Identification of uncommon chromosomal aberrations in the neuroglioma cell line H4 by spectral karyotyping.

To elucidate the reasons why mRNA expression of the LGI1 candidate tumor-suppressor gene was severely reduced in the glioma-derived cell line H4, as demonstrated in a previous study, we performed a cytogenetic analysis of this cell line using conventional methods and fluorescence in situ hybridization (FISH) techniques [spectral karyotyping (SKY), interphase- and chromosome FISH of metaphases (I- and C-FISH)]. Cell line H4 is monoclonal and near triploid (+/-3n). SKY enabled us to detect 24 structural aberrations: unbalanced translocations, n = 12; deletions, n = 10; insertion, n = 1; duplication, n = 1. The results were confirmed by I- and C-FISH analysis using chromosome-specific paints, centromer-specific probes and locus-specific probes for p53, PTEN/MMAC1, LGI1, Cyclin D1, EGR1, ETV6/TEL, AML1, and the genomic region 13q14.3 containing the Rb locus. We found loss of one copy of p53 as well as of one copy of Rb. Complete loss of PTEN/MMAC1 was detected, while all copies of LGI1 and Cyclin D1 were preserved. Interestingly, there was a gain of ETV6/TEL and EGR1, which were each present in quadruplicate. Additionally, the AML1 locus revealed mosaicism of cells with three and four copies, respectively. Additionally, a 5q-chromosome [del(5)(q13q33)] was found, which is one of the common features in hematological malignancies, and der(12)t(1;12) was found, suggesting that there might be an additional ETV6/TEL fusion protein. The combination of SKY, I- and C-FISH demonstrates that the neuroglioma cell line H4 harbors cytogenetic aberrations that are reported to occur in glioma-derived cell lines and additional chromosomal aberrations that have so far not been reported to occur in these cell lines. The complex aberrant karyotype and possibly generation of transcription factors by fusion proteins might be reasons for the impaired mRNA expression of the LGI1 candidate tumor-suppressor gene in cell line H4.

Comparison of [18F]FHPG and [124/125I]FIAU for imaging herpes simplex virus type 1 thymidine kinase gene expression.

Various radiotracers based on uracil nucleosides (e.g. [124I]2'-fluoro-2'-deoxy-5-iodo-1-beta-D-arabinofuranosyluracil, [124I]FIAU) and acycloguanosine derivatives (e.g. [18F]9-[(3-fluoro-1-hydroxy-2-propoxy) methyl] guanine, [18F]FHPG) have been proposed for the non-invasive imaging of herpes simplex virus type 1 thymidine kinase (HSV1-tk) reporter gene expression. However, these radiotracers have been evaluated in different in vitro and in vivo models, precluding a direct comparison. Therefore, we directly compared [18F]FHPG and radioiodinated FIAU to assess their potential for PET imaging of transgene expression. The uptake of [125I]FIAU, [18F]FHPG and [3H]acyclovir was determined in vitro using four different HSV1-tk expressing cell lines and their respective negative controls. The in vitro tracer uptake was generally low in non-transduced parental cell lines. In HSV1-tk expressing cells, [3H]acyclovir showed approximately a twofold higher tracer accumulation, the [18F]FHPG uptake increased by about sixfold and the [125I]FIAU accumulation increased by about 28-fold after 120-min incubation of T1115 human glioblastoma cells. Similar results were found in the other cell lines. In addition, biodistribution and positron emission tomography (PET) studies with [18F]FHPG and [124/125I]FIAU were carried out in tumour-bearing BALB/c mice. Significantly higher specific accumulation of radioactivity was found for [125I]FIAU compared with [18F]FHPG. The ratio of specific tracer accumulation between [125I]FIAU and [18F]FHPG increased from 21 (30 min p.i.) to 119 (4 h p.i.). PET imaging, using [124I]FIAU, clearly visualised and delineated HSV1-tk expressing tumours, whereas only a negligible uptake of [18F]FHPG was observed. This study demonstrated that in vitro and in vivo, the radioiodinated uracil nucleoside FIAU has a significantly higher specific accumulation than the acycloguanosine derivative [18F]FHPG. This suggests that [124I]FIAU should be the preferred reporter probe for PET imaging of HSV1-tk gene expression. Thus, further attempts to develop suitable PET tracers for the assessment of HSV1-tk gene expression should also focus on 18F-labelled uracil derivatives.

[Gene therapy of malignant tumors]. / Gentherapie maligner Tumoren.

New techniques in molecular biology and increasing insights in the genetic basis of tumorigenesis led to the development of therapeutic agents composed of genetic information. The majority of gene therapy trials is targeted towards cancer therapy. The most frequently used strategy is immunotherapy which comprises transfer of genes encoding cytokines, costimulatory molecules or tumorspecific antigens to enable cells of the immune system to identify and destroy tumor cells. The second important strategy makes use of suicide genes to selectively kill tumor cells. Compensation of genetic defects by the downregulation of activated oncogenes or the restoration of tumor suppressor gene functions is the third concept to revert the malignant phenotype of cancer cells.

Gene therapy and gastrointestinal cancer: concepts and clinical facts.

BACKGROUND: Principles of the treatment of gastrointestinal cancer with gene therapy evolved from the advent of techniques in molecular biology, from increasing insights into the molecular basis of tumorigenesis and from the need to develop more efficient treatment modalities. Any gene therapy approach has to take two major tasks into consideration: the therapeutic gene has to be delivered into the target cell population with high efficiency, specificity and safety, and has to act in a way that provides a benefit to the patient. DISCUSSION: Data on 22 clinical trials on malignancies of the gastrointestinal tract are available. They utilize a variety of gene-delivery methods and target cell populations, and there is considerable variety among their strategies. Gene transfer is performed by injection of naked plasmid DNA and by use of DNA-liposome complexes and viral vectors. In some cases, the gene transfer is carried out ex vivo and the patients receive genetically modified cells, whereas other approaches deliver the vector to the target cell population in vivo. The theoretical concepts of gene therapy can be divided into three groups. One approach makes use of suicide genes comprising bacterial or viral genes that convert a nontoxic prodrug into a highly cytotoxic chemotherapeutic agent at the tumor site. This approach aims at higher therapeutic specificity and fewer side effects than with the systemic delivery of cytotoxic agents. The second strategy makes an attempt to invoke the immune system to destroy malignant cells. Different strategies, such as immunization with genetically modified tumor cells or transfer of new genes to T cells, are considered to have clinical benefits. The major advantage of these immunotherapeutic approaches is the systemic effect both on the primary tumor and on metastases. The third strategy evolved from the insight that cancer is a genetic disease caused by activation of oncogenes or inactivation of tumor-suppressor genes. Compensation of genetic defects by the downregulation of activated oncogenes or the restoration of tumor-suppressor-gene functions may be able to revert the malignant phenotype of cancer cells. Of the 22 gene-therapy trials, 17 trials focus on immunotherapy. Only two trials make use of suicide genes and, in three trials, a functional copy of the p53 tumor-suppressor gene was reintroduced into malignant cells. Modalities for gene transfer and the strategies underlying gene therapy will be discussed in the context of gastrointestinal malignancies and the potential benefits for patients.

The transcription factor Sp1 regulates the myeloid-specific expression of the human hematopoietic cell kinase (HCK) gene through binding to two adjacent GC boxes within the HCK promoter-proximal region.

The human hemopoietic cell kinase (HCK) is a member of the src family of protein tyrosine kinases specifically expressed in myeloid cells and to a minor extent in B-lymphoid cells. HCK expression is up-regulated at the transcriptional level during myeloid differentiation of hematopoietic cells. To elucidate the molecular basis of the differential HCK gene expression, the genomic region containing the HCK promoter was isolated and functionally characterized. A DNA fragment containing 101 base pairs of the 5'-flanking sequence showed strong promoter activity in the macrophage cell line RAW264 but was inactive in the non-monocytic cell lines HUT-78 and NIH-3T3. Site-directed mutagenesis of the proximal promoter region showed that two GC-rich sequence elements are essential for transcriptional activity in myeloid cells. Electrophoretic mobility shift analysis using nuclear extracts obtained from RAW264 cells and from the promonocytic cell line U-937 revealed the formation of at least three distinct protein-DNA complexes at each of these sites, one of which was found to contain the transcription factor Sp1. Expression of a reporter gene linked to the -101 HCK promoter region was up-regulated by Sp1, but not by other members of the Sp1 family of transcription factors, in Drosophila Schneider cells. A synergistic effect on HCK promoter activity was observed at high concentrations of Sp1. Our results show that Sp1 plays an essential role in the regulation of the differential gene expression of the HCK gene.

Correction of respiratory burst activity in X-linked chronic granulomatous cells to therapeutically relevant levels after gene transfer into bone marrow CD34+ cells.

Chronic granulomatous disease (CGD) is a disorder of the lymphohematopoietic system, whereby phagocytes of affected patients are unable to kill microorganisms. CGD is caused by a functional defect in the phagocytic nicotinamide adenine dinucleotide phosphatase (NADPH) oxidase (phox) enzyme complex, leading to a lack of microbicidal metabolites. As a therapeutic approach toward the predominant X-linked form of CGD, we have developed a bicistronic retroviral vector containing the coding sequences of gp91-phox and a cytoplasmically truncated version of the human low-affinity receptor for nerve growth factor (deltaLNGFR). Full reconstitution of superoxide-generating activity was achieved with this vector in a gp91-phox-deficient cell line. Using an optimized gene transfer protocol, up to 85% of the CD34+ cells obtained from the bone marrow of X-CGD patients were transduced. CD15+ cells differentiated in vitro from transduced X-CGD CD34+ cells showed correction of NADPH oxidase activity to 45-52% of normal levels whereas deltaLNGFR expression was found in 40-67% of the CD15+ cells. Moreover, immunoblots prepared from extracts of transduced CD15+ cells revealed gp91-phox protein levels similar to those found in neutrophils derived from normal CD34+ cells. Taking into consideration that superoxide production in only 5 to 10% of wild-type neutrophils is sufficient to protect X-CGD heterozygotes from serious infections, the results achieved in this study shows that for X-CGD patients a curative approach based on the genetic modification of hematopoietic stem/progenitor cells is feasible.