Normal Tissue Protection Research Articles

Overexpression of extracellular superoxide dismutase protects mice from radiation-induced lung injury

Purpose/Objective: Superoxide dismutase (SOD) is a key enzyme in the metabolism of reactive oxygen species (ROS). Transgenic mice overexpressing extracellular-SOD (EC-SOD) have demonstrated protection of normal tissue from ROS mediated injury in several models, including cerebral ischemia, cardiac ischemia/reperfusion injury, and lung injury from hyperoxia, influenza, and hemorrhage. Therefore, we hypothesized that overexpression of EC-SOD in the mouse lung would be protective against radiation injury by ameliorating damage due to oxidative stress. Materials/Methods: Fourteen B6C3 transgenic (TG) mice that overexpress EC-SOD in the lungs were compared to 9 wild-type (WT) counterparts. Whole lung radiation was delivered to 9 TG mice (XRT-TG) and 6 WT mice (XRT-WT). The remaining mice were not irradiated (Con-TG and Con-WT). Whole lung radiation was given with a single dose of 15 Gy using 4MV photons through a dorsal-ventral field. Pulmonary function was assessed by measuring breathing frequency every two weeks for 10 weeks, then every week up to 24 weeks. Animals displaying evidence of respiratory distress were sacrificed. The remaining animals, including all controls, were sacrificed at the predetermined time of 6 months post-radiation. Upon sacrifice, the entire right lung wet weight was measured as a gross indication of lung consolidation and edema. Histopathology was used to assess tissue fibrosis and immunohistochemistry to assess macrophage activity. ELISA was performed using lung tissue to measure the percentage of the activated fibrogenic cytokine, TGF-β1. Oxidative stress was assessed indirectly by measuring lipid peroxidation in the form of malondialic acid (MDA) using HPLC. Results: Four of 6 XRT-WT mice required euthanasia at 15-20 weeks post-radiation due to respiratory distress. During the same time period, none of the 9 XRT-TG mice displayed evidence of respiratory distress or increased breathing frequency, but 3 mice were randomly sacrificed for pathologic comparison to the WT mice. The mice sacrificed in this time period of 15-20 weeks comprise the pathologic data presented in this study. At the peak respiratory response (14 weeks), the mean breathing frequency (breaths per minute ±SEM) was significantly higher for XRT-WT mice than XRT-TG mice (380 ±34 vs. 286 ±2, p<0.05). Breathing frequency between XRT-TG and Con-TG was not significantly different (286 ±2 vs. 279 ±3, p>0.05). Similarly, right lung weights (mg ±SEM) were higher for XRT-WT mice (228 ±44) than XRT-TG mice (113 ±7) with a trend toward statistical significance, p=0.08. Histologic evaluation of the XRT-WT mice demonstrated greater fibrosis, consolidation, and collagen deposition compared to the XRT-TG and control mice. The XRT-WT mice (48 ±1) had a ten-fold higher number of activated macrophages per 40X field compared to the XRT-TG mice (4.9 ±0.5). The macrophage count for the XRT-TG mice was not significantly different than the control mice, p>0.05. Fibrogenic cytokine activity, measured by percent activated TGF-β1, was higher in the XRT-WT mice than in the XRT-TG mice (37% vs. 11%, p<0.05). Percent of activated TGF-β1 in the XRT-TG mice was not significantly different than the Con-TG mice (11% vs. 12%, p>0.05). Similar results were obtained for lipid oxidation: XRT-WT mice showed a 1.9-fold increase in MDA levels compared to XRT-TG mice (624 ±123 vs. 323 ±24, p<0.05), and a 2.0-fold increase compared to Con-WT mice (264 ±42, p<0.05). Conclusions: Overexpression of EC-SOD in transgenic mice confers significant protection against radiation-induced lung injury. This is supported by several assessments of lung injury, including pulmonary function, gross pathology, histopathology, immunohistochemistry, and TGF-β1 activity. The high level of radioprotection is demonstrated by the fact that the degree of damage in the irradiated transgenic mice was not significantly different than the unirradiated control mice. Finally, as hypothesized, the mechanism appears to be related to a decrease in oxidative stress, as demonstrated by the levels of lipid oxidation.

Plan space: representation of treatment plans in multidimensional space

Purpose : Treatment planning balances the need to provide adequate radiation coverage of the target with the need to reduce against the risk of overdosing normal tissues. An acceptable plan fulfills minimum dose-volume criteria for irradiation of tumor and normal tissues. However, multiple plans can satisfy these minimum criteria, and some plans provide for better protection of normal tissue than others. Here, we present a method to help the planner compare plans and decide whether a particular plan is the “best” plan on the basis of a set of certain dose-volume conditions. Methods and Materials : Treatment plans are represented as points in multidimensional space. One dimension is assigned to the target and one to normal tissues of each anatomic structure under consideration. Minimum target dose is used as the target axis coordinate and the percentage volume of each normal structure receiving more than a specified dose as each normal-tissue coordinate. Images of plan space are developed for model phantom anatomy as well as for two clinical cases in the thorax and abdomen. Results : When a sufficient number of plans have been plotted, a feasibility boundary becomes evident. This hypersurface in plan space represents the limit of the given treatment technique. By using a plan on this boundary, the benefits of a given treatment modality are maximized, providing assurance that the selected plan is the “best” plan. The beam angles and relative weights of a plan can be changed to alter its position in plan space, allowing improvements in an existing plan. Frequently, plans with normal-tissue dose distributions superior to the minimum acceptable criteria can be selected. The benefits of using plan-space images have been demonstrated at sites in the thorax and abdomen. Conclusion : Instead of defining dose-volume criteria at the outset, it is possible to select the best achievable plans by first evaluating the space of possible plans for a particular patient’s unique anatomy and then choosing the plan with the optimum dose-volume characteristics. No attempt is made to arrive at a single plan score so that explicit judgments about the relative worth of individual structures are avoided. Visualization of plan-space images allows physicians to make choices based on their assessment of the relative significance of irradiation of each normal structure.

Efficacy of Radioprotection in the Prevention of Radiation-Induced Craniofacial Bone Growth Inhibition

It has been reported that radiotherapy-induced craniofacial deformities can occur in 66 to 100 percent of survivors of childhood head and neck cancers. Recent interest in the effectiveness of radioprotectors in the protection of normal tissue against radiation injury led us to investigate a possible role of radioprotection in the prevention of radiation-induced craniofacial bone growth inhibition. Therefore, the objective of this study was to use the radioprotective agent amifostine (Ethyol, WR-2721) as a probe to determine the effectiveness of radioprotection in the prevention of radiation-induced craniofacial bone growth inhibition after single-dose orthovoltage radiation to the infant rabbit orbital-zygomatic complex. Seven-week-old male New Zealand white rabbits were randomized into three groups (n = 10 each): group 1, 0 Gy (sham radiation); group 2, 35-Gy single-dose orthovoltage radiation; and group 3, 35-Gy single-dose orthovoltage radiation and amifostine (300 mg/kg intravenously, given 20 minutes before radiation). Serial radiographs and computed tomographic scans were obtained for cephalometric analysis, bone volume, and bone density measurements until skeletal maturity at 21 weeks. Significant (p < 0.05) reductions in orbital-zygomatic complex linear bone growth, bone volume, and bone density were observed after 35-Gy radiation compared with nonirradiated controls. No significant differences were noted between groups in cephalometric analysis of the nontreated (nonirradiated) left orbital-zygomatic complex, indicating no crossover effect from the radiation beam. However, pretreatment with amifostine, 20 minutes before 35-Gy radiation, resulted in significant (p < 0.05) preservation of linear bone growth, bone volume, and bone mineral density in the rabbit orbital-zygomatic complex compared with controls. This study demonstrated for the first time the effectiveness of a radioprotector in the prevention of radiation-induced craniofacial bone growth inhibition, and it paves the way for investigation into the pathogenic mechanism and prevention of radiotherapy-induced craniofacial deformities.

Efficient and selective gene transfer into primary human brain tumors by using single-chain antibody-targeted adenoviral vectors with native tropism abolished.

The application of adenoviral vectors in cancer gene therapy is hampered by low receptor expression on tumor cells and high receptor expression on normal epithelial cells. Targeting adenoviral vectors toward tumor cells may improve cancer gene therapy procedures by providing augmented tumor transduction and decreased toxicity to normal tissues. Targeting requires both the complete abolition of native tropism and the addition of a new specific binding ligand onto the viral capsid. Here we accomplished this by using doubly ablated adenoviral vectors, lacking coxsackievirus-adenovirus receptor and alpha(v) integrin binding capacities, together with bispecific single-chain antibodies targeted toward human epidermal growth factor receptor (EGFR) or the epithelial cell adhesion molecule. These vectors efficiently and selectively targeted both alternative receptors on the surface of human cancer cells. Targeted doubly ablated adenoviral vectors were also very efficient and specific with primary human tumor specimens. With primary glioma cell cultures, EGFR targeting augmented the median gene transfer efficiency of doubly ablated adenoviral vectors 123-fold. Moreover, EGFR-targeted doubly ablated vectors were selective for human brain tumors versus the surrounding normal brain tissue. They transduced organotypic glioma and meningioma spheroids with efficiencies similar to those of native adenoviral vectors, while exhibiting greater-than-10-fold-reduced background levels on normal brain explants from the same patients. As a result, EGFR-targeted doubly ablated adenoviral vectors had a 5- to 38-fold-improved tumor-to-normal brain targeting index compared to native vectors. Hence, single-chain targeted doubly ablated adenoviral vectors are promising tools for cancer gene therapy. They should provide an improved therapeutic index with efficient tumor transduction and effective protection of normal tissue.

Radioprotection of Lung and Esophagus by Overexpression of the Human Manganese Superoxide Dismutase Transgene

Protection of normal tissue from radiation-induced damage has been demonstrated in the murine lung and esophagus using human manganese superoxide dismutase (MnSOD) plasmid/liposome complex (PL). C57BL/6J mice were injected intratracheally with MnSOD-PL, LacZ-PL, or metallothionein (MT2)-PL and irradiated 24 hours later at 2,000 cGy to the pulmonary cavity. Mice injected with MnSOD-PL had significantly increased survival compared with control, LacZ-PL, or MT2-PL irradiated mice. In an esophagitis model, male C3H/HeJ mice were injected intraesophageally with either MnSOD-PL or LacZ-PL and irradiated 24 hours later at 3,500 cGy to the pulmonary cavity. Mice injected with MnSOD-PL had significantly increased survival after irradiation at 3,500 cGy compared with control or LacZ-PL injected mice. However, orthotopic 3LL lung tumors in C57BL/6J mice were not protected from radiation following injection of MnSOD-PL, as seen by increased survival of mice with these tumors following irradiation. MnSOD-PL gene therapy protected normal lung and esophagus but not 3LL orthotopic lung tumors from radiation-induced damage.

BNP7787, a novel protector against platinum-related toxicities, does not affect the efficacy of cisplatin or carboplatin in human tumour xenografts

BNP7787 (2′,2′-dithio-bis-ethane sulphonate sodium), a water-soluble disulphide, is chemically and mechanistically different from other sulphur-containing chemoprotective agents. Presently, BNP7787 is under investigation for its protective properties with regard to the side-effects of platinum compounds. In this study, we evaluated BNP7787, mesna and amifostine for their effects on the antitumour activity of platinum compounds. Continuous exposure to BNP7787 did not affect the antiproliferative effects of cisplatin or carboplatin, but the efficacy of both compounds was reduced in the presence of mesna in vitro in two human ovarian cancer cell lines. BNP7787 or amifostine combined with cisplatin or carboplatin given in standard schedules for the treatment of nude mice bearing well-established OVCAR-3 xenografts did not interfere with platinum-induced inhibition of tumour growth. Of interest, BNP7787 or amifostine co-administered with carboplatin was significantly more effective than carboplatin alone (P<0.01). In the presence of amifostine, doses of cisplatin and carboplatin could be safely increased by factors of 1.6 and 1.5, respectively. Unlike in a previous study of BNP7787 in tumour-bearing rats, BNP7787 did not protect against additional weight loss following treatment with higher doses of cisplatin in OVCAR-3-bearing mice. Pharmacokinetics of (mixed) disulphides including BNP7787 and extractable mesna in deproteinised plasma revealed a rapid disappearance of BNP7787 and an AUC5-60 value of mesna 9-fold lower than that calculated after an equivalent dose of mesna by weight. We can conclude that BNP7787 does not interfere with the antitumour activity of platinum compounds in vitro and in vivo. Clinical trials are underway to evaluate the protection of normal tissues by BNP7787 when combined with cisplatin.

Nitroxides as Radiation Protectors

Selective protection of normal tissues from the damaging effects of ionizing radiation is an important objective in cancer treatment research. Likewise, radioprotective agents may be useful in protecting the human population in the event of radiation-related accidents or military conflicts. Over the past decade, we have identified stable nitroxide compounds as a unique class of antioxidants with demonstrated radioprotective properties. We have shown that nitroxides at nontoxic concentrations are effective as in vitro and in vivo antioxidants when oxidation is induced by superoxide, hydrogen peroxide, organic hydroperoxides, ionizing radiation, or specific DNA-damaging anticancer agents. Studies have shown that nitroxides protect against radiation-induced DNA damage, chromosome aberrations, mutation induction, cell killing, and lethality in mice that receive whole-body irradiation. Whether these agents provide radioprotection for low-level radiation doses remains to be determined.

Hypofractionated and accelerated radiotherapy with cytoprotection (HypoARC): a short, safe, and effective postoperative regimen for high-risk breast cancer patients

Purpose: Although the role of radiotherapy (RT) after mastectomy in reducing the local relapse rate is well established, its impact on overall survival is strongly questioned. Up to 70% of patients will not benefit from additional RT, and a “wait and see” policy is often adopted. Establishment of short, still safe, and effective RT regimens would render adjunctive radiotherapy more appealing. We evaluated the toxicity and efficacy of a Hypofractionated and intensively Accelerated RT regimen supported with amifostine Cytoprotection (HypoARC) in a cohort of 72 high-risk breast cancer patients treated with modified mastectomy or conservative surgery and FEC (5-fluorouracil/epirubicin/cyclophosphamide) chemotherapy. Patients and Methods: A high dose of amifostine, 1,000 mg, was given as a 5-min i.v. infusion before each of the 12 consecutive fractions of RT (4 × 3.5 Gy/fraction and 8 × 4 Gy/fraction, 1 fraction/day, 5 fractions/week). The breast or chest wall, as well as supraclavicular and axillary area, was included in the RT fields. The follow-up of patients ranged from 18 to 42 months (median, 28 months). Alkaline phosphatase (AF) expression was assessed immunohistochemically in normal and cancerous breast tissues. Results: Ninety-two percent of patients successfully completed the regimen, the only side effects being mild nausea and asthenia. In 7% of patients, amifostine was interrupted because of a rash/fever reaction. A dramatic reduction in acute skin toxicity was noted ( p < 0.0001). Acute pneumonitis, as well as late toxicity in breast, chest wall, axillary, and lung tissue, was lower with the HypoARC regimen, although not significantly, than with the standard fractionation regimen used to treat two matched control cohorts. Both HypoARC and standard RT significantly reduce the local relapse rate ( p < 0.0001), although the local relapse-free and overall survival times were marginally better for the HypoARC group of patients ( p > 0.09). AF showed a mixed nuclear/cytoplasmic pattern of expression in the epithelial, endothelial, and stromal component of the normal breast and benign lesions, whereas an impressive loss of AF expression was noted in in situ and invasive breast cancer and tumoral stroma. Conclusions: The HypoARC regimen is convenient for both patients and radiotherapy departments. The regimen is well tolerated and shows a significantly better profile in terms of early toxicity; a reduced rate of late sequel may be expected. The local relapse rate is as low as that expected from conventional RT. The absence of AF expression in cancer cells and tumoral stroma is probably a major reason for the selective protection of normal breast tissue by amifostine.

Gene therapy of cancer.

Gene therapy was initially thought of as a means to correct single gene defects in hereditary disease. In the meantime, cancer has become by far the most important indication for gene therapy in clinical trials. In the foreseeable future, the best way to achieve reasonable intratumoral concentrations of a transgene with available vectors is direct intratumoral injection with or without the aid of various techniques such as endoscopy or CT-guidance. At present, viral and non-viral methods of gene transfer are used either in vivo or ex vivo/in vitro. The most important viral vectors currently in use in clinical trials comprise retroviruses, adenoviruses, adeno-associated viruses, and herpes viruses. None of the available vectors satisfies all the criteria of an ideal gene therapeutic system, and vectors with only minimal residues of their parent viruses ("gutless vectors") as well as completely "synthetic viral vectors" will gain more and more importance in the future. Non-viral gene therapy methods include liposomes, injection of vector-free DNA ("naked DNA"), protein-DNA complexes, delivery by "gene gun," calcium-phosphate precipitation, electroporation, and intracellular microinjection of DNA. The first clinical trial of gene therapy for cancer was performed in 1991 in patients with melanoma, and since then more than 5000 patients have been treated worldwide in more than 400 clinical protocols. With the exception of a case of fatal toxicity in a young man with hereditary liver disease treated intrahepatically with high doses of adenovirus, side effects have been rare and usually mild in all these studies and expression of the transgene could be demonstrated in patients in vivo. However, despite anecdotal reports of therapeutic responses in some patients, unequivocal proof of clinical efficacy is still lacking for most of the varied approaches to gene therapy in humans. As well as our only fragmentary understanding of the molecular pathophysiology of many diseases, the principal reason for the present lack of clinical success of gene therapy is the very low transduction and expression efficiency in vivo of available vectors. Despite the complexities of gene therapy for cancer, the numerous different approaches can be subdivided into three basic concepts: (1) strengthening of the immune response against a tumour, (2) repair of cell cycle defects caused by losses of tumour suppressor genes or inappropriate activation of oncogenes, and (3) suicide gene strategies. In addition, the importance of gene marker studies and gene therapeutic protection of normal tissue are briefly covered in this review.

Gene therapy of cancer.

Gene therapy was initially thought of as a means to correct single gene defects in hereditary disease. In the meantime, cancer has become by far the most important indication for gene therapy in clinical trials. In the foreseeable future, the best way to achieve reasonable intratumoral concentrations of a transgene with available vectors is direct intratumoral injection with or without the aid of various techniques such as endoscopy or CT-guidance. At present, viral and non-viral methods of gene transfer are used either in vivo or ex vivo/in vitro. The most important viral vectors currently in use in clinical trials comprise retroviruses, adenoviruses, adeno-associated viruses, and herpes viruses. None of the available vectors satisfies all the criteria of an ideal gene therapeutic system, and vectors with only minimal residues of their parent viruses ("gutless vectors") as well as completely "synthetic viral vectors" will gain more and more importance in the future. Non-viral gene therapy methods include liposomes, injection of vector-free DNA ("naked DNA"), protein-DNA complexes, delivery by "gene gun," calcium-phosphate precipitation, electroporation, and intracellular microinjection of DNA. The first clinical trial of gene therapy for cancer was performed in 1991 in patients with melanoma, and since then more than 5000 patients have been treated worldwide in more than 400 clinical protocols. With the exception of a case of fatal toxicity in a young man with hereditary liver disease treated intrahepatically with high doses of adenovirus, side effects have been rare and usually mild in all these studies and expression of the transgene could be demonstrated in patients in vivo. However, despite anecdotal reports of therapeutic responses in some patients, unequivocal proof of clinical efficacy is still lacking for most of the varied approaches to gene therapy in humans. As well as our only fragmentary understanding of the molecular pathophysiology of many diseases, the principal reason for the present lack of clinical success of gene therapy is the very low transduction and expression efficiency in vivo of available vectors. Despite the complexities of gene therapy for cancer, the numerous different approaches can be subdivided into three basic concepts: (1) strengthening of the immune response against a tumour, (2) repair of cell cycle defects caused by losses of tumour suppressor genes or inappropriate activation of oncogenes, and (3) suicide gene strategies. In addition, the importance of gene marker studies and gene therapeutic protection of normal tissue are briefly covered in this review.

Terapêutica citoprotetora em pacientes tratados com quimio e/ou radioterapia anti neoplásica

In recent years, cytoprotective agents have been developed to protect normal cells from the toxic effects of chemotherapy and radiotherapy. The ideal cytoprotectant is that which is able to allow intensification of chemotherapy; protects a broad spectrum of normal tissues and organs when used with a variety of chemotherapeutic agents; confers specific protection for normal tissues; preserves anti tumour activity and has little or manageable toxicity of its own. A cytoprotectant is administered prior to cytotoxic therapy, in contrast to the colony stimulant factors and Leucovorin, which are administered after chemotherapy to rescue the bone marrow and stimulate haematological recovery. Currently there are three cytoprotectors: two chemotherapy-specific (Dexrazoxane and Mesna) and one broad-spectrum (Amifostine). The authors discuss the main properties and usefulness of these drugs in Oncohematology.

Acute effects of whole-body proton irradiation on the immune system of the mouse.

The acute effects of proton whole-body irradiation on the distribution and function of leukocyte populations in the spleen and blood were examined and compared to the effects of photons derived from a (60)Co gamma-ray source. Adult female C57BL/6 mice were exposed to a single dose (3 Gy at 0.4 Gy/min) of protons at spread-out Bragg peak (SOBP), protons at the distal entry (E) region, or gamma rays and killed humanely at six different times thereafter. Specific differences were noted in the results, thereby suggesting that the kinetics of the response may be variable. However, the lack of significant differences in most assays at most times suggests that the RBE for both entry and peak regions of the Bragg curve was essentially 1.0 under the conditions of this study. The greatest immunodepression was observed at 4 days postexposure. Flow cytometry and mitogenic stimulation analyses of the spleen and peripheral blood demonstrated that lymphocyte populations differ in radiosensitivity, with B (CD19(+)) cells being most sensitive, T (CD3(+)) cells being moderately sensitive, and natural killer (NK1.1(+)) cells being most resistant. B lymphocytes showed the most rapid recovery. Comparison of the T-lymphocyte subsets showed that CD4(+) T helper/inducer cells were more radiosensitive than the CD8(+) T cytotoxic/suppressor cells. These findings should have an impact on future studies designed to maximize protection of normal tissue during and after proton-radiation exposure.

Tumor dose enhancement and normal tissue protection using magnetic field modulated photon beams in radiation therapy

Tumor dose enhancement and normal tissue protection using magnetic field modulated photon beams in radiation therapy

Gene therapy of cancer.

Gene therapy was initially thought of as a means to correct single gene defects in hereditary disease. Since then, cancer has become the most important indication for gene therapy in clinical trials. In the foreseeable future, the best way to achieve reasonable intratumoral concentrations of a transgene with available vectors will be direct intratumoral injection with or without the help of various techniques such as endoscopy or computed tomography guidance. At present, viral and nonviral methods of gene transfer are used either in vivo or ex vivo/in vitro. The most important viral vectors currently used in clinical trials are retroviruses, adenoviruses, adeno-associated viruses and herpes viruses. However, none of them satisfies all the criteria of an ideal gene therapeutic system, and vectors with only minimal residues of their parent viruses (gutless vectors) and completely synthetic viral vectors are gaining importance. Nonviral methods of gene therapy include liposomes, injection of vector-free (naked) DNA, protein-DNA complexes, delivery by gene gun, calcium-phosphate precipitation, electroporation and intracellular microinjection of DNA. The first clinical trial of human gene therapy was performed in 1990 and since then more than 5000 patients have been treated worldwide in over 400 clinical protocols. Side effects were rare and mostly mild in all of these studies and expression of the transgene was demonstrated in patients in vivo. Despite anecdotal reports of therapeutic responses in some patients, there is still no unequivocal proof of clinical efficacy of most approaches to gene therapy in cancer, primarily due to very low transduction and expression efficacy in vivo of available vectors. Strategies for gene therapy of cancer can be subdivided into four basic concepts: 1) strengthening of the immune response against a tumor, 2) repair of cell cycle defects caused by loss of tumor suppressor genes or inappropriate activation of oncogenes, 3) suicide gene strategies and 4) inhibition of tumor angiogenesis. Gene marker studies and gene protection of normal tissue are also discussed.

Lewis blood group and CEA related antigens; coexpressed cell-cell adhesion molecules with roles in the biological progression and dissemination of tumours.

The Lewis blood group and carcinoembryonic antigen (CEA) related antigens have adhesive functions in human tissues, with roles in embryonic sorting and migration of cells (organogenesis), differentiation and protection of...

Discussion

Growing interest in the MRP (multidrug resistance protein) gene stems from its importance in multidrug resistance to chemotherapy, its possible use in gene therapy, and its relationship with the glutathione system. The recent generation of mrp gene knockout models in vitro and in vivo is providing information on the mechanism of action and the physiological function(s) of mrp. The importance of mrp in protection of normal tissues from the toxicity of the anticancer agent etoposide has been established. A total block of mrp has been found to be compatible with life, suggesting that MRP inhibitors can be safely used for treating cancer patients. In some sub-classes of leukocytes, mrp contributes to the transport of leukotriene C4, an endogenous glutathione-S-conjugate. However, the baseline expression of mrp does not appear to contribute to the export of glutathione-S-conjugates of alkylating agents, and thus does not exert a protective role against their toxicity. Besides being capable of exporting certain glutathione-S-conjugates, mrp also catalyzes the co-transport of GSH and drug and, presumably, a presently unknown endogenous metabolite(s).

Improving enzymes for cancer gene therapy.

New techniques now make it feasible to tailor enzymes for cancer gene therapy. Novel enzymes with desired properties can be created and selected from vast libraries of mutants containing random substitutions within catalytic domains. In this review, we first consider genes for the ablation of tumors, namely, genes that have been mutated (or potentially can be mutated) to afford enhanced activation of prodrugs and increased sensitization of tumors to specific chemotherapeutic agents. We then consider genes that have been mutated to provide better protection of normal host tissues, such as bone marrow, against the toxicity of specific chemotherapeutic agents. Expression of the mutant enzyme could render sensitive tissues, such as bone marrow, more resistant to specific cytotoxic agents.

Gene therapy of viral hepatitis and hepatocellular carcinoma.

Gene therapy represents an attractive approach to treat a great variety of diseases, both inherited and acquired, and it is moving slowly from a proof-of-principle phase to a wide application in most medical fields. Liver cancer and viral hepatitis are natural targets for this new therapeutic alternative due to the lack of success of conventional antitumoral and antiviral treatments and the ominous prognosis related with liver tumours. Gene therapy for viral hepatitis is aimed to boost the patient immune response against viral antigens or to make cells resistant to infection by blocking the viral life cycle. Gene transfer techniques applied to the treatment of hepatocellular carcinoma include drug sensitization by suicide genes, genetic immunotherapy, normal tissue protection by transfer of the multidrug resistance gene, replacement of tumour suppressor genes, inhibition of oncogenes and modifications of the biology of the tumour (antiangiogenesis). However, major advances in our understanding of the regulation of gene expression, design of the expression cassettes and development of more efficient gene transfer vectors are mandatory before gene therapy can become a widely used therapeutic modality.

Chemoprotectants: a review of their clinical pharmacology and therapeutic efficacy.

Dose-limiting toxicity secondary to antineoplastic chemotherapy is due to the inability of cytotoxic drugs to differentiate between normal and malignant cells. The consequences of this may include impairment of patient quality of life, because of toxicity, and reduced tumour control because of the inability to deliver adequate dose-intensive therapy against the cancer. Specific examples of toxicity against normal tissues include cisplatin-related neurotoxicity and nephrotoxicity, myelotoxicity secondary to treatment with alkylating agents and carboplatin, oxazaphosphorine-induced haemorrhagic cystitis, and cumulative dose-related cardiac toxicity secondary to anthracycline treatment. Chemoprotectants have been developed as a means of ameliorating the toxicity associated with cytotoxic agents by providing site-specific protection for normal tissues, without compromising antitumour efficacy. Clinical trials with toxicity protectors must include sufficient dose-limiting events for study, and assessment of both toxicity (allowing for measurement of efficacy of protection) and antitumour effect. Several chemoprotective compounds have now been extensively investigated, including dexrazoxane, amifostine, glutathione, mesna and ORG 2766. Dexrazoxane appears to complex with metal co-factors including iron, to reduce the incidence of anthracycline-induced cardiotoxicity, allowing the delivery of higher cumulative doses of anthracyclines without the expected consequence of cardiomyopathy. Numerous studies have demonstrated that sulfur-containing nucleophiles, including amifostine, glutathione, and mesna can specifically bind cisplatin- or alkylating agent-generated free radicals or alkylating agent metabolites to reduce the incidence of cisplatin-associated neurotoxicity and nephrotoxicity, or alkylating agent-associated myelosuppression and urothelial toxicity. These studies, in the majority of instances, have not revealed any evidence of reduction in antitumour efficacy. Further randomised trials are required to identify the optimal role of chemoprotectants when used alone or in combination with other toxicity modifiers including haemopoietic growth factors.

Chemoprotective gene transfer II: multilineage in vivo protection of haemopoiesis against the effects of an antitumour agent by expression of a mutant human O6-alkylguanine-DNA alkyltransferase.

Murine bone marrow cells were transduced ex vivo with a retrovirus encoding an O6-benzylguanine (O6-beG) insensitive, double mutant form of the human DNA repair protein O6-alkylguanine-DNA alkyltransferase (hATPA/GA). In animals reconstituted with the transduced bone marrow, about 50% of cells in the multipotent spleen colony-forming cells (CFU-S) and lineage restricted granulocyte-macrophage (GM-CFC) haemopoietic progenitor populations were found to be carrying the transgene and this correlated with the frequency of bone marrow cells and spleen colonies which stained positive for hATPA/GA by immunocyto-chemistry. Expression of hATPA/GA was associated with significant in vivo protection of both CFU-S (P = 0.001) and GM-CFC (P < 0.024) against the toxicity of the antitumour methylating agent, temozolomide, given in combination with O6-beG. Expression of hATPA/GA also led to a reduction in the frequency of combined O6-beG/temozolomide-induced micronuclei seen in polychromatic erythrocytes (P < 0.003). This study is the first to demonstrate in vivo protection of multipotent haemopoietic progenitors against the toxic and clastogenic effects of an O6-alkylating agent in the presence of O6-beG. It also represents the first report of reduced clastogenesis as a consequence of expression of an O6-beG-resistant ATase. In the accompanying article we report hATPA/GA-mediated resistance of human CD34+ haemopoietic progenitors to combined O6-beG/O6-alkylating agent toxicity. Together these two reports suggest that a gene therapy strategy whereby protection of normal haemopoietic tissue may be combined with O6-beG-mediated tumour sensitisation may be efficacious in achieving an increase in therapeutic index.