A potential strategy to increase the efficacy of topotecan to treat central nervous system (CNS) malignancies is modulation of the activity of ATP-binding cassette (ABC) transporters at the blood-brain and blood-cerebrospinal fluid barriers to enhance topotecan CNS penetration. This study focused on topotecan penetration into the brain extracellular fluid (ECF) and ventricular cerebrospinal fluid (CSF) in a mouse model and the effect of modulation of ABC transporters at the bloodbrain and blood-cerebrospinal fluid barriers by a tyrosine kinase inhibitor (gefitinib). After 4 and 8 mg/kg topotecan i.v., the brain ECF to plasma AUC ratio of unbound topotecan lactone was 0.21 F 0.04 and 0.61 F 0.16, respectively; the ventricular CSF to plasma AUC ratio was 1.18 F 0.10 and 1.30 F 0.13, respectively. To study the effect of gefitinib on topotecan CNS penetration, 200 mg/kg gefitinib was administered orally 1 hour before 4 mg/kg topotecan i.v. The brain ECF to plasma AUC ratio of unbound topotecan lactone increased by 1.6-fold to 0.35 F 0.04, which was significantly different from the ratio without gefitinib (P < 0.05). The ventricular CSF to plasma AUC ratio significantly decreased to 0.98 F 0.05 (P < 0.05). Breast cancer resistance protein 1 (Bcrp1), an efficient topotecan transporter, was detected at the apical aspect of the choroid plexus in FVB mice. In conclusion, topotecan brain ECF penetration was lower compared with ventricular CSF penetration. Gefitinib increased topotecan brain ECF penetration but decreased the ventricular CSF penetration. These results are consistent with the possibility that expression of Bcrp1 and P-glycoprotein at the apical side of the choroid plexus facilitates an influx transport mechanism across the blood-cerebrospinal fluid barrier, resulting in high topotecan CSF penetration. (Cancer Res 2006; 66(23): 11305-13)

Topotecan is a substrate of the ATP-binding cassette transporters P-glycoprotein (P-gp/MDR1) and breast cancer resistance protein (BCRP). To define the role of these transporters in topotecan penetration into the ventricular cerebrospinal fluid (vCSF) and brain parenchymal extracellular fluid (ECF) compartments, we performed intracerebral microdialysis on transporter-deficient mice after an intravenous dose of topotecan (4 mg/kg). vCSF penetration of unbound topotecan lactone was measured as the ratio of vCSF-to-plasma area under the concentration-time curves. The mean F SD ratios for wild-type, Mdr1a/b À/À , Bcrp1

In these studies we show that introduction of a normal human chromosome 6 or 6q can suppress the immortal phenotype of simian virus 40-transformed human fibroblasts (SV/HF). Normal human fibroblasts have a limited life span in culture. Immortal clones of SV/HF displayed nonrandom rearrangements in chromosome 6. Single human chromosomes present in mouse/human monochromosomal hybrids were introduced into SV/HF via microcell fusion and maintained by selection for a dominant selectable marker gpt, previously integrated into the human chromosome. Clones of SV/HF cells bearing chromosome 6 displayed limited potential for cell division and morphological characteristics of senescent cells. The loss of chromosome 6 from the suppressed clones correlated with the reappearance of immortal clones. Introduced chromosome 6 in the senescing cells was distinguished from those of parental cells by the analysis for DNA sequences specific for the donor chromosome. Our results further show that suppression of immortal phenotype in SV/HF is specific to chromosome 6. Introduction of individual human chromosomes 2, 8, or 19 did not impart cellular senescence in SV/HF. In addition, introduction of chromosome 6 into human glioblastoma cells did not lead to senescence. Based upon these results we propose that at least one of the genes (SEN6) for cellular senescence in human fibroblasts is present on the long arm of chromosome 6.

A method to rapidly measure dopamine (DA), dihydroxyindolphenylacetic acid, homovanillic acid, serotonin (5-HT) and 5-hydroxyindoleacetic acid concentrations in cerebrospinal fluid (CSF) has not yet been reported. A rapid, sensitive, and specific HPLC method was therefore developed using electrochemical detection. CSF was mixed with an antioxidant solution prior to freezing to prevent neurotransmitter degradation. Separation of the five analytes was obtained on an ESA MD-150 x 3.2 mm column with a flow rate of 0.37 mL/min and an acetonitrile-aqueous (5 : 95, v/v) mobile phase with 75 mM monobasic sodium phosphate buffer, 0.5 mM EDTA, 0.81 mM sodium octylsulfonate and 5% tetrahydrofuran. The optimal electrical potential settings were: guard cell +325 mV, E1 -100 mV and E2 +300 mV. Within-day and between-day precisions were <10% for all analytes and accuracies ranged from 91.0 to 106.7%. DA, 5-HT, and their metabolites were stable in CSF with antioxidant solution at 4 degrees C for 8 h in the autoinjector. This method was used to measure neurotransmitters in CSF obtained from children enrolled on an institutional medulloblastoma treatment protocol.

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Normal cells show a limited lifespan in culture and the phenotype of cellular senescence. Tumors and tumor cell lines have typically overcome this form of growth suppression and grow continuously as immortal cell lines in culture. We have exploited the DNA virus SV40 to study the mechanism by which human ®broblasts overcome senescence and become immortal. Multiple steps have now been identi®ed, including inactivation of cellular growth suppressors through direct interaction with SV40 large T antigen and through mutation of a gene on chromosome 6 (designated SEN6). In this study, we sublocalize the site of SEN6 to 6q26-27 based on molecular genetic analysis. Twelve SV40-immortalized ®broblast cell lines share a deletion in this area based on assessment for loss of heterozygostiy (LOH) for seven informative markers on 6q. Two immortal cell lines (AR5 and HALneo) appeared to have retained separate single copies of chromosome 6 despite the fact that they are both derived from the same preimmortal SV40-transformant and should share the same mutated allele of SEN6 (Hubbard-Smith et al., 1992). Detailed analysis by polymerase chain reaction, restriction fragment length polymorphism and¯uorescence in situ hybridization shows, however, that although they di er for 17 markers from the centromere to 6q26, they share AR5 derived sequences (eight markers) distal to 6q26 including the minimal deletion region, further supporting the assignment of SEN6 to this region. Since human tumors including non-Hodgkins lymphoma, mammary carcinoma and ovarian carcinoma show LOH in 6q26-27, inactivation of SEN6 may be responsible for immortalization of these tumors as well.