Aptamers targeting a tumor-associated extracellular matrix component: The human mature collagen XIα1.

The extracellular matrix (ECM) plays an essential role in tumor progression and invasion through its continuous remodeling. The growth of most carcinomas is associated with an excessive collagen deposition that provides the proper environment for tumor development and chemoresistance. The α1 chain of a minor human collagen, type XI, is overexpressed in some tumor stroma, but not found in normal stroma. To test the clinical utility of this collagen as a cancer biomarker, specific receptors are needed. Available antibodies do not show enough selectivity or are directed toward the propeptide region that is cleaved when the protein is released to the ECM. Here we show the selection of an aptamer for the specific C-telopeptide region using a 16-mer peptide as the target for the SELEX. The aptamer selected with a Kd of ∼25 nM was able to capture the collagen XI from cell lysates. It was also used for target detection in a mixed antibody-aptamer sandwich assay showing it can be useful for diagnostic purposes in biological fluids.

Collagen, type XI, alpha 1: an accurate marker for differential diagnosis of breast carcinoma invasiveness in core needle biopsies.

Accurate diagnosis of invasive breast lesions, when analyzed by Core Needle Biopsy, may suppose a major challenge for the pathologist. Various markers of invasiveness such as laminin, S-100 protein, P63 or calponin have been described; however, none of them is completely reliable. The use of a specific marker of the infiltrating tumor microenvironment seems vital to support the diagnosis of invasive against in situ lesions. At this point, Collagen, type XI, alpha 1 (COL11A1), might be helpful since it has been described to be associated to cancer associated fibroblasts in other tumors such as lung, pancreas or colorectal. This paper aims to analyze the role of COL11A1 as a marker of invasiveness in breast tumor lesions. Two hundred and one breast Core Needle Biopsy samples were analyzed by immunohistochemistry against pro-COL11A1. The results show a significant difference (p < 0.0001) when comparing the expression in infiltrative tumors (93%) versus immunostaining of non-invasive lesions (4%). Forty cases of underestimated DCIS were also stained for COL11A1, presenting a sensitivity of 90% when compared with p63 and calponin which not tagged invasion. In conclusion, pro-COL11A1 expression is a promising marker of invasive breast lesions, and may be included in immunohistochemical panels aiming at identifying infiltration in problematic breast lesions.

The effect of cyclic mechanical strain on the expression of adhesion-related genes by periodontal ligament cells in two-dimensional culture.

BACKGROUND AND OBJECTIVE: Cell adhesion plays important roles in maintaining the structural integrity of connective tissues and sensing changes in the biomechanical environment of cells. The objective of the present investigation was to extend our understanding of the effect of cyclic mechanical strain on the expression of adhesion-related genes by human periodontal ligament cells. MATERIAL AND METHODS: Cultured periodontal ligament cells were subjected to a cyclic in-plane tensile deformation of 12% for 5 s (0.2 Hz) every 90 s for 6-24 h in a Flexercell FX-4000 Strain Unit. The following parameters were measured: (i) cell viability by the MTT assay; (ii) caspase-3 and -7 activity; and (iii) the expression of 84 genes encoding adhesion-related molecules using real-time RT-PCR microarrays. RESULTS: Mechanical stress reduced the metabolic activity of deformed cells at 6 h, and caspase-3 and -7 activity at 6 and 12 h. Seventy-three genes were detected at critical threshold values < 35. Fifteen showed a significant change in relative expression: five cell adhesion molecules (ICAM1, ITGA3, ITGA6, ITGA8 and NCAM1), three collagen α-chains (COL6A1, COL8A1 and COL11A1), four MMPs (ADAMTS1, MMP8, MMP11 and MMP15), plus CTGF, SPP1 and VTN. Four genes were upregulated (ADAMTS1, CTGF, ICAM1 and SPP1) and 11 downregulated, with the range extending from a 1.76-fold induction of SPP1 at 12 h to a 2.49-fold downregulation of COL11A1 at 24 h. CONCLUSION: The study has identified several mechanoresponsive adhesion-related genes, and shown that onset of mechanical stress was followed by a transient reduction in overall cellular activity, including the expression of two apoptosis 'executioner' caspases.

Proteomic analysis of Col11a1-associated protein complexes.

Cartilage plays an essential role during skeletal development within the growth plate and in articular joint function. Interactions between the collagen fibrils and other extracellular matrix molecules maintain structural integrity of cartilage, orchestrate complex dynamic events during embryonic development, and help to regulate fibrillogenesis. To increase our understanding of these events, affinity chromatography and liquid chromatography/tandem mass spectrometry were used to identify proteins that interact with the collagen fibril surface via the amino terminal domain of collagen α1(XI) a protein domain that is displayed at the surface of heterotypic collagen fibrils of cartilage. Proteins extracted from fetal bovine cartilage using homogenization in high ionic strength buffer were selected based on affinity for the amino terminal noncollagenous domain of collagen α1(XI). MS was used to determine the amino acid sequence of tryptic fragments for protein identification. Extracellular matrix molecules and cellular proteins that were identified as interacting with the amino terminal domain of collagen α1(XI) directly or indirectly, included proteoglycans, collagens, and matricellular molecules, some of which also play a role in fibrillogenesis, while others are known to function in the maintenance of tissue integrity. Characterization of these molecular interactions will provide a more thorough understanding of how the extracellular matrix molecules of cartilage interact and what role collagen XI plays in the process of fibrillogenesis and maintenance of tissue integrity. Such information will aid tissue engineering and cartilage regeneration efforts to treat cartilage tissue damage and degeneration.

Differential gene expression in the perichondrium and cartilage of the neonatal mouse temporomandibular joint.

Our goal was to discover genes differentially expressed in the perichondrium (PC) of the mandibular condylar cartilage (MCC) that might enhance regenerative medicine or orthopaedic therapies directed at the tissues of the temporomandibular joint. We used targeted gene arrays (osteogenesis, stem cell) to identify genes preferentially expressed in the PC and the cartilaginous (C) portions of the MCC in 2-day-old mice. Genes with higher expression in the PC sample related to growth factor ligand-receptor interactions [FGF-13 (6.4x), FGF-18 (4x), NCAM (2x); PGDF receptors, transforming growth factor (TGF)-beta and IGF-1], the Notch isoforms (especially Notch 3 and 4) and their ligands or structural proteins/proteoglycans [collagen XIV (21x), collagen XVIII (4x), decorin (2.5x)]. Genes with higher expression in the C sample consisted mostly of known cartilage-specific genes [aggrecan (11x), procollagens X (33x), XI (14x), IX (4.5x), Sox 9 (4.4x) and Indian hedgehog (6.7x)]. However, the functional or structural roles of several genes that were expressed at higher levels in the PC sample are unclear [myogenic factor (Myf) 9 (9x), tooth-related genes such as tuftelin (2.5x) and dentin sialophosphoprotein (1.6x), VEGF-B (2x) and its receptors (3-4x) and sclerostin (1.7x)]. FGF, Notch and TGF-beta signalling may be important regulators of MCC proliferation and differentiation; the relatively high expression of genes such as Myf6 and VEGF-B and its receptors suggests a degree of unsuspected plasticity in PC cells.

Early-onset osteoarthritis due to otospondylomegaepiphyseal dysplasia in a family with a novel splicing mutation of the COL11A2 gene.

OBJECTIVE: To evaluate an approach to the clinical, radiographic, and molecular diagnosis of an underlying skeletal dysplasia in adults presenting with early-onset polyarticular osteoarthritis (OA). METHODS: We identified a family with 2 adults with polyarticular OA and a child with generalized arthralgia. General, musculoskeletal, ocular, and auditory evaluations were undertaken. Investigations included radiographs of symptomatic joints, analysis of serum inflammatory markers and joint fluid, and mutational analyses of the COL11A2 gene. RESULTS: The 3 affected individuals had normal stature, mild mid-face hypoplasia, and hearing impairment, but normal eyes. Radiographs of the affected adults showed severe polyarticular OA but did not reveal diagnostic evidence of an underlying skeletal dysplasia. However, the child's radiographs showed enlarged epiphyses with an advanced bone age. The combination of skeletal, facial, and auditory features together with the absence of ocular features indicated that they had otospondylomegaepiphyseal dysplasia, also known as Stickler syndrome type III. The diagnosis was confirmed by identifying a mutation in the COL11A2 gene that encodes the pre-pro-alpha2(XI) chain of type XI collagen that is involved in type II collagen fibrillogenesis. CONCLUSION: Early-onset polyarticular OA may occur in adults without a known or obvious underlying skeletal dysplasia. This study provides an approach to the diagnosis of an underlying skeletal dysplasia in such individuals.

Tumor vascular proteins as biomarkers in ovarian cancer.

PURPOSE: This study aimed to identify novel ovarian cancer biomarkers and potential therapeutic targets through molecular analysis of tumor vascular cells. METHODS: Immunohistochemistry-guided laser-capture microdissection and genome-wide transcriptional profiling were used to identify genes that were differentially expressed between vascular cells from human epithelial ovarian cancer and healthy ovaries. Tumor vascular markers (TVMs) were validated through quantitative real-time polymerase chain reaction (qRT-PCR) of immunopurified tumor endothelial cells, in situ hybridization, immunohistochemistry, and Western blot analysis. TVM expression in tumors and noncancerous tissues was assessed by qRT-PCR and was profiled using gene expression data. RESULTS: We identified a tumor vascular cell profile of ovarian cancer that was distinct from the vascular profile of normal ovary and other tumors. We validated 12 novel ovarian TVMs. These were expressed by immunopurified tumor endothelial cells and localized to tumor vasculature. Select TVMs were found to be specifically expressed in ovarian cancer and were absent in all normal tissues tested, including female reproductive tissues with physiologic angiogenesis. Many ovarian TVMs were expressed by a variety of other solid tumors. Finally, overexpression of any one of three ovarian TVMs by vascular cells was associated with decreased disease-free interval (all P < .005). CONCLUSION: We have identified for the first time the molecular profile of ovarian tumor vasculature. We demonstrate that TVMs may serve as potential biomarkers and molecular targets for ovarian cancer and a variety of other solid tumors.

Oxindole alkaloids from Uncaria tomentosa induce apoptosis in proliferating, G0/G1-arrested and bcl-2-expressing acute lymphoblastic leukaemia cells.

Natural products are still an untapped source of promising lead compounds for the generation of antineoplastic drugs. Here, we investigated for the first time the antiproliferative and apoptotic effects of highly purified oxindole alkaloids, namely isopteropodine (A1), pteropodine (A2), isomitraphylline (A3), uncarine F (A4) and mitraphylline (A5) obtained from Uncaria tomentosa, a South American Rubiaceae, on human lymphoblastic leukaemia T cells (CCRF-CEM-C7H2). Four of the five tested alkaloids inhibited proliferation of acute lymphoblastic leukaemia cells. Furthermore, the antiproliferative effect of the most potent alkaloids pteropodine (A2) and uncarine F (A4) correlated with induction of apoptosis. After 48 h, 100 micromol/l A2 or A4 increased apoptotic cells by 57%. CEM-C7H2 sublines with tetracycline-regulated expression of bcl-2, p16ink4A or constitutively expressing the cowpox virus protein crm-A were used for further studies of the apoptosis-inducing properties of these alkaloids. Neither overexpression of bcl-2 or crm-A nor cell-cycle arrest in G0/G1 phase by tetracycline-regulated expression of p16INK4A could prevent alkaloid-induced apoptosis. Our results show the strong apoptotic effects of pteropodine and uncarine F on acute leukaemic lymphoblasts and recommend the alkaloids for further studies in xenograft models.

In vitro analysis of differential expression of collagens, integrins, and growth factors in cultured human chondrocytes.

OBJECTIVES: Tissue engineering represents a promising method for the construction of autologous chondrogenic grafts for reconstructive surgery. In cultured chondrocytes, the dedifferentiation and proliferation of the cells are critical factors that influence the generation of transplants. The aim of our study was to find and characterize markers for cell proliferation and dedifferentiation in cultured chondrocytes. STUDY DESIGN AND SETTING: Human chondrocytes were isolated from septal cartilage and held in primary cell culture. Cells were harvested after 1, 6, and 21 days. The differentiation of the cells was investigated with bright-field microscopy, the expression patterns of various proteins using immunohistochemistry, and the expression of distinct genes with the microarray technique. RESULTS: The chondrocytes showed a strong proliferation. After 6 and 21 days, collagen 9 and 10 were downregulated; collagen 11 was activated. Collagen 1 and 2 were downregulated after 6 days but were reactivated after 21 days. Tumor growth factor beta (TGF-beta)1 was strongly expressed on days 1, 6, and 21, TGF-beta2 was never expressed, and TGF-beta3 and -beta4 were upregulated from day 1 to day 21. The TGF-beta receptor III was expressed on days 1, 6, and 21. Integrin beta1, beta5, and alpha5 were upregulated from day 1 to day 21; integrin beta3 was downregulated. CONCLUSION AND SIGNIFICANCE: Collagens 3, 4, 8, 9, and 11 might be new markers for the dedifferentiation of chondrocytes. Collagen 2 might be a marker for the synthetic activity of the cells rather than the dedifferentiation. TGF-beta3 and -beta4 might influence the dedifferentiation, which is fortified by the expression of TGF-beta receptor III. Integrin beta1, beta5, and alpha5 might be involved in signal transmission for the dedifferentiation.

Anchorage on fibronectin via VLA-5 (alpha5beta1 integrin) protects rheumatoid synovial cells from Fas-induced apoptosis.

BACKGROUND: Rheumatoid synovial cells are resistant to apoptosis induction in vivo, whereas, fibroblast-like synovial cells in rheumatoid arthritis (RA-FLS) are vulnerable to Fas-induced apoptosis in vitro. OBJECTIVE: To clarify this discrepancy by studying the contribution of the interaction between cellular integrin and matrix fibronectin (Fn), which is significantly increased in the rheumatoid joints, to the induction of apoptosis in RA-FLS. METHODS: Integrin and Fas mRNAs were measured by reverse transcription-polymerase chain reaction in RA-FLS. Integrins expressed in rheumatoid synovial tissues were analysed by immunohistochemistry. RA-FLS plated either on Fn or on control poly-L-lysine were incubated with agonistic anti-Fas monoclonal antibodies (mAbs). Apoptosis induction was evaluated using terminal deoxynucleotidyl transferase mediated UTP nick end labelling (TUNEL) and immunoblotting for caspase-3 and poly (ADP-ribose) polymerase in the presence or absence of anti-VLA-5 mAb. RESULTS: VLA-5 (alpha5beta1 integrin), a major integrin expressed on RA-FLS, was required for the adhesion of RA-FLS on Fn. RA-FLS plated on Fn were more resistant to Fas-induced apoptosis than those plated on control poly-L-lysine. This protection by Fn was reversed by anti-VLA-5 mAb. CONCLUSION: Anchorage of RA-FLS on matrix Fn via VLA-5 protects RA-FLS from Fas-induced apoptosis, and Fn abundantly present in rheumatoid synovium appears to afford RA-FLS resistance against apoptosis induction in vivo.

A study of cytotoxic synergy of UCN-01 and flavopiridol in syngeneic pair of cell lines.

Flavopiridol and UCN-01 are two novel protein kinase inhibitors with diverse cellular effects that may complement each other with regards to induction of apoptosis. HeLa cells engineered to overexpress human survivin (HeLa-S) were at least approximately 4.8-fold resistant to UCN-01 relative to proliferation observed in control HeLa cells (HeLa-V). Flavopiridol cytotoxicity as measured using the MTT assay was unaffected in HeLa-S cells when compared with HeLa-V cells. Similarly, simultaneous treatment of HeLa-V cells with flavopiridol and UCN-01 for 72 hours did not result in synergistic inhibition of proliferation; however, in HeLa-S cells, this combination resulted in synergistic inhibition of cell proliferation. Flavopiridol and UCN-01 augmented apoptosis in HeLa-S cells (as compared with HeLa-V cells) as measured by caspase-3 cellular activity assay, DNA fragmentation and PARP cleavage by western blot. In HeLa-V and -S cells, combination treatment resulted in caspase-8 cleavage. Caspase-9 was expressed in HeLa-V cells; however, there was a marked reduction of caspase-9 content in HeLa-S cells only. Combination treatment resulted in a significant reduction in survivin abundance in HeLa-S and SKBR3-UR cells, but not in their respective parental lines. The synergy of Flavopiridol and UCN-01 are selectively toxic to survivin-overexpressing cell lines and the mechanism of toxicity involves caspase-dependent cell death.

Articular cartilage repair using a tissue-engineered cartilage-like implant: an animal study.

OBJECTIVE: Because articular cartilage has limited ability to repair itself, treatment of (osteo)chondral lesions remains a clinical challenge. We aimed to evaluate how well a tissue-engineered cartilage-like implant, derived from chondrocytes cultured in a novel patented, scaffold-free bioreactor system, would perform in minipig knees with chondral, superficial osteochondral, and full-thickness articular defects. DESIGN: For in vitro implant preparation, we used full-thickness porcine articular cartilage and digested chondrocytes. Bioreactors were seeded with 20x10(6) cells and incubated for 3 weeks. Subsequent to culture, tissue cartilage-like implants were divided for assessment of viability, formaldehyde-fixed and processed by standard histological methods. Some samples were also prepared for electron microscopy (TEM). Proteoglycans and collagens were identified and quantified by SDS-PAGE gels. For in vivo studies in adult minipigs, medial parapatellar arthrotomy was performed unilaterally. Three types of defects were created mechanically in the patellar groove of the femoral condyle. Tissue-engineered cartilage-like implants were placed using press-fit fixation, without supplementary fixation devices. Control defects were not grafted. Animals could bear full weight with an unlimited range of motion. At 4 and 24 weeks postsurgery, explanted knees were assessed using the modified ICRS classification for cartilage repair. RESULTS: After 3-4 weeks of bioreactor incubation, cultured chondrocytes developed a 700-microm- to 1-mm-thick cartilage-like tissue. Cell density was similar to that of fetal cartilage, and cells stained strongly for Alcian blue and safranin O. The percentage of viable cells remained nearly constant (approximately 90%). Collagen content was similar to that of articular cartilage, as shown by SDS-PAGE. At explantation, the gross morphological appearance of grafted defects appeared like normal cartilage, whereas controls showed irregular fibrous tissue covering the defect. Improved histologic appearance was maintained for 6 months postoperatively. Although defects were not always perfectly level upon implantation at explanation the implant level matched native cartilage levels with no tissue hypertrophy. Once in place, implants remodelled to tissues with decreased cell density and a columnar organization. CONCLUSIONS: Repair of cartilage defects with a tissue-engineered implant yielded a consistent gross cartilage repair with a matrix predominantly composed of type II collagen up to 6 months after implantation. This initial result holds promise for the use of this unique bioreactor/tissue-engineered implant in humans.