Hubert E. Appert

Identification of the full-length coding sequence for human galactosyltransferase (β-N-acetylglucosaminide: β1,4-galactosyltransferase)

A λgt11 human placenta cDNA library was screened using a cDNA probe encoding the COOH-terminal region of human β 1,4-galactosyltransferase and with a synthetic oligonucleotide having a sequence corresponding to that of the 5′ end of the cDNA probe. The newly isolated cDNA was found to code for the NH 2 -terminal and the 5′-untranslated region, primed at an (A) 8 region in the coding sequence. A complete amino acid sequence has been deduced which shows only one membrane anchoring domain near the NH 2 -terminus. Comparison of the sequence to the soluble enzyme suggests proteolytic cleavage at Arg 77. Presently obtained information of human β 1,4-galactosyltransferase makes it possible to study DNA mutations responsible for genetic defects such as the altered expression of galactosyltransferase found in a variant of congenital dyserythropoietic anemia type II (HEMPAS).

Prognostic value of serum CA 19-9 levels in pancreatic adenocarcinoma.

Thirty-eight patients with histologically proven pancreatic adenocarcinoma were investigated to establish the utility of serum CA 19-9 as a prognostic indicator. CA 19-9 assays were performed serially during the course of the disease. In four patients with negative Lewis blood type, the CA 19-9 levels remained essentially normal throughout the disease course. In the remaining 34 patients, (1) CA 19-9 levels were significantly lower in patients with tumor size no larger than 5 cm in diameter, and in patients with resectable tumors than in those with tumor size larger than 5 cm or with unresectable tumors (p less than 0.01). 2) CA 19-9 levels dropped sharply after resection in all 11 resectable patients, whereas no significant change was found after laparotomy without resection. (3) The average survival time in seven patients whose CA 19-9 levels returned to normal after resection was significantly longer than in those four patients with postoperative CA 19-9 levels that decreased but did not return to normal (21.9 versus 8.7 months, p less than 0.05). (4) In 6 of 11 patients who underwent resection, recurrent elevation of CA 19-9 preceded changes detectable by computed tomography or clinical examination by 2 to 9 months. (5) In 23 patients who died of pancreatic carcinoma, 15 (65%) had an obvious rise in CA 19-9 level before death. There was a correlation between the doubling time of the CA 19-9 serum level and survival time (r = 0.5, p less than 0.05). Because it can be demonstrated that the reduction of tumor burden by resection lowers serum CA 19-9 levels, serum CA 19-9 levels may be a useful indicator of whether other forms of treatment such as radiation therapy or chemotherapy also reduce the tumor burden.

Overexpression of HER2/neu oncogene in pancreatic cancer correlates with shortened survival

SummaryFor the purpose of determining the prognostic significance of HER2/neu oncogene in pancreatic and ampullary cancers, 21 pancreatic cancers of ductal origin and six cancers of the ampulla of Vater were studied immunohistochemically using the monoclonal antibody (MAb) CB11, specifically reactive with HER2/neu product. Staining of the epithelium of the normal duct and acini was negative or weakly positive. Moderately and strongly positive reactions indicated the overexpression of this gene, and were found in 10 of 21 (47.6%) pancreatic cancers of ductal origin and in 2 of 6 (33.3%) ampullary adenocarcinomas. Overexpression of HER2/neu was closely and inversely related to the survival of the patients with pancreatic cancer of ductal origin: 19.1±11.7 mo for those not overexpressing vs 7.3±3.8 mo for the overexpressors (p<0.01). Among the pancreatic cancer group, 11 patients underwent cancer resection. The average survival for the 7 with nonoverexpressing cancer was 21.4±14.3 mo vs 10.5±3.6 mo for those with the overexpressing tumor. Among those not undergoing resection, the average survival for the 4 with nonoverexpressing cancer was 15.0 ±3.8 mo as contrasted to 5.2±2.1 mo for the overexpressors (p<0.01). Although the number of patients is small, these findings duggest that the overexpression of HER2/neu gene product may be frequently found in pancreatic cancer of ductal origin and may be one of the useful prognostic biomarkers for this cancer.

Matrix metalloproteinase expression in breast cancer

BACKGROUND Matrix metalloproteinases (MMPs) have been implicated as possible mediators of invasion and metastasis in some cancers. Our objective was to investigate which MMPs were constitutively expressed in breast tumor cells versus those that could be up-regulated by a number of agents known to affect MMP expression in other cell systems. METHODS We evaluated expression of MMPs 1-16 in breast tumor cell lines MDA-MB-231, T47D, and MCF-7 using semiquantitative RT-PCR and gelatin zymography. Exposure to 12-O-tetradecanoylphorbal-3-acetate (TPA), concanavalin-A (Con-A), the fibronectin-mimetic peptide GRGDSP (RGD), extracellular matrix (ECM) components, and anti-integrin antibodies was used to test for possible MMP up-regulation. Mitogen-activated protein kinase inhibitors (MAPK-I) were used to evaluate signal transduction pathways and regulation of MMP expression. RESULTS MMPs 1, 2, 7-11, 13, 14, and 16 were constitutively expressed in some tumor cell lines but not in normal breast epithelial cells. Administration of TPA, Con-A, and RGD increased the expression of MMPs 1, 2, 9, and 10. No MMP up-regulation was seen in MDA-MB-231 or MCF-7 after exposure to ECM components or after exposure to anti-integrin antibodies. MAPK-I had no effect on constitutive MMP expression but reduced or abolished the TPA up-regulation of MMP-9 in MDA-MB-231 and MCF-7. CONCLUSIONS Breast tumor cell lines constitutively express a number of MMPs. Because MMP expression can be up-regulated by Con-A, the fibronectin-mimetic peptide RGD, and TPA while being susceptible to inhibition by MAPK antagonists, MAPK signaling appears to play a role in this expression.

Adhesion and Migration of Extracellular Matrix-Stimulated Breast Cancer

BACKGROUND Extracellular matrix (ECM) components, such as vitronectin and fibronectin, have been shown to enhance the metastatic potential of breast cancer cells. We hypothesized that ECM binding to integrin receptors on breast cancer cells influenced cellular adhesion and migration. MATERIALS AND METHODS Adhesion assays were performed using breast cancer cell lines MDA-MB-435 and MDA-MB-231 and various concentrations of vitronectin or fibronectin. Migration assays were performed using the same cell lines and invasion chambers with 8 microm pore polycarbonate membranes. Blocking antibodies and specific peptidomimetic inhibitors to integrin receptors were used to identify the integrin subunits reacting with vitronectin and fibronectin. RESULTS While both breast cancer cell lines adhered to and migrated toward vitronectin and fibronectin, MDA-MB-435 had a higher maximum binding to vitronectin and MDA-MB-231 had a higher maximum binding to fibronectin. Anti-beta1 antibody inhibited the adhesion and migration of MDA-MB-231 to fibronectin and the adhesion of MDA-MB-231 to vitronectin but had no effect on vitronectin-induced adhesion or migration of MDA-MB-435. The alpha(v)beta3/alpha(v)beta5 antagonist, SB 265123, inhibited MDA-MB-231 and MDA-MB-435 adhesion and migration to vitronectin but had no effect on migration to fibronectin in either cell line. CONCLUSIONS We conclude that the integrin subunits beta1, alpha(v)beta3, and alpha(v)beta5 can be involved in breast cancer cell adhesion and migration to vitronectin and fibronectin. Because more than one integrin inhibitor was required to block adhesion or migration in the cell lines studied, breast cancer therapy based on integrin antagonists would most likely require concomitant use of multiple agents.

Adenocarcinoma of duodenum and ampulla of Vater: Clinicopathology study and expression of p53, c-neu, TGF-α, CEA, and EMA

Oncogenes, tumor suppressor genes, and growth factors are being explored as to their role in the initiation and progression of most neoplasms, but little information exists on the expression of oncoproteins or growth factors in adenocarcinoma of the duodenum or ampulla of Vater. This report covers expressions of p53, c‐neu, TGF‐α, CEA, and EMA in duodenal adenocarcinoma and ampullary adenocarcinoma, as well as correlations between expressions and tumor stage, histological grade and patient survival. The expression of p53, c‐neu, TGF‐α, CEA, and EMA has been studied in 15 duodenal adenocarcinomas and in eight ampullary adenocarcinomas by avidin‐biotin‐peroxidase complex indirect immunoperoxidase technique. The positive reaction for p53, c‐neu, TGF‐α, CEA, and EMA in duodenal adenocarcinoma was 20%, 60%, 60%, 73%, and 100%, respectively, and in ampullary adenocarcinoma, 13%, 100%, 50%, 63%, and 100%. Among the duodenal tumors, C‐neu and p53 expression was noted more frequently in groups with high histological grades. Patients with c‐neu positive duodenal adenocarcinoma had a shorter survival than the patients with c‐neu negative duodenal adenocarcinoma (P < 0.01). C‐neu product may serve as an unfavorable prognostic indicator in duodenal adenocarcinoma. No statistically significant correlation was found between the expressions of CEA, EMA, p53, and TGF‐α and patient survival, tumor stage, or histological grade in either duodenal or ampullary adenocarcinomas.

Chromosomal localization of the gene for a human galactosyltransferase (GT-1)

The gene for human galactosyltransferase (EC 2.4.1.22) has been localized to the short arm of chromosome 9 by in situ hybridization to human metaphase chromosomes of a 985 bp cDNA probe for the gene.

3H-methyl scopolamine binding to dispersed pancreatic acini

SummaryMaximal amylase release occurred with 10-5 M carbachol and slightly greater than half maximal response occurred with 3×10-7 M carbachol in dispersed pancreatic acini. The preparation released more than 45% of its initial amylase content after 60 min of maximal carbachol stimulation. Electron microscopy revealed depletion of zymogen granules and the presence of secretory material in the ductules after carbachol stimulation. At 37° C, maximal binding of methyl scopolamine occurred in about 45 min with 3×10-10 M 3H-methyl scopolamine. The dissociation constant for 3H-methyl scopolamine was 6.8×10-10 M and saturation occurred at 109 pm/g protein. The I.C. 50 for 3H-methyl scopolamine inhibition of carbachol-induced amylase secretion was 7 × 10-10 M.

Studies of paralytic lleus

Abstract This study is significant in demonstrating that the small intestine of the dog is extremely resistant to paralytic ileus. The various types of intra-abdominal irritation studied were quite severe. After a transient period of inhibition, however, in most instances motility of the small intestine returned and continued until near the time of death. Various types of intra-abdominal irritation were used to study paralytic ileus in dogs, including intraperitoneal injection of gastric juice, gastroperitoneal fistula, appendiceal ligation, intraperitoneal injection of Lugols iodine solution, retroperitoneal injection of blood, and mechanical and thermal irritation of the intestine and peritoneum. The electrical and mechanical activity of the small intestine was observed by means of a Thomas cannula implanted in the jejunum. The presence or absence of fluid accumulation within the intestinal lumen or peritoneal cavity was noted at autopsy. Intra-abdominal chemical irritation caused a transient inhibition of intestinal motility, which was reversed when the irritation was stopped. Repeated irritation did not appear to cause progressive, irreversible inhibition of intestinal motility. When intestinal motility was depressed, spike potentials were absent in the recordings of electrical activity of the intestine. The “slow” electrical waves were distinguishable at all times. With the exception of the gastroperitoneal fistulas, the procedures were tolerated with only transient inhibition of intestinal motility. Accumulation of intraperitoneal fluid occurred in dogs subjected to gastroperitoneal fistulas. A small amount of intraluminal fluid accumulated in dogs subjected to repeated thermal and mechanical irritation of the intestines and peritoneum. In the other groups of dogs no significant increase in intestinal or intraperitoneal fluid was observed.

The effects of distention and obstruction on the accumulation of fluid in the lumen of small bowel of dogs.

Fluid accumulation in either the obstructed upper or lower intestinal segments of the dog was found in most animals to be negligible. Distention pressures of 25 cm of water tended to reduce fluid accumulation within the intestinal lumen. These studies suggest that if the dog is comparable to man, the intraluminal accumulation of fluid in the obstructed small bowel of man might be due to alterations in blood supply to the intestine, rather than to obstruction per se, or the accumulated fluid originates proximal to the jejunum.