Immortalization of conditionally transformed chicken cells: loss of normal p53 expression is an early step that is independent of cell transformation.

Clones of mortal chicken fibroblasts and erythroblasts transformed by temperature-sensitive v-src and v-erb B oncoproteins have been developed into immortal cell lines that retain the conditional transformed phenotype. The expressions of two tumor suppressor genes, the retinoblastoma (Rb) gene and the p53 gene, were investigated during senescence, crisis, and cell line establishment. In temperature-sensitive (ts)-v-erb B erythroblasts and ts-v-src fibroblasts (as well as in v-myc macrophages), loss of p53 mRNA or expression of a mutated p53 gene invariably occurred in the early phase of immortalization. In contrast, expression of the Rb gene was unchanged at all stages of immortalization. Inactivation of the original temperature-sensitive oncogene led to loss of the transformed phenotype in fibroblasts and to differentiation in erythroblasts, even in lines that were immortal and lacked p53. The results demonstrate that the process of immortalization is distinct from cell transformation, probably requiring different mutational events.     

Interferon-γ induces cellular senescence through p53-dependent DNA damage signaling in human endothelial cells

Cellular senescence is a stress-response phenomenon in which cells lose the ability to proliferate; it is induced by telomere shortening, activation of oncogenes or tumor suppressor genes, or exposure to a sub-lethal dose of DNA damaging agents or oxidative stresses. cDNA microarray analysis reveals that the levels of interferons (IFNs) and IFN-inducible genes were altered during replicative senescence in human umbilical vascular endothelial cells (HUVECs). However, the role of IFNs in cellular senescence of HUVECs remains unidentified. This study demonstrated that prolonged treatment with IFN-γ induced cellular senescence in HUVECs, as confirmed by G0/G1 cell cycle arrest, up-regulation of p53 and p21 protein levels, increased SA-β-gal staining, and the accumulation of phospho-H₂AX foci. IFN-γ-induced cellular senescence was observed only in p16-knockdown cells or p16-null mouse embryonic fibroblasts (MEFs), but not in p53-knockdown cells or p53-null MEFs. IFN-γ treatment increased ROS production, and an antioxidant, N-acetylcysteine, inhibited IFN-γ-induced cellular senescence. Knockdown of ATM kinase or IFI16 rescued IFN-γ-induced cellular senescence. Therefore, these results suggest that IFN-γ might play an important role in cellular senescence through a p53-dependent DNA damage pathway and contribute to the pathogenesis of atherosclerosis via its pro-senescent activity.     

UBTD1 induces cellular senescence through an UBTD1–Mdm2/p53 positive feedback loop

The tumour suppressor p53 plays an important role in tumourigenesis. Besides inducing apoptosis, it regulates cellular senescence, which constitutes an important barrier to tumourigenesis. The mechanism of regulation of cellular senescence by p53 and its downstream pathway are poorly understood. Here, we report that the ubiquitin domain‐containing 1 (UBTD1) gene, a new downstream target of p53, induces cellular senescence and acts as a novel tumour suppressor by a mechanism that depends on p53. Expression of UBTD1 increased upon cellular senescence induced by serial passageing of cultures, as well as by exposure to DNA‐damageing drugs that induce premature senescence. Over‐expression of UBTD1 induces senescence in human fibroblasts and cancer cells and attenuation of the transformed phenotype in cancer cells. UBTD1 is down‐regulated in gastric and colorectal cancer tissues, and its lower expression correlates with a more aggressive phenotype and worse prognosis. Multivariate analysis revealed that UBTD1 expression was an independent prognostic factor for gastric cancer patients. Furthermore, UBTD1 increased the stability of p53 protein, by promoting the degradation of Mdm2 protein. Importantly, UBTD1 and p53 function mutually depend on each other in regulating cellular senescence and proliferation. Thus, our data suggest that, upon DNA damage, p53 induction by UBTD1 creates a positive feedback mechanism to further increase p53 expression. Our results establish UBTD1 as a regulator of cellular senescence that mediates p53 function, and provide insights into the mechanism of Mdm2 inhibition that impacts p53 dynamics during cellular senescence and tumourigenesis. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Genetic selection for aflatoxin B1 resistance influences chicken T-cell and thymocyte proliferation

Studies were conducted with two lines of chickens that were selected for high and low plasma protein concentrations in response to aflatoxin B₁ (AFB₁) exposure. The experiments were designed to determine genetic differences in the responses of T cells and thymocytes to the toxin. Chicks were orally administered AFB₁ at a rate of 0, 100, or 500 μg/kg body weight up to 21 days of age. At 4 weeks of age, concanavalin A (Con A, 2.5 μg/mL) stimulated T-cell proliferation was similar for untreated chicks from the low line (LL) and the high line (HL). However, AFB₁ reduced the responses of T cells with HL cells being more sensitive. In a second experiment, immature chickens were bled and peripheral blood lymphocytes were cultured with Con A and either 0, 3.125, 6.25, 12.5, or 25 μg/mL AFB₁. T cells from LL had greater responses to Con A than those from HL, and LL T-cells were also more resistant to in vitro AFB₁ exposure. Furthermore, thymocyte proliferation was greater for LL chicks; but when thymocytes were cultured with 25 μg/mL AFB₁, ³H-thymidine incorporation was similarly reduced in both lines. Cell cycle analysis indicated that there were more LL thymocytes in S phase, and the percentages for both lines decreased with AFB₁ treatment. Although there were no differences between the lines for percent G₂/M cells, AFB₁ treatment increased the percentages of thymocytes in G₂/M. These studies showed that selection for plasma protein response also changed T-cell and thymocyte proliferative activity.     

GST-M1 is transcribed moreso than AKR7A2 in AFB1-exposed human monocytes and lymphocytes

Abstract

Glutathione-S-transferases (GST) and aldo-keto reductases (AKR) are key aflatoxin (AF)-detoxifying enzymes. In this study, the expression of GST-M1, GST-T1, AKR-7A2, and AKR-7A3 genes in human monocytes and lymphocytes was analyzed after in vitro exposure to 10 or 100?ng AFB₁/ml for 2?h. Unlike in pilot studies that showed that all four examined genes were present in HepG2 cells, in lymphocytes and monocytes, only GST-M1 and AKR-7A2 were detected. In fact, the induced expression of both GST-M1 and AKR-7A2 genes in human monocytes was moreso than that seen in AFB₁-exposed lymphocytes. In addition, analyses of the effects of the exposures on cell cycle status were performed as, in cells lacking adequate detoxification capacities, it would be expected the cells would arrest at checkpoints in the cell cycle or progress to apoptotic/necrotic states. The results here indicated that only the high dose of AFB₁ led to a change in cell cycle profiles and only in the monocytes (i.e. cells in S phase were significantly reduced). In general, the results here strongly suggest that human immune cell lineages appear to be able to increase their expression of AFB₁-detoxifying enzymes (albeit to differing degrees) and, as a result, are able to counter potential toxicities from AFB₁ and (likely) its metabolites.     

Translational repression of p53 by RNPC1, a p53 target overexpressed in lymphomas

The p53 pathway is critical for tumor suppression, as the majority of human cancer has a faulty p53. Here, we identified RNPC1, a p53 target and a RNA-binding protein, as a critical regulator of p53 translation. We showed that ectopic expression of RNPC1 inhibited, whereas knockdown of RNPC1 increased, p53 translation under normal and stress conditions. We also showed that RNPC1 prevented cap-binding protein eIF4E from binding p53 mRNA via its C-terminal domain for physical interaction with eIF4E, and its N-terminal domain for binding p53 mRNA. Consistent with this, we found that RNPC1 directly binds to p53 5′ and 3′untranslated regions (UTRs). Importantly, we showed that RNPC1 inhibits ectopic expression of p53 in a dose-dependent manner via p53 5′ or 3′ UTR. Moreover, we showed that loss of RNPC1 in mouse embryonic fibroblasts increased the level of p53 protein, leading to enhanced premature senescence in a p53-dependent manner. Finally, to explore the clinical relevance of our finding, we showed that RNPC1 was frequently overexpressed in dog lymphomas, most of which were accompanied by decreased expression of wild-type p53. Together, we identified a novel p53–RNPC1 autoregulatory loop, and our findings suggest that RNPC1 plays a role in tumorigenesis by repressing p53 translation.     

A senescence rescue screen identifies BCL6 as an inhibitor of anti-proliferative p19(ARF)-p53 signaling

Senescence limits the proliferative capacity of primary cells in culture. We describe here a genetic screen to identify genes that allow bypass of this checkpoint. Using retroviral cDNA expression libraries, we identify BCL6 as a potent inhibitor of senescence. BCL6 is frequently activated in non-Hodgkin's lymphoma, but its mechanism of action has remained unclear. BCL6 efficiently immortalizes primary mouse embryonic fibroblasts and cooperates with RAS in oncogenic transformation. BCL6 overrides the senescence response downstream of p53 through a process that requires induction of cyclin D1 expression, as cyclin D1 knockout fibroblasts are specifically resistant to BCL6 immortalization. We show that BCL6 expression also dramatically extends the replicative lifespan of primary human B cells in culture and induces cyclin D1 expression, indicating that BCL6 has a similar activity in lymphoid cells. Our results suggest that BCL6 contributes to oncogenesis by rendering cells unresponsive to antiproliferative signals from the p19(ARF)-p53 pathway.     

Cdk4 disruption renders primary mouse cells resistant to oncogenic transformation,leading to Arf/p53-independent senescence

A large number of human cancers display alterations in the Ink4a/cyclin D/Cdk4 genetic pathway, suggesting that activation of Cdk4 plays an important role in oncogenesis. Here we report that Cdk4-null mouse embryonic fibroblasts are resistant to transformation in response to Ras activation with dominant-negative (DN) p53 expression or in the Ink4a/Arf-null background, judged by foci formation, anchorage-independent growth, and tumorigenesis in athymic mice. Cdk4-null fibroblasts proliferate at normal rates during early passages. Whereas Cdk4(+/+)Ink4a/Arf(-/-) cells are immortal in culture, Cdk4(-/-)Ink4a/Arf(-/-) cells undergo senescence during continuous culture, as do wild-type cells. Activated Ras also induces premature senescence in Cdk4(-/-)Ink4a/Arf(-/-) cells and Cdk4(-/-) cells with DNp53 expression. Thus, Cdk4 deficiency causes senescence in a unique Arf/p53-independent manner, which accounts for the loss of transformation potential. Cdk4-null cells express high levels of p21(Cip1/Waf1) with increased protein stability. Suppression of p21(Cip1/Waf1) by small interfering RNA (siRNA), as well as expression of HPV-E7 oncoprotein, restores immortalization and Ras-mediated transformation in Cdk4(-/-)Ink4a/Arf(-/-) cells and Cdk4(-/-) cells with DNp53 expression. Therefore, Cdk4 is essential for immortalization, and suppression of Cdk4 could be a prospective strategy to recruit cells with inactive Arf/p53 pathway to senescence.     

Effect of calcitonin on the regulation of intracellular pH in primary cultures of rabbit early distal tubule

To examine the intracellular pH (pH_i) regulation in primary cultures of rabbit distal convoluted tubules (DCTb) we used the pH-sensitive dye 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF/AM) and a video-microscopy technique. DCTb segments were microdissected from rabbit kidney cortex and cultured in a hormonally defined medium. The culture epithelia were grown on semi-transparent permeable supports. Before pH_i measurement, DCTb primary cultures were maintained for 48–96 h in growth-factor-free medium to obtain quiescent cells. We had previously shown that two mechanisms are involved in the regulation of intracellular pH: a basolateral Na⁺/H⁺ exchanger and an apical Cl^–/HCO ₃ ^– exchanger [1]. The pH_i of DCTb cells was significantly decreased by the addition of 60 nM human calcitonin (from 7.30±0.04 to 7.08±0.04). This response to calcitonin was dose-dependent and mimicked by both forskolin and permeant cyclic AMP derivatives. An initial acidification (of 0.25 pH unit in 7–8 min) was observed after the addition of basolateral amiloride (1 mM). The persistence of the effect induced by human calcitonin in these conditions, suggests that the Na⁺/H⁺ exchanger is not involved in the response. However, the acidification response was blocked in both the absence of chloride at the apical side and by the apical addition of 0.1 mM 4,4-diisothiocyanostilbene-2,2-disulphonic acid (DIDS). These experiments suggest that the target for the human calcitonin effect on pH_i is the Cl^–/HCO ₃ ^– exchanger. This study confirms the importance of this transporter in pH_i regulation within the physiological pH_i range and the influence of calcitonin in the regulation of DCTb cell function.     

Endogenous oxidative stress prevents telomerase-dependent immortalization of human endothelial cells

Introduction

With aging, oxidative stress accelerates vascular endothelial cell (EC) telomere shortening-induced senescence, and may promote atherosclerosis in humans. Our aim was to investigate whether an antioxidant treatment combined with telomerase (hTERT) over-expression would prevent senescence of EC isolated from patients with severe atherosclerosis.

Methods

Cells were isolated from internal mammary arteries (n = 11 donors), cultured until senescence with or without N-acetylcystein (NAC) and infected, or not, with a lentivirus over-expressing hTERT.

Results

Compared to control EC, hTERT-NAC cells had increased telomerase activity, longer telomeres and underwent more cell divisions. According to the donor, hTERT-NAC either delayed (n = 5) or prevented (n = 4) EC senescence, the latter leading to cell immortalization. Lack of cell immortalization by hTERT-NAC was accompanied by an absence of beneficial effect of NAC alone in paired EC. Accordingly, lack of EC immortalization by hTERT-NAC was associated with high endogenous susceptibility to oxidation. In EC where hTERT-NAC did not immortalize EC, p53, p21 and p16 expression increased with senescence, while oxidative-dependent DNA damage associated with senescence was not prevented.

Conclusion

Our data suggest that irreversible oxidative stress-dependent damages associated with cardiovascular risk factors are responsible for senescence of EC from atherosclerotic patients.     

Prostaglandin F2α can modulate the growth and the differentiation of bovine corneal epithelial cells cultured in vitro

The effects of PGF_2α on the growth, morphology, morphometry and keratinization pattern of bovine corneal epithelial cells cultured in vitro were studied. The cells were grown with a basal medium or, in the presence of keratocytes and/or their products, using a keratocyte-conditioning medium. Cell growth was evaluated by MTT assay. Daily treatments with exogenous PGF_2α at concentrations equal to or lower than 10⁻⁶ M induced significant increases in cell proliferation when the epithelial cells were cultured on a keratocyte feeder-layer or with the conditioning medium. No variations were observed in cultures grown with the basal medium. 10⁻⁵ M PGF_2α induced a decrease in cell growth under all culturing conditions. PGF_2α did not affect cell morphology and modified only nuclear dimensions among the cells grown under different culturing conditions. No variations of any parameters were observed between cells cultured on feeder-layer, with conditioning or basal medium and the corresponding cultures supplemented with the autacoid. Moreover, PGF_2α induced only the disappearance of 43 kDa keratin in cells grown on basal medium, while the keratin pattern of epithelial cells cultured on feeder-layer or with the conditioning medium was not modified by the autacoid. From these data we can suppose that a cooperation could exist between PGF_2α and fibroblasts and their products for the modulation of cell growth. Finally, it was observed that the autacoid had no effect on cell morphology and morphometry, except for nuclear dimensions, despite the presence of other prostaglandins, such as PGE₂.     

Ability of Lactobacillus plantarum MON03 to mitigate aflatoxins (B1 and M1) immunotoxicities in mice

Abstract

Aflatoxin B₁ (AFB₁) and M₁ (AFM₁) are mycotoxins produced by numerous Aspergillus species in pre- or post-harvest cereals and milk. AFB₁ and AFM₁ display a potent economic loss in livestock and also cause severe immunological problems. The aims of this study were to: evaluate a new AFB₁ and AFM₁-binding/degrading micro-organism for biological detoxification; examine its ability to degrade AFB₁ and AFM₁ in liquid medium; and evaluate its potential for in vivo preventative effects against AFB₁- and AFM₁-induced immunomodulation in mice. Lactobacillus plantarum MON03 (LP) isolated from Tunisian artisanal butter was found to display significant binding ability to AFB₁ and AFM₁ in PBS (i.e. 82% and 89%, respectively) within 24?h of incubation and able to tolerate gastric acidity, have strongly hydrophilic cells surface properties, and adhere efficacy to Caco-3 cells in vitro. The in vivo study was conducted using Balb/c mice that received by oral gavage vehicle (control), LP only (2?×?10⁹ CFU/L, ～2?g/kg BW), AFB₁ or AFM₁ alone (0.25 and 0.27?mg/kg, respectively), or AFB₁?+?LP or AFM₁?+?LP daily for 15 days. Compared to in control mice, treatments with AFB₁ and AFM₁ led to significantly decreased body weight gains, histopathological changes, and decrements in all hematologic and immune parameters assessed. Co-treatment with LP strongly reduced the adverse effects of each mycotoxin. In fact, the mice receiving AFB₁?+?LP or AFM₁?+?LP co-treatment displayed no significant differences in the assayed parameters as compared to the control mice. By itself, the bacteria alone had no adverse effects in the mice. From these data, it is concluded that the tested bacteria could be beneficial in biotechnology detoxification of contaminated food and feed for humans and animals.     

Overcoming cellular senescence in human cancer pathogenesis

Elevation of p16, the CDKN2/p16 tumor suppressor gene (TSG) product, occurs at senescence in normal human uroepithelial cells (HUC). Immortal HUCs and bladder cancer cell lines show either alteration of p16 or pRb, the product of the retinoblastoma (RB) TSG. In addition, many human cancers show p16 or pRb alteration along with other genetic alterations that we associated with immortalization, including +20q and −3p. These observations led us to hypothesize that p16 elevation plays a critical role in senescence cell cycle arrest and that overcoming this block is an important step in tumorigenesis in vivo, as well as immortalization in vitro. Using a novel approach, we tested these hypotheses in the present study by examining p16 and pRb status in primary culture (P0) and after passage in vitro of transitional cell carcinoma (TCC) biopsies that represented both superficial bladder tumors and invasive bladder cancers. We demonstrated that all superficial TCCs showed elevated p16 after limited passage in vitro and then senesced, like normal HUCs. In contrast, all muscle invasive TCCs contained either a p16 or a pRb alteration at P0 and all spontaneously bypassed senescence (P=0.001). Comparative genomic hybridization (CGH) was used to identify regions of chromosome loss or gain in all TCC samples. The application of a statistical model to the CGH data showed a high probability of elevated alteration rates of +20q11−q12 (0.99) and +8p22−pter (0.94) in the immortal muscle invasive TCCs, and of −9q (0.99) in the superficial TCCs. Three myoinvasive TCCs lost 3p13−p14. In this study, four of six myoinvasive TCCs also showed TP53 mutation that associated well with genome instability (P=0.001), as previously hypothesized. Notably, TP53 mutation, which has been used as a marker of tumor progression in many human cancers, was less significant in associating with progression in this study (P=0.04) than was p16 or pRb alteration (P=0.001). Thus, these data support a new model in which overcoming senescence plays a critical role in human cancer pathogenesis and requires at least two genetic changes that occur in several combinations that can include either p16 or pRb loss and at least one additional alteration, such as +20q11−q12, −3p13−p14, or −8p21−pter.     

Frequent deletions in nine newly immortal human cell lines.

Nine newly immortal lines of human fibroblasts transfected with SV40 T antigen were examined for recurrent chromosome losses. In order of decreasing frequency, all nine lines had three or more of the following minimal deletions specifically associated with the immortalization event: del(6)(q21), del(3)(p24), del(1)(p34), del(4)(p25), del(5)(p14), del(11)(p11), del(11)(q14), del(12)(p12), and del(14)(p?). Many other chromosome changes were not clearly associated with immortalization, but were acquired during other stages of this multistep model of neoplastic transformation. We propose that these chromosome loci associated with immortalization are candidates for the location of genes involved in cellular senescence.     

Senescence of human fibroblasts induced by oncogenic Raf

The oncogenes RAS and RAF came to view as agents of neoplastic transformation. However, in normal cells, these genes can have effects that run counter to oncogenic transformation, such as arrest of the cell division cycle, induction of cell differentiation, and apoptosis. Recent work has demonstrated that RAS elicits proliferative arrest and senescence in normal mouse and human fibroblasts. Because the Raf/MEK/MAP kinase signaling cascade is a key effector of signaling from Ras proteins, we examined the ability of conditionally active forms of Raf-1 to elicit cell cycle arrest and senescence in human cells. Activation of Raf-1 in nonimmortalized human lung fibroblasts (IMR-90) led to the prompt and irreversible arrest of cellular proliferation and the premature onset of senescence. Concomitant with the onset of cell cycle arrest, we observed the induction of the cyclin-dependent kinase (CDK) inhibitors p21^Cip1 and p16^Ink4a. Ablation of p53 and p21^Cip1 expression by use of the E6 oncoprotein of HPV16 demonstrated that expression of these proteins was not required for Raf-induced cell cycle arrest or senescence. Furthermore, cell cycle arrest and senescence were elicited in IMR-90 cells by the ectopic expression of p16^Ink4a alone. Pharmacological inhibition of the Raf/MEK/MAP kinase cascade prevented Raf from inducing p16^Ink4a and also prevented Raf-induced senescence. We conclude that the kinase cascade initiated by Raf can regulate the expression of p16^Ink4a and the proliferative arrest and senescence that follows. Induction of senescence may provide a defense against neoplastic transformation when the MAP kinase signaling cascade is inappropriately active.     

Estimation of<Emphasis Type="Italic"> V̇</Emphasis>O<Subscript>2max</Subscript> from the ratio between HR<Subscript>max </Subscript>and HR<Subscript>rest</Subscript> – the Heart Rate Ratio Method

The effects of ̇raining and/or ageing upon maximal oxygen uptake (O_2max) and heart rate values at rest (HR_rest) and maximal exercise (HR_max), respectively, suggest a relationship between O_2max and the HR_max-to-HR_rest ratio which may be of use for indirect testing of O_2max. Fick principle calculations supplemented by literature data on maximum-to-rest ratios for stroke volume and the arterio-venous O₂ difference suggest that the conversion factor between mass-specific O_2max (ml·min^–1·kg^–1) and HR_max·HR_rest ^–1 is ~15. In the study we experimentally examined this relationship and evaluated its potential for prediction of O_2max. O_2max was measured in 46 well-trained men (age 21–51 years) during a treadmill protocol. A subgroup (n=10) demonstrated that the proportionality factor between HR_max·HR_rest ^–1 and mass-specific O_2max was 15.3 (0.7) ml·min^–1·kg^–1. Using this value, O_2max in the remaining 36 individuals could be estimated with an SEE of 0.21 l·min^–1 or 2.7 ml·min^–1·kg^–1 (~4.5%). This compares favourably with other common indirect tests. When replacing measured HR_max with an age-predicted one, SEE was 0.37 l·min^–1 and 4.7 ml·min^–1·kg^–1 (~7.8%), which is still comparable with other indirect tests. We conclude that the HR_max-to-HR_rest ratio may provide a tool for estimation of O_2max in well-trained men. The applicability of the test principle in relation to other groups will have to await direct validation. O_2max can be estimated indirectly from the measured HR_max-to-HR_rest ratio with an accuracy that compares favourably with that of other common indirect tests. The results also suggest that the test may be of use for O_2max estimation based on resting measurements alone.An erratum to this article can be found at     

Characterization of aeroallergen-induced dyspnea in unrestrained guinea pigs by bias-flow-ventilated whole body plethysmography

Aeroallergen-induced dyspnea in guinea pigs was associated with an increase in amplitude in the box pressure fluctuations (212%) and pseudo-flow signal (604%) and an 80% decline (from 0.19 to 0.04 s) in relaxation time (the time it takes the box pressure signal to drop from its peak to 1/3 of its peak value). All of these lung dysfunction changes were highly significant (P<0.001). Pyrilamine (1 mg/kg, p.o., –2 h) inhibited dyspnea (P and F) by 50–53%. This technique allows quantitative analysis of allergic dyspnea in conscious, unrestrained guinea pigs.