Development of monoclonal antibodies recognizing 7-(2-hydroxyethyl)guanine and imidazole ring-opened 7-(2-hydroxyethyl)guanine

7-(2-Hydroxyethyl)guanine (7HEG) is of biological interest becauseit is formed in vivo by reaction of DNA with ethylene oxide(EO). Furthermore, the major DNA adduct of vinyl chloride, 7-(2-oxyethyl)guanine,can be converted to this adduct by reduction. Two monoclonalantibodies (9E2, 4A5) recognizing 7HEG have been developed fromBALB/c mice immunized with the adduct coupled to keyhole limpethemocyanin. In addition, another antibody (8E10) was developedagainst the imidazole ring-opened form of the adduct (ro-7HEG).ELISAs were used to determine the sensitivity and specificityof these antibodies. With antibody 9E2, 50% inhibition of antibodybinding in the competitive ELISA was at 54 pmol of the modifiedbase 7HEG/well and 67 pmol 7HEGR/well, while with antibody 4A5,the values were 3.6 pmol 7HEG/well and 6.7 pmol 7HEGR/well.Antibody 8E10 gave 50% inhibition at 48 pmol ro-7HEGR/well.Neither antibody 9E2 nor 8E10 cross-reacted with unmodifiedDNA or with the normal nudeosides at the highest concentrationtested. However, antibody 4A5 had a low affinity for deoxyguanosine(50% inhibition at 31 000 pmol). Sensitivity of adduct measurementcan be increased 3- to 10-fold using an ELISA with fluorescenceendpoint detection. These antibodies have been used to determinethe level of adducts in DNA modified in vitro with [³H]- or[¹⁴C]EO. Because of the cross-reactivity of the most sensitiveantibody, 4A5, with deoxyguanosine, a combined HPLC/immunoassaymethod was developed to quantitate 7HEG in DNA. The limit ofsensitivity of this method is dependent upon the amount of DNAavailable for analysis. Using 30 fmol as the lowest detectableamount (20% inhibition) in the fluorescent ELISA with antibody4A5 and 100 µg of DNA assayed per well, adduct levelsof 1/10⁷ nucleotide can be determined. This method was appliedto DNA adduct detection in EO-treated myeloma cells and wholeblood. Antibody 8E10 was also used in immunohistochemical studiesto visualize ring-opened adducts in cells treated with EO followedby high pH. These antibodies will be used for the detectionand quantitation of adducts in human samples.     

The isolation of monoclonal antibodies selected for the detection of imidazole ring-opened N7-ethylguanine in purified DNA and in cells in situ. Crossreaction with methyl, 2-hydroxyethyl and sulphur mustard adducts

Monoclonal antibodies have been obtained against imidazole ring-opened N7-ethylguanine (RON7-EtGua) in DNA. The antibodies were selected for good performance in the ELISA with either DNA or nucleated blood cells as immobilized antigen. Antibodies thus selected were studied for their suitability for the in situ detection of RON7-EtGua in the nuclei of cells by means of immunofluorescence microscopy (IFM). Two antibodies have been characterized in detail with respect to specificity and sensitivity. Competitive ELISA demonstrated that the antibodies recognize not only RON7-EtGua but also the corresponding methyl and 2-hydroxyethyl components, with efficiencies that vary with the chemical environment (base, nucleoside or DNA), the nature of the alkyl group and the antibody. They have a clear specificity for the ring-opened alkyl adducts and show an at least 100-fold stronger preference for such structures in DNA when compared to the free nucleoside adducts. Furthermore, they hardly bind to non-alkylated DNA, and do not bind to guanosine or N1- or O6-ethylguanosine. Analysis by DNA-ELISA showed that the binding preference of antibody N7E-026 for ring-opened alkyl adducts is methyl approximately ethyl greater than 2-hydroxyethyl much greater than sulphur mustard, while that of N7E-102 is 2-hydroxyethyl greater than ethyl greater than methyl approximately sulphur mustard. Analysis of RON7-EtGua in DNA with competitive ELISA, DNA-ELISA and IFM showed that in all cases the lowest detection limit can be reached with antibody N7E-026. Competitive ELISA was the most sensitive method, followed by DNA-ELISA and IFM, with detection limits of 2.2, 16 and 23 RON7-EtGua/10(6) nucleotides respectively. In the DNA-ELISA, 12 methyl adducts/10(6) nucleotides can be detected with N7E-026 and 11 2-hydroxyethyl adducts/10(6) nucleotides with N7E-102.     

The isolation of monoclonal antibodies selected for the detection of imidazole ring-opened N7-ethylguanine in purified DNA and in cells in situ. Crossreaction with methyl, 2-hydroxethyl and sulphur mustard adducts

Monoclonal antibodies have been obtained against imidazole ring-openedN7-ethylguanine (RON7-EtGua) in DNA. The antibodies were selectedfor good performance in the ELISA with either DNA or nucleatedblood cells as immobilized antigen. Antibodies thus selectedwere studied for their suitability for the in situ detectionof RON7-EtGua in the nuclei of cells by means of immunofluorescencemicroscopy (IFM). Two antibodies have been characterized indetail with respect to specificity and sensitivity. CompetitiveELISA demonstrated that the antibodies recognize not only RON7-EtGuabut also the corresponding methyl and 2-hydroxyethyl components,with efficiencies that vary with the chemical environment (base,nucleoside or DNA), the nature of the alkyl group and the antibody.They have a clear specificity for the ring-opened alkyl adductsand show an at least 100-fold stronger preference for such structuresin DNA when compared to the free nucleoside adducts. Furthermore,they hardly bind to non-alkylated DNA, and do not bind to guanosineor N1- or O⁶-ethylguanosine. Analysis by DNA-ELISA showed thatthe binding preference of antibody N7E-026 for ring-opened alkyladducts is methyl = ethyl > 2-hydroxyethyl sulphur mustard,while that of N7E-102 is 2-hydroxyethyl > ethyl > methyl= sulphur mustard. Analysis of RON7-EtGua in DNA with competitiveELISA, DNA-ELISA and IFM showed that in all cases the lowestdetection limit can be reached with antibody N7E-026. CompetitiveELISA was the most sensitive method, followed by DNA-ELISA andIFM, with detection limits of 2.2, 16 and 23 RON7-EtGua/10⁶nudeotides respectively. In the DNA-ELISA, 12 methyl adducts/10⁶nucleotides can be detected with N7E-026 and 11 2-hydroxyethyladducts/10⁶ nudeotides with N7E-102.     

Molecular dosimetry of endogenous and ethylene oxide-induced N7-(2-hydroxyethyl) guanine formation in tissues of rodents.

The formation of N7-(2-hydroxyethyl)guanine (7-HEG) in DNA was investigated previously in target and non-target tissues of F-344 rats and B6C3F1 mice exposed to >/=ISOdia>/=10 p.p.m. concentrations of ethylene oxide (EO) using fluorescence-linked high-performance liquid chromatography [V.E.Walker et al. (1992) Cancer Res., 52, 4238-4334]. In order to study the dose-responses for 7-HEG at lower exposures, a highly sensitive and specific gas chromatography coupled with high-resolution mass spectrometry (GC-HRMS) assay was developed. DNA was extracted from liver, brain, lung and spleen of B6C3F1 mice and F-344 rats exposed to 0, 3, 10, 33 or 100 p.p.m. EO for 4 weeks (6 h/day, 5 days/week). Analysis of DNA from control rodents showed that endogenous 7-HEG varied from 0.2 +/- 0.1 to 0.3 +/- 0.2 pmol/micromol guanine in tissues of rats and mice. 7-HEG exhibited tissue- and species-specific dose-response relationships in EO-exposed animals. Linear dose-response relationships were evident in mouse liver, brain and spleen at exposures between 3 and 100 p.p.m. Mouse lung exhibited a slightly sublinear response between 33 and 100 p.p.m. EO. The relationships were linear in liver and spleen of rats between 3 and 100 p.p.m. EO, but were slightly sublinear in brain and lung between 33 and 100 p.p.m. EO. The number of 7-HEG adducts present in rats exposed to 3 p.p.m. EO was 5.3-12.5 times higher than endogenous 7-HEG in unexposed controls. In contrast, mice exposed to 3 p.p.m. EO only had 1.3- to 2.5-fold greater numbers of 7-HEG adducts. The factors driving the exposure-response relationships are also likely to affect carcinogenic and mutagenic responses of rodents to EO. Likewise, a better understanding of the relationships between 7-HEG derived from low exposures to EO and endogenously formed 7-HEG may be important for the accurate extrapolation of risk to humans.     

Separation of 7-methyl- and 7-(2-hydroxyethyl)-guanine adducts in human DNA samples using a combination of TLC and HPLC

We have used a combination of thin-layer chromatography (TLC)and high pressure liquid chromatography (HPLC) to achieve separationof ³²P-postlabelled 7-methylguanine and 7-(2-hydroxyethyl)-guanineadducts. The level of these two adducts was determined in humantotal white blood cells (mean values 0.7 to 1.5 adducts per10⁷ normal nucleotides) and isolated lymphocytes (mean values1.1 to 12 adducts per 10⁷ normal nucleotides). The separationof these two adducts revealed that the level of 7-(2-hydroxyethyl)-guaninewas twice the level of 7-methylguanine adducts in total whiteblood cells, whereas, in isolated lymphocytes it was at leastfour times more than the 7-methylguanine adduct. The combinedlevel of these two adducts in the lymphocytes of non-smokerswas 1.1 to 8.4 adducts per 10⁷ normal nucleotides and in thelymphocytes of smokers, the level was 5.6 to 12 adducts per10⁷ normal nucleotides. We also report detection of three unidentifiedadducts in the samples analysed, and at least one of these adductsseemed to be related to smoking. The chromatographic behaviourand depurination at neutral pH indicated the probable 7-alkylguanineor 3-alkyladenine nature of these unidentified adducts.     

Characterisation of the imidazole ring-opened forms of trans-8,9-dihydro-8,9-dihydro-8-(7-guanyl)9-hydroxy aflatoxin B1

Hydroxyl ion attack on aflatoxin B₁ (AFB₁)-adducted DNA followedby acid hydrolysis releases two AFB₁ derivatives, 8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-ylformamido) 9-hydroxy AFB₁ (major compound) and 8, 9-dihydro-8-(2-ammino-6-formamido-4-oxo-3,4dihydropyrimid-5-ylamino)9-hydroxy AFB₁. The minor compound converts into the majorisomer in aqueous dimethylsulphoxide.     

Formation and persistence of O6-(2-hydroxyethyl)-2'-deoxyguanosine in DNA of various rat tissues following a single dose of N-nitroso-N-(2-hydroxyethyl)urea. An immuno-slot-blot study

Rabbit antibodies against O⁶-(2-hydroxyethyl)-2'-deoxyguanosine(O⁶-HEdG) were used to develop a highly sensitive immuno-slot-blotassay for this promutagenic base which enabled the quantitationof 3.6 µmol O⁶-HEdG/mol deoxy-guanosine, correspondingto 5 fmol in a 3-µg DNA sample. This assay was used tostudy DNA hydroxyethylation by N-nitroso-N-(2-hydroxyethyl)urea(HENU) in adult male F344 rats. Initial amounts of O⁶-HEdG 2h after a single i.v. dose of 50 mg/kg were highest in kidney(81 µmol O⁶HEdG/mol deoxyguanosine), followed by lungand liver (67 and 55 µmol/mol dG respectively). Formationof O⁶-HEdG in cerebral DNA was considerably lower (18 µmolO⁶-HEdG/mol deoxyguanosine), probably reflecting delayed crossingof the blood—brain barrier by HENU due to its hydrophilicity.The formation of O⁶-HEdG in liver and kidney was strictly proportionalto dose over a range of 5–50 mg HENU/kg. Repair of O⁶-HEdGwas very rapid in liver (apparent half-life, 12 h), and somewhatslower in kidney and lung (approximate half-life, 40 h and 48h respectively). In contrast, 62% of the initial amount of O⁶-HEdGin cerebral DNA was still present after 7 days. Saturation ofthe hepatic O⁶-alkyl-guanine-DNA alkyltransferase by pretreatmentwith N-nitrosodimethylamine (20 mg/kg) almost completely inhibitedthe removal of O⁶-HEdG, indicating that O⁶-HEdG is predominantlyrepaired by this repair enzyme.     

Levels of N7-(2-hydroxyethyl)guanine as a molecular dosimeter of drug delivery to human brain tumors

The level of N7-(2-hydroxyethyl)guanine (N7-HOEtG), one of the DNA alkylation products formed by 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) treatment, was measured in human brain tumor samples by high performance liquid chromatography with electrochemical detection. The tumors from 6 recurrent chemotherapy-naive patients with recurrent glioblastoma multiforme were analyzed as controls. The mean level of N7-HOEtG in DNA of these specimens was 0.42 pmol/mg DNA. Samples were also obtained from a patient with a recurrent glioblastoma multiforme after direct intratumoral therapy with BCNU in ethanol (DTI-015). The levels of N7-HOEtG in the samples distal, medial, and adjacent to the site of injection were 0.8, 2.6, and 369.5 pmol/mg DNA, respectively. Comparison of the level of N7-HOEtG detected in the distal sample after injection with BCNU in ethanol with the mean level of the untreated samples indicated that it was not sufficiently different to be ruled out as a chance occurrence. Comparison of the levels of N7-HOEtG in the medial and adjacent brain tumor samples with the mean level of the control samples showed values that were approximately 6- and 879-fold higher. These results demonstrate that intratumoral administration of BCNU in ethanol produces significant levels of DNA alkylation and suggest that DNA adduct measurements provide a unique molecular dosimeter to evaluate delivery of alkylating agents to brain tumors.     

Experimental study of tumor detection using 131I-labeled F (ab)2 fragments of monoclonal antibodies to CA 19-9 and CEA (IMACIS-1)

To determine if IMACIS-1 might have the capability of specifically pinpointing an adenocarcinoma in the human lung, xenografts of adenocarcinomatous nude mice that were injected with IMACIS-1 have been evaluated by scintigraphy, and the IMACIS-1 biodistribution in the tissue measured after sacrifice. Results have revealed that scintigraphic images obtained twenty-four hours after the IMACIS-1 injection showed some activity in the area of the tumor. At seventy-two hours after the IMACIS-1 injection, tumoral radioactivity was only seen in mice with a large tumor. At ninety-two hours after injection, the animals were sacrificed and a biodistribution study was performed. The IMACIS-1 uptake was expressed in counts per minute per gram of tissue. The tissue-to-blood uptake ratio in mice with a large tumor was 8.4 in the viable part of the tumor, 27.2 in the necrotic part of the tumor, 3.0 in the liver, and 1.5 in the spleen, respectively. In contrast, the ratio in mice with a small tumor was 3.4 in the viable part and 18.3 in the necrotic part. Immunoperoxidase staining with either the anti CEA or the anti CA 19-9 antibody was strongly visible in the necrotic part of the tumor.     

The in vivo levels of DNA alkylation products in human lymphocytes are not age dependent: an assay of 7-methyl- and 7-(2-hydroxyethyl)-guanine DNA adducts

Endogenous DNA damage is assumed to be a major contributor to aging and cancer. This study compares the steady-state levels of 7-methyl- and 7-(2-hydroxyethyl)-guanine DNA adducts in lymphocytes isolated from the younger (mean age 39.8 years) and the older (mean age 82.8 years) healthy subjects. Using a 32P-post-labelling method, these adducts were measured in lymphocyte DNA from a total of 34 subjects. The results show that the amount of both 7-methyl- and 7-(2-hydroxyethyl)-guanine adducts in the younger age group was similar to that in the older age group. Our findings show that at steady-state the levels of DNA alkylation products are independent of age, suggesting that endogenous DNA damage, through methylation or lipid peroxidation, and the repair of such damage may not be deficient in lymphocytes of older individuals.     

Preclinical evaluation of the antineoplastic efficacy of 7-(2-hydroxyethyl)theophylline on melanoma cancer cells

Differentiation-based therapeutics are an underutilized but potentially a significant option for cancer treatment. The effect of methylxanthines on melanoma cell differentiation has been well documented. We report the in-vitro and in-vivo anticancer potential of a theophylline analogue, 7-(2-hydroxyethyl)theophylline (HET), on murine B16-F10 and human Sk-Mel 110 metastatic melanoma cell lines. The effects on cell proliferation were related to the induction of differentiation, demonstrated as increased intracellular transglutaminase activity. The involvement of this methylxanthine in the control of the in-vitro adhesion and in the in-vivo metastastic spread of melanoma cells was further investigated. HET oral administration of C57BL6/N mice intravenously injected with B16-F10 cells markedly reduced lung metastases frequency. The overall results demonstrated that HET possesses a remarkable in-vivo antimetastatic capability.     

Molecular dosimetry of ethylene oxide: formation and persistence of 7-(2-hydroxyethyl)guanine in DNA following repeated exposures of rats and mice.

The formation of 7-(2-hydroxyethyl)guanine (7-HEG) in DNA of target and nontarget tissues was investigated in male B6C3F1 mice (20/group) and F344 rats (10/group) exposed to 0, 3, 10, 33, 100, or 300 (rats only) ppm ethylene oxide (ETO) by inhalation for 6 h/day for 4 weeks (5 days/week) and mice exposed to 100 ppm ETO for 1 or 3 days or 1, 2, or 4 weeks (5 days/week). The persistence of 7-HEG was studied in mice killed up to 7 days after cessation of the 4-week time-course study. In addition, the formation of O6-(2-hydroxyethyl)guanine and 3-(2-hydroxyethyl)adenine was evaluated in rats exposed to 300 ppm ETO. DNA samples from control and treated animals were analyzed for 7-HEG using neutral thermal hydrolysis, microconcentration, and high-performance liquid chromatography separation with fluorescence detection. Fluorescence-linked high-performance liquid chromatography was used for O6-(2-hydroxyethyl)guanine quantitation, and immunochromatography and gas chromatography-mass spectrometry were used for 3-(2-hydroxyethyl)adenine detection. Analysis of DNA from tissues of control mice and rats revealed the presence of peaks equivalent to 2-6 pmol 7-HEG/mg DNA. In mice exposed to 100 ppm ETO, 7-HEG accumulated to a similar extent in target and nontarget tissues, with adduct concentrations ranging from 17.5 +/- 3.0 (SE) (testis) to 32.9 +/- 1.9 (lung) pmol adduct/mg DNA after 4 weeks of exposure. Concurrent exposures of mice and rats to 100 ppm ETO for 4 weeks led to 2- to 3-fold lower concentrations of 7-HEG in mouse DNA in all tissues compared to rat DNA. 7-HEG disappeared slowly in a nearly linear fashion from the DNA of mouse kidney (t1/2 = 6.9 days) and rat brain and lung (t1/2 = 5.4-5.8 days), which was consistent with the loss of adduct mainly by chemical depurination. In contrast, a more rapid removal of 7-HEG from other mouse (t1/2 = 1.0-2.3 days) and rat (t1/2 = 2.9-4.8 days) tissues was consistent with adduct loss by depurination and DNA repair. Dose-response relationships for 7-HEG were nonlinear in both mice and rats, with the alkylating efficiency of ETO increasing at high exposures.(ABSTRACT TRUNCATED AT 400 WORDS)     

Vinyl chloride-induced DNA adducts. II: Formation and persistence of 7-(2'-oxoethyl)guanine and N2,3-ethenoguanine in rat tissue DNA

The formation and persistence of the DNA adducts 7-(2'-oxoethyl)guanine (OEG) and N2,3-ethenoguanine (EG) were investigated in preweanling Sprague-Dawley rats exposed to vinyl chloride (VC). Lactating female CD rats with 10 day old pups were exposed to 600 p.p.m. VC by inhalation for 5 days, 4 h/day. Groups of rats were killed immediately and 3, 7 and 14 days after exposure. The concentrations of OEG and EG were measured in liver, lung, kidney, brain and spleen. HPLC with fluorescence detection was used for OEG detection, and gas chromatography-negative ion chemical ionization mass spectrometry was used for EG detection. In tissues of neonatal rats, the concentrations of both DNA adducts, expressed as pmol/mumols unmodified guanine, were highest in liver (OEG 162 +/- 36, EG 1.81 +/- 0.25), followed by kidney (OEG 29 +/- 1, EG 0.31 +/- 0.02), and lung (OEG 20 +/- 7, EG 0.21 +/- 0.08). No adducts were found in brain or spleen. DNA adducts were detected only in liver (OEG 43 +/- 7, EG 0.47 +/- 0.14) and lung (OEG 20 +/- 5, EG 0.27 +/- 0.03) of the dams. The ratio between EG and OEG was approximately 1:100 in all tissues immediately after exposure. In the liver of the preweanling rats, this ratio increased to 1:14 1 week after exposure, reflecting a greater persistence of EG. A half-life of 62 h was calculated for OEG, and the estimated half-life for EG was greater than 30 days. In view of the slow loss of EG and its high efficiency for causing base-pair mismatch, these results suggest that EG may be an important DNA adduct in VC-induced carcinogenesis.     

Excretion of the mercapturic acid S-[2-(N7-guanyl)ethyl]-N-acetylcysteine in urine following administration of ethylene dibromide to rats

Administration of the carcinogen ethylene dibromide (EDB) to rats resulted in the urinary excretion of S-[2-(N7-guanyl)ethyl]-N-acetylcysteine, which is derived from the nucleic acid adduct S-[2-(N7-guanyl)ethyl]glutathione. This mercapturic acid was isolated from urine by reversed-phase and propylamino high-performance liquid chromatography and was quantitated by measurement of fluorescence intensity. The urinary mercapturic acid was identified as S-[2-(N7-guanyl)ethyl]-N-acetylcysteine on the basis of cochromatography and UV, fluorescence, 1H nuclear magnetic resonance, and fast atom bombardment mass spectra, all of which were identical with the authentic synthesized material. The excretion of mercapturic acid into urine of rats given injections of various doses of EDB occurred in a dose-dependent, linear manner over the range of 0.5-37 mg EDB/kg body weight. A good correlation was found between the excretion of mercapturic acid and the (in vivo) formation of DNA adducts in liver and kidney DNA. The higher level of urinary mercapturic acid compared to the level of hepatic DNA adduct indicates that extra-hepatic DNA adducts and RNA adducts may contribute to the mercapturic acid production. The measurement of the mercapturic acid may provide a means of noninvasive estimation of DNA adducts derived from EDB exposure.     

Cytotoxic activity against a series of human tumour cell lines of some diorganotin(iv) 1,2-ethylenediamine N,N'-diacetates, N-(2-hydroxyethyl)- and N-(carbamoylmethyl)-iminodiacetates, and ortho-aminobenzoates.

The di-n-butyltin(IV) derivatives of N-(2-hydroxyethyl)- and N-(carbamoylmethyl)- iminodiacetic acid, compounds 1 and 2, the di-n-butyltin-, di-n-octyltin and dimethyltin-(IV) derivatives of 1,2-ethylene diamine N,N'-diacetic acid, compounds 3, 4 and 5, and di-n-butyltin bis-o-aminobenzoate, compound 6, were tested in vitro at the National Cancer Institute, Bethesda, Maryland, USA, for cytotoxic activity against a series of human tumor cell lines. Compounds 1 and 2 score globally satisfactorily since four of the five relevant selectivity or sensitivity parameters [DGI50, DTGI or DLC50 greater than 50, DH and MGDH greater than 75)] are statistically significant; compounds 4 and 6 score satisfactorily for only three of these parameters and compound 3, for only two. Compound 5, the dimethyltin derivative, is inactive.     

Stimulation of proliferation in human colon cancer cells by human monoclonal antibodies against the TF antigen (galactose β1-3 N-acetyl-galactosamine)

In many tissues, the TF (Thomsen-Friedenreich) blood group antigen (Galβ1-3GalNAcα-) behaves as an onco-foetal carbohydrate antigen, showing increased expression in malignancy and hyperplasia. Dietary lectins which bind the TF antigen have marked effects on proliferation of epithelial cells without cytotoxicity. This led us to speculate that anti-TF antibodies, including those that naturally occur in humans, might have similar effects. Five anti-TF antibodies, TF2 (human), TF5 (human), 5A8 (mouse), 8D8 (mouse) and BM22 (mouse), but not TF1 (human) or 49H.9 (mouse), showed marked dose-dependent stimulation (95–192%) of [³H]thymidine incorporation by HT29 human colon cancer cells. Similar stimulation of proliferation of HT29 cells by these monoclonal antibodies (MAbs) was found when cell count assessment was used. Antibody-stimulated proliferation was inhibited by co-incubation with glycoproteins expressing Galβ1-3GalNAcα- (asialo glycophorin or [Galβ1-3GalNAcα-O-p-aminophenyl]_n-human serum albumin). A proliferative effect of these antibodies was also demonstrated on human colon cancer cell lines LS174T and HT29-MTX but not on Caco-2 cells. Although immunoblotting showed similar binding patterns of all the antibodies on HT29 cell membrane extracts, there was little correlation between cell surface binding assessed by immunofluorescence and proliferative response, and internalization of the biotinylated antibody TF5 was demonstrated by confocal microscopy. Our results provide further evidence that cell surface glycoproteins which express TF antigen may play an important role in the regulation of cell proliferation and also suggest that human anti-TF antibodies may have proliferative effects on cells which express TF antigen. Int. J. Cancer 73:424–431, 1997. © 1997 Wiley-Liss, Inc.     

Antitumor activity of 7-N-(2-((2-(gamma-L-glutamylamino) ethyl) dithio) ethyl) mitomycin C (KW2149) against human tumor xenografts serially transplanted into nude mice

The antitumor activity and toxicity of 7-N-(2-((2-(gamma-L-glutamylamino) ethyl) dithio) ethyl) mitomycin C (KW2149) were evaluated using a human tumor xenograft--nude mouse system, and compared with those of the maternal compound, mitomycin C. The maximum tolerated dose of KW2149 was estimated to be 15 mg/kg by bolus intraperitoneal or intravenous injection, at which a remarkable reduction of spleen weight was observed, suggesting bone marrow suppression by this agent. A bolus injection of KW2149 seemed to be more effective than a divided injection schedule, when a total of 15 mg KW2149/kg was administered to mice bearing breast (MX-1) and colon (Co-4) carcinomas. The antitumor activity of KW2149 was dose-dependent, and the difference in antitumor effect according to route of administration was minimal. The antitumor spectrum of KW2149 was essentially identical to that of mitomycin C administered intraperiotoneally as a bolus at a dose of 6 mg/kg.     

Formation of N-7-(2-carbamoyl-2-hydroxyethyl)guanine in DNA of the mouse and the rat following intraperitoneal administration of [14C]acrylamide

Acrylamide is an alkylating agent which reacts very slowly indirect reactions with DNA and is negative in the Ames test,but is carcinogenic in mice and rats. In order to explain thecancer-initiating properties of acrylamide we have studied DNAadduct formation in vitro with a metabolizing system and invivo in mice and rats following i.p. administration of ¹⁴C-labeledacrylamide. A major adduct found in both species was N-7-(2-carbamoyl-2-hydroxy-ethyl)guanine,formed by reaction of the DNA with the epoxide metabolite glycidamide.The levels of this adduct were similar in the different organsof the two rodent species, which supports the notion that glycidamideis relatively evenly distributed among tissues and that theorgan-specificity in acrylamide carcinogenesis cannot be explainedby a selective accumulation of the DNA-reactive metabolite intarget organs.     

Comparison of adherent lymphokine-activated killer (a-lak) cells generated by IL-2 and IL-7 - cellular modifications induced by IL-7

At present, the clinical application of plastic-adherent-lymphokine-activated killer (A-LAK) cells shows limited success in the immunotherapy of patients with advanced cancer because of a low responder rate, severe side effects and failures in yielding sufficient numbers of cells for adoptive transfers. Since interleukin-7 (IL-7) is able to induce LAK activity independently of IL-2, we investigated the ability of IL-7 to improve the yield and the properties of A-LAK cells. A-LAK cells from 7 healthy donors generated in the presence of IL-2, IL-7 or combinations of IL-2 plus IL-7 (each 1000 U/ml) were compared with regard to plastic adherence, expansion rate, immunophenotype, cytokine secretion and cytotoxicity against malignant melanoma cells and non-malignant target cells. Our results demonstrate that A-LAK cells generated by a simultaneous stimulation of IL-2 plus IL-7 displayed a significantly higher expansion rate (10.7-fold vs. 9.0-fold), but showed no difference in the cytolytic activity compared to A-LAK cells generated by IL-2 alone. A-LAK cells generated by IL-7 alone demonstrated a low expansion rate (1.1-fold vs. 8.8-fold), and decreased in other properties like plastic adherence, CD56(+)/CD3(+) cell-ratio and cytolytic activity compared to A-LAK cells generated by IL-2 alone. A-LAK cells generated by IL-7 or a sequential stimulation of IL-2 and IL-7, on the other hand, exhibited a more selective cytotoxicity for malignant melanoma cells compared to the non-malignant keratinocyte target cell line (HaCaT) and normal fibroblasts. A sequential replacement of LL-2 by IL-7 might help to reduce the severe side effects of IL-2. In vivo experiments are necessary to evaluate the potential value of IL-7 in adoptive immunotherapy.