Biomedical subjects

J A Gallagher

Publications and source records attributed to J A Gallagher.

At least 37 records · Page 2Linked to original sources

PubMed2000

Parathyroid hormone-related protein (1-34) and urothelial redifferentiation in the neuropathic urinary bladder.

STUDY DESIGN: A comparative study of immunostaining for parathyroid hormone-related protein (1-34) (PTHrP (1-34)) in the vesical epithelium of biopsies obtained from patients with non-neuropathic bladder and those with neuropathic bladder. OBJECTIVES: To investigate the immunostaining for PTHrP (1-34) in the control cases and in neuropathic bladders showing (1) normal transitional epithelium, (2) hyperplastic transitional epithelium, and (3) squamous metaplasia. SETTING: Regional Spinal Injuries Centre, and Department of Cellular Pathology, Southport & Ormskirk Hospitals NHS Trust, Southport, Department of Pathology, Royal Liverpool University Hospital and the Departments of Clinical Chemistry and Cell Biology, The University of Liverpool, Liverpool, England. METHODS: Cold cup biopsies of bladder mucosa were taken from patients suffering from neuropathic urinary bladder when they were undergoing a therapeutic procedure in the urinary tract. Immunohistochemistry was performed on these biopsy specimens using a rabbit polyclonal antibody raised to a synthetic peptide corresponding to human PTHrP (1-34). Control group (n=10) consisted of archival biopsies taken from non-neuropathic bladders. RESULTS: In the control group, the transitional epithelium showed no immunostaining, or at the most, very faint positive staining was seen in the transitional epithelium of non-neuropathic bladder. Positive immunostaining to PTHrP (1-34) was seen in the normal transitional epithelium of neuropathic bladder in nine of 13 cases. Hyperplastic transitional epithelium showed positive immunostaining for PTHrP (1-34) in 11 of 13 biopsies from patients with neuropathic bladder. Immunostaining for PTHrP (1-34) was observed in the metaplastic squamous epithelium in 14 of 17 cases with neuropathic bladder. CONCLUSION: The transitional epithelium of non-neuropathic bladder showed no immunostaining, or at the most, very faint positive staining for PTHrP (1-34). In contrast to this, positive immunostaining for PTHrP (1-34) was observed more frequently in the vesical epithelium of neuropathic bladder. This observation opens up avenues for innovative therapy with PTHrP or its analogues for possible modulation of urothelial differentiation in the neuropathic bladder.

Adult↗

PubMed2000

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

OBJECTIVE: 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 D(3) (1,25(OH)(2)D(3)). DESIGN: The effects of 1,25(OH)(2)D(3) on PTHrP production and mRNA expression were determined in vitro. 1,25(OH)(2)D(3) therapy was administered to three patients with MAS. PATIENTS: Clinical data from four MAS patients (MAS1, 2, 3 and 4), and in vitro studies using bone from three MAS patients (MAS1, 2, and 3), are presented. MEASUREMENTS: Immunoradiometric assay and low-cycle number reverse transcriptase-linked PCR were used to determine PTHrP production and mRNA expression in vitro. Standard clinical biochemistry was recorded pre and post commencement of 1,25(OH)(2)D(3) treatment. RESULTS: We report the elevated secretion of PTHrP, and a concomitant rise in PTHrP mRNA expression, in cultured osteoblasts from three MAS patients. Treatment with 1,25(OH)(2)D(3) produced a dose-dependent decrease in PTHrP protein secretion and mRNA expression. Marked improvement in bone biochemistry in MAS1, 2 and 3 post treatment with 1,25(OH)(2)D(3) is documented. CONCLUSION: This study provides the first evidence suggesting that PTHrP may contribute to the aetiology of fibrous dysplasia in MAS. In addition, the therapeutic administration of 1,25(OH)92)D(3) may provide clinicians with an important new regime for symptomatic relief of bone pain and fracture in some patients with MAS.

Adolescent↗

PubMed2000

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-1beta, IL-3, IL-6, IL-8, TNF-alpha and -beta, and the three TGF-beta 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.

Adolescent↗

PubMed2000

Extracellular ATP activates multiple signalling pathways and potentiates growth factor-induced c-fos gene expression in MCF-7 breast cancer cells.

In the human breast cancer cell line MCF-7, the nucleotides ATP gamma S and UTP, acting extracellularly through the purinergic receptor P2Y(2), lead to elevated intracellular calcium levels and increased proliferation. ATP gamma S and UTP treatment of MCF-7 cells activated transcription of the immediate early gene c-fos, an important component in the response to proliferative stimulation. c-fos induction was enhanced by co-treatment with ATP gamma S and a variety of proliferative agents including growth factors, tumour promoters and stress. Stimulation with ATP gamma S or epidermal growth factor (EGF) led to extracellular signal-regulated kinase (ERK) activation and phosphorylation of the transcription factors CREB and Elk-1. Co-stimulation synergistically activated fos expression and notably led to increased levels of ERK, CREB and EGF receptor phosphorylation, as well as hyperphosphorylation of ternary complex factor. Nevertheless, the ERK pathway does not fully account for this synergy, since fos induction was differentially sensitive to the MEK inhibitor U0126, indicating that these two agonists signal differently to this immediate early gene. Thus, extracellular nucleotides co-operate with growth factors to activate genes linked to the proliferative response in MCF-7 cells through activation of specific purinergic receptors, which thereby represent important potential targets for arresting the neoplastic progression of breast cancer cells.

Adenosine Triphosphate↗

PubMed1999

Expression of members of the thrombospondin family by human skeletal tissues and cultured cells.

We have previously shown that the multifunctional platelet glycoprotein thrombospondin-1 (TSP-1) promotes resorption in an in vitro resorption assay. However, TSP-1 is one of a family of multifunctional TSP molecules, and the current study was undertaken to investigate whether it is TSP-1 or another TSP family member which may be involved in regulation of resorption in vivo. RT-PCR was performed on cultured human bone cells, cultured human chondrocytes, and three separate samples of human osteoclastoma tissue using primers specific for each TSP family member. mRNA for TSP-2 was detected in almost all samples, and significantly in all osteoclastomas in the above tissues, while TSP-1 was detected less frequently and was only seen in one of three osteoclastomas. TSP-3, -4, and COMP were detected only in a minority of cases. These results indicate that TSP-2 is the most common TSP family member found in skeletal tissues and that TSP-2, rather than TSP-1, may be the molecule responsible for promoting resorption in vivo.

Bone Resorption↗

PubMed1999

Signaling in human osteoblasts by extracellular nucleotides. Their weak induction of the c-fos proto-oncogene via Ca2+ mobilization is strongly potentiated by a parathyroid hormone/cAMP-dependent protein kinase pathway independently of mitogen-activated protein kinase.

Extracellular nucleotides acting through specific P2 receptors activate intracellular signaling cascades. Consistent with the expression of G protein-coupled P2Y receptors in skeletal tissue, the human osteosarcoma cell line SaOS-2 and primary osteoblasts express P2Y1 and P2Y2 receptors, respectively. Their activation by nucleotide agonists (ADP and ATP for P2Y1; ATP and UTP for P2Y2) elevates [Ca2+]i and moderately induces expression of the c-fos proto-oncogene. A synergistic effect on c-fos induction is observed by combining ATP and parathyroid hormone, a key bone cell regulator. Parathyroid hormone elevates intracellular cAMP levels and correspondingly activates a stably integrated reporter gene driven by the Ca2+/cAMP-responsive element of the human c-fos promoter. Nucleotides have little effect on either cAMP levels or this reporter, instead activating luciferase controlled by the full c-fos promoter. This induction is reproduced by a stably integrated serum response element reporter independently of mitogen-activated protein kinase activation and ternary complex factor phosphorylation. This novel example of synergy between the cAMP-dependent protein kinase/CaCRE signaling module and a non-mitogen-activated protein kinase/ternary complex factor pathway that targets the serum response element shows that extracellular ATP, via P2Y receptors, can potentiate strong responses to ubiquitous growth and differentiative factors.

Adenosine Triphosphate↗

PubMed1999

Regulation of epidermal homeostasis through P2Y2 receptors.

1. Previous studies have indicated a role for extracellular ATP in the regulation of epidermal homeostasis. Here we have investigated the expression of P2Y2 receptors by human keratinocytes, the cells which comprise the epidermis. 2. Reverse transcriptase-polymerase chain reaction (RT - PCR) revealed expression of mRNA for the G-protein-coupled, P2Y2 receptor in primary cultured human keratinocytes. 3. In situ hybridization studies of skin sections revealed that P2Y2 receptor transcripts were expressed in the native tissue. These studies demonstrated a striking pattern of localization of P2Y2 receptor transcripts to the basal layer of the epidermis, the site of cell proliferation. 4. Increases in intracellular free Ca2+ concentration ([Ca2+]i) in keratinocytes stimulated with ATP or UTP demonstrated the presence of functional P2Y receptors. 5. In proliferation studies based on the incorporation of bromodeoxyuridine (BrdU), ATP, UTP and ATPgammaS were found to stimulate the proliferation of keratinocytes. 6. Using a real-time firefly luciferase and luciferin assay we have shown that under static conditions cultured human keratinocytes release ATP. 7. These findings indicate that P2Y2 receptors play a major role in epidermal homeostasis, and may provide novel targets for therapy of proliferative disorders of the epidermis, including psoriasis.

Adenosine Triphosphate↗

PubMed1999

Immunohistochemical study of parathyroid hormone-related protein in vesical transitional epithelium of patients with spinal cord injury.

INTRODUCTION: Parathyroid hormone-related protein (PTHrP), in addition to the well-established role in endochrondral bone development, is believed to be an important mediator of cellular growth and differentiation in a number of non-bony tissues. OBJECTIVES: To compare the immunohistochemical staining of vesical transitional epithelium to antibodies raised to synthetic peptides of PTHrP composed of amino acid sequences 43 - 52 and 127 - 138 in patients with spinal cord injury (SCI) and neuropathic bladder (n=14), and control patients with intact neuraxis and no history of bladder cancer (n=10). SETTING: Male SCI patients registered with Regional Spinal Injuries Centre, Southport, England. INTERVENTION: Endoscopic cold cup biopsy from the trigone of the urinary bladder was taken from patients with SCI while they were undergoing a therapeutic procedure in the urinary bladder. The control samples of bladder biopsies were taken from the archives of the Department of Histopathology, District General Hospital, Southport. Immunohistochemistry was performed using rabbit antibodies raised against synthetic peptides of human PTHrP (43 - 52) and PTHrP (127 - 138). The biopsies were examined for immunostaining of transitional epithelium. RESULTS: Of the 14 biopsies of SCI patients, positive immunostaining using antibodies to both the PTHrP peptides was found in four cases; five biopsies showed positive immunostaining only to anti-PTHrP (43 - 52); and five biopsies showed no immunostaining with either of the PTHrP peptides. In contrast, transitional epithelium in the biopsy specimens of ten control subjects with no history of bladder cancer showed no immunostaining with either of the PTHrP peptides. CONCLUSION: This study revealed that the transitional epithelium of neuropathic urinary bladder exhibits increased predilection for positive immunohistochemical staining for PTHrP (43 - 52), and to a lesser extent, to PTHrP (127 - 138), as compared to the vesical transitional epithelium of able bodied individuals with no history of vesical malignancy. The possible role of PTHrP in the cellular differentiation of urothelium of neuropathic bladder, and thereby, in the pathogenesis of cystitis in SCI patients, needs to be explored.

Humans↗

PubMed1998

G-protein coupled receptors in bone.

The skeleton is a dynamic structure that undergoes continuous remodeling, a prerequisite to meeting the constant loading demands placed upon it. This process is controlled by a multitude of systemic and local factors which interact with receptors presented on the surface of both osteoblasts and osteoclasts; the osteogenic and osteolytic cells of bone. The seven transmembrane G-protein coupled superfamily of receptors are amongst the most important expressed by bone cells. Many local and systemic factors, including prostaglandins and parathyroid hormone, initiate cellular processes via interaction with members of this receptor family. The diversity of signals and signaling cross talk generated by activated G-protein receptor complexes, facilitates a huge range of downstream responses essential in the remodeling of the skeleton. Indeed, agonist-activated signaling crosstalk provides a mechanism for integrating the activities of local and systemic factors, an essential requirement of focal remodeling. This review has focused on those currently known seven transmembrane receptors expressed by bone cells that couple to G-proteins, and describes the nature of receptor-G protein interaction and the resultant functional consequences of effector activation within bone cells.

Animals↗

PubMed1998

P2Y2 receptors are expressed by human osteoclasts of giant cell tumor but do not mediate ATP-induced bone resorption.

Extracellular nucleotides acting through P2 receptors elicit a range of responses in many cell types. Previously, we have cloned the G-protein coupled P2Y2 receptor from a human osteoclastoma complementary deoxyribonucleic acid (cDNA) library and demonstrated its expression by reverse transcription linked (RT)-PCR and Southern analysis in a number of skeletal tissues, including a purified population of giant cells. In this study we have localized the expression of P2Y2 receptor transcripts to osteoclasts of giant cell tumor of bone by in situ hybridization. In osteoblasts and other cell types, the P2Y2 receptor is coupled to Ins(1,4,5)P3-mediated Ca2+ release from intracellular stores. In this study, the P2Y2 receptor agonists adenosine triphosphate (ATP) and uridine triphosphate (UTP) did not increase cytosolic free calcium concentration ([Ca2+]i) in giant cells isolated from osteoclastoma, while the G-protein coupled calcium sensing receptor agonist, Ni2+, elevated [Ca2+]i in the same cells. These data indicate that P2Y2 receptor transcripts expressed by giant cells are not presented at the surface of cells as functional receptors, or alternatively, functional receptors are coupled to an effector other than [Ca2+]i. ATPgammaS (10 micromol/L), but not UTP (10 micromol/L), significantly stimulated resorption by an enriched giant cell population. These results indicate that ATP-induced effects on resorption, following direct osteoclastic activation, are mediated by a P2 receptor other than the P2Y2 subtype. Nucleotides, released locally in the bone microenvironment in response to acute trauma or transient physical stress, will interact with a complement of P2 receptors expressed by both osteoclasts and osteoblasts to influence the remodeling process.

Adenosine Triphosphate↗

PubMed1998

Human keratinocytes express transcripts for three isoforms of parathyroid hormone-related protein (PTHrP), but not for the parathyroid hormone/PTHrP receptor: effects of 1,25(OH)2 vitamin D3.

Parathyroid hormone-related protein (PTHrP) is strongly expressed in the epidermis and has been implicated in the regulation of growth and differentiation of keratinocytes. PTHrP has N-terminal sequence homology with parathyroid hormone (PTH) and binds to the type I PTH/PTHrP receptor, but earlier reports suggest that keratinocytes do not possess this cell surface receptor. In order to determine which PTHrP mRNA isoforms are expressed by keratinocytes and whether the type I receptor mRNA is present, we designed specific primers for reverse transcriptase-polymerase chain reaction. The interaction of PTHrP with other promoters of keratinocyte differentiation is unclear. In particular, 1,25(OH)2D3 is also fundamental in calcium homeostasis and induces changes in intracellular calcium. We therefore investigated the effect of 1,25(OH)2D3 on PTHrP mRNA expression and protein production in cultured human keratinocytes. Cells were incubated for 3 days at concentrations of 1.25(OH)2D3 of 10(-10)-10(-6) mol/L. PTHrP in culture supernatant, measured by two site immunoradiometric assay, was 915 +/- 98 PTHrP fmol/mg of cell layer protein in untreated cultures decreasing to 570 +/- 113 with 10(-8) mol/L and 402 +/- 24 with 10(-6) mol/L 1,25(OH)2D3 (mean +/- SEM, P < 0.01, n = 6). Transcripts for all three PTHrP isoforms (139, 141 and 173 amino acids) were detectable in keratinocyte mRNA. Corresponding to the decrease in PTHrP protein we demonstrated a reduction in all three PTHrP mRNA transcripts after 3 days' incubation with 1,25(OH)2D3 over a concentration range 10(-10)-10(-6) mol/L. Repeated studies failed to detect type I PTH/PTHrP receptor mRNA in human keratinocytes, either in control cultures or in the presence of 1,25(OH)2D3. We have shown that keratinocytes produce abundant PTHrP and that this is modulated by 1,25(OH)2D3, suggesting a physiological role. Further studies are required to investigate the relative expression of PTHrP isoforms, their role in keratinocyte signalling and the receptors involved.

Calcifediol↗

PubMed1997

The osteoclast-associated protease cathepsin K is expressed in human breast carcinoma.

Human cathepsin K is a novel cysteine protease previously reported to be restricted in its expression to osteoclasts. Immunolocalization of cathepsin K in breast tumor bone metastases revealed that the invading breast cancer cells expressed this protease, albeit at a lower intensity than in osteoclasts. In situ hybridization and immunolocalization studies were subsequently conducted to demonstrate cathepsin K mRNA and protein expression in samples of primary breast carcinoma. Expression of cathepsin K mRNA was confirmed by reverse transcription PCR and Southern analysis in a number of human breast cancer cell lines and in primary human breast tumors and their metastases. As this protease is known to degrade extracellular matrix, including bone matrix proteins, it is possible that cathepsin K may contribute to the invasive potential of breast cancer cells, including those that metastasize to bone. Thus, cathepsin K may be a potential target leading to the design of novel drugs for cancer therapy.

Blotting, Southern↗

PubMed1997

Effects of PTH on PTHrP gene expression in human osteoblasts: up-regulation with the kinetics of an immediate early gene.

Parathyroid hormone-related peptide (PTHrP) is a local regulator of human bone turnover, which shares some sequence homology with the systemic hormone parathyroid hormone (PTH). The two proteins exert cellular effects through interaction with a common G protein-coupled PTH/PTHrP receptor on target cells. Whilst the PTH gene has a relatively simple structure the PTHrP gene is complex, alternative splicing of which can generate multiple mRNA species encoding PTHrP of 139, 141 and 173 amino acids. To date little is known regarding the extent to which PTH and PTHrP interact to modulate bone cell function. In this study we have used the quantitative technique of Real-Time polymerase chain reaction (PCR) to investigate the ability of PTH to induce PTHrP expression in SaOS-2 cells and in primary human osteoblasts. In addition, we have used the semi-quantitative techniques of PCR followed by Southern analysis and scanning densitometry to investigate the effects of PTH(1-34) on expression of mRNA species encoding the three PTHrP isoforms. We report a 50 fold increase in PTHrP mRNA expression 30 min after treatment with 100 ng/ml human recombinant PTH (1-34) in SaOS-2 cells, and a 38 fold rise in human osteoblasts 45-90 min post-PTH treatment. mRNA species encoding for PTHrP 1-139, 1-141 and 1-173 were all induced in human osteoblasts 45 min after exposure to PTH. Whilst the 1-139 mRNA species exhibited a sustained expression, both the 1-141 and 1-173 isoforms showed a biphasic induction with a second peak 6 hr post PTH treatment. These data demonstrate that PTH induces expression of the PTHrP gene in both SaOS-2 and primary human osteoblasts with the kinetics of an immediate early gene. Up-regulation of the PTHrP gene in response to PTH may be an important physiological mechanism by which this systemic factor effects a localised response in bone.

Cyclic AMP Response Element-Binding Protein↗

PubMed1997

Localization of cathepsin K in human osteoclasts by in situ hybridization and immunohistochemistry.

We have recently cloned cathepsin K from a human bone cDNA library. Since cathepsins are proposed to be involved in the degradation of mineralized bone matrix, we have investigated, by in situ hybridization and immunocytochemistry, the expression of the cathepsin K mRNA transcripts and protein in sections of bone and giant cell tumor to determine which cells express this enzyme. Within all tissues studied, cathepsin K was highly expressed in osteoclasts. Furthermore, the expression of cathepsin K mRNA in giant cell tumor tissue appeared to be confined to the periphery of the osteoclast indicating a compartmentalization of the mRNA. Immunohistochemistry confirmed the specific localization of cathepsin K to the osteoclast. In actively resorbing osteoclasts, the immunostaining was localized at the ruffled border, whereas in osteoclasts in sections of giant cell tumor, staining was observed in lysosomal vacuoles, which in some cases were seen to fuse with the cell membrane. Other cells within the bone, such as osteoblasts and osteocytes, did not express either the cathepsin K transcript or protein. However, there were very low levels of cathepsin K detected in a population of mononuclear cells, possibly representing osteoclast progenitor cells, within the marrow/stromal layer. The specific localization of cathepsin K within osteoclasts would therefore indicate the potential role of this enzyme in the bone resorptive process.

Blotting, Northern↗

PubMed1997

PTHrP and the PTH/PTHrP receptor are co-expressed in human breast and colon tumours.

Using RNA extracted from human tumour samples removed during surgery, we have analysed expression of mRNA for parathyroid hormone-related protein (PTHrP) and for the PTH/PTHrP receptor by RT-PCR in a panel of human breast and colon tumours. All but 1 of 18 breast tumour samples expressed PTHrP, whereas receptor expression was detected in 11 of these. Expression of the PTH/PTHrP receptor was found in three out of four metastatic lesions, including one sample in which no receptor was detected in the primary tumour. PTHrP expression was also detected in five colon tumours, and receptor expression detected in two of these. These results demonstrate that PTHrP and the PTHrP receptor are also co-expressed in breast tumours in vivo and provide further evidence that PTHrP may be an important autocrine/paracrine growth factor in breast cancer.

Breast Neoplasms↗

PubMed1997

Extracellular nucleotides stimulate proliferation in MCF-7 breast cancer cells via P2-purinoceptors.

Nucleotides such as ATP can act as extracellular effector molecules by interaction with specific cellular receptors known as P2-purinoceptors. Recently, we cloned the human P2U purinoceptor from osteoclastoma and demonstrated its expression in skeletal tissues. In the current study we have investigated the expression of P2U purinoceptors in human breast tumour cell lines and examined functional effects of extracellular nucleotides on these cells. By reverse transcription-linked polymerase chain reaction (RT-PCR) the expression of mRNA for P2U purinoceptors was demonstrated in four human breast cancer cell lines, Hs578T, MCF-7, SK-Br3 and T47-D. In MCF-7 cells, extracellular ATP (1-100 microM) elevated intracellular free calcium concentration [Ca2+]i, indicating that these cells express functional P2-purinoceptors. UTP elevated [Ca2+]i in an identical manner to ATP, whereas 2-methylthioATP was completely ineffective, and ADP only partially effective. This pharmacological profile suggests that the P2U subtype may be the only P2-purinoceptor expressed by these cells. The functional significance of P2U purinoceptor expression by MCF-7 cells was investigated by analysing the effects of extracellular ATP on cell proliferation. The slowly hydrolysed analogue of ATP, ATPgammaS (which was also shown to elevate [Ca2+]i), induced proliferation of MCF-7 cells when added daily to serum-free cultures over a period of 3 days. ATPgammaS-induced proliferation was demonstrated by three separate methods, detection by scintillation counting of [3H]thymidine incorporation, immunocytochemical detection of 5-bromo-2-deoxyuridine incorporation and direct counting of cell numbers. These data suggest that ATP, possibly released at sites of tissue injury or inflammation, may be capable of growth factor action in promotion of tumour proliferation or progression.

Adenosine Triphosphate↗

PubMed1997

Effects of extracellular nucleotides on single cells and populations of human osteoblasts: contribution of cell heterogeneity to relative potencies.

1. Human osteoblasts responded to the application of extracellular nucleotides, acting at P2-receptors, with increases in cytosolic free calcium concentration ([Ca2+]i). 2. In populations of human osteoblasts, adenosine 5'-diphosphate (ADP) evoked a rise in [Ca2+]i with less than 40% of the amplitude of that induced by adenosine 5'-triphosphate (ATP). 3. ATP and uridine 5'-triphosphate (UTP) were applied to single human osteoblasts and induced [Ca2+]i rises of comparable amplitude in every cell tested. 4. However, from the results of single cell studies with ADP (and 2-methylthioATP (2-meSATP)) two groups of cells were delineated; one group responded to ADP (or 2-meSATP) with a rise in [Ca2+]i indistinguishable from that evoked by ATP; whereas the second group failed completely to respond to ADP (or 2-meSATP). 5. Therefore heterogeneity of receptor expression exists within this population of human osteoblasts. The limited distribution of the ADP-responsive receptor underlies the small response to ADP, compared with ATP, recorded in populations of human osteoblasts. This heterogeneity may reflect differences in the differentiation status of individual cells.

Adenine Nucleotides↗

PubMed1997

Clonal heterogeneity of the growth and invasive response of a human breast carcinoma cell line to parathyroid hormone-related peptide fragments.

It has been previously reported that 8701-BC cells, derived from a primary carcinoma of the breast, constitutively express parathyroid hormone (PTH)-related peptide (PTHrP) and PTH/PTHrP receptor (PTH/PTHrP-R) genes, that N-terminal, mid-regional and C-terminal immunoreactive PTHrP can be found in cell conditioned medium and, furthermore, that exogenously added PTHrP (1-34), (67-86) and, to a minor extent, (107-139) are anti-mitogenic but promote Matrigel invasion by this cell line. It has also been reported that PTHrP gene expression is selectively switched on in those 8701-BC clonal lines endowed with a higher proliferation rate and invasive ability in vitro. Here we have first examined the presence of PTH/PTHrP-R transcript in the different 8701-BC clones by PCR and Southern blot analysis. Second, we have studied the growth and invasive response in vitro to PTHrP fragments by some of these clones, i.e. BC-3A, BC-61 and BC-66, selected on the basis of their lower (BC-3A) or higher (BC-1 and BC-66) Matrigel invasion ability and their expression of PTHrP (positive for BC-61 and BC-66) and PTH/PTHrP-R (positive for BC-61). Our data show the existence of clonal heterogeneity for PTH/PTHrP-R mRNA and for the proliferative and invasive responses elicited by treatment with diverse PTHrP fragments. In particular: (i) the sensitivity to PTHrP (1-34) is restricted due to the uneven expression of PTH/PTHrP-R; (ii) BC-3A cells (the less 'aggressive' clone) are resistant to the anti-mitogenic effect of the PTHrP domains and, most noticeably, exhibit a growth-potentiating response to PTHrP (67-86) opposite to that found for both the parental 8701-BC cells and the two other clones; (iii) all PTHrP fragments tested induced the expression of a growth-restraining and invasion-promoting phenotype by BC-61 cells (one of the more 'aggressive' clones). Present data in vitro support the hypothesis that in vivo PTHrP may be a key element in local control of the invasive process during breast carcinoma development and that its role may be, in turn, dependent upon the biological characteristics and the level of malignancy of the target cells within the multiclonal population of a primary tumour.

Breast Neoplasms↗