Biochemical and genetic analysis of toxic effect of HOE 15030 in mammalian cells

1989 ◽  
Vol 15 (4) ◽  
pp. 279-288 ◽  
Author(s):  
Ryoji Ishida ◽  
Miwako Nishizawa ◽  
Fumiko Kohtani ◽  
Taijo Takahashi
Keyword(s):  
Genetic Analysis ◽  
Toxic Effect ◽  
Mammalian Cells

Genetic analysis of intracellular aminoglycerophospholipid traffic

2004 ◽  
Vol 82 (1) ◽  
pp. 156-169 ◽  
Author(s):  
Dennis R Voelker
Keyword(s):  
Genetic Analysis ◽  
Mammalian Cells ◽  
Molecular Genetic ◽  
Family Members ◽  
Molecular Genetic Analysis ◽  
Transport Processes ◽  
Membrane Biogenesis ◽  
Independent Manner ◽  
Multiple Genes ◽  
Vacuolar Protein

Inter- and intramembrane phospholipid transport processes are central features of membrane biogenesis and homeostasis. Relatively recent successes in the molecular genetic analysis of aminoglycerophospholipid transport processes in both yeast and mammalian cells are now providing important new information defining specific protein and lipid components that participate in these reactions. Studies focused on phosphatidylserine (PtdSer) transport to the mitochondria reveal that the process is regulated by ubiquitination. In addition, a specific mutation disrupts PtdSer transport between mitochondrial membranes. Analysis of PtdSer transport from the endoplasmic reticulum to the locus of PtdSer decarboxylase 2 demonstrates the requirement for a phosphatidylinositol-4-kinase, a phosphatidylinositol-binding protein, and the C2 domain of the decarboxylase. Examination of NBD-phosphatidylcholine transport demonstrates the involvement of the prevacuolar compartment and a requirement for multiple genes involved in regulating vacuolar protein sorting for transport of the lipid to the vacuole. In intramembrane transport, multiple genes are now identified including those encoding multidrug resistant protein family members, DNF family members, ATP binding cassette transporters, and pleiotropic drug resistance family members. The scramblase family constitutes a collection of putative transmembrane transporters that function in an ATP-independent manner. The genetic analysis of lipid traffic is uncovering new molecules involved in all aspects of the regulation and execution of the transport steps and also providing essential tools to critically test the involvement of numerous candidate molecules.Key words: lipid transport, lipid sorting, membrane biogenesis, organelles, flippase.

scholarly journals Reversal of Mefloquine and Quinine Resistance in Plasmodium falciparum with NP30

2003 ◽  
Vol 47 (8) ◽  
pp. 2393-2396 ◽  
Author(s):  
Michelle Ciach ◽  
Kathleen Zong ◽  
Kevin C. Kain ◽  
Ian Crandall
Keyword(s):  
Plasmodium Falciparum ◽  
Multidrug Resistance ◽  
Genetic Analysis ◽  
Resistance Genes ◽  
Mammalian Cells ◽  
Point Mutations ◽  
In Vitro Assays ◽  
Drug Efflux ◽  
P Glycoprotein

ABSTRACT Quinoline resistance in malaria is frequently compared with P-glycoprotein-mediated multidrug resistance (mdr) in mammalian cells. We have previously reported that nonylphenolethoxylates, such as NP30, are potential Plasmodium falciparum P-glycoprotein substrates and drug efflux inhibitors. We used in vitro assays to compare the ability of verapamil and NP30 to sensitize two parasite isolates to four quinolines: chloroquine (CQ), mefloquine (MF), quinine (QN), and quinidine (QD). NP30 was able to sensitize (reversal, >80%) P. falciparum to MF, QN, QD, and, to a lesser extent, CQ. The presence of 2 μM verapamil had no effect on mefloquine resistance; however, the presence of verapamil modulated the activities of QN and QD in a manner parallel to that observed for CQ. Genetic analysis of putative quinoline resistance genes did not suggest an association between known point mutations in pfcrt and pfmdr1 and NP30 sensitization activity. We conclude that the sensitization action of NP30 is distinct both phenotypically and genotypically from that of verapamil.

Regulation and mechanisms of gene amplification

1995 ◽  
Vol 347 (1319) ◽  
pp. 49-56 ◽  
Keyword(s):  
Dna Damage ◽  
Genetic Analysis ◽  
Mammalian Cells ◽  
Chromosome Breakage ◽  
Cellular Responses ◽  
Human Cells ◽  
Aspartate Transcarbamylase ◽  
Tumour Suppressor Function ◽  
Damage Recognition ◽  
Sister Chromatids

Amplification in rodent cells usually involves bridge-breakage-fusion (bbf) cycles initiated either by endto-end fusion of sister chromatids, or by chromosome breakage. In contrast, in human cells, resistance to the antimetabolite A-(phosphonacetyl)-L-aspartate (PALA) can be mediated by several different mechanisms that lead to overexpression of the target enzyme carbamyl-P synthetase, aspartate transcarbamylase, dihydro-orotase (CAD). Mechanisms involving bbf cycles account for only a minority of CAD amplification events in the human fibrosarcoma cell line HT 1080. Here, formation of a 2p isochromosome and overexpression of CAD by other types of amplification events (and even without amplification) are much more prevalent. Broken DNA is recognized by mammalian cells with intact damage-recognition pathways, as a signal to arrest or to die. Loss of these pathways by, for example, loss of p53 or pRb tumour suppressor function, or by increased expression of ras and myc oncogenes, causes non-permissive rat and human cells to become permissive both for amplification and for other manifestations of DNA damage. In cells that are already permissive, amplification can be stimulated by overexpressing oncogenes such as c-myc or ras , or by damaging DNA in a variety of ways. To supplement genetic analysis of amplification in mammalian cells, an amplification selection has been established in Schizosaccharomyces pombe . Selection with LiCl yields cells with amplified sod2 genes in structures related to those observed in mammalian cells. The effect on amplification in S. pombe can now be tested for any mutation in a gene involved in repair of damaged DNA or in normal cellular responses to DNA damage.

scholarly journals Molecular and genetic analysis of the toxic effect of RAP1 overexpression in yeast.

Genetics ◽  
1995 ◽  
Vol 141 (4) ◽  
pp. 1253-1262 ◽  
Author(s):  
K Freeman ◽  
M Gwadz ◽  
D Shore
Keyword(s):  
Dna Binding ◽  
Genetic Analysis ◽  
Toxic Effect ◽  
Transcriptional Activation ◽  
Ribosomal Proteins ◽  
Regulatory Protein ◽  
Point Mutations ◽  
Binding Domain ◽  
Dna Binding Domain

Abstract Rap1p is a context-dependent regulatory protein in yeast that functions as a transcriptional activator of many essential genes, including those encoding ribosomal proteins and glycolytic enzymes. Rap1p also participates in transcriptional silencing at HM mating-type loci and telomeres. Overexpression of RAP1 strongly inhibits cell growth, perhaps by interfering with essential transcriptional activation functions within the cell. Here we report a molecular and genetic analysis of the toxic effect of RAP1 overexpression. We show that toxicity does not require the previously defined Rap1p activation and silencing domains, but instead is dependent upon the DNA-binding domain and an adjacent region of unknown function. Point mutations were identified in the DNA-binding domain that relieve the toxic effect of overexpression. Two of these mutations can complement a RAP1 deletion yet cause growth defects and altered DNA-binding properties in vitro. However, a small deletion of the adjacent (downstream) region that abolishes overexpression toxicity has, by itself, no apparent effect on growth or DNA binding. SKO1/ACR1, which encodes a CREB-like repressor protein in yeast, was isolated as a high copy suppressor of the toxicity caused by RAP1 overexpression. Models related to the regulation of Rap1p activity are discussed.

scholarly journals Aberrant replication licensing drives Copy Number Gains across species

2020 ◽  
Author(s):  
Patroula Nathanailidou ◽  
Michalis Petropoulos ◽  
Styliani Maxouri ◽  
Eirini Kasselimi ◽  
Ioanna Eleni Symeonidou ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Genetic Analysis ◽  
Genome Sequencing ◽  
Genetic Heterogeneity ◽  
Copy Number ◽  
Mammalian Cells ◽  
Whole Genome ◽  
Genome Wide ◽  
Repair Pathways ◽  
Replication Intermediates

AbstractCopy Number Gains (CNGs) lead to genetic heterogeneity, driving evolution and carcinogenesis. The mechanisms promoting CNG formation however remain poorly characterized. We show that abnormal expression of the replication licensing factor Cdc18 in fission yeast, which leads to genome-wide re-replication, drives the formation of CNGs at different genomic loci, promoting the acquisition of new selectable traits. Whole genome sequencing reveals Mb long, primarily extrachromosomal amplicons. Genetic analysis shows that homology-mediated repair is required to resolve re-replication intermediates into heritable CNGs. Consistently, we show that in mammalian cells overexpression of CDC6 and/or CDT1 leads to CNGs and promotes drug resistance. In human cells, multiple repair pathways are activated upon rereplication and act antagonistically, with RAD52 promoting and 53BP1 inhibiting CNG formation. In tumours, CDT1 and/or CDC6 overexpression correlates with copy number gains genome-wide. We propose re-replication as an evolutionary-conserved driver of CNGs, highlighting a link between aberrant licensing, CNGs and cancer.

scholarly journals Methylthioadenosine toxicity and metabolism to methionine in mammalian cells

1988 ◽  
Vol 255 (1) ◽  
pp. 145-152 ◽  
Author(s):  
L Christa ◽  
J Kersual ◽  
J Augé ◽  
J L Pérignon
Keyword(s):  
Total Synthesis ◽  
Toxic Effect ◽  
Mammalian Cells ◽  
Resting Cells ◽  
Free Medium ◽  
Raji Cells ◽  
Polyamine Synthesis ◽  
Ccl39 Cells ◽  
Growth Experiments ◽  
Metabolic Cycle

5′-Deoxy-5′-methylthioadenosine, a by-product of polyamine synthesis, can support the growth of Raji cells in a methionine-free medium, but not the growth of CCL39 cells, although these cells are also able to incorporate radiolabelled 5′-deoxy-5′-methylthioadenosine (MeSAdo) into methionine, S-adenosyl-L-methionine (AdoMet) and proteins [Christa, Kersual, Augé & Pérignon (1986) Biochem. Biophys. Res. Commun. 135, 131-138]. We first tested the hypothesis of a toxic effect of MeSAdo in the conditions of growth experiments: we could not demonstrate any toxic effect of MeSAdo on the synthesis of macromolecules, nor any toxicity mediated by polyamines or pyrimidine starvation, and we found that the growth of CCL39 cells was strictly dependent on the supply of exogenous methionine. We then tried to determine whether the ability of CCL39 cells to metabolize MeSAdo to methionine and AdoMet was modulated by the proliferation state of CCL39 cells, which is dependent on the supply of exogenous methionine. Studies of the incorporation of radiolabelled MeSAdo show that: (i) the total synthesis of methionine from MeSAdo is twice as high in subconfluent cells (grown in 100 microM-methionine) as in resting cells (cultured in 0 microM-methionine); (ii) the incorporation into proteins does not parallel the total protein synthesis, and the methionine derived from MeSAdo mostly flows out of the cell; (iii) addition of methionine to resting cells immediately leads to a transient and marked increase in metabolism of MeSAdo to AdoMet, presumably reflecting the rapid replenishment of the AdoMet pool of the cells. Taken together, these results suggest that the methionine derived from MeSAdo is preferentially used to synthesize AdoMet rather than proteins, and that this synthesis of AdoMet depends on the ability of the CCL39 cells to grow, and hence on the supply of exogenous methionine. It is proposed that, in CCL39 cells, the metabolic pathway leading from MeSAdo (a by-product of polyamine synthesis) to methionine and to AdoMet (a precursor of polyamine synthesis) is part of a metabolic cycle the activity of which depends, like polyamine synthesis itself, on cell proliferation.

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