Molecular cloning of complementary DNAs encoding alkaline phosphatase in human colon cancer cells.

We have isolated a set of complementary DNA (cDNA) clones that together encode the alkaline phosphatase of human colon cancer LS174T cells. These clones include two cDNAs isolated from a conventionally prepared oligodeoxythymidylate-primed lambda ZAP cDNA library and three cDNA clones prepared by using the polymerase chain reaction. The deduced amino acid sequence of the alkaline phosphatase primary transcript contains 532 amino acids. This enzyme is similar to, but not identical with, placental alkaline phosphatase (PLAP); it exhibits 12-19 amino acid substitutions when compared to the various alleles of PLAP. Also, it is similar to PLAP in that it is apparently attached to the cell membrane by a phosphatidylinositol-containing anchor as judged by the ability of phosphatidylinositol-specific phospholipase C to release it from membranes. It is different from PLAP however, in terms of its signal sequence which only contains 19 amino acids as compared to 22 for PLAP. Moreover, the 3'-untranslated region of the LS174T cell alkaline phosphatase message diverges considerably from the PLAP message. The LS174T cell alkaline phosphatase cDNAs are actually much more similar to the "germ cell" alkaline phosphatase gene than they are to PLAP. Only 7 amino acid substitutions exist between the LS174T cell enzyme and the alkaline phosphatase encoded by the germ cell alkaline phosphatase genomic DNA clone isolated by Millan and Manes (Proc. Natl. Acad. Sci. USA, 85: 3024-3028, 1988). Furthermore, the 3'-untranslated region of the LS174T cell alkaline phosphatase message is very similar to the sequence immediately downstream of the coding region of the germ cell alkaline phosphatase genomic DNA clone. Thus, these results indicate that this colon cancer cell alkaline phosphatase is likely to represent an allelic variant encoded at the germ cell alkaline phosphatase locus.

Heterogeneity in the induction and expression of carcinoembryonic antigen-related antigens in human colon cancer cell lines.

Sodium butyrate induces morphological and biochemical changes consistent with a more differentiated phenotype in some colon cancer cell lines. These changes include increased expression of carcinoembryonic antigen (CEA) and other oncodevelopmental markers. We utilized domain-specific probes and polyclonal antibodies against CEA-related antigens to study sodium butyrate-induced expression of the CEA gene family in a villous adenoma-derived cell line, which is nontumorigenic in nude mice (VACO 235), and two colonic carcinoma cell lines known to respond to sodium butyrate exposure by phenotypic differentiation (HT-29 and LS 174T). The induction begins as quickly as 24 h after exposure and occurs primarily at a transcriptional level, although some translational control is also evident. No evidence was found for gene amplification, rearrangement, or methylation to account for the mechanism of this transcriptional control. [35S]Cysteine pulse-labeled cell lysate immunoblots and polyadenylated RNA blot hybridization suggest that increases in mRNA transcript and CEA-related glycoprotein levels are primarily due to increased synthesis rather than decreased degradation. A considerable amount of heterogeneity is seen in the biosynthesis of the CEA-related glycoproteins, with each cell line showing a distinct pattern of CEA-related antigen expression from a limited number of mRNA transcripts.

Release of carcinoembryonic antigen from human colon cancer cells by phosphatidylinositol-specific phospholipase C.

Carcinoembryonic antigen (CEA) is released from colon cancer cells into the circulation where it is monitored clinically as an indicator of the recurrence or progression of cancer. We have studied the mechanism of CEA membrane attachment and release using the human colonic adenocarcinoma cell line LS-174T, specimens of human colon cancers, and serum from colon cancer patients. CEA release by cells in vitro and in vivo is associated with the conversion of CEA from a membrane-bound, hydrophobic molecule to a soluble, hydrophilic form with no apparent decrease in molecular mass. When LS-174T cell membranes were incubated with various buffers, proteases, and phospholipases, the only agents that released CEA and converted it to the hydrophilic form were preparations of phosphatidylinositol-specific phospholipase C (PI-PLC). Both [3H]ethanolamine and [3H]palmitate could be incorporated metabolically into CEA but only palmitate was released by treatment with PI-PLC, consistent with the presence of a glycosyl-phosphatidylinositol linkage. PI-PLC treatment also release significant quantities of CEA from living monolayers and from seven human colon cancer specimens. These experiments suggest that cellular CEA is anchored to membranes by a covalent linkage to a membrane phosphatidylinositol molecule, and that an endogenous phospholipase may be important for releasing CEA in vitro and in vivo.

Cyclic-AMP-stimulated synthesis and release of carcinoembryonic antigen by pancreatic cancer cells.

The effect of cyclic adenosine 3':5'-monophosphate (cAMP) upon the synthesis and release of carcinoembryonic antigen (CEA) was studied in the human pancreatic ductal cancer cell line, SW-1990. Incubation for up to 24 h with forskolin, an activator of adenylate cyclase, or isobutylmethyl xanthine, a theophylline analog, increased cellular cAMP levels by over 100-fold and significantly increased CEA release and cellular CEA content. Whereas cAMP levels were augmented within 10 min of exposure to these agents, CEA release and CEA cell content were not increased until 90 min and 24 h, respectively. Similar results were obtained using dibutyryl-cAMP, a cAMP analog, but not using sodium butyrate, a metabolite of dibutyryl-cAMP. Cells were incubated with 35S-cysteine and 3H-glucosamine in the presence or absence of forskolin in order to compare the effects of high cAMP levels upon the synthesis and release of total proteins, total glycoproteins, and immunoprecipitable CEA. Both CEA synthesis and release were enhanced by forskolin, but these effects were not specific to CEA since the release of labeled proteins and glycoproteins also increased. In addition, altered CEA expression caused by forskolin was consistently associated with a cessation of cell division, an effect which was reversible upon removing the agent. There was no effect upon cell morphology or viability. The data indicate that increased levels of cellular cAMP in pancreatic cancer cells is associated with decreased cell proliferation and increased expression of CEA and other glycoproteins.

Effects of cyclic adenosine 3':5'-monophosphate upon glycoprotein and carcinoembryonic antigen synthesis and release by human colon cancer cells.

The role of cyclic adenosine 3':5'-monophosphate (cAMP) in the regulation of the synthesis and release of glycoproteins and of carcinoembryonic antigen by colon cancer cells was studied using LS174T cells in vitro. Adenylate cyclase and cAMP phosphodiesterase activities were assessed by measuring cellular cAMP in response to forskolin and cholera toxin (adenylate cyclase activators) and to 3-isobutyl-1-methylxanthine (a phosphodiesterase inhibitor). Each agent increased cAMP levels significantly. Dibutyryl-cAMP (1 mM) stimulated glycoprotein synthesis and release when [3H]fucose was used as a precursor. The synthesis and release of carcinoembryonic antigen, a membrane-associated glycoprotein antigen, was also significantly increased by these test agents. A close dose-response relationship existed for forskolin and for cholera toxin between cAMP generation and carcinoembryonic antigen release. cAMP may play a role in regulating the synthesis and release of glycoprotein antigens by colon cancer cells.