Showing papers by "Eppley Institute for Research in Cancer and Allied Diseases published in 1994"

DNA bending by asymmetric phosphate neutralization.

Abstract: DNA is often bent when complexed with proteins. Understanding the forces responsible for DNA bending would be of fundamental value in exploring the interplay of these macromolecules. A series of experiments was devised to test the hypothesis that proteins with cationic surfaces can induce substantial DNA bending by neutralizing phosphates on one DNA face. Repulsions between phosphates in the remaining anionic helix are predicted to result in an unbalanced compression force acting to deform the DNA toward the protein. This hypothesis is supported by the results of electrophoretic experiments in which DNA spontaneously bends when one helical face is partially modified by incorporation of neutral phosphate analogs. Phasing with respect to a site of intrinsic DNA curvature (hexadeoxyadenylate tract) permits estimation of the electrostatic bend angle, and demonstrates that such modified DNAs are deformed toward the neutralized surface, as predicted. Similar model systems may be useful in exploring the extent to which phosphate neutralization can account for DNA bending by particular proteins.

N-Acetylgalactosamine Glycosylation of MUC1 Tandem Repeat Peptides by Pancreatic Tumor Cell Extracts

Abstract: Synthetic peptides corresponding to the human mucin MUC1 tandem repeat domain (20 residues) were glycosylated in vitro by using UDP- N -[3H]acetyl-d-galactosamine (GalNAc) and lysates of pancreatic tumor cell lines Results obtained with peptides of different lengths (from one to five repeats) suggest that increasing the number of tandem repeats has neither a positive nor a negative effect on the density of glycosylation along the MUC1 tandem repeat protein backbone Purified glycopeptides were sequenced on a gas-phase sequencer, and glycosylated positions were determined by measuring the incorporated radioactivity in fractions collected following each round of Edman degradation The results showed that two of three threonine residues on the MUC1 tandem repeat peptides were glycosylated by pancreatic tumor cell lysates at the following positions: GVTSAPDTRPAPGSTAPPAH (underlined T indicates position of GalNAc attachment) None of the serine residues were glycosylated Determination of the mass of the glycopeptides by mass spectrometry confirmed that a maximum of two molecules of GalNAc were covalently linked to each 20-residue repeat unit in the peptides The data presented here show that acceptor substrate specificity of the UDP-GalNAc:polypeptide N -acetylgalactosaminyltransferase detected in lysates of pancreatic and breast tumor cell lines is identical and is limited to some but not all threonines in the MUC1 tandem repeat peptide sequence The influence of primary amino acid sequence on acceptor substrate activity was evaluated by using several peptides that contain single or double amino acid substitutions (relative to the native human MUC1 sequence) These included substitutions in the residues that were glycosylated and substitutions of the surrounding primary amino acid sequence The results of these studies suggest that primary amino acid sequence, length, and relative position of the residue to be glycosylated dramatically affect the ability of peptides to serve as acceptor substrates for the UDP-GalNAc:polypeptide N -acetylgalactosaminyltransferase

Selective cytotoxicity to human leukemic myeloblasts produced by oligodeoxyribonucleotide phosphorothioates complementary to p53 nucleotide sequences.

Abstract: Cells were treated in vitro with oligodeoxyribonucleotide phosphorothioates (ODNs) complementary to sites common to both wild-type and mutant p53 nucleotide sequences. Acute myelogenous leukemia (AML) blasts from peripheral blood were exposed to four different p53 ODNs and showed anti-leukemic effects in suspension culture. This effect continued after removal of the ODN from the medium. Blocking of self-renewal of the leukemic blast stem cells in secondary plating of cells from cloning assays by two of the p53 ODNs was also observed. Control ODNs had no effect on leukemic blasts. Treatment of normal bone marrow cells with the four p53 ODNs did not influence their growth, nor was there any effect by the p53 ODNs on the leukemic cell-line, HL60, that does not express p53. These data suggest that p53 ODNs are selectively toxic to primary myelogenous blasts and may be therapeutically useful in AML.

Decapeptide Agonists of Human C5a: The Relationship between Conformation and Spasmogenic and Platelet Aggregatory Activities

Abstract: A series of decapeptide analogues corresponding to the C-terminal region of human C5a anaphylatoxin (C5a) was synthesized with residue substitutions to restrict conformational flexibility in the C-terminus. These conformationally constrained peptides behaved as agonists of C5a in spasmogenic assays (smooth muscle contraction in human fetal artery, guinea pig ileum, and guinea pig lung parenchyma) as well as guinea pig platelet aggregation. There were significant correlations in the potencies of these peptides between the various assays. A structure-function analysis led to the identification of a preferred backbone conformation that correlated with the expression of these biological responses. These backbone structural motifs were consistent with a helix-like conformation for residues 65-69, an elongated structure for residues 70-71, and a β-turn of either type II or type V for residues (71)72-74. The most potent of these agonists expressed almost 5% if the potency of natural C5a.

Bacterial Expression and in Vitro Folding of the Beta-Subunit of Human Chorionic Gonadotropin (hCG Beta) and Functional Assembly of Recombinant hCG Beta With hCG Alpha

Abstract: A bacterial expression system for the beta-subunit of hCG (hCG beta) has been developed to produce suitable amounts of this protein for structural and biological studies. To produce hCG beta in Escherichia coli, the nucleotide sequence that encodes the amino acid leader sequence was removed from the hCG beta complementary DNA, and the gene was cloned into a pET expression vector. After induction of protein synthesis in host bacteria, recombinant hCG beta (rhCG beta) accumulated in inclusion bodies in an unfolded state. The inclusion bodies were purified from induced cultures of E. coli, solubilized in urea, and fractionated by reverse phase HPLC. In this way, 6-7 mg unfolded hCG beta were recovered from 1 liter culture, rhCG beta was folded in the presence of 6.4 mM cysteamine and 3.6 mM cystamine at pH 8.7 at a final concentration of 0.02 mg/ml protein. The folded protein assembled with urinary hCG alpha and the purified rhCG beta/urinary alpha dimer bound to and activated the human LH/CG receptor permanently expressed in a cell line, indicating that it was a functional hormone. The rhCG beta/urinary alpha dimer also stimulated in vivo ovulation in rats, thus confirming the biological activity of bacterially expressed hCG beta. Because E. coli lacks the ability to glycosylate proteins, these activity results indicate that the N-linked and O-linked oligosaccharides of hCG beta are not required for protein folding, subunit assembly, or full biological activity. The success of producing hCG beta in bacteria and of folding it in vitro implies that the beta-subunits of the other members of the glycoprotein hormone family, LH, FSH, and TSH, can also be produced in this manner. This may facilitate structural studies of these hormones as well as lead to the production of recombinant hormones for biological studies and clinical use.

Pancreatic centroacinar cells. The regulator of both exocrine and endocrine function.

Abstract: The relationship between pancreatic centroacinar cells (CAC), the acinar cells, and the endocrine cells was examined in fetuses and newborn Syrian hamsters histologically, immunohistochemically, and electron microscopically. Pancreatic anlage, composed of undifferentiated cells and a few α cells, were found at day 12, δ cells at day 13, acinar cells at day 14, and β cells at day 15 of the gestation. Intermediate cells (hybrid cells with both zymogen and endocrine granules) were also found after day 14. In the late-gestational period and after birth, two types of acini could be distinguished: one was composed exclusively of acinar cells and the second of acinar and CAC. In the latter type, some CAC covered the surface, lateral, and basal portion of the acinar cells, which showed a relative reduction of zymogens and increased autophagic vacuoles, a finding that indicated that CAC control the zymogen release from the acinar cells. Two types of CAC were encountered: dark cells with cytoplasmic processes located on the surface of acinar cells and larger light cells located between the acinar cells. The transitional forms between the light CAC and endocrine cells were found frequently at day 15 and a day after birth. During the endocrine cell differentiation, the committed cells lost their connection to the lumen by the force of the cytoplasmic processes of the dark CAC, which then overlaid the differentiated endocrine cells. From these findings, it can be concluded that CAC control both the pancreatic exocrine secretion and endocrine cell function.

Cryptic promoter activity within the backbone of a plasmid commonly used to prepare promoter/reporter gene constructs

Abstract: This report demonstrates that the plasmids, pBLCAT2 and pBLCAT3, which are used widely for the preparation of promoter reporter gene constructs, exhibit cryptic promoter activity when expressed in embryonal carcinoma (EC) cells and their differentiated cells. The promoterless plasmid pBLCAT3 is used widely because it has two multiple cloning sites. We demonstrate that the activity of the cryptic promoter present in pBLCAT3 is increased dramatically by an enhancerlike region of the murine k-FGF gene. However, the basal cryptic promoter activity and the enhanced cryptic promoter activity can be silenced effectively by the insertion of three tandemly arranged polyadenylation sequences. To characterize the influence of the cryptic promoter in pBLCAT3, we tested its effects on two promoters. Our findings suggest that the cryptic promoter increases by several fold the expression of the reporter gene in pBLCAT2, which contains the thymidine kinase promoter. In contrast, the cryptic promoter present in pBLCAT3 does not seem to influence the expression of the k-FGF promoter. Last, we observed cryptic promoter activity when pBLCAT3 was expressed transiently in EC-differentiated cells. Together, our findings argue that transcription silencing sequences should be used when examining weak promoters in these plasmids, especially in combination with enhancers.

Analysis of duplex DNA by triple helix formation: application to detection of a p53 microdeletion.

Abstract: Conventional methods for mutation detection include Southern hybridization, direct sequencing of PCR products and single-strand conformation polymorphism analysis. We present an additional screening method that employs oligonucleotide-directed DNA triple helix formation to detect mutations within homopurine sequences. The proposed strategy is simple and may be of particular value when screening many DNA samples for changes involving particular homopurine sites. We have applied the method to the analysis of a clinically relevant 8-bp micro-deletion in the human p53 tumor suppressor gene. Affinities of oligonucleotide probes toward radiolabeled wild-type and mutant p53 DNA duplexes were quantitated by electrophoretic mobility shift assays. Recombinant plasmids carrying wild-type or microdeleted forms of the p53 homopurine sites of interest were created. Dimethyl sulfate footprinting was used to verify intended probe specificities. Duplex PCR products amplified from plasmid constructs were directly probed by incubation with labeled oligonucleotides. After electrophoresis and autoradiography, patterns of triple helix formation allowed discrimination between the mutant and wild-type p53 sequences. Direct DNA analysis by triple helix formation may simplify other procedures that normally require DNA denaturation and hybridization.

Redox conditions for stimulation of in vitro folding and assembly of the glycoprotein hormone chorionic gonadotropin

Abstract: The formation of native disulfide bonds during in vitro protein folding can be limiting in obtaining biologically active proteins. Thus, optimization of redox conditions can be critical in maximizing the yield of renatured, recombinant proteins. We have employed a folding model, that of the beta subunit of human chorionic gonadotropin (hCG- beta), to investigate in vitro oxidation conditions that facilitate the folding of this protein, and have compared the in vitro rates obtained with the rate of folding that has been observed in intact cells. Two steps in the folding pathway of hCG-beta were investigated: the rate-limiting events in the folding of this protein, and the assembly of hCG-beta with, hCG-alpha. The rates of these folding events were determined with and without protein disulfide isomerase (PDI) using two different types of redox reagents: cysteamine and its oxidized equivalent, cystamine, and reduced and oxidized glutathione. Rates of the rate-limiting folding events were twofold faster in cysteamine/cystamine redox buffers than in glutathione buffers in the absence of PDI. Optimal conditions for hCG-beta folding were attained in a 2 mM glutathione buffer, pH 7.4, that contained 1 mg/mL PDI and in 10 microM cysteamine/cystamine, pH 8.7, without PDI. Under these conditions, the half-time of the ratelimiting folding event was 16 to 20 min and approached the rate observed in intact cells (4 to 5 min). Moreover, folding of the beta subunit under these conditions yields a functional protein, based on its ability to assemble with the alpha subunit. The rates of assembly of hCG-beta with hCG-alpha in the cysteamine/cystamine or glutathione/PDI redox buffers were comparable (t(1/2/sb> = 9 to 12 min)). These studies show that rates of folding and assembly events that involve disulfide bond formation can be optimized by a simple buffer system composed of cysteamine and cystamine.

Consumption of reduced-energy/low-fat diet or constant-energy/high-fat diet during mezerein treatment inhibited mouse skin tumor promotion

Abstract: Previous studies in our laboratory have shown that promotion of two-stage skin carcinogenesis in the SENCAR mouse model was inhibited in mice fed energy-restricted/low-fat diets, and elevated in mice fed high-fat diets. Studies reported here describe the influence of dietary energy restriction from fat and carbohydrate (ER) or high-fat (HF) diet on early promotion by 12-O-tetradecanoylphorbol-13-acetate (TPA) and on late promotion by mezerein (MEZ). Female SENCAR mice were initiated topically with 10 nmol 7,12-dimethylbenz[a]anthracene (DMBA) in 0.2 ml acetone at 9 weeks of age. For the following 2 weeks they received 3.2 nmol TPA in 0.2 ml acetone twice weekly, and for the next 16 weeks they received 10 nmol MEZ in 0.2 ml acetone twice weekly. All mice were fed control diet before TPA began and following the final MEZ treatment. Control mice received the control diet (c) throughout TPA and MEZ (C/C). The six experimental groups received: (1) ER diet throughout TPA and MEZ treatment (ER/ER); (2) HF diet throughout TPA and MEZ treatment (HF/HF); (3) ER during TPA (ER/C); (4) ER during MEZ (C/ER); (5) HF diet during TPA (HF/C); or (6) HF diet during MEZ (C/HF). Papilloma incidence and multiplicity, and carcinoma incidence were similarly reduced in the mice fed ER diet during MEZ (ER/ER and C/ER groups). In comparing the HF groups, papilloma multiplicity was highest in the HF/C group, intermediate in the C/C and lowest in the C/HF groups, but papilloma and carcinoma incidences were not modified by the HF diet protocols. Papilloma regression was greater in the C/HF group (27%, 4 regressions/15 tumor-bearing mice) than in the controls (0/18) during weeks 21-23, immediately following the end of MEZ treatment (P < 0.05).

Involvement of cytochrome P450 2E1-like isoform in the activation of N-nitrosobis(2-oxopropyl)amine in the rat nasal mucosa.

Abstract: Induction of tumours in the nasal olfactory region of MRC rats by N -nitrosobis(2-oxopropyl)amine (BOP) is inhibited by orchiectomy and restored by testosterone. These results suggest the involvement of a sex-specific enzyme in BOP bioactivation in rat nasal mucosa. The present study was undertaken to identify this enzyme. Enzyme-linked immunosorbent assay (ELISA) and the metabolism of known substrates ( P -nitrophenol) pointed to a microsomal cytochrome P450 (P450) 2E1-like isoform as a candidate enzyme. A correlation was found between the enzyme activity in nasal mucosal microsomes and serum testosterone levels. Four times more activity was detected in the nasal mucosa than in the liver of male rats. Vanillin inhibited the activity of the nasal mucosal enzyme to a greater extent than that of the liver enzyme. The overall results suggest that a nasal mucosal P450 2E1-like isoform is involved in BOP metabolism.

The patterns of coexpression of tumor-associated antigens CA 19-9, TAG-72, and DU-PAN-2 in human pancreatic cancer.

Abstract: Coexpression of CA 19-9, DU-PAN-2, and TAG-72 was examined by a multilabeling immunohistochemical procedure in 31 surgically resected human pancreatic carcinomas. CA 19-9 was expressed in 74%, DU-PAN-2 in 84%, and TAG-72 in 65% of the cases. CA 19-9 and DU-PAN-2 were coexpressed in 16 cases (52%), CA 19-9 and TAG-72 in 10 cases (32%), DU-PAN-2 and TAG-72 in 8 cases (26%), and all three antigens in 10 tumors (32%). With the combination of the three antibodies, all 31 tumors were labeled. However, heterogeneity in antigen expression existed and the antibodies against CA 19-9, DU-PAN-2, and TAG-72 depicted 55%, 49%, and 35% of the tumor cells, respectively. The average number of cells coexpressing CA 19-9 and DU-PAN-2, CA 19-9 and TAG-72, DU-PAN-2, and TAG-72 was 22%, 11%, and 10%, respectively. Only about 3% of tumor cells coexpressed all three antigens, whereas 8% of tumor cells did not express any of the antigens. There was no correlation between the patterns of antigen expression and age or sex. However, there was a tendency of reduced CA 19-9, DU-PAN-2, and TAG-72 expression in less differentiated tumor areas. The results show that: 1) pancreatic cancer cells coexpress two or three antigens in different proportions; and 2) although the sensitivity for pancreatic cancer reaches 100% by all three antibodies, a remarkable heterogeneity exists and a minor fraction of tumor cells does not seem to produce any of these antigens.