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Biomedical subjects

U Stein

Publications and source records attributed to U Stein.

At least 19 recordsLinked to original sources
PubMed2003

Genetic imbalances with impact on survival in colorectal cancer patients.

AIMS: To investigate the prognostic significance of chromosomal alterations in colorectal cancer patients. Histopathological tumour classification is still considered to be the gold standard for the characterization of solid tumours. However, it is well known that such established parameters do not satisfactorily predict the clinical outcome in individual cases. Markers that reliably predict survival are needed. These markers should guide the clinical treatment of neoplastic disease. METHODS AND RESULTS: Chromosomal imbalances in 61 colorectal carcinoma specimens in 37 patients determined by comparative genomic hybridization were correlated with patient survival using custom-made computer software which enabled the assessment of individual chromosomal loci. Kaplan-Meier analysis revealed that over-representations of 2p14-15, 6q23-6q24, 15q22-15q23, 22q11.2 and deletions of 1p36.1-36.2, 4q31.3, 4q35, 8q12-q21, 8p11.2 and 9p22 were significantly associated with shorter disease-specific survival, whereas over-expression of 20q13.3 and deletion of 18q11.2 were significantly associated with longer disease-specific survival in this collection of colorectal cancers. Multivariate Cox proportional hazards regression models consistently identified gains of 2p14-15, 15q22-23, 22q11.2 and losses of 1p36.1-36.2 and 4q35 as independent markers of shorter patient survival carrying greater significance than the classical clinicopathological parameters of nodal status and tumour grade. CONCLUSIONS: These five markers allow a molecular categorization of patients into high and low clinical risk groups. Thus, the genomic data have refined the histopathological classification highlighting the necessity for a supplementary genetically based stratification of colorectal cancer.

Adenocarcinoma↗
PubMed2002

Pentoxifylline influences drug transport activity of P-glycoprotein and decreases mdrl gene expression in multidrug resistant mouse leukemic L1210/VCR cells.

The effects of pentoxifylline (PTX) on intracellular accumulation of doxorobicin (DOX), DOX cytotoxicity and expression of Pgp in multidrug resistant L1210/VCR cell line were investigated. PTX (100 mg/l) was able to enhance the DOX accumulation in resistant cells. The maximum intracellular levels of DOX were reached after treatment with PTX for 24 hours (total duration of PTX-treatment was 72 hours). The levels of mdrl mRNA (measured by RT-PCR method) were decreased 2-fold in the presence of 100 mg/l PTX (minimum reached within 48 hours) in comparison to control cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2001

Hyperthermia-induced nuclear translocation of transcription factor YB-1 leads to enhanced expression of multidrug resistance-related ABC transporters.

Genotoxic stress leads to nuclear translocation of the Y-box transcription factor YB-1 and enhanced expression of the multidrug resistance gene MDR1. Because hyperthermia is used for the treatment of colon cancer in combination with chemoradiotherapy, we investigated the influence of hyperthermia on YB-1 activity and the expression of multidrug resistance-related genes. Here we report that hyperthermia causes YB-1 translocation from the cytoplasm into the nucleus of human colon carcinoma cells HCT15 and HCT116. Nuclear translocation of YB-1 was associated with increased MDR1 and MRP1 gene activity, which is reflected in strong efflux pump activity. However, a combination of hyperthermia and drug treatment effectively reduced cell survival of the HCT15 and HCT116 cells. These results demonstrate that activation of MDR1 and MRP1 gene expression and increased efflux pump activity after hyperthermia were insufficient to cause an increase in drug resistance in colon cancer cell lines. The ability of hyperthermia to abrogate drug resistance in the presence of an increase in functional MDR proteins may provide an explanation for the efficacious results seen in the clinic in colon cancer patients treated with a combination of hyperthermia and chemotherapy.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2001

Nutmeg (myristicin) poisoning--report on a fatal case and a series of cases recorded by a poison information centre.

In literature, cases of nutmeg abuse have been described repeatedly, but only one fatal case of poisoning was reported [1]. In the present case, myristicin (4 microg/ml) was detected for the first time in the postmortal serum of a 55-year-old woman. Identification was achieved with the aid of UV-VIS spectroscopy and TLC; for quantification, HPLC was used. Because also flunitrazepam (0.072 microg/ml) was found, death had probably been due to the combined toxic effect of both substances. From 1996 to 1998, in a series of cases, seven poisonings with nutmeg were recorded by the Erfurt Poison Information Centre. Even where higher doses (20-80 g of powder) had been ingested, a life-threatening situation was never observed. In one of these cases, a myristicin blood level of 2 microg/ml was measured 8h after ingestion of two to three tablespoonful of nutmeg powder (approx. 14-21 g, or 280-420 mg/kg).

Adolescent↗
PubMed2001

Nonviral in vivo gene delivery into tumors using a novel low volume jet-injection technology.

The jet-injection technology has developed as an applicable alternative to viral or liposomal gene delivery systems. In this study a novel, low-volume, 'high-speed jet injector' hand-held system was used for the direct gene transfer of naked DNA into tumors. Lewis-lung carcinoma bearing mice were jet-injected with the beta-galactosidase (LacZ), the green fluorescence (GFP) or the human tumor necrosis factor alpha (TNF-alpha) gene carrying vector plasmids. The animals received five jet injections into the tumor at a pressure of 3.0 bar, delivering 3--5 microl plasmid DNA (1 microg DNA/microl in water) per single jet injection. The jet injection of DNA leads to a widespread expression pattern within tumor tissues with penetration depths of 5--10 mm. Analysis of tumor cryosections revealed moderate LacZ or GFP expression at 48 h and strong reporter gene expression 72 h and 96 h after jet injection. The simultaneous jet injection of the TNF-alpha and LacZ carrying vectors demonstrated efficient expression and secretion of both the cytokine, as well as LacZ expression within the tumor 24 h, 48 h, 72 h, 96 h and 120 h after jet injection. These studies demonstrate the applicability of jet injection for the efficient in vivo gene transfer into tumors for nonviral gene therapy of cancer using minimal amounts of naked DNA.

Animals↗
PubMed2001

Magnesium deficiency-induced anorexia in hyperphagic obese Zucker rats.

In clinical practice diabetes mellitus is the most significant cause of hypomagnesemia and Mg depletion. The obese Zucker rat approaches non-insulin-dependent type II diabetes; lean Zucker rats being suitable controls. Using this disease model the influence of dietary Mg deficiency was studied: animals received a diet providing only approximately 25 per cent of the Mg requirement; controls received drinking water fortified with Mg (16 mmol/L). During 125 days ad libitum feeding, Mg-deficient obese rats consumed nearly 50 per cent less feed pellets and gained 50 per cent less body weight than their obese counterparts. This effect was not fully reversible indicating Mg depletion. Blood glucose reflected food consumption, no glucosuria was detectable using test strips. In the heart muscle Mg was decreased and Ca increased in Mg-deficient rats indicating increased cardiac risk. When the rats were pair fed with lean Mg-deficient controls the development of obesity was prevented. Despite pronounced Mg deficiency blood glucose remained unaffected and no glucosuria was detectable. In future experiments the production of marginal Mg deficiency not inducing anorexia should be applied to study the pathogenetic role of Mg depletion in obese Zucker rats.

Animals↗
PubMed2000

Cloning and initial analysis of the human multidrug resistance-related MVP/LRP gene promoter.

The lung resistance-related protein (LRP) was identified as the human major vault protein (MVP), and is overexpressed in various multidrug-resistant cancer cell lines and clinical samples. We characterized DNA sequences upstream to the transcription initiation site of the MVP gene in the human non-small cell lung cancer cell line SW-1573. A 1.9-kb and a shortened 0.7-kb fragment of the 5'-upstream genomic region show strong promoter activity in chloramphenicol acetyltransferase (CAT) reporter assays. The promoter is TATA-less and contains an inverted CCAAT-box and a Sp1 site located near to a p53 binding motif. An alternative 3'-splice site of intron 1 results in a splicing variant within the 5'-untranslated region of MVP mRNA.

5' Untranslated Regions↗
PubMed2000

Mdr1 promoter-driven tumor necrosis factor-alpha expression for a chemotherapy-controllable combined in vivo gene therapy and chemotherapy of tumors.

Cancer gene therapy approaches are often designed as single-agent treatments; however, greater therapeutic effect might be obtained if combined with an established conventional treatment regimen such as chemotherapy. In this context, conditional promoters are useful tools, because they may be induced by therapeutic modalities. The human multidrug resistance gene (mdr1) promoter is inducible by cytostatic drugs and can be employed for the chemotherapy-regulated expression of therapeutic genes. In this in vivo study, the human mdr1 promoter fragment (-207 to +158) was used for drug-inducible expression of human tumor necrosis factor-alpha (TNF-alpha) in the vector construct pM3mdr-p-hTNF. The single doxorubicin and vincristine treatment of nude mice xenografted with pM3mdr-p-hTNF-transduced MCF-7 mammary tumors resulted in drug-induced and time-dependent elevation of intratumoral TNF-alpha expression at the mRNA and protein level. The highest drug induction was achieved at 2 days after drug application, as reflected by a maximum 25-fold increase in TNF-alpha secretion in the tumor. This drug-induced TNF-alpha expression is more effective in inhibiting tumor growth compared with the growth of tumors transduced with constitutively TNF-alpha-expressing vectors in combination with chemotherapy.

Antineoplastic Agents↗
PubMed2000

Malignant odontogenic myxoma of the maxilla: case with cytogenetic confirmation.

An odontogenic myxoma of the maxilla with an aggressive clinical course is presented. The tumour arose in a 53-year-old patient, recurred two times after extended maxillectomy and ultimately caused the patient's death by uncontrollable local disease with infiltration of the cranial cavity. Microscopically, the tumour showed histological features of a low grade malignant myxosarcoma with cellular areas, enhanced mitotic activity and nuclear pleomorphism. Cytogenetic analysis revealed an unexpectedly aberrant hypertetraploid chromosome complement, that was considered as incompatible with the usual karyotypic patterns of benign tumours.

Humans↗
PubMed2000

High-copy cDNA amplification of minimal total RNA quantities for gene expression analyses.

This protocol describes a PCR-based cDNA amplification technique of small total RNA quantities, optimized for determination and verification of gene expression variations in cells or tissue specimen. A proportional amplification of rare and abundant transcripts is thereby achieved by initial random hexamer-primed reverse transcription of total RNA. Compared to established oligo(dT)-primed techniques, this approach generates shorter than full length copies of long RNAs which leads to a normalized cDNA pool for a more adequate PCR-amplification. Subsequent double oligo(dA) tailing of the synthesized total cDNA strands and the utilization of heteropolymeric primers allow a highly specific, up to 500-fold PCR-amplification of the total cellular RNA amount. Thus, obstacles in availability of RNA from limited sources, such as human biopsies or microdissected histological sections, can be overcome. The amplified total cDNA (atcDNA) is shown to be applicable for confirmation of differential gene expression, as demonstrated in this protocol by expression analysis of the multidrug resistance-associated genes mdr1, mrp1 and lrp, using human cell lines as well as microdissected human tissue sections.

Base Sequence↗
PubMed2000

Viral vectors for gene transfer: a review of their use in the treatment of human diseases.

The efficient delivery of therapeutic genes and appropriate gene expression are the crucial issues for clinically relevant gene therapy. Viruses are naturally evolved vehicles which efficiently transfer their genes into host cells. This ability made them desirable for engineering virus vector systems for the delivery of therapeutic genes. The viral vectors recently in laboratory and clinical use are based on RNA and DNA viruses processing very different genomic structures and host ranges. Particular viruses have been selected as gene delivery vehicles because of their capacities to carry foreign genes and their ability to efficiently deliver these genes associated with efficient gene expression. These are the major reasons why viral vectors derived from retroviruses, adenovirus, adeno-associated virus, herpesvirus and poxvirus are employed in more than 70% of clinical gene therapy trials worldwide. Among these vector systems, retrovirus vectors represent the most prominent delivery system, since these vectors have high gene transfer efficiency and mediate high expression of therapeutic genes. Members of the DNA virus family such as adenovirus-, adeno-associated virus or herpesvirus have also become attractive for efficient gene delivery as reflected by the fast growing number of clinical trials using these vectors. The first clinical trials were designed to test the feasibility and safety of viral vectors. Numerous viral vector systems have been developed for ex vivo and in vivo applications. More recently, increasing efforts have been made to improve infectivity, viral targeting, cell type specific expression and the duration of expression. These features are essential for higher efficacy and safety of RNA- and DNA-virus vectors. From the beginning of development and utilisation of viral vectors it was apparent that they harbour risks such as toxicities, immunoresponses towards viral antigens or potential viral recombination, which limit their clinical use. However, many achievements have been made in vector safety, the retargeting of virus vectors and improving the expression properties by refining vector design and virus production. This review addresses important issues of the current status of viral vector design and discusses their key features as delivery systems in gene therapy of human inherited and acquired diseases at the level of laboratory developments and of clinical applications.

Adenoviridae↗
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↗