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Biomedical subjects

U Stein

Publications and source records attributed to U Stein.

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

GLI gene expression in bone and soft tissue sarcomas of adult patients correlates with tumor grade.

The GLI gene encodes a transcription factor harboring five zinc finger motifs that bind to DNA in a sequence-specific manner. The gene was originally identified because of its amplification in a human glioblastoma, and previous studies have shown it to be amplified in a significant proportion of mesenchymal tumors, such as childhood sarcomas. Here we evaluate GLI gene expression in bone and soft tissue sarcomas of adult patients. Samples from 40 patients (37 sarcomas and 3 benign mesenchymal tumors) and samples of 15 normal mesenchymal tissues were examined for GLI gene amplification and expression by Southern hybridization, reverse transcription-PCR of tissue RNA, and immunohistochemistry, using a new polyclonal GLI antibody developed against an epitope outside of the zinc finger region. In contrast to childhood sarcomas, amplification of the GLI gene was not observed in sarcomas of adult patients. Although GLI gene expression in sarcomas was significantly higher than that in normal mesenchymal tissues (P < 0.0001), the levels were very variable. Attempts to correlate the expression data with different pathophysiological parameters only showed a significant relationship to tumor grade. Based on these data, increased levels of GLI gene expression may be indicative of the aggressiveness of the tumor.

Adult↗
PubMed1999

Hyperthermia for treatment of rectal cancer: evaluation for induction of multidrug resistance gene (mdr1) expression.

Environmental stress factors, such as heat, may induce multidrug resistance gene (mdr1) expression, which could result in the disadvantageous multidrug resistance (MDR) phenotype. To evaluate this possibility in a clinical situation, we investigated mdr1 gene expression in patients with locally advanced rectal cancer who underwent preoperative radio-chemo-thermo-therapy (RCTT). Patients were classified into groups according to the treatment schedule of RCTT vs. radio-chemo-therapy (RCT) without hyperthermia (control group). Expression of the mdr1 gene was analyzed in tumors and normal rectal tissues prior to and post-treatment (RCTT or RCT, respectively) by means of semi-quantitative and quantitative reverse transcription-polymerase chain reaction (RT-PCR). The data were correlated with therapeutic response and survival parameters. Based on our evaluation criteria, in 2 of 19 tumors of the RCTT group, mdr1 gene expression was increased more than 2-fold; in 3 of 19 tumors of this group, however, mdr1 expression was decreased more than 2-fold. In the patient control group, levels of mdr1 gene expression were reduced in 2 of 8 tumors. Thus, hyperthermia combined with RCT (RCTT) in comparison with RCT alone does not lead to an increase in mdr1 gene expression in patients with locally advanced rectal cancer within the preoperative treatment schedule. The risk of inducing the classical multidrug resistance phenotype by hyperthermia was thus minimal in this clinical setting. Subsequent adjuvant chemotherapy should thus not be hindered.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1999

Therapeutic genes for cancer gene therapy.

Cancer still represents a disease of high incidence and is therefore one major target for gene therapy approaches. Gene therapy for cancer implies that ideally selective tumor cell killing or inhibition of tumor cell growth can be achieved using nucleic acids (DNA and RNA) as the therapeutic agent. Therefore, the majority of cancer gene therapy strategies introduce foreign genes into tumor cells which aim at the immunological recognition and destruction, the direct killing of the target cells or the interference with tumor growth. To achieve this goal for gene therapy of cancer, a broad variety of therapeutic genes are currently under investigation in preclinical and in clinical studies. These genes are of very different origin and of different mechanisms of action, such as human cytokine genes, genes coding for immunostimulatory molecules/antigens, genes encoding bacterial or viral prodrug-activating enzymes (suicide genes), tumor suppressor genes, or multidrug resistance genes.

Gene Transfer Techniques↗
PubMed1997

Development and characterisation of novel human multidrug resistant mammary carcinoma lines in vitro and in vivo.

Clinical chemotherapy of breast carcinomas must be considered insufficient, mainly due to the appearance of drug resistance. The multidrug resistance (MDR) phenotype, either intrinsically occurring or acquired, e.g., against a panel of different antineoplastic drugs, is discussed in relation to several MDR-associated genes such as the MDR-gene mdr1 encoding the P-glycoprotein (PGP), the MRP gene (multidrug resistance protein) encoding an MDR-related protein or the LRP gene encoding the lung resistance protein. Numerous experimental and clinical approaches aiming at reversing resistance require well-characterised in vitro and in vivo models. The aim of our work was to develop multidrug resistant sublines from human xenotransplanted breast carcinomas, in addition to the broadly used line MCF-7 and its multidrug resistant subline MCF-7/AdrR. MDR was induced in vitro with increasing concentrations of Adriablastin (ADR) for several weeks, resulting in a 3.5- to 35-fold increase in IC50 values using the MTT-test. Cell lines were cross-resistant toward another MDR-related drug, vincristine, but remained sensitive to non-MDR-related compounds such as cisplatin and methotrexate. The resistance toward Adriamycin and vincristine was confirmed in vivo by a lack of tumour growth inhibition in the nude mouse system. Gene expression data for the mdr1/PGP, MRP/MRP and LRP/LRP on both the mRNA (RT-PCR) and the protein levels (immunoflow cytometry) demonstrated that induction of mdr1 gene expression was responsible for the acquired MDR phenotype. Rhodamine efflux data, indicated by PGP overexpression, underlined the development of this MDR mechanism in the newly established breast carcinoma lines MT-1/ADR, MT-3/ADR and MaTu/ADR.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

Tumor necrosis factor-alpha and expression of the multidrug resistance-associated genes LRP and MRP.

BACKGROUND AND PURPOSE: Cancer cells that express P-glycoprotein, multidrug resistance-associated protein (MRP), or lung resistance protein (LRP) have demonstrated resistance to a wide variety of chemotherapeutic drugs. Recently, we reported that human colon carcinoma cells that express all three proteins exhibit reduced P-glycoprotein gene expression and a loss of multidrug resistance after exposure to tumor necrosis factor-alpha, a hormone-like protein produced by cells of the immune system. In this study, we examined the effects of tumor necrosis factor-alpha on MRP and LRP gene expression in the same colon carcinoma cells. METHODS: HCT15 and HCT116 colon carcinoma cells were incubated with tumor necrosis factor-alpha at 100 U/mL for 2, 12, 24, 48, or 72 hours; alternatively, cells transfected with an expression vector containing a human tumor necrosis factor-alpha complementary DNA were studied. The effects of tumor necrosis factor-alpha on MRP and LRP messenger RNA expression were evaluated by means of reverse transcription and the polymerase chain reaction; effects on MRP and LRP protein expression were examined by use of specific monoclonal antibodies and flow cytometry. The flow cytometry data were analyzed by use of the two-sided, nonparametric Mann-Whitney rank sum test. RESULTS: Treatment with exogenous tumor necrosis factor-alpha reduced the level of LRP messenger RNA in both cell types in an apparently time-dependent fashion; in HCT15 cells, almost no LRP messenger RNA was detected after 48 hours of treatment. In contrast, the level of MRP messenger RNA was increased in HCT116 cells by such treatment, but the level in HCT15 cells was unchanged. Treatment with exogenous tumor necrosis factor-alpha induced changes in LRP and MRP protein expression in the two cell types that paralleled the changes found for messenger RNA. In transfected cells, the endogenous production of tumor necrosis factor-alpha reduced LRP gene expression (both messenger RNA and protein) and increased MRP gene expression (both messenger RNA and protein), regardless of cell type. CONCLUSION: In human colon carcinoma cells, tumor necrosis factor-alpha influences MRP and LRP gene expression in opposite ways. The findings for LRP gene expression parallel our earlier findings for P-glycoprotein expression in these cells. IMPLICATION: In developing strategies for overcoming multidrug resistance in tumor cells, the possibility that an agent can suppress one or more mechanisms of drug resistance and enhance others should be considered.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

Employment of the mdr1 promoter for the chemotherapy-inducible expression of therapeutic genes in cancer gene therapy.

Numerous approaches in gene therapy of human cancers are focused on the establishment of cell type specific or inducible expression vectors allowing the targeted and regulated expression of therapeutic genes. Various conditionally active vectors have been created carrying promoters responding to certain factors or therapeutic modalities (eg hormones, irradiation). The promoter of the multidrug resistance gene (mdr1) harbors such responsive elements and two of these elements have been related to drug responsiveness. In earlier studies we and others have characterized the mdr1 drug responsive-element in CAT reporter assays demonstrating its inducibility by MDR-associated drugs. To exploit this property, we linked the mdr1 promoter sequence to the human tumor necrosis factor alpha (TNF) cDNA in a retroviral vector and transduced the vector into human mammary and colon carcinoma cell lines. These cells were treated with various mdr1-associated drugs to induce TNF expression in vitro. We have shown that the mdr1 promoter-driven TNF expression is drug-inducible and that this induction is drug concentration and time dependent. The studies demonstrate the feasibility of the novel vector system for a chemotherapy-inducible expression of a chemosensitizing cytokine that is successful at enhancing cytotoxicity of drugs in cancer therapy.

Antineoplastic Agents↗
PubMed1996

Reversal of multidrug resistance by transduction of cytokine genes into human colon carcinoma cells.

BACKGROUND: Multidrug resistance can be a major obstacle to successful cancer chemotherapy and is often associated with increased expression of the mdr1 (also known as P-glycoprotein) gene. Some of the proteins produced by the body's immune system, i.e., cytokines such as tumor necrosis factor-alpha (TNF) and interleukin 2 (IL-2), have been shown to modulate multidrug resistance. However, cytokines administered by the conventional intravenous method can cause severe side effects. Transduction of cytokine genes into tumor cells constitutes an alternative approach for production and release of the cytokine proteins in the local tumor microenvironment, which may reduce problems of toxicity associated with systemic administration. PURPOSE: In this study, we investigated the therapeutic potential of a combination of gene therapy and chemotherapy on the basis of cytokine-mediated modulation of multidrug resistance in human colon carcinoma cells. METHODS: Human colon carcinoma cell lines HCT15 and HCT116 were transduced with TNF or IL-2 carrying murine leukemia virus (MLV)-based retroviral vectors. Tumor cell clones were analyzed for cytokine expression by reverse transcriptase-polymerase chain reaction (RT-PCR) and by cytokine-specific enzyme-linked immunosorbent assays (TNF-ELISA or IL-2-ELISA). Expression of mdr1 messenger RNA (mRNA) was investigated using RT-PCR, and P-glycoprotein (Pgp) expression was determined by immunoflow cytometry with the monoclonal antibodies MRK16 and C219. The function of Pgp was analyzed by measuring accumulation of the fluorescent drug doxorubicin by flow cytometry. The XTT-(i.e., [2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)]-5-[(phenylamino)-carbon yl-2H- tetrazolium hydroxide]-colorimetric cytotoxicity assay was used to determine chemosensitivity of cytokine gene-transfected tumor cells to doxorubicin and vincristine. Statistical significance was determined by the nonparametric Mann-Whitney rank sum test for the flow cytometry experiments (Pgp detection as well as drug uptake assays) and the parametric Student's t test for the chemosensitivity assay (XTT cytotoxicity assay). All P values reported were derived from two-sided statistical tests. RESULTS: Transduction and expression of human TNF and IL-2 in HCT15 and HCT116 human colon carcinoma cell lines were found to reverse multidrug resistance. Both TNF and IL-2 secretion reduced mdr1 expression on the mRNA and Pgp levels (P < .0243). This result was associated with enhancement of doxorubicin accumulation within the cells (P < .0001). The cytokine-mediated effects on mdr1 expression resulted in increased chemosensitivity of the transduced cells to doxorubicin and vincristine (P < .0460). CONCLUSIONS AND IMPLICATIONS: We show that endogenous expression of cytokine genes in tumor cells and after transduction secretion of the related proteins, such as TNF and IL-2, can modulate multidrug resistance in vitro. This modulation enhances the susceptibility of the cells to the cytotoxic drugs. Our findings suggest the potential value of combined treatment of resistant tumors with gene therapy and chemotherapy.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

Differential expression of 5 alpha-reductase isoenzymes in the human prostate and prostatic carcinomas.

BACKGROUND: Steroid 5 alpha-reductase is essential for the intracellular accumulation of dihydrotestosterone (DHT), which mediates androgen effects on target tissue. METHODS: In the present study, we describe the differential expression and cellular localization of 5 alpha-reductase 1 and 2 isoenzymes in the human prostate, and untreated and hormone-resistant prostatic carcinomas. The secretory epithelium of normal and hyperplastic glands showed strong nuclear 5 alpha-reductase 1 reactivity. Accordingly, the DHT forming 5 alpha-reductase process in secretory luminal cell types may be mediated predominantly by the type 1 isoenzyme. The androgen-independent basal cell layer variably expressed type 1 and 2 isoenzymes in nuclear and cytoplasmatic compartments. This suggests that circulating androgens are involved to control the basal cell layer, which represents the proliferative compartment of the human prostate. RESULTS: When compared with benign prostate tissue, increased 5 alpha-reductase reactivity was detected in prostate cancer, particularly in high-grade tumors and androgen-insensitive states of the disease. In cancerous lesions, the type 1 isoenzyme tended to shift to the cytoplasm, while the nuclear staining remained unchanged or slightly increased. Referring to the type 2 isoenzyme, increased cytoplasmatic and nuclear enzyme activity was detected in malignant cells when compared with adjacent benign prostate tissue. Even endocrine differentiated tumor cells that consistently lacked the nuclear androgen receptor variably expressed 5 alpha-reductase immunoreactivity. CONCLUSIONS: Although the functional significance of the differential subcellular localization of type 1 and 2 isoenzymes is currently unknown, the present data suggest that prostate cancer retains the DHT forming 5 alpha-reductase process in high-grade lesions and recurrent disease. Accordingly, circulating androgens may be still significant in these hormone-refractory malignancies.

Androgens↗
PubMed1996

Cell type specific and inducible promoters for vectors in gene therapy as an approach for cell targeting.

Gene therapy is used to correct genetic defects or to deliver new therapeutic functions to the target cells. Viral vectors are employed mainly as a gene delivery system. A great variety of viral expression systems have been developed and assessed for their ability to transfer genes into somatic cells. In particular, retroviral and adenoviral mediated gene transfer have been extensively studied and improved. Preclinical and clinical studies covering a large range of genetic disorders are currently underway to solve basic issues dealing with gene transfer efficiencies, regulation of gene expression, and potential risks of the use of viral vectors. The majority of clinical gene therapy trials that employ viral vectors perform exvivo gene transfer into target cells. The main issue in potential clinical application of gene therapy is the need for increased gene transfer efficiency and target specificity associated with regulated gene expression at therapeutically relevant levels in vivo. Gene regulatory elements, such as promoters and enhancers, possess cell type specific activities and can be activated by certain induction factors (e.g., hormones, growth factors, cytokines, cytostatics, irradiation, heat shock) via responsive elements. A controlled and restricted expression of these genes can be achieved using such regulatory elements as internal promoters to drive the expression of therapeutic genes in viral vector constructs. In addition to high level and efficient gene expression, minimizing or excluding inappropriate gene expression in surrounding nontarget cells is of great importance for numerous gene therapeutic approaches. This contribution furnishes insight into the field of cell type specific promoter and enhancer systems which have been used for targeted and inducible expression of therapeutic genes in certain genetic disorders, viral infections, and malignancies. We also discuss promoters that represent attractive candidates for the construction of viral vectors.

Gene Expression Regulation↗
PubMed1996

Vincristine induction of mutant and wild-type human multidrug-resistance promoters is cell-type-specific and dose-dependent.

To investigate multidrug-resistance gene (MDR1) promoter efficacy and drug inducibility in cells with different multidrug-resistance phenotypes, multidrug-resistant HCT15 and drug-sensitive KM12 human colon carcinoma cell lines were transfected with constructs incorporating the chloramphenicol acetyltransferase (CAT) reporter gene, driven by wild-type and point-mutated MDR1 promoter regions. The basal CAT expression level in HCT15 cells was markedly elevated compared to KM12 cells. CAT induction by vincristine was dose-dependent over a broad concentration range (40-500 ng/ml) in both lines. The induction levels were related to the cells' MDR phenotype, with the multidrug-resistant HCT15 cells showing the greater effect. In both cell types, basal and drug-induced CAT expression were significantly enhanced by the point-mutated promoter regions. The findings support the possible exploitation of the MDR1 promoter for construction of drug-inducible and MDR-cell-targeted expression vectors for use in gene therapy.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

Targeted vectors for gene therapy of cancer and retroviral infections.

Gene therapy has developed to a technology which rapidly moved from the laboratory bench to the bedside in the clinic. This implies safe, efficient and targeted gene transfer systems for suitable application to the patient. Beside the development of such gene transfer vectors of viral or nonviral origin, improvement of cell type specific and inducible gene expression is pivotal for successful gene therapy leading to targeted gene action. Numerous gene therapy approaches for treatment of cancer and retroviral infections utilize cell type specific and/or regulatable promoter and enhancer sequences for the selective expression of therapeutic genes in the desired cell populations and tissues. In this article the recent developments and the potential of expression targeting are reviewed for gene therapy approaches of cancer and retroviral infections.

Animals↗
PubMed1996

MDR1 gene expression: evaluation of its use as a molecular marker for prognosis and chemotherapy of bone and soft tissue sarcomas.

Successful chemotherapeutic treatment of malignant tumours is often limited by the intrinsic or acquired multidrug resistance (MDR). The classical MDR phenotype is characterised by reduced drug accumulation within the cell, caused by overexpression of the MDR1 gene encoded P-glycoprotein. Some reports have been published evaluating MDR1 expression as a molecular marker for response to chemotherapy in human bone and soft tissue sarcomas. In this review, an attempt is made to summarise the accuracy of the measurement of MDR1 expression for use in prognosis, as well as in decisions on chemotherapeutic treatment of sarcomas. In addition, general problems for the performance of such studies is discussed.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

Modulation of mdr1 expression by cytokines in human colon carcinoma cells: an approach for reversal of multidrug resistance.

Reversal of multidrug resistance (MDR) may offer a means of increasing the effectiveness of tumour chemotherapy. A variety of recent evidence indicates that cytokines may be particularly useful in this endeavour. To investigate the molecular mechanism by which cytokines may sensitise multidrug-resistant colon carcinoma cells, HCT15 and HCT116, to treatment with MDR-related drugs, we evaluated the effects of the human cytokines tumour necrosis factor alpha (TNF alpha), interleukin 2 (IL-2) and interferon gamma (IFN gamma) on mdr1 gene expression at the mRNA level by reverse transcription-polymerase chain reaction (RT-PCR) and at the protein level with monoclonal antibodies by immuno flow cytometry. P-glycoprotein function was examined after accumulation of the fluorescent drug, doxorubicin, by flow cytometry. Chemosensitivity to doxorubicin and vincristine was analysed using the XTT assay. All three cytokines were found to modulate the MDR characteristics on mdr1 expression levels, P-glycoprotein function and measured chemosensitivity to MDR-associated anti-cancer drugs. This cytokine-induced reversal of MDR was strongly time dependent, with maximal effects after 48 and 72 h of cytokine treatment. If similar modulation of MDR phenotype can be obtained in in vivo models, it may be possible to verify the time course for modulation by cytokine treatment and to design appropriate clinical trials of this strategy for MDR reversal.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

Gene transfer of human TNF alpha into glioblastoma cells permits modulation of mdr1 expression and potentiation of chemosensitivity.

Despite substantial advances in the surgery, radiotherapy and chemotherapy of gliomas, the prognosis of patients with glioblastomas has still not improved. Disappointing results in chemotherapy of glioblastomas resulting from multi-drug resistance (MDR) prompted us to investigate the influence of cytokine gene transfer in glioblastoma cells on the expression of P-glycoprotein and on chemosensitivity of transduced cells. Several investigations have shown that malignant gliomas express P-glycoprotein at high levels. The P-glycoprotein is a product of the multi-drug resistance gene (mdr1) and functions as an energy-dependent efflux pump which decreases drug accumulation and cytotoxicity. Since tumour necrosis factor alpha (TNF alpha) is a powerful anti-cancer agent used in clinical trials and gene therapy protocols, this cytokine gene was chosen for the present investigations. Transduction of the human TNF alpha (hTNF) gene carrying retroviral vector pN2tk-hTNF into U373MG human glioblastoma cells resulted in expression and secretion of biologically active hTNF. Release of transduced hTNF reduces P-glycoprotein expression and is associated with enhanced rhodamine-123 uptake and potentiation of cytotoxicity of the MDR-relevant drugs vincristine and doxorubicin. Furthermore, the transfected cell clones showed a reduced growth rate compared to the parental cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗