Metabolic Silencing via Methionine-Based Amino Acid Restriction in Head and Neck Cancer.

In recent years, various forms of caloric restriction (CR) and amino acid or protein restriction (AAR or PR) have shown not only success in preventing age-associated diseases, such as type II diabetes and cardiovascular diseases, but also potential for cancer therapy. These strategies not only reprogram metabolism to low-energy metabolism (LEM), which is disadvantageous for neoplastic cells, but also significantly inhibit proliferation. Head and neck squamous cell carcinoma (HNSCC) is one of the most common tumour types, with over 600,000 new cases diagnosed annually worldwide. With a 5-year survival rate of approximately 55%, the poor prognosis has not improved despite extensive research and new adjuvant therapies. Therefore, for the first time, we analysed the potential of methionine restriction (MetR) in selected HNSCC cell lines. We investigated the influence of MetR on cell proliferation and vitality, the compensation for MetR by homocysteine, the gene regulation of different amino acid transporters, and the influence of cisplatin on cell proliferation in different HNSCC cell lines.

Mass Spectrometric Metabolic Fingerprinting of 2-Deoxy-D-Glucose (2-DG)-Induced Inhibition of Glycolysis and Comparative Analysis of Methionine Restriction versus Glucose Restriction under Perfusion Culture in the Murine L929 Model System.

All forms of restriction, from caloric to amino acid to glucose restriction, have been established in recent years as therapeutic options for various diseases, including cancer. However, usually there is no direct comparison between the different restriction forms. Additionally, many cell culture experiments take place under static conditions. In this work, we used a closed perfusion culture in murine L929 cells over a period of 7 days to compare methionine restriction (MetR) and glucose restriction (LowCarb) in the same system and analysed the metabolome by liquid chromatography mass spectrometry (LC-MS). In addition, we analysed the inhibition of glycolysis by 2-deoxy-D-glucose (2-DG) over a period of 72 h. 2-DG induced very fast a low-energy situation by a reduced glycolysis metabolite flow rate resulting in pyruvate, lactate, and ATP depletion. Under perfusion culture, both MetR and LowCarb were established on the metabolic level. Interestingly, over the period of 7 days, the metabolome of MetR and LowCarb showed more similarities than differences. This leads to the conclusion that the conditioned medium, in addition to the different restriction forms, substantially reprogramm the cells on the metabolic level.

Repurposing of quinoline alkaloids identifies their ability to enhance doxorubicin-induced sub-G0/G1 phase cell cycle arrest and apoptosis in cervical and hepatocellular carcinoma cells.

The ability of quinoline alkaloids (cinchonine, cinchonidine, quinine, and quinidine) to sensitize different human cancer cell lines to doxorubicin (DOX)-induced cell death was evaluated. Cell viability was analyzed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and the alkaloids ability to enhance DOX-induced apoptosis was explored using Western blotting analysis. Also, flow cytometry was applied to analyze cell fractions in the different cell cycle phases. All alkaloids showed a significant enhancement of DOX-induced cell death in HeLa and HepG2 cell lines. The chemosensitizing activity of the quinoline alkaloids was attributed to the induction of apoptosis as indicated by splitting of caspase-3 and its substrate poly (ADP-ribose) polymerase (PARP). In addition, there was an increase in the cell fractions in sub-G0/G1 phase in case of DOX combination with the alkaloids. This study proves the ability of the quinoline alkaloids to enhance DOX-induced apoptotic cell death in human cervical and hepatocellular carcinoma cells.

Metabolic Fingerprinting of Murine L929 Fibroblasts as a Cell-Based Tumour Suppressor Model System for Methionine Restriction.

Since Otto Warburg reported in 1924 that cancer cells address their increased energy requirement through a massive intake of glucose, the cellular energy level has offered a therapeutic anticancer strategy. Methionine restriction (MetR) is one of the most effective approaches for inducing low-energy metabolism (LEM) due to the central position in metabolism of this amino acid. However, no simple in vitro system for the rapid analysis of MetR is currently available, and this study establishes the murine cell line L929 as such a model system. L929 cells react rapidly and efficiently to MetR, and the analysis of more than 150 different metabolites belonging to different classes (amino acids, urea and tricarboxylic acid cycle (TCA) cycles, carbohydrates, etc.) by liquid chromatography/mass spectrometry (LC/MS) defines a metabolic fingerprint and enables the identification of specific metabolites representing normal or MetR conditions. The system facilitates the rapid and efficient testing of potential cancer therapeutic metabolic targets. To date, MS studies of MetR have been performed using organisms and yeast, and the current LC/MS analysis of the intra- and extracellular metabolites in the murine cell line L929 over a period of 5 days thus provides new insights into the effects of MetR at the cellular metabolic level.

Cysteine Restriction in Murine L929 Fibroblasts as an Alternative Strategy to Methionine Restriction in Cancer Therapy.

Methionine restriction (MetR) is an efficient method of amino acid restriction (AR) in cells and organisms that induces low energy metabolism (LEM) similar to caloric restriction (CR). The implementation of MetR as a therapy for cancer or other diseases is not simple since the elimination of a single amino acid in the diet is difficult. However, the in vivo turnover rate of cysteine is usually higher than the rate of intake through food. For this reason, every cell can enzymatically synthesize cysteine from methionine, which enables the use of specific enzymatic inhibitors. In this work, we analysed the potential of cysteine restriction (CysR) in the murine cell line L929. This study determined metabolic fingerprints using mass spectrometry (LC/MS). The profiles were compared with profiles created in an earlier work under MetR. The study was supplemented by proliferation studies using D-amino acid analogues and inhibitors of intracellular cysteine synthesis. CysR showed a proliferation inhibition potential comparable to that of MetR. However, the metabolic footprints differed significantly and showed that CysR does not induce classic LEM at the metabolic level. Nevertheless, CysR offers great potential as an alternative for decisive interventions in general and tumour metabolism at the metabolic level.

HGF-Induced PD-L1 Expression in Head and Neck Cancer: Preclinical and Clinical Findings.

Head and neck squamous cell carcinoma (HNSCC) is a widespread disease with a low survival rate and a high risk of recurrence. Nowadays, immune checkpoint inhibitor (ICI) treatment is approved for HNSCC as a first-line treatment in recurrent and metastatic disease. ICI treatment yields a clear survival benefit, but overall response rates are still unsatisfactory. As shown in different cancer models, hepatocyte growth factor/mesenchymal-epithelial transition (HGF/Met) signaling contributes to an immunosuppressive microenvironment. Therefore, we investigated the relationship between HGF and programmed cell death protein 1 (PD-L1) expression in HNSCC cell lines. The preclinical data show a robust PD-L1 induction upon HGF stimulation. Further analysis revealed that the HGF-mediated upregulation of PD-L1 is MAP kinase-dependent. We then hypothesized that serum levels of HGF and soluble programmed cell death protein 1 (sPD-L1) could be potential markers of ICI treatment failure. Thus, we determined serum levels of these proteins in 20 HNSCC patients before ICI treatment and correlated them with treatment outcomes. Importantly, the clinical data showed a positive correlation of both serum proteins (HGF and sPD-L1) in HNSCC patient's sera. Moreover, the serum concentration of sPD-L1 was significantly higher in ICI non-responsive patients. Our findings indicate a potential role for sPD-L1 as a prognostic marker for ICI treatment in HNSCC.

The Influence of Met Receptor Level on HGF-Induced Glycolytic Reprogramming in Head and Neck Squamous Cell Carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is known to overexpress a variety of receptor tyrosine kinases, such as the HGF receptor Met. Like other malignancies, HNSCC involves a mutual interaction between the tumor cells and surrounding tissues and cells. We hypothesized that activation of HGF/Met signaling in HNSCC influences glucose metabolism and therefore substantially changes the tumor microenvironment. To determine the effect of HGF, we submitted three established HNSCC cell lines to mRNA sequencing. Dynamic changes in glucose metabolism were measured in real time by an extracellular flux analyzer. As expected, the cell lines exhibited different levels of Met and responded differently to HGF stimulation. As confirmed by mRNA sequencing, the level of Met expression was associated with the number of upregulated HGF-dependent genes. Overall, Met stimulation by HGF leads to increased glycolysis, presumably mediated by higher expression of three key enzymes of glycolysis. These effects appear to be stronger in Methigh-expressing HNSCC cells. Collectively, our data support the hypothesized role of HGF/Met signaling in metabolic reprogramming of HNSCC.

The Selection of NFκB Inhibitors to Block Inflammation and Induce Sensitisation to FasL-Induced Apoptosis in HNSCC Cell Lines Is Critical for Their Use as a Prospective Cancer Therapy.

Inflammation is a central aspect of tumour biology and can contribute significantly to both the origination and progression of tumours. The NFκB pathway is one of the most important signal transduction pathways in inflammation and is, therefore, an excellent target for cancer therapy. In this work, we examined the influence of four NFκB inhibitors-Cortisol, MLN4924, QNZ and TPCA1-on proliferation, inflammation and sensitisation to apoptosis mediated by the death ligand FasL in the HNSCC cell lines PCI1, PCI9, PCI13, PCI52 and SCC25 and in the human dermal keratinocyte cell line HaCaT. We found that the selection of the inhibitor is critical to ensure that cells do not respond by inducing counteracting activities in the context of cancer therapy, e.g., the extreme IL-8 induction mediated by MLN4924 or FasL resistance mediated by Cortisol. However, TPCA1 was qualified by this in vitro study as an excellent therapeutic mediator in HNSCC by four positive qualities: (1) proliferation was inhibited at low µM-range concentrations; (2) TNFα-induced IL-8 secretion was blocked; (3) HNSCC cells were sensitized to TNFα-induced cell death; and (4) FasL-mediated apoptosis was not disrupted.

MAGE-A11 expression contributes to cisplatin resistance in head and neck cancer.

OBJECTIVE: The objective of this study is to investigate the roles of melanoma-associated antigens (MAGEs) in the cisplatin treatment of head and neck cancer. MATERIALS AND METHODS: We assessed the efficacy of cisplatin in a set of four head and neck cancer cell lines using a crystal violet assay. The MAGE-A expression in all cell lines was measured with RT-qPCR. The correlation between MAGE-A expression and cisplatin efficacy was investigated using Spearman's correlation analysis. Furthermore, we established a cell line with stable overexpression of MAGE-A11 and determined influence on proliferation, cisplatin efficacy and cell apoptosis. In this cell line, the effects of cisplatin were assessed using either crystal violet assays or flow cytometry (Annexin V). RESULTS: For MAGE-A11, we observed the highest correlation (r = 1.000, p = 0.0417) with low cisplatin efficacy. Stable overexpression of MAGE-A11 resulted in no changes in proliferation, but in lower cisplatin cytotoxicity and lower rates of apoptosis. Also, mouse double minute 2 homolog (MDM2) expression was induced by MAGE-A11 overexpression. CONCLUSION: We provide evidence that MAGE-A11 expression contributes to cisplatin resistance in head and neck cancer. CLINICAL RELEVANCE: Our study underscores the negative predictive role of MAGE-A11 expression in head and neck cancer.

Blocking TWEAK-Fn14 interaction inhibits hematopoietic stem cell transplantation-induced intestinal cell death and reduces GVHD.

Inhibition of the tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK)/fibroblast growth factor-inducible 14 (Fn14) system reduces intestinal cell death and disease development in several models of colitis. In view of the crucial role of TNF and intestinal cell death in graft-versus-host disease (GVHD) and the ability of TWEAK to enhance TNF-induced cell death, we tested here the therapeutic potential of Fn14 blockade on allogeneic hematopoietic cell transplantation (allo-HCT)-induced intestinal GVHD. An Fn14-specific blocking human immunoglobulin G1 antibody variant with compromised antibody-dependent cellular cytotoxicity (ADCC) activity strongly inhibited the severity of murine allo-HCT-induced GVHD. Treatment of the allo-HCT recipients with this monoclonal antibody reduced cell death of gastrointestinal cells but neither affected organ infiltration by donor T cells nor cytokine production. Fn14 blockade also inhibited intestinal cell death in mice challenged with TNF. This suggests that the protective effect of Fn14 blockade in allo-HCT is based on the protection of intestinal cells from TNF-induced apoptosis and not due to immune suppression. Importantly, Fn14 blockade showed no negative effect on graft-versus-leukemia/lymphoma (GVL) activity. Thus, ADCC-defective Fn14-blocking antibodies are not only possible novel GVL effect-sparing therapeutics for the treatment of GVHD but might also be useful for the treatment of other inflammatory bowel diseases where TNF-induced cell death is of relevance.

The prognostic value of GLUT-1 staining in the detection of malignant transformation in oral mucosa.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) ranks as the sixth most common tumor entity worldwide. Unfortunately, the multimodal treatment consisting of surgery, radiation, and chemotherapy does not show the desired efficacy. The intent of this study was to evaluate the sensitivity and specificity of an oral brush biopsy in combination with glucose transporter (GLUT)-1 staining in identifying premalignant and malignant lesions. METHODS: A total of 72 patients were included in the study, divided into four diagnostic subgroups (24 healthy, 15 carcinoma, 18 leukoplakia, 15 oral lichen planus). Oral brush biopsies were taken and analyzed for GLUT-1 expression by immunocytologic staining. Incisional biopsy served as the gold standard. RESULTS: Twelve (80 %) of the 15 carcinomas, nine (50 %) of the 18 leukoplakia, nine (60 %) of the 15 oral lichen planus, and none of the healthy specimens stained positive for GLUT-1. This resulted in a sensitivity rate of 80 % and a specificity rate of 68.42 %. Diagnostic accuracy was 70.83 % based on the correct diagnoses in 51 of 72 patients. CONCLUSION: An oral brush biopsy can easily be performed throughout the entire oral cavity, is noninvasive, and shows high sensitivity and specificity rates with conventional cytology or computer-assisted analysis. CLINICAL RELEVANCE: The significance of GLUT-1-specific staining with an oral brush biopsy is more limited than expected but could be used as an additional tool in detecting malignant transformation in the oral cavity.

Erlotinib and gefitinib responsiveness in head and neck cancer cell lines--a comparing analysis with cetuximab.

OBJECTIVE: The objective of this study is to examine the efficacy of erlotinib and gefitinib with respect to epidermal growth factor (EGF) and cetuximab response in head and neck cancer cell lines. MATERIALS AND METHODS: Five human head and neck carcinoma cell lines were treated with EGF, cetuximab, erlotinib, and gefitinib, and the effects were measured with a crystal violet assay. The efficacies of cetuximab, erlotinib, and gefitinib in clinically relevant concentrations were statistically analyzed. The expression of the epidermal growth factor receptor (EGFR) and phosphorylation patterns were detected with fluorescence-activated cell sorting (FACS) analysis and western blot analysis. The endogenous production of EGF by the cells was detected with an enzyme-linked immunosorbent assay. Finally, EGFR, KRAS, BRAF, and PI3K mutation analyses were performed. RESULTS: All of the cell lines had a poor or no response to EGF but exhibited distinct EGFR phosphorylation and EGFR expression. Compared to cetuximab, erlotinib and gefitinib demonstrated a greater impact on the majority of the cell lines. The only cell line that showed a concentration-dependent behavior toward EGF and strong EGFR phosphorylation was entirely resistant to cetuximab, erlotinib, and gefitinib. The production of EGF in all cell lines was very low. Mutational analysis of all cell lines revealed wild-type EGFR, KRAS, BRAF, and PI3K. CONCLUSIONS: The prediction of anti-EGFR treatment cannot be based on responsiveness to EGF or EGFR activation. CLINICAL RELEVANCE: Erlotinib and gefitinib show good response in EGF-independent cell lines and might be useful drugs in tumors that are less responsive to cetuximab.

Cytotoxic effects of SMAC-mimetic compound LCL161 in head and neck cancer cell lines.

OBJECTIVES: Head and neck squamous cell carcinoma (HNSCC) is one of the most common tumor entities worldwide. Unfortunately, recent drug developments in other fields of oncology have yielded no efficacy in the treatment of oral squamous cell carcinoma. As a new starting point, we investigated the impact of Fas ligand (FasL) and the SMAC-mimetic compound LCL161 in mono- and combination treatment in HNSCC cell lines. METHODS: Five different cell lines of HNSCC were treated with FasL and LCL161 in mono- and combination treatment. Cytotoxicity was measured via a crystal violet assay. The cell lines were characterized for CD95 (FasL receptor) expression via flow cytometry. The degradation of cellular inhibitor of apoptosis protein 1 (cIAP1) was detected via Western blot. RESULTS: Incubation with FasL led to a significant decrease in three out of five cell lines. Combination treatment with LCL161 enhanced cytotoxicity significantly. Two cell lines were FasL resistant, but one of them could be resensitized with LCL161. In all cell lines, Western blot analysis showed degradation of cIAP1 after LCL161 application. However, one cell line showed only minor vulnerability to the FasL and LCL161 combination. CONCLUSION: This is the first study investigating combination treatment of FasL and LCL161 in head and neck cancer cell lines. Pro-apoptotic effects of the combination were detected in the majority of the cell lines. Interestingly, one of two FasL-resistant cell lines was sensitive to the combination therapy with FasL and LCL161. CLINICAL RELEVANCE: SMAC-mimetic compounds show promising results in the treatment of other tumor entities in vitro and might be useful drugs to improve HNSCC therapy.

Fibroblast growth factor inducible (Fn14)-specific antibodies concomitantly display signaling pathway-specific agonistic and antagonistic activity.

BACKGROUND: Fn14 is a therapeutic target in various diseases. RESULTS: Anti-Fn14 antibodies activate the alternative NFκB pathway but not other Fn14-related activities induced by soluble or membrane-bound TWEAK. FcγR-bound anti-Fn14 antibodies, however, activate the full spectrum of Fn14-associated activities. CONCLUSION: Anti-Fn14 antibodies elicit agonistic activities differing from those of the natural Fn14 ligand TWEAK. SIGNIFICANCE: These findings influence the rationale of designing Fn14-targeted therapies. The Fn14-specific monoclonal antibodies PDL192 and P4A8, which are under consideration in clinical trials, showed no agonistic activity with respect to IL8 production and cell death induction. However, oligomerization with protein G or binding to Fcγ receptors converted both anti-Fn14 antibodies into potent agonists. TNF-like weak inducer of apoptosis (TWEAK), the ligand of Fn14, occurs naturally in two forms with partly different signaling capabilities, as a membrane-bound ligand and as a soluble trimeric molecule. Although membrane TWEAK strongly triggers all Fn14-associated pathways, soluble TWEAK predominately triggers the alternative nuclear factor κB (NFκB) pathway and enhances TNF-induced cell death but has only a poor effect on the classical NFκB pathway and chemokine production. Thus, the oligomerized and FcγR-bound anti-Fn14 mAbs mimicked the activity of membrane TWEAK. Notably, both anti-Fn14 antibodies significantly triggered p100 processing, the hallmark of the alternative NFκB pathway, and therefore resembled soluble TWEAK. In contrast to the latter, however, the anti-Fn14s showed no effect on TNF receptor 1-induced cell death and P4A8 even blocked the corresponding TWEAK response. Thus, we showed that Fn14 antibodies display an alternative NFκB pathway-specific agonistic activity but fail to phenocopy other activities of soluble TWEAK, whereas oligomerized or FcγR-bound Fn14 antibodies fully mimic the activity of membrane TWEAK. In view of the trivalent nature of the TWEAK-Fn14 interaction, this suggests that the alternative NFκB pathway is uniquely responsive already to Fn14 dimerization enabling antibodies to elicit an unnatural response pattern distinct from that of the naturally occurring Fn14 ligands.

Studies of binding of tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) to fibroblast growth factor inducible 14 (Fn14).

To perform highly sensitive cellular binding studies with TNF-like weak inducer of apoptosis (TWEAK), we developed a bioluminescent variant of soluble TWEAK (GpL-FLAG-TNC-TWEAK) by fusing it genetically to the C terminus of the luciferase of Gaussia princeps (GpL). Equilibrium binding studies on human (HT1080 and HT29) and murine (Renca and B16) cell lines at 37 °C revealed high affinities of human TWEAK from 53 to 112 pm. The dissociation rate constant of the TWEAK-Fn14 interaction was between 0.48×10(-3) s(-1) (HT29) and 0.58×10(-3) s(-1) (HT1080) for the human molecules, and the association rate constant obtained was 3.3×10(6) m(-1) s(-1) for both cell lines. It has been shown previously that oligomerization of soluble TWEAK trimers results in enhanced Fn14-mediated activation of the classical NFκB pathway. Binding studies with GpL-FLAG-TNC-TWEAK trimers oligomerized by help of a FLAG tag-specific antibody gave no evidence for a major increase in Fn14 occupancy by oligomerized ligand despite strongly enhanced induction of the NFκB target IL8. Thus, aggregated complexes of soluble TWEAK and Fn14 have a higher intrinsic activity to stimulate the classical NFκB pathway and qualitatively differ from isolated trimeric TWEAK-Fn14 complexes. Furthermore, determination of IL8 induction as a function of occupied activated receptors revealed that the intrinsic capability of TNFR1 to stimulate the classical NFκB pathway and IL8 production was ∼100-fold higher than Fn14. Thus, although ∼25 activated TNFR1 trimers were sufficient to trigger half-maximal IL8 production, more than 2500 cell-bound oligomerized TWEAK trimers were required to elicit a similar response.

Crystal structure analysis reveals a spring-loaded latch as molecular mechanism for GDF-5-type I receptor specificity.

Dysregulation of growth and differentiation factor 5 (GDF-5) signalling, a member of the TGF-beta superfamily, is strongly linked to skeletal malformation. GDF-5-mediated signal transduction involves both BMP type I receptors, BMPR-IA and BMPR-IB. However, mutations in either GDF-5 or BMPR-IB lead to similar phenotypes, indicating that in chondrogenesis GDF-5 signalling seems to be exclusively mediated through BMPR-IB. Here, we present structural insights into the GDF-5:BMPR-IB complex revealing how binding specificity for BMPR-IB is generated on a molecular level. In BMPR-IB, a loop within the ligand-binding epitope functions similar to a latch allowing high-affinity binding of GDF-5. In BMPR-IA, this latch is in a closed conformation leading to steric repulsion. The new structural data now provide also a molecular basis of how phenotypically relevant missense mutations in GDF-5 might impair receptor binding and activation.

Soluble and transmembrane TNF-like weak inducer of apoptosis differentially activate the classical and noncanonical NF-kappa B pathway.

TNF-like weak inducer of apoptosis, TWEAK, is a typical member of the TNF ligand family. Thus, it is initially expressed as a type II transmembrane protein from which a soluble variant can be released by proteolytic processing. In this study, we show that membrane TWEAK is superior to soluble variant of TWEAK (sTWEAK) with respect to the activation of the classical NF-kappaB pathway, whereas both TWEAK variants are potent inducers of TNFR-associated factor-2 depletion, NF-kappaB-inducing kinase accumulation and p100 processing, hallmarks of activation of the noncanonical NF-kappaB pathway. Like other soluble TNF ligands with a poor capability to activate their corresponding receptor, sTWEAK acquires an activity resembling those of the transmembrane ligand by oligomerization or cell surface-immobilization. Blockade of the Fn14 receptor inhibited NF-kappaB signaling irrespective of the TWEAK form used for stimulation, indicating that the differential activities of the two TWEAK variants on classical and noncanonical NF-kappaB signaling is not related to the use of different receptors.

Low Energy Status under Methionine Restriction Is Essentially Independent of Proliferation or Cell Contact Inhibition.

Nonlimited proliferation is one of the most striking features of neoplastic cells. The basis of cell division is the sufficient presence of mass (amino acids) and energy (ATP and NADH). A sophisticated intracellular network permanently measures the mass and energy levels. Thus, in vivo restrictions in the form of amino acid, protein, or caloric restrictions strongly affect absolute lifespan and age-associated diseases such as cancer. The induction of permanent low energy metabolism (LEM) is essential in this process. The murine cell line L929 responds to methionine restriction (MetR) for a short time period with LEM at the metabolic level defined by a characteristic fingerprint consisting of the molecules acetoacetate, creatine, spermidine, GSSG, UDP-glucose, pantothenate, and ATP. Here, we used mass spectrometry (LC/MS) to investigate the influence of proliferation and contact inhibition on the energy status of cells. Interestingly, the energy status was essentially independent of proliferation or contact inhibition. LC/MS analyses showed that in full medium, the cells maintain active and energetic metabolism for optional proliferation. In contrast, MetR induced LEM independently of proliferation or contact inhibition. These results are important for cell behaviour under MetR and for the optional application of restrictions in cancer therapy.

Gene expression profiling of connective tissue growth factor (CTGF) stimulated primary human tenon fibroblasts reveals an inflammatory and wound healing response in vitro.

PURPOSE: The biologic relevance of human connective tissue growth factor (hCTGF) for primary human tenon fibroblasts (HTFs) was investigated by RNA expression profiling using affymetrix(TM) oligonucleotide array technology to identify genes that are regulated by hCTGF. METHODS: Recombinant hCTGF was expressed in HEK293T cells and purified by affinity and gel chromatography. Specificity and biologic activity of hCTGF was confirmed by biosensor interaction analysis and proliferation assays. For RNA expression profiling HTFs were stimulated with hCTGF for 48h and analyzed using affymetrix(TM) oligonucleotide array technology. Results were validated by real time RT-PCR. RESULTS: hCTGF induces various groups of genes responsible for a wound healing and inflammatory response in HTFs. A new subset of CTGF inducible inflammatory genes was discovered (e.g., chemokine [C-X-C motif] ligand 1 [CXCL1], chemokine [C-X-C motif] ligand 6 [CXCL6], interleukin 6 [IL6], and interleukin 8 [IL8]). We also identified genes that can transmit the known biologic functions initiated by CTGF such as proliferation and extracellular matrix remodelling. Of special interest is a group of genes, e.g., osteoglycin (OGN) and osteomodulin (OMD), which are known to play a key role in osteoblast biology. CONCLUSIONS: This study specifies the important role of hCTGF for primary tenon fibroblast function. The RNA expression profile yields new insights into the relevance of hCTGF in influencing biologic processes like wound healing, inflammation, proliferation, and extracellular matrix remodelling in vitro via transcriptional regulation of specific genes. The results suggest that CTGF potentially acts as a modulating factor in inflammatory and wound healing response in fibroblasts of the human eye.

Receptor oligomerization and beyond: a case study in bone morphogenetic proteins.

BACKGROUND: Transforming growth factor (TGF)beta superfamily members transduce signals by oligomerizing two classes of serine/threonine kinase receptors, termed type I and type II. In contrast to the large number of ligands only seven type I and five type II receptors have been identified in mammals, implicating a prominent promiscuity in ligand-receptor interaction. Since a given ligand can usually interact with more than one receptor of either subtype, differences in binding affinities and specificities are likely important for the generation of distinct ligand-receptor complexes with different signaling properties. RESULTS: In vitro interaction analyses showed two different prototypes of binding kinetics, 'slow on/slow off' and 'fast on/fast off'. Surprisingly, the binding specificity of ligands to the receptors of one subtype is only moderate. As suggested from the dimeric nature of the ligands, binding to immobilized receptors shows avidity due to cooperative binding caused by bivalent ligand-receptor interactions. To compare these in vitro observations to the situation in vivo, binding studies on whole cells employing homodimeric as well as heterodimeric bone morphogenetic protein 2 (BMP2) mutants were performed. Interestingly, low and high affinity binding sites were identified, as defined by the presence of either one or two BMP receptor (BMPR)-IA receptor chains, respectively. Both sites contribute to different cellular responses in that the high affinity sites allow a rapid transient response at low ligand concentrations whereas the low affinity sites facilitate sustained signaling but higher ligand concentrations are required. CONCLUSION: Binding of a ligand to a single high affinity receptor chain functioning as anchoring molecule and providing sufficient complex stability allows the subsequent formation of signaling competent complexes. Another receptor of the same subtype, and up to two receptors of the other subtype, can then be recruited. Thus, the resulting receptor arrangement can principally consist of four different receptors, which is consistent with our interaction analysis showing low ligand-receptor specificity within one subtype class. For BMP2, further complexity is added by the fact that heterooligomeric signaling complexes containing only one type I receptor chain can also be found. This indicates that despite prominent ligand receptor promiscuity a manifold of diverse signals might be generated in this receptor limited system.