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Posts Tagged ‘Proceedings of the National Academy of Sciences of the United States of America’


Reporter/Curator: Stephen J. Williams, Ph.D.

Picture of a human melanoma cell line growing in tissue culture

Cultured human melanocytes .

Nitric oxide (NO), a gas with many biological functions in healthy cells, has also been implicated in the development of pathologies such as cancer.  Nitric oxide may also play a role in chemotherapeutic reisitance. For example it had been known (in the 1996 Melanoma study by Joshi et al. curated below) that nitric oxide synthase activity (the enzyme system which produces NO) was significantly elevated in cultured melanoma cell lines versus normal melanocytes.   Although it is known that many protein and enzymes systems could be directly covalently-modified by nitric oxide, either by S-nitrosylation or NO-NAD+ modifications (one of my earlier postings described one such protein modified by nitric oxide, GAPDH, and the effect these NO-modifications of GAPDH has on the etiology of various pathologies.), the molecular mechanisms by which these modifications affect cellular processes, lead to disease etiology, the proteins which are affected, and mechanisms related to chemotherapeutic sensitivity need to be further characterized. A new study from MIT reveals how NO-induced modifications may reduce cisplatin sensitivity in melanoma cells.  This study focuses on how decreasing nitric oxide levels in melanoma cells increases their cisplatin sensitivity.  The study also describes a possible mechanism for this effect: NO-induced modifications of the proapoptotic enzyme caspace-3 and prolyl-hdroxylase-2 (responsible for targeting prosurvival HIF-1α for proteosomal degradation).  Also, for a description of other cancer-related targets of nitric oxide please see the posting by Dr. Saxena at Crucial role of Nitric Oxide in Cancer on this site.

To read more background on nitric oxide and its role in disease etiology please see our e-book Perspectives on Nitric Oxide in Disease Mechanisms (Biomed e-Books) available on Amazon at:

http://www.amazon.com/Perspectives-Nitric-Disease-Mechanisms-ebook/dp/B00DINFFYC

      It is important, however, to note that most of these relationships between NO-induced protein modification and its relationship to disease mechanisms are causal, meaning that, in general, one notices a nitric-induced modification of a protein/enzyme with concomitant alteration of protein/enzyme function occurring in a disease/phenotype.  However, unlike reversible modifications, which have a cadre of pharmacologic inhibitors, nitric oxide induced modifications are covalent and nonenzymatic, therefore hindering easy cause/effect relationships.

With that said, the following was adapted from the MIT site at http://web.mit.edu/newsoffice/2013/how-melanoma-evades-chemotherapy-0407.html.

  

 

The findings from Dr. Luiz Godoy’s PNAS paper ENDOGENOUSLY PRODUCED NITRIC OXIDE MITIGATES SENSITIVITY OF MELANOMA CELLS TO CISPLATIN,  were presented at the 2013 annual meeting of the American Association for Cancer Research. The prognosis is generally worse for patients whose tumors have high levels of NO, said Luiz Godoy, an MIT research associate and lead author of the study.

Godoy and his colleagues have unraveled the mechanism behind melanoma’s resistance to cisplatin, a commonly used chemotherapy drug, and, in ongoing studies, have found that cisplatin treatment also increases NO levels in breast and colon cancers.

“This could be a mechanism that is widely shared in different cancers, and if you use the drugs that are already used to treat cancer, along with other drugs that could scavenge or decrease the production of NO, you may have a synergistic effect,” said Godoy, who works in the lab of Gerald Wogan, an MIT professor emeritus of biological engineering and senior author of the study.

NO has many roles within living cells. At low concentrations, it helps regulate processes such as cell death and muscle contraction. NO, which is a free radical, is also important for immune-system function. Immune cells, such as macrophages, produce large amounts of NO during infection, helping to kill invading microbes by damaging their DNA or other cell components.

“It’s really a molecule that has a dual effect,” Godoy said. “At low concentrations it can act as a signaling molecule, while high concentrations will be toxic.”

Knocking out NO

In the new study, the researchers treated melanoma cells grown in the lab with drugs that capture NO before it can act. They then treated the cells with cisplatin and tracked cell-death rates. The NO-depleted cells became much more sensitive to the drug, confirming earlier findings.

The MIT team then went a step further, investigating how NO confers its survival benefits. It was already known that NO can alter protein function through a process known as S-nitrosation, which involves attaching NO to the target protein. S-nitrosation can affect many proteins, but in this study the researchers focused on two that are strongly linked with cell death and survival, known as caspase-3 and PHD2.

The role of caspase-3 is to stimulate cell suicide, under the appropriate conditions, but adding NO to the protein deactivates it. This prevents the cell from dying even when treated with cisplatin, a drug that produces massive DNA damage.

PHD2 is also involved in cell death; its role is to help break down another protein called HIF-1 alpha, which is a pro-survival protein. When NO inactivates PHD2, HIF-1 alpha stays intact and keeps the cell alive.

“Now we have a mechanistic link between nitric oxide and the increased aggressiveness of melanoma,” said Douglas Thomas, an assistant professor of medicinal chemistry and pharmacognosy at the University of Illinois at Chicago, who was not part of the research team. “It certainly would be worth exploring whether this mechanism is also present in different tumor types as well.”

The MIT researchers also found in some cancer cells, NO levels were five times higher than normal following cisplatin treatment. Godoy is now investigating how cisplatin stimulates that NO boost, and is also looking for other proteins that NO may be targeting.

Source: http://web.mit.edu/newsoffice/2013/how-melanoma-evades-chemotherapy-0407.html

Melanoma Res. 1996 Apr;6(2):121-6.

Nitric oxide synthase activity is up-regulated in melanoma cell lines: a potential mechanism for metastases formation.

Joshi M, Strandhoy J, White WL.

Source

Department of Dermatology, Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, NC 27157, USA.

Abstract

Nitric oxide (NO) may be an important mediator of tumour angiogenesis and metastasis formation. Tumour cell derived NO may be important in the regulation of angiogenesis and vasodilatation of the blood vessels surrounding a tumour. The aims of the present study were, firstly, to determine whether malignant melanoma cells and normal melanocytes had nitric oxide synthase (NOS) activity (measured by the conversion of L-arginine to L-citrulline) and, secondly, to determine whether there was a difference in NOS activity between malignant and normal cell types. This paper assays NOS activity directly in lysates from normal human melanocyte and malignant melanoma cell lines. The enzyme activity was not inducible with bacterial lipopolysaccharide and could be heat denatured. The activity of NOS was demonstrated to be both NADPH- and calcium-dependent and it was inhibitable in a dose-dependent manner by the NOS inhibitor Nw-nitro-L-arginine methyl ester. We conclude that melanoma and melanocyte cells express a constitutive form of NOS. Finally, nitric oxide synthase activity in melanoma cell lines was found to be significantly greater than in normal melanocytes. These findings suggest that NO synthesis is elevated in malignant melanoma. An elevated NO concentration in melanoma is expected to promote metastases by maintaining a vasodilator tone in the blood vessels in and around the melanoma.

Proc Natl Acad Sci U S A. 2012 Dec 11;109(50):20373-8. doi: 10.1073/pnas.1218938109. Epub 2012 Nov 26.

Endogenously produced nitric oxide mitigates sensitivity of melanoma cells to cisplatin.

Godoy LC, Anderson CT, Chowdhury R, Trudel LJ, Wogan GN.

Source

Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

Abstract

Melanoma patients experience inferior survival after biochemotherapy when their tumors contain numerous cells expressing the inducible isoform of NO synthase (iNOS) and elevated levels of nitrotyrosine, a product derived from NO. Although several lines of evidence suggest that NO promotes tumor growth and increases resistance to chemotherapy, it is unclear how it shapes these outcomes. Here we demonstrate that modulation of NO-mediated S-nitrosation of cellular proteins is strongly associated with the pattern of response to the anticancer agent cisplatin in human melanoma cells in vitro. Cells were shown to express iNOS constitutively, and to generate sustained nanomolar levels of NO intracellularly. Inhibition of NO synthesis or scavenging of NO enhanced cisplatin-induced apoptotic cell death. Additionally, pharmacologic agents disrupting S-nitrosation markedly increased cisplatin toxicity, whereas treatments favoring stabilization of S-nitrosothiols (SNOs) decreased its cytotoxic potency. Activity of the proapoptotic enzyme caspase-3 was higher in cells treated with a combination of cisplatin and chemicals that decreased NO/SNOs, whereas lower activity resulted from cisplatin combined with stabilization of SNOs. Constitutive protein S-nitrosation in cells was detected by analysis with biotin switch and reduction/chemiluminescence techniques. Moreover, intracellular NO concentration increased significantly in cells that survived cisplatin treatment, resulting in augmented S-nitrosation of caspase-3 and prolyl-hydroxylase-2, the enzyme responsible for targeting the prosurvival transcription factor hypoxia-inducible factor-1α for proteasomal degradation. Because activities of these enzymes are inhibited by S-nitrosation, our data thus indicate that modulation of intrinsic intracellular NO levels substantially affects cisplatin toxicity in melanoma cells. The underlying mechanisms may thus represent potential targets for adjuvant strategies to improve the efficacy of chemotherapy.

Other posts on this site regarding Nitric Oxide and Cancer include:

Crucial role of Nitric Oxide in Cancer

Nitric Oxide Covalent Modifications: A Putative Therapeutic Target?

Nitric Oxide has a ubiquitous role in the regulation of glycolysis -with a concomitant influence on mitochondrial function

Nitric Oxide Signalling Pathways

In focus: Melanoma therapeutics

Combined anti-CTLA4 and anti-PD1 immunotherapy shows promising results against advanced melanoma

Whole exome somatic mutations analysis of malignant melanoma contributes to the development of personalized cancer therapy for this disease

In focus: Melanoma therapeutics

In focus: Melanoma Genetics

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IRF-1 Deficiency Skews the Differentiation of Dendritic Cells

Reporter: Larry H Bernstein, MD, FCAP

 

 

IFN Regulatory Factor-1 Negatively Regulates CD4+CD25+ Regulatory T Cell Differentiation by Repressing Foxp3 Expression1

 

Alessandra Fragale*, Lucia Gabriele†, Emilia Stellacci*, Paola Borghi†,…. and Angela Battistini2,*
The Journal of Immunology   Aug 1, 2008; 181(3): 1673-1682

Regulatory T (Treg) cells are critical in inducing and maintaining tolerance. Despite progress in understanding the basis of immune tolerance,

  • mechanisms and molecules involved in the generation of Treg cells remain poorly understood.

IFN regulatory factor (IRF)-1 is a pleiotropic transcription factor implicated in the regulation of various immune processes. In this study, we report that IRF-1 negatively regulates CD4+CD25+ Treg cell

  • development and function by specifically repressing Foxp3 expression.

IRF-1-deficient (IRF-1−/−) mice showed a selective and marked increase of highly activated and differentiated CD4+CD25+Foxp3+ Treg cells in thymus and in all peripheral lymphoid organs. Furthermore,

  • IRF-1−/− CD4+CD25− T cells showed extremely high bent to differentiate into CD4+CD25+Foxp3+ Treg cells, whereas
  • restoring IRF-1 expression in IRF-1−/− CD4+CD25− T cells
    • impaired their differentiation into CD25+Foxp3+ cells.

Functionally, both isolated and TGF-β-induced CD4+CD25+ Treg cells from IRF-1−/− mice

  • exhibited more increased suppressive activity than wild-type Treg cells.

Such phenotype and functional characteristics were explained at a mechanistic level by the finding that

  • IRF-1 binds a highly conserved IRF consensus element sequence (IRF-E) in the foxp3 gene promoter in vivo and
  • negatively regulates its transcriptional activity.

We conclude that IRF-1 is a key negative regulator of CD4+CD25+ Treg cells

  • through direct repression of Foxp3 expression.
Introduction

Tolerance is critical for prevention of autoimmunity and maintenance of immune homeostasis by active suppression of inappropriate immune responses. Suppression has a dedicated population of  T cells that

  • control the responses of other T cells.

This cell population, referred to as regulatory T (Treg)3 cells, actually comprises several subsets, including naturally occurring CD4+CD25+ Treg cells that arise in thymus. Once generated,

  • thymic Treg cells are exported to peripheral tissues, and
  • comprise 5–10% of peripheral CD4+ T cells (1, 2, 3).

CD4+CD25+ Treg cells are characterized by

  • constitutive expression of IL-2Rα (CD25), CTLA-4, and glucocorticoid-induced TNFR family-related gene; moreover,
  • they express CD62 ligand (CD62L) and are mainly CD45RBlow (4).

In contrast to cell surface markers, which can be shared with other T cells populations,

  • the forkhead/winged-helix family transcriptional repressor Foxp3 is
  • specifically expressed in CD4+CD25+ Treg cells and
  • rigorously controls their development and function (5, 6, 7).

Functionally after TCR stimulation, CD4+CD25+ Treg cells can

  • mediate strong suppression of proliferation and
  • IL-2 production by CD4+ T cells both in vivo and in vitro (8).

Although mechanisms of suppression are not fully understood,

  • they appear to be cell contact-mediated, whereas
  • the relative contribution of soluble cytokines remains controversial
    • with differences between in vitro and in vivo results (1, 8, 9).

Indeed, the involvement of cytokines in the suppressor function of CD4+CD25+ Treg cells has been proposed in vivo,

  • where they are able to produce IL-10 and TGF-β (10, 11, 12), and
  • importantly, IL-10 activity has been recently associated with the function of TGF-β-induced CD4+CD25−CD45RBlow cells (13).

Beside naturally occurring CD4+CD25+ Treg cells, CD4+CD25+ Treg cells can also be

  • induced (inTreg) in vivo or in vitro after TCR stimulation and TGF-β treatment,
  • acquiring expression of CD25 and Foxp3 both in mice (14, 15, 16) and humans (17, 18, 19, 20),
    • although with characteristic functional differences (20).

Despite extensive studies on the role of Foxp3 in inducing and maintaining tolerance, little information on regulation of its expression is available. Transcription factors of the IFN regulatory factor (IRF) family participate in

  • the early host response to pathogens,
  • in immunomodulation and
  • hematopoietic differentiation (21).

Nine members of this family have been identified based on a unique helix-turn-helix DNA binding domain, located at

  • the N terminus that is responsible for binding to the IRF consensus element (IRF-E) (21).
The first member of the family, IRF-1, was originally identified as a protein that binds
  • the cis-acting DNA elements in the ifnβ gene promoter and the IRF-E (also referred to as the IFN-stimulated response element; ISRE),
  • in the promoters of IFN-αβ-stimulated genes (22).

IRF-1 is expressed at low basal levels in all cell types examined, but

  • accumulates in response to several stimuli and cytokines including IFN-γ, the strongest IRF-1 inducer (22).
Intensive functional analyses conducted on this transcription factor have revealed a remarkable functional diversity in the
  • regulation of cellular responses through the
  • modulation of different sets of genes,
  • depending on
    1. cell type,
    2. state of the cell, and/or
    3. nature of the stimuli (21).
We and others have shown that IRF-1 affects the differentiation of both lymphoid and myeloid lineages (22, 23, 24, 25, 26, 27, 28). In particular, studies in knockout (KO) mice have implicated IRF-1
in the regulation of various immune processes:
  1. impairment of CD8+ T cell and NK cell maturation,
  2. impaired IL-12 macrophage production,
  3. exclusive Th2 differentiation, and
  4. defective Th1 responses…………. have all been observed (22, 23, 24, 25, 26).
As a result, IRF-1−/− mice are highly susceptible to infections, for which effective host control
    • is associated with a Th1 immune response (24).
In contrast, these mice are characterized by
  • increased resistance to several autoimmune diseases such as
  1. collagen-induced arthritis,
  2. experimental autoimmune encephalomyelitis,
  3. Helicobacter pylori-induced gastritis,
  4. induced lymphocytic thyroiditis,
  5. insulitis, or
  6. diabetes (29, 30, 31, 32).
Recently, we reported that IRF-1−/− mice display a prevalence of
  • dendritic cell (DC) subsets with immature and tolerogenic features that were
    • unable to undergo full maturation after stimulation.
Moreover, IRF-1−/− DC conferred
    • increased suppressive activity to CD4+CD25+ Treg cells (33).
Because there is growing evidence that immature or partially matured DC can induce tolerance (34, 35), we hypothesized that IRF-1 could play a role in
  • Treg development and function.
In this study, we analyzed the CD4+CD25+ compartment in IRF-1−/− mice and
  • we found that in vivo IRF-1 deficiency resulted in a
  • selective and marked increase in highly differentiated and activated CD4+CD25+Foxp3+ Treg cells, whereas
reintroduction of IRF-1 by retrovirus transduction
    • impaired TGF-β-mediated differentiation of IRF-1−/− CD4+CD25− T cells into CD4+CD25+Foxp3+ Treg cells.
At molecular level, we show that IRF-1 plays a direct role in the generation and expansion of CD4+CD25+ Treg cells
    • specifically repressing Foxp3 transcriptional activity.
Our results, therefore, highlight a unique role for IRF-1 as regulator of Foxp3, thus pointing to IRF-1 as a specific tool to control altered tolerance.
Results
CD4+CD25+ Treg from IRF-1−/− mice are increased and functionally more suppressive than WT Treg cells
The distribution and the phenotype of CD4+CD25+Foxp3+ Treg in lymphoid organs of IRF-1−/− mice were determined by flow cytometry.
the number of ex vivo double positive CD4+CD25+ cells was significantly increased in spleens and skin draining and mesenteric lymph nodes (2.8-, 2.3-, and 2.1-fold increase, respectively), and to a lesser extent, in thymus (1.6-fold increase) of IRF-1−/− mice as compared with WT mice. Consistently with previous reports (23, 41), no differences in CD4+ T cell and total cell numbers in all lymphoid organs from WT or IRF-1−/− mice were found (data not shown). Strikingly, intracellular analysis of Foxp3 expression showed that this factor was increasingly expressed in CD4+CD25+ Treg cells from spleens as well as from other lymphoid organs of IRF-1−/− mice
FACS analysis of splenic magnetically sorted CD4+CD25+ Treg cells was performed to evaluate the expression of activation markers.  IRF-1−/− Treg cells were to a large extent characteristic of a marked activated and differentiated phenotype.
Because there is accumulating evidence that activity of CD4+CD25+ Treg cells in vivo involves some immunosuppressive cytokines (9, 10, 11, 12), we also compared the cytokine profile of IRF-1−/− CD4+CD25+ Treg cells with the profile of WT counterparts . Lower levels of proinflammatory cytokines, such as TNF-α and IFN-γ, whereas higher levels of IL-4 were expressed in CD4+CD25+ Treg cells as well as in CD4+CD25− T lymphocytes from KO as compared with WT cells. Notably, only IRF-1−/− Treg cells showed a clear-cut increase in the expression of IL-10. By contrast, TGF-β was expressed at similar levels in CD4+CD25+ Treg cells from both IRF-1−/− and WT mice. Accordingly with mRNA data, IL-10 secretion in supernatants of TCR-stimulated CD4+CD25+ cocultures from IRF-1−/− mice was significantly increased (3-fold), whereas
    • IFN-γ secretion was decreased (2.5-fold) compared with cocultures from WT mice (Fig. 2⇑C).
As the functional hallmark of Treg cells is their ability to suppress the expansion of effector T cells, we next evaluated this activity performing suppression assays (1, 2, 3, 8). Importantly, CD4+CD25+ Treg cells from IRF-1−/− mice were found significantly more efficient than WT Treg cells in suppressing the proliferation of syngeneic CD4+CD25− responder T cells in a dose-dependent fashion. Next, to verify whether IRF-1−/− Treg cells suppression ability was retained vs WT responder T cells, we performed suppression assays using IRF-1−/− Treg and WT responders and vice versa. The suppressive activity of IRF-1−/− Treg cells toward WT responders was dose-dependently increased, as well.
IRF-1−/− CD4+CD25− T cells show high bent to convert into CD4+CD25+ Treg cells
It has been reported in mice and human that TGF-β promotes the induction of peripheral CD4+CD25− T cells into CD4+CD25+ Treg cells (inTreg), that acquire Foxp3 expression and regulatory functions.
In presence of TGF-β, 44.2% of CD4+CD25+ inTreg cells were generated in the coculture of CD4+CD25− T cells from IRF-1−/− mice, whereas
  • only 24% of double positive cells were detected in the corresponding coculture from WT mice.
Notably, even in absence of TGF-β, 25.4% CD4+CD25+ inTreg were generated in the coculture of CD4+CD25− T cells from IRF-1−/− mice, as
  • compared with 16.5% of Treg cells generated in WT cocultures.
Importantly, an increased number of CD4+CD25+-gated Foxp3+ cells were observed in IRF-1−/− inTreg cells in the presence (4.5-fold increase) or in the absence (8-fold increase) of TGF-β compared with WT inTreg cells. Next, to evaluate quantitatively Foxp3 expression levels in TGF-β-induced Treg vs ex vivo freshly purified Treg cells, quantitative real-time PCR was performed. A clear-cut
induction of Foxp3 mRNA (4.5-fold increase) was detected in TGF-β-treated IRF-1−/− cells compared with WT cells. Of note, these levels were comparable with those present in freshly isolated IRF-1−/− CD4+CD25+ cells. Strikingly, also untreated IRF-1−/− T cells showed higher levels of Foxp3 mRNA than WT untreated cells (6-fold increase) and similar to levels present in freshly purified WT CD4+CD25+ Treg cells.
The functionality of CD4+CD25+Foxp3+ inTreg cells was then assessed by suppression assays. TGF-β-treated IRF-1−/− inTreg cells were significantly more effective than the WT counterpart cells
  • in suppressing proliferation of effector T cells in a dose-dependent way.
Interestingly, a saturating amount of anti-IL-10 m Abs neutralized the suppression ability of  inTreg cells from both IRF-1−/− and WT mice even though the effect was much more marked in IRF-1−/− inTreg cells. Control Abs did not exhibit any effect.
Restoring IRF-1 expression in IRF-1−/− CD4+CD25− T cells impairs their differentiation into CD4+CD25+Foxp3+ cells
To address the specificity of IRF-1 role in differentiation of CD4+CD25+ Treg cells from CD25− cells, we investigate whether
  • forced expression of IRF-1 in CD4+CD25− IRF-1−/− T cells could rescue the WT phenotype.
  • bicistronic retroviral vectors expressing murine IRF-1 and human CD8 protein as surface marker (MigR1 IRF-1-CD8) or CD8 alone (MigR1 EV-CD8) were generated.
Splenic CD4+CD25− cells from IRF-1−/− mice were stimulated with plate-bound anti-CD3 and anti-CD28 Abs and infected with either retrovirus.
  • 31.6% of MigR1 EV-CD8 CD4+ retrovirus-infected cells were CD25+, by contrast
  • only 17.7% of MigR1 IRF-1-CD8 retrovirus-infected cells were double positive.
Consistently, Foxp3 expression in CD8+-gated cells was significantly decreased in MigR1 IRF-1-CD8-infected cells as compared with
  • those infected with MigR1 EV-CD8 vectors,
  • strongly supporting the evidence that IRF-1 specifically impairs CD4+CD25+ cell differentiation.
IRF-1 binds an IRF-E on the Foxp3 core promoter and inhibits its transcriptional activity
To shed light on the molecular mechanisms responsible for the striking effect exerted by IRF-1 on the development and function of CD4+CD25+ Treg cells, we investigated whether IRF-1, which is a regulator of key immunomodulatory genes (21), could directly regulate the foxp3 gene promoter activity. The proximal promoter of human foxp3 gene has been recently characterized and localized at −511/+176 bp upstream of the 5′ untranslated region (38). By the Genomatix software, we analyzed this region and found an IRF-E spanning from −234 to −203 bp . This region has been found highly homologous to mouse and rat foxp3 promoter, and of note, the IRF-E is perfectly conserved between humans and these species (38). To determine whether IRF-1 could bind this sequence, DNA affinity purification assays were performed with cell extracts from Jurkat T cells, which display discrete basal levels of IRF-1, and from the same cells treated with IFN-γ to maximally stimulate IRF-1 expression. A total of 200 μg of nuclear extracts was incubated with oligonucleotides containing the WT or the a mutated version of IRF-E. The isolated complexes were then examined by immunoblotting against IRF-1. A specific binding of IRF-1 to Foxp3 oligonucleotide was evident. The binding was strongly stimulated by IFN-γ treatment and, interestingly, it was comparable to that obtained when the same extracts were incubated with a synthetic oligonucleotide corresponding to C13, the canonical IRF-1 consensus sequence (21). IRF-1 binding was highly specific because a mutated version of the Foxp3/IRF-E, or an unrelated oligonucleotide corresponding to the STAT binding site present on the β-casein gene promoter, did not retain any protein from the same extracts. To functionally characterize the specific binding of IRF-1 to the foxp3 gene promoter, we cloned the encompassing part of the proximal promoter containing the IRF-E from −296 to +7 bp of foxp3 gene promoter upstream the luciferase reporter gene. The effect of IRF-1 was evaluated in Jurkat T cells transiently cotransfected with the luciferase reporter gene and increasing doses of an IRF-1-expressing vector.
The results indicated that the basal transcriptional activity of the foxp3 gene promoter
    • was substantially reduced in the presence of IRF-1 and the effect was dose-dependent.
Conversely, the basal activity of the foxp3 gene promoter construct mutated in the IRF-E
    • was not affected by IRF-1 overexpression.
Interestingly, IRF-2, a repressor of IRF-1 transcriptional activity on most promoters (21), neither affected the promoter activity nor counteracted the inhibitory effect exerted by IRF-1.  IRF-1, IRF-2, as well as the IFN-γ treatment drastically reduced the transcriptional activity of the il4 gene promoter, whereas
  • the low molecular mass polypeptide lmp2 construct was stimulated by IRF-1 and by IFN-γ treatment, but it was not affected by IRF-2.

All together these results demonstrate the specificity and functional relevance of IRF-1 binding to the foxp3 proximal promoter.

Foxp3 is a direct target of IRF-1 in human and mouse primary CD4+CD25− T cells and CD4+CD25+ Treg cells
To assess the biological relevance of the the reported effects of IRF-1 on Treg development and on the regulation of Foxp3 expression, we performed experiments with primary cells. We first assessed by Western blot IRF-1 expression levels in CD4+CD25+ Treg cells vs CD4+CD25− T cells magnetically sorted from PBMC of healthy donors or from mice spleens. Strikingly, we found that IRF-1 was down-regulated in double positive cells as compared with CD4+CD25− T cells both in mouse and human primary cells. To determine whether IRF-1 binds the Foxp3 oligonucleotides in primary Treg cells, pull-down assays with the same extracts were then performed. IRF-1 binding to Foxp3 oligonucleotide was significantly decreased in primary CD4+CD25+ Treg cells compared with CD4+CD25− T cells from both species. Foxp3 staining of CD4+CD25− T cells and CD4+CD25+ human Treg cells confirmed that these cells expressed low and high levels of Foxp3, respectively, and
  • Foxp3 expression was further increased by IL-2 treatment.
To test whether IRF-1 expression was also down-modulated during the acquisition of Treg cell phenotype upon TGF-β treatment, freshly purified TCR-activated CD4+CD25− T cells from both species were cultured with TGF-β, or left untreated, for 3 days and Western blot analysis was performed. When cells were cultured in presence of TGF-β, IRF-1 expression was substantially decreased, as compared with untreated cells. Pull-down assays revealed that IRF-1 binding to Foxp3 oligonucleotide was decreased in TGF-β-treated primary cells compared with untreated cells, as well. Consistently, FACS analysis of these cultures indicated that ∼35% of TGF-β-treated CD4+ cells were Foxp3+ in human and ∼10% in mouse TGF-β treated cultures, respectively. By contrast, even though 46.3% of human untreated cells were CD25+ only 5% were Foxp3+.
Next, we assessed the in vivo IRF-1 binding to foxp3 gene in human and mouse primary magnetically sorted CD4+CD25− T cells and CD4+CD25+ Treg cells, using ChIP assay with anti-IRF-1 Abs. After DNA immunoprecipitation, subsequent real-time PCR amplification of the foxp3 gene surrounding the IRF-E site showed significant IRF-1 binding to Foxp3 promoter in CD4+CD25−Foxp3− T cells, and by contrast, a 5-fold decrease of IRF-1 binding in CD4+CD25+Foxp3high human Treg cells (Fig. 6⇑C). Similarly, the binding of IRF-1 to the Foxp3 promoter in the mouse Treg cells was decreased by ∼50%.
Finally, to assess the functionality of the in vivo IRF-1 binding, negatively selected primary human and mouse CD4+ T lymphocytes were nucleofected with the Foxp3 luciferase reporter gene along with expression vector for IRF-1. Fig. 6⇑E shows the results obtained with T cells from three different healthy donors and Fig. 6⇑F shows a representative experiment with mouse T cells from three independent experiments. In all samples, a discrete basal activity of foxp3 gene promoter was present and this activity was significantly repressed by IRF-1.
Discussion
The identification of molecules controlling Treg differentiation and function is important not only in understanding host immune responses in malignancy and autoimmunity but also in shaping immune response.
In this study, we have shown that IRF-1, a transcription factor involved in the IFN signaling, selectively affects CD4+CD25+ Treg cell development and function, unraveling a novel immunoregulatory function of IRF-1 in addition to its well-established role in balancing Th1 vs Th2 type immune responses. Several lines of evidence support this conclusion:
1) IRF-1−/− mice show a selective and marked increase in all lymphoid organs of CD4+CD25+Foxp3+ Treg cells; 2) CD4+CD25+ from IRF-1−/− mice are characterized by a highly activated and differentiated  phenotype and higher levels of Foxp3 that make them to be functionally more suppressive than WT Treg cells;
3) after TGF-β treatment, and importantly also in its absence, CD4+CD25− T cells from KO mice promptly converted into CD4+CD25+Foxp3+ Treg with a higher suppressive activity than WT cells;
4) forced retrovirus-mediated expression of IRF-1 in IRF-1−/− CD4+CD25− T cells impairs their differentiation into CD25+Foxp3+ cells; and 5) IRF-1 directly regulates transcriptional activity of the foxp3 gene promoter.
The phenotypical and functional characteristics of IRF-1−/− Treg cells strongly support the conclusion that IRF-1 can be considered a key negative regulator of CD4+CD25+ Treg cells.
The increased frequency of differentiated and activated CD4+CD25+ Treg cells characterized by an immunosuppressive cytokine profile described in this study
    • may provide a mechanistic base for the reduced incidence and severity of several autoimmune diseases characterizing IRF-1−/− mice .
In this regard, it has been recently shown that CD4+CD25+ Treg cells were increased in IRF-1−/− mice backcrossed with the MRL/lpr mice, which showed reduced glomerulonephritis.
The increased production of the immunosuppressive cytokine IL-10 by isolated Treg cells from IRF-1−/− mice and the reverted suppression ability of inTreg by anti-IL-10 Abs suggest that this cytokine could play a key role in their suppressor function. Consistently, IL-10 activity has been recently associated with the function of TGF-β-induced CD4+CD25−CD45RBlow cells because their suppressive activity was abrogated with anti-IL-10R Ab treatment (13). Moreover, several reports focused on the in vivo IL-10 role in peripheral CD4+CD25+ Treg cell function in various autoimmunity models (10, 11, 12), although IL-10 seems not required for the functions of thymically derived Treg cells (1). In contrast with the increased IL-10 production, T cells from IRF-1−/− mice failed to produce significant amounts of proinflammatory cytokines such as IFN-γ or TNF-α. Accordingly, an inverse relationship between in vivo IFN-γ administration and generation or activation of CD4+CD25+ Treg cells has been recently shown (45). Moreover, in humans, it has been reported that TNF-α inhibits the suppressive function of both naturally occurring CD4+CD25+ Treg and TGF-β-induced Treg cells, and an anti-TNF Ab therapy reversed their suppressive activity by down-modulating the expression of Foxp3 (46). These latter and our results are apparently in contrast with what was recently reported on the stimulating role of IFN-γ on Foxp3 induction and conversion of CD4+CD25− T cells to CD4+ Treg cells in the IFN-γ KO model (47). In this regard, it is noteworthy to underline that, as it has been also suggested, although knocking down genes involved in up-regulation of IFN-γ expression do not significantly influence autoimmunity, by contrast the absence of genes expressed in response to IFN-γ, including IRF-1, lead to greatly reduced autoimmunity (48). Thus, although the exact mechanism underlying IFN-γ and TNF-α interference with the elicitation of Treg cells remains to be defined, we can speculate that induction of IRF-1 expression, which is up-regulated by IFN-γ and TNF-α, may represent a mechanism through which proinflammatory cytokines negatively affect Foxp3 expression, thereby influencing generation or activation of CD4+CD25+ Treg cells.
It is well known that Foxp3 plays a pivotal role in the regulatory functions of CD4+CD25+ T cells both in humans and in animal models. Thus, the key question in the field of Treg biology is which are molecules and signals that govern Foxp3 transcription.
We identify Foxp3 as specific target of IRF-1 and we show
    • that it binds to foxp3 gene promoter in vitro and in vivo and represses its expression.
Structure of the human foxp3 gene promoter and elements necessary for its induction in T cells have been reported. We have identified an IRF-E sequence at 203 bp upstream of the transcriptional start site that is highly conserved. This element is bound by IRF-1 as proven by pull-down experiments and by ChIP analysis in intact cells, and IRF-1 binding resulted in a specific,
  • dose-dependent repression of the foxp3 proximal promoter.
Notably, treatments with IFN-γ, a major IRF-1 inducer, significantly inhibited foxp3 gene promoter transcriptional activity, whereas IRF-2 did not have any effects. It is noteworthy that the foxp3 gene is highly conserved between mouse and man species, and in particular, the core promoter and the IRF-E identified in this study are perfectly conserved between mouse and human. Such conservation underscores the importance of this motif as regulatory element and provides additional evidence for the role of IRF-1 in regulating foxp3 gene expression.  IRF-1 binds this sequence and negatively regulates its expression in both human and mouse cells. The molecular interactions enabling IRF-1 to inhibit Foxp3 are not yet identified, although our preliminary results show that IRF-1 may compete with c-Myb for the binding to the same overlapping consensus sequence on the foxp3 gene promoter.
In summary, the current study provides evidence that IRF-1 affects CD4+CD25+ development and function by Foxp3 repression. Thus, our data demonstrate a new important contribution by which IRF-1 affects T cell differentiation and provide new important insights into molecular mechanisms controlling immune homeostasis.


Th1-Th2-Th17-Treg origin

Th1-Th2-Th17-Treg origin (Photo credit: Wikipedia)

 

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RNA Virus Genome as Bacterial Chromosome

Reporter: Larry H Bernstein, MD, FCAP

 

Engineering the largest RNA virus genome as an infectious bacterial artificial chromosome
F Almazan, JM Gonzalez, Z Penzes, A Izeta, E Calvo, J Plana-Duran, and L Enjuanes

PNAS  May 9, 2000; 97(10): 5516–5521.

the application of two strategies,

  • cloning of the cDNAs into a bacterial artificial chromosome and
  • nuclear expression of RNAs that are typically produced within the cytoplasm

is useful for the engineering of large RNA molecules.
A cDNA encoding an infectious coronavirus RNA genome

  • has been cloned as a bacterial artificial chromosome.

The rescued coronavirus

  • conserved all of the genetic markers introduced throughout the sequence and
  • showed a standard mRNA pattern and

the antigenic characteristics expected for the synthetic virus.
The cDNA was transcribed

  • within the nucleus, and
  • the RNA translocated to the cytoplasm.
Interestingly, the recovered virus had
  • essentially the same sequence as the original one, and
      • no splicing was observed.

During the engineering of the infectious cDNA,

  • the spike gene of the virus was replaced by
  • the spike gene of an enteric isolate.

The synthetic virus

  • replicated abundantly in the enteric tract and was fully virulent, demonstrating that
  • the tropism and virulence of the recovered coronavirus can be modified.

the application of two strategies,

  • cloning of the cDNAs into a bacterial artificial chromosome and
  • nuclear expression of RNAs that are typically produced within the cytoplasm,
    • is useful for the engineering of large RNA molecules.

A cDNA encoding an infectious coronavirus RNA genome has been cloned as a bacterial artificial chromosome. The rescued coronavirus

  • conserved all of the genetic markers introduced throughout the sequence and
  • showed a standard mRNA pattern and
  • the antigenic characteristics expected for the synthetic virus.
    • The cDNA was transcribed within the nucleus, and
    • the RNA translocated to the cytoplasm.

Interestingly, the recovered virus had essentially the same sequence as the original one, and no splicing was observed. During the engineering of the infectious cDNA, the spike gene of the virus was replaced by the spike gene of an enteric isolate. The synthetic virus

  • replicated abundantly in the enteric tract and
  • was fully virulent,

demonstrating that the tropism and virulence of the recovered coronavirus can be modified.}
http://www.PNAS.org/Engineering_the_largest_RNAvirus_genome_as_an_infectious_bacterial_artificial_chromosome/

Description: The interaction of mRNA in a cell...

Description: The interaction of mRNA in a cell. Source: http://www.genome.gov/Pages/Hyperion/DIR/VIP/Glossary/Illustration/mrna.shtml (file) License: “All of the illustrations in the Talking Glossary of Genetics are freely available and may be used without special permission.” http://www.genome.gov/page.cfm?pageID=10003803 (Photo credit: Wikipedia)

RNA Protein Virus

RNA Protein Virus (Photo credit: Wikipedia)

This image was created as part of the Philip G...

This image was created as part of the Philip Greenspun illustration project. (Photo credit: Wikipedia)

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Cloning the Vaccinia Virus Genome as a Bacterial Artificial Chromosome

Curator: Larry H Bernstein, MD, FCAP

Cloning the vaccinia virus genome as a bacterial artificial chromosome in Escherichia coli and recovery of infectious virus in mammalian cells

A Domi and B Moss
PNAS  Sep 17, 2002; 99(19):12415–12420     http://www.pnas.org/dx.cgi.doi/10.1073/pnas.192420599
The ability to manipulate the vaccinia virus (VAC) genome,
  • as a plasmid in bacteria,
  • would greatly facilitate genetic studies and
  • provide a powerful alternative method of making recombinant viruses.
VAC, like other poxviruses, has a linear, double-stranded DNA genome with covalently closed hairpin ends that are resolved
  • from transient head-to-head and tail-to-tail concatemers
  • during replication in the cytoplasm of infected cell.
Our strategy to construct a nearly 200,000-bp VAC-bacterial artificial chromosome (BAC) was based on
  • circularization of head-to-tail concatemers of VAC DNA.
Cells were infected with a recombinant VAC containing inserted sequences for plasmid replication and maintenance in Escherichia coli; DNA concatemer resolution was inhibited
  • leading to formation and accumulation of head-to-tail concatemers,
in addition to the usual head-to-head and tail-to-tail forms;
  • the concatemers were circularized
    • by homologous or Cre–loxP-mediated recombination; and
  • E. coli were transformed with DNA from the infected cell lysates.
Stable plasmids containing the entire VAC genome, with an intact concatemer junction sequence, were identified. Rescue of infectious VAC was consistently achieved
  • by transfecting the VAC–BAC plasmids into mammalian cells that were infected with a helper nonreplicating fowlpox virus.
The plasmids used to implement the repressilat...

The plasmids used to implement the repressilator in Escherichia coli. (Photo credit: Wikipedia)

There are two types of plasmid integration int...

There are two types of plasmid integration into a host bacteria: Non-integrating plasmids replicate as with the top instance, whereas episomes, the lower example, integrate into the host chromosome. (Photo credit: Wikipedia)

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Rewriting the Mathematics of Tumor Growth[1]; Teams Use Math Models to Sort Drivers from Passengers[2]:  Two JNCI Reviews by Mike Martin Regarding Genomics, Cancer, and Mutation

Curator: Stephen J. Williams, Ph.D.

Recently, there has been extensive interest in the cancer research and oncology community on detecting those mutations responsible for the initiation and propagation of a neoplastic cell (driver mutations) versus those mutations that are randomly (or by selective pressures) acquired due to the genetic instability of the transformed cell.  The impact of either type of mutation has been a topic for debate, with a recent article showing that some passenger mutations may actually be responsible for tumor survival.  In addition many articles, highlighted on this site (and referenced below) in recent years have described the importance of classifying driver and passenger mutations for the purposes of more effective personalized medicine strategies directed against tumors. Two review articles by Mike Martin in the Journal of the National Cancer Institute (JCNI) shed light on the current efforts and successes to discriminate between these passenger and driver mutations and determine impact of each type of mutation to tumor growth.  However, as described in the associated article, the picture is not as clear cut as previously thought and highlights some revolutionary findings. In Rewriting the Mathematics of Tumor Growth, researchers discovered that driver mutations may confer such a small growth advantage that, multiple mutations, including the so called passenger mutations are necessary in order to sustain tumor growth. In fact, much experimental evidence has suggested at least six defined genetic events may be necessary for the in-vitro transformation of human cells.  The following table shows some of the genetic events required for in-vitro transformation in cell culture systems.

Genetic events required for transformation

 Species  Cell type  # of genes required for tumor formation*  Genes used  Reference Events required for priming
Human FibroblastsEmbryonic kidney 3 hTERTH-rasLarge T (a)Hahn(Weinberg) 2LT+hTERT
Mammary epithelialMyoblastsEmbryonic kidney 6 hTERTH-rasP53DDc-myc

cyclin D1CDK4

(b)Kendall(Counter) Hras required for tumorigenesis so probably 5 events needed
Fibroblasts 4 Large TSmall TH-rashTERT (c)Sun(Hornsby) 2Large T + H-ras
Fibroblasts 4 Large TSmall ThTERTRas (d)Rangarajan(Weinberg) 3hTERT, Ras and either small or largeT
Keratinocytes 4 CyclinD1

dnp53

EGFR

c-myc

(e)Goessel(Opitz) 3 for anchorage independence (cyclin D1, dnp53, EGFR),Cyclin D1+dnp53 for immortalization
HOSE 6 CDK4, cyclin D, hTERT plus combination of either P53DD, myrAkt, and H-ras or P53DD, H-ras, c-myc Bcl2 (f)Sasaki(Kiyono) 5
HOSE 3 hTERTSV40 earlyH-ras orK-ras (g)Liu(Bast) 2hTERT+ SV40 early
HOSE 3 Large ThTERTH-ras orc-erB-2 (h)Kusakari(Fujii) 2hTERT+large T
Rat Fibroblasts 2 Large TH-ras (i)Hirakawa Did not analyze
Fibroblasts 2 Large TH-ras (d)Rangarajan(Weinberg) Large T
Mouse MOSEIn p53-/- background 3 c-mycK-rasAkt (j)Orsulic
Pig Fibroblasts 6 p53DDhTERT

CDK4H-ras c-myc

cyclin D1

(k)Adam(Counter) 5 need all butp53DD

Note: priming means events required to immortalize but not fully transform.  * Note that both ability to form colonies in soft agarose and subsequently tested for tumor formation in immunocompromised mice.

a.         Hahn, W. C., Counter, C. M., Lundberg, A. S., Beijersbergen, R. L., Brooks, M. W., and Weinberg, R. A. (1999) Creation of human tumour cells with defined genetic elements, Nature 400, 464-468.

b.         Kendall, S. D., Linardic, C. M., Adam, S. J., and Counter, C. M. (2005) A network of genetic events sufficient to convert normal human cells to a tumorigenic state, Cancer Res 65, 9824-9828.

c.         Sun, B., Chen, M., Hawks, C. L., Pereira-Smith, O. M., and Hornsby, P. J. (2005) The minimal set of genetic alterations required for conversion of primary human fibroblasts to cancer cells in the subrenal capsule assay, Neoplasia 7, 585-593.

d.         Rangarajan, A., Hong, S. J., Gifford, A., and Weinberg, R. A. (2004) Species- and cell type-specific requirements for cellular transformation, Cancer Cell 6, 171-183.

e.         Goessel, G., Quante, M., Hahn, W. C., Harada, H., Heeg, S., Suliman, Y., Doebele, M., von Werder, A., Fulda, C., Nakagawa, H., Rustgi, A. K., Blum, H. E., and Opitz, O. G. (2005) Creating oral squamous cancer cells: a cellular model of oral-esophageal carcinogenesis, Proc Natl Acad Sci U S A 102, 15599-15604.

f.          Sasaki, R., Narisawa-Saito, M., Yugawa, T., Fujita, M., Tashiro, H., Katabuchi, H., and Kiyono, T. (2009) Oncogenic transformation of human ovarian surface epithelial cells with defined cellular oncogenes, Carcinogenesis 30, 423-431.

g.         Liu, J., Yang, G., Thompson-Lanza, J. A., Glassman, A., Hayes, K., Patterson, A., Marquez, R. T., Auersperg, N., Yu, Y., Hahn, W. C., Mills, G. B., and Bast, R. C., Jr. (2004) A genetically defined model for human ovarian cancer, Cancer Res 64, 1655-1663.

h.         Kusakari, T., Kariya, M., Mandai, M., Tsuruta, Y., Hamid, A. A., Fukuhara, K., Nanbu, K., Takakura, K., and Fujii, S. (2003) C-erbB-2 or mutant Ha-ras induced malignant transformation of immortalized human ovarian surface epithelial cells in vitro, Br J Cancer 89, 2293-2298.

i.          Hirakawa, T., and Ruley, H. E. (1988) Rescue of cells from ras oncogene-induced growth arrest by a second, complementing, oncogene, Proc Natl Acad Sci U S A 85, 1519-1523.

j.          Orsulic, S., Li, Y., Soslow, R. A., Vitale-Cross, L. A., Gutkind, J. S., and Varmus, H. E. (2002) Induction of ovarian cancer by defined multiple genetic changes in a mouse model system, Cancer Cell 1, 53-62.

k.         Adam, S. J., Rund, L. A., Kuzmuk, K. N., Zachary, J. F., Schook, L. B., and Counter, C. M. (2007) Genetic induction of tumorigenesis in swine, Oncogene 26, 1038-1045.

However it may be argued that the aforementioned experimental examples were produced in cell lines with a more stable genome than that which is seen in most tumors and had used traditional assays of transformation, such as growth in soft agarose and tumorigenicity in immunocompromised mice, as endpoints of transformation, and not representative of the tumor growth seen in the clinical setting.

Therefore Bert Vogelstein, M.D., along with collaborators around the world developed a model they termed the “sequential driver mutation theory”, in which they describe that driver mutations multiply over time with each mutation “slightly increasing the tumor growth rate through a process that depends on three factors”:

  1. Driver mutation rate
  2. The 0.4% selective growth advantage
  3. Cell division time

This model was based on a combination of experimental data and computer simulations of gliobastoma multiforme and pancreatic adenocarcinoma.  Most tumor models follow a Gompertz kinetics, which show how tumor growth is exponential but eventually levels off over time.

This new theory shows though that a tumor cell with only one driver mutation can only grow so much, until a second driver mutation is required.  Using data for the COSMIC database (Catalog of Somatic Mutations in Cancer) together with analysis software CHASM (Cancer-specific High-throughput Annotation of Somatic Mutations) the researchers analyzed 713 mutations sequenced from 14 glioma patients and 562 mutations in nine pancreatic adenocarcinomas, revealing at least 100 tumor suppressor genes and 100 oncogenes altered.  Therefore, the authors suggested these may be possible driver mutations, or at least mutations required for the sustained growth of these tumors.  Applying this new model to data obtained from Dr. Giardiello’s publication concerning familial adenopolypsis in New England Journal of medicine in 19993 and 2000, the sequential driver mutation model predicted age distribution of FAP patients, number and size of polyps, and polyp growth rate than previous models.  This surprising number of required driver mutations for full transformation was also verified in a study led by University of Texas Southwestern Medical Center biologist Jerry Shay, Ph.D., who noted “this team’s surprise nearly 45% of all colorectal candidate oncogenes (65 mutations) drove malignant proliferation”[3].

However, some investigators do not believe the model is complex enough to account for other factors involved in oncogenesis, such as epigenetic factors like methylation and acetylation.  In addition the review also discusses host and tissue factors which may complicate the models, such as location where a tumor develops.  However, most of the investigators interviewed for this review agreed that focusing on this long-term progression of the disease may give us clues to other potential druggable targets.

Teams Use Math Models to Sort Drivers From Passengers

A related review from Mike Martin in JNCI [2] describes a statistical method, published in 2009 Cancer Informatics[4], which distinguishes chromosomal abnormalities that can drive oncogenesis from passenger abnormalities.  Chromosomal abnormalities, such as deletions, additions, and translocations are common in cancer.  For instance, the well-known Philadelphia chromosome, a translocation between chromosome 9 and 22 which results in the BCR-ABL tyrosine kinase fusion protein is the molecular basis of chronic myelogenous leukemia.

In the report, Eytan Domany, Ph.D., from Weizmann Institute and several colleagues from University of Lausanne, University of Haifa and the Broad Institute were analyzing chromosomal aberrations in a subset of medulloblastoma, which had more gain and losses in chromosomes than had been attributed to the disease.  Using a statistical method they termed a “volumetric sieve”, the investigators were able to identify driver versus passenger aberrations based on three filters:

  • Fraction of patients with the abnormality
  • Length of DNA involved in the aberrant chromosome
  • Abnormality’s copy number

Another method to sort the most “important” chromosomal aberrations from less relevant alterations is termed GISTIC[5], as the website describes is: a tool to identify genes targeted by somatic copy-number alterations (SCNAs) that drive cancer growth (at the Broad Institute website http://www.broadinstitute.org/software/cprg/?q=node/31).  The method allows for comparison across multiple tumors so noise is eliminated and improves consistency of analysis.  This method had been successfully used to determine driver aberrations is mesotheliomas, leukemias, and identify new oncogenes in adenocarcinomas of the lung and squamous cell carcinoma of the esophagus.

Main references for the two Mike Martin articles are as follows:

1.         Martin M: Rewriting the mathematics of tumor growth. Journal of the National Cancer Institute 2011, 103(21):1564-1565.

2.         Martin M: Aberrant chromosomes: teams use math models to sort drivers from passengers. Journal of the National Cancer Institute 2010, 102(6):369-371.

3.         Eskiocak U, Kim SB, Ly P, Roig AI, Biglione S, Komurov K, Cornelius C, Wright WE, White MA, Shay JW: Functional parsing of driver mutations in the colorectal cancer genome reveals numerous suppressors of anchorage-independent growth. Cancer research 2011, 71(13):4359-4365.

4.         Shay T, Lambiv WL, Reiner-Benaim A, Hegi ME, Domany E: Combining chromosomal arm status and significantly aberrant genomic locations reveals new cancer subtypes. Cancer informatics 2009, 7:91-104.

5.         Beroukhim R, Getz G, Nghiemphu L, Barretina J, Hsueh T, Linhart D, Vivanco I, Lee JC, Huang JH, Alexander S et al: Assessing the significance of chromosomal aberrations in cancer: methodology and application to glioma. Proceedings of the National Academy of Sciences of the United States of America 2007, 104(50):20007-20012.

Further posts on CANCER and GENOMICS and Sequencing published on the site include:

The Initiation and Growth of Molecular Biology and Genomics

Inaugural Genomics in Medicine – The Conference Program, 2/11-12/2013, San Francisco, CA

LEADERS in Genome Sequencing of Genetic Mutations for Therapeutic Drug Selection in Cancer Personalized Treatment: Part 2

Paradigm Shift in Human Genomics – Predictive Biomarkers and Personalized Medicine – Part 1

Breast Cancer: Genomic profiling to predict Survival: Combination of Histopathology and Gene Expression Analysis

Computational Genomics Center: New Unification of Computational Technologies at Stanford

GSK for Personalized Medicine using Cancer Drugs needs Alacris systems biology model to determine the in silico effect of the inhibitor in its “virtual clinical trial”

arrayMap: Genomic Feature Mining of Cancer Entities of Copy Number Abnormalities (CNAs) Data

Comprehensive Genomic Characterization of Squamous Cell Lung Cancers

Mosaicism’ is Associated with Aging and Chronic Diseases like Cancer: detection of genetic mosaicism could be an early marker for detecting cancer.

http://onlinelibrary.wiley.com/doi/10.1111/j.1755-148X.2011.00905.x/full

https://pharmaceuticalintelligence.com/2013/02/05/winning-over-cancer-progression-new-oncology-drugs-to-suppress-driver-mutations-vs-passengers-mutations/

Additional references:

[1] Michor F, Iwasa Y, and Nowak MA (2004) Dynamics of cancer

progression. Nature Reviews Cancer 4, 197-205.

[2] Crespi B and Summers K (2005) Evolutionary biology of cancer.

Trends in Ecology and Evolution 20, 545-552.

[3] Merlo LMF, et al. (2006) Cancer as an evolutionary and ecological

process. Nature Reviews Cancer 6, 924-935.

[4] McFarland C, et al. “Accumulation of deleterious passenger mutations

in cancer,” in preparation.

[5] Birkbak NJ, et al. (2011) Paradoxical relationship between

chromosomal instability and survival outcome in cancer. Cancer

Research 71,3447-3452.

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Curator: Aviva Lev-Ari, PhD, RN

Chaperon Protein Mechanism inspired MIT Team to Model the Role of Genetic Mutations on Cancer Progression, proposing the next generation of Oncology drugs to aim at Suppression of Passenger Mutations. Current drug, in clinical trials, use the Chaperon Protein Mechanism to suppress Driver Mutations.

Deleterious Mutations in Cancer Progression

Kirill S. Korolev1, Christopher McFarland2, and Leonid A. Mirny3

1Department of Physics, MIT, Cambridge, MA.

E-mail: papers.korolev@gmail.com

2Graduate Program in Biophysics, Harvard University, Cambridge, MA.

3Health Sciences and Technology, MIT, Cambridge, MA

The research was funded by the National Institutes of Health/National Cancer Institute Physical Sciences Oncology Center at MIT.

SOURCE:

http://cnls.lanl.gov/q-bio/wiki/images/4/40/Abstract.pdf

Deleterious passenger mutations significantly affect evolutionary dynamics of cancer. Including passenger mutations in evolutionary models is necessary to understand the role of genetic diversity in cancer progression and to create new treatments based on the accumulation of deleterious passenger mutations.

Evolutionary models of cancer almost exclusively focus on the acquisition of driver mutations, which are beneficial to cancer cells. The driver mutations, however, are only a small fraction of the mutations found in tumors. The other mutations, called passenger mutations, are typically neglected because their effect on fitness is assumed to be very small. Recently, it has been suggested that some passenger mutations are slightly deleterious. We find that deleterious passengers significantly affect cancer progression. In particular, they lead to a critical tumor size, below which tumors shrink on average, and to an optimal mutation rate for cancer evolution.

ANCER is an outcome of somatic evolution [1-3]. To outcompete their benign sisters, cancer cells need to acquire many heritable changes (driver mutations) that enable proliferation. In addition to the rare beneficial drivers, cancer cells must also acquire neutral or slightly deleterious passenger mutations [4]. Indeed, the number of possible passengers exceeds the number of possible drivers by orders of magnitude. Surprisingly, the effect of passenger mutations on cancer progression has not been explored. To address this problem, we developed an evolutionary model of cancer progression, which includes both drivers and passengers. This model was analyzed both numerically and analytically to understand how mutation rate, population size, and fitness effects of mutations affect cancer progression.

RESULTS

Upon including passengers in our model, we found that cancer is no longer a straightforward progression to malignancy. In particular, there is a critical population size such that smaller populations accumulate passengers and decline, while larger populations accumulate drivers and grow. The transition to cancer for small initial populations is, therefore, stochastic in nature and is similar to diffusion over an energy barrier in chemical kinetics. We also found that there is an optimal mutation rate for cancer development, and passengers with intermediate fitness costs are most detrimental to cancer. The existence of an optimal mutation rate could explain recent clinical data [5] and is in stark contrast to the predictions of the models neglecting passengers. We also show that our theory is consistent with recent sequencing data.

SOURCE:

http://cnls.lanl.gov/q-bio/wiki/images/4/40/Abstract.pdf

Just as some mutations in the genome of cancer cells actively spur tumor growth, it would appear there are also some that do the reverse, and act to slow it down or even stop it, according to a new US study led by MIT.

Senior author, Leonid Mirny, an associate professor of physics and health sciences and technology at MIT, and colleagues, write about this surprise finding in a paper to be published online this week in the Proceedings of the National Academy of Sciences.

In a statement released on Monday, Mirny tells the press:

“Cancer may not be a sequence of inevitable accumulation of driver events, but may be actually a delicate balance between drivers and passengers.”

“Spontaneous remissions or remissions triggered by drugs may actually be mediated by the load of deleterious passenger mutations,” he suggests.

Cancer Cell‘s Genome Has “Drivers” and “Passengers”

Your average cancer cell has a genome littered with thousands of mutations and hundreds of mutated genes. But only a handful of these mutated genes are drivers that are responsible for the uncontrolled growth that leads to tumors.

Up until this study, cancer researchers have mostly not paid much attention to the “passenger” mutations, believing that because they were not “drivers”, they had little effect on cancer progression. 

Now Mirny and colleagues have discovered, to their surprise, that the “passengers” aren’t there just for the ride. In sufficient numbers, they can slow down, and even stop, the cancer cells from growing and replicating as tumors. 

New Drugs Could Target the Passenger Mutations in Protein Chaperoning

Although there are already several drugs in development that target the effect of chaperone proteins in cancer, they are aiming to suppress driver mutations.

Recently, biochemists at the University of Massachusetts Amherst“trapped” a chaperone in action, providing a dynamic snapshot of its mechanism as a way to help development of new drugs that target drivers.

But Mirny and colleagues say there is now another option: developing drugs that target the same chaperoning process, but their aim would be to encourage the suppressive effect of the passenger mutations.

They are now comparing cells with identical driver mutations but different passenger mutations, to see which have the strongest effect on growth.

They are also inserting the cells into mice to see which are the most likely to lead to secondary tumors (metastasize).

Written by Catharine Paddock PhD
Copyright: Medical News Today

SOURCE:

http://www.medicalnewstoday.com/articles/255920.php

After proteins are synthesized, they need to be folded into the correct shape, and chaperones help with that process. In cancerous cells, chaperones help proteins fold into the correct shape even when they are mutated, helping to suppress the effects of deleterious mutations.
Several potential drugs that inhibit chaperone proteins are now in clinical trials to treat cancer, although researchers had believed that they acted by suppressing the effects of driver mutations, not by enhancing the effects of passengers.

In current studies, the researchers are comparing cancer cell lines that have identical driver mutations but a different load of passenger mutations, to see which grow faster. They are also injecting the cancer cell lines into mice to see which are likeliest to metastasize.

Drugs that tip the balance in favor of the passenger mutations could offer a new way to treat cancer, the researchers say, beating it with its own weapon — mutations. Although the influence of a single passenger mutation is minuscule, “collectively they can have a profound effect,” Mirny says. “If a drug can make them a little bit more deleterious, it’s still a tiny effect for each passenger, but collectively this can build up.”

In natural populations, selection weeds out deleterious mutations. However, Mirny and his colleagues suspected that the evolutionary process in cancer can proceed differently, allowing mutations with only a slightly harmful effect to accumulate.

If enough deleterious passengers are present, their cumulative effects can slow tumor growth, the simulations found. Tumors may become dormant, or even regress, but growth can start up again if new driver mutations are acquired. This matches the cancer growth patterns often seen in human patients.

“Spontaneous remissions or remissions triggered by drugs may actually be mediated by the load of deleterious passenger mutations.”

When they analyzed passenger mutations found in genomic data taken from cancer patients, the researchers found the same pattern predicted by their model — accumulation of large quantities of slightly deleterious mutations.

REFERENCE

Massachusetts Institute of Technology (2013, February 4). Some cancer mutations slow tumor growth. ScienceDaily. Retrieved February 4, 2013, from http://www.sciencedaily.com­/releases/2013/02/130204154011.htm

Biochemists Trap A Chaperone Machine In Action

Main Category: Biology / Biochemistry
Article Date: 11 Dec 2012 – 0:00 PST

Molecular chaperones have emerged as exciting new potential drug targets, because scientists want to learn how to stop cancer cells, for example, from using chaperones to enable their uncontrolled growth. Now a team of biochemists at the University of Massachusetts Amherst led by Lila Gierasch have deciphered key steps in the mechanism of the Hsp70 molecular machine by “trapping” this chaperone in action, providing a dynamic snapshot of its mechanism.

She and colleagues describe this work in the current issue of Cell. Gierasch’s research on Hsp70 chaperones is supported by a long-running grant to her lab from NIH’s National Institute for General Medical Sciences.

Molecular chaperones like the Hsp70s facilitate the origami-like folding of proteins, made in the cell’s nanofactories or ribosomes, from where they emerge unstructured like noodles. Proteins only function when folded into their proper structures, but the process is so difficult under cellular conditions that molecular chaperone helpers are needed. 

The newly discovered information about chaperone action is important because all rapidly dividing cells use a lot of Hsp70, Gierasch points out. “The saying is that cancer cells are addicted to Hsp70 because they rely on this chaperone for explosive new cell growth. Cancer shifts our body’s production of Hsp70 into high gear. If we can figure out a way to take that away from cancer cells, maybe we can stop the out-of-control tumor growth. To find a molecular way to inhibit Hsp70, you’ve got to know how it works and what it needs to function, so you can identify its vulnerabilities.”

Chaperone proteins in cells, from bacteria to humans, act like midwives or bodyguards, protecting newborn proteins from misfolding and existing proteins against loss of structure caused by stress such as heat or a fever. In fact, the heat shock protein (Hsp) group includes a variety of chaperones active in both these situations.

As Gierasch explains, “New proteins emerge into a challenging environment. It’s very crowded in the cell and it would be easy for them to get their sticky amino acid chains tangled and clumped together. Chaperones bind to them and help to avoid this aggregation, which is implicated in many pathologies such as neurodegenerative diseases. This role of chaperones has also heightened interest in using them therapeutically.”

However, chaperones must not bind too tightly or a protein can’t move on to do its job. To avoid this, chaperones rapidly cycle between tight and loose binding states, determined by whether ATP or ADP is bound. In the loose state, a protein client is free to fold or to be picked up by another chaperone that will help it fold to do its cellular work. In effect, Gierasch says, Hsp70s create a “holding pattern” to keep the protein substrate viable and ready for use, but also protected.

She and colleagues knew the Hsp70’s structure in both tight and loose binding affinity states, but not what happened between, which is essential to understanding the mechanism of chaperone action. Using the analogy of a high jump, they had a snapshot of the takeoff and landing, but not the top of the jump. “Knowing the end points doesn’t tell us how it works. There is a shape change in there that we wanted to see,” Gierasch says.

To address this, she and her colleagues postdoctoral fellows Anastasia Zhuravleva and Eugenia Clerico obtained “fingerprints” of the structure of Hsp70 in different states by using state-of-the-art nuclear magnetic resonance (NMR) methods that allowed them to map how chemical environments of individual amino acids of the protein change in different sample conditions. Working with an Hsp70 known as DnaK from E. coli bacteria, Zhuravleva and Clerico assigned its NMR spectra. In other words, they determined which peaks came from which amino acids in this large molecule.

The UMass Amherst team then mutated the Hsp70 so that cycling between tight and loose binding states stopped. As Gierasch explains, “Anastasia and Eugenia were able to stop the cycle part-way through the high jump, so to speak, and obtain the molecular fingerprint of a transient intermediate.” She calls this accomplishment “brilliant.”

Now that the researchers have a picture of this critical allosteric state, that is, one in which events at one site control events in another, Gierasch says many insights emerge. For example, it appears nature uses this energetically tense state to “tune” alternate versions of Hsp70 to perform different cellular functions. “Tuning means there may be evolutionary changes that let the chaperone work with its partners optimally,” she notes.

“And if you want to make a drug that controls the amount of Hsp70 available to a cell, our work points the way toward figuring out how to tickle the molecule so you can control its shape and its ability to bind to its client. We’re not done, but we made a big leap,” Gierasch adds. “We now have a idea of what the Hsp70 structure is when it is doing its job, which is extraordinarily important.” 

Article adapted by Medical News Today from original press release. Click ‘references’ tab above for source.
Visit our biology / biochemistry section for the latest news on this subject.
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progression. Nature Reviews Cancer 4, 197-205.

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process. Nature Reviews Cancer 6, 924-935.

[4] McFarland C, et al. “Accumulation of deleterious passenger mutations

in cancer,” in preparation.

[5] Birkbak NJ, et al. (2011) Paradoxical relationship between

chromosomal instability and survival outcome in cancer. Cancer

Research 71,3447-3452.

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Drugging the Epigenome

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Reporter: Prabodh Kandala, PhD

A typical cancer cell has thousands of mutations scattered throughout its genome and hundreds of mutated genes. However, only a handful of those genes, known as drivers, are responsible for cancerous traits such as uncontrolled growth. Cancer biologists have largely ignored the other mutations, believing they had little or no impact on cancer progression.

But a new study from MIT, Harvard University, the Broad Institute and Brigham and Women’s Hospital reveals, for the first time, that these so-called passenger mutations are not just along for the ride. When enough of them accumulate, they can slow or even halt tumor growth.

The findings, reported in this week’sProceedings of the National Academy of Sciences, suggest that cancer should be viewed as an evolutionary process whose course is determined by a delicate balance between driver-propelled growth and the gradual buildup of passenger mutations that are damaging to cancer, says Leonid Mirny, an associate professor of physics and health sciences and technology at MIT and senior author of the paper.

Furthermore, drugs that tip the balance in favor of the passenger mutations could offer a new way to treat cancer, the researchers say, beating it with its own weapon — mutations. Although the influence of a single passenger mutation is minuscule, “collectively they can have a profound effect,” Mirny says. “If a drug can make them a little bit more deleterious, it’s still a tiny effect for each passenger, but collectively this can build up.”

Lead author of the paper is Christopher McFarland, a graduate student at Harvard. Other authors are Kirill Korolev, a Pappalardo postdoctoral fellow at MIT, Gregory Kryukov, a senior computational biologist at the Broad Institute, and Shamil Sunyaev, an associate professor at Brigham and Women’s.

Power struggle

Cancer can take years or even decades to develop, as cells gradually accumulate the necessary driver mutations. Those mutations usually stimulate oncogenes such as Ras, which promotes cell growth, or turn off tumor-suppressing genes such as p53, which normally restrains growth.

Passenger mutations that arise randomly alongside drivers were believed to be fairly benign: In natural populations, selection weeds out deleterious mutations. However, Mirny and his colleagues suspected that the evolutionary process in cancer can proceed differently, allowing mutations with only a slightly harmful effect to accumulate.

To test this theory, the researchers created a computer model that simulates cancer growth as an evolutionary process during which a cell acquires random mutations. These simulations followed millions of cells: every cell division, mutation and cell death.

They found that during the long periods between acquisition of driver mutations, many passenger mutations arose. When one of the cancerous cells gains a new driver mutation, that cell and its progeny take over the entire population, bringing along all of the original cell’s baggage of passenger mutations. “Those mutations otherwise would never spread in the population,” Mirny says. “They essentially hitchhike on the driver.”

This process repeats five to 10 times during cancer development; each time, a new wave of damaging passengers is accumulated. If enough deleterious passengers are present, their cumulative effects can slow tumor growth, the simulations found. Tumors may become dormant, or even regress, but growth can start up again if new driver mutations are acquired. This matches the cancer growth patterns often seen in human patients.

“Cancer may not be a sequence of inevitable accumulation of driver events, but may be actually a delicate balance between drivers and passengers,” Mirny says. “Spontaneous remissions or remissions triggered by drugs may actually be mediated by the load of deleterious passenger mutations.”

When they analyzed passenger mutations found in genomic data taken from cancer patients, the researchers found the same pattern predicted by their model — accumulation of large quantities of slightly deleterious mutations.

Tipping the balance

In computer simulations, the researchers tested the possibility of treating tumors by boosting the impact of deleterious mutations. In their original simulation, each deleterious passenger mutation reduced the cell’s fitness by about 0.1 percent. When that was increased to 0.3 percent, tumors shrank under the load of their own mutations.

The same effect could be achieved in real tumors with drugs that interfere with proteins known as chaperones, Mirny suggests. After proteins are synthesized, they need to be folded into the correct shape, and chaperones help with that process. In cancerous cells, chaperones help proteins fold into the correct shape even when they are mutated, helping to suppress the effects of deleterious mutations.

Several potential drugs that inhibit chaperone proteins are now in clinical trials to treat cancer, although researchers had believed that they acted by suppressing the effects of driver mutations, not by enhancing the effects of passengers.

In current studies, the researchers are comparing cancer cell lines that have identical driver mutations but a different load of passenger mutations, to see which grow faster. They are also injecting the cancer cell lines into mice to see which are likeliest to metastasize.

Ref:

Massachusetts Institute of Technology (2013, February 4). Some cancer mutations slow tumor growth. ScienceDaily. Retrieved February 4, 2013, from http://www.sciencedaily.com­/releases/2013/02/130204154011.htm

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