Differential regulation of trypsinogen mRNA translation: full-length mRNA sequences encoding two oppositely charged trypsinogen isoenzymes in the dog pancreas.

In the absence of changes in functional mRNA levels, stimulation of the pancreas with caerulein, a peptide analog of cholecystokinin, has been previously shown to increase the synthesis of anionic but not cationic trypsinogen. To look for structure-function correlations, a high-yield, full-length cDNA library has been constructed from canine pancreatic poly(A)+ mRNA. Full-length clones coding for the two major trypsinogen isoenzyme forms have been identified by colony hybridization and verified by in vitro translation of hybrid-selected mRNA in the presence of microsomal membranes and an optimal redox potential. Disulfide-bonded translation products were separated and identified by two-dimensional isoelectric focusing-sodium dodecyl sulfate-gel electrophoresis. Nucleotide sequence analysis allowed us to deduce the amino acid sequences for the anionic and cationic forms of canine trypsinogen, which contain 232 and 231 residues, respectively (77% amino acid identity), and the 15-residue amino terminal signal sequences (53% amino acid identity) associated with the two presecretory forms. Measurements of relative and absolute mRNA levels, when related to relative protein synthesis values, indicated that the translational efficiency of anionic trypsinogen mRNA exceeded that of cationic trypsinogen mRNA by 1.5- to 2.9-fold under basal conditions. Analysis of the 5' noncoding regions of trypsinogen mRNAs revealed a striking conservation of sequence (10 of 12 bases) between dog and rat anionic trypsinogen forms. This contrasted markedly with the divergence of the 5' noncoding regions observed between dog anionic and cationic trypsinogen mRNAs.

Identification of cDNA clones encoding secretory isoenzyme forms: sequence determination of canine pancreatic prechymotrypsinogen 2 mRNA.

A cDNA library has been constructed from canine poly(A)+ mRNA. Clones containing cDNA inserts coding for prechymotrypsinogen 2 (isoelectric point = 7.1; Mr = 27,500), one of three canine pancreatic isoenzyme forms, were selected by colony hybridization using a cDNA probe synthesized from immunoselected prechymotrypsinogen 2 mRNA. To verify that cDNA clones code for prechymotrypsinogen 2 forms that translocate across rough endoplasmic reticulum membranes and fold into stable and identifiable secretory proteins, we conducted in vitro translation of hybrid-selected mRNA in the presence of microsomal membranes and optimal concentrations of glutathione and analyzed nascent translation products in their nonreduced state by two-dimensional isoelectric focusing/NaDodSO4 gel electrophoresis and fluorography. A near full-length chymotrypsinogen 2 cDNA and its primed extension were used to determine the nucleotide sequence for the entire coding region of prechymotrypsinogen 2 mRNA and 87 residues, including a poly(A) addition signal, in the 3' nontranslated region. The deduced amino acid sequence shows a 263-residue presecretory protein containing an 18-residue amino-terminal transport peptide (Met-Ala-Phe-Leu-Trp-Leu-Leu-Ser-Cys-Phe-Ala-Leu-Leu-Gly-Thr-Ala-Phe-Gly ), which we have previously shown to mediate the translocation of chymotrypsinogen 2 across the rough endoplasmic reticulum membrane. Following the transport peptide is a 245-residue proenzyme, which shows 82% and 80% sequence identity with bovine chymotrypsinogens A and B, respectively. Conserved among the three zymogens are 10 Cys residues that form five disulfide bonds in bovine chymotrypsinogens A and B and the residues that are required for zymogen activation, substrate binding, and catalytic activity.

Correlation of increased acetate binding with alkylating agent resistance in Walker and Yoshida tumor cells.

The utilization of acetate is fundamental to numerous cellular processes. At the level of chromatin, histone acetylation is thought to regulate transcription, and acetate is a major source of cellular energy as it is the substrate for the citric acid cycle. The present work investigates the incorporation of [1-14C]sodium acetate in alkylating agent sensitive (WS) and resistant (WR) Walker 256 carcinosarcoma cells. WR bound the labeled acetate four to six times faster than WS cells, as determined by incorporation of [1-14C]sodium acetate into trichloroacetic acid precipitable material. This difference was consistently observed in both nuclear and cytoplasmic fractions of the cells and in cells permeabilized prior to incubation with radioactive acetate. WS and WR cells did not differ from each other in content of either reduced or acetylated CoA. Since adding exogenous CoA-SH to cell lysates did not alter acetate binding or reduce the differences between WS and WR, increased acetylation in WR cells was independent of CoA levels. Using [1-14C]acetyl CoA to label lysolecithin-permeabilized WS and WR cells revealed no difference between the sensitive and resistant lines, in contrast to the 5-fold greater binding of [1-14C]sodium acetate in permeabilized WR cells. This suggests that WR cells formed acetyl CoA more rapidly than did the WS cells from acetate plus endogenous CoA. Chlorambucil treatment (24 hr) did not affect acetylation of nuclear proteins in log phase cells. Finally, 3-fold greater acetylation in a line of Yoshida sarcoma cells that is resistant to alkylating agents, compared to the sensitive line, supported the generality of the phenomenon.

Distribution and binding of the carcinogens 1-methyl-1-nitrosourea and 1-methyl-3-nitro-1-nitrosoguanidine in the guinea pig after oral administration.

Guinea pigs were given radioisotopic 1-methyl-1-nitrosourea (MNU) or 1-methyl-3-nitro-1-nitrosoguanidine (MNNG) as a single oral dose of 0.1 mmol/kg and uptake into TCA-precipitable material, whole tissue protein and 7-methyl-guanine and O6-methylguanine were determined in brain, kidney, liver, pancreas, small intestine and stomach after 2, 4 and 24 h. The labeling of TCA-precipitable material and the formation of methylated bases by methyl-labelled MNU clearly exceeded that by MNNG in extra-intestinal tissues such as brain, liver, kidney and pancreas. In the stomach, there was greater DNA methylation by MNNG than by MNU. Labelling of TCA-precipitable material and whole tissue protein by the carbonyl group of MNU was greater than that by the guanidino group of MNNG in brain, liver, kidney and pancreas, while MNNG exceeded MNU in the stomach. These data are consistent with the concept that orally administered MNU would be an effective carcinogen in extra-intestinal tissues, while MNNG would be a locally acting gastric carcinogen.

Binding of N-nitroso carcinogens in pancreatic tissue.

Carcinoma of the pancreas can be produced in guinea pigs by the oral administration of 1-methyl-1-nitrosourea (MNU). Our present studies have examined the distribution of this compound in these animals following parenteral and oral administration and also its specific sites of binding within the pancreatic cell. Emphasis has been placed upon the comparison of the uptake and binding of MNU with the related compound 1-methyl-3-nitro-1-nitrosoguanidine (MNNG), which does not seem to be carcinogenic to the pancreas after oral administration. The distribution data indicate uptake into pancreas that is comparable to that of other organs. Binding data show both MNU and MNNG to extensively modify subcellular organelles and the DNA and proteins of chromatin, but that MNU does this to a somewhat greater extent than MNNG.

Uptake and binding of 1-methyl-1-nitrosourea (MNU) and 1-methyl-3-nitro-1-nitrosoguanidine (MNNG) by the isolated guinea pig pancreas.

1-Methyl-1-nitrosourea (MNU) and 1-methyl-3-nitro-1-nitrosoguanidine (MNNG) are carcinogens which methylate nucleic acids and proteins and covalently modify proteins by carbamoylation (MNU) or guanidination (MNNG). Using MNU and MNNG labeled with carbon-14 in the individual carbon positions, the above reactions were quantitated in the isolated guinea pig pancreas, an organ susceptible to tumorigenesis by MNU. Freshly prepared pancreatic lobules were incubated with the labeled drugs (0.03, 0.3 and 1.0 mM) for one hour at 37 degrees C. Alkylated purines from hydrolyzed DNA were separated on Sephadex G10 and acid-soluble nuclear proteins were extracted and separated on polyacrylamide gels. Total uptake of all four labels into lobules was linear with concentration. Acid insoluble radioactivity also increased linearly except for MNNG methylation which plateaued between 0.3 and 1.0 mM. 7-Methylguanine formation by both compounds was approximately ten fold greater than 06-methylation. However, DNA modification by MNU exceeded that by MNNG, especially at the higher drug concentrations. No carbamoylation or guanidination of DNA was detected. Total binding (methylation plus carbamoylation/guanidination) to acid-extractable chromatin proteins was equivalent to DNA modification on a molar basis (approximately 0.35 and 0.08 pmol/microg for exposure to 1.0 mM MNU and MNNG, respectively). All histones were labeled by all drug preparations, with H2A being the principal site of methylation and H2B, H3 and H1 being the major targets of carbamoylation and guanidination. H4 was the least modified histone. Drug binding to cytoplasmic organelles also occurred. These results show a broad spectrum of nuclear and cytoplasmic modification of pancreatic cells by MNU and, to a smaller extent, MNNG.