Growth-permissive Conditions Research Articles

Growth pattern of de novo small clusters of colorectal cancer is regulated by Notch signaling at detachment.

Cancer cell clusters have a higher capacity for metastasis than single cells, suggesting cancer cell clusters have biological properties different from those of single cells. The nature of de novo cancer cell clusters that are newly formed from tumor masses is largely unknown. Herein, we generated small cell clusters from colorectal cancer organoids and tracked the growth patterns of the clusters up to four cells. Growth patterns were classified into actively growing and poorly growing spheroids (PG). Notch signaling was robustly activated in small clusters immediately after dissociation, and Notch signaling inhibition markedly increased the proportion of PG spheroids. Only a limited number of PG spheroids grew under growth-permissive conditions invitro, but xenograft tumors derived from Notch inhibited clusters showed growth rates comparable to those of untreated spheroids. Thus, de novo clusters are composed of cells with interchangeable growth fates, which are regulated in a context-dependent manner by Notch signaling.

Neuroprotection and neuroregeneration of retinal ganglion cells after intravitreal carbon monoxide release.

PurposeRetinal ischemia induces apoptosis leading to neurodegeneration and vision impairment. Carbon monoxide (CO) in gaseous form showed cell-protective and anti-inflammatory effects after retinal ischemia-reperfusion-injury (IRI). These effects were also demonstrated for the intravenously administered CO-releasing molecule (CORM) ALF-186. This article summarizes the results of intravitreally released CO to assess its suitability as a neuroprotective and neuroregenerative agent.MethodsWater-soluble CORM ALF-186 (25 μg), PBS, or inactivated ALF (iALF) (all 5 μl) were intravitreally applied into the left eyes of rats directly after retinal IRI for 1 h. Their right eyes remained unaffected and were used for comparison. Retinal tissue was harvested 24 h after intervention to analyze mRNA or protein expression of Caspase-3, pERK1/2, p38, HSP70/90, NF-kappaB, AIF-1 (allograft inflammatory factor), TNF-α, and GAP-43. Densities of fluorogold-prelabeled retinal ganglion cells (RGC) were examined in flat-mounted retinae seven days after IRI and were expressed as mean/mm2. The ability of RGC to regenerate their axon was evaluated two and seven days after IRI using retinal explants in laminin-1-coated cultures. Immunohistochemistry was used to analyze the different cell types growing out of the retinal explants.ResultsCompared to the RGC-density in the contralateral right eyes (2804±214 RGC/mm2; data are mean±SD), IRI+PBS injection resulted in a remarkable loss of RGC (1554±159 RGC/mm2), p<0.001. Intravitreally injected ALF-186 immediately after IRI provided RGC protection and reduced the extent of RGC-damage (IRI+PBS 1554±159 vs. IRI+ALF 2179±286, p<0.001). ALF-186 increased the IRI-mediated phosphorylation of MAP-kinase p38. Anti-apoptotic and anti-inflammatory effects were detectable as Caspase-3, NF-kappaB, TNF-α, and AIF-1 expression were significantly reduced after IRI+ALF in comparison to IRI+PBS or IRI+iALF. Gap-43 expression was significantly increased after IRI+ALF. iALF showed effects similar to PBS. The intrinsic regenerative potential of RGC-axons was induced to nearly identical levels after IRI and ALF or iALF-treatment under growth-permissive conditions, although RGC viability differed significantly in both groups. Intravitreal CO further increased the IRI-induced migration of GFAP-positive cells out of retinal explants and their transdifferentiation, which was detected by re-expression of beta-III tubulin and nestin.ConclusionIntravitreal CORM ALF-186 protected RGC after IRI and stimulated their axons to regenerate in vitro. ALF conveyed anti-apoptotic, anti-inflammatory, and growth-associated signaling after IRI. CO’s role in neuroregeneration and its effect on retinal glial cells needs further investigation.

Small molecule modulation of splicing factor expression is associated with rescue from cellular senescence

BackgroundAltered expression of mRNA splicing factors occurs with ageing in vivo and is thought to be an ageing mechanism. The accumulation of senescent cells also occurs in vivo with advancing age and causes much degenerative age-related pathology. However, the relationship between these two processes is opaque. Accordingly we developed a novel panel of small molecules based on resveratrol, previously suggested to alter mRNA splicing, to determine whether altered splicing factor expression had potential to influence features of replicative senescence.ResultsTreatment with resveralogues was associated with altered splicing factor expression and rescue of multiple features of senescence. This rescue was independent of cell cycle traverse and also independent of SIRT1, SASP modulation or senolysis. Under growth permissive conditions, cells demonstrating restored splicing factor expression also demonstrated increased telomere length, re-entered cell cycle and resumed proliferation. These phenomena were also influenced by ERK antagonists and agonists.ConclusionsThis is the first demonstration that moderation of splicing factor levels is associated with reversal of cellular senescence in human primary fibroblasts. Small molecule modulators of such targets may therefore represent promising novel anti-degenerative therapies.

Regulation of YAP by mTOR and autophagy reveals a therapeutic target of tuberous sclerosis complex.

Genetic studies have shown that the tuberous sclerosis complex (TSC) 1-TSC2-mammalian target of Rapamycin (mTOR) and the Hippo-Yes-associated protein 1 (YAP) pathways are master regulators of organ size, which are often involved in tumorigenesis. The crosstalk between these signal transduction pathways in coordinating environmental cues, such as nutritional status and mechanical constraints, is crucial for tissue growth. Whether and how mTOR regulates YAP remains elusive. Here we describe a novel mouse model of TSC which develops renal mesenchymal lesions recapitulating human perivascular epithelioid cell tumors (PEComas) from patients with TSC. We identify that YAP is up-regulated by mTOR in mouse and human PEComas. YAP inhibition blunts abnormal proliferation and induces apoptosis of TSC1-TSC2-deficient cells, both in culture and in mosaic Tsc1 mutant mice. We further delineate that YAP accumulation in TSC1/TSC2-deficient cells is due to impaired degradation of the protein by the autophagosome/lysosome system. Thus, the regulation of YAP by mTOR and autophagy is a novel mechanism of growth control, matching YAP activity with nutrient availability under growth-permissive conditions. YAP may serve as a potential therapeutic target for TSC and other diseases with dysregulated mTOR activity.

Genome-Wide Identification of Genes Essential for the Survival of Streptococcus pneumoniae in Human Saliva

Since Streptococcus pneumoniae transmits through droplet spread, this respiratory tract pathogen may be able to survive in saliva. Here, we show that saliva supports survival of clinically relevant S. pneumoniae strains for more than 24 h in a capsule-independent manner. Moreover, saliva induced growth of S. pneumoniae in growth-permissive conditions, suggesting that S. pneumoniae is well adapted for uptake of nutrients from this bodily fluid. By using Tn-seq, a method for genome-wide negative selection screening, we identified 147 genes potentially required for growth and survival of S. pneumoniae in saliva, among which genes predicted to be involved in cell envelope biosynthesis, cell transport, amino acid metabolism, and stress response predominated. The Tn-seq findings were validated by testing a panel of directed gene deletion mutants for their ability to survive in saliva under two testing conditions: at room temperature without CO2, representing transmission, and at 37°C with CO2, representing in-host carriage. These validation experiments confirmed that the plsX gene and the amiACDEF and aroDEBC operons, involved in respectively fatty acid metabolism, oligopeptide transport, and biosynthesis of aromatic amino acids play an important role in the growth and survival of S. pneumoniae in saliva at 37°C. In conclusion, this study shows that S. pneumoniae is well-adapted for growth and survival in human saliva and provides a genome-wide list of genes potentially involved in adaptation. This notion supports earlier evidence that S. pneumoniae can use human saliva as a vector for transmission.

Microbial Scout Hypothesis, Stochastic Exit from Dormancy, and the Nature of Slow Growers

We recently proposed a scout model of the microbial life cycle (S. S. Epstein, Nature 457:1083, 2009), the central element of which is the hypothesis that dormant microbial cells wake up into active (so-called scout) cells stochastically, independently of environmental cues. Here, we check the principal prediction of this hypothesis: under growth-permissive conditions, dormant cells initiate growth at random time intervals and exhibit no species-specific lag phase. We show that a range of microorganisms, including environmental species, Escherichia coli, and Mycobacterium smegmatis, indeed wake up in a seemingly stochastic manner and independently of environmental conditions, even in the longest incubations conducted (months to years long). As is implicit in the model, most of the cultures we obtained after long incubations were not inherently slow growers. Of the environmental isolates that required ≥7 months to form visible growth, only 5% needed an equally long incubation upon subculturing, with the majority exhibiting regrowth within 24 to 48 h. This apparent change was not a result of adaptive mutation; rather, most microbial species that appear to be slow growers were in fact fast growers with a delayed initiation of division. Genuine slow growth thus appears to be less significant than previously believed. Random, low-frequency exit from the nongrowing state may be a key element of a general microbial survival strategy, and the phylogenetic breadth of the organisms exhibiting such exit indicates that it represents a general phenomenon. The stochasticity of awakening can also provide a parsimonious explanation to several microbiological observations, including the apparent randomness of latent infections and the existence of viable-but-nonculturable cells (VBNC).

Pathogens as potential hitchhikers on intercontinental dust

Desert dust seeds distant lands and waters with minerals as well as micro-organisms raising the question of whether this ancient phenomenon also spreads pathogens across the globe. Severe dust storms require strong winds blowing over land-masses that are largely devoid of vegetation, effectively limiting the scope for winds to raise pathogens into the air. Nevertheless, changing patterns of land-use, often driven by belligerency, result in refugees spreading to areas that were previously deemed barely habitable. With the help of the International Committee of the Red Cross, a number of sand/dust samples were collected from the Republic of Chad, some near refugee camps, others further removed from human influence. In parallel studies, we documented the micro-organisms present in these samples and used a number of the isolates here to test the effect of environmental constraints on their ability to survive intercontinental flight. We also added traditional pathogens to the palette of microbes and tested the effects of UV irradiation, desiccation and temperature on survival of both bacteria and fungi. A clear trend was obvious—those microbes that are coloured or able to form conidia or spores (in other words, those that are native to deserts) were well able to resist the imposed stresses. On the other hand, most pathogens were more sensitive to stresses than the environmental isolates. Toxin production in two species of Aspergillus was also investigated. Short-term desiccation (simulating environmental conditions during intercontinental travel) of sand amended with fungal spores containing sterigmatocystin leads to increased mycotoxin contents, but significant mycotoxin production was only possible under growth-permissive conditions, e.g. at higher humidity. It thus seems likely that an ever-decreasing fraction of the initial pathogen load survives as the dust recedes from its desert source and that those organisms that land on other continents are highly enriched in desert dwellers.

Cell-cell contact regulates gene expression in CDK4-transformed mouse podocytes

We transformed mouse podocytes by ectopic expression of cyclin-dependent kinase 4 (CDK4). Compared with podocytes transformed with a thermo-sensitive SV40 large T antigen mutant tsA58U19 (tsT podocytes), podocytes transformed with CDK4 (CDK4 podocytes) exhibited significantly higher expression of nephrin mRNA. Synaptopodin mRNA expression was significantly lower in CDK4 podocytes and in tsT podocytes under growth-permissive conditions (33°C) compared with tsT podocytes under growth-restricted conditions (37°C), which suggests a role for cell cycle arrest in synaptopodin mRNA expression. Confluent CDK4 podocytes showed significantly higher mRNA expression levels for nephrin, synaptopodin, Wilms tumor 1, podocalyxin, and P-cadherin compared with subconfluent cultures. We carried out experiments to clarify roles of various factors in the confluent podocyte cultures; our findings indicate that cell-cell contact promotes expression of five podocyte marker genes studied, that cellular quiescence increases synaptopodin and podocalyxin mRNA expression, and that soluble factors play a role in nephrin mRNA expression. Our findings suggest that CDK4 podocytes are useful tools to study podocyte biology. Furthermore, the role of cell-cell contact in podocyte gene expression may have relevance for podocyte function in vivo.

Quantifying growth and transformation frequency of oncogene‐expressing mouse hepatocytes in vivo†

Gene changes can affect cancer cells in many ways, but changes that increase disease severity--by allowing cells to proliferate when they should be quiescent, by enhancing their rate of growth under growth permissive conditions, or by increasing the risk that they will accumulate additional carcinogenic alterations--must be identified so that strategies to counter their effects can be developed. We describe a novel in vivo assay system based on hepatocyte transplantation that permits us to accomplish this objective for genetically modified hepatocytes. We find that the oncogenes c-myc and transforming growth factor alpha, but not simian virus 40 T-antigen, increase the rate of hepatocyte growth under growth permissive conditions. However, no single oncogene can induce hepatocyte growth in quiescent liver. In contrast, at least one oncogene combination, transforming growth factor alpha/T-antigen, was sufficient to direct cell autonomous growth even in this nonpermissive environment. Furthermore, we could quantify risk for progression to neoplasia associated with oncogene expression; increased transformation frequency was the principal carcinogenic effect of T-antigen. This system identifies biological mechanistic role(s) in carcinogenesis for candidate genetic changes implicated in development of human liver cancer. The quantitative and comparative evaluation of gene effects on liver cancer allows us to prioritize targets for therapeutic intervention.

Farnesol‐mediated inhibition of Candida albicans yeast growth and rescue by a diacylglycerol analogue

During Candida albicans yeast cell growth to early stationary phase, metabolites accumulate in the medium, including the quorum-sensing molecule farnesol. We found that besides germ tube inhibition, 40 microM farnesol also inhibited C. albicans yeast growth under yeast growth permissive conditions. Consistent with this observation, transcriptional analysis of yeast cells resuspended in fresh medium with 40 microM farnesol revealed that genes involved in hyphal formation, GTPase activation, mitosis and DNA replication were downregulated many-fold. Farnesol-mediated inhibition of yeast growth was dependent on the growth phase of the C. albicans cells. The growth defect was relieved by addition of a diacylglycerol analogue, implicating phosphatidylinositol signalling in the delay. Although diacylglycerol is an activator of protein kinase C (PKC) in mammalian cells, there is some question about activation of fungal PKCs. A mutant strain deleted for PKC1 responded to farnesol and the diacylglycerol analogue similar to wild-type, suggesting that PKC is not the target of the diacylglycerol analogue.

Role of Jun N-terminal Kinase (JNK) signaling in the wound healing and regeneration of a Drosophila melanogaster wing imaginal disc

When a fragment of a Drosophila imaginal disc is cultured in growth permissive conditions, it either regenerates the missing structures or duplicates the pattern present in the fragment. This kind of pattern regulation is known to be epimorphic, i.e. the new pattern is generated by proliferation in a specialized tissue called the blastema. Pattern regulation is accompanied by the healing of the cut surfaces restoring the continuous epithelia. Wound healing has been considered to be the inductive signal to commence regenerative cell divisions. Although the general outlines of the proliferation dynamics in a regenerating imaginal disc blastema have been well studied, little is known about the mechanisms driving cells into the regenerative cell cycles. In this study, we have investigated the role of Jun N-terminal Kinase (JNK) signaling in the wound healing and regeneration of a Drosophila wing imaginal disc. By utilizing in vivo and in vitro culturing of incised and fragmented discs, we have been able to visualize the dynamics in cellular architecture and gene expression involved in the healing and regeneration process. Our results directly show that homotypic wound healing is not a prerequisite for regenerative cell divisions. We also show that JNK signaling participates in imaginal disc wound healing and is regulated by the physical dynamics of the process, as well as in recruiting cells into the regenerative cell cycles. A model describing the determination of blastema size is discussed.

Geminin Is Targeted for Repression by the Retinoblastoma Tumor Suppressor Pathway through Intragenic E2F Sites

The geminin protein is a critical regulator of DNA replication. It functions to control replication fidelity by blocking the assembly of prereplication complexes in the S and G(2) phases of the cell cycle. Geminin protein levels, which are low in G(0)/G(1) and increase at the G(1)/S transition, are controlled through coordinate transcriptional and proteolytic regulation. Here we show that geminin is regulated transcriptionally by the retinoblastoma tumor suppressor (RB)/E2F pathway. Initially, we observed that the activation of RB led to the repression of geminin transcription. Conversely, Rb-null mouse embryonic fibroblasts have enhanced the expression of geminin relative to wild type mouse embryonic fibroblasts. Similarly, an acute loss of Rb in mouse adult fibroblasts deregulated geminin RNA and protein levels. To delineate the responsible regulatory motifs, luciferase reporter constructs containing fragments of the geminin promoter were generated. An analysis of the critical regulatory cis-acting elements in the geminin promoter indicated that intragenic E2F sites down-stream of the first exon were responsible for RB-mediated repression of geminin. The direct analysis of the endogenous geminin promoter revealed that these intragenic E2F sites are occupied by E2F proteins, and the mutation of these sites eliminates responsiveness to RB. Together, these data link the expression of geminin to the RB/E2F pathway and represent the first promoter analysis of this important regulator of DNA replication.

Modulation of radiation-induced strand break repair by cisplatin in mammalian cells

Purpose: To investigate the repair of ionizing radiation-induced DNA lesions in human skin fibroblasts in the presence of cisplatin-DNA adducts and to determine the persistence of DNA repair inhibition by cisplatin. Materials and methods: Normal human fibroblasts (AG1522) treated with cisplatin were exposed to 4 Gy 60Co gamma-radiation and assayed for repair of radiation-induced damage under growth-permissive conditions. DNA damage was measured by the fluorescence analysis of DNA unwinding (FADU) and cytokinesis-blocked micronucleus assays. Results: Rejoining of strand breaks caused by 4 Gy radiation in cells without cisplatin pre-treatment appeared to be biphasic with an initial fast component (up to 15 min of repair time) followed by a slower component, and was completed by 90 min. Cisplatin treatment (10 mu g/ml, 30 min) immediately before irradiation had no effect on the fast rejoining component, but inhibited the slow component (p 0.01). The same cisplatin treatment 24 h prior to irradiation inhibited both slow and fast components (p 0.01). In contrast, decreasing the cisplatin exposure to 1.0 mu g/ml for 30 min, 24 h prior to irradiation, resulted in an increased amount of strand break repair at each time point measured compared with irradiated control cells. This mild cisplatin treatment (95% survival) also resulted in a reduction of radiation-generated micronuclei indicating an adaptive response. Conclusions: Cisplatin used in combination with ionizing radiation can produce differential cellular responses depending upon the severity of the cisplatin treatment and the time interval between cisplatin and radiation exposures.

Direct actions of kit-ligand on theca cell growth and differentiation during follicle development.

The direct actions of kit-ligand/stem cell factor (KL) in developing ovarian follicles were investigated. Previous studies have shown that granulosa cells express KL that can support oocyte development. The current study demonstrates that KL can also act directly on theca cells to promote cellular growth and differentiation. Through RT-PCR analysis it was shown that bovine granulosa cells express KL, and theca cells express the receptor c-kit. Bovine theca interna cells were isolated and cultured in serum-free conditions to study KL actions. KL stimulated theca cell growth in a dose-dependent manner as measured by [3H]thymidine incorporation into DNA when cells were cultured under subconfluent conditions. KL had no effect on theca cell androstenedione or progesterone production under these growth-permissive conditions. In contrast, KL stimulated theca cell androstenedione production but had no effect on progesterone production when theca cells were cultured under confluent (non-growth-permissive) conditions. Estradiol (10(-7) M) and human CG (100 ng/ml) were used as controls and regulated theca cell steroid production at any cell density. These results demonstrate that KL can directly stimulate theca cell growth and steroid production during follicular development. The observation that KL stimulated androstenedione production but not progesterone production suggests that KL promotes a follicular phase differentiated state in theca cells. The potential regulation of KL and c-kit expression during follicular development was studied using a specific quantitative RT-PCR procedure. Total RNA from granulosa cells (for KL) and theca cells (for c-kit) was examined from small (<5 mm), medium (5-10 mm), and large (>10 mm) size follicles. Steady state levels of KL messenger RNA were highest in granulosa cells from large size follicles and lowest in small and medium size follicles. No differences were observed in the steady state levels of c-kit messenger RNA in theca cells from small, medium, or large size follicles. The observation that KL expression is highest in large size follicles suggests that KL may be important for increased growth and steroid production in large and dominant follicles. Observations demonstrate that KL can dramatically alter theca cell function and support the hypothesis that local granulosa-theca cell interactions play an important role in regulating cellular function within ovarian follicles. This study identifies KL as the first granulosa cell-derived growth factor that can directly stimulate theca cell growth and androstenedione production in the absence of gonadotropins.