Characterization of a complementary DNA for rat liver aryl sulfotransferase IV and use in evaluating the hepatic gene transcript levels of rats at various stages of 2-acetylaminofluorene-induced hepatocarcinogenesis.
A complementary DNA (cDNA) for rat hepatic aryl sulfotransferase IV (AST IV) was isolated, characterized, and used as a hybridization probe to evaluate the molecular basis for the differential expression of AST IV during 2-acetylaminofluorine (2AAF)-induced hepatocarcinogensis. The AST IV cDNA clone was obtained by immunochemical screening of a male Sprague-Dawley rat liver cDNA library. The AST IV cDNA was found to be 1.3 kilobases long and to encode a fusion protein which was reactive with an antibody to AST IV and enzymatically able to generate the sulfuric acid ester of N-hydroxy-2AAF. Sequence analysis of the AST IV cDNA showed it to be 1127 residues in length and to have essentially complete homology with PST-I cDNA, a previously reported (S. Ozawa, et al., Nucleic Acids Res., 18: 4001, 1990), 1028-base cDNA for an uncharacterized rat liver aryl sulfotransferase. Comparison of the PST-I/AST IV cDNA-deduced amino acid sequence with data from a partial (51%) amino acid sequence analysis of purified AST IV showed complete amino acid homology, confirming the identity of the cDNA and establishing that AST IV was an N-blocked, 291-amino acid protein with a molecular mass of 33,909 daltons. The AST IV cDNA sequence differed from the PST-I cDNA in two principal ways: the 5' end lacked 18 coding bases, and the 3' end contained a 190-base extention in the untranslated region, including a consensus sequence for signalling polyadenylation. Studies of AST IV gene transcript levels showed that the livers of rats fed 2AAF for 3 wk (early stage hepatocarcinogenesis) and hyperplastic nodules from the livers of rats fed 2AAF for 19 wk (intermediate stage hepatocarcinogenesis) displayed transcript levels similar to those of livers from normal rats. This contrasted with the 60 to 70% lower than normal capacity of the mRNA fractions to express AST IV observed during in vitro translation. These results indicated that modulation of AST IV expression at early and intermediate stages of hepatocarcinogenesis involved regulatory mechanisms at the translational level. In contrast, mRNA fractions isolated from some 2AAF-induced liver tumors or from known chemical carcinogen-derived rat hepatoma cell lines showed losses of both AST IV transcript level and in vitro translation capacity, suggesting that regulation at the transcriptional level may become important at late stages of 2AAF-induced hepatocarcinogenesis. These results indicated that the molecular mechanisms for the 2AAF-mediated down regulation of AST IV expression during 2AAF-induced hepatocarcinogenesis involved alterations in regulation at both translational and transcriptional levels.
Yerokun
T
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Samuel Roberts Noble Foundation, Inc., Ardmore, Oklahoma 73402.
Etheredge
J L
JL
Norton
T R
TR
Carter
H A
HA
Chung
K H
KH
Birckbichler
P J
PJ
Ringer
D P
DP
D015073:2-Acetylaminofluorene / Q000633:toxicity; D000595:Amino Acid Sequence; D000818:Animals; D015239:Arylsulfotransferase / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D015870:Gene Expression; D008099:Liver / Q000201:enzymology; D008113:Liver Neoplasms / Q000201:enzymology / Q000235:genetics*; D008114:Liver Neoplasms, Experimental / Q000139:chemically induced / Q000235:genetics*; D008297:Male; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D011230:Precancerous Conditions / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D012334:RNA, Neoplasm / Q000235:genetics; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000276:immunology / Q000378:metabolism; D016415:Sequence Alignment
D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D011993:Recombinant Fusion Proteins; D004247:DNA; D015073:2-Acetylaminofluorene; D015239:Arylsulfotransferase