Alkaline phosphatase activity and the regulation of growth in transformed mammalian cells (original) (raw)
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Expression of alkaline phosphatase in murine lymphoma cells
Biochemical and Biophysical Research Communications, 1991
Alkaline phosphatase (ALP) was secreted and expressed at the cell surface of the lymphoma A/63-2 cell line but not on another clone A/63-1 deriving from a single thymoma (A/63) induced by a wild-type Abelson-Moloney viral complex. The enzyme was heat-sensitive and strongly inhibited by L-p-bromotetramisole and L-homoarginine but not by L-phenylalanine. All these data indicated that this enzyme was most likely identical to the L/B/K ALP isoenzyme. Southern blot analysis showed that neither amplification nor polymorphism were responsible for the high expression of the ALP gene observed in A/63-2 cells. On the opposite, the mRNA transcripts of ALP were only detected in A/63-2 cells indicating that a modulation of the ALP gene transcription occurred which could be due to the insertion of the v-abl gene within or near the 5'-flanking region of the ALP promotor in A/63-2 cells. Butyrate strongly increased both the secretion and the expression of the enzyme on A/63-2 cell surface. This induction was strongly inhibited by cordycepin, an RNA biosynthesis inhibitor, and at a lesser degree by cycloheximide, a translation inhibitor suggesting that butyrate induction occurs both at the transcriptional and the translational level.
World Journal of Gastroenterology, 1998
AIM To explore the expression and changes of hepatoma specific alkaline phospatase (ALP) in rats during canceration. METHODS The ALPs and isoenzymes of rat livers and sera were investigated in SD hepatomas induced with 0.05% 2-fluoenylacetamide (2-FAA). RESULTS By pathological examination and biochemical analysis. ALPs were overexpressed in rat livers during canceration and then were secreted into blood. Serum total ALP activities, liver ALP specific activities (U/g) including soluble and membrane-combined ALP activities of each group were all significantly higher (P<0.01)than those of control group. The average ratios of soluble ALP to membrane-combined ALP were increased significantly after 6 weeks. ALP isoenzymes of rat sera and livers showed 5 bands on PAGE: ALP-I and ALP-II were specific for normal liver and rat hepatoma tissues, the ALP-II appeared in rat liver after 6 weeks and in sera after 8 weeks. CONCLUSIONS ALP with carcino-embryonic protein was overexpressed in hepatoma tissues; the abnormal ALP-II of ALP isoenzymes in sera and liver of rats can be used as a tumor marker for early diagnosis of rat hepatoma.
Effect of cellular determination on oncogenic transformation by chemicals and oncogenes
Molecular and cellular biology, 1988
Three developmentally determined myogenic cell lines derived from C3H 10T1/2 C18 (10T1/2) mouse embryo cells treated with 5-azacytidine were compared with the parental 10T1/2 line for their susceptibility to oncogenic transformation by 3-methylcholanthrene or the activated human c-Ha-ras oncogene. Neither the 10T1/2 cells nor the myogenic derivatives grew in soft agar or formed tumors in nude mice. In contrast to 10T1/2 cells, the three myogenic derivatives were not susceptible to transformation by 3-methylcholanthrene, so that cellular determination altered the response of 10T1/2 cells to chemical carcinogen. On the other hand, all cell types were transformed to a tumorigenic phenotype following transfection with the activated c-Ha-ras gene. The transfected myogenic cells expressed both the c-Ha-ras gene and the muscle determination gene MyoD1. In contrast to other reports, the presence of as many as six copies of the c-Ha-ras gene per genome did not prevent the formation of striat...
FEBS Letters, 1994
We measured the level of reduced glutathione (GSH) and oxidized glutathione (GSSG) in normal and oncogene-transformed NIH/3T3 fibroblasts and 32D hematopoietic cells. NIH/3T3 cells transformed by the activated oncogenes erbB, src, and raf, showed increased levels of GSH with concomitant alterations in the levels of GSH-related enzymes. Transfection and over-expression of a synthetic gene coding for a phosphotyrosine protein phosphatase (PTPase), which inhibited the proliferation of normal and transformed NIH/3T3 cells, was accompanied by a decrease in GSH levels in normal and erbB-transformed fibroblasts, and by an increase in src and raf transformants. Among GSH-related enzymes, only y-glutamylcysteine synthetase was altered in normal and erbB-transformed NIH/3T3 fibroblasts following PTPase transfection. Therefore, tyrosine phosphorylation could be selectively involved in the regulation of GSH metabolism in normal and oncogene-transformed NIH/3T3 fibroblasts, possibly by a dual-type effect on receptor/oncoprotein-mediated mitogenic signal transduction. However, no relationship was observed between the GSH and PTPase effect on cell growth, either after oncogene transfection or PTPase transfection. Moreover, the changes in GSH metabolism were specifically related to cell lineage. In fact GSH and related enzymes did not change in 32D hematopoietic cells transformed by the same activated erbB oncogene and in those -normal or transformed -over-expressing the PTPase: in these cells also, over-expression of the PTPase gene was not accompanied by growth inhibition.
Regulation of the expression of alkaline phosphatase in a human breast-cancer cell line
The Biochemical journal, 1994
Treatment of the cultured human breast-cancer cells BC-M1 with dexamethasone induced a placental-type alkaline phosphatase (ALP). Both the ALP activity and the mRNA level in the cells were increased. The induction of ALP activity by dexamethasone was time- and dose-dependent. The accumulation of ALP mRNA was inhibited by both actinomycin D and cycloheximide, indicating that its induction is a complex event and may involve other regulatory proteins. Retinoic acid showed opposing effects with dexamethasone on the expression of alkaline phosphatase. Retinoic acid (RA) and phorbol 12-myristate 13-acetate also substantially reduced the dexamethasone-induced expression of ALP. Studies on thermostability and sensitivity to various amino acid inhibitors indicated that the BC-M1 ALP is most similar to the placental form. Northern hybridization analysis also revealed that ALP mRNA transcripts in BC-M1 and term placenta are similar in size and are distinct from that of the placental-like mRNA ...
Mutation research, 1996
Cell transformation models have been established for studying the cellular and molecular basis of the neoplastic process. Transformation models have also been utilized extensively for studying mechanisms of chemical carcinogenesis and, to a lesser degree, screening chemicals for their carcinogenic potential. Complexities associated with the conduct of cell transformation assays have been a significant factor in discouraging broad use of this approach despite their reported good predictivity for carcinogenicity. We previously reported that many of the experimental difficulties with the Syrian hamster embryo (SHE) cell transformation assay could be reduced or eliminated by culturing these cells at pH 6.7 culture conditions compared to the historically used pH 7.1-7.3. We and others have shown that morphological transformation (MT), the earliest recognizable phenotype in the multi-step transformation process and the endpoint used in the standard assay to indicate a chemical's trans...
FEBS Letters, 1993
Having determined the complete amino acid sequence of a cytosolic phosphatase purified from bovine liver, we studied the role of this enzyme (referred to as 'PTPase') in the control of cell proliferation. We used NIH/3T3 fibroblasts, both normal and transformed by the oncogenes v-erbB, v-src, and v-raf: a synthetic gene coding for PTPase was transfected into, and overexpressed in, normal and transformed NIH/3T3 cells with resulting inhibition of cell growth. Inhibition of proliferation correlated with the level of foreign PTPase; growth in soft agar was also inhibited in transformants overexpressing the enzyme. However, PTPase overexpression did not inhibit the rapid turnover of inositol lipids stimulated by platelet-derived growth factor. We conclude that this novel PTPase is active on cell type-specific signalling substrates that control normal and transformed fibroblast proliferation.
Archives of Biochemistry and Biophysics, 2001
The expression and regulation of alkaline phosphatase (AP) was studied in the human gastric cancer cell line TMK-1. Biochemical analysis, reverse transcription-polymerase chain reaction, and Northern blot analysis demonstrated that the cells express placental, germ cell, and intestinal AP isozymes constitutively. Dexamethasone (Dex), a synthetic glucocorticoid, was shown to specifically induce the placental AP activity to about 10-fold and sodium butyrate (NaBu) induced germ cell AP activity to about 4-fold, respectively. In contrast, these two agents showed little effect on the level of intestinal isozymes. Dex and NaBu also differentially induced the mRNA levels of the placental and germ cell APs. Northern blot analysis of the placental AP transcript in the presence of the transcription inhibitor, 5, 6-dichloro-1--D-ribofuranosyl benzimidazole, revealed that the half-life of placental AP mRNA is about 27 h for both the Dex-treated and untreated cells. Nuclear run-on transcription analysis indicated an apparent increase in the rate of placental AP gene transcription in Dex-treated cells. These results indicated that the effect of Dex occurred primarily by activation of the placental AP gene transcription in the cells. In order to study the direct Dex and NaBu effect on AP gene expression, the proximal promoter regions of AP genes were fused to luciferase reporter vectors. Despite the high similarity in nucleotide sequences of these two genes, transient transfection analysis demonstrated that Dex and NaBu exerted a specific stimulation only through the respective placental and germ cell AP gene promoter. Taken together, this study indicates that the expression of PAP and GCAP isozymes have specific regulatory mechanisms that can be differentially controlled by signals including glucocorticoid and NaBu.
Proceedings of the National Academy of Sciences, 1986
Cell hybrids between normal, early-passage Syrian hamster embryo cells and a highly tumorigenic, chemically transformed hamster cell line, BP6T, were formed, selected, and analyzed. Tumorigenicity andanchorage-independent growth were suppressed in the hybrid cells compared to the tumorigenic BP6T cells. These two phenotypes segregated coordinately in these cells. To determine at what stage in the neoplastic process this tumor-suppressive function was lost, two chemically induced immortal cell lines were examined at different passages for the ability to suppress the tumorigenic phenotype of BP6T cells following hybridization. Hybrids of BP6T cells with the immortal, nontumorigenic cell lines at early passages were suppressed for tumorigenicity and anchorageindependent growth. This tumor-suppressive ability was reduced in the same cells at later passages and in some cases nearly completely lost, prior to the neoplastic transformation of the immortal cell lines. Subclones of the cell lines were heterogeneous in their ability to suppress tumorigenicity in cell hybrids; some clones retained the tumor-suppressive ability and others lost this function. The susceptibility to neoplastic transformation of these cells following DNA transfection with the viral ras oncogene or BP6T DNA inversely correlated with the tumor-suppressive ability of the cells. These results suggest that chemically induced neoplastic progression of Syrian hamster embryo cells involves at least three steps: (i) induction of immortality, (ii) activation of a transforming oncogene, and (iii) loss of a tumor-suppressive function. The conversion of a normal cell into a malignant cell is recognized as a multistep process (1-3); however, the number of genetic changes involved is not known. A major advance in our understanding was the discovery of oncogenes that are capable of transforming immortal cells as well as normal, primary rodent cells when certain combinations of oncogenes are transfected into the cells (4-7). These experiments indicate that at least two cooperating, apparently dominantly acting, oncogenes are required for neoplastic transformation ofnormal, diploid cells. It has been proposed that one oncogene is involved in the immortalization process and the second in the expression of various transformed phenotypes, such as focus formation or anchorage-independent growth (3, 4, 7). However, certain observations suggest that changes in addition to these two steps are also needed. One of the most compelling lines of evidence comes from experiments involving hybridization of normal and malignant cells. Many, but not all, of these experiments indicate that tumorigenicity is a recessive trait (8-14). A major paradox in cancer biology, therefore, exists: DNA transfection experiments have identified dominantly acting cancer genes (oncogenes), whereas cell hybridization experiments suggest that tumorigenicity is recessive in nature.
FEBS Letters, 1993
Many human tumors contain high concentrations of ethanolamine phosphate (EtnP). An important question is whether increased formation of EtnP is merely the consequence of cell transformation, or is it associated with the process of carcinogenesis. Here we show that in C,I-I/lOTlR embryonic fibroblasts, an established cellular model for the study of carcinogenesis, the environmental carcinogens, 7,12dimethylbenz[a]anthracene (DMBA) and benzo[a]pyrene (B[a]P) (0.1-l &ml concentration; 24 h treatment), stimulate phosphorylation of ethanolamine derived from increased hydrolysis of phosphatidylethanolamine.