Mechanisms of angiogenesis impairment by radiation therapy

Summary : Clinical evidence indicates that tumors recurring within previously irradiated fields are highly invasive and metastatic, suggesting a role of the tumor stroma in this effect. Angiogenesis plays a critical role in tumor progression. Ionizing radiation is known to induce apoptosis of angiogenic endothelial cells, while the effect on quiescent endothelial cells and de novo angiogenesis is not well characterized. We recently observed that irradiation of normal tissue prevents tumor- and growth factor-induced angiogenesis. The main aim of my thesis work was to characterize the mechanisms of radiation-mediated inhibition of angiogenesis. To this purpose we used a combination of in vivo and ex vivo studies on irradiated healthy tissue, and in vitro irradiation experiments using angiogenesis models and isolated endothelial cells. We found that irradiation did not induce endothelial cell apoptosis and did not disrupt quiescent vessels within irradiated skin. Radiation reduced the recruitment of leukocytes to angiogenic Matrigel plugs, but this effect was rather secondary to decreased angiogenesis, as exogenous addition of leucocytes to Matrigel plugs did not rescue the angiogenesis defects. To ascertain the direct effect of radiation on endothelial cells, we used the mouse aortic ring assay to test the sprouting capacity of irradiated endothelial cells ex vivo and in vitro, and found that irradiation completely suppressed endothelial cell sprouting. Using HUVEC cells, we showed that irradiation of quiescent confluent endothelial cells did not induce cell death but suppressed subsequent migration and cell proliferation and induced senescence. By Western blotting, we observed a rapid and sustained increase in p21 levels, previously shown to be activated by p53 in response to double strand break, and mediating senescence in human cells. Current experiments focus on the mechanism of sustained p21 upregulation and its role in reduced migration. Inhibition of endothelial cell migration and proliferation by radiation may explain reduced angiogenesis in tumors growing in previously irradiated fields.

Nucleotide sequence of the 5' noncoding region and part of the gag gene of mouse mammary tumor virus; identification of the 5' splicing site for subgenomic mRNAs.

We have determined the sequence of the first 1371 nucleotides at the 5' end of the genome of mouse mammary tumor virus using molecularly cloned proviral DNA of the GR virus strain. The most likely initiation codon used for the gag gene of mouse mammary tumor virus is the first one, located 312 nucleotides from the 5' end of the viral RNA. The 5' splicing site for the subgenomic mRNA's is located approximately 288 nucleotides downstream from the 5' end of the viral RNA. From the DNA sequence the amino acid sequence of the N-terminal half of the gag precursor protein, including p10 and p21, was deduced (353 amino acids).

Identification et caractérisation de gènes suppresseurs de tumeurs dans le glioblastome

Introduction: Glioblastoma (WHO Grade IV glioma) is the most frequent and most¦malignant primary tumor of the brain. With a mean survival of 15 months despite¦multidisciplinary management combining surgery, chemo- and radiotherapy, the prognosis¦is poor. Different studies measured a down-regulation of Wnt Inhibitory Factor 1 (WIF1)¦expression in a majority of gliobastoma due to genetic and epigenetic regulation. Recently,¦a focus on chromosome 12 identified WIF1 as a potential tumor suppressor gene. In¦previous results, transfected glioblastoma cells with ectopic expression of WIF1 had a¦decreased growth rate and adopted a senescence-like phenotype. In this report, we first¦investigated the effect of WIF1 re-expression in glioblastoma cell lines to see if Wnt¦inhibition by WIF1 can lead to senescence. To look further, we assessed p21 and c-Myc¦expression. p21 has a key role in senescence onset and is directly inhibited by c-Myc,¦itself a target of Wnt-pathway. We thus looked if a variation of expression of these genes is¦triggered by WIF1 activity. Finally, as autophagy is thought to play a role in senescence¦onset, we analyzed the expression of different autophagy genes. We therefore looked for¦an association between autophagy activity and senescent phenotype in WIF1-¦overexpressing cell lines.¦Methods: WIF1-overexpressing clones were selected after transfection of stable¦glioblastoma cell lines. Analysis were made through quantitative Polymerase Chain¦Reaction (qPCR), Fluorescence-activated Cell Sorting (FACS) and histochemistry.¦IGFBP7 and ALDH1A3 have been selected to reflect senescence. ATG5, ATG7 and ULK3¦have been selected to reflect autophagy activity.¦Results: Using FACS analysis, we found a higher percentage of large cells with increased¦granularity amongst WIF1-overexpressing cell lines, which are characteristics of¦senescence. In addition, histochemistry showed a higher percentage of multi-nucleated,¦beta-galactosidase positive cells in the same cell lines. An increased expression of genes¦associated with senescence was found as well. All characteristics were correlated with¦levels of WIF1 expression. We did not find any association between p21 and WIF1¦expression. No correlation between WIF1 and c-Myc expression was noticed either. In one¦of the two cell lines analyzed, the expression of autophagy genes showed some¦correlation with expression of WIF1 and expression of genes associated with senescence.¦Discussion: After investigations and characterizations on multiple levels, we have¦evidence for a senescence phenotype upon WIF1-overexpressing cell lines. This gives a¦role to Wnt pathway in the tumorigenicity of glioblastoma. Further experiments are¦required to investigate how Wnt inhibition leads to senescence. The role of autophagy in¦our senescent cells is here still unclear. Some correlations can be found, letting us think¦that there is indeed some involvement of autophagy. However, it is yet to soon to explain¦this relationship. Further experiments are required again to confirm the preliminary results¦and analyze the variations of autophagy activity within time.

Bortezomib reverses the proliferative and antiapoptotic effect of neuropeptides on prostate cancer cells.

Objectives:  Neuropeptides are important signal initiators in advanced prostate cancer, partially acting through activation of nuclear factor kappa B. Central to nuclear factor kappa B regulation is the ubiquitin-proteasome system, pharmacological inhibition of which has been proposed as an anticancer strategy. We investigated the putative role of the proteasome inhibitor bortezomib in neuropeptides signaling effects on prostate cancer cells. Methods:  Human prostate cancer cell lines, LNCaP and PC-3, were used to examine cell proliferation, levels of proapoptotic (caspase-3, Bad) and cell cycle regulatory proteins (p53, p27, p21), as well as total and phosphorylated Akt and p44/42 mitogen-activated protein kinase proteins. Furthermore, 20S proteasome activity, subcellular localization of nuclear factor kappa B and transcription of nuclear factor kappa B target genes, interleukin-8 and vascular endothelial growth factor, were assessed. Results:  Neuropeptides (endothelin-1, bombesin) increased cell proliferation, whereas bortezomib decreased proliferation and induced apoptosis, an effect maintained after cotreatment with neuropeptides. Bad, p53, p21 and p27 were downregulated by neuropeptides in PC-3, and these effects were reversed with the addition of bortezomib. Neuropeptides increased proteasomal activity and nuclear factor kappa B levels in PC-3, and these effects were prevented by bortezomib. Interleukin-8 and vascular endothelial growth factor transcripts were induced after neuropeptides treatment, but downregulated by bortezomib. These results coincided with the ability of bortezomib to reduce mitogen-activated protein kinase signaling in both cell lines. Conclusions:  These findings are consistent with bortezomib-mediated abrogation of neuropeptides-induced proliferative and antiapoptotic signaling. Thus, the effect of the drug on the neuropeptides axis needs to be further investigated, as neuropeptide action in prostate cancer might entail involvement of the proteasome.

IRF6 is a mediator of Notch pro-differentiation and tumour suppressive function in keratinocytes.

While the pro-differentiation and tumour suppressive functions of Notch signalling in keratinocytes are well established, the underlying mechanisms remain poorly understood. We report here that interferon regulatory factor 6 (IRF6), an IRF family member with an essential role in epidermal development, is induced in differentiation through a Notch-dependent mechanism and is a primary Notch target in keratinocytes and keratinocyte-derived SCC cells. Increased IRF6 expression contributes to the impact of Notch activation on growth/differentiation-related genes, while it is not required for induction of 'canonical' Notch targets like p21(WAF1/Cip1), Hes1 and Hey1. Down-modulation of IRF6 counteracts differentiation of primary human keratinocytes in vitro and in vivo, promoting ras-induced tumour formation. The clinical relevance of these findings is illustrated by the strikingly opposite pattern of expression of Notch1 and IRF6 versus epidermal growth factor receptor in a cohort of clinical SCCs, as a function of their grade of differentiation. Thus, IRF6 is a primary Notch target in keratinocytes, which contributes to the role of this pathway in differentiation and tumour suppression.

IRF6 is a mediator of Notch pro-differentiation and tumour suppressive function in keratinocytes.

While the pro-differentiation and tumour suppressive functions of Notch signalling in keratinocytes are well established, the underlying mechanisms remain poorly understood. We report here that interferon regulatory factor 6 (IRF6), an IRF family member with an essential role in epidermal development, is induced in differentiation through a Notch-dependent mechanism and is a primary Notch target in keratinocytes and keratinocyte-derived SCC cells. Increased IRF6 expression contributes to the impact of Notch activation on growth/differentiation-related genes, while it is not required for induction of 'canonical' Notch targets like p21(WAF1/Cip1), Hes1 and Hey1. Down-modulation of IRF6 counteracts differentiation of primary human keratinocytes in vitro and in vivo, promoting ras-induced tumour formation. The clinical relevance of these findings is illustrated by the strikingly opposite pattern of expression of Notch1 and IRF6 versus epidermal growth factor receptor in a cohort of clinical SCCs, as a function of their grade of differentiation. Thus, IRF6 is a primary Notch target in keratinocytes, which contributes to the role of this pathway in differentiation and tumour suppression.

Modulation of cdk2, cyclin D1, p16INK4a, p21WAF and p27Kip1 expression in endothelial cells by TNF/IFN gamma.

BACKGROUND: Regional administration of high doses of tumor necrosis factor (TNF) and interferon gamma (IFN gamma) to metastatic melanoma patients causes selective disruption of the tumor vasculature. This effect is paralleled by decreased endothelial cell proliferation and suppressed integrin alpha V beta 3-mediated adhesion in vitro. Overexpression of the cyclin-dependent kinase (cdk) inhibitory protein p16INK4a was reported to interfere with integrin alpha V beta 3-dependent melanoma cell adhesion. MATERIALS AND METHODS: TNF- and IFN gamma-treated HUVEC were analyzed for cell cycle progression and for protein expression by flow cytometry and Western blotting, respectively. p16INK4a was overexpressed by transient transfection, and HUVEC adhesion was tested in short-term adhesion assays. RESULTS: TNF and IFN gamma synergistically induced a G1 arrest associated with reduced levels of cyclin D1 and cdk2, and increased expression of the cdk inhibitors p16INK4a, p21WAF and p27Kip1. p16INK4a overexpression, however, had no effect on alpha V beta 3-mediated adhesion. CONCLUSION: These results implicate the down-regulation of cyclin D1 and cdk-2, and up-regulation of p16INK4a, p21WAF and p27Kip1 in the suppression of endothelial cell proliferation induced by TNF/IFN gamma and demonstrate that increased p16INK4a levels are not sufficient to suppress alpha V beta 3-mediated endothelial cell adhesion.

Study of the mechanisms regulating the proliferative potential of human CD8+T lymphocytes over-expressing telomerase reverse transcriptase (hTERT)

Résumé La plupart des cellules issues du sang ont une durée de vie limitée. Dans les cellules somatiques humaines, y incluant les lymphocytes T, la taille des télomères diminue progressivement à chaque division cellulaire, pouvant aboutir à des instabilités chromosomiques. L'expression ectopique du gène de la transcriptase réverse de la télomérase (hTERT) dans les cellules restaure l'activité de la télomérase, et permet un rallongement de leur vie réplicative. Malgré l'absence de signes caractéristiques de transformation, nous ne savons pas encore si les cellules somatiques qui surexpriment hTERT sont physiologiquement indiscernables des cellules normales. Certaines études récentes proposent que la télomérase joue plusieurs rôles additionnels dans d'autres phénomènes biologiques tels que la réparation de l'ADN, la survie et la croissance des cellules. Dans notre étude, nous avons utilisé des clones issus de lymphocytes T cytotoxiques surexprimant la télomérase afin d'étudier les mécanismes moléculaires qui règlent leur prolifération et leur sénescence. Nous avons montré que les «jeunes » cellules T exprimant ou non hTERT révèlent des taux de croissance identiques suite à des réponses de stimulation induites par des mitogènes. De plus, aucun changement global dans leur expression des gènes n'a pu être mis en évidence. Curieusement, nous avons observé des réponses réduites dans la prolifération des cellules transduites avec la télomérase qui présentaient une élongation des télomères et une durée de vie prolongée. Ces cellules, malgré le maintien d'un niveau élevé de l'expression de gènes impliqués dans la progression du cycle cellulaire, ont également montré une expression accrue de plusieurs gènes trouvés en commun avec nos lymphocytes T vieillissants n'exprimant pas de télomérase. En particulier, les cellules ayant une durée de vie prolongée grâce à l'expression de la télomérase accumulaient également certains inhibiteurs du cycle cellulaire tels que p16Ink4a et p21Cip1, associés à l'arrêt de la croissance cellulaire. En résumé, nos résultats indiquent la présence fonctionnelle de mécanismes alternatifs pouvant contrôler la croissance réplicative de ces cellules; ils sont donc encourageants dans l'optique d'une utilisation à moindre risque de lymphocytes T «immortalisés » à des fins thérapeutiques pour traiter les tumeurs malignes ou les infections. Summary Most mature blood cells have a finite life span. In human somatic cells, including T lymphocytes, telomeres progressively shorten with each cell division eventually leading to chromosomal instability. Ectopic expression of the human telomerase reverse transcriptase (hTERT) gene in cells restores telomerase activity and results in the extension of their replicative life span. Despite lack of transformation characteristics, it is yet unknown whether somatic cells that over-express telomerase are biologically and physiologically indistinguishable from normal cells. Recent data suggest that telomerase might mediate additional functions in DNA repair, cell survival and cell growth. Using CD8+ T lymphocyte clones over-expressing telomerase we investigated the molecular mechanisms that regulate T cell proliferation and senescence. Here we show that early-passage T cell clones transduced or not with hTERT displayed identical growth rates upon mitogenic stimulation and no marked global changes in gene expression. Surprisingly, reduced proliferative responses were observed in hTERT-transduced cells with elongated telomeres and extended life span. These cells, despite maintaining high expression level of genes involved in cell cycle division and progression, also showed increased expression of several genes associated with normal aging T lymphocytes. In particular, late passage T cells over-expressing telomerase accumulated the cyclin-dependent inhibitors p16INK4a and p21CIP1 that have largely been associated with in vitro growth arrest. Whether tumor-reactive CD8+ T cells that ectopically express telomerase could now be used for adoptive transfer therapy in cancer patients remains unclear at this point. Nevertheless, our results regarding the safe and effective use of hTERT-transduced lymphocytes are encouraging, since they indicate that alternative growth arrest mechanisms such as p 16 and p21 are still functional in these cells and regulate to some extend their growth potential.

p21WAF1/Cip1 is a negative transcriptional regulator of Wnt4 expression downstream of Notch1 activation.

In keratinocytes, the cyclin/CDK inhibitor p21(WAF1/Cip1) is a direct transcriptional target of Notch1 activation; loss of either the p21 or Notch1 genes expands stem cell populations and facilitates tumor development. The Notch1 tumor-suppressor function was associated with down-regulation of Wnt signaling. Here, we show that suppression of Wnt signaling by Notch1 activation is mediated, at least in part, by down-modulation of Wnts gene expression. p21 is a negative regulator of Wnts transcription downstream of Notch1 activation, independently of effects on the cell cycle. More specifically, expression of the Wnt4 gene is under negative control of endogenous p21 both in vitro and in vivo. p21 associates with the E2F-1 transcription factor at the Wnt4 promoter and causes curtailed recruitment of c-Myc and p300, and histone hypoacetylation at this promoter. Thus, p21 acts as a selective negative regulator of transcription and links the Notch and Wnt signaling pathways in keratinocyte growth control.

Radiotherapy suppresses angiogenesis in mice through TGF-betaRI/ALK5-dependent inhibition of endothelial cell sprouting.

BACKGROUND: Radiotherapy is widely used to treat cancer. While rapidly dividing cancer cells are naturally considered the main target of radiotherapy, emerging evidence indicates that radiotherapy also affects endothelial cell functions, and possibly also their angiogenic capacity. In spite of its clinical relevance, such putative anti-angiogenic effect of radiotherapy has not been thoroughly characterized. We have investigated the effect of ionizing radiation on angiogenesis using in vivo, ex vivo and in vitro experimental models in combination with genetic and pharmacological interventions. PRINCIPAL FINDINGS: Here we show that high doses ionizing radiation locally suppressed VEGF- and FGF-2-induced Matrigel plug angiogenesis in mice in vivo and prevented endothelial cell sprouting from mouse aortic rings following in vivo or ex vivo irradiation. Quiescent human endothelial cells exposed to ionizing radiation in vitro resisted apoptosis, demonstrated reduced sprouting, migration and proliferation capacities, showed enhanced adhesion to matrix proteins, and underwent premature senescence. Irradiation induced the expression of P53 and P21 proteins in endothelial cells, but p53 or p21 deficiency and P21 silencing did not prevent radiation-induced inhibition of sprouting or proliferation. Radiation induced Smad-2 phosphorylation in skin in vivo and in endothelial cells in vitro. Inhibition of the TGF-beta type I receptor ALK5 rescued deficient endothelial cell sprouting and migration but not proliferation in vitro and restored defective Matrigel plug angiogenesis in irradiated mice in vivo. ALK5 inhibition, however, did not rescue deficient proliferation. Notch signaling, known to hinder angiogenesis, was activated by radiation but its inhibition, alone or in combination with ALK5 inhibition, did not rescue suppressed proliferation. CONCLUSIONS: These results demonstrate that irradiation of quiescent endothelial cells suppresses subsequent angiogenesis and that ALK5 is a critical mediator of this suppression. These results extend our understanding of radiotherapy-induced endothelial dysfunctions, relevant to both therapeutic and unwanted effects of radiotherapy.

Divergent effect of cobalt and beryllium salts on the fate of peripheral blood monocytes and T lymphocytes.

Occupational exposure to metals such as cobalt and beryllium represents a risk factor for respiratory health and can cause immune-mediated diseases. However, the way they act may be different. We show here that the two metals have a divergent effect on peripheral T lymphocytes and monocytes: BeSO(4) induces cell death in monocytes but not in T lymphocytes, which instead respond by producing Interferon gamma (IFN-γ); conversely, CoCl(2) induces apoptosis in T lymphocytes but not in monocytes. Interestingly, both metals induce p53 overexpression but with a dramatic different outcome. This is because the effect of p53 in CoCl(2)-treated monocytes is counteracted by the antiapoptotic activity of cytoplasmic p21(Cip1/WAF1), the activation of nuclear factor κB, and the inflammasome danger signaling pathway leading to the production of proinflammatory cytokines. However, CoCl(2)-treated monocytes do not fully differentiate into macrophage or dendritic cells, as inferred by the lack of expression of CD16 and CD83, respectively. Furthermore, the expression of HLA-class II molecules, as well as the capability of capturing and presenting the antigens, decreased with time. In conclusion, cobalt keeps monocytes in a partially activated, proinflammatory state that can contribute to some of the pathologies associated with the exposure to this metal.

Selective requirement for c-Myc at an early stage of V(alpha)14i NKT cell development.

Valpha14 invariant (Valpha14i) NKT cells are a subset of regulatory T cells that utilize a semi-invariant TCR to recognize glycolipids associated with monomorphic CD1d molecules. During development in the thymus, CD4(+)CD8(+) Valpha14i NKT precursors recognizing endogenous CD1d-associated glycolipids on other CD4(+)CD8(+) thymocytes are selected to undergo a maturation program involving sequential expression of CD44 and NK-related markers such as NK1.1. The molecular requirements for Valpha14i NKT cell maturation, particularly at early developmental stages, remain poorly understood. In this study, we show that CD4-Cre-mediated T cell-specific inactivation of c-Myc, a broadly expressed transcription factor with a wide range of biological activities, selectively impairs Valpha14i NKT cell development without perturbing the development of conventional T cells. In the absence of c-Myc, Valpha14i NKT cell precursors are blocked at an immature CD44(low)NK1.1(-) stage in a cell autonomous fashion. Residual c-Myc-deficient immature Valpha14i NKT cells appear to proliferate normally, cannot be rescued by transgenic expression of BCL-2, and exhibit characteristic features of immature Valpha14i NKT cells such as high levels of preformed IL-4 mRNA and the transcription factor promyelocytic leukemia zinc finger. Collectively our data identify c-Myc as a critical transcription factor that selectively acts early in Valpha14i NKT cell development to promote progression beyond the CD44(low)NK1.1(-) precursor stage.

Notch signaling is a direct determinant of keratinocyte growth arrest and entry into differentiation.

The role of Notch signaling in growth/differentiation control of mammalian epithelial cells is still poorly defined. We show that keratinocyte-specific deletion of the Notch1 gene results in marked epidermal hyperplasia and deregulated expression of multiple differentiation markers. In differentiating primary keratinocytes in vitro endogenous Notch1 is required for induction of p21WAF1/Cip1 expression, and activated Notch1 causes growth suppression by inducing p21WAF1/Cip1 expression. Activated Notch1 also induces expression of 'early' differentiation markers, while suppressing the late markers. Induction of p21WAF1/Cip1 expression and early differentiation markers occur through two different mechanisms. The RBP-Jkappa protein binds directly to the endogenous p21 promoter and p21 expression is induced specifically by activated Notch1 through RBP-Jkappa-dependent transcription. Expression of early differentiation markers is RBP-Jkappa-independent and can be induced by both activated Notch1 and Notch2, as well as the highly conserved ankyrin repeat domain of the Notch1 cytoplasmic region. Thus, Notch signaling triggers two distinct pathways leading to keratinocyte growth arrest and differentiation.

FANCD2 binds MCM proteins and controls replisome function upon activation of s phase checkpoint signaling.

Proteins disabled in Fanconi anemia (FA) are necessary for the maintenance of genome stability during cell proliferation. Upon replication stress signaling by ATR, the FA core complex monoubiquitinates FANCD2 and FANCI in order to activate DNA repair. Here, we identified FANCD2 and FANCI in a proteomic screen of replisome-associated factors bound to nascent DNA in response to replication arrest. We found that FANCD2 can interact directly with minichromosome maintenance (MCM) proteins. ATR signaling promoted the transient association of endogenous FANCD2 with the MCM2-MCM7 replicative helicase independently of FANCD2 monoubiquitination. FANCD2 was necessary for human primary cells to restrain DNA synthesis in the presence of a reduced pool of nucleotides and prevented the accumulation of single-stranded DNA, the induction of p21, and the entry of cells into senescence. These data reveal that FANCD2 is an effector of ATR signaling implicated in a general replisome surveillance mechanism that is necessary for sustaining cell proliferation and attenuating carcinogenesis.

A dynamic model of keratinocyte stem cell renewal and differentiation: role of the p21WAF1/Cip1 and Notch1 signaling pathways.

We present here a dynamic model of functional equilibrium between keratinocyte stem cells, transit amplifying populations and cells that are reversibly versus irreversibly committed to differentiation. According to this model, the size of keratinocyte stem cell populations can be controlled at multiple levels, including relative late steps in the sequence of events leading to terminal differentiation and by the influences of a heterogeneous extra-cellular environment. We discuss how work in our laboratory, on the interconnection between the cyclin/CDK inhibitor p21WAF1/Cip1 and the Notch1 signaling pathways, provides strong support to this dynamic model of stem cell versus committed and/or differentiated keratinocyte populations.