A comprehensive model for the proliferation-quiescence decision in response to endogenous DNA damage in human cells.

Human cells that suffer mild DNA damage can enter a reversible state of growth arrest known as quiescence. This decision to temporarily exit the cell cycle is essential to prevent the propagation of mutations, and most cancer cells harbor defects in the underlying control system. Here we present a mechanistic mathematical model to study the proliferation-quiescence decision in nontransformed human cells. We show that two bistable switches, the restriction point (RP) and the G1/S transition, mediate this decision by integrating DNA damage and mitogen signals. In particular, our data suggest that the cyclin-dependent kinase inhibitor p21 (Cip1/Waf1), which is expressed in response to DNA damage, promotes quiescence by blocking positive feedback loops that facilitate G1 progression downstream of serum stimulation. Intriguingly, cells exploit bistability in the RP to convert graded p21 and mitogen signals into an all-or-nothing cell-cycle response. The same mechanism creates a window of opportunity where G1 cells that have passed the RP can revert to quiescence if exposed to DNA damage. We present experimental evidence that cells gradually lose this ability to revert to quiescence as they progress through G1 and that the onset of rapid p21 degradation at the G1/S transition prevents this response altogether, insulating S phase from mild, endogenous DNA damage. Thus, two bistable switches conspire in the early cell cycle to provide both sensitivity and robustness to external stimuli.

Nutrient sensing, growth and senescence.

Cell growth is dictated by a wide range of mitogenic signals, the amplitude and relative contribution of which vary throughout development, differentiation and in a tissue-specific manner. The ability to sense and appropriately respond to changes in mitogens is fundamental to control cell growth, and reduced responsiveness of nutrient sensing pathways is widely associated with human disease and ageing. Cellular senescence is an important tumour suppressor mechanism that is characterised by an irreversible exit from the cell cycle in response to replicative exhaustion or excessive DNA damage. Despite the fact that senescent cells can no longer divide, they remain metabolically active and display a range of pro-growth phenotypes that are supported in part by the mTORC1-autophagy signalling axis. As our understanding of the basic mechanisms of controlling mTORC1-autophagy activity and cell growth continues to expand, we are able to explore how changes in nutrient sensing contribute to the acquisition and maintenance of cellular senescence. Furthermore, while the protective effect of senescence to limit cellular transformation is clear, more recently, the age-related accumulation of these pro-inflammatory senescent cells has been shown to contribute to a decline in organismal fitness. We will further discuss whether dysregulation of nutrient sensing pathways can be targeted to promote senescent cell death which would have important implications for healthy ageing.

Uncoupling the Mitogenic and Metabolic Functions of FGF1 by Tuning FGF1-FGF Receptor Dimer Stability.

The recent discovery of metabolic roles for fibroblast growth factor 1 (FGF1) in glucose homeostasis has expanded the functions of this classically known mitogen. To dissect the molecular basis for this functional pleiotropy, we engineered an FGF1 partial agonist carrying triple mutations (FGF1ΔHBS) that diminished its ability to induce heparan sulfate (HS)-assisted FGF receptor (FGFR) dimerization and activation. FGF1ΔHBS exhibited a severely reduced proliferative potential, while preserving the full metabolic activity of wild-type FGF1 in vitro and in vivo. Hence, suboptimal FGFR activation by a weak FGF1-FGFR dimer is sufficient to evoke a metabolic response, whereas full FGFR activation by stable and sustained dimerization is required to elicit a mitogenic response. In addition to providing a physical basis for the diverse activities of FGF1, our findings will impact ongoing drug discoveries targeting FGF1 and related FGFs for the treatment of a variety of human diseases.

Mitogen-induced B-cell proliferation activates Chk2-dependent G1/S cell cycle arrest.

B-cell activation and proliferation can be induced by a variety of extracellular stimuli. The fate of an activated B cell following mitogen stimulation can be dictated by the strength or duration of the signal, the expression of downstream signaling components necessary to promote proliferation, and the cell intrinsic sensors and regulators of the proliferative program. Previously we have identified the DNA damage response (DDR) signaling pathway as a cell intrinsic sensor that is activated upon latent infection of primary human B cells by Epstein-Barr virus (EBV). Here we have assessed the role of the DDR as a limiting factor in the proliferative response to non-viral B-cell mitogens. We report that TLR9 activation through CpG-rich oligonucleotides induced B-cell hyper-proliferation and an ATM/Chk2 downstream signaling pathway. However, B-cell activation through the CD40 pathway coupled with interleukin-4 (IL-4) promoted proliferation less robustly and only a modest DDR. These two mitogens, but not EBV, modestly induced intrinsic apoptosis that was independent from the DDR. However, all three mitogens triggered a DDR-dependent G1/S phase cell cycle arrest preventing B-cell proliferation. The extent of G1/S arrest, as evidenced by release through Chk2 inhibition, correlated with B-cell proliferation rates. These findings have implications for the regulation of extra-follicular B-cell activation as it may pertain to the development of auto-immune diseases or lymphoma.

Pro- and anti-mitogenic actions of pituitary adenylate cyclase-activating polypeptide in developing cerebral cortex: potential mediation by developmental switch of PAC1 receptor mRNA isoforms.

During corticogenesis, pituitary adenylate cyclase-activating polypeptide (PACAP; ADCYAP1) may contribute to proliferation control by activating PAC1 receptors of neural precursors in the embryonic ventricular zone. PAC1 receptors, specifically the hop and short isoforms, couple differentially to and activate distinct pathways that produce pro- or anti-mitogenic actions. Previously, we found that PACAP was an anti-mitogenic signal from embryonic day 13.5 (E13.5) onward both in culture and in vivo and activated cAMP signaling through the short isoform. However, we now find that mice deficient in PACAP exhibited a decrease in the BrdU labeling index (LI) in E9.5 cortex, suggesting that PACAP normally promotes proliferation at this stage. To further define mechanisms, we established a novel culture model in which the viability of very early cortical precursors (E9.5 mouse and E10.5 rat) could be maintained. At this stage, we found that PACAP evoked intracellular calcium fluxes and increased phospho-PKC levels, as well as stimulated G1 cyclin mRNAs and proteins, S-phase entry, and proliferation without affecting cell survival. Significantly, expression of hop receptor isoform was 24-fold greater than the short isoform at E10.5, a ratio that was reversed at E14.5 when short expression was 15-fold greater and PACAP inhibited mitogenesis. Enhanced hop isoform expression, elicited by in vitro treatment of E10.5 precursors with retinoic acid, correlated with sustained pro-mitogenic action of PACAP beyond the developmental switch. Conversely, depletion of hop receptor using short-hairpin RNA abolished PACAP mitogenic stimulation at E10.5. These observations suggest that PACAP elicits temporally specific effects on cortical proliferation via developmentally regulated expression of specific receptor isoforms.

Oxidized LDL enhances stretch-induced smooth muscle cell proliferation through alterations in nuclear protein import.

Mechanical stress contributes to hypertension and atherosclerosis partly through the stimulation of vascular smooth muscle cell (VSMC) proliferation. Oxidized low density lipoprotein (oxLDL) is another important atherogenic factor that can increase VSMC proliferation. The purpose of this study was to investigate whether oxLDL could further enhance the proliferative action of mechanical stretch on VSMC, and to determine the mechanism responsible for this interaction. Because nuclear protein import is critical in regulating gene expression, transcription, and cell proliferation, its involvement in the mitogenic effects of oxLDL and mechanical stress was studied. OxLDL enhanced the proliferative effects of mechanical stretch on its own in rabbit aortic VSMC, and induced increases in the expression of HSP60 in an additive manner. Adenoviral-mediated overexpression of HSP60 induced increases in cell proliferation compared with uninfected VSMC. Mechanical stretch and oxLDL stimulated the rate of nuclear protein import in VSMC and increased the expression of nucleoporins. These effects were sensitive to inhibition of the MAPK pathway. We conclude that oxLDL and mechanical stretch have a synergistic effect on VSMC proliferation. This synergistic effect is induced through a stimulation of nuclear protein import via HSP60 and an activation of the MAPK pathway.

Mitotic phosphorylation activates hepatoma-derived growth factor as a mitogen.

BACKGROUND: Hepatoma-derived growth factor (HDGF) is a nuclear protein that is a mitogen for a wide variety of cells. Mass spectrometry based methods have identified HDGF as a phosphoprotein without validation or a functional consequence of this post-translational modification. RESULTS: We found that HDGF in primary mouse aortic vascular smooth muscle cells (VSMC) was phosphorylated. Wild type HDGF was phosphorylated in asynchronous cells and substitution of S103, S165 and S202 to alanine each demonstrated a decrease in HDGF phosphorylation. A phospho-S103 HDGF specific antibody was developed and demonstrated mitosis-specific phosphorylation. HDGF-S103A was not mitogenic and FACS analysis demonstrated a G2/M arrest in HDGF-S103A expressing cells, whereas cells expressing HDGF-S103D showed cell cycle progression. Nocodazole arrest increased S103 phosphorylation from 1.6% to 29% (P = 0.037). CONCLUSIONS: Thus, HDGF is a phosphoprotein and phosphorylation of S103 is mitosis related and required for its function as a mitogen. We speculate that cell cycle regulated phosphorylation of HDGF may play an important role in vascular cell proliferation.

Quantitative assessment of AKAP12 promoter methylation in human prostate cancer using methylation-sensitive high-resolution melting: correlation with Gleason score.

OBJECTIVES: To quantitatively investigate the A kinase anchoring protein 12 (AKAP12) gene promoter methylation and its association with clinicopathologic variables in human prostate cancer (PCa). The AKAP12 gene has shown reduced expression and marked hypermethylation in a variety of cancers. METHODS: The percentage levels of DNA methylation were measured in 78 PCa, 22 benign prostatic hyperplasia, and 22 normal adjacent tissue samples using an AKAP12 methylation-sensitive high-resolution melting assay. AKAP12 gene expression was also examined in 4 human prostate carcinoma cell lines, PC-3, DU145, LNCaP, and 22RV1, using quantitative reverse transcriptase-polymerase chain reaction and methylation-sensitive high-resolution melting analysis and after DNA methyltransferase inhibition with 5-aza-2'-deoxycytidine. RESULTS: Methylation (>1%) of the AKAP12 promoter region was present in 47 (60.2%) of the 78 PCa, 5 (22.7%) of the 22 benign prostatic hyperplasia, and 2 (9.1%) of the 22 adjacent normal tissue samples. AKAP12 methylation was significantly greater in the PCa than in the benign prostatic hyperplasia or adjacent tissue samples (P < .01). AKAP12 methylation was significantly greater in the PCa samples with higher Gleason scores (P = .03); however, no correlation was found with age, pT category, or serum prostate-specific antigen level. Reverse transcriptase-polymerase chain reaction demonstrated that PC-3 and DU-145 cells expressed AKAP12 RNA and LNCaP and 22RV1 did not. The AKAP12 locus was methylated in the LNCaP and 22RV1 cells. Treatment of LNCaP cells with 5-aza-2'-deoxycytidine markedly decreased the methylation levels and increased the expression of AKAP12. CONCLUSIONS: The results of the present study have demonstrated that AKAP12 promoter methylation is a frequent event in human PCa. AKAP12 methylation represents a potential molecular biomarker for predicting the malignancy of PCa.

Hypoxia-induced EMAP-II transcription in colorectal cancer.

Endothelial monocyte-activating polypeptide-II (p431EMAP-II) is a proinflammatory cytokine and a chemoattractant for mononuclear phagocytes and polymorphonuclear leucocytes, found in culture supernatants of many tumour cell lines. It was demonstrated that p43/EMAP-II induces apoptosis in mitogen-stimulated lymphocytes, and suggested that it may be a constituent of a novel immune evasion mechanism employed by tumour cells. Quantitative real-time reverse transcription- polymerase chain reaction (qRT-PCR) analysis for EMAP-II mRNA was performed for colorectal adenocarcinoma cell lines, DLD-1, HT 29; human umbilical vein endothelial cells (HUVEC); and normal colon under normal and hypoxic conditions. Under hypoxic conditions, EMAP-II transcript expression increased up to 22-fold over normoxia in tumour cells, while there was 1-fold increase due to hypoxia in HUVEC and no increase in normal colon. These results demonstrate that EMAP-II transcripts are upregulated in tumour cells in hypoxic conditions and support the notion that EMAP-II plays a complex and important role in human cancer.

Fractalkine has anti-apoptotic and proliferative effects on human vascular smooth muscle cells via epidermal growth factor receptor signalling.

AIMS: Fractalkine (CX3CL1) is a membrane-bound chemokine that signals through the G protein-coupled receptor CX3CR1 that is implicated in the development of atherosclerosis. We have previously reported that CX3CR1 is expressed by primary human coronary artery smooth muscle cells (CASMC), where it mediates chemotaxis towards CX3CL1. We sought to determine the effect of CX3CL1 on CASMC survival and proliferation and elucidate the signalling mechanisms involved. METHODS AND RESULTS: CX3CL1 significantly reduces staurosporine-induced apoptosis of CASMC, as quantified by caspase 3 immunostaining and Annexin-V flow cytometry. Furthermore, CX3CL1 is a potent mitogen for primary CASMC and induces phosphorylation of extracellular signal-regulated kinase (ERK) and Akt, measured by western blotting. Inhibition of either ERK or phosphoinositide 3-kinase (PI3K) signalling abrogates proliferation, while only PI3K signalling is involved in the anti-apoptotic effects of CX3CL1. We describe a novel and specific small molecule antagonist of CX3CR1 (AZ12201182) which abrogates the mitogenic and anti-apoptotic effects of CX3CL1 on CASMC. Pharmacological inhibition of the epidermal growth factor receptor (EGFR) blocks CASMC survival and DNA synthesis, indicating a previously undocumented role for EGFR signalling in response to CX3CL1 involving release of a soluble EGFR ligand. Specifically, CX3CL1 induces shedding of epiregulin and increases epiregulin mRNA expression 20-fold within 2 h. Finally, antibody neutralization of epiregulin abrogates the mitogenic effect of CX3CL1. CONCLUSION: We have demonstrated two novel and important functions of CX3CL1 on primary human SMCs: anti-apoptosis and proliferation, both mediated via epiregulin-induced EGFR signalling. Our data have important implications in vascular pathologies including atherosclerosis, restenosis, and transplant accelerated arteriosclerosis, where the balance of SMC proliferation and apoptosis critically determines both plaque stability and vessel stenosis.

Mitogenic effects of cytokines on smooth muscle are critically dependent on protein kinase A and are unmasked by steroids and cyclooxygenase inhibitors.

Excessive smooth muscle growth occurs within the context of inflammation associated with certain vascular and airway diseases. The inflammatory cytokines interleukin (IL)-1beta and tumor necrosis factor-alpha (TNF-alpha) have been shown previously to inhibit mitogen-stimulated smooth muscle growth through a mechanism presumed to be dependent on the induction of cyclooxygenase-2, prostaglandins, and activation of the cAMP-dependent protein kinase (PKA). Using both molecular and pharmacological strategies, we demonstrate that the mitogenic effects of IL-1beta and TNF-alpha on cultured human airway smooth muscle (ASM) cells are tightly regulated by PKA activity. Suppression of induced PKA activity by either corticosteroids or cyclooxygenase inhibitors converts the cytokines from inhibitors to enhancers of mitogen-stimulated ASM growth, and biological variability in the capacity to activate PKA influences the modulatory effect of cytokines. Promitogenic effects of IL-1beta are associated with delayed increases in p42/p44 and phosphoinositide-3 kinase activities, suggesting a role for induced autocrine factors. These findings suggest a mechanism by which mainstream therapies such as corticosteroids or cyclooxygenase inhibitors could fail to address or exacerbate the pathogenic smooth muscle growth that occurs in obstructive airway and cardiovascular diseases.

Relevance of plant lectins in human cell biology and immunology.

Protein-carbohydrate interactions are used for intercellular communication. Mammalian cells are known to bear a variety of glycoconjugates. Lectins, first discovered in plants, are proteins which can specifically bind carbohydrates. Given the high affinity of plant lectins for carbohydrates, they have always been important as molecular tools in the identification, purification and stimulation of specific glycoproteins on human cells. Lectins have provided important clues to the repertoire of carbohydrate structures in animal cells. The discovery of plant lectins gave a great impulse to modern glycobiology. They represent important biochemical reagents for numerous applications in the biomedical field and in research. Sequence determinations and structural characterization helped to understand the mechanism of action in many biologic systems. Plant lectins have been fundamental in human immunological studies because some of them are mitogenic/activating to lymphocytes. Understanding the molecular basis of lectin-carbohydrate interactions and of the intracellular signalling evoked holds promise for the design of novel drugs for the treatment of infectious, inflammatory and malignant diseases. It may also be of help for the structural and functional investigation of glycoconjugates and their changes during physiological and pathological processes.

An agglutinin with mitogenic and antiproliferative activities from the mushroom Flammulina velutipes.

A hemagglutinin with a molecular mass of 12 kDa was isolated from the fruiting bodies of the mushroom Flammulina velutipes. Its molecular mass is similar to that of the fungal immunomodulatory protein isolated from F. velutipes (FIP-fve) with ice-cold 5% acetic acid and 50 mM 2-mercaptoethanol as extraction medium and to that of the larger 12 kDa subunit of F. velutipes lectin isolated with phosphate buffer as extraction medium. Its hemagglutinating activity cannot be inhibited by a variety of carbohydrates tested. The activity is stable between pH 4 and pH 11. Loss in activity occurred when the temperature is raised to 60 C and 70 C. Activity is indiscernible at and above 80 C. Its N-terminal sequence shows differences from that of FIP-fve. F. velutipes hemagglutinin stimulates [3H-methyl] thymidine uptake by mouse splenocytes. It inhibits proliferation of leukemia L1210 cells with an IC50 of 13 microM.

The mitogenic function of hepatitis B virus X protein resides within amino acids 51 to 140 and is modulated by N- and C-terminal regulatory regions.

The hepatitis B virus (HBV) X protein (pX) is implicated in hepatocarcinogenesis by an unknown mechanism. pX variants encoded by HBV genomes found integrated in genomic DNA from liver tumors of patients with hepatocellular carcinoma (HCC) generally lack amino acids 134 to 154. Since deregulation of mitogenic pathways is linked to oncogenic transformation, herein we define the pX region required for mitogenic pathway activation. A series of pX deletions was used to construct tetracycline-regulated pX-expressing cell lines. The activation of the mitogenic pathways by these pX deletions expressed in the constructed cell lines was measured by transient transreporter assays, effects on endogenous cyclin A expression, and apoptosis. Conditional expression of pX51-140 in AML12 clone 4 cell line activates the mitogenic pathways, induces endogenous cyclin A expression, and sensitizes cells to apoptosis, similar to wild-type (WT) pX. By contrast, pX1-115 is inactive, supporting the idea that amino acids 116 to 140 are required for mitogenic pathway activation. Moreover, this pX deletion analysis demonstrates that WT pX function is modulated by two regions spanning amino acids 1 to 78 and 141 to 154. The N-terminal X1-78, expressed via a retroviral vector in WT pX-expressing 4pX-1 cells, coimmunoprecipitates with WT pX, indicating this pX region participates in protein-protein interactions leading to pX oligomerization. Interestingly, pX1-78 interferes with WT pX in mediating mitogenic pathway activation, endogenous gene expression, and apoptosis. The C-terminal pX region spanning amino acids 141 to 154 decreases pX stability, determined by pulse-chase studies of WT pX and pX1-140, suggesting that increased stability of naturally occurring pX variants lacking amino acids 134 to 154 may play a role in HCC development.

The aryl hydrocarbon receptor directly regulates expression of the potent mitogen epiregulin.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is known to cause a large number of adverse effects, mediated largely by its binding to the aryl-hydrocarbon receptor (AhR) and subsequent modulation of gene expression. It is thought that AhR mediates these effects through the untimely and disproportionate expression of specific genes. However, the exact mechanism, or the genes involved, through which TCDD leads to these effects is still unknown. This study reports the discovery of a novel target gene, epiregulin, which is regulated by TCDD-activated AhR. Epiregulin is a growth regulator which belongs to the epidermal growth factor (EGF) family. Using real time quantitative PCR (qPCR), it was established that TCDD upregulates epiregulin gene expression. The promoter region of epiregulin has a dioxin responsive element (DRE) 56 nucleotides upstream of the transcription start site, along with three potential Sp1 binding sites. Chromatin immunoprecipitation (ChIP) assays with an anti-AhR antibody showed promoter occupancy upon TCDD treatment. Luciferase reporter assays using a vector harboring the first 125 base pairs of the epiregulin rat promoter revealed an increase in signal on TCDD treatment, which was lost upon mutation of the DRE. Epiregulin and TCDD treatment mediated a dose-dependent increase in primary mouse keratinocyte growth. These results demonstrate that AhR directly increases epiregulin expression, which could play an important role in TCDD mediated tumor promotion observed in rodent models.

Generation of inhibitory DNA aptamers against human hepatocyte growth factor.

Hepatocyte growth factor (HGF), a multifunctional cytokine, can act on many cell types. It is involved in cancer growth and metastasis by enhancing the motility of cancer cells and stimulating angiogenesis. The development of effective inhibitors for HGF is an important issue in cancer therapy. In this study, we isolated DNA aptamers against human HGF using the systematic evolution of ligands by exponential enrichment method. The selected DNA aptamers had a highly conserved consensus sequence, and could be divided into two major classes (classes I and II). The consensus motif of classes I and II might contribute to the formation of a hairpin loop structure and a G-quartet structure, respectively. These DNA aptamers bound to human HGF with high affinity and specificity. The dissociation constants of typical aptamers H38-15 and H38-21, representative of the two classes, were calculated to be approximately 20 nM. H38-15 and H38-21 inhibited the biological activities of HGF including the stimulation of scattering, migration, and invasion of pancreatic cancer KP-3 cells. Furthermore, both aptamers inhibited HGF-induced tube formation by human umbilical vein endothelial cells. These results suggested that the isolated DNA aptamers will be useful as therapeutic and diagnostic reagents for cancers.

High-affinity CD25-binding IL-2 mutants potently stimulate persistent T cell growth.

We have used directed evolution to construct IL-2 mutants that bind the IL-2 alpha receptor subunit (IL-2Ralpha, CD25) with affinities comparable to that of the IL-15-IL-15 alpha receptor subunit (IL-15Ralpha) interaction. T cells proliferate for up to 6 days following a 30 minute incubation with these IL-2 mutants, which may lead to potential applications for cancer and viral immunotherapy. Several alternative mechanisms have been proposed to explain the contrasting effects of IL-2 and IL-15 on T cell proliferation and death. These IL-2 mutants exhibit T cell growth response-receptor occupancy curves indistinguishable from that for IL-15, suggesting that much of the difference between wild-type IL-2 and IL-15 effects arises simply from their 1000-fold differing affinities for their private alpha receptor subunits.

Prokineticins (endocrine gland-derived vascular endothelial growth factor and BV8) in the bovine ovary: expression and role as mitogens and survival factors for corpus luteum-derived endothelial cells.

A highly vascular endocrine gland, the corpus luteum (CL) is an excellent model for the study of angiogenic factors. Prokineticins (PK-1 and -2), also termed endocrine-gland-derived vascular endothelial growth factor (VEGF) and BV8 are newly identified proteins described as selective angiogenic mitogens. We previously identified PK binding sites, two closely homologous G protein-coupled receptors (PK-R1 and PK-R2) in human and bovine ovarian cells, but their function remained unknown. In this study we examined the presence and effects of PK in CL-derived endothelial and steroidogenic cell types (LEC and LSC, respectively). PK-1 mRNA was identified in CL and follicles by real-time PCR, using primers specific for the bovine PK-1 sequence (retrieved from Bos taurus whole genome shotgun database). PK were potent angiogenic mitogens for LEC; they enhanced cell proliferation, elevated [3H]thymidine incorporation, MAPK activation, and c-jun/fos mRNA expression. The effects of PK proteins on cell survival were examined by nuclear morphology (4',6-diamidino-2-phenylindole dihydrochloride staining), measurement of DNA fragmentation (terminal dUTP nucleotide end labeling assay), and caspase-3 cleavage. Results obtained by these techniques demonstrated that PK protected LEC from serum starvation-induced apoptosis. Stress conditions such as serum withdrawal, TNF-alpha, and hypoxia markedly increased PK-R2 expression, whereas mRNA levels of PK-R1 remained unchanged. These suggest that the antiapoptotic effect of PK-1 on LEC may be mediated via PK-R2. PK-1 increased VEGF mRNA expression by LSC, implying that it could also indirectly, via VEGF, affect luteal angiogenesis. Together, these findings suggest an important role for PK-1 in luteal function by acting as a mitogen and survival factor in LEC.

Nonviral HVJ (hemagglutinating virus of Japan) liposome-mediated retrograde gene transfer of human hepatocyte growth factor into rat nervous system promotes functional and histological recovery of the crushed nerve.

Hepatocyte growth factor (HGF) is well known to be involved in many biological functions, such as organ regeneration and angiogenesis, and to exert neurotrophic effects on motor, sensory, and parasympathetic neurons. In this study, we gave repeated intramuscular injections of the human HGF gene, using nonviral HVJ (hemagglutinating virus of Japan) liposome method, to examine whether transfection of the rat nervous system with this gene is able to exert neurotrophic effects facilitating recovery of a crushed nerve. The expression of HGF protein and HGF mRNA indicated that gene transfer into the nervous system did occur via retrograde axonal transport. At 4 weeks after crush, electrophysiological examination of the crushed nerve showed a significantly shorter mean latency and a significantly greater mean maximum M-wave amplitude with repeated injections of HGF gene. Furthermore, histological findings showed that the mean diameter of the axons, the axon number and the axon population were significantly larger in the group with repeated injections of HGF gene. The above results show that repeated human HGF gene transfer into the rat nervous system is able to promote crushed-nerve recovery, both electrophysiologically and histologically, and suggest that HGF gene transfer has potential for the treatment of crushed nerve.

Suppression of metastasis of human pancreatic cancer to the liver by transportal injection of recombinant adenoviral NK4 in nude mice.

NK4, a 4-kringle fragment of hepatocyte growth factor (HGF), is an HGF antagonist that also acts as an angiogenesis inhibitor. NK4 strongly inhibits the infiltration, metastasis, and tumor growth of pancreatic cancer. The aim of our study was to evaluate the antitumor effect of adenovirus-mediated NK4 gene transfer to the liver on hepatic metastasis of pancreatic cancer in vivo. We constructed recombinant adenoviral NK4 (Ad-NK4), which encodes a secreted form of human NK4. Intrasplenic injection of Ad-NK4 induced high and relatively maintained expression of NK4 protein in the liver and suppressed the number and growth of metastatic foci in the liver in a nude mouse model. Microscopically, central necrosis was found even in small metastatic foci in Ad-NK4 treated mice. Immunohistochemical analysis of metastatic tumors showed a remarkable decrease in microvessel density and an increase in the number of apoptotic tumor cells after treatment with Ad-NK4. These results indicate that intraportal injection of Ad-NK4 may be a useful therapeutic modality for the clinical control of hepatic metastasis in pancreatic cancer.