MDR1 promoter activity increases in response to various environmental stimuli, including anticancer drugs and in a manner that is dependent on the inverted CCAAT box. The binding activity of a nuclear factor MDR-NF1 that interacts with this promoter region was augmented when the nuclear extract was prepared from cells that had been treated with either UV or anticancer drugs. In an attempt to understand the molecular basis for the stress-dependent induction of MDR1 promoter activity, the cDNA for MDR-NF1 have been cloned. The amino acid sequence encoded by the cloned cDNA was identical to that of YB-1. The human Y-Box binding protein (YB-1) is a member of a DNA-binding protein family with a structurally and functionally conserved cold shock domain. YB-1 was found to be much higher in all cisplatin-resistant cell lines than that in the respective drug-sensitive parental counterparts. Two transfectants with a YB-1 antisense construct showed increased sensitivity to cisplatin, mitomycin C and UV irradiation, but not to vincristine, doxorubicin, camptothecin or etoposide. Thus, YB-1 may protect cells from the cytotoxic effects of agents that induce cross-linking of DNA, suggesting a novel function of this ancestor DNA binding protein. Regulatory mechanism of the YB-1 gene expression and the cellular functions of YB-1 are currently under investigation in relation to drug resistance.

Chemosensitivity test is useful in evaluating the appropriate cancer chemotherapy for solid tumors which are less sensitive to conventionally available antitumor agents. Most of assays used in clinics are in vitro assay, in which the cutoff concentration and contact time should be determined to mimic the clinical response of the chemosensitivity test. Since the antitumor effect of most of antitumor agents depends on their concentration x contact time, and the contact time will be determined according to the assay, the cutoff concentration will be calculated from the cumulative efficacy curve or 50% inhibitory concentration and in vivo response of human tumor xenografts in nude mouse. The clinical result obtained from chemosensitivity test was discussed.

There are several major groups of multidrug resistance mechanisms. 1) The multidrug resistant phenotype. 2) Glutathione S-transferences (GST) and detoxification mechanisms. 3) Topoisomerase I and II. 4) DNA repair. 5) Drug activation by cytochrome P450 (P450). In this article the biochemical functions of GST and P450 are described to show how individual enzymes contribute to resistance to carcinogens and anti-tumor drugs. Cancer cell lines indicated resistant to anti-cancer drugs, such as mitomycin C, doxorubicin, tamoxifen, cyclophosphamide and their derivatives, by a high activity of GST and a low activity of P450 in general. However, the mechanism of change of these enzyme activities is complicated and different in each drug. We show the study on the mechanism of multidrug resistance using cancer cell lines.

Some of anticancer drugs must undergo bioactivation in order to exert their anticancer activity. Those include most of antimetabolites, mitomycin C, cyclophosphamide and a camptothecin analog CPT-11. The latter two drugs are usually activated in liver or plasma, and the others are converted to active metabolites in the target cells. A number of biochemical mechanisms of resistance have been proposed and for the drugs to be activated in cancer cells impairment of activation enzymes were reported as one of the mechanisms; for example, deoxycytidine kinase for cytarabine and gemcitabine, orotate phosphoribosyl transferase for 5-fluorouracil, hypoxanthine-guanine phosphoribosyl transferase for 6-mercaptopurine, folylpolyglutamate synthetase for methotrexate and DT-diaphorase for mitomycin C were concerned.

Antimetabolic anticancer agents possess their own target enzymes: that of methotrexate is dihydrofolate reductase; 5-fluorouracil and ZD1604, thymidylate synthase; hydroxyurea, ribonucleotide reductase; 2'-deoxycoformycin, adenosine deaminase; N-(phosphonacetyl)-L-aspartate, aspartate transcarbamylase. Overproduction of each target enzyme has been observed with various animal and human cell lines which acquired resistance to all these agents. These facts suggest that this is a common mechanism for resistance to these agents. Most of these resistant cells showed amplification of the corresponding genes in double minute chromosome or homogeneously stained region of the chromosome. The relation between the degree of resistance and those of enzyme overproduction, the expression and amplification of the gene coding for each enzyme protein in various resistant cell lines are demonstrated and discussed.

ATP-binding cassette(ABC) superfamily transporters, including P-glycoprotein and MRP, actively transport various structurally dissimilar chemotherapeutic compounds out of cancer cells and confer multidrug resistance. Members of ABC superfamily which may extrude anti-cancer drugs are still expanding, thus the importance of these proteins are further increasing for cancer chemotherapy. Multidrug resistance will be acquired either by the induction of expression of ABC superfamily transporters or by mutations of ABC superfamily genes which cause amino acids substitutions. We recently found that amino acid substitutions in the first predicted transmembrane domain of P-glycoprotein increase the ability to confer resistance to important anti-cancer drugs adriamycin and VP-16. The mechanisms for drug recognition and transport of human P-glycoprotein and MRP are discussed.

Anticancer drugs are traditionally classified either by their mechanism of action or by their origins. Alkylating agents are reactive to DNA and cellular proteins and the primary mode of action is mostly through cross-linking of DNA strands, inhibiting replication of DNA and transcription of RNA. Some antimetabolites are structural analogs of normal molecular essentials for cell growth. After intaking into cells the analogues change to substances to interfere with DNA or RNA synthesis. Drugs derived from microorganism are called antitumor antibiotics. Some plants alkaloids blind to tublin and inhibit the formation of microtubules causing metaphase arrest, while camptothecins inhibit topoisomelase 1. Other compounds which are not classified to any categories expects characteristic mode for action to induce cell death or differentiation.

Individualizing the doses of cancer chemotherapy agents and progress in supportive therapy have improved the prognosis for elderly patients with non-Hodgkin's lymphoma. Prolonged hospitalization elderly patients has adverse effects, which include dementia, difficulty in walking, and depression. We treated 10 elderly patients (> or = 85 years) with non-Hodgkin's lymphoma as outpatients with oral etoposide, 25 mg or 50 mg daily for as long as possible, or until the white blood cell count decreased to < or = 2,000/microliter or the platelet count decreased to < or = 5 x 10(4)/microliter. Complete remission was achieved in 4 patients and partial remission in 4; the median duration of survival was 19 months. Adverse effects included leukopenia in 1 patient (< or = 1,000 cells/microliter), thrombocytopenia in 1 patient (< or = 5 x 10(4) cells/microliter), and anorexia in 1 patient. These results indicate that prolonged oral administration of low-dose etoposide is effective and safe for the treatment of non-Hodgkin's lymphoma in elderly patients. This outpatient chemotherapy caused no serious adverse reactions.

To estimate the relationship between the expression of thymidylate synthase (TS) in tumors and clinical response and prognosis in cancer patients treated with 5-fluorouracil (5-FU), the anti-TS polyclonal antibody against recombinant human TS (rhTS) was prepared and purified. At the initial stage, we attempted to clarify the relationships between the expression of TS protein and the cytotoxicity of 5-FU in established human cancer cells in vitro. Our purified anti-TS antibody was demonstrated to react specifically with intracellular TS as revealed by western blot analysis and immunohistochemistry. Furthermore, it was revealed that the expression of TS proteins correlated with cytotoxicity (IC50 value) of 5-FU against human colorectal tumor cells, both sensitive and those with acquired resistance to 5-fluoropyrimidines, and other cancer cell lines as well. These results suggest that our anti-TS polyclonal antibody (IgG) is suitable for clinical prospective and retrospective studies on TS expression in various cancers as a prognostic factor and 5-FU response-predicting parameter.

We report a 45-year-old male with chronic myelogenous leukemia (CML) who experienced skin ulcers of the left lateral malleolus and dorsum of both feet. He had been treated with hydroxyurea (HU) for 2 years. His leg ulcers improved after HU was discontinued. Skin biopsy of the ulcerated lesion revealed that the lesion is compatible with small vessel vasculitis, but circulating immune complexes (C1q, anti-C3d antibody) were negative. Although the precise mechanism of the skin ulcer is unknown, we must take into consideration the skin changes were secondary to hydroxyurea therapy in myeloproliferative disorders.

Echocardiographic reports on 144 adults receiving anthracycline therapy and 18 controls were reviewed for the possible relationship between dosage and ejection fractions. The cardiotoxicity of each anthracycline drug was evaluated as follows: Pirarubicin = 0.8, Mitoxantrone = 3.4, Daunorubicin = 0.5, Aclarubicin = 0, and Epirubicin = 0.6 with Doxorubicin = 1 as a control. As a whole, the ejection fractions, which decreased subsequently compared with increasing amount of dosage, showed a remarkable decrease at the dosage level of 600 mg/m2. However, the ejection fractions differed among individual patients. It was predicted that heart failure would not develop when the ejection fractions exceeded 55%. It is desirable to stop anthracycline therapy when the ejection fractions drop to 55%.

Ifosfamide chemotherapy was studied in 20 patients with advanced pancreatic carcinoma. It was administered at a dose of 1.2-1.5 g/body/day for 5 consecutive days every 3-4 weeks, and the patients treated over 2 courses were registered in our trial. Eleven patients had not received prior chemotherapy. Results achieved were as follows: PR in one patient, NC in 9 patients, and PD in 10 patients. The response rate was 5% and the median survival time was 17 weeks. Toxic effects included anorexia (80%), nausea and vomiting (65%), alopecia (20%), mental disturbance (20%), granulocytopenia (20%), thrombocytopenia (5%), and no renalurological disturbance. We concluded that ifosfamide was not effective for chemotherapy of advanced pancreatic carcinoma.

In order to establish an effective cancer chemotherapy, theoretical analyses of chemoresistance factors in recurrent patients and possible approaches to curative chemotherapy were undertaken. Multidrug resistance of tumor cells in recurrent patients may hamper the development of new antitumor agents. Thus, we developed a new screening method (FACS method) which employs fresh clinical specimens. By this method, TAS-103 was found to be far more effective than 10 standard drugs and to be one of the most effective drugs among 20 or more new antitumor agents under study. Development of several effective drugs, such as TAS-103, might lead to a curative cancer chemotherapy.

A chemosensitivity test using growth assay with MTT endpoint was recently evaluated in a retrospective clinical trial and was found to correlate to drug sensitivity, resistance and patient survival. In addition, there were no significant differences in the survival rates between "Resistant" group and surgery-alone group. In order to further investigate the potential of chemosensitivity test (Histoculture Drug Response Assay or HDRA) to contribute to patient survival, 215 patients with gastric cancer from 45 medical centers were tested with the HDRA in a blinded study after resection of the primary lesion. One hundred and sixty-eight patients received at least 20 mg/m2 of mitomycin C (MMC) and a minimum of 30 g/body of UFT, thereby making them eligible for the study. Of these cases 128 were evaluable by HDRA. The evaluable patient tumors were tested by HDRA with the [3H] thymidine incorporation endpoint measured by micro-autoradiography to be Drug "Sensitive" or "Resistant". Most importantly in the blinded study, the overall and disease-free survival rates of the HDRA "Sensitive" Group were found to be significantly higher than that of the HDRA "Resistant" Group tested under the above conditions. The results demonstrate that the HDRA response correlates to patient survival, which suggests the potential of the HDRA to contribute to patient survival in gastric cancer when used prospectively.

The antitumor activity of 1-(3-C-ethynyl-beta-D-ribo-pentofuranosyl) cytosine (ECyd) and 1-(3-C-ethynyl-beta-D-ribo-pentofuranosyl) uracil (EUrd), designed as a potential multifunctional antitumor nucleoside to inhibit RNA and DNA syntheses, was examined. ECyd and EUrd inhibited the growth of 47 kinds of cultured human cells in vitro and also showed strong antitumor effects on 15 human solid cancers xenografted into nude mice at a dose of 0.25 mg/kg (ECyd) or 2 mg/kg (EUrd) by intravenous administration for 10 consecutive days. The in vitro cytotoxic effect of ECyd and EUrd was prevented dose dependently by cytidine and uridine, suggesting that ECyd and EUrd may require phosphorylation by uridine/cytidine kinase for antitumor activity. ECyd and EUrd strongly inhibited RNA synthesis and slightly inhibited DNA synthesis. ECyd and EUrd have shown potent antitumor activity against human experimental solid type tumors with minimal toxic effects in vivo, suggesting that ECyd and EUrd is a promising agent with a unique mechanism of action for the treatment of cancer.

A NCS-chromophore (molecular weight: approximately 695) can be extracted from an antitumor antibiotic neocarzinostatin (NCS) (approximately 11,000, pI 3.3) with methanol as a DNA cleavaging molecule. Recently, the Goldberg research group (Harvard University, USA) proposed two distinct mechanisms of DNA cleavages (thiol-dependent DNA cleavage and thiol-independent base-catalyzed (bc) cleavage of ssDNA) by NCS-chromophore in vitro. Therefore, it is concluded that the ability of NCS-chromophore to cleave DNA is its primary action, which selectively inhibits DNA synthesis in cultured cells. Furthermore, we found that NCS-chromophore inhibits protein phosphorylation by CK-II (casein kinase II) involved in transcriptional regulation in a dose-dependent manner. Disruption of the repair systems of the NCS-chromophore-induced biological damages results in the positive induction of apoptosis, because the drug is greatly activated by thiols at the intracellular level, and it inhibits the activities of several transcriptional factors through their specific phosphorylation by nuclear kinases, such as CK-II. Taken together, all these biological and biochemical data suggest that the NCS-chromophore could be an effective chemotherapeutic drug for human cancer if its toxicity can be appropriately controlled.

Although evidence exists that intracellular signaling through oncogene products may play a role in the resistance of anticancer drugs, the underlying mechanism remains to be clarified. Using a HAG-1 cell line as a human epithelial model, we have studied the alteration of drug sensitivity and determined the mechanism of resistance induced in HAG-1 cells transfected with various oncogenes, which are highly expressed and activated in human cancers. Non-tumorigenic HAG-1 cells acquired fully neoplastic phenotype and resistance to Cisplatin (CDDP) by transfection with v-src but not by activated ras oncogene. The src-transfected cells showed a significant decrease in the formation of CDDP-induced DNA interstrand cross-links with rapid removal of these lesions. Upon treatment with src kinase inhibitors, the level of CDDP resistance and the rate of removal were both reduced in src-transformed cells. These results suggest the ability of v-src oncogene product but not ras to induce CDDP resistance by modulating the certain DNA repair pathway. Thus, intracellular signaling may be the appropriate target for strategies to reverse the drug resistance. In this review, we discuss the role of oncogenes important for signal transduction such as ras and those with protein tyrosine kinase activity in the drug resistance.

The gene transfectant with gamma-glutamylcysteine synthetase (gamma-GCS) gene, a rate limiting enzyme in GSH biosynthesis, exerted increased GSH content and ATP-dependent glutathione S-conjugate export pump (GS-X pump) decreased intracellular platinum and sensitivity against cisplatin. It is suggested that GS-X pump expression is related to cellular GSH metabolism and involved in cisplatin resistance. gamma-GCS transfectant also showed the resistance to MMC with the decreased DNA-MMC adduct. On the other hand, a MMC derivative, KW-2149, which is activated by thiol molecule, exhibited high cytotoxicity against the transfectant with an equal amount of DNA-MMC adduct compared with parental cells. It is suggested that KW-2149 could circumvent the GSH-mediated drug resistance.

The demonstration that RNA can be cleaved by cis ribozyme (catalytic RNAs, RNA enzymes) has potentially important therapeutic implications. Ribozymes are effective for modulation of gene expression because of their simple structure, site-specific cleavage activity, and catalytic potential. The targets of ribozyme-mediated gene modulation have ranged from cancer cells to foreign genes that cause infectious diseases. Additional target sites for ribozymes are in initial phases of development and design. Ribozymes have been targeted against myriad genes, including oncogenes (ras, BCR-ABL) and drug resistance genes (MDR-1, c-fos). These ribozymes have cleaved the target RNAs in culture system and developed to the in vivo system. We reported that fos ribozyme has altered the expression of c-fos and DNA repair genes in drug resistance cancer cells, and reversed the sensitivity to cisplatin. Furthermore, we have developed high efficiency by the transfer system in vivo.

Ramosetron hydrochloride as a 5-HT3 receptor antagonist-type antiemetic, which was developed in Japan. It has an indole ring which is the mother nucleus of serotonin (5-HT) and a tetrahydrobenzimidazol radical. These components are linked by a carbonyl radical. It was reported in non-clinical studies that ramosetron hydrochloride exhibited more potent and sustained antagonistic activities against 5-HT3 receptors than existing 5-HT3 receptor antagonist-type antiemetics. It was also reported that ramosetron hydrochloride inhibited vomiting by anticancer drugs in a potent and sustained manner. The phase I trial was initiated in May, 1991. Phase II and phase III trials were then conducted in a total of 357 patients at 121 institutions in Japan. The symptoms targeted in these trials were nausea and vomiting induced by anticancer drugs, such as cisplatin. Based on the results of the phase II trial, it was recommended that ramosetron hydrochloride be infected once daily at a dose of 0.3 mg. In phase III trial, a placebo-controlled double-blind study and an open trial was performed. The utility of the drug seemed to be confirmed by the results of these studies. Ramosetron hydrochloride shown an efficacy rate of 79.8% (178/223 patients) against nausea and vomiting induced by anticancer drugs, such as cisplatin when administered at a dose of 0.3 mg. The efficacy rate was 85.1% (40/47 patients) when given at a dose of 0.3 mg before the administration of anticancer drugs, such as cisplatin. The incidence of adverse effects was 2.0% (7/352 patients). Main adverse effects reported were feeling of heat, headache, and heavy feeling in the head. These adverse effects were of no clinical importance.