Metallothionein (MT), a metal binding protein induced by bismuth and other heavy metals, has been proved to have a potential to prevent toxic side effects of several antitumor drugs. It has also been demonstrated that the induction of MT in tumor tissues diminishes the antitumor activity of the drugs as well. These facts suggest a possibility that MT may play an important role in acquiring multi-drug-resistance in tumor cells. Thus, we were encouraged to examine the sensitivity of cultured cells overexpressing MT to various antitumor drugs which differ from each other in the mechanism of action. Cadmium-resistant cells (HeLa-R) were obtained from HeLa S3 cells (HeLa-S) by successive cultivation in a medium containing 100 microM CdCl2. MT level in HeLa-R was 180 times higher than that of HeLa-S. Comparison of the sensitivity of these two strains to the antitumor drugs, such as, cis-diamminedichloroplatinum (cis-DDP), adriamycin (ADR), peplomycin (PEP) and melphalan (MEL), revealed that HeLa-R was significantly more resistant to these drugs than HeLa-S. Further, there was no significant discrepancy between these strains, either in the level of major radical scavenging factors other than MT or in the cellular uptake of the drugs. The experimental results described above appear to support the hypothesis that MT may act as a multi-drug-resistance factor in tumor tissues.

Peripheral blood stem cells released following chemotherapy were examined in 21 children with malignancies in early remission. In order to obtain more than 1 x 10(5) CFU-GM per liter which is the minimal concentration to achieve autologous blood stem cell transplantation by cytapheresis, myelosuppressive chemotherapy which reduced leukocyte count below 1,000/microliters or neutrophil count below 200/microliters was necessary. Because the repetition of chemotherapy reduced the release of CFU-GM in peripheral blood, blood stem cells should be collected early after the beginning of chemotherapy. By long term culture method, peripheral blood stem cells seemed to contain pluripotent stem cells. Using our therapeutic protocol, more than 1 x 10(5) CFU-GM per liter could be obtained in malignant lymphoma and acute non-lymphoblastic leukemia, thus autologous blood stem cell transplantation seemed to be possible in these diseases.

Muroctasin, a derivative of muramyl dipeptide (MDP), increases WBC count through induction of the colony stimulating factor (CSF). This action of muroctasin is useful to treat cancer patients with leukopenia on chemotherapy. Urogenital cancer patients were randomized to either a 50 micrograms or 100 micrograms dose group of muroctasin when the WBC count dropped below 3,000/cmm in the first cycle of chemotherapy. The patients were then subcutaneously treated with muroctasin once daily for 6 consecutive days, and restoration of their WBC count determined. Out of the 32 patients who were enrolled in the study, 25, 24 and 23 patients were assessed for safety, usefulness and efficacy, respectively. The 100 micrograms dose was more effective in restoring the WBC count than the 50 micrograms dose based on the evaluation by the physicians. This result was also confirmed when we used the criteria for evaluation of adverse reactions as recommended by the Japanese guidelines for evaluation of efficacy of chemotherapy on solid tumors. Mild fever and reaction at the injection site were noted in 16% (4/25). In conclusion, muroctasin is useful for leukopenia after chemotherapy.

Cytochrome p-450 is a product of a multigene family, and catalyzes the activation and the detoxication of a wide variety of exogenous as well as endogenous compounds. Recent studies have the purified forms of cytochrome p-450 and provided evidence that some anticancer agents are metabolically activated by the cytochrome. In general, cancer cells express lower amounts of cytochrome p-450 as compared to normal liver cells. We recently succeeded in purifying P-450 HFLa, a form of cytochrome P-450 in human fetal livers. Examinations using antibodies to P-450 HFLa, however, showed that proteins cross-reactive with antibodies to P-450 HFLa existed in gynecologic malignancies. Development of multiple drug resistance is usually associated with a decrease in the content of cytochrome P-450, which is in contrast with glutathione S-transferase and a few other enzymes. The mechanisms responsible for such altered enzyme activity by multiple drug resistance are unclear as yet.

Glutathione S-transferase (GST) is a family of multimolecular forms with multi-functions for detoxication of drugs, and certain GST forms have been reported to concern multidrug resistance (MDR) mechanisms of neoplastic cells to anticancer drugs. In this paper, recent studies of GSTs concerning MDR are briefly reviewed, and the problems to be clarified are discussed. The reduced glutathione (GSH) is known to play important roles in the inactivation (detoxication) of the anticancer drugs. Most of them, especially alkylating agents, are conjugated with GSH by GSTs and detoxified, and the peroxides from drugs such as adriamycin are also reduced with GSH and detoxified by the GSH peroxidase activity of certain GST forms. Rat GST-P (GST 7-7) and human GST-pi, both of which belong to Class pi in the species-independent classification of GST, have been known as a marker enzyme for rat and human (pre) neoplastic lesions, respectively. GST-P is increased in rat hepatic preneoplastic foci resistant to cytotoxic agents. GST-pi is also increased not only in cancer cells such as colon carcinoma and non-small cell lung carcinoma, which exhibit "natural resistance" to anticancer drugs, but also increased in breast, ovarian and other tissue carcinomas with increased "acquired resistance" to certain drugs. A few research groups have attempted to confirm by transfection of a vector expressing a GST form such as GST-pi into non-resistant cell lines whether there is a direct relationship between the expression of a specific GST form and the appearance of MDR. However, MDR did not always appear. Recently, it was found in our laboratory that GST-P, GST-pi and even mouse GST II in Class pi, all are very strongly inactivated by SH-modifiers and active oxygens, indicating that these properties may be useful for overcoming MDR, if the forms really are involved with MDR.

Two monoclonal antibodies of F (ab')2 form, MRK 16 and MRK 20 that recognize P-glycoprotein and P85 kD protein respectively, were useful to detect multidrug resistant cells in human lymphoma, leukemia and gastrointestinal cancer cell lines. They were classified into 4 groups: Group I (4 cell lines) was insensitive to vinca alkaloids, anthracyclines, etoposide (VP-16) and actinomycin-D (ACT-D), and reactive to MRK 16 and MRL 20. Group II (2 cell lines) was insensitive to vincristine (VCR), but not reactive to both antibodies. Group III (3 cell lines) was insensitive to anthracyclines and VP-16, but sensitive to vinca alkaloids and ACT-D, and reactive to MRK 20 but not to MRK 16. Group IV (all other cell lines) was sensitive to these drugs, and not reactive to both antibodies. MRK 16 detects P-glycoprotein-associated multidrug resistance (MDR), while MRK 20 detects P 85kd-associated novel MDR. These monoclonal antibodies were useful for detection of MDR cells in clinical samples.

We have identified the proteins specifically expressed in multidrug-resistant tumor cells and studied the functions of these proteins. The 170-to 180-kDa membrane glycoprotein (P-glycoprotein) is an ATPase which works as a pump molecule transporting chemotherapeutic drugs outside the resistant cells. The 22-kDa soluble protein (sorcin) is a calcium binding protein of unknown function. The 85-kDa membrane protein is specifically expressed in adriamycin-resistant cells and induced by treatment with adriamycin, suggesting a mechanism unique for adriamycin resistance. Our monoclonal antibodies to these proteins may well become useful tools for the diagnosis of clinical drug resistance.

Since the discovery of several potent anticancer agents which are specific inhibitors of DNA topoisomerases, the key enzymes in the DNA metabolism, efforts to elucidate the mechanism of acquired resistance to these agents and to overcome them have been focused on these enzymes, the cellular targets of the anticancer agents. These trends were briefly reviewed.

Thirty two patients with hematologic malignancies and solid tumors were treated with intensive therapy and autologous bone marrow transplantation. In nine out of 32 patients, it took more than 50 days to achieve a sustained platelet count of 50,000/microliter or greater. Significant associations with poor platelet recovery were found for patient age, diseases, period of cryopreservation, the kinds of eradicative therapy and in vitro purging. But most of these factors overlapped each other in the same patients. No correlation was found between platelet recovery and number of cells or CFU-GM infused.

A fact that the Second International Conference of Intracavitary Chemotherapy was held at Sandiego in 1988 clearly suggests that the intraperitoneal chemotherapy attracts again the attention as a new administration route aimed at complete cure and useful modality against refractory intraabdominal cancer. Basic problems (peritoneal circulation, importance of peritoneal clearance, conditions required for ip infusion and penetration depth from the tumor surface) are described. As to ip infusion of CDDP, pharmacokinetic characteristics (iv vs ip), the difference of ultrafiltrable Pt. value (chemical assay vs biological assay) and clinical notes are reviewed. Several clinical trials now on going (CDDP-STS combination, CDDP-STS-Angiotensin II, CHPP and IPCP) are introduced. Finally the future direction of intraperitoneal chemotherapy is discussed.

Many drugs are applied in local treatment for skin malignant tumors. These drugs are living-BCG, OK-432, MY-1, WPG, interferon preparation (alpha, beta and gamma), TNF, IL-2, peplomycin, bleomycin and others. Some of them already have completed clinical trials and others are under clinical observation. In local administration of these drugs, skin lesions (malignant melanoma, CTL-mainly mycosis fungoides, carcinoma in situ and others) show good improvement. The effects were more observed in the tumors with diameters of 1 cm or less and appeared 3 to 10 injections in most cases. As complications, there are fever, general fatigue, vomiting, anorexia, leucopenia and others. Among them, the fever was most observed immediately after injections without any more severe complications. It may be concluded that treatment by intratumoral administration is useful for skin malignant tumors.

Intrathecal chemotherapy is inevitable method for the treatment of seeding of malignant brain tumors which appears in high incidence. The therapy is performed by means of lumbar tap, intraventricular or intratumorous administration, or ventriculo-cisternal perfusion. The same methods are applied to the treatment of original tumor mass as well. The main drugs used for the therapy are methotrexate and cytosine arabinoside. The causes of limitations of the method are the adverse effects of drugs and their short distance of diffusion penetrating into the tumor tissue. Local or intrathecal administration of Interferon could be reached high levels in the cerebrospinal fluid without severe side-effect. 7 out of 22 cases (31.8%) of malignant glioma responded to the local therapy with Interferon. Intrathecal Interferon therapy also revealed marked effect on the disseminated medulloblastoma. Intrathecal therapy should become much more important method including application to the immunotherapy in the future.

A human salivary adenocarcinoma cell line, HSGc, was tested for the chemosensitivity and compared with a human squamous cell carcinoma cell line, KB. From IC50 values of anti-cancer drugs on the cells, it was found that HSGc was sensitive to CDDP, 5-FU and DTIC but resistant to ADM, MTX, VCR, PEP and MMC as compared to KB. The chemosensitivity of HSGc was in agreement with previously reported clinical data on the therapeutic results of salivary gland tumors. This suggests that HSGc may be an useful model for understanding the biological response of salivary adenocarcinoma cells to anti-cancer drugs. We further examined the combined effects of filipin and verapamil with these anti-cancer drugs. Filipin was found to enhance cell-killing effect of 5-FU and PEP on HSGc, while the combination of filipin with either DTIC or PEP was also effective on KB. Verapamil was effective in combination with 5-FU, VCR or PEP on HSGc and with DTIC, VCR or PEP on KB. Especially the most predominant enhancement on HSGc was observed in combination of filipin or verapamil with PEP. These findings suggest that even low-sensitive drug, PEP, is also useful when combined with either filipin or verapamil.

We studied an antiproliferative effect of cancer chemotherapeutic agents, interferon (IFN) and, in particular, their combination effect on renal cell carcinoma. Either of colony formation assay (CFA) or [3H]-thymidine incorporation assay ([3H]-TdR assay) was employed as an in vitro chemosensitivity testing system. When compared, these two systems produced similar results with a good correlation (r = 0.97, p less than 0.01), in the antiproliferative effect of 30 drugs for 4 primary renal cell carcinomas and 5 xenotransplantable renal cell carcinomas. In vitro chemosensitivity test (CFA or [3H]-TdR assay) screened successfully only 5 "sensitive" drugs (7.9%) out of a total 63 cancer chemotherapeutic agents for 24 human renal cell carcinoma. This confirmed the findings reported by others. In the study of the antiproliferative effect of a cancer chemotherapeutic agent, human lymphoblastoid interferon (HLBI) and their combination on human renal cel carcinoma cell line (SMK-R2). Each of VBL, MTX or HLBI tended to suppress [3H]-TdR uptake in a dose-dependent manner. The combination of VBL (0.05 microgram/ml) and HLBI (10(2) or 10(3) IU/ml) produced a subadditive effect, and that of MTX (0.1 microgram/ml) and HLBI 10(2) IU/ml produced a synergistic effect on the human renal cell carcinoma cell line, the effect which is evaluated by Valeriote and Lin's criteria of combination. In particular, the synergistic effect by MTX and HLBI under the clinically achievable drug concentration seems important, when its clinical application is considered.

We took cultures of many human renal cell cancers and established two cell lines in vitro which were derived from the spindle cell carcinoma of a 67-year-old woman (MR-1) and from the dark cell carcinoma of a 74-year-old woman (MR-2). Using a nude mouse, we established the cell line in vivo (MRV) which was derived from the same patient as that for MR-1. MR-2 was maintained through 140 passages and MR-2 110 passages. MRV was maintained through 7 passages. With passaging, the characteristics, the doubling times and the chromosomal numbers of these cell lines changed, these changes were caused by the transformation, the mutation and the selection of the passaging cells. The study of the sensitivity test of the anti-cancer drugs to the established cell lines was done using the radioisotope uptake assay in vitro and the tumor volume changes in nude mice in vivo. We considered there were differences between the effective drugs used in vitro assay and in vivo assay. As to radioisotope uptake assay, it is difficult to judge the exact effect of the plant alkaloids, which inhibit the cell division and are dependent on the contact time, and we may overestimate the effect of the cytotoxic antibiotics. Further investigation on the methodology of the sensitivity test of the anti-cancer drugs would be necessary for the clinical application.

We studied in rats the anticancer drugs which can utilize more the advantage of bone marrow transplantation (BMT) and on their administration ways in high-dose chemotherapy (HC) with BMT. Among six anticancer drugs tested (ACNU, ADR, CY, MMC, VDS, VP-16), a beneficial effect of BMT was observed only with CY and ACNU. In order to increase the beneficial effect of BMT observed with CY and ACNU, the methods of administering these two drugs were carefully designed, and better survival curves were obtained in the following administration groups: 1) (CY 200 mg/kg, days 0 & 1) + BMT greater than (CY 400 mg/kg, day 0) + BMT, (ACNU 20 mg/kg, day 0 & 1) + BMT greater than (ACNU 40 mg/kg, day 0) + BMT. 2) (CY 200 mg/kg + ACNU 20 mg/kg, day 0) + BMT greater than (CY 400 mg/kg or ACNU 40 mg/kg, day 0) + BMT. 3) (CY 200 mg/kg, day 0) + (ACNU 20 mg/kg, day 1) + BMT greater than (ACNU 20 mg/kg, day 0) + (CY 200 mg/kg, day 1) + BMT. Further studies on anticancer drugs more appropriate for HC-BMT and on their administration methods were considered to be very necessary.