RAD001 targeted HUVECs reverses 12-lipoxygenase-induced angiogenesis in oesophageal squamous cell carcinoma

12-LOX plays an important role in the progression of various malignancies. However, the underlying mechanisms of the action of 12-LOX and tumour treatment strategies remain not fully defined. In this study, we investigated the possible roles of 12-LOX in ESCC and explored the new therapeutic target. Approximately 73% of ESCC tissues showed marked up-regulation of 12-LOX, which was associated with poor prognosis. 12-LOX overexpression was positively correlated with the malignant progression of ESCC as demonstrated both in vitro and in vivo. Up-regulation of 12-LOX significantly increased the proliferation of ESCC cells and the xenograft volume. Moreover, 12-LOX up-regulation promoted tube formation of HUVECs and tumour angiogenesis in xenografts. Mechanism investigation indicated that 12-LOX overexpression led to activation of the PI3K/AKT/mTOR pathway and the up-regulation of VEGF in ESCC cells. Subsequent analysis indicated that the RAD001 could reverse the 12-LOX-induced promoting effect on ESCC. Specifically, the application of RAD001 inhibited the proliferation of ESCC cells and the tube-forming ability of HUVECs. In the drug group, the xenografts exhibited significant volume reduction and angiogenesis inhibition. We demonstrated that RAD001 could inhibit HUVEC migration. These findings presented the evidence that RAD001 had distinct roles on HUVECs and could exert anti-tumour effects by targeting not only the PI3K/AKT/mTOR pathway but the angiogenesis in ESCC.     

Reexpression of ARHI inhibits tumor growth and angiogenesis and impairs the mTOR/VEGF pathway in hepatocellular carcinoma

The Ras-related tumor suppressor gene aplasia Ras homolog member I (ARHI) is frequently downregulated in many types of cancer, including hepatocellular carcinoma (HCC). In this study, we sought to explore the therapeutic implications of ARHI reconstitution in the treatment of HCC. We generated stable cell lines overexpressing ARHI in Hep3B and SK-Hep1 cells, both of which lack endogenous ARHI. The effects of ARHI reexpression on tumor growth and angiogenesis were assessed. Given the key role of mammalian target of rapamycin (mTOR) signaling in HCC progression, we also tested whether ARHI overexpression affected the mTOR pathway. Forced expression of ARHI resulted in a significant inhibition of the proliferation of both Hep3B and SK-Hep1 cells compared to control cells (P<0.01). Cell cycle analysis revealed a G0-G1 arrest induced by ARHI reexpression. Moreover, ARHI reexpression significantly retarded Hep3B xenograft growth in vivo, and caused a marked reduction in tumor angiogenesis assessed by CD31-stained microvessel count. Western blot analysis of the xenografts showed that ARHI overexpression substantially reduced the phosphorylation of two mTOR substrates, S6K1 and 4E-BP1, indicative of an inactivation of the mTOR pathway. Accompanying with the mTOR inactivation, the angiogenic factors, hypoxia-inducible factor 1 alpha and vascular endothelial growth factor, were significantly downregulated. These data highlighted an important role for ARHI in controlling HCC growth and angiogenesis, therefore offering a possible therapeutic strategy against this malignancy.     

Inhibitory effects of the HDAC inhibitor valproic acid on prostate cancer growth are enhanced by simultaneous application of the mTOR inhibitor RAD001

AimsTo analyze the combined impact of the histone deacetylase (HDAC) inhibitor valproic acid (VPA) and the mammalian target of rapamycin (mTOR) inhibitor RAD001 on prostate cancer cell growth.Main methodsPC-3, DU-145 and LNCaP cells were treated with RAD001, VPA or with an RAD001–VPA combination for 3 or 5 days. Tumor cell growth, cell cycle progression and cell cycle regulating proteins were then investigated by MTT assay, flow cytometry and Western blotting, respectively. Effects of drug treatment on cell signaling pathways were determined.Key findingsSeparate application of RAD001 or VPA distinctly reduced tumor cell growth and impaired cell cycle progression. Significant additive effects were evoked when both drugs were used in concert. Particularly, the cell cycle regulating proteins cdk1, cdk2, cdk4 and cyclin B were reduced, whereas p21 and p27 were enhanced by the RAD001–VPA combination. Signaling analysis revealed deactivation of EGFr, ERK1/2 and p70S6k. Phosphorylation of Akt was diminished in DU-145 but elevated in PC-3 and LNCaP cells.SignificanceThe RAD001–VPA combination exerted profound antitumor properties on a panel of prostate cancer cell lines. Therefore, simultaneous blockage of HDAC and mTOR related pathways should be considered when designing novel therapeutic strategies for treating prostate carcinoma.     

The Combination of RAD001 and MK-2206 Exerts Synergistic Cytotoxic Effects against PTEN Mutant Gastric Cancer Cells: Involvement of MAPK-Dependent Autophagic,but Not Apoptotic Cell Death Pathway

In the current study, we showed that the combination of mammalian target of rapamycin (mTOR) inhibitor RAD001 (everolimus) and Akt inhibitor MK-2206 exerted synergistic cytotoxic effects against low-phosphatase and tensin homolog (PTEN) gastric cancer cells (HGC-27 and SNU-601 lines). In HGC-27 cells, RAD001 and MK-2206 synergistically induced G1/S cell cycle arrest, growth inhibition, cell death but not apoptosis. RAD001 and MK-2206 synergistically induced light chain 3B (LC3B) and beclin-1 expression, two important autophagy indicators. Meanwhile, the autophagy inhibitor 3-methyladenine (3-MA) and chloroquine inhibited the cytotoxic effects by RAD001 and MK-2206, suggesting that autophagic, but not apoptotic cell death was important for the cytotoxic effects by the co-administration. We observed that the combination of RAD001 and MK-2206 exerted enhanced effects on Akt/mTOR inhibition, cyclin D1 down-regulation and ERK/MAPK(extracellular signal-regulated kinase/mitogen-activated protein kinases) activation. Intriguingly, MEK/ERK inhibitors PD98059 and U0126 suppressed RAD001 plus MK-2206-induced beclin-1 expression, autophagy induction and cytotoxicity in HGC-27 cells. In conclusion, these results suggested that the synergistic anti-gastric cancer cells ability by RAD001 and MK-2206 involves ERK-dependent autophagic cell death pathway.     

Molecular analysis of sunitinib resistant renal cell carcinoma cells after sequential treatment with RAD001 (everolimus) or sorafenib

Sequential application of target drugs is standard procedure after renal cell carcinoma (RCC) patients develop resistance. To optimize the sequence, antitumour effects of the mTOR inhibitor RAD001 or the tyrosine kinase inhibitor (TKI) sorafenib on RCC cells with acquired resistance to the TKI sunitinib was evaluated. RCC cells were exposed to 1 μM sunitinib for 24 hrs (as control) and for 8 weeks (to induce resistance) and then switched to RAD001 (5 nM) or sorafenib (5 μM) for a further 8 weeks. Tumour cell growth, cell cycle progression, cell cycle regulating proteins and intracellular signalling were then investigated. Short‐term application of sunitinib (24 hrs) induced cell growth blockade with accumulation in the G2/M phase. RCC cells became resistant to sunitinib after 8 weeks, demonstrated by accelerated cell growth along with enhanced cdk1, cdk2, loss of p27, activation of Akt, Rictor and Raptor. Switching to sorafenib only slightly reduced growth of the sunitinib resistant RCC cells and molecular analysis indicated distinct cross‐resistance. In contrast, full response was achieved when the cancer cells were treated with RAD001. p19 and p27 strongly increased, phosphorylated Akt, Rictor and Raptor decreased and the tumour cells accumulated in G0/G1. It is concluded that an mTOR‐inhibitor for second‐line therapy could be the strategy of choice after first‐line sunitinib failure.     

The combination of RAD001 and NVP-BEZ235 exerts synergistic anticancer activity against non-small cell lung cancer in vitro and in vivo

The phosphoinositide 3-kinase (PI3K)-mammalian target of rapamycin (mTOR) signaling axis has emerged as a novel target for cancer therapy. Agents that inhibit PI3K, mTOR or both are currently under development. The mTOR allosteric inhibitor, RAD001, and the PI3K/mTOR dual kinase inhibitor, BEZ235, are examples of these agents. We were interested in developing strategies to enhance mTOR-targeted caner therapy. In this study, we found that BEZ235 alone effectively inhibited the growth of rapamycin-resistant cancer cells. Interestingly, the combination of sub-optimal concentrations of RAD001 and BEZ235 exerted synergistic inhibition of the growth of human lung cancer cells along with induction of apoptosis and G1 arrest. Furthermore, the combination was also more effective than either agent alone in inhibiting the growth of lung cancer xenografts in mice. The combination showed enhanced effects on inhibiting mTOR signaling and reducing the expression of c-Myc and cyclin D1. Taken together, our results suggest that the combination of RAD001 and BEZ235 is a novel strategy for cancer therapy.     

Cell proliferation and polyamine metabolism in 9L cells treated with (2R,5R)-6-heptyne-2,5-diamine or α-difluoromethylornithine

Abstract We studied the effects of the ornithine decarboxylase inhibitors (2R,5R)-6-heptyne-2,5-diamine (R,R,-MAP) and α-difluoromethylornithine (DFMO) on cell proliferation and polyamine metabolism in 9L rat brain tumour cells. Treatment with 5 μM R,R-MAP inhibited cell proliferation to the same extent as did treatment with 1 mM DFMO. Both inhibitors depleted putrescine and spermidine concentrations to less than detectable levels within 24 h and 48 h of drug treatment, respectively; spermine levels were not affected significantly by either inhibitor. The effects of DFMO on 9L cell cycle kinetics were similar to those of R,R-MAP. During the first 3 days of treatment, both drugs caused an accumulation of cells in G₁ and a reduction of cells in S phase, as compared with control cells with a slowing in the rate of cell cycle traverse. In cultures seeded at low (1 × 10⁵), medium (5 × 10⁵), or high (2 × 10⁶) cell densities in a 25 cm² flask, inhibition of cell proliferation and polyamine depletion by both R,R-MAP and DFMO was more pronounced at the lower densities relative to the density-matched control cells. Thus, R,R-MAP was a more potent inhibitor of ornithine decarboxylase than was DFMO in 9L cells, and the inhibitory effects of both compounds on cell proliferation and polyamine biosynthesis were greater in actively proliferating cells.     

mTOR,A New Therapeutic Target in Acute Myeloid Leukemia

The mTOR (mammalian target of rapamycin) serine threonine kinase is involved in the regulation of the cell cycle, apoptosis and angiogenesis. mTOR inhibitors (rapamycin, or analogues such as CCI-779, RAD001, AP23573), which have been shown to have a potent anti-neoplastic effect in many solid tumour models, are now being used in clinical trials. Recent data have shown that the mTOR pathway is also aberrantly activated in hematological malignancies including acute myeloid leukemia (AML). This disease still has a bad prognosis and new therapeutic strategies are required. Rapamycin, used at low concentrations, induces the profound inhibition of AML cell clonogenic properties in 60% of cases while sparing their normal counterparts. Moreover, clinical responses have been achieved in poor-risk AML patients. In this review, we discuss the possible mechanisms of mTOR activation, the mechanisms involved in the inhibition of cell proliferation by rapamycin, the possible resistance mechanisms and ways of improving rapamycin efficacy in the context of AML.     

Autophagy upregulation by inhibitors of caspase-3 and mTOR enhances radiotherapy in a mouse model of lung cancer

Autophagy has been reported to be increased in irradiated cancer cells resistant to various apoptotic stimuli. We therefore hypothesized that induction of autophagy via mTOR inhibition could enhance radiosensitization in apoptosis-inhibited H460 lung cancer cells in vitro and in a lung cancer xenograft model. To test this hypothesis, combinations of Z-DEVD (caspase-3 inhibitor), RAD001 (mTOR inhibitor) and irradiation were tested in cell and mouse models. The combination of Z-DEVD and RAD001 more potently radiosensitized H460 cells than individual treatment alone. The enhancement in radiation response was not only evident in clonogenic survival assays, but also was demonstrated through markedly reduced tumor growth, cellular proliferation (Ki67 staining), apoptosis (TUNEL staining) and angiogenesis (vWF staining) in vivo. Additionally, upregulation of autophagy as measured by increased GFP-LC3-tagged autophagosome formation accompanied the noted radiosensitization in vitro and in vivo. The greatest induction of autophagy and associated radiation toxicity was exhibited in the tri-modality treatment group. Autophagy marker, LC-3-II, was reduced by 3-methyladenine (3-MA), a known inhibitor of autophagy, but further increased by the addition of lysosomal protease inhibitors (pepstatin A and E64d), demonstrating that there is autophagic induction through type III PI3 kinase during the combined therapy. Knocking down of ATG5 and beclin-1, two essential autophagic molecules, resulted in radiation resistance of lung cancer cells. Our report suggests that combined inhibition of apoptosis and mTOR during radiotherapy is a potential therapeutic strategy to enhance radiation therapy in patients with non-small cell lung cancer.     

Autophagy upregulation by inhibitors of caspase-3 and mTOR enhances radiotherapy in a mouse model of lung cancer

Autophagy has been reported to be increased in irradiated cancer cells resistant to various apoptotic stimuli. We therefore hypothesized that induction of autophagy via mTOR inhibition enhances radiosensitization in apoptosis-inhibited H460 lung cancer cells in vitro and in a lung cancer xenograft model. To test this hypothesis, combinations of Z-DEVD (caspase-3 inhibitor), RAD001 (mTOR inhibitor) and irradiation were tested in cell and mouse models. The combination of Z-DEVD and RAD001 more potently radiosensitized H460 cells than individual treatment alone. The enhancement in radiation response was not only evident in clonogenic survival assays, but also was demonstrated through markedly reduced tumor growth, cellular proliferation (Ki67 staining), apoptosis (TUNEL staining), and angiogenesis (vWF staining) in vivo. Additionally, upregulation of autophagy as measured by increased GFP-LC3-tagged autophagosome formation accompanied the noted radiosensitization in vitro and in vivo. The greatest induction of autophagy and associated radiation toxicity was exhibited in the tri-modality treatment group. Autophagy marker, LC-3-II, was reduced by 3-methyladenine (3-MA), a known inhibitor of autophagy, but further increased by the addition of lysosomal protease inhibitors (pepstatin A and E64d), demonstrating that there is autophagic induction through type III PI3 kinase during the combined therapy. Knocking down of ATG5 and beclin-1, two essential autophagic molecules, resulted in radiation resistance of lung cancer cells. Our report suggests that combined inhibition of apoptosis and mTOR during radiotherapy is a potential therapeutic strategy to enhance radiation therapy in patients with non-small cell lung cancer.     

Novel role for polycystin-1 in modulating cell proliferation through calcium oscillations in kidney cells

Abstract.   Objectives : Polycystin-1 (PC1), a signalling receptor regulating Ca²⁺-permeable cation channels, is mutated in autosomal dominant polycystic kidney disease, which is typically characterized by increased cell proliferation. However, the precise mechanisms by which PC1 functions on Ca²⁺ homeostasis, signalling and cell proliferation remain unclear. Here, we investigated the possible role of PC1 as a modulator of non-capacitative Ca²⁺ entry (NCCE) and Ca²⁺ oscillations, with downstream effects on cell proliferation. Results and discussion : By employing RNA interference, we show that depletion of endogenous PC1 in HEK293 cells leads to an increase in serum-induced Ca²⁺ oscillations, triggering nuclear factor of activated T cell activation and leading to cell cycle progression. Consistently, Ca²⁺ oscillations and cell proliferation are increased in PC1-mutated kidney cystic cell lines, but both abnormal features are reduced in cells that exogenously express PC1. Notably, blockers of the NCCE pathway, but not of the CCE, blunt abnormal oscillation and cell proliferation. Our study therefore provides the first demonstration that PC1 modulates Ca²⁺ oscillations and a molecular mechanism to explain the association between abnormal Ca²⁺ homeostasis and cell proliferation in autosomal dominant polycystic kidney disease.     

Inhibition of transient receptor potential canonical channels impairs cytokinesis in human malignant gliomas

Abstract.  Objectives : Glial-derived primary brain tumours, gliomas, are among the fastest growing malignancies and present a huge clinical challenge. Research suggests an important, yet poorly understood, role of ion channels in growth control of normal and malignant cells. In this study, we sought to functionally characterize Transient Receptor Potential Canoncial (TRPC) channels in glioma cell proliferation. TRPC channels form non-selective cation channels that have been suggested to represent a Ca²⁺ influx pathway impacting cellular growth. Materials and Methods : Employing a combination of molecular, biochemical and biophysical techniques, we characterized TRPC channels in glioma cells. Results : We showed consistent expression of four channel family members (TRPC-1, -3, -5, -6) in glioma cell lines and acute patient-derived tissues. These channels gave rise to small, non-voltage-dependent cation currents that were blocked by the TRPC inhibitors GdCl₃, 2-APB, or SKF96365. Importantly, TRPC channels contributed to the resting conductance of glioma cells and their acute pharmacological inhibition caused an ~10 mV hyperpolarization of the cells' resting potential. Additionally, chronic application of the TRPC inhibitor SKF96365 caused near complete growth arrest. A detailed analysis, by fluorescence-activated cell sorting and time-lapse microscopy, showed that growth inhibition occurred at the G₂+ M phase of the cell cycle with cytokinesis defects. Cells underwent incomplete cell divisions and became multinucleate, enlarged cells. Conclusions : Nuclear atypia and enlarged cells are histopathological hallmarks for glioblastoma multiforme , the highest grade glioma, suggesting that a defect in TRPC channel function may contribute to cellular abnormalities in these tumours.     

Role of PI3K/AKT/mTOR signaling in the cell cycle progression of human prostate cancer

Prostate cancer is one of the most common cancers among men. Recent studies demonstrated that PI3K signaling is an important intracellular mediator which is involved in multiple cellular functions including proliferation, differentiation, anti-apoptosis, tumorigenesis, and angiogenesis. In the present study, we demonstrate that the inhibition of PI3K activity by LY294002, inhibited prostate cancer cell proliferation and induced the G(1) cell cycle arrest. This effect was accompanied by the decreased expression of G(1)-associated proteins including cyclin D1, CDK4, and Rb phosphorylation at Ser780, Ser795, and Ser807/811, whereas expression of CDK6 and beta-actin was not affected by LY294002. The expression of cyclin kinase inhibitor, p21(CIP1/WAF1), was induced by LY294002, while levels of p16(INK4) were decreased in the same experiment. The inhibition of PI3K activity also inhibited the phosphorylation and p70(S6K), but not MAPK. PI3K regulates cell cycle through AKT, mTOR to p70(S6K). The mTOR inhibitor rapamycin has similar inhibitory effects on G(1) cell cycle progression and expression of cyclin D1, CDK4, and Rb phosphorylation. These results suggest that PI3K mediates G(1) cell cycle progression and cyclin expression through the activation of AKT/mTOR/p70(S6K) signaling pathway in the prostate cancer cells.     

Genotype-dependent efficacy of a dual PI3K/mTOR inhibitor, NVP-BEZ235, and an mTOR inhibitor, RAD001, in endometrial carcinomas

The PI3K (phosphatidylinositol-3-kinase)/mTOR (mammalian target of rapamycin) pathway is frequently activated in endometrial cancer through various PI3K/AKT-activating genetic alterations. We examined the antitumor effect of NVP-BEZ235--a dual PI3K/mTOR inhibitor--and RAD001--an mTOR inhibitor--in 13 endometrial cancer cell lines, all of which possess one or more alterations in PTEN, PIK3CA, and K-Ras. We also combined these compounds with a MAPK pathway inhibitor (PD98059 or UO126) in cell lines with K-Ras alterations (mutations or amplification). PTEN mutant cell lines without K-Ras alterations (n?=?9) were more sensitive to both RAD001 and NVP-BEZ235 than were cell lines with K-Ras alterations (n?=?4). Dose-dependent growth suppression was more drastically induced by NVP-BEZ235 than by RAD001 in the sensitive cell lines. G1 arrest was induced by NVP-BEZ235 in a dose-dependent manner. We observed in vivo antitumor activity of both RAD001 and NVP-BEZ235 in nude mice. The presence of a MEK inhibitor, PD98059 or UO126, sensitized the K-Ras mutant cells to NVP-BEZ235. Robust growth suppression by NVP-BEZ235 suggests that a dual PI3K/mTOR inhibitor is a promising therapeutic for endometrial carcinomas. Our data suggest that mutational statuses of PTEN and K-Ras might be useful predictors of sensitivity to NVP-BEZ235 in certain endometrial carcinomas.     

Growth suppression of human hepatocellular carcinoma xenografts by a monoclonal antibody CH12 directed to epidermal growth factor receptor variant III

Human hepatocellular carcinoma (HCC) is considered difficult to cure because it is resistant to radio- and chemotherapy and has a high recurrence rate after curative liver resection. Epidermal growth factor receptor variant III (EGFRvIII) has been reported to express in HCC tissues and cell lines. This article describes the efficacy of an anti-EGFRvIII monoclonal antibody (mAb CH12) in the treatment of HCC xenografts with EGFRvIII expression and the underlying mechanism of EGFRvIII as an oncogene in HCC. The results demonstrated that CH12 bound preferentially to EGFRvIII with a dissociation constant (K(d)) of 1.346 nm/liter. In addition, CH12 induces strong antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity in Huh7-EGFRvIII (with exogenous expression of EGFRvIII) and SMMC-7721 (with endogenous expression of EGFRvIII) cells. Notably, CH12 significantly inhibited the growth of Huh7-EGFRvIII and SMMC-7721 xenografts in vivo with a growth inhibition ratio much higher than C225, a U. S. Food and Drug Administration-approved anti-EGFR antibody. Treatment of the two HCC xenografts with CH12 significantly suppressed tumor proliferation and angiogenesis. Mechanistically, in vivo treatment with CH12 reduced the phosphorylation of constitutively active EGFRvIII, Akt, and ERK. Down-regulation of the apoptotic protectors Bcl-x(L), Bcl-2, and the cell cycle regulator cyclin D1, as well as up-regulation of the cell-cycle inhibitor p27, were also observed after in vivo CH12 treatment. Collectively, these results indicate that the monoclonal antibody CH12 is a promising therapeutic agent for HCC with EGFRvIII expression.     

microRNA‐1914, which is regulated by lncRNA DUXAP10, inhibits cell proliferation by targeting the GPR39‐mediated PI3K/AKT/mTOR pathway in HCC

Increasing studies have confirmed that abnormally expressed microRNAs (miRNAs) take part in the carcinogenesis as well as the aggravation of hepatocellular carcinoma (HCC). However, little information is currently available about miR‐1914 in HCC. Here, we first confirmed that miR‐1914 inhibition in HCC cell lines and tumour specimens correlates with tumour size and histological grade. In a series of functional experiments, miR‐1914 inhibited tumour proliferation and colony formation, resulting in cell cycle arrest and increased apoptosis. Moreover, miR‐1914 mediated its functional effects by directly targeting GPR39 in HCC cells, leading to PI3K/AKT/mTOR repression. Restoring GPR39 expression incompletely counteracted the physiological roles of miR‐1914 in HCC cells. In addition, down‐regulation of AKT phosphorylation inhibited the effects of miR‐1914 in HCC. Furthermore, the overexpression of lncRNA DUXAP10 negatively correlated with the expression of miR‐1914 in HCC; thus, lncRNA DUXAP10 regulated miR‐1914 expression and modulated the GPR39/PI3K/AKT‐mediated cellular behaviours. In summary, the present study demonstrated for the first time that lncRNA DUXAP10–regulated miR‐1914 plays a functional role in inhibiting HCC progression by targeting GPR39‐mediated PI3K/AKT/mTOR pathway, and this miRNA represents a novel therapeutic target for patients with HCC.     

New molecular mediators in tumor angiogenesis

Angiogenesis is essential for tumor growth and progression. It has been demonstrated that tumor growth beyond a size 1 to 2 mm³ requires the induction of new vessels. Angiogenesis is regulated by several endogenous stimulators and inhibitors of endothelial cell migration, proliferation and tube formation. Under physiological conditions these mediators of endothelial cell growth are in balance and vessel growth is limited. In fact, within the angiogenic balance endothelial cell turnover is sufficient to maintain a functional vascular wall but does not allow vessel growth. Tumor growth an progression has successfully been correlated to the serum concentration of angiogenic mediators. Furthermore, the vascular density of tumor tissues could be correlated to the clinical course of the disease in several tumor entities. Within the last years several new mediators of endothelial cell growth have been isolated e.g. angiopoietin 1, angiopoietin 2, midkine, pleiotropin, leptin and maspin. In this review we discuss the mechanisms leading to tumor angiogenesis and describe some of the newer mediators of endothelial cell stimulation and inhibition.     

Cyclic AMP-Elevating Agents Inhibit Proliferation of Keratinizing Guinea Pig Epidermal Cells

Cultured guinea pig epidermal cells and dermal fibroblasts were chosen as model systems to study possible growth inhibition by cyclic AMP (cAMP)-elevating drugs. The rate of DNA synthesis was used to assay growth rate in control cultures and those treated with agents which increase intracellular cAMP, including dibutyryl cAMP, the phosphodiesterase inhibitors papaverine and theophylline and agents which stimulate adenylate cyclase, iso-proterenol and prostaglandin E₂ methyl ester. Treatment for 24 h with dibutyryl cAMP (10⁻⁴ to 10⁻² M) inhibited cell growth by 50 to 95%, whereas butyrate(10⁻⁴M) showed essentially no effect. This inhibition could not be attributed to decreased precursor transport or to drug toxicity. Papaverine (10⁻⁶ to 10⁻⁴ M) and theophylline (10⁻⁴ to 10⁻³ M) also gave dose-dependent growth inhibition as did isoproterenol and prostaglandinE₂methyl ester. Radioautographic analysis of grain density after dibutyryl cAMP treatment and ³H-thymidine incorporation indicated no S-phase inhibition. Cyclic AMP-elevating drugs appear to inhibit growth of guinea-pig epidermal cells and dermal flbroblasts by blocking the cell cycle in G₋₂, M₁, or G. ₋₁     

Co-ordination between localized wound-induced Ca2+ signals and pre-wound serum signals is required for proliferation after mechanical injury

Abstract. The signals which initiate proliferation of endothelial cells after injury are important for selective blood vessel growth during wound healing or tumour growth. Upon mechanically wounding quiescent cells, a transient [Ca²⁺]_; increase was induced in cells at the wound edge. These same cells proliferated 18-24 h post wounding, as measured by bromodeoxyuridine incorporation. The localized Ca²⁺ signal was required specifically during wounding since blocking Ca²⁺ influx reduced proliferation by 40-50%. Proliferation also required serum since starvation reduced proliferation by 80%. Serum-starved cells proliferated if briefly primed with serum prior to wounding. The signals derived from serum and [Ca²⁺]j combined at least additively to induce proliferation. Therefore, serum priming followed by a single, transient Ca²⁺ signal induced by mechanical injury must occur in a temporally and spatially regulated manner for normal proliferation. Co-ordination between signalling cascades induced by growth factors and release from contact inhibition might be obligatory for localized re-endothelialization after injury.