The metabolic and mitogenic properties of basal insulin analogues.

CONTEXT: Retrospective, observational studies have reported an association between diabetes treatment with insulin and a higher incidence of cancer. OBJECTIVE: Overview the literature for in vitro and in vivo studies of the metabolic and mitogenic properties of basal insulin analogues and assess the implications for clinical use. METHODS: Relevant studies were identified through PubMed and congress abstract database searches; data on metabolic and mitogenic signalling in relation to insulin treatment of diabetes are included in this review. RESULTS: The balance of evidence shows that although some analogues have demonstrated mitogenic potency in some in vitro studies in cancer cell lines, these findings do not translate to the in vivo setting in animals or to the clinical setting in humans. CONCLUSIONS: The current consensus is that there is no clinical or in vivo evidence to indicate that any commercially available insulin analogue has carcinogenic effects. Large-scale, prospective clinical and observational studies will further establish any potential link.

Impact of bone harvesting techniques on cell viability and the release of growth factors of autografts.

BACKGROUND: Autogenous bone grafts obtained by different harvesting techniques behave differently during the process of graft consolidation; the underlying reasons are however not fully understood. One theory is that harvesting techniques have an impact on the number and activity of the transplanted cells which contribute to the process of graft consolidation. MATERIALS AND METHODS: To test this assumption, porcine bone grafts were harvested with four different surgical procedures: bone mill, piezosurgery, bone drilling (bone slurry), and bone scraper. After determining cell viability, the release of molecules affecting bone formation and resorption was assessed by reverse transcription polymerase chain reaction and immunoassay. The mitogenic and osteogenic activity of the conditioned media was evaluated in a bioassay with isolated bone cells. RESULTS: Cell viability and the release of molecules affecting bone formation were higher in samples harvested by bone mill and bone scraper when compared with samples prepared by bone drilling and piezosurgery. The harvesting procedure also affected gene expression, for example, bone mill and bone scraper samples revealed significantly higher expression of growth factors such as bone morphogenetic protein-2 and vascular endothelial growth factor compared with the two other modalities. Receptor activator of nuclear factor kappa B ligand expression was lowest in bone scraper samples. CONCLUSION: These data can provide a scientific basis to better understand the impact of harvesting techniques on the number and activity of transplanted cells, which might contribute to the therapeutic outcome of the augmentation procedure.

Immunomodulatory effects of dietary exposure to selected polycyclic aromatic hydrocarbons in the bluegill (Lepomis macrochirus).

Polycyclic aromatic hydrocarbons (PAH) have been demonstrated to affect immune system modulation. The freshwater species of fish, Lepomis macrochirus (bluegill), was employed to investigate the effects of a 14-day dietary exposure to PAH including 2-aminoanthracene (2-AA), 2-methylnaphthalene (2-MN), and 9,10-dimethylanthracene (9,10-DMA) and a mixture of these 3 compounds at a total dose of 3.1 ± 0.01 mg on lymphocyte proliferation stimulated with 3 mitogens (concanavalin A [Con A], phorbol ester, and calcium ionophore). 2-Aminoanthracene was mitogenic itself and with added mitogens. 2-Methylnaphthalene induced some stimulatory and some inhibitory effects upon cell proliferation by Con A. 9,10-DMA and the mixture each suppressed cell proliferation. The mixture was highly suppressive to lymphocytes. Intracellular baseline calcium levels were reduced, possibly as a step prior to cell death. All PAH compounds tested were immunomodulatory to bluegill lymphocytes. Bluegill were demonstrated to have utility as a biomarker species for investigation of immunotoxicity.

El neuron-derived orphan receptor-1 previene la apoptosis inducida por la hipoxia en las celulas endoteliales vasculares / NOR-1 prevents vascular endothelial cell apoptosis induced by hypoxia

Background NOR-1 is a nuclear receptor that is over expressed in human atherosclerotic plaques. This receptor is involved in endothelial cell (EC) growth induced by vascular endothelial growth factor (VEGF) and other mitogens. Our aim was to analyze NOR-1 regulation by hypoxia in EC. Results Hypoxia increased NOR-1 mRNA levels in a time- and dose-dependent manner. Hypoxia-induced NOR-1 expression was prevented by BAPTA-AM (a calcium chelator) but not by inhibitors of p38 MAPK, MEK1/2 (..) (AU)

Background NOR-1 is a nuclear receptor that is over expressed in human atherosclerotic plaques. This receptor is involved in endothelial cell (EC) growth induced by vascular endothelial growth factor (VEGF) and other mitogens. Our aim was to analyze NOR-1 regulation by hypoxia in EC. Results Hypoxia increased NOR-1 mRNA levels in a time- and dose-dependent manner. Hypoxia-induced NOR-1 expression was prevented by BAPTA-AM (a calcium chelator) but not by inhibitors of p38 MAPK, MEK1/2 or protein kinase C (PKC) pathways. Incubation of EC with blocking antibodies against VEGF or SU5614 (a VEGF-R2 inhibitor) did not prevent hypoxia-induced NOR-1 expression. Hypoxia did not significantly induce early activation of cAMP response element binding protein (CREB), a key transcription factor involved in the up-regulation of NOR-1 expression by VEGF. In contrast, reduction of hypoxia-induced HIF-1¦Á protein levels by inhibitors of the PI3K/Akt/m TOR pathway or by siRNA against HIF-1, significantly counteracted hypoxia-induced NOR-1 up-regulation. In transient transfection assays, the activity of the NOR-1 promoter construct was increased by hypoxia or by co-transfection with an HIF-1¦Á expression plasmid. We identified a hypoxia response element in the NOR-1 promoter responsible for this hypoxia-mediated effect as shown by site-directed mutagenesis and serial deletions. Finally, over-expression of NOR-1 (using a bicistronic recombinant plasmid) decreased the rate of cells undergoing apoptosis (annexin V positive/propidium iodide negative), while inhibition of NOR-1 (using specific siRNA) increased cell apoptosis. Conclusions Therefore, HIF-1 modulates the expression of NOR-1, a transcription factor that regulates the survival response of EC to hypoxia (..) (AU)

Antecedentes El neuron-derived orphan receptor-1 (NOR-1) es un receptor nuclear que esta sobreexpresado en las lesiones ateroscleraticas humanas y que participa en la proliferacion de las celulas endoteliales (CE) en respuesta al factor de crecimiento del endotelial vascular (VEGF) y a otros (..) (AU)

Enhanced stability of recombinant keratinocyte growth factor by mutagenesis.

Native sequence keratinocyte growth factor (KGF) is fairly unstable, as manifested by the loss of the monomeric native protein accompanied by the accumulation of aggregated species during storage at moderate temperatures. Several different types of analogs were generated and the storage stability of the protein assessed. In the first type of analog one or more of the five cysteinyl residues in KGF were replaced; in the second class the N-terminal residues that included the first disulfide bond were deleted. Both of these types of analogs involved removal of the disulfide bond between cysteines 1 and 15. The third group involved mutating one of the basic amino acids located in a cluster of positive charges (involved in heparin binding) around Arg144 to a neutral or acidic amino acyl residue. Among the cysteine replacement analogs, the double mutation of Cys1 and 15 to Ser resulted in significantly increased stability without compromising the mitogenic activity, while Cys to Ser mutations at other positions were either destabilizing or had no effect. Deletion of the 15, 23 or 27 N-terminal amino acyl residues also increased the stability of the protein. The activity of the analogs was not affected by the deletion of 15 or 23 amino acids, but it was significantly decreased upon removal of the 27 N-terminal amino acyl residues. Much greater stability was achieved by mutation of the basic amino acids, especially Arg144, to Glu or Gln, but this increase in stability was accompanied by large decrease in activity. The analog with the 23 N-terminal amino acyl residues deleted represents one of the best compromises between increased stability and retention of activity.

Rat hepatoproliferin revealed the status of a complete hepatomitogen in human hepatoma cells.

Hepatoproliferin (HPF), a liver regeneration factor isolated from rat hepatocytes, was assessed for its mitogenic status in the human hepatoma cell line PLC/PRF-5. HPF was able to enhance hepatoma cell growth on its own without the aid of the established complete mitogens EGF and TGF-alpha or the hepato-priming factor TNF-alpha. HPF therefore acted as a complete hepatomitogen and had no co-mitogenic properties since it did not augment proliferation when combined with EGF or TGF-alpha but showed only an additive effect in the presence of TGF-alpha. Rat HPF was phylogenetically unrestricted, because it was found active in human cells. When each of the established growth factors (GFs) was used alone, the hepatoma cells responded with the same kind of response profile, namely a bi-phasic bell-shaped dose-dependent response due to stimulation at low levels and inhibition at higher levels. However, hepatocyte growth factor (HGF) was an exception since it did not induce a growth response in hepatoma cells. On the contrary HPF, on its own, showed a progressive enhanced linear dose response at the levels used for the GFs (ie 1.0-15 ng/5 x 10(5) cells). The comparative potency (CP) (dpm x 10(3)/microg DNA/ng GF) of HPF (CP = 13) was in the same range as for the complete hepatomitogens EGF (CP = 12) and TGF-alpha (CP = 14), revealing that HPF has indeed the status of a complete mitogen.

NOV (CCN3) regulation in the growth plate and CCN family member expression in cartilage neoplasia.

Growth plate chondrocytes undergo a coordinated differentiation process resulting in terminal differentiation and new bone formation. Enchondromas are pre-malignant, benign cartilaginous lesions that arise from growth plate chondrocytes that fail to undergo terminal differentiation. NOV (nephroblastoma overexpressed) is a member of the CCN family of proteins, which share a common multi-modular organization. While the role of NOV in chondrocyte development and cartilage neoplasia is not known, other CCN family members play a role in chondrocyte differentiation, or are differentially regulated in cartilage neoplasia. In embryonic murine growth plates, NOV was expressed in pre-hypertrophic and early hypertrophic chondrocytes. PTHrP treatment (which inhibits terminal differentiation) decreased NOV expression in murine femurs maintained in organ culture, and decreased the activity of a NOV reporter construct in vitro. Expression of the CCN family members NOV, CTGF, CYR61, and WISP-1 was examined in 15 chondrosarcomas of various grades and in three enchondromas. Expression of all of the family members was lower in the higher-grade tumours. As identification of the grade of cartilage neoplasia can sometimes be difficult using histology alone, the level of expression of CCN family members could be a useful adjunct in the determination of tumour grade.

Peptide fragments of AMP-18, a novel secreted gastric antrum mucosal protein, are mitogenic and motogenic.

Antrum mucosal protein (AMP)-18 is a novel 18-kDa protein synthesized by cells of the gastric antrum mucosa. The protein is present in secretion granules of murine gastric antrum epithelial cells and is a component of canine antrum mucus, suggesting that it is secreted into the viscoelastic gel layer on the mucosal surface. Release of the protein appears to be regulated because forskolin decreased the amount of immunoreactive AMP-18 in primary cultures of canine antrum mucosal epithelial cells, and indomethacin gavaged into the stomach of mice reduced AMP-18 content in antrum mucosal tissue before inducing histological injury. A functional domain of the protein was identified by preparing peptides derived from the center of human AMP-18. A 21-mer peptide stimulated growth of gastric and intestinal epithelial cells, but not fibroblasts, and increased restitution of scrape-wounded gastric epithelial monolayers. These functions of AMP-18 suggest that its release onto the apical cell surface is regulated and that the protein and/or peptide fragments may protect the antral mucosa and promote healing by facilitating restitution and proliferation after injury.

Pleiotrophin, an embryonic differentiation and growth factor, is expressed in osteoarthritis.

OBJECTIVE: Pleiotrophin (PTN) is a 15.3 kDa heparin-binding peptide, which is expressed in mesodermal and neuroectodermal cells during development, but rarely in adult tissues. In fetal or juvenile cartilage, PTN is an abundant protein and appears to be involved in chondrocyte differentiation. Since developmentally regulated factors often re-appear in the disease state, we examined PTN expression in cartilage and synovial fluid of patients with osteoarthritis (OA). METHODS: PTN mRNA and protein expression was assayed by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot, the protein was localized by immunohistochemistry and quantified by enzyme-linked immunoassay (ELISA). RESULTS: PTN was undetectable in normal adult cartilage, but PTN mRNA and protein were found in OA. In cartilage from the tibial plateaus of OA patients, PTN could be immunostained in clusters of superficial chondrocytes. In the synovial fluids of OA patients, PTN concentrations were elevated in earlier OA stages, but rarely in late OA stages. Chondrosarcomas were PTN-immunonegative. CONCLUSIONS: In addition to certain types of cancer, the embryonic growth and differentiation factor PTN is found also in adults in inflammatory diseases. In OA, PTN is especially expressed in early stages, and PTN concentrations in the synovial fluid could serve as a marker for the progress of the disease. PTN might be involved in cartilage repair in OA, in particular, in earlier stages.

An intramyocardial catheter for repeated in vivo sampling of interstitial fluid.

INTRODUCTION: Coronary collateral development is an important adaptive response to chronic myocardial ischemia. Characterization of mitogenic factors responsible for collateral formation has been an elusive goal because these substances are difficult to sample from the myocardial interstitium at multiple times. We report the implantation of an exchange catheter capable of in vivo sampling of myocardial interstitial fluid in chronically instrumented dogs. METHODS: The catheter consisted of multiple perforations within a 2-cm segment of Micro-Renathane tubing that was implanted into the left ventricular myocardium between the left anterior descending (LAD) and left circumflex coronary artery (LCCA) perfusion territories and secured to the epicardium with a Silastic disk. Dogs (n=5) underwent brief (2 min) LAD occlusions once per hour, 8 times/day, 7 days/week for 2 weeks to stimulate coronary collateral growth. Another group of dogs (n=6) without repetitive coronary occlusions served as controls. Myocardial interstitial fluid was collected daily, and mitogenic activity was evaluated by the proliferative responses of growth-arrested, cultured vascular smooth muscle and endothelial cells. RESULTS: All dogs tolerated catheter implantation without complication. Each catheter functioned well throughout the duration of the experiment. Myocardial interstitial fluid obtained using the exchange catheter in this model of repetitive coronary occlusion produced marked proliferation of vascular smooth muscle and endothelial cells in vitro. DISCUSSION: The exchange catheter enables chronic in vivo sampling of myocardial interstitial fluid and may facilitate identification of mitogens involved in coronary collateral development.

Amphiregulin is a mitogen for adult neural stem cells.

Neurons are continuously generated from stem cells in the hippocampus and along the lateral ventricles in the adult brain. Neural stem cells can be propagated in vitro in the presence of epidermal growth factor (EGF) or fibroblast growth factor-2. We report here that amphiregulin, a growth factor related to EGF, is a mitogen for adult mouse neural stem cells in vitro and displays potency similar to that of EGF. Neural stem cell cultures can be initiated and the cells propagated as efficiently in the presence of amphiregulin only as with EGF. Furthermore, we show that amphiregulin is expressed in the choroid plexus of the ventricular system and in the hippocampus in the adult brain, suggesting that amphiregulin may participate in the regulation of neural stem cell proliferation and neurogenesis in the adult brain.

A single cell density-sensing factor stimulates distinct signal transduction pathways through two different receptors.

In Dictyostelium discoideum, cell density is monitored by levels of a secreted protein, conditioned medium factor (CMF). CMFR1 is a putative CMF receptor necessary for CMF-induced G protein-independent accumulation of the SP70 prespore protein but not for CMF-induced G protein-dependent inositol 1,4,5-trisphosphate production. Using recombinant fragments of CMF, we find that stimulation of the inositol 1,4,5-trisphosphate pathway requires amino acids 170-180, whereas SP70 accumulation does not, corroborating a two-receptor model. Cells lacking CMFR1 do not aggregate, due to the lack of expression of several important early developmentally regulated genes, including gp80. Although many aspects of early developmental cAMP-stimulated signal transduction are mediated by CMF, CMFR1 is not essential for cAMP-stimulated cAMP and cGMP production or Ca(2+) uptake, suggesting the involvement of a second CMF receptor. Exogenous application of antibodies against either the region between a first and second or a second and third possible transmembrane domain of CMFR1 induces SP70 accumulation. Antibody- and CMF-induced gene expression can be inhibited by recombinant CMFR1 corresponding to the region between the first and third potential transmembrane domains, indicating that this region is extracellular and probably contains the CMF binding site. These observations support a model where a one- or two-transmembrane CMFR1 regulates gene expression and a G protein-coupled CMF receptor mediates cAR1 signal transduction.

SSeCKS immunolabeling in rat primary sensory neurons.

SSeCKS (src suppressed C kinase substrate) is a protein kinase C substrate that may play a role in tumor suppression. Recently described in fibroblasts, testes and mesangial cells, SSeCKS may have a function in the control of cell signaling and cytoskeletal arrangement. To investigate the distribution of SSeCKS throughout the nervous system, representative sections of brain, spinal cord and dorsal root ganglia were processed using immunofluorescence. Labeling of central axonal collaterals of primary sensory neurons was observed in the dorsal horn at all spinal levels. SSeCKS-immunoreactivity was also observed in the cerebellum, medulla and sensory ganglia (including trigeminal ganglia). The pattern and distribution of anti-SSeCKS labeling in dorsal root ganglia and the dorsal horn of the spinal cord was similar to that observed for other markers of small primary sensory neurons. Therefore, the coexistence of SSeCKS with substance P, CGRP and acid phosphatase was examined in sections of sensory ganglia, spinal cord and medulla using double immunofluorescent labeling for SSeCKS and substance P/CGRP or sequential SSeCKS immunofluorescence and acid phosphatase/fluoride-resistant acid phosphatase enzyme histochemistry. A small portion of the SSeCKS-labeled cell bodies appeared to represent a subpopulation of substance P (4.8%) and CGRP (4.7%) containing neurons, while 45.0% contained fluoride-resistant acid phosphatase reactivity. These results indicate that SSeCKS has a restricted distribution within the nervous system and that expression of this protein may reflect the specific signaling requirements of a distinct population of nociceptive sensory neurons.

Connective tissue growth factor in drug-induced gingival overgrowth.

BACKGROUND: Drug-induced gingival overgrowth is a known side effect of certain chemotherapeutic agents used for the treatment of systemic disorders. The pathogenesis and mechanisms responsible for this condition are not fully understood. This study assesses for the presence and localization of connective tissue growth factor (CTGF) in drug-induced gingival overgrowth tissues. CTGF immunostaining was compared with sections stained with transforming growth factor (TGF)-beta1 and CD31 antibodies in order to investigate possible pathogenic mechanisms. METHODS: Gingival overgrowth samples were obtained from patients undergoing therapy with phenytoin (n = 9), nifedipine (n = 4), cyclosporin A (n = 5), and control tissues from systemically healthy donors (n = 9). Tissue sections were subjected to peroxidase immunohistochemistry and were stained with CTGF and TGF-beta1 polyclonal primary antibodies. Possible relationships between CTGF staining and angiogenesis were also studied using an anti-CD31 antibody as a marker for endothelial cells. Staining was analyzed by computer-assisted quantitative and semiquantitative methodology at 5 defined sites in all samples based on the location of specific landmarks including epithelium and underlying connective tissues. RESULTS: Cellular and extracellular CTGF content in phenytoin gingival overgrowth tissues was significantly (P<0.05) higher compared to the other gingival overgrowth tissues and the controls. Higher CTGF staining in phenytoin gingival overgrowth tissues was accompanied by an increased abundance of fibroblasts and connective tissue fibers. No strong association of CTGF staining with TGF-beta1 or CD31 staining was found. CONCLUSIONS: The data from the present study show significantly higher CTGF staining in phenytoin-induced gingival overgrowth tissues compared to controls, cyclosporin A-, or nifedipine-induced gingival overgrowth. Moreover, semiquantitative analyses of histologic samples support the concept that the phenytoin overgrowth tissues are fibrotic. These associations suggest a possible role for CTGF in promoting development of fibrotic lesions in phenytoin-induced gingival overgrowth.

Mitogenic effects of neutrophins on a periodontal ligament cell line.

Nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) are three representative neurotrophins responsible for the differentiation and survival of neurons, and their high-affinity receptors are tropomyosin-receptor-kinase (TRK)A, TRKB, and TRKC, respectively. In this study, we investigated the expression of neurotrophins in a mouse periodontal ligament cell line (MPL), by reverse transcription-polymerase chain-reaction (RT-PCR) and enzyme-linked immunoabsorbent assay (ELISA). We also studied the expression of TRK receptors on MPL by immunostaining and the effects of neurotrophins on the proliferation of MPL, with a hypothesis of autocrine mechanism of neurotrophins. Each neurotrophin and TRK receptor was expressed, and neurotrophins enhanced the proliferation of MPL. These findings suggest that the MPL has functional neurotrophin receptors involved in an autocrine function of neurotrophins. The expression level of neurotrophins and TRKs showed the reverse pattern, and we propose an auto-regulatory mechanism of ligands and receptors in accordance with the level of synthesized neurotrophins.

Immunocytochemical localization of NOVH protein and ultrastructural characteristics of NCI-H295R cells.

In this study we present biochemical and immunocytochemical results on NOVH protein secretion and localization in NCI-H295R cells, as well as results on the ultrastructural characteristics of NCI-H295R cells. NCI-H295R cells were characterized by small quantities of rough and smooth endoplasmic reticulum, many free ribosomes, large nuclei with prominent nucleoli, numerous elongated mitochondria, a few Golgi complexes, and a small number of lipid droplets. Large numbers of coated pits and coated vesicles were present, but no secretory granules or exocytotic profiles were seen. Best ultrastructural preservation of NCI-H295R cells was achieved when fixation was done directly on the culture dishes and the cells were detached by scraping. Our biochemical results showed that NCI-H295R cells secreted large amounts of NOVH protein. The immunocytochemical localization of NOVH protein showed that the protein was localized in the cytoplasm, the plasma membrane and the nuclear envelope. This localization pattern, along with the ultrastructural and biochemical findings raise interesting questions on the function(s) and the mode of secretion of NOVH protein.

Localization of connective tissue growth factor in human uterine tissues.

Connective tissue growth factor (CTGF) is a recently described heparin-binding mitogen for fibroblasts and smooth muscle cells. The aim of this study was to investigate the production of CTGF by human uterine tissues using immunohistochemical and Northern blotting analyses. For immunohistochemistry, formalin-fixed human proliferative (n = 5), early secretory (n = 5; days 15-19), mid-secretory (n = 5; days 20-23), late secretory (n = 5; days 24-28) endometrial, and decidual (n = 5) tissues were stained using a highly specific affinity-purified polyclonal antibody raised against residues 81-94 of human CTGF. Myometrial (n = 5) and leiomyoma (n = 5) tissues were also used for CTGF immunochemistry. In proliferative endometrium, epithelial and vascular endothelial cells showed strong CTGF immunoreactivity, whereas stromal cells were negative or only weakly positive for the CTGF protein. Throughout the entire secretory stage, CTGF was detected in epithelial and endothelial cells of endometrium. Stromal cells showed strong immunoreactivity to CTGF only in oedematous areas for early and mid-secretory endometrium, and in decidualized regions of late secretory endometrium. During pregnancy, the decidual, epithelial and endothelial cells of the endometrium were all immunoreactive to CTGF. In myometrial and leiomyoma samples, CTGF immunoreactivity was found only in the endothelial cells. Northern blotting of mRNA from normal uterus (n = 2) or leiomyoma (n = 6) using a 320 bp human CTGF cDNA probe revealed a single 2.4 kb transcript. This study is the first to demonstrate CTGF gene expression and localization of its encoded protein in human uterine tissues. The cell- and cycle-specific localization of CTGF support a role for this molecule in regulating aspects of uterine cell growth, migration, and/or matrix production during the menstrual cycle and pregnancy.

Expression of connective tissue growth factor in cartilaginous tumors.

BACKGROUND: Connective tissue growth factor (CTGF) predominantly is expressed in hypertrophic chondrocytes and its specific receptors are demonstrated on chondrocytic cells. Therefore, CTGF may be involved in the proliferation and/or differentiation of cartilage cells. In the current study, CTGF expression was examined both in chondrosarcoma and enchondroma to clarify the relation between the expression of CTGF and the grade of malignancy. METHODS: The expression of CTGF and proliferating cell nuclear antigen (PCNA) were analyzed immunohistochemically in 34 cartilaginous tumor specimens. Eighteen tumors were determined to be chondrosarcoma including 8 Grade 1 tumors, 6 Grade 2 tumors, and 4 Grade 3 tumors. The percentage of CTGF positive and PCNA positive cells was quantified using at least 500 cells. RESULTS: CTGF was expressed in 70.1% of enchondroma cells, 84.0% of Grade 1 chondrosarcoma cells, 53.7% of Grade 2 tumor cells, and 26.8% of Grade 3 tumor cells (rho = -0.501; P = 0.0053). In chondrosarcoma cases, CTGF expression was correlated closely with tumor grade (rho = -0.920; P = 0.0001). There was a strong correlation between PCNA expression and tumor grade (rho = 0.907; P < 0.0001) and a strong negative correlation between CTGF and PCNA expression (rho = -0.493; P = 0.0061). In chondrosarcoma cases, patients with high expression of CTGF (>/= 30%) showed higher overall survival compared with those with low expression (< 30%) (P = 0.004). CONCLUSIONS: The current study revealed a correlation between the histologic grade of chondrosarcoma and prognosis, and the concomitant association between CTGF immunostaining and tumor grade and prognosis. Therefore, immunohistochemical staining with CTGF is a useful procedure for assessing the tumor grade and clinical course in patients with chondrosarcoma.