25 research outputs found
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 μg/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
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
Selection and characterisation of a phage-displayed human antibody (Fab) reactive to the lung resistance-related major vault protein
The major vault protein is the main component on multimeric vault particles, that are likely to play an essential role in normal cell physiology and to be associated with multidrug resistance of tumour cells. In order to unravel the function of vaults and their putative contribution to multidrug resistance, specific antibodies are invaluable tools. Until now, only conventional major vault protein-reactive murine monoclonal antibodies have been generated, that are most suitable for immunohistochemical analyses. The phage display method allows for selection of human antibody fragments with potential use in clinical applications. Furthermore, cDNA sequences encoding selected antibody fragments are readily identified, facilitating various molecular targeting approaches. In order to obtain such human Fab fragments recognising major vault protein we used a large non-immunized human Fab fragment phage library. Phages displaying major vault protein-reactive Fabs were obtained through several rounds of selection on major vault protein-coated immunotubes and subsequent amplification in TG1 E coli bacteria. Eventually, one major vault protein-reactive clone was selected and further examined. The anti-major vault protein Fab was found suitable for immunohistochemical and Western blot analysis of tumour cell lines and human tissues. BIAcore analysis showed that the binding affinity of the major vault protein-reactive clone almost equalled that of the murine anti-major vault protein Mabs. The cDNA sequence of this human Fab may be exploited to generate an intrabody for major vault protein-knock out studies. Thus, this human Fab fragment should provide a valuable tool in elucidating the contribution(s) of major vault protein/vaults to normal physiology and cellular drug resistance mechanisms