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Results for “mutational profiling”

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PubMed1999

Rapid F508del and F508C assay using fluorescent hybridization probes.

Amplification and fluorescent genotyping of the cystic fibrosis F508del locus was achieved from human genomic DNA in less than 30 min. The hybridization of adjacent fluorescent probes at the mutation site was monitored by resonance energy transfer between fluorescein and Cy5 during heating or cooling. Characteristic curves were obtained for each genotype; the first derivative of these fluorescent curves has a maximum at an apparent hybridization temperature (Tm) that is specific for each probe/allele duplex. The direction and rate of temperature change determines the difference between the apparent Tm and the true equilibrium Tm. One hundred and five sample were genotyped for the F508del cystic fibrosis mutation by heating and cooling curve profiles. These genotypes were validated by allele-specific amplification. Two fluorescein hybridization probes were designed to match the wild-type sequence perfectly from either codons 502 to 513 or from 504 to 511 on the cystic fibrosis transconductance regulator gene of chromosome 7. While genotyping for the F508del, an allele with the F508C base change was detected. For both F508del and F508C variants, the Tm shift from wild type was greater with a 24-mer probe than with a 35-mer probe. Fluorescent monitoring of hybridization probes is a versatile technique that can detect unexpected sequence alterations.

Alleles↗

PubMed1998

Sensitivity to cisplatin and platinum-containing compounds of Schizosaccharomyces pombe rad mutants.

The role of genes that affect response to radiation in determining sensitivity to platinum-containing compounds was studied using a panel of 23 strains of the yeast Schizosaccharomyces pombe. The radiation-hypersensitive mutants all had the same genetic background and most of them contained mutations that disabled either cell cycle checkpoints or DNA repair. The tested platinum compounds included cisplatin and two complexes containing diaminocyclohexane (oxaliplatin and tetraplatin), two ammine/cyclohexylamine complexes with different orientation of the leaving groups (JM216 and JM335) and a multinuclear platinum complex (BBR 3464). The cytotoxic effect of the selected platinum complexes was evaluated by using a microtiter growth inhibition assay with a 48 hr exposure to drug. The mutants fell into three groups with respect to sensitivity to cisplatin: four mutants (rad2, -7, -11, -15) exhibited minimal change in sensitivity; fifteen mutants (rad4-6, -8-10, -12-14, -16-17, -19-21, and -22) were 5.1-21.7-fold hypersensitive; only rad1 and -3 mutants, defective in checkpoints, and rad18, defective in repair, displayed a marked hypersensitivity. None of the mutants demonstrated appreciable change in sensitivity to JM216 presumably as a consequence of a lack of resistance of the wild-type strain, whereas a moderate increase in sensitivity to JM335 was observed for most of the mutants, and hypersensitivity to BBR3464 was observed only in rad1 and -3. No relevant changes in sensitivity to tetraplatin were observed. Most of the mutants, with the exception of rad2, -7, and -15, were hypersensitive to oxaliplatin. These findings demonstrate that specific mutations have disparate effects on the profile of sensitivity to different members of the same class of cytotoxic agents, which provides genetic evidence that different mechanisms are involved in differential cytotoxicity induced by Pt compounds. The results also demonstrate the utility of such a panel of mutants, constructed on the same genetic background, for detecting specific cellular response; presumably, this reflects the recognition or processing of specific DNA adducts. In conclusion, because the rad1 and rad3 gene products are determinants of cellular response to a large number of platinum-containing compounds, the present results support a critical role of genes involved in cell cycle control in cellular sensitivity to these agents.

Cell Cycle↗

PubMed2001

The islet amyloid polypeptide (amylin) gene S20G mutation in Chinese subjects: evidence for associations with type 2 diabetes and cholesterol levels.

BACKGROUND AND OBJECTIVES: There has been evidence that the S20G mutation in the islet amyloid polypeptide (amylin) gene may be associated with type 2 diabetes. In the present study, we investigated the distribution of the mutation in Hong Kong Chinese, and examined whether there was evidence for associations between the mutation and type 2 diabetes and/or metabolic profiles. SUBJECTS AND METHODS: This study involved 227 early and 235 late-onset (defined as onset age < or = 40 and > 40 years, respectively) type 2 diabetic patients, as well as 126 nondiabetic subjects. The mutation was detected using a PCR-RFLP method. RESULTS: We identified six (2.6%) and one (0.4%) patients heterozygous for the mutation from the early and late-onset groups, respectively (P = 0.05). None of the nondiabetic subjects had the mutation. Insulin deficiency and poor glycaemic control were not common findings amongst carriers of the mutation. In the early onset group, the patients with the mutation had lower plasma levels of total (4.3 +/- 0.9 mmol/l vs. 5.3 +/- 1.1 mmol/l, P = 0.02) and low density lipoprotein (LDL)-cholesterol (2.3 +/- 0.7 mmol/l vs. 3.2 +/- 0.9 mmol/l, P = 0.01) than those without the mutation. CONCLUSIONS: Our data suggest that the islet amyloid polypeptide gene mutation might be associated with early occurrence of type 2 diabetes and lower plasma levels of total and low density lipoprotein-cholesterol in the Chinese population.

Adolescent↗

PubMed2004

Selection and properties of Streptococcus thermophilus mutants deficient in urease.

Natural variations of the urea content of milk have a detrimental effect on the regularity of acidification by Streptococcus thermophilus strains used in dairy processes. The aim of the present study was to select urease-deficient mutants of S. thermophilus and to investigate their properties. Using an improved screening medium on agar plates, mutants were selected from 4 different parent strains after mutagen treatment and by spontaneous mutation. Most mutants were stable and had a phage sensitivity profile similar to that of their parent strain. Some of them contained detrimental secondary mutations, as their acidifying activity was lower than that of the parent strain cultivated in the presence of the urease inhibitor flurofamide. The proportion of this type of mutant was much lower among spontaneous mutants than among mutants selected after mutagen treatment. Utilization of urease-deficient mutants in dairy processes may have several advantages, such as an increase in acidification, an improved regularity of acidification, and a lower production of ammonia in whey.

Base Sequence↗

PubMed2006

C-kit gene mutation: common and widely distributed in intracranial germinomas.

OBJECT: Of the intracranial germ cell tumors (IGCTs), 10% of germinomas and most nongerminomatous tumors remain refractory to multimodality therapy. The authors investigated the mutation of c-kit and the expression of its product KIT in IGCTs to identify tumors susceptible to imatinib mesylate, a synthetic agent targeting KIT. METHODS: The authors investigated 26 IGCTs, including 13 germinomas, five mixed germ cell tumors (MGCTs), four immature teratomas (ITs), and two each of yolk sac tumors and choriocarcinomas. These tumors were examined for the expression of KIT and CD34 by immunohistochemical analysis, and for mutations in exons 2, 8 to 11, 13, and 17 of c-kit. Strong KIT expression was found in the cell membrane of germinomas (100%) and germinomatous cells of MGCTs (80%), as well as in the cytoplasm of epithelial and smooth-muscle cells of ITs. The membranous expression of CD34 was found in the nongerminomatous tumor cells and the chondrocytes of MGCTs (60%), ITs (100%), and a choriocarcinoma (50%), but not in germinomas and germinomatous cells. A total of five missense mutations distributed in exons 2, 11, 13, and 17 of c-kit were detected in three (23%) of the 13 germinomas. The novel mutations E73K, T96M (both in exon 2), and A636V (in exon 13) were detected in a single tumor. The presence or type of c-kit mutation was not correlated with patient prognosis. CONCLUSIONS: Immunohistochemical analysis of KIT expression is useful for the diagnosis of germinoma. This study may help in clarifying the pathogenesis of IGCTs and in identifying tumors susceptible to drugs targeting KIT.

Adolescent↗

PubMed2000

Nucleotide-induced conformational changes in P-glycoprotein and in nucleotide binding site mutants monitored by trypsin sensitivity.

Limited trypsin digestion was used to monitor nucleotide-induced conformational changes in wild-type P-glycoprotein (Pgp) as well as in nucleotide binding domain (NBD) Pgp mutants. Purified and reconstituted wild-type or mutant mouse Mdr3 Pgps were preincubated with different hydrolyzable or nonhydrolyzable nucleotides, followed by limited proteolytic cleavage at different trypsin:protein ratios. The Pgp tryptic digestion products were separated by SDS-PAGE followed by immunodetection with the mouse monoclonal anti-Pgp antibody C219, which recognizes a conserved epitope (VVQE/AALD) in each half of the protein. Different trypsin digestion patterns were observed for wild-type Pgp incubated with MgCl(2) alone, MgADP, MgAMP.PNP, MgATP, and MgATP + vanadate. A unique trypsin digestion profile suggestive of enhanced resistance to trypsin was observed under conditions of vanadate-induced trapping of nucleotides (MgATP + vanadate). The trypsin sensitivity profiles of Pgp mutants bearing either single or double mutations in Walker A (K429R, K1072R) and Walker B (D551N, D1196N) sequence signatures of NBD1 and NBD2 were analyzed under conditions of vanadate-induced trapping of nucleotides. The proteolytic cleavage pattern observed for the double mutants K429R/K1072R and D551N/D1196N, and for the single mutants K429R, K1072R, and D1196N were similar and clearly distinct from wild-type Pgp under the same conditions. This is consistent with the absence of ATP hydrolysis and of vanadate-induced trapping of 8-azido-ADP previously reported for these mutants [Urbatsch et al. (1998) Biochemistry 37, 4592-4602]. Interestingly, the trypsin digestion profiles observed under vanadate-induced trapping for the D551N and D1196N mutants were quite different, with the D551N mutant showing a profile resembling that seen for wild-type Pgp. The different sensitivity profiles of Pgp mutants bearing mutations at the homologous residue in NBD1 (D551N) and NBD2 (D1196N) suggest possible structural and functional differences between the two sites.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2006

Identification of 16 new disease-causing mutations in the CPT2 gene resulting in carnitine palmitoyltransferase II deficiency.

The exonic regions of the carnitine palmitoyltransferase 2 (CPT2) gene were characterized from 101 patients with defined clinical and biochemical evidence for the adult onset form of CPT II deficiency and in 2 patients detected as newborns with abnormal acylcarnitine profiles. Twenty-seven disease-causing mutations within the CPT2 gene were identified in this cohort, 16 of which were novel. A total of 60 disease-causing mutations have been identified to date in CPT2 and 41 of these are predicted to produce amino acid substitution/deletions. The implications of these mutations are described in light of recent advances in our understanding of the molecular structure of members of the carnitine acyltransferase family.

Amino Acid Sequence↗

PubMed2026

Age-related genomic characterization and therapeutic targets in Chinese breast cancer: insights from prospective targeted sequencing and clinical data analysis.

BACKGROUND: In China, breast cancer occurs at a much younger age and has a higher recurrence and mortality rate. However, with changes in lifestyle, there has been a trend towards an older age of breast cancer incidence in Chinese women. There is a paucity of large-scale next-generation sequencing cohorts for the analysis of genomic characterization in these populations and the identification of potential therapeutic targets. METHODS: To address this gap, we performed prospective targeted sequencing of tumor and blood samples from Chinese patients and collected detailed clinical information. We then categorized patients into two groups based on age (< 40 years, n = 637; ≥ 40 years, n = 3442) and proceeded to provide comprehensive descriptions of somatic and germline mutations in both groups. RESULTS: The somatic mutation analysis revealed that PIK3CA, FOXA1, and TBX3 mutations were more prevalent in elderly patients. By leveraging the aforementioned mutational characteristics, we employed our institution's FUTURE-SUPER clinical trial, an umbrella study targeting metastatic breast cancer, to confirm the potential benefits of PI3K-AKT-mTOR pathway inhibitors among elderly patients with breast cancer. Furthermore, TP53 and ERBB2 were more likely to be co-mutated in young women. Patients with TP53 and ERBB2 co-mutation tend to have a poorer prognosis, but through investigation of the SPARK cohort, patients carrying the TP53 and ERBB2 co-mutation are more likely to benefit from immune checkpoint inhibitor combination with tyrosine kinase inhibitor therapy. In our study, we observed a higher frequency of mutations in the DNA homology-dependent recombination pathway in young patients with breast cancer, which was associated with an elevated Ki67 index. Additionally, we confirmed a significant prevalence of germline breast cancer susceptibility gene 1 (gBRCA1) mutations in young patients, whereas germline checkpoint kinase 2 (gCHEK2) mutations are more common in elderly patients. CONCLUSIONS: Our study, which makes use of the largest Chinese breast cancer sequencing cohort, sought to characterize the age-related genomic profile of breast cancer patients and identify novel therapeutic opportunities for individuals with breast cancer.

Adult↗

PubMed1992

Phenotypic heterogeneity associated with defective apolipoprotein B-100 and occurrence of the familial hypercholesterolemia phenotype in the absence of an LDL-receptor defect within a Canadian kindred.

Of 163 individuals with a diagnosis of heterozygous familial hypercholesterolemia (FH), only one subject was found to be positive for familial defective apo B-100 (FDB). The eight-member kindred ascertained through this subject who presented with both a clinical phenotype of FH and the FDB apo B-100 (Arg3500----Gln) mutation was studied. Plasma lipid and lipoprotein profiles, apo E phenotypes, apo B gene markers at the 3' hypervariable region and LDL-receptor haplotypes (ApaLI, PvuII, NcoI), were determined, together with LDL-receptor activity on freshly isolated blood lymphocytes. The FDB mutation, present in four relatives, was associated with three different phenotypes: FH and severe hypercholesterolemia, moderate hypercholesterolemia and normolipidemia. The FH phenotype occurred in the absence of any functional LDL-receptor defect. In homozygotes for the absence of the PvuII cutting site who had the apo B mutation, LDL-cholesterol levels were low in the presence of the apo E3/2 phenotype and high in the presence of the apo E4/4 phenotype. None of the major known environmental influences accounted for the wide range of variation in LDL-cholesterol among the affected members. Further observations in the spouse and offspring of the normolipidemic FDB subject confirmed the association of apo E4, the FDB mutation and the PvuII(-/-) genotype with high cholesterol levels. It is concluded that the phenotypic expression of the FDB mutation may vary widely as a function of the genetic environment within a family.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed2006

Mutations within the kinase domain and truncations of the epidermal growth factor receptor are rare events in bladder cancer: implications for therapy.

PURPOSE: It has previously been reported that the patient response to gefitinib depends on the presence of mutations within the kinase domain of epidermal growth factor receptor (EGFR) or the expression of its truncated form, EGFR variant III (EGFRvIII). The focus of this study was to determine if these alterations are present within the tyrosine kinase and ligand-binding domain of EGFR in urothelial carcinoma. EXPERIMENTAL DESIGN: The kinase domain found within exons 18 to 21 of the EGFR from 11 bladder cancer cell lines and 75 patient tumors were subjected to automated sequencing. EGFRvIII expression was determined by immunohistochemistry using a urothelial carcinoma tissue microarray, and its expression was subsequently verified by reverse transcription PCR, real-time PCR, and Western blot analysis, using an EGFRvIII-transfected glioblastoma cell line and glioblastoma tumors as positive controls. RESULTS: Our analysis failed to detect mutations within the tyrosine kinase domain of EGFR in the 11 cell lines and 75 patients tested. The initial analysis of EGFRvIII expression by immunohistochemistry revealed that at least 50% of the patient tumors expressed EGFRvIII in a urothelial carcinoma tissue microarray. Conflicting reports exist, however, regarding the extent of EGFRvIII expression in tissues owing to the specificity of the antibodies and the methodologies used. Therefore, we sought to validate this observation by reverse transcription PCR, real-time PCR, and Western blot analysis. In these assays, none of the samples were positive for EGFRvIII except for control transfectants and glioblastomas. CONCLUSIONS: When our results are taken together, we conclude that alterations within the tyrosine kinase domain and expression of EGFRvIII are rare events in bladder cancer. The present study has clinical implications in selecting tyrosine kinase inhibitors for the therapy of urothelial carcinoma.

Blotting, Western↗

PubMed2003

Methods for the molecular analysis of cancer. An overview.

Cancer arises as a result of complex and interacting abnormalities. However, over the past 20-25 yr numerous technological advances in molecular biology have led to a dramatic increase in the identification of the molecular processes involved in the development of cancers. The analysis of these molecular changes can be done on different levels: at the DNA or RNA level, or by assessment of posttranscriptional events. This overview discusses the merits of different methods for the analysis of DNA such as SSCP or DGCE. The exciting methods of RNA expression analysis using oligo or cDNA gene chips are also discussed. The importance of methods to analyze posttranscriptional or the effect that altered telomerase activity or methylation status contributes to the phenotype of the cancer cell is emphasized. These techniques will contribute to a better understanding of cancer initiation and progression and will eventually lead toward the development of new molecular-targeted drug therapies.

Animals↗

PubMed1995

Novel pattern of P53 mutation in breast cancers from Austrian women.

Since mutagens produce an extraordinary diversity of mutational patterns, differential mutational exposures among populations are expected to produce different patterns of mutation. Classical epidemiological methods have been successful in implicating specific mutagens in cancers such as those of lung and skin in which one mutagen predominates. In breast cancer, however, no mutagens have been implicated in an unequivocal manner. In an attempt to facilitate epidemiological studies, we have been studying the pattern of p53 gene mutations in breast cancers from multiple populations with high and low breast cancer incidences. We previously reported that breast cancers from Midwest United States, predominantly rural Caucasian women, have a different pattern of p53 gene mutation from populations of Western European women. Herein, we analyze patterns of p53 mutations from Graz, Austria, another population with a high incidence of breast cancer. Among the 60 Austrian breast cancers analyzed, 14 (23%) have a p53 gene mutation in exons 5-9 or in adjacent splice junctions. Analysis of the patterns of mutation shows differences between the "Western European" profile and the Austrian and Midwest United States groups (P = 0.027 and 0.024, respectively). The Austrian pattern is characterized by a high frequency of A:T-->T:A transversions (P = 0.006). The presence of distinct patterns of mutation among the limited number of analyzed populations of Western European origin supports the idea that differential mutagenic exposure and/or genetic differences contribute to breast cancer mutagenesis among geographically distinct Caucasians of Western European origin.

Aged↗

PubMed1991

Characterization of the spontaneous mutant H-2Kbm29 indicates that gene conversion in H-2 occurs at a higher frequency than detected by skin grafting.

A spontaneous mutation of H-2Kb, Kbm29, was discovered among the progeny of F1 hybrid parents. Unlike other characterized spontaneous class I variants, this mutant was detected with the use of antibody, rather than tissue grafting. Although Kbm29 is serologically indistinguishable from the previously described mutant molecule Kbm3, it is identical to the parental Kb by skin grafting and CTL assays. A full length cDNA of Kbm29 was amplified by polymerase chain reaction with locus-specific primers, cloned, and sequenced. Two nucleotides were found to be mutated, resulting in a single amino acid change (Lys----Ala) at amino acid 89 of the mature glycoprotein. This is consistent with the observed serologic changes, as the same amino acid substitution is responsible for the serologic profile of Kbm3. The occurrence of a mutation which is not detectable by the methods normally used to screen for H-2 mutants provides evidence that the high spontaneous rate of structural mutation described for the Kb molecule is underestimated.

Amino Acid Sequence↗

PubMed2005

A CLN5 mutation causing an atypical neuronal ceroid lipofuscinosis of juvenile onset.

Three related patients from Colombia presented with a juvenile-onset neuronal ceroid lipofuscinosis. Electron microscopy of one case showed condensed fingerprint profiles, and genetic analyses identified a novel missense mutation in CLN5. The authors demonstrate the existence of pathogenic CLN5 mutations outside northern Europe and that mutations in this gene can lead to an atypical late-onset neuronal ceroid lipofuscinosis disease, in addition to the late infantile form first described in Finland.

Amino Acid Sequence↗

PubMed2000

[Cloning of cancer-related genes of Rat6 fibroblasts by using an improved differential display method].

The p53 gene is the most frequently mutated gene identified so far in human cancers. When a mutant p53(135)-val gene was allowed to be over-expressed in Rat6(R6) cells, a high incidence of spontaneous transformation was observed in long-term culture of this cell line(R6 # 13-8). To identify genes involved in cell transformation, parental p53 over-expressing cell, R6 # 13-8, and its spontaneous transformant T2, were analyzed by an improved mRNA differential display technique, which was reproducible, simpler, and was able to clone cDNA longer than 500 bp, and was with less false positives. When 33 10-mer or 12-mer single primers with arbitrary but defined sequence were used for PCR, over 90 discrete cDNAs were obtained from R6 # 13-8 and T2 cells. Three differentially expressed cDNAs were identified, one of them is highly expressed in T2 cells, while the other two, 0.8 kb and 0.9 kb long, are highly expressed in R6 # 13-8 cells. The latter were cloned and confirmed by Northern hybridization. Both cloned fragment were not homologous with any published sequence. Our results suggest that the activation and inactivation of genes are involved in the process of the spontaneous transformation from R6 # 13-8 to T2.

Animals↗

PubMed2003

High affinity for farnesyltransferase and alternative prenylation contribute individually to K-Ras4B resistance to farnesyltransferase inhibitors.

Farnesyltransferase inhibitors (FTIs) block Ras farnesylation, subcellular localization and activity, and inhibit the growth of Ras-transformed cells. Although FTIs are ineffective against K-Ras4B, the Ras isoform most commonly mutated in human cancers, they can inhibit the growth of tumors containing oncogenic K-Ras4B, implicating other farnesylated proteins or suggesting distinct functions for farnesylated and for geranylgeranylated K-Ras, which is generated when farnesyltransferase is inhibited. In addition to bypassing FTI blockade through geranylgeranylation, K-Ras4B resistance to FTIs may also result from its higher affinity for farnesyltransferase. Using chimeric Ras proteins containing all combinations of Ras background, CAAX motif, and K-Ras polybasic domain, we show that either a polybasic domain or an alternatively prenylated CAAX renders Ras prenylation, Ras-induced Elk-1 activation, and anchorage-independent cell growth FTI-resistant. The polybasic domain alone increases the affinity of Ras for farnesyltransferase, implying independent roles for each K-Ras4B sequence element in FTI resistance. Using microarray analysis and colony formation assays, we confirm that K-Ras function is independent of the identity of the prenyl group and, therefore, that FTI inhibition of K-Ras transformed cells is likely to be independent of K-Ras inhibition. Our results imply that relevant FTI targets will lack both polybasic and potentially geranylgeranylated methionine-CAAX motifs.

3T3 Cells↗

PubMed2002

Effects of mutations of a glutamine residue in loop D of the alpha7 nicotinic acetylcholine receptor on agonist profiles for neonicotinoid insecticides and related ligands.

1. Neonicotinoid insecticides are agonists of insect nicotinic acetylcholine receptors (AChRs) and show selective toxicity for insects over vertebrates. To elucidate the molecular basis of the selectivity, amino acid residues influencing neonicotinoid sensitivity were investigated by site-directed mutagenesis of the chicken alpha7 nicotinic AChR subunit, based on the crystal structure of an ACh binding protein (AChBP). 2. In the ligand binding site of AChBP, Q55 in loop D is close to Y164 in loop F that corresponds to G189 of the alpha7 nicotinic receptor. Since Q55 of AChBP is preserved as Q79 in the alpha7 nicotinic receptor and the G189D and G189E mutations have been found to reduce the neonicotinoid sensitivity, we investigated effects of Q79E, Q79K and Q79R mutations on the neonicotinoid sensitivity of the alpha7 receptor expressed in Xenopus laevis oocytes to evaluate contributions of the glutamine residue to nicotinic AChR-neonicotinoid interactions. 3. The Q79E mutation markedly reduced neonicotinoid sensitivity of the alpha7 nicotinic AChR whereas the Q79K and Q79R mutations increased sensitivity, suggesting electronic interactions of the neonicotinoids with the added residues. 4. By contrast, the Q79E mutation scarcely influenced responses of the alpha7 nicotinic receptor to ACh, (-)-nicotine and desnitro-imidacloprid (DN-IMI), an imidacloprid derivative lacking the nitro group, whereas the Q79K and Q79R mutations reduced the sensitivity to these ligands. The results indicate that the glutamine residue of the alpha7 nicotinic receptor is likely to be located close to the nitro group of the insecticides in the nicotinic receptor-insecticide complex.

Amino Acid Sequence↗

PubMed2004

Apoptotic signaling pathways induced by nitric oxide in human lymphoblastoid cells expressing wild-type or mutant p53.

Loss of p53 function by inactivating mutations results in abrogation of NO*induced apoptosis in human lymphoblastoid cells. Here we report characterization of apoptotic signaling pathways activated by NO* in these cells by cDNA microarray expression and immunoblotting. A p53-mediated transcriptional response to NO* was observed in p53-wild-type TK6, but not in closely related p53-mutant WTK1, cells. Several previously characterized p53 target genes were up-regulated transcriptionally in TK6 cells, including phosphatase PPM1D (WIP1), oxidoreductase homolog PIG3, death receptor TNFRSF6 (Fas/CD95), and BH3-only proteins BBC3 (PUMA) and PMAIP1 (NOXA). NO* also modulated levels of several gene products in the mitochondria-dependent and death-receptor-mediated apoptotic pathways. Inhibitors of apoptosis proteins X-chromosome-linked inhibitor of apoptosis, cellular inhibitor of apoptosis protein-1, and survivin were significantly down-regulated in TK6 cells, but not in WTK1 cells. Smac release from mitochondria was induced in both cell types, but release of apoptosis-inducing factor and endonuclease G was detected only in TK6 cells. Fas/CD95 was increased, and levels of the antiapoptotic proteins Bcl-2 and Bcl-x/L were reduced in TK6 cells. Activation of procaspases 3, 8, 9, and 10, as well as Bid and poly(ADP-ribose) polymerase cleavage, were observed only in TK6 cells. NO* treatment did not alter levels of death receptors 4 and 5, Fas-associated death domain or proapoptotic Bax and Bak proteins in either cell line. Collectively, these data show that NO* exposure activated a complex network of responses leading to p53-dependent apoptosis via both mitochondrial and Fas receptor pathways, which were abrogated in the presence of mutant p53.

Apoptosis↗