MDR1 gene encodes a membrane glycoprotein (P-glycoprotein) that acts as a energy-dependent pump to transport antitumor drugs out of the cells. P-glycoprotein, 1280 amino acids long, consists of two homologous parts of approximately equal length. The protein has binding sites for ATP, antitumor drugs and calcium channel blockers. MDR1 gene is expressed tissue-specific in human normal adrenal, kidney, liver and colon. The normal function and transcriptional regulation of this gene are also discussed.

SRCA was performed using surgically removed fresh tissues in 26 cases of lung cancer. The histological examination of day 6 xenograft showed that only a few xenografts contained tumor cells because of the host cell infiltration and stroma. For the assay considered as evaluable, we defined that control mice needed to show delta tumor size larger than -0.5 OMU and histological presence of tumor cell more than 50% in the day 6 xenograft. The evaluable assay rate (EAR) was no more than 25% (4/16). By the pretreatment with X-ray irradiation of 3 Gy, host cell infiltration was significantly suppressed. With this procedure however, only 40% (4/10) of EAR was gained. The reason for this low EAR was due to the difficulty to obtain the specimens enriched with tumor cells because of stroma and necrotic tissue. Then we used specimen obtained from the human lung cancer line xenografted in nude mice, which resulted in a high EAR of 84% (16/19). We concluded that SRCA for fresh surgical materials was still difficult. However SRCA for lung cancer line was feasible, especially for in vivo preclinical chemosensitivity test for new agents and decision for new drug combination. SRCA as a disease oriented chemosensitivity test is expected to develop in the future.

In order to determine some important cytological behavioral characteristics of endometrial cancer, I tried to establish endometrial cancer cell lines. Three endometrial cancer cell lines (KKNS-1, KKNS-2, KKNS-3) derived from the same patient have been established and successfully maintained in vitro for more than one year. The cells formed a monolayer in a mosaic fashion and pile up. Pathological findings for the tumor induced in athymic nude mice were: KKNS-1 was undifferentiated, KKNS-2 was differentiated and KKNS-3 was undifferentiated. Population doubling time was calculated to be about 35(KKNS-1), 60(KKNS-2), and 28(KKNS-3) hours. The modal chromosomal number for the cells fell in the diploid range. Estrogen receptor was demonstrated only in KKNS-2 by the ER-EIA and ER-ICA methods. HLA-expression was demonstrated, HLA-ABC was positive in all three lines and HLA-DR was positive only in KKNS-2. Anticancer drug sensitivity was demonstrated only in KKNS-3. Hormone sensitivity was demonstrated only in KKNS-2. We must therefore carefully treat endometrial cancer patients with anticancer drug or hormonal therapy whose pathological findings are heterogeneous.

When the plasma membrane capacity to maintain an ionic gradient is correlated with the cell viability, the anticancer drug-induced-cation (K+, Ca2+) mobilization, an early event associated with cell death, might be used as a rapid in vitro drug sensitivity test. A cyanine dye, dis-C3-5, was used to determine the membrane potential (Em), which was calculated as the following formula; Em = -RT/F In ([K+] in/[K+] out) in cancer cell lines. The change in cytoplasmic free calcium ion ([Ca2+]i) mobilization induced by the drugs was measured by fluorescent dye Fura2-AM. The results suggest that the sensitive drug, which showed greater than or equal to 50% inhibition of succinate dehydrogenase activity in SDI test, induced greater than or equal to 30% fall of membrane potential after 2 hour exposure to the drugs and also induced [Ca2+]i mobilization. On the other hand, the resistant drug showed no change of Em and [Ca2+]i.

Preoperative intra-arterial continuous chemotherapy combined with surgery in the treatment of malignant bone and soft tissue sarcomas has been used since 1968 in our clinic. This approach offers a theoretical advantage over systemic chemotherapy in that it delivers higher concentrations of chemotherapeutic agents to the tumor bed without increasing systemic toxicity. Tumor response depends on tumor sensitivity and the maximum amount of agent which is deliverable to the tumor tissues, so it is important to maintain a proper catheter placement to obtain good tumor response. 99mTc-Labeled macroaggregated albumin (99mTc-MAA) perfusion studies were carried out 19 patients who had histologically proven malignant bone and soft tissue sarcomas. The use of 99mTc-MAA perfusion studies in the treatment of intra-arterial chemotherapy offers an excellent way to evaluate catheter placement and tumor perfusion, in addition to providing a way to evaluate tumor A-Vshunts with lung scanning. The dynamic images of 99mTc-MAA obtained at each stage of the chemotherapy period also demonstrate the degree of tumor tissue response to intra-arterial chemotherapy. Therefore we conclude that 99mTc-MAA perfusion combined with preoperative intra-arterial infusion is a very useful method in cases involving limb salvage surgery.

The pharmacological properties of MCNU, methyl 6-[3-(2-chloroethyl)-3-nitrosoureido]-6-deoxy-alpha-D-glucopyranoside, were investigated in laboratory animals. MCNU had no effects on the central nervous, respiratory or the cardiovascular systems, but dilation of isolated auricular vessel was seen. No local anesthetic activity was demonstrated. Treatment with MCNU had practically no influence on the contraction of the isolated phrenic nerve-diaphragm, ileum, vas deferens or uterus. Furthermore, no effects on the passage of charcoal meal, size of the pupil and the contraction of nictitating membrane were observed. MCNU caused a reduction of leucocyte counts, suppression of immune responses, local irritation, suppression of blood coagulating activity and slight inhibition of gastric secretion. No definite effects were observed on blood glucose level or renal and liver functions. MCNU had no antiinflammatory and diuretic activities and did not cause hemolysis. Vomiting and diarrhea were observed by the administration of MCNU. In conclusion, the major pharmacological effects of MCNU are reduction of leukocyte counts, local irritation and immuno-suppression. The reduction of leukocyte counts induced by MCNU was more significant than that by chlorozotocin, but less than that by CCNU. Other effects may be considered somewhat weak or almost the same extent compared with these agents.

We developed a new method to rapidly measure the effectiveness of certain anticancer drugs by analyzing their effects on cell kinetics and cell cycle progression of labeled cells using simultaneous flow cytometric BrdU/DNA analysis. Three anticancer drugs, adriamycin (ADR), vincristine (VCR), and cisplatin (CDDP) were tested using the cultured cell line (MBT-2) originated from FANFT-induced mouse bladder tumor. The effects of these drugs were compared with percent colony survivals calculated by colony assay. The anticancer effects (IC90 levels) of ADR and CDDP could be determined by analyzing the effect of the drug on cell kinetics and cell cycle progression using the aforementioned method. However, in the case of VCR no relationship was found between the effect of the drug on cell kinetics and its anticancer effectiveness. Therefore, we must search for a better parameter for VCR, such as the inhibition rate of BrdU uptake.

A simple in vitro sensitivity test of oncolytic drugs has been applied in 60 cases of human ovarian cancer. Tumor tissue blocks were minced with a razor blade, after which the cell clumps were poured into a tissue culture medium, containing an anticancer drug with a certain concentration, and incubated at 37 degrees C. Nine to 16 kinds of drugs were tested for each specimen. After incubation, the cell clumps were dispersed for providing smear specimens. Typical morphological changes appeared in the nucleus, characterized by karyorrhexis and karyopyknosis. The individual tumors displayed different sensitivities to the various drugs. The positive rate of alkylating agents ranged around 30%, and specimens examined after oncolytic treatment displayed low sensitivity.

Forty-one patients with advanced gastric cancer underwent gastrectomy, and the correlation between tissue uptake of the adjuvant drug and the prognosis were studied. The patients were preoperatively administered Tegafur and samples of tissue were obtained intraoperatively. 5-FU levels in the tumor and lymphnodes were measured by gas chromato-mass fragmentography (GCMF). The patients measured for 5-FU tissue uptake were given more than 60 g of tegafur as postoperative adjuvant chemotherapy, and divided into two groups; one in which the 5-FU uptake by tumor tissue and lymphnode was over 0.05 microgram/g and the other lower than 0.05 microgram/g. In both groups there were no significant differences in background factors. Each survival rate was calculated by the Kaplan-Meier method, and the generalized Wilcoxon method was used for statistical analysis. There was no statistically significant correlation between 5-FU uptake by the tumor and the prognosis, but the 5-year survival rate in the group with over 0.05 microgram/g uptake by lymphnodes was statistically significant (p = 0.018).

Interactions between fluorinated Pyrimidine derivatives and anticoagulants were studied experimentally and clinically, using transplanted tumor tissues of Donryu rats and tissues from lung cancer patients. In rats, which were given 5-FU, uracil, tegafur and UFT respectively, the higher tumor concentrations of 5-FU and uracil were observed when given warfarin and ticlopidine beforehand, on the other hand, the concentrations of tegafur were almost the same between rats with and without anticoagulants. In patients with lung cancer, who were given UFT and anticoagulants, the higher concentrations of 5-FU and uracil in plasma, tumor and lymph nodes were observed than those who were given UFT alone. The 5-FU concentrations in normal lung tissues were about a half of those in tumor. These results suggest a existence of the synergistic effects between fluorinated pyrimidine derivatives and anticoagulants in plasma and tissue concentrations of 5-FU.

The ability of CSF-HU (P-100) to inhibit and improve the leukocytopenia and granulocytopenia which occur following cancer chemotherapy was investigated in a double-blind study which included an inactive placebo. The drug was administered, 2 vials/day (8 X 10(6)U of P-100), by intravenous drip infusion consecutively for 7 days starting from the 5th day after cancer chemotherapy was initiated. The total cases included in the study numbered 261. The efficacy rate was 53.0% for the P-100 group and 38.9% for the placebo group, while the usefulness rate was 54.8% for the former and 38.1% for the latter. In either case, statistically significant differences were observed in favor of the P-100 group. The reduction in the number of days before the leukocyte count returned to 4,000/mm3 was statistically significant in the P-100 group. While side effects appeared in both the P-100 (4.8%) and the placebo (4.4%) groups, none of them were considered to be serious. Judging from these results, p-100 was considered to be a useful therapeutic drug for the treatment of leukocytopenia and granulocytopenia following cancer chemotherapy.