Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.30.2 (endonuclease)
18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Upon exposures of Ehrlich ascites carcinoma cells to heat shock (44 degrees, 1 hr), oxidative stress or energy deprivation, their DNA undergoes fragmentation (35-45% after 5 hrs of incubation) which is considered as a hallmark of apoptosis. Prior to DNA fragmentation the cells exhibited blebbing (55-90% after 1 hr), thus being suggestive of cytoskeletal damage and a 1.5-2-fold increase in the Triton-insoluble protein concentration (protein aggregation) after 3 hrs. Rapid cell death (75% after 4 hrs) occurred only under oxidative stress. Electrophoresis of the Triton-insoluble protein fraction revealed that the common feature of all stress exposures used in this study was a dramatic increase in the aggregation of cytoskeletal proteins--actin and the 57 kDa protein. No dependence of DNA fragmentation on intracellular Ca2+ increase was found. Both DNA fragmentation and protein aggregation were suppressed by glucose, whereas Zn2+, an endonuclease inhibitor, suppressed only DNA fragmentation without any effect on protein aggregation. It is suggested that cytoskeletal damage may trigger tumor cell apoptosis.
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PMID:[Fragmentation of Ehrlich ascites carcinoma DNA during influences causing aggregation of cytoskeletal proteins]. 801 77

To date, apoptosis has been characterized biochemically by the production of 180-200 bp internucleosomal DNA fragments resulting from the activation of an endonuclease(s). The principal morphological feature of apoptosis is the condensation of chromatin and it has been assumed that this may reflect the oligonucleosomal fragmentation pattern. We have re-examined this dogma by comparing the biochemical and morphological features of cell death in several epithelial cell types (HT-29-I1 colon adenocarcinoma, CC164 mink lung, DU-145 human prostatic carcinoma and MCF-7 human breast adenocarcinoma) and one mesenchymal cell line (H11ras-R3 ras-transformed rat fibroblasts). Cell death was induced either by serum deprivation, TGF-beta 1 or etoposide, or by leaving cells to reach confluence. Cell death was assessed with respect to detachment from monolayers, morphological changes and DNA integrity. The DNA-binding fluorophore Hoechst 33258 revealed chromatin condensation patterns consistent with apoptotic cell death in all cell types except MCF-7 cells. Using field inversion gel electrophoresis in conjunction with conventional 2% agarose gel electrophoresis, cleavage of DNA to 50 kbp fragments was observed in all cases except MCF-7 cells. This preceded the appearance of oligonucleosomal fragments in HT-29-I1, CC164 and H11ras-R3 cells. Although the DNA of DU-145 cells fragmented into 50 kbp units, and although the cells exhibited classical apoptotic morphology, no subsequent internucleosomal cleavage was observed. These results suggest that changes in the integrity of DNA indicative of the release of chromatin loop domains occur before cleavage at internucleosomal sites is initiated and that the latter is not an essential step in the apoptotic process.
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PMID:Apoptotic death in epithelial cells: cleavage of DNA to 300 and/or 50 kb fragments prior to or in the absence of internucleosomal fragmentation. 825 89

The cytologic diagnosis of pancreatic carcinoma is notoriously difficult, particularly in distinguishing benign atypia from well-differentiated adenocarcinoma. Mutation of codon 12 in the K-ras oncogene is frequently found with pancreatic cancers. Detection by polymerase chain reaction (PCR) followed by restriction endonuclease digestion can provide a powerful tool to improve and confirm diagnosis. The authors examined the utility of PCR-based detection in the diagnosis of pancreatic carcinoma using routinely obtained cytology smears that could be collected at most hospitals. Pancreatic cytology smears were collected retrospectively from 60 patients. DNA was extracted from the slides and amplified by PCR using mismatched primers that generated a Bst-N1 recognition site with the wild type codon 12 but not with the mutant allele. Results were compared with clinical follow-up. K-ras codon 12 mutations were observed in 44 of 46 (95.7%) cases of pancreatic cancer, but not in 12 benign cases nor in 2 cases of islet cell tumor. The amplification and digestion steps proved robust and sensitive, capable of detecting mutant K-ras alleles from cytology smears that contained only small foci of suspicious cells. Our results indicate that K-ras mutation analysis can be done reliably within 1 to 2 days from routine cytology slides without special handling, increasing the sensitivity of diagnosis in ambiguous cases while maintaining cost-effective and relatively noninvasive sampling strategy.
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PMID:Polymerase chain reaction-based K-ras mutation detection of pancreatic adenocarcinoma in routine cytology smears. 860 3

The beta chain of human chorionic gonadotropin (hCG) hormone is produced by fetal cells, gonadal cell tumors and several types of non-gonadal carcinoma. hCG is composed of an alpha and a beta chain, the latter of which can be used to distinguish the molecule from other related gonadotropin hormones. Detection of beta-hCG mRNA transcripts can be potentially useful as a marker to identify tumor cells. We devised a highly specific and sensitive assay to detect the atavistic expression of beta-hCG in cutaneous melanoma by RT-PCR. Twenty-four melanoma cell lines and 43 melanoma biopsies were evaluated for beta-hCG mRNA expression. An RT-PCR assay was developed to specifically distinguish beta-hCG poly-A mRNA from other related gonadotropin beta chains. This was performed by endonuclease digestion of a unique Sty 1 site in the beta chain, followed by Southern blot analysis with a beta-hCG cDNA probe. Of the 24 melanoma cell lines analyzed, 18 expressed beta-hCG mRNA. Analysis of melanoma biopsy specimens revealed beta-hCG mRNA expression in 17/25 melanoma-positive TDLN, and in only 5/15 non-lymphoid melanoma metastases. Beta-hCG mRNA expression had a 53% correlation to tyrosinase mRNA, a predominant melanoma marker. Beta-hCG mRNA was not detected in normal donor PBL and normal lymph nodes. Detection of beta-hCG mRNA expression may be a useful molecular marker to define a subset of malignant melanoma.
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PMID:Detection of beta-human chorionic gonadotropin mRNA as a marker for cutaneous malignant melanoma. 862 Dec 27

Higher order DNA fragmentation may be an essential signal in apoptosis. We found that etoposide (VP-16) induced apoptosis in human DU-145 prostatic carcinoma cells in a time- and concentration-dependent manner. Chromatin condensation was morphologically evident only when cells detached from the monolayer; untreated or VP-16-treated attached cells retained a normal morphology. We describe a radiolabeled alu-I sequence-based quantitative field inversion gel electrophoresis (QFIGE) method that permitted observation and quantification of discrete high molecular weight DNA fragments in detached (apoptotic) and attached (preapoptotic) DU-145 cells. The DNA fragments generated during the apoptotic death of these cells were > or = 1 (mega-base pairs) mbp, 450-600 (kilo-base pairs) kbp, and 30-50 kbp; we observed that these DNA fragments increased 9 +/- 2-, 8 +/- 2-, and 25 +/- 11-fold versus control, respectively, with a 24-hr exposure to 30 microM VP-16 in attached cell populations. In detached VP-16-treated cells, there was accrual of 30-50-kbp DNA fragments with a concomitant loss of the > or = 1-mbp and 450-600-kbp fragments; internucleosomal DNA cleavage was never observed. This pattern of high molecular weight DNA fragmentation was inhibited by cycloheximide treatment and was common to other apoptotic agents, including melphalan and bleomycin. These findings suggest that the > or = 1-mbp and 450-600-kbp DNA fragments are products of endonuclease activation and are not topoisomerase II/DNA interactions. Finally, the generation of the 30-50-kbp DNA fragments may mediate chromatin condensation, which characterizes apoptosis.
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PMID:Genesis of discrete higher order DNA fragments in apoptotic human prostatic carcinoma cells. 863 56

The role of G protein mutations in the pathogenesis of adrenal cortex neoplasms is controversial. Two published studies disagree on the existence of a cysteine or histidine for arginine substitution at position 179 (R179C/H) of the GTP binding region of the alpha chain of an inhibitory G protein (Gi2alpha) in these tumors. Prior studies using detection by mutation-specific oligonucleotide hybridization showed either 3 of 11 or 0 of 56 tumors harbored mutations. To resolve this discrepancy and ascertain the importance of the R179C/H Gi2alpha mutation in the development of adrenal cortex tumors, we screened tumors from 29 patients (24 with adenoma, 5 with carcinoma) using a more sensitive assay employing polymerase chain reaction (PCR) and examination for restriction fragment length polymorphisms (RFLP). Detection of the potential R179C/H mutation by this technique was possible because the wild-type coding sequence includes the BSTU-1 restriction endonuclease recognition site CGCG, whereas the mutated gene does not. Results showed complete digestion of the amplified DNA samples from all 29 patients and the negative control DNA by BSTU-1, indicating that all tumor samples exhibited only the wild-type sequence. Direct sequencing of PCR product from four tumor samples confirmed the presence of only the wild-type sequence. The 0 of 29 rate of R179C/H mutations we found in Gi2alpha is different than the 3 of 11 positive rate (p < 0.05, Fishers' exact) previously reported but agrees with the report showing 0 of 56 mutations. We conclude a mutation at position 179 of Gi2alpha is not important in the pathogenesis of most adrenal cortical tumors.
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PMID:Gip-2 codon 179 oncogene mutations: absent in adrenal cortical tumors. 867 43

The induction of cell death along with cell-cycle arrest is one of the foremost mechanisms regulating cell growth. In the human breast carcinoma cell line MCF-7 we investigated two chemotherapeutic agents, the antiestrogen tamoxifen and the DNA-damaging drug cisplatin, for the relative contribution of these mechanisms to growth inhibition in culture. Growth kinetics and flow cytometry confirmed that tamoxifen at 1 microM acts mainly by arresting cells in the G0/G1 phase of the cell cycle. Compared to untreated controls, only a few more cells were detached from the monolayer and dead after a 5-day incubation. On the other hand, cisplatin at 1 microM did not induce the well-defined G2/M-arrest reported for other cell types, but resulted in a marked increase in the rate of cell death. A morphological feature observed, especially with cisplatin-treated MCF-7 cells, was the formation of numerous micronuclei (in up to 30% of the cells) and an increase in the number of binucleate cells (up to 20%). In both tamoxifen- and cisplatin- treated cultures, cell death appeared to occur by apoptosis, as indicated morphologically by cellular and nuclear shrinkage accompanied by DNA-condensation and ultimately the formation of DNA containing apoptotic bodies. However, no internucleosomal DNA degradation or endogenous endonuclease activity could be detected in the cells of the monolayer or in the mainly dead and detached cells of the culture supernatant. DNA fragmentation was only observed when isolated MCF-7 nuclei were incubated with exogenous endonucleases. However, as determined by reverse transcriptase/polymerase chain reaction amplification, MCF-7 cells do express the mRNA for DNase I, an endonuclease known to be involved in apoptosis. Thus, apoptosis is part of the growth-inhibitory process and occurs without apparent internucleosomal DNA fragmentation in MCF-7 cell cultures.
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PMID:Cell-cycle arrest, micronucleus formation, and cell death in growth inhibition of MCF-7 breast cancer cells by tamoxifen and cisplatin. 887 58

Curcumin (diferuloyl methane) is the major active yellow pigment of turmeric and curry. Studies in recent years have indicated that curcumin is a potent inhibitor of the initiation and promotion of chemical carcinogen-induced skin carcinogenesis in mice. When COLO205 colorectal carcinoma cells were treated with curcumin (60 microM), the appearance of apoptotic DNA ladders was delayed about 5 h, and G1 arrest was detected. Further analysis of the endonuclease activities in these cells revealed that the activity of Ca(+2)-dependent endonuclease in COLO205 cells was profoundly inhibited and that the extent of inhibition depended on the degree of calcium depletion. The reduction of p53 gene expression was accompanied by the induction of HSP70 gene expression in the curcumin-treated cells. These findings suggest that curcumin may induce the expression of the HSP70 gene through the initial depletion of intracellular Ca(+2), followed by the suppression of p53 gene function in the target cells.
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PMID:Induction of HSP70 gene expression by modulation of Ca(+2) ion and cellular p53 protein by curcumin in colorectal carcinoma cells. 898 16

We report 8 newly established gastric-carcinoma cell lines (SNU-216, 484, 520, 601, 620, 638, 668, 719) from Korean patients. Morphologic study was carried out using light and electron microscopes. CEA, alpha FP, and CA 19-9 and TPA in supernatant and in cell lysate were measured by radioimmunoassay. p53 and c-Ki-ras gene mutations were screened and confirmed by sequencing. The cell lines, derived from tumors with moderate differentiation, grew as a diffuse monolayer, and those from tumors with poor differentiation and minimal desmoplasia grew exclusively as non-adherent. Out of the 8 gastric-cancer cell lines, 5 had detectable levels of CEA both in supernatant and in cell lysate; there was no expression or secretion of alpha FP in these cells; 4 cell lines showed high levels of CA 19-9 in cell pellets. All cell lines except SNU-484 had high concentrations of TPA both in cell lysate and in supernatants. p53 mutation was found in 6 cell lines (75%): 2 (SNU-216 and SNU-668) had mutations in exon 6, and other 3 in exon 8. The c-Ki-ras mutation was found in 2 cell lines (25%), SNU-601 and SNU-668. The former showed GGT-to-GAT transition mutation at codon 12, while the latter showed CAA-to-AAA transversion mutation at codon 61. DNA profiles using restriction endonuclease HinfI and polymorphic DNA probes ChdTC-15 and ChdTC-114 showed different unique patterns; which suggests that these cell lines are unique and not cross-contaminated. We believe that the newly characterized gastric-cancer cell lines presented in this paper will provide a useful in vitro model for studies related to human gastric cancer.
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PMID:Establishment and characterization of human gastric carcinoma cell lines. 903 53

Although molecular alterations involved in the development of squamous cell carcinoma of the cervix have been extensively described, these genetic changes have not been as well characterized in the development of cervical adenocarcinoma. Twenty-seven paraffin-embedded adenocarcinomas of the cervix, including three cases of adenoma malignum, were analyzed for molecular alterations associated with other gynecologic malignancies. The presence of human papillomavirus (HPV) was assessed by polymerase chain reaction (PCR) using internally nested consensus primers. HPV types were identified by restriction endonuclease digestion of the PCR products, using DNA sequencing to confirm each digestion pattern. The presence of HPV was correlated with immunohistochemical expression of the p53 gene product, the presence of mutations in codon 12 of Ki-ras, and allelic deletion of markers associated with the development of other gynecologic carcinomas. HPV was identified in 16 (59%) of 27 cases, including type 18 in 7 tumors, type 16 in 7 tumors, and type 45 in 2 tumors. HPV types 16 and 45 were always identified in adjacent uninvolved cervical epithelium, but HPV type 18 was absent from the adjacent non-neoplastic epithelium in four of the seven positive cases. HPV was not identified in any of three cases of adenoma malignum. Diffuse immunohistochemical staining of the p53 gene product was present in only one (HPV-negative) tumor. A mutation in codon 12 of Ki-ras was observed in one endocervical adenocarcinoma (with an endometrioid pattern). Loss of heterozygosity was identified only for a marker on chromosome 6p in one mucinous endocervical carcinoma. Most endocervical adenocarcinomas lack molecular alterations characteristic of other histologically similar gynecologic malignancies, as well as those described in cervical squamous cell carcinomas.
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PMID:Analysis of human papillomavirus infection and molecular alterations in adenocarcinoma of the cervix. 1061 75


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