Microclaw Array Fabricated by Single Exposure of Femtosecond Airy Beam and Self-Assembly for Regulating Cell Migratory Plasticity.

The highly aligned extracellular matrix of metastatic breast cancer cells is considered to be the "highway" of cancer invasion, which strongly promotes the directional migration of cancer cells to break through the basement membrane. However, how the reorganized extracellular matrix regulates cancer cell migration remains unknown. Here, a single exposure of a femtosecond Airy beam followed by a capillary-assisted self-assembly process was used to fabricate a microclaw-array, which was used to mimic the highly oriented extracellular matrix of tumor cells and the pores in the matrix or basement membrane during cell invasion. Through the experiment, we found that metastatic breast cancer MDA-MB-231 cells and normal breast epithelial MCF-10A cells exhibit three major migration phenotypes on microclaw-array assembled with different lateral spacings: guidance, impasse, and penetration, whereas guided and penetrating migration are almost completely arrested in noninvasive MCF-7 cells. In addition, different mammary breast epithelial cells differ in their ability to spontaneously perceive and respond to the topology of the extracellular matrix at the subcellular and molecular levels, which ultimately affects the cell migratory phenotype and pathfinding. Altogether, we fabricated a microclaw-array as a flexible and high-throughput tool to mimic the extracellular matrix during invasion to study the migratory plasticity of cancer cells.

Brain targeted delivery of carmustine using chitosan coated nanoparticles via nasal route for glioblastoma treatment.

This study aims to develop chitosan-coated PLGA nanoparticles intended for nose-to-brain delivery of carmustine. Formulations were prepared by the double emulsion solvent evaporation method and optimized by using Box-Behnken Design. The optimized nanoparticles were obtained to satisfactory levels in terms of particle size, PDI, entrapment efficiency, and drug loading. In vitro drug release and ex-vivo permeation showed sustained release and enhanced permeability (approx. 2 fold) of carmustine compared to drug suspension. The AUC0-t of brain obtained with carmustine-loaded nanoparticles via nasal administration in Albino Wistar rats was 2.8 and 14.7 times that of intranasal carmustine suspension and intravenous carmustine, respectively. The MTT assay on U87 MG cell line showed a significant decrease (P < 0.05) in the IC50 value of the formulation (71.23 µg ml-1) as compared to drug suspension (90.02 µg ml-1).These findings suggest chitosan coated nanoparticles could be used to deliver carmustine via intranasal administration to treat Glioblastoma multiforme.

Targeting Inhibition of Notch1 Signaling Pathway on the Study of Human Gastric Cancer Stem Cells with Chemosensitization.

Background: Gastric cancer is the second most frequent cause of cancer death worldwide, although much geographical variation in incidence exists. Prevention and personalized treatment are regarded as the best options to reduce gastric cancer mortality rates (Hartgrink et al., 2009). Numerous studies have suggested that Notch1 and its ligands are overexpressed in gastric cancer, and its knockdown can inhibit the proliferation and survival of gastric cancer cells. Objective: To investigate the effect of Notch1 on the stemness and drug sensitivity of human gastric cancer SGC-7901 cells. Methods: Highly expressed Notch1 intracellular domain (NICD1) and Notch1-shRNA lentiviral expression vector were used to infect human gastric cancer SGC-7901 cells cultured in vitro, and western blot and immunofluorescence staining were used to identify highly expressed NICD and Notch1 silenced cells. The percentage of CD133+ cells was analyzed by flow cytometry, the expression of nestin and CFAP by immunofluorescence staining, the formation rate of tumor cell spheres and the tumorigenicity of SCID mice in vivo, and the regulation of cell stemness by Notch1. The sensitivity of each group of cells to the chemotherapeutic drugs teniposide (VM-26) and carmustine (BCNU) was also detected by the MTT method. Results: The stemness phenotype of tumor cells with the increased NICD expression was enhanced, such as an increased proportion of CD133+ cells, enhanced nestin expression, decreased GFAP expression, increased tumor cell sphere formation rate and tumorigenic rate of SCID mice implantation, and decreased sensitivity to VM-26 and BCNU. In contrast, the stemness phenotype of tumor cells with downregulated Notch1 gene expression was significantly suppressed, while the sensitivity to VM-26 and BCNU was increased. Conclusion: High Notch1 expression increased the stemness of SGC-7901 cells and decreased the sensitivity of SGC-7901 cells to chemotherapeutic drugs.

Covalent inhibition of P. falciparum ferredoxin-NADP+ reductase: Exploring alternative strategies for the development of new antimalarial drugs.

The protozoan Plasmodium falciparum is the main aetiological agent of tropical malaria. Characteristic of the phylum is the presence of a plastid-like organelle which hosts several homologs of plant proteins, including a ferredoxin (PfFd) and its NADPH-dependent reductase (PfFNR). The PfFNR/PfFd redox system is essential for the parasite, while mammals share no homologous proteins, making the enzyme an attractive target for novel and much needed antimalarial drugs. Based on previous findings, three chemically reactive residues important for PfFNR activity were identified: namely, the active-site Cys99, responsible for hydride transfer; Cys284, whose oxidation leads to an inactive dimeric form of the protein; and His286, which is involved in NADPH binding. These amino acid residues were probed by several residue-specific reagents and the two cysteines were shown to be promising targets for covalent inhibition. The quantitative and qualitative description of the reactivity of few compounds, including a repurposed drug, set the bases for the development of more potent and specific antimalarial leads.

Mathematical Modelling of Convection Enhanced Delivery of Carmustine and Paclitaxel for Brain Tumour Therapy.

PURPOSE: Convection enhanced delivery (CED) is a promising method of anticancer treatment to bypass the blood-brain barrier. This paper is aimed to study drug transport under different CED operating conditions. METHODS: The convection enhanced delivery of chemotherapeutics to an intact and a remnant brain tumour after resection is investigated by means of mathematical modelling of the key physical and physiological processes of drug transport. Realistic models of brain tumour and its holding tissue are reconstructed from magnetic resonance images. Mathematical modelling is performed for the delivery of carmustine and paclitaxel with different infusion rates, solution concentrations and locations of infusion site. RESULTS: Modelling predications show that drug penetration can be improved by raising the infusion rate and the infusion solution concentration. The delivery of carmustine with CED is highly localised. High drug concentration only can be achieved around the infusion site. The transport of paclitaxel is more sensitive to CED-enhanced interstitial fluid as compared to carmustine, with deeper penetration into tumour interior. Infusing paclitaxel in the upstream of interstitial fluid flow leads to high spatial averaged concentration and relatively uniform distribution. CONCLUSION: Results obtained in this study can be used to guide the design and optimisation of CED treatment regimens.

Solid lipid nanoparticles carrying chemotherapeutic drug across the blood-brain barrier through insulin receptor-mediated pathway.

Carmustine (BCNU)-loaded solid lipid nanoparticles (SLNs) were grafted with 83-14 monoclonal antibody (MAb) (83-14 MAb/BCNU-SLNs) and applied to the brain-targeting delivery. Human brain-microvascular endothelial cells (HBMECs) incubated with 83-14 MAb/BCNU-SLNs were stained to demonstrate the interaction between the nanocarriers and expressed insulin receptors (IRs). The results revealed that the particle size of 83-14 MAb/BCNU-SLNs decreased with an increasing weight percentage of Dynasan 114 (DYN). Storage at 4 °C for 6 weeks slightly deformed the colloidal morphology. In addition, poloxamer 407 on 83-14 MAb/BCNU-SLNs induced cytotoxicity to RAW264.7 cells and inhibited phagocytosis by RAW264.7 cells. An increase in the weight percentage of DYN from 0% to 67% slightly reduced the viability of RAW264.7 cells and promoted phagocytosis. Moreover, the transport ability of 83-14 MAb/BCNU-SLNs across the blood-brain barrier (BBB) in vitro enhanced with an increasing weight percentage of Tween 80. 83-14 MAb on MAb/BCNU-SLNs stimulated endocytosis by HBMECs via IRs and enhanced the permeability of BCNU across the BBB. 83-14 MAb/BCNU-SLNs can be a promising antitumor drug delivery system for transporting BCNU to the brain.

BCNU-sequestration by metallothioneins may contribute to resistance in a medulloblastoma cell line.

PURPOSE: Resistance of neoplastic cells to the alkylating drug BCNU [1,3-bis(2-chloroethyl)-1-nitrosourea] has been correlated with expression of O (6)-methylguanine-DNA methyltransferase, which repairs the O (6)-chloroethylguanine produced by the drug. Other possible mechanisms of resistance include raised levels of glutathione or increased repair of the DNA interstrand cross-links formed by BCNU. Transcriptional profiling revealed the upregulation of several metallothionein (MT) genes in a BCNU-resistant medulloblastoma cell line [D341 MED (OBR)] relative to its parental line. Previous studies have shown that MTs, through their reactive thiol groups can quench nitrogen mustard-derived alkylating drugs. In this report, we evaluate whether MTs can also quench BCNU. METHODS: To demonstrate the binding of BCNU to MT, we used an assay that measured the release of the MT-bound divalent cations (Zn(2+), Cd(2+)) upon their displacement by the drug. We also measured the decomposition rates of BCNU at those reaction conditions. RESULTS: The rate of release of the cations was higher in pH 7.4 than at pH 7.0, which is likely a result of more rapid decomposition of BCNU (thus faster release of MT-binding intermediate) at pH 7.4 than at pH 7.0. CONCLUSION: We demonstrate that resistance to BCNU may be a result of elevated levels of MTs which act by sequestering the drug's decomposition product(s).

Levels and distribution of BCNU in GBM tumors following intratumoral injection of DTI-015 (BCNU-ethanol).

The alkylation products formed by in vitro treatment of DNA with tritium-labeled 1,3-bis(2-chloroethyl)-1-nitrosourea ((3)H-BCNU) were identified and quantified. Twelve adducts were resolved by high-performance liquid chromatography (HPLC). The principal DNA adducts formed by BCNU treatment corresponded to N-7-(2-hydroxyethyl)guanine (N7-HOEtG) (26%), N-7-(2-chloroethyl)guanine (15%), and phosphotriesters (19%). In addition, several minor products were identified as 1,2-(diguan-7-yl)ethane, N-1-(2-hydroxyethyl)-2-deoxyguanosine, 1-(N-1-2-deoxyguanosinyl), 2-(N-3-2-deoxycytidyl)ethane cross-link, and O-6-(2-hydroxyethyl)-2-deoxyguanosine, and individually they represented 1% to 5% of the total alkylation. An HPLC-electrochemical method was applied to quantify the levels of N7-HOEtG in samples treated with BCNU. Treatment of either purified DNA or U87MG cells with various amounts of BCNU produced a linear increase in the amount of N7-HOEtG. These results demonstrated that the levels of N7-HOEtG formed by BCNU treatment could be used as a molecular dosimeter of BCNU treatment dose. We measured the levels of N7-HOEtG in DNA isolated from tumor samples taken from four patients with GBM tumors following stereotactic intratumoral injection with DTI-015 (BCNU-ethanol). The level of N7-HOEtG in these samples ranged from 14.7 to 121.9 micromol N7-HOETG/mol DNA within 1 cm of the site of injection. As the distance from the site of injection increased, the levels of N7-HOEtG in tumor DNA decreased. In two of the samples, the levels of N7-HOEtG were 0.2 to 0.3 micromol N7-HOETG/mol DNA at 3.5 to 3.9 cm from the site of injection, demonstrating significant distribution of BCNU in the tumor. The levels of N7-HOEtG in these tumor samples corresponded to BCNU treatment concentrations of 0.02 to 43.0 mM. These studies demonstrate that stereotactic intratumoral injection of DTI-015 into human GBM tumors produces high concentrations of BCNU up to 2.5 cm from the site of injection in some of the tumors. These observations suggest that intratumoral injection of DTI-015 may be of benefit in the treatment of primary and recurrent GBM tumors.

Differential inhibition of cellular glutathione reductase activity by isocyanates generated from the antitumor prodrugs Cloretazine and BCNU.

The antitumor, DNA-alkylating agent 1,3-bis[2-chloroethyl]-2-nitrosourea (BCNU; Carmustine), which generates 2-chloroethyl isocyanate upon decomposition in situ, inhibits cellular glutathione reductase (GR; EC 1.8.1.7) activity by up to 90% at pharmacological doses. GR is susceptible to attack from exogenous electrophiles, particularly carbamoylation from alkyl isocyanates, rendering the enzyme unable to catalyze the reduction of oxidized glutathione. Evidence implicates inhibition of GR as a cause of the pulmonary toxicity often seen in high-dose BCNU-treated animals and human cancer patients. Herein we demonstrate that the prodrug Cloretazine (1,2-bis[methylsulfonyl]-1-[2-chloroethyl]-2-[(methylamino)carbonyl]hydrazine; VNP40101M), which yields methyl isocyanate and chloroethylating species upon activation, did not produce similar inhibition of cellular GR activity, despite BCNU and Cloretazine being equally potent inhibitors of purified human GR (IC(50) values of 55.5 microM and 54.6 microM, respectively). Human erythrocytes, following exposure to 50 microM BCNU for 1h at 37 degrees C, had an 84% decrease in GR activity, whereas 50 microM Cloretazine caused less than 1% inhibition under the same conditions. Similar results were found using L1210 murine leukemia cells. The disparity between these compounds remained when cells were lysed prior to drug exposure and were partially recapitulated using purified enzyme when 1mM reduced glutathione was included during the drug exposure. The superior antineoplastic potential of Cloretazine compared to BCNU in animal models could be attributed in part to the contribution of the methyl isocyanate, which is synergistic with the co-generated cytotoxic alkylating species, while at the same time unable to significantly inhibit cellular GR.

In vivo selection of MGMT(P140K) lentivirus-transduced human NOD/SCID repopulating cells without pretransplant irradiation conditioning.

Infusion of transduced hematopoietic stem cells into nonmyeloablated hosts results in ineffective in vivo levels of transduced cells. To increase the proportion of transduced cells in vivo, selection based on P140K O6-methylguanine-DNA-methyltransferase (MGMT[P140K]) gene transduction and O6-benzylguanine/1,3-bis(2-chloroethyl)-1-nitrosourea (BG/BCNU) treatment has been devised. In this study, we transduced human NOD/SCID repopulating cells (SRCs) with MGMT(P140K) using a lentiviral vector and infused them into BG/BCNU-conditioned NOD/SCID mice before rounds of BG/BCNU treatment as a model for in vivo selection. Engraftment was not observed until the second round of BG/BCNU treatment, at which time human cells emerged to compose up to 20% of the bone marrow. Furthermore, 99% of human CFCs derived from NOD/SCID mice were positive for provirus as measured by PCR, compared with 35% before transplant and 11% in untreated irradiation-preconditioned mice, demonstrating selection. Bone marrow showed BG-resistant O6-alkylguanine-DNA-alkyltransferase (AGT) activity, and CFUs were stained intensely for AGT protein, indicating high transgene expression. Real-time PCR estimates of the number of proviral insertions in individual CFUs ranged from 3 to 22. Selection resulted in expansion of one or more SRC clones containing similar numbers of proviral copies per mouse. To our knowledge, these results provide the first evidence of potent in vivo selection of MGMT(P140K) lentivirus-transduced human SRCs following BG/BCNU treatment.

Methylguanine methyltransferase-mediated in vivo selection and chemoprotection of allogeneic stem cells in a large-animal model.

Clinical application of gene therapy for genetic and malignant diseases has been limited by inefficient stem cell gene transfer. Here we studied in a clinically relevant canine model whether genetic chemoprotection mediated by a mutant of the DNA-repair enzyme methylguanine methyltransferase could circumvent this limitation. We hypothesized that genetic chemoprotection might also be used to enhance allogeneic stem cell transplantation, and thus we evaluated methylguanine methyltransferase-mediated chemoprotection in an allogeneic setting. We demonstrate that gene-modified allogeneic canine CD34+ cells can engraft even after low-dose total body irradiation conditioning. We also show that cytotoxic drug treatment produced a significant and sustained multilineage increase in gene-modified repopulating cells. Marking in granulocytes rose to levels of up to 98%, the highest in vivo marking reported to date to our knowledge in any large-animal or human study. Increases in transgene-expressing cells after in vivo selection provided protection from chemotherapy-induced myelosuppression, and proviral integration site analysis demonstrated the protection of multiple repopulating clones. Drug treatment also resulted in an increase in donor chimerism. These data demonstrate that durable, therapeutically relevant in vivo selection and chemoprotection of gene-modified cells can be achieved in a large-animal model and suggest that chemoprotection can also be used to enhance allogeneic stem cell transplantation.

Miscoding properties of 1,N6-ethanoadenine, a DNA adduct derived from reaction with the antitumor agent 1,3-bis(2-chloroethyl)-1-nitrosourea.

1,N(6)-Ethanoadenine (EA) is an exocyclic adduct formed from DNA reaction with the antitumor agent, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). To understand the role of this adduct in the mechanism of mutagenicity or carcinogenicity by BCNU, an oligonucleotide with a site-specific EA was synthesized using phosphoramidite chemistry. We now report the in vitro miscoding properties of EA in translesion DNA synthesis catalyzed by mammalian DNA polymerases (pols) alpha, beta, eta and iota. These data were also compared with those obtained for the structurally related exocyclic adduct, 1,N(6)-ethenoadenine (epsilonA). Using a primer extension assay, both pols alpha and beta were primarily blocked by EA or epsilonA with very minor extension. Pol eta, a member of the Y family of polymerases, was capable of catalyzing a significant amount of bypass across both adducts. Pol eta incorporated all four nucleotides opposite EA and epsilonA, but with differential preferences and mainly in an error-prone manner. Human pol iota, a paralog of human pol eta, was blocked by both adducts with a very small amount of synthesis past epsilonA. It incorporated C and, to a much lesser extent, T, opposite either adduct. In addition, the presence of an A adduct, e.g. epsilonA, could affect the specificity of pol iota toward the template T immediately 3' to the adduct. In conclusion, the four polymerases assayed on templates containing an EA or epsilonA showed differential bypass capacity and nucleotide incorporation specificity, with the two adducts not completely identical in influencing these properties. Although there was a measurable extent of error-free nucleotide incorporation, all these polymerases primarily misincorporated opposite EA, indicating that the adduct, similar to epsilonA, is a miscoding lesion.

Pharmacokinetics of the carmustine implant.

Controlled release delivery of carmustine from biodegradable polymer wafers was approved as an adjunct to surgical resection in the treatment of recurrent glioblastoma multiforme after it was shown in clinical trials to be well tolerated and effective. Given the localised nature of the drug in the brain tissue, no direct pharmacokinetic measurements have been made in humans after implantation of a carmustine wafer. However, drug distribution and clearance have been extensively studied in both rodent and non-human primate brains at various times after implantation. In addition, studies to characterise the degradation of the polymer matrix, the release kinetics of carmustine and the metabolic fate of the drug and polymer degradation products have been conducted both in vitro and in vivo. GLIADEL wafers have been shown to release carmustine in vivo over a period of approximately 5 days; when in continuous contact with interstitial fluid, wafers should degrade completely over a period of 6 to 8 weeks. Metabolic elimination studies of the polymer degradation products have demonstrated that sebacic acid monomers are excreted from the body in the form of expired CO(2), whereas 1,3-bis-(p-carboxyphenoxy)propane monomers are excreted primarily through the urine. Carmustine degradation products are also excreted primarily through the urine. Pharmacokinetic studies in animals and associated modelling have demonstrated the capability of this modality to produce high dose-delivery (millimolar concentrations) within millimetres of the polymer implant, with a limited penetration distance of carmustine from the site of delivery. The limited spread of drug is presumably due to the high transcapillary permeability of this lipophilic molecule. However, the presence of significant convective flows due to postsurgical oedema may augment the diffusive transport of drug in the hours immediately after wafer implantation, leading to a larger short-term spread of drug. Additionally, in non-human primates, the presence of significant doses in more distant regions of the brain (centimetres away from the implant) has been shown to persist over the course of a week. The drug in this region was presumed to be transported from the implant site by either cerebral blood flow or cerebrospinal fluid flow, suggesting that although drug is able to penetrate the blood-brain barrier at the site of delivery, it may re-enter within the confines of the brain tissue.

The polymorphic human glutathione transferase T1-1, the most efficient glutathione transferase in the denitrosation and inactivation of the anticancer drug 1,3-bis(2-chloroethyl)-1-nitrosourea.

A member of the Theta class of human glutathione transferases (GST T1-1) was found to display the greatest catalytic activity towards the cytostatic drug 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) of the GSTs studied. In this investigation (the most extensive to date), enzymes from four classes of the soluble human GSTs were heterologously expressed, purified, and kinetically characterized. From the 12 enzymes examined, only GST M2-2, GST M3-3 and GST T1-1 had significant activities with BCNU. This establishes that the activity is not a characteristic of a particular class of GSTs. Although GST M3-3 was previously reported to have the greatest activity with BCNU, the current investigation demonstrates that GST M2-2 is equally active and that GST T1-1 has an approximately 20-fold higher specific activity than either of the Mu class enzymes. A more rigorous kinetic analysis of GST T1-1 gave the following parameters with BCNU: a k(cat) of 0.035 +/-0.003s(-1) and a K(M) of 1.0 +/- 0.1mM. The finding that GST T1-1 has the highest activity towards BCNU is significant since GST T1-1 is expressed in the brain, a common target for BCNU treatment. Furthermore, the existence of a GST T1-1 null allele in up to 60% in some populations, may influence both the sensitivity of tumors to chemotherapy and the severity of adverse side-effects in patients treated with this agent.

Modulation of 1,3-bis-(2-chloroethyl)-1-nitrosourea resistance in human tumor cells using hammerhead ribozymes designed to degrade O6-methylguanine DNA methyltransferase mRNA.

O6-Methylguanine DNA Methyltransferase (MGMT) protects tumor cells from the cytotoxic effects of the DNA alkylating agent 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU). To improve the therapeutic index of BCNU, biochemical strategies to deplete MGMT activity have been developed. In the present study, a molecular strategy for modulating BCNU resistance was explored using hammerhead ribozymes (Rz) designed to degrade the long-lived MGMT mRNA. The ribozymes were designed against eight GUC sites within the MGMT mRNA. cDNAs of these ribozymes were cloned into an expression vector and then all eight vectors were pooled and stably transfected into HeLa cells. Several HeLa/Rz clones sensitive to a sublethal dose of BCNU were identified using a short-term cell proliferation assay. The ribozyme inserts were amplified from genomic DNA by polymerase chain reaction and sequenced in the BCNU-sensitive clones. The ribozyme inserts Rz161, 178, and 212, targeted against nucleotide 161, 178, and 212, respectively, in the MGMT mRNA, were found to be present in these clones. MGMT activity, Western, and Northern blot analyses revealed that two of the HeLa/Rz clones contained very low levels of MGMT activity, protein, and mRNA. Investigation of CpG methylation within the MGMT promoter indicated that the lack of MGMT expression in these HeLa/Rz clones was not likely due to methylation silencing of the MGMT gene. By colony formation, the cell killing induced by 100 microM BCNU was increased by 2 to 3 logs in the HeLa/Rz clones compared with wild-type HeLa cells.

Modulation of the antiproliferative activity of anticancer drugs in hematopoietic tumor cell lines by the poly(ADP-ribose) polymerase inhibitor 6(5H)-phenanthridinone.

Poly (ADP-ribose) polymerase (PARP) is involved in the cellular responses to genotoxic damage and its inhibition has been proposed as potentiating anticancer drug activity. Here, we evaluated the ability of the PARP inhibitor, 6(5H)-phenanthridinone, to modulate the antiproliferative activity of bleomycin, carmustin and doxorubicin in a murine (RDM4) and a human (U937) lymphoma cell lines. 6(5H)-phenanthridinone was shown to suppress PARP activity with the same potency in both cell lines. At 25 microM, this compound potentiated the activity of carmustin in RDM4 but not in U937 cells. In contrast, 6(5H)-phenanthridinone failed to affect the doxorubicin toxicity in murine lymphoma cells, whereas it prevented the cytotoxicity of this drug in the human cell line. Altogether, these findings indicated that 6(5H)-phenanthridinone modulates the cytotoxicity of anticancer agents differently according to the cell type and the drug. Therefore, this PARP inhibitor could be considered as the prototype of a new class of adjuncts in cancer chemotherapy.

Direct demonstration in synthetic oligonucleotides that N,N'-bis(2-chloroethyl)-nitrosourea cross links N1 of deoxyguanosine to N3 of deoxycytidine on opposite strands of duplex DNA.

The sequence specificity and covalent structure of the lesion caused by the DNA interstrand cross-linking reaction of N,N'-bis(2-chloroethyl)-nitrosourea (BCNU) were investigated using synthetic oligonucleotides. The efficiency of interstrand cross-linking was found to parallel the efficiency of monoadduct formation, preferring deoxyguanosine-deoxycytidine-rich duplexes and, particularly, runs of deoxyguanosine. No explicit sequence specificity was observed. Enzymatic digestion of purified, interstrand cross-linked DNA returned primarily the unmodified deoxynucleosides, along with 1-[N3-deoxycytidyl]-2-[N1-deoxyguanosyl]ethane. This substance was characterized by comparison of its mass spectrum, high-pressure liquid chromatography retention time, and UV spectrum to an authentic standard prepared by chemical synthesis. These studies provide the first direct evidence that BCNU has no strong sequence preference for interstrand cross-linking and that substance 4, which has been previously isolated from BCNU-treated DNA, derives from alkylation on opposite strands of DNA. The lack of sequence preference and lesion structure together suggest that one source of BCNU interstrand cross-links is linkage of deoxyguanosine and deoxycytidine partners from a single bp.

DNA interstrand cross-linking and cytotoxicity induced by chloroethylnitrosoureas and cisplatin in human glioma cell lines which vary in cellular concentration of O6-alkylguanine-DNA alkyltransferase.

Fifteen human glioma cell lines were examined for their sensitivity to 1,3-bis(chloroethyl)-nitrosourea (BCNU, carmustine) and cis-dichlorodiamminoplatinum (cisplatin), the induction of DNA interstrand cross-linking (DNA-ISC) induced by the two agents and cellular O6-alkylguanine alkyltransferase (ATase) activity. Cell lines differed in their sensitivities to BCNU by up to 12-fold and to cisplatin by up to 21-fold. For both drugs, the extent of DNA-ISC was related to the drug sensitivity. There was a wide range of cellular ATase levels. Increasing ATase levels correlated with increased resistance to BCNU and with decreased formation of DNA-ISC following treatment with BCNU. In contrast, following treatment with cisplatin, there was no correlation between cellular ATase content and cytotoxicity or between ATase and DNA-ISC. Four sublines of varying ATase activity were prepared from one of the cell lines. These sublines showed a sensitivity to BCNU in inverse proportion to ATase activity, while sensitivity to cisplatin was more uniform. The experiments confirm the direct relationship between ATase concentration and sensitivity to BCNU in glioma cells. Although there was some correlation between cisplatin cytotoxicity and BCNU cytotoxicity, this was not mediated through ATase.

Formulation of BCNU-loaded microspheres: influence of drug stability and solubility on the design of the microencapsulation procedure.

The design of a microencapsulation procedure for the preparation of biodegradable BCNU-loaded microspheres used as intracerebral implants is the aim of this work. This approach will give sustained high local concentrations of the anti-tumour drug in the brain without the associated significant systemic toxicity. The microencapsulation technique used is a solvent-evaporation process based on the formation of an oil-in-water emulsion. The stability of BCNU in methylene chloride saturated with water and its high value of partition coefficient between methylene chloride and water justifies the selection of this organic solvent as the dispersed phase in the methodology. A spectrophotometric method for the quantification of BCNU in mixtures containing PLAGA is developed which allows the evaluation of drug photodecomposition. The volume of methylene chloride and the concentration of PVA in the external aqueous phase are the two variables that induce the largest variations of the microsphere size. The two main process parameters leading to the highest microencapsulation yield are the polymer concentration in the organic phase and the volume of the external aqueous phase; whereas the pH of the external aqueous phase and the use of co-solvents in the organic phase lead only to a small improvement in microsphere payload.