Effect of pH on the induction of competence and progression to the S-phase in mouse fibroblasts

The pH dependence of competence induction and progression to the S-phase in quiescent stimulated cells has been studied. The results show that: (i) induction of competence by fibroblast growth factor in these cells is relatively independent of the external pH between pH 5.6-7.6; (ii) progression of cells to the S-phase is highly sensitive to pH and shows a dramatic increase between pH 6.8-7.2. These observations suggest that the intracellular alkalinization triggered by growth factors is fundamental for progression but not for competence induction.     

The novel putative transporter NPT1 plays a critical role in early stages of Plasmodium berghei sexual development

Transmission of Plasmodium species from a mammalian host to the mosquito vector requires the uptake, during an infected blood meal, of gametocytes, the precursor cells of the gametes. Relatively little is known about the molecular mechanisms involved in the developmental switch from asexual development to sexual differentiation or the maturation and survival of gametocytes. Here, we show that a gene coding for a novel putative transporter, NPT1, plays a crucial role in the development of Plasmodium berghei gametocytes. Parasites lacking NPT1 are severely compromised in the production of gametocytes and the rare gametocytes produced are unable to differentiate into fertile gametes. This is the earliest block in gametocytogenesis obtained by reverse genetics and the first to demonstrate the role of a protein with a putative transport function in sexual development. These results and the high degree of conservation of NPT1 in Plasmodium species suggest that this protein could be an attractive target for the development of novel drugs to block the spread of malaria.     

Catalase plays a critical role in the CSF-independent survival of human macrophages via regulation of the expression of BCL-2 family

M-colony-stimulating factor (M-CSF)-induced monocyte-derived macrophages (M-Mphi) required continuous presence of M-CSF for their survival, and depletion of M-CSF from the culture induced apoptosis, whereas human alveolar macrophages (A-Mphi) and granulocyte-macrophage (GM)-CSF-induced monocyte-derived macrophages (GM-Mphi) survived even in the absence of CSF. The expression of BCL-2 was higher in M-Mphi, and M-CSF withdrawal down-regulated the expression. The expression of BCL-X(L) was higher in A-Mphi and GM-Mphi, and the expression was CSF-independent. The expression of MCL-1 and BAX were not different between M-Mphi and GM-Mphi and were CSF-independent. Down-regulation of the expression of BCL-2 and BCL-X(L) by RNA interference showed the important role of BCL-2 and BCL-X(L) in the survival of M-Mphi and GM-Mphi, respectively. Human erythrocyte catalase (HEC) and conditioned medium obtained from GM-Mphi or A-Mphi cultured in the absence of GM-CSF prevented the M-Mphi from apoptosis and restored the expression of BCL-2. The activity of the conditioned medium was abrogated by pretreatment with anti-HEC antibody. Anti-HEC antibody also induced the apoptosis of M-Mphi cultured in the presence of M-CSF and GM-Mphi and A-Mphi cultured in the presence or absence of GM-CSF and down-regulated the expression of BCL-2 and BCL-X(L) in these Mphis. GM-Mphi and A-Mphi, but not M-Mphi, can produce both extracellular catalase and cell-associated catalase in a CSF-independent manner. Intracellular glutathione levels were kept equivalent in these Mphis, both in the presence or absence of CSF. These results indicate a critical role of extracellular catalase in the survival of human macrophages via regulation of the expression of BCL-2 family genes.     

Cdk2 plays a critical role in hepatocyte cell cycle progression and survival in the setting of cyclin D1 expression in vivo

Cdk2 was once believed to play an essential role in cell cycle progression, but cdk2-/- mice have minimal phenotypic abnormalities. In this study, we examined the role of cdk2 in hepatocyte proliferation, centrosome duplication, and survival. Cdk2-/- hepatocytes underwent mitosis and had normal centrosome content after mitogen stimulation. Unlike wild-type cells, cdk2-/- liver cells failed to undergo centrosome overduplication in response to ectopic cyclin D1 expression. After mitogen stimulation in culture or partial hepatectomy in vivo, cdk2-/- hepatocytes demonstrated diminished proliferation. Cyclin D1 is a key mediator of cell cycle progression in hepatocytes, and transient expression of this protein is sufficient to promote robust proliferation of these cells in vivo. In cdk2-/- mice and animals treated with the cdk2 inhibitor seliciclib, cyclin D1 failed to induce hepatocyte cell cycle progression. Surprisingly, cdk2 ablation or inhibition led to massive hepatocyte and animal death following cyclin D1 transfection. In a transgenic model of chronic hepatic cyclin D1 expression, seliciclib induced hepatocyte injury and animal death, suggesting that cdk2 is required for survival of cyclin D1-expressing cells even in the absence of substantial proliferation. In conclusion, our studies demonstrate that cdk2 plays a role in liver regeneration. Furthermore, it is essential for centrosome overduplication, proliferation, and survival of hepatocytes that aberrantly express cyclin D1 in vivo. These studies suggest that cdk2 may warrant further investigation as a target for therapy of liver tumors with constitutive cyclin D1 expression.     

Excision repair in u.v. (254 nm) damaged non-dividing human skin fibroblasts: a major biological role for DNA polymerase alpha

We have used the eukaryotic DNA polymerase alpha inhibitor, aphidicolin, and the polymerase beta inhibitor, dideoxythymidine, to examine the role of these enzymes in excision repair of ultraviolet (u.v., 254 nm) damage induced in non-dividing (arrested) human skin fibroblasts. The effects of these drugs on u.v.-treated cells have been monitored using a simple and reproducible repair synthesis assay in parallel with viability measurements to determine the degree of inhibition of repair of potentially lethal damage. In agreement with previous studies using density gradients, repair synthesis induced by low fluences of u.v. (less than 3 J m-2) is relatively insensitive to inhibition by aphidicolin compared to high fluences where approximately 85 per cent inhibition is observed at the highest (20 micrograms/ml) aphidicolin concentration employed. However, repair of potentially lethal damage is inhibited by at least 90 per cent over the entire fluence range. Although dideoxythymidine led to considerable inhibition of repair synthesis, the result is probably an artifact under these in vivo conditions. The polymerase beta inhibitor was not toxic to u.v.-treated cells nor did it add to the toxicity of aphidicolin when the drugs were used in combination. We conclude that if the beta polymerase is involved in excision repair then its temporary (4 h) inhibition by dideoxythymidine is entirely reversible. In contrast, polymerase alpha appears to be an enzyme essential to the majority of biologically effective excision repair over the entire u.v. fluence range tested.     

Gap junction communication between uterine stromal cells plays a critical role in pregnancy-associated neovascularization and embryo survival

In the uterus, the formation of new maternal blood vessels in the stromal compartment at the time of embryonic implantation is critical for the establishment and maintenance of pregnancy. Although uterine angiogenesis is known to be influenced by the steroid hormones estrogen (E) and progesterone (P), the underlying molecular pathways remain poorly understood. Here, we report that the expression of connexin 43 (Cx43), a major gap junction protein, is markedly enhanced in response to E in uterine stromal cells surrounding the implanted embryo during the early phases of pregnancy. Conditional deletion of the Cx43 gene in these stromal cells and the consequent disruption of their gap junctions led to a striking impairment in the development of new blood vessels within the stromal compartment, resulting in the arrest of embryo growth and early pregnancy loss. Further analysis of this phenotypical defect revealed that loss of Cx43 expression resulted in aberrant differentiation of uterine stromal cells and impaired production of several key angiogenic factors, including the vascular endothelial growth factor (Vegf). Ablation of CX43 expression in human endometrial stromal cells in vitro led to similar findings. Collectively, these results uncovered a unique link between steroid hormone-regulated cell-cell communication within the pregnant uterus and the development of an elaborate vascular network that supports embryonic growth. Our study presents the first evidence that Cx43-type gap junctions play a critical and conserved role in modulating stromal differentiation, and regulate the consequent production of crucial paracrine signals that control uterine neovascularization during implantation.     

Micronuclei and gene mutations in transgenic big Blue((R)) mouse and rat fibroblasts after exposure to the epoxide metabolites of 1, 3-butadiene

1,3-Butadiene (BD) is a commodity compound and by-product in the manufacture of synthetic rubber that elicits a differential carcinogenic response in rodents after chronic exposure. Mice are up to approximately 1000-fold more sensitive to the tumorigenicity of inhaled BD than rats, thereby confounding human risk assessment analyses. Rodent transgenic in vivo and in vitro models have been recently utilized for generating genetic toxicology data in support of risk assessment studies. However, studies have not been extended to investigate multiple endpoints of genetic damage using in vitro transgenic models. The goal of this study was to evaluate possible differences in the production of genetic damage in transgenic Big Blue((R)) mouse (BBM1) and rat (BBR1) fibroblasts exposed to three predominant epoxide metabolites of BD. Analyses of cytotoxicity, micronucleus (MN) formation, cII mutant frequency (MF) and apoptosis were assessed after in vitro exposure of BBM1 and BBR1 cells exposed to various concentrations of butadiene monoepoxide (BMO), diepoxybutane (DEB) and butadiene diolepoxide (BDE). Both BMO and DEB reduced cell survival in BBM1 and BBR1 cells. However, BDE decreased cell survival only in BBM1 cells at the concentrations evaluated. Concentration-dependent increases in the formation of MN was observed in both BBM1 and BBR1 cells, with DEB being the most potent followed by BDE and then BMO. The dose-response for mutations induced at the cII locus was essentially equal after DEB exposure of BBM1 and BBR1 fibroblasts. In contrast, the cII MF was significantly increased only in BBM1 cells after exposure to either BMO or BDE. These data demonstrate a differential genetic response for gene mutations but not for MN formation in transgenic BBM1 and BBR1 fibroblasts and suggest a rodent species-specific difference in the persistence of DNA damage that results in gene mutations. In addition, apoptosis was observed in BBR1 cells but not in BBM1 cells when treated with any of the three BD epoxide metabolites. This response may partially explain the differential response to mutations induced by BMO and BDE. These data offer insight into specific differences in mouse and rat cells with respect to their response to BD epoxide metabolites. Such data may help to explain the different tumorigenicity results observed in rodent BD carcinogenicity studies.     

Tumor suppressor p53 plays a key role in induction of both tristetraprolin and let-7 in human cancer cells

Tristetraprolin (TTP) and let-7 microRNA exhibit suppressive effects on cell growth through down-regulation of oncogenes. Both TTP and let-7 are often repressed in human cancers, thereby promoting oncogenesis by derepressing their target genes. However, the precise mechanism of this repression is unknown. We here demonstrate that p53 stimulated by the DNA-damaging agent doxorubicin (DOX) induced the expression of TTP in cancer cells. TTP in turn increased let-7 levels through down-regulation of Lin28a. Correspondingly, cancer cells with mutations or inhibition of p53 failed to induce the expression of both TTP and let-7 on treatment with DOX. Down-regulation of TTP by small interfering RNAs attenuated the inhibitory effect of DOX on let-7 expression and cell growth. Therefore, TTP provides an important link between p53 activation induced by DNA damage and let-7 biogenesis. These novel findings provide a mechanism for the widespread decrease in TTP and let-7 and chemoresistance observed in human cancers.     

Rev7/Mad2B plays a critical role in the assembly of a functional mitotic spindle

The spindle assembly checkpoint (SAC) acts as a guardian against cellular threats that may lead to chromosomal missegregation and aneuploidy. Mad2, an anaphase-promoting complex/cyclosome-Cdc20 (APC/C^Cdc20) inhibitor, has an additional homolog in mammals known as Mad2B, Mad2L2 or Rev7. Apart from its role in Polζ-mediated translesion DNA synthesis and double-strand break repair, Rev7 is also believed to inhibit APC/C by negatively regulating Cdh1. Here we report yet another function of Rev7 in cultured human cells. Rev7, as predicted earlier, is involved in the formation of a functional spindle and maintenance of chromosome segregation. In the absence of Rev7, cells tend to arrest in G2/M-phase and display increased monoastral and abnormal spindles with misaligned chromosomes. Furthermore, Rev7-depleted cells show Mad2 localization at the kinetochores of metaphase cells, an indicator of activated SAC, coupled with increased levels of Cyclin B1, an APC^Cdc20 substrate. Surprisingly unlike Mad2, depletion of Rev7 in several cultured human cell lines did not compromise SAC activity. Our data therefore suggest that besides its role in APC/C^Cdh1 inhibition, Rev7 is also required for mitotic spindle organization and faithful chromosome segregation most probably through its physical interaction with RAN.     

Atypical protein kinase Ciota plays a critical role in human lung cancer cell growth and tumorigenicity

Atypical protein kinase C (aPKC) isozymes function in epithelial cell polarity, proliferation, and survival and have been implicated in cellular transformation. However, the role of these enzymes in human cancer is largely unexplored. Here, we report that aPKCiota is highly expressed in human non-small cell lung cancer cell lines, whereas the closely related aPKC isozyme PKCzeta is undetectable in these cells. Disruption of PKCiota signaling reveals that PKCiota is dispensable for adherent growth of non-small cell lung cancer cells but is required for transformed growth in soft agar in vitro and for tumorigenicity in vivo. Molecular dissection of signaling down-stream of PKCiota demonstrates that Rac1 is a critical molecular target for PKCiota-dependent transformation, whereas PKCiota is not necessary for NFkappaB activation in vitro or in vivo. Expression of the PB1 domain of PKCiota (PKCiota-(1-113)) blocks PKCiota-dependent Rac1 activity and inhibits cellular transformation indicating a role for this domain in the transforming activity of PKCiota. Taken together, our data demonstrate that PKCiota is a critical lung cancer gene that activates a Rac1-->Pak-->Mek1,2-->Erk1,2 signaling pathway required for transformed growth. Our data indicate that PKCiota may be an attractive molecular target for mechanism-based therapies for treatment of lung cancer.     

Autophagy plays a critical role in kidney tubule maintenance, aging and ischemia-reperfusion injury

Autophagy is responsible for the degradation of protein aggregates and damaged organelles. Several studies have reported increased autophagic activity in tubular cells after kidney injury. Here, we examine the role of tubular cell autophagy in vivo under both physiological conditions and stress using two different tubular-specific Atg5-knockout mouse models. While Atg5 deletion in distal tubule cells does not cause a significant alteration in kidney function, deleting Atg5 in both distal and proximal tubule cells results in impaired kidney function. Already under physiological conditions, Atg5-null tubule cells display a significant accumulation of p62 and oxidative stress markers. Strikingly, tubular cell Atg5-deficiency dramatically sensitizes the kidneys to ischemic injury, resulting in impaired kidney function, accumulation of damaged mitochondria as well as increased tubular cell apoptosis and proliferation, highlighting the critical role that autophagy plays in maintaining tubular cell integrity during stress conditions.     

Excision repair in permeable arrested human skin fibroblasts damaged by UV (254 nm) radiation: evidence that alpha- and beta-polymerases act sequentially at the repolymerisation step

We have characterised far-ultraviolet-radiation-induced DNA-repair synthesis in permeabilised arrested (non-dividing) primary human skin fibroblasts. Approximately half the maximum repair synthesis is seen after a UV fluence of 4.0 Jm-2 and little additional incorporation was observed at fluences above 20.0 Jm-2. UV-damaged permeable cells were treated with specific inhibitors of DNA polymerase alpha and beta, both alone and in combination. The degree of inhibition of repair incorporation by aphidicolin indicates that polymerase alpha is involved in the majority (85-90%) of repair synthesis after both high and low (less than 4.0 Jm-2) UV fluences. Dideoxythymidine triphosphate seems able to inhibit DNA-repair synthesis only when polymerase alpha is fully or almost fully functional, indicating that polymerase beta is unable to substitute in repair for an alpha polymerase blocked by aphidicolin. These data suggest that the two enzymes may act sequentially to complete repair patches rather than acting independently.     

PTPN2 regulates the activation of KRAS and plays a critical role in proliferation and survival of KRAS-driven cancer cells

RAS genes are the most commonly mutated in human cancers and play critical roles in tumor initiation, progression, and drug resistance. Identification of targets that block RAS signaling is pivotal to develop therapies for RAS-related cancer. As RAS translocation to the plasma membrane (PM) is essential for its effective signal transduction, we devised a high-content screening assay to search for genes regulating KRAS membrane association. We found that the tyrosine phosphatase PTPN2 regulates the plasma membrane localization of KRAS. Knockdown of PTPN2 reduced the proliferation and promoted apoptosis in KRAS-dependent cancer cells, but not in KRAS-independent cells. Mechanistically, PTPN2 negatively regulates tyrosine phosphorylation of KRAS, which, in turn, affects the activation KRAS and its downstream signaling. Consistently, analysis of the TCGA database demonstrates that high expression of PTPN2 is significantly associated with poor prognosis of patients with KRAS-mutant pancreatic adenocarcinoma. These results indicate that PTPN2 is a key regulator of KRAS and may serve as a new target for therapy of KRAS-driven cancer.     

Caveolin-1-mediated negative signaling plays a critical role in the induction of regulatory dendritic cells by DNA and protein coimmunization

Induction of Ag-specific regulatory T cells (iTregs) by vaccination is a promising strategy for treating autoimmune diseases. We previously demonstrated that DNA and protein covaccination converted naive T cells to Ag-specific iTregs by inducing CD11c(+)CD40(low)IL-10(+) regulatory dendritic cells (DCregs). However, it is unclear how coimmunization induces the DCregs. In this paper, we report that the event is initiated by coentry of sequence-matched DNA and protein immunogens into the same DC via caveolae-mediated endocytosis, which leads to inhibition of phosphorylation of caveolin-1 (Cav-1), the main component of caveolae, and upregulation of Tollip. This triggers downstream signaling that upregulates suppressor of cytokine signaling 1 and downregulates NF-κB and STAT-1α. Silencing either Cav-1 or Tollip blocks the negative signaling, leading to upregulated expression of CD40, downregulated production of IL-10, and loss of iTreg-inducing function. We further show that DCregs can be induced in culture from primary DCs and JAWS II DC lines by feeding them sequence-matched DNA and protein immunogens. The in vitro-generated DCregs are effective in ameliorating autoimmune and inflammatory diseases in several mouse models. Our study thus suggests that DNA and protein coimmunization induces DCregs through Cav-1- and Tollip-mediated negative signaling. It also describes a novel method for generating therapeutic DCregs in vitro.     

Emerging role of LRRK2 in human neural progenitor cell cycle progression, survival and differentiation

The γ-secretase complex is a major therapeutic target for the prevention and treatment of Alzheimer's disease. Previous studies have shown that treatment of young APP mice with specific inhibitors of γ-secretase prevented formation of new plaques. It has not yet been shown directly whether existing plaques would be affected by γ-secretase inhibitor treatment. Similarly, alterations in neuronal morphology in the immediate vicinity of plaques represent a plaque-specific neurotoxic effect. Reversal of these alterations is an important endpoint of successful therapy whether or not a treatment affects plaque size. In the present study we used longitudinal imaging in vivo with multiphoton microscopy to study the effects of the orally active γ-secretase inhibitor LY-411575 in 10–11 month old APP:PS1 mice with established amyloid pathology and neuritic abnormalities. Neurons expressed YFP allowing fluorescent detection of morphology whereas plaques were labelled with methoxy-XO4. The same identified neurites and plaques were followed in weekly imaging sessions in living mice treated daily (5 mg/kg) for 3 weeks with the compound. Although LY-411575 reduced Aβ levels in plasma and brain, it did not have an effect on the size of existing plaques. There was also no effect on the abnormal neuritic curvature near plaques, or the dystrophies in very close proximity to senile plaques. Our results suggest that therapeutics aimed at inhibition of Aβ generation are less effective for reversal of existing plaques than for prevention of new plaque formation and have no effect on the plaque-mediated neuritic abnormalities, at least under these conditions where Aβ production is suppressed but not completely blocked. Therefore, a combination therapy of Aβ suppression with agents that increase clearance of amyloid and/or prevent neurotoxicity might be needed for a more effective treatment in patients with pre-existing pathology.     

PI-3K/Akt signal pathway plays a crucial role in arsenite-induced cell proliferation of human keratinocytes through induction of cyclin D1

Exposure of arsenite can induce hyperproliferation of skin cells, which is believed to play important roles in arsenite-induced carcinogenesis by affecting both promotion and progression stages. However, the signal pathways and target genes activated by arsenite exposure responsible for the proliferation remain to be defined. In the present study, we found that: (1) exposure of human keratinocytic HaCat cells to arsenite caused an increase in cell proliferation, which was significantly inhibited by pretreatment of wortmannin, a specific chemical inhibitor of PI-3K/Akt signal pathway; (2) arsenite exposure was also able to activate PI-3K/Akt signal pathway, which thereby induced the elevation of cyclin D1 expression level in both HaCat cells and human primary keratinocytes based on that inhibition of PI-3K/Akt pathway by either pretreatment of wortmannin or the transfection of their dominant mutants, significantly inhibited cyclin D1 expression upon arsenite exposure; (3) PI-3K/Akt pathway is implicated in arsenite-induced proliferation of HaCat cells through the induction of cyclin D1 because either knockdown of cyclin D1 by its siRNA or inhibition of PI-3K/Akt signal pathway by their dominant mutants markedly impaired the proliferation of HaCat cells induced by arsenite exposure. Taken together, we provide the direct evidence that PI-3K/Akt pathway plays a role in the regulation of cell proliferation through the induction of cyclin D1 in human keratinocytes upon arsenite treatment. Given the importance of aberrant cell proliferation in cell transformation, we propose that the activation of PI-3K/Akt pathway and cyclin D1 induction may be the important mediators of human skin carcinogenic effect of arsenite.     

BMP7 plays a critical role in TMEM100-inhibited cell proliferation and apoptosis in mouse metanephric mesenchymal cells in vitro

Kidney mainly arises from the induction of metanephric mesenchymal cells (MM cells) and the ureteric bud (UB). Transmembrane protein-100 (Tmem100) consists of two transmembrane regions with strong temporal and spatial expression characteristics during renal development. However, the function of Tmem100 in mouse embryonic kidney-derived cells remained unclear. We provided qPCR to verify the relationship between Tmem100 and the BMP signal pathway. To clarify the role of Tmem100 in cell proliferation and apoptosis, we carry out EdU incorporation, annexin V- fluorescein isothiocyanate (FITC) apoptosis assay. Here, we find that the knockdown of Tmem100 increases the proliferation and apoptosis of mouse embryonic kidney-derived cells, and this promotion can be inhibited by knockdown of BMP7 at the same time; these results suggest that BMP7 plays a crucial role in Tmem100-regulated cell proliferation and apoptosis. qRT-PCR results further demonstrate that the deficiency of Tmem100 leads to BMP7 upregulation and overexpression could get opposite results. In BMP7-depleted MK3 cells, Tmem100 is highly upregulated and BMPR-II is downregulated. And in BMP7-overexpressed MK3 cells, the expression of Tmem100 is decreased. In BMPR-II-depleted MK3 cells, Tmem100 is downregulated and BMP7 expression remains still. These findings indicate that both BMP7 and BMPR-II can regulate Tmem100 and vice versa, and BMPR-II expression is regulated by BMP7. However, BMP7 has no association with BMPR-II in MK3 cells. Our data demonstrated the significant role of BMP7 in Tmem100-regulated cell proliferation and apoptosis and revealed the complicated regulation network among Tmem100, BMP7, and BMPR-II in mouse embryonic kidney-derived cells.     

Casein kinase II motif-dependent phosphorylation of human papillomavirus E7 protein promotes p130 degradation and S-phase induction in differentiated human keratinocytes

The E7 proteins of human papillomaviruses (HPVs) promote S-phase reentry in differentiated keratinocytes of the squamous epithelia to support viral DNA amplification. In this study, we showed that nuclear p130 was present in the differentiated strata of several native squamous epithelia susceptible to HPV infection. In contrast, p130 was below the level of detection in HPV-infected patient specimens. In submerged and organotypic cultures of primary human keratinocytes, the E7 proteins of the high-risk mucosotrophic HPV-18, the benign cutaneous HPV-1, and, to a lesser extent, the low-risk mucosotropic HPV-11 destabilized p130. This E7 activity depends on an intact pocket protein binding domain and a casein kinase II (CKII) phosphorylation motif. Coimmunoprecipitation experiments showed that both E7 domains were important for binding to p130 in extracts of organotypic cultures. Metabolic labeling in vivo demonstrated that E7 proteins were indeed phosphorylated in a CKII motif-dependent manner. Moreover, the efficiencies of the E7 proteins of various HPV types or mutations to induce S-phase reentry in spinous cells correlated with their relative abilities to bind and to destabilize p130. Collectively, these data support the notion that p130 controls the homeostasis of the differentiated keratinocytes and is therefore targeted by E7 for degradation to establish conditions permissive for viral DNA amplification.