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M Fornaro

Publications and source records attributed to M Fornaro.

At least 37 records · Page 2Linked to original sources
PubMed1999

Prostatic carcinoma cell migration via alpha(v)beta3 integrin is modulated by a focal adhesion kinase pathway.

The highly invasive human prostate cancer PC3 cell line was found to express the alpha(v)beta3 integrin; in contrast, the noninvasive LNCaP prostate cancer cell line did not express alpha(v)beta3. PC3 cells adhered to and migrated on vitronectin (VN), an alpha(v)beta3 ligand expressed in mature bone where prostate cancer cells preferentially metastasize. In contrast, LNCaP cells did not adhere to or migrate on VN. Analysis of primary human prostate cancer cells isolated from 16 surgical specimens, showed that these cells expressed alpha(v)beta3, whereas normal prostate epithelial cells did not. In addition, only primary prostate cancer cells adhered to and migrated on VN. The role of alpha(v)beta3 in mediating prostate epithelial cell migration was confirmed using LNCaP cell transfectants expressing beta3 (beta3-LNCaP). Exogenous expression of alpha(v)beta3 induced LNCaP cells to adhere to and migrate on VN. In response to alpha(v)beta3 engagement, increased tyrosine phosphorylation of focal adhesion kinase (FAK), a signaling molecule activated by integrins and able to modulate cell migration, was detected. Transfection of FAK-related nonkinase, known to compete with FAK for its correct localization and phosphorylation, caused inhibition of beta3-LNCaP cell migration, specifically on VN. These data indicate that de novo expression of alpha(v)beta3 integrin in prostate cancer cells generates a migratory phenotype that is modulated by a FAK signaling pathway. This study points to alpha(v)beta3 as potential target in prostate cancer cell invasion and metastasis.

Cell Adhesion Molecules↗
PubMed1999

p27(kip1) acts as a downstream effector of and is coexpressed with the beta1C integrin in prostatic adenocarcinoma.

Integrins are a large family of transmembrane receptors that, in addition to mediating cell adhesion, modulate cell proliferation. The beta1C integrin is an alternatively spliced variant of the beta1 subfamily that contains a unique 48-amino acid sequence in its cytoplasmic domain. We have shown previously that in vitro beta1C inhibits cell proliferation and that in vivo beta1C is expressed in nonproliferative, differentiated epithelium and is selectively downregulated in prostatic adenocarcinoma. Here we show, by immunohistochemistry and immunoblotting analysis, that beta1C is coexpressed in human prostate epithelial cells with the cell-cycle inhibitor p27(kip1), the loss of which correlates with poor prognosis in prostate cancer. In the 37 specimens analyzed, beta1C and p27(kip1) are concurrently expressed in 93% of benign and 84%-91% of tumor prostate cells. Forced expression of beta1C in vitro is accompanied by an increase in p27(kip1) levels, by inhibition of cyclin A-dependent kinase activity, and by increased association of p27(kip1) with cyclin A. beta1C inhibitory effect on cell proliferation is completely prevented by p27(kip1) antisense, but not mismatch oligonucleotides. beta1C expression does not affect either cyclin A or E levels, or cyclin E-associated kinase activity, nor the mitogen-activated protein (MAP) kinase pathway. These findings show a unique mechanism of cell growth inhibition by integrins and point to beta1C as an upstream regulator of p27(kip1) expression and, therefore, a potential target for tumor suppression in prostate cancer.

Adenocarcinoma↗
PubMed1998

Cloning of the murine TROP2 gene: conservation of a PIP2-binding sequence in the cytoplasmic domain of TROP-2.

Trop-2 is a novel calcium signal transducer expressed at high levels by most human carcinomas. To develop an animal model to study the function of this molecule in vivo, we have cloned the murine Trop2 gene. Using human TROP2 primers, we amplified by PCR a segment of murine Trop2. This was used as a probe to clone a full-length gene by hybridization of a genomic library. The cloned murine Trop2 gene is functional, as indicated by sequencing and by expression after transfection. The murine Trop2 is 87.4% similar to its human homologue, with the highest conservation in the extracellular region between residues 86 and 157. Essentially all cysteines are conserved between the human and the murine genes, suggesting conservation of the Trop2 disulfide bridges and of its overall structure. Intriguingly, the cytoplasmic tail of Trop2 shows a highly conserved phosphatidylinositol 4,5-bisphosphate (PIP2)-binding sequence, which overlaps with a protein kinase C phosphorylation site. Thus, we speculate that PIP2 might regulate the phosphorylation state of Trop2 and play a role in its signal transduction. Murine Trop2 mRNA is detected in normal kidney, lung, ovary and testis, similarly to the human gene. Interestingly, the highest levels of expression are found in immortalized keratinocytes. Since Trop2 is undetectable in undifferentiated spindle cell carcinomas, this suggests a preferential expression at early stages of tumor progression.

Amino Acid Sequence↗
PubMed1998

Beta1C integrin in epithelial cells correlates with a nonproliferative phenotype: forced expression of beta1C inhibits prostate epithelial cell proliferation.

The expression of the beta1C integrin, an alternatively spliced variant of the beta1 subunit, was investigated in human adult and fetal tissues. In the adult, beta1C immunoreactivity was found in nonproliferative, differentiated simple, and/or pseudostratified epithelia in prostate glands and liver bile ducts. In contrast, beta1C was undetectable in stratified squamous epithelium of the epidermis and/or in hepatocytes. Luminal prostate epithelial cells expressed beta1C in vivo and in vitro, but no beta1C was seen in basal cells, which are proliferating cells. Fetal prostate expressed beta1C in differentiated glands that had a defined lumen, but not in budding glands, indicating that beta1C is a marker of prostate epithelium differentiation. The beta1C and the common beta1A variants are differentially distributed: beta1A was found in luminal and basal epithelial as well as in stromal cells in the prostate. In the liver, beta1C and beta1A were coexpressed in biliary epithelium, whereas vascular cells expressed only beta1A. Because we found beta1C in nonproliferative and differentiated epithelium, we investigated whether beta1C could have a causal role in inhibiting epithelial cell proliferation. The results showed that exogenous expression of a beta1C, but not of a beta1A, cytoplasmic domain chimeric construct, completely inhibited thymidine incorporation in response to serum by prostate cancer epithelial cells. Consistent with these in vitro results, beta1C appeared to be downregulated in prostate glands that exhibit regenerative features in benign hyperplastic epithelium. These data show that the presence of beta1C integrins in epithelial cells correlates with a nonproliferative, differentiated phenotype and is growth inhibitory to prostate epithelial cells in vitro. These findings indicate a novel pathophysiological role for this integrin variant in epithelial cell proliferation.

Alternative Splicing↗
PubMed1998

Integrin laminin receptor profile of pulmonary squamous cell and adenocarcinomas.

The differential expression of laminin receptors has been shown to modulate the invasive capability of malignant cells. We have investigated the reactivity of human pulmonary squamous carcinomas (SSC, n = 20) and adenocarcinomas (ADC, n = 20) with monoclonal antibodies to the cytoplasmic and extracellular domains of the integrin subunits alpha3 and alpha6. Integrins containing these subunits are laminin receptors. Monoclonal antibodies to beta1 and beta4 subunits, the beta1C splice variant of beta1, as well as to Ki-67, were also used. Reverse transcription polymerase chain reaction (PCR) single-strand conformational polymorphism analysis was done to detect possible mutations in the cytodomains. All carcinomas expressed alpha3 extensively; alpha3 expression predominated (40 of 40) over alpha6 (25 of 40). In all alpha6-positive carcinomas, alpha6A was expressed, whereas alpha6B was weakly expressed only in some of them. No mutations of the intracytoplasmic domain A of alpha3 and of the A or B intracytoplasmic domains of alpha6 were shown. Notably, in normal bronchial epithelium, alpha6 colocalized with beta4, whereas in the tumors, alpha6A frequently overlapped with beta1 in a circumferential pattern; alpha6beta1 coexpression was also shown by coprecipitation experiments. Strong and extensive beta4 reactions were invariably polarized at the cell/stroma interface in SCC and ADC. An inverse correlation was found between the expression of beta1C and Ki-67. The prevalence of alpha6A in pulmonary SCC and ADC is in contrast with previous results in colonic ADC in which alpha6B prevails, and alpha6 predominates over alpha3. The absence of mutations of the cytodomains suggests that the integrin subunits of these carcinomas are potentially active. Predominance of alpha3 over alpha6 and of alpha6A over alpha6B may contribute to explain the aggressive and metastatic behavior of lung carcinomas.

Adenocarcinoma↗
PubMed1997

Alternatively spliced variants: a new view of the integrin cytoplasmic domain.

A large number of studies have underscored a major role for the integrin alpha beta cytoplasmic domains in the modulation of cell functions. Cytoplasmic domain variants of the beta 1, beta 3, beta 4, alpha 3, alpha 6 and alpha 7 subunits have been described. These molecules are generated by alternative splicing events and are expressed in a cell- or tissue-type specific manner. Some of these variants (beta 1C, beta 1D, alpha 6A and alpha 7A) are predominantly expressed upon differentiation and have been shown to be regulated during development. The studies on the structure-function relationship of the integrin variant subunits, published between 1989 and now, will be reviewed here for the first time. The results demonstrate that differences in the cytoplasmic domain do not affect either the alpha beta heterodimer formation or the ligand specificity. Instead, alternatively spliced integrin cytoplasmic domains appear to be essential modulators of receptor localization, cell proliferation and migration, as well as phosphorylation of signaling molecules. These observations lead to the current hypothesis that cell-type specific regulation of alternatively spliced integrin cytoplasmic domains may provide a highly specialized mechanism to control cell growth and intracellular signaling pathways.

Alternative Splicing↗
PubMed1997

Modulation of cell proliferation by the integrin cytoplasmic domain.

Integrin adhesion receptors modulate cell functions, including cell proliferation and survival. The beta 1C integrin, an alternatively spliced form of beta 1A, containing a unique cytoplasmic domain sequence, inhibits cell growth in vitro. In vivo, the expression of beta 1C correlates with a benign, nonproliferative phenotype in epithelial cells. The studies discussed in this article indicate that modulation of cell proliferation, in normal or pathological conditions, might be achieved by the regulated expression of variant integrin subunits.

Amino Acid Sequence↗
PubMed1996

High-efficiency expression gene cloning by flow cytometry.

Our goal was to develop a convenient and widely applicable procedure for gene cloning based on flow cytometry. To this purpose, we have developed an efficient protocol for DNA transfection and selection of rare transfectants. Transfection by calcium phosphate co-precipitation was extensively investigated. The use of specific batches of calcium chloride, of carrier DNA purified in guanidinium thiocyanate, and of plasmid DNA banded in cesium chloride proved crucial for high efficiency of transfection. Several tissue culture parameters were also found critical. With the optimized procedure we can transfect almost 100% of the COS-7 cells with cDNA encoding cell surface antigens or green fluorescent protein. Moreover, we routinely obtain high average levels of expression. Efficient cell sorting in flow cytometry was achieved by subtracting the cell autofluorescence background, by displacing stained cells in the red dimension, and by combining fluorescein-conjugated primary and secondary antibodies. Efficient recovery of the transfected DNA constructs was obtained from 2500-3000 cells directly sorted in Hirt lysis buffer. Using the above protocol we have cloned by expression the gene encoding Trop-2, a cell surface glycoprotein expressed by human carcinomas.

Animals↗
PubMed1996

Down-regulation of beta 1C integrin, an inhibitor of cell proliferation, in prostate carcinoma.

The beta 1C integrin, a member of the cell adhesion receptor superfamily, is an alternatively spliced variant of the beta 1A subunit and, in contrast to its wild-type counterpart, inhibits cell proliferation in vitro. The expression of beta 1C integrin in tumor cell growth was investigated. In benign and neoplastic human prostate tissues, immunohistochemical analysis performed using affinity-purified antibodies specific for beta 1C demonstrated a predominant epithelial expression of beta 1C in benign prostate glands with marked staining of the apical, basal, and lateral surfaces. In the adjacent prostate adenocarcinoma glands, the beta 1C variant was dramatically down-regulated in 27 of 34 (79%) analyzed cases, whereas the expression and distribution of its wild-type counterpart, beta 1A, remained unchanged. Tumors exhibiting different Gleason's patterns showed that beta 1C was down-regulated in comparison with the benign tissue regardless of the histological grade. Immunoblotting analysis, using affinity-purified antibodies specific for beta 1C, was performed, in a quantitative manner, to compare beta 1C expression in benign and tumor prostate tissue. The results showed that beta 1C was expressed in benign prostate tissue whereas it was undetectable in prostate adenocarcinoma. Taken together, these data show that beta 1C integrin down-regulation in prostate tissues correlates with a neoplastic phenotype consistent with its in vitro growth-inhibitory properties. These findings indicate a novel pathophysiological role for this integrin variant in tumorigenesis.

Adenocarcinoma↗
PubMed1995

The novel structural motif Gln795-Gln802 in the integrin beta 1C cytoplasmic domain regulates cell proliferation.

Alternative splicing of the integrin beta 1 subunit mRNA generates a variant form, beta 1C, with a unique cytoplasmic domain that differs from beta 1 for a 48-amino acid COOH-terminal sequence. The potential role of this unique sequence in modulating cellular functions was investigated using Chinese hamster ovary (CHO)1 cells transiently transfected with cDNAs coding for human integrin beta 1C or beta 1 subunits or mutants containing truncated forms of the beta 1C cytoplasmic domain. A differential effect of beta 1C and beta 1 on cell proliferation was observed. Expression of wild type beta 1 was associated with a 6-10-fold increase in cell proliferation in response to serum, as measured by [3H]thymidine incorporation. In contrast, only a 2-fold increase in cell proliferation was observed in transfectants expressing comparable levels of beta 1C. Cells expressing the beta 1C mutant truncated at Leu794 and lacking the last 31 amino acids of the cytoplasmic domain showed a 12-fold proliferation increase in response to serum. However, three beta 1C deletion mutants, lacking the COOH-terminal 23, 13, and 8 amino acids, which all contained residues Gln795-Gln802 of the variant cytoplasmic domain responded to serum stimulation with a 2-fold increase in [3H]thymidine uptake. The effect of beta 1C expression on cell proliferation was not associated with changes in exposure of integrin functional epitopes, as judged by the finding that CHO transfectants expressing beta 1C, full-length or deletion mutants, or beta 1 equally adhered to a functionally inhibitory monoclonal antibody against human beta 1 integrin. Expression of beta 1C inversely correlated with the mitogenic potential of vascular cells. Absent on growing cultured endothelial cells, surface expression of beta 1C was induced in growth-arrested, tumor necrosis factor-stimulated endothelial cells. These findings suggest that integrin alternative splicing may provide an accessory mechanism to modulate cell type-specific growth regulatory pathways during vascular cell injury in vivo.

Alternative Splicing↗
PubMed1995

Inhibition of cell cycle progression by the alternatively spliced integrin beta 1C.

Integrins regulate cell growth, differentiation, and behavior in many systems. Integrin beta 1C (beta 1S) is an alternatively spliced variant of integrin beta 1 with a specific cytoplasmic domain and is expressed in several human tissues. Human beta 1c transiently expressed in mouse 10T1/2 fibroblasts showed a diffuse pattern of cell surface staining, whereas beta1 localized to focal adhesions. Moderate concentrations of beta 1C had no effect on actin stress fibers or focal adhesions, but markedly inhibited DNA synthesis. Inhibition by beta 1C mapped to the late G1 phase of the cell cycle, near the G1-S boundary. Thus, alternative splicing of beta1 results in transmission of distinct signals that may regulate growth in vivo.

Alternative Splicing↗
PubMed1995

Cloning of the gene encoding Trop-2, a cell-surface glycoprotein expressed by human carcinomas.

We have cloned by expression the cDNA encoding Trop-2, a cell-surface glycoprotein expressed by most human carcinomas. Formal proof of the identity of the clone is the hybridization to DNA and RNA from genomic TROP2 transfectants. TROP2 is a single-copy gene in human cells, hybridizes to a single 1.8-kb mRNA from expressing sources and encodes a 35,709 Da type-1 transmembrane protein with a single transmembrane domain. TROP2 is essentially identical to GA733-1. Thus, we have proven that GA733-1, for which a protein product had not been identified, is a functional gene. TROP2 is also homologous to TROP1/KSA/GA733-2, confirming the serological similarities between the 2 molecules. The homology between the Trop-1 and Trop-2 peptides is clustered in 2 extracytoplasmic domains and in the transmembrane/cytoplasmic region. Twelve cysteines and a potential cytoplasmic tyrosine phosphorylation site are also conserved. Trop-1 and Trop-2 are homologous to serum IGF-II-binding proteins and appear as signal transducers. Thus, they likely represent novel cell-surface receptors and may play a role in regulating the growth of carcinoma cells. On the other hand, we have found no evidence for a role of Trop-2 and Trop-1 as homophilic adhesion molecules.

Amino Acid Sequence↗
PubMed1995

[Gastric lipoma as an unusual cause of upper gastrointestinal bleeding].

This is a case report of a gastric lipoma causing a severe upper gastrointestinal bleeding. About 200 cases of this very rare benign gastric tumor have been reported so far. Symptoms are not characteristic, but may also mimic malignancy when occurring with bleeding, obstruction or weight loss. Malignant transformation is possible, but extremely rare. Because the tumor is situated under the submucosal layer in 90%, preoperative diagnosis by endoscopic biopsy is almost never possible. The tumor has to be treated by resection. A diagnosis by frozen section during the operation is recommended.

Adult↗
PubMed1995

Regulation of leukocyte-endothelium interaction and leukocyte transendothelial migration by intercellular adhesion molecule 1-fibrinogen recognition.

Although primarily recognized for its role in hemostasis, fibrinogen is also required for competent inflammatory reactions in vivo. It is now shown that fibrinogen promotes adhesion to and migration across an endothelial monolayer of terminally differentiated myelomonocytic cells. This process does not require chemotactic/haptotactic gradients or cytokine stimulation of the endothelium and is specific for the association of fibrinogen with intercellular adhesion molecule 1 (ICAM-1) on endothelium. Among other adhesive plasma proteins, fibronectin fails to increase the binding of leukocytes to endothelium, or transendothelial migration, whereas vitronectin promotes the binding but not the migration. The fibrinogen-mediated leukocyte adhesion and transendothelial migration could be inhibited by a peptide from the fibrinogen gamma-chain sequence N117NQKIVNL-KEKVAQLEA133, which blocks the binding of fibrinogen to ICAM-1. This interaction could also be inhibited by new anti-ICAM-1 monoclonal antibodies that did not affect the ICAM-1-CD11a/CD18 recognition, thus suggesting that the fibrinogen binding site on ICAM-1 may be structurally distinct from regions previously implicated in leukocyte-endothelium interaction. Therefore, binding of fibrinogen to vascular cell receptors is sufficient to initiate (i) increased leukocyte adhesion to endothelium and (ii) leukocyte transendothelial migration. These two processes are the earliest events of immune inflammatory responses and may also contribute to atherosclerosis.

Amino Acid Sequence↗
PubMed1992

Membrane association and shedding of the GPI-anchored Ca-MOv18 antigen in human ovary carcinoma cells.

The antigen recognized by the MOv18 MAb (Ca-MOv18) was recently shown to be a glycosylphosphatidylinositol (GPI)-linked protein. In this report we show that GPI-anchorage is not limited to IGROVI cells nor to other ovary carcinoma cell lines, but Ca-MOv18 was also found to be sensitive to phosphatidylinositol-specific phospholipase C (PI-PLC) treatment on fresh ovarian cancer cells. Furthermore, we found a heterogeneous sensitivity of Ca-MOv18 to PI-PLC cleavage, not only among the different cells studied but also in different experiments performed on the same cell line, during extended periods of time in culture. Sensitivity to PI-PLC cleavage was determined by immunofluorescence on live cells and by double-determinant radioimmunoassay of the antigen released in the supernatant. The specificity of the PI-PLC cleavage was demonstrated as follows: (a) TX114 solubilized Ca-MOv18 shifts from the detergent to the aqueous phase after treatment with PI-PLC; (b) on membrane preparations, PI-PLC specifically released a fraction of the antigen, which is distinct from the weakly associated form released by high-salt treatment; (c) Ca-MOv18 from IGROVI expressed the cross-reacting determinant (CRD), which is characteristic of GPI-linked molecules. The absence of CRD expression on the spontaneously released protein and the possibility of artificially inducing antigen shedding during the resynthesis of Ca-MOv18 which follows bacterial PI-PLC treatment are interesting points which need to be further investigated in order to understand the physiology of the Ca-MOv18 tumor antigen.

Antibodies, Monoclonal↗
PubMed1992

Biochemical characterization of Trop-2, a cell surface molecule expressed by human carcinomas: formal proof that the monoclonal antibodies T16 and MOv-16 recognize Trop-2.

Trop-2 is a cell surface structure recognized by the 162-46.2 mAb and expressed by most human carcinomas. Since the 162-46.2 mAb works poorly in immunoprecipitation, to characterize the structure of Trop-2 we searched for other mAbs directed against this molecule. Selection of candidates was performed by analyzing the characteristics of mAbs directed against epithelial cells and by comparing the staining pattern of each mAb with the one of the 162-46.2 on frozen sections of human epidermis. Two mAbs, T16 and MOv-16, were selected for further analysis. Formal proof that candidate mAbs reacted with Trop-2 was obtained by comparing their binding patterns to mouse L cells transfected with the Trop-2 gene by genomic DNA transfection and selected by FACS using the FITC-162-46.2 mAb. In immunofluorescence FACS analysis the FITC-T16 and FITC-MOv-16 mAbs specifically stained Trop-2 transfectants. The specificity of binding was confirmed by selective blocking of the staining by the respective unconjugated mAb. Interestingly, cross-blocking studies indicated that the 162-46.2, T16 and MOv-16 mAbs recognize the same epitope or closely spaced ones on the Trop-2 molecule. T16 and MOv-16 efficiently immunoprecipitate Trop-2 from Trop-2 transfectants and from the human cell line OVCA-432, indicating that it is a cell surface glycoprotein, with an apparent molecular weight of 57 kD in non-reducing conditions. A weaker band of 38 kD is often co-precipitated with the 57 kD form in an apparently specific manner.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Monoclonal↗
PubMed1992

Molecular cloning, reconstruction and expression of the gene encoding the alpha-chain of the bovine CD8--definition of three peptide regions conserved across species.

We report the cloning of a cDNA encoding the alpha-chain of the bovine CD8 (BoCD8 alpha). A bovine thymus cDNA library was hybridized at low stringency with a human CD8 alpha cDNA clone. The first round of screening of 5 x 10(4) independent colonies yielded 12 clones containing incomplete BoCD8 alpha genes. Two further rounds of colony hybridization were conducted, each using as a probe the 5' fragment from the longest BoCD8 alpha clone previously isolated. The final screening yielded a clone containing a 2 kilobase (kb) insert. We mapped and sequenced the 2 kb BoCD8 alpha clone and compared it with the published sequences of the genes encoding the human, mouse and rat CD8 alpha. Sequence analysis confirmed that the clone under study encoded the BoCD8 alpha. The overall similarity of the BoCD8 alpha coding region with the human CD8 alpha coding sequence is 74.7% at the nucleotide level and 62.1% at the protein level. Lower levels of similarity are found with the mouse and rat CD8 alpha. Interestingly, three separate highly homologous regions are clearly defined at the peptide level in bovine versus human and mouse versus rat comparisons. Two of the regions are highly conserved among all species analysed, while the most 5' region is not. We speculate that the latter region may contain the binding site of CD8 alpha to the alpha 3 domain of major histocompatibility complex (MHC) class I molecules. Sequence analysis showed that the 2 kb BoCD8 alpha clone contains an incomplete coding region, i.e. lacks six bases corresponding to the first two amino acids of the leader region. To allow efficient translation and processing of the BoCD8 alpha gene, we constructed a chimeric gene containing the coding sequence of the BoCD8 alpha clone and synthetic sequences corresponding to the first two amino acids of the human CD8 alpha leader sequence. The chimeric gene was subcloned in the pKSV10 expression vector. The pKSV10-BoCD8 alpha construct is efficiently expressed both transiently in COS cells and stably in L cells, as determined by Northern blot and by FACS analysis, using the ILA-51 monoclonal antibody to BoCD8 alpha. The latter result formally proves that the ILA-51 antibody does indeed recognize the product of the BoCD8 alpha gene, as previously suggested on serological grounds.

Amino Acid Sequence↗