The myc intron-binding polypeptide associates with RFX1 in vivo and binds to the major histocompatibility complex class II promoter region, to the hepatitis B virus enhancer, and to regulatory regions of several distinct viral genes.

We demonstrated that MIF-1, identified initially as a binding activity that associated with the intron I element of the c-myc gene, consists of two polypeptides, the myc intron-binding peptide (MIBP1) and the major histocompatibility class II promoter-binding protein, RFX1. Using a polyclonal antiserum directed against either oligonucleotide affinity-purified MIBP1 or a peptide derived from RFX1, we showed that MIBP1 and RFX1 are distinct molecules that associate in vivo and are both present in DNA-protein complexes at the c-myc (MIF-1) and major histocompatibility complex class II (RFX1) binding sites. We have also found that MIBP1 and RFX1 bind to a regulatory site (termed EP) required for enhancer activity of hepatitis B virus. In addition, we have identified MIF-1-like sequences within regulatory regions of several other viral genes and have shown that MIBP1 binds to these sites in cytomegalovirus, Epstein-Barr virus, and polyomavirus. We have also demonstrated that the MIF-1 and EP elements can function as silencers in the hepatocarcinoma HepG2 and the cervical carcinoma HeLa cell lines. These findings indicate that MIBP1 and EP/RFX1 can associate in vivo and may regulate the expression of several distinct cellular and viral genes.

Characterization of a low-molecular-weight growth inhibitor formed by density-inhibited, tumorigenic V79 Chinese hamster cells.

A novel type of low-molecular-weight growth-inhibitory factor responsible for the density inhibition of tumorigenic V79 Chinese hamster cells has been purified, if not homogenously, by a series of reverse-phase and gel filtration high-performance liquid chromatography. The factor is an acid-stable, heat-labile substance distinct from antiproliferative nucleoside analogues or polyamines and has a molecular weight of approximately 2000. The biological activity of this inhibitor was enhanced nearly 5-fold by trypsin treatment, thereby suggesting that the inhibitor may be a precursor peptide which becomes an oligopeptide with intense biological activity by proteolysis, or that trypsin treatment allows resultant small molecules to efficiently transfer across the cytoplasmic membrane. This inhibitor reversibly inhibits the growth of a broad spectrum of cell types from neoplastic and nonneoplastic cells from various species. These data suggest that this inhibitor is primarily a growth-regulatory molecule common to mammalian cells and may play an important role in regulating growth of both normal and neoplastic cells.

Establishment of Epstein-Barr virus-negative diffuse large cell lymphoma cell line with an 8;22 chromosomal translocation.

A new human B-cell lymphoma cell line was established from a pleural effusion of a patient with a diffuse large cell lymphoma which originated from an ileocecal tumor. The cell line, designated KAL-1, has been passaged 280 times over a period of 22 months. This cell line was successfully maintained in a chemically defined serum-free medium; its doubling time is approximately 24 h. Immunologically, the cells were demonstrated to express IgM lambda on the cell surface and to react with monoclonal antibodies to B-cell antigen including B1, B4, HLA-DR, and common acute lymphoblastic leukemic antigen but not with B2 and all the T-cell markers. Immunoglobulin gene analysis revealed rearrangements of both JH and C lambda. These data indicate that this cell line represents the B-cell lineage at the immature B-cell stage. This cell line was negative for Epstein-Barr virus nuclear antigen and had no detectable Epstein-Barr virus genome in cellular DNA. Chromosome analyses revealed that the cells carried an 8;22 chromosome translocation, reminiscent of variant type Burkitt's lymphoma. However, there was no histological evidence for Burkitt's lymphoma. Molecular studies showed that KAL-1 had deregulated high constitutive expression of c-myc. This cell line was demonstrated to be highly tumorigenic when injected into athymic nude mice. This tumor model should provide clues about the molecular mechanism involved in the pathogenesis of B-cell malignancy and appears to be a useful in vivo model for the study of molecular events during B-cell differentiation and therapeutic investigations.

Evidence for the involvement of endogenous thymidine in the density-inhibition of tumorigenic Chinese hamster V79 cells.

Two distinct low-molecular-weight growth inhibitory activities were isolated from supernatants of a density-inhibited, tumorigenic V79 Chinese hamster cell line. By chromatographic analyses, one of these was purified to homogeneity and eventually proved to be thymidine (dThd). In order to investigate the biological role of dThd in a density-inhibited culture of these cells, a dThd-kinase deficient (TK-) clone resistant to the excess of dThd was isolated from V79 cells and the effect of the supernatants on growth of these TK- or TK-proficient (TK+) cells was examined. As a result, the growth of TK- cells was not inhibited but enhanced by the supernatant at the concentrations which significantly inhibited the growth of TK+ cells. Such TK-dependent differential responses to supernatants suggest the presence of deoxyribonucleosides including a high level of dThd in the supernatants. Since it is unlikely that dThd might derive from denatured DNA of dead cells, an accumulation of endogenous dThd in confluent culture appears to be responsible for dThd triphosphates which are synthesized de novo, degraded and excreted into the medium rather than incorporated into DNA as a consequence of aberrant growth in the presence of certain growth inhibitors produced by density-inhibited V79 cells.

Increase of peripheral B lymphocytes committed to the production of monoreactive and high affinity antibodies to HTLV-1 in patients with HAM/TSP.

We quantitated the frequency of B lymphocytes capable of producing antibodies to HTLV-1 in the peripheral blood from patients with HAM/TSP, non-HAM/TSP HTLV-1 carriers and seronegative healthy subjects. Epstein-Barr virus (EBV) was used as a polyclonal activator of B lymphocytes in a limiting dilution condition. We found that B lymphocytes committed to the production of monoreactive-IgG and -IgA antibodies to recombinant HTLV-1 (gag + env) hybrid protein were significantly increased in a number in patients with HAM/TSP as compared to non-HAM/TSP HTLV-1 carriers and seronegative healthy subjects. By transforming these B lymphocytes with EBV and fusing them with human-mouse heteromyeloma (F3B6), a stable hybridoma producing IgG monoclonal antibody (mAb) to HTLV-1 (gag + env) protein was generated from a patient with HAM/TSP. This mAb (IgG1, kappa), designated F31.1, specifically bound to the amino acid residues from 235 to 254 of HTLV-1 envelope glycoproteins (gp46) with high affinity (Kd = 4.0 x 10(-9) mol/l). These data indicate that the antigen-driven process of B lymphocytes maturation by HTLV-1 antigens is markedly increased in patients with HAM/TSP.

Simple and efficient chemosensitivity assay for human primary tumor-cells using contact-sensitive microtiter plates.

A simple and efficient chemosensitivity assay for human primary tumors has been developed using 96-well microtiter plates covered totally with lethally irradiated 3T3 monolayers termed cell mats, on which the proliferation of normal human fibroblasts is preferentially inhibited. Two days after the inoculation of tumor cells into the microtiter cell mat plates, the cells were treated with various anticancer drugs, and the cell numbers were assessed by radioactivity using a 96-well automatic scintillation counter after subsequent radiolabeling with H-3-deoxyuridine for 24h. In this manner, complete dose response curves of many anticancer drugs were available from one plate within 5 days. Drugs tested in triplicate were adriamycin, mitomycin C, cisplatin, etoposide, and 5-fluorouracil at 0.01, 0.1, and Ix the peak tolerated drug concentrations in serum. Clinically, of the 39 primary tumor specimens of different types received, 4 were contaminated. The remaining 35 samples were successfully cultured, with 5 cultures being abandoned due to an insufficient cpm. As a result, cell survival curves were obtained from 30 (77%) specimens. This high evaluable rate might be due to the feeder effect brought by cell mats. Although optimization of the assay system has yet to be determined, the ability to have multiple drug sensitivity per plate with short-term duration would make this system an efficient assay for chemosensitivity test of human primary tumor cells.

Intravenous cyclosporine and tacrolimus caused anaphylaxis but oral cyclosporine capsules were tolerated in an allogeneic bone marrow transplant recipient.

A Japanese female patient with angioimmunoblastic T cell lymphoma underwent allogeneic bone marrow transplantation (BMT) from her brother. Cyclosporine at a dose of 3 mg/kg was started by continuous infusion over 24 h on day -1 of BMT. Within a couple of minutes after the infusion was begun, she developed diffuse pruritic erythema on her whole body and tachycardia. The infusion was immediately stopped and corticosteroid was given, resulting in disappearance of the erythema gradually. She was then switched to intravenous tacrolimus. However, she suffered urticalial erythema again. Since polyoxyethylated castor oil, a solubilizer used in the injective formulation of both cyclosporine and tacrolimus, is considered to be responsible for the reaction, she was given oral capsules of cyclosporine (Sandimmun) in which polyoxyethylated castor oil was not contained. No further anaphylactic reaction was observed. The BM cells were successfully engrafted without causing severe GVHD. She was discharged on cyclosporine capsules without any further adverse effects. Anaphylaxis to intravenous cyclosporine and tacrolimus is a very rare but a serious complication. Our present case indicates that oral capsule of Sandimmun is a safe alternative to prevent GVHD in such a case of anaphylactic reaction against intravenous formulation.

Oligoclonal expansion of alphabeta T lymphocytes in Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis with abnormal karyotypes.

We observed a fatal case of Epstein-Barr virus (EBV)-associated hemophagocytic lymphohistiocytosis (HLH) with an abnormal karyotype. Increased levels of alphabeta T cells of the patient were investigated using an inverse polymerase chain reaction (PCR) of the T-cell receptor variable region gene, followed by Jbeta-PCR and single-strand conformation polymorphism (SSCP) to confirm the clonality of specific alphabeta-T cell subsets. A high frequency (>15%) was recognized in Vbeta9 at onset, but not in any Vbeta and Valpha families 2 weeks after chemotherapy. High levels (>20%) of some Jbeta genes were detected in all Vbeta families investigated, and the predominant bias of the Jbeta2 gene relative to the Jbeta1 gene (86.1% versus 13.9% at onset, and 77.4% versus 23.5% after chemotherapy) was recognized in pan-alphabeta T cells. When each Vbeta-Jbeta fragment was compared among the samples at onset and after chemotherapy by SSCP analysis, several distinct bands were observed that indicate a clonal evolution. Thus, the findings suggest that some of the alphabeta T cell clones could be associated with abnormal karyotypes in EBV-HLH. The present findings provide molecular evidence of the presence of oligoclonal T cells in pan-alphabeta-T cells and clonal evolution during a short clinical course in EBV-HLH with abnormal karyotypes.

In vivo comparative therapeutic study of optimal administration of 5-fluorouracil and cisplatin using a newly established HST-1 human squamous-carcinoma cell line.

The efficacy and toxicity of 5-fluorouracil (5-FU) and cisplatin (CDDP) given in a sequential combination were evaluated in nude mice transplanted with HST-1, a newly established human squamous-carcinoma cell line. 5-FU and CDDP were given i.p. for 5 days and 1 day, respectively, either as single agents or in a sequential manner separated by a 24-h interval. The treatment was repeated every 30 days. Although inhibition of tumor growth was seen in all of the treated groups after two cycles, the sequence of 5-FU followed by CDDP significantly reduced the tumor burdens throughout all three courses and was more effective than the reverse sequence or either drug alone. Neither treatment-related death nor significant hematologic or nephrologic toxicities were seen, even following three cycles of therapy. Significant weight loss was observed only in mice treated with CDDP followed by 5-FU. This sequence dependence of the activity and toxicity of the 5-FU and CDDP combination should thus be incorporated into the design of a clinical trial.

Production of anti-cardiolipin antibody in AKR/J mice with streptozocin-induced insulitis and diabetes.

We herein report that anti-cardiolipin antibodies (ACA) were detected in AKR/J mice treated with multiple low doses of streptozocin (STZ)-induced insulitis and diabetes. Daily intraperitoneal (i.p.) injections of 40 mg/kg body wt. of STZ for five consecutive days in the AKR/J mice resulted in hyperglycemia and mononuclear cell infiltrations of islets (insulitis). ACA appeared on day 14, when hyperglycemia began to occur, at a rate of 13.3% (4/30). The rate increased to 83.3% (25/30) on day 21, when diabetes developed, and then fell to 10% (3/30) on day 28. Neither the diabetic AKR/J mice treated with a single high dose of STZ (200 mg/kg body wt.) nor the non-diabetic insulitis free Balb/c mice and B10.S(9R) mice treated with multiple low doses of STZ (40 mg/kg body wt.) produced ACA. The IgG subclass of the ACA belonged mainly to IgG2a. These findings suggest that ACA are produced in association with the development of insulitis, but not induced by either hyperglycemia or STZ.

Protective role of potentially lethal damage repair in the neoplastic transformation of Balb/c 3T3 cells treated with N-methyl-N'-nitro-N-nitrosoguanidine.

To determine the role of repair of potentially lethal damage (PLD) in the initiation process of neoplastic transformation, Balb/c 3T3 cells treated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) were temporarily exposed to conditioned medium obtained from density-inhibited Chinese hamster cell cultures, as a post-treatment for the induction of PLD repair. With or without this exposure, cell survival and transformation frequencies were simultaneously determined by colony-formation and focus-formation assays, respectively. Temporary exposure to conditioned medium resulted in a 20-30% increase in cell survival compared with no exposure. Post-treatment with conditioned medium resulted in a 60-65% reduction in transformation frequencies. At the molecular level, the repair of MNNG-induced single-strand breaks of DNA occurred much more rapidly in conditioned medium. These data suggest that PLD repair reduces the in vitro neoplastic transformation through excision repair operative during the cessation of DNA replication. Thus, PLD repair appears to be preventive against neoplastic fixation in initiation of neoplastic development.

Growth modulation of human tumor cells by a growth-inhibiting activity derived from tumorigenic V79 Chinese hamster cells.

A growth-inhibiting activity was identified in supernatants of the neoplastic V79 Chinese hamster cell line based on its ability to inhibit the proliferation of the same cell line. The partially purified activity, provisionally termed "growth inhibiting factor" (GIF) activity, inhibited the growth of a wide variety of human tumor cells, but not various normal human fibroblasts. This species-nonspecific activity was reversible, saturable, and highly potent in tumorigenic cell lines, and was noted in both monolayer culture and in soft agar. The inhibitory activity of GIF was also exhibited in a chemically defined serum-free medium supplemented with insulin and transferrin. GIF activity was stable to acid, heat, trypsin, and dithiothreitol but sensitive to alpha-chymotrypsin. The pattern of growth modulation by GIF on V79 cells was apparently different from those exhibited by bifunctional peptides such as transforming growth factor-beta, tumor necrosis factor-alpha, and interleukin-1-alpha. In addition, GIF activity cannot be ascribed to these cytokines based on the physicochemical and immunologic properties. Although GIF has yet to be purified to homogeneity, these data suggest that GIF might be a novel growth regulator which has a critical role in regulating growth of V79 cells. The growth modulation of tumor cells by this tumor-derived growth inhibiting activity suggested the presence of an autocrine growth regulatory mechanism even in tumor cells.