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W H Fishman

Publications and source records attributed to W H Fishman.

At least 37 records · Page 2Linked to original sources
PubMed1983

Alkaline phosphatase biosynthesis in the endoplasmic reticulum and its transport through the Golgi apparatus to the plasma membrane: cytochemical evidence.

Enzyme induction of HeLa cell placental alkaline phosphatase with various agents such as prednisolone, sodium butyrate, hyperosmolality (NaCl), or combination of these inducers resulted in the appearance of enzyme activity in the rough endoplasmic reticulum, nuclear envelope, Golgi apparatus, and plasma membrane. In the Golgi apparatus, intense reaction product deposits tended to be concentrated on its trans side, with small vesicles and granules also being positively stained. Inhibition of protein synthesis with cycloheximide was followed by the disappearance of enzyme activity from these cytoplasmic organelles but not from the plasma membrane. Treatment with monensin, a secretory protein transport inhibitor, uniformly increased activity in the rough endoplasmic reticulum while causing marked dilatation of the intensely positive Golgi cisternae. These results suggest that intracellular alkaline phosphatase is newly synthesized in the endoplasmic reticulum and then passes en route through the Golgi apparatus to the plasma membrane. Accordingly, the present system could represent the biosynthesis, transport, and incorporation of the model cell surface enzyme protein to add to the vesicular stomatitus virus glyco-1 (VSV-G) protein and acetylcholine receptor model systems for studying the dynamics of cell surface protein genesis, transport, and membrane integration.

Alkaline Phosphatase↗
PubMed1983

Newly established uterine cervical cancer cell line (SKG-III) with Regan isoenzyme, human chorionic gonadotropin beta-subunit, and pregnancy-specific beta 1-glycoprotein phenotypes.

The production of Regan isoenzyme (heat-stable, L-phenylalanine-sensitive term-placental alkaline phosphatase), human chorionic gonadotropin beta-subunit, and pregnancy-specific beta 1-glycoprotein by newly characterized human uterine cervical cancer cell lines, SKG-IIIa and SKG-IIIb, is reported. These cell lines were derived from a moderately differentiated epidermoid cancer partially mixed with epidermoid clear-cell components. At the end of the first 4 months in culture 2 sublines with different morphologies were identified. In nude mice, SKG-IIIa produce clear-cell epidermoid cancer with much glycogen, while SKG-IIIb grew as a moderately differentiated epidermoid cancer rich in tonofilaments. The presence of Regan isoenzyme was established by biochemistry, enzyme cytochemistry, immunocytochemistry, and immunoelectrophoresis. However, the copresence of small amounts of early placental alkaline phosphatase was also demonstrated. The alkaline phosphatase specific activities of SKG-IIIa cells and SKG-IIIb cells were 3.7 and 1.4 nmol per mg protein per min, respectively. The existence was proven by radioimmunoassay of human chorionic gonadotropin beta-subunit (SKG-IIIa, 5.0 mlU/mg protein; SKG-IIIb, 4.4 mlU/mg protein), pregnancy-specific beta 1-glycoprotein (SKG-IIIa, 0.7 ng/mg protein) in the culture media as a tumor cell product. The described cell lines may serve as a more representative model system for studies of regulation of oncodevelopmental genes in gynecological tumors in general and in epidermoid cervical cancer in particular.

Alkaline Phosphatase↗
PubMed1982

Characterization and use of an allotype-specific monoclonal antibody to placental alkaline phosphatase in the study of cancer-related phosphatase polymorphism.

The hybridoma technique was used to produce an allotype-specific monoclonal antibody (F11) that reacts with the products of the S, I, and D alleles of PLAP but not of the F allele. Serum and ascites samples from patients with different cancers containing high levels of PLAP were tested for reactivity with F11. These tumor-derived PLAPs were of the Nagao type as shown by their sensitivity to inhibition by L-leucine. This type of inhibition is exhibited also by the rare D allelic variant of PLAP but not by the common forms. Thus, it has been proposed that the Nagao enzyme represents reexpression of the D allele of PLAP. F11 reactive and nonreactive samples as well as samples with intermediate reactivity were found among the cancer sera and ascites. Our results show tha tumor-derived Nagao enzyme does not represent the reexpression of the D allele but instead, in spite of its distinct inhibition pattern, expresses the same genetic polymorphism that is found in the placenta.

Alkaline Phosphatase↗
PubMed1982

Placental alkaline phosphatase as a tumor marker for seminoma.

A sensitive and specific enzyme-linked immunoabsorbent assay was used in a retrospective study of serum levels of placental alkaline phosphatase (PLAP) in testicular cancer. Sixteen of 28 men with active seminoma had elevated PLAP levels, and 71% had elevated levels of either PLAP, human chorionic gonadotropin, or both. Only four of 22 men with active nonseminomatous cancer had elevated PLAP levels, and the levels were normal in all control patients, including 33 men apparently cured of testicular cancer. In six of ten serial studies, PLAP levels provided information not otherwise available that would have been useful clinically, and the levels never were elevated inappropriately. Our data suggest that PLAP is a clinically useful serum tumor marker for seminoma.

Alkaline Phosphatase↗
PubMed1981

Placental alkaline phosphatase isoenzyme expression by the non-HeLa DoT cervical-carcinoma cell line.

Expression of the oncodevelopmental protein, placental alkaline phosphatase, was observed in DoT cells, an epidermoid cell line derived from cervical carcinoma. Under normal conditions of growth in vitro, biochemical inhibition, cytochemical and immunological studies revealed that these cells express the term-placental (Regan) isoenzyme. Thus alkaline phosphatase activity was observed to be heat-stable and inhibited by L-phenylalanine. These properties, supported by immunoelectrophoretic analysis using antisera specific for liver, intestinal or term-placental isoenzymes, identified the isoenzyme as placental type. DoT cells treated with prednisolone (1 microgram/ml) increased total alkaline phosphatase specific activity. This activity was also identified as term-placental phosphatase isoenzyme. On the other hand, treatment of the same cells with sodium butyrate (1 mM) did not induce increased activity of the term-placental isoenzyme, an unexpected observation. As a result of these studies, DoT cells are proposed as a representative cell line for studies of the regulation of oncodevelopmental gene expression in human tumour cells of cervical origin.

Alkaline Phosphatase↗
PubMed1981

Alkaline phosphatase protein increases in response to prednisolone in HeLa cells.

Quantification of term-placental alkaline phosphatase isoenzyme protein in HeLa TCRC-1 cells grown in the presence and absence of prednisolone indicates that there is a net increase in amount of enzyme-specific protein in prednisolone-stimulated cells. In a similar analysis of HeLa D98AH2 cells, prednisolone treatment causes the appearance of term-placental alkaline phosphatase protein and the loss of the intestinal isoenzyme protein. These results support the interpretation that the response of these cells to corticosteroids is the net accumulation of alkaline phosphatase protein rather than the modification of pre-existing enzyme to a more active state.

Alkaline Phosphatase↗
PubMed1981

Intracellular alkaline phosphatase activity in cultured human cancer cells.

The effect of saponin treatment in demonstrating intracellular portion of alkaline phosphatase activity in human cancer cell lines was evaluated. Previous reports using standard lead-salt techniques visualized enzyme almost exclusively on the plasma membrane and sometimes in the lysosomes. However, by treating cells with saponin before or during the cytochemical incubation, intracellular alkaline phosphatase became demonstrable at the endoplasmic reticulum. Golgi apparatus, Golgi-derived vesicles and mitochondria as well as lysosomes and plasma membrane. These intracellular catalytic activities were significantly inhibited by the specific amino acid inhibitors characteristic for each cell line, and this suggested that intracellular alkaline phosphatase is the same isoenzyme as that present in the plasma membrane. The results of our current and previous studies therefore indicate that saponin reveals latent intracellular alkaline phosphatase activity by changing the membrane's physical state; thereby increasing the availability of both catalytic and antigenic sites of the enzyme to substrate and to antibody respectively.

Alkaline Phosphatase↗
PubMed1981

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.

Alkaline Phosphatase↗
PubMed1981

Demonstration of gamma-glutamyl transferase, alkaline phosphatase, CEA and HCG in human lung cancer.

In contrast to its absence in normal and hyperplastic bronchial mucosa, gamma glutamyl transferase (GGT) has been demonstrated on the luminal surface of adenocarcinoma cells of bronchogenic cancer in three cases studied. On the other hand, little or no GGT was demonstrable in four epidermoid carcinomas and one oat cell cancer. Metaplastic alveolar cells appearing as cuboidal epithelial cells were uniformly positive for GGT and L-homoarginine-sensitive alkaline phosphatase. Whereas interest in GGT has centered in the past on experimental hepatocarcinogenesis, the current results demonstrate an ectopic expression of GGT in metaplastic alveolar cells and in adenocarcinoma of the lung. These findings merit further exploration to document the extent of the expression of GGT in bronchogenic cancer and to attempt to explain its presence in alveolar cell metaplasia.

Adenocarcinoma↗
PubMed1980

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.

Alkaline Phosphatase↗
PubMed1980

Demonstration of species difference of placental alkaline phosphatase isozymes in acetone-fixed paraffin-embedded tissues.

The best preservation and discrimination of alkaline phosphatase isozymes by means of amino acid inhibition and heat treatment was obtained in sections of acetone-fixed paraffin-embedded tissues as compared to tissues fixed either in 95% ethanol or standard formalin. The results are illustrated in a study differentiating the isozymes of human and mouse placenta.

Acetone↗
PubMed1980

Presence of the rare D-variant heat-stable, placental-type alkaline phosphatase in normal human testis.

In 11 adult testes studied, about 0.3 to 4.6% of the total alkaline phosphatase activity was heat stable and L-phenylalanine sensitive but L-homoarginine insensitive. The testicular heat-stable enzyme was more susceptible to inhibition by L-leucine and ethylenediaminetetraacetate than were the normal placental and intestinal enzymes. By antibody-directed enzyme inhibition test, the testicular heat-stable enzyme cross-reacted completely with normal placental enzyme but clearly distinguished itself from a heat-stable component of normal intestinal enzyme. Thus, placental alkaline phosphatase D-variant is synthesized in testis, indicating that the gene for elaborating this placental protein is probably already active in the testicular cells. The high incidence of this protein in cancers of testis and ovary is probably due to its increased production by gonadal genes present in the genome of these particular tumors.

Alkaline Phosphatase↗
PubMed1980

Hypophosphatasia (adult form): quantitation of serum alkaline phosphatase isoenzyme activity in a large kindred.

We used heat inactivation, L-phenylalanine inhibition, and electrophoresis on polyacrylamide gel and cellulose acetate membranes--with and without use of specific antisera against the liver-bone, intestinal, and placental isoenzymes--to distinguish and quantitate the different alkaline phosphatase isoenzymes in sera from 23 adult members of a kindred affected by the adult form of hypophosphatasia. Nine subjects had values for total activity more than two standard deviations below the mean values for age- and sex-matched normal persons. Bone isoenzyme was diminished in all nine, whereas liver isoenzyme was subnormal in only four. Phosphoethanolamine and phosphoserine in the urine of eight hypophosphatasemic individuals correlated inversely with both total and liver alkaline phosphatase activity in their serum, but not with the activity of the bone isoenzyme. Total activity in the serum of adult kindred members correlated best with the circulating liver isoenzyme activity. The findings suggest that altered hepatic metabolism is responsible for the increased urinary excretion of phosphoethanolamine, and perhaps phosphoserine, in hypophosphatasia.

Adult↗
PubMed1979

Complement fixation for study of placental-type alkaline phosphatase.

Preparations of human placental alkaline phosphatase differing in specific enzyme activities were compared by microcomplement fixation assays using monospecific antisera. While both specific enzyme activity and complement fixation units increased 15,000-fold upon purification, the ratio between these units remained constant. Separation of an alkaline phosphatase preparation into 'A' and 'B' forms by ampholine isoelectric focusing indicated that these forms also possessed the same ratio of immunoreactive enzyme protein to enzyme activity. The correspondence of complement fixation units with specific enzyme activity indicates that complement fixation with monospecific antisera can be used to analyze structural differences among alkaline phosphatase isoenzymes.

Alkaline Phosphatase↗
PubMed1979

Experimentally induced autoantibody to heat-stable alkaline phosphatase in the baboon.

Experimental evidence has been collected which signifies that autoantibody has been induced against lung heat-stable alkaline phosphatase which represented 60% of the total alkaline phosphatase of that tissue. Immunization of a male baboon with highly purified human placental alkaline phosphatase (heat-stable and cross-reactive with the baboon heat-stable enzyme) resulted in production of a precipitating factor in the immune serum which reacted with the heat-stable enzyme of both the human and baboon but not the heat-labile form of alkaline phosphatase of either species. This precipitating factor is a baboon autoantibody because 1) it had a gamma mobility on immunoelectrophoresis and retarded the electrophoretic mobility of the heat-stable enzyme from both normal and immunized baboons; 2) its titer increased as more booster injections were administered; 3) it formed a well-defined precipitin rocket with the baboon heat-stable enzyme in the Lurell's antigen-antibody crossed electrophoresis; 4) in immunodiffusion it formed a discrete precipitin line with the baboon heat-stable enzyme, which fused partially with the precipitin line of human placental alkaline phosphatase (immunogen).

Alkaline Phosphatase↗