Effect of 5-azacytidine on the differentiation of human leukemia K-562 cells

The treatment of K-562 cells with 10(-5) M to 10(-7) M 5-azacytidine induced a marked increase in benzidine-positive cells. Similarly, the exposure of K-562 cells to 2 X 10(-3) M butyric acid or 5 X 10(-7) M 1-beta-arabinofuranosylcytosine or 1 X 10(-3) M hydroxyurea induced an erythroid differentiation of K-562 cells. The activity of DNA-methyltransferase and the level of methylcytosine in newly synthesized DNA were significantly decreased when the cells were treated with 5-azacytidine or butyric acid, while 1-beta-arabinofuranosylcytosine or hydroxyurea had no inhibitory effect on DNA-methylation of K-562 cells. These results suggest that the inhibition of DNA-methylation is not necessarily a specific phenomenon for erythroid differentiation of K-562 cells.     

Gamma interferon modulates myogenesis through the major histocompatibility complex class II transactivator, CIITA

Gamma interferon (IFN-γ) is an inflammatory cytokine that has complex effects on myogenesis. Here, we show that the IFN-γ-induced inhibition of myogenesis is mediated by the major histocompatibility complex (MHC) class II transactivator, CIITA, which binds to myogenin and inhibits its activity. In IFN-γ-treated myoblasts, the inhibition of muscle-specific genes includes the expression of myogenin itself, while in myotubes, myogenin expression is unaffected. Thus, CIITA appears to act by both repressing the expression and inhibiting the activity of myogenin at different stages of myogenesis. Stimulation by IFN-γ in skeletal muscle cells induces CIITA expression as well as MHC class II gene expression. The IFN-γ-mediated repression is reversible, with myogenesis proceeding normally upon removal of IFN-γ. Through overexpression studies, we confirm that the expression of CIITA, independent of IFN-γ, is sufficient to inhibit myogenesis. Through knockdown studies, we also demonstrate that CIITA is necessary for the IFN-γ-mediated inhibition of myogenesis. Finally, we show that CIITA, which lacks DNA binding activity, is recruited to muscle-specific promoters coincident with reductions in RNA polymerase II recruitment. Thus, this work reveals how IFN-γ modulates myogenesis and demonstrates a key role for CIITA in this process.     

Functional expression of a heterologous major histocompatibility complex class I gene in transgenic mice.

The regulated expression of major histocompatibility complex class I antigens is essential for assuring proper cellular immune responses. To study H-2 class I gene regulation, we have transferred a foreign class I gene to inbred mice and have previously shown that the heterologous class I gene was expressed in a tissue-dependent manner. In this report, we demonstrate that these mice expressed the transgenic class I molecule on the cell surface without any alteration in the level of endogenous H-2 class I antigens. Skin grafts from transgenic mice were rapidly rejected by mice of the background strain, indicating that the transgenic antigen was expressed in an immunologically functional form. As with endogenous H-2 class I genes, the class I transgene was inducible by interferon treatment and suppressible by human adenovirus 12 transformation. Linkage analysis indicated that the transgene was not closely linked to endogenous class I loci, suggesting that trans-regulation of class I genes can occur for class I genes located outside the major histocompatibility complex.     

Infection by polyomavirus of murine cells deficient in class I major histocompatibility complex expression.

Embryonic fibroblasts and kidney epithelial cells from beta 2-microglobulin-deficient mice were as infectible by polyomavirus as cells from normal littermates were, as judged by expression of nuclear viral capsid antigen, development of cytopathic effects, and yields of infectious virus. We conclude that expression of intact class I major histocompatibility complex molecules is not essential for polyomavirus infection.     

Human T-cell leukemia virus type I trans activator induces class I major histocompatibility complex antigen expression in glial cells

Transfection of the tax gene encoding the trans activator of human T-cell leukemia virus type I into glial line cells induced class I major histocompatibility complex (MHC) antigens on these cells. This occurred through the interaction of tax protein with the gene encoding class I MHC antigens but not through any soluble factors, such as interferons, or factors from glial cells. Since neural cells do not usually express MHC antigens, this novel mechanism may be an intermediate event between viral infection and subsequent immune-mediated pathology in the central nervous system.     

Human leukemia K-562 cells: induction of erythroid differentiation by 5-azacytidine

In this article we show that the cytidine analog 5-azacytidine is able to induce differentiation of the human leukemia K-562 cell line. Erythroid induction is associated with (a) an increase of the overall globin synthesis and globin mRNA accumulation, (b) a relative increase of fetal with respect to embryonic globins, and (c) a decrease of the proliferative capacity of hemoglobin-containing cells. In addition, we have analysed the DNA methylation pattern at the cleavage sites of MspI and HpaII restriction enzymes, which are known to cleave differently CCGG DNA sequences when 5-methylcytosine is present. These experiments indicate that in K-562 cells treated with 5-azacytidine, DNA becomes hypomethylated, suggesting that genetic programmes leading to an erythroid phenotype may be activated by a reduction of DNA methylation.     

A nonpolymorphic class I gene in the murine major histocompatibility complex

DNA sequence analysis of a class I gene (Q10), which maps to the Qa2,3 locus in the C57BL/10 (H-2b haplotype) mouse, reveals that it is almost identical to a cDNA clone (pH16) isolated from a SWR/J (H-2q haplotype) mouse liver cDNA library. Exon 5, in particular, has an unusual structure such that a polypeptide product is unlikely to be anchored in the cell membrane. Our findings suggest that the two sequences are derived from allelic class I genes, which are nonpolymorphic, in contrast to H-2K allelic sequences from the same mice, and they may encode liver-specific polypeptides of unknown function. Our previous studies indicate that the Q10 gene is a potential donor gene for the generation of mutations at the H-2K locus by inter-gene transfer of genetic information. Thus the lack of polymorphism in class I genes at the Q10 locus implies either that they are not recipients for such exchanges or that selective pressure prevents the accumulation of mutations in genes at this locus.     

Suppression of major histocompatibility complex class I and class II gene expression in Listeria monocytogenes-infected murine macrophages

Macrophage cells play a central role during infection with Listeria monocytogenes by both providing a major habitat for bacterial multiplication and presenting bacterial antigens to the immune system. In this study, we investigated the influence of L. monocytogenes infection on the expression of MHC class I and class II genes in two murine macrophage cell lines. Steady-state levels of I-Aβ chain mRNA were decreased in both resting J774A.1 and P388D₁ macrophages infected with L. monocytogenes whereas reduction of H-2K mRNA was only observed in P388D₁ cells. In addition, L. monocytogenes suppressed induction of MHC class I and class II mRNAs in response to γ-interferon as well as the maintenance of the induced state in activated P388D₁ macrophages. Exposure to the non-pathogenic species L. innocua or a deletion mutant of L. monocytogenes, which lacks the lecithinase operon, did not cause a reduction in H-2K and I-Aβ mRNA levels nor suppress expression of Ia antigens. Inhibition of MHC gene expression may represent an important part of the cross-talk between L. monocytogenes and the macrophage that probably influences the efficiency of a T cell-mediated immune response and thus the outcome of a listerial infection.     

Downregulation of major histocompatibility complex class I molecules by Kaposi's sarcoma-associated herpesvirus K3 and K5 proteins

The T-cell-mediated immune response plays a central role in the defense against intracellular pathogens. To avoid this immune response, viruses have evolved elaborate mechanisms that target and modulate many different aspects of the host's immune system. A target common to many of these viruses is the major histocompatibility complex (MHC) class I molecules. Kaposi's sarcoma-associated herpesvirus (KSHV) encodes K3 and K5 zinc finger membrane proteins which remove MHC class I molecules from the cell surface. K3 and K5 exhibit 40% amino acid identity to each other and localize primarily near the plasma membrane. While K3 and K5 dramatically downregulated class I molecules, they displayed different specificities in downregulation of HLA allotypes. K5 significantly downregulated HLA-A and -B and downregulated HLA-C only weakly, but not HLA-E, whereas K3 downregulated all four HLA allotypes. This selective downregulation of HLA allotypes by K5 was partly due to differences in amino acid sequences in their transmembrane regions. Biochemical analyses demonstrated that while K3 and K5 did not affect expression and intracellular transport of class I molecules, their expression induced rapid endocytosis of the molecules. These results demonstrate that KSHV has evolved a novel immune evasion mechanism by harboring similar but distinct genes, K3 and K5, which target MHC class I molecules in different ways.     

Pregnancy and interferon tau regulate major histocompatibility complex class I and beta2-microglobulin expression in the ovine uterus

Major histocompatibility complex (MHC) class I molecules, consisting of an alpha chain and beta2-microglobulin (beta2MG), play an important role in immune rejection responses by discriminating self and nonself and are increased by type I interferons during antiviral responses. Interferon tau (IFNtau), the pregnancy-recognition signal in ruminants, is a type I interferon produced by the ovine conceptus between Days 11 and 21 of gestation. In study 1, expression of MHC class I alpha chain and beta2MG mRNA and protein was detected primarily in endometrial luminal epithelium (LE) and glandular epithelium (GE) on Days 10 and 12 of the estrous cycle and pregnancy. On Days 14-20 of pregnancy, MHC class I and beta2MG expression increased only in endometrial stroma and GE and, concurrently, was absent in LE and superficial ductal GE (sGE). Although neither MHC class I nor beta2MG proteins were detected in Day 20 trophectoderm, beta2MG mRNA was detected in conceptus trophectoderm. In study 2, cyclic ewes were ovariectomized on Day 5, treated daily with progesterone to Day 16, received intrauterine infusions between Days 11 and 16 of either control serum proteins or recombinant ovine IFNtau, and were hysterectomized on Day 17. The IFNtau increased MHC class I and beta2MG expression only in endometrial stroma and GE. During pregnancy, MHC class I and beta2MG gene expression is inhibited in endometrial LE and sGE but, paradoxically, is stimulated by IFNtau in the stroma and GE. The silencing of MHC class I alpha chain and beta2MG genes in the endometrial LE and sGE during pregnancy recognition and establishment may be a critical mechanism preventing immune rejection of the conceptus allograft.