KF25706, a novel oxime derivative of radicicol, exhibits in vivo antitumor activity via selective depletion of Hsp90 binding signaling molecules.

Radicicol, a macrocyclic antifungal antibiotic, has been shown to bind to the heat shock protein 90 (Hsp90) chaperone, interfering with its function. Hsp90 family chaperones have been shown to associate with several signaling molecules and play an essential role in signal transduction, which is important for tumor cell growth. Because radicicol lacks antitumor activity in vivo in experimental animal models, we examined the antitumor activity of a novel radicicol oxime derivative, radicicol 6-oxime (KF25706), on human tumor cell growth both in vitro and in vivo. KF25706 showed potent antiproliferative activities against various human tumor cell lines in vitro and inhibited v-src- and K-ras-activated signaling as well as radicicol. In addition, Hsp90 family chaperone-associated proteins, such as p185erbB2, Raf-1, cyclin-dependent kinase 4, and mutant p53, were depleted by KF25706 at a dose comparable to that required for antiproliferative activity. KF25706 was also shown to compete with geldanamycin for binding to Hsp90. KF29163, which is an inactive derivative of radicicol, was less potent both in p185erbB2 depletion and Hsp90 binding. More importantly, KF25706 showed significant growth-inhibitory activity against human breast carcinoma MX-1 cells transplanted into nude mice at a dose of 100 mg/kg twice daily for five consecutive i.v. injections. KF25706 was also shown to possess antitumor activity against human breast carcinoma MCF-7, colon carcinoma DLD-1, and vulval carcinoma A431 cell lines in vivo in an animal model. Finally, we confirmed the depletion of Hsp90-associated signaling molecules (Raf-1 and cyclin-dependent kinase 4) with ex vivo Western blotting analysis using MX-1 xenografts. In agreement with in vivo antitumor activity, KF25706 depleted Hsp90-associated molecules in vivo, whereas KF29163 and radicicol did not show this activity in vivo. Taken together, these results suggest that antitumor activity of KF25706 may be mediated, at least in part, by binding to Hsp90 family proteins and destabilization of Hsp90-associated signaling molecules.

T-cell large granular lymphocytic leukemia occurring after autologous peripheral blood stem cell transplantation.

A 61-year-old man with angioimmunoblastic lymphoma in first complete remission underwent autologous peripheral blood stem cell transplantation. At 1 month post transplant, asymptomatic large granular lymphocytosis developed. The surface marker profile of the cells was CD3+CD8+CD56-CD57+. The disease course was chronic and indolent. The patient remains in complete remission from angioimmunoblastic lymphoma more than 6 months post transplant with persistent large granular lymphocytosis (lymphocyte count, 5-15 x 10(9)/l). Although post transplantation T-cell lymphoproliferative disorders have mostly occurred in allogeneic transplantation recipients and presented as aggressive lymphomas/leukemias, we suggest that chronic indolent T-cell large granular lymphocytic leukemia can occur after autologous stem cell transplantation.

Amino acid sequence of silkworm (Bombyx mori) hemolymph antitrypsin deduced from its cDNA nucleotide sequence: confirmation of its homology with serpins.

A cDNA clone of silkworm (Bombyx mori) larval hemolymph antitrypsin (sw-AT) has been isolated from a fat body cDNA library. The cDNA has an open reading frame which codes a 392-amino acid residue polypeptide comprising a 16-residue signal peptide and a 376-residue mature sw-AT of Mr 41,805. The reactive site of sw-AT for inhibition of bovine trypsin [Sasaki, T. et al. (1987) J. Biochem. 102, 433-441] was identified as Lys343-Val344. Alignment of the sw-AT amino acid sequence with those of 11 members of the serpin superfamily of proteins clearly confirmed the homology of sw-AT with serpins. The amino acid sequence of sw-AT is 56% identical with that of the proteinase inhibitor from a lepidopteron, Manduca sexta [Kanost, M.R. et al. (1989) J. Biol. Chem. 264, 965-972], but the sequence around the reactive site shows no homology and the inhibitory specificity for proteinases is very different.

Purification, some properties, and primary structure of a base non-specific ribonuclease from oyster (Crussdstrea grigus).

A ribonuclease (RNase Oy) was purified to homogeneity on SDS-PAGE from the homogenate of oyster (Crussdstrea grigus). The apparent molecular weight estimated from SDS-PAGE was ca. 28 kDa. The pH optimum of the RNase was 5.0. The RNase released mononucleotides from RNA in the order of 3'-GMP, 3'-AMP, and 3'-UMP. The complete amino acid sequence of RNase Oy was determined, mostly by analyzing the peptides generated by BrCN cleavage or digestion by lysylendopeptidase, staphylococcal V8 protease, and alpha-chymotrypsin. The molecular weight of the protein moiety of RNase Oy deduced from the sequence was 24,359. The sequence of RNase Oy contained two typical histidine residues in segments common to the active site of RNase T2 family enzymes. The locations of six half cystine residues among eight were almost superimposable on those of four known plant RNases of RNase T2 family. The sequence homology between RNase Oy and five fungal and four plant RNases amount, to 43-56 amino acid residues. The amino acid sequence of the N-terminal part of RNase Oy is more similar to those of plant RNases than to those of fungal RNases. This RNase is the first RNase T2 family RNase from mollusc whose primary structure has been elucidated.

Graft rejection and recovery of host-derived hematopoiesis after allogeneic bone marrow transplantation: relation to conditioning with high dose etoposide and total body irradiation.

Engraftment failure following allogeneic bone marrow transplantation (BMT) is rare in patients with acute leukemia, after frequent conditioning with marrow-lethal chemoradiotherapy. We evaluated the efficacy of a preparatory regimen consisting of fractionated total body irradiation (TBI) (12 Gy in six fractions) and high dose etoposide (60 mg/kg) administered as an 8-h infusion for allogeneic BMT in 16 consecutive patients with acute leukemia. Although 14 patients showed complete and sustained engraftment, the remaining two patients rejected bone marrow grafts from HLA-identical sibling donors and showed subsequent recovery of host-derived hematopoiesis. Despite the limited number of patients, this observation suggests that the immunosuppressive potential of etoposide may be inferior to that of cyclophosphamide (CY) and that etoposide as an alternative to CY as an antileukemic and immunosuppressive agent in allogeneic BMT may increase the risk of graft rejection.

Development of interferon-alpha resistant subline from human chronic myelogenous leukemia cell line KT-1.

OBJECTIVE: Interferon-alpha (IFN-alpha) is one of the most effective therapeutic agents for a number of hematological malignancies, including chronic myelogenous leukemia (CML). Nevertheless, its efficacy is limited because of the development of resistance to IFN-alpha therapy. Previously, we established the novel human CML cell line KT-1, which is sensitive to the antiproliferative effects of IFN-alpha. Here, we report the establishment of an IFN-alpha-resistant subline, KT-1/A3R alpha 1000, by culturing KT-1/A3 cells (IFN-alpha-sensitive subline of KT-1) with increasing concentrations of IFN-alpha, in order to analyze the mechanism of acquisition of IFN-alpha resistance in CML cells after IFN-alpha therapy. SUBJECTS AND METHODS: We developed an IFN-alpha-resistant tumor cell variant, KT-1/A3R alpha 1000, from the KT-1/A3 cell line by culturing cells with increasing concentrations of IFN-alpha. This subline was examined for its ability to proliferate and its resistance to apoptosis in high concentrations of IFN-alpha. The induction of the ISGF3 complex in response to IFN-alpha alpha in KT-1/A3R alpha 1000 was compared with that in the parental cell. RESULTS: The levels of interferon-stimulated gene factor 3 components (STAT1, STAT2, and p48) proteins and STAT2 tyrosine phosphorylation induced after IFN-alpha treatment were unchanged, but formation of the ISGF3 complex was remarkably reduced in KT-1/A3R alpha 1000 cells compared to parental cells. CONCLUSION: The KT-1/A3R alpha 1000 subline is a useful model for studying the mechanism of IFN-alpha resistance after IFN-alpha therapy.

Successful treatment of dark-colored epidermal nevus with ruby laser.

The pulsed ruby laser has a selective thermolytic effect. Recently, it has been available for the treatment of superficial pigmented disorders. We studied 5 cases of epidermal nevus treated with the pulsed ruby laser. In comparison with the usual methods including electrocautery, cryotherapy and skin abrasion, ruby laser therapy is an excellent tool due to technological ease and rapid improvement. Depigmentation after treatment in 2 cases was the only side effect of this therapy. Bose cases had a dark pigmentation of the skin. Despite of the risk of discoloration, the ruby laser is one of the most effective tools for therapy of pigmented epidermal nevus.

A combination of Laserthermia and chemotherapy for the treatment human gastric cancers transplanted into nude mice.

The effects of Laserthermia plus chemotherapy on a human gastric cancer transplanted into nude mice was investigated. Three different treatment regimes were tested. The relative tumor growth rate according to the Battele Columbus Laboratories protocol, was as follows. 5-FU chemotherapy, 54%; Laserthermia, 31%; and combined treatment with 5-FU and Laserthermia, 21%. The greatest degree of tumor regression was achieved in the combined therapy group. It is suggested that the combination therapy of Laserthermia plus antitumor agents may be useful in the treatment of gastric cancer in humans.

[A combination chemotherapy with cytarabine ocfosfate, low dose etoposide, and G-CSF for the treatment of high risk MDS (RAEB, RAEBt)--a pilot study].

We evaluated a combination chemotherapy with cytarabine ocfosfate (SPAC), low dose etoposide and G-CSF for the treatment of high-risk MDS (RAEB, RAEBt). Seven patients with high-risk MDS were treated with a daily combination, 200 mg/day SPAC p.o., 50 mg/day etoposide p.o. and 75 micrograms/day G-CSF s.c. One patient achieved complete response, 2 achieved good response and one patient minor response. Although all of the patients developed severe marrow hypoplasia after chemotherapy, the nonhematologic adverse effects were mild enough to be tolerated. This combination chemotherapy should be useful in the clinical management of patients with high-risk MDS.

Effects of valsartan and amlodipine on home blood pressure and cardiovascular events in Japanese hypertensive patients: a subanalysis of the VART.

The Valsartan Amlodipine Randomized Trial (VART) was performed to compare the beneficial effects of valsartan and amlodipine on cardiovascular events in Japanese hypertensive patients. In this subanalysis of the VART, we assessed the relationship between home blood pressure (HBP) levels and cardiovascular events in the enrolled patients. We enrolled 1021 patients with mild-to-moderate hypertension in the VART. The participants were allocated randomly to either the valsartan group or the amlodipine group. The primary end point was a composite of all-cause death, sudden death, cerebrovascular events, cardiac events, vascular events and renal events. A total of 621 patients (valsartan group: 305 and amlodipine group: 316) completed the measurements of HBP (morning and evening) throughout the trial. Both the agents evenly and significantly lowered morning HBP and evening HBP throughout the trial. There was no significant difference in the primary end point between the two groups. However, we observed significant decreases in the left ventricular mass index and urinary albumin to creatinine ratio in the valsartan group but not in the amlodipine group. There were no significant differences in HBP levels and the main outcome of the cardiovascular events between the valsartan and amlodipine groups. However, in the valsartan group, significant improvements in left ventricular hypertrophy and microalbuminuria were observed.

Sensitivity to interferon-alpha and interferon-stimulated gene factor 3 binding activity in human chronic myelogenous leukemia cell line KT-1.

The mechanism of responsiveness of chronic myelogenous leukemia (CML) cells to interferon (IFN)-alpha was examined by using two subclones of CML cell line KT-1 which exhibited significantly different sensitivities to the antiproliferative and apoptosis-inducing effects of IFN-alpha. IFN-stimulated gene factor 3 (ISGF3) formation by IFN-alpha was reduced in the IFN-alpha-resistant subclone compared to the IFN-alpha-sensitive subclone. We conclude that the level of ISGF3 formation is responsible for the difference in IFN-alpha responses between these subclones.

Molecular cloning of silkworm (Bombyx mori) antichymotrypsin. A new member of the serpin superfamily of proteins from insects.

The cDNA of silkworm (Bombyx mori) antichymotrypsin (sw-Achy) was cloned from larval fat body and its nucleotide sequence was determined. The deduced amino acid sequence of mature sw-Achy begins with Phe1 and ends with Phe384, with a preceding 16-amino-acid signal peptide. The amino-acid sequence similarities of sw-Achy with the serine-proteinase inhibitors (serpins) silkworm antitrypsin, tobacco hornworm alaserpin, human alpha-1-antitrypsin and human alpha-1-antichymotrypsin were 29.6%, 30.3%, 26.1%, and 25.0%, respectively. The highly conserved amino acids in other serpins are also conserved in sw-Achy. sw-Achy is thought to be a new member of the serpin family. Multiple alignment of sw-Achy with 23 other kinds of serpin by the progressive method produced a phylogenetic tree in which all four insect serpins are grouped separately within one branch. The reactive site of sw-Achy with alpha-chymotrypsin was identified as Thr343-Ser344 by direct amino-acid sequence analysis of cleaved and purified protein.

Crystallization and preliminary crystallographic analysis of recombinant abrin-a A-chain with ribosome inactivating activity.

Crystals have been obtained for a recombinant abrin-a A-chain produced by E. coli. The crystals were grown using PEG6000 as the precipitating agent. The crystals belong to an orthrhombic space group (P2(1)2(1)2(1)) and diffract to 1.7 A.

Familial occurrence of chronic neutrophilic leukaemia.

A father and son who both developed chronic neutrophilic leukaemia (CNL) are reported. The father, aged 63, had been exposed to radioactive fallout after the atomic bomb attack on Hiroshima; he presented with hepatosplenomegaly and neutrophilic leucocytosis, and died of intracerebral haemorrhage 1 month after diagnosis. 4 years later his son, then aged 44, presented with neutrophilic leucocytosis. Leukaemic transformation to acute myelogenous leukaemia (AML-M1) occurred, and he died of refractory leukaemia 4 months after the diagnosis of CNL. This is the first report of this rare disease occurring in family members; genetic effect due to radioactive poisoning was suspected in the development of CNL in these two cases.

Deleted HTLV provirus in peripheral blood cells of a patient with T-cell prolymphocytic leukaemia.

We describe the first case of T-cell prolymphocytic leukaemia (T-PLL) in which the peripheral blood cells contained a human T-lymphotropic virus (HTLV) related tax sequence. Serum screening tests for anti-HTLV-I/II antibodies were negative. Polymerase chain reaction disclosed the presence of an HTLV-I tax sequence in the peripheral blood. Other sets of oligonucleotide primers for HTLV-I gag, pol, env and the long terminal repeat regions and for the HTLV-II pol region were negative in the DNA of the cells. Although patients with T-PLL have been reported to be seronegative for HTLV-I, our findings point to the possibility that HTLV-I infection might be involved in the aetiology of at least some cases of T-PLL and that there may be alternative mechanisms involved in HTLV-associated leukaemogenesis.

Radicicol leads to selective depletion of Raf kinase and disrupts K-Ras-activated aberrant signaling pathway.

Activation of Ras leads to the constitutive activation of a downstream phosphorylation cascade comprised of Raf-1, mitogen-activated protein kinase (MAPK) kinase, and MAPK. We have developed a yeast-based assay in which the Saccharomyces cerevisiae mating pheromone-induced MAPK pathway relied on co-expression of K-Ras and Raf-1. Radicicol, an antifungal antibiotic, was found to inhibit the K-ras signaling pathway reconstituted in yeast. In K-ras-transformed, rat epithelial, and K-ras-activated, human pancreatic carcinoma cell lines, radicicol inhibited K-Ras-induced hyperphosphorylation of Erk2. In addition, the level of Raf kinase was significantly decreased in radicicol-treated cells, whereas the levels of K-Ras and MAPK remained unchanged. These results suggest that radicicol disrupts the K-Ras-activated signaling pathway by selectively depleting Raf kinase and raises the possibility that pharmacological destabilization of Raf kinase could be a new and powerful approach for the treatment of K-ras-activated human cancers.