A Role for PML in Innate Immunity.

The promyelocytic leukemia gene (PML) of acute promyelocytic leukemia is an established tumor suppressor gene with critical functions in growth suppression, induction of apoptosis, and cellular senescence. Interestingly, although less studied, PML seems to play a key role also in immune response to viral infection. Herein, we report that Pml(-/-) mice spontaneously develop an atypical invasive and lethal granulomatous lesion known as botryomycosis (BTM). In Pml(-/-) mice, BTM is the result of impaired function of macrophages, whereby they fail to become activated and are thus unable to clear pathogenic microorganisms. Accordingly, Pml(-/-) mice are resistant to lipopolysaccharide (LPS)-induced septic shock as a result of an ineffective production of cytokines and chemokines, suggesting a role for PML in the innate immune Toll-like receptor (TLR)/NF-κB prosurvival pathway. These results not only shed light on a new fundamental function of PML in innate immunity, but they also point to a proto-oncogenic role for PML in certain cellular and pathological contexts.

Modulatory effects of mycobacterial heat-shock protein 70 in DNA vaccination against lymphoma.

BACKGROUND AND OBJECTIVES: Pathogen-derived molecules are danger signals and are able to activate innate immunity that in turn controls and regulates generation of adaptive immune responses. Mycobacterium tuberculosis heat shock protein 70 (myc HSP70) has been shown to exert a potent adjuvant effect in vaccination against both infectious agents and solid tumors. Here we explore the use of myc HSP70, as an adjuvant, in DNA vaccination against lymphoma. DESIGN AND METHODS: We describe the effects of vaccination using myc HSP70 encoding plasmid (pHSP70) co-injected with idiotype encoding plasmid (pId), in the 38C13 murine lymphoma model. We dissect mechanisms of anti-tumor immune response and compared efficacy with that of other DNA vaccination strategies. RESULTS: We show that myc HSP70 plasmid prolongs survival of immunized mice challenged with a high number (2000) of tumor cells. The magnitude of the anti-tumor effect is comparable to that obtained using granulocyte-macrophage colony-stimulating factor (GM-CSF) in the same setting. Moreover, HSP-induced protection is independent from the generation of IgG1 and IgG2a antibodies. Instead, anti-idiotype antibodies of IgG2b subclass develop after vaccination with pHSP as well as with pId and Id-GM-CSF fusion plasmid (pId-GM). INTERPRETATION AND CONCLUSIONS: Co-injection of HSP70 and Id plasmids induces a specific pattern of anti-idiotype immune response able to improve survival of immunized mice.

Acute myeloid leukemia fusion proteins deregulate genes involved in stem cell maintenance and DNA repair.

Acute myelogenous leukemias (AMLs) are genetically heterogeneous and characterized by chromosomal rearrangements that produce fusion proteins with aberrant transcriptional regulatory activities. Expression of AML fusion proteins in transgenic mice increases the risk of myeloid leukemias, suggesting that they induce a preleukemic state. The underlying molecular and biological mechanisms are, however, unknown. To address this issue, we performed a systematic analysis of fusion protein transcriptional targets. We expressed AML1/ETO, PML/RAR, and PLZF/RAR in U937 hemopoietic precursor cells and measured global gene expression using oligonucleotide chips. We identified 1,555 genes regulated concordantly by at least two fusion proteins that were further validated in patient samples and finally classified according to available functional information. Strikingly, we found that AML fusion proteins induce genes involved in the maintenance of the stem cell phenotype and repress DNA repair genes, mainly of the base excision repair pathway. Functional studies confirmed that ectopic expression of fusion proteins constitutively activates pathways leading to increased stem cell renewal (e.g., the Jagged1/Notch pathway) and provokes accumulation of DNA damage. We propose that expansion of the stem cell compartment and induction of a mutator phenotype are relevant features underlying the leukemic potential of AML-associated fusion proteins.

The PML gene is not involved in the regulation of MHC class I expression in human cell lines.

The promyelocytic leukemia gene, PML, is a growth and transformation suppressor. An additional role for PML as a regulator of major histocompatibility complex (MHC) class I antigen presentation has been proposed in a murine model, which would account for evasion from host immunity of tumors bearing malfunctioning PML, such as acute promyelocytic leukemia. Here we investigated a possible role of PML for the control MHC class I expression in human cells. PML function was perturbed in human cell lines either by PML/RAR alpha transfection or by PML- specific RNA interference. Impairment of wild-type PML function was proved by a microspeckled disassembly of nuclear bodies (NBs), where the protein is normally localized, or by their complete disappearance. However, no MHC class I down-regulation was observed in both instances. We next constructed a PML mutant, PML mut ex3, that is a human homolog of the murine PML mutant, truncated in exon 3, that was shown to down-regulate murine MHC class I. PML mut ex3 transfected in human cell lines exerted a dominant-negative effect since no PML molecules were detected in NBs but, instead, in perinuclear and cytoplasmic larger dot-like structures. Nevertheless, no down-regulation of MHC class I expression was evident. Moreover, neither transfection with PML mut ex3 nor PML-specific RNA interference affected the ability of gamma-interferon to up-regulate MHC class I expression. We conclude that, in human cell lines, PML is not involved directly in the regulation of MHC class I expression.