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PubMed2006

Non-transcribed strand repair revealed in quiescent cells.

Stem cells, one of the progenitors of cancer, exist predominately in a quiescent state. Thus, understanding the mechanisms of DNA repair and mutagenesis in such arrested cells may help unravel the complex process of tumorigenesis. Two major nucleotide excision repair (NER) pathways are known to remove bulky physical or chemical lesions from DNA. Transcription-coupled repair (TCR) acts solely on the transcribed strand of expressed genes, while global genomic repair (GGR) is responsible for the ubiquitous repair of the genome. Indirectly, it has been shown that while TCR functions in quiescent cells GGR does not. To explicitly elucidate this phenomenon, we adapted a quantitative PCR (QPCR) assay to study UV-damage repair via TCR and GGR in quiescent and proliferating cells. We present evidence that repair of untranscribed silent regions of the genome and repair of the non-transcribed strand of active genes proceeds by two discrete mechanisms in quiescent cells; rather than by GGR, which was believed to encompass both. Thus, our findings suggest the existence of an alternate NER pathway in quiescent cells. The proposed subcategories of NER are as follows: (i) TCR, responsible for maintenance of transcribed strands; (ii) GGR, responsible for ubiquitous genome repair; and (iii) non-transcribed strand repair (NTSR), predominantly responsible for the repair of the NTS in arrested cells. In quiescent cells, it is evident that TCR and NTSR function and GGR are arrested. As a consequence, mutation accumulation at temporally silent genes and incomplete or imperfect repair of transcribed genes, in quiescent stem cells, may provide a source of cancer causing mutations.

Animals↗
PubMed2000

Non-target gene mutations in the development of fluoroquinolone resistance in Escherichia coli.

Mutations in loci other than genes for the target topoisomerases of fluoroquinolones, gyrA and parC, may play a role in the development of fluoroquinolone resistance in Escherichia coli. A series of mutants with increasing resistance to ofloxacin was obtained from an E. coli K-12 strain and five clinical isolates. First-step mutants acquired a gyrA mutation. Second-step mutants reproducibly acquired a phenotype of multiple antibiotic resistance (Mar) and organic solvent tolerance and showed enhanced fluoroquinolone efflux. None of the second-step mutants showed additional topoisomerase mutations. All second-step mutants showed constitutive expression of marA and/or overexpressed soxS. In some third-step mutants, fluoroquinolone efflux was further enhanced compared to that for second-step mutants, even when the mutant had acquired additional topoisomerase mutations. Attempts to circumvent the second-step Mar mutation by induction of the mar locus with sodium salicylate and thus to select for pure topoisomerase mutants at the second step were not successful. At least in vitro, non-target gene mutations accumulate in second- and third-step mutants upon exposure to a fluoroquinolone and typically include, but do not appear to be limited to, mutations in the mar or sox regulons with consequent increased drug efflux.

Anti-Infective Agents↗
PubMed2005

Genomic variability within an organism exposes its cell lineage tree.

What is the lineage relation among the cells of an organism? The answer is sought by developmental biology, immunology, stem cell research, brain research, and cancer research, yet complete cell lineage trees have been reconstructed only for simple organisms such as Caenorhabditis elegans. We discovered that somatic mutations accumulated during normal development of a higher organism implicitly encode its entire cell lineage tree with very high precision. Our mathematical analysis of known mutation rates in microsatellites (MSs) shows that the entire cell lineage tree of a human embryo, or a mouse, in which no cell is a descendent of more than 40 divisions, can be reconstructed from information on somatic MS mutations alone with no errors, with probability greater than 99.95%. Analyzing all approximately 1.5 million MSs of each cell of an organism may not be practical at present, but we also show that in a genetically unstable organism, analyzing only a few hundred MSs may suffice to reconstruct portions of its cell lineage tree. We demonstrate the utility of the approach by reconstructing cell lineage trees from DNA samples of a human cell line displaying MS instability. Our discovery and its associated procedure, which we have automated, may point the way to a future "Human Cell Lineage Project" that would aim to resolve fundamental open questions in biology and medicine by reconstructing ever larger portions of the human cell lineage tree.

Animals↗
PubMed2025

Single-cell mutational burden distributions in birth-death processes.

Genetic mutations are footprints of cancer evolution and reveal critical dynamic parameters of tumour growth, which otherwise are hard to measure in vivo. The mutation accumulation in tumour cell populations has been described by various statistics, such as site frequency spectra (SFS), single-cell division distributions (DD) and mutational burden distributions (MBD). While DD and SFS have been intensively studied in phylogenetics especially after the development of whole genome sequencing technology of bulk samples, MBD has drawn attention more recently with the single-cell sequencing data. Although those statistics all arise from the same somatic evolutionary process, an integrated understanding of these distributions is missing and requires novel mathematical tools to better inform the ecological and evolutionary dynamics of tumours. Here we introduce dynamical matrices to analyse and unite the SFS, DD and MBD and derive recurrence relations for the expectations of these three distributions. While we successfully recover classic exact results in pure-birth cases for the SFS and the DD through our new framework, we derive a new expression for the MBD and approximate all three distributions when death is introduced. We demonstrate a natural link between the SFS and the single-cell MBD, and show that the MBD can be regenerated through the DD. Counter-intuitively, the single-cell MBD is mainly driven by the stochasticity arising in the DD, rather than the extra stochasticity in the number of mutations at each cell division.

Single-Cell Analysis↗
PubMed2006

Overlapping contributions of Msh1p and putative recombination proteins Cce1p, Din7p, and Mhr1p in large-scale recombination and genome sorting events in the mitochondrial genome of Saccharomyces cerevisiae.

The mechanisms that govern mutation avoidance in the mitochondrial genome, though believed to be numerous, are poorly understood. The identification of individual genes has implicated mismatch repair and several recombination pathways in maintaining the fidelity and structural stability of mitochondrial DNA. However, the majority of genes in these pathways have not been identified and the interactions between different pathways have not been extensively studied. Additionally, the multicopy presence of the mitochondrial genome affects the occurrence and persistence of mutant phenotypes, making mitochondrial DNA transmission and sorting important factors affecting mutation accumulation. We present new evidence that the putative recombination genes CCE1, DIN7, and MHR1 have overlapping function with the mismatch repair homolog MSH1 in point mutation avoidance and suppression of aberrant recombination events. In addition, we demonstrate a novel role for Msh1p in mtDNA transmission, a role not predicted by studies of its nuclear homologs.

Amino Acid Sequence↗
PubMedDate not supplied

Molecular biology, epidemiology, and the demise of the linear no-threshold (LNT) hypothesis.

The prime concern of radiation protection policy since 1959 has been protecting DNA from damage. The 1995 NCRP Report 121 on collective dose states that since no human data provides direct support for the linear no threshold hypothesis (LNT), and some studies provide quantitative data that, with statistical significance, contradict LNT, ultimately, confidence in LNT is based on the biophysical concept that the passage of a single charged particle could cause damage to DNA that would result in cancer. Current understanding of the basic molecular biologic mechanisms involved and recent data are examined before presenting several statistically significant epidemiologic studies that contradict the LNT hypothesis. Over eons of time a complex biosystem evolved to control the DNA alterations (oxidative adducts) produced by about 10(10) free radicals/cell/d derived from 2-3% of all metabolized oxygen. Antioxidant prevention, enzymatic repair of DNA damage, and removal of persistent DNA alterations by apoptosis, differentiation, necrosis, and the immune system, sequentially reduce DNA damage from about 10(6) DNA alterations/cell/d to about 1 mutation/cell/d. These mutations accumulate in stem cells during a lifetime with progressive DNA damage-control impairment associated with aging and malignant growth. A comparatively negligible number of mutations, an average of about 10(-7) mutations/cell/d, is produced by low LET radiation background of 0.1 cGy/y. The remarkable efficiency of this biosystem is increased by the adaptive responses to low-dose ionizing radiation. Each of the sequential functions that prevent, repair, and remove DNA damage are adaptively stimulated by low-dose ionizing radiation in contrast to their impairment by high-dose radiation. The biologic effect of radiation is not determined by the number of mutations it creates, but by its effect on the biosystem that controls the relentless enormous burden of oxidative DNA damage. At low doses, radiation stimulates this biosystem with consequent significant decrease of metabolic mutations. Low-dose stimulation of the immune system may not only prevent cancer by increasing removal of premalignant or malignant cells with persistent DNA damage, but used in human radioimmunotherapy may also completely remove malignant tumors with metastases. The reduction of gene mutations in response to low-dose radiation provides a biological explanation of the statistically significant observations of mortality and cancer mortality risk decrements, and contradicts the biophysical concept of the basic mechanisms upon which, ultimately, the NCRPs confidence in the LNT hypothesis is based.

Antimutagenic Agents↗
PubMed1996

Estimation of deleterious-mutation parameters in natural populations.

The rate and average effects of spontaneous deleterious mutations are important determinants of the evolution of breeding systems and of the vulnerability of small populations to extinction. Nevertheless, few attempts have been made to estimate the properties of such mutations, and those studies that have been performed have been extremely labor intensive, relying on long-term, laboratory mutation-accumulation experiments. We present an alternative to the latter approach. For populations in which the genetic variance for fitness is a consequence of selection-mutation balance, the mean fitness and genetic variance of fitness in outbred generations can be expressed as simple functions of the genomic mutation rate, average homozygous effect and average dominance coefficient of new mutations. Using empirical estimates for the mean and genetic variance of fitness, these expressions can then be solved to obtain joint estimates of the deleterious-mutation parameters. We employ computer simulations to evaluate the degree of bias of the estimators and present some general recommendations on the application of the technique. Our procedures provide some hope for obtaining estimates of the properties of deleterious mutations from a wide phylogenetic range of species as well as a mechanism for testing the validity of alternative models for the maintenance of genetic variance for fitness.

Gene Deletion↗
PubMedDate not supplied

The sexual nature of the eukaryote genome.

This paper supports a previous conjecture that the sexual cycle of eukaryotes arose from the infection of cells by genome parasites. The finding are as follows. (1) In prokaryotes, conjugative plasmids ensure their own spread by directing partial cell fusion. (2) Conjugative plasmids permit gene transfer between prokaryotes and eukaryotes, and can be integrated into the eukaryote genome. (3) Genes can be transferred between unrelated eukaryotes, and between different genomes within eukaryotic cells. (4) Elements such as transposons and retroviruses evolve as parasites of the eukaryote sexual system. (5) The mating-type genes of bipolar fungi are idiomorphic: alternative genes directing sexual specificity are dissimilar and non-homologous. It is argued that they arose as parasitic elements directing cell fusion, which have become integrated into the genome. (6) Mating-type idiomorphs in different taxa may be dissimilar, reflecting the independent acquisition of different infectious elements by different eukaryote lineages. (7) Sexual competence is rapidly lost through mutation accumulation or antagonistic pleiotropy during vegetative proliferation, so that many lineages are sexually sterile. (8) In multipolar systems, novel mating-type alleles arising by mutation spread in a parasite-like manner by virtue of their access to lineages which have become sexually sterile. (9) It is suggested that centromeres arise as devices to enable low copy number plasmids to persist. (10) Crossing over is favored if it enables the genes that direct it to unlink themselves from inferior genomes, or from the consequences of their own transposition.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA Transposable Elements↗
PubMed2002

Contiguous deletion of the X-linked adrenoleukodystrophy gene (ABCD1) and DXS1357E: a novel neonatal phenotype similar to peroxisomal biogenesis disorders.

X-linked adrenoleukodystrophy (X-ALD) results from mutations in ABCD1. ABCD1 resides on Xq28 and encodes an integral peroxisomal membrane protein (ALD protein [ALDP]) that is of unknown function and that belongs to the ATP-binding cassette-transporter superfamily. Individuals with ABCD1 mutations accumulate very-long-chain fatty acids (VLCFA) (carbon length >22). Childhood cerebral X-ALD is the most devastating form of the disease. These children have the earliest onset (age 7.2 +/- 1.7 years) among the clinical phenotypes for ABCD1 mutations, but onset does not occur at <3 years of age. Individuals with either peroxisomal biogenesis disorders (PBD) or single-enzyme deficiencies (SED) in the peroxisomal beta-oxidation pathway--disorders such as acyl CoA oxidase deficiency and bifunctional protein deficiency--also accumulate VLCFA, but they present during the neonatal period. Until now, it has been possible to distinguish unequivocally between individuals with these autosomal recessively inherited syndromes and individuals with ABCD1 mutations, on the basis of the clinical presentation and measurement of other biochemical markers. We have identified three newborn boys who had clinical symptoms and initial biochemical results consistent with PBD or SED. In further study, however, we showed that they lacked ALDP, and we identified deletions that extended into the promoter region of ABCD1 and the neighboring gene, DXS1357E. Mutations in DXS1357E and the ABCD1 promoter region have not been described previously. We propose that the term "contiguous ABCD1 DXS1357E deletion syndrome" (CADDS) be used to identify this new contiguous-gene syndrome. The three patients with CADDS who are described here have important implications for genetic counseling, because individuals with CADDS may previously have been misdiagnosed as having an autosomal recessive PBD or SED

ATP Binding Cassette Transporter, Subfamily D, Mem↗
PubMed2001

p53 gene mutation and protein accumulation during neoplastic progression in Barrett's esophagus.

The aim of the present study was to characterize expression and mutation of p53 during the neoplastic progression from Barrett's esophagus to adenocarcinoma and to test the reliability of immunohistochemistry for p53 overexpression as an indicator of p53 mutation in this context. The association of both gene mutation and protein accumulation with clinicopathological findings and survival was also studied. A total of 77 samples from 30 esophagectomy specimens with Barrett's esophagus and adenocarcinoma of patients in longitudinal clinical follow-up were analyzed. Different lesions (intestinal metaplasia, dysplasia, and adenocarcinoma) as well as normal squamous-cell esophageal epithelia were sampled from formalin-fixed, paraffin-embedded tissues by microdissection. Mutations in p53 Exons 5 to 9 were detected by polymerase chain reaction-single-strand conformation polymorphisms (PCR-SSCP) and confirmed by direct DNA sequencing. Nuclear accumulation of p53 protein was analyzed immunohistochemically from tissue sections adjacent to those used for microdissection. p53 gene mutations were found in 17 and p53 protein accumulation were found in 20 tumor samples. Of the 17 adenocarcinomas with a p53 mutation, 16 stained positive for p53 protein. p53 mutations were detected significantly more frequently in high-grade dysplastic than in low-grade dysplastic lesions (77% versus 29%, P < 0.01). In contrast, nuclear accumulation of p53 was detected in 85% of high-grade and 71% of low-grade dysplastic lesions. In eight cases with p53 mutation, the mutation identified in the tumors was also detected in premalignant lesions, mainly in high-grade dysplasia. In four cases of p53-mutated tumors, clones with different p53 mutations were detected in premalignant lesions. Neither p53 mutations nor p53 protein accumulations were found in metaplastic lesions. In summary, we found that p53 mutations occurred mainly during the transition from low-grade to high-grade dysplasia in the neoplastic progression of Barrett's esophagus but not in the nondysplastic Barrett's mucosa. Mutational analysis of p53 by PCR-SSCP and p53 accumulation by immunohistochemistry were mostly concordant in adenocarcinoma and high-grade dysplastic lesions but frequently discordant in low-grade dysplastic lesions. No correlation between p53 gene mutation or p53 accumulation and clinicopathological findings was observed in this study.

Adenocarcinoma↗
PubMedDate not supplied

Mutations of transforming growth factor beta 1 type II receptor, BAX, and insulin-like growth factor II receptor genes in microsatellite unstable cell lines.

Inactivation of the DNA mismatch repair (MMR) system allows the genome to accumulate mutations because of failure to correct mispairing of nucleotides and slippage mistakes at microsatellite sequences (termed microsatellite instability ¿MSI¿). While most mutations are acquired in noncoding regions by virtue of its larger share of DNA, mutations may occur in exons of genes that contain microsatellite sequences. The type II receptor for TGF beta 1 (TGF beta RII), the insulin-like growth factor II receptor (IGFIIR), and the proapoptotic gene BAX have been shown to contain mononucleotide microsatellites, and in MSI tumors, mutations may occur in these sequences late in the multistep carcinogenesis pathway. Here, we characterize 9 cell lines for MSI and mutations in TGF beta RII, BAX, and IGFIIR by PCR-based assays. The MMR-proficient cell lines SW480 and HT29 demonstrate stability at microsatellite sequences and do not have mutations in TGF beta RII, BAX, or IGFIIR. The MMR-deficient cell lines LoVo, SW48, LS174t, and HCT116 all demonstrate MSI and have only mutant alleles of TGF beta RII. Additionally, SW48 cells were heterozygous for wild-type and mutant IGFIIR. While LoVo and LS174t cells possessed only mutant BAX alleles, HCT116 was heterozygous and SW48 had only the wild-type allele. The MMR-deficient ovarian cell line 2774 demonstrated MSI, but showed only wild-type TGF beta RII, IGFIIR, and BAX alleles. In HCT116+ch2 cells, there was no genotypic change from the hMLH1-mutated HCT116 cells. In HCT116+ch3 cells, MSI was corrected, and this cell line became heterozygous for mutant and wild-type TGF beta RII because wild-type hMLH1 and TGF beta RII are both located on chromosome 3. Thus, the presence of a defective MMR system correlates with MSI, and the wild-type allele of TGF beta RII was absent in all microsatellite unstable colon cell lines, whereas the absence of wild-type BAX occurred in only two colon cell lines. In ovarian cancer cells with MSI, mutations in TGF beta RII, BAX, and IGFIIR may be unimportant in the genesis of this tumor.

Base Pair Mismatch↗
PubMed1989

Adaptive eradication of methionine and cysteine from cyanobacterial light-harvesting proteins.

Sulphur is unique among the main elements of living cells in that it is covalently bound to biopolymers but does not occur in the biopolymer backbone. Indeed, most of the bacterial sulphur content resides in the methionine and cysteine side-chains of proteins. The growth yield of an organism under conditions of sulphur limitation could therefore be greatly enhanced by mutations that substitute Met and Cys in the organism's proteins for sulphur-free amino acids. Because the saving in sulphur would increase with such accumulating mutations, Met and Cys changes could be progressively selected. Abundant proteins should be the prime targets of such a selection. A few published observations give credence to this scenario. Sulphate permease, which is abundantly produced by sulphur-starved Salmonella typhimurium, lacks Met and Cys residues. Also, two species of marine purple bacteria synthesize more protein than can be expected from a limited sulphate supply. We now report that the cyanobacterium Calothrix sp. PCC 7601 (referred to here as Calothrix) encodes sulphur-depleted versions of its most abundant proteins--phycocyanin and its auxiliary polypeptides--which it specifically expresses under conditions of sulphur limitation. Although these proteins do not take part in the fixation of sulphur, their elevated synthesis affects the sulphur budget of cyanobacterial cells. Direct evidence is thus provided that the structure of macromolecules can be subject to metabolic optimization.

Amino Acid Sequence↗
PubMed2004

Age-specific acceleration of cancer.

One of the great challenges of cancer research is to explain the epidemiological patterns of cancer incidence based on the molecular processes that lead to uncontrolled cellular proliferation. The epidemiological data demonstrate that the age-specific incidence of many cancers increases in an approximately linear way with age when plotted on a log-log scale, with different slopes for different cancers. However, those epidemiological data also show that cancers of various tissues depart from log-log linearity in particular ways. Here, I illustrate those departures from log-log linearity by introducing plots of the age-specific acceleration of cancer. I then develop a very general model of cancer progression, which I use to explain the observed differences between tissues in age-specific acceleration. In one application of the model, I show that the spectacular rise and fall in age-specific acceleration observed in prostate cancer may be explained by multiple rounds of clonal expansion. In a second application, I demonstrate that the steady decline in age-specific acceleration of breast cancer may occur because precancerous mutations accumulate in many cellular lineages.

Age Factors↗
PubMed2004

Epistasis and its relationship to canalization in the RNA virus phi 6.

Although deleterious mutations are believed to play a critical role in evolution, assessing their realized effect has been difficult. A key parameter governing the effect of deleterious mutations is the nature of epistasis, the interaction between the mutations. RNA viruses should provide one of the best systems for investigating the nature of epistasis because the high mutation rate allows a thorough investigation of mutational effects and interactions. Nonetheless, previous investigations of RNA viruses by S. Crotty and co-workers and by S. F. Elena have been unable to detect a significant effect of epistasis. Here we provide evidence that positive epistasis is characteristic of deleterious mutations in the RNA bacteriophage phi 6. We estimated the effects of deleterious mutations by performing mutation-accumulation experiments on five viral genotypes of decreasing fitness. We inferred positive epistasis because viral genotypes with low fitness were found to be less sensitive to deleterious mutations. We further examined environmental sensitivity in these genotypes and found that low-fitness genotypes were also less sensitive to environmental perturbations. Our results suggest that even random mutations impact the degree of canalization, the buffering of a phenotype against genetic and environmental perturbations. In addition, our results suggest that genetic and environmental canalization have the same developmental basis and finally that an understanding of the nature of epistasis may first require an understanding of the nature of canalization.

Bacteriophage phi 6↗
PubMed2004

Influence of BCR/ABL fusion proteins on the course of Ph leukemias.

The hallmark of chronic myeloid leukemia (CML) and a subset of acute lymphoblastic leukemia (ALL) is the presence of the Philadelphia chromosome as a result of the t(9;22) translocation. This gene rearrangement results in the production of a novel oncoprotein, BCR/ABL, a constitutively active tyrosine kinase. There is compelling evidence that the malignant transformation by BCR/ABL is critically dependent on its Abl tyrosine kinase activity. Also the bcr part of the hybrid gene takes part in realization of the malignant phenotype. We supposed that additional mutations accumulate in this region of the BCR/ABL oncogene during the development of the malignant blast crisis in CML patients. In ALL patients having p210 fusion protein the mutations were supposed to be preexisting. Sequencing of PCR product of the BCR/ABL gene (Dbl, PH region) showed that along with single-nucleotide substitutions other mutations, mostly deletions, had occurred. In an ALL patient a deletion of the 5th exon was detected. The size of the deletions varied from 36 to 220 amino acids. For one case of blast crisis of CML changes in the character of actin organization were observed. Taking into account the functional role of these domains in the cell an etiological role of such mutations on the disease phenotype and leukemia progression is plausible.

Actins↗
PubMed1987

Models of nearly neutral mutations with particular implications for nonrandom usage of synonymous codons.

The population dynamics of nearly neutral mutations are studied using a single-site and a multisite model. In the latter model, the nucleotides in a sequence are completely linked and the selection schemes employed are additive, multiplicative, and additive with a threshold. Although the third selection scheme is very different from the first two, the three schemes produce identical results for a wide range of parameter values. Thus the present study provides a general theory for the population dynamics of nearly neutral mutations because the results can also be used to draw inferences about other selection schemes such as stabilizing selection and synergistic selection. It is shown that the number of slightly deleterious mutations accumulated in a sequence can be considerably larger under the multisite model than under the single-site model, particularly if the sequence is long or if the mutation rate per site is high. The results show that even a very slight selective difference between synonymous codons can produce a strong bias in codon usage. Three alternative explanations for the strong bias in codon usage in bacterial and yeast genes are considered. The implications of the present results for molecular evolution are discussed.

Biological Evolution↗
PubMed2003

5-lipoxygenase-activating protein gene expression. Key role of CCAAT/enhancer-binding proteins (C/EBP) in constitutive and tumor necrosis factor (TNF) alpha-induced expression in THP-1 cells.

We examined expression of the 5-lipoxygenase activating protein (FLAP), which is critical for inflammatory cell leukotriene synthesis. A 3.4-kb segment of the FLAP gene 5'-untranslated region accounted for a 22-fold increase in promoter activity when transfected into the monocyte-like cell line, THP-1, and demonstrated no activity in non-inflammatory cells. Virtually all of the promoter activity was mediated by the first 134 bp upstream of the transcription start site, a region that contains CCAAT/enhancer-binding proteins (C/EBP) consensus binding sites, at -36 to -28 bp (distal) and -25 to -12 bp (proximal). DNase I footprint analyses demonstrated THP-1 nuclear extract proteins bind to the proximal site. Electrophoretic mobility shift assay analyses revealed that C/EBP alpha, delta, and epsilon bind to the proximal site and C/EBP alpha and epsilon bind to the distal site, constitutively. Transfection studies indicated that mutation of both the proximal and distal sites decreased constitutive FLAP promoter activity. Overexpression of C/EBP alpha, beta, and delta transactivated promoter activity and increased native FLAP mRNA accumulation. Mutation of both C/EBP sites essentially abolished promoter induction by C/EBP overexpression. Tumor necrosis factor (TNF) alpha induced FLAP mRNA expression, FLAP promoter activity, and C/EBP alpha, delta, and epsilon binding to the proximal and distal promoter consensus sites. Chromatin immunoprecipitation assays demonstrated that C/EBP alpha, delta, and epsilon bound to this region of the 5'-untranslated region, whereas C/EBP beta does not bind even under conditions of overexpression and stimulation. We conclude that the FLAP gene is transactivated by members of the C/EBP family of transcription factors in inflammatory cells and that these factors play an important role in FLAP gene induction by TNFalpha.

5' Untranslated Regions↗
PubMed2002

Amprenavir-resistant HIV-1 exhibits lopinavir cross-resistance and reduced replication capacity.

OBJECTIVES: To evaluate protease inhibitor (PI) cross-resistance and reductions in replication capacity conferred by amprenavir-selected mutations. METHODS: HIV-1IIIB variants derived from passage in increasing concentrations of amprenavir were studied, as well as 3'Gag/protease recombinants derived from them. These strains progressively accumulated mutations at codons 10, 46, 47, 50 and 84 in the protease as well as a p1/p6 cleavage site mutation at codon 449 in Gag. Their susceptibility (IC50) to various PI and their corresponding replication capacities were evaluated by a single-cycle growth assay and compared with measures using competitive cultures and p24 antigen production. RESULTS: Amprenavir susceptibility decreased with increasing numbers of protease mutations. Changes in lopinavir susceptibility paralleled changes in amprenavir susceptibility. Certain amprenavir-selected mutants conferred greater than 10-fold cross-resistance to lopinavir, including PrL10F/M46I/I50V-GagL449F (19-fold) and PrL10F/M46I/I47V/I50V-GagL449F (31-fold). Moreover, one isolate with only two mutations in the protease (L10F/84V) and GagL449F displayed a 7.7-fold increase in lopinavir IC50. Low-level cross-resistance to ritonavir and nelfinavir was also observed. The replication capacity of viruses containing either I84V or I50V was at least 90% lower than the reference virus in the single-cycle assay. The order of relative replication capacity was wild-type > L10F > L10F/I84V > L10F/M46I/I50V > L10F/M46I/I47V/I50V. CONCLUSION: These results indicate that until more comprehensive genotype-phenotype correlations between amprenavir and lopinavir susceptibility are established, phenotypic testing may be preferable to genotyping to detect cross-resistance, and should be considered when switching patients from a failing amprenavir-containing regimen. This study also provides data on the concordance of replication capacity measurements generated using rapid single-cycle growth and competition assays.

Carbamates↗