal of this study was to determine whether the expression of interferon-beta by cells in the epidermis correlated with terminal differentiation. In situ hybridization analysis and immunohistochemical staining of formalin-fixed, paraffin-embedded specimens of normal human and murine epidermis and human and murine skin tumors of epithelial origin revealed that only differentiated, nondividing cells of the epidermis expressed interferon-beta protein. Keratinocyte cultures established from the epidermis of 3 d old mice were maintained under conditions permitting continuous cell division or induction of differentiation. Continuously dividing cells did not produce interferon-beta whereas nondividing differentiated cells expressing keratin 1 did. Growth-arrested, undifferentiated keratinocytes also expressed interferon-beta protein. Neutralizing interferon-beta in the culture medium inhibited differentiation, but the addition of exogenous interferon-beta did not stimulate differentiation. These data indicate that interferon-beta is produced by growth-arrested, terminally differentiated keratinocytes.

We determined whether the IFN-beta gene can be used to suppress angiogenesis, tumor growth, and metastasis of human prostate cancer cells growing in the prostate of nude mice. Highly metastatic PC-3M human prostate cancer cells were engineered to constitutively produce murine IFN-beta subsequent to infection with a retroviral vector containing murine IFN-beta cDNA. Parental (PC-3M-P), control vector-transduced (PC-3M-Neo), and IFN-beta-transduced (PC-3M-IFN-beta) cells were injected into the prostate (orthotopic) or subcutis (ectopic) of nude mice. PC-3M-P and PC-3M-Neo cells produced rapidly growing tumors and regional lymph node metastases, whereas PC-3M-IFN-beta cells did not. PC-3M-IFN-beta cells also suppressed the tumorigenicity of bystander nontransduced prostate cancer cells. PC-3M-IFN-beta cells produced small tumors (3-5 mm in diameter) in nude mice treated with anti-asialo GM1 antibodies and in severe combined immunodeficient/Beige mice. Immunohistochemical staining revealed that PC-3M-IFN-beta tumors were homogeneously infiltrated by macrophages, whereas control tumors contained fewer macrophages at their periphery. Most tumor cells in the control tumors were stained positive by an antibody to proliferative cell nuclear antigen; very few were positively stained by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling. In sharp contrast, PC-3M-IFN-beta tumors contained fewer proliferative cell nuclear antigen-positive cells and many terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling-positive cells. Staining with antibody against CD31 showed that control tumors contained more blood vessels than PC-3M-IFN-beta tumors. PC-3M-IFN-beta cells were more sensitive to lysis mediated by natural killer cells in vitro or to cytostasis mediated by macrophages than control transduced cells. Conditioned medium from PC-3M-IFN-beta cells augmented splenic cell-mediated cytolysis to control tumor cells, which could be neutralized by antibody against IFN-beta. Collectively, the data suggest that the suppression of tumorigenicity and metastasis of PC-3M-IFN-beta cells is due to inhibition of angiogenesis and activation of host effector cells.

We determined whether cutaneous angiogenesis induced by exposure of mice to ultraviolet-B (UVB) radiation is associated with an imbalance between positive and negative angiogenesis-regulating molecules. Unshaved C3H/HeN mice were exposed to a single dose (15 kJ per m2) of UVB. At various times, the mice were killed, and their external ears were processed for routine histology and immunohistochemistry. Antibodies against proliferating cell nuclear antigen and bromodeoxyuridine identified dividing cells. Antibodies against CD31/ PECAM-1 identified endothelial cells, and antibodies against basic fibroblast growth factor (bFGF), vascular endothelial growth factor/vascular permeability factor, and interferon-beta (IFN-beta) identified angiogenesis-regulating molecules. Epidermal hyperplasia was documented by 48 h and reached a maximum on day 7 after exposure to UVB. The expression of bFGF increased by 24 h, whereas the expression of IFN-beta decreased by 72 h after exposure to UVB. The expression of vascular endothelial growth factor/vascular permeability factor increased slightly after irradiation. The altered balance between bFGF and IFN-beta was associated with increased endothelial cell proliferation (bromodeoxyuridine + CD31 + cells) within existing blood vessels, leading to telangiectasia and new blood vessels. UV-induced epidermal hyperplasia and cutaneous angiogenesis were highest in IFN-alpha/beta receptor knockout mice. These results demonstrate that in response to UVB radiation, dividing keratinocytes produce a positive angiogenic molecule (bFGF) but not a negative angiogenic molecule (IFN-beta), and that this altered balance is associated with enhanced cutaneous angiogenesis.

The body's first line of defense against external challenge are the epithelial cells that line the skin and the respiratory, digestive, and genitourinary tracts. Inasmuch as interferon-beta (IFN-beta) participates in host defense against viral, bacterial, and parasitic infections and tumors, we hypothesized that this secreted protein is expressed in various murine epithelial cell types that line portals of entry to the body. We used immunohistochemistry and in situ hybridization techniques to measure IFN-beta expression in the various epithelial cell types and in internal murine organs sheltered from environmental stimuli. The epithelial cell types lining the skin, digestive tract, urinary tract, reproductive tract, and upper respiratory tract constitutively expressed IFN-beta. Specifically, all differentiated epithelial cells at risk of environmental exposure expressed IFN-beta (protein and mRNA) with the exception of the ciliated epithelial cells lining the lower respiratory tract. Epithelial cells of internal organs that are not directly exposed to external pathogens did not express IFN-beta.

In this study, we endeavored to determine the effectiveness of interferon beta (IFNbeta) gene therapy against highly metastatic murine UV-2237m fibrosarcoma cells. UV-2237m cells were engineered to produce murine IFNbeta constitutively following infection by a retroviral vector harboring the murine IFNbeta gene. Parental (UV-2237m-P), control-vector-transduced (UV-2237m-Neo), and IFNbeta-transduced (UV-2237m-IFNbeta) cells were injected subcutaneously (s.c.) or intravenously (i.v.) into syngeneic mice. Parental and control-transduced cells produced rapidly growing tumors, whereas IFNbeta-transduced cells did not. The tumorigenicity of IFNbeta-sensitive or -resistant parental cells was significantly suppressed when they were injected s.c. together with IFNbeta-transduced cells. The IFNbeta-transduced cells did not inhibit growth of parental cells injected s.c. at a distant site. UV-2237m-IFNbeta cells produced s.c. tumors in nude, SCID/Beige, and natural killer(NK)-cell-compromised syngeneic mice. The IFNbeta-transduced cells were more sensitive to in vitro splenic cell-mediated lysis than were the parental or control-transduced cells. Pretreatment of C3H/HeN mice with the NK-cell-selective antiserum (anti-asialoGM1) partially abrogated the cytotoxic activity of the cells. Cytotoxic activity was not observed in mixed culture of UV-2237m-IFNbeta cells and splenic cells from SCID/Beige mice. Significant cytotoxicity against UV-2237m-IFNbeta cells was mediated by macrophages activated by either IFNgamma, lipopolysaccharide, or a combination of both. Our data led us to conclude that the constitutive expression of IFNbeta can suppress tumorigenicity and metastasis of UV-2237m cells, which is due, in part, to activation of host effector cells.

The purpose of these studies was to determine whether systemic administration of IFN-alpha can inhibit the expression of basic fibroblast growth factor (bFGF) in human transitional cell carcinoma, reduce its angiogenesis, and thus inhibit its growth in the bladder wall of nude mice. In vitro incubation of the highly metastatic 253J B-V cells and the IFN-alpha-resistant 253J B-V IFNR cells with noncytostatic concentrations of IFN-alpha down-regulated the steady-state mRNA transcripts and protein production of bFGF. IFN-alpha-insensitive and IFN-alpha-resistant cells were implanted in the bladder wall of nude mice. Systemic administration of IFN-alpha decreased the in vivo expression of bFGF, decreased blood vessel density in the tumors, and inhibited tumor growth of both IFN-alpha-insensitive and IFN-alpha-resistant cells. These data suggest that in addition to its well-documented antiproliferative effects, IFN-alpha can inhibit the growth of human bladder cancer cells by inhibition of angiogenesis.

The purpose of this study was to determine whether sustained local production of murine IFN-beta (mIFN-beta) could inhibit the tumorigenicity and metastasis of human and murine tumor cells implanted into nude mice. Human melanoma cells (A375SM), renal carcinoma cells (SN12PM6), and colon carcinoma cells (KM12SM) were transfected with mIFN-beta or a control neomycin resistance vector. All cell lines grew well in culture. Tumor cells were injected into the subcutis, kidney, spleen, or lateral tail vein of nude mice. Parental or control transfected cells produced local tumors and experimental or spontaneous lung metastases, whereas mIFN-beta-transfected cells did not. In vivo survival experiments using [125I]IdUdR-labeled cells showed that by day 7 after s.c. implantation, all IFN-beta-transfected cells died. IFN-beta transfection prevented the outgrowth of parental or control-transfected cells only when they were injected together with transfected cells into one site, suggesting that IFN-beta promoted a local lysis of the bystander cells. Similar indirect antitumor activity was demonstrated in various human (KM12SM and SN12PM6) and murine (CT-26 colon carcinoma, RENCA renal cell carcinoma, and 3LL Lewis lung carcinoma) tumors. The IFN-beta-transfected tumor cells stimulated a high level of nitric oxide production by murine macrophages under in vitro and in vivo conditions, which correlated with the vigorous nonspecific antitumor activity. Collectively, these results demonstrate that local production of IFN-beta can eradicate tumor cells of different histology by inducing inducible nitric oxide synthase expression in infiltrating cells.

Recent studies have shown that the treatment of nonmetastatic K-1735 murine melanoma cells with cytokines induces the production of nitric oxide (NO) and hence cell death. The purpose of this study was to determine the mechanism of this cytokine-induced NO-mediated apoptosis. Incubation of nonmetastatic K-1735 cells with interleukin-1 alpha (IL-1alpha) and interferon-gamma (IFN-gamma) induced high NO production, Bcl-2 downregulation, and apoptotic cell death. In contrast, incubation of metastatic K-1735 cells with IL-1alpha and IFN-gamma did not induce significant production of NO, downregulation of Bcl-2, or cell death. The exposure to exogenous NO derived from the NO donors, sodium nitroprusside (SNP), or GEA5024 produced a dose-dependent apoptotic cell death in both the metastatic and nonmetastatic K-1735 cells, which was associated with downregulation of Bcl-2 at the mRNA level and, to a lesser extent, at the protein level. Nonmetastatic and metastatic K-1735 cells transfected with the Bcl-2 gene were more resistant to apoptosis mediated by both endogenous and exogenous NO. Subsequent to intravenous injection, the tumor cells transfected with the Bcl-2 gene had an increased survival rate in the lungs of nude mice and produced a higher number of experimental lung metastases. These data suggest that NO-induced apoptosis in K-1735 melanoma cells is associated with downregulation of Bcl-2.

We studied the influence of 10 synthetic glycoamine analogs on colony formation in 0.3 and 0.9% agarose by metastatic human breast carcinoma (MDA-MB-435) and melanoma (TXM-13) cells. Nine synthetic analogs significantly inhibited the colony formation in 0.9% agarose of MDA-MB-435 human breast carcinoma cells; five compounds caused a 73-83% reduction of colony formation. Seven synthetic glycoamines caused a significant inhibition of colony formation in 0.9% agarose by TXM-13 melanoma cells with the inhibitory effect ranging from 71 to 87%. The 50% inhibition (I50) doses and relative activity rank of the compounds were similar for both breast carcinoma and melanoma cell lines. The murine B16 melanoma cell aggregation assay was employed to elucidate the potential mechanism(s) of the inhibitory activity of synthetic glycoamines. The relative activity ranks of the compounds based on the independently determined I50 doses for both cell aggregation and clonogenic growth assays were very similar for the four most active synthetic analogs and clearly indicated the importance of hydrophobic amino acid in mediating the bioactivity of synthetic glycoamines. In both experimental systems (clonogenic growth in agarose and cell aggregation assay) the leading compound was N-(1-deoxy-D-fructos-1-yl)-D-leucine (Fru-D-Leu) and the least active analog was N-(l-deoxy-D-fructos-1-yl)-glycine (Fru-Gly). These results show that synthetic glycoamines may act by competing for specific carbohydrate-lectin interactions, particularly those involving beta-galactoside-specific lectins expressed on metastatic cells.

The purpose of this study was to determine whether production of liver metastasis by human colon carcinoma (HCC) cells depends on the response of tumor cells to organ-derived growth factors. HCC cells were isolated from several surgical specimens whose malignant potential differed (Dukes' stage B or D tumors), adapted to grow in culture, and assessed for expression of the epidermal growth factor receptor (EGF-R). Northern blot analyses revealed that highly metastatic HCC cells expressed >5-fold the number of EGF-R mRNA transcripts as low metastatic cells. The level of mRNA correlated with the amount of EGF-R protein as detected by Western blotting, immunohistochemistry, and Scatchard analyses. HCC growth response in vitro to picograms of transforming growth factor alpha was associated with functional cell surface EGF-Rs as determined by receptor tyrosine kinase activity assays. The EGF-R gene was not amplified or rearranged in highly metastatic cells. However, fluorescence in situ hybridization analysis showed that the copy number of chromosome 7 was higher in the highly metastatic cells. HCC cells were selected in vitro for low or high expression of EGF-R. Subsequent to injection into nude mice, only cells with high expression of EGF-R produced a high incidence of liver metastasis. These data demonstrate that expression of EGF-R by HCC cells directly correlates with their ability to produce hepatic metastasis.