Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.64 (proteinase K)
 4,071 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Androgen receptor-acceptor complexes in nuclei from rat ventral prostates were cross-linked in situ with formaldehyde and partially purified using affinity chromatography. To isolate acceptor DNA, the cross-linked receptor-acceptor complexes in formaldehyde-treated chromatin samples were adsorbed to dihydrotestosterone-17 beta-succinyl agarose, eluted with 75 microM dihydrotestosterone-1% SDS, digested with proteinase K and extracted with phenol-chloroform. After 32P end-labelling and PAGE, this DNA contained two distinct bands of DNA (about 300 and 400 base pairs respectively) which were unique relative to the total prostatic DNA. As an alternative approach for characterizing acceptor DNA, the DNA in prostatic nuclei and cross-linked chromatin was labelled with 32P by nick translation and analysed in glycerol density gradients for associations with cross-linked androgen receptors. A symmetrical 7s peak of 32P-DNA with a small amount of coincident receptor was observed in the gradients after mild trypsin treatment. In the absence of trypsin treatment, both the cross-linked receptors and the labelled DNA sedimented to the bottom of the gradients. Isolation of acceptor proteins involved iodination of cross-linked chromatin with 125I and androgen affinity chromatography. A comparison of the relative efficiency of retention and elution of 125I-proteins from different affinity columns revealed that testosterone-17 beta-succinyl agarose was potentially most suitable for purification of acceptor proteins. After electrophoresis on SDS-polyacrylamide gels, the eluates from this type of affinity matrix were found to contain two major peaks of 125I-labelled proteins--one corresponding to a protein with a similar molecular weight as the nuclear androgen receptor (33,000 Da); the other having a molecular weight of 20,000 Da. While the precise identity of this latter entity is unknown, its enrichment and retention by the affinity gel implies that it is closely associated with the androgen receptor and may be a component of the acceptor sites.
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PMID:DNA and protein components of nuclear acceptor sites for androgen receptors in the rat prostate. 369 93

Desmoid tumor is a locally aggressive, nonmetastasizing soft tissue tumor. Whether desmoid tumor is a truly neoplastic cellular proliferative process or, alternatively, an unchecked reactive process has been a subject of debate. In order to determine whether desmoid tumor is composed of a clonal cell population as opposed to being a polyclonal reactive process, analysis of patterns of X-chromosome inactivation was performed. Hematoxylin and eosin stained sections of paraffin-embedded, formalin-fixed tissues were microdissected to obtain both lesional and normal control samples, and the genomic DNAs were extracted by proteinase K digestion. Following treatment with methylation sensitive restriction endonuclease (Hha I or Hpa II), the genomic DNAs were amplified by polymerase chain reaction (PCR), using nested primers targeted to a highly polymorphic short tandem repeat (STR) of the human androgen receptor (HUMARA). In eight of 12 cases, PCR amplification of the genomic DNAs was successful, and all eight of the amplified cases were heterozygous in the size of the HUMARA target. The remaining cases could not be studied because of failure to amplify DNA. Following digestion with HhaI or Hpa II, uniform patterns of X-chromosome inactivation were found in all eight desmoid tumors, whereas normal control tissue remained heterozygous. These results confirm a clonal composition of the tumors. The demonstration of clonality in the tumors in all eight informative cases indicates that desmoid tumor is a true neoplastic process, not an unchecked polyclonal reactive process.
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PMID:Desmoid tumor is a clonal cellular proliferation: PCR amplification of HUMARA for analysis of patterns of X-chromosome inactivation. 906 Jun

The pathogenesis of carcinosarcoma is still a subject of controversy. In the present study, molecular techniques were applied to determine the pathogenesis of uterine carcinosarcomas. The patterns of chromosome X inactivation were analyzed, targeting a portion of exon 1 of the human androgen receptor (HUMARA) in malignant epithelial and mesenchymal components. The presence of p53 and K-ras mutations were also analyzed. H&E-stained sections of paraffin-embedded, formalin-fixed tissues were microdissected to obtain both epithelial and nonepithelial lesions from 25 carcinosarcomas, and DNAs were extracted by proteinase K digestion. Following treatment with methylation-sensitive restriction endonuclease (HhaI or HpaII), PCR amplification was performed using nested primers targeted to the HUMARA locus. Mutations in the p53 gene and K-ras gene were found in eight (32%) and six (24%) tumors, respectively. The patterns of chromosome X inactivation were different between the carcinomatous and sarcomatous components of three carcinosarcomas, indicating that these three tumors represent collision tumors. By contrast, the patterns of chromosome X inactivation, K-ras sequence, and p53 sequence were identical in both carcinomatous and sarcomatous components in 21 carcinosarcomas, indicating that these 21 tumors represent combination tumors. One case produced equivocal results that precluded determination of whether it represented a collision or combination tumor. These observations show that although most carcinosarcomas are combination tumors, some develop as collision tumors. The determination of histogenesis in individual cases of carcinosarcoma using molecular markers may be worthwhile, because the result could help predict the prognosis of individual cases and help guide clinical management.
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PMID:Molecular evidence that most but not all carcinosarcomas of the uterus are combination tumors. 939 63

The histogenesis of carcinosarcoma of the breast is controversial. In the current case, the demarcation between the carcinomatous and sarcomatous components was distinct in all microscopic fields. Immunohistochemical analysis was negative for epithelial membrane antigen (EMA) and keratin in the sarcomatous component and was negative for desmin in the carcinomatous component, suggesting that this tumor could be derived from the two different stem cells. To determine the histogenesis of this tumor, both carcinomatous and sarcomatous lesions were microdissected from formalin-fixed tissues and DNAs were prepared by proteinase K digestion. PCR amplification of the human androgen receptor (HUMARA) short tandem repeat (STR), after Hpa II digestion of the genomic DNA, indicated that the patterns of X-chromosome inactivation were identical in both components. Moreover, both components contained the identical TGT --> TTT transversion in codon 275 of the p53 gene. These observations strongly support the hypothesis that this tumor is derived from a single totipotent stem cell.
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PMID:Carcinosarcoma of the breast: molecular-biological study for analysis of histogenesis. 982 16

Archival cervical smears: a versatile resource for molecular investigations Archival cervical smears represent a huge resource of pathological specimens. This, together with long clinical follow-up data, makes archival smears the most valuable resource for cervical cancer research. Despite this huge potential, only a few molecular investigations have been carried out based on archival smears. It has been shown that archival smears can be used for amplification of genomic sequences by polymerase chain reaction (PCR). However, it is unknown whether PCR can be applied to minute dyskaryotic cells microdissected from archival cervical smears and whether these archival materials are suitable for reverse transcription PCR (RT-PCR). To address these issues, we prepared DNA and RNA samples from dyskaryotic cells microdissected from archival cervical smears with a storage time of 11 years and systematically tested the extent that these materials can be used for PCR-based molecular investigations at both DNA and RNA levels. Our results showed that a crude DNA preparation simply by proteinase K digestion was suitable for PCR amplification of genomic sequences. By targeting the amplified genomic sequence to 250 bp or less, most if not all archival smears could be used for PCR and are therefore suitable for screening gene mutations and loss of heterozygosity, human papillomavirus typing, etc. Purified DNA samples from microdissected dyskaryotic cells were adequate for restriction enzyme digestion and could be used for a PCR-based clonality analysis of the androgen receptor gene. Finally, RNA samples extracted from dyskaryotic cells microdissected from archival smears were adequate for RT-PCR as long as a gene-specific primer was used for the RT reaction and the target sequence was restricted to 150 bp or less. In summary, our results demonstrated that archival cervical smears are suitable for a range of molecular investigations at both DNA and RNA levels. The potential gain of knowledge on cervical cancer by the molecular study of archival smears is immense.
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PMID:Archival cervical smears: a versatile resource for molecular investigations. 1242 45

More than one neoplastic founder clone can exist in benign epithelial tumours. Although theories of clonal selection make pluriclonality appear unlikely in carcinomas, published data do not exclude this possibility. This study looked for evidence of multiclonal X inactivation in ovarian carcinoma using AR methylation as a marker. Fifteen unifocal ovarian carcinomas and 14 multifocal carcinomas all in Scottish patients were studied. One representative formalin-fixed paraffin-embedded tumour block was chosen for each of the former and two for the latter. From each of these 43 tumour blocks three samples each of approximately 10(4) carcinoma cells were obtained by microdissection (129 in all). DNA released by proteinase K digestion was subjected to PCR amplification of the androgen receptor gene AR exon I CAG repeat polymorphism with and without prior digestion with methylation-sensitive restriction enzymes HpaII and HhaI. Complex amplification patterns were consistent with mosaic X inactivation in some ovarian carcinomas but acquired anomalies of AR methylation cannot be excluded. Parallel analysis of other X-linked polymorphic loci would strengthen the inference of clonality status from DNA methylation data in tumour X studies. Strikingly, the number of CAG repeats in the 29 ovarian tumour patients (median 16, range 11 - 20) was substantially fewer than in 34 previously studied breast cancer patients from the same scottish population (median 21, range 14 - 26; P < 0.0001), and women homozygous for the AR CAG repeat were over-represented in the ovarian cancer patients but not in the breast cancer series. These findings reinforce recent suggestions that AR may have a role in ovarian carcinogenesis.
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PMID:Androgen receptor gene methylation and exon one CAG repeat length in ovarian cancer: differences from breast cancer. 1554 19