Electrophoretic heterogeneity of alkaline phosphatase isozymes in seminoma and normal testis

Electrophoretic patterns of seminoma- and normal-testis-derived alkaline phosphatase isozymes, the placental alkaline phosphatase (PLAP)-like enzyme and the tissue-nonspecific (liver) alkaline phosphatase (LAP), were studied on starch gel and isoelectric focusing (IEF). Different migration patterns of the PLAP-like enzyme were observed with respect to both seminomas and normal testes on starch gel electrophoresis. On IEF, seminomas showed different staining patterns among different tumors; however, a common main activity was focused at pIs of 4.3-4.6, corresponding to pIs of PLAP. Normal testes showed two enzyme-staining regions, at pIs of 4.1 and 5.0-5.2, which were discriminated from pIs of PLAP and the PLAP-like enzyme in seminoma. The PLAP-like enzyme in seminoma was differentiated from PLAP by digestion with neuraminidase. Neuraminidase treatment simplified the distribution patterns of the PLAP-like enzyme in normal testis, but did not alter the pattern of microheterogeneity in seminoma. Two factors other than sialylation, namely structural modification of the carbohydrate moiety and variation of hydrophobicity, were shown to contribute to the microheterogeneity of the PLAP-like enzyme in seminoma. LAP in seminoma and in normal testis also showed marked electrophoretic heterogeneity and differences in pI distributions from LAP of liver. However, the migration patterns after desialylation were very similar to each other. The findings imply that electrophoretic heterogeneity demonstrated in LAP in seminoma and in normal testis is caused by a difference in sialic acid content in the molecule, and the heterogeneity of the PLAP-like enzyme in seminoma is considerable.     

Hydrophobicity and lectin affinity of alkaline phosphatase isozymes in seminoma and normal testis

Isozymes of alkaline phosphatases (ALP) in seminoma and normal testis were separated by use of high-performance liquid chromatography and a TSK-gel phenyl-5PW column. The tissue-nonspecific (liver) ALP (LAP) was the dominating isozyme, consisting of more than 90% ALP activity. The placental ALP (PLAP)-like enzyme contributed to 4-8% of the total ALP activity. The intestinal isozyme (IAP) could not be identified. The glycosylation patterns of the isozymes were studied using concanavalin A (Con A) affinity chromatography and batch elution with competing sugar. All PLAP activity in placental extracts and LAP activity in liver extracts was bound to Con A-Sepharose. In the tumor extracts, only 50-70% of the PLAP-like enzyme and 20-50% of the LAP activity from seminomas were bound to Con A-Sepharose. A similar binding pattern of the PLAP-like enzyme and LAP was also seen in the normal testes. This variability in Con A reactivity with PLAP or the PLAP-like enzyme was also reflected in serum of seminoma patients and of pregnant women. Thus, ALP expressed in seminoma has different lectin affinity characteristics compared with the same isozyme from placenta and liver, but almost identical to ALP in the normal testes. These findings imply that the PLAP-like enzyme and LAP in the testis can be discriminated from PLAP of placenta and LAP of liver by carbohydrate lectin affinity. It also supports the concept that the increased amounts of ALP in seminomas result from the enhanced eutopic expression of enzymes normally expressed in the testis.     

Identification and characterization of alkaline phosphatase isozymes in human colorectal adenocarcinomas

Using alkaline phosphatase isozyme-specific immunocatalytical assays, the content of isozymes was determined in normal mucosas and adenocarcinomas from human colon or rectum. Tumor levels of both the tissue (liver)-unspecific and the placental-like alkaline phosphatase (PLAP-like) were elevated compared to normal mucosas of the same patients. Such elevations have been reported previously, particularly in seminomas and ovarian tumors. In several tumors, moreover, the intestinal isozyme was expressed in lesser amounts than in the adjacent mucosa. The present results indicate that the activation of two of the phosphatase isozymes, including expression of the typical germ cell line phosphatase (the PLAP-like isozyme), may occur even in nongonadal tumors. This may reflect an induction pattern of phosphatase isozymes, with implications for malignant transformation also in other tumors.     

Placental-like alkaline phosphatase and DNA flow cytometry in spermatocytic seminoma.

Immunohistochemical analysis was done on 7 testicular tumors classified as spermatocytic seminoma (SS) and 25 classic seminomas. Except for a few scattered cells, the spermatocytic seminomas were negative for placental-like alkaline phosphatase (PLAP); the classic seminomas were all positive for this enzyme. The SS also were negative for alpha-fetoprotein (AFP), beta-human chorionic gonadotropin (hCG), and leukocyte common antigen (LCA). The ploidy of the seven tumors of SS was as follows: two, diploid; two, near-diploid; one, tetraploid; one, aneuploid; and one, uninterpretable. The essentially negative staining of SS for PLAP was strikingly different from the pattern in classic seminoma. Thus, staining for this enzyme is useful for making the differential diagnosis between classic seminoma and SS. To differentiate between malignant lymphoma and SS, staining for leukocyte common antigen is helpful.     

Phenotypes of placental-type alkaline phosphatase in seminoma sera as defined by monoclonal antibodies

PLAP-like enzymes could be detected in serum of patients with primary testicular tumors, in particular seminomas. The use of a panel of monoclonal antibodies (MAbs) permitted typing into 6 different testicular serum phenotypes, of which one appeared similar to a placental type (II) and 2 have not been previously described. Most tumor sera belonged to type I, as described for seminoma tissues. With a more advanced tumor the mean serum PLAP-like levels increased. After operation, after radiotherapy or with no evidence of disease lower or non-detectable enzyme levels were found. In typing the tissue PLAP-like antigen in serum of the same patient or sera from recurrences of a seminoma in the same patient, the same phenotypes of PLAP-like antigen were usually but not always found. None of the 6 phenotypes appeared to confer a poorer prognosis. We conclude that the expression of PLAP-like antigen is eutopic and is enhanced by testicular malignancy, especially in seminoma cells.     

Significance of alkaline phosphatase isozymes in the monitoring of patients with colorectal carcinoma

The clinical course of colorectal carcinoma may be monitored by tumor markers such as carcinoembryonic antigen (CEA), carcinoma antigen (CA) 19-9 and CA-50. Alkaline phosphatase isozymes were previously used to study the clinical course of testicular and gynecologic tumors. In this study we investigated 8 patients with advanced colorectal carcinoma. Their sera were analyzed for the tumor markers CEA, CA 19-9, CA-50 and three alkaline phosphatase isozymes: the nonspecific liver isozyme LAP, the intestinal isozyme IAP and the placental isozyme PLAP. Rising levels of CEA, CA 19-9 and CA-50 were seen as expected, and PLAP also showed rising levels during tumor progression. LAP remained elevated. This indicates an association between progression of colorectal carcinoma and a raised serum content of alkaline phosphatase isozymes.     

Demonstration of placental alkaline phosphatase in human breast cancer.

The screening of a series of 11 metastatic breast tumors for the presence of the placental isoenzyme of alkaline phosphatase (EC3.1.3.1) by RIA revealed one strong producer. The alkaline phosphatase of this tumor was characterized with respect to its immunochemical cross-reactivity, inhibition by L-phenylalanine and levamisole, subunit molecular weight (Mr) and isoelectric point (pl) in two-dimensional electrophoresis, and one-dimensional peptide map. In all parameters of the characterization, the tumor alkaline phosphatase, except for subunit molecular weight which was slightly lower (60,000 versus 64,000 for the placental isoenzyme). No strong placental alkaline phosphatase producers were found among 16 primary tumors examined by RIA. The screening of patients' sera for the placental alkaline phosphatase using RIA indicated elevated levels over post-menopausal controls in 20% of the metastatic patients. Only 3% of the primary patients had elevated serum levels. These results suggest that the placental isoenzyme of alkaline phosphatase may be a useful tumor marker for recurrent breast cancer.     

Expression of human placental-type alkaline phosphatase in primary breast cancer

A monoclonal antibody (H317) with specificity for the heat-stable placental-type alkaline phosphatase (PI-ALP) isoenzyme has been used to investigate the occurrence of PI-ALP in patients with primary breast carcinoma. All pre-operative plasma samples were negative for PI-ALP in a sensitive solid-phase enzyme immunoassay with a lower limit of detection of 0.1 U/l. In contrast, using a peroxidase-anti-peroxidase staining technique on fixed tissue sections, as well as enzyme immunoassay on fresh tissue extracts, PI-ALP could be demonstrated in the carcinomatous tissue of all seven patients investigated.     

Placental alkaline phosphatase as a tumour marker in seminoma using the H17 E2 monoclonal antibody assay

Serum samples from 62 patients with seminoma were assayed for placental alkaline phosphatase-like activity using the monoclonal antibody H17 E2, in order to evaluate its utility as a serum tumour marker. Fifteen of 16 patients (94%) with active seminoma had elevated serum PLAP levels. Sixteen of 46 (35%) of patients considered to be in remission had elevated PLAP levels (false positive rate 35%). Fifteen false positive results were considered attributable to concomitant smoking, and if these patients are excluded, only one false positive case was detected. In 7 out of 7 patients sequential PLAP assays reflected clinical response to treatment.     

Serum alpha-fetoprotein and variant alkaline phosphatase in human hepatocellular carcinoma

Alpha-fetoprotein and variant alkaline phosphatase were studied in a group of patients with hepatoma. The incidence of positive alpha-fetoprotein was 67.6% in confirmed cases. Variant alkaline phosphatase was present only in patients with positive fetoprotein test, and it appeared in only 28.6% of the cases.     

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.     

A hidden antigenic determinant on membrane-bound human placental alkaline phosphatase

Two sets of monoclonal antibodies (mAbs) specific for human placental alkaline phosphatase (PLAP) were compared. One set of four mAbs was generated against solubilized and purified PLAP; the other set of seven mAbs was generated against the malignant cell line Hela TCRC-1 in which PLAP is an ectopically synthesized membrane-bound enzyme. Double immunodiffusion and competitive enzyme-linked immunosorbent assays were used to examine the relative spatial arrangement of the antigenic determinants to which each of the eleven mAbs binds. Significant differences in immunoreactivity of the antibodies were demonstrated. The mAbs to the solubilized and purified enzyme bound in either of two regions of the molecule. By contrast, all of the mAbs to PLAP as presented on the tumor cell surface bound in only one of these two regions. One of the major antigenic determinants on the solubilized enzyme is apparently unavailable for recognition by immunoreactive cells during immunization with whole cells. Furthermore, when mAbs are generated to this region using purified PLAP as the immunogen, they do not recognize membrane-bound PLAP. The 'hidden' determinant can be exposed in vitro after partial solubilization using butanol to extract the enzyme from HeLa TCRC-1 cells and subsequent treatment with 0.5% Nonidet P-40 detergent. The results of this study have implications for the potential use of mAbs in studies of other cell surface antigens and in tumor immunolocalization and drug targeting.     

Placental-type alkaline phosphatase in cervical neoplasia

Monoclonal antibodies reactive with placental-type alkaline phosphatase have formed the basis of methods for detection of this oncodevelopmental antigen in patients with pre-invasive and invasive cervical neoplasia, with or without evidence of papilloma virus infection. Disease-related elevations of placental-type alkaline phosphatase were not observed in patients' sera. Solubilised cervical smears or biopsy material, and cervical mucus swabs, often contained substantial amounts of this isoenzyme; however, there was no significant difference between any of the patient and control groups. Thus, serological and smear test assays for placental-type alkaline phosphatase were not useful in differential diagnosis of cervical lesions. However, its presence in most biopsy specimens, often at high levels, indicated possible application for in vivo radioimmunoimaging studies of invasive or metastatic cervical cancer.