M.A. Birch

PCR phenotyping of cytokines, growth factors and their receptors and bone matrix proteins in human osteoblast-like cell lines

The expression of a total of 58 cytokines, growth factors, and their corresponding receptors and bone matrix proteins was assessed using reverse transcription-linked polymerase chain reaction (RT-PCR) analysis to determine the similarity in the expression profile between clonal osteosarcoma-derived human osteoblast-like cell lines and primary human osteoblast-like cell cultures derived from human trabecular bone explants. The spectrum of cytokines, growth factors, and bone-related proteins expressed by three human osteosarcoma-derived cell lines, TE-85, MG-63, SaOS-2, and primary human osteoblast-like cells was found to be highly comparable and for the first time the expression of EGF, ECGF, FGF beta, oncostatin M, TNF beta, and SCF by human osteoblast-like cells was detected. Also the expression of several receptor types including IL-4R, IL-7R, IFN alpha/beta R, and SCFR was detected that has not been previously described for human osteoblast-like cells. For the factors examined, no qualitative variations in the expression profile were observed in the six primary human osteoblast-like cell cultures used in this study. Of the 58 factors examined, only 13 showed some degree of nonuniformity of expression between all of the three cell lines and primary cell cultures. These differences were seen especially in the expression of cytokine receptor mRNA and to a lesser extent with some cytokines. Differences in receptor expression would suggest that the possible spectrum of response to exogenously added factors, or even autocrine/ paracrine networks would be determined by the repertoire of receptors expressed by each cell type. Whether the differences are related to the status of cell maturation within the osteoblast development lineage or to their abberant regulation of expression cannot be concluded at this stage. However, this PCR-phenotyping approach rapidly provides a resource of information, which can be subsequently used for further in depth studies to facilitate the analysis of the molecular mechanisms, whereby the target gene of interest is modulated in a model cell line. In addition, this study indicates that at least based on the transcript expression profile of the factors analyzed, human osteosarcoma-derived osteoblast-like cells are useful as models for their nontransformed counterparts.

Parathyroid hormone (PTH)/PTH-related protein (PTHrP) receptor expression and mitogenic responses in human breast cancer cell lines.

Previous reports have shown the production of parathyroid hormone-related protein (PTHrP) by breast cancer cells in vivo and in vitro. We have investigated the expression of the PTH/PTHrP receptor by the human breast cancer cell lines MCF-7, ZR-75-1, T-47-D, SK-BR-3, Hs578T and MDA-MB231. Using reverse transcription-polymerase chain reaction (RT-PCR) and Southern blot analysis, we detected transcripts for the receptor in MCF-7, SK-BR-3 and MDA-MB231 cells. There was no evidence of receptor mRNA in ZR-75-1 and Hs578T cells. Furthermore, Northern blot analysis of mRNA from MCF-7 cells showed two transcripts of 1.5 and 2.4 kb which coded for the PTH/PTHrP receptor. Expression of PTH/PTHrP receptor mRNA by the breast cancer cell lines was also correlated with the detection of PTHrP transcripts. RT-PCR demonstrated PTHrP mRNA in MCF-7, ZR-75-1, T-47-D and Hs578T cells, but not in SK-BR-3 and MDA-MB231 cells. The detection of receptor transcripts was complemented by [3H]thymidine and bromodeoxyuridine incorporation studies, in which mitogenic responses to PTH and PTHrP were observed in MCF-7 cells but not in Hs578T cells. In response to both PTH(1-34) and PTHrP(1-34), quiescent MCF-7 cells proliferated in a similar dose-dependent manner (1.6-100 ng ml-1). No mitogenic effects of these peptides were observed with Hs578T cells. In addition, levels of intracellular cAMP were measured in MCF-7 and Hs578T cells in response to PTHrP(1-34). In MCF-7 cells there was a significant rise in cAMP with 100 ng ml-1 PTHrP(1-34). The expression of PTH/PTHrP receptor by breast cancer cells suggests that PTHrP may be a paracrine/autocrine regulator of breast carcinoma.

Purification of an insect derived recombinant human ADAMTS-1 reveals novel gelatin (type I collagen) degrading activities.

ADAMTS-1 (A Disintegrin And Metalloprotease with ThromboSpondin repeats) is a member of a family of secreted proteolytic enzymes with a complex modular structure. These enzymes are characterised by an N-terminal metalloproteinase domain, a disintegrin-like domain and a carboxyl terminal region containing variable numbers of a repeat sequence with homology to thrombospondin-1. The expression of the gene for ADAMTS-1 has been associated with inflammation, ovulation, angiogenesis, cellular proliferation and bone formation. ADAMTS-1 can proteolytically process large proteoglycans indicating a potential role in extracellular matrix turnover. In this study, we have tested ADAMTS-1 activity in gelatin zymogram assays. Since previous data demonstrate that ADAMTS-1 is a matrix metalloproteinase (MMP) substrate and is highly unstable in conditioned medium from eukaryotic cell types, we created an insect cell line expressing human ADAMTS-1. We isolated an epitope tagged full-length recombinant ADAMTS-1 from serum free insect cell conditioned medium. The purified protein had aggrecanase activity and appears as two major bands on the silver stained SDS-PAGE corresponding well to a pro-domain on form of 115 kDa and a pro-domain off form of 90 kDa. Using denatured type I collagen in zymographic analysis we demonstrate that ADAMTS-1 has a previously unreported gelatinolytic activity. Also, we notice that processing of its C-terminal region by an apparently autocatalytic process reveals a 27 kDa species with gelatinolytic activity. Furthermore, we show that MMP2 but not MMP13 remove ADAMTS-1 specific gelatin zymopraphic zones.

Polyhipe Polymer: A Novel Scaffold for In Vitro Bone Tissue Engineering

During the past decade exciting new approaches have emerged that could potentially revolutionise the treatment of patients suffering from failure of vital tissues and organs. Tissue engineering is a multidisciplinary field that applies the knowledge of engineering, life sciences, and the clinical sciences to solve the critical medical problems of tissue loss and organ failure. This rapidly expanding area of research is where new materials and techniques are continually being sought. The aim is to produce a biomaterial scaffold that enables the production of tissue that closely matches healthy cellular structures and has good functionality.

Parathyroid hormone‐related protein in the aetiology of fibrous dysplasia of bone in the McCune Albright syndrome

Fibrous dysplasia, observed in bone lesions in the McCune Albright syndrome (MAS), is thought to result from abnormalities in cells of the osteogenic lineage associated with over‐activation of the cAMP signalling pathway in affected cells. The aim of this study was to investigate the role of parathyroid hormone‐related protein (PTHrP) in the aetiology of MAS, and to determine a possible therapeutic role for 1,25‐dihydroxy vitamin D3 (1,25(OH)2D3).

Primary cultures of human bone-derived cells produce parathyroid hormone-related protein: a study of 40 patients of varying age and pathology

Parathyroid hormone-related protein (PTHrP), a mediator of hypercalcemia of malignancy, has been detected in many tumours and in some normal foetal and adult tissues. PTHrP has potent effects on bone turnover in vivo and in vitro. In this study we cultured cells derived from explants of bone obtained from 40 subjects (age range 2-88 years). Immunoreactive PTHrP (iPTHrP) was detected by immunoradiometric assay (IRMA) in conditioned medium from 25 of 40 cultures of bone-derived cells. PTHrP mRNA was detected in bone-derived cells by reverse transcriptase-linked polymerase chain reaction (RT-PCR). The identity of PCR products was confirmed by Southern blotting. Local production of PTHrP in vivo may be important in the regulation of bone growth and remodelling.

Cytokine expression by cultured osteoblasts from patients with osteoporotic fractures

Human osteoblasts were derived in culture from explants of bone from patients who had recently suffered osteoporotic fractures and from patients with no evidence of osteoporosis. The expression of cytokine mRNA in these osteoblasts was subsequently determined by reverse transcriptase‐linked polymerase chain reaction (RT‐PCR). We have detected mRNA for IL‐1β, IL‐3, IL‐6, IL‐8, TNF‐α and ‐β, and the three TGF‐β isoforms in the cells. The profile of cytokines expressed by osteoblasts derived from patients with osteoporotic fractures was consistent with profiles observed in osteoblasts derived from patients with no evidence of reduced bone mass — the latter included children undergoing corrective surgery and adult subjects ranging from 31 to 80 years undergoing elective surgery for osteoarthritis and other bone pathologies.

RT-PCR DETECTION OF CYTOKINE TRANSCRIPTS IN A SERIES OF CULTURED HUMAN MENINGIOMAS

The expression of cytokine transcripts has been investigated in a series of cultured human meningiomas using reverse transcriptase linked polymerase chain reaction (RT‐PCR), which allowed simultaneous analysis of a range of cytokines. The main histological subgroups of meningioma were investigated; these included transitional, fibroblastic, and syncytial as well as atypical meningiomas. Meningiomas from each of the different histological subgroups were subjected to a standard tissue culture regime. Total RNA was extracted from representative cultures and reverse‐transcribed to yield cDNA. PCR was performed using oligonucleotide primers designed to detect interleukin (IL)‐1α/β to IL‐8, transforming growth factor (TGF)β1–3, tumour necrosis factor (TNF)α/β, and interferon (IFN)γ. Transcripts for IL‐3, IL‐6, IL‐8, and TGFβ3 were detected in all cultures. Transcripts for the three isomers of TGFβ were expressed in the transitional and fibroblastic meningioma cells. TGFβ2 and TGFβ3 transcripts were expressed in the syncytial and TGFβ1 and TGFβ3 in the atypical meningioma cells. IL‐1β transcripts were expressed in fibroblastic and atypical cultures and TNFβ transcripts were expressed in syncytial and transitional cultures only. Transcripts for IL‐1α, IL‐2, IL‐4, IL‐5, IL‐7, TNFα, or IFNγ were not detected in any of the meningioma cultures. This investigation using cells cultured from a small number of tumours from each of the classic histological subtypes suggests that there is a distinct pattern of cytokine mRNA expression linked with histological classification.

A generic expression system to produce proteins that co-assemble with alkane thiol SAM.

Surface biology aims to observe and control biological processes by combining bio-, surface, and physical chemistry. Self-assembled monolayers (SAM) on gold surfaces have provided excellent methods for nanoscale surface preparation for such studies. However, extension of this work requires the specific immobilization of whole protein domains and the direct incorporation of recombinant proteins into SAM is still problematic. In this study a short random coil peptide has been designed to insert into thioalkane layers by formation of a hydrophobic helix. Surface plasmon resonance (SPR) studies show that specific immobilization via the internal cysteine is achieved. Addition of the peptide sequence to the terminus of a protein at the genetic level enables the production of a range of recombinant fusion-proteins with good yield. SPR shows that the proteins display the same gold-binding behavior as the peptide. It is shown that cell growth control can be achieved by printing the proteins using soft lithography with subsequent infilling with thioalkanes The expression plasmid is constructed so that any stable protein domain can be easily cloned, expressed, purified and immobilized.

Cytokines and Growth Factors in Paget’s Disease

Paget’s disease is a focal disorder of bone in which there is a localized increase in bone remodeling. The cellular actions which constitute normal bone remodeling are complex and require the spatially and temporally coordinated actions of several cell types.1 Remodeling commences with an initiation signal(s) leading to a phase of resorption where bone is excavated from the remodeling site by multinucleated osteoclasts. In pagetic foci, but not in unaffected sites, the osteoclasts exhibit increased multinuclearity and are overactive, resulting in excessive resorption. Normally resorption lacunae are then colonized by osteoblasts which synthesize bone matrix components (osteoid) and the bone is replaced. The mechanism(s) which ensures that the processes of bone resorption and formation remain tightly linked is termed “coupling.”2 The increased osteoclastic activity observed in Paget’s disease is matched by an increase in osteoblastic activity, so bone resorption and bone formation remain coupled.