Fluctuation of alkaline phosphatase activity in synchronized heteroploid cell cultures: effects of prednisolone

In two heteroploid cell lines synchronized with thymidine double block, activity of alkaline phosphatase decreased during the 12 hour period preceding mitotic peak. A return to high values was observed during the next 12 hours of synchronous cycle. Prednisolone (11β, 17α, 21-trihydroxy-1,4-pregnadiene-3,20-dione), when added to such cell cultures increased alkaline phosphatase activity in one of the cell lines (Henle embryonic intestine) but had the opposite effect on another line (HeLa-S3) in which the enzyme activity was decreased. Neither effect could be demonstrated if the hormone was added at the end of S phase or if cells were arrested in metaphase by vinblastine sulfate.     

Presence and activity of alkaline phosphatase in two human osteosarcoma cell lines

The presence and activity of alkaline phosphatase in SAOS-2 and TE-85 human osteosarcoma cells grown in culture were examined at the ultrastructural level. A monoclonal antibody raised against purified human bone osteosarcoma alkaline phosphatase was used to localize the enzyme in cultures of the osteosarcoma cells. Similar cultures were analyzed for alkaline phosphatase activity using an enzyme cytochemical method with cerium as the capture agent. Alkaline phosphatase was immunolocalized at the light microscopic level in an osteogenic sarcoma and ultrastructurally on the SAOS-2 cell membrane and the enclosing membrane of extracellular vesicular structures close to the cells. In contrast, the TE-85 cells were characterized by the absence of all but a few traces of immunolabeling at the cell surface. Enzyme cytochemical studies revealed strong alkaline phosphatase activity on the outer surface of the SAOS-2 cell membrane. Much lower enzyme activity was observed in the TE-85 cells. The results support biochemical data from previous studies and confirm that SAOS-2 cells have a significantly greater concentration of alkaline phosphatase at the plasma membrane.     

Non-random distribution of abnormal mitoses in heteroploid cell lines.

We have performed absorption-cytometric DNA measurements of the DNA contents of the G0/G1, G2, metaphase, and telophase cells of the heteroploid MCa-11 and HL-60 lines, as well as the WCHE-5 line which has a narrowly restricted number of chromosomes. We found that morphologically unbalanced mitoses occurred much more frequently in telophase-cell pairs of the heteroploid MCa-11 and HL-60 lines than in those of the chromosomally stable WCHE-5 line. Furthermore, the morphologically unbalanced mitoses represented unequal segregation of DNA into each of the daughter telophase nuclei. Such mitotic segregation errors (MSE) occurred almost exclusively in telophase cells with DNA contents which were above, or below, the DNA content of the modal telophase population. The net effect of these non-random, unblanced divisions of heteroploid cells with non-modal DNA contents is to produce one daughter cell with a DNA content that tends to return to the modal DNA content peak.     

Characterization of human foetal intestinal alkaline phosphatase. Comparison with the isoenzymes from the adult intestine and human tumour cell lines.

The molecular structure of human foetal intestinal alkaline phosphatase was defined by high-resolution two-dimensional polyacrylamide-gel electrophoresis and amino acid inhibition studies. Comparison was made with the adult form of intestinal alkaline phosphatase, as well as with alkaline phosphatases isolated from cultured foetal amnion cells (FL) and a human tumour cell line (KB). Two non-identical subunits were isolated from the foetal intestinal isoenzyme, one having same molecular weight and isoelectric point as placental alkaline phosphatase, and the other corresponding to a glycosylated subunit of the adult intestinal enzyme. The FL-cell and KB-cell alkaline phosphatases were also found to contain two subunits similar to those of the foetal intestinal isoenzyme. Characterization of neuraminidase digests of the non-placental subunit showed it to be indistinguishable from the subunits of the adult intestinal isoenzyme. This implies that no new phosphatase structural gene is involved in the transition from the expression of foetal to adult intestinal alkaline phosphatase, but that the molecular changes involve suppression of the placental subunit and loss of neuraminic acid from the non-placental subunit. Enzyme-inhibition studies demonstrated an intermediate response to the inhibitors tested for the foetal intestinal, FL-cell and KB-cell isoenzymes when compared with the placental, adult intestinal and liver forms. This result is consistent with the mixed-subunit structure observed for the former set of isoenzymes. In summary, this study has defined the molecular subunit structure of the foetal intestinal form of alkaline phosphatase and has demonstrated its expression in a human tumour cell line.     

Establishment of heteroploid cell lines from mouse peritoneal exudate cells.

Proliferation was observed during in vitro cultivation of peritoneal exudate cells that had been educed from a C3H mouse with Freund's incomplete adjuvant. These cells were successfully subcultured by release with trypsin-EDTA solution and are now at passage 108 after 22 months in culture. Using this technique, 12 other rapidly growing peritoneal exudate cultures were obtained, whereas 10 cultures not educed with adjuvant did not proliferate. Characteristics of four adjuvant-induced cell lines established in culture include: rapid attachment to glass, doubling time in culture of 18 to 19 hr, phagocytosis of colloidal carbon, enhanced phagocytosis of specifically sensitized bacteria, epithelium-like morphology, and retention of C3H histocompatible specificities. These cell lines had widely varying chromosome distributions with modes from 37.3 +/- 2.4 to 82.6 +/- 2.30, but inoculation of 10(7) cultured cells into syngeneic animals did not produce tumors. Procedures described for the reproducible establishment of peritoneal exudate cell lines did not require use of conditioned media or exogenous viral infection.     

Establishment and characterization of tartrate-resistant acid phosphatase and alkaline phosphatase double positive cell lines

Morphologically macrophage-like cells were cloned from hamster bone marrow cells by coculturing bone marrow cells with hamster chondrocytes. One of the clones (CCP-2) was characterized in the present study. CCP-2 cells were positive in an osteoclast marker enzyme, tartrate-resistant acid phosphatase (TRAP), alkaline phosphatase (ALP) and non-specific esterase (NSE). We showed CCP-2 cells degraded cartilage matrix and hydroxyapatite coated on Osteologic disks. A gelatinase secreted from CCP-2 cells was observed and purified from serum-free conditioned medium of the cells. N-terminal amino acid sequencing of the purified enzyme revealed it was matrix metalloproteinase-9. However, CCP-2 cells failed to express calcitonin receptors, a mature osteoclast marker, even after coculture with osteoblast ST2 cells in the presence of 1alpha, 25-dihydroxyvitamin D3 [1alpha, 25-(OH)2D3]. The cells showed high affinity to types X and I but not to type II collagen. In addition, histochemical studies have shown the presence of tartrate-resistant acid phosphatase and alkaline phosphatase double positive cells at the secondary ossification site of the hamster humerus. From these observations, we concluded that CCP-2 cells are similar to osteoclast but not the same. CCP-2 cells are therefore important tools for investigating chondroclastogenesis/osteoclastogenesis and endochondral ossification.     

Characterization of KB cell alkaline phosphatase. Evidence of similarity to placental alkaline phosphatase.

The alkaline phosphatase from KB cells was purified, characterized, and compared to placental alkaline phosphatase, which it resembles immunologically. Two nonidentical nonomeric subunits of the KB phosphatase were found. The two subunits, which have apparent molecular weights of 64,000 and 72,000, can be separated on polyacrylamide gels containing sodium dodecyl sulfate. The Mr = 64,000 KB subunit appears to be identical in protein structure to the monomer of placental alkaline phosphatase. The Mr = 72,000 KB subunit, while differing in the NH2-terminal amino acid, appears also to be very similar to the placental alkaline phosphatase monomer. Both KB phosphatase subunits bind (32P)phosphate, and bind to Sepharose-bound anti-placental alkaline phosphatase. Native KB phosphatase is identical to the placental isozyme in isoelectric point, pH optimum, and inhibition by amino acids, and has a very similar peptide map. The data presented support the hypothesis that the Mr = 64,000 KB phosphatase subunit may the the same gene product as the monomer of placental alkaline phosphatase. This paper strengthens the evidence that the gene for this fetal protein, normally repressed in all cells but placenta, is derepressed in the KB cell line. In addition, this paper presents the first structural evidence that there are two different subunit proteins comprising the placental-like alkaline phosphatase from a human tumor cell line.     

Expression of intestinal alkaline phosphatase in human organs.

Human intestinal alkaline phosphatase was immunohistochemically identified and localized in the pancreas, liver and kidney by use of a monoclonal antibody specific for intestinal alkaline phosphatase isozyme and by amplified biotin-streptavidin staining. In all the examined organs, the intestinal isozyme was found to be localized in the epithelial cells of ducts: bile ducts in the liver, distal convoluted tubules and collecting tubules in the kidney and ducts in the secretory epithelium in the pancreas. In the liver the antibody also stained some sinus-lining cells. In all the examined organs the endothelial cells of the capillaries and some vessels were stained. By use of immunoelectron microscopy, intestinal alkaline phosphatase was, as expected, found to be localized to the microvillar region of the small intestine. The isozyme was abundantly expressed in the apical area of the microvilli and in membrane remnants in the fuzzy coat. Capillaries and vessels in the submucosa were also stained, as well as small vesicles in the endothelial cells. The present investigation demonstrates the expression and localization of the intestinal alkaline phosphatase in several organs, though previously believed to be expressed only in the intestine.     

Identification of protein-tyrosine phosphatase 1B as the major tyrosine phosphatase activity capable of dephosphorylating and activating c-Src in several human breast cancer cell lines

c-Src tyrosine kinase activity is elevated in several types of human cancer, and this has been attributed to elevated c-Src expression levels, increased c-Src specific activity, and activating mutations in c-Src. We have found a number of human breast cancer cell lines with elevated c-Src specific activity that also possess elevated phosphatase activity directed against the carboxyl-terminal negative regulatory domain of Src family kinases. To identify this phosphatase, cell extracts from MDA-MB-435S cells were chromatographed and the fractions were assayed for phosphatase activity. Four peaks of phosphatase activity directed against the nonspecific substrate poly(Glu/Tyr) were detected. One peak also dephosphorylated a peptide modeled against the c-Src carboxyl-terminal negative regulatory domain and intact human c-Src. Immunoblotting and immunodepletion experiments identified the phosphatase as protein-tyrosine phosphatase 1B (PTP1B). Examination of several human breast cancer cell lines with increased c-Src activity showed elevated levels of PTP1B protein relative to normal control breast cells. In vitro c-Src reactivation experiments confirmed the ability of PTP1B to dephosphorylate and activate c-Src. In vivo overexpression of PTP1B in 293 cells caused a 2-fold increase of endogenous c-Src kinase activity. Our findings indicate that PTP1B is the primary protein-tyrosine phosphatase capable of dephosphorylating c-Src in several human breast cancer cell lines and suggests a regulatory role for PTP1B in the control of c-Src kinase activity.     

Apolipoprotein B genetic polymorphisms in several human hepatoma derived liver cell lines.

The genetic polymorphism of apoB EcoRI and XbaI restriction sites and the 3' VNTR hypervariable region was examined in nine human hepatoma derived liver cell lines and related to the cells' ability to secrete lipids and apoB. EcoRI and XbaI genotypes appeared to be unrelated to triglyceride, cholesterol and apoB accumulating in the medium. The VNTR consisted of alleles with 47 to 67 repeats; however, these repeats were not associated with elevated concentrations of lipid or apoB. Data suggest that in the hepatoma cell lines, apoB polymorphisms in EcoRI, XbaI and the VNTR hypervariable region are not sufficient in themselves to account for triglyceride, cholesterol and apoB in the medium. It is possible that intracellular apoB synthesis and/or degradation as well as postsecretory apoB binding and uptake are responsible for the variability of apoB and lipid accumulation in the culture medium.