Linked Life Data

7812966

Source:http://linkedlifedata.com/resource/pubmed/id/7812966

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
2
pubmed:dateCreated
1995-2-3
pubmed:abstractText
An association between the resistance to mitomycin C (MMC) and a decrease of NAD(P)H:quinone oxidoreductase (NQO1) activity was reported for a MMC-resistant subline, HCT 116-R30A, derived from MMC-sensitive HCT 116 cells. Eight NQO1 cDNA clones were isolated from these two sublines by reverse transcription-PCR. Two clones, pDT9 from HCT 116 and pDT20 from HCT 116-R30A, are the full length of 274 amino acids. These two clones differ by a T to C substitution at nucleotide 464, which results in a replacement of arginine 139 by tryptophan in the enzyme. NQO1 of pDT9 and pDT20 was expressed in Escherichia coli, purified, and shown to have a protein subunit of M(r) 30,000. The change of amino acid 139 resulted in a shift of isoelectric pH from 9.5 to 8.35 and a 60% decrease of activity in reducing MMC. All of the other six clones differ from pDT9 by a deletion of exon 4. On Northern blot, we detected two mRNA species of NQO1 (1.2 and 2.7 kilobases) due to alternative polyadenylation in all sublines. MMC-resistant sublines showed 75-90% mRNA expression relative to HCT 116 cells. Reverse transcription-PCR amplification of cDNA fragment of nucleotide 298-617 revealed two full-length mRNAs in HCT 116 cells but only one full-length mRNA in HCT 116-R30A cells. An exon 4 deletion mRNA was detected in both sublines. The two full-length mRNAs may be from either alleles or chimeras of the same gene and the exon 4 deletion mRNA is a result of alternative splicing. On Western blot, we detected only one M(r) 30,000 protein in all sublines. A substantial decrease of this protein in MMC-resistant sublines (5% of HCT 116) explained the 95% decrease of their NQO1 activity. Transcriptional regulation and posttranscriptional modification may be responsible for the disparity of gene expression of NQO1 and the low concentration of NQO1 protein in MMC-resistant sublines. Reversal of MMC resistance and the recovery of NQO1 in two revertants further supports the hypothesis that cellular control of NQO1 can modulate the cytotoxicity of MMC.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Jan
pubmed:issn
0008-5472
pubmed:author
pubmed:issnType
Print
pubmed:day
15
pubmed:volume
55
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
330-5
pubmed:dateRevised
2006-11-15
pubmed:meshHeading
pubmed:year
1995
pubmed:articleTitle
NAD(P)H:quinone oxidoreductase expression and mitomycin C resistance developed by human colon cancer HCT 116 cells.
pubmed:affiliation
Division of Developmental Therapeutics, University of Maryland Cancer Center, Baltimore 21201.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't