phosphorus-radioisotopes and Colonic-Neoplasms

The rapidly growing field of targeted tumor therapy often utilizes an antibody, sometimes tagged with a tumor-ablating material such as radioisotope, directed against a specific molecule.. This report describes the discovery of nine novel decapeptides which can be radioactively labeled, bind to, and deliver (32)P to colon cancer cells. The decapeptides vary from one another by one to three amino acids and demonstrate vastly different binding abilities. The most avidly binding decapeptide can permanently deliver very high levels of radioisotope to the adenocarcinoma cancer cell lines at an efficiency 35 to 150 times greater than to a variety of other cell types, including cell lines derived from other types of cancer or from normal tissue.. This experimental approach represents a new example of a strategy, termed peptide binding therapy, for the potential treatment of colorectal and other adenocarcinomas.

Topics: Adenocarcinoma; Caco-2 Cells; Cell Line, Tumor; Chemistry, Pharmaceutical; Colonic Neoplasms; Drug Delivery Systems; Drug Design; Humans; Immunotherapy; Medical Oncology; Peptides; Pharmaceutical Preparations; Phosphorus Radioisotopes; Protein Binding; Technology, Pharmaceutical

For the systemic treatment of colorectal cancer, 5-fluorouracil (FU)-based chemotherapy is the standard. However, only a subset of patients responds to chemotherapy. Breathing of carbogen (95% O2 and 5% CO2) may increase the uptake of FU through changes in tumor physiology. This study aims to monitor in animal models in vivo the effects of carbogen breathing on tumor blood plasma volume, pH, and energy status, and on FU uptake and metabolism in two colon tumor models C38 and C26a, which differ in their vascular structure and hypoxic status. Phosphorus-31 magnetic resonance spectroscopy (MRS) was used to assess tumor pH and energy status, and fluorine-19 MRS was used to follow FU uptake and metabolism. Advanced magnetic resonance imaging methods using ultrasmall particles of iron oxide were performed to assess blood plasma volume. The results showed that carbogen breathing significantly decreased extracellular pH and increased tumor blood plasma volume and FU uptake in tumors. These effects were most significant in the C38 tumor line, which has the largest relative vascular area. In the C26a tumor line, carbogen breathing increased tumor growth delay by FU. In this study, carbogen breathing also enhanced systemic toxicity by FU.

Topics: Animals; Antimetabolites, Antineoplastic; Carbon Dioxide; Cell Line, Tumor; Colonic Neoplasms; Disease Models, Animal; Fluorine Radioisotopes; Fluorouracil; Hydrogen-Ion Concentration; Magnetic Resonance Spectroscopy; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Neovascularization, Pathologic; Oxygen; Phosphorus Radioisotopes; Plasma; Radiation-Sensitizing Agents; Respiration

To find the mechanisms of the ongoing clinical trials in intralesional colloidal chromic 32P (32P-CP) brachytherapy, the cellular uptake of 32P-CP, changes in tumor interstitial fluid pressure (TIFP), and tumor blood flow (TBF) using two (AsPC-1, Ls174T) human tumors were measured.. After exposure to 32p-CP using exponential and plateau-phase cells, cells were trypsinized at various time intervals, then measured for the levels of radioactivity using a y-counter. Also measured were TIFP using the WIN technique and TBF with laser Doppler flowmetry.. The plateau growth-phase of both tumors showed the maximal uptake of 32P-CP at approximately 100 min. TBF decreased within 10 min after an intratumoral (i.t.) injection of 32P-CP, and reached 75% of control value by 1 h.. If 32P-CP was introduced i.t., it maintained highly efficient tumor targeting, mainly due to two physiological mechanisms: the high adherence of 32P-CP to the infused regions and the reduction in TBF by this therapeutic colloid.

Topics: Adenocarcinoma; Animals; Brachytherapy; Colloids; Colonic Neoplasms; Female; Humans; Injections, Intralesional; Laser-Doppler Flowmetry; Mice; Mice, Nude; Neoplasm Transplantation; Pancreatic Neoplasms; Phosphorus Radioisotopes; Radiotherapy

Examination of microsatellites is frequent in the diagnosis of cancer. Microsatellites are repeat DNA sequences scattered throughout the human genome. These repeat regions are very frequent and highly polymorphic elements. In this study we focus on dinucleotide repeats. We compared three different methods for the detection of microsatellites: use of the ABI Prism 377 fluorescence sequencer, autoradiography and silver-stained gels. DNA was extracted from various clinical samples and amplified by different polymerase chain reaction (PCR) protocols. DNA from normal and tumor tissues was analysed using each method. The fluorescence method was more sensitive than the two other methods; however, this technology is very expensive. It seems possible, when examining microsatellites on a low budget, to avoid radioactivity by using silver-stained gels as an alternative. In conclusion, we observed identical results when comparing autoradiography with the fluorescence technique. However, we observed variability in the results when interpreting a single locus comparing silver staining with autoradiography and the fluorescence technique. Classification of the tumors based on several microsatellite loci was always identical.

Topics: Autoradiography; Colonic Neoplasms; DNA Primers; DNA, Neoplasm; DNA, Satellite; Fluorescent Dyes; Genetic Markers; Humans; Phosphorus Radioisotopes; Polymerase Chain Reaction; Repetitive Sequences, Nucleic Acid; Sequence Analysis, DNA; Silver Staining; Urinary Bladder Neoplasms

The major limitations of monoclonal antibody conjugates as therapeutic agents have been their poor tumour targeting, inadequate tumour penetration and immunogenicity. More even and deeper tissue penetration has been demonstrated with smaller antibody fragments. The smaller size and absence of an Fc segment may contribute to a lowered immunogenicity with single-chain antibodies (scFv) and also permit their recombinant engineering and bacterial expression. We describe the successful engineering, expression and pre-clinical characterisation of a phosphorylatable "kemptide" (Leu-Arg-Arg-Ala-Ser-Gly) anti-carcinoembryonic antigen (anti-CEA) scFv (PKS-scFv), for use as a radioimmunotherapeutic agent. Specifically, a yield of 6 mg/l induced culture was obtained. Site-specific phosphorylation was demonstrated without loss of specificity. In vitro assays revealed a selective cytotoxicity of 32P-PKS-scFv for high-CEA-expressing LS-174T cells compared to the low-CEA-expressing HT-29 cells, with a rapid internalisation rate.

Topics: Binding Sites; Carcinoembryonic Antigen; Cloning, Molecular; Colonic Neoplasms; Drug Stability; Escherichia coli; Genetic Engineering; Humans; Immunoglobulin Fragments; Immunoglobulin Variable Region; Immunotoxins; Oligopeptides; Phosphorus Radioisotopes; Phosphorylation; Radioimmunotherapy; Recombinant Proteins; Tumor Cells, Cultured

The adenomatous polyposis coli (APC) gene is etiologically associated with familial adenomatous polyposis and gastrointestinal malignancies, but its cellular function and role in tumorigenesis are unclear. Recent reports indicate that wild-type, but not mutant, APC gene product (APC) is associated with and promotes the assembly of cytoskeletal microtubules in vitro, suggesting that this mechanism has importance in tumor development. Because other microtubule-associated proteins (MAPs) undergo phosphorylation in their normal functioning, we postulated that APC is a phosphoprotein. HCT116 cells, containing full-length APC protein, were [32P]-prelabeled, and a 300-kDa band corresponding to phosphorylated APC was immunoprecipitated using each of three different anti-APC antibodies. High voltage electrophoresis of [32P]-labeled APC showed the presence of phospho-serine and phospho-threonine residues. Further immunoprecipitation analyses showed phosphorylation of i) full-length APC in human lymphoblastoid cells and ii) carboxyl-truncated APC in SW480 and DiFi colon carcinoma cells. Thus, APC is probably a phosphoprotein in normal and malignant tissues. We hypothesize a mechanism whereby phosphorylation of APC may play a regulatory role in its interaction with microtubules. This may involve phosphorylation of (Ser/Thr)-Pro amino acid motifs in APC's basic domain. We propose that deletion of this domain disrupts APC binding to microtubules, explaining how APC mutations are linked to cancer development.

Topics: Adenomatous Polyposis Coli Protein; Amino Acid Sequence; Cell Line; Colonic Neoplasms; Cytoskeletal Proteins; Dipeptides; Electrophoresis, Polyacrylamide Gel; Genes, APC; Humans; Lymphocytes; Phosphates; Phosphorus Radioisotopes; Phosphorylation; Phosphoserine; Phosphothreonine; Tumor Cells, Cultured

The human ras gene plays a fundamental role in the transduction of extracellular signals to the nucleus, thereby regulating cell growth and differentiation. Point mutations in the ras gene convert it into a transforming oncogene that has been found in many solid and hematologic malignancies. We describe a rapid and sensitive assay based on a radiolabeled polymerase chain reaction followed by restriction enzyme digestion that we have adapted for differentiating between the wild-type and mutant ras genes. This assay should prove useful in the analysis of ras gene point mutations in clinical tumor specimens in which ras oncogene activation is an early event in carcinogenesis.

Topics: Base Sequence; Blotting, Southern; Codon; Colonic Neoplasms; Deoxyribonucleases, Type II Site-Specific; DNA, Neoplasm; Genes, ras; Humans; Molecular Sequence Data; Nucleic Acid Hybridization; Phosphorus Radioisotopes; Point Mutation; Polymerase Chain Reaction; Tumor Cells, Cultured

There are conflicting data about the effect of the epidermal growth factor (EGF) on protein kinase C (PKC) enzyme activity. The aim of our study was to find out which type of phospholipids [phosphatidylinositol 4,5-bisphosphate PI4,5P2 or the other phospholipids-phosphatidylcholine (PC) or phosphatidic acid (PA)] could be the source of 1,2-diacylglycerol (1,2-DAG) in PKC activation. In colon carcinoma cells (HT29) we observed a more than 2-fold increase in the PC pool and at the same time decreased tyrosine kinase activity (50%). With increasing incubation time EGF affects the pools of both phosphatidylinositols and other phospholipids parallel with the activation of the tyrosine kinase activity. EGF increases the activity of PKC in the HT29 cell line and PC could be the source of 1,2-DAG which may stimulate PKC activity.

Topics: Carcinoma; Colonic Neoplasms; Epidermal Growth Factor; Humans; Phospholipids; Phosphorus Radioisotopes; Protein Kinase C; Protein-Tyrosine Kinases; Signal Transduction; Time Factors; Tumor Cells, Cultured

2-Amino-1-methyl-6-phenylimidazo[4,5-b]-pyridine (PhIP) is known to induce colon tumors in male Fischer-344 rats. Using 32P-postlabeling assays, we have examined PhIP-DNA adduct formation in various organs and white blood cells (WBCs) of the male Fischer-344 rat 24 h after a single oral dose of 0, 0.5, 5 or 50 mg PhIP/kg. Three PhIP-DNA adducts were detected in WBCs and in all organs, except in the liver and stomach which had only two adducts. The extent of adduct formation was dose-related, but at 0.5 mg/kg no adducts could be detected in any of the organs. At 50 mg/kg, adduct levels, expressed as relative adduct labeling values (RAL x 10(7), or adducts per 10(7) nucleotides assuming complete labeling) were highest in the large intestine (5.66), followed by WBCs (5.04), stomach (1.44), small intestine (1.32), kidney (1.16), liver (0.67) and lungs (0.52). It is concluded that orally administered PhIP forms high levels of specific DNA adducts in the large intestine, the target organ in PhIP carcinogenesis in the male Fischer-344 rat, and that the high level of adducts in WBCs indicates that significant amounts of the ultimate carcinogenic form of PhIP are present in the circulation.

Topics: Animals; Colonic Neoplasms; DNA; Imidazoles; Male; Mutagens; Phosphorus Radioisotopes; Rats; Rats, Inbred F344

Analogues of somatostatin (SS) and luteinizing hormone-releasing hormone (LH-RH) activate tyrosine phosphatases in MIA PaCa-2 human pancreatic cancer cell line membranes and inhibit growth. We compared the substrates phosphorylated by epidermal growth factor (EGF) to those dephosphorylated by the SS analogue RC-160 (D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Trp-NH2) and [D-Trp6]LH-RH in cancer cell lines such as MIA PaCa-2 (human pancreatic cancer), HCPC (hamster cheek pouch carcinoma), A-549 (human lung cancer), HT-29 (human colon cancer), and R3230AC (breast cancer). EGF phosphorylated proteins of 170, 65, and 60 kDa and analogues of SS and LH-RH promoted the dephosphorylation of these proteins in MIA PaCa-2 and HCPC cell lines. The EGF receptor is 170 kDa. pp60src (60 kDa) is known to be a substrate for EGF receptor. The LH-RH receptor is also 60 kDa. The effects of RC-160 and [D-Trp6]LH-RH were quantitatively different. Examinations of HT-29, A-549, and R3230AC cancer cell lines revealed no phosphorylation by EGF or dephosphorylation by RC-160 and [D-Trp6]LH-RH. In addition to the 170-, 65-, and 60-kDa proteins, 35-kDa proteins were also phosphorylated in some cancer cell lines. This work demonstrates that analogues of SS and LH-RH can reverse the effects of EGF biochemically as well as functionally.

Topics: Amino Acid Sequence; Animals; Antineoplastic Agents; Autoradiography; Breast Neoplasms; Cell Line; Colonic Neoplasms; Epidermal Growth Factor; Gonadotropin-Releasing Hormone; Humans; Kinetics; Lung Neoplasms; Membrane Proteins; Molecular Sequence Data; Pancreatic Neoplasms; Phosphorus Radioisotopes; Phosphorylation; Protein Kinases; Protein-Tyrosine Kinases; Somatostatin; Triptorelin Pamoate; Tyrosine

Molecular alterations of cellular proteins were assessed in three previously described subpopulations of human colon carcinoma cells that were originally isolated from a single human primary colon carcinoma. The subpopulations designated HCT 116b, HCT 116, and HCT 116a exhibited significant differences in their expression of several biological properties. Immunoautoradiographic analysis of membrane components, fractionated by SDS-PAGE and electrophoretically transferred onto nitrocellulose sheets, distinguished the most aggressive tumor cell subpopulation from the intermediate and least aggressive subpopulations. An elevated expression of antigens of molecular weight in the range of 116-120 kd, 92 kd, 55-66 kd, and 31 kd was found to be associated with the most aggressive subpopulation of HCT 116a cells. The binding pattern of RCA I and WGA lectins to membrane components, fractionated by SDS-PAGE and fixed on nitrocellulose, also distinguished the three subpopulations of cells. Elevated bindings of RCA I to 80-92 kd components of HCT 116b cells and elevated bindings of WGA to 120 kd components of HCT 116a cells distinguished the three subpopulations from one another. Analysis of cellular phosphoprotein profiles, following labeling of the cells with 32Pi in the presence of phosphate-free medium, further distinguished the most aggressive subpopulation from the intermediate and least aggressive subpopulations. High level of phosphorylation of 120 kd and 250 kd nuclear components, 30 and 90 kd cytosolic components, and low level of phosphorylation of lower molecular weight membrane components were found to be associated with the most aggressive subpopulation. It is concluded that differences in the expression of membrane antigens, differences in the glycosylation of membrane components, and the selective phosphorylation and/or dephosphorylation of cellular proteins exist in subpopulations of intratumoral colonic carcinoma cells with different biological properties. These biochemical alterations of cellular proteins may play an important role in the generation of phenotypic diversity and heterogeneity of malignant cells.

Topics: Antigens, Neoplasm; Antigens, Surface; Cell Line; Colonic Neoplasms; Electrophoresis, Polyacrylamide Gel; Humans; Immune Sera; Lectins; Membrane Proteins; Molecular Weight; Neoplasm Proteins; Phosphoproteins; Phosphorus Radioisotopes; Phosphorylation; Subcellular Fractions

Radionuclide peritoneoscintigraphy has been used prior to chromic phosphate P-32 (P-32CP) intraperitoneal therapy to assure proper placement of the catheter in the peritoneal cavity, to exclude loculation, and to predict inadequate distribution of P-32CP. This is a case report of the detection of a peritoneal catheter improperly placed into the bowel lumen by pretherapy radionuclide peritoneoscintigraphy, and this case demonstrates the distinguishing characteristics of the radiocolloid distribution secondary to an intraluminal injection relative to an intraperitoneal injection.

Topics: Adenocarcinoma; Brachytherapy; Catheterization; Chromium; Chromium Compounds; Colonic Neoplasms; Female; Humans; Middle Aged; Peritoneal Cavity; Peritoneal Neoplasms; Phosphates; Phosphorus Radioisotopes; Radionuclide Imaging; Technetium Tc 99m Sulfur Colloid

Topics: Colonic Neoplasms; Colonoscopy; Humans; Neoplasm Recurrence, Local; Phosphorus Radioisotopes; Radiometry; Rectal Neoplasms

Established cancer in the liver can, in selected patients who have a good arterial circulation in these tumors, be effectively treated by intrahepatic artery radioactive yttrium-90 resin microspheres. Even in unselected patients treated in the last five years by the author, 17 of 25 patients treated have had good objective regression of cancers, improvement of symptoms and prolongation of life. Treatment is relatively simple and associated with few side effects. For adjuvant therapy of colon cancer having positive nodes (Dukes C), internal radiation therapy of the liver is best done with Phosphorus-32 Colloid passed through the circulation of the gut to be effectively and homogeneously trapped by the Kupffer cells of the liver. Four such patients have been subjected to a pilot study--three of the four are doing well without significant side effects and no evidence of liver cancer after two years. When the fourth died of brain metastases, he too had less liver cancer than would be expected.

Topics: Aged; Animals; Brachytherapy; Colloids; Colonic Neoplasms; Female; Humans; Liver Neoplasms; Male; Microspheres; Middle Aged; Phosphorus Radioisotopes; Pilot Projects; Radiotherapy Dosage; Rats; Yttrium Radioisotopes