W. J. Zeller

QuickMap: a public tool for large-scale gene therapy vector insertion site mapping and analysis

Several events of insertional mutagenesis in pre-clinical and clinical gene therapy studies have created intense interest in assessing the genomic insertion profiles of gene therapy vectors. For the construction of such profiles, vector-flanking sequences detected by inverse PCR, linear amplification-mediated-PCR or ligation-mediated-PCR need to be mapped to the host cells genome and compared to a reference set. Although remarkable progress has been achieved in mapping gene therapy vector insertion sites, public reference sets are lacking, as are the possibilities to quickly detect non-random patterns in experimental data. We developed a tool termed QuickMap, which uniformly maps and analyzes human and murine vector-flanking sequences within seconds (available at www.gtsg.org). Besides information about hits in chromosomes and fragile sites, QuickMap automatically determines insertion frequencies in +/− 250 kb adjacency to genes, cancer genes, pseudogenes, transcription factor and (post-transcriptional) miRNA binding sites, CpG islands and repetitive elements (short interspersed nuclear elements (SINE), long interspersed nuclear elements (LINE), Type II elements and LTR elements). Additionally, all experimental frequencies are compared with the data obtained from a reference set, containing 1 000 000 random integrations (‘random set’). Thus, for the first time a tool allowing high-throughput profiling of gene therapy vector insertion sites is available. It provides a basis for large-scale insertion site analyses, which is now urgently needed to discover novel gene therapy vectors with ‘safe’ insertion profiles.

Chemoprotection of human hematopoietic stem cells by simultaneous lentiviral overexpression of multidrug resistance 1 and O 6 -methylguanine-DNA methyltransferase P140K

Myelotoxicity is a dose-limiting effect of many chemotherapeutic regimens. Thus, there is great interest in protecting human hematopoietic stem cells by the transfer of drug resistance genes. The main focus of this study was the simultaneous overexpression of multidrug resistance 1 (MDR1) and the O6-benzylguanine (O6-BG)-resistant mutant MGMTP140K (O6-methylguanine-DNA methyltransferase) with a bicistronic lentiviral vector (HR′SIN–MDR1–IRES–MGMTP140K), with regard to the capability to convey chemoprotection in the leukemia cell line, HL60, and human hematopoietic stem cells (CD34+). Combination therapy with O6-BG/1-(2-chloroethyl)-3-(4-amino-2-methylpyrimidine-5-yl)methyl-1-nitrosourea) (ACNU) plus paclitaxel showed a significant survival advantage of HL60 cells transduced with this combination vector. In CD34+ cells, monotherapy with O6-BG/temozolomide (TMZ) resulted in an increased percentage of MGMT-positive cells (vs untreated cells) after transduction with HR′SIN–MDR1–IRES–MGMTP140K (28.3%). For combination therapy with O6-BG/temozolomide plus paclitaxel the increase was higher with the combination vector (52.8%) than with a vector expressing MGMTP140K solely (29.1%). With regard to MDR1-positive cells the protective effect of the combination vector (88.5%) was comparable to the single vector HR′SIN–MDR1 (90.0%) for monotherapy with paclitaxel and superior for combination therapy with O6-BG/temozolomide plus paclitaxel (84.6 vs 69.7%). In conclusion, the combination vector presents simultaneous protective effects of two drug-resistance genes, offering an opportunity to increase the cancer therapeutic index.

Combination effects of poly(ADP-ribose) polymerase inhibitors and DNA-damaging agents in ovarian tumor cell lines —with special reference to cisplatin

The effects of the poly(ADP-ribose) polymerase inhibitors 4-amino-1,8-naphthalimide (4-ANI), 6(5H)-phenanthridinone (PHD), 1,5-isoquinolinediol (IQD), 3-aminobenzamide (3-AB) or 4-hydroxyquinazoline (4-HYA) on the cytotoxicity of cisplatin were investigated. The human ovarian tumor cell lines SK-OV-3 and OAW 42 and the rat ovarian tumor cell line O-342 as well as its cisplatin (DDP)-resistant subline O-342/DDP were used. Cytotoxicity was determined with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. 1-Methyl-3-nitro-1-nitrosoguanidine (MNNG) plus its respective combinations with poly(ADP-ribose) polymerase inhibitors served as positive controls. In addition, the alkylating agentsl-threitol-1,4-bismethanesulfonate (DHB) and 1,3-bis(2-chloroethyl)-1-nitrosourea (carmustine) as well as two other DNA-repair inhibitors caffeine and theophylline were included in the investigations. The cytotoxicity of cisplatin could not be increased by 4-ANI, PHD, IQD, 4-HYA or 3-AB in any cell line investigated., while it was increased by caffeine in lines O-342/DDP and SK-OV-3 as well as by theophylline in lines O-342/DDP, SK-OV-3 and OAW 42. The cytotoxicity of MNNG was increased by combination with 4-ANI, PHD, IQD, 4-HYA, 3-AB or theophylline for all lines except OAW42; in the latter line, only 4-ANI, PHD and IQD increased MNNG cytotoxicity. The cytotoxicity of DHB was increased by 4-ANI, PHD, 4-HYA, theophylline and caffeine in line O-342/DDP; by 4-HYA, theophylline and caffeine in line SK-OV-3; and by theophylline and caffeine in line OAW42. The cytotoxicity of carmustine was increased only by 3-AB in two lines (SK-OV-3 and OAW 42). Results are discussed with regard to different DNA-repair mechanisms.

No Evidence of Clonal Dominance in Primates up to 4 Years Following Transplantation of Multidrug Resistance 1 Retrovirally Transduced Long‐Term Repopulating Cells

Previous murine studies have suggested that retroviral multidrug resistance 1 (MDR1) gene transfer may be associated with a myeloproliferative disorder. Analyses at a clonal level and prolonged long‐term follow‐up in a model with more direct relevance to human biology were lacking. In this study, we analyzed the contribution of individual CD34‐selected peripheral blood progenitor cells to long‐term rhesus macaque hematopoiesis after transduction with a retroviral vector either expressing the multidrug resistance 1 gene (HaMDR1 vector) or expressing the neomycin resistance (NeoR) gene (G1Na vector). We found a total of 122 contributing clones from 8 weeks up to 4 years after transplantation. One hundred two clones contained the G1Na vector, whereas only 20 clones contained the HaMDR1 vector. Here, we show for the first time real‐time polymerase chain reaction based quantification of individual transduced cell clones constituting 0.0008% ± 0.0003% to 0.0041% ± 0.00032% of primate peripheral blood cells. No clonal dominance was observed.

In vitro evaluation of platinum, titanium and ruthenium metal complexes in cisplatin-sensitive and-resistant rat ovarian tumors

SummaryThe antitumor activity of eight new metal complexes (three platinum, one titanium, four ruthenium derivatives) was investigated in a cisplatin (DDP)-sensitive (O-342) and a DDP-resistant (O-342/DDP) ovarian tumor line using the bilayer soft-agar assay. A continuous exposure set up at logarithmically spaced concentrations was used to test the drugs; to uncover possible pharmacokinetic features, a short-term exposure was additionally included for selected compounds. DDP served as the reference drug. The following compounds were investigated: 18-crown-6-tetracarboxybis-diammineplatinum(II) (CTDP),cis-aminotrismethylenephosphonato-diammineplatinum(II) (ADP),cis-diamminecyclohexano-aminotrismethylenephosphonato-platinum(II) (DAP), diethoxybis(1-phenylbutane-1,3-dionato)titanium(IV) (DBT, budotitane),trans-imidazolium-bisimidazoletetrachlororuthenate(III) (ICR),trans-indazolium-tetrachlorobisindazoleruthenate(III) (IndCR),cis-triazolium-tetrachlorobistriazoleruthenate(III) (TCR) andtrans-pyrazolium-tetrachlorobispyrazoleruthenate(III) (PCR). Of the new metal complexes, CTDP was the most active compound in O-342, resulting in a percentage of control plating efficiency (±SE) of 1±1, 12±8 and 40±21 following continuous exposure to 10, 1 and 0.1 μm, respectively, and was thus comparable to DDP at equimolar concentrations. In the resistant line, 10 μm CTDP reduced colony growth to 18%±8%, whereas an equimolar concentration of DDP effected a reduction to 26%±9%. During short-term exposure, CTDP was inferior to DDP, which may be ascribed to the stability of the bis-dicarboxylate platinum ring system. The titanium compound DBT, in contrast, showed promising effects at its highest concentration (100 μm) during short-term exposure in both lines; at this concentration the activity in O-342/DDP was higher than that in O-342 (7%±7% vs 34%±17% of control plating efficiency at 100 μm). All ruthenium complexes showed higher activity in the resistant line O-342/DDP than in the sensitive counterpart. ICR was the most active compound. Following continuous exposure of O-342/DDP cells to 10 μm ICR, colony growth was reduced to 18%±4% that of controls. Further studies should concentrate on CTDP and ICR for the following reasons: the activity of CTDP was equal to that of DDP at equimolar concentrations during continuous exposure; considering that the in vivo toxicity of DDP was 3-fold that of CTDP, an increase in the therapeutic index of CTDP would be expected. ICR showed the best effect of all ruthenium complexes; it was superior to DDP in the resistant line.

In vitro investigations on induction and reversal of cisplatin resistance in a rat ovarian tumor cell line

SummaryResistance of a rat ovarian tumor cell line (O-342/DDP) to cisplatin was induced in vitro by stepwise increase of cisplatin concentrations. Both chemosensitive parental cells (O-342) and resistant O-342/DDP cells grow in a monolayer and enter log-phase growth about 24 h after seeding (cell population doubling time in log-phase growth is about 24 h). O-342/DDP cells show a 33-fold resistance to cisplatin as compared to 0-342 cells (ID50=33 μM in O-342/DDP vs 1 μM in O-342 cells). The intracellular total glutathione (GSH+GSSG) of O-342/DDP cells was twice as high as that of O-342 cells (3.04 vs 1.37 nmol/106 cells), while the intracellular GSSG was increased by 26% in O-342/DDP cells compared to O-342 cells. DNA interstrand crosslinks were found to be 8.5 times higher in O-342 cells than in O-342/DDP cells (204 vs 24 rad equiv.), following cisplatin treatment. DNA single-strand breaks were approximately doubled in the sensitive line as compared to the resistant line following exposure to cisplatin. Chromosome analysis uncovered a change in the karyotype of O-342/DDP cell as compared to O-342 cells. In the sensitive line hyperploid (3n) clones, in the resistant line near-diploid clones predominated. Both DL-buthionine-(S,R)-sulfoximine and 3-aminobenzamide were able to sensitize the resistant line towards cisplatin. Thus, the present results suggest that mechanisms for cisplatin resistance in this tumor line apparently are multifactorial, and include a higher intracellular GSH content and an increased repair activity.

Anticancer Activity of New Nitrosoureas against Walker Carcinosarcoma 256 and DMBA-Induced Mammary Cancer of the Rat

Three new nitrosoureas and chlorozotocin were screened for anticancer activity against Walker carcinoma 256 and DMBA-induced mammary cancer of the rat. High single doses (80% of LD50) of water-soluble 1-(2-chloroethyl)-1-nitroso-3-(methylencarboxamido)urea and 2-[3-(2-chloroethyl)-3-nitrosoureido]ethylmethansulfonate effected a similar tumor weight inhibition than BCNU in treatment of subcutaneously implanted Walker 256. In dose-response studies low doses of the new analogs effected a higher tumor weight inhibition than BCNU in the treatment of subcutaneously implanted Walker 256. The therapeutic index calculated as LD50/ED50, was 2.7 and 2.4 for the new compounds in comparison to BCNU with 2.1. Chlorozotocin and 1-(methylenecarboxyethyl)-1-nitroso-3-phenylurea were not active. Against DMBA-induced mammary cancer the new BCNU analogs yielded a greater tumor weight inhibition than adriamycin and BCNU. At a dose-level of approximately LD10, the remission rates of individual tumors, which were at least 0.6 g at the beginning of treatment were 46% (13/28) for therapy with 2-[3-(2-chloroethyl)-3-nitrosoureido]ethylmethanesulfonate, 32% (9/28) for 1-(2-chloroethyl)-1-nitro-3-(methylenecarboxamido)urea, 26% (6/23) for chlorozotocin, 21% (7/33) for adriamycin and 17% for BCNU, respectively. As single agents the 2 new analogs 2[3-(1-chloroethyl)-3-nitrossureido[ethylmethanesulfonate, the water-soluble 1-(2-chloroethyl)-1-nitroso-3-(methylenecarboxamido)urea and chlorozotocin were the most effective compounds against DMBA-induced mammary cancer of the rat.

Nicotine and estrogen metabolism — possible implications of smoking for growth and outcome of treatment of hormone-dependent cancer? Discussion of experimental results

SummaryThe combination treatment of hormone-dependent autochthonous mammary carcinomas in the rat with nicotine plus HECNU, a water-soluble nitrosourea, resulted in a potentiation of antitumor action. Nicotine and its metabolite continine are strong inhibitors of the aromatase. With regard to investigations in smoking women, suggesting a decreased endogenous estrogen production, our results indicate that smoking might influence growth and treatment results of hormone-dependent human cancer.

Examination of four newly synthesized 2-chloroethylnitrosoureas in comparison with BCNU, CCNU, MeCCNU, chlorozotocin and hydroxyethyl-CNU in preterminal rat leukemia L 5222

SummaryThe newly synthesized nitrosoureas 1-(2-chloroethyl)-1-nitroso-3-(methylenecarboxamido)-urea (Acetamido-CNU), 1-(2-chloroethyl)-1-nitroso-3-(4-morpholino)-urea (Morpholino-CNU), 1-(2-chloroethyl)-1-nitroso-3-(1-piperidino)-urea (Piperidino-CNU), and 2-[3-(2-chloroethyl)-3-nitrosoureido]-ethylmethanesulfonate (Ethylmethanesulfonato-CNU) were compared in their chemotherapeutic activity against preterminal rat leukemia L 5222 with BCNU, CCNU, MeCCNU, Chlorozotocin, and Hydroxyethyl-CNU.With respect to the dose range effecting a median survival time of more than 90 days, MeCCNU was superior to the other substances. Chlorozotocin, on the other hand, was the only substance which achieved no cures in this experimental arrangement.From the four newly introduced substances the water-soluble substances Acetamido-CNU and Morpholino-CNU were approximately comparable to CCNU with regard to the dose range effecting a median survival time of >90 days. Piperidino-CNU and Ethylmethanesulfonato-CNU also effected cures; however, only Piperidino-CNU in one dosage effected a median survival time of >90 days.ZusammenfassungDie therapeutische Aktivität der vier neusynthetisierten Nitrosoharnstoffe 1-(2-Chlorethyl)-1-nitroso(3-methylencarboxamido)-harnstoff (Acetamido-CNU), 1-(2-Chlorethyl)-1-nitroso-3-(4-morpholino)-harnstoff (Morpholino-CNU), 1-(2-Chlorethyl)-1-nitroso-3-(1-piperidino)-harnstoff (Piperidino-CNU) und 2-[3-(2-Chlorethyl)-3-nitrosoureido]-ethylmethansulfonat (Ethylmethansulfonato-CNU) wurde an der präterminalen Rattenleukämie L 5222 mit der Wirkung von BCNU, CCNU, MeCCNU, Chlorozotocin und Hydroxyethyl-CNU verglichen.MeCCNU war den anderen Substanzen im Hinblick auf den breiten Dosisbereich, in dem eine mediane Überlebenszeit von >90 Tagen bewirkt wurde, überlegen. Chlorozotocin dagegen bewirkte als einzige Substanz in dieser Versuchsanordnung keine Heilungen. Von den neu vorgestellten Substanzen waren die wasserlöslichen Verbindungen Acetamido-CNU und Morpholino-CNU im Hinblick auf die Breite des Dosisbereiches, in dem eine mediane Überlebenszeit von >90 Tagen erzielt wurde, annähernd dem CCNU vergleichbar. Piperidino-CNU und Ethylmethansulfonato-CNU bewirkten auch Heilungen; jedoch wurde nur mit einer Dosierung von Piperidino-CNU eine mediane Überlebenszeit von >90 Tagen erreicht.

Antiproliferative activity of the non-myelotoxic antitumour agent of plant origin, Thaliblastine, on two human glioma cell lines.

SummaryThe antiproliferative activity of the non-myelotoxic antitumour agent of plant origin, Thaliblastine, on two human glioma cell lines is described. Thaliblastine was added once one day following start of culture; proliferation was monitored over 7 days. The anti-proliferative activity of Thaliblastine was strongly dependent on concentration and time of incubation. The ID50 of Thaliblastine in T406 and GW27 glioma lines was 5.1 μg/ml and 8.2 μg/ml (7.0 μM and 11.2 μM), respectively.