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Biomedical subjects

M K Magnusson

Publications and source records attributed to M K Magnusson.

10 recordsLinked to original sources
PubMed2005

Tumor cell targeted gene delivery by adenovirus 5 vectors carrying knobless fibers with antibody-binding domains.

Most human carcinoma cell lines lack the high-affinity receptors for adenovirus serotype 5 (Ad5) at their surface and are nonpermissive to Ad5. We therefore tested the efficiency of retargeting Ad5 to alternative cellular receptors via immunoglobulin (Ig)-binding domains inserted at the extremity of short-shafted, knobless fibers. The two recombinant Ad5's constructed, Ad5/R7-Z(wt)-Z(wt) and Ad5/R7-C2-C2, carried tandem Ig-binding domains from Staphylococcal protein A (abbreviated Z(wt)) and from Streptococcal protein G (C2), respectively. Both viruses bound their specific Ig isotypes with the expected affinity. They transduced human carcinoma cells independently of the CAR pathway, via cell surface receptors targeted by specific monoclonal antibodies, that is, EGF-R on A549, HT29 and SW1116, HER-2/neu on SK-OV-3 and SK-BR-3, CA242 (epitope recognized by the monoclonal antibody C242) antigen on HT29 and SW1116, and PSMA (prostate-specific membrane antigen) expressed on HEK-293 cells, respectively. However, Colo201 and Colo205 cells were neither transduced by targeting CA242 or EGF-R nor were LNCaP cells transduced by targeting PSMA. Our results suggested that one given surface receptor could mediate transduction of certain cells but not others, indicating that factors and steps other than cell surface expression and virus-receptor interaction are additional determinants of Ad5-mediated transduction of tumor cells. Using penton base RGD mutants, we found that one of these limiting steps was virus endocytosis.

Adenoviruses, Human↗
PubMed2002

Genetic modification of adenovirus 5 tropism by a novel class of ligands based on a three-helix bundle scaffold derived from staphylococcal protein A.

The use of adenovirus (Ad) as an efficient and versatile vector for in vivo tumor therapy requires the modulation of its cellular tropism. We previously developed a method to genetically alter the tropism of Ad5 fibers by replacing the fiber knob domain by an extrinsic trimerization motif and a new cellular ligand. However, fibers carrying complex ligands such as single-chain antibody fragments did not assemble into functional pentons in vitro in the presence of penton base, and failed to be rescued into infectious virions because of their inability to fold correctly within the cytoplasm of Ad-infected cells. Here we show that the coding sequence for a disulfide bond-independent three-helix bundle scaffold Z, derived from domain B of Staphylococcal protein A and capable of binding to the Fc portion of immunoglobulin (Ig) G1, could be incorporated into modified knobless Ad fiber gene constructs with seven shaft repeats. These fiber gene constructs could be rescued into viable virions that were demonstrated to enter 293 cells engineered for IgG Fc surface expression but not unmodified 293 cells, via a mechanism that could be specifically blocked with soluble Fc target protein. However, the tropism modified viruses showed a slightly impaired cellular entry and a lower infectivity than wildtype (WT) virus. In addition, we generated recombinant fibers containing an IgA binding Affibody ligand, derived from combinatorial specificity-engineering of the Z domain scaffold. Such fiber constructs also showed the expected target specific binding, indicating that the affibody protein class is ideally suited for genetic engineering of Ad tropism.

Adenoviridae↗
PubMed2001

Rabaptin-5 is a novel fusion partner to platelet-derived growth factor beta receptor in chronic myelomonocytic leukemia.

Chromosomal translocations involving the platelet-derived growth factor beta receptor (PDGFbetaR) gene have been reported in some patients with chronic myelomonocytic leukemia (CMML). The resultant fusion proteins have constitutive PDGFbetaR tyrosine kinase activity, but the partner genes previously reported (tel, Huntingtin interacting protein 1 [HIP-1], H4/D10S170) have poorly understood roles in the oncogenic activity of the fusion proteins. A novel PDGFbetaR fusion protein has been characterized in a patient with CMML and an acquired t(5;17)(q33;p13). Southern blot analysis on patient leukemia cells demonstrated involvement of the PDGFbetaR gene. Using 5' rapid amplification of complementary DNA ends-polymerase chain reaction (RACE-PCR) on patient RNA, rabaptin-5 was identified as a novel partner fused in-frame to the PDGFbetaR gene. The new fusion protein includes more than 85% of the native Rabaptin-5 fused to the transmembrane and intracellular tyrosine kinase domains of the PDGFbetaR. Transduction with a retroviral vector expressing rabaptin-5/PDGFbetaR transformed the hematopoietic cell line Ba/F3 to growth factor independence and caused a fatal myeloproliferative disease in mice. Rabaptin-5 is a well-studied protein shown to be an essential and rate-limiting component of early endosomal fusion through interaction with the Ras family GTPases Rab5 and Rab4. The fusion protein includes 3 of 4 coiled-coil domains (involved in homodimerization of native rabaptin-5), 2 caspase-3 cleavage sites, and a binding site for the tumor suppressor gene tuberin (tuberous sclerosis complex-2). Early endosomal transport is critical in regulation of various growth factor receptors, through ligand-induced clathrin-mediated endocytosis, and thus this new fusion protein links together 2 important pathways of growth regulation.

Adult↗
PubMed2001

Modulation of cell interactions with extracellular matrix by lysophosphatidic acid and sphingosine 1-phosphate.

Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (SPP) are lipid mediators released upon platelet activation. The concentration of LPA in serum is estimated at 1-10 microM whereas the concentration in plasma is considerably less. The SPP concentration in serum is 0.5 microM, approximately two-fold higher than the plasma concentration. The lipids are present during tissue injury and promote cellular processes involved in wound repair. LPA and SPP have multiple effects on cells, many of which are pertinent to wound healing and require that the cells interact in some fashion with components of the extracellular matrix. This review focuses on modulation of cell adhesion, cell migration, collagen gel contraction, and fibronectin matrix assembly by LPA and SPP.

Cell Adhesion↗
PubMed2001

Genetic retargeting of adenovirus: novel strategy employing "deknobbing" of the fiber.

For efficient and versatile use of adenovirus (Ad) as an in vivo gene therapy vector, modulation of the viral tropism is highly desirable. In this study, a novel method to genetically alter the Ad fiber tropism is described. The knob and the last 15 shaft repeats of the fiber gene were deleted and replaced with an external trimerization motif and a new cell-binding ligand, in this case the integrin-binding motif RGD. The corresponding recombinant fiber retained the basic biological functions of the natural fiber, i.e., trimerization, nuclear import, penton formation, and ligand binding. The recombinant fiber bound to integrins but failed to react with antiknob antibody. For virus production, the recombinant fiber gene was rescued into the Ad genome at the exact position of the wild-type (WT) fiber to make use of the native regulation of fiber expression. The recombinant virus Ad5/FibR7-RGD yielded plaques on 293 cells, but the spread through the monolayer was two to three times delayed compared to WT, and the ratio of infectious to physical particles was 20 times lower. Studies on virus tropism showed that Ad5/FibR7-RGD was able to infect cells which did not express the coxsackie-adenovirus receptor (CAR), but did express integrins. Ad5/FibR7-RGD virus infectivity was unchanged in the presence of antiknob antibody, which neutralized the WT virus. Ad5/FibR7-RGD virus showed an expanded tropism, which is useful when gene transfer to cells not expressing CAR is needed. The described method should also make possible the construction of Ad genetically retargeted via ligands other than RGD.

Adenoviridae↗
PubMed1999

Sphingosine 1-phosphate stimulates fibronectin matrix assembly through a Rho-dependent signal pathway.

Fibronectin matrix assembly is a cell-dependent process mediated by cell surface binding sites for the 70-kD N-terminal portion of fibronectin. We have shown that Rho-dependent cytoskeleton reorganization induced by lysophosphatidic acid (LPA) or the microtubule-disrupting agent nocodazole increases fibronectin binding (Zhang et al, Mol Biol Cell 8:1415, 1997). Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid implicated in mitogenesis and cytoskeletal remodelling. Both LPA and S1P are present in increased amounts in serum as compared with plasma as a result of platelet activation. Addition of S1P to human osteosarcoma MG63 cells or human foreskin fibroblasts increased cell-mediated binding and assembly of fibronectin. MG63 cells expressed the Edg-2 and Edg-4 G-protein-coupled receptors for bioactive lipids, whereas foreskin fibroblasts expressed Edg-2, Edg-3, and Edg-4. The stimulatory effect of S1P on the binding of fibronectin or the N-terminal 70-kD fragment of fibronectin was dynamic and due to increases in both the number and affinity of binding sites. The stimulation of 70-kD fragment binding by nanomolar S1P, like stimulation of binding by LPA or nocodazole, was blocked by inactivation of Rho with C3 exotoxin but not by pertussis toxin-mediated inactivation of Gi. These results indicate a common signal pathway leading to control of cellular fibronectin matrix assembly by bioactive lipids generated during blood coagulation.

Binding Sites↗
PubMed1997

Lysophosphatidic acid and microtubule-destabilizing agents stimulate fibronectin matrix assembly through Rho-dependent actin stress fiber formation and cell contraction.

Fibronectin (FN) matrix assembly is a cell-dependent process mediated by cell surface-binding sites for the 70-kDa amino-terminal region of FN. We have shown recently that lysophosphatidic acid (LPA) is a stimulator of FN matrix assembly. Disruption of microtubules has been shown to mimic some of the intracellular effects of LPA including the formation of actin stress fibers and myosin light chain phosphorylation. We compared the effects of microtubule disruption and LPA on FN binding and actin cytoskeleton organization. The disruption of microtubules by nocodazole or vinblastine increased FN binding to adherent cells. The modulation of binding sites was rapid, dynamic, and reversible. Enhanced binding was due to increases in both the number and affinity of binding sites. These effects are similar to the effects of LPA on FN binding. Binding induced by nocodazole was inhibited by the microtubule-stabilizing agent Taxol but not by pretreatment with a concentration of phospholipase B that totally abolished the stimulatory effect of LPA. Fluorescence microscopy revealed a close correlation among actin stress fiber formation, cell contraction, and FN binding. Blockage of the small GTP binding protein Rho or actin-myosin interactions inhibited the effects of both nocodazole and LPA on FN binding. These observations demonstrate that Rho-dependent actin stress fiber formation and cell contraction induce increased FN binding and represent a rapid labile way that cells can modulate FN matrix assembly.

Animals↗
PubMed1994

Low iron-binding capacity as a risk factor for myocardial infarction.

BACKGROUND: In a recent Finnish study, ferritin was suggested to be an independent risk factor for acute myocardial infarction. This study suggested that high levels of iron stores might thus be atherogenic and possibly explain partly the sex difference in the incidence of ischemic heart disease. METHODS AND RESULTS: A randomly selected group (n = 2036), men and women aged 25 to 74 years, were examined between June and September 1983. All classic risk factors for coronary artery disease were measured as well as basic hematologic parameters and the parameters of iron metabolism, ie, iron, total iron-binding capacity (TIBC), and ferritin. During the follow-up for 8.5 years, 81 subjects experienced acute myocardial infarction (63 men and 18 women). The differences in the iron parameters between men and women were almost exclusively seen in ferritin values (198 micrograms/L in men and 91 micrograms/L in women), whereas small differences were seen in TIBC. The Cox proportional hazards model was used to estimate the contribution of independent variables to the risk of myocardial infarction. TIBC was found to be a strong independent negative risk factor in men (RR = 0.95; 95% CI, 0.92 to 0.98), whereas ferritin (RR = 0.999; 95% CI, 0.997 to 1.001) or other iron parameters had no significant predictive power. Each increase in TIBC of 1 mumol/L was associated with a 5.1% decrease in the risk of myocardial infarction. The classic major risk factors, ie, blood pressure, smoking, total cholesterol, and high-density lipoprotein, had significant independent correlation with myocardial infarction. When Cox multivariate analysis was carried out on both sexes combined, TIBC was still an independent negative risk factor, and the logarithmic transform of ferritin had a weak negative correlation but was not statistically significant. Sex was in this group still a very strong risk factor after taking into account all classic risk factors as well as the parameters of iron metabolism. CONCLUSIONS: This study suggests that transferrin, measured as TIBC, is an independent negative risk factor for myocardial infarction. Other parameters of iron metabolism, including ferritin, were not found to contribute to the risk.

Adult↗