MDR1 P-gp expression and activity in intact human placental tissue; upregulation by retroviral transduction.

This study investigates P-gp activity in placental villous fragments and the possibility of upregulating its expression and function by retroviral transduction. In fresh fragments, cyclosporin A caused a significant increase in 3H-vinblastine accumulation (187+/-48% at 180 min n=4), consistent with multi-drug resistance activity. After 7 days in culture, villous fragments showed a similar increase in 3H-vinblastine accumulation (143+/-10% at 180 min n=4), which was not significantly different from that in fresh tissue. Following transduction, immunohistochemistry revealed increased P-gp expression. However, the distribution of the protein differed from that in controls, with P-gp being located throughout the tissue as opposed to the normal specific location on the maternal facing plasma membrane. Transduced explants showed a significantly larger increase in 3H-vinblastine accumulation in the presence of cyclosporin A than control explants (245+/-15.5% at 180 min, n=4), suggesting reduced capacity to efflux vinblastine. This study demonstrates P-gp activity in intact placental tissue which is maintained in explant culture. Retroviral transduction of P-gp to such tissue leads to increased but undirected expression of the protein. The consequent increased activity at sites such as the basal, fetal facing, plasma membrane probably explains the increased substrate accumulation within the tissue.

Retroviral transfer and expression of human MDR-1 in a murine haemopoietic stem cell line does not alter factor dependence, growth or differentiation characteristics.

In view of the recent report of a myeloproliferative syndrome in mice that had received an MDR-1-transduced haemopoietic graft, we have investigated the potential effects of MDR-1 expression on primitive haemopoietic cell growth and differentiation. Retroviral gene transfer was used to achieve exogenous expression of either MDR-1 or truncated nerve growth factor receptor (tNGFR) in the multipotent murine haemopoietic progenitor cell line, FDCP-mix. Following gene transfer, clonal lines were derived and FACS analysis confirmed appropriate expression of each transgene. MDR-1 (but not tNGFR) expression was associated with verapamil-sensitive rhodamine efflux and resistance to killing by etoposide. When growth factor responsiveness, proliferative capacity and differentiation capacity were examined, MDR-1 expressing FDCP-mix cells exhibited a normal phenotype and mimicked the response of tNGFR-expressing or untransduced FDCP-mix cells. Thus, in the model system we have used, MDR-1 does not perturb haemopoietic cell growth and development and our data do not support a myeloproliferative role for MDR-1.

The effects of dose, route of administration, drug scheduling and MDR-1 gene transfer on the genotoxicity of etoposide in bone marrow.

We have used the bone marrow micronucleus assay (BMMN) as a measure of clastogenicity, in response to etoposide exposure in murine bone marrow. Oral delivery of etoposide resulted in a reduced number of micronucleated polychromatic erythrocytes (MPE) relative to the same dose delivered intraperitoneally (P < 0.001). Daily fractionation of the oral schedule of etoposide led to a more than six-fold increase in cumulative MPE frequency over that observed with the same total, unfractionated dose, with the potency of the response increasing with serial exposure (r = 0.79). Retrovirally-mediated expression of MDR1 in murine bone marrow resulted in partial protection against the clastogenic activity of etoposide relative to mock transduced control mice. The model system developed has indicated a variety of factors able to influence the genotoxicity of etoposide. It should now be possible to further exploit this model in order to define other factors governing haemopoietic sensitivity to etoposide.

O6-benzylguanine increases the sensitivity of human primary bone marrow cells to the cytotoxic effects of temozolomide.

The sensitivity of human primary bone marrow granulocyte/macrophage precursor cells to the cytotoxic effects of the methylating antitumor agent temozolomide (8-carbamoyl-3- methylimidazo[5,1-d]-1,2,3,5-tetrazin-4-[3H]-1) was investigated using an in vitro colony-forming assay. In the eight samples examined, there was a range of sensitivities with D37 values from 18.2 to > 55 microM. When cells were simultaneously exposed to the O6-alkylguanine-DNA alkyltransferase (ATase) inactivating agent, O6-benzylguanine (O6BeG; 10 microM), the cytotoxicity of temozolomide was substantially increased with D37 values between 5 and 38.5 microM. O6BeG also increased temozolomide sensitivity in the human colon carcinoma cell line, WiDr, and this was shown to correlate with the O6BeG-mediated depletion of ATase activity. Where the extent of sensitization produced by O6BeG could be calculated, there was a correlation between this and the D37 value in the absence of O6BeG (R = 0.996); thus, sensitization was more extensive in the cells that were inherently more resistant to temozolomide. These data have implications for possible increased hematological toxicity in clinical protocols designed to exploit O6BeG or other agents to deplete ATase activity in tumors cells prior to treatment of patients with temozolomide or related agents.

Recombinant adeno-associated virus-mediated expression of O6-alkylguanine-DNA-alkyltransferase protects human epithelial and hematopoietic cells against chloroethylating agent toxicity.

Recombinant adeno-associated virus (rAAV) encoding the human O6-alkylguanine-DNA-alkyltransferase (hAT) protein and a selectable marker (Neo(r)) was used to transduce human cervical carcinoma (HeLa) cells and erythroleukemic (K562) cells and clones were selected using G418 (0.4 mg/ml). Thirteen HeLa clones were isolated, 9 of which survived for 2-3 months before cell death ensued, presumably owing to the loss of G418 resistance. Northern blot analysis of the remaining four clones, using a neo probe, showed high levels of RNA equivalent in size to the bicistronic RNA expected to be produced from this construct. Analysis of hAT activity showed that 2000-5000 fmol/mg protein was expressed relative to untransduced cells (800-900 fmol/mg protein). Cell survival analysis following exposure to the chloroethylating agent mitozolomide revealed that expression of hAT at levels two- to fourfold higher than background conferred significant resistance (p < 0.001) to the toxic effects of this drug. Two days following infection of K562 cells with the rAAV vector, immunoblot analysis showed that hAT protein was being produced. Three K562 clones, isolated using G418 selection, were studied in detail and were shown to express hAT activities of 1500, 1010, and 890 fmol/mg protein, respectively, at 40 days posttransduction (mock-transduced K562 cells contain <2 fmol of hAT/mg protein). As with HeLa cells, Northern blot analysis showed the production of an appropriately sized transcript and immunoblot analysis indicated that hAT protein was being produced. These clones were assayed for cell survival following exposure to mitozolomide. Expression of hAT at levels 800- to 1500-fold higher than background conferred significant resistance (p < 0.001) to the toxic effects of mitozolomide. We have therefore successfully conferred a protective advantage against mitozolomide toxicity to cells by rAAV-mediated hAT expression.

Enhancing hemopoietic drug resistance: a rationale for reconsidering the clinical use of mitozolomide.

Retroviral gene transfer was used to achieve expression in mouse bone marrow of a mutant form of the DNA repair protein O6-alkylguanine-DNA alkyltransferase (hATPA/GA), which exhibits resistance to inactivation by O6-benzylguanine (O6-beG). After reconstitution of mice with transduced bone marrow, approximately 50% of the bipotent granulocyte-macrophage colony-forming cell (GM-CFC) and multipotent spleen colony-forming unit (CFU-S) hemopoietic populations showed expression of the transgene; this expression was associated with resistance to either mitozolomide or to a combination of O6-beG and mitozolomide, relative to mock-transduced controls. Thus, at a dose of mitozolomide in vivo that allowed only 70% and 62% survival of mock-transduced GM-CFC and CFU-S, respectively, the hATPA/GA CFC were totally resistant to the same dose of mitozolomide (P < .05 and .001, respectively). In the presence of O6-beG, the toxicity of mitozolomide was greatly potentiated. Only 24% and 18%, respectively, of mock-transduced GM-CFC and CFU-S survived combination treatment, whereas 45% (P < .05) and 37% (P < .01) of GM-CFC and CFU-S, respectively, from hATPA/GA mice survived the same combination of doses. Furthermore, as a result of transgene expression, the number of micronucleated polychromatic erythrocytes induced by mitozolomide was significantly reduced (P < .05) by 40% relative to mock-transduced controls, indicating the potential of this approach to reduce the frequency of mutation associated with chemotherapy exposure. The protection against the toxic and clastogenic effects of mitozolomide in both primitive and more mature hemopoietic cells suggests that the severe myelosuppression that halted further clinical investigation of this drug could be substantially ameliorated by the exogenous expression of O6-alkylguanine-DNA alkyltransferase. Therefore, these data raise the prospect for the reinvestigation of mitozolomide and other proscribed drugs in the context of genetically protected hemopoiesis.

Genetic chemoprotection with mutant O6-alkylguanine-DNA-alkyltransferases.

One of the main barriers to more efficacious use of modern chemotherapeutic agents, is the collateral toxicity exhibited in normal, highly proliferative tissues, primarily the haemopoietic, gastrointestinal and pulmonary tissues. Drug resistance of tumours to these drugs compounds this problem. This review discusses the role of O6-alkylguanine-DNA alkyltransferase (ATase) in conferring protection against O6-alkylating agents in normal tissue, focusing mainly on the haemopoietic compartment. The development of mutant forms of ATase, which are resistant to the effects of soluble analogues of O6-alkylation such as O6-benzylguanine, is examined and the gene therapy approach of combining these two strategies to confer chemoprotection to vulnerable tissues whilst sensitising malignant tissue is reviewed.

New perspectives for cancer chemotherapy by genetic protection of haematopoietic cells.

The effectiveness of anti-cancer chemotherapy can be limited by acute suppression of the bone marrow (myelosuppression). There is also a risk of therapy-related secondary haematopoietic malignancy as well as acute and longer term effects in other tissues. Clinical strategies have been established to address some of these problems, particularly toxic effects on the bone marrow (acute myelotoxicity); however, there is still substantial scope for improving the management of chronic toxicity and mutagenicity to haematopoietic cells and collateral damage to non-haematopoietic cells during chemotherapy. In this review, we have discussed a novel strategy that involves the transfer and expression of drug-resistance functions into haematopoietic stem cells and more-mature blood progenitor cells, to overcome both the acute and long-term deleterious effects of anti-tumour treatment in bone marrow. The potential advantages of this approach include: (1) the in vivo selection of protected cell populations, which offers the possibility of intensification or escalation of chemotherapeutic drug doses; (2) a reduction in the frequency of therapy-related leukaemia and (3) tumour sensitisation to chemotherapy at the same time as haematopoietic protection.

Prospects for gene therapy using haemopoietic stem cells.

Gene therapy has thus far promised much and delivered little. Much of this has been due to deficiencies in the reagents and methodologies employed in early clinical trials. Recent technological advances in vectors and haemopoietic stem cell manipulation, coupled with improved pre-clinical assays of gene transfer and expression in re-populating stem cells give cause for greater optimism. Here we review these advances and indicate areas requiring further development before clinical gene therapy in the haemopoietic system becomes a widely applicable treatment modality.

Engineering drug resistance in human cells.

Many of the problems with current anti-tumour therapies stem from a lack of specificity for tumour as opposed to normal tissues. To address the problem of collateral toxicity during anti-tumour chemotherapy we have been developing a gene therapy approach to protect normal tissues from the toxic and potentially mutagenic effects of chemotherapeutic agents. As a paradigm for this we have been examining the potential of the DNA repair protein O6-alkylguanine-DNA-alkyltransferase (ATase) to confer genetic chemoprotection to the bone marrow. By transfer and expression of a mutant form of this protein, which is resistant to inactivation by the tumour sensitising agent O6-benzylguanine (O6-beG), we have been able to demonstrate protection of murine bone marrow in vitro from the cytotoxic and clastogenic effects of O6-beG in combination with the anti-tumour agent temozolomide. This protection is seen in multiple lineages, including erythroid and granulocyte/macrophage progenitors, as well as more primitive cells. Importantly, significant protection of the platelet lineage is also seen, with faster recovery of platelets. The multi-lineage protection seen has encouraged us to take this approach forward to clinical trial in the near future.

Genetic modification of haematopoietic cells for combined resistance to podophyllotoxins, other agents covered by MDR1-mediated efflux activity and nitrosoureas.

Genetic transfer and expression of drug-resistance functions into haematopoietic stem and progenitor cells is a promising means to overcome both the acute and longterm side-effects of cytotoxic drugs in bone marrow. Here, we describe a functional analysis of a retroviral vector that co-expresses human cDNAs for multidrug resistance 1/P-glycoprotein (MDR1) and a double mutant of O(6)-alkylguanine-alkyltransferase (hATPA/GA) to high levels. The hATPA/GA protein contains two amino acid substitutions that render it resistant to compounds such as O(6)-benzylguanine that inhibit the wild-type protein which is often overexpressed in resistant tumour cells. Evidence for simultaneous drug resistance of genetically modified primary murine progenitor cells to colchicine or the podophyllotoxin etoposide, both covered by MDR1-mediated efflux activity, and the nitrosourea BCNU, which is counteracted by hATPA/GA, is presented using in vitro colony assays.

Modulation of O6-alkylating agent induced clastogenicity by enhanced DNA repair capacity of bone marrow cells.

The murine bone marrow micronucleus assay has been used to examine (1) the potentiation of fotemustine and streptozotocin induced-clastogenicity by the O6-alkylguanine-DNA alkyltransferase (ATase) inactivator O6-benzylguanine (O6-beG) and (2) the level of protection afforded against this potentiation by retrovirus-mediated expression of an O6-beG-resistant mutant of human ATase (haTPA/GA) in mouse bone marrow. Both fotemustine and streptozotocin induced significantly higher levels of micronucleated polychromatic erythrocytes (p < 0.001 for the highest doses studied) compared to those seen in vehicle-treated animals. The number of micronuclei produced by either agent was dramatically elevated by pretreatment with O6-beG (p < 0.001). Furthermore, in myeloablated mice reconstituted with bone marrow expressing the O6-beG-resistant hATPA/GA as a result of retroviral gene transfer, the frequency of micronucleus formation following exposure of mice to otherwise clastogenic doses of fotemustine or streptozotocin, in the presence of O6-beG, wash highly significantly reduced (p < 0.001 for both agents) relative to that in mock transduced controls. These data clearly implicate O6-chloroethyl- and O6-methylguanine as clastogenic lesions in vivo and establish ATase as a major protective mechanism operating to reduce the frequency of such damage. The potentiation of drug induced clastogenicity by O6-beG suggests that the clinical use of this inactivator in combination with O6-alkylating agents, could substantially increase the risk of therapy related malignancy. Nevertheless the use of hATPA/GA as a protective mechanism via gene therapy may overcome this risk.

Assays to predict the genotoxicity of the chromosomal mutagen etoposide -- focussing on the best assay.

The topoisomerase II inhibitor etoposide is used routinely to treat a variety of cancers in patients of all ages. As a result of its extensive use in the clinic and its association with secondary malignancies it has become a compound of great interest with regard to its genotoxic activity in vivo. This paper describes a series of assays that were employed to determine the in vivo genotoxicity of etoposide in a murine model system. The alkaline comet assay detected DNA damage in the bone marrow mononuclear compartment over the dose range of 10--100mg/kg and was associated with a large and dose dependent rise in the proportion of cells with severely damaged DNA. In contrast, the bone marrow micronucleus assay was found to be sensitive to genotoxic damage between the doses of 0.1--1mg/kg without any corresponding increases in cytotoxicity. An increase in the mutant frequency was undetectable at the Hprt locus at administered doses of 1 and 10mg/kg of etoposide, however, an increase in the mutant frequency was seen at the Aprt locus at these doses. We conclude that the BMMN assay is a good short-term predictor of the clastogenicity of etoposide at doses that do not result in cytotoxic activity, giving an indication of potential mutagenic effects. Moreover, the detection of mutants at the Aprt locus gives an indication of the potential of etoposide to cause chromosomal mutations that may lead to secondary malignancy.

Genotoxicity studies on the azo dye Direct Red 2 using the in vivo mouse bone marrow micronucleus test.

The clastogenicity of the azo dye Direct Red 2 (DR2) has been investigated using the murine bone marrow micronucleus assay. A potent dose-dependent response was observed following oral gavage of DR2 up to 4 mg/kg, after which significant toxicity to the erythroid compartment was observed. The route of administration had a significant effect on the frequency of micronucleus formation: intraperitoneal injection was approximately two-fold less clastogenic than the equivalent dose delivered orally (p<0.05). The requirement for activation of DR2 by intestinal microflora was indicated by the fact that mice given acid-treated water prior to administration of DR2 showed a significant reduction (40%; p<0.001) in micronucleated polychromatic erythrocyte formation. The implications of these findings for the health and safety of occupationally exposed workers are discussed.

[Will transfer of cytostatic drug resistance genes increase hematopoiesis resistance in the treatment of malignant tumors?]. / Pomuze prenos genu pro rezistenci k cytostatikum zvýsit odolnost krvetvorby pri lécbe zhoubných nádoru?

The aim of aggressive antitumor chemotherapy is to kill the tumor with the largest possible dose of a cytotoxic drug. The maximum dose tolerated by the patient is limited by the toxicity to normal tissue, hematopoiesis being frequently the most sensitive system. Transfer of drug resistance genes to hematopoietic cells could protect them against chemotherapy-related toxicity and thus could be a way of gene therapy in cancer. Methylating and chloroethylating derivatives of nitrosourea are effective anticancer drugs, however, acute hematopoietic toxicity and late risk of leukemia are serious side effects. The major lesion responsible for toxic and mutagenic effects of alkylnitrosoureas is O6-alkylation of guanine in DNA. This lesion is specifically repaired by O6-alkylguanine-DNA-alkyltransferase and hematopoietic cells can be protected against toxic and mutagenic effect of nitrosoureas by alkyltransferase gene transfer. Endogenous alkyltransferase in tumor tissue could be inactivated by administration of O6-benzylguanine, while hematopoietic cells could still be chemoprotected by inhibitor-resistant alkyltransferase gene transfer. This approach could increase the therapeutic efficacy of nitrosoureas in gene therapy augmented cancer treatment.

Towards gene therapy of Hurler syndrome.

Allogeneic bone marrow transplantation is the most effective treatment for Hurler's syndrome. However, due to a lack of matched related donors and unacceptable morbidity of matched unrelated transplants, this therapy is not available to all patients. Therefore we have been developing an alternative approach based on transfer and expression of the normal gene in autologous bone marrow. A retroviral vector carrying the full length cDNA for alpha-L-iduronidase has been constructed and used to transduce bone marrow from patients with this disorder. A number of different gene transfer protocols have been assessed including the effect of intensive schedules of exposure of bone marrow to viral supernatant and the influence of growth factors. With these protocols we have demonstrated successful gene transfer into primitive CD34+ cells and subsequent enzyme expression in their maturing progeny. Also, using long-term bone marrow cultures, we have demonstrated high levels of enzyme expression sustained for several months. The efficiency of gene transfer has been assessed by PCR analysis of haemopoietic colonies as around 50%. No advantage has been demonstrated for the addition of growth factors or intensive viral exposure schedules. Indeed a possible disadvantage has been identified for the use of intensive transduction procedures. The enzyme is secreted into the medium and functional localisation has been demonstrated by reversal of the phenotypic effects of lysosomal storage in macrophages. This pre-clinical work forms the basis for a clinical trial of gene therapy for Hurler syndrome.

Human monocytes expressing a CEA-specific chimeric CD64 receptor specifically target CEA-expressing tumour cells in vitro and in vivo.

Antibody-dependent cellular cytotoxicity (ADCC) is one means by which macrophages (as well as natural killer cells and granulocytes) elicit a cytotoxic response. This is achieved via interaction of the Fc-gamma-receptor (CD64) with the Fc portion of antibody bound to target cells. We have created a chimeric CD64 molecule that incorporates a single chain Fv molecule, targeted against human carcinoembryonic antigen (CEA), fused to the membrane spanning and cytosolic domains of human CD64. Following adenoviral transfer to primary human monocytes, this chimeric CD64 receptor induced antigen-specific cytokine secretion during culture on immobilised CEA protein or on CEA-expressing tumour cells. Moreover, CEA targeted, but not control, monocytes effectively retarded CEA-positive tumour cell growth in vitro. Importantly, targeted monocyte cultures significantly reduced in vivo tumour growth rates in xenograft studies resulting in improved survival rates over that of control monocyte cultures. These data suggest that genetically directing monocytes against tumour antigens may be a useful means of achieving an immunotherapeutic response.

Marrow stromal cells from patients affected by MPS I differentially support haematopoietic progenitor cell development.

Bone marrow transplantation is the therapy of choice in patients affected by MPS I (Hurler syndrome), but a high incidence of rejection limits the success of this treatment. The deficiency of alpha-L-iduronidase (EC 1.2.3.76), one of the enzymes responsible for the degradation of glycosaminoglycans, results in accumulation of heparan and dermatan sulphate in these patients. Heparan sulphate and dermatan sulphate are known to be important components of the bone marrow microenvironment and critical for haematopoietic cell development. In this study we compared the ability of marrow stromal cells from MPS I patients and healthy donors to support normal haematopoiesis in Dexter-type long term culture. We found an inverse stroma/supernatant ratio in the number of clonogenic progenitors, particularly the colony-forming unit granulocyte-machrophage in MPS I cultures when compared to normal controls. No alteration in the adhesion of haematopoietic cells to the stroma of MPS I patients was found, suggesting that the altered distribution in the number of clonogenic progenitors is probably the result of an accelerated process of differentiation and maturation. The use of alpha-L-iduronidase gene-corrected marrow stromal cells re-established normal haematopoiesis in culture, suggesting that correction of the bone marrow microenvironment with competent enzyme prior to transplantation might help establishment of donor haematopoiesis.

The molecular control of hematopoiesis.

Experiments both in vitro and in vivo have increased our understanding of how cytokines can act to influence the maintenance of the primitive hemopoietic populations and the production of mature blood cells. Such understanding helps us to use cytokines as therapeutic agents in order to manipulate the hemopoietic response to disease and its treatment.