Integration of human herpesvirus 6 in a Burkitt's lymphoma cell line

Human herpesvirus 6 (HHV-6) genome has been found in several human lymphoid malignancies, but configuration of the HHV-6 genome has not been well delineated. We established the HHV-6-positive, Epstein-Barr virus-negative Burkitt's lymphoma cell line Katata. In this study we investigated the status of the HHV-6 genome in Katata cells. Neither linear nor circular HHV-6 DNA was detected by Gardella gel analysis. The fluorescence in situ hybridization technique enabled us to directly visualize the integrated HHV-6 DNA at the single-cell level. Only one integrated site of viral DNA was detected in metaphase chromosomes and it was preferentially located at the long arm of chromosome 22 (22q13). Treatment of the cells with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or with calcium ionophore A23187 led to induction of the HHV-6 immediate-early gene as well as the late gene. Sodium n-butyrate also gave rise to expression of the HHV-6 genes. The TPA inducibility was synergistically enhanced when combined with A23187 or n-butyrate. Our study provides, for the first time, an in vitro model system of latent HHV-6 infection whose genome is integrated into host DNA of lymphoma cells.     

Modulation of homeobox B6 and B9 genes expression in human leukemia cell lines during myelomonocytic differentiation

Homeobox genes (HOX) may have a regulatory function in the differentiation process of hematopoiesis. We examined the change of HOX B6 and HOX B9 mRNA expressions during the in vitro differentiation of four myeloid leukemia cell lines because HOX B6 may be involved closely in myeloid differentiation. HL-60, NB4, NKM-1 and NOMO-1 were established from acute leukemia of M2, M3, M2 and M5 subtype of the French-American-British classification, respectively. All-trans retinoic acid (ATRA), TPA, and G-CSF were used as differentiation inducers. Each cell line was cultured with each inducer and total RNA was isolated on day 1, 2, 3, or 5. HOX B mRNA was detected by Northern blotting and RT-PCR methods. HOX B6 and HOX B9 mRNAs were constitutively expressed in NB4, NKM-1 and NOMO-1, but were expressed at very low levels in HL-60. HOX B6 and HOX B9 mRNAs were also expressed in fresh acute myelocytic leukemia blasts. HOX B6 mRNA expression in HL-60, NB4, and NKM-1 cultured with ATRA increased on day 3 and decreased on day 5. HOX B6 mRNA expression in NB4 and NKM-1 cultured with TPA decreased on day 3. HOX B9 mRNA expression displayed changes similar to those of HOX B6 mRNA in NB4 and NKM-1. These results indicate that myeloid leukemia cell lines express HOX B6 and HOX B9, and that their respective mRNA expressions in NB4 and HL-60 increase at a mid stage of myeloid differentiation by ATRA induction and then decrease during a late stage. HOX B6 mRNA expression decreased in monocytoid differentiation by TPA induction in NB4, HL-60 and NKM-1. HOX B6 antisense-oligonucleotide inhibited the proliferation of NB4 and NKM-1. These results suggest that HOX B gene expression is related to simultaneous activation of cellular proliferation and differentiation in leukemic cells.     

Successful replication of parvovirus B19 in the human megakaryocytic leukemia cell line MB-02

The pathogenic human parvovirus B19 has been shown to undergo productive replication in the erythroid lineage in primary normal human hematopoietic progenitor cells. However, none of the established erythroleukemia cell lines has allowed B19 virus replication in vitro. The remarkable erythroid tissue tropism of B19 virus was evaluated with a human megakaryocytic leukemia cell line, MB-02, which is dependent on the growth factor granulocyte-macrophage colony-stimulating factor but can be induced to undergo erythroid differentiation following treatment with erythropoietin (Epo). Whereas these cells did not support B19 virus DNA replication in the presence of granulocyte-macrophage colony-stimulating factor alone, active viral DNA replication was observed if the cells were exposed to Epo for 5 to 10 days prior to B19 virus infection, as detected by the presence of the characteristic B19 virus DNA replicative intermediates on Southern blots. No replication occurred if the cells were treated with Epo for 3 days or less. In addition, complete expression of the B19 virus genome also occurred in Epo-treated MB-02 cells, as detected by Northern blot analysis. B19 progeny virions were released into culture supernatants that were biologically active in secondary infection of normal human bone marrow cells. The availability of the only homogeneous permanent cell line in which induction of erythroid differentiation leads to a permissive state for B19 virus replication in vitro promises to yield new and useful information on the molecular basis of the erythroid tissue tropism as well as parvovirus B19-induced pathogenesis.     

Down-regulation of human sialyltransferase gene expression during in vitro human keratinocyte cell line differentiation

We studied cytokines and anti-cytokine autoantibodies (Aabs) during T.b.brucei infections in IFN-gamma-/-, IFN-gammaR-/- and wild-type mice. Increased serum levels of IFN-gamma, TNF-gamma and IL-4 with decreased Aabs to these cytokines were recorded early during infections in all mice (except IFN-gamma in IFN-gamma-/- mice). Later, these responses were reversed, and surprisingly Aabs reacting to IFN-gamma in the IFN-gamma -/- mice were detected. To examine the possibility that an IFN-? immunoreactive molecule might be expressed due to infections and upon gene deletion, anti-IFN-gamma antibody was inoculated and resulted in abrogation of such Aabs. The scenario was different for IL-10 and TGF- since IFN-gammaR-/- and wild-type mice showed low cytokines and high Aabs early during infections, but later high cytokines and low Aabs were registered. Interestingly, IFN-gamma-/- mice exhibited reversed levels of both IL-10 and TGF-beta, and also of their Aabs. Fab fragments of purified serum immunoglobulins showed binding and neutralizing effects in biological assays. Pre-absorption of the Fab fragments with a cytokine inhibited the binding and neutralization effects of this cytokine, but not of other cytokines. These results highlight an important role for autoimmunity in cytokine regulation, and that genomic deletion of IFN-gamma modulates cytokines and their Aab responses in experimental African trypanosomiasis.     

Basophilic differentiation of the human leukemia cell line KU812 upon treatment with interleukin-4

To assess the effect of right ventricular pacing on rate regularity during exercise and daily life activities, 16 patients with sinoatrial disease and chronic atrial fibrillation (AF) were studied. Incremental ventricular pacing was commenced at 40 beats/min until > 95% of ventricular pacing were achieved during supine, sitting, and standing. Thirteen patients also underwent randomized paired submaximal exercise tests in either a fixed rate mode. (VVI) or a ventricular rate stabilization (VRS) mode in which the pacing rate was set manually at 10 beats/min above the average AF rate during the last minute of each exercise stage. The pacing interval for rate regularization was shortest during standing (692 +/- 26 ms) compared with either supine or sitting (757 +/- 30 and 705 +/- 26 ms, respectively, P < 0.05). During exercise VRS pacing significantly increased the maximum rate (119 +/- 5.2 vs 106 +/- 4.2 ms, P < 0.05), percent of ventricular pacing (85% +/- 5% vs 23% +/- 7%, P < 0.05), rate regularity index (5.8% +/- 1.6% vs 13.4% +/- 1.9%, P < 0.05), and maximum level of oxygen consumption (12.4 +/- 0.5 vs 11.3 +/- 0.5 mL/kg, P < 0.05) compared with VVI pacing. There was no change in oxygen pulse or difference in symptom scores in this acute study between the two pacing modes. It is concluded that right ventricular pacing may significantly improve rate regularity and cardiopulmonary performance in patients with chronic AF. This may be incorporated in a pacing device for rate regularization of AF using an algorithm that is rate adaptive to postural and exercise stresses.     

Transfer of the IL-6 gene into a human colorectal carcinoma cell line and consequent enhancement of tumor antigen expression

As part of the FINMONICA project, serum total cholesterol (TC) and high density lipoprotein cholesterol (HDLC) concentrations were determined in 1216 AMI patients (937 men, 279 women) aged 35-64 years in the province of Kuopio in eastern Finland during the 5-year period 1983-87. The distributions were compared with the corresponding distributions in a representative sample of the general population of the same area (1026 men, 1021 women). The mean serum TC levels did not differ between the AMI patients and the normal population. Only the prevalence of a very high serum TC level (> 8.0 mmol l-1) among women was significantly higher in the AMI group than in the population sample. On the other hand, in both sexes the age-adjusted mean HDLC was significantly lower in the AMI group than in the population sample. Our findings emphasize the importance of HDLC measurement as a part of the assessment of the lipid risk factor profile in patients with AMI.     

Copy number-dependent expression of a YAC-cloned human CFTR gene in a human epithelial cell line

INTRODUCTION: The reference values (RV) of biological indicators are used in the interpretation of the results of such indicators in individuals occupationally exposed to chemical agents. The Brazilian Group for the Establishment of Reference Values has worked on these definitions for the purpose of establishing RVs for several bioindicators in various regions of the country. In the present study, the RV for carboxyhemoglobin (COHb) was determined for the South of Minas Gerais. MATERIAL AND METHOD: The COHb was analyzed by the Beutler and West (1984) spectrophotometric method, optimized in our laboratory. In all the samples, analyses of some biochemical and hematological parameters were made to evaluate the health condition of a population of 200 volunteer non-smokers occupationally not exposed to CO. Each individual answered a questionnaire to obtain data pertinent to the interpretation of the results. The reference values were expressed as mean values +/- standard deviation, with a 95% confidence interval, and an upper reference value. The statistical distribution of the results was made so as to enable comparisons between the results of groups of workers, rather than individual evaluations, to be made. RESULTS AND CONCLUSIONS: The mean value +/- standard deviation was 1.0% +/- 0.75; the 95% confidence interval was 0.9-1.1% and the upper reference value was 2.5%. By the t Student test (p < or = 0.05), no difference was detected between the values related to sex, age or ingestion of alcoholic beverages. The reference values obtained were close to those reported for others countries.     

Absence of topoisomerase IIbeta in an amsacrine-resistant human leukemia cell line with mutant topoisomerase IIalpha

Numerous chemotherapeutic agents act via stabilization of a topoisomerase (topo) II-DNA complex. HL-60/AMSA, a human leukemia cell line, is resistant to intercalator-mediated DNA complex formation and cytotoxicity. HL-60/AMSA contains a mutant form of topo IIalpha that was thought to explain this resistance. However, our present data show that expression of topo IIbeta RNA in HL-60/AMSA is only 10% of that in HL-60, and topo IIbeta protein levels are undetectable. Southern analysis of topo IIbeta shows no differences in gene dosage between the two cell lines but does show differences in the restriction patterns. These data suggest that decreased topo IIbeta expression may contribute to the intercalator resistance of HL-60/AMSA cells.     

Use of Mono Mac 6 human monocytic cell line and J774 murine macrophage cell line in parallel antimycobacterial drug studies

The Mono Mac 6 (MM6) human monocytic cell line was evaluated with the established J774 murine macrophage cell line to ascertain its effectiveness in determining the intracellular activities of antimycobacterial drugs. Cells were infected with Mycobacterium tuberculosis H37Ra and treated with drug concentrations corresponding to the MICs, as well as to threefold higher than and threefold less than the MICs. Changes in CFU were compared after 7 days to determine significant differences between treated and nontreated groups. The results suggest that MM6 will make a useful model for testing the intracellular activities of antituberculosis drugs.     

Cellular and karyotypic characterization of two doxorubicin resistant cell lines isolated from the same parental human leukemia cell line

Separate mechanisms underlying the multidrug resistant (MDR) phenotype were identified in 2 independent approaches to select tumour cells resistant to low concentrations of doxorubicin (Dox) from the sensitive T cell leukemia cell line CCRF-CEM. The CEM/A7 cell line was selected at an initial concentration of 0.005 microgram/ml of Dox and maintained at 0.07 microgram/ml. In contrast, the CEM/A5 line was selected using an initial concentration of 0.01 microgram/ml and maintained in Dox at a concentration of 0.05 microgram/ml. P-glycoprotein expression was demonstrated in the CEM/A7 line but not the CEM/A5 line. Amplification of the mdrI gene was not observed in the CEM/A7 cell line. Both cell lines showed cross-resistance to a number of structurally unrelated cytotoxic drugs including anthracyclines and etoposide (VP-16), although only the CEM/A7 line was cross resistant to Vinca alkaloids. Immunoblots of total cell lysates of the CEM/A5 line have revealed almost undetectable levels of topoisomerase II alpha and beta in this line. Cytogenetic analyses of both lines revealed numerous karyotypic abnormalities which were present in the parental cell line as well as both resistant cell lines. The CEM/A7 line also demonstrated a duplication of part of the long arm of chromosome 7 which included the region containing the mdrI gene, a finding not seen in the parental or CEM/A5 line. CEM/A5, however, demonstrated an abnormality of chromosome 7, outside the region of the mdrI gene, and it also contained a deletion of the short arm of chromosome 2. Abnormalities in this latter region of genome have been associated with non-P-glycoprotein-mediated MDR.     

Induction of resistance in the human leukemia cell line HL60 towards hexadecylphosphocholine and other ether phospholipid analogues

Hexadecylphosphocholine (HePC) is a new ether lipid analogue with remarkable antineoplastic activity in vitro and in vivo. As the precise molecular mechanism by which this substance and probably other ether lipids exert their biological effects is still not defined, we tried to approach this problem by generating a cell line resistant to the antiproliferative properties of HePC. This was successfully accomplished by slow adaptation over a period of 14 months, of the very sensitive human leukemia cell line HL60. HePC resistant HL60 cells (HL60R) tolerate 8- to 10-fold higher doses of HePC and are continuously cultured in medium containing 10 micrograms/ml of HePC. An immunophenotypic and karyotypic characterization of HL60 and HL60R cells showed only marginal differences between the two cell lines. Total phospholipids, total cholesterol, protein and vinyl ether lipid content were equal in both cells. A down-regulation of the ether lipid mass in HL60R of about 40% could reflect one mechanism of tolerance induction. Though HePC uptake in HL60R cells was significantly lower than in the parental line, steady state measurements of cellular HePC content revealed similar HePC content in the membranes at HePC concentrations that were cytotoxic for HL60 but did not affect HL60R. This observation indicates that uptake and cellular accumulation of HePC do not determine HePC resistance. The resistant HL60R cells also showed a considerable degree of cross-resistance to ether phospholipids ET-18-OCH3 and BM 41.440, suggesting a common mode of action for HePC and other ether lipid analogues.     

Differential human multiple myeloma cell line responsiveness to interferon-alpha. Analysis of transcription factor activation and interleukin 6 receptor expression

Although IFN-alpha is commonly used as maintenance treatment for multiple myeloma patients, its effectiveness is varied. In this study, we have used a panel of IL-6 responsive myeloma cell lines that vary remarkably in responsiveness to IFN-alpha. Three cell lines were growth arrested by IFN-alpha; however, IFN-alpha significantly stimulated growth of the fourth cell line, KAS-6/1. Our studies have focused on elucidating the mechanism of differential IFN-alpha responsiveness. First, we have shown that IFN-alpha-stimulated growth of the KAS-6/1 cells did not result from induction of autocrine IL-6 expression. Second, analysis of Stats 1, 2, and 3 and IFN regulatory factor-1 (IRF-1) and IRF-2 activation failed to reveal differences between the IFN-alpha growth-arrested or growth-stimulated cells. Third, although IFN-alpha treatment of the IFN-alpha growth-inhibited cell lines reduced IL-6 receptor (IL-6R) expression, IFN-alpha also reduced KAS-6/1 IL-6R expression. Finally, although IFN-alpha treatment reduced IL-6R numbers on each cell line, analysis of Stat protein activation revealed that the receptors were still functional. We conclude that myeloma cell responsiveness to IFN-alpha is heterogeneous and that mechanisms of IFN-alpha-mediated growth inhibition other than IL-6R downregulation must exist in myeloma. Identification of these mechanisms may allow development of agents that are more universally effective than IFN-alpha.     

TNF-alpha mediates inducible nitric oxide synthase expression in human neuroblastoma cell line by cisplatin

The human neuroblastoma cell line NB-39-nu expressed inducible nitric oxide synthase (iNOS) mRNA following treatment with a combination of interferon-gamma (IFN-gamma) and cis-diamminedichloroplatinum(II) (cisplatin). The level of iNOS mRNA peaked at 48 hr after treatment, and dexamethasone inhibited the induction of iNOS mRNA expression. Cisplatin induced tumor necrosis factor-alpha (TNF-alpha) mRNA expression, and an anti-TNF-alpha neutralizing antibody inhibited the induction of iNOS expression by a combination of cisplatin and IFN-gamma in NB-39-nu cells. Thus, iNOS expression in NB-39-nu cells by a combination of cisplatin and IFN-gamma involves in the TNF-alpha-mediated signal transduction mechanism.