ABSTRACT
Endothelin (ET)-1 can act as an autocrine/paracrine growth factor or an antiapoptotic factor in human cancers. To study the role of ET-1 in human colon cancer, proliferation and apoptosis of colon carcinoma cells was investigated using human HT-29 and SW480 colon carcinoma cells. ET-1 was secreted by these cells. Treatment of cells with bosentan, a dual ET(A/B)-receptor antagonist, decreased cell number. Inhibition of DNA synthesis by bosentan was observed only in the presence of serum. Exogenously added ET-1 did not increase DNA synthesis in serum-deprived cells. SW480 cells were sensitive and HT-29 cells were resistant to FasL-induced apoptosis. Bosentan sensitised resistant HT-29 cells to FasL-induced, caspase-mediated apoptosis, but not to TNF-alpha-induced apoptosis. Bosentan and/or FasLigand (FasL) did not modulate the expression of caspase-8 or FLIP. Bosentan sensitisation to apoptosis was reversed by low concentrations (10(-13)-10(-10) M), but not by high concentrations (10(-9)-10(-7) M) of ET-1. These results suggest that the binding of ET-1 to high-affinity sites inhibits FasL-induced apoptosis, while the binding of either ET-1 or receptor antagonists to low-affinity sites promotes FasL-induced apoptosis. In conclusion, endothelin signalling pathways do not induce human colon cancer cell proliferation, but are survival signals controling resistance to apoptosis.
Subject(s)
Apoptosis/drug effects , Cell Division/drug effects , Colonic Neoplasms/pathology , Endothelin Receptor Antagonists , Sulfonamides/pharmacology , Base Sequence , Bosentan , Caspase 8 , Caspase 9 , Caspases/metabolism , Colonic Neoplasms/metabolism , DNA Primers , Endothelin-1/metabolism , Flow Cytometry , Humans , Immunohistochemistry , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
The carboxy-terminus of p53 contains a basic region which represses DNA binding, and this repression can be relieved by PAb421, an antibody against the basic region. The EB-1 human cell line contains wild type p53 protein which fails to express the PAb421 epitope and is highly active both in biological assays and in DNA binding assays. We show by wheat germ agglutinin chromatography and galactosyl-transferase labelling that this p53 is O-glycosylated, and that at least one of the sugar residues masks the PAb421 epitope, as demonstrated by recovery of reactivity with PAb421 after digestion of Western blots of EB-1 cell extract with hexosaminidase. A minor population of p53 molecules in EB-1 cells lacks the modification, and there is a correlation between the ability to bind DNA with high affinity and masking of the PAb421 epitope. We also show that strongly positively charged peptides, including short peptides from the basic region of p53, can derepress DNA binding, probably by disruption of an intramolecular interaction involving the basic region. We propose that any intervention which prevents this intramolecular interaction, including addition of bulky residues such as sugar groups, can activate DNA binding by p53.
Subject(s)
DNA-Binding Proteins/metabolism , DNA/metabolism , Tumor Suppressor Protein p53/metabolism , Amino Acid Sequence , Base Sequence , Blotting, Western , Cell Line , Cloning, Molecular , DNA Primers , DNA-Binding Proteins/isolation & purification , Epitopes/analysis , Epitopes/chemistry , Glycosylation , Humans , Molecular Sequence Data , Oligonucleotide Probes , Polymerase Chain Reaction , Recombinant Proteins/biosynthesis , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Saccharomyces cerevisiae , Sequence Homology, Amino Acid , Tumor Suppressor Protein p53/biosynthesis , Tumor Suppressor Protein p53/isolation & purificationABSTRACT
A wild-type p53 gene under control of the metallothionein MT-1 promoter was stably transfected into human colon tumor-derived cell line EB. Repeated inductions of the metallothionein wild-type p53 gene with zinc chloride results in progressive detachment of wild-type p53 cells grown on culture dishes. Examination at both the light and electron microscopic level revealed that cells expressing wild-type p53 developed morphological features of apoptosis. DNA from both attached and detached cells was degraded into a ladder of nucleosomal-sized fragments. Expression of wild-type p53 inhibited colony formation in soft agar and tumor formation in nude mice. Furthermore, established tumors in nude mice underwent regression if wild-type p53 expression was subsequently induced. Regressing tumors showed histological features of apoptosis. Thus, regression of these tumors was the result of apoptosis occurring in vivo. Apoptosis may be a normal part of the terminal differentiation program of colonic epithelial cells. Our results suggest that wild-type p53 could play a critical role in this process.
Subject(s)
Cell Death/genetics , Colonic Neoplasms/genetics , Genes, p53 , Transfection , Base Sequence , Cell Line , Colonic Neoplasms/pathology , Colonic Neoplasms/ultrastructure , DNA, Neoplasm/genetics , DNA, Neoplasm/metabolism , Gene Expression , Humans , Kinetics , Metallothionein/genetics , Molecular Sequence Data , Oligodeoxyribonucleotides , Polymerase Chain Reaction/methods , Promoter Regions, Genetic , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Neoplasm/genetics , RNA, Neoplasm/metabolism , Transcription, GeneticABSTRACT
The number of active Amy-2 genes has been estimated in strain CE/J mice which produce four distinct electrophoretic forms of alpha-amylase in their pancreas. cDNA cloning and DNA sequence analysis discloses five distinct mRNA sequences which differ by approximately 1% of their nucleotides. Two of these mRNAs specify the same protein. Changes in the nucleotide sequences result in amino acid replacements that alter the net charges of the deduced proteins. This has allowed a tentative assignment of individual mRNAs to isozymes detected by electrophoresis. Quantitative Southern blot hybridization using a DNA probe specific for the first exon of Amy-2 reveals the presence of greater than 10 Amy-2 related sequences per haploid CE/J genome. Models which could account for the mouse strain-specific differences with respect to the number of pancreatic alpha-amylase isozymes and their variable but genetically determined quantitative ratios are discussed.
Subject(s)
Genes , Genetic Variation , Pancreas/enzymology , alpha-Amylases/genetics , Alleles , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , DNA Restriction Enzymes , Homozygote , Mice , Mice, Inbred Strains , Nucleic Acid Hybridization , Species SpecificityABSTRACT
The sequence of 1,773-nucleotide major and 1,806-nucleotide minor mouse liver alpha-amylase mRNAs differ only with respect to approximately 30 additional residues at the extreme 5' terminus of the minor species. Comparison of the liver alpha-amylase mRNAs with their salivary gland counterpart reveals that these mRNAs share identical coding and 3'-noncoding sequences, but contain distinct 5'-terminal residues. These data suggest that all three mRNAs might be transcribed from the same gene.
Subject(s)
Amylases/genetics , RNA, Messenger/genetics , alpha-Amylases/genetics , Animals , Base Sequence , Liver/physiology , Mice , Salivary Glands/physiology , Transcription, GeneticABSTRACT
We have determined the nucleotide sequence of two different mouse alpha-amylase mRNAs, one found in the pancreas and the other in the salivary gland. The 1577 and 1659 nucleotide mRNAs from pancreas and salivary gland, respectively are the major alpha-amylase species which accumulate in each tissue. Differences in mRNA length are primarily in the 5' noncoding regions. Comparable portions of the mRNAs are 89% homologous. The mRNA sequences predict alpha-amylase precursor proteins of 508 and 511 amino acid residues, accounting for nearly the entire coding capacity of the mRNAs; differences in protein length occur as a result of a nine nucleotide segment present within the translated portion of salivary gland, but not pancreas, mRNA. The lengths and amino acid compositions of the predicted proteins concur with those determined empirically by others. These proteins differ 12% in amino acid sequence, explaining previously observed differences in net charge and antigenic properties. Finally, translation of the salivary gland alpha-amylase mRNA is not initiated at the AUG codon nearest the 5' terminus since that codon is almost immediately followed by the termination triplet UAA. This observation may have implications for the mechanism of translation initiation in eucaroytes.
