Biomedical subjects

B Iacopetta

Publications and source records attributed to B Iacopetta.

46 records · Page 3Linked to original sources

PubMed1997

p53 alterations are associated with improved prognosis in distal colonic carcinomas.

Alterations of the p53 gene and the p53 protein are common in a wide spectrum of human malignancies. In several tumor types, p53 gene mutation and/or p53 protein overexpression correlate with a more clinically aggressive phenotype as judged by worse patient survival. This has not been clearly demonstrated to be the case in colorectal cancer. Herein, we report results of the prognostic significance of p53 protein accumulation and gene mutation in a large series of colorectal cancers (n = 541) with long patient follow-up (mean, 87 months). The large majority of patients (95%) received no postoperative systemic adjuvant therapy. The incidence of p53 accumulation detected by immunohistochemistry with the monoclonal antibody DO-7 was 30%, whereas the incidence of p53 gene mutation in exons 5-8 detected using PCR-single strand conformation polymorphism was 36%. Accumulation of p53 protein was associated with improved patient survival independent of tumor stage or grade (hazard ratio, 0.66; 95% confidence interval, 0.47-0.93; P = 0.017). A marked difference was observed depending on the location of the tumor: tumors originating in the distal colon showed a strong association between the presence of p53 accumulation and improved patient survival (P = 0.003), but this was not the case for those located in the proximal colon. Dukes' stage C tumors, but not stage B, also showed an association between p53 accumulation and better outcome (P = 0.013). Mutation of the p53 gene was associated with a trend toward improved survival, particularly in the distal tumors. Our results demonstrate that in some tumor types, the presence of p53 abnormalities can correlate with better prognosis.

Aged↗

PubMed1996

Nested PCR-SSCP assay for the detection of p53 mutations in paraffin wax embedded bone tumours: improvement of sensitivity and fidelity.

DNA extraction and PCR amplification from paraffin wax embedded bone tumour specimens present several difficulties, firstly, because of the abundant matrix they contain and, secondly, because decalcification often causes degradation of DNA. In this report, comparative studies were carried out to determine the most efficient method for DNA extraction and PCR amplification from such specimens. The results indicated that nested PCR produced appropriate strong reaction products with minimal background contamination. A method for DNA extraction from paraffin wax embedded bone tissue and a nested PCR-SSCP technique have been developed for use in such diagnostic specimens.

Journal Article↗

PubMed1994

Loss of heterozygosity of tumour suppressor gene loci in human colorectal carcinoma.

We used Southern blot analysis and polymerase chain reaction-based techniques to examine deletions of tumour suppressor gene loci in 91 primary colorectal tumours. The tumour suppressor genes studied were MCC and APC on chromosome 5q, p53 on chromosome 17p, DCC on chromosome 18q, and the putative suppressor gene nm23-H1 on chromosome 17q. The most frequent allelic loss observed was in chromosome 17p with 76% (68/89) of informative tumours showing loss of heterozygosity at this locus, followed by 34% (19/55) for DCC, 31% (12/39) for MCC, 17% (9/53) for APC and 16% (3/19) for nm23. No significant differences in the frequency of these suppressor gene allelic losses were observed between Dukes B and C stage adenocarcinomas.

Adenocarcinoma↗

PubMed1994

Comparison of p53 gene mutation and protein overexpression in colorectal carcinomas.

Immunocytochemistry (ICC) has been used routinely to stain for p53 overexpression in a range of human tumours. The underlying assumption has been that positive staining indicates a mutation in the p53 coding sequence. Recently, however, discordancy has been observed and the accuracy of ICC as a marker of p53 gene mutation has been questioned. In this study of 109 colorectal adenocarcinomas, we compared ICC staining with p53 gene mutations detected by single-strand conformation polymorphism (SSCP) analysis. Concordancy between the two techniques was found in 69% of tumours. ICC-positive/SSCP-negative cases accounted for 20% of tumours and ICC-negative/SSCP-positive cases for the remaining 11%. These results caution against the assumption that p53 protein overexpression is always associated with a gene mutation. Epigenetic phenomena may account for a significant proportion of ICC-positive tumours.

Antibodies, Monoclonal↗

PubMed1993

The internalization signal and the phosphorylation site of transferrin receptor are distinct from the main basolateral sorting information.

Wild-type human transferrin receptor (hTfR), like endogenous canine receptor, is expressed almost exclusively (97%) at the basolateral membrane of transfected Madin-Darbey canine kidney (MDCK) cells. We investigated the role of two distinct features of the hTfR cytoplasmic domain, namely the endocytic signal and the unique phosphorylation site, in polarized cell surface delivery. Basolateral location was not altered by point mutation of Ser24-->Ala24, indicating that phosphorylation is not involved in vectorial sorting of hTfR. The steady state distribution of hTfR was partially affected by a deletion of 36 cytoplasmic residues encompassing the internalization sequence. However, 80% of the receptors were still basolateral. As assessed by pulse-chase experiments in combination with biotinylation, newly synthesized wild-type and deletion mutant receptors were directly sorted to the domain of their steady state residency. Although both receptors could bind human transferrin, endocytosis of the deletion mutant was strongly impaired at either surface. These data indicate that the predominant basolateral targeting signal of hTfR is independent of the internalization sequence.

Amino Acid Sequence↗

PubMed1991

Internalization pathway of C3b receptors in human neutrophils and its transmodulation by chemoattractant receptors stimulation.

On the surface of phagocytes, C3b receptors (CR1) bind C3b-coated particles and promote their ingestion after activation by appropriate stimuli such as lymphokines or the chemoattractant formyl methionyl leucyl phenylalanine (fMLP) and fibronectin. The aims of the present study were 1) to define at the electron microscopic level the nature of the process responsible for CR1 internalization and 2) to dissect the mechanism by which a physiological activator (fMLP) stimulates this process. CR1 was visualized either by the immunogold technique or by quantitative electron microscopic autoradiography using a monoclonal anti-CR1 antibody. Both techniques revealed that after anti-CR1 binding, CR1 cluster on the neutrophil surface in a time-, temperature-, and antibody-dependent fashion, but do not concentrate in coated pits. CR1 internalization requires receptor cross-linking (does not occur in the presence of Fab fragments of anti-CR1) and intact microfilaments. It results in the association of the internalized material with large flattened vacuoles, organized in stacks. Together with the surface localization of CR1 close to cytoplasmic projections (ruffles), these observations suggest that uptake of CR1 occurs through a macropinocytotic process. Eventually, CR1 concentrate in lysosomal structures. fMLP markedly stimulates this pattern of CR1 internalization without affecting their clustering or their lack of association with coated pits. Stimulation by fMLP is inhibited by pertussis toxin, unaffected by preventing receptor-triggered cytosolic free calcium [Ca2+]i elevations, and mimicked by phorbol myristate acetate. Taken together our data demonstrate 1) that, in neutrophils, CR1 is internalized via a coated pit independent macropinocytotic process, dependent on intact microfilaments and receptor cross-linking; 2) that, in the same cells, fMLP is internalized via the classical coated pits pathway; and 3) that fMLP amplifies CR1 uptake possibly via protein kinase C stimulation.

Autoradiography↗

PubMed1986

Role of intracellular calcium and protein kinase C in the endocytosis of transferrin and insulin by HL60 cells.

The role of the cytosolic free calcium concentration ([Ca2+]i) and of protein kinase C on the internalization of transferrin and insulin in the human promyelocytic cell line HL60 was investigated. [Ca2+]i was selectively monitored and manipulated by the use of the fluorescent Ca2+ indicator and buffer quin2, while receptor-ligand internalization was studied directly by quantitative electron microscope autoradiography. Decreasing the [Ca2+]i up to 10-fold below resting level had no effect on the internalization of transferrin or insulin. Similarly, a 10-fold elevation of the [Ca2+]i using the calcium ionophore ionomycin caused little or no change in the endocytosis of the two ligands. In contrast, activation of protein kinase C by phorbol myristate acetate markedly stimulated the internalization of both occupied and unoccupied transferrin receptors, even in cells with very low [Ca2+]i. The insulin receptor was found to behave differently in response to phorbol myristate acetate, however, in that only the occupied receptors were stimulated to internalize. We conclude that the [Ca2+]i plays only a minor role in regulating receptor-mediated endocytosis, whereas protein kinase C can selectively modulate receptor internalization depending on receptor type and occupancy.

Calcium↗

PubMed1986

Diacylglycerol modulation of insulin receptor from cultured human mononuclear cells. Effects on binding and internalization.

Tumor-promoting phorbol esters alter binding of growth factors and hormones to their specific receptors. Action of diacylglycerols, endogenous phorbol ester analogues, on 125I-labeled insulin binding to its receptor from human cells was therefore investigated. A variety of 1,2-diacylglycerols and 1,3-diacylglycerols inhibited 125I-insulin binding to intact human monocyte-like (U-937) and lymphoblastoid (IM-9) cells in a dose-, time-, and temperature-dependent manner within 30 sec at 37 degrees C in a fashion analogous to that of the tumor-promoting phorbol diester 12-O-tetradecanoylphorbol-13-acetate (TPA). Inhibition of insulin binding by diacylglycerols, analyzed by Scatchard plot, seems to be due to altered binding affinity of the insulin receptor. Diacylglycerol effects were reversible, were seen regardless of the order of addition of 125I-insulin and diacylglycerols, and were demonstrated only with occupied insulin receptors. Corresponding fatty acids or phospholipids did not affect specific insulin binding to the intact U-937 cells. Diacylglycerols also inhibited binding of 125I-insulin-like growth factor (IGF) I but not that of 125I-human growth hormone (HGH) to the human cells. The non-tumor-promoting phorbols (phorbol, 4-alpha-phorbol, phorbol-12,13-distearate) did not affect insulin binding to intact cells. Both diacylglycerols and TPA stimulated internalization of 125I-insulin by U-937 and IM-9 cells. The ability of diacylglycerol to mimic the effects of TPA on the insulin receptor supports the concept of diacylglycerols as endogenous phorbol diester analogues even though the sole role of protein kinase C in our system is doubtful.(ABSTRACT TRUNCATED AT 250 WORDS)

Cells, Cultured↗

PubMed1985

Detection of surface-bound ligands by freeze-fracture autoradiography.

This article describes a new freeze-fracture autoradiographic technique for the detection of radioactive ligands associated with the surface of cells in monolayer or suspension culture. Since freeze-fracture replicas are produced in the conventional way, all membrane features normally seen in freeze-fracture are retained, and autoradiographic grains produced by the labeled ligands are seen superimposed on unaltered exoplasmic membrane fracture faces. To assess the feasibility and resolution of this technique, we compared the surface distribution of alpha 2-macroglobulin and cholera toxin, labeled either with 125I or with colloidal gold, on 3T3-L1 fibroblasts. Both by autoradiography and cytochemical gold labeling, alpha 2-macroglobulin was associated specifically with coated pits, whereas cholera toxin was preferentially found over smaller, apparently non-coated membrane invaginations. Together with data on the surface localization of 125I-transferrin on HL-60 myelomonocytic cells, these results demonstrate the application of this technique for the accurate determination of ligand distribution over large areas of plasma membrane. The simplicity and reproducibility of the method should now allow freeze-fracture autoradiography to become a standard technique for investigating the distribution of both endogenous and exogenous cell surface-associated molecules, as well as the redistribution of such molecules under different experimental conditions.

Animals↗

PubMed1984

Heme inhibits transferrin endocytosis in immature erythroid cells.

The inhibitory effect of heme on iron uptake from transferrin by rat and rabbit reticulocytes and erythroid cells from the fetal rat liver was studied in vitro. Addition of hemin was shown to cause a decrease in the rate of transferrin endocytosis, the degree of inhibition being proportional to the reduction in iron uptake. The heme synthesis inhibitors, isoniazid and succinylacetone, stimulated the rate of transferrin endocytosis by 15-30% and caused a proportional increase in the rate of iron uptake, possibly by reducing the intracellular free heme concentration. It is concluded from these results that heme affects iron uptake by influencing the rate of transferrin endocytosis and recycling.

Animals↗