Martijn Schoester

MDR 1 expression is an independent prognostic factor for response and survival in de novo acute myeloid leukaemia

The Multidrug Resistance gene (MDR 1) is frequently expressed in acute myeloid leukaemia (AML). MDR 1 is associated with resistance to chemotherapy in vitro and with a poor response rate in AML. We have investigated the prognostic value of MDR 1 expression in relation to other patient characteristics with respect to response and survival.

Clinical modulation of multidrug resistance in multiple myeloma: effect of cyclosporine on resistant tumor cells.

PURPOSE In multiple myeloma (MM) refractory to doxorubicin (DXR) and/or vincristine (VCR), myeloma cells frequently express the multidrug resistance (MDR) phenotype, associated with overexpression of P-glycoprotein (Pgp), which acts as a drug efflux pump. Recently, studies have shown that clinical resistance can be modulated by drug resistance modifiers. The present study was performed to investigate if MDR modulation in vivo is caused by a direct effect of cyclosporine (CSA) on resistant myeloma plasma cells (PC). PATIENTS AND METHODS Eight patients with VAD-refractory MM were treated with DXR, VCR, and dexamethasone (VAD) plus CSA. Pgp expression in PC was determined by flow cytometry/immunocytochemistry before and after clinical treatment. Functional Pgp expression was determined by the effect of CSA on the intracellular accumulation of DXR and VCR. RESULTS Five of eight patients responded to VAD/CSA. The percentage of Pgp-positive (Pgp+) PC was 30% to 100% (median, 90%) before treatment and 4 to 90% (median, 40%) after treatment. CD56+/- or CD38+/- PC had identical Pgp expression. CSA, as well as SDZ PSC 833, but not dexamethasone, increased pretreatment intracellular accumulation of DXR and VCR in Pgp+ PC in three of four and six of six patients, respectively. After clinical treatment, in vitro drug accumulation in residual Pgp-negative (Pgp-) PC of four of four responding patients was not further modulated by CSA or SDZ PSC 833. At later relapse, PC of two of four patients remained Pgp-. CONCLUSION These data indicate that Pgp overexpression is functional in refractory myeloma and that clinical modulation of MDR by CSA is mediated through an inhibition of Pgp-associated drug efflux. Pgp-expressing PC can be eliminated by clinical treatment with VAD/CSA.

Reversal of typical multidrug resistance by cyclosporin and its non-immunosuppressive analogue SDZ PSC 833 in Chinese hamster ovary cells expressing the mdr1 phenotype

SummaryThe 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 <100 ng/ml at 82 and 820nm PSC. In the CHO-A3 cell line that was resistant to 200 ng colchicine/ml, the LC50 values decreased from >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.

Analysis of multidrug-resistance (MDR-1) glycoprotein and CD56 expression to separate monoclonal gammopathy from multiple myeloma

Summary Monoclonal gammopathy of undetermined significance (MGUS) is different from multiple myeloma (MM) by a low proliferation and by its indolent clinical course. In this study, two biological parameters were investigated which mark the transition from MGUS to MM, i.e. expression of the P‐170 glycoprotein associated with the multidrug resistance phenotype (MDR‐1) and expression of the natural killer cell antigen, CD56. Strong MDR‐1 expression was found in plasma cells of 32/38 untreated MGUS as compared with 33/105 untreated MM stage I–III (84%v 32%, P<0.001) and in 0/10 normal plasma cell samples. CD56 expression in high density was present in 43/57 analysed untreated MM but in none of 23 MGUS (78%v 0%P<0.0001). Plasma cells did characteristically show a low Ki‐67 proliferation index in 14/15 MGUS patients (mean 0.05%, range 0–0.2%) and a higher index in 25 analysed MM patients (mean 2.31%, range 1–7%, P<0.03).

Uptake of transcobalamin II-bound cobalamin by HL-60 cells: Effects of differentiation induction

Binding and uptake of transcobalamin II-bound cobalamin by HL-60 promyelocytic leukemia cells proceed through receptor-mediated endocytosis. The affinity constant of the receptor for transcobalamin II-cobalamin was found to be 6.1 liter/nmol and the maximal rate of uptake 12 pmol/10(9) cells/h. This uptake is mediated by about 3000 receptor sites per cell. Evidence is presented that the receptor recirculates from the cell surface to the lysosomes and vice versa. Upon differentiation induction of the cells by either DMSO in granulocytic direction or by 1,25-dihydroxy-vitamin D3 in monocytic direction a rapid decline in cellular uptake and cell surface binding of the protein-bound vitamin ensues. In particular the internalization of the complex decreases faster than all other observed signs of the ongoing differentiation process, such as reduction in the OKT9-reactive transferrin receptor, increase in lineage-specific surface markers, and decrease in [3H]thymidine incorporation and actual cell proliferation. The transcobalamin II receptor on the cell surface appears to be a proliferation-associated membrane component in human leukemic cells.

Efflux Kinetics and Intracellular Distribution of Daunorubicin Are Not Affected by Major Vault Protein/Lung Resistance-Related Protein (Vault) Expression

Vaults may contribute to multidrug resistance by transporting drugs away from their subcellular targets. To study the involvement of vaults in the extrusion of anthracyclines from the nucleus, we investigated the handling of daunorubicin by drug-sensitive and drug-resistant non-small lung cancer cells, including a green fluorescent protein (GFP)-tagged major vault protein (MVP)-overexpressing transfectant (SW1573/MVP-GFP). Cells were exposed to 1 μm daunorubicin for 60 min, after which the cells were allowed to efflux the accumulated drug. No significant differences in daunorubicin efflux kinetics were observed between the sensitive SW1573 and SW1573/MVP-GFP transfectant, whereas the drug-resistant SW1573/2R120 cells clearly demonstrated an increased efflux rate. It was noted that the redistribution of daunorubicin from the nucleus into distinct vesicular structures in the cytoplasm was not accompanied by changes in the intracellular localization of vaults. Similar experiments were performed using mouse embryonic fibroblasts derived from wild-type and MVP knockout mice, which were previously shown to be devoid of vault particles. Both cell lines showed comparable drug efflux rates, and the intracellular distribution of daunorubicin in time was identical. Reintroduction of a human MVP tagged with GFP in the MVP−/− cells results in the formation of vault particles but did not give rise an altered daunorubicin handling compared with MVP−/− cells expressing GFP. Our results indicate that vaults are not directly involved in the sequestration of anthracyclines in vesicles nor in their efflux from the nucleus.

Application of a simple immunoadsorption assay for the measurement of saturated and unsaturated transcobalamin II and R-binders

In this paper an immunoadsorption method for the selective measurement of the concentrations of saturated and unsaturated transcobalamin II and R-binders in human plasma is presented. With this assay the concentrations of saturated transcobalamin II found in the plasma of 70 normal individuals ranged from 20-220 pmol per litre, with a mean of 70 pmol/l. The concentration of R-binders, carrying cobalamin, ranged from 87-491 pmol/l (mean 195 pmol/l). The 33-203 pmol/l (mean 111 pmol/l) of cobalamin analogues were found to be almost exclusively bound to the R-binder fraction. The level of saturated binding proteins is not, or only marginally, influenced by the absorption of ingested cobalamin, even with an oral dose of 15 micrograms. The concentration of transcobalamin II-bound cobalamin is apparently not determined by the availability of unsaturated binding protein. On the contrary, a positive correlation was found between the R-binder-cobalamin concentration and total R-binder level. These observations suggest that the concentration of transcobalamin II-bound cobalamin is primarily determined by the concentration of cobalamin in the tissues.

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°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°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.

In vitro Ig‐synthesis and proliferative activity in multiple myeloma are stimulated by different growth factors

Plasma cells isolated from bone marrow (BM) aspirates of 15 patients with active multiple myeloma (MM) were cultured and analysed for in vitro proliferative response and Ig‐synthesis upon stimulation with interleukin‐3 (IL‐3), interleukin‐4 (IL‐4) and interleukin‐6 (IL‐6). The proliferative response, determined as Ki‐67 positivity in concentrated plasma cells, was increased by IL‐6 (Stimulation Index, SI=1.77±0.21 (M±SEM)) but not by IL‐3 or IL‐4. This proliferation could be blocked by anti‐IL‐6. In vitro Igsynthesis was stimulated by IL‐4 (SI = 1.62±0.12 (M±SEM) P<0.05) but not by IL‐6 or IL‐3. This effect was not antagonized by anti‐IL‐6. An inverse correlation was found in this group of patients between the IL‐6 induced stimulation of plasma cell proliferative activity and the IL‐4 induced increase of Ig‐synthesis (P=0.027).

Unimpaired dendritic cell functions in MVP/LRP knockout mice.

Dendritic cells (DCs) act as mobile sentinels of the immune system. By stimulating T lymphocytes, DCs are pivotal for the initiation of both T‐ and B‐cell‐mediated immune responses. Recently, ribonucleoprotein particles (vaults) were found to be involved in the development and/or function of human DCs. To further investigate the role of vaults in DCs, we examined the effects of disruption of the major vault protein (MVP/LRP) on the development and antigen‐presenting capacity of DCs, using our MVP/LRP knockout mouse model. Mononuclear bone marrow cells were isolated from wild‐type and knockout mice and stimulated to differentiate to DCs. Like human DCs, the wild‐type murine DC cultures strongly expressed MVP/LRP. Nevertheless, the MVP/LRP‐deficient DCs developed normally and showed similar expression levels of several DC surface markers. No differences were observed in in vitro studies on the antigen uptake and presenting capacities of the wild‐type and MVP/LRP knockout DCs. Moreover, immunization of the MVP/LRP‐deficient mice with several T‐cell antigens led to responses similar to those observed in the wild‐type mice, indicating that the in vivo DC migration and antigen‐presentation capacities are intact. Moreover, no differences were observed in the induction of the T cell‐dependent humoral responses and orally induced peripheral T‐cell tolerance. In conclusion, vaults are not required for primary DC functions. Their abundance in DCs may, however, still reflect basic roles in myeloid cell proliferation and DC development.