Regulation of manganese superoxide dismutase and other antioxidant genes in normal and leukemic hematopoietic cells and their relationship to cytotoxicity by tumor necrosis factor

Myeloid cells are a major source of superoxide and other oxygen metabolites. As a protective mechanism, cells express antioxidant enzymes including manganese superoxide dismutase (Mn-SOD), copper-zinc SOD (Cu/Zn-SOD), and glutathione peroxidase (GSX-PX). Even though hematopoietic cells are a major source of oxidants, little is known of their expression of antioxidants. We found that seven myeloid leukemic cell lines blocked at different stages of differentiation constitutively expressed Mn-SOD, Cu/Zn-SOD, and GSX-PX RNAs. Level of Mn-SOD activities paralleled levels of Mn-SOD RNA. Terminal differentiation of native HL-60 cells to either granulocytes or macrophages did not alter levels of Mn-SOD RNA but markedly decreased cell division. Myeloid leukemic lines sensitive to cytotoxic effects of tumor necrosis factor (TNF) as well as normal peripheral blood lymphocytes and monocytes, dramatically increased their levels of Mn- SOD RNA in the presence of TNF. In contrast, Cu/Zn-SOD and GSX-PX RNA levels did not increase in these same cells. TNF-resistant leukemic lines had higher constitutive levels of Mn-SOD RNA and activity; and these levels did not change in the presence of TNF. Antisense but not random oligonucleotides to Mn-SOD markedly increased the sensitivity to the inhibitory effects of TNF for both the native HL-60 (TNF-sensitive) and K562 (TNF-resistant) cell lines. Further studies showed that the antisense oligonucleotides entered the cells and resulted in decreased levels of Mn-SOD RNA. The data suggest that Mn-SOD may provide protection against cytotoxicity of TNF in hematopoietic cells.     

Somatic cell hybrid analyses of hematopoietic differentiation

A differentiated cell expresses an entire set of specialized features. Somatic cell hybridization provides a method to examine control of gene regulation. We studied the expression of tissue-specific features in hybrids between human promyelocytes (HL-60) and human Burkitt's lymphoma cells (P3HR-1). Two hybrid lines, HP-1 and HP-2, and 18 hybrid clones were established and confirmed by karyotype, isozyme, and surface antigen analyses. The hybrids extinguished the 10 myeloid (HL- 60) features that we examined including myeloid morphology, histochemistry, and functions that included response to colony- stimulating factor and ability to differentiate to granulocytes or macrophages. In contrast, the hybrids synthesized immunoglobulin and expressed Epstein-Barr nuclear, early, and viral capsid antigens similar to the P3HR-1 lymphoid parental line. Results are contrasted to the findings when P3HR-1 lymphocytes are fused to human erythroid- myeloid cells (K562). Taken together, our results suggest that phenotypic differences between human myeloid and lymphoid cells in the hematopoietic lineage involve mutually exclusive programs and may possibly be mediated by the activity of diffusible, transacting molecules.     

Mutation and protein expression of p53 in acquired immunodeficiency syndrome-related lymphomas

p53 mutations are found in a variety of neoplasia. B-immunoblastic lymphoma (BIBL) is a rapidly progressive, aggressive lymphoma. As patients with acquired immunodeficiency syndrome (AIDS) live longer, BIBL is becoming an increasing problem. We asked three questions in our study. What is the frequency of p53 mutations in BIBL? Is it more frequent in patients with AIDS? Can immunohistochemical staining of lymph nodes for expression of p53 substitute for mutational analysis of p53 to detect lymphomas with mutated p53? Exons 5, 6, 7, 8 of the p53 gene (hot-spots for mutations) were amplified and examined for mutations by single-strand conformation polymorphism (SSCP) analysis. Altered migration was observed in 7 of 52 BIBL samples. Of these, 4 of 25 were from individuals infected with human immunodeficiency virus (HIV) and 3 of 27 were not infected with HIV. Direct sequencing of amplified material confirmed the presence of mutations in exons 5, 7, 8 of p53. A total of 26 BIBL as well as other lymphoma/leukemia samples, stained strongly by immunohistochemistry with three antibodies directed against human p53. Five of 6 BIBL samples with p53 mutations stained strongly for p53, but 20 lymphoma samples with no detectable p53 mutations also stained strongly for p53. Of note, however, 10 hyperplastic, nonmalignant lymph nodes from individuals either infected or not infected with HIV had negligible staining for p53 protein. In conclusion, p53 mutations occur in about 14% BIBL samples; the frequency of p53 mutations in BIBL in individuals with and without AIDS was similar. Positive p53 immunohistochemistry did not correlate with detectable p53 mutations in the same tissue, but positive immunohistochemical staining for p53 was only found in neoplastic lymph nodes. This latter finding provides a strong warning that p53 immunochemistry with available reagents cannot be used to determine which tumors have mutations of p53.     

An undifferentiated variant derived from the human acute myelogenous leukemia cell line (KG-1)

A variant subline (KG-1a) of the human acute myelogenous leukemia (AML) cell line (KG-1) has been isolated. The cells retain the same constitutive markers as the parent line, including HLA antigens, isoenzymes, and karyotype. The cells from the subline are morphologically and histochemically undifferentiated blast cells, while the parent cells and several of its clones are at the myeloblast and promyelocyte stages of development. The variant cells do not respond to colony-stimulating factor (CSF), and they do not express the human la antigen, nor a recently characterized AML antigen. The parent KG-1 cells are stimulated to proliferate in the presence of CSF and the cells express the la and AML antigen. Variant AML cell lines, such as KG-1a, will be useful in vitro models for investigating cellular response to CSF and for studying antigen expression in leukemic cells.     

Randomized study of 13-cis retinoic acid v placebo in the myelodysplastic disorders

A double-blind, placebo-controlled randomized trial of 13-cis retinoic acid was performed to determine if the drug has a therapeutic effect in patients with myelodysplastic syndromes (MDS). Sixty-eight evaluable patients with MDS were randomized to receive a single, daily oral dose of either 13-cis retinoic acid (13-CRA, 100 mg/m2) or matching placebo. Treatment was continued, when possible, for a period of 6 months. Determination of response to treatment was based on clinical course, repeat bone marrow biopsies, and aspirates and blood counts (CBC) with WBC differential, platelet, and reticulocyte numbers at specified intervals. No significant difference was noted between the two treatment groups in response to test drug (P = .66). One patient (3%) in the 13-CRA group and two patients (6%) in the placebo group had a minor response. Approximately 30% of patients in both groups had progression of their disease, and progression-free survival was nearly identical. Greater than 90% of the patients receiving 13-CRA developed mild or moderate skin toxicity that was reversible with decreasing or discontinuing the drug. Our study did not find that 13-CRA exerts a beneficial therapeutic effect in patients with MDS.     

Deletions of the cyclin-dependent kinase inhibitor genes p16INK4A and p15INK4B in non-Hodgkin's lymphomas

The tumor suppressor genes p16INK4A and p15INK4B map to the 9p21 chromosomal locus and are either homozygously deleted or mutated in a wide range of human cancer cell lines and tumors. Although chromosome 9 abnormalities have been described in non-Hodgkin's lymphomas (NHLs), to date, the mutational status of these genes has not been determined for these malignancies. A total of five cell lines and 75 NHLs were examined for homozygous deletions or point mutations in the coding regions of both the p15 and p16 genes using Southern blot and/or polymerase chain reaction-single-strand conformation polymorphism analyses. Homozygous deletions of either the p16 gene or both the p15 and p16 genes were observed in one diffuse large B-cell lymphoma cell line and two uncultured lymphomas consisting of one large B-cell and one mixed T-cell lymphoma. In contrast, point mutations were not detected in either the cell lines or lymphomas. These results indicate that the rate of alterations in the p15 and p16 genes is low for lymphomas, but loss of p16 and/or p15 may be involved in the development of some lymphomas.     

N-ras gene point mutations in childhood acute lymphocytic leukemia correlate with a poor prognosis

Ras genes can be altered by point mutations at critical portions of their coding regions to acquire transforming ability in vitro. These point mutations have been detected in a variety of human malignancies. However, their relevance for the clinical and biologic behavior of the subgroups of patients exhibiting these mutations in unclear. We analyzed 100 patients with childhood acute lymphocytic leukemias (ALLs) for point mutations of exons 1 and 2 of all three ras genes (H-ras, K- ras, and N-ras) by polymerase chain reaction and a combination of oligonucleotide hybridization and direct DNA sequencing. A 6% incidence of N-ras gene mutations was detected, all of which occurred at different nucleotides of codons 12 or 13 of N-ras. When correlating presence of ras mutations with the clinical and biologic features and the clinical outcome of these cases, a significantly higher risk for hematologic relapse (P = .01) and a trend toward a lower rate of complete remission (P = .07) was noted. The two groups did not differ in any of the known high-risk factors of ALL. These results suggest that presence of an N-ras mutation in children with ALL may be an independent predictor for worse clinical outcome and therefore may have therapeutic implications; further studies to confirm these findings are required because of the small number of patients with N-ras mutations.     

Chronic myelocytic leukemia: eosinophils involved in the malignant clone

Chronic myelocytic leukemia (CML) is a clonal disorder involving neutrophil, monocyte, erythrocyte, and platelet precursors. In order to determine if the eosinophils are also involved in the leukemic clone, we purified the eosinophils from a woman heterozygous for the common electrophoretic variants of the G6PD gene. Only type B enzyme was demonstrable in the eosinophils, neutrophils, and red cells, but both A and B enzymes were found in the fibroblasts. The data provide evidence that the eosinophil is involved in the malignant clone.