The overall process of DNA biosynthesis can be divided into two major steps, one consisting essentially of nucleotide synthesis from low-molecular-weight metabolites and the other of polymerization of the nucleotides to form the duplicated DNA. Some antineoplastic agents are structural analogues of bases or nucleosides of intermediate metabolites, and are converted to their ribotides by enzymes catalyzing nucleotide metabolism. With some of these agents, the resulting ribotides then act as inhibitors of nucleotide synthesis. With others the resulting ribotides are subjected to stepwise enzymatic reactions and are then incorporated into DNA during its synthesis, thus rendering it inactive. Some antineoplastic agents, on the other hand, affect the DNA chain apparently through intercalation in double-stranded DNA, binding to DNA or nuclear protein, or interstrand linkage, or else through activation of endonuclease or inhibition of topoisomerase. The former effects result in inhibition of DNA double-strand dissociation, while the latter result in double-stranded DNA scission and apurinic acid formation. Antineoplastic agents thus vary widely, with respect to both the processes of their activation and inactivation and their effects on DNA synthesis. Their mechanisms of action and effects also tend to differ among various types of tumor cells and host organs. Investigation of the action mechanisms of these agents and determination of their appropriate utilization will be required in order to achieve better results in cancer chemotherapy.

In case of chemotherapy against brain tumors, it is most important to choose suitable drugs for brain tumors, since human tumors have different drug sensitivity and growth. Heretofore, the nitrosourea-induced rat glioma cell, such as C6, or immunodeficient mice were usually used for predicting the drug sensitivity of brain tumors. We took notice of Murphy's system for the chemosensitivity test, in which a human tumor is transplanted into the chorioallantoic membrane (CAM) of a chick embryo. By modifying the conventional Murphy's system, we studied the efficiency of this system in predicting the drug sensitivity of brain tumors. First, we compared the result of a drug sensitivity test using CAM of a chick embryo with that using nude mice. Next we studied the effect of chemotherapeutic agents against brain metastasis of a chick embryo caused by the intravenous injection of mouse B16 melanoma cells. The tumor reduction rate of the sensitivity test using a chick embryo tended to agree with that using nude mice. In the drug sensitivity test against brain metastasis, ACNU was the most effective. This result supports the result of the clinical study. In conclusion, the drug sensitivity test using a chick embryo is thought to be useful and the advantages or disadvantages of this system are discussed.

Recombinant human granulocyte colony-stimulating factor (Re Hu G-CSF) was prepared and its stimulating effect on granulocytopoiesis was examined in mice. Human G-CSF was purified to homogeneity from conditioned media of a G-CSF-producing cell line. The amino-terminal sequence was determined. By using oligonucleotides as probes, determined by the amino acid sequence, a cDNA library prepared from human macrophages was screened. The cloned G-CSF cDNA was expressed in E. coli K12MM294, and the mature protein was purified to homogeneity. Mice were given intraperitoneal injections of Re Hu G-CSF every day for 14 days. Peripheral blood granulocyte counts were examined 4, 8, 12 and 14 days after injection. Mice were sacrificed on the 14th day for histologic examinations of the bone marrow and spleen. Granulocyte counts began to increase on the 4th day and reached about 80,000/mm3 on the 14th day. Cells of granulocyte lineage were markedly increased in the bone marrow and spleen. Granulocyte precursors (CFU-C) were remarkably increased in the spleen. When mice were treated with 5-fluorouracil, cyclophosphamide or irradiation, the period of granulocytopenia was significantly shortened by subcutaneous injection of Re Hu G-CSF. These results suggest that human G-CSF play a central role in granulocyte production in vivo. The ability of Re Hu G-CSF to stimulate granulocyte production implies that this factor will be clinically useful in neutropenic patients treated with anti-cancer agents or irradiation.

The nature, type and mechanism of action of various colony-stimulating factors (CSFs) have been described. Among these CSFs, injection of recombinant human granulocyte CSF(rhG-CSF) caused a marked increase in neutrophils in mice as well as in monkeys. This neutrophilia in injected mice was preceded by a marked increase in hematopoietic precursors in hematopoietic organs. Injection of monkeys with rh granulocyte-macrophage CSF(rhGM-CSF) also induced a marked increase in peripheral blood neutrophils as well as eosinophilia and monocytosis. Injection of recombinant mouse interleukin 3(rmIL-3) caused a significant increase in peripheral blood eosinophils, neutrophils and lymphocytes. With rmIL-3, however, a remarkable increase was observed in various hematopoietic precursor cells in hematopoietic organs. In this study, both G-CSF and GM-CSF were shown to significantly shorten the period of neutropenia after irradiation and autologous bone marrow transplantation in monkeys. rhG-CSF was demonstrated to accelerate the recovery from neutropenia induced in mice and monkeys by 5-fluorouracil or cyclophosphamide. M-CSF purified from human urine, which has been reported to stimulate monocyte-macrophages to produce G-CSF, was demonstrated to be effective in accelerating the recovery from neutropenia in patients with various kinds of gynecological and urological malignancies after chemotherapy. It also accelerated the recovery from neutropenia after allogeneic as well as autologous bone marrow transplantation. These results indicate that CSFs are very effective for the treatment of neutropenia after cancer chemotherapy and bone marrow transplantation.

This is the histopathological analysis of 18 post-irradiated brains with metastases of breast cancers. There was no evidence of radiation necrosis, except for one with demyelinization and one with degeneration of nerve cells. There was no radiation damage in re-irradiated group. Whole brain irradiation of about 40 Gy may be safe, but that of 60 Gy or more may be not so safe. It may be more useful for preventing of radiation damage to take split-course-method or shrinking-technique at doses of 40 Gy or more. The combination of chemotherapy and radiation therapy seems to aggravate the course of radiation damage.

Six-day SRCA using normal mice developed by Bogden et al. is a promising in vivo chemosensitivity test. However, this method has a problem on the influence of the host reaction. We compared the tumor growth kinetics and host reaction between normal and nude mice. The tumor diameter increased until day 6 in normal and day 16 in nude mice, but the histological finding revealed many host reactive cells and few viable tumour cells on day 6 in normal mice, and well preserved tumour cells on day 16 in nude mice. These results were supported by flow cytometrical analysis. Autoradiogram using 3H-TdR showed a recovery of labeling index to the steady label by day 1. This index was similar between normal and nude mice. When antitumor activity of adriamycin, cisplatin or mitomycin C was compared with nude mice system, the order of effectiveness was the same as the system using nude mice implanted tumor cells subcutaneously and given the drugs intraperitoneally, but different in normal mice.

Six-day SRC assay as a chemosensitivity test has an advantage of high predictive rate for clinical response. However, it is pointed out that very few viable tumor cells are observed at the end of the assay, so that it may make the assay results unreliable. In this paper, we tested the effect of immunosuppressants on SRC assay using Walker carcinosarcoma originated from Wistar rat xenografted under the renal capsule of BDF1 mice. The changes of tumor size, pathological features and proliferative ability of xenografted tumor under the renal capsule of mice treated with cyclophosphamide, mizolibine or cyclosporin A are examined. Only cyclosporin A treatment could maintain the viable tumor cells and proliferative ability of the tumor grafted under the renal capsule 21 days after transplantation. In order to compare the original 6-day SRC assay developed by Bogden et al, we applied immunosuppressants to the 6-day assay. It is suggested that cyclosporin A and mizolibine amplify the sensitivity of tumor in 6-day SRC assay.

Chemosensitivity of liver cell carcinoma was studied by subrenal capsule assay. The method of assay was based on Bogden's one, but the antitumor activity was evaluated by tumor growth inhibition rate (TG-IR). The anticancer agent with more than 50% TG-IR was judged as positive in the chemosensitivity test. Of 3 human hepatoma cell lines transplanted in the subcutaneous space of nude mice, all of 3 were evaluable. The positive rates of ADR, MMC, CDDP, 5-FU and CPA were 66.7%, 100%, 66.7%, 100% and 0%, respectively. Of 24 patients who provided fresh tumor specimens for the assay, 12 (50%) were evaluable. The positive rates of ADR, MMC, CDDP, 5-FU and CPA were 25%, 16.7%, 16.7%, 33.3% and 8.3%, respectively. Our study suggested that 5-FU, MMC and ADR were comparatively active against the hepatoma cell, CDDP was less active than these 3 agents, CPA was inactive. These results seem to justify the use of current anticancer agents against hepatic cell carcinoma and indicate the usefulness of SRC assay for selecting chemotherapeutic agents against liver cell carcinoma.

Effort looking for new antitumor antibiotics useful for the treatment of curing cancer resulted to the discovery of a number of new compounds with newer action mechanism as well as newer structural feature. The antibiotics which have been discovered since 1984 are discussed under classifications of action mechanism and structural feature, as well. The first group, which belong to a novel class of antibiotics containing a bicyclodiynene carbon skeleton in the molecules exhibited the most strong anti-tumor activity comparing with the antitumor antibiotics so far discovered. The action mechanism of this was explained by the diradical formation of diynene-cyclization, which led to the scission of double strand DNA. Amongst, esperamicin A seems of great interest in view of the therapeutic development. Moreover, elsamicin A, a member of chatarin antibiotics, and FR-900482 compound, an antibiotic having a polycyclic alkalodal skeleton are under development for the new chemotherapeutic agents. Rhizoxin, the metabolite of Rhizopus chinensis is also a promising candidate as anticancer agent. Its action mechanism was classified as an inhibitor of mitosis by binding to the microtibline proteins. Rhizoxin A shows no cross resistance with vincristine. MX2 (KRN 8602), the morpholino derivative of 13-deoxo-10-hydroxy-carminomycin, shows anticancer activity against tumor cells resistant to P388/ADM as well as low cardial toxicity. Miscellaneous compounds whose action mechanism are unknown are described.

With the progress in surgical technique, remarkable improvement has been noted in the treatment of bile duct carcinoma. However, in the cholangiocarcinoma at porta hepatis or in the progressive carcinoma, many cases have been reported, for which radical surgery is not achievable. In recent years, discussion has been concentrated on the necessity of multidisciplinary treatment for the bile duct carcinoma, but fundamental research has not been done enough. In the present paper, the process for obtaining CHGS strain implantable to the nude mouse derived from a human cholangiocarcinoma as achieved in our department was discussed, and its biological characteristics-above all, the sensitivity to carcinostatic agents and to radiation-were evaluated. The doubling time of CHGS strain is 6.2 days, and nude mice showed stable proliferation with 100% viability. Histologically, it was tubular adenocarcinoma similar to the primary tumor. It has high mucin producing ability, and necrosis hardly occurs. The search for DNA ploidy by flow cytometry revealed the presence of two types of cells: The cells of diploid pattern and aneuploid pattern. In the tests to determine the sensitivity of CHGS strains to carcinostatic in MMC, ADR, 5-FU and CDDP groups, and to radiation according to the Battele Columbus Laboratories Protocol, the regression of tumor was observed in MMC, ADR, CDDP groups. Particularly, in MMC group, some of the tumors had disappeared. Recurrence was also noted in this case, but the survival, was still recognized nearly four years after the operation through the postoperative auxiliary therapy. This was regarded as the case, where the sensitivity test using the nude mouse implantable tumor strain was reflected well in clinical application.

DNA damages caused by various anticancer nitrosourea compounds such as ACNU and MCNU were studied. Reiterated fragments of 167 and 203 base pairs (bp) were obtained after Hind III and Hae III restriction endonuclease digestion of 9L rat brain tumor DNA. The end-labeled reiterated fragments were reacted with ACNU and MCNU, which resulted in the scission breaks corresponding to the locations of guanine on an extended Maxam-Gilbert sequencing gel. Subsequent piperidine hydrolysis yielded scission products more frequently. These results indicate that nitrosoureas such as ACNU and MCNU generate DNA scission breaks and/or alkali-labile sites preferentially at the position of guanine moieties in rat brain tumor DNA.

We carried out a total of 36 in vivo chemosensitivity tests in 33 cases of human malignant tumor using the subrenal capsule assay, developed by A.E. Bogden et al. Of the 36 assays, 31 were evaluable. The chemosensitivity of each tumor varied individually. UFT, 5-fluorouracil, mitomycin-C and adriamycin were administered to gastrointestinal cancer patients regularly, but our SRC-assay showed a high sensitivity rate for UFT and 5-fluorouracil but a low sensitivity rate for mitomycin-C and adriamycin. Nine patients had clinically evaluable lesions and a correlation between the assay results and clinical response existed in 6 cases. The true positive rate was 50% (3/6), the true negative rate 100% (3/3), and the overall predictive accuracy 66% (6/9). This study suggested that 6-day SRC assay is useful for selecting effective anti-tumor agents for the treatment of cancer patients.