The presence of single nucleotide instability in human breast cancer cell lines.

The presence of single nucleotide instability, an increase of spontaneous point mutation rates (MR: number of mutations per cell division) without microsatellite instability, was demonstrated previously in two rat mammary carcinoma cell lines. In this study, spontaneous point MRs were analyzed in human breast cancer cell lines by the fluctuation test using the hypoxanthine-guanine phosphoribosyltransferase (hprt) marker gene. MRs obtained for six breast cancer cell lines, MCF-7, ZR-75-1, T-47D, MDA-MB-231, MDA-MB-468, and BT-474, all of which were proficient in G/T mismatch binding and reported to be negative for microsatellite instability, were 7.6, 4.6, 6.3, 2.2, 5.6, and 19 x 10(-7) mutations/hprt/cell division. Those in normal human mammary epithelial cells and in a colon cancer cell line with proficient mismatch repair, SW480, were 1.6 and 1.4 x 10(-7) mutations/hprt/cell division, respectively. These findings showed that single nucleotide instability was also present in five of the six human breast cancer cell lines and strongly indicates it has important roles in human and rat mammary carcinogenesis.

LacI transgenic animal study: relationships among DNA-adduct levels, mutant frequencies and cancer incidences.

In the processes of carcinogenesis caused by genotoxic carcinogens, DNA-adduct formation and resultant genetic changes are crucially important. In this report, the relationship between DNA-adduct levels and mutant frequencies (MFs), DNA-adduct levels and cancer incidences, and MFs and cancer incidences induced by heterocyclic amines (HCAs), to which humans are exposed on daily basis were investigated. There was no direct correlation between adduct levels and MFs detected after feeding Big Blue mice with 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), in a comparison among various organs. Further, there was no direct correlation between DNA-adduct levels and cancer incidences, in a comparison among various organs of F344 rats. Since DNA-adducts are fixed as mutations after cell proliferation, and mutations in cancer-related genes result in cancer development, it is expected that MFs directly correlate with cancer incidences. However, there was no direct correlation between MFs and cancer incidences. Possible mechanisms involved in the discordance between DNA damage markers and cancer incidences are discussed.

Single nucleotide instability without microsatellite instability in rat mammary carcinomas.

Mutation frequencies (MnFs) of the lacI transgene and mutation rates (MRs) of the endogenous hprt gene were analyzed in two mammary carcinoma cell lines that we established from mammary carcinomas that had been induced by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in female lacI-transgenic rats. Using the lacI transgene, corrected MnF, which is the number of independent lacI mutations that occurred while 102 cells expanded into 10(7) cells and which reflect the dynamic increase of point mutations, was measured. The corrected MnFs in the two mammary carcinoma cell lines (59 x 10(-6) and 72 x 10(-6) mutations) were significantly higher than that in the primary culture of normal mammary epithelium (4.7 x 10(-6)). MRs of the hprt gene in the two mammary carcinoma cell lines (8.2 x 10(-7) and 11 x 10(-7) mutations/hprt/cell division) were also higher than the same control (1.4 x 10(-7)). A:T to C:G transversion was observed at significantly higher frequencies in the two cell lines (6 of 24 and 6 of 25 for lacI; 10 of 67 and 19 of 92 for hprt) than in the control (0 of 6 for lacI; 0 of 4 for hprt). Taking advantage of the lacI transgene, high frequencies of A:T to C:G transversion (6 of 38 and 8 of 33, respectively) was also confirmed in the primary carcinomas of the two cell lines, which indicated the presence of a common abnormality in the cell lines and in the primary carcinomas. Both the established cell lines and their primary carcinomas were negative for microsatellite instability, which is known to be caused mainly by mismatch repair insufficiency and to increase point mutations, and for p53 mutations. These findings showed that the two cell lines, and possibly their primary carcinomas, had increases in the MRs of point mutations attributable to a mechanism(s) different from mismatch repair insufficiency, and we would suggest that such a state be designated as single nucleotide instability (SNI).

Interpretation of mutational spectra from different genes: analyses of PhIP-induced mutational specificity in the lacI and cII transgenes from colon of Big Blue rats.

The cII assay provides an alternative choice to the lacI transgene for mutational studies involving Big Blue(R) transgenic mice and rats, or permits the evaluation of mutational responses in both genes. Here, we compare the mutational response of the cII gene from colon of Big Blue(R) F344 rats treated with a dietary mutagen and animal carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), to those previously determined in the lacI transgene from colon of the same group of animals. A cursory inspection of PhIP-induced mutational spectra (MS) in cII and lacI suggests that the two transgenes respond differently to PhIP-induced mutation. However, a more thorough analysis of the MS in the two transgenes, including consideration of the number of mutational target sequences in each gene and nearest neighbor analyses of mutated nucleotides, indicates that PhIP-induced mutational specificity is similar in both genes. The evaluation of PhIP-induced mutational responses in these two transgenes serves as a model for intergenic mutational analyses.

Mutation induction by mechanical irritation caused by uracil-induced urolithiasis in Big Blue rats.

Some chronic mechanical irritations induce cancers, and it is speculated that mutations are induced by increased rate of cell proliferation caused by the irritation. In this study, it was investigated using chronic mechanical irritation to urothelium caused by urolithiasis, whether mutations are really induced by such cell proliferation or not. Male rats transgenic for lacI (Big Blue(R) rats), in which lacI mutations accumulated in tissue can be measured, were fed 3% uracil, a component of RNA, to induce urolithiasis associated with papillomatosis, and eventually with bladder cancers. The frequency of independent mutations in the bladders of the treated rats showed 3-5 fold increases at weeks 10, 20, and 51 (P=0.01 at week 51) while the frequency was not elevated at week 2. The mutation frequencies in the control bladders ranged from 3 to 9x10(-6). In both groups, G to A transitions at CpG sites, indicative of spontaneous mutations, constituted the most prevalent mutations. Mechanical irritation caused by uracil was shown to induce a 3-5 fold increase of mutations, possibly through an elevation of spontaneous mutations by vigorous cell proliferation.

Preferential induction of guanine deletion at 5'-GGGA-3' in rat mammary glands by 2-amino- 1-methyl-6-phenylimidazo[4,5-b]pyridine.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is known to induce a characteristic mutation, G deletion at the 5'-GGGA-3' site, preferentially in the lacI transgene of the colonic mucosa of Big Blue((R)) rats (BBR) and mice and specifically in the Apc gene of rat colon tumors. In this study, lacI mutations of the mammary glands in PhIP-treated rats were investigated. Six-week-old female (BBRxSprague-Dawley)F(1) rats were administered 10 gavages of 65 mg/kg/day PhIP. Mammary ducts were collected from the macroscopically normal mammary tissue of PhIP-treated and untreated rats at 56-69 weeks of age by collagenase treatment. The mutant frequencies were 25 +/- 2.1x10(-6) in control rats and 323 +/- 44x10(-6) in the PhIP-treated rats. By sequencing 40 and 177 mutants in the control and PhIP-treated groups, respectively, 34 and 149 mutations were considered independent mutations. In the control group, G:C-->A:T transitions at CpG sites dominated and no G:C deletions were detected. In the PhIP-treated group, G:C-->T:A transversions were most frequent (43%), followed by single base pair deletions of G:C (21%). A total of nine deletions were at 5'-GGGA-3' sites, accounting for 29% of the G:C deletions and 6% of the 149 total mutations. Clusters of more than three mutations at one nucleotide position were observed at 12 positions and two were G deletions at 5'-GGGA-3' sites. Comparison of the PhIP-induced mutations in the mammary glands with those previously reported in the colon revealed that G:C-->T:A transversions occurred at a significantly higher frequency in the mammary glands and that G:C deletions occurred at a significantly lower frequency. However, the signature mutation, G deletion at the 5'-GGGA-3' site, was commonly observed in both tissues.

Perfluorooctanoic acid, a peroxisome-proliferating hypolipidemic agent, dissociates apolipoprotein B48 from lipoprotein particles and decreases secretion of very low density lipoproteins by cultured rat hepatocytes.

The hypolipidemic effect is evoked by various peroxisome proliferators. Modulation of gene transcription via peroxisome proliferator-activated receptor (PPAR) is generally responsible for this effect. In addition, we have found a PPAR-independent mechanism in which fibrates, known peroxisome proliferators, decrease hepatic secretion of very low density lipoproteins (VLDL) through inhibition of phosphatidylcholine synthesis via methylation of phosphatidylethanolamine (PE) (T. Nishimaki-Mogami et al., Biochim. Biophys. Acta 1304 (1996) 21-31). In the present study, we show a novel mechanism by which perfluorooctanoic acid (PFOA), a potent peroxisome proliferator and inhibitor of PE methylation, exerts its hypolipidemic effect. PFOA (100 microM) added to the medium rapidly decreased the secretion of triglyceride by cultured rat hepatocytes, which was independent of the activity of cellular PE methylation. Analysis of the density of apoB secreted into the medium showed that PFOA decreased apoB48 in VLDL, but increased apoB48 in the bottom d>1.21 fraction. This lipid-poor apoB48 was also generated by incubating medium that had been harvested from control cells with PFOA, indicating that PFOA has the ability to dissociate apoB48 from lipoprotein particles. Exposure of cells to PFOA for 2 h prior to the experiment was sufficient to generate lipid-poor apoB48, indicating that PFOA exerted its effect intracellularly. Taken together, the data suggest that a strong interaction of PFOA with apoB48 disturbs the association of apoB48 with lipids in the process of intracellular VLDL assembly, thereby inhibiting VLDL secretion. This study shows that the mechanisms of hypolipidemic effect caused by various classes of peroxisome proliferators are diverse.

Monoclonal antibodies specific for P-glycoprotein.

Multidrug resistance (MDR) is one of the major obstacles to successful cancer chemotherapy. Since P-glycoprotein (P-gp) encoded by the MDR-1 gene plays a key role in MDR, many P-gp-specific monoclonal antibodies (MAbs) have been generated for characterization and analysis of P-gp. Among those antibodies, MRK16 has been widely used not only for elucidation of the mechanisms of P-gp-mediated MDR but also for diagnostic and therapeutic studies. Two types of magnetic cell sorting assays, termed MRK16-MACS and MRK16-MACS-FACS, have been established by us and may offer a useful tool to quantitate low levels of P-gp expression. This article describes the characteristics of the antibodies against P-gp and discuss the diagnostic implications of the antibodies.

Retroviral coexpression of two different types of drug resistance genes to protect normal cells from combination chemotherapy.

Drug resistance genes can protect normal hematopoietic cells from the toxicity of anticancer agents. Because chemotherapeutic agents are often used in combination in current clinical protocols, coexpression of two different drug resistance genes should be useful in protecting normal bone marrow cells from the hematotoxicities caused by combination chemotherapy. In this study, we have combined the human multidrug resistance gene (MDR1) and human O6-methylguanine DNA methyltransferase (MGMT) gene as drug resistance genes. For the coexpression of two drug resistance genes, we have constructed two bicistronic retrovirus vectors. One vector is Ha-MDR-IRES-MGMT, in which translation of the MDR1 cDNA is cap-dependent and MGMT translation is dependent on an internal ribosome entry site (IRES). The other is Ha-MGMT-IRES-MDR, which has cap-dependent MGMT translation and IRES-dependent MDR1 translation. MGMT-negative HeLa derivative (MR) cells transduced with these retroviruses showed resistance to vincristine (from MDR1) and 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosou rea (ACN; from MGMT). Cells transduced with Ha-MDR-IRES-MGMT showed higher resistance to vincristine and lower resistance to ACNU than those transduced with Ha-MGMT-IRES-MDR. In any case, the resistance levels of cells transduced with either vector were high enough to select transduced cells with vincristine or ACNU. The expression levels of P-glycoprotein or MGMT in the transduced cells determined by FACS and Western blot analysis correlated well with the extent of resistance to vincristine and ACNU, respectively. All of the MGMT-transduced cells expressed higher amounts of MGMT than the MGMT-expressing parental cell line HeLa S3. Murine bone marrow cells transduced with Ha-MDR-IRES-MGMT and selected with vincristine also showed simultaneous resistance to vincristine and ACNU. These results suggest that bicistronic retroviral vectors allow the functional coexpression of two different types of drug resistance genes. This strategy could be applicable to any combination of drug resistance genes.

Coexpression of a multidrug resistance gene (MDR1) and herpes simplex virus thymidine kinase gene in a bicistronic retroviral vector Ha-MDR-IRES-TK allows selective killing of MDR1-transduced human tumors transplanted in nude mice.

Ha-MDR-IRES-TK is a bicistronic vector that coexpresses the MDR1 gene and the herpes simplex virus thymidine kinase (HSV-TK) gene. In the present study we examined the effect of ganciclovir on MDR1-positive tumors that have been transduced with Ha-MDR-IRES-TK. To establish a human tumor xenograft model of MDR1-transduced recurrent tumors, human KB-3-1 carcinoma cells were transduced with HaMDR or Ha-MDR-IRES-TK, and one each of representative clones, termed KB/MDR and KB/MDR-TK, respectively, were isolated. KB/MDR and KB/MDR-TK showed similar levels of multidrug resistance in vitro. Vinblastine strongly inhibited the growth of the parental KB-3-1 tumors in nude mice but showed little or no effect against KB/MDR-TK tumors. Ganciclovir inhibited the in vivo growth of KB/MDR-TK tumors almost completely under conditions that did not affect the growth of KB-3-1 tumors. Coadministration of vinblastine and ganciclovir inhibited the in vivo growth of KB/MDR-TK premixed with KB-3-1 at any ratio. Long-term, high-level expression of human P-glycoprotein was observed in peripheral blood cells of mice transplanted with Ha-MDR-IRES-TK-transduced bone marrow cells. Ganciclovir eliminated the P-glycoprotein-positive normal blood cells. However, no systemic toxicity was observed. These results clearly demonstrate that it is possible to use ganciclovir to treat MDR1-positive tumors that have been unintentionally transduced with Ha-MDR-IRES-TK. This safety-modified vector should be useful for introducing the MDR1 gene into bone marrow cells to protect normal cells from the toxic effects of cancer chemotherapy.

Detection of mutagenicity in Ames test using a metalloporphyrin/oxidant model system for cytochrome P450.

A chemical model system for cytochrome P450, a porphyrin and an oxidant, was used in Ames assay as a substitute for S9 mix. In the presence of tetrakis(pentafluorophenyl)porphyrinatoiron(III) chloride [Fe(F5P)Cl] and tert-butyl hydroperoxide (t-BuOOH), mutagenicity of N-nitrosodibutylamine (NDB) in Salmonella typhimurium TA1535 was detected. The mutagenicity depended on the pre-incubation period, and also on the concentration of an oxidant and of bacteria. In the chemical model system, pH affected the mutagenicity of NDB, which suggested that as observed in an enzymatic activating system, the mutagenicity was due to the labile alkylating species which was derived from NDB activated in the chemical activation system and was sensitive to pH. Under the optimum conditions; a higher concentration of an oxidant, a higher concentration of bacterial culture, and a weakly acidic medium, mutagenicity of N-nitrosodipropylamine in S. typhimurium TA1535 was also detected. Besides N-nitrosodialkylamines, 2-aminofluorene (2-AF) and benzo[a]pyrene (BaP) were also used as mutagens. Mutagenicity of 2-AF and BaP in S. typhimurium TA1538 were both detected in the same system as used in detecting the mutagenicity of N-nitrosodialkylamines. Ames test using a metalloporphyrin/oxidant model system makes it possible to detect mutagenicity derived from both base pair substitution mutagens and frameshift mutagens without using enzymatic activating system. These results demonstrate that the assay with the chemical model system is useful in detecting unstable unknown active mutagens or investigating the mechanisms of the metabolic pathway of mutagens or carcinogens in a protein-free medium.

Effects of gender and species on spectra of mutation induced by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine in the lacI transgene.

Feeding of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) to F344 rats induces colon tumors specifically in male rats. Mutant frequencies and mutational spectra of the lacI transgene were studied in male and female Big Blue transgenic rats after feeding 400 ppm of PhIP in the diet for 60 days. Mutant frequencies in the colon mucosa were increased 20-25 times compared with those of the control rats, being 661.4 +/- 33.3 x 10(-6) and 718.2 +/- 16.9 x 10(-6) in males and females, respectively. No significant differences in types and distribution of the mutations were detected between males and females. One-base deletions were the most frequent mutation, including the characteristic guanine deletion at 5'-GGGA-3' which is also seen in the Apc gene of rat colon cancers induced by PhIP. Comparison of the lacI mutations in the rat colon with those previously identified in the mouse colon showed that the rate of G to T transversions was significantly higher in the mouse. This is the first report stating that there exist differences in the mutation specificity on the same gene, among mammalian species. However, the characteristic guanine deletion was recovered in both the mouse and the rat. These findings do not offer a mechanistic explanation of the gender specificity of PhIP-induced colon cancer in rats, though the universality of the guanine deletion suggests that this alteration may prove a useful indicator of human exposure.

Bezafibrate and clofibric acid are novel inhibitors of phosphatidylcholine synthesis via the methylation of phosphatidylethanolamine.

The effects of bezafibrate and clofibric acid, fibrate hypolipidemic agents, on phosphatidylcholine (PC) synthesis via the phosphatidylethanolamine (PE) methylation pathway were studied. In cultured rat hepatocytes, bezafibrate and clofibric acid added to the medium rapidly and markedly reduced the conversion of ethanolamine-labeled PE to PC (IC50 30 and 150 microM, respectively). Furthermore, the methylation of PE derived from serine was also blocked by bezafibrate, as was the secretion of PC derived from either serine or ethanolamine. The microsomal activity of PE N-methyltransferase was inhibited by these agents. Perfluorooctanoic acid but not DCQVA, though both are potent peroxisome proliferators comparable to fibrates, produced this inhibition. The inhibitory effects produced by these agents were diminished by dithiothreitol (DTT) added to the assay or alkaline pH assay condition. Inhibition by oleic acid was also attenuated under these conditions, suggesting a common mechanism of inhibition. However, unlike fatty acids, fibrates did not have rapid stimulatory effects on the CDP-choline pathway in hepatocytes. These results suggest that fibrates may mimic fatty acids in regulating PC synthesis from the PE methylation pathway but not the CDP-choline pathway.

Dealkylation of N-nitrosodibenzylamine by metalloporphyrin/oxidant model systems for cytochrome P450.

N-Nitrosodialkylamines are alkylating carcinogens which are metabolically activated to alpha-hydroxy nitrosamines through oxidative dealkylation by cytochrome P450. In this study, the dealkylation step in the activation of N-nitrosodibenzylamine (NDBz) was investigated with metalloporphyrin/oxidant model systems under non-aqueous conditions, as biomimetic chemical models of cytochrome P450. In the model systems, NDBz was dealkylated and benzaldehyde was released. The catalytic activity required a porphyrin ring with a central metal that can interact with an oxidant. The oxidative activity of the model varied with the oxidant used in the order of tert-butyl hydroperoxide > cumene hydroperoxide > iodosobenzene, and also with the central metal of porphyrin used in the order of tetraphenylporphyrinatoiron(III) chloride > its manganese derivative. It was confirmed that N-nitrosodialkylamine undergoes oxidation to alpha-hydroxy nitrosamine in chemical model systems which are free of protein. These biomimetic models should be useful in elucidating the mechanisms of the metabolism of N-nitrosodialkylamines.

Establishment and evaluation of MRK16-magnetic cell sorting assays for detecting low expression of multidrug resistance P-glycoprotein using human leukemia cell lines and peripheral blood cells from healthy donors.

Two types of magnetic cell sorting assays, termed MRK16-MACS and MRK16-MACS-FACS, have been established to detect low expression level of P-glycoprotein (P-gp) using a monoclonal antibody MRK16, which recognizes a cell surface epitope of P-gp. With K-562 and U-937 cell lines, which are known to express low levels of P-gp and hence routinely used as negative control cell lines in conventional flow cytometry, both assays gave significantly positive reactivities indicating improved specificity and sensitivity of these assays. The findings in the dilution test, where P-gp-positive cells were added to P-gp-negative cells at various ratios, demonstrated that the MRK16-MACS assay is quantitative and capable of detecting small numbers of P-gp-positive cells as few as 2.5% of the total cells tested. Furthermore, specific enrichment of P-gp-expressing cells in magnetic cell sorting assays was verified by reverse transcription-polymerase chain reaction (RT-PCR) analysis and functional assay for P-gp with Rhodamine 123. The availability of such magnetic cell sorting assays may offer an approach to quantitate low level of P-gp expression.

Activation of N-nitrosodialkylamines to mutagens by a metalloporphyrin/oxidant model system for cytochrome P450.

N-Nitrosodialkylamines are environmental alkylating carcinogens which are metabolically activated to alpha-hydroxy nitrosamines by cytochrome P450. The precise mechanism of their activation is not well understood, and a simplified chemical model for cytochrome P450 as a non-enzymatic system is useful for investigating the mechanism. In the present study, a chemical model was used in a bacterial mutation assay as a substitute of S9 mix, and its ability in the activation of N-nitrosodialkylamines was elucidated. In the presence of tetrakis(pentafluorophenyl)porphyrinatoiron(III) chloride and tert-butyl hydroperoxide, the mutagenicity of N-nitrosodialkylamines [alkyl = methyl (NDM), ethyl (NDE), propyl (NDP) and butyl (NDB)] in Salmonella typhimurium YG7108 was detected. The mutagenicity increased by the pre-incubation and was dependent on the concentration of mutagens. The mutagenic activity of N-nitrosodialkylamines in Salmonella typhimurium YG7108 was in the following order: NDB > NDM > NDE approximately NDP. The formation of aldehydes derived from dealkylation in the present model was exemplified by the formation of acetaldehyde from NDE. These results showed that N-nitrosodialkylamines were activated by the model system, and consequent mutagenicity was observed. The oxidation by the model can mimic the metabolic activation of chemical carcinogens by cytochrome P450, and the biomimetic catalyst is useful in investigating the mechanisms of the metabolic pathway of N-nitrosodialkylamines.

Specific targeting and killing activities of anti-P-glycoprotein monoclonal antibody MRK16 directed against intrinsically multidrug-resistant human colorectal carcinoma cell lines in the nude mouse model.

Anti-P-glycoprotein (P-gp) monoclonal antibody, MRK16, and its F(ab')2 fragment were evaluated for its therapeutic efficacy to P-gp-mediated multidrug resistant human colorectal carcinoma cell lines in a nude mouse model. In a blood clearance experiment, 125I-labeled MRK16 had a half-life (16 h) 7 times longer than its F(ab')2 fragment (half-life of 1.8 h) in circulation in nude mice, and approximately 16 and 5% of MRK16 were retained on days 10 and 20 after injection, respectively. In biodistribution experiments using nude mice bearing HCT-15, an intrinsically resistant cell line, 125I-labeled MRK16 accumulated at the tumor site significantly higher than its F(ab')2 fragment as revealed by the percentage of injected dose/g of tissue values (7.4 versus 0.6%) on day 3 after injection. In contrast, the tissue to blood ratio at the tumor site of the MRK16 was significantly lower than that of its F(ab')2 fragment (1.2 versus 10.5). Specific targeting of the MRK16 F(ab')2 fragment to the P-gp-positive tumor (HCT-15) but not to the P-gp-negative tumor (COLO 205) was observed in the nude mice bearing both tumors. In the therapeutic efficacy tests, when administered i.v. 3 times on days 1, 4, and 7 after tumor s.c. inoculation, MRK16 alone showed the significant inhibition of tumor growth of P-gp-positive cell lines, HCT-15, DLD-1, SW480, and SW1417 in contrast to cases of P-gp-negative cell lines, COLO 205 and KM20L2. This inhibitory effect of MRK16 was enhanced in combination with Adriamycin, which alone hardly inhibited the tumor growth. However, MRK16 F(ab')2 fragment alone, even at 1 mg/mouse, had little inhibitory effect on the growth of HCT-15 in the same treatment schedule. When administered at early palpable stage, the degree of HCT-15 tumor growth suppression depended on the number of MRK16 injections. At more progressed stages, treatment with MRK16 alone showed little antitumor activity but when combined with Adriamycin resulted in significant suppression of tumor growth. The present results suggest that MRK16 may be useful for in vivo immunoscintigraphy and immunotherapy of multidrug-resistant colorectal carcinoma.

The analysis of internal image-bearing anti-idiotypic monoclonal antibody in relation to carcinoembryonic antigen.

Five anti-Id mAb (Ab2) were prepared from a BALB/c mouse immunized with anti-carcinoembryonic Ag (CEA) mAb MA208 (Ab1) in a syngeneic system. These anti-Id mAb appear to recognize unique idiotopes at the combining site of mAb MA208, because they were specifically reactive with mAb MA208 and showed the inhibitory activity against the binding of mAb MA208 to CEA. These anti-Id mAb were divided into three groups: group 1 (M7-625), group 2 (M7-413, M7-914), and group 3 (M7-049, M7-418), according to the analysis of anti-anti-Id antibodies (Ab3) induced with each anti-Id mAb (Ab2). Anti-anti-Id mAb M7-625 antisera (Ab3) reacted with purified CEA in binding assay and in Western blot analysis, and competed with Ab1 binding to CEA. Furthermore, the binding of anti-Id mAb M7-625 (Ab2) to mAb MA208 (Ab1) was inhibited with CEA, indicating that Ab2 mimicks the structure of the epitope in CEA which was recognized with Ab1. These serologic findings suggest that anti-Id mAb M7-625 carries the internal image of the Ag. According to the amino acid sequences of CDR 1, 2, and 3 of the mAb M7-625 variable region, there exists a homology of amino acid sequences between CDR2 in the H chain (5 amino acids of 10) and CDR3 in the L chain (3 amino acids of 9) of mAb M7-625 and domain III of CEA (545-554).

Establishment of multidrug resistant human colorectal carcinoma HCT-15 cell lines and their properties.

A series of MDR cell lines with various levels of P-glycoprotein have been established from a human colorectal carcinoma cell line, HCT-15, by stepwise exposure to adriamycin. The relative drug resistance of these cell lines correlated directly with both MDR1 mRNA levels and P-glycoprotein expression levels. Intracellular accumulation of adriamycin decreased inversely to their resistance. Drug sensitivities of these lines were reversed using verapamil. Since these cell lines are transplantable to nude mice, they may provide a useful animal model of MDR solid tumors for therapeutic experiments.

Detection of 300-kilodalton membrane protein in adriamycin-resistant human tumor cells by a monoclonal antibody MRK18.

To characterize the membrane changes related to adriamycin (ADM) resistance in tumor cells, we have developed monoclonal antibodies against an ADM-resistant subline of human myelogenous leukemia K562 (K562/ADM), and reported the overexpression of P-glycoprotein and 85-kDa protein as determined by the antibodies. In the present study, we have established a monoclonal antibody, MRK18, with higher reactivity to K562/ADM than to K562. MRK18 also showed higher reactivity to other human ADM-resistant lines, 2780AD and Hattori/ADM, than the corresponding parental lines. MRK18 also reacted to human breast cancer MCF-7 and human T-lymphoma CCRF-CEM which have never been exposed to anticancer agents in culture. MRK18 recognized a 300-kDa membrane protein of K562/ADM and MCF-7 and inhibited the growth of these cell lines in culture. These results indicate an induction of the 300-kDa protein during the development of ADM resistance.