Biomedical subjects

M Wierdl

Publications and source records attributed to M Wierdl.

10 recordsLinked to original sources

PubMed2001

Sensitization of human tumor cells to CPT-11 via adenoviral-mediated delivery of a rabbit liver carboxylesterase.

Irinotecan, 7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin (CPT-11) is activated by carboxylesterases (CE) to yield the potent topoisomerase I inhibitor, SN-38. We have demonstrated previously that a rabbit liver CE is approximately 100-1000-fold more efficient at drug activation than a highly homologous human CE. In an attempt to use rabbit CE expression in combination with CPT-11 for gene therapy approaches for the treatment of cancer, we have developed an adenoviral vector expressing this intracellular CE. After transduction, this virus produces very high levels of CE activity in a panel of human tumor cell lines and results in marked sensitization to CPT-11 of all of the transduced cells. Reductions in IC(50) values for this drug ranged from 11-127-fold. Additionally, comparison with an adenovirus expressing a secreted form of the rabbit CE indicated that a collateral effect could be achieved with reductions in the IC(50) values ranging from 4-19-fold. These data suggest that the described reagents may be suitable for use in vivo in a viral-directed enzyme prodrug therapy approach using CPT-11.

Adenoviridae↗

PubMed2001

A virus-directed enzyme prodrug therapy approach to purging neuroblastoma cells from hematopoietic cells using adenovirus encoding rabbit carboxylesterase and CPT-11.

Tumor cells that contaminate hematopoietic cell preparations contribute to the relapse of neuroblastoma patients who receive autologous stem cell rescue as a component of therapy. Therefore, effective purging methods are needed. This study details in vitro experiments to develop a viral-directed enzyme prodrug purging method that specifically targets neuroblastoma cells. The approach uses an adenovirus to deliver the cDNA encoding a rabbit liver carboxylesterase that efficiently activates the prodrug irinotecan,7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin (CPT-11). The data show that an adenoviral multiplicity of infection of 50 transduces 100% of cultured neuroblastoma cells and primary tumor cells, irrespective of the level of tumor cell line contamination. Exposure of neuroblastoma cell lines or of mixtures of these cell lines with CD34(+) cells at a ratio of 10:90 to replication-deficient AdRSVrCE for 24 h and subsequent exposure of cells to 1-5 microM CPT-11 for 4 h increased the toxicity of CPT-11 to three neuroblastoma cell lines (SJNB-1, NB-1691, and SK-N-SH) from approximately 20-50-fold and eradicated their clonogenic potential. Also, after "purging," RNA for neuroblastoma cell markers (tyrosine hydroxylase, synaptophysin, and N-MYC) was undetectable by reverse transcription-PCR. In contrast, the purging protocol did not affect the number or type of colonies formed by CD34(+) cells in an in vitro progenitor cell assay. No bystander effect on CD34(+) cells was observed. The method described is being investigated for its potential clinical utility, particularly its efficacy for use with patients having relatively high tumor burdens, because no published methods have been shown to be efficacious when the tumor burden exceeds 1%.

Adenoviridae↗

PubMed2001

Structural constraints affect the metabolism of 7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin (CPT-11) by carboxylesterases.

7-Ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin [CPT-11 (irinotecan)] is a water-soluble camptothecin-derived prodrug that is activated by esterases to yield the potent topoisomerase I poison SN-38. We identified a rabbit liver carboxylesterase (CE) that was very efficient at CPT-11 metabolism; however, a human homolog that was more than 81% identical to this protein activated the drug poorly. Recently, two other human CEs have been isolated that are efficient in the conversion of CPT-11 to SN-38, yet both demonstrate little homology to the rabbit protein. To understand this phenomenon, we have characterized a series of esterases from human and rabbit, including several chimeric proteins, for their ability to metabolize CPT-11. Computer predictive modeling indicated that the ability of each enzyme to activate CPT-11 was dependent on the size of the entrance to the active site. Kinetic studies with a series of nitrophenyl and naphthyl esters confirmed these predictions, indicating that activation of CPT-11 by a CE is constrained by size-limited access of the drug to the active site catalytic amino acid residues.

Amino Acid Sequence↗

PubMed2000

Activation of CPT-11 in mice: identification and analysis of a highly effective plasma esterase.

The camptothecin prodrug CPT-11 (irinotecan, 7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin) is converted by esterases to yield the potent topoisomerase I poison SN-38 (7-ethyl-10-hydroxycamptothecin). Recently, a mouse strain (Es1(e)) has been identified that demonstrates reduced plasma esterase activity, and we have monitored the ability of plasma from these mice to metabolize CPT-11. Total plasma esterase activity was reduced 3-fold in Esl(e)mice in comparison to control mice, and this resulted in a 200-fold reduction in SN-38 production after incubation with CPT-11 in vitro. In addition, pharmacokinetic studies of CPT-11 and SN-38 in these animals demonstrated approximately 5-fold less conversion to SN-38. However, extracts derived from tissues from Es1(e) animals revealed total esterase activities similar to those of control mice, and these extracts metabolized CPT-11 with equal efficiency. Northern analysis of RNA isolated from organs indicated that the liver was the primary source of Es-1 gene expression and that very low levels of Es-1 RNA were present in Es1(e) mice. These results suggest that the reduced levels of Es-1 esterase present in Es1(e) mice are due to down-regulation of gene transcription, and that this plasma esterase is responsible for the majority of CPT-11 metabolism in mice.

Animals↗

PubMed2000

Isolation and characterization of a cDNA encoding a horse liver butyrylcholinesterase: evidence for CPT-11 drug activation.

Butyrylcholinesterases (BuChEs; acylcholine acylhydrolase; EC 3.1.1.8) have been demonstrated to convert the anticancer agent CPT-11 (irinotecan, 7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin) into its active metabolite SN-38 (7-ethyl-10-hydroxycamptothecin). In addition, significant differences in the extent of drug metabolism have been observed with BuChEs derived from different species. In an attempt to understand these differences, we have isolated the cDNA encoding a horse BuChE. Based upon the NH2-terminal amino acid sequence of a purified horse BuChE, we designed degenerate primers to amplify the coding sequence from horse liver cDNA. Following polymerase chain reaction and rapid amplification of the cDNA ends, we generated an 1850-bp DNA fragment, containing an 1806-bp open reading frame. The cDNA encodes a protein of 602 amino acid residues, including a 28-amino-acid NH2-terminal signal peptide. Furthermore, the DNA sequence and the deduced amino acid sequence revealed extensive homology to butyrylcholinesterase genes from several other species. In vitro transcription-translation of the cDNA produced a 66-kDa protein, identical to the size of native horse serum BuChE following removal of carbohydrate residues with endoglycosidase F. Additionally, transient expression of the cDNA in Cos-7 cells yielded extracts that exhibited cholinesterase activity and demonstrated a Km value for butyrylthiocholine of 106+/-9 nM. This extract converted the anticancer drug CPT-11 into SN-38, demonstrating that this drug can be activated by enzymes other than carboxylesterases.

Amino Acid Sequence↗

PubMed1998

Cellular localization domains of a rabbit and a human carboxylesterase: influence on irinotecan (CPT-11) metabolism by the rabbit enzyme.

Enzyme activation of prodrugs to improve the therapeutic index of specific anticancer agents is an attractive alternative to current chemotherapy regimens. This study addresses the potential for activating irinotecan (CPT-11) with recombinant carboxylesterases (CEs). CEs are a ubiquitous class of enzymes thought to be involved in the detoxification of xenobiotics. Their primary amino acid sequence indicates that these proteins should be localized to the endoplasmic reticulum. By PCR-mediated mutagenesis of a rabbit liver and a human alveolar macrophage CE cDNA, expression in Cos7 cells, and subsequent immunohistochemical localization, we have determined that an 18-amino acid NH2-terminal hydrophobic signal peptide is responsible for the localization of these proteins to the endoplasmic reticulum. By similar approaches, we have demonstrated that the COOH-terminal amino acids HIEL prevent secretion of the proteins from the cell. Enzymatic activity was lost by removing the NH2-terminal domain; however, active enzyme could be detected in the culture media of cells expressing the COOH-terminally truncated proteins. Secretion of CEs lacking the six COOH-terminal amino acids could be prevented with brefeldin A, confirming that these truncated enzymes were processed and released from cells by endoplasmic reticulum-mediated exocytosis. Double-truncation mutant enzymes lacking both NH2- and COOH-terminal sequences demonstrated immunostaining patterns similar to those of the NH2-terminally truncated proteins and also lacked CE activity. In all cases, metabolism of the classic esterase substrate o-nitrophenyl acetate predicted the sensitivity of cells expressing the rabbit CE to the anticancer agent CPT-11. In addition, the secreted enzyme sensitized Cos7 cells to this drug, indicating that protein association with a lipid bilayer is not required for substrate metabolism.

Animals↗

PubMed1997

Microsatellite instability in yeast: dependence on the length of the microsatellite.

One of the most common microsatellites in eukaryotes consists of tandem arrays [usually 15-50 base pairs (bp) in length] of the dinucleotide GT. We examined the rates of instability for poly GT tracts of 15, 33, 51, 99 and 105 bp in wild-type and mismatch repair-deficient strains of Saccharomyces cerevisiae. Rates of instability increased more than two orders of magnitude as tracts increased in size from 15 to 99 bp in both wild-type and msh2 strains. The types of alterations observed in long and short tracts in wild-type strains were different in two ways. First, tracts > or = 51 bp had significantly more large deletions than tracts < or = 33 bp. Second, for the 99- and 105-bp tracts, almost all events involving single repeats were additions; for the smaller tracts, both additions and deletions of single repeats were common.

DNA, Fungal↗

PubMed1996

Ergodic theorems along sequences and Hardy fields.

Let a(x) be a real function with a regular growth as x --> infinity. [The precise technical assumption is that a(x) belongs to a Hardy field.] We establish sufficient growth conditions on a(x) so that the sequence ([a(n)])(infinity)(n=1) is a good averaging sequence in L2 for the pointwise ergodic theorem. A sequence (an) of positive integers is a good averaging sequence in L2 for the pointwise ergodic theorem if in any dynamical system (Omega, Sigma, m, T) for f [symbol, see text] in L2(Omega) the averages [equation, see text] converge for almost every omicron in. Our result implies that sequences like ([ndelta]), where delta > 1 and not an integer, ([n log n]), and ([n2/log n]) are good averaging sequences for L2. In fact, all the sequences we examine will turn out to be good averaging for Lp, p > 1; and even for L log L. We will also establish necessary and sufficient growth conditions on a(x) so that the sequence ([a(n)]) is good averaging for mean convergence. Note that for some a(x) (e.g., a(x) = log2 x), ([a(n)]) may be good for mean convergence without being good for pointwise convergence.

Journal Article↗

PubMed1996

Destabilization of simple repetitive DNA sequences by transcription in yeast.

Simple repetitive DNA sequences in the eukaryotic genome frequently alter in length. In wild-type strains, we find that transcription through a repetitive poly GT tract destabilizes the tract four- to ninefold. In mismatch repair-deficient yeast strains, simple repeats are very unstable. High levels of transcription in such strains destabilize repetitive tracts an additional two- to threefold.

DNA Damage↗

PubMed1991

DNA-binding protein RAP1 stimulates meiotic recombination at the HIS4 locus in yeast.

In the yeast Saccharomyces cerevisiae, as in other eukaryotes, some regions of the genome have a much higher rate of meiotic recombination than others. We show below that the binding of the RAP1 protein to a site upstream of the HIS4 gene is necessary for a high rate of meiotic (but not mitotic) recombination at this locus. A mutation in the RAP1 binding site at HIS4 results in a decrease in recombination; overproduction of RAP1 causes an increase in recombination at HIS4 above wild-type levels.

Base Sequence↗