Alkaline phosphatase isozymes in human testicular germ cell tumors, their precancerous stage, and three related cell lines

The four known isozymes of the human alkaline phosphatase (ALP) were detected by isoelectric focusing in extracts of various types of germ cell tumors, three related cell lines, and their precancerous elements (atypical germ cells). In seminoma, placental alkaline phosphatase (PLAP) and germ cell alkaline phosphatase (PLAP-like) could be separated by isoelectric focusing following isolation by immunoaffinity. The occurrence of both isozymes in seminoma could explain partial heat sensitivity and variation in electrophoretic patterns of the seminoma isozyme frequently observed upon starch gels, in comparison to the normal placental phenotype. The four ALP isozymes are produced not only in germ cell tumors, but already in precancerous tissues. Quantitative analysis showed that the amount of the four isozymes varies in parallel in the tumors tested. Maximal expression was found in seminoma. The relation between ALP gene overexpression and gene amplification by polyploidy of chromosomes 1 and 2 in these lesions is discussed. On the other hand, the ectopic expression of intestinal alkaline phosphatase and PLAP associated with overexpression of PLAP-like in tumor cells as well as in their precancerous stage indicates gene activation by some unknown mechanisms, probably a regulatory process affecting the three tissue-specific ALP genes simultaneously.     

Identification of testicular atypical germ cells by an immunohistochemical technique for placental alkaline phosphatase

The identification of atypical testicular germ cells is often difficult by by routine histologic examination. By immunohistochemical detection of placental alkaline phosphatase (PLAP) and by periodic acid Schiff staining of glycogen, atypical germ cells were easily identified in testicular samples. Forty-one fetal and adult testes were used for a preliminary study, and 121 testes from infants and adults with either cryptorchidism or germ cell tumors were studied for the presence of atypical germ cells. Two types of clear germ cells were differentiated histochemically, and one with PLAP-positive cell surfaces and glycogen-rich cytoplasm was considered to be atypical. The alkaline phosphatase of atypical germ cells appeared to be similar to that found in a few germ cells of early fetal testes. The atypical germ cells seemed to be multi-potential malignant cells capable of developing not only into seminoma but also into other germ cell tumors. Only in yolk sac tumor of infants were the atypical germ cells absent from tumor-adjacent seminiferous tubules.     

Expression of a Nagao-type, phosphatidylinositol-glycan anchored alkaline phosphatase in human choriocarcinomas

The alkaline phosphatase (AP) synthesized by human tumor cells closely resembles human placental AP (PLAP). Little is known about the molecular events that lead to the expression of a placenta-like AP in tumor cells. The complementary DNA encoding the AP expressed by a choriocarcinoma cell line, BeWo, was isolated and characterized. The complementary DNA is the product of the germ cell AP (Nagao isozyme) gene and not of the term PLAP gene. Like placental AP, the tumor AP can be released from the cell membrane by a phosphaditylinositol-specific phospholipase C and has a phosphaditylinositol-glycan (PI-glycan) moiety at the COOH terminus. Immunoprecipitation of phosphaditylinositol-specific phospholipase C-treated AP and analysis by polyacrylamide gel electrophoresis or isoelectric focusing demonstrates that at least 95% of the AP contains PI-glycan. Two-dimensional gel electrophoresis reveals two precursors of the mature AP. One of these does not bind an antibody against the Trypanosoma variable surface glycoprotein cross-reacting determinant and probably does not contain PI-glycan. This precursor had a shorter half-life than the more prominent PI-glycan-containing precursor in pulse-chase experiments, suggesting a precursor-product relationship between the two proteins. These data demonstrate that BeWo AP is the product of a gene normally expressed in testis, thymus, and germ cells, but not in placenta. Thus, the expression of BeWo AP results from the repression of the PLAP gene and derepression of the germ cell AP gene and, as such, the expression is ectopic. The BeWo AP (Nagao isozyme) is modified with PI-glycan that is added soon after translation, not cotranslationally.     

Monoclonal antibody assay of serum placental alkaline phosphatase in the monitoring of testicular tumours

A monoclonal antibody (H17E2) recognising both placental alkaline phosphatase (PLAP) and testicular PLAP-like alkaline phosphatase was incorporated in a solid phase immunoassay. This was used to measure levels of PLAP in 257 sera from 148 patients with germ cell neoplasms of the testis. High levels of PLAP were found in all patients with active seminomas (mean 0.85 O.D.) compared to those in clinical remission (mean 0.20 O.D.) (P less than 0.0001). More importantly, changing levels of PLAP correlated with the course of disease in 79 samples from 33 patients with seminoma (P less than 0.0001). Elevated PLAP levels were also noted in patients in remission who were smokers (mean 0.32 O.D.) compared to non-smokers (mean 0.15 O.D.) (P less than 0.001). These data demonstrate that determination of PLAP levels using this sensitive immunoassay is an important new adjunct in the monitoring of the response to treatment in patients with seminoma.     

Folate-binding protein is a marker for ovarian cancer.

We describe the isolation of a complementary DNA (cDNA) sequence encoding the ovarian cancer-associated antigen recognized by monoclonal antibody MOv18 and its identification as a high-affinity folate-binding protein (FBP). Functional cDNA clones were isolated using mRNA from the ovarian carcinoma cell line SKOV3 and colon carcinoma cell line HT29, by transient expression in WOP cells and selection of expressing cells by adhesion to antibody-coated magnetic beads. The cDNAs differed in the lengths of 5'- and 3'-noncoding regions, but they encoded identical peptides. A database search clearly showed them to be adult high-affinity FBPs with amino acid sequences identical with those isolated from normal placenta and several carcinoma cell lines. Reactivity of cell lines with MOv18 was quantitatively consistent with the expression of FBP mRNA. Southern hybridizations show evidence of a family of related genes and/or pseudogenes and were mapped to chromosome 11q13.3-14.1 by fluorescent in situ hybridization using cosmid clones containing part of this region. Also identified were two PstI polymorphisms of four and three alleles, respectively, and a two-allele MspI polymorphism. The folate-binding protein locus was not amplified in any of the 16 carcinoma cell lines tested and in only 1 of 10 serous adenocarcinomas, indicating that overexpression of FBP in ovarian cancer cannot, in general, be due to gene amplification.     

Evidence for homology of normal and neoplastic human placental alkaline phosphatases as determined by monoclonal antibodies to the cancer-associated enzyme

The HeLa TCRC-1 human adenocarcinoma cell line expresses a form of alkaline phosphatase that is similar to the common S-variant of placental alkaline phosphatase (PLAP) on the basis of electrophoretic mobility, catalytic properties, and reactivity with polyclonal antibodies. More sensitive probes of changes in protein structure than polyclonal antibodies are monoclonal antibodies (MAbs) which recognize individual antigenic sites on molecules. Therefore, we produced MAbs to HeLa TCRC-1 cells and selected those which bound to the alkaline phosphatase expressed by the cancer cells. Seven MAbs were obtained and characterized by (a) fine specificity analysis using allelic variants of PLAP and other human alkaline phosphatase isozymes, (b) immunoglobulin isotype, and (c) relative binding affinities to PLAP from two sources, placental tissue and HeLa TCRC-1 cells. The seven MAbs bind the enzymes from both sources with equal affinity indicating a high degree of structural homology if not identity between the normal S-variant of PLAP and its cancer-associated counterpart. We note that most of the MAbs to cancer cell surface-bound PLAP express either Ig (immunoglobulin) G2a or IgG2b heavy-chain isotypes, a higher incidence of these classes of IgG than has been observed with the purified and soluble PLAP immunogen which yields MAbs predominantly of the IgG1 isotype. Finally, some of these antibodies, like the ones prepared from purified PLAP, recognize differences between allelic variants.     

Production of placental alkaline phosphatase (PLAP) and PLAP-like material by epithelial germ cell and non-germ cell tumours in vitro.

Placental and placental-like alkaline phosphatase (PLAP) levels in the culture media of 87 cell lines of neoplastic and ''normal'' origin were measured by a conventional immunosorbent enzymatic assay (IAEA) and by a new immunoradiometric assay (IRMA). The IRMA detected immunoreactive PLAP in 37 of 80 (46%) human epithelial and germ cell cultures, while the IAEA detected PLAP in only 25 (33%). Of the 52 non-germ cell tumour cultures, the IRMA detected expression in 24 (46%) and the IAEA in only 16 (31%). In 17 cases (21%) the IRMA recorded levels double that of the IAEA, while in five cultures (6%) the reverse was true. The IRMA was much more robust than the IAEA and had considerably lower inter- and intra-assay coefficients of variation (3.75-8.5% vs 5.2-46%). Detection of PLAP(-like) expression by IAEA is dependent on neoplastic expression of enzymatically functional molecules and quantification assumes constant enzyme kinetics. PLAP-like material has a higher catalytic rate constant than PLAP and thus will give higher values on a stoichiometric basis in an IAEA. The higher detection rate and levels of PLAP-like material in neoplastic cultures when measured by the IRMA clearly demonstrate ectopic expression of non-enzymatic PLAP and PLAP-like genes. The incidence of PLAP(-like) expression by non-germ cell and possible germ cell tumours has been underestimated and its utility as a tumour marker should be re-examined using assays which measure antigen mass rather than phosphatase activity.     

DNA sequencing of a mouse liver protein that binds selenium: implications for selenium's mechanism of action in cancer prevention

Complementary DNA clones for liver protein 56K (SLP-56) were isolated by screening a lambda Zap mouse liver library. The cloned cDNAs represented the complete message. The correct reading frame was verified by alignment of the deduced amino acid sequence with that of peptides sequenced from the purified protein. The primary sequence has not been reported previously since homologous DNA sequences were not found in GenBank. Most importantly, the DNA sequence did not contain an in-frame TGA codon that would code for seleno-cysteine, as occurs in the prototypic selenoprotein, glutathione peroxidase. Hydropathy analysis suggested the protein was not a membrane-spanning protein. SLP-56 was previously localized as a cytosolic-soluble protein on the basis of cell fractionation experiments. The results suggest that SLP-56 is different from proteins whose synthesis and concentration are dependent upon selenium and require TGA to encode for selenocysteine. In this respect, SLP-56 appears to be similar to liver fatty acid binding protein (SLP-14) for which selenium is a ligand. Our working hypothesis is that selenium exerts its inhibitory effects on cell growth by modulating the properties of existing growth regulatory proteins. The two proteins that are readily labeled by selenium in many rodent tissues, SLP-56 and SLP-14, would fit into this category.     

Growth Inhibition, Enhancement of Intercellular Adhesion, and Increased Expression of Carcinoembryonic Antigen by Overexpression of Phosphoinositides-specific Phospholipase C β1 in LS174T Human Colon Adenocarcinoma Cell Line

By using a retrovirus-derived system we generated derivatives of the human colon adenocarcinoma cell line LS174T (ATCC CL 188) that stably overexpress a full-length cDNA encoding the β1 isoform of bovine phosphoinositides-specific phospholipase C (PI-PLC). This was confirmed by the elevated levels of catalytic activity to release phosphoinositides from phosphatidylinositol (PI-PLC) or phosphatidylinositol-bis-phosphate (PIP₂-PLC), and the enhanced expressions of messenger RNA and protein. PI-PLC β1 overexpresser clones grew to form cell clumps floating in liquid medium, whereas the pMV7-introduced control clones displayed morphologic characteristics that were very similar to those of the parent LS174T cell line. Three individual PI-PLC β1 overexpresser cell lines displayed increased doubling time (18.0 h, 21.5 h, and 23.8 h) when compared with 4 individual pMV7-introduced control cell lines (13.1 h, 10.7 h, 12.9 h, and 9.3 h). Anchorage-independent growth ability in soft agar medium was dramatically suppressed by overexpression of PLC β1, and the ability of PLC-overproducer clones to form aggregates when cultured in liquid medium was dramatically enhanced when compared with that of pMV7-introduced control clones. Tumorigenicity of PLC β1-overproducers was much weaker than that of vector-transduced control clones. The spontaneous release of carcinoembryonic antigen from PLC β1-overproducer clones was much higher than that from pMV7 control clones. The ability of PLC β1-overproducer clones to form aggregates during suspension culture was much stronger than that of the control clones. These results provide the first evidence that elevated levels of endogenous PI-PLC β1 suppress tumor cell growth, but enhance the ability to form cell aggregates and to release carcinoembryonic antigen, an intercellular adhesion molecule.     

Growth inhibition, enhancement of intercellular adhesion, and increased expression of carcinoembryonic antigen by overexpression of phosphoinositides-specific phospholipase C beta 1 in LS174T human colon adenocarcinoma cell line.

By using a retrovirus-derived system we generated derivatives of the human colon adenocarcinoma cell line LS174T (ATCC CL 188) that stably overexpress a full-length cDNA encoding the beta 1 isoform of bovine phosphoinositides-specific phospholipase C (PI-PLC). This was confirmed by the elevated levels of catalytic activity to release phosphoinositides from phosphatidylinositol (PI-PLC) or phosphatidylinositol-bis-phosphate (PIP2-PLC), and the enhanced expressions of messenger RNA and protein. PI-PLC beta 1 overexpresser clones grew to form cell clumps floating in liquid medium, whereas the pMV7-introduced control clones displayed morphologic characteristics that were very similar to those of the parent LS174T cell line. Three individual PI-PLC beta 1 overexpresser cell lines displayed increased doubling time (18.0 h, 21.5 h, and 23.8 h) when compared with 4 individual pMV7-introduced control cell lines (13.1 h, 10.7 h, 12.9 h, and 9.3 h). Anchorage-independent growth ability in soft agar medium was dramatically suppressed by overexpression of PLC beta 1, and the ability of PLC-overproducer clones to form aggregates when cultured in liquid medium was dramatically enhanced when compared with that of pMV7-introduced control clones. Tumorigenicity of PLC beta 1-overproducers was much weaker than that of vector-transduced control clones. The spontaneous release of carcinoembryonic antigen from PLC beta 1-overproducer clones was much higher than that from pMV7 control clones. The ability of PLC beta 1-overproducer clones to form aggregates during suspension culture was much stronger than that of the control clones. These results provide the first evidence that elevated levels of endogenous PI-PLC beta 1 suppress tumor cell growth, but enhance the ability to form cell aggregates and to release carcinoembryonic antigen, an intercellular adhesion molecule.     

Serum marker potential of placental alkaline phosphatase-like activity in testicular germ cell tumours evaluated by H17E2 monoclonal antibody assay

A monoclonal antibody (H17E2) was used in a solid-phase localisation of enzyme activity (ILEA) assay to evaluate placental-like alkaline phosphatase (PLAP) as a serum marker of testicular germ cell tumours. Single or repeated assays were performed on 213 normal blood donor and a smaller number of term pregnancy and testicular cancer sera. The detection limit of PLAP by this system was 0.14 O.D. units equivalent to 0.04iul-1. Of 50 patients with established metastatic disease tested before treatment, 88% of 16 with seminoma, 54% of 13 with mixed seminoma and malignant teratoma and 33% of 21 with malignant teratoma had serum PLAP greater than 0.2 O.D. units. This compared to an incidence of 2% in non-smokers and of 29% in smokers who had been free of disease for more than 12 months. In 15 of 22 successfully treated patients, pre-treatment serum PLAP exceeded 0.2 O.D. units (mean 0.69 O.D.) and varying (53-97%) reductions in the initial levels occurred with treatment. These results with monoclonal antibody ILEA assay suggest that measurement of PLAP levels will be useful in the management of patients with germ cell tumours, particularly seminoma.     

Isolation and characterization of colon cancer mucin from xenografts of LS174T cells

The structure of colonic mucin, which is thought to be important in several diseases, including ulcerative colitis and colon cancer, is poorly understood. Mucin was isolated from nude mouse xenografts of the LS174T colonic adenocarcinoma cell line by gel filtration and CsCl density gradient centrifugation. The isolated mucin had a high content of threonine, serine, and proline, with 28% of the total amino acids O-glycosylated. The carbohydrates present were fucose, sialic acid, galactose, N-acetyl-glucosamine, and N-acetyl-galactosamine in the ratio of 0.4:1.5:1.0:0.9:1.4. Rabbit antibodies were prepared that recognized primarily protein-dependent determinants. By DEAE-cellulose chromatography, the purified mucin was found to be heterogeneous, with three major components that had small differences in carbohydrate composition. LS174T was antigenically and chromatographically similar to mucins in colon cancer tissue specimens and in nonmalignant colonic mucosae.     

Isolation and sequencing of cDNA clones homologous to the v-fgr oncogene from a human B lymphocyte cell line, IM-9

Two c-fgr cDNA clones were isolated from a cDNA library derived from a human B lymphocyte cell line, IM-9. Sequence analysis of the clones showed that they contained inserts corresponding to nearly full-length human c-fgr mRNA, which could encode a polypeptide of 529 amino acids with a calculated molecular weight of 59,478. Although the amino acid sequence between Gly-78 and the carboxy-terminus of the c-fgr is highly homologous to the corresponding sequence of the c-yes protein, the homology between the two proteins is low in the amino-terminal proximal region. Northern blot hybridization analysis using the c-fgr specific sequence showed that the c-fgr mRNA was expressed at higher level in the liver than in the brain, lung, or kidney of a human fetus.