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Biomedical subjects

D Larocca

Publications and source records attributed to D Larocca.

18 recordsLinked to original sources
PubMed2002

Regulation of fibroblast growth factor 2 and fibroblast growth factor receptors by transforming growth factor beta in human osteoblastic MG-63 cells.

Fibroblast growth factor 2 (FGF-2) and its receptors (FGFRs) are important regulators of bone cell function. Although FGF-2 is a major modulator of bone cell function, its expression and regulation in human osteoblasts have not been investigated. We examined FGF-2 messenger RNA (mRNA) expression and regulation in the human osteosarcoma MG-63 cells. Northern analysis revealed that MG-63 cells expressed FGF-2 mRNA transcripts of 7, 4, 2.2, and 1.3 kilobases (kb). In the absence of serum, treatment with transforming growth factor beta (TGF-beta; 0.1-10 ng/ml) increased all FGF-2 mRNA transcripts. Maximal increase was seen with 1 ng/ml of TGF-beta. TGF-beta increased FGF-2 mRNA expression within 2 h and this was sustained for 24 h. Phorbal myristate acetate (PMA; 1 microM) also increased FGF-2 mRNA at 6 h. Time course studies showed that TGF-beta did not significantly alter FGFR1 or FGFR2 mRNA expression in MG-63 cells. Western blotting with anti-human FGF-2 revealed that MG-63 cells synthesize three isoforms of FGF-2 protein of approximately 18, 22/23, and 24 kDa, which were increased after either 6 h or 24 h of treatment with TGF-beta. Increased FGF-2 mRNA and protein expression in response to TGF-beta was markedly reduced by the protein kinase A (PKA) inhibitor H-89. Immunogold labeling of MG-63 cells treated with TGF-beta showed increased labeling for FGF-2 and FGFR2 in the nuclei. In contrast, TGF-beta treatment significantly decreased FGFR1 labeling in the nuclei. These data show that TGF-beta regulates FGF-2 gene expression in human osteosarcoma cells. Furthermore, TGF-beta modulates the cellular localization of FGF-2 and its receptors.

Cell Line↗
PubMed2001

Receptor-mediated gene transfer by phage-display vectors: applications in functional genomics and gene therapy.

Recent studies have demonstrated targeted gene-delivery to mammalian cells using modified phage-display vectors. Specificity is determined by the choice of the genetically displayed targeting ligand. Without targeting, phage particles have virtually no tropism for mammalian cells. Thus, novel ligands can be selected from phage libraries by their ability to deliver a reporter gene to targeted cells. Together with advances in cDNA display technologies, these findings offer new opportunities for the use of phage-display technology in functional genomics. In addition, targeted phage particles have potential as alternative gene therapy vectors that can be further improved using directed evolution.

Journal Article↗
PubMed2001

Receptor-targeted gene delivery using multivalent phagemid particles.

Although growth factor- and antibody-targeted filamentous phage have recently been demonstrated to transduce mammalian cells, there is a significant need to increase transduction efficiency so as to improve the usefulness of targeted phage vectors for gene therapy and ligand discovery. Here, we describe the use of multivalent phagemid vectors that are specifically designed for ligand-targeted mammalian cell transduction. This phagemid system has certain advantages over phage vectors, such as larger insert size and vector stability, and it retains the multivalent display necessary for efficient cell binding and internalization. Immunoblotting revealed that the most efficient multivalent display (exceeding that of a phage vector) was achieved in the phagemid system when epidermal growth factor (EGF) was fused to the C-terminal domain of the pIII coat protein. We compared phagemid particles displaying EGF at high or low valence by rescuing the vector with R408d3 (pIII deleted) or wild-type R408 helper phage, respectively. More efficient display of EGF correlated with increased internalization, vector potency, and transduction efficiency ( approximately 9%). The findings described here support our original hypothesis that phage-based vectors can be modified for more efficient gene transfer and suggest that directed evolution may be applied to increase their potential even further.

Bacteriophages↗
PubMed1999

Genetic selection of phage engineered for receptor-mediated gene transfer to mammalian cells.

Although phage display is a powerful way of selecting ligands against purified target proteins, it is less effective for selecting functional ligands for complex targets like living cells. Accordingly, phage display has had limited utility in the development of targeting agents for gene therapy vectors. By adapting a filamentous bacteriophage for gene delivery to mammalian cells, however, we show here that it is possible to screen phage libraries for functional ligands capable of delivering DNA to cells. For example, when targeted with epidermal growth factor (EGF), M13 bacteriophage were capable of delivering a green fluorescent protein (GFP) gene to EGF receptor bearing cells in a ligand-, time-, and phage concentration-dependent manner. The EGF-targeted phage transduced COS-1 cells in a highly specific manner as demonstrated by competition with excess free EGF or alternatively with anti-EGF receptor antibodies. We further demonstrate that EGF-phage can be selected, by their ability to transduce EGF receptor bearing cells from libraries of peptide display phage. When phage were incubated with COS-1 cells, EGF ligand-encoding sequences were recovered by PCR from FACsorted, GFP-positive cells and the EGF-displaying phage were enriched 1 million-fold by four rounds of selection. These data suggest the feasibility of applying molecular evolution to phage gene delivery to select novel cell-specific DNA-targeting ligands. The same approach could be used to select genetically altered phage that are specifically designed and evolved as gene therapy vectors.

Animals↗
PubMed1999

Gene transfer to mammalian cells using genetically targeted filamentous bacteriophage.

We have genetically modified filamentous bacteriophage to deliver genes to mammalian cells. In previous studies we showed that noncovalently attached fibroblast growth factor (FGF2) can target bacteriophage to COS-1 cells, resulting in receptor-mediated transduction with a reporter gene. Thus, bacteriophage, which normally lack tropism for mammalian cells, can be adapted for mammalian cell gene transfer. To determine the potential of using phage-mediated gene transfer as a novel display phage screening strategy, we transfected COS-1 cells with phage that were engineered to display FGF2 on their surface coat as a fusion to the minor coat protein, pIII. Immunoblot and ELISA analysis confirmed the presence of FGF2 on the phage coat. Significant transduction was obtained in COS-1 cells with the targeted FGF2-phage compared with the nontargeted parent phage. Specificity was demonstrated by successful inhibition of transduction in the presence of excess free FGF2. Having demonstrated mammalian cell transduction by phage displaying a known gene targeting ligand, it is now feasible to apply phage-mediated transduction as a screen for discovering novel ligands.

Animals↗
PubMed1998

Targeting bacteriophage to mammalian cell surface receptors for gene delivery.

Filamentous bacteriophages represent one of nature's most elegant ways of packaging and delivering DNA. In an effort to develop novel methods for ligand discovery via phage gene delivery, we conferred mammalian cell tropism to filamentous bacteriophages by attaching basic fibroblast growth factor (FGF2), transferrin, or epidermal growth factor (EGF) to their coat proteins and measuring CMV promoter-driven reporter gene expression in target cells. In this system, FGF2 was a more effective targeting agent than transferrin or EGF. The detection of green fluorescent protein (GFP) or beta-galactosidase (beta-Gal) activity in cells required FGF2 targeting and was phage concentration dependent. Specificity of the targeting for high-affinity FGF receptors was demonstrated by competing the targeted phage with FGF2, by the failure of FGF2-targeted bacteriophage to transduce high-affinity FGF receptor-negative cells, and by their ability to transduce these same cells when stably transfected with FGFR1, a high-affinity FGF receptor. Long-term transgene expression was established by selecting colonies for G418 resistance, suggesting that with the appropriate targeted tropism, filamentous bacteriophage can serve as a vehicle for targeted gene delivery to mammalian cells.

Animals↗
PubMed1998

Establishment of epitope-defined monoclonal antibodies with specificity for fibroblast growth factor receptor types 1 and 2.

The development of specific antibody probes for characterizing the expression of the family of 4 fibroblast growth factor receptor (FGFR) types has been difficult because of their close homology to each other and high degree of evolutionary conservation. Of the existing anti-FGFR monoclonal antibodies (MAbs), there are few that are useful for staining paraffin-embedded tissues. We have raised MAbs against human FGFR1 and FGFR2 in both rats and mice using bacterial recombinant receptor fusion proteins as immunogens. We used peptide epitope mapping to characterize the immune sera and the selected MAbs. Immunized animals were selected that displayed the broadest reactivity against epitopes unique to the immunizing receptor type. We produced FGFR1 specific MAbs that bind epitopes in immunoglobulin domain I (Ig-I) and FGFR2 specific MAbs that bind epitopes in Ig-I, Ig-II, and the acid box. The specificity of the antibodies was demonstrated by ELISA and immunoblot analysis of purified recombinant FGFR1 and FGFR2 extracellular domains produced both in E. coli and in eucaryotic cells. Based on the lack of epitope homology, these MAbs would not be expected to cross-react with FGFR3 or FGFR4. We isolated MAbs that bound to paraffin embedded tissue and immunoblots of recombinant receptor. These epitope-defined MAbs can distinguish between members of the FGF receptor family and should be useful as tools for assessing FGF receptor expression in a variety of normal and diseased tissues.

Amino Acid Sequence↗
PubMed1994

Localization of functionally important epitopes within the second C-type domain of coagulation factor V using recombinant chimeras.

Coagulation factor V, an integral component of the prothrombinase complex, possesses two C-type domains at the carboxyl-terminal end of the molecule. Homologous C-type domains are present in factor VIII as well as several non-coagulation proteins. Deletion of the second C-type domain of factor V results in the loss of procoagulant activity and the ability to bind phosphatidylserine. We now report the effect of substitution of all or a portion of the C2 domain of factor V with the corresponding regions of factor VIII or the human breast carcinoma protein BA46. Substitution of the entire domain with a heterologous C2 domain does not restore significant procoagulant activity, although smaller, exon-size substitutions do result in chimeras with partial activity (approximately 10% of factor Va). Using chimeras with partial substitutions, we determined that the amino-terminal region of the domain is involved in binding to phosphatidylserine. In contrast, the central region of the domain is not involved in phosphatidylserine binding, but an antibody binding at or near this site inhibits procoagulant activity, suggesting that this region is involved in a separate function. Lastly, the molecular basis for the light chain doublet, which is important in the expression of full procoagulant activity, is located within the carboxyl-terminal region of the C2 domain.

Animals↗
PubMed1992

A novel serum assay for breast epithelial antigen using a fusion protein.

Serum levels of breast epithelial antigens (BrE-Ags) are presently used in the follow-up of breast cancer patients. Available assays do not have optimal sensitivity and rely on reagents that could vary in their source and purity. A novel competitive solid-phase radioimmunoassay was developed for BrE-Ags that consists of the NP5 fusion protein, produced in Escherichia coli, that is composed of beta-galactosidase and polypeptide sequence obtained from a breast carcinoma cell line cDNA library, and anti-human milk fat globule monoclonal antibody Mc5. The fusion protein carries an altered epitope sequence (mimotope) that is similar, but not identical, to that found in the native antigen. This new competitive assay configuration has two essential features, a solid-phase affinity step that purifies the fusion protein carrying the mimotope for Mc5 and a competitive step that provides quantitation. Serum values for this assay show high specificity and sensitivity for breast cancer patients when compared to normal subjects and post-surgical breast cancer patients during their disease-free period.

Adult↗
PubMed1992

High level expression in E. coli of an alternate reading frame of pS2 mRNA that encodes a mimotope of human breast epithelial mucin tandem repeat.

The high molecular weight mucin found in human milk fat globule and on the surface of mammary and other epithelial cells contains a 20 amino acid tandem repeat sequence that is highly immunogenic. We have immunoscreened lambda gt11 cDNA expression libraries from MCF7 cells and lactating breast tissue with 5 anti-mucin monoclonal antibodies. We isolated a group of cDNA clones that had the repeat sequence (HB11-2, HB11-6, HB11-10) and a group that had little or no homology with the repeat sequence (NP4, NP5, HB11-4). A fusion protein produced by NP4 bound preferentially BrE2, while HB11-4 bound only BrE2 and BrE3, NP5 produced a fusion protein that bound only Mc5 and not the other MAbs. Sequencing of the NP5 cDNA revealed it to be distinct from the mucin sequence and instead to have 97% identity with the estrogen induced transcript, pS2. An alternate reading frame was translated by the lambda gt11 fusion gene yielding a 44 amino acid protein having no homology with pS2 protein. Only a short region of homology (5 amino acids) with the breast mucin tandem repeat was found which was shown to be a mimotope for the Mc5 epitope on the breast mucin. High level expression of the NP5 cDNA was achieved by subcloning it into pEX2. The NP5 fusion protein has been useful for developing an assay for the presence of mucin derived antigen in patient serum.

Amino Acid Sequence↗
PubMed1991

A Mr 46,000 human milk fat globule protein that is highly expressed in human breast tumors contains factor VIII-like domains.

The human milk fat globule has proved to be a good source of antigenic material for production of antibodies against surface components of breast epithelial cells. Monoclonal antibodies against one of the major components of the human milk fat globule, which identify a glycoprotein with an apparent molecular weight of 46,000, have been found to be useful for both breast cancer diagnosis and therapy. In order to characterize this Mr 46,000 glycoprotein, specific monoclonal antibodies were used to select complementary DNAs from a lambda gt11 expression library from lactating breast. The largest complementary DNA insert (BA46-1) was 1270 base pairs and encoded 217 amino acids. A single 2.2-kilobase RNA was specifically detected in a variety of carcinoma cell lines, using this complementary DNA probe, and it was overexpressed in some carcinoma lines. The mRNA levels correlated with the level of expression of the antigen in these cell lines as detected by Western blot analysis. Sequence analysis revealed strong homology of the Mr 46,000 glycoprotein with serum factors VIII and V, in the region implicated in phospholipid binding.

Amino Acid Sequence↗
PubMed1990

Cloning and sequencing of a complementary DNA encoding a Mr 70,000 human breast epithelial mucin-associated antigen.

The human milk fat globule (HMFG) membrane contains several glycoproteins that have been referred to as breast differentiation antigens and that are expressed in normal breast, breast tumors, breast tumor-derived cell lines, and are found in breast cancer patient serum. These antigens include a high molecular weight mucin and several smaller components including Mr 150,000; 70,000; and 46,000 glycoproteins. We have used 2 monoclonal antibodies (McR2 and Mc13) that bind the Mr 70,000 component of HMFG to immunoscreen a lambda gt11 expression library prepared from human lactating breast tissue. We report here the sequence of a complementary DNA clone (BA70-1) that codes for a peptide that binds both McR2 and Mc13 but not monoclonal antibodies to the breast mucin or other components of HMFG.A 1.8-kilobase RNA was detected in 9 of 9 breast tumor cell lines using 32P-labeled BA70-1 as probe. The BA70-1 RNA was highly expressed in 6 of 9 cells lines of breast and several other carcinomas lines compared with a lymphoblastoid cell line (Raji). The BA70-1 complementary DNA sequence has no extensive homology with previously reported sequences including the high-molecular weight mucin complementary DNA. Since the Mr 70,000 molecule appears to be associated with the breast mucin by disulfide bonds, its study could help elucidate the structure of this latter complex and how it is organized in the cell membrane, and prove useful in diagnosis and therapy of breast cancer.

Antibodies, Monoclonal↗
PubMed1989

Human T-cell leukemia virus minus strand transcription in infected T-cells.

The plus strand of the Human T-cell Leukemia Virus RNA genome encodes proteins critical for infectivity, replication, and transformation. Thus far, gene products encoded on the minus strand of the HTLV-I provirus have not been found. We have identified several open reading frames, located between transcription start and poly (A) signal sequences on the minus strand of HTLV-I. The 3' terminus of HTLV-I was shown to promote test gene expression in either orientation. RNA blots probed with single stranded RNA complementary to the predicted transcribed region revealed 2.5 and 2.9 kilobase minus strand RNAs in HTLV-I infected T-cells but not in an uninfected control. The effect of mutations on minus strand gene products should also be considered when studying human retroviral protein function by mutational analysis.

Amino Acid Sequence↗
PubMed1986

Ecdysterone and heat shock induction of transfecting and endogenous heat shock genes in cultured Drosophila cells.

Various combinations of the heat shock genes of Drosophila melanogaster are transcribed in response to elevated temperature and also during normal development and in certain ecdysterone-treated Drosophila cell lines. Here I describe an homologous transient expression system using Schneider 3 cells, a Drosophila line responsive to ecdysterone. I compare the constitutive, ecdysterone and heat shock-induced accumulation of transcripts of five transfecting heat shock genes (hsp82, hsp70, hsp28, hsp26 and hsp23) to the accumulation of transcripts of their endogenous counterparts. The pattern of expression of the transfecting genes under these various conditions is generally similar to that of the endogenous genes.

Animals↗
PubMed1982

Hypomethylation of Epstein-Barr virus DNA in the nonproducer B-cell line EBR.

The conversion of the Epstein-Barr virus-negative Ramos cell line has previously been shown to result in an Epstein-Barr virus-positive non-virus-producer cell line, EBR. We report here that Epstein-Barr virus DNA from EBR alone among several cell lines examined was totally unmethylated at three of four sites containing guanine plus cytosine which were tested. This is in direct contrast to reports of high degrees of methylation in the DNAs of other animal viruses, including herpesviruses, isolated from cells in which the viral genome is expressed at a low level.

5-Methylcytosine↗
PubMed1977

Bacterial growth kinetics on diphenylmethane and naphthalene-heptamethylnonane mixtures.

Experiments were carried out to determine if diphenylmethane is utilized by a species of Pseudomonas (Hydrogenomonas) in the dissolved state regardless of the physical state (liquid or solid) of the undissolved diphenylmethane suspended in the medium. Bacterial growth rates in the presence of various amounts of solid or liquid diphenylmethane indicate that liquid diphenylmethane is utilized at the aqueous-diphenylmethane interface but that solid diphenylmethane is not. A Pseudomonas sp. that was isolated on naphthalene (solid), but could not utilize heptamethylnonane, was grown in the presence of various amounts of a naphthalene-heptamethylnonane mixture (liquid). The growth rates indicate that the bacterium could utilize naphthalene at the aqueous-hydrocarbon interface, which is not the case in the absence of the heptamethylnonane.

Journal Article↗