24 research outputs found
Effect of nitrous oxide on folate coenzyme distribution and de novo synthesis of thymidylate in human bone marrow cells
Abstract
The effect of nitrous oxide on intracellular folate metabolism of the human bone marrow was studied in vitro. Bone marrow cells, obtained from healthy volunteers, were incubated with 5 Ă 10â8m-[3H]5-formyltetrahydrofolate (5-formylTHF) for 18 hr to label intracellular folate pools. Subsequently the cells were exposed to nitrous oxide for up to 10 hr, and the intracellular folate coenzyme levels were quantitated by HPLC. The dU suppression test was carried out on part of the bone marrow samples in order to measure folate-dependent synthesis of the DNA precursor thymidylate (dTMP). After 5 hr exposure to nitrous oxide the de novo dTMP synthesis of the bone marrow cells was significantly decreased (P < 0.05), and this reduced synthesis persisted at 10 hr. After both 5 and 10 hr of exposure to nitrous oxide the amount of 10-formylTHF was reduced (P < 0.05) while that of 5-methylTHF was increased (P < 0.05). At 10 hr the level of THF was also decreased (P < 0.05). This study shows that nitrous oxide exposure of human bone marrow cells causes a redistribution of the various folate coenzymes which supports the idea of âfunctional cobalamin deficiencyâ. Moreover it seems probable that following prolonged exposure to nitrous oxide, not only folate-dependent dTMP synthesis but also de novo purine synthesis is reduced
Reversal of typical multidrug resistance by cyclosporin and its non-immunosuppressive analogue SDZ PSC 833 in Chinese hamster ovary cells expressing the mdr1 phenotype
Summary
The new non-immunosuppressive cyclosporin derivative SDZ PSC 833 (PSC) is a potent agent used to overcome typical multidrug resistance (MDR) associated with overexpression of themdr1 gene encoding for a P-170 glycoprotein. In the present study, the efficacy of PSC as compared with cyclosporin was determined in Chinese hamster ovary cell lines exhibiting different levels of resistance to colchicine (0, 0.1, 0.2 and 10 ÎŒg/ml, respectively). Low concentrations of PSC (8.2nm) increased the cytotoxicity of colchicine in cell lines expressing low levels of drug resistance. The concentration resulting in 50% cell survival (LC50 value) found for colchicine alone or in combination with PSC in the CHO-A3 cell line that was resistant to 100 ng colchicine/ml decreased from >500 to 200 ng/ml at 8.2nm PSC and to 500 ng/ml for colchicine alone to 500 ng/ml for colchicine used in combination with 8.2nm PSC and to <100 ng/ml for colchicine combined with 82 or 820nm PSC. At a concentration of 82nm PSC, the maximal effect in MDR reversal was observed in the cell lines exhibiting moderate resistance. In the highly resistant cell line, PSC (820nm) also reversed colchicine resistance. In drug-accumulation experiments, we obtained a 4-fold increase in intracellular doxorubicin accumulation using 820nm PSC. A comparison of PSC with cyclosporin revealed that a cyclosporin concentration 20-fold that of PSC was required to obtain the same sensitising effect. On the basis of these data, it may be concluded that PSC is a most promising chemosensitiser
Anti-leukemic potential of methyl-cobalamin inactivation by nitrous oxide
Myeloâcytotoxicity of extended nitrous oxide (N2O) inhalation was described almost forty years ago and then incidentally applied already with temporary success for suppressing leukemia. In 1948 the accompanying megaloblastic maturation arrest was explained by inactivation of the methylcobalamin coenzyme and subsequent folate deficiency. We studied the antiâleukemic effect of N2O on a transplantable acute leukemia in B(rown) N(orway) rats. Progression of this B,N,M(yelocytic)L(eukemia) was measured as spleen and liver weights, and leukemic blood cell counts. The deoxyuridine (dU)âsuppression test provided in vitro indication of the functional folate activity of leukemic cells. Breathing of N2Oâoxygen considerably reduced but did not eradicate, BNMLâproliferation. Addition of antiâmetabolites, interfering with some enzyme in the folate metabolism beyond the methylcobalamin coâenzyme dependent methionine synthase step, acted at least synergistically. The antiâleukemic effect of cycloleucine, which reduces Sâadenosylâmethionine synthesis by inactivation of methionine adenosyltransferase, was moderate but became much stronger with N2O inhalation. Methotrexate, a potent antiâleukemic agent by inhibiting tetrahydrofolate (THF) generation through inactivation of diâHF reductase, became highly antiâBNML, even in low dosage when combined with or preceded by N2O. 5âFluorouracil, which inhibits methyleneâTHF dependent thymidilate synthase, itself was surprisingly antiâBNML, but also became much more potent with previous or concomittant N2O exposure. Preliminary dUâsuppression test results with human acute leukemia cells, exposed to N2O and/or folate antagonists in vitro, correlated well with the in vivo BNMLâexperiments. Combining the anticobalamin activity of N2O with an antiâfolate therefore seems to be a promising chemotherapeutic approach. Copyrigh
The formation of vault-tubes: a dynamic interaction between vaults and vault PARP
Vaults are barrel-shaped cytoplasmic ribonucleoprotein particles that are
composed of a major vault protein (MVP), two minor vault proteins
[telomerase-associated protein 1 (TEP1), vault poly(ADP-ribose) polymerase
(VPARP)] and small untranslated RNA molecules. Not all expressed TEP1 and
VPARP in cells is bound to vaults. TEP1 is known to associate with the
telomerase complex, whereas VPARP is also present in the nuclear matrix
and in cytoplasmic clusters (VPARP-rods). We examined the subcellular
localization and the dynamics of the vault complex in a non-small cell
lung cancer cell line expressing MVP tagged with green fluorescent
protein. Using quantitative fluorescence recovery after photobleaching
(FRAP) it was shown that vaults move temperature independently by
diffusion. However, incubation at room temperature (21 degrees C) resulted
in the formation of distinct tube-like structures in the cytoplasm.
Raising the temperature could reverse this process. When the vault-tubes
were formed, there were fewer or no VPARP-rods present in the cytoplasm,
suggesting an incorporation of the VPARP into the vault-tubes. MVP
molecules have to interact with each other via their coiled-coil domain in
order to form vault-tubes. Furthermore, the stability of microtubules
influenced the efficiency of vault-tube formation at 21 degrees C. The
dynamics and structure of the tubes were examined using confocal
microscopy. Our data indicate a direct and dynamic relationship between
vaults and VPARP, providing further clues to unravel the function of
vaults
Disruption of the murine major vault protein (MVP/LRP) gene does not induce hypersensitivity to cytostatics
Vaults are ribonucleoprotein particles with a distinct structure and a
high degree of conservation between species. Although no function has been
assigned to the complex yet, there is some evidence for a role of vaults
in multidrug resistance. To confirm a direct relation between vaults and
multidrug resistance, and to investigate other possible functions of
vaults, we have generated a major vault protein (MVP/lung
resistance-related protein) knockout mouse model. The MVP(-/-) mice are
viable, healthy, and show no obvious abnormalities. We investigated the
sensitivity of MVP(-/-) embryonic stem cells and bone marrow cells derived
from the MVP-deficient mice to various cytostatic agents with different
mechanisms of action. Neither the MVP(-/-) embryonic stem cells nor the
MVP(-/-) bone marrow cells showed an increased sensitivity to any of the
drugs examined, as compared with wild-type cells. Furthermore, the
activities of the ABC-transporters P-glycoprotein, multidrug
resistance-associated protein and breast cancer resistance protein were
unaltered on MVP deletion in these cells. In addition, MVP wild-type and
deficient mice were treated with the anthracycline doxorubicin. Both
groups of mice responded similarly to the doxorubicin treatment. Our
results suggest that MVP/vaults are not directly involved in the
resistance to cytostatic agents