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U Rodeck

Publications and source records attributed to U Rodeck.

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

A central role of Bcl-X(L) in the regulation of keratinocyte survival by autocrine EGFR ligands.

The epidermal growth factor receptor has multiple roles in epidermal biology relating to growth, migration, and, as shown recently, survival of keratinocytes. In cultured keratinocytes activation of the epidermal growth factor receptor upregulates expression of Bcl-x(L), an anti-apoptotic Bcl-2 homolog. The functional contribution of epidermal growth factor receptor-dependent Bcl-x(L) expression to keratinocyte survival is poorly understood. Here we demonstrate that inhibition of the epidermal growth factor receptor tyrosine kinase activity with either an epidermal growth factor receptor antagonistic monoclonal antibody (MoAb 425) or an epidermal growth factor receptor-selective tyrosine kinase inhibitor (AG 1478) downregulated Bcl-x(L) expression in normal human keratinocytes but had no effect on expression of the pro-apoptotic Bcl-2 homologs Bad, Bak, and Bax. Bovine pituitary extract and insulin partially alleviated both, downregulation of Bcl-x(L) expression and cell death upon epidermal growth factor receptor inhibition. Forced expression of Bcl-x(L) attenuated cell death of immortalized keratinocytes (HaCaT) induced by either forced suspension (anoikis) or by epidermal growth factor receptor blockade. These results demonstrate that epidermal growth factor receptor-dependent signaling pathways control the balance of pro-apoptotic and anti-apoptotic Bcl-2 family members expressed in normal keratinocytes. Inappropriate survival supported by aberrant signaling through the epidermal growth factor receptor may contribute to the pathogenesis of psoriasis and of squamous cell carcinomas.

Apoptosis↗
PubMed1999

Loss of insulin-like growth factor I receptor-dependent expression of p107 and cyclin A in cells that lack the extracellular matrix protein secreted protein acidic and rich in cysteine.

The extracellular matrix-associated glycoprotein secreted protein acidic and rich in cysteine (SPARC) has been implicated in the control of cell proliferation during tissue remodeling, wound healing, and malignant development. Here, we describe a novel mechanism through which SPARC influences cell cycle progression in embryonic fibroblasts derived from Sparc-nullizygous (-/-) mice. SPARC-deficient cells were indistinguishable from wild-type cells in their ability to initiate DNA synthesis after treatment with either fetal bovine serum or platelet-derived growth factor. In contrast, Sparc -/- cells responded poorly to activation of the insulin-like growth factor receptor (IGFI-R) by insulin. This defect was traced to reduced expression of the IGFI-R in Sparc -/- cells. Consistent with impaired cell cycle progression through S-phase, insulin-stimulated Sparc -/- cells also revealed reduced expression of two key regulators of S phase progression (cyclin A and thymidine kinase), whereas expression of the G1 phase progression regulators cmyc or cyclin D1 was unaffected. An examination of the status of retinoblastoma family pocket proteins in Sparc -/- cells revealed a selective and dramatic reduction in levels of the retinoblastoma-related protein p107. Exogenous platelet-derived growth factor restored expression of the IGFI-R and IGFI-R dependent DNA synthesis as well as induction of cyclin A, thymidine kinase, and p107 in insulin-stimulated Sparc -/- cells. These results suggest that SPARC-dependent matrix to cell interactions contribute to the regulation of p107 and cyclin A through IGFI-R dependent pathway(s).

Animals↗
PubMed1998

Adenoviral transduction of melanoma cells with B7-1: antitumor immunity and immunosuppressive factors.

Previous studies in experimental models have demonstrated that the transduction of human or murine melanoma cells with the co-stimulatory B7-1 molecule induces effective antitumor immune responses. In order to develop B7-1 gene transfer as a therapeutic tool in the clinical management of melanoma, efficient means of in vivo gene transfer must be used. To this end we evaluated in vitro and in vivo immune responses associated with adenoviral transduction of murine and human melanoma cells with B7-1. Adenovirus-mediated transduction of human and murine melanoma cells with B7-1 leads to high-level transgene expression in vitro and in vivo and does not affect MHC class I and II expression. Adenovirus-delivered B7-1 induced antitumor immune responses, on the basis of observations that human melanoma cells transduced to express human B7-1 were able to co-stimulate allogeneic and autologous T cells to proliferate and that murine melanoma K1735 cells transduced to express murine B7-1 were rejected by syngeneic, immunocompetent mice. By contrast, intratumoral injection of an adenovirus encoding murine B7-1 failed to eliminate established murine melanoma (K1735) despite high-level transgene expression in tumor cells. Potent T cell inhibitory factor(s) secreted by both K1735 cells and select human melanoma cells may contribute to the failure to achieve protection in this setting. Thus, immune inhibitory melanoma-derived factors need to be taken into account when considering the clinical use of B7-1 immunotherapy.

Adenoviridae↗
PubMed1998

Characterization of monoclonal antibodies directed to the amino-terminus of the WT1, Wilms' tumor suppressor protein.

We have produced and characterized three monoclonal antibodies (MAbs) directed to the amino terminus of the WT1 Wilms' tumor suppressor transcription factor and compared their properties to rabbit polyclonal sera raised to the same immunogen. A recombinant protein consisting of amino acids 1-181 of human WT1 was overexpressed in E. coli, purified, and used as the immunogen. Three MAbs designated 6F-H2, 6F-H7, and 6F-HC-17--all of the IgG1 subclass--were selected and further characterized. Each recognized all isoforms of the full-length WT1 protein in Western blot assays and immunoprecipitated WT1 in both physiologic buffers and under high detergent/high salt (RIPA) conditions. Preliminary epitope mapping suggests that all three MAbs recognize a region in the amino terminal 84 amino acids of WT1 and that the MAbs do not recognize the polyglycine or polyproline regions of the protein. The WT1 antibodies do not recognize the structurally and functionally related early growth response (EGR)1, EGR2, EGR3, or EGR4 proteins. All WT1 MAbs recognize the murine WT1 protein and immunohistochemical staining of murine embryonic and newborn kidney sections show strong staining of condensing metanephric mesenchyme and primitive podocytes in developing glomeruli. These WT1-specific MAbs should be useful in characterizing the biochemical and developmental roles of WT1 and in defining the emerging role of WT1 as a diagnostic and/or prognostic marker in mesothelioma, leukemias, and breast cancer.

Amino Acid Sequence↗
PubMed1997

An episomal vector for stable tetracycline-regulated gene expression.

The recently introduced tetracycline (Tc)-regulatable eukaryotic gene expression system based on the Escherichia coli Tn 10 tetracycline operon has proven to be a powerful tool for controlled expression of a variety of genes in vitro as well as in vivo . Control elements of this expression system are contained in two separate plasmid vectors. The tTA vector encodes a transactivator protein and the tetP vector contains a responsive operator-promoter element (tetP) that controls gene expression depending on tTA binding. Establishment of cell lines expressing a gene of interest under tetP control requires two subsequent rounds of transfection and clonal selection after each transfection. Here we describe a modification of this system in which the tetP element is placed in an episomal EBNA-based plasmid that can be stably maintained in primate but not in rodent cells. Using HeLa and human melanoma cells, we show that upon transient or stable transfection a reporter gene is expressed in a Tc-regulated manner similar to the original system. Thus, this expression system combines the advantages of episomal vectors, such as high efficiency of transfection and time-efficient selection of mass cultures, with tight control of gene expression provided by the Tc-regulatable system.

Dose-Response Relationship, Drug↗
PubMed1997

Regulation of Bcl-xL expression in human keratinocytes by cell-substratum adhesion and the epidermal growth factor receptor.

Cell-substratum adhesion is an essential requirement for survival of human neonatal keratinocytes in vitro. Similarly, activation of the epidermal growth factor receptor (EGF-R) has recently been implicated not only in cell cycle progression but also in survival of normal keratinocytes. The mechanisms by which either cell-substratum adhesion or EGF-R activation protect keratinocytes from programmed cell death are poorly understood. Here we describe that blockade of the EGF-R and inhibition of substratum adhesion share a common downstream event, the down-regulation of the cell death protector Bcl-xL. Expression of Bcl-xL protein was down-regulated during forced suspension culture of keratinocytes, concurrent with large-scale apoptosis. Similarly, EGF-R blockade was accompanied by down-regulation of Bcl-xL steady-state mRNA and protein levels to an extent comparable to that observed in forced suspension culture. However, down-regulation of Bcl-xL expression by EGF-R blockade was not accompanied by apoptosis; in this case, a second signal, generated by passaging, was required to induce rapid and large-scale apoptosis. These findings are consistent with the conclusions that (i) Bcl-xL represents a shared molecular target for signaling through cell-substrate adhesion receptors and the EGF-R, and (ii) reduced levels of Bcl-xL expression through EGF-R blockade lower the tolerance of keratinocytes for cell death signals generated by cellular stress.

Animals↗
PubMed1997

Detection by monoclonal antibodies of the Wilms' tumor (WT1) nuclear protein in patients with acute leukemia.

The WT1 gene encodes a transcriptional regulator which during embryogenesis is involved in growth control and differentiation of diverse tissues. It is also expressed in few human malignancies, including acute leukemia. We tested 3 different monoclonal antibodies (MAbs H2, H7, HCl7) and the polyvalent serum WTC-19 for WT1 protein detection in mononuclear cell (MNC) preparations of 104 newly diagnosed acute leukemia patients. Using RT-PCR, these MNC preparations were also analyzed for WT1 gene expression. MAbs H2, H7 and HCl7 and the polyclonal WTC-19 exhibited nuclear immunoreactivity in 63 of 99, 28 of 56, 38 of 60 and 22 of 43 WT1 gene-expressing leukemia samples, respectively. With these antibodies, no WT1 immunoreactivity was found in MNCs from blood of healthy volunteers, from CD34+ progenitor cell-enriched leukapheresis products of patients conditioned for peripheral stem cell harvest or from reactive bone marrow. Contrary to WTC-19, all MAbs reacted highly specifically with the WT1 protein (0.71 vs. 1.0). The WT1 protein was heterogeneously detected in leukemia blast preparations by all antibodies, irrespective of cell morphology. Very few HL60 cells and blasts from newly diagnosed leukemia patients interspersed among normal blood MNCs (50 blasts among 5 x 10(5) MNCs) were easy to identify by indirect immunofluorescence using MAbs H2 and HCl7. Taken together, MAbs H2 and HCl7 were superior to MAb H7 and the polyvalent WTC-19 in detecting the WT1 nuclear protein.

Antibodies, Monoclonal↗
PubMed1997

WT1 mutation in malignant mesothelioma and WT1 immunoreactivity in relation to p53 and growth factor receptor expression, cell-type transition, and prognosis.

The Wilms tumour 1 (WT1) gene is believed to contribute to the growth and differentiation of certain tissues, including mesothelium. This study assessed WT1 gene status by mutational screening in 42 malignant mesotheliomas (MMs) and 3 MM cell lines and detected two tumours with identical heterozygous single nucleotide deletions in intron 7, with no apparent consequence for WT1 function. Furthermore, the expression pattern of the WT1 gene was studied in MMs and related lesions using three anti-WT1 monoclonal antibodies (MAbs). Strong to moderate nuclear immunoreactivity was noted in MM in situ (54/56), cultured mesothelioma cells (4/5), and hyperplastic and normal pleural (non-neoplastic, NNM) specimens. WT1 immunoreactivity was absent in all primary tumours of lung and in pleural metastases from adenocarcinomas of breast and colon; immunoreactivity was present in pleural metastases from renal carcinomas, melanomas, and papillary carcinomas of the ovary. Expression of the WT1 protein in MM was not correlated with survival. Coordinate expression of the WT1 protein and its putative transcriptional target genes was determined by correlating WT1 immunostaining with epidermal growth factor receptor (EGF-R) and insulin-like growth factor 1 receptor (IGF-1R) expression on MM and NNM; no significant correlation was found, irrespective of p53 expression status. Finally, the putative involvement of WT1 in cell-type transition was supported by this study, in that epithelial mesothelioma showed the strongest WT1 immunoreactivity while sarcomatous mesothelioma showed the least.

Adult↗
PubMed1997

Growth stimulation of colorectal carcinoma cells via the c-kit receptor is inhibited by TGF-beta 1.

Activation of the receptor tyrosine kinase c-kit by the kit-ligand, also known as stem cell factor (SCF), is essential to melanocyte and germ cell development and during the early stages of hematopoiesis. Deregulated expression of c-kit has been reported in malignancies affecting these lineages, i.e., myeloid leukemias, melanomas, and germ cell tumors. In addition, c-kit and SCF are coexpressed in some breast and colorectal cancer (CRC) cells, raising the question of whether c-kit serves an autocrine role in normal or malignant epithelial tissues. In this study, we demonstrate that human colorectal carcinomas, but not normal colorectal mucosa cells, coexpress SCF and c-kit in situ. Expression of c-kit was also observed in mucosa adjacent to colorectal tumor tissue. Consistent with a growth-regulatory role of SCF in CRC cells, exogenous SCF stimulated anchorage-dependent and anchorage-independent growth in four out of five CRC cell lines. Exogenous transforming growth factor (TGF)-beta 1 added at nanomolar concentrations to HT-29 CRC cells, which express the type I, II, and III TGF-beta receptors, downregulated c-kit expression to background levels and inhibited c-kit-dependent proliferation. Similarly, TGF-beta 1 inhibited SCF-dependent proliferation of three first-passage CRC cell lines. In summary, expression of the potential autocrine SCF/ c-kit axis is a tumor-associated phenomenon in colorectal cancer that can be suppressed by TGF-beta 1 in TGF-beta-responsive CRC cells.

Adult↗
PubMed1997

EGF-R dependent regulation of keratinocyte survival.

Tissue organization and maintenance within multicellular organisms is in part dependent on the ability of cells to undergo programmed cell death or apoptosis. Conversely, disruption of cell death pathways often is associated with tumor development. At present, the molecular control of apoptosis in epithelial cells is poorly understood. Here we describe evidence linking epidermal growth factor-receptor (EGF-R) activation to survival of normal human keratinocytes in culture. Inhibition of EGF-R activation by an anti-EGF-R antagonistic monoclonal antibody (mAb 425), followed by detachment of keratinocytes from the substratum, induced extensive death with several features of apoptosis in keratinocyte cultures. Other, non-epithelial normal human cells including melanocytes and fibroblasts, did not show this effect. Similar to EGF-R blockade by mAb 425, inhibition of the EGF-R tyrosine kinase activity using tyrphostin AG1478 resulted in lack of attachment and extensive cell death upon passaging. Attachment to keratinocyte-derived ECM partially resuced mAb 425-treated keratinocytes from cell death, indicating that adhesion-dependent and EGF-R-dependent signal transduction pathways serve partially overlapping but not redundant roles in supporting keratinocyte survival.

Apoptosis↗
PubMed1996

Identification of differentially expressed messenger RNAs in human melanocytes and melanoma cells.

The phenotype of malignant lesions is a reflection of genetic events altering the RNA and protein expression patterns of normal cells. We have investigated RNA expression patterns distinguishing normal melanocytes (FM 902), a primary melanoma cell line (WM 793), and its variant cell line (1205-LU), selected for metastatic phenotype in athymic mice. Using mRNA differential display, we identified 42 different cDNA PCR products with cell line-specific expression patterns. Direct sequence analysis matched approximately 50% of the cDNA PCR products with gene sequences accessible in DNA databases. Among the known genes, two functionally distinct groups were recognized: (a) genes encoding ribosomal and mitochondrial proteins that were predominantly up-regulated in the malignant cells; and (b) genes encoding modulators of the immune response. Among the immunomodulators, the T-cell antigen MART-1 and the protease inhibitor alpha2-macroglobulin were detected in the melanocyte cell line but not in the tumor cells. By contrast, mRNAs for the complement inhibitor CD59 and the cytokine IL-1beta were found to be overexpressed in the malignant melanoma cells. RNA slot blot hybridization on a larger panel of melanocyte and melanoma cell lines confirmed differential expression of 15 of 42 genes including MART-1, alpha2-macroglobulin, and CD59. This molecular screening approach identified also three partially characterized and three novel sequences with differential expression patterns in normal and malignant melanocytes.

Animals↗
PubMed1996

Melanoma-associated expression of transforming growth factor-beta isoforms.

Melanocytic neoplasia is characterized by the aberrant overproduction of multiple cytokines in vitro. However, it is largely unknown how cytokine expression relates to melanoma progression in vivo. Transforming growth factor beta (TGF-beta) is a multifunctional cytokine commonly produced by cultured melanoma cells. The in situ expression of all three TGF-beta isoforms (TGF-beta1, -2, and -3) was determined immunohistochemically in melanocytes and in 51 melanocytic lesions using isoform-specific antibodies. Significant linear trends of expression were observed from melanocytes through nevi and primary and metastatic melanomas for all three isoforms. TGF-beta1 was expressed by some melanocytes and almost uniformly by nevi and melanomas. TGF-beta2 and -3 were not expressed in normal melanocytes but were expressed in nevi and early and advanced primary (radial and vertical growth phase) and metastatic melanomas in a tumor-progression-related manner. TGF-beta2 was heterogeneously expressed in advanced primary and metastatic melanomas, whereas TGF-beta3 was uniformly and highly expressed in these lesions. Thus, expression of TGF-beta isoforms in melanocytes and melanocytic lesions is heterogeneous and related to tumor progression, and expression of TGF-beta2 and TGF-beta3 commences at critical junctures during progression of nevi to primary melanomas.

Biomarkers, Tumor↗
PubMed1995

Regulation of NK cell functions by TGF-beta 1.

The contribution of exogenous and endogenous TGF-beta 1 to human peripheral blood NK cell proliferation and activity in vitro was investigated. Exogenous bioactive TGF-beta 1 inhibited NK cell DNA synthesis and production of IFN-gamma, TNF-alpha, and granulocyte-macrophage CSF (GM-CSF) by NK cultures consisting of > 98% CD56+ cells. The cytotoxic activity of NK cells was also weakly inhibited by exogenous TGF-beta 1. All TGF-beta 1-induced inhibitory effects occurred in the absence and presence of the NK cell-activating cytokines IFN-alpha, IL-2, and IL-12. Unstimulated NK cell cultures expressed steady state TGF-beta 1 mRNA detected by Northern blot analysis and produced TGF-beta protein (1.6 ng/ml), as determined by ELISA. When NK cell proliferation was induced by IL-2, IL-12, IFN-alpha, or a combination of IL-2 and IL-12, expression of TGF-beta 1 mRNA and protein was moderately and consistently reduced by approximately 20%, as compared with unstimulated control cultures. Unstimulated and rapidly proliferating NK cell cultures secreted primarily latent TGF-beta into their culture medium, as determined by the Mv1Lu bioassay. These results indicate that, in vitro, endogenous NK cell-derived TGF-beta 1 has no negative autocrine effect upon activation of NK cells by various cytokines.

Animals↗
PubMed1995

Induction of autocrine epidermal growth factor receptor ligands in human keratinocytes by insulin/insulin-like growth factor-1.

Autocrine activation of the epidermal growth factor (EGF) receptor on keratinocytes has been recognized as an important growth regulatory mechanism involved in epithelial homeostasis, and, possibly, hyperproliferative diseases. Insulin-like growth factor (IGF)-1 and insulin have been shown to be paracrine keratinocyte mitogens that bind to the type I IGF receptor which is expressed on actively proliferating keratinocytes in situ. In this report, we demonstrate that IGF-1/insulin induced production of keratinocyte-derived autocrine growth factors that bind to the EGF receptor. Increased steady-state mRNA levels for transforming growth factor alpha (TGF-alpha) and for amphiregulin (AR) were observed upon incubation of keratinocytes with mitogenic concentrations of IGF-1. IGF-1 also induced production and secretion of TGF-alpha and AR proteins as detected by immunoassays. An EGF receptor antagonistic monoclonal antibody abolished the mitogenic effect of IGF-1 on cultured keratinocytes. These results suggest that stimulation of keratinocyte growth of IGF-1 requires activation of an EGF receptor-mediated autocrine loop.

Amphiregulin↗
PubMed1995

Antiproliferative effects of tyrosine kinase inhibitors (tyrphostins) on human bladder and renal carcinoma cells.

Growth factor receptors with tyrosine kinase activity mediate paracrine and autocrine growth regulation of normal and malignant cells. The epidermal growth factor receptor (EGF-R) is a tyrosine kinase transmembrane protein that is overexpressed by many epithelial malignancies, including transitional cell and renal cell carcinoma. Ligand-induced stimulation of cell growth depends on activation of the tyrosine kinase activity of the EGF-R. Tyrphostins are small molecular weight compounds that have been shown to preferentially inhibit the EGF-R tyrosine kinase and thus may inhibit EGF-R-dependent cell growth. We examined the effect of two tyrphostins, RG14620 and AG555, on the proliferation of three transitional cell carcinoma lines (RT4, J82, and T24) and three renal cell carcinoma lines (A-198, Caki-1, and Caki-2). Both tyrphostins inhibited proliferation of all six cell lines in a dose-dependent fashion. They were equally effective with IC50s ranging between 3 and 16 microM. Complete inhibition of growth was achieved at tyrphostin concentrations between 10 and 30 microM. Although both tyrphostins inhibited proliferation of T24 transitional carcinoma cells in growth assays, only RG14620 but not AG555 was found to specifically inhibit EGF-R autophosphorylation in this cell line. These results suggest that other intracellular targets in addition to the EGF-R are affected by these agents. In summary, tyrphostins are potent growth inhibitors for urological malignancies.

Adult↗
PubMed1995

Use of a serum-free epidermal culture model to show deleterious effects of epidermal growth factor on morphogenesis and differentiation.

The presence of serum has limited the utility of many culture models for the study of cytokine effects because its complexity and variability can confound the interpretation of data. In the present study, a serum-free skin co-culture model was used to investigate the effect of exogenous epidermal growth factor (EGF) on epidermal proliferation and differentiation. Human keratinocytes cultured on collagen rafts at the air-liquid interface produced a well-differentiated epithelium that resembled normal epidermis. Keratin filaments, membrane-coating granules, and keratohyalin granules were all observed. Epidermal differentiation markers keratin K1/K10, involucrin, and transglutaminase were localized in most of the suprabasal layers, whereas profilaggrin/filaggrin was confined to the granular layers and stratum corneum. In the continual presence of 10-20 ng/mL EGF, the epidermis was less organized, thinner, and less proliferative. EGF also depressed several indicators of differentiation: The number of keratohyalin granules and membrane-coating granules was greatly decreased; antigen expression of profilaggrin/filaggrin appeared diminished by immunocytochemical staining; frequent nuclear retention was noted in the relatively thickened stratum corneum-like layers. As detected by immunohistochemical staining, the expression of EGF receptor in the epidermis was reduced by exogenous EGF. These data illustrate that EGF cannot be considered a simple mitogen. Our findings also underscore the importance of using sophisticated culture models to assess complex cytokine effects that may be dependent on the architecture of a differentiating epidermis.

Air↗
PubMed1995

Presence of Wilms' tumor gene (wt1) transcripts and the WT1 nuclear protein in the majority of human acute leukemias.

The wt1 gene is located on chromosome 11p13 and encodes a zinc finger motif-containing transcription factor involved in regulation of growth and differentiation. Its expression was shown during embryonic development in various tissues as well as in a few human malignancies including acute leukemias. Using RT-PCR, we found wt1 gene expression in blast cells of the majority of 150 acute leukemia patients. Particularly, the wt1 transcript was detected in 12 of 14 (86%) pre-pre-B-ALL patients, in 33 of 41 (80%) cALL patients, in 23 of 31 (74%) T-ALL patients, and in 53 of 57 (93%) AML patients. Additionally, mononuclear cells from CML patients expressed the wt1 gene only when diagnosed with blast crisis. In contrast to acute human leukemias, mononuclear cells from reactive bone marrow (n = 4), and peripheral blood of healthy volunteers (n = 20), as well as normal peripheral CD34+ hematopoietic progenitors (n = 6) did not express the wt1 gene at detectable levels. Using the anti-WT1 MoAb 6F-H2 in an immunofluorescence assay on single cell level, we found the translated WT1 protein only in nuclei of leukemia blast cells but not in nuclei of normal CD34+ hematopoietic progenitor cells. Blast cells of 12 of 20 leukemia patients (60%) all tested positive for the wt1 gene expression by RT-PCR displayed a strong nuclear immunofluorescence. Its expression in the majority of human acute leukemias but not in normal mononuclear blood cells and normal CD34+ hematopoietic progenitors qualifies the wt1 gene transcript as a 'pan-acute leukemic' marker probably useful in monitoring minimal residual disease after chemotherapy and in detecting leukemic blast cells in purged or unpurged hematopoietic stem cell preparations intended to be used for autologous bone marrow transplantation.

Adult↗