Keiko Nishida

Elevation of serum lipid peroxide level associated with doxorubicin toxicity and its amelioration by [dl]-alpha-tocopheryl acetate or coenzyme Q10 in mouse (doxorubicin, toxicity, lipid peroxide, tocopherol, coenzyme Q10).

SummaryElevations of serum lipid peroxide levels were demonstrated in mice after an equitoxic dose of doxorubicin. When BDF1 mice were injected with doxorubicin (20 mg/kg body weight, IP), lipid peroxide levels in sera were elevated 1 day after the injection and the levels declined on subsequent days. 5-Fluorouracil (400 mg/kg body weight, IP) never changed the peroxide levels in serum. Furthermore, it was found that the co-administration of [dl]-α-tocopheryl acetate or coenzyme Q10 IM strongly inhibited the doxorubicin-induced elevation of lipid peroxides in serum.The effectiveness of [dl]α-tocopheryl acetate or coenzyme Q10 in reducing the lethality of doxorubicin in mice was also confirmed.These results indicate that the measurement of serum 2-thiobarbituric acid-reacting substances provides a useful measurement of lipid peroxide levels, a useful measurement of lipid peroxide levels, which may be involved in some way with doxorubicin toxicity, and that the administration of antioxidants provide protection against some of the side effects of doxorubicin.

Interferon‐γ differentially regulates susceptibility of lung cancer cells to telomerase‐specific cytotoxic T lymphocytes

There is accumulating evidence that peptides derived from the catalytic subunit of human telomerase reverse transcriptase (hTERT) are specifically recognized by CD8+ cytotoxic T lymphocytes. We investigated the cytotoxicity of a human leukocyte antigen (HLA)‐A*2402‐restricted hTERT‐derived peptide 461–469 (hTERT461)‐specific CD8+ T‐cell clone, designated as K3‐1, established from a healthy donor by repetitive peptide stimulation. This clone exhibited cytotoxicity against 4 out of 6 HLA‐A24‐positive lung cancer cell lines with positive telomerase activity but not 4 HLA‐A24‐negative examples. When the target cells were pretreated with 100 U/ml of interferon (IFN)‐γ for 48 hr, the susceptibility to K3‐1 increased with PC9 cells but unexpectedly decreased with LU99 cells. However, in both cell lines, the expression of molecules associated with epitope presentation such as HLA‐A24, transporters associated with antigen processing, low molecular weight polypeptide 7 and proteasome activator 28 was similarly increased after IFN‐γ treatment. Results of CTL assays using acid‐extracted peptides indicated that the epitope increased on PC9 cells but not on LU99 cells after IFN‐γ treatment. Semi‐quantitative reverse transcriptase polymerase chain reaction disclosed that the expression of hTERT was attenuated in LU99 but not in PC9 cells, accounting for the decreased cytotoxicity mediated by K3‐1. The attenuation of the hTERT expression and K3‐1‐mediated cell lysis after IFN‐γ treatment was also observed in primary adenocarcinoma cells obtained from pulmonary fluid of a lung cancer patient. Our data underline the utility of peptide hTERT461 in immunotherapy for lung cancer, as with other malignancies reported earlier, and suggest that modulation of hTERT expression by IFN‐γ needs to be taken into account in therapeutic approach.

Serological analysis of cell surface antigens of HL-60 cells before and after treatment with a phorbol ester tumor promoter

The human HL-60 cell line derived from acute promyelocytic leukemia, consisting of promyelocytic type of cells, was able to differentiate into adherent cells with monocytemacrophage features by the treatment with 12-0-tetradecanoyl phorbol-13-acetate (TPA). Cell surface antigens of HL-60 cells before and after TPA treatment were studied with monoclonal antibodies and four hybridoma clones producing IgM antibodies were established. Two antibodies (HL-21 and HL-47) reacted only with the immunizing TPA-treated HL-60 cells, and HL-1 antibody produced against untreated cells was reactive with both TPA-treated and untreated cells, but HL-5 antibody reacted predominantly with the immunizing untreated cells. Serological reactivity against various types of normal hematopoietic cells and acute leukemias (diagnosed by the French-American-British classification) was studied by immune adherence assay and immuno-electron microscopy. HL-21 antibody was reactive with monocytes and most cases of M4 and M5 types of acute non-lymphocytic leukemia cells. HL-47 antibody did not react with the cells of myelocyte-monocyte lineage or mature lymphocytes, but it did react with one-third of acute lymphocytic leukemia (L1 and L2) cases. Since all HL-47+ cases were included in the group of common ALL antigen positive cases, it was estimated that HL-47 is a differentiation antigen present on lymphocyte precursors, from which null-cell type acute lymphocytic leukemia cells generally originate. HL-1 antibody reacted with the cells of myelocyte-monocyte lineage as well as those of most acute non-lymphocytic leukemias. HL-5 antibody reacted with granulocytes and M2 type of acute myelocytic leukemia cases, and also with M5 type of acute monocytic leukemia cases. Serological studies of these antibodies revealed that TPA can induce to differentiate HL-60 cells not only into HL-21+ macrophage-like cells, but also into HL-47+ lymphoid stem cells. In addition, these antibodies were demonstrated to be very valuable for differential diagnosis of acute leukemias.

In vivo activity on murine tumors of a novel antitumor compound, N-β-dimethylaminoethyl 9-carboxy-5-hydroxy-10-methoxybenzo[a]phenazine-6-carboxamide sodium salt (NC-190)

SummaryA novel antitumor compound, N-β-dimethylaminoethyl 9-carboxy-5-hydroxy-10-methoxybenzo[a]-phenazine-6-carboxamide sodium salt (NC-190) was evaluated for its antitumor activity in experimental murine tumor systems. In the initial studies with P388 leukemia (i.p.-i.p.), NC-190 led to an increase of >200% in life span (ILS), and 75% of the mice were alive on day 30, when the optimal dose (50 mg/kg, days 1–5) was given. Additionally, the compound had significant activities against i.p. inoculated mouse L1210 leukemia, B16 melanoma, M5076 reticulum cell sarcoma, sarcoma 180, mouse hepatoma MH134, and rat Yoshida sarcoma and Yoshida ascites hepatoma AH130. The optimal dose resulted in a >280% ILS with a 30-day survival of 50% in mice with L1210 leukemia (100 mg/kg, days 1–5), a 156% ILS in mice with B16 melanoma (50 mg/kg, days 1–5), a 98% ILS with a 90-day survival of 25% in mice with M5076 reticulum cell sarcoma (25 mg/kg, days 1, 5, 9, and 13), a >300% ILS with a 60-day survival of 50% in mice with sarcoma 180 (50 mg/kg, days 3–10), a 148% ILS with a 60-day survival of 25% in mice with MH134 (25 mg/kg, days 1–5), a 129% ILS with a 60-day survival of 12.5% in rats with Yoshida sarcoma (12.5 mg/kg, day 3–10), and a >161% ILS with a 60-day survival of 50% in rats with AH130 (6.3 mg/kg, days 3–10). In the experiments with s.c. inoculated tumors, NC-190 not only inhibited tumor growth, but also increased the life span of mice with Lewis lung carcinoma or B16 melanoma. The 60-day survivors accounted for 60% and 30% in mice with Lewis lung carcinoma and B16 melanoma, respectively. The compound significantly inhibited the spontaneous lung metastasis of Lewis lung carcinoma by more than 90% when eight daily i.v. injections were given. NC-190 was active by the i.p., s.c., and i.v. routes. Five consecutive daily i.p. doses (days 1–5) were more effective than a single dose (day 1), two doses (days 1 and 5), or three doses (days 1, 5, and 9). NC-190 warrants further study as a potential antineoplastic agent against human neoplasms, as it has a broad spectrum of antitumor activity and inhibits metastasis.

Different Susceptibilities of Postmitotic Checkpoint-proficient and -deficient Balb/3T3 Cells to ICRF-193, a Catalytic Inhibitor of DNA Topoisomerase II

Two distinct types of Balb/3T3 cells were isolated which exhibit either 4 N DNA or both 4 N and 8 N DNA after exposure to colcemid for 48 h. They were found to differ with respect to the postmi‐totic checkpoint, but not the mitotic checkpoint. Firstly, the checkpoint‐proficient and ‐deficient cells exhibited the same accumulation and subsequent decrease in the number of mitotic cells following exposure to microtubule inhibitors. Secondly, after exit from abnormal mitosis in the presence of ICRF (Imperial Cancer Research Fund)‐193, the checkpoint‐proficient cells were arrested in the next cycle Gl, while the checkpoint‐deficient cells progressed into S and G2 phase. When either mitotic or asynchronous cells were exposed to ICRF‐193, the checkpoint‐proficient cells proved more sensitive to the cytotoxic effect of this agent than the checkpoint‐deficient cells. The different susceptibilities of the two types of cells to ICRF‐193 were not caused by variation in topoisomerase (topo) II function since both the biochemical activity of this enzyme and chromosome segregation were inhibited by similar concentrations of ICRF‐193 in both checkpoint‐proficient and ‐deficient cells. We propose that the inhibition of chromosome segregation by ICRF‐193 is monitored by the next Gl checkpoint, resulting in an irreversible Gl block in the case of postmi‐totic checkpoint‐proficient cells. As the checkpoint‐deficient cells can escape this Gl block, these cells have an increased survival capacity. In summary, ICRF‐193 may prove to be a very useful drug for examination of the postmitotic checkpoint.

Chapter 24 Serological and biochemical analysis of four antigens associated with small cell lung cancer

Abstract Four IgG1 monoclonal antibodies (Moab) reactive with cell surface antigens of small cell lung cancer (SCLC) were produced and serologically analysed. NE-25 and NE-150 Moabs showed an almost identical specificity, reacting with SCLC and other tumours of neural and/or (neuro)endocrine origin. NE-25 Moab precipitated an antigen of 25 kilodaltons (kD). In addition, a band of 150 kD was also recognized on certain cell lines. In contrast, NE-150 Moab precipitated only a band of 150kD. OE-130 Moab was produced to distinguish SCLC from non-SCLC. This Moab precipitated a main band of 130kD and three other molecules. PE-35 Moab detected a 35kD pan-epithelial antigen. Sixteen SCLC and 38 non-SCLC were studied with these 4 Moabs. Typical phenotype of SCLC was NE-25(NE-150) + /PE-35 + /OE-130 − , whereas that of non-SCLC was NE-25(NE-150) − /PE-35 + /OE-130 + . These Moabs were found to be useful for studying the heterogeneity and cell lineage of SCLC.

In vitro effects of a recombinant toxin, mSCF-PE40, targeting c-kit receptors ectopically expressed in small cell lung cancers

Most small cell lung cancers (SCLCs) ectopically express high levels of the c-kit receptor. We have examined if the receptor can serve as a target for a chimeric toxin, mSCF-PE40 composed of murine stem cell factor (SCF) genetically fused to the N terminus of a modified form of Pseudomonas exotoxin (PE) lacking its cell recognition domain. Selective cytotoxicity was found for human c-kit receptor-negative cells. This agent thus warrants further evaluation for therapy of human CSLCs.

Immunity against mouse thymus‐leukemia antigen (TL) protects against development of lymphomas induced by a chemical carcinogen, N‐butyl‐N‐nitrosourea

Mouse thymus‐leukemia antigens (TL) are aberrantly expressed on T lymphomas in C57BL/6 (B6) and C3H/He (C3H) mice, while they are not expressed on normal T lymphocytes in these strains. When N‐butyl‐N‐nitrosourea (NBU), a chemical carcinogen, was administered orally to B6 and C3H strains, lymphoma development was slower than in T3b‐TL gene‐transduced counterpart strains expressing TL ubiquitously as self‐antigens, suggesting that anti‐TL immunity may play a protective role. In addition, the development of lymphomas was slightly slower in C3H than in B6, which seems to be in accordance with the results of skin graft experiments indicating that both cellular and humoral immunities against TL were stronger in C3H than B6 mice. The interesting finding that B lymphomas derived from a T3b‐TL transgenic strain (C3H background) expressing a very high level of TL were rejected in C3H, but not in H‐2Kb transgenic mice (C3H background), raises the possibility that TL‐specific effector T cell populations are eliminated and/or anergized to a certain extent by interacting with H‐2Kb molecules.

Characterization of OM-B monoclonal antibody-defined antigen associated with mucinous type human ovarian tumor.

Partial characterization of the OM‐B antigen associated with mucinous‐type ovarian tumors was conducted. This antigen was defined by OM‐B monoclonal antibody, which was raised against a mucinous‐type ovarian tumor, and was present in all the mucinous‐type tumors tested, but only a fraction of serous‐type tumors. The OM‐B crude antigen preparation fractionated from cystic fluids had a density of 1.40–1.43 g/ml, with a high neutral sugar content. Molecular mass (Mr) estimated by gel filtration was more than 2,000,000. Trypsinization of the antigen preparation under appropriate conditions resulted in two major bands and one minor band with molecular sizes of less than Mr 250,000, as detected by immunoblotting. Immunoaffinity chromatography was then conducted and the amino acid composition of the purified product was determined; the high contents of serine, threonine and proline are characteristic of a mucin. Binding inhibition enzyme‐linked immunosorbent assay was developed to measure OM‐B antigen activity in cystic fluids and sera from patients with mucinous‐type tumors. The antigen was easily detected in most cystic fluids, but not in sera, suggesting that improvement in the sensitivity of this assay is necessary before its utilization for serum diagnosis will be feasible.

Screening of Monoclonal Antibodies against T Cell Differentiation Antigens Using Transfectants or Somatic Cell Hybrids

With the development of hybridoma techniques, a large number of monoclonal antibodies have been produced against cell surface antigens of human hematopoietic cells. In order to compare the specificity of these antibodies produced by various investigators, the ideal target cells are those which express only one or a few antigens on the cell surface. In our laboratories, as a first step to study the genes coding T cell differentiation antigens, transfection of mouse L cells with high molecular weight DNA from human T cells has been carried out and a stable transfectant expressing 41-Kd pan T antigen detected by the Tp40 antibody (1) was established. In addition, to study the chromosomal regulation of T cell differentiation antigens, human-mouse T cell hybrids have been produced and two clones retaining a few human chromosomes were found to be positive with Tp120 antibody (1), detecting the 120-Kd pan T antigen.