Yasunobu Kuraishi

Activation of C3G guanine nucleotide exchange factor for Rap1 by phosphorylation of tyrosine 504

C3G is a guanine nucleotide exchange factor for Rap1 and is activated by the expression of Crk adaptor proteins. We found that expression of CrkI in COS cells induced significant tyrosine phosphorylation of C3G. To understand the mechanism by which C3G is phosphorylated and activated by Crk, we constructed a series of deletion mutants. Deletion of the amino terminus of C3G to amino acid 61 did not remarkably affect either tyrosine phosphorylation or Crk-dependent activation of C3G. When C3G was truncated to amino acid 390, C3G was still phosphorylated on tyrosine but was not effectively activated by CrkI. Deletion of the amino terminus of C3G to amino acid 579 significantly reduced the Crk-dependent tyrosine phosphorylation of C3G and increased GTP-bound Rap1 irrespective of the presence of CrkI. We substituted all seven tyrosine residues in this region, amino acids 391–579, for phenylalanine for identification of the phosphorylation site. Among the substitution mutants, the C3G-Y504F mutant, in which tyrosine 504 was substituted by phenylalanine, was remarkably less activated and phosphorylated than the wild type. All the other substitution mutants were activated and tyrosyl-phosphorylated by the expression of CrkI. Thus, CrkI activates C3G by the phosphorylation of tyrosine 504, which represses thecis-acting negative regulatory domain outside the catalytic region.

Enhancement of Guanine-Nucleotide Exchange Activity of C3G for Rap1 by the Expression of Crk, CrkL, and Grb2

Crk is an adaptor protein that consists almost entirely of SH2 and SH3 domains. We have previously demonstrated, by using in vivo and in vitro systems, that C3G, which was identified as a Crk SH3 domain-binding guanine nucleotide exchange factor, specifically activates Rap1. C3G also binds to other adaptor proteins, including CrkL and Grb2. In the present study, we analyzed the effect of Crk, CrkL, and Grb2 on the C3G-Rap1 pathway. Expression of Crk, CrkL, and Grb2 with C3G in Cos1 cells significantly increased the ratio of GTP/GDP bound to Rap1. Both the SH2 and SH3 domains of Crk were required for this activity. However, Crk did not stimulate the guanine nucleotide exchange activity of C3G for Rap1in vitro, suggesting that Crk does not activate C3G by an allosteric mechanism. The requirement of the SH2 domain of Crk for the enhancement of guanine nucleotide exchange activity for Rap1 could be compensated for by the addition of a farnesylation signal to Crk, indicating that Crk enhanced the guanine nucleotide exchange activity of C3G by membrane recruitment of C3G. These results demonstrate that Crk, CrkL, and Grb2 positively modulate the C3G-Rap1 pathway primarily by recruiting C3G to the cell membrane.

Characterization of GFR, a novel guanine nucleotide exchange factor for Rap1

Three groups of Rap1‐specific guanine nucleotide exchange factors including C3G, CalDAG‐GEFI, and Epac/cAMP‐GEFI/II have been identified to date. In the present study, we report a new Rap1 guanine nucleotide exchange factor which we have named GFR ( uanine nucleotide exchange actor for ap1). GFR shows close sequence similarity to EPAC/cAMP‐GEFI/II although GFR lacks a cAMP binding domain and contains a nuclear localization signal. We demonstrated that GFR can activate Rap1 but not H‐Ras in 293T cells and that the cdc25 domain of GFR is required for the activation of Rap1. Northern blot analysis suggested that GFR mRNA is strongly expressed in the brain. In transfected HeLa cells, GFR has been found to be localized in the nuclei.

Intracellular carboxyl esterase activity is a determinant of cellular sensitivity to the antineoplastic agent KW-2189 in cell lines resistant to cisplatin and CPT-11

KW‐2189, a novel antitumor antibiotic belonging to the duocarmycins, possesses marked DNA‐binding activity upon activation by carboxyl esterase to its active form, DU‐86. Three duocarmycins, KW‐2189, DU‐86 and duocarmycin SA, were active against the cisplatin (CDDP)‐resistant human non‐small cell lung cancer cell lines PC‐9/CDDP and PC‐14/CDDP, and the multidrug‐resistant human small cell lung cancer cell line H69/VP. However, HAC2/0.1, a CDDP‐resistant human ovarian cancer cell line which is also resistant to CPT‐11 because of decreased intracellular activation of CPT‐11, was about 12.8‐fold more resistant to KW‐2189. HAC2/0.1 was not resistant to other duocarmycins as compared to its parental cell line, HAC2. There was no difference between HAC2 and HAC2/0.1 with regard to the intracellular accumulation of KW‐2189. Addition of 130 mU/ml of carboxyl esterase to the culture medium did not influence the sensitivity of HAC2 cells to KW‐2189. However, the sensitivity of HAC2/0.1 cells to KW‐2189 was enhanced to the level of HAC2. These results suggest that HAC2/0.1 is less potent than HAC2 in activating KW‐2189. The carboxyl esterase activity of whole‐cell and microsomal extracts from HAC2/0.1 was approximately 60% of that from HAC2. The cell‐free experiment revealed that KW‐2189 bound to DNA more efficiently in the presence of HAC2 than HAC2/0.1 cell extract. It was concluded that decreased intracellular carboxyl esterase activity in HAC2/0.1 cells caused decreased intracellular conversion of KW‐2189 to its active form, thus producing resistance to KW‐2189. The decreased conversion of CPT‐11 to SN‐38 in HAC2/0.1 cells might be explained by decreased carboxyl esterase activity.

A novel antitumor antibiotic, KW-2189 is activated by carboxyl esterase and induces DNA strand breaks in human small cell lung cancer cells.

KW‐2189 has been selected as a lead compound for clinical trial among duocarmycin derivatives with structural similarity to CC‐1065, a cyclopropylpyrroloindole. The purpose of this study was to examine the DNA‐binding potency and the mechanisms of cytotoxicity of KW‐2189. In order to analyze DNA‐binding activity of KW‐2189, plasmid pBR322 was treated with KW‐2189 with or without pretreatment with carboxyl esterase, which we demonstrated to be an activating enzyme, and the products were examined by agarose gel electrophoresis and restriction enzyme analysis. Cytotoxic activity was examined by exposing a human small cell lung cancer cell line, NCI‐H69 to KW‐2189 with or without carboxyl esterase. Alkaline elution was performed to examine whether KW‐2189 induces DNA strand breaks. DNA treated with KW‐2189 and carboxyl esterase migrated faster than KW‐2189‐treated DNA, which migrated at the same rate as untreated DNA. In addition DNA treated with esterase‐activated KW‐2189 was protected from digestion by some restriction enzymes. KW‐2189 showed concentration‐ and time‐dependent growth inhibitory effect with IC50 values (drug concentration required for 50% growth inhibition) of 58 nM (96 h) to 1900 nM (1 h) in H69 cells. The IC50 values of 4‐h exposure of H69 to KW‐2189 with 0, 26, 130, 650 mU/ml carboxyl esterase were 460, 120, 30, and 7 nM, respectively. Time‐dependent enhancement of cytotoxicity by carboxyl esterase was also observed. KW‐2189 induced DNA strand breaks in H69 cells in a concentration‐dependent manner around the IC50 value. We conclude that 1) KW‐2189 is activated by carboxyl esterase to its active form(s), 2) activated KW‐2189 has a stronger DNA‐binding activity and cytotoxicity than KW‐2189, 3) DNA cleavage is one of the major mechanisms of KW‐2189‐mediated cytotoxicity.

Phase II study of paclitaxel (BMS-181339) intravenously infused over 3 hours for advanced or metastatic breast cancer in Japan

A Phase II study of paclitaxel in patients with primary advanced or metastatic breast cancer was conducted by a cooperative study group consisting of 16 institutions in Japan. Paclitaxel at a dose of 210 mg/m2 was intravenously infused over 3 hours, along with premedication to prevent hypersensitivity reactions. The course was repeated at 21-day intervals. Of 62 eligible patients, 60 were evaluable for toxicity and 59 were evaluable for efficacy. Forty-five patients were previously treated with anthracyclines. Twenty-one of 59 patients (35.6%) had a major objective response including 2 CRs and 19 PRs (95% confidence interval, 23.6–49.1%). A response rate of 35.5% (CR1, PR10) was observed in 31 patients refractory to the anthracyclines containing prior metastatic chemotherapy. Median (range) time was 41 (6–100) days to onset of and median duration of response was 125 (36–305) days. Toxicities included leukopenia (grade 3, 4: 67%), anemia (grade 1–3: 80%), thrombocytopenia (grade 1: 8%), alopecia (grade 3: 43%), peripheral neuropathy (grade 1–3: 93%), arthralgia (59%), myalgia (46%), nausea and vomiting (40%), fever (33%), allergic reaction (grade 3: 2%) and hypotension (grade 3: 5%). All toxicities were tolerable and manageable. Paclitaxel intravenously infused over 3 hours demonstrated a significant antitumor activity for metastatic breast cancer. Furthermore, paclitaxel exhibited non-cross resistance to anthracycline. Paclitaxel administered as a convenient 3-hour infusion is effective for patients with metastatic breast cancer and has an acceptable toxicity profile.

Role of daunorubicin in the induction therapy for adult acute myeloid leukemia

PURPOSE To evaluate the relationship of total-dose of daunorubicin (DNR) to the induction therapy and treatment outcome, we have administered individualized doses of DNR during induction treatment to patients with acute myelogenous leukemia (AML). PATIENTS AND METHODS Ninety-two previously untreated adult patients with AML who entered our hospital were analyzed for the dose of DNR required to achieve complete remission (CR), the CR rate, disease-free survival (DFS), and overall survival (OS). Induction therapy consisted of DNR 40 mg/m2 daily intravenously from day 1 until the marrow was hypoplastic, cytarabine (Ara-C), prednisolone (PRD), and/or 6-thioguanine (6-TG). RESULTS Eighty-three of 92 patients with adult AML were assessable for this study. Sixty-three (76%) patients achieved CR. Fifty-two of 63 CR patients achieved the CR in the first course of induction therapy, and 11 patients required the second course of induction therapy. The 5-year and 10-year DFS rates were 31.2% and 5-year and 10-year OS rates were 45.1% and 42.3%, respectively. The median total dose of DNR in the induction therapy was 280 mg/m2 (120 to 480 mg/m2). DNR dose did not influence the response to therapy and was not influenced by the initial WBC count or French-American-British (FAB) system classification. CONCLUSION These results indicated that when the dose was linked to observed tumor response, the optimal dose of DNR in the induction therapy was approximately 280 mg/m2 (40 mg/m2 for 7 days), which is greater than the conventional dose of 40 to 60 mg/m2 for 3 days.

A Pharmacokinetic Study of Idarubicin in Japanese Patients With Malignant Lymphoma: Relationship With Leukocytopenia and Neutropenia

To clarify the pharmacokinetic properties of idarubicin (IDA) in Japanese patients and to clarify the relationship between the pharmacokinetic parameters of IDA or idarubicinol (IDAol), an active metabolite of IDA, and leukocytopenia or neutropenia, we examined the pharmacokinetics of IDA in patients with malignant lymphoma. Nine of 21 patients registered in an early phase II study of IDA were enrolled in the pharmacokinetic study. IDA (12 or 15 mg/mp2) was administered by intravenous infusion for 5 minutes. The elimination half lives (tb1/2) of IDA were 11.0 hours and 12.5 hours after administration of 12 and 15 mg/mp2 IDA, respectively. IDAol appeared rapidly both in plasma and in blood cells, and its concentrations exceeded those of IDA within 4 hours. IDAol had a very long tb1/2 (69.2 hours and 70.0 hours for 12 and 15 mg/mp2, respectively). The areas under the concentra-tion curves of IDAol in plasma were 3.4 and 5.8 times higher than those of IDA after administration of 12 and 15 mg/mp2 IDA, respectively.The tb1/2 of IDAol in plasma correlated significantly with the nadir of neutrophils, and the steady-state volume of distribution of IDA in plasma and in blood cells correlated significantly with the nadirs of white blood cells and neutrophils. These results suggest that both IDA and IDAol play an important role in leukocytopenia or neutropenia. No substantial differences between Japanese and Caucasian people in the pharmacokinetics of IDA were apparent.

A phase II study employing combination regimens containing KRN8602 in drug-resistant acute myeloid leukemia and acute lymphoblastic leukemia

We conducted a phase II multicenter study in order to evaluate the efficacy and toxicity of two combination regimens containing KRN8602 (MX2) for drug-resistant acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). AML was treated with KRN8602, 15 mg/m2 i.v. push for 5 days, and cytarabine (AraC), 100 mg/m2 by 24 h coutinuous infusion for 7 days. ALL was treated with KRN8602, 15 mg/m2 i.v. push for 5 days, vincristine (VCR), 1.4 mg/m2 i.v. push, once weekly, and prednisolone (PSL), 40 mg/m2, 3 h infusion for 5 days. In AML and ALL, the complete remission (CR) rate was 36.4% (16 of 44) and 24.1% (seven of 29), and the overall response rate (CR+PR) was 52.3% (23 of 44) and 51.7% (15 of 29), respectively. Among the 29 relapsed cases of AML, a higher CR rate, 51.7% (15 of 29), was obtained. A high incidence of nausea/vomiting and anorexia was observed, and some patients experienced central nervous system disorders and peripheral neuropathy. There was a low incidence of severe neurotoxicities; all other toxicities were manageable. KRN8602 was found to overcome drug resistance clinically, confirming results based on the preclinical studies. We conclude that KRN8602 is an effective novel anthracycline for drug-resistant acute leukemias.

A COMPARATIVE PATHOLOGICAL STUDY OF LIVER INJURY AFTER DIFFERENT COMBINATION CHEMOTHERAPIES FOR LEUKEMIA

A form of liver injury charcterized by bile ductule‐like transformation of hepatocytes, prominent hemosiderin deposition and fibrosis, was occasionally encountered in autopsy cases we examined during the period from 1973 to 1981. It was found that this liver injury was irreversible and intimately related to combination chemotherapy with the DCMP regimen (daunorubicin, cytosine arabinoside, 6MP and prednisolone) for acute non‐lymphocytic leukemia (ANLL). Its correlation was reconfirmed by the fact that this liver injury disappeared after withdrawal of the DCMP regimen in 1981. After 1978, the regimen of combination chemotherapy for adult ANLL was partly changed, 6‐thioguanine being utilized (DCTP) instead of the 6MP in the DCMP regimen. The hepatic injury occurring after the change in the regimen was different from that produced with DCMP, showing reversible intrahepatic cholestasis. These facts indicated that substitution for one drug in a combination chemotherapy regimen could cause a different type of hepatic change. ACTA PATHOL JPN 38: 1417–1432, 1988.