IL-2 controls the stability of Foxp3 expression in TGF-beta-induced Foxp3+ T cells in vivo

Stimulation of naive mouse CD4(+)Foxp3(-) T cells in the presence of TGF-β results in the induction of Foxp3 expression and T suppressor function. However, Foxp3 expression in these induced regulatory T cells (iTreg) is unstable, raising the possibility that iTreg would not be useful for treatment of autoimmune diseases. To analyze the factors that control the stability of Foxp3 expression in iTreg, we generated OVA-specific iTreg from OT-II Foxp3-GFP knockin mice. Following transfer to normal C57BL/6 mice, OT-II GFP(+) cells maintained high levels of Foxp3 expression for 8 d. However, they rapidly lost Foxp3 expression upon stimulation with OVA in IFA in vivo. This unstable phenotype was associated with a strong methylation of the Treg-specific demethylated region within the Foxp3 locus. Administration of IL-2/anti-IL-2 complexes expanded the numbers of transferred Foxp3(+) iTreg in the absence of Ag challenge. Notably, when the iTreg were stimulated with Ag, treatment with IL-2/anti-IL-2 complexes stabilized Foxp3 expression and resulted in enhanced demethylation of the Treg-specific demethylated region. Conversely, neutralization of IL-2 or disruption of its signaling by deletion of Stat5 diminished the level of Foxp3 expression resulting in decreased suppressor function of the iTreg in vivo. Our data suggest that stimulation with TGF-β in vitro is not sufficient for imprinting T cells with stable expression of Foxp3. Administration of IL-2 in vivo results in stabilization of Foxp3 expression and may prove to be a valuable adjunct for the use of iTreg for the treatment of autoimmune diseases.     

A molecular mechanism of complement resistance of human melanoma cells

The susceptibility of human melanoma cells to lysis by human complement after sensitization with the R24 murine IgG3 monoclonal antibody to the GD3 ganglioside antigen was investigated. It was found that the melanoma cell lines were either susceptible (greater than or equal to 70% cytotoxicity) or resistant (less than or equal to 30% cytotoxicity) to complement-mediated killing. We determined the kinetics of binding of C3 to and its subsequent fate on the melanoma cells. We found that on susceptible cell lines, maximal binding of C3 occurred within 10 min of incubation. At that time, approximately 90% of the bound C3 was in the form of C3b. During the subsequent incubation, the C3b was slowly inactivated, apparently generating the physiologic degradation products iC3b, C3dg, and C3d. However, this degradation of C3b could be inhibited without affecting the final degree of cytotoxicity, indicating that it is of no apparent consequence for the killing of susceptible melanoma cells. Very different results were obtained with resistant melanoma cells. Bound C3b was rapidly inactivated, and C3d was the predominant form of C3 on resistant cells throughout the incubation. Therefore, rapid inactivation of C3b was identified as a protective mechanism of human melanoma cells against complement attack. In addition, we found that resistance to complement is not an inherent property of the cells but depends on the antibody used for sensitization, because the resistant cell lines could be lysed after sensitization with polyclonal antiserum.     

Impaired transport as a mechanism of resistance to thiopurines in human T-lymphoblastic leukemia cells

In order to better understand the mechanisms of resistance to thiopurines, we studied two sublines of the MOLT4 T-lymphoblastic leukemia cell line, resistant to 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG). We found that the underlying mechanism of resistance in both resistant cell lines was a markedly reduction in initial transport of 6-MP (3- and 5-fold, respectively, in 6-MP- and 6-TG-resistant cells). No significant alteration of activities of hypoxanthine-guanine phosphoribosyl transferase, thiopurine methyltransferase or inosine monophosphate dehydrogenase, the key enzymes involved in the metabolism of thiopurines was detected. We conclude that defected initial transport of thiopurines by cells may very well explain their resistance to these drugs.     

An intrinsic mechanism predisposes Foxp3-expressing regulatory T cells to Th2 conversion in vivo

Naturally occurring regulatory T (nTreg) cells express Foxp3 and were originally discovered as immune suppressors critical for self-tolerance and immune homeostasis. Through yet-to-be-defined mechanisms, nTreg cells were recently shown to convert into proinflammatory cells. Particularly, attenuation of Foxp3 expression led to Th2 conversion of nTreg cells in vivo. In this paper, we demonstrated an nTreg-specific mechanism controlling their Th2 conversion. We found that wild-type nTreg cells expressing reduced levels of Foxp3 but not those expressing no Foxp3 produced the Th2 cytokine IL-4. Intriguingly, IL-4 production by converted nTreg cells is required for Th2 differentiation of coexisting naive CD4 T cells in vivo, suggesting that Th2 conversion of nTreg cells might be critical for directing Th2 immune responses. Th2 conversion of nTreg cells was not due to their inability to become Th1 cells, because IFN-γ was produced by Foxp3-low-expressing cells when IL-4/STAT-6 signaling was abrogated. Surprisingly, however, unlike naive CD4 T cells whose IL-4 production is dependent on STAT-6, Foxp3-low-expressing cells generated IL-4 independent of STAT-6, indicating an intrinsic mechanism that favors nTreg-to-Th2 differentiation. Indeed, compared with naive CD4 T cells, nTreg expressed elevated levels of GATA-3 independent of STAT-6. And GATA-3 was required for nTreg-to-Th2 conversion. Foxp3 may account for this GATA-3 upregulation in nTreg cells, because ectopic expression of Foxp3 preferentially promoted GATA-3 but not T-bet expression. Thus, we have identified an intrinsic mechanism that imposes a Th2/Th1 imbalance and predisposes Foxp3-expressing cells to IL-4 production independent of STAT-6 signaling.     

Muscle cells enhance resistance to pro-inflammatory cytokine-induced cartilage destruction

Pro-inflammatory cytokines IL-1β and TNFα play important roles in the manifestation of arthritis by disrupting the anabolic and catabolic activities of the chondrocytes. We observed a novel mechanism of cartilage regulation by which muscle cells diminish the response of chondrocytes to IL-1β and TNFα. We found that chondrocytes cocultured with muscle cells or cultured in muscle cell-conditioned medium significantly enhanced the expression of cartilage matrix proteins (collagen II and collagen IX) and resisted IL-1β and TNFα-induced cartilage damage. Our data suggest that this effect is achieved by inhibiting the expression of key components of the signaling pathways of pro-inflammatory cytokines (including NFκB, ESE-1, Cox-2, and GADD45β), leading to attenuated expression of cartilage-degrading enzymes (MMPs and ADAMTS4). Therefore, our work unveils a potential role of muscle in regulating cartilage homeostasis and response to pro-inflammatory stimuli, and provides insights on designing treatment strategies for joint degenerative diseases such as arthritis.     

Correction of DNA methylation disorder as a possible way to modulation of malignant cells drug resistance

The role of DNA methylation in functioning of a normal and cancer cell is shown in this review. The value of methylation in functioning of the systems which are involved in the process of formation of malignant cells drug resistance phenotype (regulator proteins of cellular cycle, DNA-adducts reparation, transport systems, systems of detoxication and adhesion) is explicated. The prospects of the antineoplastic therapy directed to regulation of DNA methylation by means of homocysteine and demethylation agents are analyzed.     

Codon swapping as a possible evolutionary mechanism

Summary It is apparent in the genetic code that amino acids of similar chemical nature have similar codons. I show how through successive codon captures (multiple rounds of Osawa-Jukes type reassignments), complete codon swappings in an unfavorable genetic code are evolutionarily feasible. This mechanisms could have complemented the ambiguity reduction and the vocabulary extension processes of codon-amino acid assignments. Evolution of wobble rules is implied. Transfer RNA molecules and synthetases may still carry memories of it.     

Reversible resistance induced by FLT3 inhibition: a novel resistance mechanism in mutant FLT3-expressing cells

Objectives

Clinical responses achieved with FLT3 kinase inhibitors in acute myeloid leukemia (AML) are typically transient and partial. Thus, there is a need for identification of molecular mechanisms of clinical resistance to these drugs. In response, we characterized MOLM13 AML cell lines made resistant to two structurally-independent FLT3 inhibitors.

Methods

MOLM13 cells were made drug resistant via prolonged exposure to midostaurin and HG-7-85-01, respectively. Cell proliferation was determined by Trypan blue exclusion. Protein expression was assessed by immunoblotting, immunoprecipitation, and flow cytometry. Cycloheximide was used to determine protein half-life. RT-PCR was performed to determine FLT3 mRNA levels, and FISH analysis was performed to determine FLT3 gene expression.

Results and Conclusions

We found that MOLM13 cells readily developed cross-resistance when exposed to either midostaurin or HG-7-85-01. Resistance in both lines was associated with dramatically elevated levels of cell surface FLT3 and elevated levels of phosphor-MAPK, but not phospho-STAT5. The increase in FLT3-ITD expression was at least in part due to reduced turnover of the receptor, with prolonged half-life. Importantly, the drug-resistant phenotype could be rapidly reversed upon withdrawal of either inhibitor. Consistent with this phenotype, no significant evidence of FLT3 gene amplification, kinase domain mutations, or elevated levels of mRNA was observed, suggesting that protein turnover may be part of an auto-regulatory pathway initiated by FLT3 kinase activity. Interestingly, FLT3 inhibitor resistance also correlated with resistance to cytosine arabinoside. Over-expression of FLT3 protein in response to kinase inhibitors may be part of a novel mechanism that could contribute to clinical resistance.     

Stochastic resonance as a possible mechanism of amplification of weak electric signals in living cells

The most important but still unresolved problem in bioelectromagnetics is the interaction of weak electromagnetic fields (EMFs) with living cells. Thermal and other types of noise pose restrictions in cell detection of weak signals. As a consequence, some extant experimental results that indicate low-intensity field effects cannot be accounted for, and this renders the results themselves questionable. One way out of this dead end is to search for possible mechanisms of signal amplification. In this paper, we discuss a general mechanism in which a weak signal is amplified by system noise itself. This mechanism was discovered several years ago in physics and is known, in its simplest form, as a stochastic resonance. It was shown that signal amplification may exceed a factor of 1000, which renders existing estimations of EMF thresholds highly speculative. The applicability of the stochastic resonance concept to cells is discussed particularly with respect to the possible role of the cell membrane in the amplification process. © 1994 Wiley-Liss, Inc.     

Stabilization of enzymes by dormancy autoinducers as a possible mechanism of resistance of resting microbial forms

Alkyl-substituted hydroxybenzenes (AHBs), autoinducers of microbial dormancy (ord ₁ factors), were found to stabilize the structure of protein macromolecules, making them metabolically less active and more resistant to stresses. In vitro experiments with theBacillus intermedius ribonuclease and chymotrypsin showed that the degree of the physical and chemical stability of these enzymes treated with AHBs depends on their concentration and incubation time. Experiments with RNase, which is capable of refolding, i.e., renaturation after heat denaturation, revealed that AHBs efficiently interact with both intact and denatured proteins. The data obtained allow the inference to be made thatd ₁ factors may play the role of natural chemical chaperons, blocking metabolism in dormant cells through the formation of catalytically inactive thermostable complexes with enzymes.     

Stabilization of enzymes by anabiosis autoinducers as a possible mechanism of resistance of resting microbial forms

Alkyl-substituted hydroxybenzenes (AHBs), auto-inducers of microbial dormancy (or d1 factors), were found to stabilize the structure of protein macromolecules, making them metabolically less active and more resistant to stresses. In vitro experiments with the Bacillus intermedius ribonuclease and chymotrypsin showed that the degree of the physical and chemical stability of these enzymes treated with AHBs depends on their concentration and incubation time. Experiments with RNase, which is capable of refolding, i.e., renaturation after heat denaturation, revealed that AHBs efficiently interact with both intact and denatured proteins. The data obtained allow the inference to be made that d1 factors may play the role of natural chemical chaperons, blocking metabolism in dormant cells through the formation of catalytically inactive thermostable complexes with enzymes.     

Extracellular adenosine metabolism in immune cells in melanoma

Malignant melanoma is characterized by the development of chronic inflammation in the tumor microenvironment, which leads to a strong immunosuppression associated with a rapid tumor progression. Adenosine is considered as one of the main immunosuppressive factors in the tumor environment. It is produced via enzymatic hydrolysis of extracellular ATP by ectonucleotidases CD39 and CD73 localized on cell surface. Using the ret transgenic mouse melanoma model that closely mimics human melanoma, we demonstrated an increased frequency of ectonucleotidase-positive myeloid-derived suppressor cells (MDSCs) in melanoma lesions and lymphoid organs. Furthermore, we observed that conventional CD4⁺FoxP3^? and CD8⁺ T cells infiltrating melanoma lesions of ret transgenic mice were distinctly enriched in the CD39⁺CD73⁺ subpopulation that co-expressed also PD-1. Ectonucleotidase expression was also up-regulated in CD4⁺ and CD8⁺ T cells upon activation. In addition, these ectoenzymes were largely found to be expressed on memory T cell compartment (in particular, on effector memory cells). Our data suggest that extracellular adenosine produced by regulatory T cells (Tregs) and MDSCs can suppress T cell effector functions through paracrine signaling. Another mechanism involves its production also by effector T cells and an inhibition of their anti-tumor reactivity via autocrine signaling as a part of the negative feedback loop. This mode of adenosine signaling could be also used by Tregs and MDSCs to enhance their immunosuppressive activity.