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.

Efficient uptake and retention of iron oxide-based nanoparticles in HeLa cells leads to an effective intracellular delivery of doxorubicin.

The purpose of this study was to construct and characterize iron oxide nanoparticles (IONPCO) for intracellular delivery of the anthracycline doxorubicin (DOX; IONPDOX) in order to induce tumor cell inactivation. More than 80% of the loaded drug was released from IONPDOX within 24 h (100% at 70 h). Efficient internalization of IONPDOX and IONPCO in HeLa cells occurred through pino- and endocytosis, with both IONP accumulating in a perinuclear pattern. IONPCO were biocompatible with maximum 27.9% ± 6.1% reduction in proliferation 96 h after treatment with up to 200 µg/mL IONPCO. Treatment with IONPDOX resulted in a concentration- and time-dependent decrease in cell proliferation (IC50 = 27.5 ± 12.0 µg/mL after 96 h) and a reduced clonogenic survival (surviving fraction, SF = 0.56 ± 0.14; versus IONPCO (SF = 1.07 ± 0.38)). Both IONP constructs were efficiently internalized and retained in the cells, and IONPDOX efficiently delivered DOX resulting in increased cell death vs IONPCO.

Efficient mobilization of peripheral blood stem cells following CAD chemotherapy and a single dose of pegylated G-CSF in patients with multiple myeloma.

High-dose chemotherapy followed by autologous blood stem cell transplantation is the standard treatment for myeloma patients. In this study, CAD (cyclophosphamide, adriamycin, dexamethasone) chemotherapy and a single dose of pegfilgrastim (12 mg) was highly effective in mobilizing peripheral blood stem cells (PBSCs) for subsequent transplantation, with 88% of patients (n = 26) achieving the CD34+ cell harvest target of > or = 7.50 x 10(6) CD34+ cells/kg body weight, following a median of two apheresis procedures (range 1-4) and with first apheresis performed at a median day 13 after CAD application (range 10-20). Patients treated with pegfilgrastim showed a reduced time to first apheresis procedure from mobilization compared with filgrastim-mobilized historical matched controls (n = 52, P = 0.015). The pegfilgrastim mobilization regimen allowed for transplantation of a median of 3.58 x 10(6) CD34+ cells/kg body weight while leaving sufficient stored cells for a second high-dose regimen and back-ups in most patients. Engraftment following transplantation was comparable to filgrastim, with a median time of 14 days to leucocyte > or =1.0 x 10(9)/l (range 10-21) and 11 days to platelets > or = 20 x 10(9)/l (range 0-15). The results of this study thus provide further support for the clinical utility of pegfilgrastim for the mobilization of PBSC following chemotherapy in cancer patients scheduled for transplantation.

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.

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.

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.

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.

Prospects and RISC score of viral gene therapy for sarcoma.

Soft tissue sarcomas are a challenge for medical oncology and gene therapy. Protective and sensitising approaches that target normal and malignant tissue, respectively, both have their role for opening the therapeutic window. Recent data show that an intensive maintenance chemotherapy significantly reduces metastatic spread and improves disease-free survival in selected patient groups. However, delays of treatment due to cytopenia are frequent. Cytostatic drug resistance gene transfer to haematopoietic progenitor cells using retroviral vectors may allow further improvement of therapy results. In recent years, retroviral vector design, transduction techniques and engraftment capability of transduced cells have been optimised. Safety considerations of retroviral gene transfer have attracted public attention and can be addressed by analysis of genomic vector integration sites. A data bank project, 'retroviral insertion estimate of chromosomal integration' (RISC), containing > 200 integration sequences, has been set up by the authors' group to recognise critical genomic regions and genes involved with possible transforming capacity. Monitoring these parameters will allow the selection of the most suitable vectors for clinical application. Sarcoma cells seem to be highly susceptible to a variety of vectors, such as recombinant adeno-associated virus-2 (rAAV-2) vectors, adenoviral vectors or oncolytic herpes simplex viruses. Results from the first clinical trials with adenoviral vectors encoding for cytokines are promising. The other systems await further development towards clinical applications. Perspectives for further research are discussed in this review.

Protection of hematopoietic stem cells from chemotherapy-induced toxicity by multidrug-resistance 1 gene transfer.

An increased chemotherapeutic dose intensity is believed to translate into higher survival rates among cancer patients. Pancytopenia is the dose-limiting toxic result of most anticancer agents. Overexpression of the human multidrug resistance 1 (MDR1) gene in transgenic animals resulted in complete myeloprotection against high doses of cytostatic drugs. Stem cell research, vector development, and experimental pharmacology are uniting their efforts in an attempt to achieve a similar effect in human hematopoietic stem cells. This article gives an overview of the crucial steps involved, from retroviral vector design and optimization of viral titers to vector uptake, gene integration, and expression. The authors' own results are presented with special regard in vitro and in vivo assays for the detection of hematopoietic stem cell transduction.

Delineation of cell cycle state and correlation to adhesion molecule expression of human CD34+ cells from steady-state bone marrow and peripheral blood mobilized following G-CSF-supported chemotherapy.

Treatment with a combination of chemotherapy and G-CSF leads to the release of hematopoietic stem cells from the bone marrow (BM) to the peripheral blood (PB), where they can be harvested for transplantation. Premobilization BM CD34+ cells were reported to proliferate actively, while virtually none of the mobilized PB CD34+ cells were in the S/G2M phase. We were interested in elucidating the cell cycle state further and in investigating the role of adhesion molecule expression on marrow-adherent and circulating CD34+ cells during different phases of the cell cycle. Consecutive premobilization BM and leukapheresis product (LP) samples were obtained from 14 patients following G-CSF-supported chemotherapy. Steady-state BM and LP CD34+ selected cells were triple-stained for CD34, for DNA using the intercalating dye 7-aminoactinomycin D, and for Ki-67, cyclins, or adhesion antigens. Ki-67 is expressed in all phases of the cell cycle except G0 and was found in 69.14%+/-3.46% (mean +/- standard error [SE]) of BM CD34+ cells and 62.78%+/-3.37% of LP CD34+ cells, while in BM significantly more CD34+/Ki-67+ cells were in the S/G2M phase of the cell cycle than in LP (8.6%+/-0.9% versus 1.8%+/-0.3%, respectively, p = 0.0001). Therefore, most circulating mobilized CD34+ cells are in the G1 phase, similar to their steady-state BM counterparts. Cyclin A became detectable in the 2n DNA peak. As expected, a higher proportion of CD34+/cyclin A+/S/G2M cells was found in BM than in LP (p < 0.05). Antigen density of the cyclins D3 and D2 tended to be higher on LP than on BM CD34+ cells, while D1 was found at low levels in similar density. The adhesion antigens CD18, CD49b, CD49d, CD49e, CD58, and CD62L were expressed in a significantly higher proportion of S/G2M-phase than in G0/G1-phase CD34+ cells. The strongest association to the proliferative status was observed for CD49d, which was coexpressed by 85.9% +/-2.6% (BM) or 90.8%+/-2.5% (LP) of CD34+/S/G2M cells, whereas a distinct CD34+/CD49d-/S/G2M population could not be detected. The average coexpression of the other antigens was 57% (CD49e, CD18) or lower. Our results demonstrate that the majority of PB CD34+ cells mobilized following G-CSF-supported chemotherapy and steady-state BM CD34+ cells are in the late G1 phase of the cell cycle and show a correlation between the expression of adhesion receptors and cell cycle status of CD34+ cells in both BM and LP.

Peripheral blood progenitor cell (PBPC) counts during steady-state haemopoiesis enable the estimation of the yield of mobilized PBPC after granulocyte colony-stimulating factor supported cytotoxic chemotherapy: an update on 100 patients.

Peripheral blood progenitor cells (PBPC) can be mobilized using chemotherapy and granulocyte colony-stimulating factor (G-CSF). We and others previously reported a correlation of steady-state PBPC counts and the PBPC yield during mobilization in a small group of patients. Here we present data on 100 patients (patients: 25 non-Hodgkin's lymphoma (NHL), five Hodgkin's disease, 35 multiple myeloma (MM), 35 solid tumour) which enabled a detailed analysis of determinants of steady-state PBPC levels and of mobilization efficiency in patient subgroups. Previous irradiation (P = 0.0034) or previous chemotherapy in patients with haematological malignancies (P = 0.0062) led to a depletion of steady-state PB CD34+ cells. A correlation analysis showed steady-state PB CD34+ cells (all patients: r = 0.52, P < 0.0001; NHL patients, r = 0.69, P = 0.0003; MM patients: r = 0.66, P = 0.0001) and PB colony-forming cells can reliably assess the CD34+ cell yield in mobilized PB. In patients with solid tumour a similar trend was observed in mobilization after the first chemotherapy cycle (r = 0.51, P = 0.05) but not if mobilization occurred after the second or further cycle of a sequential dose-intensified G-CSF-supported chemotherapy regimen, when premobilization CD34+ counts were 18-fold elevated (P = 0.004). When the patients with MM (r = 0.63, P = 0.0008) or with NHL (r = 0.65, P = 0.006) were analysed separately, a highly significant correlation of the steady-state PB CD34+ cell count to the mean leukapheresis CD34+ cell yield was found, whereas no correlation was observed for patients with a solid tumour. For patients with haematological malignancies estimates could be calculated which, at a specific steady-state PB CD34+ cell count, could predict with a 95% probability a defined minimum progenitor cell yield. These results enable recognition of patients who mobilize PBPC poorly and may assist selection of patients for novel mobilization regimens.

Gene therapy for sarcoma.

Soft tissue sarcomas are mesenchymal tumors which respond poorly to systemic therapy. Recent studies suggest a higher response rate with an increased doxorubicin dosage. However, this was parallel with a profound hematotoxicity in 75% of patients. Transfer of the human multidrug resistance 1 (MDR1) gene to normal hematopoietic stem cells and transplantation may significantly reduce the hematotoxicity of anthracyclin-based chemotherapy. To test this concept of supportive gene therapy in advance of a clinical study, we transduced mobilized peripheral blood progenitor cells (PBPC) with the retroviral vector SF91m3 containing the human MDR1 gene, transplanted these cells to immune-deficient mice, allowed 6 weeks for engraftment to occur and treated the animals with MDR1-based chemotherapy. In the MDR1-transduced group the human leukocytes were significantly protected from the toxicity of chemotherapy (p < 0.05). While the gene transfer rate was in the range of 10% and thus comparable to recent clinical trials, the gene expression was 59% of transduced cells and thus significantly higher than previously reported for less-advanced vectors. On the other hand, ifosfamide, a drug which has been used successfully for stem cell mobilization, is active in soft tissue sarcoma. Due to these favorable characteristics sarcoma is an attractive target to test the efficacy of MDR1 gene therapy in a clinical setting. Gene therapeutic strategies may also be used to directly target sarcoma cells, e.g. by transfer of suicide genes. We found that adenoassociated virus 2 (AAV-2) vectors efficiently transduce human HS-1 and HT1080 sarcoma cells (>90%) while other tumor cell lines and primary human PBPC were less susceptible. The thymidine kinase (TK) suicide gene was cloned into an AAV-2 vector and a complete kill of TK-transduced HS-1 and HT1080 cells was observed following exposure to aciclovir or ganciclovir (GCV), while >90% of mock-transduced HS-1 cells survived at these dosages. Transplantation of those sarcoma cells to nonobese diabetic (NOD)/LtSz-severe-combined immunodeficient (scid)/scid (NOD/SCID) mice resulted in a survival of >5 months in the AAV-TK-transduced/GCV-treated group, while the mice in the mock-transduced/GCV-treated group had died after 3 weeks. These data show that soft tissue sarcomas are a particularly suitable model system for the development and clinical testing of new gene therapeutic concepts.