Retrovirus-mediated gene transfer of the multidrug resistance-associated protein (MRP) cDNA protects cells from chemotherapeutic agents

Transduction of hematopoietic progenitors with a multidrug resistance gene like mdr-1 or mrp aims to protect bone marrow from toxicity of chemotherapeutic agents. The interest in the use of mrp as an alternative to mdr-1 gene transfer for bone marrow protection lies in its different modulation. Indeed, classical P-gp reversal agents, tested in the clinic to decrease mdr-1 tumor resistance, have little or no effect on MRP function. This would allow, in the same patient, the use of reversal agents to decrease P-gp tumor resistance without reversing bone marrow protection of the transduced hematopoietic cells provided by multidrug resistance-associated protein (MRP). As a first step, we have constructed and tested two different mrp-containing vectors with either the Harvey retroviral long terminal repeat (LTR) or PGK as promoters and generated ecotropic producer cells. We have shown by Southern blot analysis that retroviral supernatant from these producer cells can efficiently transmit the mrp gene to target cells. Mrp expression could be detected by fluorescence-activated cell sorting (FACS) analysis in the producer cells. The transduced cells have increased resistance to doxorubicin, vincristine, and etoposide. Furthermore, chemoprotection of the transduced cells was increased after selection with chemotherapeutic agents in the presence of glutathione, a co-factor for MRP function. These data indicate that mrp retroviral vectors may be useful for chemoprotection and selection.     

Chemotherapy immediately following autologous stem-cell transplantation in patients with advanced breast cancer

Most patients relapse after high-dose chemotherapy (HDCT) with autologous stem-cell transplantation (ASCT) for metastatic breast cancer. Further chemotherapy immediately after hematopoietic recovery from ASCT is not given for fear of irreversibly damaging the newly engrafted stem cells. In a pilot chemoprotection trial, autologous CD34+ cells from patients with metastatic breast cancer were exposed to a replication-incompetent retroviral vector carrying MDR-1 cDNA and then reinfused after HDCT. Immediately on recovery, patients received multiple courses of escalating dose paclitaxel. All of the 10 patients tolerated reinfusion of modified cells without any toxicity and had myeloid engraftment within 12 days (range, 11-14). The bone marrow cells of three patients contained vector MDR-1-positive cells only at the time of the first course of posttransplant paclitaxel, indicating that the MDR-1 vector-modified cells had only short-term engrafting potential. A total of 83 courses of paclitaxel were administered starting at a median of 30 (range, 21-32) days from ASCT. The median dose of paclitaxel was 225 mg/m2 and the median interval between paclitaxel cycles of therapy was 21 (range, 20-41) days. Five of the six CR patients were able to receive all of the 12 courses of paclitaxel. Three patients who had achieved less than a complete response to the HDCT (2 patients) and partial response (1 patient) were converted to complete clinical responses during the 12 cycles of paclitaxel. No delayed toxicity or bone marrow failure was noted in these patients with a median follow-up of 2 years from ASCT. This is the first study of chemotherapy immediately after transplantation with autologous CD34+ cells. These data indicate that paclitaxel can be safely administered immediately after ASCT without any delayed toxicities. Paclitaxel given immediately after ASCT can further improve the response to pretransplant chemotherapy in patients with advanced breast cancer.     

Gene transfer for the therapy of hematologic malignancy

Gene transfer has allowed a number of biologic issues in the therapy of hematologic malignancy to be addressed. In autologous bone marrow transplantation, gene marking studies have shown that infused marrow contributes to relapse in acute myeloid leukemia, neuroblastoma, and chronic myeloid leukemia. In addition, double gene marking with distinguishable retroviral vectors has allowed comparison of purging techniques and the ability of different sources of stem cells to repopulate. In allogeneic bone marrow transplantation, genetically modified T cells have proven valuable for the prophylaxis and treatment of viral diseases and may be of use to treat disease relapse. Gene transfer is also being used to modify tumor function, enhance immunogenicity, modify function of adoptively transferred immune system cells, and confer drug resistance to normal hematopoietic stem cells.     

The clinical significance of MDR-1 gene expression in the hemopoietic cells of patients with acute leukemias in different phases of the disease

AIM: Analysis of cytostatic therapy effects on expression of gene MDR-1 in hemopoietic cells of patients with acute leukemia (AL) in complete clinicohematological remission (CCHR). MATERIALS AND METHODS: The study included 48 AL patients. 27 of them were untreated, 25 were resistant to or had recurrent AL. 4 patients were followed up. Bone marrow mononuclear fraction was investigated. Expression of MDR-1 gene in the cells was evaluated at hybridization. RESULTS: High expression of MDR-1 gene occurred in leukemic blast cells either upon achievement of CCHR or at least 6 months after its onset. When using schemes of chemotherapy containing two potential inductors of gene MDR-1, expression of this gene was registered significantly more frequently than in using schemes based on one inducing drug (p < 0.05). Frequency of occurrence of enhanced expression of gene MDR-1 in leukemic blasts significantly correlated with frequency of CCHR (p < 0.05). Correlation between occurrence of the gene's expression in normal hemopoietic cells in CCHR and occurrence of early AL recurrences was not found. CONCLUSION: The findings may facilitate design of new AL treatment programs.     

Cognitive rehabilitation of chronic alcohol abusers

Gene-based therapies for cancer in clinical trials include strategies that involve augmentation of immunotherapeutic and chemotherapeutic approaches. These strategies include ex vivo and in vivo cytokine gene transfer, drug sensitization with genes for prodrug delivery, and the use of drug-resistance genes for bone marrow protection from high-dose chemotherapy. Inactivation of oncogene expression and gene replacement for tumor suppressor genes are among the strategies for targeting the underlying genetic lesions in the cancer cell. A review of clinical trial results to date, primarily in patients with very advanced cancers refractory to conventional treatments, indicates that these treatments can mediate tumor regression with acceptably low toxicity. Vector development remains a critical area for future research. Important areas for future research include modifying viral vectors to reduce toxicity and immunogenicity, increasing the transduction efficiency of nonviral vectors, enhancing vector targeting and specificity, regulating gene expression, and identifying synergies between gene-based agents and other cancer therapeutics.     

Gene transfer into hematopoietic stem cells

The ability to insert a gene into hematopoietic stem cells and achieve lineage specific expression of the transferred gene within hematopoietic organs following bone marrow transplantation would create the potential to effectively treat many genetic and acquired diseases. The use of retroviral vectors to achieve this purpose has been investigated extensively in animal models and most recently, in humans. In the murine model, about 20-30% of repopulating stem cells can be genetically modified with a retroviral vector. Peripheral blood stem cells, mobilized by cytokine administration in splenectomized animals, are readily transduced and are capable of long-term reconstitution of transplant recipients with genetically modified cells. Similar protocols have been utilized to transduce highly purified stem cells from rhesus monkeys. Although long-term repopulation with cells that persistently express the transferred gene has been achieved, the frequency of cells containing the vector genome is only about 1-2%. Genetic marking of human bone marrow and peripheral blood cells has been utilized to investigate their potential for contributing to long-term reconstitution following autologous transplantation. Future work will focus on improving gene transfer efficiencies for specific therapeutic applications.     

Somatic gene therapy in squamous epithelial carcinomas of the head-neck area

Gene therapy is an important new approach to the treatment and prevention of human diseases. Somatic gene therapy involves the introduction of novel genetic material into somatic cells to express therapeutic gene products. Two main strategies in somatic gene therapy of cancer are applied: the genetic correction of the defect, or the elimination of cancer cells by cytotoxic drugs or the immuno system. Gene transfer can be accomplished by physical and chemical methods or nonreplicating viruses. The different transfer systems vary strongly in their efficiency of transfection, plasmid maintenance, and protein expression. The clinical application could be performed in two ways: Firstly, by in vivo application of genome modifying substances injected directly into the tumour; secondly, by ex vivo application of genetically modified tumour cells as a tumour vaccine. The following review will discuss some of the gene therapy strategies that could be effective in managing head and neck cancer.     

Impact of exogenous growth factors on proliferation and chemosensitivity of minimal residual acute myeloid leukemia

The biological heterogeneity of AML makes growth factor augmentation of cell cycle-dependent chemotherapy unlikely to be successful for all patients. Patients whose leukemic cells empirically demonstrate cytokine-induced chemosensitization in vitro might benefit from the concurrent administration of growth factors during consolidation chemotherapy. We have explored the growth factor-dependence and response of primary bone marrow samples from patients with AML at diagnosis, remission, and relapse to determine whether minimal residual leukemia remains growth factor-responsive. Most cases of AML studied at all phases of treatment were growth factor-responsive. Growth factor response of occult remission clonogenic leukemic precursors (CFU-L) was usually concordant with their response at diagnosis. Occult CFU-L were markedly resistant to cytosine arabinoside (median LD99% 20 microM); preincubation with IL-3 or GM-CSF did not significantly improve their ara-C sensitivity. While occult remission CFU-L appear to remain growth factor-responsive, it appears unlikely that growth factor augmentation of consolidation chemotherapy will overcome the important problem of drug resistance of residual leukemia.     

Retrovirus-mediated transfer of the human alpha-L-iduronidase cDNA into human hematopoietic progenitor cells leads to correction in trans of Hurler fibroblasts

Hurler syndrome (mucopolysaccharidosis IH or MPS IH) is a congenital mucopolysaccharide storage disorder resulting from a genetic deficiency of alpha-L-iduronidase (IDUA), which is required for lysosomal degradation of glycosaminoglycans heparan sulfate and dermatan sulfate. Even though histocompatible bone marrow transplantation has been applied for the treatment of Hurler syndrome, gene therapy via autologous bone marrow transplantation (BMT) may be more beneficial for this disease. Two retroviral vectors containing a full-length human IDUA cDNA were constructed using Moloney murine leukemia virus (MoMLV)-based vector backbones. High-titer vector-producing clones containing the L-HuID-SN and MFG-HuID retroviral vectors were established. The efficiency of gene transfer into primitive human CD34+ hematopoietic cells using both retroviral vectors is in the range of 18-23%. The level of enzyme expression in transduced primary bone marrow cells was increased 40- to 50-fold compared with that of sham-transduced cells. Enzyme produced by the progeny of the transduced human CD34+ cells carrying IDUA cDNA corrected Hurler fibroblasts via mannose-6-phosphate receptors. These findings suggest that genetically modified hematopoietic progenitor cells can potentially be useful for gene therapy of Hurler syndrome.     

Host immune potentiation of drug responses to a murine mammary adenocarcinoma. II. Effect of melphalan therapy on the host immune system

The role of chemotherapy in influencing tumor-specific immunity to a mouse mammary adenocarcinoma was investigated. By studying different stages of tumor growth we were able to identify several factors important to drug-induced tumor regression: (1) antibody response, (2) delayed hypersensitivity, (3) sensitivity of tumor cells to immune attack and (4) tumor burden. The presence of tumor-specific delayed hypersensitivity and circulating antibody as well as specifically armed monocytes in the tumor mass characterize the T1699 adenocarcinoma. Successful chemotherapy had previously been shown to depend on prior establishment of the above immune responses. Treatment with alkylating agents was marked in all animals by abrogation of a humoral response to the tumor when drug was given early (day 7), and was associated with poor chemotherapeutic results. Later treatment (day 10) was associated with depression of antibody titers only in the minority of animals not responding to drug and prolongation of the delayed hypersensitivity response in all treated animals. Tumors recurring following initial drug-induced regression were marked by lack of delayed hypersensitivity in the host, lack of drug response and suppression of humoral immunity following treatment. Successive passage of cells from these resistant tumors led to decreasing sensitivity to chemotherapy despite established immunity on the part of the host. The selection of tumor cells resistant to immune destruction rather than drug resistance per se appeared to pay a role. Melphalan was thus able to affect both favorably and adversely the immune factors important to drug-induced regression.     

Particle-mediated gene transfer of granulocyte-macrophage colony-stimulating factor cDNA to tumor cells: implications for a clinically relevant tumor vaccine

The necessity for prolonged tissue culture manipulations limits the clinical application of many form of gene therapy in patients with malignancies. We hypothesized that granulocyte-macrophage colony-stimulating factor (GM-CSF) cDNA in a plasmid expression vector could be effectively introduced into resting tumor cells, without the need for tissue culture propagation prior to or following transfection, and that efficient expression of transgenic GM-CSF by the transfected tumor cells would confer an effective immune response against tumors. GM-CSF cDNA in expression vectors was coated onto gold particles and accelerated with a gene gun device into mouse and human tumor cells. Human tumor tissue transfected within 4 hr of surgery produced significant levels of transgenic human GM-CSF protein in vitro. Human GM-CSF was readily detectable in serum and at the injection site following subcutaneous implantation of these transfected tumor cells into nude mice. Transfected and irradiated murine B16 melanoma cells produced > or = 100 ng/ml murine GM-CSF/10(6) cells per 24 hr in vitro for at least 10 days. The antitumor efficacy of this nonviral approach was tested using irradiated B16 tumor cells that were transfected with mGM-CSF cDNA and injected into mice as tumor "vaccine". Subsequent challenge of these mice with nonirradiated, nontransfected B16 tumor cells showed that 58% of the animals wer protected from the tumor by the prior vaccine treatment. In contrast, only 2% of control animals were protected by prior treatment with irradiated B16 cells transfected with the vector containing the luciferase gene. These results suggest that particle-mediated transfection of fresh tumor explants with cytokine cDNA is an effective and clinically attractive approach for cancer therapy.     

Disseminated tumor cells: diagnosis, prognostic relevance, phenotyping and therapeutic strategies

The incidence of local relapse after complete (R0) resection of solid tumors is largely determined by the skill of the surgeon, whereas metastatic relapse in distant organs is caused by pre- or perioperative systemic dissemination of tumor cells. The presence of individual disseminated tumor cells--e.g., in bone marrow as indicator organ--can be detected by sensitive immunocytochemical and molecular methods and is increasingly considered as a clinically relevant prognostic indicator. In contrast to solid metastases, isolated micrometastatic tumor cells are appropriate targets for intravenously applied anti-cancer therapeutics because they are easily accessible to macromolecules and immunologic effector cells. The majority of these tumor cells appear to be nonproliferating (i.e., in the G0 phase of the cell cycle), which may explain the failure of adjuvant chemotherapy. Adjuvant therapeutic strategies aimed at quiescent tumor cells are therefore of increasing interest. This therapeutic rationale has been tested and confirmed in a randomized clinical trial using antibody 17-1A in patients with non-metastatic colorectal carcinoma (UICC stage III). The antibody therapy kills also quiescent tumor cells ("dormant cells") and is independent from a potential chemotherapy resistance of the tumor cells. As treatment for minimal residual cancer, the clinical use of antibody therapy could be envisaged in conjunction with chemotherapy, applied either in parallel or sequentially. The aim of this review is to present and discuss the current state of research in the field of diagnosis and therapy of minimal residual cancer.     

Adenoviral-mediated transfer of a heat-inducible double suicide gene into prostate carcinoma cells

Tumor cells that express a fusion gene comprised of Escherichia coli cytosine deaminase (CD) and herpes simplex virus type 1 thymidine kinase (TK) sequences exhibit activation of and subsequent killing by the normally innocuous prodrugs 5-fluorocytosine and ganciclovir (Rogulski et al., Hum. Gene Ther., 8: 73-85, 1997). To target localized expression of this therapeutic gene, we have constructed a recombinant adenovirus containing the CD-TK fusion gene under the control of a human inducible heat shock protein 70 promotional sequence. Strong expression of the fusion gene product was induced by heating at 41 degrees C for 1 h. Expression levels obtained were dependent on the multiplicity of infection used and the incubation time after heat shock. Heat-induced expression of the CD-TK protein significantly reduced the survival of PC-3 cells in the presence of both 5-fluorocytosine and ganciclovir. These studies represent a novel form of gene therapy for the transduction and regulation of a double suicide gene in tumor cells and may provide a unique application for hyperthermia in cancer therapy.     

Gene transfer into hepatocytes and human liver tissue by baculovirus vectors

Gene therapy of liver diseases requires the development of efficient vectors for gene transfer in vivo. Retroviral and adenoviral vectors have been shown to deliver genes efficiently into hepatocytes in vitro and in vivo. However, these vectors do not allow for exclusive infection of the liver which would be highly advantageous for in vivo gene therapy strategies. We have recently demonstrated that genetically modified baculoviruses (Autographa californica nuclear polyhedrosis virus) efficiently deliver genes into cultured cells and have a strong preference for hepatocytes of different origin. Baculoviral gene transduction efficiency into human hepatocytes was determined to approach 100% and expression levels are high, provided that gene expression is controlled by mammalian promoters. In this report, we present further properties of baculoviruses regarding their use for hepatocyte gene transfer. Baculovirus-mediated gene expression declines rapidly in the hepatocellular carcinoma cell line Huh7 and more slowly in primary cultures of mouse hepatocytes. Direct application of baculoviruses for gene delivery to the liver in vivo is hampered by serum components, presumably by complement. However, we demonstrate here that baculoviral gene transfer is feasible in ex vivo perfused human liver tissue. This result suggests the development of a strategy using baculoviral vectors for liver-directed gene therapy.     

A physiologic function for p-glycoprotein (MDR-1) during the migration of dendritic cells from skin via afferent lymphatic vessels

P-glycoprotein (MDR-1) is a well-known transporter that mediates efflux of chemotherapeutic agents from the intracellular milieu and thereby contributes to drug resistance. MDR-1 also is expressed by nonmalignant cells, including leukocytes, but physiologic functions for MDR-1 are poorly defined. Using an initial screening assay that included >100 mAbs, we observed that neutralizing mAbs MRK16, UIC2, and 4E3 against MDR-1 specifically and potently blocked basal-to-apical transendothelial migration of mononuclear phagocytes, a process that may mimic their migration into lymphatic vessels. Antagonists of MDR-1 then were used in a model of authentic lymphatic clearance. In this model, antigen-presenting dendritic cells (DC) migrate out of explants of cultured human skin and into the culture medium via dermal lymphatic vessels. DC and T cells derived from skin expressed MDR-1 on their surfaces. Addition of anti-MDR-1 mAbs MRK16, UIC2, or the MDR-1 antagonist verapamil to skin explants at the onset of culture inhibited the appearance of DC, and accompanying T cells, in the culture medium by approximately 70%. Isotype-matched control mAbs against other DC molecules including CD18, CD31, and major histocompatibility complex I did not block. In the presence of MDR-1 antagonists, epidermal DC were retained in the epidermis, in contrast to control conditions. In summary, this work identifies a physiologic function for MDR-1 during the mobilization of DC and begins to elucidate how these critical antigen-presenting cells migrate from the periphery to lymph nodes to initiate T lymphocyte-mediated immunity.     

Natural killer cell-mediated eradication of neuroblastoma metastases to bone marrow by targeted interleukin-2 therapy

Targeted interleukin-2 (IL-2) therapy with a genetically engineered antidisialoganglioside GD2 antibody-IL-2 fusion protein induced a cell-mediated antitumor response that effectively eradicated established bone marrow and liver metastases in a syngeneic model of neuroblastoma. The mechanism involved is exclusively natural killer (NK) cell-dependent, because NK-cell deficiency abrogated the antitumor effect. In contrast, the fusion protein remained completely effective in the T-cell-deficient mice or immunocompetent mice depleted of CD8+ T cells in vivo. A strong stimulation of NK-cell activity was also shown in vitro. Immunohistology of the leukocytic infiltrate of livers from treated mice revealed a strong staining for NK cells but not for CD8+ T cells. The therapeutic effect of the fusion protein was increased when combined with NK-cell-stimulating agents, such as poly I:C or recombinant mouse interferon-gamma. In conclusion, these data show that targeted delivery of cytokines to the tumor microenvironment offers a new strategy to elicit an effective cellular immune response mediated by NK cells against metastatic neuroblastoma. This therapeutic effect may have general clinical implications for the treatment of patients with minimal residual disease who suffer from T-cell suppression after high-dose chemotherapy but are not deficient in NK cells.     

Identification of envelope protein residues required for the expanded host range of 10A1 murine leukemia virus

The 10A1 murine leukemia virus (MuLV) is a recombinant type C retrovirus isolated from a mouse infected with amphotropic MuLV (A-MuLV). 10A1 and A-MuLV have 91% amino acid identity in their envelope proteins yet display different host ranges. For example, CHO-K1 cells are resistant to A-MuLV but susceptible to infection by 10A1. We have now determined that retroviral vectors bearing altered A-MuLV envelope proteins containing 10A1-derived residues at positions 71 (A71G), 74 (Q74K), and 139 (V139M) transduce CHO-K1 cells at efficiencies similar to those achieved with 10A1 enveloped vectors. A-MuLV enveloped retroviral vectors with these three 10A1 residues were also able to transduce A-MuLV-infected NIH 3T3 cells. This observation is consistent with the ability of vectors bearing this altered A-MuLV envelope protein to recognize the 10A1-specific receptor present on NIH 3T3 cells and supports the possibility that residues at positions 71, 74, and 139 of the 10A1 envelope SU protein account for the expanded host range of 10A1.     

Chemosensitization of human prostate carcinoma cell lines to anti-fas-mediated cytotoxicity and apoptosis

Androgen ablation has been an effective treatment in patients with advanced prostate cancer. However, most treated patients develop hormonally resistant disease and do not respond to conventional chemotherapy. Immunotherapy against prostate cancer is an alternative approach in overcoming hormonal/drug-resistant prostate cancer. Cytotoxic immune lymphocytes kill target cells via the perforin/granzyme and the Fas-ligand (Fas-L) pathways. We hypothesize that tumor cells respond poorly to immunotherapy by developing resistance to killing by the Fas-L mechanism. This study investigated whether prostate tumor cells are sensitive to Fas-mediated killing. The human prostate carcinoma cell lines DU145, PC-3, and LnCAP were examined for their sensitivity to killing and apoptosis by the Fas-L agonist anti-Fas antibody and CTLs. All three lines moderately expressed the Fas antigen on the cell surface; however, all three lines were relatively resistant to cytotoxicity mediated by anti-Fas (CH-11) antibody. Pretreatment of DU145 and PC-3 with subtoxic concentrations of drugs followed by anti-Fas antibody resulted in synergistic cytotoxicity and apoptosis, whereas only an additive effect was obtained with LnCAP. Chemosensitization with drugs and anti-Fas was completely blocked by the addition of neutralizing anti-Fas antibody. The murine CTL hybridoma, PMMI, which kills only via the Fas-L pathway, was able to kill chemosensitized PC-3 and DU145 but not LnCAP cells. Furthermore, this cytotoxicity was blocked by anti-Fas neutralizing antibody. Chemosensitization of PC-3 and DU145 prostate tumor cells was not due to up-regulation of Fas-receptor antigen expression. Treatment of tumor cells with cisplatin did not down-regulate the antiapoptotic genes bcl-2, FAP-1, and c-myc. Further, there was no induction by cisplatin of Fas-L on the tumor cells, thus ruling out Fas/Fas-L-mediated autologous killing. These findings demonstrate that pretreatment of drug-resistant/CTL-resistant prostate DU145 and PC-3 tumor cells with subtoxic concentrations of certain chemotherapeutic drugs sensitizes the tumor cells to Fas-mediated cytotoxicity. These findings suggest that chemosensitization of tumor cells should optimize the response to immunotherapeutic interventions in the treatment of hormone-resistant/drug-resistant prostate cancer.     

MHC class II-transfected tumor cells directly present antigen to tumor-specific CD4+ T lymphocytes

We have developed and shown to be efficacious an immunotherapeutic strategy to enhance the generation of tumor-specific CD4+ T helper lymphocytes. The approach uses autologous tumor cells genetically modified to express syngeneic MHC class II genes as cell-based immunogens and is based on the hypothesis that tumor cells directly present tumor Ags to CD4+ T cells. Since the conventional pathway for CD4+ T cell activation is indirect via professional APC, induction of immunity following immunization with class II-transfected tumor cells was examined in bone marrow chimeric mice. Both tumor and host-derived cells are APC for tumor Ags, suggesting that the efficacy of tumor cell vaccines can be significantly improved by genetic modifications that enhance tumor cell Ag presentation.