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Biomedical subjects

L Connolly

Publications and source records attributed to L Connolly.

14 recordsLinked to original sources
PubMed2001

Selection with melphalan or paclitaxel (Taxol) yields variants with different patterns of multidrug resistance, integrin expression and in vitro invasiveness.

A melphalan-resistant variant (Roswell Park Memorial Institute (RPMI)-2650Ml) and a paclitaxel-resistant variant (RPMI-2650Tx) of the drug-sensitive human nasal carcinoma cell line, RPMI-2650, were established. The multidrug resistance (MDR) phenotype in the RPMI-2650Tx appeared to be P-glycoprotein (PgP)-mediated. Overexpression of multidrug resistant protein (MRP) family members was observed in the RPMI-2650Ml cells, which were also much more invasive in vitro than the parental cell line or the paclitaxel-resistant variant. Increased expression of alpha(2), alpha(5), alpha(6), beta(1) and beta(4) integrin subunits, decreased expression of alpha(4) integrin subunit, stronger adhesion to collagen type IV, laminin, fibronectin and matrigel, increased expression of MMP-2 and MMP-9 and significant motility compared with the parental cells were observed, along with a high invasiveness in the RPMI-2650Ml cells. Decreased expression of the alpha(2) integrin subunit, decreased attachment to collagen type IV, absence of cytokeratin 18 expression, no detectable expression of gelatin-degrading proteases and poor motility may be associated with the non-invasiveness of the RPMI-2650Tx variant. These results suggest that melphalan exposure can result in not only a MDR phenotype, but could also make cancer cells more invasive, whereas paclitaxel exposure resulted in MDR without increasing the in vitro invasiveness in the RPMI-2650 cells.

Antigens, CD↗
PubMed2001

A new monoclonal antibody, P2A8(6), that specifically recognizes a novel epitope on the multidrug resistance-associated protein 1 (MRP1), but not on MRP2 nor MRP3.

Multidrug resistance (MDR) is a major problem in the chemotherapeutic treatment of cancer. Overexpression of the multidrug resistance-associated protein 1 (MRP1), is associated with MDR in certain tumors. A number of MRP1-specific MAbs, which facilitate both clinical and experimental investigations of this protein, are available. To add to this panel of existing antibodies, we have now generated an additional MRP1-specific monoclonal antibody (MAb), P2A8(6), which detects a unique heat stable epitope on the MRP1 molecule. Female Wistar rats were immunized via footpad injections with a combination of two short synthetic peptides corresponding to amino acids 235-246 (peptide A) and 246-260 (peptide B) of the MRP1 protein. Immune reactive B cells were then isolated from the popliteal lymph nodes for fusion with SP2/O-Ag14 myeloma cells. Resultant hybridoma supernatants were screened for MRP1-specific antibody production. Antibody P2A8(6) was characterized by Western blotting and immunocytochemistry on paired multidrug resistant (MRP1 overexpressing) and sensitive parental cell lines. The antibody detects a protein of 190 kDa in MRP1-expressing cell lines but not in MRP2- or MRP3-transfected cell lines. P2A8(6) stains drug-selected and MRP1-transfected cell lines homogeneously by immunocytochemistry and recognizes MRP1 by immunohistochemistry on formalin-fixed paraffin wax-embedded tissue sections. Peptide inhibition studies confirm that P2A8(6) reacts with peptide B (amino acids 246-260), therefore recognizing a different epitope from that of all currently available MRP1 MAbs. This new MAb, chosen for its specificity to the MRP1 protein, may be a useful addition to the currently available range of MRP1-specific MAbs.

Animals↗
PubMed1999

A new monoclonal antibody that specifically recognises the MDR-3-encoded gene product.

The MDR-3-encoded P-glycoprotein (Pgp) is highly expressed in liver and is thought to function as a hepatic transporter of phospholipids into bile. However its role, if any, in other tissues remains undefined. Although transfection experiments have indicated that it may be unable to confer drug resistance, there is evidence that it may be involved in drug resistance in certain B-cell leukaemias. To date, most work on clinical samples has been performed at the mRNA level; limited work has been performed using polyclonal antibodies raised to MDR-3 and mdr-2 (the murine equivalent of MDR-3). We have generated a new monoclonal antibody, termed 6/1G, which specifically recognises the human MDR-3 gene-encoded product. Antibody 6/1G was produced by in vitro immunisation of spleen cells from BALB/c mice with a synthetic 12-amino acid peptide. Cells from MDR-3 transgenic mice showed consistent membranous staining with antibody 6/1G. Immunoblotting with 6/1G identified a band at 170 kDa on lysates of MDR-3 transgenic cells. Preliminary results with a range of B-cell leukaemias suggest that MDR-3 Pgp positivity may be a marker for a more malignant phenotype in B-CLL. Antibody 6/1G may be useful in defining a role for MDR-3 in malignancy and drug resistance, as well as in certain liver diseases such as progressive familial intracholeostasis.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed1997

The response to extracytoplasmic stress in Escherichia coli is controlled by partially overlapping pathways.

The activity of the alternate sigma-factor sigmaE of Escherichia coli is induced by several stressors that lead to the extracytoplasmic accumulation of misfolded or unfolded protein. The sigmaE regulon contains several genes, including that encoding the periplasmic protease DegP, whose products are thought to be required for maintaining the integrity of the cell envelope because cells lacking sigmaE are sensitive to elevated temperature and hydrophobic agents. Selection of multicopy suppressors of the temperature-sensitive phenotype of cells lacking sigmaE revealed that overexpression of the lipoprotein NlpE restored high temperature growth to these cells. Overexpression of NlpE has been shown previously to induce DegP synthesis by activating the Cpx two-component signal transduction pathway, and suppression of the temperature-sensitive phenotype by NlpE was found to be dependent on the Cpx proteins. In addition, a constitutively active form of the CpxA sensor/kinase also fully suppressed the temperature-sensitive defect of cells lacking sigmaE. DegP was found to be necessary, but not sufficient, for suppression. Activation of the Cpx pathway has also been shown to alleviate the toxicity of several LamB mutant proteins. Together, these results reveal the existence of two partially overlapping regulatory systems involved in the response to extracytoplasmic stress in E. coli.

Bacterial Outer Membrane Proteins↗
PubMed1997

Isolation from a human MDR lung cell line of multiple clonal subpopulations which exhibit significantly different drug resistance.

The heterogeneous nature of an adriamycin-selected human MDR squamous lung cell line, DLKP-A, was investigated by isolating and characterising 9 of its clonal subpopulations. The DLKP-A cell line exhibits resistance to the classical MDR drugs, overexpresses P-glycoprotein and displays reduced topoisomerase II amounts. The clonal cell lines exhibit a wide range of resistance extents, with the most resistant clone displaying 9 times the extent of adriamycin resistance observed in the least resistant clone. A number of clones exhibit sensitivity to the concentration of adriamycin in which the parental cell line was selected, possibly indicating cooperation between the more and less resistant cells. Detailed analysis of 4 of the clonal subpopulations revealed broadly similar drug resistance mechanisms. Alterations in expression of the MDR-associated genes MDR1 and Topo IIalpha were observed, with no detectable changes in the expression of MDR3, MRP, GSTpi, Topo IIbeta, Topo I and CYP1A1 noted. However, each clonal cell line displayed a distinct extent of expression of MDR1 and Topo IIalpha and further characterisation of the clones indicated that other modes of drug resistance may exist in at least one of the cell lines. In particular, 2 of the clones (DLKPA6B and DLKPA11B) which have almost identical drug resistance profiles appear to have quite different mechanisms of resistance. The clonal subpopulations possess individual growth rates, amounts of adriamycin accumulation and susceptibility to toxicity-enhancement by MDR-modulating agents. It was possible to generate a cell line with a drug toxicity profile similar to DLKP-A by mixing some of the clonal subpopulations. Our results provide evidence of heterogeneity within an MDR human cell population with respect to resistance and expression of MDR-associated genes.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

The sigmaE-mediated response to extracytoplasmic stress in Escherichia coli is transduced by RseA and RseB, two negative regulators of sigmaE.

The extracytoplasmic stress response in Escherichia coli is controlled by the alternative sigma factor, sigma(E). sigma(E) activity is uniquely induced by the accumulation of outer membrane protein precursors in the periplasmic space, and leads to the increased production of several proteins, including the periplasmic protease DegP, that are thought to be required for maintaining cellular integrity under stress conditions. Genetic and biochemical experiments show that sigma(E) activity is under the control of three genes, rseABC (for regulator of sigma E), encoded immediately downstream of the sigma factor. Deletion of rseA leads to a 25-fold induction of sigma(E) activity. RseA is predicted to be an inner membrane protein, and the purified cytoplasmic domain binds to and inhibits sigma(E)-directed transcription in vitro, indicating that RseA acts as an anti-sigma factor. Deletion of rseB leads to a slight induction of sigma(E), indicating that RseB is also a negative regulator of sigma(E). RseB is a periplasmic protein and was found to co-purify with the periplasmic domain of RseA, indicating that RseB probably exerts negative activity on sigma(E) through RseA. Deletion of rseC, in contrast, has no effect on sigma(E) activity under steady-state conditions. Under induction conditions, strains lacking RseB and/or C show wild-type induction of sigma(E) activity, indicating either the presence of multiple pathways regulating sigma(E) activity, or the ability of RseA alone to both sense and transmit information to sigma(E).

Bacterial Outer Membrane Proteins↗
PubMed1997

SigmaE is an essential sigma factor in Escherichia coli.

SigmaE is an alternative sigma factor that controls the extracytoplasmic stress response in Escherichia coli. SigmaE is essential at high temperatures but was previously thought to be nonessential at temperatures below 37 degrees C. We present evidence that sigmaE is an essential sigma factor at all temperatures. Cells lacking sigmaE are able to grow at low temperatures because of the presence of a frequently arising, unlinked suppressor mutation.

Escherichia coli↗
PubMed1994

A soluble form of a receptor for subgroup A avian leukosis and sarcoma viruses (ALSV-A) blocks infection and binds directly to ALSV-A.

A receptor that confers susceptibility to infection by subgroup A avian leukosis and sarcoma viruses (ALSV-A) has been described (P. Bates, J. A. T. Young, and H. E. Varmus, Cell 74:1043-1051, 1993). A soluble form of the receptor was generated to determine whether this protein interacts directly with virus particles in the absence of other cell surface factors. The soluble protein comprised the extracellular region of the ALSV-A receptor fused to an antibody epitope tag and six histidine residues. Preincubating this protein with virus led to an efficient block to infection of avian cells by ALSV-A but had no effect on infection by ALSV-B, ALSV-C, or ALSV-D. Furthermore, an antibody directed against the introduced epitope tag immunoprecipitated ALSV-A particles bound to the soluble receptor. In contrast, other ALSV subgroups were not immunoprecipitated by this procedure. These data demonstrate that the cloned receptor interacts directly with ALSV-A and discriminates between different ALSV subgroups at the level of virus binding.

Alpharetrovirus↗
PubMed1988

The human X-linked steroid sulfatase gene and a Y-encoded pseudogene: evidence for an inversion of the Y chromosome during primate evolution.

The mammalian X and Y chromosomes are thought to have evolved from a common, nearly homologous chromosome pair. Although there is little sequence similarity between the mouse or the human X and Y, there are several regions in which moderate to extensive sequence homologies have been found, including, but not limited to, the so-called pseudoautosomal segment, in which X-Y pairing and recombination take place. The steroid sulfatase gene is in the pseudoautosomal region of the mouse, but not in man. We have cloned and characterized the human STS X-encoded locus and a pseudogene that is present on the long arm of the Y chromosome. Our data in humans and other primates suggest that there has been a pericentric inversion of the Y chromosome during primate evolution that has disrupted the former pseudoautosomal arrangement of these genes. These results provide additional insight into the evolution of the sex chromosomes and into the nature of this interesting portion of the human genome.

Animals↗
PubMedDate not supplied

Biosensor-based micro-affinity purification for the proteomic analysis of protein complexes.

A biosensor-based micro-affinity purification method to recover protein binding partners and their complexes for down stream proteomics analysis has been developed using the BIAcore 3000 fitted with a prototype Surface Prep Unit (SPU). The recombinant GST-intracellular domain of E-cadherin or the recombinant GST-beta-catenin binding domain of Adenomatous Polyposis Coli (APC) were immobilized onto the SPU and used to affinity purify binding partners from chromatographically enriched SW480 colon cancer cell lysates. A GST- immobilized surface was used as a control. Samples recovered from the SPU were subjected to SDS-PAGE with sensitive Coomassie staining followed by automated in-gel digestion and LC-MS/MS. The results obtained using the SPU were compared with similar experiments performed using Sepharose beads.

Biosensing Techniques↗
PubMedDate not supplied

Treatment of ras-induced cancers by the F-actin-bundling drug MKT-077.

A rhodacyanine dye called MKT-077 has shown a highly selective toxicity toward several distinct human malignant cell lines, including bladder carcinoma EJ, and has been subjected to clinical trials for cancer therapy. In the pancreatic carcinoma cell line CRL-1420, but not in normal African green monkey kidney cell line CV-1, it is selectively accumulated in mitochondria. However, both the specific oncogenes responsible for its selective toxicity toward cancer cells, and its target proteins in these cancer cells, still remain to be determined. This study was conducted using normal and ras-transformed NIH 3T3 fibroblasts to determine whether oncogenic ras mutants such as v-Ha-ras are responsible for the selective toxicity of MKT-077 and also to identify its targets, using its derivative called "compound 1" as a specific ligand. We have found that v-Ha-ras is responsible for the selective toxicity of MKT-077 in both in vitro and in vivo. Furthermore, we have identified and affinity purified at least two distinct proteins of 45 kD (p45) and 75 kD (p75), which bind MKT-077 in v-Ha-ras-transformed cells but not in parental normal cells. Microsequencing analysis has revealed that the p45 is a mixture of beta- and gamma-actin, whereas the p75 is HSC70, a constitutive member of the Hsp70 heat shock adenosine triphosphatase family, which inactivates the tumor suppressor p53. MKT-077 binds actin directly, bundles actin filaments by cross-linking, and blocks membrane ruffling. Like a few F-actin-bundling proteins such as HS1, alpha-actinin, and vinculin as well as F-actin cappers such as tensin and chaetoglobosin K (CK), the F-actin-bundling drug MKT-077 suppresses ras transformation by blocking membrane ruffling. These findings suggest that other selective F-actin-bundling/capping compounds are also potentially useful for the chemotherapy of ras-associated cancers.

3T3 Cells↗