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1.
A major hurdle to adenovirus (Ad)-mediated gene transfer is that the target issue lacks sufficient levels of receptors to mediate vector attachment via its fiber coat protein. Endothelial and smooth muscle cells are primary targets in gene therapy approaches to prevent restenosis following angioplasty or to promote or inhibit angiogenesis. However, Ad poorly binds and transduces these cells because of their low or undetectable levels of functional Ad fiber receptor. The Ad-binding deficiency of these cells was overcome by targeting Ad binding to alpha v integrin receptors that are sufficiently expressed by these cells. In order to target alpha v integrins, a bispecific antibody (bsAb) that comprised a monoclonal Ab to the FLAG peptide epitope, DYKDDDDK, and a monoclonal Ab to alpha v integrins was constructed. In conjunction with the bsAb, a new vector, AdFLAG, which incorporated the FLAG peptide epitope into its penton base protein was constructed. Complexing AdFLAG with the bsAb increased the beta-glucuronidase transduction of human venule endothelial cells and human intestinal smooth muscle cells by seven- to ninefold compared with transduction by AdFLAG alone. The increased transduction efficiency was shown to occur through the specific interaction of the complex with alpha v integrins. These results demonstrate that bsAbs can be successfully used to target Ad to a specific cellular receptor and thereby increase the efficiency of gene transfer.  相似文献   

2.
 This report summarizes our experimental data concerning the use of bispecific antibodies (bsAb) for the treatment of the murine BCL1 B cell lymphoma model. Initially we used a hybrid-hybridoma-derived bsAb with specificity for the TcR/CD3 complex on T cells and the idiotype of the membrane-bound IgM on the tumor cells. The bsAb used as a single agent could cure animals with a low tumor load (resembling minimal residual disease). However, in experiments aimed at increasing the therapeutic effect in animals with a higher tumor burden, we could demonstrate the importance of additional T-cell-costimulatory signals and the careful timing of the bsAb administration. Recently we have generated a bispecific single-chain Fv (bsscFv) fusion protein with the same dual specificity as the hybrid-hybridoma-derived bsAb. Immunotherapy with this smaller molecule also resulted in tumor elimination in BCL1-bearing mice. A second bsscFv (α-CDl9×α-CD3) with a broader applicability is now being characterized and tested in vivo. Accepted: 14 October 1997  相似文献   

3.
 The bispecific monoclonal antibody (bsAb) BIS-1 combines a monoclonal-antibody(mAb)-defined specificity for the CD3 complex, as present on all T lymphocytes, with a mAb-defined specificity for the pancarcinoma/epithelium associated glycoprotein EGP-2. In vitro studies indicate that BIS-1 can direct T lymphocytes to kill EGP-2-positive tumour target cells. T cell pre-activation is necessary for this activity and can be obtained either via incubation of isolated peripheral blood mononuclear cells with CD3 mAb, followed by short culturing in recombinant interleukin-2-containing medium, or via costimulation with CD5- and CD28-based bsAb. Clinical application of BIS-1 was started in a pilot study in which carcinoma patients suffering from malignant ascites or intrapleural effusion were treated. In this study, ex vivo activated autologous lymphocytes were applied locally, i.e. intraperitoneally or intrapleurally, in the presence of BIS-1. Local inflammation and antitumour activity were observed, whereas no or only minor systemic toxicity was seen in these patients. Intravenous administration of BIS-1 F(ab′)2 in combination with subcutaneously given recombinant interleukin-2 (i.v. bsAb/rIL-2 treatment) induced transient but considerable toxicity including peripheral vasoconstriction, dyspnoea and fever with a maximal tolerated dose of 5–8 μg/kg. High plasma concentrations of the inflammatory cytokines tumor necrosis factor α and interferon γ were observed at this dose. Whereas bsAb-dictated antitumour activity could be demonstrated to be present in blood samples of these patients in an in vitro assay, no clear clinical responses were observed. In a rat model it was found that i.v. bsAb/rIL-2 treatment of EGP-2-positive tumours was effective when a low systemic tumour burden was present, suggesting that systemic bsAb/rIL-2 treatment might be effective in situations of minimal residual disease. Accepted: 14 October 1997  相似文献   

4.
 The class I IgG receptor (FcγRI) on cytotoxic effector cells has been reported to initiate destruction of tumour cells by effector cells in vitro. We are aiming at developing an immunocompetent model to evaluate the cytotoxic capacity of human FcγRI for the rejection of tumour cells in vivo. Therefore, we recently generated a transgenic mouse strain expressing human FcγRI on monocytes, macrophages, and neutrophils. In these mice, the human receptor is up-regulated by granulocyte-colony-stimulating factor (G-CSF) and is able to trigger cellular responses. Subsequently, in the present study the B cell lymphoma IIA1.6 cell line is selected as a tumour target, and a human FcγRI-directed antitumour bispecific antibody (bsAb) is constructed and characterized. Fab′ fragments of mAb 22, which bind hFcγRI at an epitope that is distinct from the ligand binding site, were chemically linked to Fab′ fragments of rat anti-(mMHC class II antigens) mAb M5/114, yielding bsAb 22×M5/114. This bsAb was able to bind simultaneously to hFcγRI and mMHC class II antigens in a dose-dependent fashion. Binding of 22×M5/114 to FcγRI was not inhibited in the presence of human IgG. It is important to note that, MHC-class-II-expressing IIA1.6 lymphoma cells were lysed by whole blood from G-CSF-treated transgenic mice in the presence of bsAb 22×M5/114. No lysis by whole blood from non-transgenic mice or from transgenic animals that had not received G-CSF was observed. These results indicate that human FcγRI is able to mediate lysis of murine IIA1.6 lymphoma cells by transgenic effector cells via bsAb 22×M5/114. A trial with transgenic mice, evaluating the efficacy of these hFcγRI-directed bsAb in combination with G-CSF for treatment of IIA1.6 B cell lymphoma, is currently in progress. Accepted: 14 October 1997  相似文献   

5.
 The aim of this work was to test for cure and immunity in a micrometastatic tumor model using in vivo T cell activation with staphylococcal enterotoxin B (SEB) and retargeting with antitumor×anti-CD3 F(ab′)2 bispecific antibodies (bsAb). All studies were performed in C3H/HeN mice using syngeneic tumor cell lines. For survival studies, mice were injected intravenously on day 0 with CL62 (a p97-transfected clone of the K1735 murine melanoma tumor). Day-3 treatments included saline (control), SEB (50 γg intraperitoneal) with or without bsAb (5 μg i.v.). Cured mice, surviving beyond 60 days, were rechallenged with subcutaneous CL62, K1735, or a nonmelanoma control, AG104. SEB activation studies were performed with pulmonary tumor-infiltrating lymphocytes isolated from 10-day established CL62 tumors. Maximal tumor-infiltrating lymphocyte cytotoxicity was demonstrated 24 h following SEB injection, therefore bsAb treatments were administered 24 h after SEB. When survival was examined at 60 days, there were significantly more survivors in the group receiving SEB plus bsAb (70%) compared to the group receiving SEB alone (30%), and the controls (0%) (P=0.02 and P<0.01, respectively). Mice cured of CL62 using SEB alone or with bsAb demonstrated equal immunity to CL62, however, mice treated with SEB plus bsAb were more often immune to the p97 parental cell line, K1735(P=0.001). Ag104 consistently grew in all mice. Results of these studies demonstrate that SEB plus bsAb can be effective, not only in curing tumors but also in providing protective immunity against targeted and nontargeted tumor antigens. Accepted: 14 October 1997  相似文献   

6.
The αvβ3 integrin has emerged as a key mediator in angiogenesis. Its role in tumor-induced angiogenesis is supported by its up-regulation in vivo in the vasculature of a number of different types of carcinoma. The potential clinical significance of αvβ3 expression on blood vessels in carcinomas is suggested by its association with tumor progression. Currently no information is available about the clinical significance of αvβ3 expression on the vasculature of lesions of melanocytic origin. Since we have previously found that αvβ3 expression on melanoma cells in primary lesions is associated with a poor prognosis, in the present study we have compared αvβ3 expression on blood vessels and on cells of melanocytic origin in nevi and in malignant melanoma lesions. In addition we have examined the lesions for expression of the αv subunit to gain information on the regulation of αvβ3 expression on endothelial cells and on cells of the melanocyte lineage. αvβ3 expression on endothelial cells and on melanocytic cells was a relatively sensitive and specific marker for malignant lesions. However, αvβ3 expression on endothelial cells in primary melanoma lesions was not associated with the prognosis of the disease. The αv subunit and the αvβ3 complex were differentially expressed on endothelial cells and on melanocytic cells, implying that different regulatory pathways control their expression. This finding may account for the differential clinical significance of αvβ3 expression on tumor vasculature and on melanoma cells we observed in our patient cohort. Lastly, αvβ3 may be a useful target for immunotherapeutic approaches in melanoma because of its high expression on the vasculature of all metastatic lesions tested and its restricted distribution in normal tissues. Received: 18 February 2000 / Accepted: 12 April 2000  相似文献   

7.
Despite the success of mAb and bispecific (bs)Ab in the treatment of certain malignancies, there is still considerable uncertainty about the most appropriate format in which they should be used. In the current work we have investigated a panel of bsAb [IgG and F(ab)2] with dual specificity for T cells and neoplastic B cells. Throughout this work, anti-CD2 or anti-CD3 were used to bind the mouse T cells, and antibodies to surface IgM idiotype (Id), CD19, CD22, or MHC class II were used to target mouse B cell lymphomas BCL1 or A31. In vitro, killing was measured in a conventional cytotoxicity assay using 51Cr-labelled A31 and BCL1 cells as targets and activated mouse splenocytes as effectors. bsAb showed a wide range of cytotoxic activities, which could be ranked in the following order: [anti-CD3×anti-class-II]>[anti-CD3×anti-CD19] >[anti-CD3×anti-Id]>[anti-CD3×anti-CD22], with the [anti-CD2×anti-Id] derivative showing relatively little cytotoxic activity. This hierarchy of activity indicates some correlation with the binding activity of the bsAb on target cells, but showed a much stronger parallel with the tendency of the anti-(target cells) mAb to undergo antigenic modulation (less modulation, more killing). In vivo, the situation was completely different and only the anti-ld derivatives, [anti-CD3×anti-ld] and [anti-CD2×anti-ld], were effective in prolonging the survival of tumour-bearing animals. Under optimal conditions Id-positive tumour was eradicated with a single treatment of bsAb. We conclude from this work that the target cell specificity of a bsAb is critical in determining therapeutic outcome and that in vitro cytotoxicity assays do not predict in vivo activity. Accepted: 14 October 1997  相似文献   

8.
9.
 Unlike monoclonal antibodies, clinical application of bispecific antibodies has so far lagged behind because of difficult, low-yield production techniques as well as considerable toxicity attributed to bispecific antibody preparations containing immunoglobulin-Fc parts and anti-CD3 homodimers [10, 2]. These difficulties were overcome by recombinant generation of a bispecific single-chain antibody (bscAb) joining two single-chain Fv fragments via a five-amino-acid glycine-serine linker. The anti-CD3 specificity directed against human T cells was combined with another specificity against the epithelial 17-1A antigen. The following domain arrangement was critical in this individual case to obtain a fully functional bscAb: VL17-1A-VH17-1A-VHCD3-VLCD3. The bscAb was expressed in chinese hamster ovary cells with a yield of 15 mg/l culture supernatant whereas numerous attempts to obtain a functional protein expression in Escherichia coli failed. The low-molecular-mass bispecific construct (60 kDa) could easily be purified by its C-terminal histidine tail. The antigen-binding properties are indistinguishable from those of the corresponding univalent single-chain Fv fragments as shown by enzyme immunoassay and flow cytometry. We could show that the bscAb, which lacks Fc parts and anti-CD3 homodimers is highly cytotoxic for 17-1A positive tumor cells in nanomolar concentrations and in the presence of human T cells. Most remarkably, it does not stimulate T lymphocyte proliferation in the absence of tumor cells and, moreover, does not induce CD25 up-regulation and the secretion of potentially toxic lymphokines such as tumor necrosis factor α, interleukin-6 and interferon γ. Maximal cytotoxicity (51Cr release) was achieved without notable costimulation and was not further enhanced by adding costimulatory signals such as those delivered by anti-CD28 antibodies. CD8+ as well as CD4+ T cell subpopulations were recruited to exert cytotoxicity against tumor cells with different kinetics. CD8+ cells induced high 51Cr release within 4 h while CD4+ cells required a 20-h incubation. The systemic application of the 17-1A/CD3-bscAb could be a major improvement in therapy against disseminated micrometastatic tumor cells. A prospective, randomized clinical trial showing that an anti-17-1A monoclonal antibody could prolong survival of colorectal cancer patients after 5 and 7 years, warrants an assessment of the clinical efficacy of this bscAb exhibiting a 1000-fold higher specific cytotoxicity against tumor cells in virto. Accepted: 14 October 1997  相似文献   

10.
Targeted adenovirus-mediated gene delivery to T cells via CD3.   总被引:5,自引:2,他引:3       下载免费PDF全文
T cells are primary targets in numerous gene therapy protocols. However, the use of subgroup C adenovirus serotype 2 or 5 (Ad2 or Ad5) as a vector to transduce T cells is limited by its poor transduction efficiency for these cells. In this report we show that poor T-cell transduction results from these cells lacking both the primary Ad2-Ad5 receptor, used in attachment, and the secondary Ad receptor, which mediates entry of most adenovirus serotypes. These deficiencies were overcome by using a bispecific antibody (bsAb) with specificities for human CD3 and for a FLAG epitope genetically introduced into Ad5 (Ad.FLAG) to redirect the virus to human T cells. The anti-FLAG x anti-CD3 bsAb increased Ad.FLAG binding 30-fold, induced the efficient uptake of Ad.FLAG into the cells, and led to a 100- to 500-fold increase in the transduction of resting T cells. Moreover, fluorescence-activated cell sorter analysis showed that 25 to 90% of the T cells were transduced by the bsAb-complexed Ad.FLAG at multiplicities of infection between 20 and 100 active particles per cell. These results demonstrate that bsAbs can target Ad to non-Ad receptors on cells that are normally resistant to Ad, resulting in their efficient and specific transduction.  相似文献   

11.
Kang HW  Weissleder R  Bogdanov A 《Amino acids》2002,23(1-3):301-308
Summary.  Targeted diagnostic agents are expected to have a significant impact in molecular imaging of cell-surface associated markers of proliferation, inflammation and angiogenesis. In this communication, we describe a new class of targeted polyamino acid-based protected graft copolymers (PGC) of poly-(L-lysine) and methyl poly-(ethylene glycol) (PGC) covalently conjugated with a monoclonal antibody fragment, F(ab′)2. We utilized targeted PGC conjugates as carriers of near-infrared indocyanine fluorophores (Cy5.5) for optical imaging of endothelial cell populations expressing IL-1β inducible proinflammatory marker E-selectin. We compared two conjugation chemistries, involving either introduction of sulfhydryl group to F(ab′)2, or via direct attachment of the antibody fragment directly to the chemically activated PGC. Both PGC-based targeted agents demonstrated high binding specificity (20–30 fold over non-specific uptake) and were utilized for imaging E-selectin expression on human endothelial cells activated with IL-1β. Received June 29, 2001 Accepted August 8, 2001 Published online August 9, 2002  相似文献   

12.
 A new type of cancer vaccine for therapeutic application in cancer patients is described. It consists of three components. (1) autologous tumor cells, (2) Newcastle Disease Virus (NDV), to be used for infection and (3) bispecific antibodies (bsAb) which attach to the viral hemagglutinin neuraminidase (HN) molecule on the infected tumor cells. A standardized procedure has been developed for generating virus infected human autologous tumor cell vaccines (ATV-NDV) which includes cell dissociation, removal of leukocytes and cell debris, gamma-irradiation and cryopreservation. Infection with the non-virulent strain NDV Ulster is performed within 30 min of co-incubation. While virus infection already increased immunogenicity of the tumor vaccine, further augmentation of T cell stimulatory capacity is achieved by attachment of specially designed bi-specific antibodies (bs HN × CD28 or bs HN × CD3). Received: 6 August 1996 / Accepted: 20 September 1996  相似文献   

13.
Integrins, a family of transmembrane heterodimeric polypeptides, mediate various biological responses including cell adhesion and migration. In this report, we show that sphingosine-1-phosphate (S1P) activates integrin αvβ3 in endothelial cells (ECs) via the sphingosine-1-phosphate receptor subtype 1 (S1P1)-mediated signaling pathway. S1P treatment results in the activation of integrin αvβ3 in the lamellipodia region of ECs, suggesting that integrin αvβ3 plays a critical role in the S1P-stimulated chemotactic response of ECs. Indeed, S1P treatment induces the association of focal adhesion kinase (FAK) and cytoskeletal proteins with integrin αvβ3, the ligation of αv and β3 subunits, as well as enhances endothelial migration on vitronectin-coated substrata. Knockdown endothelial S1P1 receptor, treatments with pertussis toxin or dominant-negative-Rho family GTPases abrogates the S1P-induced integrin αvβ3 activation in ECs. Consequently, these treatments markedly inhibit the S1P-induced endothelial migratory response on vitronectin-coated substrata. Collectively, these data indicate that the S1P-mediated signaling via the S1P1/Gi/Rho GTPases pathway activates integrin αvβ3, which is indispensable for S1P-stimulated chemotactic response of ECs.  相似文献   

14.
 Cytokine-induced killer cells (CIK), generated in vitro from peripheral blood mononuclear cells (PBMC) by addition of interferon γ (IFNγ), interleukin-2 (IL-2), IL-1 and a monoclonal antibody (mAb) against CD3, are highly efficient cytotoxic effector cells with the CD3+CD56+ phenotype. In this study, we evaluated whether the cytotoxicity of these natural-killer-like T lymphocytes against the colorectal tumor cell line HT29 can be enhanced by the addition of a bispecific single-chain antibody (bsAb) directed against EpCAM/CD3. For determination of bsAb-redirected cellular cytotoxicity we used a new flow-cytometric assay, which directly counts viable tumor cells and can assess long-term cytotoxicity. We found that this bsAb induced distinct cytotoxicity at a concentration above 100 ng/ml with both PBMC and CIK at an effector-to-target cell ratio as low as 1:1. CIK cells revealed higher bsAb-redirected cytotoxicity than PBMC. Cellular cytotoxicity appeared after 24 h whereas PBMC showed the highest bsAb-redirected cytotoxicity after 72 h. The addition of the cytokines IL-2 and IFNα but not granulocyte/macrophage-colony-stimulating factor enhanced bsAb-redirected cytotoxicity of both PBMC and CIK. When the bsAb was combined with the murine mAb BR55-2, which recognizes the Lewisy antigen, bsAb-redirected cytotoxicity was partly augmented, whereas murine mAb 17-1A, which binds to EpCAM as well, slightly suppressed bsAb-redirected cytotoxicity induced by the bsAb. We conclude that CIK generated in vitro or in vivo combined with this new EpCAM/CD3 bsAb and the cytokine IL-2 should be evaluated for the treatment of EpCAM-expressing tumors. Received: 9 December 1999 / Accepted: 18 May 2000  相似文献   

15.
 T cells play a key role in the control of abnormal B cell proliferation. Factors that play a role in inadequate T cell responses include absence of expression of costimulatory and adhesion molecules by the malignant B cells and lack of cytotoxic T cells specific for tumor-associated antigens. A number of approaches have been used to enhance T cell response against malignant B cells. Agents such as soluble CD40 ligand can enhance expression of costimulatory molecules by the malignant B cells and improve their ability to activate T cells. Anti-CD3-based bispecific antibodies can retarget T cells toward the tumor cells irrespective of T cell specificity. We used the V 38C13 murine lymphoma model to assess whether the combination of soluble CD40 ligand and anti-CD3-based bispecific antibody can enhance T cell activation induced by malignant B cells more effectively than either approach alone. Expression of CD80, CD86, and ICAM-1 on lymphoma cells was up-regulated by soluble CD40 ligand. Syngeneic T cells were activated more extensively by lymphoma cells when the lymphoma cells were pre-treated with soluble CD40 ligand. Bispecific-antibody induced T cell activation was more extensive when lymphoma cells pretreated with soluble CD40 ligand were present. The combination of soluble CD40 ligand plus bispecific antibody enhanced the median survival of mice compared to mice treated with bispecific anibody alone. We conclude that pretreatment of tumor cells with agents capable of inducing costimulatory molecule expression, such as soluble CD40 ligand can enhance the ability of malignant B cells to activate T cells. This effect is enhanced by the addition of bispecific antibody. The combination of enhanced expression of costimulatory molecules and retargeting of T cells by bispecific antibody may allow for a more effective T-cell-based immunotherapy. Accepted: 14 October 1997  相似文献   

16.
 The bispecific monoclonal antibody (bsmAb) 2B1, targeting the extracellular domain of c-erbB-2, the protein product of the HER-2/neu proto-oncogene, and FcγRIII (CD16), expressed by human natural killer cells, neutrophils and differentiated monocytes, mediates the specific cytotoxic activity of these effector cells to tumor cells. A group of 24 patients with c-erbB-2-overexpressing tumors were treated with intravenously administered 2B1 in a phase I clinical trial and followed after treatment to evaluate the diversity and extent of the 2B1-induced humoral immune responses. As expected, 17 of 24 patients developed human anti-(murine Ig) antibodies (HAMA) to whole 2B1 IgG in a range from 100 ng/ml to more than 50 000 ng/ml; 10 of these patients (42%) had strong (at least 1000 ng/ml) HAMA responses, some of which were still detectable at day 191. These responses were usually associated with similar reactivity to the F(ab′)2 fragments of the parental antibodies 520C9 (anti-c-erbB-2) and 3G8 (anti-CD16). We sought evidence of an idiotypic cascade induction, indicating a prolonged specific treatment-induced effect on at least one selected target of 2B1. Using competition-based enzyme-linked immunosorbent assays, specific anti-idiotypic antibodies (Ab2) were detectable against 520C9 in 11 patients and against 3G8 in 13 patients. Peak anti-idiotypic antibodies generally occurred 3–5 weeks from treatment initiation, with a downward trend thereafter. There was a statistically significant correlation among the induction of significant HAMA responses, anti-idiotypic antibody production and the development of antibodies to c-erbB-2. The anti-c-erbB-2 responses, which were distinct from anti-anti-idiotypic (Ab3) antibodies, were detected in the post-treatment sera of 6/16 patients examined. No obvious correlation could be made between the development of humoral immune responses, the dose received, and the clinical response. Future investigations involving 2B1 therapy will concentrate on investigating an association of these humoral responses to any c-erbB-2-specific cellular responses. Manipulations of 2B1 therapy effects that augment immunity to c-erbB-2 could provide additional avenues for immunotherapy with this and other bispecific antibodies. Received: 1 August 1996 / Accepted: 28 March 1997  相似文献   

17.
 Tumor necrosis factor α (TNFα) and interferon γ (IFNγ) are important immunomodulators. They are capable of acting in a synergistic manner on tumor cells in vitro and in vivo. In a clinical phase I study 13 patients with malignant ascites due to abdominal spread of different primary tumors received intraperitoneally (i. p.) TNFα and IFNγ once weekly over 3 – 8 weeks in order to evaluate the effect of locoregionally administered TNFα/IFNγ on ascites formation. Therefore some peripheral and local immunological functional parameters of peripheral blood and malignant ascites were investigated. Mononuclear lymphocytes and natural killer (NK) cell activity of peripheral blood and ascites, TNF-inhibitory activity, soluble p55 and p75 TNF receptors, and prostaglandin E2 values in ascites were measured immediately before and 24 h after each TNFα/IFNγ infusion. Peripheral mononuclear lymphocytes and NK activity decreased significantly 24 h after i. p. TNFα/IFNγ application. However, over the entire treatment schedule, peripheral NK activity in all responders showed a continuous increase, when compared to pre TNFα/IFNγ treatment levels. In contrast, NK activity in non-responders constantly decreased. In contrast to non-responders, TNF-inhibitory activity and soluble p55 TNF receptor levels, determined in ascites, decreased in responders. Taken together, our findings suggest, that successful locoregional i. p. TNFα/IFNγ therapy induces systemic immunological reactions possibly after saturation of soluble p55 TNF receptors in ascites, which leads to an increase of peripheral NK activity. Received: 28 September 1995 / Accepted: 16 November 1995  相似文献   

18.
Summary Alpha-smooth muscle actin is currently considered a marker of smooth muscle cell differentiation. However, during various physiologic and pathologic conditions, it can be expressed, sometimes only transiently, in a variety of other cell types, such as cardiac and skeletal muscle cells, as well as in nonmuscle cells. In this report, the expression of actin mRNAs in cultured rat capillary endothelial cells (RFCs) and aortic smooth muscle cells (SMCs) has been studied by Northern hybridization in two-dimensional cultures seeded on individual extracellular matrix proteins and in three-dimensional type I collagen gels. In two-dimensional cultures, in addition to cytoplasmic actin mRNAs which are normally found in endothelial cell populations, RFCs expressed α-smooth muscle (SM) actin mRNA at low levels. α-SM actin mRNA expression is dramatically enhanced by TGF-β1. In addition, double immunofluorescence staining with anti-vWF and anti-α-SM-1 (a monoclonal antibody to α-SM actin) shows that RFCs co-express the two proteins. In three dimensional cultures, RFCs still expressed vWF, but lost staining for α-SM actin, whereas α-SM actin mRNA became barely detectable. In contrast to two-dimensional cultures, the addition of TGF-β1 to the culture media did not enhance α-SM actin mRNA in three-dimensional cultures, whereas it induced rapid capillary tube formation. Actin mRNA expression was modulated in SMCs by extracellular matrix components and TGF-β1 with a pattern very different from that of RFCs. Namely, the comparison of RFCs with other cell types such as bovine aortic endothelial cells shows that co-expression of endothelial and smooth muscle cell markers is very unique to RFCs and occurs only in particular culture conditions. This could be related to the capacity of these microvascular endothelial cells to modulate their phenotype in physiologic and pathologic conditions, particularly during angiogenesis, and could reflect different embryologic origins for endothelial cell populations. Supported by a Post-Doctoral Fellowship from the Swiss National Science Foundation (OK) and grant HL-RO1-28373 (JAM) from the Department of Human Services, Public Health Service, Washington, D.C.  相似文献   

19.
Summary The epigenetic modulation by histone deacetylase (HDAC) inhibitors including trichostatin A (TSA) has been known to block cell proliferation, induce apoptosis and inhibit cell migration in human cancer cells that represents the potential therapeutic agents for cancers and fibrosis. However, more than 55% of Hep3B cells remained alive after our initial study of 100 nM TSA treatment. To further study the epigenetic modulation and the biological function of newly activated genes by HDAC inhibitor involved in HCC progression and metastasis, we profiled 23 integrin genes including 15α and 8β in TSA-treated Hep3B cells. Six integrins including three down-regulated α6, α10, β8 and three significant up-regulated α4, β2, β6 integrins were revealed after semi-quantitative RT-PCR. To confirm the epigenetic modulation and explore their biological functions, we selected the three significantly up-regulated integrins for confirmation of protein up-regulation, hyperacetylated-histones by ChIP assays, and functional inhibition by specific neutralizing antibodies of integrins. Our results indicated that epigenetic modulation in TSA-treated Hep3B cells up-regulated new integrins including α4, β2 and β6 and reduced migration activities by specific neutralizing antibodies to 61.3%, 42.4% and 34.5%, respectively. Our novel findings provided a better understanding of the epigenetic modulation of integrins and suggested that targeting the epigenetic up-regulated integrins to abrogate the migration activity might be a promising strategy to prevent HCC progression.  相似文献   

20.
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