Multiple myeloma is an incurable disease requiring the development of effective therapies which can be used clinically. We have elucidated the potential for manipulating the cAMP signaling pathway as ...a target for inhibiting the growth of multiple myeloma cells.
As a model system, we primarily used the murine multiple myeloma cell line MOPC315 which can be grown both in vivo and in vitro. Human multiple myeloma cell lines U266, INA-6 and the B-cell precursor acute lymphoblastic leukemia cell line Reh were used only for in vitro studies. Cell death was assessed by flow cytometry and western blot analysis after treatment with cAMP elevating agents (forskolin, prostaglandin E2 and rolipram) and cAMP analogs. We followed tumor growth in vivo after forskolin treatment by imaging DsRed-labelled MOPC315 cells transplanted subcutaneously in BALB/c nude mice.
In contrast to the effect on Reh cells, 50 μM forskolin more than tripled the death of MOPC315 cells after 24 h in vitro. Forskolin induced cell death to a similar extent in the human myeloma cell lines U266 and INA-6. cAMP-mediated cell death had all the typical hallmarks of apoptosis, including changes in the mitochondrial membrane potential and cleavage of caspase 3, caspase 9 and PARP. Forskolin also inhibited the growth of multiple myeloma cells in a mouse model in vivo.
Elevation of intracellular levels of cAMP kills multiple myeloma cells in vitro and inhibits development of multiple myeloma in vivo. This strongly suggests that compounds activating the cAMP signaling pathway may be useful in the field of multiple myeloma.
In the present study, we aimed at identifying the mechanisms whereby the vitamin A metabolite all-trans retinoic acid (RA) promotes the formation of plasma cells upon stimulation of B cells via the ...innate immunity receptors TLR9 and RP105. Most often, differentiation of B cells involves the sequential events of class switch recombination and somatic hypermutations characteristic of germinal center reactions, followed by plasma cell formation. By studying the regulatory networks known to drive these reactions, we revealed that RA enhances the expression of the plasma cell-generating transcription factors IFN regulatory factor (IRF)4 and Blimp1, and paradoxically also activation-induced deaminase (AID) involved in somatic hypermutations/class switch recombination, in primary human B cells. IRF4 was identified as a particularly important protein involved in the RA-mediated production of IgG in TLR9/RP105-stimulated B cells. Based on kinetic studies, we present a model suggesting that the initial induction of IRF4 by RA favors AID expression. According to this model, the higher level of IRF4 that eventually arises results in sustained elevated levels of Blimp1. Regarded as a master regulator of plasma cell development, Blimp1 will in turn suppress AID expression and drive the formation of IgG-secreting plasma cells. Notably, we demonstrated IRF4 to be deregulated in B cells from common variable immunodeficiency patients, contributing to the observed aberrant expression of AID in these patients. Taken together, the present study both provides new insight into the mechanisms whereby RA induces differentiation of B cells and identifies IRF4 as a key to understand the defective functions of B cells in common variable immunodeficiency patients.
Bacterial lipopolysaccharide (LPS) is a major inducer of systemic inflammatory reactions and oxidative stress in response to microbial infections and may cause sepsis. In the present study, we ...demonstrate that retinoic acid inhibits LPS-induced activation in transgenic reporter mice and human monoblasts through inhibition of nuclear factor κB (NF-κB). By using noninvasive molecular imaging of NF-κB luciferase reporter mice, we showed that administration of retinoic acid repressed LPS-induced whole-body luminescence, demonstrating in vivo the dynamics of retinoic acid's ability to repress physiologic response to LPS. Retinoic acid also inhibited LPS-induced NF-κB activity in the human myeloblastic cell line U937. Retinoic-acid-receptor-selective agonists mimicked — while specific antagonists inhibited — the effects of retinoic acid, suggesting the involvement of nuclear retinoic acid receptors. Retinoic acid also repressed LPS-induced transcription of NF-κB target genes such as
IL-6,
MCP-1 and
COX-2. The effect of retinoic acid was dependent on new protein synthesis, was obstructed by a deacetylase inhibitor and was partly eliminated by a signal transducer and activator of transcription-1 (STAT1)/methyltransferase inhibitor, indicating that retinoic acid induces a new protein, possibly STAT1, that is involved in inhibiting NF-κB. This provides more evidence for retinoic acid's anti-inflammatory potential, which may have clinical implications in terms of fighting microbial infections.
Common variable immunodeficiency (CVID) is a disease that is characterized primarily by low levels of serum Igs, resulting in a high incidence of infections. It also has been associated with impaired ...B cell signaling via TLR9 and reduced serum levels of vitamin A. Given the established link between vitamin A deficiency and increased susceptibility to infections, we investigated the ability of the vitamin A metabolite all-trans retinoic acid (RA) to restore the defective immune responses in CVID-derived B cells activated through the TLRs TLR9 and RP105. We demonstrate that RA almost normalizes proliferation and IL-10 secretion in patient-derived B cells. IgG secretion is also partially restored, but to a more moderate extent. This can be explained by impaired RA-mediated isotype switching in TLR9/RP105-stimulated CVID-derived B cells owing to reduced induction of activation-induced deaminase. Accordingly, these B cells secreted higher levels of IgM than did normal B cells, and RA augmented IgM secretion. The ability of RA to improve critical immune parameters in CVID-derived B cells stimulated through TLR9 and RP105 support the possibility of combining RA with TLR stimulation for the treatment of CVID.
With cAMP signaling having a profound inhibitory effect on DNA damage-induced apoptosis in B-cell precursor acute lymphoblastic leukemia (BCP-ALL) cells, understanding how this signaling pathway ...affects the survival capacity of the cell has important implications for cancer therapy. We have recently shown that p53 is critical for the inhibitory effect of cAMP on genotoxic agents-mediated apoptosis in BCP-ALLs. Here, we show that elevation of cAMP levels in cells exposed to DNA damage enhances the nuclear translocation and DNA binding of NF-κB by accelerating the phosphorylation of IKKβ and thereby phosphorylation and degradation of IκBα. Furthermore, we show that the ability of cAMP to potentiate the ionizing radiation-induced activation of NF-κB requires the activity of MEK. Importantly, pharmacological or genetic ablation of NF-κB reversed the inhibitory effect of cAMP on DNA damage-induced apoptosis, demonstrating that, in addition to p53, cAMP relies on the activity of NF-κB to provide cells with a survival advantage in the face of DNA damage. Collectively, our results uncover a novel and important interaction between the cAMP and NF-κB pathways that may have implications for the targeted treatment of lymphoid malignancies, such as BCP-ALL, in which aberrant NF-κB activity functions as a driving force for treatment resistance.
Summary
Vitamin A is an essential anti‐infective agent with pleiotropic effects on cells of the immune system. The goal of the present study was to unravel the impact of the vitamin A metabolite ...retinoic acid (RA) on B‐cell survival related both to normal B‐cell homeostasis and to the detrimental effects imposed by DNA‐damaging agents. By combining RA with Toll‐like receptor 9 (TLR9) ligands, we show that RA prevents spontaneous, irradiation‐ and doxorubicin‐induced apoptosis of human B cells in an RA receptor‐dependent manner. RA‐mediated survival involved up‐regulation of the anti‐apoptotic protein myeloid cell leukemia 1 (MCL1) at the transcriptional level, and knock down of MCL1 by small interfering RNA partially reversed the effects of RA. To ensure that the combination of TLR9‐ligands and RA would not promote the survival of malignant B cells, the combined effects of stimulation with RA and TLR9 ligands was assessed on cells from patients with B‐cell malignancies. In contrast to the effects on normal B cells, the combination of TLR9 stimulation and RA neither enhanced the MCL1 levels nor inhibited the death of malignant B cells challenged by DNA‐damaging agents. Taken together, the present results reveal a vital role of MCL1 in RA‐mediated survival of normal B cells. Moreover, the findings suggest that RA in combination with TLR9 ligands might be useful adjuvants in the treatment of B‐cell malignancies by selectively protecting normal and not malignant B cells from DNA‐damage‐induced cell death.
In the present study we have shown that spontaneous and DNA damage‐induced apoptosis of human B cells is inhibited by retinoic acid (RA) in combination with ligands activating Toll‐like receptor 9 (TLR9). RA prevents the cell death in an RA receptor‐dependent manner involving up‐regulation of the anti‐apoptotic protein MCL1 at the transcription level. In contrast to the effects on normal B cells, the combination of TLR9 stimulation and RA neither enhanced the MCL1 levels nor inhibited the death of malignant B cells challenged by DNA‐damaging agents, suggesting that RA in combination with TLR9 ligands might be useful adjuvants in treatment of B‐cell malignancies.
Antimicrobial peptides produced by multicellular organisms protect against pathogenic microorganisms, whereas such peptides produced by bacteria provide an ecological advantage over competitors. ...Certain antimicrobial peptides of metazoan origin are also toxic to eukaryotic cells, with preference for a variety of cancerous cells. Plantaricin A (PlnA) is a peptide pheromone with membrane permeabilizing strain-specific antibacterial activity, produced by
Lactobacillus plantarum C11. Recently, we have reported that PlnA also permeabilizes cancerous rat pituitary cells (GH
4 cells), whereas normal rat anterior pituitary cells are resistant. To investigate if preferential effect on cancerous cells is a general feature of PlnA, we have studied effects of the peptide on normal and cancerous lymphocytes and neuronal cells. Normal human B and T cells, Reh cells (from human B cell leukemia), and Jurkat cells (from human T cell leukemia) were studied by flow cytometry to detect morphological changes (scatter) and viability (propidium iodide uptake), and by patch clamp recordings to monitor membrane conductance. Ca
2+ imaging based on a combination of fluo-4 and fura-red was used to monitor PlnA-induced membrane permeabilization in normal rat cortical neurons and glial cells, PC12 cells (from a rat adrenal chromaffin tumor), and murine N2A cells (from a spinal cord tumor). All the tested cell types were affected by 10–100
μM PlnA, whereas concentrations below 10
μM had no significant effect. We conclude that normal and cancerous lymphocytes and neuronal cells show similar sensitivity to PlnA.
B cell precursor acute lymphoblastic leukaemia (BCP-ALL) is the most common paediatric cancer. BCP-ALL blasts typically retain wild type p53, and are therefore assumed to rely on indirect measures to ...suppress transformation-induced p53 activity. We have recently demonstrated that the second messenger cyclic adenosine monophosphate (cAMP) through activation of protein kinase A (PKA) has the ability to inhibit DNA damage-induced p53 accumulation and thereby promote survival of the leukaemic blasts. Development of BCP-ALL in the bone marrow (BM) is supported by resident BM-derived mesenchymal stromal cells (MSCs). MSCs are known to produce prostaglandin E(2) (PGE(2)) which upon binding to its receptors is able to elicit a cAMP response in target cells. We hypothesized that PGE(2) produced by stromal cells in the BM microenvironment could stimulate cAMP production and PKA activation in BCP-ALL cells, thereby suppressing p53 accumulation and promoting survival of the malignant cells.
Primary BCP-ALL cells isolated from BM aspirates at diagnosis were cocultivated with BM-derived MSCs, and effects on DNA damage-induced p53 accumulation and cell death were monitored by SDS-PAGE/immunoblotting and flow cytometry-based methods, respectively. Effects of intervention of signalling along the PGE(2)-cAMP-PKA axis were assessed by inhibition of PGE(2) production or PKA activity. Statistical significance was tested by Wilcoxon signed-rank test or paired samples t test.
We demonstrate that BM-derived MSCs produce PGE(2) and protect primary BCP-ALL cells from p53 accumulation and apoptotic cell death. The MSC-mediated protection of DNA damage-mediated cell death is reversible upon inhibition of PGE(2) synthesis or PKA activity. Furthermore our results indicate differences in the sensitivity to variations in p53 levels between common cytogenetic subgroups of BCP-ALL.
Our findings support our hypothesis that BM-derived PGE(2), through activation of cAMP-PKA signalling in BCP-ALL blasts, can inhibit the tumour suppressive activity of wild type p53, thereby promoting leukaemogenesis and protecting against therapy-induced leukaemic cell death. These novel findings identify the PGE(2)-cAMP-PKA signalling pathway as a possible target for pharmacological intervention with potential relevance for treatment of BCP-ALL.
Summary
Vitamin A is an essential anti‐infective agent with pleiotropic effects on cells of the immune system. The goal of the present study was to unravel the impact of the vitamin A metabolite ...retinoic acid (
RA
) on B‐cell survival related both to normal B‐cell homeostasis and to the detrimental effects imposed by
DNA
‐damaging agents. By combining
RA
with Toll‐like receptor 9 (
TLR
9) ligands, we show that
RA
prevents spontaneous, irradiation‐ and doxorubicin‐induced apoptosis of human B cells in an
RA
receptor‐dependent manner.
RA
‐mediated survival involved up‐regulation of the anti‐apoptotic protein myeloid cell leukemia 1 (
MCL
1) at the transcriptional level, and knock down of
MCL
1
by small interfering
RNA
partially reversed the effects of
RA
. To ensure that the combination of
TLR
9‐ligands and
RA
would not promote the survival of malignant B cells, the combined effects of stimulation with
RA
and
TLR
9 ligands was assessed on cells from patients with B‐cell malignancies. In contrast to the effects on normal B cells, the combination of
TLR
9 stimulation and
RA
neither enhanced the
MCL
1 levels nor inhibited the death of malignant B cells challenged by
DNA
‐damaging agents. Taken together, the present results reveal a vital role of
MCL
1 in
RA
‐mediated survival of normal B cells. Moreover, the findings suggest that
RA
in combination with
TLR
9 ligands might be useful adjuvants in the treatment of B‐cell malignancies by selectively protecting normal and not malignant B cells from
DNA
‐damage‐induced cell death.
ABSTRACT
Our study aimed to investigate, in vivo, the relationship between vitamin A status and NF‐κ B activity, a transcription factor central in regulating inflammatory and immune responses. We ...used a novel transgenic murine NF‐κ B‐luciferase reporter model that enabled molecular imaging of NF‐κ B activity in live mice via an intensified image‐capture apparatus. Whole‐body luminescence, which reflects overall NF‐κ B activity, was elevated 2.2‐fold in vitamin A‐ deficient (VAD) mice compared with control mice. Specifically, NF‐κ B activity in VAD mice was increased 1.8‐fold in the lymph nodes and 1.4‐fold in the thymus and, NF‐κ B induction in UVB radiation‐exposed skin was also enhanced in VAD mice compared with control mice. The administration of all‐trans retinoic acid to VAD mice resulted in a transient reduction in NF‐κ B activity and, conversely, a single dose of the RAR‐pan‐antagonist, AGN 194310, administered to control mice, led to a marked, transient induction of whole‐body luminescence. Our results suggest that vitamin A status, and vitamin A itself, affects NF‐κ B activity in vivo and that the elevated NF‐κ B activity in VAD may be a mechanism underlying some of the features of VAD syndrome.
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