Regan Isoenzyme Research Articles

Regan isoenzyme of alkaline phosphatase as a tumour marker for renal cell carcinoma

Alkaline phosphatase is an enzyme present in all tissues of the human body. Several isoforms of this enzyme have been described with different catalytic nature, stability and antigenic structure. Rises in the activity of alkaline phosphatase are recognised in various states including bone diseases, liver disease, pregnancy, hyperthyroidism and malignant processes. The Regan isoenzyme, a rare variant of placental alkaline phosphatase, has been identified circulating in association with various tumours. The reported case describes a rising Regan isoform of alkaline phosphatase concentrations that led to a new diagnosis of occult renal cell carcinoma and persistently elevated activity postoperatively signposting persistent or recurrent disease.

The placental alkaline phosphatase variant D and natural selection. Relationship to complications of pregnancy and to "the Regan isoenzyme".

The placental alkaline phosphatase variant D is controlled by a gene which in most populations is rare (0.001–0.007). A significant increase of abortions in previous pregnancies was found in a series of deliveries from Sweden where the placenta (fetus) showed the D-variant. Among deliveries after various complications during pregnancy the D-variant was found in an increased frequency compared to a series of unselected, consecutive deliveries. In 1973 Inglis et al. claimed that the D-variant occurs in a frequency of 40–50% among cancer patients displaying ectopic (derepressed) heat-stable alkaline phosphatase (“the Regan isozyme”). A reexamination of their data suggests that the D-variant is not associated with cancer and that the identity between “the Regan isozyme” and placental alkaline phosphatase is still an open question.

Isozymes, Tumor Markers and Oncodevelopmental Biology

This is the history of discoveries of several enzyme tumor markers in the awardees laboratory. The first, beta-glucuronidase, was originally related to the physiological actions of estrogens and androgens. Perfection of histochemical techniques based on new substrates demonstrated the dual localization of beta-glucuronidase in endoplasmic reticulum and lysosomes. Tumor tissues, in general, are enriched with beta-glucuronidase. Next, acid phosphatase of the prostate gland possesses the distinctive property of undergoing inhibition by L-tartrate. This organ-specific inhibitor was incorporated into the Fishman-Lerner method for measuring serum acid phosphatase of prostatic origin. This significantly increased the specificity of the measurement of serum acid phosphatase for prostatic cancer. Finally, the discovery of the Regan Isoenzyme, placental alkaline phosphatase (PLAP) in a patient with disseminated lung cancer provided a tumor marker useful in the management of gonadal tumors, in particular. Closely related to PLAP is germ cell alkaline phosphatase which is eutopically expressed in seminoma. Finally, radioimmunolocalization and radioimmunotherapy of PLAP in these tumors have been achieved by others.

Tumor Markers in Leukemia: Evaluation of Serum Levels of Different Forms of Sialic Acid, Regan Isoenzyme and Lactate Dehydrogenase

A sensitive and specific serum marker can greatly help in the early diagnosis of malignancy as well as in monitoring the treatment of cancer patients. The present work was initiated for determining serum levels of Total Sialic Acid (TSA), Lipid Bound Sialic Acid (LSA), Free Sialic Acid (FSA). Regan Isoenzyme (RI) and Lactate Dehydrogenase (LDH), so as to evaluate their value as potential tumor markers. Fifty patients with anemia and 78 patients with leukemia were studied. The leukemia group consisted of 32 cases of Acute Myeloid Leukemia (AML), 29 cases of Chronic Myeloid Leukemia (CML) and 17 cases of Acute Lymphatic Leukemia (ALL). The levels were compared with the values obtained from 88 healthy individuals. Compared to the healthy controls, all the biomarkers were significantly elevated in patients with anemia as well as in those with leukemia. However, in leukemia patients significantly higher levels of TSA, LSA, FSA and LDH were observed compared to anemia patients. TSA levels were significantly higher in AML patients compared to CML and ALL patients. LSA levels were also significantly higher in AML patients compared to ALL patients. LSA was the most sensitive (84.6%) while FSA and RI levels were the most specific (78.0%) markers for leukemia. The combined use of the markers showed increased sensitivity and specificity (100.0% and 98.0%, respectively). The study suggested that the biomarkers investigated might be used for differentiating anemic from leukemic conditions, however, more in-depth studies are indicated to assess their utility in classifying various leukemias.

Placental Alkaline Phosphatase‐like Isoenzymes Produced by Human Gastric Cancer Cells

The human gastric cancer cell lines, MKN1 and SCH, were biochemically and biologically characterized according to the monophenotypic expression of placental alkaline phosphatase (PLAP)-like enzymes. The MKN1 cell line, derived from adenosquamous carcinoma, showed the same enzyme properties as the Regan isoenzyme, whereas the SCH cell line, derived from primary gastric choriocarcinoma, had properties identical with the Nagao isoenzyme. Regan isoenzyme activity expressed by MKN1 cells was stimulated by glucocorticoid and suppressed by retinoic acid. Both agents had no significant effect on SCH cells. On the other hand, Nagao isoenzyme activity expressed by SCH cells was stimulated by sodium butyrate, which had no stimulatory effect on MKN1 cells. Moreover, the PLAP-like activity of MKN1 cells showed no observable relationship to human chorionic gonadotropin (hCG)-producibility. Whereas expression of the Nagao isoenzyme by the SCH cell line is presumably a result of functional differentiation in the trophoblastic direction, that of the Regan isoenzyme by the MKN1 cell line is probably not. Perhaps the Regan isoenzyme is related to carcinogenesis.

Immunoperoxidase investigation of Regan isoenzyme of alkaline phosphatase in maxillary sinus carcinomas

The Regan isoenzyme of alkaline phosphatase is known to be produced ectopically by various malignant tumors. To assess whether this isoenzyme is expressed in maxillary sinus carcinomas, immunostaining for Regan isoenzyme was carried out on 35 maxillary sinus carcinomas comprising 27 squamous cell carcinomas, 6 adenocarcinomas, and 2 adenoid cystic carcinomas. The enzyme was shown to be present in 11 of 27 cases of squamous cell carcinoma, 2 of 6 cases of adenocarcinoma, and 1 of 2 cases of adenoid cystic carcinoma. Regan isoenzyme was identified in the cytoplasm and/or cell membrane of squamous cell carcinoma cells, whereas it was demonstrated mainly on the luminal membrane and in the secretions of both adenocarcinoma and adenoid cystic carcinoma cells. These findings show that Regan isoenzyme does, indeed, occur in maxillary sinus carcinomas.

Clinical significance of heat labile Regan isoenzyme variant in Hodgkin's disease.

The activity of a heat labile variant of Regan type alkaline phosphatase (HLRI) was studied in the serum of 38 histologically proved patients with Hodgkin's disease. The activity was also studied in 40 normal healthy adults matched for age and sex as control. HLRI was significantly elevated in patients with Hodgkin's disease in comparison with normal controls (1.12 +/- 0.12 units/ml vs 0.09 +/- 0.01 units/ml; P less than 0.001). The mean values of the isoenzyme tended to increase progressively with increasing stages of disease, reflecting a significant linear trend (P less than 0.001). The serial measurement of this variant may serve as a useful parameter in assessing disease activity and response to chemotherapy.

Studies of the Regan alkaline phosphatase isoenzyme in plasma from a lung-carcinoma patient.

We compared an alkaline phosphatase (EC 3.1.3.1) isoenzyme from a plasma sample of a patient with carcinoma of the lung, who was suspected of having the Regan-type isoenzyme, with placental serum alkaline phosphatase and with tissue preparations from placenta, liver, bone, and intestine. The placental and Regan phosphatases responded similarly to heat treatment and modification by various inhibitors, except that the Regan isoenzyme was more resistant to EDTA inactivation than was the placental serum alkaline phosphatase, but more sensitive than placental tissue alkaline phosphatase. Isoelectric focusing and Cellogel electrophoresis showed the Regan isoenzyme to have a mobility similar to that of the placental type. We conclude that the isoenzyme was of the Regan type and support the contention that the placental and Regan isoenzymes are products of the same structural gene, with small differences due to post-genetic modification.

Sensitive fluorometry of heat-stable alkaline phosphatase (Regan enzyme) activity in serum from smokers and nonsmokers.

We developed a simple, sensitive enzymatic assay involving the fluorogenic substrate naphthol AS-MX phosphate [(3-hydroxy-2-naphthoic acid 2,4-dimethylanilide) phosphate] to measure heat-stable alkaline phosphatase (EC 3.1.3.1), the Regan isoenzyme, in human serum. The day-to-day CV was 5.7% for a serum activity of 0.080 arbitrary units/L. Measurable amounts of enzyme were detected in most normal individuals. The mean for 51 nonsmokers was 0.068 (SD 0.037) arb. units/L; for 25 smokers it was 0.440 (SD 0.360) arb. units/L. Activity of this isoenzyme in smokers was as much as 10-fold the upper normal limit for nonsmokers. Activation of this tumor marker by smoking has not received attention hitherto. We conclude that a truly normal range can only be established among nonsmokers. The isoenzymes in smokers, nonsmokers, and pregnant women were similar in their heat stability, immunologic cross reactivity, and inhibition by L-phenylalanine.

Serum-sialyltransferase activity in cancer patients.

Serum-sialyltransferase activity was measured in serum samples of 116 patients with malignant tumors of various origins and different clinical stages using asialo-fetuin as the acceptor and cytidine-5'-mono-phospho[14C]sialic acid as the donor. Only patients with metastatic tumors had significantly elevated serum-sialyltransferase levels. Increased enzyme activity was also associated with rheumatoid arthritis and with acute hepatitis, whereas no significant alteration of enzyme activity was observed in cystic fibrosis patients. In a group of tumor patients, various additional tumor markers were determined (carcinoembryonic antigen, alkaline phosphatase: Regan isoenzyme, creatinekinase: BB-isoenzyme, lactatedehydrogenase: isoenzyme 5) and the data compared to the clinical diagnoses. The sensitivity and specificity of serum-sialyltransferase as a tumor marker is assessed.

Drug-induced biochemical markers of cancer in cervical carcinoma cells

The elevation in the serum level of CEA in cancer patients undergoing treatment with 5-FU and other antitumor drugs has been reported. In the present study, the ectopic synthesis of multiple carcinoplacental markers has been observed to be induced (10- to 264-fold) simultaneously in the same cervical carcinoma cells (HeLa65, HeLa71 and HeLa2.2) by hydroxyurea and sodium butyrate. Among the drug-induced biochemical markers observed in HeLa cells are four sialopeptides. Regan Isoenzyme (Placental Isoenzyme of Alkaline Phosphatase), HCT-Beta, FSH-Beta, HCG-Alpha and also a steroid hormone, Progesterone. The peptide and steroid hormones were quantitated by specific radioimmunoassays (RIA), in cultured cells, media, and homogenates of tumor tissues. The induction of biochemical markers was observed also with lung carcinoma cells. That multiple polypeptides, or steroids regulated by them, are simultaneously inducible in the same cancer cells, suggest the proximity on the DNA strand of several oncofetal and oncoplacental genes derepressed by antineoplastic drugs. This fundamental study has had important clinical ramifications. The results may be used to recognize the retention by cancer patients of occult malignancy after radiotherapy or surgery. The unsuspected metastasis may be reflected by a transient rise in the serum level of these markers during chemotherapy with anticancer drugs, which specifically inhibit DNA replication without interfering with the transcription of messenger-RNA and subsequent translation of proteins. The drug-induced protein-hormones, observed in this study, are the products of activated trophoblastic/pituitary genes in the nondividing DNA of neoplastic cells.

Immunoperoxidase study of alkaline phosphatase in testicular tumor.

Indirect immunoperoxidase staining was carried out on human testicular tumors using monospecific antibodies against placental (Regan) and intestinal isoenzymes of alkaline phosphatase (ALPase). The very high incidence of seminoma (approximately 90%) revealed positive staining of placental ALPase mainly on the cell membrane of tumor cells, whereas none of the seminoma showed presence of intestinal isoenzyme. Placental isoenzyme was not recognized in any embryonal carcinoma and interstitial cell tumor. The epithelial cells of the glandular elements of teratoma occasionally exhibited strong staining for intestinal ALPase and weak staining for placental ALPase. The appearance of Regan isoenzyme in seminomas might be considered possible conversion of hepatic to placental isoenzyme, a consequence of malignant transformation of spermatogenic cells. Regan isoenzyme appears to be a new tumor marker for seminoma and the frequent identification of Regan isoenzyme in seminoma may disclose a unique biologic characteristic of this germinal tumor.

Immunocytochemical demonstration of intracytoplasmic alkaline phosphatase in HeLa TCRC-1 cells.

The ultrastructural localization of alkaline phosphatase has been examined in cells of a HeLa subline (TCRC-1) that are monophenotypic for Regan isoenzyme expression. Enzyme activity was demonstrated at the cell surface plasma membrane and in certain lysosomes as revealed by the lead citrate method. The regular direct immunoperoxidase procedure utilizing antibodies in IgG or Fab' form showed the same distribution patterns of alkaline phosphatase. However, when the cell surface antigen was blocked in advance with specific unlabeled antibodies and direct immunocytochemistry performed in the presence of saponin, intracellular alkaline phosphatase antigen was observed in the perinuclear space, endoplasmic reticulum, and Golgi apparatus. The results appeared to be concordant with the current concept that membrane glycoproteins are formed in the endoplasmic reticulum, modified in the Golgi apparatus and then transported to the cell surface. Intracellular alkaline phosphatase was observed predominantly in some cell populations especially mitotic cells, suggesting that the enzyme protein was synthesized in and around the mitotic phase. Accordingly, this technique of differential membrane immunocytochemistry appears to provide an opportunity to follow ectopic gene expression as a function of cell cycle and enzyme induction.

Carcinoplacental isoenzyme (Regan) in carcinoma of the prostate. National Prostatic Cancer Project.

Serum samples were examined for the placental-like "Regan" isoenzyme of alkaline phosphatase in two groups of patients with advanced cancer of the prostate from clinical trials of the National Prostatic Cancer Project. In the first group, 98 samples from 76 patients over three consecutive months revealed elevated isoenzyme activity in 14 (18%) of the patients, a frequency similar to those reported for some other tumor types. Four of these patients whose serum demonstrated the "Regan" isoenzyme had samples taken more than once, and their sera had negative readings when taken a month or more before or after the sample that was positive for the isoenzyme. Comparisons of general disease and patient characteristics for patients whose sera were positive or negative for Regan isoenzyme revealed no demonstrable relationships. The second group consists of 72 samples received from 52 study patients with metastatic lesions under treatment from August 1, 1979 to November 16, 1979. Fifteen patients had sufficient serial values. In this study to date, the relationship with clinical response to treatment is inconclusive, although elevated levels of the isoenzyme appear to occur in patients with metastatic disease with bony lesions that are clinically in progression, as well as in those apparently responding to chemotherapy.

HEAT-STABLE ALKALINE PHOSPHATASE IN THE NORMAL FEMALE GENITAL ORGAN

The Regan isoenzyme, a heat-stable alkaline phosphatase, which has been thought to be derived only from cancer or the placenta, has been reported in non-cancer, non-pregnant patients. We performed histochemical studies to determine whether the heat-stable alkaline phosphatase exists in normal cells of the female reproductive organ. For this purpose, the heat-stability test and the L-phenylalanine inhibition test were used. Heat-stable and L-phenylalanine-sensitive alkaline phosphatase was found in normal cells of Muellerian duct origin, e.g., uterine cervical reserve cells, cervical columnar cells and epithelial cells of the endometrial surface. Our findings suggest that heat-stable alkaline phosphatase, such as the Regan enzyme, may be produced by normal cells of Muellerian duct origin.

Immunology and biochemistry of the Regan isoenzyme.

The Regan isoenzyme is a placental-type alkaline phosphatase that is expressed in a number of human tumors, particularly in gonadal and urologic cancers. Attention is given to the unique gene that codes for placental alkaline phosphatase and the similarities and differences in the tumor and placental gene products. The separation and identification of individual organ-specific isoenzymes is accomplished by a variety of biochemical, immunologic, and electrophoretic techniques and the correlation of the Regan isoenzyme, non-Regan isoenzyme, and Nagao isoenzyme, and the Kasahara isoenzyme is made with their developmental counterparts. The L-leucine-sensitivity phenotypes of placental and tumor alkaline phosphatases and the non-Regan early placental type alkaline phosphatases appear to be developmental phase-specific. Oncotrophoblast gene expression has been investigated with monophenotypic cell culture lines as a consequence of modulation by prednisolone and hyperosmolarity. Finally, general discussion of oncodevelopmental proteins as tumor markers precedes a current opinion of Regan isoenzyme as a tumor marker. Evidence now points to seminoma as a consistent producer of Regan isoenzyme although much more work will be required to establish its clinical utility.

Stauffer's syndrome in renal cell carcinoma evidence for intravascular coagulation.

In 40 patients with non-metastasising (n = 31) and metastasising (n = 9) renal cell carcinoma, evidence of Stauffer's syndrome (increase in alkaline serum phosphatase and prolongation of prothrombin time) was found in 18 patients. Prolongation of prothrombin time was not due to depletion of vitamin K-dependent coagulation factors or manifest fibrinolysis, but due to the presence of circulating fibrinogen fibrinmonomer-FDP complexes. Ethanol gelation test was found to be positive in 28/40 subjects and soluble fibrin monomer complexes were increased in 38/40 patients. The resulting disturbance of fibrinogen-fibrin conversion was reflected by an increase in thrombin coagulase time and reptilase time. These findings suggests a state of latent compensated intravascular coagulation (presumably triggered within the vascular tumor). For diagnostic purposes the most sensitive indicator is thrombin coagulase time. Thrombin coagulase time normalised after tumor resection and was positive in patients with recurrent metastases. The increase in alkaline serum phosphatase was due to an increase in the hepatic isoenzyme. Such an increase was much more common than the elevation of total alkaline serum phosphatase. Regan's isoenzyme was only found in 1 subject. In parallel, gamma-GT was elevated in 24 patients. The study shows that Stauffer's syndrome occurs more frequently than commonly assumed when thrombin coagulase time, gamma-GT and the hepatic isoenzyme of alkaline serum phosphatase are determined in patients with renal cell carcinoma. DIC and low grade fibrinolysis may account for the coagulation abnormalities of the syndrome.

Fetal isoenzyme expression of heterotransplanted HeLa cells in nude mice.

The nude mouse has been successfully employed for the propagation of human tumors, without the need for immunosuppression. In light of the limited data on embryonic gene expression in such tumors, we undertook a study of fetal isoenzyme expression during tumor growth. HeLa TCRC-1 which has been shown to produce the placental Regan isoenzyme was used in these studies. The isoenzyme produced by these cells in culture is initially replaced by an isoenzyme referred to as chorionic. In the later stages of tumor growth, the so-called oncoamniotic (FL) isoenzyme then becomes the dominant enzyme form. The chorionic isoenzyme is produced by the early chorionic membranes of the developing conceptus, while the oncoamniotic (FL) isoenzyme is most similar to that found in the fetal human intestine. The alteration in the expression of fetal isoenzymes in tumors growing in the nude mouse is similar to that seen in the immunosuppressed rat and hamster host animals, indicating that the phenomenon is not related to immunosuppression per se.

Expression of KB Cell Alkaline Phosphatase Isoenzymes During Growth in Immunosuppressed LEW Rats<xref ref-type="fn" rid="FN2">2</xref>

Alkaline phosphatase isoenzyme expression of human tumor xenografts was studied by the growing of KB cells in immunosuppressed neonatal LEW rats. In culture these cells produced the oncoamniotic (FL) isoenzyme as the major form and the Regan isoenzyme as a minor fraction as well as a "hybrid" that shared properties of both of the other isoenzymes. Despite a reduction in specific activity, this isoenzyme pattern was essentially unchanged during in vivo growth. KB cells "pretreated" in culture with the glucocorticoid prednisolone in hyperosmolal medium exhibited a decrease in the levels of the oncoamniotic (FL) isoenzyme and an increase in the Regan isoenzyme. During growth of pretreated cells in vivo, a time-dependent resumption in the expression of the oncoamniotic (FL) isoenzyme was associated with the disappearance of the Regan isoenzyme. This shows that the expression of the oncoamniotic (FL) isoenzyme is not restricted to human tumor cells monophenotypic with respect to alkaline phosphatase.

Determination of Placental Alkaline Phosphatase‐Regan Isoenzyme in Cancer Sera by a Sensitive Radioimmunoassay

Three common genetically classified placental alkaline phosphatase variants (F, I, S) were purified to complete homogeneity. Specific antisera were raised against all three types. Complete fusion reactions in double diffusion tests were obtained for all combinations of antigen and antibody, indicating close immunological relatedness.A sensitive and specific double antibody solid phase (DASP) radioimmunoassay with a least detectable dose of 9 ng/ml was developed.Sera from 241 patients with different malignant disorders were assayed for the presence of placental alkaline phosphatase, also called the Regan isoenzyme. Significantly elevated values were obtained in only one case of embryonal carcinoma. This corresponds to a frequency of 0.4%. Three selected sera from cancer patients with a known content of Regan isoenzyme were tested in the same assay and had elevated values.