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P L Devine

Publications and source records attributed to P L Devine.

13 recordsLinked to original sources
PubMed1992

Second-generation monoclonal antibodies to intestinal MUC2 peptide reactive with colon cancer.

BACKGROUND: Antitumor antibodies have traditionally been made to whole tumors or tumor extract. The use of defined synthetic antigens would be desirable for producing monoclonal antibodies. PURPOSE: Our purpose was to determine if antipeptide antibody to MUC2 had antitumor activity and specificity. METHODS: A 29-amino-acid peptide to MUC2 was synthesized and monoclonal antibodies were produced after immunizing BALB/c mice with peptide-keyhole-limpet hemocyanin in complete Freund's adjuvant, and the monoclonal antibodies were tested on peptides and human tissues. RESULTS: CCP31, CCP37, and CCP58 monoclonal antibodies were produced using MUC2 MI-29 (KYPTTTPISTTTMVTPTPTPTGTQTPTTT) containing one repeat unit of 23 amino acids and part of the next repeat of four amino acids. These antibodies reacted with the MUC2-derived peptide but not with MUC1- or MUC3-derived peptides. One of the monoclonal antibodies, CCP58, reacted strongly with human colon cancer and normal intestine in both fresh and formalin-fixed tissues; two other antibodies, CCP37 and CCP31, reacted only with fresh human tissues of normal colon and malignant colon tumors by immunoperoxidase staining. In addition, CCP37 and CCP58 reacted strongly with human gastric cancer; all antibodies reacted weakly with human salivary gland, and none reacted with tissues from normal human lung, kidney, stomach, pancreas, or endometrium. By analysis of mucin molecules by Western blotting, the antigen detected by monoclonal antibodies CCP37 and CCP58 was found to be of a high relative molecular mass (520 kd). CONCLUSIONS: Anti-MUC2 peptide antibodies appear to be relatively tissue specific and represent a new method of producing antitumor antibodies.

Amino Acid Sequence↗
PubMed1992

Mucins: structure, function, and associations with malignancy.

Mucins are a family of high molecular weight, highly glycosylated glycoproteins found in the apical cell membrane of human epithelial cells from the mammary gland, salivary gland, digestive tract, respiratory tract, kidney, bladder, prostate, uterus and rete testis. Increased synthesis of the core protein and alterations in the carbohydrates attached to these glycoproteins are believed to play important roles in the function and proliferation of tumour cells. Aberrant glycosylation leads not only to the production of novel carbohydrate structures, but also to the exposure of the core peptide. These novel epitopes may be candidates for diagnosis or therapy, by using either synthetic mucin fragments as vaccines, or monoclonal antibody-based reagents which detect these structures.

Amino Acid Sequence↗
PubMed1992

Expression of MUC1 and MUC2 mucins by human tumor cell lines.

The secretion and nature of mucins produced from a panel of recently available new gastric and colon carcinoma cell lines (LIM1839, LIM1215, LIM1863, LIM1899, LIM2099, LIM2405, LIM2408, LIM2412, LIM2463), as well as other colon (LS174T, HT29, HT29-SB, COLO533, COLO206), breast (T47D, MCF-7, BT20, ZR75-1) and ovarian (COLO316) tumor cell lines, was investigated. ELISA and Western blotting of the culture supernatants with novel anti-MUC1 and anti-MUC2 monoclonal antibodies (MAbs) specific for mucin core proteins showed their secretion by most of these cell lines. In addition, mucins produced by these cell lines expressed the tumor-associated carbohydrate detected by MAb 3E1.2 (glycolylsialyl-Tn, mammary serum antigen or MSA) and the Tn or T antigens reactive with lectin SSA-M. SSA-M detected MUC1 or MUC2 captured by MAbs BC2 or CCP58, while 3E1.2 only detected MUC1-associated carbohydrate, indicating that the MAb may react with a conformationally dependent epitope, or that the sialyl/glycolyl-transferases involved in MSA production may be sequence specific. In addition, the BC2/SSA-M and CCP58/SSA-M assays detected mucins in some samples which were not detected by BC2/BC2 or CCP58/CCP58 dual determinant assays, indicating that this format may be more appropriate for the detection of tumor-associated mucins in body fluids. These new cell lines and assays should be of use in the investigation of mucin core proteins, particularly LIM2463 and LIM1839 which express significant quantities of both MUC1 and MUC2.

Amino Acid Sequence↗
PubMed1992

A sensitive microplate assay for glycoproteins that utilizes an immunological digoxigenin-based detection system.

A sensitive microplate assay for glycoproteins, based on an immunological digoxigenin-based detection system, is described. When log absorbance was plotted against log concentration, linear standard curves were obtained over the range 25 ng/ml to 25 microliters/ml with bovine submaxillary mucin, porcine stomach mucin, fetal calf fetuin, asialofetuin and human transferrin. This detection limit and range are far superior to other assays that have been described. The use of the microtiter plate format means that a large number of samples can be assayed with ease. Due to the broad range of the assay, the concentration of the sample is not a particular problem, and samples above the upper cutoff value can be serially diluted in the microtiter plate to find an appropriate value.

Animals↗
PubMed1991

The breast tumor-associated epitope defined by monoclonal antibody 3E1.2 is an O-linked mucin carbohydrate containing N-glycolylneuraminic acid.

The breast cancer-associated epitope (mammary serum antigen or MSA) defined by monoclonal antibody (Mab) 3E1.2 is a neuraminidase-sensitive carbohydrate expressed on MUC-1-encoded molecules. However, the reactivity of Mab 3E1.2 is also reduced by protease treatment of the mucin, which suggests that 3E1.2 binds to multimers of the sialylated carbohydrate in a protein conformation-dependent manner. The common N-acetyl derivative of neuraminic acid (5-acetylneuraminic acid) is not involved in the epitope, since lectins specific for 5-acetylneuraminic acid (linked to GalNAc or Gal) are nonreactive with MSA-positive molecules. However, the N-glycolyl derivative, 5-glycolylneuraminic acid (Neu5Gc), forms a major part of the epitope since both free Neu5Gc and porcine stomach mucin (greater than 90% neuraminic acid as Neu5Gc) inhibit the binding of Mab 3E1.2, while bovine or ovine submaxillary mucins, fetuin, bovine gangliosides, and other carbohydrates do not. Indeed, the presence of Neu5Gc on human tumor mucin was confirmed by electrospray mass spectrometry. Neu5Gc is attached to an O-linked carbohydrate, since the expression of MSA by MCF-7 breast cancer cells is inhibited by the O-glycosylation inhibitor phenyl-N-acetyl-alpha-D-galactosaminide, but not by the N-glycosylation inhibitor tunicamycin, and the epitope is removed by treatment with O-glycanase but not N-glycanase F, endoglycosidase F, or endoglycosidase H, which are specific for N-linked glycans. This is likely to be a core glycan since 3E1.2 reacts after treatment of the mucin with trifluoromethanesulfonic acid, which removes most backbone and peripheral carbohydrates. Treatment with galactosidase or N-acetyl glucosaminidase enhances the binding of Mab 3E1.2, indicating that the Neu5Gc is not attached to galactose or N-acetyl galactosamine. Furthermore, the susceptibility of MSA to treatment with Arthrobacter urea-faciens neuraminidase [which is specific for alpha (2-6)-linked NeuNAc] and the loss in reactivity of GalNAc-specific lectins after periodate oxidation [alpha (2-3)-linked but not alpha (2-6)-linked NeuNAc protects GalNAc from periodate oxidation] indicate that the Neu5Gc may be attached alpha (2-6) to peptide-linked GalNAc. These results show that MSA is a Neu5Gc-containing O-linked core glycan, which represents a unique tumor-associated epitope not previously identified on human mucins.

Animals↗
PubMed1991

Production of MUC1 and MUC2 mucins by human tumor cell lines.

A mucus secreting, clonal derivative (HT29-SB) of the human colonic adenocarcinoma cell line HT29, and the LS174T colon cancer cell line, secrete mucin into the culture medium as a viscoelastic gel. Mab BC2, which defines a peptide epitope present in the variable number of tandem repeats (VNTR) of the MUC1 core protein, reacted with this material after deglycosylation. Two high molecular weight bands were detected in TFMSA treated gel-formed mucin from HT29-SB and LS174T by western blotting (Mr 580 kDa and 420 kDa). A similar pattern of reactivity was seen with the culture supernatants from HT29-SB, the ovarian tumor cell line COLO-316, and the breast cancer cell line MCF-7. Mab CCP58 (anti-MUC2 VNTR) reacted with a 580 kDa band in gel-formed mucin produced by LS174T, but was not reactive with mucin produced by the other cell lines. The findings indicate that human colonic cell lines, in addition to breast and ovarian cell lines, may both express and secrete the MUC1 protein core, and that the LS174T cell line expresses and secretes both the MUC1 and MUC2 core proteins.

Adenocarcinoma↗
PubMed1991

Reactivity of mucin-specific lectin from Sambucus sieboldiana with simple sugars, normal mucins and tumor-associated mucins. Comparison with other lectins.

Mucin-specific lectin from Sambucus sieboldiana (SSA-M) reacts in Western blotting and ELISA with mucins from porcine stomach, bovine and ovine submaxillary glands, the human milk fat globule membrane, in vitro human ovarian, breast and colonic tumor cell lines, and mucins produced in vivo in the ascites of patients with endometrial and ovarian tumors, but not with fetal bovine fetuin or human transferrin. Sialidase treatment of these mucins led to an increase in the binding of SSA-M, suggesting that sialic acid is not part of the binding site for this lectin. Furthermore, sialic acid did not inhibit lectin binding. Treatment of asialomucin with O-glycanase decreased the binding of SSA-M, confirming the reactivity of the lectin with an O-linked carbohydrate. Treatment of mucins with trifluoromethanesulfonic acid, which removes all but core carbohydrate, led to an increase in the binding of SSA-M, suggesting that the lectin reacts with O-linked core glycans. Indeed, the increased reactivity after sialidase treatment of ovine submaxillary mucin suggests the lectin reacts with peptide-linked N-acetylgalactosamine (GalNAc), since more than 98% of the glycan chains attached to this mucin are sialylated GalNAc. The binding of SSA-M to sialidase-treated porcine mucin was inhibited strongly by GalNAc and disaccharides containing galactose (lactose, melibiose, and N-acetyllactosamine) but not by free galactose (Gal), suggesting that the glycan for optimum binding is Gal beta(1-3)GalNAc. This pattern of inhibition was different to other core glycan-reactive lectins tested, indicating that SSA-M is distinct, and should be of use in the isolation and characterisation of mucins and O-linked glycans.

Animals↗
PubMed1990

Monoclonal antibodies reactive with the breast carcinoma-associated mucin core protein repeat sequence peptide also recognise the ovarian carcinoma-associated sebaceous gland antigen.

The monoclonal antibody (Mab) OM-1, which defines the ovarian carcinoma-associated sebaceous gland antigen (SGA), and Mab F36/22, which defines the ductal carcinoma antigen (DCA), were tested for reactivity against a synthetic peptide representing the repeat twenty amino acid sequence of the human polymorphic epithelial mucin core protein plus four amino acids of the adjacent sequence (p1-24). OM-1 bound strongly to the peptide by direct dot blot assay and ELISA and the minimum epitope for OM-1 was shown to be APDTRP(A) by inhibition assay. F36/22 reacted weakly with the peptide under the same conditions and its affinity for peptide in solution was relatively very low. Mab OC125, which defines the ovarian cancer-associated antigen CA125, did not react with the p1-24 peptide. Five other anti-mucin Mabs (HMFG1, HMFG2, BC1, BC2, BC3), previously shown to bind to the p1-24 peptide, reacted strongly with SGA by direct binding and in a sandwich assay with OM-1 as the capture Mab. F36/22 was weakly positive under the same conditions suggesting that both peptide and SGA do not express the optimal epitope for F36/22 binding. These results indicate that SGA and possibly DCA have the repeat sequence core protein of the breast carcinoma-associated human polymorphic epithelial mucin.

Amino Acid Sequence↗
PubMed1986

3 beta-hydroxysteroid isomerase dehydrogenase in guinea-pig kidney: possible involvement in 11-deoxycorticosterone formation in situ.

3 beta-Hydroxysteroid isomerase dehydrogenase, capable of acting on C21- and C19-3 beta-hydroxy-5-ene-steroids has been found in guinea-pig kidney at equivalent levels to those in guinea pig testes. Of the 3 beta-hydroxy-5-ene-steroids present in guinea pig serum, 21-hydroxypregnenolone occurs in highest concentration (17 nM) followed by pregnenolone (10 nM), whereas 17 alpha-hydroxy-pregnenolone and dehydroepiandrosterone occur in very low concentrations (less than 0.5 nM). Furthermore, the concentration of 21-hydroxypregnenolone relative to 11-deoxycorticosterone (the mineralocorticoid of the guinea pig), is 10:1 (Nishikawa and Strott, Steroids 41 (1983) 105-120). The apparent Km value for 21-hydroxypregnenolone, for the reaction yielding 11-deoxycorticosterone as catalysed by guinea pig kidney microsomes, was 85 nM and the Vmax 33 pmol/min per mg protein. Pregnenolone was a competitive inhibitor (apparent Ki = 5 microM) in the above reaction. A sex difference in the level of the enzyme in the kidney was found (activity in the female was one-third of that in the male) which may indicate that the enzyme is under partial androgen control. 3 beta-Hydroxysteroid isomerase dehydrogenase activity was also detected in guinea pig liver and again it was lower in the female. Whilst the exact role of 3 beta-hydroxysteroid isomerase dehydrogenase in guinea-pig kidney remains uncertain, the data suggest that it may utilise blood-borne 21-hydroxypregnenolone, the later then playing the role of a prohormone.

17-Hydroxysteroid Dehydrogenases↗
PubMedDate not supplied

Circulating mucins as tumor markers in ovarian cancer (review).

Because a highly sensitive and specific serum marker for ovarian carcinoma has not been reported, it is unlikely that there will be an application of serum markers for screening for this disease in asymptomatic women. However, many oncologists use serum tumor markers initially to differentiate epithelial ovarian carcinoma from benign gynecological conditions prior to surgery, so as to ensure appropriate surgical referral, and then to monitor the clinical course of disease during and after adjuvant therapy. The most commonly performed tumor marker assay in ovarian cancer (CA125) has been extremely valuable in patient management, but this marker is also elevated in a considerable proportion of patients with benign gynecologic diseases and endometriosis, and a relatively small proportion of patients with early stage disease. A new class of serum tumor markers, the highly glycosylated, high molecular weight mucins, have enormous potential in the management of ovarian cancer patients, since the use of assays for these markers may overcome many of the problems associated with CA125. Indeed, when used in combination with CA125, some mucin-based assays have increased the sensitivity and specificity of detection, thereby eliminating many false positive results seen with patients with benign disease and endometriosis, and also predicted disease recurrence in the majority of patients before clinical symptoms became apparent. These markers are the subject of this review, with particular attention to the commercially-available mucin-based assays.

Antibodies, Monoclonal↗