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PubMed1991

Tomographic myocardial perfusion imaging with technetium-99m-teboroxime at rest and after dipyridamole.

This study was done to determine whether the rapidly clearing myocardial perfusion agent 99mTc-teboroxime (SQ 30217, Cardiotec) could be combined with tomographic imaging to accurately quantify regional myocardial blood flow distribution in anesthetized dogs. Following stenosis of the anterior descending (LAD, n = 10) or circumflex (LCX, n = 5) coronary arteries, teboroxime was administered simultaneously with radioactive microspheres, at rest and following infusion of dipyridamole (0.15 mg/kg/min x 4 min). Tomographic imaging began 1 min after each teboroxime injection and continued for 12 min. For LAD stenosis, when the dipyridamole study was performed first, teboroxime activity in the center of the ischemic region was closely correlated with tissue microsphere content. However, the severity of the dipyridamole-induced flow deficit was underestimated by teboroxime when the rest study was performed first. Our results show that despite rapid myocardial clearance, tomographic imaging of 99mTc-teboroxime provides reasonably accurate quantitation of dipyridamole-induced anterior wall perfusion defects, but that the flow deficit is underestimated when a rest study is performed first or when the defect is located in the inferior wall.

Animals↗

PubMed1998

The nucleus is the target for radiation-induced chromosomal instability.

We have previously described chromosomal instability in cells of a human-hamster hybrid cell line after exposure to X rays. Chromosomal instability in these cells is characterized by the appearance of novel chromosomal rearrangements multiple generations after exposure to ionizing radiation. To identify the cellular target(s) for radiation-induced chromosomal instability, cells were treated with 125I-labeled compounds and frozen. Radioactive decays from 125I cause damage to the cell primarily at the site of their decay, and freezing the cells allows damage to accumulate in the absence of other cellular processes. We found that the decay of 125I-iododeoxyuridine, which is incorporated into the DNA, caused chromosomal instability. While cell killing and first-division chromosomal rearrangements increased with increasing numbers of 125I decays, the frequency of chromosomal instability was independent of dose. Chromosomal instability could also be induced from incorporation of 125I-iododeoxyuridine without freezing the cells for accumulation of decays. This indicates that DNA double-strand breaks in frozen cells resulting from 125I decays failed to lead to instability. Incorporation of an 125I-labeled protein (125I-succinyl-concanavalin A), which was internalized into the cell and/or bound to the plasma membrane, neither caused chromosomal instability nor potentiated chromosomal instability induced by 125I-iododeoxyuridine. These results show that the target for radiation-induced chromosomal instability in these cells is the nucleus.

Animals↗

PubMed1996

Modulation of [5-125I]iododeoxyuridine incorporation into tumour and normal tissue DNA by methotrexate and thymidylate synthase inhibitors.

A potentially useful method for imaging of micrometastases and in situ radiotherapy, would be the incorporation of radioactive labelled iododeoxyuridine (IdU) into tumour DNA. However, there are two main problems: incorporation of the radioactive IdU into normal cells and low incorporation into tumour cells. The aim of this study was to attempt to augment the incorporation of [5-125I]iododeoxyuridine (125IdU) into tumour DNA and to improve the tumour/normal tissue ratio by the use of inhibitors (methotrexate, 5-fluorouracil, AG337, ZD 1694, benzyloxybenzyl uracil) which would prolong the metabolic half-life of the compound. Mammary tumours were induced in GR mice, which were then treated with the inhibitors and the 125IdU. The tumours and representative normal tissue were removed following sacrifice of the animals, and radioactivity within the tissues measured. Pretreatment of mammary carcinoma-bearing GR mice with methotrexate caused approximately a 3-fold increase in the incorporation of 125IdU into tumour DNA, and approximately a > or = 10-fold increase in the tumour/small intestine ratio of incorporated radioactivity. Inhibition of thymidylate synthase, the enzyme involved in IdU dehalogenation, by 5-fluorouracil plus folic acid, or by novel inhibitors AG337 and ZD1694 led to a 3- to 5-fold increase in the 125IdU incorporation. Benzyloxybenzyl uracil, an inhibitor of dihydrouracil dehydrogenase, had little effect. Treatment of tumour-bearing mice with methotrexate plus ZD1694 significantly reduced the rate of tumour growth, but addition of 125IdU (70 microCi/mouse, three daily injections) had no additional antitumour activity. In conclusion, these results do not support the hypothesis that systemic administration of 125IdU can be used for cancer therapy or for imaging purposes unless better methods are found to boost its incorporation into tumour DNA.

Animals↗

PubMed2000

Improved sensitivity proteomics by postharvest alkylation and radioactive labelling of proteins.

We describe approaches to improve the detection of proteins by postharvest alkylation and subsequent radioactive labeling with either [3H]iodoacetamide or 125I. Database protein sequence analysis suggested that cysteine is not suitable for detection of the entire proteome, but that cysteine alkylating reagents can increase the number of proteins able to be detected by iodination chemistry. Proteins were alkylated with beta-(4-hydroxyphenyl)ethyl iodoacetamide, or with 1,5-l-AEDANS (the Hudson Weber reagent). Subsequent iodination using the Iodo-Gen system was found to be most efficient. The enhanced sensitivity obtainable by using these approaches is expected to be sufficient for visualization of the lowest copy number proteins from human cells, such as from clinical samples. However, we argue that significantly improved methods of protein separation will be necessary to resolve the large number of proteins expected to be detectable with this sensitivity.

Acetamides↗

PubMedDate not supplied

Renal protein degradation: a biochemical target of specific nephrotoxicants.

Protein degradation in the kidney occurs mainly in lysosomes, organelles which may also accumulate nephrotoxic chemicals. The goal of this study was to evaluate the effects of intracellular accumulation of gentamicin, cephaloridine and cisplatin on lysosomal digestion of the protein lysozyme. Gentamicin (15 or 30 mg/kg/day for 3 or 5 days), cisplatin (2.5 or 5 mg/kg) or cephaloridine (500, 1000, 2000 or 2500 mg/kg) was administered ip to male Wistar rats. The main site of the nephrotoxic effects of these compounds was the proximal tubule where these agents differentially affected S1, S2 and/or S3 segments. A 2- and 4-fold increase of the excretion of N-acetyl-beta-D-glucosaminidase (NAG) was observed in the urine from cisplatin- and gentamicin-treated rats, respectively; no change in enzyme excretion occurred after cephaloradine. One hour prior to sacrifice, rat were given 0.3 mg of unlabelled lysozyme in combination with 125I-lysozyme in 0.3 mL saline. Renal cortical slices were prepared and incubated for 15, 30, 60 and 90 min. Release of trichloroacetic acid (TCA) soluble radioactivity into the medium was assumed to quantify lysosomal degradation of lysozyme. Accumulation of p-amino-hippurate (PAH) in renal cortical slices and changes in blood urea nitrogen (BUN) concentration were used as indices of renal damage. TCA-soluble radioactivity increased in the medium from kidney slices from control rats to 50% of the total radioactivity after 90 min incubation. In gentamicin-treated rats, lysozyme degradation was significantly decreased by doses of 15 and 30 mg/kg/day after 3 and 5 days of exposure in the absence of any changes in BUN or PAH accumulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed2004

Nicotinic acetylcholine receptor subtypes in the rat sympathetic ganglion: pharmacological characterization, subcellular distribution and effect of pre- and postganglionic nerve crush.

Nicotinic acetylcholine receptors (nAChRs) mediate fast synaptic transmission in autonomic ganglia, which innervate and control the activity of most visceral organs. By combining ultrastructural, immunocytochemical, and pharmacological analyses, we characterized the nAChR subtypes in the rat superior cervical ganglion (SCG) and the effect of pre- and postganglionic nerve crush on their number in the ganglion and their distribution at the intraganglionic synapses. Binding with radioactive nicotinic ligands, immunoprecipitation, and immunolocalization experiments revealed the presence of different nAChR subtypes: those containing the alpha3 subunit associated with beta4 and/or beta2 subunits that bind 3H-Epibatidine with high affinity, and those containing the alpha7 subunit that bind 125I-alphaBungarotoxin. After postganglionic nerve crush, the number of nicotinic receptors and immunopositive intraganglionic synapses for each nAChR subunit strongly decreased. Both the number of nAChRs and immunoreactivity recovered 26 days after injury, when regenerating postganglionic fibers had reinnervated the peripheral target organs, as shown by the restoration of tyrosine hydroxylase immunoreactivity in the iris. This observation and the lack of any effect of preganglionic nerve crush on the number of nicotinic receptors suggest that the peripheral targets affect the organization of intraganglionic synapses in adult SCG.

Animals↗

PubMed1993

Chemical methods for reduction of the transfer of radionuclides to farm animals in semi-natural environments.

The same chemicals can be used for reduction of radionuclide transfer to animals whether kept on farms or grazing in semi-natural and natural habitats. However, different techniques are required for administration of the active compounds. Dairy ruminants may be treated effectively by inclusion of chemicals in supplemental concentrates. Practical experience gained after the Chernobyl accident has shown that both clay minerals and hexacyanoferrates are effective in preventing high radiocaesium levels in animal products. Chemicals such as bentonite clays and CaCO3, used for reduction of 137Cs and 90Sr transfer respectively, must be fed in hectogram quantities and are only practical for dairy animals in semi-natural ecosystems. Salt licks and sustained release boli with hexacyanoferrates as caesium binders have been developed and used successfully after the Chernobyl accident for meat producing cattle, sheep and reindeer which graze freely for extended periods. Daily doses of 25-300 mg in sheep and 250-2000 mg in cows reduces 137Cs accumulation 2-10-fold. Binders for 90Sr have not been tested in grazing animals. Stable iodine could be provided in salt licks and indwelling rumen boli at rates required to block radioiodine uptake by the thyroid gland. Boli and salt licks are highly cost effective in reducing doses to man when compared to interdiction of food from farm animals.

Aluminum Silicates↗

PubMed2002

In vitro and in vivo characteristics of a human colon cancer cell line, SNU-C5N, expressing sodium-iodide symporter.

Rat NIS (rNIS) genes were transfected into a human colon cancer cell line (SNU-C5) by lipofection. The transfected cells (SNU-C5N) exhibited an increase 125I uptake to a level 10 times higher than the untransfected SNU-C5 cells. The addition of 300 microM DIDS, an anion channel blocker, to the culture media led to a 2.35 times increase of 125I uptake in the cells. For the first 10 minutes, up to 70% of the cellular radioactivity was released into the medium. In the biodistribution study using SNU-C5N-xenografted mice, the %ID/g of the SNU-C5N tumors at 1, 3, 6, and 12 h after the 125I injection were 4.43%, 1.09%, 1.05%, and 0.05%, respectively, which were significantly higher than those for the SNU-C5 tumors (P<0.05). In tumor imaging, the SNU-C5N-xenografted tumor was clearly visible. In this study, NIS lipofection is efficient for triggering significant iodide uptake by a nonthyroidal tumor. However, for an increased therapeutic effect, the key issue is iodide retention in the target tissue.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1995

Evaluation of stereoisomers of 4-fluoroalkyl analogues of 3-quinuclidinyl benzilate in in vivo competition studies for the M1, M2, and M3 muscarinic receptor subtypes in brain.

To develop a subtype selective muscarinic acetylcholine receptor (mAChR) antagonist for PET, fluorine-19 labeled alkyl analogues of quinuclidinyl benzilate (QNB) were synthesized by stereoselective reactions. To investigate these analogues for tissue subtype specificity, in vivo competitive binding studies were performed in rat brain using (R)-3-quinuclidinyl (R)-4-[125I]iodobenzilate (IQNB). Five, fifty, or five-hundred nmol of the non-radioactive ligands were coinjected intravenously with 8 pmol of the radioligand, Cold (R,R)-IQNB blocked (R,R)-[125I]IQNB in a dose-dependent manner, without showing regional specificity. For the (R,S)-fluoromethyl, -fluoroethyl and -fluoropropyl derivatives, a higher percent blockade was seen at 5 and 50 mmol levels in M2 predominant tissues (medulla, pons, and cerebellum) than in M1 predominant tissues (cortex, striatum and hippocampus). The blockade pattern of the radioligand also correlated qualitatively with the percentage of M2 receptors in the region. The S-quinuclidinyl analogues showed M2 selectivity but less efficient blockade of the radioligand, indicating lower affinities. Radioligand bound to the medulla was inversely correlated to the M2 relative binding affinity of the fluoroalkyl analogues. These results indicate that the nonradioactive ligand blocks the radioligand based on the affinity of the nonradioactive ligand for a particular receptor subtype compared to the affinity of the radioligand for the same receptor subtype. Of the seven compounds evaluated, (R,S)-fluoromethyl-QNB appears to show the most selectivity for the M2 subtypes in competition studies in vivo.

Animals↗

PubMed2000

Uptake and intracellular fate of polyethylenimine in vivo.

Branched polyamines are extensively used as nonviral vectors for plasmid DNA in transfection experiments. Moreover, recently it has been shown that these compounds are able to eliminate prions from infected cells in cultures. It has been proposed that in both cases endosomes or lysosomes are the site of action. This raises the question of how these molecules are taken up by the cells and what is their intracellular fate. In the work presented here, the question has been addressed by investigating the uptake and the intracellular distribution of branched polyethyleneimine (25 kD) by centrifugation methods. The polyamine was labelled with (125)I-tyramine cellobiose and injected to the rat. The radioactive polymer is taken up after injection into the liver, kidney, spleen, and lungs and remains in these organs for many days. In the liver, it is found mainly in the hepatocytes. Intracellular distribution of radioactivity present in that organ was investigated by differential and isopycnic centrifugations. Early after injection, radioactivity exhibits a distribution pattern similar to that of alkaline phosphodiesterase, a plasma membrane marker. Later, the distribution pattern becomes similar to that of cathepsin C, a lysosomal enzyme. Radioactivity and hydrolase distributions in a sucrose gradient are similarly modified by a pretreatment of the rat with Triton-WR1339, a specific density perturbant of lysosomes. These results indicate that polyethyleneimine is endocytosed and reaches lysosomes. For many days it persists in these organelles probably due to its resistance to lysosomal hydrolases.

Animals↗

PubMed1992

In-vivo blood-brain barrier transport of a novel adrenocorticotropic hormone analogue, ebiratide, demonstrated by brain microdialysis and capillary depletion methods.

The transport of ebiratide, a novel adrenocorticotropic hormone (ACTH) analogue, [H-Met-(O2)-Glu-His-Phe-D-Lys-Phe-NH(CH2)8-NH2], through the blood-brain barrier was directly demonstrated in-vivo. [125I]Ebiratide (16.9 MBq mL-1) or [14C]sucrose (29.2 MBq mL-1) known to be restrictively transported through the blood-brain barrier was infused into the rat internal carotid artery at a flow rate of 50 microL min-1 for 10 min, and after 15 min infusion the distribution volume of each compound in the brain parenchyma was determined by the capillary depletion method. The distribution volume of [125I]ebiratide was 167.8 +/- 62.2 microL (g brain)-1, which was about seven times higher than that of [14C]sucrose (24.9 +/- 4.0 microL g brain)-1, indicating the uptake of ebiratide into brain parenchymal cells. During the infusion into the internal carotid artery, brain microdialysis was simultaneously performed to directly collect the brain interstitial fluid as the dialysate. Radioactivity was detected in the dialysate during the [125I]ebiratide infusion and HPLC analysis of the dialysate revealed that the intact ebiratide accounted for greater than or equal to 80% total radioactivity. The concentrations of [125I]ebiratide and [14C]sucrose in the brain interstitial fluid were estimated based on the relative recovery obtained in the in-vitro recovery study. The brain interstitial fluid/internal carotid arterial blood concentration ratio for [125I]ebiratide was determined to be 1.47 x 10(-2) +/- 0.17 x 10(-2) and was about eight times higher than that for [14C]sucrose (1.92 x 10(-3) +/- 0.36 x 10(-3)), indicating significant transport of ebiratide to the brain interstitial fluid.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenocorticotropic Hormone↗

PubMed1995

Single photon emission tomography (SPET) imaging of dopamine D2 receptors in the course of dopamine replacement therapy in patients with nocturnal myoclonus syndrome (NMS).

Single photon emission tomography (SPET) permits the in vivo measurements of regional cerebral radioactivity in the human brain following the administration of compounds labeled with photon-emitting isotopes. According to our SPET findings of a reduced binding of [123I]labeled (S)-2-hydroxy-3-iodo-6-methoxy-([1-ethyl-2-pyrrolidinyl]methyl) benzamide (IBZM) (a highly selective CNS D2 dopamine receptor ligand) to D2 dopamine receptors in striatal structures in untreated patients with nocturnal myoclonus syndrome (NMS) it seemed to be of interest to investigate whether there are changes in D2 receptor binding under dopamine replacement therapy or not. We studied the uptake and distribution of [123I]IBZM before and in the course of dopamine replacement therapy in four patients with severe insomnia caused by a nocturnal myoclonus syndrome (NMS). We found an increase of the IBZM binding to D2 receptors in the course of treatment, which was associated with an improvement of sleep quality. Reasons for this are discussed. The [123I]IBZM SPET technique in conclusion offers an intersting tool for in vivo investigations of functional changes in the dopaminergic neurotransmitter system in longitudinal studies.

Aged↗

PubMed1964

A RADIOIODINATED AZO DYE WITH AFFINITY FOR AMYLOID: A PRELIMINARY REPORT.

Investigation was undertaken to produce a radioactive material with affinity for amyloid tissue. Studies of a number of major dyes using paraffin histological sections of an amyloid spleen as test material showed that the only compounds with marked affinity for amyloid were watersoluble disazo dyes of the type E<--D-->E, where D is usually benzidine, o-tolidine or o-dianisidine, and E is a naphthol or aminonaphthol sulfonic acid. Substitution of an amino or a hydroxyl group in position 1 of the naphthalene nucleus caused loss of staining affinity. It was found that trypan blue could be radioiodinated at position 8 using the Sandmeyer reaction, and the labelled dye showed excellent staining and radioautographs of the test amyloid section. Metabolism of the tagged dye using 10-200 mug./kg. in animals showed minimal organ uptake, except for the liver which accumulated 30-40% of the injected dose by the end of four days. Use of this material in the diagnosis of human amyloid disease is being explored.

Amyloid↗

PubMed1999

Validation of K-edge 125I brachytherapy enhancement with silver compounds.

Brachytherapy with radioactive seeds implanted within the tumour volume has demonstrated good success rates in treating certain cancers. In an effort to improve the curative rates in cancer patients, ongoing research is being conducted to enhance the amount of radiation dose that is absorbed within the tumour volume while minimizing the dose absorbed by the surrounding normal tissue. One method for enhancing tumour dose absorption with 125I brachytherapy seeds is to increase the number of photoelectric atomic interactions within the tumour volume by introducing small quantities of a silver compound, taking advantage of the K-edge effect. Because low-energy electrons and Auger electrons are the primary sources of brachytherapy dose enhancement, acquiring accurate experimental measurements of absorbed dose increases is a major challenge. To circumvent this problem, an x ray fluorescence excitation spectroscopy dosimetry technique supplemented with clinically accepted dosimetry calculations was developed to estimate relative absorbed dose increases in a water phantom containing up to 7.5 mM of silver. Excellent agreement was observed between theoretically derived Monte Carlo dosimetric predictions and experimental measurements. These results successfully demonstrated that K-edge enhanced 125I brachytherapy is indeed possible with future development of a non-toxic silver chelate.

Algorithms↗

PubMed1996

Influence of insulin-like growth factor-binding protein-2 on plasma clearance and transfer of insulin-like growth factors-I and -II from plasma into mammary-derived lymph and milk of goats.

Plasma clearance of insulin-like growth factors-I and -II (IGF-I and -II) and insulin-like growth factor-binding protein-2 (IGFBP-2) from lactating goats (n = 4) was determined following a single intravenous injection of the corresponding 125I-labelled human protein. Transfer of these proteins out of the vascular space was monitored by their subsequent appearance in mammary-derived lymph and milk. Clearance of 125I-IGFBP-2 from circulation was 0.37 +/- 0.06 ml/min/kg, which is markedly greater than that of 125I-IGF-I or -II (0.11 +/- 0.01 and 0.12 +/- 0.01 ml/min/kg respectively). This was also reflected in longer elimination half-lives for IGF-I (353 +/- 6 min) and -II (254 +/- 8 min) compared with IGFBP-2 (110 +/- 9 min). Three hours after injection of the 125I-labelled protein, the plasma:lymph ratio of trichloroacetic acid-precipitable radioactivity was 1.54 +/- 0.04, 3.3 +/- 0.6 and 4.1 +/- 0.4 for IGFBP-2, IGF-I and -II respectively. The form of 125I-IGFBP-2 in lymph was not different from that of plasma. Elevation of plasma concentrations of IGFBP-2 by its intravenous infusion significantly decreased plasma half-life of both IGF-I and -II (251 +/- 8 and 198 +/- 7 min respectively). Although the amount and rate of transfer of IGF into mammary-derived lymph was decreased slightly by IGFBP-2, concentrations eventually obtained were not different from control. However, secretion of IGFs into milk was significantly reduced by IGFBP-2, particularly in the case of IGF-I. These results are consistent with the ability of all three compounds to cross the vascular endothelium intact and of IGFBP-2 to decrease the uptake of IGF by mammary epithelium and subsequent secretion into milk. IGFBP-2 may well have acted to target plasma IGF towards non-mammary tissues, thus explaining the more rapid plasma clearance of IGFs in the presence of elevated IGFBP-2.

Animals↗

PubMed1992

Uptake of tyramine cellobiose by rat liver.

The uptake of 125I-tyramine cellobiose (TC) by isolated rat hepatocytes and by total rat liver is markedly higher than that of 14C-sucrose and 125I-PVP, suggesting that TC does not enter the cells by fluid phase endocytosis. The distribution of radioactivity after differential centrifugation shows that the compound is shared out amongst sedimentable structures and unsedimentable fraction. Analysis by isopycnic centrifugation indicates that quickly after its penetration into the cells, most of sedimentable 125I-TC is associated with lysosomes. Such an intracellular localization is confirmed by the distributions observed after free flow electrophoresis and by the fact that radioactivity and cathepsin C, a lysosomal hydrolase, are simultaneously released from a mitochondrial fraction treated with glycyl-L-phenylalanine-2-naphthylamide. Pretreatment of the rats with chloroquine, an acidotropic drug that accumulates in lysosomes, prevents to some extent the entry of 125I-TC into these organelles. Experiments performed with purified lysosomes show that 14C-sucrose does not cross the lysosomal membrane when 125I-TC accumulates linearly with time in the fractions. These results are explained by supposing that the linkage of tyramine to cellobiose allow the disaccharide to diffuse through the plasma and the lysosome membranes, and that the accumulation of the molecule in these organelles results from its weak basic properties. 125I-TC could be an interesting molecule with which to study acidotropism in the whole animal and in isolated and cultured cells.

Animals↗

PubMed1993

Determination of serum biotinidase activity with biotinyl derivatives of lodotyramines as substrates.

We synthesized biotinylated mono- and di-iodotyramine and their radioactive counterparts and used these substances as substrates to estimate serum biotinidase activity in a radioassay system. The Km values determined for mono- and di-iodobiotinyl derivatives were 15.8 and 25.9 microM, respectively, whereas, the maximum velocities of the enzymatic reaction were 27.0 and 8.7 nmol.min-1.mL-1, respectively. Both substrates competed with biocytin for the same active site of the enzyme and the Ki values were 7.30 and 9.56 microM for the mono- and di-iodinated substrate, respectively. Higher assay sensitivity was obtained using [125I]biotinyl-monoiodotyramine as substrate, and the values obtained were directly related with those determined with the well-established colorimetric method (r = 0.9377, n = 31). However, for routine use, the assay may be accomplished by diluting the radiotracer with biocytin instead of its "cold" counterpart, because it is a commercially available reagent. The values obtained in this case were very well correlated with those determined by the colorimetric assay as well (r = 0.9289, n = 31).

Amidohydrolases↗

PubMed1989

Attenuated processing of epidermal growth factor in the face of marked degradation of transforming growth factor-alpha.

T3M4 human pancreatic carcinoma cells avidly bound and internalized 125I-labeled epidermal growth factor (EGF) but did not readily degrade the ligand. Pulse-chase experiments in which the cell-bound radioactivity was allowed to dissociate into the incubation medium in the presence of unlabeled EGF indicated that the majority of the released 125I-EGF consisted of intact EGF and a slightly processed species that readily bound to the cell. Omission of unlabeled EGF during the chase period markedly decreased the amount of radioactivity in the incubation medium, mainly as a result of the rebinding of EGF to the cells. In contrast, T3M4 cells readily degraded 125I-labeled transforming growth factor-alpha (TGF-alpha), and the released radiolabeled products did not rebind to the cells. Both ligands were released from T3M4 cells under acidic conditions, complete dissociation occurring at a pH of 4.5 for EGF, and a pH of 6.5 for TGF-alpha. A 431 human epidermoid carcinoma cells and ASPC-1 human pancreatic carcinoma cells also failed to extensively degrade 125I-EGF, whereas Rat-1 fibroblasts markedly degraded the growth factor. As in the case of T3M4 cells, ASPC-1 cells extensively degraded 125I-TGF-alpha. Degradation of either ligand was blocked by the lysosomotropic compound methylamine in all the tested cell lines. Immunoprecipitation of the EGF receptor with specific polyclonal antibodies and Western blot analysis revealed the anticipated 170-kDa protein in T3M4 cells. Both EGF and TGF-alpha enhanced EGF receptor degradation, but TGF-alpha was less effective than EGF. These findings indicate that in certain cell types EGF and TGF-alpha may be differentially processed.

Animals↗