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

Bifunctional alkylating agents are known to produce cross-links between DNA and protein and between paired DNA strands. The possibility of discriminating these two classes of cross-links in L1210 cells treated with haloethylnitrosoureas or nitrogen mustard was explored with the alkaline elution technique. Two classes of cross-links were demonstrated, based on sensitivity to proteinase K; the proteinase-sensitive cross-links appear to be DNA-protein cross-links, and the proteinase-resistant class may include interstrand cross-links. Proteinase-sensitive cross-links form more rapidly than do proteinase-resistant cross-links in cells treated with chloroethylnitrosoureas, perhaps because these agents can chloroethylate protein sulfhydryl or amino groups followed by rapid reaction of these chloroethylated groups with DNA. Although both types of cross-links produced by nitrogen mustard disappeared or were repaired after 24 hr, the removal of cross-links produced by chloroethylnitrosoureas either did not occur or was incomplete in 24 hr. In addition to cross-links, cells treated with haloethylnitrosoureas exhibited DNA strand breaks; a method is suggested for estimating the apparent frequencies of strand breaks and cross-links in the DNA.
Cancer Res 1978 Oct
PMID:DNA-protein cross-linking and DNA interstrand cross-linking by haloethylnitrosoureas in L1210 cells. 15 Sep 40

The effects of cis- and trans-platinum(II) diamminedichloride on L1210 cells were investigated using the technique of DNA alkaline elution. Both agents produced reductions in alkaline elution rates which were partially or, in some cases, almost completely reversed by incubation of the cell lysates with proteinase K. The proteinase-sensitive component of this effect is taken to reflect DNA-protein cross-linking, while the proteinase-resistant component of this effect may include interstrand cross-links. The cytotoxicity of the two agents did not correlate with the extent of DNA-protein cross-linking but did appear to correlate with the extent of proteinase-resistant cross-linking; therefore, there may be a relationship between cytotoxicity and interstrand cross-linking.
Cancer Res 1979 Feb
PMID:DNA-protein and DNA interstrand cross-linking by cis- and trans-platinum(II) diamminedichloride in L1210 mouse leukemia cells and relation to cytotoxicity. 57 92

The DNA alkaline elution technique provides a sensitive assay for the effects of DNA-damaging drugs in mammalian cells. We have adapted this method to permit measurements of effects on DNA in solid tumors. Human colon carcinoma xenografts in nude mice were treated with a single i.p. injection of 1-(2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitrosourea, and the effects on the DNA were followed for 19 hr. Drug doses in the pharmacological range produced significant reductions in DNA alkaline elution rates in assays in which X-ray was used to introduce a standard frequency of single-strand breaks. These changes in alkaline elution rate were attribute to the production of both DNA interstrand and DNA-protein cross-links, which were distinguished from each other on the basis of the extent to which the effect on elution could be reversed by proteinase K. Crosslinking increased for about 8 hr after treatment with little change thereafter up to 19 hr. A drug-resistant tumor line exhibited substantially less cross-linking than did a drug-sensitive line at all time points examined.
Cancer Res 1978 Aug
PMID:DNA cross-linking by in vivo treatment with 1-(2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitrosourea of sensitive and resistant human colon carcinoma xenograms in nude mice. 66 42

Novel derivatives of K-252a, (8R*,9S*,11S*)-(-)-9-hydroxy-9-methoxycarbonyl- 8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H,8H,11H-2,7b,11a-triazadibe nzo[a,g]-cycloocta[cde]trinden-1-one, an inhibitor of protein kinases and calmodulin-dependent phosphodiesterase, were synthesized and evaluated for their antitumor activity in vitro and in vivo. Of ten derivatives tested, four were active against the P388 murine leukemia i.p.-i.p. system, although K-252a was inactive. Among these derivatives, KT6124 was selected for further biological evaluation studies because its efficacy was the highest. KT6124 was also active against sarcoma 180 and B16 melanoma. It exerted a relatively broad spectrum of antiproliferative activity against 20 human tumor cell lines in vitro. To determine the mechanism(s) of action underlying the antitumor activity of KT6124, we tested the drug for inhibition of protein kinases, including Ca(2+)- and phospholipid-dependent protein kinase (PKC), in intact A431 human epidermoid carcinoma cells in comparison with the PKC-inhibitory activity of K-252a. KT6124 did not antagonize the action of phorbol 12-myristate 13-acetate (PMA) in A431 cells, whereas K-252a did, suggesting that KT6124 may not act on protein kinases in the cells. The interaction of KT6124 with DNA in living cells was examined by the alkaline elution method. KT6124 apparently exhibited DNA scission both dose- and time-dependently in the target cells. The DNA breakage was dependent on proteinase K treatment, suggesting its possible interaction with DNA-related enzyme(s). These results indicate that KT6124 exerts antitumor activity by acting on DNA or on DNA-related enzyme(s) in tumor cells rather than via the inhibition of protein kinases.
Cancer Chemother Pharmacol 1992
PMID:Antitumor effect of KT6124, a novel derivative of protein kinase inhibitor K-252a, and its mechanism of action. 153 71

Benzamide (BA) enhances the cytotoxicity of 1,2:5,6-dianhydrogalactitol (DAG) in resistant P388 leukemia cell lines but not in the sensitive parent line. To examine the reason for this difference in response, we carried out an alkaline elution assay using proteinase K to study DNA interstrand cross-linking. At early time points, equal concentrations of DAG produced the same level of interstrand cross-linking (ICL) in the resistant and sensitive P388 leukemic cells, although marked differences were observed in their cytotoxicity toward the two cell lines. In the sensitive cells, neither the amount of DNA cross-linking nor the cytotoxicity changed during the observation period (38 h) in either the presence or the absence of BA. In contrast, the elution rate of the DNA of DAG-treated resistant cells increased with time and had reached the control levels by 38 h. However, when these cells were postincubated with BA for 38 h, the elution rate of DNA was much faster than that observed for the untreated resistant cells, indicating an accumulation of DNA single-strand breaks (SSB). The SSB accumulation caused by BA was associated with an inhibition of the activity of ligase II enzyme, which was stimulated when resistant cells were treated with DAG alone. The potentiating effect of BA on the resistant cells can thus be related to the inhibiting action of BA on the DNA-rejoining enzyme, ligase II. The lack of sensitization by BA of the DAG-treated parent cell line may be attributable to the absence of DNA-SSB formation, which is necessary for ligase II activation through the stimulation of poly(ADP-ribose) synthesis.
Cancer Chemother Pharmacol 1992
PMID:Benzamide potentiation of the cytotoxicity of bifunctional galactitol [correction of galacticol] in resistant P388 leukemia correlates with inhibition of DNA ligase II. 164 2

A new immunohistochemical method using a monoclonal estrogen receptor (ER) antibody and a progesterone receptor (PR) antibody has been performed to determine hormonal receptors on Bouin's liquid fixed and paraffin embedded tissue. This technique has been evaluated by comparison with conventional immunohistochemistry on frozen sections for 100 surgical biopsy specimens of breast carcinoma Erica (C) Prica (C); in 77 of these cases it was compared with enzyme immunoassay analyses (ER-EIA, PR-EIA). After a pretreatment by proteinase K for ER, the two specific monoclonal antibodies ER or PR were incubated overnight and revealed with a streptavidin biotin complex. The specific staining for ER and PR were exclusively located in the nuclei of cancer cells in both paraffin for ER and PR were exclusively located in the nuclei of cancer cells in both paraffin and frozen sections. The results on paraffin-embedded material were compared successively with ERICA (C) and ER-EIA and with PRICA (C) and PR-EIA. In 100 breast infiltrating carcinomas studied simultaneously on both paraffin and frozen sections, the concordance was 82% for ER and 85% for PR. In 77 breast carcinoma studied simultaneously on paraffin sections and EIA, the concordance was 74% for ER and 79.2% for PR. These results suggest that in the absence of frozen material, an immunohistochemical method on paraffin-embedded material can be used with a lower sensibility than on frozen sections. However, the detection of both ER and PR allows the identification of the hormondependent cases.
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PMID:[Human breast cancer embedded in paraffin, fixed in Bouin's fluid. Immunohistochemical analysis of estrogen and progesterone receptors]. 195 56

Pre-treatment with low, non-toxic concentrations (0.04 microM) of methotrexate (MTX) for 16 hr increased etoposide (VP16)-induced growth inhibition and cytotoxicity in the U937 human histiocytic lymphoma cell line. VP16 cytotoxicity was significantly potentiated when the drug was given for 2 hr immediately after MTX pre-treatment or between 2 and 4 hr or 4 and 6 hr after recovery from MTX pre-treatment. By 24 hr after recovery from MTX, no potentiation was evident. The increased cytotoxicity of VP16 was associated with an increase in drug-induced DNA breaks as assessed by the alkaline elution method after proteinase K digestion. The amount of DNA single-strand breaks (DNA SSB) increased when the drug was given 0, 2, and 4 hr after MTX pre-treatment. DNA SSBs induced by the drug between 6 and 24 hr after MTX pre-treatment were similar to those seen in cells without pretreatment. The amount of DNA double-strand breaks (DNA DSB) caused by VP16 increased significantly when the drug was given 4 hr after recovery from MTX pre-treatment. VP16-induced DNA DSBs were still higher 6 hr after MTX pre-treatment, but by 24 hr they were similar to those observed in MTX-untreated cells. Flow cytometric analysis showed that MTX pre-treatment was causing an accumulation of U937 cells at the G1-S boundary of the cell cycle. When MTX was removed, a wave of synchronization followed. Using Western blot electrophoresis and polyclonal antibodies to antitopoisomerase II, we found that MTX pre-treatment raised the cellular topoisomerase II content. Our findings suggest that the potentiation of VP16 cytotoxicity on U937 cells by low, non-toxic MTX pre-treatment is due to a larger fraction of S-phase cells containing a higher concentration of topoisomerase II, which is the putative target of VP16 action.
Int J Cancer 1990 Jan 15
PMID:Increase in topoisomerase-II-mediated DNA breaks and cytotoxicity of VP16 in human U937 lymphoma cells pretreated with low doses of methotrexate. 215 35

The cyanomorpholino analog of doxorubicin (MRA-CN) is a potent cytotoxic agent which is known to cross-link DNA. A human ovarian carcinoma cell line, ES-2, was grown in increasing concentrations of MRA-CN from 0.1 to 0.5 nM. The resultant resistant subline, ES-2R, was 4-fold resistant to MRA-CN. DNA damage and repair in response to MRA-CN were compared in the parental and resistant cell lines using alkaline elution. DNA cross-links were detectable after 3-h incubation of the cells at 37 degrees C in MRA-CN at concentrations greater than or equal to 1.0 nM. Paradoxically, 2-fold more cross-links were detected in the ES-2R cells as compared with the ES-2 cells. This paradoxical difference in cross-links between the 2 cell lines was observed to increase with time of exposure to 2.5 nM of MRA-CN. Non-protein-associated DNA strand breaks were also detected in the 2 cell lines after exposure to 2.5 nM of the drug. The ES-2 cells consistently showed twice as many breaks as the ES-2R cells, which could explain the paradoxical higher apparent DNA cross-linking observed with the ES-2R cells after exposure to MRA-CN. Studies of the time course of cross-link repair after exposure to MRA-CN revealed that 75% of the DNA cross-links disappeared in the ES-2R cells by the end of 8 h in drug-free medium. In contrast, cross-links in the ES-2 cells were undetectable after 4 h, which coincided with a progressive increase in DNA strand breaks. The topoisomerase II level in the ES-2 cells was 2- to 4-fold higher than that in the ES-2R cells. However, proteinase K treatment of the lysed cells did not increase the number of apparent strand breaks produced by MRA-CN, suggesting that topoisomerase II may not be involved. These findings indicate that, in addition to DNA cross-linking, MRA-CN causes DNA strand breakage. Resistance to MRA-CN in the ES-2R cells is associated with more apparent DNA cross-linking and less DNA strand breakage, which may be a consequence of differences in DNA repair and/or nonspecific DNA degradation between the resistant and the sensitive cell lines.
Cancer Res 1990 Jul 01
PMID:Paradoxical increase in DNA cross-linking in a human ovarian carcinoma cell line resistant to cyanomorpholino doxorubicin. 216 51

1. The combined application of DNA strand-scission agents (bleomycin) and inhibitors of recovery from lethal damage (calmodulin antagonist W-13) could be a novel and potentially important approach to cancer therapy. 2. As determined by alkaline elution, both DNA-DNA and DNA-protein cross-links in bleomycin-treated cells were revealed by the presence of the proteinase K assay. 3. This lethal effect could be potentiated by the addition of calmodulin antagonist W-13 that prevents the repair of DNA strand breaks and DNA cross-links caused by bleomycin. 4. The results indicated that combinations of bleomycin and W-13 were more effective than treatment with either single agent. Isobologram analysis suggests synergistic effect of these drugs. 5. Therefore, the rational use of combinations of DNA-strand-scission agents and inhibitors of recovery from lethal damage based on mechanistic considerations should result with improved therapeutic regimens for the treatment of cancer.
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PMID:Calmodulin antagonist W 13 prevents DNA repair after bleomycin treatment of human urological tumor cells growing on extracellular matrix. 244 75

The tumor antigen capable of inducing tumor resistance (tumor rejection antigen; TRA) was separated and some of its physicochemical properties were characterized. Cytosol and plasma membrane fractions were separated from Rous sarcoma virus (RSV)-induced CSA1M tumor cells. Immunization with membrane but not cytosol fraction of these tumor cells together with complete Freund's adjuvant resulted in complete protection against subsequent challenge with viable CSA1M cells. The TRA activity contained in the membrane fraction was recovered in the sodium dodecyl sulfate (SDS)-solubilized fraction after the SDS-extraction of CSA1M membranes. This CSA1M SDS-solubilized preparation gave protection against syngeneic RSV-induced CSA9F tumor cells as well as the homologous tumor cell type, but failed to induce resistance to RSV-unrelated tumor cells. The membrane or SDS-solubilized fraction from RSV-unrelated tumor cells was unable to generate anti-CSA1M protective immunity. Physicochemical analyses have demonstrated that TRA activity in the SDS-solubilized fraction was completely abolished by treatment with proteinase K but was only marginally affected after treatment with glycosidase mixture. When the SDS-solubilized preparation was applied to a Sephacryl S-300 superfine column, TRA activity was recovered in the range of molecular weight of 50-90 kD. Further fractionation of this TRA-positive fraction by SDS-polyacrylamide gel electrophoresis revealed that the molecular size of TRA is 56-68 kD. These results indicate that membrane proteins which were isolated from CSA1M tumor cells and have a molecular size of about 60 kD are capable of inducing RSV-induced tumor-specific in vivo protective immunity.
Jpn J Cancer Res 1988 Mar
PMID:Separation of the tumor rejection antigen of Rous sarcoma virus-induced murine fibrosarcoma. 245 98


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