phosphorus-radioisotopes and Carcinoma--Squamous-Cell

The efficacy of DNA-targeting radionuclide therapies might be strongly enhanced by employing short range particle-emitters. However, the gain of effectiveness is not yet well substantiated. We compared the Auger electron emitter I-125 to the ß. TFO were labeled with P-32 or I-125 using the primer extension method. Cell survival was analyzed by colony-forming assay and DNA damage was assessed by microscopic quantification of protein 53 binding protein 1 (53BP1) foci in SCL-II cells.. I-125-TFO induced a pronounced decrease of cell survival (D. I-125-TFO proved to be much more radiotoxic than P-32-TFO per decay and per unit dose although targeting the same sequence in the GAPDH gene. This might be well explained by the high number of low energy Auger electrons emitted by I-125 per decay, leading to a high ionization density in the immediate vicinity of the decay site, probably producing highly complex DNA lesions overcharging DNA repair mechanisms.

Topics: Apoptosis; Carcinoma, Squamous Cell; Cell Line, Tumor; DNA; DNA Damage; Humans; Iodine Radioisotopes; Male; Phosphorus Radioisotopes; Radiopharmaceuticals; Radiotherapy Dosage; Treatment Outcome

Adjunct treatments for conjunctival malignancies are needed when standard therapy provides limited benefits or fails.. To describe the results of patients with diffuse conjunctival neoplasms treated with radioactive phosphorus 32 (32P)-impregnated flexible film.. This retrospective case series between January 1, 2010, and January 1, 2013, was conducted at Memorial Sloan-Kettering Cancer Center, a tertiary referral center. The study was conducted on 7 eyes of 6 patients treated for diffuse conjunctival squamous cell carcinoma, sebaceous carcinoma, or lymphoma that had recurrent or residual disease after primary treatment.. Patients underwent mapping biopsies and detailed conjunctival drawings to delineate the pathologic extent of the disease. The brachytherapy film used for treatment was the RIC Conformal Source Model 100 (RIC-100, RI Consultants). The RIC-100 is a flexible, thin (approximately 0.5-mm) film made of a polymer chemically bound to 32P. The radioactive 32P film was placed intraoperatively, allowed to stay in place until the prescription dose was reached, and then removed. The median dose at the prescription point (1 mm from the surface of the film) was 15 Gy (range, 5-17 Gy).. Patients were tested for best-corrected visual acuity, recurrence-free survival, and adverse events scored by using the Adult Comorbidity Evaluation-27 scale.. Between 2010 and 2013, 7 eyes of 6 patients were treated. The median age of patients was 70 years. All patients had a recurrent or persistent neoplasm. Four patients with squamous cell carcinoma, 1 with sebaceous carcinoma, and 1 with metachronous bilateral lymphomas were treated. The median treatment time was 19 minutes (range, 10-52 minutes). The median follow-up was 24.9 months (range, 3.1-38.2 months). Recurrence-free survival 24 months after brachytherapy was 75% (95% CI, 19-89.1). Two moderate adverse events and 1 severe adverse event occurred. Visual acuity was stable or improved in 5 of the 7 eyes (ie, better than 20/70 in the 5 patients who retained their treated eye).. Our results show the use of an intraoperative high-dose rate of 32P brachytherapy in selected cases of recalcitrant diffuse conjunctival neoplasms. This technique offers a novel adjunct in the treatment of these cancers. Further follow-up and study are warranted.

Topics: Adenocarcinoma, Sebaceous; Aged; Aged, 80 and over; Brachytherapy; Carcinoma, Squamous Cell; Conjunctival Neoplasms; Disease-Free Survival; Female; Humans; Intraoperative Care; Lymphoma, T-Cell; Male; Middle Aged; Phosphorus Radioisotopes; Radiotherapy Dosage; Radiotherapy, Adjuvant; Retrospective Studies; Visual Acuity

To study whether the main malondialdehyde-DNA adduct (M1-dG) produced by lipid peroxidation is involved in the carcinogenesis of esophagus.. DNA samples were isolated from normal esophageal epithelium (n = 32) obtained by biopsy and esophageal squamous cell carcinoma specimens (n = 30) obtained by surgery. All tissue samples came from individuals living in Linxian, Henan, a high-risk area of esophageal cancer. Contents of M1-dG adducts were detected by 32P-postlabeling method.. M1-dG adducts were detectable both in the normal and cancerous tissue samples. However, normal esophageal epithelial tissues exhibited significantly lower levels of M1-dG adducts (median 3.4, range 1.7/10(8)-55.4/10(8) nucleotides) than those found in esophageal cancer tissues (median 14.1, range 1.4/10(8)-59.0/10(8) nucleotides, P < 0.0001). The adduct levels were neither associated with gender, age, tobacco smoking status or genetic polymorphism in the CYP2E1, an enzyme participating in the oxidation of ethanol to form reactive free radicals.. Our findings provide evidence that DNA damage, resulted from lipid peroxidation, can accumulate in the normal human esophageal tissue and reach relatively high level in cancer tissue which suggests that M1-dG adducts may be involved in the initiation and progression of cancer with its mutagenic and carcinogenic effects.

Topics: Carcinoma, Squamous Cell; Cytochrome P-450 CYP2E1; DNA Adducts; DNA, Neoplasm; Epithelium; Esophageal Neoplasms; Esophagus; Humans; Isotope Labeling; Malondialdehyde; Phosphorus Radioisotopes

This study investigated the role of histologic tumor characteristics, in comparison with a normal tissue, and of tumor vascularization on the uptake and retention of colloidal 32P used in infusional brachytherapy of solid cancers. The cytotoxicity of colloidal 32P was also evaluated for two tumors of different radiosensitivity, a melanoma, and a squamous cell carcinoma.. An in vitro analysis of colloidal 32P uptake was carried out on a human melanoma cell line, HBL, a human squamous cell carcinoma, SCC1, and normal fibroblasts, F-NBB. Tumor retention of colloidal 32P was studied in vivo for the HBL and the SCC1 tumors implanted subcutaneously in nude mice. Tumor vascular density was determined by microscopic study of Masson's trichrome slides of HBL and SCC1 tumors of about 1 cm diameter.. In vitro studies showed that the time required for maximal cell uptake of colloidal 32P was only 10-20 min for the SCC1 and HBL tumors, while it took at least 60 min for the fibroblasts. After intratumoral injection of macroaggregated albumin (MAA), followed by 50 microCi of colloidal 32P, Bremsstrahlung imaging performed at 6 and 24 h showed that the activity remained in the HBL tumor while some of the radiocolloids leaked from the SCC1 tumor and was trapped in the reticuloendothelial system of the liver. Organ activity counting confirmed this finding: 32P activity was three to four times higher in the HBL than in the SCC1 tumor, whereas the activity in the liver, insignificant in the HBL mice (less than 0.1 microCi/g), was as high as 24 microCi/g in the SCC1 mice. This phenomenon may be explained by the difference in tumor vascular density, estimated for the HBL to be about four times less than that of the SCC1 tumor (5.7 vs. 21.4 blood vessels per mm2 for the HBL and the SCC1 tumors, respectively).. Intratumoral infusion of colloidal 32P may be a useful complement of radiation therapy in the treatment of nonresectable but accessible solid tumors. Tumor vascularization must be taken into account for a successful vascular blockade by MAA prior to the infusion of colloidal 32P.

Topics: Animals; Carcinoma, Squamous Cell; Colloids; Dose-Response Relationship, Radiation; Humans; Male; Melanoma; Mice; Mice, Nude; Phosphorus Radioisotopes; Serum Albumin; Tumor Cells, Cultured

The binding of epidermal growth factor (EGF) to epidermal growth factor receptor (EGF receptor) results in the dimerization and self-phosphorylation of the receptor. Both of these responses were followed as a function of time and the concentration of EGF receptor. Dimerization of EGF receptor was monitored by immunoblotting the protein after it had been cross-linked with glutaraldehyde. The capacity for self-phosphorylation was followed by measuring the relative level of incorporation of [32P]phosphate into EGF receptor on autoradiograms of the same immunoblots used for the assay of its dimerization. When these two properties were followed as a function of time, it was found that dimerization preceded the appearance of the capacity for self-phosphorylation. Both dimeric and monomeric forms of EGF receptor were self-phosphorylated in the presence of EGF, but the dimeric form was phosphorylated preferentially to the monomeric form. When the dimerization and the capacity for self-phosphorylation were followed as a function of the concentration of dimeric EGF receptor, it was observed that the self-phosphorylation of dimeric EGF receptor increased as the concentration of dimeric EGF receptor increased. An equation including terms representing both intramolecular and intermolecular rates of self-phosphorylation was fit to the plots of self-phosphorylation as a function of concentration of EGF receptor. These fits demonstrate that intramolecular self-phosphorylation within dimers of EGF receptor is insignificant and that self-phosphorylation is an intermolecular process between dimers of EGF receptor.

Topics: Adenosine Triphosphate; Carcinoma, Squamous Cell; Cross-Linking Reagents; Dimerization; Epidermal Growth Factor; ErbB Receptors; Glutaral; Humans; Kinetics; Phosphorus Radioisotopes; Phosphorylation; Radioisotope Dilution Technique; Tumor Cells, Cultured

The physiological and therapeutic effects of the bioreductive agent RSU1069 (80 mg/kg i.p.) and its prodrug RB6145 (240 mg/kg i.p.) were investigated in the SCCVII tumour. Using laser Doppler flowmetry it was found that RSU1069 produced a significant 30% reduction in tumour blood flow 30 min after administration, while RB6145 had no effect. Tumour oxygenation, measured with an Eppendorf oxygen electrode, was unchanged by either agent except for a reduction in values less than 2.5 mmHg at 30 min after injection. Neither agent significantly altered tumour energy metabolism, assessed by 31P magnetic resonance spectroscopy. Both agents significantly increased tumour glucose content by a factor of 1.6-1.7 at 30 min after injection, but had no effect on glucose-6-phosphate or lactate levels. Tumour growth was significantly delayed by heating (42.5 degrees C, 60 min), and although neither RSU1069 nor RB6145 alone had any effect on tumour growth they produced a similar enhancement of the tumour response to heat. The therapeutic effects are consistent with the known conversion in vivo of one third of the pro-drug RB6145 to its active product RSU1069, however the physiological effects of the two agents in the SCCVII tumour are not identical.

Topics: Animals; Antineoplastic Agents; Carcinoma, Squamous Cell; Disease Models, Animal; Female; Magnetic Resonance Spectroscopy; Male; Mice; Mice, Inbred C3H; Misonidazole; Neovascularization, Pathologic; Nitroimidazoles; Oxygen; Phosphorus Radioisotopes

We have previously shown that the 180-kD bullous pemphigoid antigen (BPAII), which is a transmembrane collagenous protein of hemidesmosomes, is distributed at adhesion sites on glass coverslips on the basal membrane forming a concentric ring, or arch pattern, in a human squamous cell carcinoma cell line (DJM-1), when studied by immunofluorescence microscopy using monoclonal antibodies to BPA II. This concentric ring/arch pattern of "footsteps" of BPA II has been shown to be collapsed in association with a transient activation of protein kinase C by treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA). In the present study, therefore, the effects of TPA on the phosphorylation of BPA II was examined. DJM-1 cells, which were metabolically labelled with [32Pi], were lysed and the extracts were subjected to immunoprecipitation with anti-BPAII and anti-230 kDa bullous pemphigoid antigen (BPAI) monoclonal antibodies. The results showed that only BPA II, but not BPA I, was phosphorylated at serine residues before TPA treatment. After TPA treatment phosphorylation was prominently increased so as to generate a 190 kDa-phosphorylated peptide. This 190-kDa peptide was reacted with anti-BPA II monoclonal antibodies by immunoblotting, and it was not detected when cells were pretreated with a specific protein kinase C inhibitor (H7) before TPA treatment, suggesting that the 190 kDa peptide is phosphorylated BPAII with TPA. Prolonged treatment with TPA abolished both of 180- and 190-kDa BPA II from Triton X-100-soluble fractions. These findings suggest that the BPA II, but not BPA I, is a substrate of protein kinase C, and the generation of 190-kDa-phosphorylated BPA II has a key role in the TPA-induced collapse of the assembly of BPA II on the basal plasma membrane, probably, at hemidesmosomes.

Topics: Amino Acids; Animals; Antibodies, Monoclonal; Autoantigens; Autoradiography; Blotting, Western; Carcinoma, Squamous Cell; Carrier Proteins; Cattle; Cell Adhesion; Collagen; Collagen Type XVII; Cornea; Cytoskeletal Proteins; Desmosomes; Dystonin; Epithelial Cells; Epithelium; Humans; Nerve Tissue Proteins; Non-Fibrillar Collagens; Pemphigoid, Bullous; Phosphorus Radioisotopes; Phosphorylation; Precipitin Tests; Skin Neoplasms; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

In squamous cell carcinomas (SCCs), the tumor suppressor protein p53 is frequently overexpressed. The overexpression of p53 is often due to a mutation in the p53 gene; however, increased levels of p53 protein can be observed in the tumors without p53 gene mutations. In normal human keratinocytes, p53 is a multiconformational protein. The different conformations of p53 can be identified by their reactivity with epitope-specific, anti-p53 monoclonal antibodies. This study provides evidence that the different p53 conformations seen in human keratinocytes bind to distinct cellular proteins. Proteins that bind p53 in normal human keratinocytes were compared with p53-binding proteins from cells derived from SCC tumors by immunoprecipitation of [35S]methionine-labeled and 32P(i)-labeled cell lysates using a panel of anti-p53 monoclonal antibodies. In one tumor, the SCC cells contained a protein of Mr 30,000 bound to p53 that was not seen in normal human keratinocytes. Cells derived from a separate SCC did not have the Mr 30,000 protein but did contain two proteins of Mr 15,000 and Mr 16,000, which were not seen in normal human keratinocytes. The immunofluorescent staining pattern of cultured normal human keratinocytes, cells derived from two SCCs, as well as the original tumors from which the cells were derived, was also examined. The immunofluorescent staining of the cells derived from the tumors and the tumors themselves was different from that seen in normal cultured keratinocytes and normal epidermis. These studies suggest that there are alterations in the proteins that bind to p53 in SCCs.

Topics: Carcinoma, Squamous Cell; Carrier Proteins; Cells, Cultured; Electrophoresis, Polyacrylamide Gel; Fluorescent Antibody Technique; Humans; Infant, Newborn; Keratinocytes; Male; Methionine; Molecular Weight; Phosphates; Phosphorus Radioisotopes; Reference Values; Skin; Skin Neoplasms; Sulfur Radioisotopes; Tumor Suppressor Protein p53

Samples of clinically normal oral tissue were obtained from 17 tobacco smokers and 7 non- or ex-smokers undergoing surgery for intra-oral squamous cell carcinoma. Isolated DNA was analysed for the presence of aromatic DNA adducts using the 32P-postlabelling technique with adduct enhancement by either butanol extraction or nuclease P1 enrichment. DNA adduction detected following butanol extraction was more diverse and at a higher level than obtained with the P1 method. Adduct levels in the smokers and non- or ex-smokers were 1163 +/- 375 and 774 +/- 318 amol/micrograms, respectively. This difference is statistically significant (p < 0.05). The differential enhancement of adducts with the two protocols suggested that arylamines may be the source of at least a proportion of the DNA adduction detected. These data indicate that oral tissues are likely to be a suitable source of material for human biomonitoring and furthermore they highlight the importance of utilizing more than one enhancement procedure when examining DNA adduction induced by complex mixtures such as tobacco smoke or those encountered at industrial plants.

Topics: Butanols; Carcinoma, Squamous Cell; DNA; DNA Damage; DNA, Neoplasm; Environmental Monitoring; Evaluation Studies as Topic; Humans; Mouth Mucosa; Mouth Neoplasms; Phosphorus Radioisotopes; Single-Strand Specific DNA and RNA Endonucleases; Smoking

DNA from human brain tumor samples was analysed by the 32P-postlabeling technique for the presence of aromatic DNA adducts. Thirteen out of 16 samples showed low levels of adducts at 0.14-3.53 adducts per 10(9) nucleotides. Inter-individual variations in the patterns of these aromatic adducts were observed. On the other hand, none of 5 brain samples from epilepsy patients revealed any evidence of such adducts. The data demonstrated the presence of low level, large molecule aromatic DNA adducts in malignant brain tissues and these adducts may either result from environmental exposure to an undetermined genotoxic agent or from the aging process.

Topics: Adenoma; Adult; Aged; Brain Chemistry; Brain Neoplasms; Carcinogens; Carcinoma, Squamous Cell; Chromatography, Thin Layer; Cytochrome P-450 Enzyme System; DNA Damage; DNA, Neoplasm; Epilepsy; Female; Glioma; Humans; Male; Meningioma; Middle Aged; Neurilemmoma; Phosphorus Radioisotopes; Pituitary Neoplasms; Polycyclic Compounds

Samples of clinically normal oral tissue were obtained from patients undergoing surgery for intraoral squamous cell carcinoma. DNA was extracted from samples obtained from 20 tobacco smokers, four exsmokers, and nine nonsmokers and analyzed for the presence of aromatic DNA adducts using two distinct modifications of the 32P postlabeling assay. 32P postlabeling following butanol extraction enhancement revealed a much wider range and substantially higher levels of DNA adducts than obtained following nuclease P1 enrichment. Adduct levels in smokers, exsmokers, and nonsmokers were 1133 +/- 354, 785 +/- 251, and 660 +/- 317 amol/microgram of DNA (+/- SD), respectively. The elevation of adduct levels in smokers compared with either nonsmokers or non- and exsmokers combined is statistically significant (P < 0.005). These observations are consistent with epidemiological evidence linking tobacco smoking with oral cancer. The differential enhancement of DNA adducts with the two 32P postlabeling protocols indicate that aromatic amines and nitroaromatics may be important sources of the DNA adducts detected in human oral tissue.

Topics: Adolescent; Adult; Age Factors; Aged; Aged, 80 and over; Alcohol Drinking; Butanols; Carcinoma, Squamous Cell; DNA; DNA Damage; Female; Humans; Male; Middle Aged; Mouth; Mouth Neoplasms; Phosphorus Radioisotopes; Single-Strand Specific DNA and RNA Endonucleases; Smoking

Topics: Adenosine Triphosphate; Amino Acid Sequence; Annexins; Calcium-Binding Proteins; Carcinoma, Squamous Cell; Cell Line; Cyanogen Bromide; Electrophoresis, Polyacrylamide Gel; ErbB Receptors; Glycoproteins; Humans; Methionine; Molecular Sequence Data; Peptide Mapping; Phosphates; Phosphopeptides; Phosphorus Radioisotopes; Phosphorylation; Protein-Tyrosine Kinases; Radioisotope Dilution Technique; Trypsin

Topics: Amino Acid Sequence; Carcinoma, Squamous Cell; Cell Line; Chromatography, High Pressure Liquid; ErbB Receptors; Humans; Molecular Sequence Data; Peptide Fragments; Phosphates; Phosphopeptides; Phosphorus Radioisotopes; Phosphorylation; Protein Kinases; Radioisotope Dilution Technique

Intact A431 cells were labeled with [gamma-32P]ATP. The major phosphorylation product of the ecto-kinase activity of A431 cells had the molecular mass of 170 kd and was identified as EGF receptor by specific immunoprecipitation. This phosphorylation was not stimulated by EGF added to the reaction buffer, but replacement of MgCl2 by MnCl2 in the buffer remarkably stimulated phosphorylation. An exogenous protein substrate, alpha-casein, was also phosphorylated by intact A431 cells. The analyses for phospho-amino acids of both EGF receptor and alpha-casein revealed that phosphorylation occurred mainly at phosphotyrosine residues. Tryptic phospho-peptides of the EGF receptor of intact A431 cells labeled with [gamma-32P]ATP were fractionated by HPLC. The elution patterns were essentially the same as that of the autophosphorylated EGF receptor, indicating that the phosphorylation sites of EGF receptor labeled in vivo with [gamma-32P]ATP are located in three tyrosine residues in the carboxyl terminus. These results indicate that the carboxyl-terminal tyrosine kinase domain of a small fraction of the EGF receptor molecules of an A431 cell is exposed on the outer surface of the cells.

Topics: Adenosine Triphosphate; Carcinoma, Squamous Cell; Caseins; Chromatography, High Pressure Liquid; ErbB Receptors; Humans; Molecular Structure; Phosphorus Radioisotopes; Phosphorylation; Precipitin Tests; Protein-Tyrosine Kinases; Tumor Cells, Cultured

Recent investigations have identified a signal-transduction system involving sphingomyelin and derivatives. In this paradigm, sphingomyelin hydrolysis by a sphingomyelinase generates ceramide, which may be converted to the protein kinase C inhibitor sphingosine or to ceramide 1-phosphate. Ceramide may have second-messenger function because it induces epidermal growth factor receptor phosphorylation, presumably on Thr-669 in A-431 cells. The present studies describe a kinase that may mediate ceramide action. With a 19-amino acid epidermal growth factor receptor peptide containing Thr-669, a membrane-bound activity that phosphorylated the peptide was detected in A-431 cells. Activity was linearly related to ATP (0.3-300 microM) and peptide concentration (0.02-1 mg/ml), possessed a physiologic pH optimum (pH 7.0-7.4), and was Mg(2+)-dependent. Other cations--Ca2+, Mn2+, and Zn(2+)--were ineffective. Natural and synthetic ceramide induced time- and concentration-dependent enhancement of kinase activity. Ceramide (0.5 microM) increased kinase activity 2-fold by 30 s, and activity remained elevated for at least 15 min. As little as 0.001 microM ceramide was effective, and 1 microM ceramide induced maximal phosphorylation. Sphingosine was similarly effective. Because tumor necrosis factor (TNF) alpha rapidly induces sphingomyelin hydrolysis to ceramide during monocytic differentiation of HL-60 cells, its effects on kinase activity were assessed. Kinase activity was increased 1.5-fold at 5 min and 2-fold at 2 hr in membranes derived from TNF-stimulated cells. The effective concentration range was 3 pM-30 nM TNF. Exogenous ceramide induced a similar effect. In sum, these studies demonstrate the existence of an unusual Mg(2+)-dependent ceramide-activated protein kinase that may mediate some aspects of TNF-alpha function.

Topics: Amino Acid Sequence; Carcinoma, Squamous Cell; Cell Line; Cell Membrane; Ceramides; ErbB Receptors; Humans; Kinetics; Leukemia, Promyelocytic, Acute; Magnesium; Molecular Sequence Data; Peptides; Phosphates; Phosphopeptides; Phosphorus Radioisotopes; Protein Kinases; Sphingosine; Tumor Necrosis Factor-alpha

Production of PtdIns(4)P and PtdIns(4,5)P2 by plasma-membrane preparations from A431 cells was selectively stimulated in a dose-dependent manner by epidermal growth factor (EGF) in the presence of Na3VO4. Na3VO4 itself mimicked this effect, which was overcome after treatment by a specific phosphotyrosyl phosphatase isolated from A431 cells. PtdIns and PtdIns(4)P kinase activities were present in phosphotyrosyl-proteins isolated from EGF- and/or Na3VO4-stimulated A431 cells by immunoaffinity using an anti-phosphotyrosine antibody. These data suggest for the first time an EGF-dependent regulation of PtdIns 4-kinase and PtdIns(4)P 5-kinase activities by a mechanism involving a tyrosine-phosphorylation process.

Topics: 1-Phosphatidylinositol 4-Kinase; Adenosine Triphosphate; Carcinoma, Squamous Cell; Cell Line; Chromatography, High Pressure Liquid; Humans; Kinetics; Membrane Proteins; Molecular Weight; Neoplasm Proteins; Phosphatidylinositols; Phosphoprotein Phosphatases; Phosphoproteins; Phosphorus Radioisotopes; Phosphorylation; Phosphotransferases; Protein Tyrosine Phosphatases; Protein-Tyrosine Kinases

Transforming growth factor-alpha (TGF-alpha) stimulates (in a dose-dependent manner) the incorporation of [32P]Pi into phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP), phosphatidylinositol 4,5-bisphosphate (PIP2), and phosphatidic acid (PA) in the human epidermoid carcinoma cell line (A431). The effect of TGF-alpha on the incorporation was found to be similar to that of EGF. On the other hand, a striking difference in the activation of diacylglycerol (DG) kinase activity was seen between TGF-alpha and EGF. At least 100 times more TGF-alpha was required to achieve maximal stimulation of DG kinase activity relative to EGF. These results suggest that the activation of DG kinase by TGF-alpha may involve a mechanism independent from or subsequent to activation of the EGF receptor.

Topics: Carcinoma, Squamous Cell; Cell Line; Growth Substances; Humans; Kinetics; Peptides; Phosphates; Phosphatidic Acids; Phosphatidylinositols; Phosphorus Radioisotopes; Transforming Growth Factors

Three biologically active monoclonal antibodies against the human epidermal growth factor (EGF) receptor (2E9, 2D11 and 2G5) have been used to analyse the interrelationship between various cellular responses to EGF. Antibody 2E9 (IgG1) is directed against the protein core of the receptor, close to or at the EGF binding site, while 2D11 (IgG3) and 2G5 (IgG2a) recognize blood-group A-related carbohydrate determinants of the receptor. These antibodies have EGF-like effects in that they can activate the receptor tyrosine kinase both in vitro and in vivo. Cross-linking of the receptor-bound antibodies by a second antibody mimics EGF in inducing a rapid aggregation of receptors on the cell surface. However, all three antibodies fail to mimic EGF in raising cytoplasmic pH and free Ca2+ and do not stimulate DNA synthesis in quiescent fibroblasts, even after external cross-linking of the occupied receptors. It is concluded that EGF-R tyrosine kinase activity as well as substrate specificity can be modulated by ligands other than EGF, even if they bind to sites distinct from the EGF binding domain; activation of the receptor tyrosine kinase, receptor clustering and induction of the ionic signals are causally unrelated events; and tyrosine kinase activation and receptor cross-linking are not sufficient for stimulation of DNA synthesis.

Topics: Amino Acids; Antibodies, Monoclonal; Antigen-Antibody Complex; Carcinoma, Squamous Cell; Cell Line; DNA Replication; Epidermal Growth Factor; ErbB Receptors; HeLa Cells; Humans; Phosphorus Radioisotopes; Phosphorylation; Receptors, Cell Surface

This study investigated a possible role for the epidermal growth factor (EGF) receptor protein tyrosine kinase in phosphoinositide metabolism with plasma membrane vesicles from human epidermoid carcinoma (A431) cells. We found a novel chloroform-soluble product radiolabeled with [gamma-32P]ATP that did not migrate from the origin in the thin layer system designed to separate the phosphoinositides, appeared as a single band of Mr = 3500 on polyacrylamide gels in the presence of dodecyl sulfate, had an ultraviolet absorbance spectrum with a maximum at 275 nm and stained with Coomassie dye. Based on these properties this phosphorylation product is referred to as a proteolipid. The 32P label was not detected in phosphotyrosine [Tyr(P)], phosphoserine [Ser(P)] or phosphothreonine [Thr(P)] and was lost during acid or base hydrolysis. Phosphorylation of proteolipid was increased significantly by EGF, whereas phosphorylation of phosphatidic acid was decreased and labeling of phosphoinositides was unaffected. Thus, it appears that in A431 membranes the EGF receptor/kinase does not utilize phosphatidylinositol as a substrate, but does phosphorylate a membrane proteolipid.

Topics: Adenosine Triphosphate; Carcinoma, Squamous Cell; Cell Line; Cell Membrane; Chloroform; Chromatography, Thin Layer; Epidermal Growth Factor; Humans; Kinetics; Membrane Lipids; Phosphatidylinositols; Phosphorus Radioisotopes; Phosphorylation; Solubility

We have isolated and partially purified an intracellular vesicle fraction from A-431 cells that contains both epidermal growth factor (EGF) and enzymatically active EGF:receptor/kinase. Exposure of intact A-431 cells to EGF leads to an accumulation of both EGF and kinase activity in this vesicle fraction. The accumulation is time- and temperature-dependent and is blocked by inhibitors of energy production. The EGF receptor in internalized vesicles is capable of autophosphorylation and, in the presence of Ca2+, of phosphorylation of the previously isolated 35-kDa protein (Fava, R. A., and Cohen, S. (1984) J. Biol. Chem. 259, 2636-2645). The demonstration of an EGF-induced increase in kinase activity of an internalized vesicle fraction lends credence to the hypothesis that EGF-induced endocytosis of the receptor is of physiological significance in the response of cells to this ligand. In addition, these results are consistent with the suggestion that the phosphorylation of the 35-kDa protein is associated with internalization of the EGF:receptor/kinase complex.

Topics: Carcinoma, Squamous Cell; Cell Line; Cell Membrane; Electrophoresis, Polyacrylamide Gel; Epidermal Growth Factor; ErbB Receptors; Humans; Kinetics; Molecular Weight; Peptide Fragments; Phosphorus Radioisotopes; Phosphorylation; Protein Kinases; Trypsin

Incubation of membranes prepared from A431 cells with either epidermal growth factor (EGF) or phorbol 12-myristate 13-acetate (PMA) stimulates the transfer of 32phosphate from [gamma-32P]ATP into 8-10 membrane proteins. The major phosphorylated protein migrates on NaDodSO4/polyacrylamide gels with an apparent Mr of 180,000, corresponding to the previously identified EGF receptor. Stimulation of EGF receptor phosphorylation by PMA does not require Ca2+, suggesting that prior activation of protein kinase C is not a prerequisite for phosphate transfer. PMA-enhanced phosphorylation proceeds at 4 degrees C and requires Mn2+, both properties of tyrosine-specific protein kinases. Phospho amino acid analysis of the Mr 180,000 receptor band shows that only tyrosine residues are phosphorylated when A431 membranes are treated with either EGF or PMA. Moreover, proteolysis reveals that these residues are located in the same peptides of the receptor. These results demonstrate that a potent tumor-promoting phorbol ester can mimic a critical early response usually elicited by EGF.

Topics: Adenosine Triphosphate; Carcinoma, Squamous Cell; Cell Line; Cell Membrane; Epidermal Growth Factor; ErbB Receptors; Humans; Kinetics; Molecular Weight; Peptide Fragments; Phorbols; Phosphorus Radioisotopes; Phosphorylation; Receptors, Cell Surface; Tetradecanoylphorbol Acetate; Tyrosine

Biodistribution and tumor uptake studies were carried out with intravenously injected tracer doses of Gpp(NH)p labeled with 3H, 32P or 99mTc . Syrian golden hamsters with cheek pouch carcinomas, induced by repeated topical applications of DMBA, were used as a tumor model. The biodistributions of these three radionuclides were different, indicating significant molecular cleavage of this nucleotide analog. It was also apparent that this compound labeled with 99mTc may not be useful for tumor imaging due to low tumor-to-blood specific activity ratio. The cheek pouch carcinoma tumor model may be valuable for the evaluation of tumor localizing radiopharmaceuticals.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Carcinoma, Squamous Cell; Cricetinae; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Male; Mesocricetus; Mouth Neoplasms; Organotechnetium Compounds; Phosphorus Radioisotopes; Technetium; Tissue Distribution; Tritium

Topics: Animals; Carcinoma, Squamous Cell; Cell Line; Cell Membrane; Epidermal Growth Factor; ErbB Receptors; Humans; Mice; Molecular Weight; Phosphorus Radioisotopes; Phosphorylation; Phosphotyrosine; Protein Kinases; Protein-Tyrosine Kinases; Radioisotope Dilution Technique; Receptors, Cell Surface; Tyrosine

Chromic colloidal phosphate labeled with 32P, which has been proposed for the treatment of several articular diseases, was injected intra-articularly in the knee joint of adult Wistar rats. After a 270 days minimum latent period, tumors began to appear in the injected zone, to a 70% frequency. Ten lung metastases were detected. In five cases, squamous cell carcinomas were induced in the injected area. The relevance of a sound evaluation of the risk involved in treatments with radioactive isotopes, is discussed.

Topics: Animals; Carcinoma, Squamous Cell; Hindlimb; Injections, Intra-Articular; Joint Diseases; Joints; Lung Neoplasms; Male; Neoplasms, Experimental; Neoplasms, Radiation-Induced; Osteosarcoma; Phosphorus Radioisotopes; Rats; Rats, Inbred Strains

The authors have determined the distribution of 32P in normal skin and in the case of certain skin tumours (basalioma and spinalioma). In both groups a lognormal distribution was found. It may be assumed that the enrichment is governed by similar distribution in the case of melanoma malignum.

Topics: Carcinoma, Basal Cell; Carcinoma, Squamous Cell; Humans; Mathematics; Melanoma; Phosphorus Radioisotopes; Skin; Skin Neoplasms

In 21 patients with a variety of skin tumors (squamous cell carcinomas, malignant melanomas, basal cell epitheliomas and mycosis fungoides) or pre-cancerous lesions (Bowen's disease, actinic keratosis, junctional nevus cell nevus) the radioactive phosphorus uptake test demonstrates a significantly increased concentration of P32 in those tumors. There were no false negative tests. The possibility of differentiation of malignant melanoma from benign nevus cell nevus and the early recognition of cutaneous metastases is described. Furthermore recurrence of previously irradiated or excised basal cell epitheliomas can be detected without a biopsy. No hematological side-effects were observed.

Topics: Aged; Bowen's Disease; Carcinoma, Basal Cell; Carcinoma, Squamous Cell; Diagnosis, Differential; False Negative Reactions; Female; Humans; Keratosis; Male; Melanoma; Middle Aged; Mycosis Fungoides; Neoplasm Metastasis; Neoplasm Recurrence, Local; Nevus, Pigmented; Phosphorus; Phosphorus Radioisotopes; Precancerous Conditions; Radionuclide Imaging; Skin Neoplasms

A particles with the diameter of 70 to 80 nm were isolated from the cytoplasm of HEp-2, HeLa, and AO cells producing oncornavirus of Mason-Pfizer-like type. Most of the A particles banded at 1.23 to 1.24 g/ml, whereas 3 to 10% banded at 1.29 g/ml in equilibrium sucrose gradients. They banded at 1.30 g/ml in CsCl gradients suggesting that they contained 8% RNA. Individual A particles sedimented at 200 to 250S in velocity sucrose gradients, but their significant part was found aggregated and sedimented at more than 300S. They were resistant to RNase digestion. A particles possessed polymerase activity which was preferentially activated by Mn(2+) rather than by Mg(2+), the RNA template being 60S RNA. Cross-hybridization with two DNA products and immunoassay showed that A particles and Mason-Pfizer-like oncornavirus produced by the same cells contained neither homological RNA sequences nor common antigens, suggesting that A particles are not intracellular precursors of Mason-Pfizer-like oncornavirus but represent an independent oncornavirus. Hybridization of A particle RNA with excess of cellular DNA revealed about 20 proviral copies per HEp-2 cell genome and no proviral copies in human embryo and placenta cell genomes.

Topics: Antigens, Viral; Carcinoma, Squamous Cell; Cell Fractionation; Cell Line; Cells, Cultured; Centrifugation, Density Gradient; Cytoplasm; DNA, Viral; HeLa Cells; Humans; Immunodiffusion; Inclusion Bodies, Viral; Laryngeal Neoplasms; Lung; Microscopy, Electron; Nucleic Acid Hybridization; Oncogenic Viruses; Phosphorus Radioisotopes; RNA-Directed DNA Polymerase; RNA, Viral; Templates, Genetic; Tritium; Uridine

Topics: Carcinoma, Squamous Cell; Cell Line; Centrifugation, Density Gradient; Dactinomycin; DNA, Viral; HeLa Cells; Humans; Kinetics; Laryngeal Neoplasms; Microscopy, Electron; Nucleic Acid Hybridization; Oncogenic Viruses; Phosphorus Radioisotopes; RNA Viruses; RNA-Directed DNA Polymerase; Templates, Genetic; Tritium; Ultracentrifugation; Uridine; Virus Replication

Topics: Adenosine Triphosphatases; Adenylyl Cyclases; Amidohydrolases; Animals; Carcinoma; Carcinoma, Squamous Cell; Cell Line; Cell Membrane; Clone Cells; Female; Fibroblasts; Guinea Pigs; HeLa Cells; Humans; Kinetics; Laryngeal Neoplasms; Liver; Mice; Naphthols; Nasopharyngeal Neoplasms; Neuroblastoma; Nucleotidases; Phosphoric Diester Hydrolases; Phosphoric Monoester Hydrolases; Phosphorus Radioisotopes; Pyrophosphatases; Uterine Cervical Neoplasms

Topics: Amnion; Carbon Radioisotopes; Carcinoma, Squamous Cell; Cell Line; Centrifugation, Density Gradient; Distemper Virus, Canine; Electrophoresis, Polyacrylamide Gel; Humans; Laryngeal Neoplasms; Measles virus; Microscopy, Electron; Molecular Weight; Nucleoproteins; Peptides; Phosphorus Radioisotopes; RNA, Viral; Spectrophotometry, Ultraviolet; Tritium; Viral Proteins

Topics: Amino Acids; Animals; Carbon Radioisotopes; Carcinoma, Squamous Cell; Cell Line; Chick Embryo; Cycloheximide; Dactinomycin; Depression, Chemical; Diphtheria Toxin; Fibroblasts; Insulin; L Cells; Laryngeal Neoplasms; Methionine; Molecular Weight; Phosphorus Radioisotopes; Protein Biosynthesis; Puromycin; RNA; Stimulation, Chemical; Tritium; Uracil Nucleotides; Uridine

Topics: Animals; Carcinoma, Squamous Cell; Fibrosarcoma; Mice; Neoplasms, Experimental; Phosphorus; Phosphorus Radioisotopes; Sarcoma, Experimental; Skin; Skin Neoplasms; Strontium; Strontium Radioisotopes