NF-kappaB and the regulation of hematopoiesis.

This review will focus on the role of nuclear factor kappaB (NF-kappaB) signaling in hematopoietic differentiation. We will also discuss several hematopoietic pathologies associated with deregulation of NF-kappaB and their potential therapies.

Replication-defective recombinant Semliki Forest virus encoding GM-CSF as a vector system for rapid and facile generation of autologous human tumor cell vaccines.

This paper describes the production of recombinant Semliki Forest virus encoding murine or human granulocyte-macrophage colony-stimulating factor (GM-CSF) and the capacity of these vectors to transduce murine and human tumor cells ex vivo. High-titer stocks (up to 3 x 10(9) particles/ml) of conditionally infective, replication-defective, recombinant SFV particles were generated using the SFV Helper-2 system. It is shown that the recombinant SFV/GM-CSF virus, as well as recombinant SFV carrying the beta-galactosidase reporter gene, efficiently transduce both murine tumor cell lines as well as primary human renal carcinoma cells. Using ELISA's specific for GM-CSF, levels of GM-CSF production by the cells were determined. Levels of murine GM-CSF (mGM-CSF) produced by SFV/mGM-CSF transduced renal cell cancer cultures were equal to or higher than corresponding levels reported in the literature after transduction of similar renal carcinoma cell cultures using a retroviral vector system. The biological activity of GM-CSF was demonstrated by using cells which are dependent on GM-CSF for growth and by using primary bone marrow cells. All the transduced cell cultures (including the human renal cell carcinoma samples) produced GM-CSF for up to at least 4 days after transduction. The results imply that the recombinant SFV system can be used for rapid and facile preparation of autologous cancer cell vaccines.

Characterization of BIS20x3, a bi-specific antibody activating and retargeting T-cells to CD20-positive B-cells.

This paper describes a bi-specific antibody, which was called BIS20x3. It retargets CD3varepsilon-positive cells (T-cells) to CD20-positive cells and was obtained by hybrid-hybridoma fusion. BIS20x3 could be isolated readily from quadroma culture supernatant and retained all the signalling characteristics associated with both of its chains. Cross-linking of BIS20x3 on Ramos cells leads to DNA fragmentation percentages similar to those obtained after Rituximab-cross-linking. Cross-linking of BIS20x3 on T-cells using cross-linking F(ab')2-fragments induced T-cell activation. Indirect cross-linking of T-cell-bound BIS20x3 via Ramos cells hyper-activated the T-cells. Furthermore, it was demonstrated that BIS20x3 effectively re-targets T-cells to B-cells, leading to high B-cell cytotoxicity. The results presented in this paper show that BIS20x3 is fully functional in retargeting T-cells to B-cells and suggest that B-cell lymphomas may represent ideal targets for T-cell retargeting bi-specific antibodies, because the retargeted T-cell is maximally stimulated in the presence of B-cells. Additionally, since B-cells may up-regulate CD95/ Fas expression upon binding of CD20-directed antibodies, B-cells will become even more sensitive for T-cell mediated killing via CD95L/ Fas L, and therefore supports the intention to use T-cell retargeting bi-specific antibodies recognizing CD20 on B-cell malignancies as a treatment modality for these diseases.

Activation of peritoneal cells upon in vivo transfection with a recombinant alphavirus expressing GM-CSF.

In this study we determined the in vivo localization of recombinant proteins expressed by intraperitoneally (i.p.) injected recombinant Semliki Forest virus (SFV) particles. Subsequently, we investigated the influence of i.p. administered SFV particles encoding recombinant murine granulocyte-macrophage colony-stimulating factor (rmGM-CSF) on intraperitoneal recruitment and activation of cells. Finally, the therapeutic effect of SFV-GM-CSF treatment on an i.p. growing ovarian tumor was determined. Intraperitoneal injections of recombinant SFV particles encoding the reporter protein luciferase resulted in a high level of luciferase activity in cells of the peritoneal lining and tumor cells in the peritoneal cavity. Low levels of luciferase activity were found in liver, spleen and lungs. Injection of SFV-GM-CSF particles resulted in a slight increase in the number of peritoneal macrophages and in a significant increase in the number of neutrophils. In contrast to multiple i.p. injections with commercially available recombinant GM-CSF, i.p. injected SFV-GM-CSF particles activated the macrophages to tumor cytotoxicity. Although treatment of tumor-bearing mice with SFV-GM-CSF particles did not result in prolonged survival, tumor growth was inhibited for 2 weeks. Our findings indicate that macrophage-activating cytokines expressed by the efficient and safe recombinant SFV system when administered i.p. may provide an immunotherapeutic treatment modality additional to current chemotherapeutic treatment of intraperitoneally growing cancers.

Bi-specific antibody therapy for the treatment of cancer.

Bi-specific antibodies (BsAbs) combine immune cell activation with tumor cell recognition as a result of which tumor cells are killed by pre-defined effector cells. In this review a brief introduction to monoclonal antibodies will precede a more in-depth presentation of the current status of BsAb therapy for cancer. Target molecules and effector mechanisms aimed at tumor cells or aimed at tumor vasculature, and the application of recombinant DNA technology in the construction of antibodies, will be discussed. The lessons learned from the last decade will be discussed in consideration of the potential future development of BsAbs for cancer therapy.

Differential regulation of IL-6 promoter activity in a human ovarian-tumor cell line transfected with various p53 mutants: involvement of AP-1.

In human ovarian carcinomas, the p53 tumor-suppressor gene is frequently mutated. Interleukin-6 (IL-6) in these tumors is known to stimulate tumor-cell proliferation. In order to evaluate the effect of several p53 phenotypes on the IL-6 promoter activity, the human ovarian wild-type (wt)-p53 cell line A2780 was stably transfected with an empty plasmid (CMV) or (m)-175-, m-248- or m-273-p53. Electrophoretic mobility-shift assays revealed differences in activator protein-1 (AP-1) DNA-binding activity in the various clones. The CMV and m-273 clone had comparable amounts of AP-1. The m-175 clone displayed the least and m-248 the most pronounced AP-1 binding. Supershift analysis of AP-1/DNA complexes with antibodies against the AP-1 sub-units, c-Fos, FosB, Fra-1, Fra-2, c-Jun, JunB, and JunD, revealed that the AP-1/DNA complexes in the various clones had different compositions. Fra proteins were basically present only in m-175 and m-248 AP-1. IL-6-promoter activity was evaluated in the presence and absence of the AP-1 binding site which showed that the m-175-transfected clone has a transcriptional suppressing AP-1, whereas the CMV and the m-273 clones have an activating AP-1. Exposure of the p53 clones to tumor-necrosis factor-alpha (TNF-alpha) clearly altered the AP-1/DNA complex composition. IL-6-promoter activity was enhanced by TNF-alpha irrespective of the presence of an AP-1 binding site, while the degree of activation differed in the various clones, being most pronounced in the m-175 and m-248 clones. The results demonstrate that the basic and activated IL-6-promoter activity is differently regulated in the various p53 clones, possibly due to alterations in the AP-1 composition.

DNA topoisomerase IIalpha and -beta expression in human ovarian cancer.

To study DNA topoisomerase IIalpha (Topo-IIalpha) and -beta expression and regulation in human ovarian cancer, 15 ovarian tumour samples were investigated. To compare different levels of expression, the samples were screened for topo IIalpha and -beta mRNA with Northern blotting and a quantitative reverse transcriptase polymerase chain reaction (RT-PCR) assay for Topo-IIalpha mRNA. Additionally, protein levels were determined with Western blotting and topoisomerase II activity levels with the decatenation assay. The results obtained were compared with each other and with the tumour volume index of the samples. In tumours with a tumour volume index > or = 50%, the mRNA levels (as determined by Northern blotting) and protein levels for each isozyme were in accordance. Additionally, correlations were found between Topo-IIalpha RT-PCR data and Topo-IIalpha Northern blot results, and between Topo-IIalpha RT-PCR data and Topo-IIalpha protein levels. Interestingly, Topo-IIbeta protein levels correlated better with Topo-II activity than Topo-IIalpha protein levels. In eight ovarian cystadenoma samples, no Topo-IIalpha protein could be found. In only three out of eight of these cystadenomas, Topo-IIbeta protein could be detected. These findings suggest that Topo-IIalpha and Topo-IIbeta protein levels are up-regulated in ovarian cancer and may indicate that Topo-IIbeta is an interesting target for chemotherapy in ovarian tumours.

Combined cytotoxic effects of tumor necrosis factor-alpha with various cytotoxic agents in tumor cell lines that are drug resistant due to mutated p53.

Several studies suggest that tumor necrosis factor-alpha (TNF) is able to overcome drug resistance in tumors. Whether TNF is able to do so in tumor cell lines that are drug resistant due to a mutation in the tumor suppressor gene p53 is unclear. Therefore, we studied the in vitro cytotoxic effects of TNF combined with various cytotoxic agents in a model consisting of a human ovarian cancer cell line containing endogenous wild-type p53 (wtp53) and sublines that were made drug resistant against various cytotoxic agents by transfection of several forms of mutated p53 (mtp53). Using the microculture tetrazolium assay, the cytotoxic effects of TNF alone, the cytotoxic agents VM-26, melphalan, cisplatin, vinblastine, paclitaxel, and mitoxantrone, plus the combined effects of 10 ng/ml TNF added 30 min before various concentrations of the cytotoxic agents were established. Compared with the control cell line (A2780/cmv), two cell lines transfected with mtp53 (A2780/m248 and A2780/m273) showed increased resistance against several cytotoxic agents but also an enhanced sensitivity to TNF. Interaction of TNF with the cytotoxic drugs was additive in the drug-sensitive control cell line as well as in the drug-resistant sublines. However, because of the increased sensitivity of A2780/m248 to TNF at the dose used for the combinations, the combination of TNF with several cytotoxic drugs reduced the level of resistance in A2780/m248 compared with the control cell line A2780/cmv. In conclusion, this study shows that addition of TNF can ameliorate resistance to cytotoxic agents in a subline that is drug-resistant because of mutated p53. This reduction in resistance by TNF is not due to synergistic interaction, but to collateral sensitivity to TNF.

Differential expression of DNA topoisomerase II alpha and -beta in P-gp and MRP-negative VM26, mAMSA and mitoxantrone-resistant sublines of the human SCLC cell line GLC4.

Sublines of the human small-cell lung carcinoma (SCLC) cell line GLC4 with acquired resistance to teniposide, amsacrine and mitoxantrone (GLC4/VM20x, GLC4/AM3x and GLC4/MIT60x, respectively) were derived to study the contribution of DNA topoisomerase II alpha and -beta (TopoII alpha and -beta) to resistance for TopoII-targeting drugs. The cell lines did not overexpress P-glycoprotein or the multidrug resistance-associated protein but were cross-resistant to other TopoII drugs. GLC4/VM20x showed a major decrease in TopoII alpha protein (54%; for all assays presented in this paper the GLC4 level was defined to be 100%) without reduction in TopoII beta protein; GLC4/AM3x showed only a major decrease in TopoII beta protein (to 18%) and not in TopoII alpha. In GLC4/MIT60x, the TopoII alpha and -beta protein levels were both decreased (TopoII alpha to 31%; TopoII beta protein was undetectable). The decrease in TopoII alpha protein in GLC4/VM20x and GLC4/MIT60x, was mediated by decreased TopoII alpha mRNA levels. Loss of TopoII alpha gene copies contributed to the mRNA decrease in these cell lines. Only in the GLC4/MIT60x cell line was an accumulation defect observed for the drug against which the cell line was made resistant. In conclusion, TopoII alpha and -beta levels were decreased differentially in the resistant cell lines, suggesting that resistance to these drugs may be mediated by a decrease in a specific isozyme.

Selection of a subpopulation with fewer DNA topoisomerase II alpha gene copies in a doxorubicin-resistant cell line panel.

A panel of doxorubicin-resistant sublines of the human small-cell lung carcinoma cell line GLC4 displays decreasing DNA topoisomerase II alpha (TopoII alpha) mRNA levels with increasing resistance. In the present study we describe how this decrease may be regulated. No significant differences in TopoII alpha mRNA stability or gene arrangement were found, using mRNA slot-blotting and Southern blotting, in the most resistant cell line compared with the parental cell line. To investigate if TopoII alpha gene copy loss contributed to the mRNA decrease, fluorescence in situ hybridisation using a TopoII alpha-specific probe was performed. During doxorubicin resistance development, the composition of the population in each cell line shifted with increasing resistance, from a population in which most cells contain three TopoII alpha gene copies (GLC4) to a population in which most cells contain only two copies. A partial revertant of the most resistant cell line displayed a shift back to the original situation. We conclude that the TopoII alpha gene copy number decrease per cell line is in good agreement with the decreased TopoII alpha mRNA and protein levels, and TopoII activity levels in these cell lines which were described previously.

Human DNA topoisomerase II: biochemistry and role in chemotherapy resistance (review).

Recently, it has been discovered that DNA topoisomerases are the target of many anti-cancer drugs which were already widely used in the clinic. Using the latest molecular biological techniques much has been learned about the function of DNA topoisomerases in normal cells and in cancer cells, and about how anticancer drugs inhibit these enzymes. In this review we present an overview of the function of human type II DNA topoisomerases, how they are inhibited by certain drugs and on how cancer cells may become resistant to these drugs.

Resistance-associated factors in human small-cell lung-carcinoma GLC4 sub-lines with increasing adriamycin resistance.

Previous studies have shown that the in vitro-selected adriamycin-resistant human small-cell lung-carcinoma cell line GLC4-ADR150 displays multidrug resistance as the result of 3-fold decreased DNA-topoisomerase II (topo II) activity and a 6-fold reduction in adriamycin accumulation. Not the MDR1 gene, but the MRP gene, was over-expressed in this cell line. The aim of our study was to establish which of these drug-resistance-associated factors are already involved in drug resistance occurring at early steps of selection with adriamycin. To address this question, changes in expression of topo II alpha/topo II beta, MRP and drug accumulation were measured along with adriamycin resistance (from 2- to 10- to 150-fold) and in a partial revertant cell line (10-fold resistant). Topo II alpha and II beta mRNA and protein levels were decreased in the resistant sub-lines, except in the 10-fold-resistant cell line. Cellular daunorubicin accumulation was decreased 1.2- to 5-fold with increasing resistance. MRP mRNA was over-expressed in all resistant sub-lines, with a marked increase in the 10-fold-resistant cells (level of expression as high as in the GLC4-ADR150 cells). Expression of an ATP-binding 190-kDa membrane protein and Western-blot analysis with anti-MRP anti-serum ASPKE, was in accordance with the expression of MRP mRNA in all cell lines. Expression of MRP mRNA and protein, however, was not proportional with the decrease in drug accumulation in all resistant sub-lines. This study also shows that drug accumulation, topo II and MRP expression were all changed at the earliest stage of resistance development of GLC4 cells upon adriamycin selection.

VP-16-mediated cytotoxicity is modulated by interleukin-3 in a growth factor-dependent leukemic cell line.

A growth factor-dependent cell line (TF-1) was treated with interleukin-3 (IL-3) or medium in combination with variable doses of VP-16 to test whether the latter's cytotoxicity can be modulated by IL-3. The results demonstrated that an augmented cell death occurred with TF-1 cells when pre-incubated for 24 h with IL-3 followed by treatment with VP-16 (10 micrograms/ml) for 1 h. The increased cell death could not be ascribed to an increased number of apoptotic cells as measured with the acridine orange method. However, the IL-3 treatment coincided with an upregulation of DNA topoisomerase II alpha (Topo II alpha) at mRNA and protein level after 24 h, which was preceded by an upregulation of c-myc mRNA. In contrast, Topo II beta did not demonstrate an upregulation at mRNA level in response to IL-3 stimulation. In addition, it was shown that cells treated with IL-3 followed by VP-16 demonstrated a higher number of cleavable DNA complexes which was not due to an increased uptake of VP-16, since cellular concentrations of VP-16 in the presence of IL-3 or medium were 16.8 +/- 7.8 ng/10(6) cells and 19.8 +/- 7.8 ng/10(6) cells (mean +/- SD, n = 3), respectively. These data indicate that IL-3 pretreatment followed by VP-16 results in an increased cell death due to cytotoxicity which may be ascribed to an upregulation of Topo II alpha.

Quantitation of DNA topoisomerase II alpha messenger ribonucleic acid levels in a small cell lung cancer cell line and two drug resistant sublines using a polymerase chain reaction-aided transcript titration assay.

BACKGROUND: We have modified a polymerase chain reaction (PCR)-aided transcript titration assay (1) in order to allow quantitation of low amounts of DNA topoisomerase II alpha mRNA in small RNA samples. EXPERIMENTAL DESIGN: The titration assay was used to quantitate the amount of DNA topoisomerase II alpha mRNA in a human small cell lung carcinoma cell line, GLC4 and its drug-resistant sublines, GLC4/ADR and GLC4/CDDP. These cell lines show differences in DNA topoisomerase II alpha protein level and DNA topoisomerase II enzyme activity. To validate the titration assay, the results were compared with the results of a DNA topoisomerase II enzyme activity assay and DNA topoisomerase II alpha northern and western blotting assays. RESULTS: Using the titration assay, we were able to quantitate DNA topoisomerase II alpha mRNA on a picogram level starting with less than 1 micrograms of total RNA/cell line. GLC4/ADR showed a markedly decreased DNA topoisomerase II alpha mRNA level that seemed to be unchanged in GLC4/CDDP when compared with the parental cell line. The results obtained with this assay are confirmed by the western blot data and are not in contradiction with the northern blot results obtained for the three cell lines. CONCLUSIONS: The DNA topoisomerase II alpha titration assay is a highly sensitive new technique to study the role of DNA topoisomerase II alpha in drug resistance and may help to identify cancer types and patients most likely to respond to DNA topoisomerase II targeted drugs. The decrease in DNA topoisomerase II alpha protein level and DNA topoisomerase II activity in GLC4/ADR may result from transcriptional down regulation of DNA topoisomerase II alpha.

Characterization of the srfA locus of Bacillus subtilis: only the valine-activating domain of srfA is involved in the establishment of genetic competence.

srfA is a locus required for the production of the lipopeptide antibiotic surfactin. This locus is also necessary for efficient sporulation and competence development. Mutations in the 5' portion of the srfA operon affect all three of these processes, whereas mutations in the 3' portion of srfA only affect sporulation and surfactin production. Analysis of the proteins encoded by the srfA locus revealed seven large domains which are likely to be responsible for the activation and binding of the seven amino acids of surfactin. Identification of the amino acid that is activated by the srfA domains was determined by amino acid-dependent pyrophosphate exchange reactions on partially purified cell extracts of strains carrying different srfA mutations. These results indicate colinearity between the order of the domains in the srfA locus and the amino acid sequence of surfactin. The minimal genetic element of srfA required for the establishment of competence was shown to be the 5' region of the second open reading of srfA, which encodes the valine activation domain. This portion of srfA, when cloned on a plasmid, complemented the competence deficiency of a srfA deletion mutant in trans.

Isolation and characterization of comL, a transcription unit involved in competence development of Bacillus subtilis.

Using the transformation-deficient mutant M465, which was previously isolated by means of insertional mutagenesis with plasmid pHV60, a transcription unit comL required for genetic competence of Bacillus subtilis was identified. A chromosomal DNA fragment flanking the inserted pHV60 was isolated and used to screen two different libraries of B. subtilis DNA in phage lambda EMBL4 and lambda EMBL12, respectively. With the aid of six recombinant phages that hybridize with this chromosomal fragment a restriction map of about 23 kb of B. subtilis chromosomal DNA was constructed. Using small adjoining pieces of this chromosomal DNA in Campbell integrations, the size of the transcription unit involved in competence development could be delimited to about 15 kb. By insertion of a promoterless lacZ gene into comL, the transcriptional regulation of comL was analysed and epistatic interactions among various other com genes were determined. The results of these experiments indicated that comL is optimally expressed in glucose-based minimal medium when the culture enters the stationary phase of growth and that the expression of late competence genes is dependent on previous transcription of comL, which in turn is dependent on the gene products of comA and comB.