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Erasmus Schneider

Publications and source records attributed to Erasmus Schneider.

10 recordsLinked to original sources
PubMed2006

Gamma-glutamyl hydrolase and drug resistance.

Gamma-glutamyl hydrolase (GGH) is a lysosomal enzyme involved in the metabolism of folates and anti-folates. It acts as an endo- and/or exo-peptidase to cleave gamma-polyglutamate chains that are attached to folates and anti-folates after they enter a mammalian cell. Whereas the addition of multiple glutamates is necessary to enable the cell to retain folates and anti-folates, hydrolysis of the polyglutamate tails by GGH has the opposite effect of making (anti)-folates exportable again. Thus, GGH plays an important role in the cellular homeostasis of folate. Furthermore, high levels of GGH have been associated with cellular resistance to anti-folates, in particular methotrexate. Consequently, GGH also has pharmacological importance. In addition to the intracellular GGH, carboxypeptidase II (also called intestinal folate conjugase, prostate specific membrane antigen or N-acetyl-alpha-linked acidic dipeptidase) is another enzyme with gamma-glutamyl hydrolase activity; it resides, however, in the cellular membrane. Although genetically and biochemically distinct, this enzyme too appears to play a major role in folate homeostasis, by cleaving polyglutamates from extracellular folate-polyglutamates, so that they can be imported into the cell. Finally, there have been reports suggesting that gamma-glutamyl hydrolase plays a role as a tumor marker in breast and lung cancer.

Animals↗
PubMed2005

Increased lysosomal uptake of methotrexate-polyglutamates in two methotrexate-resistant cell lines with distinct mechanisms of resistance.

Methotrexate (MTX) resistance in mitoxantrone-selected MCF7/MX cells and in MTX-selected CEM/MTX cells is associated with reduced drug accumulation, albeit caused by different mechanisms. In addition, in both resistant cell lines the proportion of active long-chain MTX-polyglutamate (MTX-PG) metabolites is reduced relative to that in the respective parental cell line. Previous studies by others have implied that increased lysosomal uptake could affect the rate of MTX-PG hydrolysis, and hence the length distribution of the polyglutamate chains. However, in the two cell line pairs studied, the number of lysosomes per cell was not different between the corresponding parental and resistant cells. Instead, we observed a two- to three-fold increased facilitative uptake of MTX-Glu4 by the lysosomes from these two independently derived MTX-resistant cell lines, compared to uptake by lysosomes from their corresponding parental cells. Enhanced lysosomal uptake of MTX-Glu4 was reflected in an increased maximal uptake velocity, without a change in the apparent substrate affinity. In addition, the rate of MTX efflux from lysosomes from CEM/MTX cells was two-fold faster than from lysosomes from CEM cells. Consistent with this observation, the relative amount of short-chain MTX-Glu(1+2) species, as a fraction of the total amount of all MTX-Glu(1-4) species combined, was only half as large in lysosomes from CEM/MTX cells as in lysosomes from CEM cells. Together, these results suggest the possibility that increased lysosomal uptake, and hence enhanced sequestration of MTX-PGs in resistant cells, contributes to the development of high-level MTX resistance by decreasing the cytosolic levels of MTX-PGs.

Biological Transport↗
PubMed2005

Lack of an effect of breast cancer resistance protein (BCRP/ABCG2) overexpression on methotrexate polyglutamate export and folate accumulation in a human breast cancer cell line.

Accumulation of methotrexate (MTX) and its polyglutamates (PGs) has been recognized as an important factor in MTX efficacy. We have previously described a multidrug-resistant human breast cancer cell line, MCF7/MX, that exhibits reduced accumulation of total MTX as well as MTX-PGs, and that is resistant to continuous MTX exposure [Volk EL, Rohde K, Rhee M, McGuire JJ, Doyle LA, Ross DD, et al. Methotrexate cross-resistance in a mitoxantrone selected multidrug-resistant MCF7 breast cancer cell line is due to enhanced energy-dependent drug efflux. Cancer Res 2000;60:3514-21]. These cells express high levels of the breast cancer resistance protein (BCRP/ABCG2) that has been shown to actively transport MTX and short-chain MTX-PGs in vitro. However, the effect of BCRP on MTX-PG accumulation in intact cells was unclear. Here, we show that MTX transport by BCRP is required for the observed lower levels of MTX-PGs in the resistant cells. When BCRP was inhibited with fumitremorgin C, or in cells expressing a mutated form of BCRP that is unable to transport MTX, MTX-PG accumulation was similar or even higher than that in the parental cells that do not express BCRP. Concomitantly, there was increased inhibition of thymidylate synthase. It had previously been suggested that BCRP-mediated efflux of MTX-PGs contributed to the reduced MTX-PG accumulation. However, we found no evidence of BCRP-mediated efflux of MTX-PGs from intact cells, suggesting that direct efflux of MTX-PGs does not play a major role in MTX resistance. Together, these data show that BCRP overexpression can cause a reduction in total MTX accumulation as well as a reduction in the proportion of long-chain MTX-PGs. In contrast, BCRP overexpression did not affect natural folate accumulation or the relative distribution of folylpolyglutamates in the resistant, as compared to the parental, cells. Thus, it appears that BCRP overexpression affects the metabolism of the antifolate MTX, but not that of natural folates, although indirect effects cannot be excluded.

ATP Binding Cassette Transporter, Subfamily G, Mem↗
PubMed2005

Developing a sustainable process to provide quality control materials for genetic testing.

PURPOSE: To provide a summary of the outcomes of two working conferences organized by the Centers for Disease Control and Prevention (CDC), to develop recommendations for practical, sustainable mechanisms to make quality control (QC) materials available to the genetic testing community. METHODS: Participants were selected to include experts in genetic testing and molecular diagnostics from professional organizations, government agencies, industry, laboratories, academic institutions, cell repositories, and proficiency testing (PT)/external Quality Assessment (EQA) programs. Current efforts to develop QC materials for genetic tests were reviewed; key issues and areas of need were identified; and workgroups were formed to address each area of need and to formulate recommendations and next steps. RESULTS: Recommendations were developed toward establishing a sustainable process to improve the availability of appropriate QC materials for genetic testing, with an emphasis on molecular genetic testing as an initial step. CONCLUSIONS: Improving the availability of appropriate QC materials is of critical importance for assuring the quality of genetic testing, enhancing performance evaluation and PT/EQA programs, and facilitating new test development. To meet the needs of the rapidly expanding capacity of genetic testing in clinical and public health settings, a comprehensive, coordinated program should be developed. A Genetic Testing Quality Control Materials Program has therefore been established by CDC in March 2005 to serve these needs.

Centers for Disease Control and Prevention, U.S.↗
PubMed2003

Wild-type breast cancer resistance protein (BCRP/ABCG2) is a methotrexate polyglutamate transporter.

The existence of an ATP-dependent methotrexate (MTX) efflux mechanism has long been postulated; however, until recently, the molecular components were largely unknown. We have previously demonstrated a role for the ATP-binding cassette transporter breast cancer resistance protein (BCRP) in MTX resistance (Volk et al., Cancer Res., 62: 5035-5040, 2002). Resistance to this antifolate directly correlated with BCRP expression, and was reversible by the BCRP inhibitors fumitremorgin C and GF120918. Here, we provide evidence for BCRP as a MTX-transporter using an in vitro membrane vesicle system. Inside-out membrane vesicles were generated from both drug-selected and stably transfected cell lines expressing either wild-type (Arg482) or mutant (Gly482) variants of BCRP. In the presence of the wild-type variant of BCRP, transport of MTX into vesicles was ATP-dependent, osmotically sensitive, and inhibited by fumitremorgin C. In contrast, no transport was observed in vesicles containing the mutant form of BCRP. Wild-type BCRP appeared to have low affinity, but high capacity, for the transport of MTX, with an estimated K(m) of 680 micro M and a V(max) of 2400 pmol/mg/min. MTX accumulation was greatly decreased by mitoxantrone, a known BCRP substrate, suggesting competition for transport. Furthermore, and in contrast to the multidrug resistance-associated proteins, BCRP also transported significant amounts of polyglutamylated MTX. Although transport gradually decreased as the polyglutamate chain length increased, both MTX-Glu(2) and MTX-Glu(3) were substrates for BCRP. Together, these data demonstrate that BCRP is a MTX and MTX-polyglutamate transporter and reveal a possible mechanism by which it confers resistance.

ATP Binding Cassette Transporter, Subfamily G, Mem↗
PubMed2003

Reactive oxygen species mediate the down-regulation of mitochondrial transcripts and proteins by tumour necrosis factor-alpha in L929 cells.

In this study, we show that reactive oxygen species production induced by tumour necrosis factor alpha (TNF-alpha) in L929 cells was associated with a decrease in the steady-state mRNA levels of the mitochondrial transcript ATPase 6-8. Simultaneously, the transcript levels of two nuclear-encoded glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphofructokinase, were increased. These changes were associated with decreased protein levels of the ATPase subunit a (encoded by the mitochondrial ATPase 6 gene) and cytochrome c oxidase subunit II, and increased protein levels of phosphofructokinase. Since TNF-alpha had no effect on the amount of mitochondrial DNA, the results suggested that TNF-alpha acted at the transcriptional and/or post-transcriptional level. Reactive oxygen species scavengers, such as butylated hydroxianisole and butylated hydroxytoluene, blocked the production of free radicals, prevented the down-regulation of ATPase 6-8 transcripts, preserved the protein levels of ATPase subunit a and cytochrome c oxidase subunit II, and attenuated the cytotoxic response to TNF-alpha, indicating a direct link between these two phenomena.

Animals↗
PubMed2002

Overexpression of wild-type breast cancer resistance protein mediates methotrexate resistance.

Previously, we have reported that a multidrug-resistant, mitoxantrone (MX)-selected cell line, MCF7/MX, is highly cross-resistant to the antifolate methotrexate (MTX), because of enhanced ATP-dependent drug efflux (E. L. Volk et al., Cancer Res., 60: 3514-3521, 2000). These cells overexpress the breast cancer resistance protein (BCRP), and resistance to MTX as well as to MX was reversible by the BCRP inhibitor, GF120918. These data indicated that BCRP causes the multidrug-resistance phenotype. To further examine the role of this transporter in MTX resistance, and in particular the role of amino acid 482, we analyzed a number of BCRP-overexpressing cell lines. MTX resistance correlated with BCRP expression in all of the cell lines expressing the wild-type transporter, which contains an Arg at position 482. In contrast, little or no cross-resistance was found in the MCF7/AdVp1000 and S1-M1-3.2 and S1-M1-80 cell lines, which contain acquired mutations at this position, R482T and R482G, respectively. Concomitantly, the greatest reduction in MTX accumulation was observed in the MCF7/MX cells (BCRP(Arg)) as compared with cells expressing the Thr and Gly BCRP variants. Furthermore, the reduction in drug accumulation was sensitive to BCRP inhibition by GF120918. In conclusion, we have demonstrated a novel role for BCRP as a mediator of MTX resistance and have provided further evidence for the importance of amino acid 482 in substrate specificity.

ATP Binding Cassette Transporter, Subfamily G, Mem↗
PubMed2002

Validation of sixteen leukemia and lymphoma cell lines as controls for molecular gene rearrangement assays.

BACKGROUND: Assays for rearrangement of the immunoglobulin, T-cell receptor, bcr/abl, and bcl-2 genes are valuable tools to aid in the diagnosis of leukemias and lymphomas and are now offered by many pathology laboratories. However, there is a lack of well-characterized and validated calibrators and positive controls for these assays. We therefore evaluated 16 readily available leukemia and lymphoma cell lines for their potential use as controls. METHODS: DNA and RNA were isolated from each cell line and analyzed by Southern blot and PCR or reverse transcription-PCR (RT-PCR). Rearrangements in the IgJ(H), IgJ(kappa), TcR-beta or TcR-gamma, bcr/abl, and bcl-2 genes were detected by commercially available probes and primers. Cell lineages were confirmed by immunophenotyping. RESULTS: Immunoglobulin and T-cell receptor gene rearrangements were identified in five B- and three T-cell lines, respectively. Two cell lines tested positive for the bcr/abl gene, and one was positive for the bcl-2 gene rearrangement by Southern blot. CONCLUSIONS: The 16 cell lines studied can be used as positive controls in molecular detection assays for gene rearrangements. The parallel processing of these cell lines with clinical samples can serve to quality control the experimental procedures from the first step of DNA or RNA extraction to the final step of result analysis.

B-Lymphocytes↗
PubMedDate not supplied

Effective knock down of very high ABCG2 expression by a hammerhead ribozyme.

BACKGROUND: Ribozymes are an effective tool to reduce the mRNA levels of specific target genes. Overexpression of the drug transport protein, ABCG2, has been associated with multidrug resistance in cancer cells. MATERIALS AND METHODS: An expression plasmid encoding a hammerhead ribozyme against the ABCG2 gene was stably transfected into multidrug-resistant MCF7/MX cells that express very high levels of the ABCG2 protein. The effect of the ribozyme was determined by quantitative real-time RT-PCR, Western blot and cytotoxicity assays. RESULTS: The ribozyme reduced ABCG2 mRNA levels to less than 10% of control values, which resulted in the concomitant reduction of ABCG2 protein levels and sensitization of the cells to mitoxantrone and methotrexate. CONCLUSION: The ribozyme used was highly effective in reducing the expression of its target gene, ABCG2, and was able to modulate the associated multidrug-resistant phenotype.

ATP Binding Cassette Transporter, Subfamily G, Mem↗