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Biomedical subjects

M Falzon

Publications and source records attributed to M Falzon.

At least 19 recordsLinked to original sources
PubMed2004

Imprint cytology of the sentinel lymph node in the assessment of axillary node status in breast carcinoma.

BACKGROUND: Intra-operative assessment of the sentinel lymph node (SLN) status may allow the surgeon to complete the breast cancer surgery in most patients in one sitting. We have studied the role of imprint cytology in the assessment of SLN status. PATIENTS AND METHODS: Imprint cytology of the SLN in 132 patients with invasive breast carcinoma was correlated with the histopathological assessment of the SLN and overall axillary nodal status. In 26 patients, the cytology was reported intra-operatively. RESULTS: Imprint cytology reflected the status of the parent node well (sensitivity 86%, specificity 97%, positive predictive value (PPV) 92%, negative predictive value (NPV) 93%). Its ability to reflect the axillary status was also good (sensitivity 70%, specificity 97%, PPV 95% and NPV 83%) but somewhat diminished by the relatively high number of false-negative SLN in the study. Intra-operative assessment (sensitivity 86%, specificity 100%, PPV 100% and NPV 95%) did not reduce the accuracy of imprint cytology in predicting the SLN status and took a mean of 24.5 min. CONCLUSIONS: Imprint cytology is an accurate and relatively simple method for the assessment of the SLN and can be a useful intra-operative tool.

Adult↗
PubMed2003

Why abolish C3?

Explore the source record for details and available documents.

Biopsy, Fine-Needle↗
PubMed2001

Overexpression of parathyroid hormone-related protein promotes cell growth in the rat intestinal cell line IEC-6.

The rat intestinal cell line, IEC-6, was used as a model to study effects of parathyroid hormone-related protein (PTHrP) on crypt cell growth. Studies showed that addition of PTHrP analogs (1-34), (67-86), or (107-139) to growth medium did not affect proliferation of cells grown in either high (10% Nu-Serum) or low serum (1% Nu-Serum). However, studies on clonal lines of IEC-6 cells stably transfected with PTHrP cDNA and overexpressing PTHrP showed that increased PTHrP production enhanced cell growth and 3H-thymidine incorporation in high, but not low, serum. Additional studies examined the role of the nuclear localization sequence (NLS) of PTHrP in mediating the growth effect. In three clonal IEC-6 lines transfected with PTHrP cDNA bearing a mutated NLS, the ability of PTHrP to stimulate 3H-thymidine incorporation and cell growth was lost. The results suggest that endogenously produced PTHrP can promote proliferation of IEC-6 cells and that the integrity of the NLS of PTHrP is required for its growth effects.

Animals↗
PubMed2001

Overexpression of parathyroid hormone-related protein enhances apoptosis in the rat intestinal cell line, IEC-6.

We used the rat intestinal cell line, IEC-6, to study potential effects of overexpression of PTH-related protein (PTHrP) on apoptosis. A clonal line of PTHrP-overexpressing cells was established by stably transfecting parental cells with PTHrP complementary DNA in a sense orientation (sense). A similarly transfected line stably, transfected with empty vector, served as control (vector). Immunoreactive PTHrP, measured in culture medium, showed that sense cells secreted approximately 30 times as much PTHrP as did vector control cells. Apoptosis induced by serum withdrawal was evaluated by several methods. DNA laddering was demonstrable in sense-transfected cells as early as 12 h after serum withdrawal but not until later time points in vector-transfected control cells. Flow cytometric analysis of propidium iodide-stained cells showed a greater increase in the sub-G1 (apoptotic) population in sense cells, compared with vector. Fluorescent microscopy with Hoechst 33258 dye showed increased nuclear fragmentation and condensation in sense cells. Studies of apoptotic gene expression by ribonuclease protection assay, and protein by Western blot analysis, showed an enhanced ratio of Bax to Bcl-x(L) in sense cells. Mutation of the PTHrP nuclear localization amino acid sequence negated the ability of PTHrP to enhance apoptosis.

Animals↗
PubMed2000

Single-column purification and bio-characterization of recombinant human parathyroid hormone-related protein (1-139).

Recombinant human parathyroid hormone-related protein (hPTHrP) (1-139) was expressed using the IMPACT T7 (intein-mediated purification with an affinity chitin-binding tag) system, allowing purification of free recombinant peptide in a single chromatographic step. This system utilizes an intein, which is a protein splicing element from the Saccharomyces cerevisiae VMA1 gene. The intein has been modified so that it undergoes a self-cleavage reaction at its N-terminus at low temperatures in the presence of 1,4-dithiothreitol (DTT). The cDNA encoding hPTHrP (1-139) was cloned into the pTYB1 vector to create an in-frame fusion at the N-terminus of the intein gene. The cDNA for the chitin-binding domain from Bacillus circulans is present at the C-terminus of intein for affinity purification of the three-part fusion protein on a chitin column. The recombinant plasmid was transfected into E. coli ER2566 cells and synthesis of the PTHrP fusion protein was induced with isopropyl-beta-D-thiogalactopyranoside (IPTG). This system produced pure hPTHrP (1-139) and an N-terminally truncated analogue, hPTHrP (27-139), as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis, Western blot analysis, N-terminal sequence analysis and mass spectroscopy. hPTHrP (1-139) stimulated cAMP accumulation in ROS 17/2.8 osteoblastic bone cells, whereas hPTHrP (27-139) failed to elicit a response. hPTHrP (1-139) also inhibited the growth of the breast cancer cell line MDA-MB-231; the magnitude of the response was comparable with that of synthetic hPTHrP (1-34) and (1-86). Neutralization of endogenous PTHrP and added hPTHrP (1-139) and N-terminal species with an anti-PTHrP antiserum completely abolished the growth inhibitory effects. These results indicate that the added peptides modulate cell growth by acting at the cell surface. Availability of recombinant hPTHrP (1-139) will allow further study of its biological function, as well as its structure.

Breast Neoplasms↗
PubMed2000

Characterization of human and rat glucagon-like peptide-1 receptors in the neurointermediate lobe: lack of coupling to either stimulation or inhibition of adenylyl cyclase.

Glucagon-like peptide-1 (GLP-1) has been shown to bind to the posterior pituitary in the rat. We examined GLP-1 binding sites in human postmortem and rat pituitaries. Dense [125I]GLP-1 binding was seen in both human and rat posterior pituitary. In rat neurointermediate lobe membranes the binding site showed a Kd of 0.2 +/- 0.01 nM and a binding capacity of 600 +/- 33 fmol/mg protein (n = 3). In human pituitary membranes the binding site showed a Kd of 0.82 +/-0.05 nM and a binding capacity of 680 +/- 93 fmol/mg protein (n = 3). Chemical cross-linking showed a relative mol wt for the receptor-ligand complex of 73,100 +/- 1,400 (n = 3) in man and 59,300 +/- 900 (n = 3) in rat. GLP-1 (1 microM) failed to increase cAMP levels measured in rat neurointermediate lobes, whereas pituitary adenylate cyclase-activating polypeptide (100 nM) increased cAMP from a basal level of 14 +/-1 to 80 +/- 4 pmol/neurointermediate lobe 15 min (n = 5; P < 0.01). GLP-1 (up to 1 microM) did not affect the pituitary adenylate cyclase-activating polypeptide-stimulated cAMP levels. GLP-1 (up to 1 microM) also did not stimulate release of vasopressin or oxytocin from isolated rat neurointermediate lobes. The posterior pituitary shows the highest density of GLP-1-binding sites yet seen, but their function and signal transduction mechanism remain unknown.

Adenylyl Cyclase Inhibitors↗
PubMed2000

Enhanced growth of MCF-7 breast cancer cells overexpressing parathyroid hormone-related peptide.

PTH-related peptide (PTHrP) is a secreted protein produced by breast cancer cells both in vivo and in vitro. Because of its structural similarity to PTH at the amino terminus, the two proteins interact with a common cell surface receptor, the PTH/PTHrP receptor. When overproduced by tumor cells, PTHrP enters the circulation, giving rise to the common paraneoplastic syndrome of humoral hypercalcemia of malignancy. Although initially discovered in malignancies, PTHrP is now known to be produced by most cells and tissues in the body. It acts as an autocrine and paracrine mediator of cell proliferation and differentiation, effects which are mediated via the PTH/PTHrP receptor. Recent evidence also has shown that, directly after translation, PTHrP is able to enter the nucleus and/or nucleolus and influence cell cycle progression and apoptosis. In this study, we have either overproduced PTHrP or inhibited endogenous PTHrP production in the breast cancer cell line, MCF-7. Overexpression of PTHrP was associated with an increase in mitogenesis, whereas inhibiting endogenous PTHrP production resulted in decreased cell proliferation. The overexpressed peptide targeted to the perinuclear space. In contrast, PTHrP interaction with the cell surface PTH/PTHrP receptor resulted in decreased cell proliferation in the same cell line. This latter effect is dependent on interaction with the receptor, in that exogenously added PTHrP moieties known not to interact with the receptor had no effect on cell growth. Furthermore, neutralization of added peptide with an anti-PTHrP antiserum completely abolished the growth inhibitory effects. In contrast, this antibody has no effect on the increased proliferation rate of the MCF-7 transfectants that overexpress PTHrP, compared with control cells. The net effect of autocrine/paracrine and intracrine effects of PTHrP in MCF-7 cells overproducing the peptide is accelerated cell growth. These findings have critical implications regarding the role of PTHrP in breast cancer, and they suggest that controlling PTHrP production in breast cancer may be useful therapeutically.

Antibodies, Monoclonal↗
PubMed1998

The noncalcemic vitamin D analogs EB1089 and 22-oxacalcitriol suppress serum-induced parathyroid hormone-related peptide gene expression in a lung cancer cell line.

PTH-related peptide (PTHrP) mediates the syndrome of humoral hypercalcemia of malignancy, a frequent complication of squamous cell carcinomas of the lung. This study was undertaken to determine whether 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] and two nonhypercalcemic analogs, EB1089 and 22-oxa-1,25-(OH)2D3 (22-oxacalcitriol), suppress serum- and epidermal growth factor (EGF)-induced PTHrP gene expression in a human lung squamous cancer cell line, NCI H520. PTHrP expression was up-regulated by serum and EGF in a concentration- and time-dependent manner. Nuclear run-on analysis showed that this induction was mediated via a transcriptional mechanism, and that sequences within promoter 1 were responsible. All three vitamin D3 compounds decreased both basal and serum- and EGF-induced steady state PTHrP messenger RNA and secreted peptide levels. These effects were again mediated via a transcriptional mechanism through sequences within promoter 1. All three vitamin D3 compounds also decreased the proliferation of NCI H520 cells in a concentration- and time-dependent manner. 1,25-(OH)2D3 is hypercalcemic in vivo. However, the noncalcemic analogs EB1089 and 22-oxa-1,25-(OH)2D3 have therapeutic potential, as they suppress not only the basal but also the growth factor-stimulated levels of PTHrP in a cancer cell line associated with hypercalcemia.

Blood Physiological Phenomena↗
PubMed1997

The noncalcemic vitamin D analogues EB1089 and 22-oxacalcitriol interact with the vitamin D receptor and suppress parathyroid hormone-related peptide gene expression.

Humoral hypercalcemia of malignancy, a frequent complication of squamous cell carcinomas of the lung, is mediated by the parathyroid hormone-related peptide (PTHrP). This study was undertaken to determine whether 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] and two nonhypercalcemic analogues. EB1089 and 22-oxa-1,25(OH)(2)D(3) (OCT), suppress PTHrP gene expression in a human lung squamous cancer cell line, NCI H520. All three compounds (1) decreased steady-state PTHrP mRNA and secreted peptide levels via a transcriptional mechanism; (2) modulated promoter activity of 1,25(OH)(2)D(3)-responsive DNA sequences; and (3) activated the vitamin D receptor (VDR) both in vitro and in vivo. Thus, EB1089 and OCT inhibit PTHrP gene expression in NCI H520 cells and modulate gene expression through the same mechanism as 1,25(OH)(2)D(3), namely, activation of the VDR. 1,25(OH)(2)D(3) is hypercalcemic in vivo. However, the noncalcemic analogues EB1089 and OCT have a therapeutic potential through suppression of PTHrP gene transcription.

Antineoplastic Agents↗
PubMed1996

Serum stimulation of parathyroid hormone-related peptide gene expression in ROS 17/2.8 osteosarcoma cells through transcriptional and posttranscriptional mechanisms.

The gene encoding PTH-related peptide (PTHrP), a protein that plays a primary role in the development of humoral hypercalcemia of malignancy, is widely expressed in normal and neoplastic tissues. This study demonstrates that expression of the PTHrP gene has features of early response genes, including up-regulation after serum repletion of serum-starved ROS 17/2.8 (rat osteosarcoma) cells. The PTHrP messenger RNA (mRNA) levels were induced within 30 min and peaked at 4 h. Elevated mRNA levels were accompanied by an increase in secreted PTHrP. The serum effects on PTHrP mRNA levels were blocked by actinomycin D, suggesting a requirement for gene transcription. Nuclear run-on assays revealed a 3-fold increase in PTHrP gene transcription 4 h after exposure to serum. Deletions of the 5' flanking sequence of the rat PTHrP gene fused to the chloramphenicol acetyltransferase gene and transfected into ROS 17/2.8 cells showed that the serum-responsive region is located between -1.05 kb and -0.3 kb upstream of the transcription start site. PTHrP mRNA levels were also induced by cycloheximide, another feature common to early response genes. The PTHrP mRNA half-life in serum-starved cells was 56 min. Serum treatment prolonged the half-life 2.7-fold, suggesting serum-induced stabilization of the mRNA. Insulin and epidermal growth factor also induced PTHrP mRNA expression in a time-dependent manner analogous to serum, indicating that the effects of serum may be mediated, at least partially, through these agents. In summary, serum up-regulated PTHrP mRNA expression through both transcriptional and posttranscriptional mechanisms. This rapid stimulation by growth factors suggests that PTHrP may contribute to the early cellular response after growth factor stimulation.

Animals↗
PubMed1996

DNA sequences in the rat parathyroid hormone-related peptide gene responsible for 1,25-dihydroxyvitamin D3-mediated transcriptional repression.

Expression of the gene encoding PTH-related peptide (PTHrP), a protein that plays a primary role in the development of humoral hypercalcemia of malignancy, is down-regulated at the transcriptional level by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. Deletions of the 5'-flanking region of the rat PTHrP gene, when fused to the chloramphenicol acetyl-transferase gene and transfected into ROS 17/2.8 (rat osteosarcoma) cells, showed that the 1,25-(OH)2D3 responsive region is located between -1.05 and -0.71 kb upstream of the transcription start site. Further mapping of this region revealed that a 123-bp fragment is able to confer 1,25-(OH)2D3 responsiveness to a heterologous (SV40) promoter. This region contains two potential vitamin D response elements (VDREs). One of these motifs resembles the negative VDRE (nVDRE) from the PTH gene, which is also down-regulated by vitamin D3. The other element resembles the canonical VDRE (two hexanucleotide motifs separated by three nucleotides), which has been characterized in a number of genes whose expression is modulated by vitamin D3. Electrophoretic mobility shift assays using nuclear extracts from ROS 17/2,8 cells and from vitamin D receptor. (VDR)-enriched COS 1 cells revealed that both elements interact with the VDR. This protein-DNA interaction is disrupted by an anti-VDR antibody. Therefore, modulation of PTHrP gene transcription by 1,25-(OH)2D3 is mediated by the VDR interacting with one or both of the identified motifs in the 5'-flanking sequence of the gene.

Animals↗
PubMed1994

DNA-dependent protein kinase is activated by nicks and larger single-stranded gaps.

DNA-PK is a DNA-activated serine/threonine protein kinase capable of phosphorylating a number of nuclear DNA-binding proteins. Purified human DNA-PK has two subunits, a 350-kDa polypeptide, Prkdc, which binds ATP and is presumed to contain the catalytic site, and the Ku autoantigen which mediates DNA binding and activation. Previous studies have shown that DNA-PK is activated in vitro by linear double-stranded DNA fragments; however, the Ku subunit binds a broader range of DNA structures. Here we show that EBP-80, a protein originally isolated as a transcription factor for a retroviral long terminal repeat element and subsequently found to be similar to if not identical with Ku, also mediates kinase activation. The EBP-80-Prkdc complex is activated by nanomolar concentrations of DNA constructs containing single-to-double strand transitions, including a closed stem-loop structure and single strand gaps of 0 (nick), 6, and 30 nucleotides. Kinase activation parallels the ability of EBP-80 to bind these and other constructs. Our results extend the range of DNA configurations known to activate DNA-PK and are consistent with the participation of this enzyme complex in several nuclear functions.

Adenosine Triphosphate↗
PubMed1993

EBP-80, a transcription factor closely resembling the human autoantigen Ku, recognizes single- to double-strand transitions in DNA.

We have previously reported the purification and characterization of the transcription factor EBP-80 (Falzon, M., and Kuff, E. L. (1989) J. Biol. Chem. 264, 21915-21922). EBP-80 mediates the DNA methylation effect on transcription from an endogenous proviral long terminal repeat. Here we show that EBP-80 is very similar if not identical to the Ku autoantigen, a heterodimeric nuclear protein first detected by antibodies from autoimmune patients (Mimori, T., Akizuki, M., Yamagata, H., Inada, S., Yoshida, S., and Homma, M. (1981) J. Clin. Invest. 68, 611-620). A number of laboratories have shown that the Ku protein complex binds to free double-stranded DNA ends. In this study, we have examined the binding properties of EBP-80. EBP-80 binds single-stranded DNA with low affinity. Binding to random sequence double-stranded DNA depends on the length of the duplex and is optimal with oligomers of 30 and 32 base pairs; the protein complexes formed with these oligomers have Kd values of 15-20 pM. It binds with comparable high affinities to blunt-ended duplex DNA, to duplex DNA ending in hairpin loops, and to constructs in which an internal segment of duplex DNA is flanked by single-strand extensions. EBP-80 also interacts effectively with circular duplex molecules containing a 30-nucleotide single-stranded region (gap) or a double-stranded segment of nonhomology (bubble), but only weakly with the corresponding closed circular construct made up entirely of duplex DNA. EBP-80 prefers A/T to G/C ends. The binding properties of EBP-80 are consistent with the hypothesis that is recognizes single- to double-strand transitions in DNA. A model is presented for the interaction of EBP-80 with its target sequence in the proviral long terminal repeat.

Adenoviruses, Human↗
PubMed1992

Urinary tract metastasis from renal carcinoma.

Five patients with urinary tract metastases from renal carcinoma are presented. In 4, urinary tract metastasis occurred in the absence of spread elsewhere. The initial presentation in 1 patient was ureteric obstruction; 3 others re-presented with haematuria and the fifth patient was diagnosed post mortem. Metastatic sites included ipsilateral ureter, contralateral ureter, ureteric stump, bladder and prostatic fossa. After previous nephrectomy for renal carcinoma, urinary metastases, although rare, should be considered and investigation of unexplained haematuria should include the ureteric stump. Useful palliation and possibly cure can be achieved by resection of these lesions.

Aged↗