Comparison of bone marrow extracellular matrices.

We have compared the structure and composition of adult and fetal bovine bone marrow extracellular matrices. In contrast to fetal bone marrow, adult bone marrow has more oval fenestration and accumulation of adipocytes as well as lower protein content. These differences could be due to remodeling of bone marrow tissue as it develops. Zymogram analysis of matrix metalloproteinase (MMP) and tissue inhibitor of MMP (TIMP) activities showed that fetal, but not adult bone marrow extract contained a 96-kDa MMP and TIMP-1 and -2. These activities may contribute to the structural differences between adult and fetal bone marrow tissues.     

Chromatographic analysis of purine precursors in mouse L1210 leukemia

A number of antagonists of nucleotide metabolism with anti-cancer activity affect the de novo purine pathway. To determine the biochemical mechanisms of cytotoxicity of these drugs, assay procedures have been developed for measurement of the levels of intermediates proximal to IMP in the pathway for de novo purine biosynthesis in mouse L1210 leukemia cells. Purine precursors have been synthesized in vitro from [14C]glycine using enzymes from chicken liver. These 14C-labeled intermediates have been used as marker compounds to define retention times for metabolites of leukemia cells separated by HPLC and the chromatographic mobilities of these intermediates after two-dimensional thin-layer chromatography. These new chromatographic procedures have been used in combination to determine the steady-state concentrations for purine precursors in mouse L1210 leukemia cells in the exponential phase of growth: N-formylglycineamide ribotide (16 microM); N-formylglycineamidine ribotide (4.7 microM); 5-aminoimidazole ribotide (4.0 microM); 4-carboxy-5-aminoimidazole ribotide (0.46 microM); N-succino-5-aminoimidazole-4-carboxamide ribotide (11 microM); 5-aminoimidazole-4-carboxamide ribotide (16 microM); 5-formamidoimidazole-4-carboxamide ribotide (2.7 microM); and IMP (57 microM). The metabolic effects of tiazofurin (25 microM) upon mouse L1210 leukemia cells growing in culture define a "metabolic crossover point" at the reaction catalyzed by IMP dehydrogenase (EC 1.1.1.205) which confirms previous reports of inhibition of this enzyme.     

Mercaptan and dicarboxylate inhibitors of hamster dihydroorotase

In mammals, dihydroorotase is part of a trifunctional protein, dihydroorotate synthetase, which catalyzes the first three reactions of de novo pyrimidine biosynthesis. Dihydroorotase catalyzes the formation of a peptide-like bond between the terminal ureido nitrogen and the beta-carboxyl group of N-carbamyl-L-aspartate to yield heterocyclic L-dihydroorotate. A variety of evidence suggests that dihydroorotase may have a catalytic mechanism similar to that of a zinc protease [Christopherson, R. I., & Jones, M. E. (1980) J. Biol. Chem. 255, 3358-3370]. Tight-binding inhibitors of the zinc proteases, carboxypeptidase A, thermolysin, and angiotensin-converting enzyme have been synthesized that combine structural features of the substrates with a thiol or carboxyl group in an appropriate position to coordinate a zinc atom bound at the catalytic site. We have synthesized (4R)-2-oxo-6-thioxohexahydropyrimidine-4-carboxylate (L-6-thiodihydroorotate) and have found that this analogue is a potent competitive inhibitor of dihydroorotase with a dissociation constant (Ki) in the presence of excess Zn2+ ion of 0.17 +/- 0.02 microM at pH 7.4. The potency of inhibition by L-6-thiodihydroorotate in the presence of divalent metal ions decreases in the order Zn2+ greater than Ca2+ greater than Co2+ greater than Mn2+ greater than Ni2+; L-6-thiodihydroorotate alone is less inhibitory and has a Ki of 0.85 +/- 0.14 microM. 6-Thioorotate has a Ki of 82 +/- 8 microM which decreases to 3.8 +/- 1.4 microM in the presence of Zn2+. Zn2+ alone is a moderate inhibitor of dihydroorotase and does not enhance the potency of other inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of acivicin and dichloroallyl lawsone upon pyrimidine biosynthesis in mouse L1210 leukemia cells

Acivicin (NSC 163501) and dichloroallyl lawsone (NSC 126771) are potent inhibitors of nucleotide biosynthesis with consequent anti-cancer activity against certain experimental tumors. To determine in detail the metabolic events induced by each inhibitor, we have devised a new two-dimensional chromatographic procedure for measurement of the concentrations of all pyrimidine intermediates and some purine nucleotides from 100 microliter of an extract of cells grown in the presence of [14C]bicarbonate. Addition of acivicin (25 microM) to mouse L1210 leukemia cells causes severe depletion in the cellular levels of CTP and GTP, accumulation of uridine nucleotides, and abrupt but transient increases in the concentrations of the early intermediates of both the pyrimidine and purine pathways. Addition of dichloroallyl lawsone (25 microM) results in a rapid depletion of uridine and cytidine nucleotides; carbamyl aspartate and dihydroorotate accumulate to high levels in an equilibrium ratio of 20.5:1, and orotate, orotidine, and UMP increase transiently before decreasing to levels approaching their original steady states. The predominant inhibitory effects of acivicin are upon the reactions UTP----CTP and XMP----GMP, but there is also an initial transient activation of both the pyrimidine and purine pathways by acivicin. The data obtained with dichloroallyl lawsone are consistent with inhibition of the conversion of UMP----UDP initially followed by potent inhibition of dihydroorotate----orotate.     

Identification of the replication terminator protein binding sites in the terminus region of the Bacillus subtilis chromosome and stoichiometry of the binding

DNase I footprinting of the interaction between the replication terminator protein (RTP) of Bacillus subtilis and the inverted repeat region (IRR) at the chromosome terminus, to which it binds to block the clockwise replication fork, showed that two major regions of 41 base pairs (bp) were protected from cleavage. These regions corresponded approximately to the imperfect inverted repeats (IRI and IRII) identified previously. Band retardation analyses of the interaction between RTP and portions of the IRR established that each inverted repeat (IRI or IRII) contained two RTP binding sites. By sedimentation equilibrium in the ultracentrifuge, RTP was found to exist as a dimer of 29 kDa at neutral pH and concentrations above 0.2 g/l. Quantitative studies of the RTP-IRR interaction using [3H]RTP and [32P]IRR showed that the fully saturated complex contained eight RTP monomers per IRR. It is concluded that a dimer of RTP binds to each of the four sites in IRR. The apparent dissociation constant for the interaction was estimated (in the presence of 50% glycerol) to be 1.2 x 10(-11) M (dimer of RTP). Glycerol was found to have a marked effect on the affinity of RTP for the IRR and on the relative amounts of the interaction complexes formed; in the absence of glycerol the dissociation constant was approximately 50-fold higher and there was pronounced co-operative binding of RTP dimers to adjacent sites in each inverted repeat. Examination of the DNA sequence in IRI and IRII identified two 8 bp direct repeats in each. The regions protected from DNase I cleavage in each inverted repeat and the protection afforded by a core sequence spanning just one of the 8 bp direct repeats were consistent with each 8 bp repeat representing a recognition sequence for the RTP dimer. A model describing the binding of RTP to the IRR is presented.     

Effects of brequinar and ciprofloxacin on de novo nucleotide biosynthesis in mouse L1210 leukemia

Exposure of mouse L1210 leukemia cells to 25 microM brequinar for 4 h results in large accumulations of N-carbamyl-L-aspartate and L-dihydroorotate to cellular concentrations of 8.5 mM and 0.8 mM, respectively, while UTP and CTP decrease to 4% of their initial levels; incorporation of [14C]bicarbonate into nucleic acids (DNA and RNA) was decreased to 47%. These data provide direct evidence for inhibition of DHO dehydrogenase by brequinar in growing cells. Exposure of leukemia cells to 200 microM ciprofloxacin for 4 h did not affect de novo pyrimidine nucleotide biosynthesis or the incorporation of [14C]bicarbonate into nucleic acids but resulted in a general decrease in nucleoside triphosphates, with concomitant accumulation of nucleoside mono- and diphosphates (the adenylate energy charge decreased from 0.89 to 0.69), consistent with inhibition of the electron transport chain or uncoupling of oxidative phosphorylation.     

Purification of hamster dihydroorotate synthetase using Procion blue-Sepharose

Dihydroorotate (DHO) synthetase is a trifunctional protein that catalyzes the first three reactions of de novo pyrimidine biosynthesis. A single-step procedure for purification of DHO synthetase from mutant hamster cells that overproduce this protein has been developed. The synthetase is adsorbed from a postmitochondrial supernatant to a column of Procion blue-Sepharose 4B and, after the column is washed, the synthetase is eluted as a single peak with 0.4 M KCl. Pooled fractions from the trailing side of this peak yield DHO synthetase with a specific activity for aspartate transcarbamylase of 14 mumol/min/mg protein, representing a purification factor of 8.5-fold and a recovery of 28% from the postmitochondrial supernatant. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the DHO synthetase was of high purity. A further 34% of the DHO synthetase from the leading side of the eluted peak contained a minor proportion of a proteolytic fragment. Similar results were obtained with an established four-step purification procedure.     

Profiling of apoptotic changes in human breast cancer cells using SELDI-TOF mass spectrometry.

Apoptosis is a key process in the response of tumours to chemotherapeutic agents. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in many tumor cells, while sparing most normal cells. Several chemotherapeutic drugs synergize with TRAIL in reducing tumor growth and inducing apoptosis. Because some tumour cells respond poorly to these treatments, biomarkers that predict clinical responsiveness are needed. This study used surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) to identify novel apoptotic markers in TRAIL and etoposide (T+E)-treated MDA-MB-231 and ZR-75-1 breast cancer cells and MCF-10A non-transformed breast cells. T+E induced apoptosis, increasing caspase-3 activity at 4-8h, in all cell lines. Protein profiles revealed two prominent peaks, m/z 10090 and 8560, which decreased significantly during apoptosis. Mass spectrometry sequencing of tryptic peptides identified these proteins as S100A6 (confirmed immunologically) and ubiquitin (confirmed against a purified standard), respectively. Caspase inhibition prevented the decrease in both proteins during T+E-induced apoptosis whereas proteasome inhibition combined with T+E further decreased ubiquitin, possibly by preventing its recycling. Using SELDI-TOF MS we have identified S100A6 and ubiquitin as potential protein markers of apoptosis. Further validation using patient samples is required to confirm their potential utility in monitoring the effectiveness of anti-cancer drugs in inducing tumour cell apoptosis.