Site-directed chromosome rearrangements in skin fibroblasts from persons carrying genes for hereditary neoplasms.

Chromosomal variability was studied in cultured skin fibroblasts in members of two unrelated families associated with hereditary neoplasms, one with familial childhood leukemia and the other with medullary thyroid cancer syndrome. Nonconstitutional chromosome rearrangements occurred with consistent frequency in the patients and obligate carriers. The G-banding analysis showed that th chromosome rearrangements were not random, and site of rearrangements tended to cluster to band p22 of chromosome 1 in the carriers of childhood leukemia gene and to band q23 of chromosome 17 in the patient with medullary thyroid cancer. The de novo rearrangements of chromosomes and their tendency to cluster to particular chromosomal sites strongly point to the possibility that the procancer type-dominant mutations responsible for these diseases have a mutator function analogous to the property of some insertion mutations or transposable elements.

Stability of human interferon-beta 1: oligomeric human interferon-beta 1 is inactive but is reactivated by monomerization.

Human interferon-beta 1 is extremely stable is a low ionic strength solution of pH 2 such as 10 mM HCl at 37 degrees C. However, the presence of 0.15 M NaCl led to a remarkable loss of antiviral activity. The molecular-sieve high-performance liquid chromatography revealed that, whereas completely active human interferon-beta 1 eluted as a 25 kDa species (monomeric form), the inactivated preparation eluted primarily as a 90 kDa species (oligomeric form). The specific activity (units per mg protein) of the oligomeric form was approx. 10% of that of the monomeric form. This observation shows that oligomeric human interferon-beta 1 is apparently in an inactive form. When the oligomeric eluate was resolved by polyacrylamide gel containing sodium dodecyl sulphate (SDS), it appeared to be monomeric under non-reducing conditions. Monomerization of the oligomeric human interferon-beta 1 by treatment with 1% SDS, fully regenerated its antiviral activity. These results suggest that the inactivation of the human interferon-beta 1 preparation was caused by its oligomerization via hydrophobic interactions without the formation of intermolecular disulphide bonds. These oligomers can be dissociated by SDS to restore biological activity.

Structural, functional and evolutionary implications of the three-dimensional crystal structure of murine interferon-beta.

The crystal structure of recombinant murine interferon-beta as elucidated by Senda et al. (Proc. Jap. Acad. 66B: 77-80 (1990); EMBO J. 11: 3193-3201 (1992)) appears to represent the basic structural framework of all Type I interferons including interferons-beta and all subtypes of interferons-alpha of various mammalian origin. Now the huge accumulated data on the structure-activity relationship of Type I interferons using various chemical and genetic techniques can be systematically evaluated in terms of the three-dimensional structure. Structural comparison with other cytokines, for which three-dimensional structures have been established, including interferon-gamma and considerations on the evolution of cytokines and cytokine receptors are also given.

Purification and N-terminal sequence of a novel interferon-induced 100 kD protein secreted by mouse Ehrlich ascites tumor cells.

Mouse Ehrlich ascites tumor cells secrete a 100 kD protein when they are stimulated by murine interferon-beta (2000 IU/ml). This 100 kD protein was purified from conditioned medium by chromatography on phenyl-Sepharose CL-4B, phosphocellulose, DEAE-Sephacel, and Vydac C4 columns and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A single species of 100 kD protein was isolated, and its N-terminal sequence analysis suggested that mouse Ehrlich ascites tumor cell-derived interferon-induced 100 kD protein is a novel protein.

Pharmacokinetics and biologic activities of human native and asialointerferon-beta s.

Glycoproteins are metabolized through an asialoglycoprotein metabolic pathway in vivo. They are desialylated and taken up by the liver via an asialoglycoprotein receptor. Fibroblast-derived natural human interferon-beta is a glycoprotein having a single asparagine-linked sugar chain. Although natural human interferon-beta may also be metabolized through this pathway, there is very little information about the biologic features of human asialointerferon-beta. We evaluated the pharmacokinetics and biologic activities of human native and asialointerferon-beta s. After intravenous administration to rabbits, human asialointerferon-beta was cleared from the blood circulation faster than the human native interferon-beta. More asialoprotein was distributed to the liver than the native type, but it induced less 2'5'-oligoadenylate synthetase. The human asialointerferon-beta had less activity than the human native interferon-beta on cell growth inhibition and 2'5'-oligoadenylate synthetase induction in Hep-G2 and HuH6 human hepatoblastoma cells. Southern blotting using a hepatitis B virus-transfected HuH6 cell line, HB611, revealed that the inhibition of hepatitis B virus DNA replication by the asialoprotein was weaker than that by the native protein. The results showed that the different effects exerted by the human native and asialointerferon-beta s may be a result of recognition of the sugar chains by rabbit hepatocytes or by human hepatoblastoma cells. The results also suggested that the terminal sialic acid of the sugar chains in natural human interferon-beta significantly affects its pharmacokinetics and biologic activities.

Disulfide bond interchange in Escherichia coli-derived recombinant human interferon-beta 1 under denaturing conditions.

Disulfide bond interchange has been pointed out as a considerable problem in preparing recombinant proteins from Escherichia coli cells. This has been reported in the system of reducing denaturation followed by a refolding process, where incorrectly folded molecules are sometimes produced. As the possibility of disulfide bond interchange may also arise in the cytoplasm of E. coli cells, the state of sulfhydryl groups of recombinant proteins obtained from a nonreducing and nondenaturing process should be examined. The state of sulfhydryl groups of E. coli-derived recombinant human interferon-beta 1, which had been purified under nonreducing and nondenaturing conditions, was examined by using the N-(7-dimethylamino-4-methylcoumarinyl)maleimide (DACM) labeling technique. Among the three cysteine residues in E. coli-derived human interferon-beta 1, the 17th cysteine was identified as being unpaired, as in the natural molecule. However, it was found that three isomers of the recombinant protein could be formed when the protein was denatured with 6 M guanidine hydrochloride. These three isomers were identified as having unpaired cysteine residues at positions 17, 31, and 141, respectively. These results indicate that disulfide bond interchange occurs in E. coli-derived recombinant human interferon-beta 1 under denaturing conditions in spite of the absence of a reducing agent.

Conformations of fibroblast and E. coli-derived recombinant human interferon-beta s as studied by nuclear magnetic resonance and circular dichroism.

The conformations of fibroblast and E. coli-derived recombinant human interferon-beta s were studied by circular dichroism and nuclear magnetic resonance spectroscopy in the acidic pH region of 4.6 to 1.6. Both interferons have very similar conformations with high alpha-helix contents (approximately 70%). These results suggest that glycosylation does not appreciably change the conformation of human interferon-beta. Moreover, a slow conformational change is observed below pH 2.0, which induces the disruption of beta-sheets.

Characterization of E. coli-derived recombinant human interferon-beta as compared with fibroblast human interferon-beta.

Homogeneous E. coli-derived recombinant human interferon-beta (E. coli-rHuIFN-beta) was characterized in order to elucidate its physicochemical properties, as compared with those of fibroblast human interferon-beta (fibroblast HuIFN-beta). Purified E. coli-rHuIFN-beta and fibroblast HuIFN-beta exhibited a single band of Mr 19,000 and 23,000, respectively, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The primary structure of E. coli-rHuIFN-beta was identical to the prediction from the cDNA sequence. Furthermore, both the circular dichroism (CD) spectra and the 1H nuclear magnetic resonance (NMR) spectra of E. coli-rHuIFN-beta and fibroblast HuIFN-beta at pH 6.8 were closely similar to each other. On the other hand, on reverse-phase high-performance liquid chromatography (HPLC) using a C18 column, the retention time of E. coli-rHuIFN-beta was longer than that of fibroblast HuIFN-beta. Moreover, although the isoelectric point of E. coli-rHuIFN-beta was pH 8.9, purified fibroblast HuIFN-beta exhibited multiple isoelectric points, probably due to heterogeneity of the carbohydrate moiety. These results indicate that the E. coli-rHuIFN-beta polypeptide folds similarly to fibroblast HuIFN-beta, and the carbohydrate moiety of natural HuIFN-beta has little influence on higher-order structure but does influence the hydrophobic and the electrostatic properties of the molecule.

Improved treatment results of non-Hodgkin's lymphoma in children: a report from the Children's Cancer and Leukemia Study Group of Japan.

From 1985 to 1989, 69 patients with non-Hodgkin's lymphoma (NHL) were treated by members of the Children's Cancer and Leukemia Study Group of Japan with a protocol consisting of vincristine, prednisolone, cyclophosphamide, doxorubicin, high-dose methotrexate (HD-MTX), mercaptopurine and cytarabine; central nervous system (CNS) prophylaxis with intrathecal MTX and hydrocortisone (NHL855). The 4-year event-free survival (EFS) was 78% (S.E., 10%) for patients with localized disease (n = 18) and 38% (S.E., 7%) for those with advanced disease (n = 51). Among the patients with advanced disease, those with non-lymphoblastic lymphoma tended to have a better 4-year EFS than those with lymphoblastic lymphoma (52% vs. 25%). Based on these findings, we initiated a new protocol NHL890 in which patients were assigned to two different chemotherapies according to the histology. Non-lymphoblastic subtype was treated almost identically to NHL855 while asparaginase and VP-16 were newly added in the consolidation-maintenance phase in advanced-stage lymphoblastic lymphoma. Sixty-seven patients with advanced disease were assessable. The overall 4-year EFS for advanced disease improved to 69% (S.E., 6%). A significant improvement was gained in the lymphoblastic lymphoma with a 4-year EFS of 56% (S.E., 11%) as compared with 25% (S.E., 9%) in the preceding study (P < 0.05). These findings suggest the importance of histology in the treatment of advanced-stage non-Hodgkin's lymphoma in childhood.

Assessment of testicular biopsy after cessation of maintenance chemotherapy in childhood acute lymphoblastic leukemia: a report from the Children's Cancer and Leukemia Study Group.

Among 484 male patients with acute lymphoblastic leukemia (ALL) registered into the protocols CCLSG 811, 841 and 874, from 1981 through 1990, 246 patients completed their protocols and were in continuous complete remission (CCR) for more than 3 years. One hundred and seven patients received bilateral testicular biopsies at the time of cessation of maintenance chemotherapy. Eight patients (7.5%) were found to have occult testicular leukemia (TL). Three of them did not receive any additional therapy and all suffered subsequent relapses; one bone marrow relapse and two testicular relapses. The other 3 patients received testicular radiation combined with an additional 2 years of chemotherapy, resulting in CCR for more than 6 years 10 months, 7 years 6 months, and 8 years 6 months. One with chemotherapy alone and another with radiation alone showed subsequent relapse. Overt TL after negative initial biopsy was developed in 3 (3.0%) of the 99 patients. All of them received testicular radiation with chemotherapy, resulting again in CCR for more than 1 year 0 months and 5 years 3 months; one patient showed relapse in testes and bone marrow after 3 years 8 months of CCR. These studies suggested that occult TL has an adverse prognostic significance unless retrieval chemotherapy is given and that performance of testicular biopsy at completion of maintenance chemotherapy is not contributory to prolongation of disease-free survival for males with ALL because the treatment employing testicular radiation plus retrieval chemotherapy for both occult TL and isolated overt TL after off-therapy is similarly very effective.

Retrospective analysis of late intensification therapy with high-dose methotrexate for standard-risk acute lymphoblastic leukemia in childhood (CCLSG-S811 study). The Children's Cancer and Leukemia Study Group.

Using the CCLSG-S811 protocol for children with standard-risk acute lymphoblastic leukemia (ALL), late intensification therapy (LIT) with high-dose methotrexate (HD-MTX) was conducted without randomization. Of 118 eligible patients, 114 attained complete remission and 82 maintained continuous complete remission (CCR) for at least 3 years, completing the entire S811 regimen. Among the latter, 74 patients received LIT with HD-MTX between 2-3 years after CCR onset. MTX (2,000 mg/m2 per dose per week) was administered by 24 h infusion and three doses were given every 12 weeks. Leucovorin rescue (15 mg/m2 i.v.) every 6 h was initiated 12 h after the end of MTX infusion for seven doses. As regular maintenance chemotherapy, intermittent (Regimen A) or continuous (Regimen B) MTX plus 6-mercaptopurine (6MP) combined with pulses of prednisolone and vincristine was administered (Koizumi S, Fujimoto T, Takeda T, et al. Cancer 1988; 61: 1292-1300). Retrospective analysis revealed that patients on Regimen A who started LIT earlier (within 2 years of CCR onset (n = 23)) showed a higher rate of event-free survival (EFS) at 8 years (95.5% +/- 4.4%, mean +/- S.E.) than patients who started LIT later (2.5 years after CCR onset (n = 18, 66.2% +/- 11.3%, p less than 0.01)). In addition, the superiority of four or five courses of the LIT (n = 39) as compared to 2 or 3 courses (n = 35) was noted for both regimens. The data suggest that early and aggressive LIT with HD-MTX may improve the long-term survival of childhood ALL patients.

Nitrous oxide depresses somatocardiac sympathetic A- and C-reflexes in anesthetized rats.

Effect of nitrous oxide (N2O) on the somatosympathetic A- and C-reflexes was investigated using artificially ventilated rats anesthetized with alpha-chloralose and urethane. Somatocardiac sympathetic A- and C-reflexes were elicited in the inferior cardiac nerve by electrical stimulation of A and C afferent fibers of the tibial nerve, respectively. Both reflexes were depressed by inhalation of N2O for 20 min. The depression was greater in the C-reflex than in the A-reflex. The depressive effects of N2O on both reflexes were unchanged after pretreatment with intravenous naloxone (0.2 or 2.0 mg/kg) or by prolongation of the inhalation of N2O for 2 h. These results suggest that the opioid receptor is not involved and that acute tolerance is not developed in the depressive action of N2O on the somatosympathetic A- and C-reflexes.

What is the benefit of aggressive chemotherapy for advanced neuroblastoma with N-myc amplification? A report from the Japanese Study Group for the Treatment of Advanced Neuroblastoma.

In 1985, a nationwide single protocol (cyclophosphamide, vincristine, tetrahydropyranyl Adriamycin, and cisplatin) for the treatment of advanced neuroblastoma was begun in Japan and was found to significantly increase the 3-year survival rate--to 70% for stage III, and to 45% for stage IV. In this study, the authors investigated the efficacy of this protocol for advanced neuroblastoma with or without N-myc amplification. In 159 of the 233 patients with advanced neuroblastoma treated with this protocol (between January 1985 and March 1993), genomic amplification of N-myc was determined. These 159 patients were divided into two groups according to the number of N-myc copies, ie, those with fewer than 10 copies (105 patients) and those with 10 or more copies (54 patients). The survival curves for the two groups were significantly different. The 5-year survival rate for patients with 10 or more copies was 43.9%; this is surprisingly high in comparison to results of previous studies in which no survivors were expected in cases of advanced neuroblastoma with highly amplified N-myc. Persistent bone marrow suppression was common, but there were no deaths attributable to drug side effects. Five patients with fewer than copies of N-myc amplification died more than 3 years after initial treatment. Three of the five had tumors with an unfavorable Shimada classification, and two had diploid nuclear DNA content. The authors conclude that the protocol resulted in dramatic improvement in the patients with advanced neuroblastoma, even with high N-myc amplification.(ABSTRACT TRUNCATED AT 250 WORDS)

Monoclonal antibody to human beta interferon: characterization and application.

Seven stable mouse hybridomas secreting monoclonal antibodies to human fibroblast beta interferon (IFN-beta) were isolated, all seven of which belonged to IgGl subclass and kappa type. While neutralizing the antiviral activity of human fibroblast IFN-beta, they failed to neutralized both that of human IFN-alpha and human IFN-gamma. These monoclonal antibodies neutralized the antiviral activity of human fibroblast IFN-beta but not that of human IFN-alpha and IFN-gamma. Two of the seven monoclonal antibodies, YSB-1 and YSB-2, showed particularly high neutralization titers in the ascitic fluid. Monoclonal antibodies were purified from cultures of hybridoma grown in a serum-free medium. The purified monoclonal antibodies, YSB-1(5 micrograms/ml) and YSB-2(1 microgram/ml), neutralized IFN-beta from 10EU/ml to 1EU/ml. Human fibroblast IFN-beta was purified to 3.5 X 10(7) IU/mg protein (1.0 X 10(8) IU/mg protein in the peak fraction) by the monoclonal antibody (YSB-2) affinity column chromatography, whereas human recombinant IFN-beta obtained from E.coli was also purified to 6.5 X 10(7) IU/mg protein (1.1 X 10(8) IU/mg protein in the peak fraction) by the monoclonal antibody (YSB-1) affinity column chromatography.

Growth inhibition of human glioma cells by superinduced human interferon-beta.

Superinduction of human interferon-beta (HuIFN-beta) from human glioma cells has greater cytotoxicity than purified HuIFN-beta derived from fibroblasts. However, superinduction requires several reagents like polyI:polyC, cycloheximide, and actinomycin D, which may contaminate the conditioned medium and obscure the effect of superinduced HuIFN-beta. The present study used minimum doses of polyI:polyC and cycloheximide without actinomycin D to superinduce HuIFN-beta. The superinduced HuIFN-beta was purified by passing the medium through molecular sieve column chromatography. Fractionation of the eluate provided semipurified superinduced HuIFN-beta which demonstrated a growth inhibitory effect against both the U251-MG autologous human glioma cell line and the SK-MG-1 homologous glioma cell line. This effect was neutralized by addition of anti-HuIFN-beta monoclonal antibody (YSB-1). In a separate experiment, combinations of superinduction reagents were found not to have growth inhibitory effects because all inhibition in superinduced medium was completely neutralized by YSB-1. Superinduced HuIFN-beta has a pure growth inhibitory effect on both autologous and homologous glioma cells, so may affect autocrine secretion of cytokines.

[Clinical characteristics and treatment results of acute promyelocytic leukemia in children (Children's Cancer and Leukemia Study Group)].

The clinical characteristics and treatment outcome in 40 children with acute promyelocytic leukemia (APL) treated at institutions participating in the Children's Cancer and Leukemia Study Group (CCLSG) were studied retrospectively. The median age at diagnosis was 8 years old. Bleeding diathesis was the predominant presenting symptom (90%), associated with laboratory findings of disseminated intravascular coagulation. Hepatomegaly, splenomegaly and lymphadenopathy were observed in 35%, 10%, and 15% of the cases, respectively. The median WBC count was 4.25 x 10(9)/l. Anemia (hemoglobin < 8 g/dl) and thrombocytopenia (< 30 x 10(9)/l) were present in more than half of the patients. Cytogenetic studies demonstrated the characteristic 15; 17 translocation in about 90% of the patients analyzed. Induction therapy consisted of cytosine arabinoside and an anthracycline, with or without other agents. Twenty-nine patients (73%) achieved complete remission (CR) while early fatal hemorrhage was the predominant cause of induction failure. The survival rates continued to decrease (28% at 3 years, 24% at 5 years, and 7.9% at 10 years) due to late marrow relapses. Anthracycline cardiotoxicity was fatal in three patients in remission. These clinical features of childhood APL should be taken into account in the development of new protocols.

[Analysis of cytoplasmic antigens in acute leukemia by flow cytometry].

The expression of cytoplasmic antigens in 77 cases of acute leukemia were analyzed by flow cytometry using the following monoclonal antibodies: CD3, CD22, anti-myeloperoxidase (MPO-7) and anti-mu-heavy chain. CD22 antigen was detected in the cytoplasm of all non-T-ALL patients excluding one not-tested patient. In two patients with unclassified ALL, surface CD22 antigen was not expressed but cytoplasmic CD22 antigen was strongly expressed. Three out of 9 patients with common ALL were cytoplasmic mu-heavy chain-positive, so these patients were diagnosed as Pre-B ALL. In four out of 8 patients with T-ALL, CD3 antigen was not expressed on the cell surface membrane. However all of T-ALL patients excluding one non-tested patient were cytoplasmic CD3-positive. The cytoplasmic expression of myeloperoxidase antigen was detected in twenty out of 21 patients with acute non-lymphoblastic leukemia (ANLL). One megakaryocytic leukemia patient was MPO-negative. In two ANLL patients, the percentage of MPO for conventional cytochemical staining was undetectable or low, but MPO antigens were positive (77% and 70%) for flow cytometric analysis. All of 46 non-T ALL patients were cytoplasmic MPO-negative, however 4 out of 10 T-ALL patients were cytoplasmic MPO-positive. The study proved that the analysis of cytoplasmic CD3, CD22, mu-chain and MPO antigens were very useful to define the cell lineage of leukemia and to classify ALL and ANLL. It is necessary to study further whether the expression of MPO in the cytoplasm of T-ALL was non-specific reaction or whether MPO precursors are expressed in the cytoplasm of T-ALL.

[Myelogenous leukemia in children. ANLL9205 study by Children's Cancer and Leukemia Study Group (CCLSG)].

Treatment results were evaluated in 45 children with acute myeloblastic leukemia (AML) treated on the ANLL-9205 protocol of the Children's Cancer Leukemia Study Group (CCLSG, Japan). In this protocol, terarubicin (THP-ADR), vincristine and continuous infusion of cytosine arabinoside (Ara C) were applied for remission induction therapy (AVC), and VP16+ high dose Ara C were used sequentially for 32 or 48 weeks. Eleven patients received stem cell transplantation. Thirty-eight out of the 43 eligible patients (88.4%) achieved complete remission, and the overall 3-year event-free survival (EFS) was 55.6% (S.E.,10%). This favorable response was attributed mainly to the high induction rate of patients with the M5, M7 FAB subtypes and higher WBC counts (> or = 10 x 10(9)/L). There was no difference in the 3-year EFS of these patients who discontinued treatment between 32 weeks and 48 weeks. Serious toxicities were not observed in this study. These findings suggest that the ANLL-9205 protocol is an effective and safe treatment regimen for childhood AML. When comparing the treatment period of 32 or 48 weeks, the difference was not statistically significant.

[Transient subacute encephalopathy induced by high-dose methotrexate treatment in children with acute lymphoblastic leukemia and malignant lymphoma].

Transient subacute encephalopathy was detected in 4 of 83 patients undergoing treatment with high-dose methotrexate (HD-MTX) and citrovorum factor rescue for childhood acute lymphoblastic leukemia and malignant lymphoma from 1984 to 1991. Subacute encephalopathy occurred in relatively older patients and early in the course of treatment with HD-MTX. The average interval between the HD-MTX course and the onset of the neurologic disturbance was 6.5 days. All 4 patients treated had no neurological sequelae. Laboratory evaluations disclosed nontoxic plasma MTX levels at onset of symptom and not detected in liquor. CT in 4 patients disclosed no abnormality, but MR images revealed abnormal signal intensity patterns of cerebral white matter in 2 cases. In one case the abnormal MR finding resolved after 3 months. The pathogenesis of this neurologic symptom remains unknown, but further HD-MTX treatment may be acceptable in follow-up of MR image, because the prognosis of subacute encephalopathy seems favorable.

[Evaluation of myocarditis using T1-201 myocardial scintigraphy with bull's eye functional map].

In order to evaluate myocardial damage in a patient with myocarditis, rest thallium-201 myocardial single photon emission computed tomography (SPECT) was performed in 15 patients with myocarditis. For qualitative and semiquantitative analysis, Bull's eye functional maps were made up with SPECT images. In the functional map, the abnormal area, where T1 uptake is less than mean-2SD of the T1 uptake of normal subjects, is generally distributed in the myocarditis group. But focal and sequential abnormal areas were recognized more often in the clinically severe cases. Abnormal area tended to be observed commonly at the antero-septal wall, but it was uncommon at the lateral wall. Extent score, i.e. degree of extension of abnormal area, and severity score, i.e. degree of abnormality, were in good negative correlation with left ventricular ejection fraction (r = 0.6, r = 0.7). Furthermore, existence of abnormal area was in good correlation with the left ventricular regional wall motion. Abnormal area existed 100% in the akinetic region, 71% in the region of severe hypokinesis, and 27% in the region of hypokinesis. Abnormal area occupied 30% of the normokinetic region in the myocarditis group, which was a higher rate than in the normal control group (p less than 0.05). It was suggested that latent myocardial damage existed in the normokinetic myocardium with myocarditis. Thus, rest T1-201 SPECT with Bull's eye map is useful for clinical diagnosis in patients with myocarditis.