From stones to bones: the biology of ClC chloride channels.

Chloride (Cl(-)) is the most abundant extracellular anion in multicellular organisms. Passive movement of Cl(-) through membrane ion channels enables several cellular and physiological processes including transepithelial salt transport, electrical excitability, cell volume regulation and acidification of internal and external compartments. One family of proteins mediating Cl(-) permeability, the ClC channels, has emerged as important for all of these biological processes. The importance of ClC channels has in part been realized through studies of inherited human diseases and genetically engineered mice that display a wide range of phenotypes from kidney stones to petrified bones. These recent findings have demonstrated many eclectic functions of ClC channels and have placed Cl(-) channels in the physiological limelight.

A reverse genetic analysis of components of the Toll signaling pathway in Caenorhabditis elegans.

BACKGROUND: Both animals and plants respond rapidly to pathogens by inducing the expression of defense-related genes. Whether such an inducible system of innate immunity is present in the model nematode Caenorhabditis elegans is currently an open question. Among conserved signaling pathways important for innate immunity, the Toll pathway is the best characterized. In Drosophila, this pathway also has an essential developmental role. C. elegans possesses structural homologs of components of this pathway, and this observation raises the possibility that a Toll pathway might also function in nematodes to trigger defense mechanisms or to control development. RESULTS: We have generated and characterized deletion mutants for four genes supposed to function in a nematode Toll signaling pathway. These genes are tol-1, trf-1, pik-1, and ikb-1 and are homologous to the Drosophila melanogaster Toll, dTraf, pelle, and cactus genes, respectively. Of these four genes, only tol-1 is required for nematode development. None of them are important for the resistance of C. elegans to a number of pathogens. On the other hand, C. elegans is capable of distinguishing different bacterial species and has a tendency to avoid certain pathogens, including Serratia marcescens. The tol-1 mutants are defective in their avoidance of pathogenic S. marcescens, although other chemosensory behaviors are wild type. CONCLUSIONS: In C. elegans, tol-1 is important for development and pathogen recognition, as is Toll in Drosophila, but remarkably for the latter rôle, it functions in the context of a behavioral mechanism that keeps worms away from potential danger.

211At-methylene blue for targeted radiotherapy of human melanoma xenografts: treatment of cutaneous tumors and lymph node metastases.

The next stage of our preclinical investigations of targeted radiotherapy for melanoma with 3,7-(dimethylamino)phenazathionium chloride [methylene blue (MTB)] labeled with 211At (alpha particle emitter) concerns the treatment of cutaneous tumors and their metastases. Fragments of two human melanoma xenografts, highly pigmented HX118 and poorly pigmented HX34, implanted s.c. into nude mice, were treated with four doses of 211At-MTB injected i.v. The growth rate of cutaneous tumors and the appearance and size of their lymph node metastases served as criteria of treatment effectiveness. 211At-MTB inhibited the growth of cutaneous tumors in a manner dependent on their pigmentation and initial size. Highly pigmented smaller melanomas were affected by 211At-MTB to a greater extent than poorly pigmented and larger ones. Growth of the smallest HX118 lesions investigated was completely inhibited for 65 days, whereas the growth inhibition of HX34 tumors of the same size lasted 7 days only. 211At-MTB exhibited similar pigmentation-dependent effects toward lymph node metastases. The size of metastatic lesions derived from HX118 xenografts never reached that in control animals during the period of observation, whereas those grown from HX34 xenografts attained control values after a 50-day delay. The results demonstrated the capacity of 211At-MTB to control the growth of cutaneous melanomas and their metastases.

211At-methylene blue for targeted radiotherapy of human melanoma xenografts: treatment of micrometastases.

Treatment of micrometastases of HX34 human melanoma grown as xenografts in nude mice represents an advanced stage of preclinical investigations concerning targeted radiotherapy of this neoplasm using 3,7-(dimethylamino)phenazathionium chloride [methylene blue (MTB) labeled with astatine-211 (211At) (alpha-particle emitter). The therapeutic effectiveness of 211At-MTB administered i.v. was determined by a lung colony assay combined with a search for metastases to organs other than the lungs. A single dose of 211At-MTB lowered the HX34 cell surviving fraction in lungs to below 10% almost independently of the time interval between cell inoculation and radioisotope injection and of 211At-MTB radioactivity within its investigated range. Radiation dose and the time of its administration did, however, influence the size of lung colonies. In contrast, the efficacy of 211At-MTB treatment as assessed by both surviving fraction and colony size was significantly dependent on a number of HX34 cells inoculated initially into mice. These results are explained by a short range of alpha-particles emitted by 211At and a mechanism of growth of lung colonies from tumor cells circulating with blood and blocking lung capillaries. Metastases in organs other than lungs and characteristic of control animals were not found in mice treated with 211At-MTB. The high therapeutic efficacy achieved proved that 211At-MTB is a very efficient scavenger of single melanoma cells distributed through blood and micrometastases with sizes below the limit of clinical detection.

Uptake and therapeutic effectiveness of 125I- and 211At-methylene blue for pigmented melanoma in an animal model system.

The investigations concerning a targeted radiotherapy for pigmented melanoma with a radiolabeled phenothiazine derivative, 3,7-(dimethylamino)phenazathionium chloride [methylene blue (MTB)], were continued using melanotic and amelanotic sublines of B16 melanoma. Two radionuclides, 125I and 211At, emitting Auger electrons and alpha particles, respectively, replaced 35S previously studied since their biological effectiveness is significantly higher. In vitro autoradiography revealed a selective accumulation of methylene blue labeled with either of the radioisotopes in pigmented melanoma cells but its absence in nonpigmented cells. Treatments with [125I]MTB and [211At]MTB were performed both in vitro and in vivo, with their effectiveness determined by lung clonogenic assay. [125I]MTB proved to be relatively ineffective when incorporated into melanosomes distributed in the cytoplasm, i.e., too far away from the genome. Conspicuous therapeutic effects were achieved with [211At]MTB for pigmented melanoma only. 211At itself did not affect either of the investigated sublines of B16 melanoma confirming once again the high affinity of methylene blue to melanin. Calculations of cumulative radiation doses from [211At]MTB deposited in melanotic melanoma tumors and pigmented normal organs which would be at a particular risk led to the conclusion that [211At]MTB could be used for a highly selective and very efficient targeted radiotherapy of pigmented melanomas without damaging normal tissues.

A transmembrane guanylyl cyclase (DAF-11) and Hsp90 (DAF-21) regulate a common set of chemosensory behaviors in caenorhabditis elegans.

Caenorhabditis elegans daf-11 and daf-21 mutants share defects in specific chemosensory responses mediated by several classes of sensory neurons, indicating that these two genes have closely related functions in an assortment of chemosensory pathways. We report that daf-11 encodes one of a large family of C. elegans transmembrane guanylyl cyclases (TM-GCs). The cyclic GMP analogue 8-bromo-cGMP rescues a sensory defect in both daf-11 and daf-21 mutants, supporting a role for DAF-11 guanylyl cyclase activity in this process and further suggesting that daf-21 acts at a similar step. daf-11::gfp fusions are expressed in five identified pairs of chemosensory neurons in a pattern consistent with most daf-11 mutant phenotypes. We also show that daf-21 encodes the heat-shock protein 90 (Hsp90), a chaperone with numerous specific protein targets. We show that the viable chemosensory-deficient daf-21 mutation is an unusual allele resulting from a single amino acid substitution and that the daf-21 null phenotype is early larval lethality. These results demonstrate that cGMP is a prominent second messenger in C. elegans chemosensory transduction and suggest a previously unknown role for Hsp90 in regulating cGMP levels.

[211At]methylene blue for targeted radiotherapy of human melanoma xenografts: dose fractionation in the treatment of cutaneous tumours.

3,7-(dimethylamino) phenazathionium chloride [methylene blue (MTB)] labelled with alpha-particle emitter astatine-211 (211At) selectively accumulates in melanoma cells due to an exceptionally high affinity of MTB to melanin, and proves to be a very effective agent in targeting radiotherapy for pigmented human melanoma grown in mice. This study aimed at a selection of the most advantageous [211At]MTB dose fractionation leading to irreversible regression of the treated lesions. Nude mice bearing subcutaneous human melanoma xenografts of either highly pigmented HX118 or poorly pigmented HX34 human melanoma were treated with [211At]MTB administered intravenously. The treatment was performed using three different schedules of [211At]MTB fractionation: a single large dose, five fractions delivered sequentially every 48 h and two to five fractions given with a mean frequency of one per week. The effectiveness of [211At]MTB treatment was assessed by determination of the growth rate of cutaneous tumours and length of time between tumour implantation and killing of moribund mice. [211At]MTB applied with a mean frequency of one fraction per week appeared to be the most efficient treatment for highly pigmented HX118 melanomas. Its effectiveness was dependent on [211At]MTB activity used per fraction and the size of the cutaneous tumours at the beginning of the treatment. A total dose of [211At]MTB seemed of less importance. An irreversible regression of the lesions was achieved. Poorly pigmented cutaneous melanoma xenografts were affected most significantly by [211At]MTB applied as five fractions given every 48 h. The treatment caused a temporary inhibition of tumour growth after which the lesions regained the control growth rate. These and previous results suggest that [211At]MTB could successfully control the growth of already formed lesions of pigmented melanoma, as well as prevent metastatic spread of the tumour, provided an appropriate fractionation régime of the radiolabelled compound is employed.

211At-methylene blue for targeted radiotherapy of disseminated melanoma: microscopic analysis of tumour versus normal tissue damage.

The present stage of our preclinical investigations of targeted radiotherapy for melanoma with 3,7-(dimethylamino)phenazathionium chloride [methylene blue (MTB)] labelled with astatine-211 (211At), an alpha-particle emitter, concerns toxicity of the treatment, as well as macro- and microscopic evaluation of its efficacy. Fragments of two human melanoma xenografts, pigmented HX118 and non-pigmented HX34 (used as a control), were implanted s.c. into nude mice subsequently treated with two doses of 211At-MTB injected i.v. Alterations in tumour growth rate were related to microscopic damage caused by 211At-MTB to the lesions, as determined by light microscopy using histopathological techniques. 211At-MTB-dependent growth inhibition of pigmented melanoma occurred either instantly or as a gradual reduction in the tumour growth rate. At a later stage, lesions that ceased to grow immediately consisted of quiescent, heavily pigmented tumour cells, as well as advanced fibrosis, and were extensively infiltrated by melanin-laden phagocytes. Large, unresorbed and often calcified necrotic deposits characterised the tumours responding gradually to the treatment. 211At-MTB remained non-toxic in normal organs. Only a relative number of small lymphocytes in the groin lymph nodes in a minority of animals was temporarily reduced, most often in conjunction with the treatment of pigmented tumours. The data demonstrated a high therapeutic effectiveness of 211At-MTB towards pigmented melanoma at the expense of negligible injury to normal tissues, and revealed that the macroscopic determination of tumour growth rate often underestimated an efficacy of the applied treatment.

Low-doses of ionising radiation induce melanoma metastases and trigger the immune system--adrenal axis feedback loop.

Low-doses of ionising radiation are frequently implicated in triggering and/or accelerating the growth of skin and other malignancies. It seemed probable that the radiation at similar dose levels might initiate metastasis from already existing tumours. Highly pigmented human melanoma xenograft that had lost its ability for a spontaneous metastasising and grown subcutaneously in athymic mice was exposed to very low and well-defined doses of ionising radiation to determine whether low linear energy transfer radiation can restore metastatic potential of the tumour. To ensure that all effects derived from radiation-activated neoplastic cells only, I was delivered selectively to the cutaneous melanoma instead of using the external beam. The direct response of these tumours to radiation was monitored by determining the growth rate of the lesions. Histopathological methods were employed to detect metastases. The lowest radiation dose of approximately 6 cGy deposited in the tumours initiated metastatic spread in all animals. Gradual increase of the radiation doses diminished both the frequency of the appearance of metastases and their distance from the primary lesions. There were no metastases from non-irradiated melanomas. The highest dose used (60 cGy) did not affect significantly the growth of cutaneous (primary) tumours, but lower doses that enhanced inflammatory infiltration of the lesions reduced tumour growth. Such radiation-stimulated immune responses were accompanied by increased pigmentation in cutaneous lesions and activation of the adrenal cortex indicating that the immune system-adrenal axis feedback loop had been triggered. The results demonstrate that very low-doses of ionising radiation induce melanoma metastases. The phenomenon is accompanied by the stimulation of the immune system-adrenal axis feedback loop that regulates eicosanoid synthesis, thereby suggesting an involvement of these molecules in the process. Radiation doses approaching the therapeutic level do not initiate melanoma dissemination.

Therapeutic target discovery using Caenorhabditis elegans.

Use of the human genome sequence in disease therapy will require efficient identification of disease-causing and disease-associated genes with functions that are amenable to pharmacological manipulation. The validation and development of such genes as therapeutic targets requires information about both the genes' functions and the biochemical pathways in which they participate. One powerful means of obtaining such information is the study of homologous genes in model organisms amenable to laboratory manipulation. Among model organisms the nematode Caenorhabditis elegans offers several advantages, including well-established techniques for genetic and experimental manipulation and the first completed genome sequence for a multicellular organism. Molecular genetic experiments using C. elegans can contribute at several levels to drug discovery programs, from elucidation of genetic functions and pathways to the validation of candidate targets. Additionally, the ease of culture allows adaptation of the nematode for use in high-throughput chemical screens for the identification of lead compounds in drug development.

Radioiodinated methylene blue for melanoma targeting: chemical characterisation and tumour selectivity of labelled components.

Radioiodinated methylene blue contains a mixture of components showing selective uptake in human pigmented melanoma, and it has potential for imaging and therapy. Nuclear magnetic resonance and mass spectroscopic studies show that the majority of the radioactivity (85%) is in the form of monoiodinated methylene blue, 4-iodo-3-methylamino-7-dimethylaminophenaza thionium chloride. The amino group ortho-to iodine has become demethylated to a mono-methylamino group. The remainder (15%) of the mixture is the doubly labelled 4,5-diiodo-3,7-bis(methylamino) phenazathionium chloride. The separated components show similar tumour selectivity in athymic mice bearing human pigmented melanomas.

Targeting melanoma with 211At/131I-methylene blue: preclinical and clinical experience.

The increasing incidence of melanoma and a lack of effective therapy have stimulated a search for new methods of early detection and treatment of the disease. Melanin synthesized in melanoma cells presents a unique target to which the treatment can be selectively addressed, provided the pigment is recognized by a suitable drug. Methylene blue possesses a high affinity for melanin and, therefore, accumulates preferentially in melanoma cells. Since not directly toxic to the tumor, methylene blue serves as a carrier for radioisotopes and, once taken up by melanoma cells, acts as a selectively localized source of radiation. Hence, radioderivatives of the compound can be used for diagnosis and therapy of disseminated melanoma. 131I-methylene blue in conjunction with gamma camera imaging has already proved in clinical studies to be a useful tool for the detection of early melanoma dissemination. 211At-methylene blue exceptional efficacy in treating melanoma and preventing its metastatic spread without damaging normal structures when administered systemically to human melanoma-bearing mice led to the approval of this alpha-particle emitting methylene blue derivative for the Phase I clinical trial.

The mechanism of pH-dependent hydrogen peroxide cytotoxicity in vitro.

The present paper is concerned with the influence of hydrogen ion concentration and composition of the medium on clonogenic survival of epithelial cells exposed to hydrogen peroxide in vitro. The survival of cells incubated with H2O2 in phosphate-buffered saline at pH 6.5 was 1 x 10(-2) and increased abruptly to 9 x 10(-2) at pH 7.0. The pH dependence of the cytocidal effect was particularly conspicuous when Eagle's minimum essential medium (SFMEM) was used for cell exposure to H2O2: the survival was characterized by exponential pH dependence and varied from 1 x 10(-1) to 9 x 10(-1) for pH 6.5 and 7.5, respectively, with a superimposed sharp peak value of 9 x 10(-1) at pH 7.0. The enhanced pH dependence of the H2O2 cytotoxicity in SFMEM was found to result from the additive action of glucose and histidine present in this medium. Glucose alone protected the cells with the efficiency decreasing with increasing hydrogen ion concentration. Histidine was responsible for the intermediate maximum in the pH-dependent survival spectrum. In addition, the changes in cell survival were accompanied by pH-dependent release of GSSG from the exposed cells. The GSSG efflux was inhibited by glucose in the medium. The influence of glucose on both the pattern of cell survival and the associated GSSG release indicate that the glutathione peroxidase activity supported by the pentose phosphate pathway is crucial in cell protection against extracellular H2O2 toxicity.

Enzymic pathways involved in cell response to H2O2.

An influence of possible interaction of glutathione peroxidase and cyclooxygenase on the clonogenic survival of epithelial cells exposed in vitro to H2O2 was investigated. Indomethacin served as the inhibitor of cyclooxygenase, and the use of alkaline (7.5) or acidic (6.5) pH combined with controlled supply of glucose modified glutathione peroxidase activity. Indomethacin affected survival of cells exposed to H2O2 in a biphasic manner, enhancing cytotoxicity at lower hydrogen peroxide concentrations, and diminishing it at higher concentrations. The turning point moved gradually to higher concentrations of H2O2 corresponding to the augmented decomposition of hydrogen peroxide caused by increased activity of glutathione peroxidase. The data revealed that both enzymic pathways interact in the presence of H2O2, resulting in the overall cell survival different from that obtained after inhibition of either.

Role of hydrogen peroxide in the cytotoxicity of the xanthine/xanthine oxidase system.

1. The survival of mammalian epithelial cells exposed in vitro to the xanthine/xanthine oxidase system in phosphate-buffered saline (PBS) or serum-containing medium (SCMEM) was investigated. 2. The cytotoxic effect observed depended on the composition of the medium in which the enzymic reaction was carried out; a surviving fraction of 5 x 10(-5) was found for cells exposed in PBS and 5.2 x 10(-1) for those in SCMEM. 3. The cytotoxic product(s) formed by the xanthine/xanthine oxidase system was relatively stable in PBS; survival of cells incubated after completion of the enzymic reaction was always less than that found for cells exposed during the reaction in the same system. 4. Superoxide dismutase or mannitol present during the enzymic reaction did not inhibit the cytotoxic effect. 5. NaN3 (a single-oxygen quencher and a catalase inhibitor) added to the system in SCMEM caused a reduction in survival to the level observed for cells exposed to the enzymic reaction in PBS. 6. Catalase completely protected cells, but no protection was observed when both catalase and NaN3 were present in the reaction mixture. 7. A similar cytotoxic effect was produced when cells were treated with H2O2 alone. 8. The rate of H2O2 decomposition in medium was accelerated by the presence of serum, but this was completely inhibited by NaN3. 9. It is concluded that H2O2 is the major cytotoxic product formed by the xanthine/xanthine oxidase system.

211At-methylene blue in targeted radiotherapy of disseminated melanoma.

Targeted radiotherapy with 211At-methylene blue (211At-MTB) is a systemic treatment selectively directed at melanoma due to a high affinity of MTB to melanin synthesized in the tumor cells. Since MTB forms a strong complex with melanin, it is an effective carrier for a number of radioisotopes to be addressed to the tumor deposits of any size including individually dispersed melanoma cells. Thus, appropriately radiolabeled MTB can be used for either diagnosis or therapy of the neoplasm. As predicted and found in animal experiments, 211At-MTB is most effective therapeutically. Histopathological investigations showed that the highly pigmented 211At-MTB-treated tumors were characterized initially by perivascular oedema and hydropic degeneration of tumor cells followed by gradual development of extensive areas of coagulative necrosis. The necrotic tumor areas contained microvessels occluded by thrombi and tended to undergo microfocal calcification. Although melanoma-bearing animals successfully treated with 211At-MTB did not reveal any adverse effects of the therapy, detailed toxicological studies were undertaken. No serious macro- or microscopic lesions were observed in normal organs of 211At-MTB treated mice. Only the relative number of small lymphocytes in the groin lymph nodes in a minority of animals was variably reduced, most often in conjunction with the treatment of highly, but not poorly, pigmented tumors.

Targeting disseminated melanoma with radiolabelled methylene blue: Comparative bio-distribution studies in man and animals.

Targeted radiotherapy for pigmented melanoma with 3,7-(dimethylamino) phenazathionium chloride [methylene blue (MTB)] labelled with Astatine-211 (211At; alpha-particle emitter) proved to be very effective in animal model systems. Since the results justified an introduction of the treatment to the clinic, the aim of the bio-distribution studies using [123I]-MTB and [131I]-MTB in patients was to confirm selectiveness of radiolabelled MTB uptake in melanoma lesions. The investigations were carried out using planar and SPECT (single photon emission computed tomography) gamma-cameras. A stable uptake of radioiodinated MTB was found in pigmented melanomas in man, with tumour/surrounding tissue and tumour/blood ratios amounting to 9 at 19 h after a single i.v. injection. A time-dependent kinetics of radioiodinated MTB distribution was similar to that observed in human melanoma-bearing athymic mice. Blood radioactivity decreased by about 90% during the first 2.5 min after i.v. injection of the compound (T1/2biol = 0.58 min). Its retention time in various organs was either the same or very similar to that characteristic of the blood. A rapid uptake of radioiodinated MTB in the liver and kidneys confirmed the importance of these organs in excreting the compound: 25-30% of the radioactivity administered was expelled with urine over the first 24 h after the injection. There was no obvious retention of radioiodinated MTB in the brain over the observation period and in the eyes for at least the first 14 h.

Early detection of melanoma metastases with radioiodinated methylene blue.

Melanin synthesised in melanoma cells presents a unique target to which the treatment can be selectively addressed, provided the pigment is recognised by a suitable drug. Methylene blue (MTB) possesses a high affinity for melanin and, therefore, accumulates preferentially in melanoma cells. Since not directly toxic to the tumour, MTB serves as a carrier for radioisotopes and, once taken up by melanoma cells, acts as a selectively localised source of radiation. Hence, radioderivatives of the compound can be used for both diagnosis and therapy of disseminated melanoma. Eleven patients with confirmed metastatic melanoma and one with a recent local recurrence were studied using radioiodinated (iodine-123 or iodine-131) MTB and a gamma camera. Biopsies of cutaneous lesions were taken to determine directly the compound uptake in tumours. This first clinical investigation concerning the diagnostic potential of radioiodinated MTB in patients with disseminated melanoma confirmed the existence of approximately 80% of internal lesions previously identified by routine methods and, additionally, enabled detection of unknown secondaries in 6 of 12 patients studied. There were no false-positive gamma camera images regardless of whether 123I or 131I was used. 131I proved to be more suitable than 123I for detecting melanoma metastases with radioiodinated MTB. Hazy images of the lesions treated with external beam radiation and/or some drugs suggest that initial radio- and chemotherapy might affect MTB uptake in melanoma metastases and reduce the clarity of the scintigrams obtained from a gamma camera. However, small, untreated internal lesions that cannot be visualised easily with the standard diagnostic methods are revealed with 131I-MTB regardless of their localisation. It is concluded that use of radioiodinated MTB in conjunction with gamma camera or positron emission tomographic imaging might prove to be a useful and accessible tool for the detection of early melanoma dissemination.