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 O(6)-benzylguanine (O(6)-BG)-resistant mutant MGMT(P140K) (O(6)-methylguanine-DNA methyltransferase) with a bicistronic lentiviral vector (HR'SIN-MDR1-IRES-MGMT(P140K)), with regard to the capability to convey chemoprotection in the leukemia cell line, HL60, and human hematopoietic stem cells (CD34(+)). Combination therapy with O(6)-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 O(6)-BG/temozolomide (TMZ) resulted in an increased percentage of MGMT-positive cells (vs untreated cells) after transduction with HR'SIN-MDR1-IRES-MGMT(P140K) (28.3%). For combination therapy with O(6)-BG/temozolomide plus paclitaxel the increase was higher with the combination vector (52.8%) than with a vector expressing MGMT(P140K) 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 O(6)-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.

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 cell's 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.

Multiple displacement amplification enables large-scale clonal analysis following retroviral gene therapy.

Analysis of the fate of retrovirally transduced cells after transplantation is often hampered by the scarcity of available DNA. We evaluated a promising method for whole-genome amplification, called multiple displacement amplification (MDA), with respect to even and accurate representation of retrovirally transduced genomic DNA. We proved that MDA is a suitable method to subsequently quantify engraftment efficiencies by quantitative real-time PCR by analyzing retrovirally transduced DNA in a background of untransduced DNA and retroviral integrations found in primary material from a retroviral transplantation model. The portion of these retroviral integrations in the amplified samples was 1.02-fold (range 0.2, to 2.1-fold) the portion determined in the original genomic DNA. Integration site analysis by ligation-mediated PCR (LM-PCR) is essential for the detection of retroviral integrations. The combination of MDA and LM-PCR showed an increase in the sensitivity of integration site analysis, as a specific integration site could be detected in a background of untransduced DNA, while the transduced DNA made up only 0.001%. These results show for the first time that MDA enables large-scale sensitive detection and reliable quantification of retrovirally transduced human genomic DNA and therefore facilitates follow-up analysis in gene therapy studies even from the smallest amounts of starting material.

New bioinformatic strategies to rapidly characterize retroviral integration sites of gene therapy vectors.

OBJECTIVE: Increasing use of retroviral vector-mediated gene transfer created intense interest to characterize vector integrations on the genomic level. Techniques to determine insertion sites, mainly based on time-consuming manual data processing, are commonly applied. Since a high variability in processing methods hampers further data comparison, there is an urgent need to systematically process the data arising from such analysis. METHODS: To allow large-scale and standardized comparison of insertion sites of viral vectors we developed two programs, IntegrationSeq and IntegrationMap. IntegrationSeq can trim sequences, and valid integration sequences get further processed with IntegrationMap for automatic genomic mapping. IntegrationMap retrieves detailed information about whether integrations are located in or close to genes, the name of the gene, the exact localization in the transcriptional units, and further parameters like the distance from the transcription start site to the integration. RESULTS: We validated the method using 259 files originating from integration site analysis (LM-PCR). Sequences processed by IntegrationSeq led to an increased yield of valid integration sequence detection, which were shown to be more sensitive than conventional analysis and 15 times faster, while the specificities are equal. Output files generated by IntegrationMap were found to be 99.8% identical with results retrieved by much slower conventional mapping with the ENSEMBL alignment tool. CONCLUSION: Using IntegrationSeq and IntegrationMap, a validated, fast and standardized high-throughput analysis of insertion sites can be achieved for the first time.

Retroviral vector insertions in T-lymphocytes used for suicide gene therapy occur in gene groups with specific molecular functions.

Graft-versus-host disease (GvHD) is a severe complication in the context of allogeneic stem cell transplantation and adoptive immunotherapy. The transfer of a suicide gene into donor T-lymphocytes (TLCs) allows selective elimination of GvHD-causing cells. As retroviral gene transfer into hematopoietic stem cells can induce leukaemia, there is an urgent need also to analyze retroviral integration sites in TLCs. We examined suicide gene-transduced TLCs in four grafts and from four transplanted patients. One-hundred and fifteen integration sites were detected in vitro. Of these 90 could be mapped to the human genome; 50% (45) were located in genes and 32% (29) were detected 10 kb upstream or downstream of transcription start sites. We found a significant overrepresentation of genes encoding for proteins with receptor activity, signal transducer activity, transcription regulator activity, nucleic acid binding activity and translation regulator activity. Similar data were obtained from patient samples. Our results point to preferred vector integration patterns, which are specific for the target cell population and probably independent of selection processes. Thus, future preclinical analysis of the integration repertoire with abundant amounts of transduced cells could allow a prediction also for the in vivo situation, where target cells are scarce.

Synergistic activity of imatinib and 17-AAG in imatinib-resistant CML cells overexpressing BCR-ABL--Inhibition of P-glycoprotein function by 17-AAG.

Overexpression of BCR-ABL and P-glycoprotein (Pgp) are two of the known mechanisms of imatinib resistance. As combination therapy may allow to overcome drug resistance, we investigated the effect of combination treatment with imatinib and 17-allylamino-17-demethoxygeldanamycin (17-AAG), a heat-shock protein 90 (Hsp90) inhibitor, on different imatinib-sensitive and imatinib-resistant CML cell lines. In imatinib-sensitive cells, combination index (CI) values obtained using the method of Chou and Talalay indicated additive (CI=1) or marginally antagonistic (CI>1) effects following simultaneous treatment with imatinib and 17-AAG. In imatinib-resistant cells both drugs acted synergistically (CI<1). In primary chronic-phase CML cells additive or synergistic effects of the combination of imatinib plus 17-AAG were discernible. Annexin V/propidium iodide staining showed that the activity of imatinib plus 17-AAG is mediated by apoptosis. Combination treatment with imatinib plus 17-AAG was more effective in reducing the BCR-ABL protein level than 17-AAG alone. Monotherapy with 17-AAG decreased P-glycoprotein activity, which may increase intracellular imatinib levels and contribute to the sensitization of CML cells to imatinib. The results suggest that combination of imatinib and 17-AAG may be useful to overcome imatinib resistance in a clinical setting.

Chemotherapeutic activity of new 2-chloroethylnitrosoureas in rat L5222 leukemia: comparison of bifunctional and water-soluble derivatives with 1,3-bis(2-chloroethyl)-1-nitrosourea.

The chemotherapeutic activity of eight nitrosourea derivatives was compared with that of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) in transplantable rat leukemia L5222 cells. Bifunctional 1,1'-polymethylenebis-3-(2-chloroethyl)-3-nitrosoureas effected cure rates between 30 and 75% in single equitoxic doses in the therapy of advanced ip implanted L5222 [staging of L5222 leukemia development (hr before median day of death in controls): early = greater than 120; advanced = 120--61; late = 60--25; and preterminal = 24--0]. Of three water-soluble monofunctional alkylating 1-(omega-hydroxyalkyl)-3-nitrosoureas, 1-(2-hydroxyethyl)-3-(2-chloroethyl)-3-nitrosourea yielded more cures (90%) than did BCNU (cure rate, 70%) and was also superior to the other substances. Against preterminal ip implanted and late intracerebrally implanted L5222, the hydroxyethyl compound was significantly superior to BCNU.

New platinum, titanium, and ruthenium complexes with different patterns of DNA damage in rat ovarian tumor cells.

DNA protein cross-links (DPC), DNA interstrand cross-links (ISCL), and DNA single strand breaks following treatment of experimental ovarian tumor cells (O-342) with five new metal complexes (three platinum, one titanium, one ruthenium compounds) were investigated at 6, 24, and 48 h after drug exposure and compared with their in vitro growth inhibitory potential. cis-Diamminedichloroplatinum(II) (cisplatin, DDP) served as reference drug. The following new compounds were tested: 18-crown-6-tetracarboxybis-diammineplatinum(II) (CTDP), cis-aminotrismethylenephosphonato-diammineplatinum(II) (AMDP), cis-diamminecyclohexano-aminotrismethylenephosphonato-platin um(II) (DAMP), diethoxybis-(1-phenylbutane-1,3-dionato)-titanium(IV) (budotitane), and trans-indazolium-tetrachlorobisindazole-ruthenate(III) (IndCR). At equimolar concentrations DNA cross-linking activity of the platinum agents decreased in the order cisplatin, CTDP, AMDP, DAMP; this was paralleled by growth inhibition in a cell proliferation assay. CTDP-induced interstrand cross-linking occurred more slowly compared to cisplatin (DDP) (6 h: CTDP, 73 +/- 15 versus DDP, 365 +/- 72 rad equivalents), but reached a peak similar to cisplatin 24 h after exposure (CTDP, 317 +/- 68 versus DDP, 392 +/- 116 rad equivalents). At this time point in contrast to DDP no DNA protein cross-links were observed for CTDP (total cross-links: CTDP 310 +/- 71, DDP 1987 +/- 436 rad equivalents). Thus, at 24 h, CTDP was found to be distinctly less reactive to proteins than DDP, and it is suggested that CTDP might be similar in its toxicity pattern to the structurally related compound carboplatin which was also reported to be less reactive to protein than DDP. By 48 h, CTDP- and DDP-induced interstrand cross-links were 65 +/- 21 and 180 +/- 33 rad equivalents, respectively. Although at a lower level, by 24 h, AMDP showed a ratio of ISCL to total cross-links (179 +/- 39 versus 213 +/- 31 rad equivalents), which was comparable to CTDP. The second biphosphonate complex DAMP was the least active platinum compound in terms of DNA damage, effecting only 16 +/- 7 rad equivalents ISCL and 63 +/- 28 rad equivalents total cross-links; similar to DDP, DAMP displayed a higher DPC fraction at 24 h. The titanium complex diethoxybis-(1-phenylbutane-1,3-dionato)-tita-++ +nium(IV) showed dose-dependent inhibition of cell proliferation, while no significant DNA damage could be detected with the alkaline elution technique. These results, together with observations from other authors, indicating that space-filling planar aromatic ring systems are important for its antitumor activity, suggest as possible mechanism of action of diethoxybis-(1-phenylbutane-1,3-dionato)-titanium(IV) intercalation into the DNA.(ABSTRACT TRUNCATED AT 400 WORDS)

Synergistic action of vincristine and adriamycin in the treatment of experimental rat leukemia L5222.

The therapeutic effect of different ratios of a combination of vincristine and Adriamycin was compared to monotherapy with both substances in advanced rat leukemia L5222. The combination therapy proved to by synergistic. In the upper dose range, no additive toxicity was observed. The combination of 25% vincristine plus 75% Adriamycin effected the best results. In a clinically relevant dose range, the superiority of this ratio was observed independent of the application sequence of the two substances and independent of the time interval between the applications within an 11-hr investigation period.

Chemotherapy of the transplantable acute leukemia L5222 in rats.

This study presents results of single-drug and combination chemotherapy of the transplantable acute leukemia L5222 in BD IX rats. In leukemia L5222 there is a direct relationship between the number of transplanted cells and mean life expectancy. After single-drug therapy with L-asparaginase, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), cyclophosphamide, cytosine arabinoside, daunomycin, 6-mercaptopurine, methylglyoxal bis(guanylhydrazone) dihydrochloride, prednisolone, or vincristine, the best therapeutic effect was observed with BCNU and cyclophosphamide. A massive-dose therapy with BCNU repeated twice or a conbination of vincristine with cyclophosphamide or BCNU with cyclophosphamide yielded a high percentage of cures. Morever, leukemia L5222 seems to be suitable for studying the influence of drugs on the proliferation kinetics of leukemia cells.

More than additive toxicity of the combination of 1-methyl-1-nitrosourea plus 1,3-bis(2-chloroethyl)-1-nitrosourea in the rat.

The combination toxicity index of 1-methyl-1-nitrosourea plus 1,3-bis (2-chloroethyl)-1-nitrosourea determined in the rat was 0.32. This overadditive combination toxicity appears mainly to be due to severe damage of the intestinal mucosa as diagnosed by histological examination and to damage of pluripotent stem cells in the bone marrow as could be assessed by the spleen colony technique. The molar ratio of 1-methyl-1-nitrosourea to 1,3-bis(2-chloroethyl)-1-nitrosourea resulting in maximum mortality was about 6. Concomitantly measured DNA interstrand cross-linking in bone marrow cells revealed a slight increase in DNA interstrand cross-linking following both drugs compared to 1, 3-bis(2-chloroethyl)-1-nitrosourea alone.

Biological activity of hydroxylated chloroethylnitrosoureas.

1-Nitroso-1-(2-hydroxyethyl)-3-(2-chloroethyl)urea (Compound I) and 1-nitroso-1-(2-hydroxypropyl)-3-(2-chloroethyl)urea (Compound II) display significantly reduced antitumor activity compared to the corresponding isomeric derivatives 1-nitroso-1-(2-chloroethyl)-3-(2-hydroxyethyl) urea (Compound III) and 1-nitroso-1-(2-chloroethyl)-3-(2-hydroxypropyl) urea (Compound IV). Their low therapeutic activity is paralleled by low toxicity while mutagenicity and carcinogenicity are high. A comparative investigation of the genotoxicity of Compounds I and III using primary cultures of fetal hamster lung cells revealed an about 14-fold higher rate of DNA single-strand breaks following exposure (100 microM, 1 h) to Compound I as compared to Compound III. The rate of DNA interstrand cross-links, on the other hand, was 11-fold higher following Compound III as compared to Compound I. The results underline that the therapeutic activity of chloroethylnitrosoureas is mainly attributable to their cross-linking potential while induction of DNA single-strand breaks plays a decisive role for mutagenicity and carcinogenicity but appears not to be relevant for antineoplastic effectiveness.

Synergistic activity of the new ABL-specific tyrosine kinase inhibitor STI571 and chemotherapeutic drugs on BCR-ABL-positive chronic myelogenous leukemia cells.

The ABL-specific tyrosine kinase inhibitor STI571 (formerly CGP57148B) induced cytogenetic remissions in 33% of chronic myelogenous leukemia (CML) patients in a phase I trial (Druker et al 1999). Combination therapy may increase this proportion. We tested whether combinations of STI571 and cytarabine or other chemotherapeutic agents such as hydroxyurea, mafosfamide or etoposide would display synergistic activity in BCR-ABL-positive chronic myelogenous leukemia (CML) cell lines derived from patients in blast crisis. In addition, the toxicity of these combinations on BCR-ABL-negative cells was investigated. A tetrazolium-based MTT assay was used to quantity growth inhibition after 48 h of exposure to cytotoxic agents alone and in simultaneous combination with STI571. The drug interactions were analyzed using the median-effect method of Chou and Talalay. The combination index (CI) was calculated according to the classic isobologram equation. At growth inhibition levels of over 50%, STI571 + cytarabine as well as STI571 + etoposide were significantly synergistic (CI < 1, P < 0.05) in the BCR-ABL-positive cell lines evaluated. At 60% inhibition or higher, a similar synergistic pattern became apparent for STI571 + mafosfamide (P < 0.05), while STI571 + hydroxyurea showed ambiguous, cell line-dependent synergism (BV173), additivity (EM-3) or antagonism (K562) in CML cell lines. Furthermore, the BCR-ABL-negative HL-60, KG1a and normal CD34+ progenitor cells were not affected by 0.8 microM STI571, a concentration which produced more than 50% growth inhibition in all BCR-ABL-positive cells tested, and no potentiation of growth inhibition was observed in these BCR-ABL-negative cells when STI571 was combined with chemotherapeutic agents. Our in vitro data with CML blast crisis cell lines strongly suggest that combinations of STI571 with cytarabine or etoposide be rapidly considered for clinical testing.

Imatinib restores expression of CD62L in BCR-ABL-positive cells.

Chronic myelogenous leukemia (CML) is characterized by aberrant trafficking of malignant hematopoietic progenitor cells in the peripheral blood. Expression of the cell adhesion molecule CD62L was reported to be significantly lower in CML patients than in normal controls. We studied whether the transcription of CD62L in CML cells is dependent on the activity of the BCR-ABL tyrosine kinase. Following addition of the Abelson (ABL) tyrosine kinase inhibitor imatinib (formerly STI571) to two BCR-ABL-positive cell lines (BV173, SD-1), we observed a dose-dependent increase in CD62L RNA levels of up to 45-fold by a quantitative, real-time polymerase chain reaction and an increase in the amount of cell surface-bound CD62L of up to 18-fold by quantitative flow cytometry, respectively. These data are validated by an increased CD62L expression in the bone marrow of patients (n=6) with advanced CML who received imatinib. Restoration of defective cell adhesion mediated via the CD62L pathway may be one mechanism of action of imatinib in BCR-ABL-positive leukemias.

Soluble bone marrow stroma factors improve the efficiency of retroviral transfer of the human multidrug resistance 1 gene to human mobilized peripheral blood progenitor cells.

Hematopoietic stem cells (HSCs) are a potential target for the retrovirus-mediated transfer of chemotherapeutic drug resistance genes. For integration of the proviral DNA in the HSC genome cell division is required. In the bone marrow (BM) hematopoiesis occurs in the vicinity of stroma cells. Soluble stroma components were shown to play a permissive role for the proliferation of lineage-committed and primitive hematopoietic progenitors in conjunction with cytokines. We investigated the effect of stroma-conditioned medium (SCM) of the FBMD1 cell line on the gene transfer rate of the human multidrug resistance 1 (MDR1) gene contained in the retroviral SF-MDR vector into human mobilized peripheral blood progenitor cells (PBPCs) from tumor patients (n = 14) during transwell transduction in the presence of the recombinant fibronectin fragment CH-296. Addition of SCM during transduction increased the gene transfer efficiency into myeloid lineage-committed colony-forming cells by an average of 1.5-fold (p = 0.02) as detected by an SF-MDR provirus-specific polymerase chain reaction (PCR). These data were paralleled by significantly (p = 0.04 to p = 0.007) higher proportions of MDR1-expressing myelo-monocytic progeny after transduction in SCM plus interleukin 3 (IL-3), IL-3/Flt3 ligand (FL), IL-3/IL-6/FL, or IL-3/IL-6/stem cell factor (SCF) when compared with transductions without SCM as measured by rhodamine-123 exclusion. A similar trend was observed for SCM employed in combination with IL-3/IL-6/SCF/FL or FL/thrombopoietin (TPO)/SCF during transduction. The latter combination plus SCM yielded the highest proportion, 19.16 +/- 3.10% Rh-123dull cells. The beneficial effect of SCM on transduction efficiency was confirmed in additional four patients' samples, using a serum-free viral supernatant transduction protocol. As soluble BM stroma factors are able to increase the efficiency of retrovirus-mediated gene transfer into committed progenitor cells, beyond that achieved with fibronectin fragment CH-296, their effect on gene transfer into primitive repopulating hematopoietic cells may also prove beneficial.

Interstitial chemotherapy of experimental gliomas.

Two new approaches of interstitial (intratumoral) chemotherapy of gliomas are presented. Using s.c.-transplanted rat gliomas (G616) the therapeutic activity of biologically degradable polylactide rods as carriers for methotrexate was investigated. Carrier-mediated intratumoral chemotherapy was superior both to a systemic treatment and an intratumoral treatment with the free drug. The activity of the alkyllysophospholipids Et-18-OCH3 and BM 41.440 and of the alkylphosphocholine He-PC was investigated in a human glioma xenograft (T 406). All three compounds were highly active following intratumoral administration.

Differential expression of a recombinant adeno-associated virus 2 vector in human CD34+ cells and breast cancer cells.

The use of autologous hematopoietic stem cell (HSC) grafts after high-dose chemotherapy protocols may be hampered by contamination of the grafts with tumor cells. Because epithelial cells seem to be the natural hosts of adeno-associated virus 2 (AAV-2), we speculated that epithelial tumor cells in HSC grafts might be selective targets for AAV-2-based vectors. To test this hypothesis, the breast cancer cell lines T47D and MCF-7 were infected with a recombinant AAV-2 vector expressing the green fluorescent protein (GFP) gene; in addition, human CD34+ mobilized peripheral progenitor cells were infected with the same vector. At a multiplicity of infection of 100, only 1.39% +/- 0.51% CD34+ cells expressed the GFP gene whereas, 36.06% +/- 6.53% of the infected T47D cells and 41.52% +/- 3.16% of the infected MCF-7 cells expressed the transduced GFP gene. After further optimizing the transduction procedure by using higher multiplicities of infection (100-500) and preincubation of samples with the tyrosine kinase inhibitor genistein, up to 82.52% and 85.35% GFP+ T47D and MCF-7 cells, respectively, were observed. The GFP fluorescence intensity in transduced mammary tumor cells was up to 3 logs higher than that of transduced CD34+ cells. The differential expression of recombinant AAV-2 vectors in hematopoietic and epithelial tumor cells warrants further research with this vector system, including the use of suicide genes for the purging of autologous HSC grafts.

The FBMD-1 stroma cell line secretes a unique moiety which can increase retroviral transduction of lineage-committed and primitive human peripheral blood progenitor cells.

Peripheral blood progenitor cells are a prime target for gene therapy approaches. As recent data point to the relevance of soluble stroma factors for the efficient transduction of progenitor cells, we tested the stroma-conditioned medium (SCM) of the two cell lines FBMD-1 and L88/5 as well as desulfated and O-sulfated heparin (HS dS and HS OS) for their effect on transduction of peripheral blood progenitor cells. We transduced CD34+ cells of nine tumor patients with the retroviral SF-MDR vector containing the human multidrug resistance 1 (MDR1) gene under serum-free conditions on the fibronectin fragment CH-296 with or without SCM. Provirus-specific polymerase chain reaction showed a median 1.6-fold higher integration rate of the transgene into committed progenitor cells for the group with added FBMD-1 SCM (P=.008). This was maintained after 2 (P=.02) and, as a trend, after 5 weeks of stroma-dependent long-term culture. We found a median 1.5-fold increase in rhodamine-123 (Rh-123) exclusion in myeloid lineage-committed progeny cells following transduction in the presence of FBMD-1 SCM (P=.0004). After 2 or 5 weeks of long-term culture, a significantly higher proportion of Rh-123(dull) cells could still be detected in the FBMD-1 SCM transduction group (P=.003 and P=.04, respectively). L88/5 SCM or HS OS or HS dS was not effective as supplement for improving gene transfer. The FBMD-1 stroma cell line appears to secrete a unique moiety, which can increase retroviral transduction of lineage-committed and primitive progenitor cells. The FBMD-1 stroma activity is not attributable to heparan sulfate.

Superior gene transfer into solid tumour cells than into human mobilised peripheral blood progenitor cells using helpervirus-free adeno-associated viral vector stocks.

Autologous peripheral blood progenitor cell (PBPC) grafts can be contaminated with tumour cells that potentially give rise to relapse following myeloablative therapy and PBPC transplantation. Adeno-associated virus (AAV)-based vectors produced by a new adenovirus-free technique are a gene delivery system which may be applicable for tumour cell purging. To test for the host range of these vectors, solid tumours of clinical relevance and normal CD34+ PBPC were selected as target cells for an AAV-vector, encoding the green-fluorescent protein (GFP) as the indicator gene. At a multiplicity of infection (MOI) of 100: 79.94% +/- 14.36% (mean +/- SEM) of the connective tissue sarcoma cell line (HS-1) and 64.84% +/- 6.91% of the cervical carcinoma cell line cells (HeLa-RC) expressed GFP while the other cell lines tested (1 ovarian tumour, 1 germ cell tumour, 1 osteosarcoma, 2 small cell lung cancer) ranged between 2.82% and 11.94%. Optimising the transduction protocol by use of higher MOIs of up to 500 and by pretreatment with the tyrosine kinase inhibitor, genistein, resulted in up to 95.97% and 94.10% green-fluorescent HS-1 and HeLa-RC cells, respectively. In contrast, only 1.39% +/- 0.51% of the normal haematopoietic CD34+ progenitor cells expressed GFP at a MOI of 100. The differential infectivity between HS-1 and CD34+ cells was maintained after tumour cell spiking in leucapheresis products. Our observations suggest that AAV-based vectors may prove useful for purging of autologous PBPC grafts from solid tumour cells.

Chemoresistance in rat ovarian tumours.

In a cisplatin resistant subline (O-342/DPP) of an intraperitoneally growing transplantable rat ovarian tumour (O-342), intracellular glutathione (GSH) was approximately doubled (mean [S.E.] 1.5 [0.26] vs. 0.8 [0.2] nmol/10(6) cells). GSH reductase activity was higher (30.64 [4.07] vs. 20 [0.92] nmol/min per mg protein), although no difference was found for GSH-S-transferase. 24 h after exposure to cisplatin, formation of DNA interstrand cross-links was at a maximum in both lines and significantly higher in O-342 (162 [23] vs. 88 [22] rad eq). Combination treatment of O-342/DDP with buthionine sulphoximine plus cisplatin resulted in a marginal increase in survival compared with cisplatin treatment; treatment of this line with 3-aminobenzamide plus cisplatin was also superior to cisplatin alone. In the sensitive line both combinations were likewise superior to cisplatin alone. In vitro, at equimolar concentration, a new platinum complex (CTDP) was at least as active as cisplatin in both lines, which suggests a superior therapeutic index because its LD50 in mice is threefold higher than that of cisplatin. A ruthenium complex (ICR) had a higher activity in the resistant line. A titanium complex (budotitane) was not active.