Correlation between interferon (IFN) alpha resistance and deletion of the IFN alpha/beta genes in acute leukemia cell lines suggests selection against the IFN system.

Homozygous and hemizygous deletions of the interferon A (IFNA) and IFNB genes have been frequently observed in acute leukemia cell lines, primary acute leukemia cases, and gliomas. Because IFNs have an antiproliferative effect, selection against the IFN alpha/beta system could play a role or accompany the development of the malignant phenotype. Although the deletion of the IFNA/B genes could interrupt an autocrine loop that controls cell proliferation, cells would still respond to exogenous IFN alpha/beta and, thus, lesions at the receptor or signal transduction level should also be present to render cells resistant to exogenous IFN alpha/beta. To test if selection against the IFN system was operating in acute leukemias, the sensitivity to the antiproliferative effect of IFN alpha 2 was studied in acute leukemia cell lines with and without alterations of the IFNA/B genes. We found that 10 of 11 acute leukemia cell lines with alterations of the IFNA/B genes were resistant to the antiproliferative effect of IFN alpha 2, whereas only two of eight cell lines with normal IFNA/B genes were IFN-resistant. We then examined the possibility that an alteration of the receptor expression could account for the lack of response to IFN alpha 2. No significant alteration in the expression or structure of the IFN alpha receptor was observed. We also studied the downmodulation of the alpha subunit of the IFN alpha receptor upon IFN alpha 2 binding. One cell line with deletion of the IFNA/B genes showed impaired downmodulation of the IFN alpha receptor. The data presented here suggest that selection against the IFN alpha/beta system could play a role or accompany the development of the malignant phenotype.     

Upregulation of hepcidin by interleukin-1beta in human hepatoma cell lines.

Anemia of chronic disease (ACD) is commonly observed in chronic inflammation, although its pathogenesis is poorly understood. Hepcidin is thought to be a key regulator in iron metabolism and has been implicated in ACD. Although the induction of hepcidin by an inflammatory cytokine interleukin-6 (IL-6) seems to have been confirmed, it is still controversial whether interleukin-1beta (IL-1beta), also known as an inflammatory cytokine, regulates hepcidin expression. We demonstrated that hepcidin mRNA was upregulated by IL-1beta in human hepatoma-derived HuH-7 cells, particularly at low concentrations of IL-1beta, while high concentrations of IL-6 were needed for the upregulation of hepcidin mRNA. Therefore, IL-1beta might be more important for the upregulation of hepcidin in physiological conditions than IL-6. Although IL-1beta induces IL-6 production in hepatocytes, our data indicate that the effect of IL-1beta on hepcidin expression is independent from that of IL-6. In conclusion, IL-1beta might have an important role in ACD.     

Homozygous deletion scanning in hepatobiliary tumor cell lines reveals alternative pathways for liver carcinogenesis

Despite high rates of loss of heterozygosity affecting various chromosomes, the number of tumor suppressor genes (TSGs) found to be consistently involved in primary liver cancer is low. In the past decade, characterization of homozygous deletions (HDs) in tumors has become instrumental to identify new TSGs or to reveal the influence of a particular TSG on the development of a specific tumor type. We performed a detailed HD profiling at 238 critical loci on a collection of 57 hepatobiliary tumor cell lines (hepatocellular, cholangiocellular, and bile duct carcinomas, hepatoblastomas, and immortalized hepatocytes). We identified HDs at 9 independent loci, the analysis of which was extended to 17 additional hepatobiliary tumor cell lines. In total, 34 homozygous losses involving 9 distinct genes were detected in the 74 cell lines analyzed. Besides expected deletions at the p16-INK4A/p14-ARF, FHIT, AXIN1, and p53 genes, we detected HDs at the PTEN, NF2, STK11, BAX, and LRPDIT genes that were formerly not known to be implicated in human liver tumorigenesis. In conclusion, our data suggest that these genes may represent novel liver tumor suppressive targets. Additional tumorigenic pathways should be carefully considered in hepatocarcinogenesis.     

T-lymphocyte cell lines derived from patients with acute lymphoblastic leukemia.

Several lymphoid cell lines with thymus-dependent lymphocyte (T-cell) characteristics have been established from 2 patients with acute lymphoblastic leukemia (ALL). These cell lines are considered to be leukemic T-cells on the basis of their abnormal chromosome constitution, growth in heterologous animals, ability to form rosettes with sheep red blood cells and the absence of immunoglobulin production, and destruction by antithymus cell sera. Sera from patients with leukemia did not react with these cultured cells but there was a strong reaction with infectious mononucleosis sera despite the fact that the cultured leukemia T-cells had no detectable Epstein-Barr virus (EBV) nor its genome.     

Suppression of normal human erythropoiesis by human recombinant DNA-produced alpha-2-interferon in vitro

Interferons (IFN) have been shown to suppress the proliferation of human erythroid progenitors (BFU-E, CFU-E) in vitro. We have previously demonstrated that the inhibition of erythroid colony formation by gamma-IFN in vitro is mediated, in part, through the activation of monocytes and T-lymphocytes. In order to examine the mechanism(s) underlying the inhibitory action of one type of recombinant alpha-IFN (alpha-2-IFN) on erythropoiesis, the effect of different doses (80-10,000 U) of alpha-2-IFN on erythroid colony formation by normal human bone marrow cells in the presence or absence of monocytes and/or T cells was studied. The addition of alpha-2-IFN to whole marrow caused the suppression of BFU-E (10%-68%) and CFU-E (5%-75%) in a dose-dependent fashion. This inhibition occurred with the direct addition of alpha-2-IFN to culture plates but not with brief preincubation of marrow cells with alpha-2-IFN followed by washing of the cells. By contrast, brief exposure of marrow cells to gamma-IFN resulted in significant suppression of erythroid colony formation. The inhibitory action of alpha-2-IFN was not influenced by erythropoietin. Removal of monocytes and/or T cells prior to the addition of alpha-2-IFN failed to significantly reduce the suppressive effects of this molecule (BFU-E: 21%-66%; CFU-E: 20%-83%). Coculture of purified monocytes or T-lymphocytes preexposed to alpha-2-IFN with autologous bone marrow cells did not cause suppression of erythropoiesis; monocytes or T cells similarly treated with gamma-IFN, however, inhibited autologous BFU-E and CFU-E in vitro. These results demonstrate that, unlike gamma-IFN, the inhibitory effect of alpha-2-IFN on erythroid colony formation in vitro is not mediated to any significant degree through monocytes and T-lymphocytes. The suppressive effect of alpha-2-IFN occurs either directly at the erythroid progenitor(s) level and/or through accessory cell(s) other than monocytes and T cells.     

Mouse model of human beta zero thalassemia: targeted deletion of the mouse beta maj- and beta min-globin genes in embryonic stem cells.

beta zero-Thalassemia is an inherited disorder characterized by the absence of beta-globin polypeptides derived from the affected allele. The molecular basis for this deficiency is a mutation of the adult beta-globin structural gene or cis regulatory elements that control beta-globin gene expression. A mouse model of this disease would enable the testing of therapeutic regimens designed to correct the defect. Here we report a 16-kb deletion that includes both adult beta-like globin genes, beta maj and beta min, in mouse embryonic stem cells. Heterozygous animals derived from the targeted cells are severely anemic with dramatically reduced hemoglobin levels, abnormal red cell morphology, splenomegaly, and markedly increased reticulocyte counts. Homozygous animals die in utero; however, heterozygous mice are fertile and transmit the deleted allele to progeny. The anemic phenotype is completely rescued in progeny derived from mating beta zero-thalassemic animals with transgenic mice expressing high levels of human hemoglobin A. The beta zero-thalassemic mice can be used to test genetic therapies for beta zero-thalassemia and can be bred with transgenic mice expressing high levels of human hemoglobin HbS to produce an improved mouse model of sickle cell disease.     

Diversity of the effect of recombinant tumor necrosis factors alpha and beta on human myelogenous leukemia cell lines

In vitro characteristics of response to recombinant tumor necrosis factors alpha (rTNF-alpha) and beta (rTNF-beta) were studied in six human myelogenous leukemia cell lines. Heterogeneity of the response to rTNF was observed, and one line (K562) was resistant. No enhancement of cell growth was noted in any cell line. The dose-response curves for rTNF-alpha were characteristically sigmoid, the maximum inhibitory effect occurring between 25 and 200 ng/mL. Nonresponsiveness within this range indicated resistance that could not be overcome, even with very high doses of rTNF alpha. A similar response of sensitive and resistant lines occurred after exposure to rTNF-beta. The clonogenic cells were more sensitive than the overall population to the action of rTNF alpha, and prolonged exposure was necessary for manifestation of the effect. Concomitant exposure to recombinant interferon-alpha (rIFN- alpha) increased the response of two cell lines to rTNF-alpha, but no clear synergistic action could be demonstrated. The addition of rIFN- gamma was without effect. Variations in the rTNF-alpha-induced proliferative response could not be explained by differences in the number of binding sites per cell or their affinity for rTNF-alpha. That the clonogenic cells showed a higher sensitivity than the whole population might indicate a preferential effect on more primitive, actively proliferating cells with high growth potential.     

Gene encoding the human beta-hexosaminidase beta chain: extensive homology of intron placement in the alpha- and beta-chain genes.

Lysosomal beta-hexosaminidase (EC 3.2.1.52) is composed of two structurally similar chains, alpha and beta, that are the products of different genes. Mutations in either gene causing beta-hexosaminidase deficiency result in the lysosomal storage disease GM2-gangliosidosis. To enable the investigation of the molecular lesions in this disorder and to study the evolutionary relationship between the alpha and beta chains, the beta-chain gene was isolated, and its organization was characterized. The beta-chain coding region is divided into 14 exons distributed over approximately 40 kilobases of DNA. Comparison with the alpha-chain gene revealed that 12 of the 13 introns interrupt the coding regions at homologous positions. This extensive sharing of intron placement demonstrates that the alpha and beta chains evolved by way of the duplication of a common ancestor.     

Establishment and characterization of 10 cell lines derived from patients with adult T-cell leukemia.

By using human T-cell growth factor (TCGF), 10 cell lines were established from tissue samples of 10 patients with adult T-cell leukemia (ATL). Three cell lines were adapted to growth in medium lacking TCGF. The surface markers of all cell lines were characteristic of inducer/helper T cells, i.e., OKT3+, OKT4+, OKT6-, OKT8-, OKIa1+, and human Lyt2+ and Lyt3+, except that one cell line was OKT3-. The expression of the viral antigen was examined during establishment of 8 of the 10 cell lines. The viral antigen was not expressed in leukemic cells before cultivation. In 5 lines, the viral antigen was detected by immunofluorescent staining after a short period of cultivation. However, 3 cell lines, ATL-6A, ATL-9Y, and ATL-1K did not express the viral antigen during short-term culture: the ATL-6A and ATL-9Y cell lines became positive for the viral antigen after 5 and 2 months of cultivation, respectively; the ATL-1K cell line remained antigen-negative throughout a culture period of 13 months. Southern blot hybridization assay showed that all of the cell lines, including the viral antigen-negative ATL-1K cell line, contained the viral genome. Thus, the retrovirus was associated with all 10 cell lines established from ATL patients, but there was a heterogeneity in the expression time of the retroviral antigen in leukemic cells maintained in vitro. Our findings suggested that the expression of the viral antigen was not required for maintenance of the leukemic state in vivo and for growth of leukemic cells in vitro.     

Marker profiles of human leukemia and lymphoma cell lines

Summary By means of the multiple marker analysis, a total of 55 human leukemia-lymphoma cell lines which included 15 T-cell, 30 B-cell, four myelomonocytic-cell, and six non-T, non-B cell lines was characterized for their marker profiles. The multiple markers used included a number of cell surface markers as detected by either rosette or immunofluorescence tests, enzyme assays, cytogenetic analysis, and certain functional assay. Based on the criteria previously defined it was found that all the cell lines were proved to represent original leukemia-lymphoma of ALL, AML, CLL, CML in blastic phase or variety of lymphomas. The monoclonality, a frozen state at a specific stage of differentiation-maturation, and cytogenetic marker in each leukemia-lymphoma cell line were remarkable common properties and were stable for years of cultivation. Similar, if not identical, general characteristics were observed in the study on 344 cases of uncultured fresh leukemia-lymphomas by the multiple marker analysis. While no single marker specific to any type of tumor was found, the study offers not only a basis for better understanding of the biology of leukemia-lymphoma but also an insight into normal hematopoietic cell differentiation in man.Supported by USPHS grants CA-14413, CA-17609, CA-25865, AI-08899, and contract 31-CB74165 from the National Cancer Institute and National Institute of Allergy and Infectious Diseases     

DOCK2 associates with CrkL and regulates Rac1 in human leukemia cell lines

The CDM (ced-5 of Caenorhabditis elegans, DOCK180 [downstream of Crk with molecular weight of 180 kDa] of humans, and myoblast city of Drosophila melanogaster) family of proteins has been shown to play a pivotal role in the integrin-mediated signaling pathway under the regulation of an adaptor molecule c-CT10-related kinase II (c-Crk-II) in adherent cells. Recently, hematopoietic cell-specific CDM protein DOCK2 has been shown to be indispensable for lymphocyte migration. However, the regulatory mechanism for DOCK2 is still unknown because DOCK2 lacks a c-Crk-II binding consensus motif. In this study, we demonstrated that DOCK2 bound to CrkL, which is present exclusively in hematopoietic cells both in vivo and in vitro, and we also found that 2 separate regions of DOCK2 contributed to its binding to Src homology 3 (SH3) domain of CrkL. Colocalization of DOCK2 with Crk-like (CrkL) and F-actin was shown by immunocytochemical analysis with the use of Jurkat cells. We also found that CrkL-induced activation of small guanine triphosphatase (GTPase) Rac1 was significantly inhibited by the DOCK2-dCS mutant in 293T cells. Furthermore, the association of DOCK2 and Vav, the guanine-nucleotide exchanging factor (GEF) for Rac1, was demonstrated in Jurkat cells. Finally, the stable expression of DOCK2-dCS mutant in Jurkat cells was shown to reduce cell attachment. These data suggest the presence of a novel protein complex of CrkL, DOCK2, and Vav to regulate Rac1 in leukemia cell lines.     

Homozygous deletion in KVLQT1 associated with Jervell and Lange-Nielsen syndrome

BACKGROUND: Long-QT (LQT) syndrome is a cardiac disorder that causes syncope, seizures, and sudden death from ventricular arrhythmias, specifically torsade de pointes. Both autosomal dominant LQT (Romano-Ward syndrome) and autosomal recessive LQT (Jervell and Lange-Nielsen syndrome, JLNS) have been reported. Heterozygous mutations in 3 potassium channel genes, KVLQT1, KCNE1 (minK), and HERG, and the cardiac sodium channel gene SCN5A cause autosomal dominant LQT. Autosomal recessive LQT, which is associated with deafness, has been found to occur with homozygous mutations in KVLQT1 and KCNE1 in JLNS families in which QTc prolongation was inherited as a dominant trait. METHODS AND RESULTS: An Amish family with clinical evidence of JLNS was analyzed for mutations by use of single-strand conformation polymorphism and DNA sequencing analyses for mutations in all known LQT genes. A novel homozygous 2-bp deletion in the S2 transmembrane segment of KVLQT1 was identified in affected members of this Amish family in which both QTc prolongation and deafness were inherited as recessive traits. This deletion represents a new JLNS-associated mutation in KVLQT1 and has deleterious effects on the KVLQT1 potassium channel, causing a frameshift and the truncation of the KVLQT1 protein. In contrast to previous reports in which LQT was inherited as a clear dominant trait, 2 parents in the JLNS family described here have normal QTc intervals (0.43 and 0.44 seconds, respectively). CONCLUSIONS: A novel homozygous KVLQT1 mutation causes JLNS in an Amish family with deafness that is inherited as an autosomal recessive trait.