Induction in vivo of resistance of C6 rat glioma and 9L rat glioma to ACNU [1-(4-amino-2-methyl-5-pyrimidinyl) methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride] was studied and ACNU-resistant rat meningeal gliomatosis models were developed by using these resistant glioma sublines. Rapid acquisition of resistance to the agent was present at 2nd transplant generation in both glioma lines. Cellular resistance to ACNU remained unchanged in the absence of drug over 5 transplant generations in vivo in spite of the fact that the drug treatment was discontinued at the 5th generation after a complete resistance was induced. On the other hand, the degree of resistance of 9L resistant subline established by only once ACNU treatment was found to be decreased after 5 transplant generations in vitro. Degree of resistance at the cellular level was observed with each subline by in vitro technique and compared with each other. Each subline was found to have different degree of resistance: 9L resistant subline showed higher resistance than C6 resistant subline, and the differences in degree of resistance between 9L resistant subline and C6 resistant subline were approximately 750 and 20, respectively, when they were expressed as ratios of IC50 (drug concentration for 50% growth inhibition) for the resistant subline to the original one.

The relationship between cell growth inhibitory effects and terminal differentiation-inducing effects of nine clinically active chemotherapeutic agents was evaluated using HL-60, a human acute myelogenous leukemia cell line. Continuous exposure to HL-60 cells of adriamycin, daunorubicin, mitoxantrone, m-AMSA, cytosine arabinoside, 6-thioguanine (6-TG), actinomycin-D (Act-D) and vincristine resulted in percentage increase of morphologically differentiated cells and phagocytic cells. Hydrocortisone at a concentration of up to 10(-4) M failed to produce this effect. Examination of actual numbers per flask of morphologically differentiated cells and phagocytic cells revealed, however, that all these agents except Act-D and 6-TG failed to produce a numerical increase in differentiated cells in comparison with those of control groups at day 6. Only Act-D and 6-TG were able to produce increases in the percentage, as well as actual number, of differentiated cells. These data indicate that the percentage increase in terminal differentiation-induced cells by certain chemotherapeutic agents is due not to their ability to increase differentiated cells but to their ability to inhibit the growth of primitive cells preferentially.

The antitumor effect of a new synthetic cord factor analogue, 6, 6'-Di-O-dacanoyl-alpha, alpha-trehalose (SS 554), was examined in vivo and in vitro. Remarkable life prolongation effects were observed after the intraperitoneal administration of SS554 at a dosage of 150 mg/kg in mice implanted with Ehrlich ascitic tumor cells. Using in vitro tests, the growth of various tumor cell lines i.e. Meth-A, EL-4, RL male-1, X5563, Raji and Jurkat were inhibited by SS554 at a 25 mcg/ml concentration. The mechanism of the direct antitumor activity of SS554 was studied using these tumor cell lines and bacterial cells. After treatment with SS554 at a 50 mcg/ml dose for 30 minutes, tumor cells and bacterial protoplasts but not intact bacterial cells were destroyed, indicating that SS554 causes the lysis of the membranes of these cells.

Two kinds of, serially transplantable human tumor cell lines were established in nude mice in ascitic form. One is breast cancer cell line, poorly differentiated adenocarcinoma, Hattori strain, and the other is acute lymphocytic leukemia cell line, T-cell type, Ichikawa strain. There exists a distinct correlation between the survival time of nude mice and the number of tumor cells transplanted. Clinically established antitumor agents which showed over 200% increase in life span were MMC, ADM and 5FU in Hattori strain, and VCR, VLB, VDS, ADM and BH-AC in Ichikawa strain. These results were considered to be consistent with the clinical effect of these drugs in breast cancer or in acute lymphocytic leukemia. Both strains can serve as the reliable screening system of antitumor agents.

The method of the human tumor clonogenic assay and its clinical correlations have been reported here. True positive rate for predicting of sensitivity to cytostatic drugs has been characteristically low. Results of our studies indicate that discrepancy of in vitro and in vivo results in clinical trials with the tumor clonogenic assay may be due to therapeutic heterogeneity among tumor colony-forming units within a primary tumor and between primary tumor and metastases.

In forty-eight samples from ovarian cancer patients, in vitro colony assay was carried out. The success rate of growing tumor colonies was 63% and the plating efficiencies was 0.06%. The in vitro sensitivity to standard drugs for the treatment of ovarian cancer was seemed to be compatible to that in clinical use. The predictive accuracy for sensitivity of this assay was 20% and 73% for resistance. Ovarian cancer cells can be formed colonies well and this assay method can be used for evaluation of various anticancer drugs in vitro.

Accurate in vitro anticancer drug sensitivity is helpful for successful chemotherapy of cancer patients. The purpose of the present study was to evaluate the tumor stem cell assay for the selection of effective drugs for individual patients with cancer and for the in vitro phase II screening of new anticancer drugs. Sixty-six specimens obtained from cancer patients were tested drug sensitivity by this assay. The results showed that this assay as an excellent technique for testing in vitro sensitivity of anticancer drugs against tumor cells from specimens removed from individual patients. The in vitro results correlated with in vivo response data in 93% of the instances observed in vitro-in vivo associations and a true negative rate was 100%.

An in vitro drug sensitivity test was developed to estimate the lethal effects of drugs on cancer cells. Cancer cells we incubated in the presence of radioactive precursors and anticancer drugs. The drug effects were estimated from the changes in rates of incorporation of precursors into DNA, RNA, and protein. Handling of a large number of samples became possible using a microtiter plate and a multiple automatic cell harvester. Incorporation of radioactive precursor showed linear correlation with drug-induced lethality. The results of this test were also correlated well with in vivo regression of tumors of mice. This test appeared to be more reliable than other similar test of cell lethality in vitro. For utilization in clinical studies, a test plate was simplified and 25 human cancers were tested. The results demonstrated the generally good correlation between in vitro and in vivo tumor sensitivities.

In vitro chemosensitivity test of human solid cancers, serially transplanted into nude mice was performed by detecting the inhibitory rate of 3H-thymidine incorporation to cancer cells. And the results of in vitro chemosensitivity test were compared with those in nude mice. This in vitro chemosensitivity test could present the quantitative results from a relatively small number of cancer cells within a short term (48 hours). Additionally, many samples could be handled at the same time by using microtestplates and a semiautomatic multiple cell harvester. The predictability of this in vitro chemosensitivity test to the in vivo effects was 80.6% (29/36) with 3 false positive and 4 false negative cases.

In order to improve the response rate of chemotherapy, we have investigated to the method for rapid and accurate prediction of the most appropriate anticancer agents. Thus, a rapid method using nude mice has been established as in vivo model for assessing chemosensitivity of individual human tumors, in which the final evaluation was made with 3H-thymidine incorporation and histological changes of tumor cells treated. Correlation between the sensitivity of anticancer agents and retrospective results of gastrectomy in stage IV gastric cancer was investigated. Our sensitivity test indicated a good correlation with clinical therapeutic and immunological effects. From these results, it seems reasonable to conclude that the sensitivity test using human tumor/nude mouse system may be useful for selection of appropriate agents to treat patients with cancer.

Pneumonitis-fibrosis which was induced by the treatment with antineoplastic agent(s) and/or irradiation was encountered in 37 (14.1%) of a total of 515 patients with lung cancer who had been treated in our institute during a period of seven years from 1976 through 1982. Of 251 patients who had been treated with bleomycin or pepleomycin alone or in combination with other antineoplastic agent(s) or irradiation, 46 (18.3%) had pneumonitis-fibrosis and 19 (7.6%) died therefrom. It was revealed that the patients over 50 years of age, whose PaO2 and % VC prior to the treatment with bleomycin were less than 79 mmHg and 79% respectively appeared to be predisposed to bleomycin pulmonary toxicity. Most of the pneumonitis which developed in these patients was progressive and fatal. Daily oral administration of 10 mg of prednisolone was in effective for the prevention of bleomycin-induced pneumonitis-fibrosis. A sudden decrease of PaO2 and a sharp elevation at a certain point in time during treatment were indicative of the fatal outcome of toxic pulmonary complications. Thoracic irradiation prior to, concomitant with or after bleomycin therapy enhanced the pulmonary toxicity of bleomycin. Therefore, combination therapy should be avoided. A continuous intravenous infusion may be the most effective and least toxic method to administer bleomycin.

Fourteen cases of adenocarcinoma with pleuritis carcinomatosa (lung cancer 10 cases, ovarian cancer 2 cases, colon cancer 2 cases) were treated with intra-pleural instillation of 7-N-(p-hydroxyphenyl)-mitomycin C (KW-2083), a derivative of mitomycin C. KW-2083 was administered at a dose of 40 mg once or twice weekly. In 14 evaluable patients, the rate of decrease of pleural fluid was 79%, and that of disappearance of tumor cells in pleural fluid was 64%. Median survival time (MST) from the beginning of treatment in all cases was 163 days. The toxicities of intrapleural instillation of KW-2083 were chest pain (86%), transient fever (64%), anorexia (64%), fatigability (29%), nausea (21%), vomiting (21%) and thrombocytopenia (nadir: 3.5 X 10(4)/mm3; 7%). Serum and pleural fluid KW-2083 concentrations have been measured in 3 patients after intrapleural administration of KW-2083. The mean half life of KW-2083 in serum and pleural fluid was 74 min (69-78 min) and 56 min (33-70 min), respectively. The peak serum KW-2083 concentration was 0.18 microgram/ml (0.06-0.24 microgram/ml).

Cancer chemotherapy is now the major modality to increase cure rate of cancer. In the past two decades systemic chemotherapy markedly improved the curability of several advanced malignancies in both adults and children. Several other cancers are also considered as curable by adjuvant setting. As for the low response group we must continue comprehensive approaches to improve response rate and duration. The knowledges of newer cancer biology provide us many ideas and targets of chemotherapy. The fundamental difficulties lie in cancer treatment are tumor side heterogeneity and host side individuality. Heterogeneity is now interpreted as the results of gene-instability and multi-oncogene involvement is of current interests. Thus in order to overcome these difficulties pharmacology-directed chemotherapy is now indispensable.

The relationship between the antitumor activity and the inhibition of thymidylate synthase after oral administration of 5-FU, FT-207 or UFT was examined. The inhibition of tumor growth on sarcoma-180 after oral administration of 5-FU (20 mg/kg), FT-207 (120 mg/kg) or UFT (30 mg/kg) was 40%, 50% or 70%, respectively. It was found that the inhibition of thymidylate synthase in sarcoma-180 tumor tissue after single oral administration of 5-FU (20 mg/kg), FT-207 (30 or 120 mg/kg) or UFT (30 mg/kg) were about 45%, 20%, 50% or 65%, respectively, but the activities of other enzymes involved in DNA synthesis were not almost inhibited. Thymidylate synthase was inhibited more severely by oral administration of UFT (30 mg/kg). The activities of other enzymes were not inhibited even by continuous oral administration of these drugs for 6 or 11 days. The extent of inhibition of thymidylate synthase seemed to be parallel to that of inhibition of tumor growth. These results suggest that the inhibition of thymidylate synthase by FdUMP converted from 5-FU, FT-207 or UFT plays a major role in their antitumor actions.

Certain chemotherapeutic agents against cancer have been used clinically as an immunosuppressive drug. In recent immunological advances it has been demonstrated that some chemotherapeutic agents can modify the antibody production and delayed-type hypersensitivity. In connection with modification of anticancer immune responses by the agents, many investigators have reported the increased production of NK cells in normal animals and of effector T cells in tumor-bearing animals, after administration of agents. Some authors have emphasized the possible role of the agent given before the tumor-inoculation for enhancement of host-mediated anti-tumor response on subsequent tumor growth. In general, these beneficial effects associated with the agents have been attributed to the abrogation of either suppressor T cells or macrophages. On the other hand, some reports including recent ones from this laboratory have described the production of tumoricidal macrophages by i.p. injection of the agent. The underlying mechanism (s), though not yet apparent, seems to be related to activation of macrophages. A small body of clinical suggests evidence suggesting immunoaugmentation by chemotherapy, but the therapeutic significance remains obscure. Though the immunoresponses , response components and the agents examined are heterogeneous and some results are restricted in a special experimental model, the immune response increase by anticancer chemotherapeutic agent in generally conceivable, and it should be further studied from the viewpoint of the host-mediated therapeutic effect.