Low-noise and low-power cryogenic readout electronics are developed for a focal plane instrument of the IR Imaging Surveyor. We measured the static characteristics and the noise spectra of several types of silicon MOSFETs at the cryogenic temperature where silicon JFETs do not work well due to the carrier freeze-out. The 'kink' behavior of n- channel MOSFETs was observed below the carrier freeze-out temperature, but it was not obvious for the p-channel MOSFET. It was demonstrated the p-channel MOSFETs can be used for the cryogenic readout electronics of the IRIS's far-IR array with an acceptable performance. The amplifier integrated with these MOSFETs showed low-noise at 2K under a low power consumption of 1 (mu) W per MOSFET. We now design and evaluate several circuits that are fabricated by the CMOS process for cryogenic readout.
Runt-related transcription factor 2 (RUNX2) has been considered to be one of master regulators for osteoblast differentiation and bone formation. Recently, we have described that RUNX2 attenuates p53/TAp73-dependent cell death of human osteosarcoma U2OS cells bearing wild-type p53 in response to adriamycin. In this study, we have asked whether RUNX2 silencing could enhance gemcitabine (GEM) sensitivity of p53-deficient human pancreatic cancer AsPC-1 cells. Under our experimental conditions, GEM treatment increased the expression level of p53 family TAp63, whereas RUNX2 was reduced following GEM exposure, indicating that there exists an inverse relationship between the expression level of TAp63 and RUNX2 following GEM exposure. To assess whether TAp63 could be involved in the regulation of GEM sensitivity of AsPC-1 cells, small interfering RNA-mediated knockdown of TAp63 was performed. As expected, silencing of TAp63 significantly prohibited GEM-dependent cell death as compared with GEM-treated non-silencing cells. As TAp63 was negatively regulated by RUNX2, we sought to examine whether RUNX2 knockdown could enhance the sensitivity to GEM. Expression analysis demonstrated that depletion of RUNX2 apparently stimulates the expression of TAp63, as well as proteolytic cleavage of poly ADP ribose polymerase (PARP) after GEM exposure, and further augmented GEM-mediated induction of p53/TAp63-target genes, such as p21 (WAF1) , PUMA and NOXA, relative to GEM-treated control-transfected cells, implying that RUNX2 has a critical role in the regulation of GEM resistance through the downregulation of TAp63. Notably, ablation of TAp63 gave a decrease in number of γH2AX-positive cells in response to GEM relative to control-transfected cells following GEM exposure. Consistently, GEM-dependent phosphorylation of ataxia telangiectasia-mutated protein was remarkably impaired in TAp63 knockdown cells. Collectively, our present findings strongly suggest that RUNX2-mediated repression of TAp63 contributes at least in part to GEM resistance of AsPC-1 cells, and thus silencing of RUNX2 may be a novel strategy to enhance the efficacy of GEM in p53-deficient pancreatic cancer cells.
It has been well-known that human pancreatic cancer represents the fourth and fifth leading causes of cancer-related deaths in the United States and Japan, respectively.1, 2 Notably, pancreatic cancer is characterized by high metastatic potential, resistance to chemotherapy and thus its prognosis is extremely poor with 5-year survival <5%. At diagnosis, more than 80% cases are already advanced and non-resectable.3 Therefore, chemotherapy and/or radiotherapy is the only option. Despite improvements in the treatments, the survival rate has not been significantly ameliorated over the last few decades. For chemotherapy, a deoxycytidine analog termed gemcitabine (GEM) is the first line of standard treatment given to most of the patients bearing advanced pancreatic cancer.4 Unfortunately, GEM treatment provides limited clinical benefits, especially in advanced and metastatic disease.5 Hence, the extensive efforts to clarify the precise molecular mechanisms behind GEM-resistant phenotype of malignant pancreatic cancer and also to develop the promising strategies to enhance the efficacy of GEM should be required.
RUNX2 (Runt-related transcription factor 2) is one of the RUNX family members implicated in the induction of osteoblast differentiation and bone formation.6 Recently, we have found for the first time that RUNX2 attenuates p53/TAp73-dependent proper DNA damage response in p53-proficient human osteosarcoma-derived U2OS cells.7, 8 On the basis of our results, RUNX2 prohibited the transcriptional as well as pro-apoptotic activity of p53 through the complex formation with p53 in response to adriamycin (ADR). In addition, RUNX2 trans-repressed the transcription of TAp73 following ADR exposure. Thus, our recent studies strongly suggest that RUNX2 has an oncogenic potential through the inhibition of DNA damage-dependent cell death pathway mediated by pro-apoptotic p53/TAp73. Consistent with the above-mentioned our notion, it has been described that RUNX2 has an ability to transactivate a subset of its target genes involved in cancer cell migration and invasion.9
In the current study, we have focused on human pancreatic cancer cells. According to our present observations, p53-deficient pancreatic cancer AsPC-1 cells exhibited a much more higher resistance to GEM as compared with p53-proficient pancreatic cancer SW1990 cells. Intriguingly, GEM treatment in AsPC-1 cells resulted in an induction and a reduction of pro-apoptotic TAp63 and pro-oncogenic RUNX2, respectively, indicating that there exists an inverse relationship between the expression levels of TAp63 and RUNX2 in response to GEM. Thus, it is likely that RUNX2 is capable to trans-repress TAp63 transcription. Indeed, forced expression of RUNX2 in AsPC-1 cells markedly suppressed the transcription of TAp63. Of note, close inspection of 5′-upstream region of TAp63 gene revealed that there exists a putative RUNX2-binding site (−553 to −548).10 In addition, forced expression of TAp63α in AsPC-1 cells significantly reduced the number of G418-resistant colonies as compared with control cells transfected with the empty plasmid. These observations raised a possibility that RUNX2-mediated repression of TAp63 transcription might contribute to the acquisition and/or maintenance of GEM-resistant phenotype of ASPC-1 cells. To address this issue, siRNA-mediated knockdown of TAp63 in AsPC-1 cells was performed. Our siRNA against TAp63 efficiently reduced the expression of TAp63 but not of transactivation-deficient ΔNp63. As expected, silencing of TAp63 remarkably reduced the sensitivity of AsPC-1 cells to GEM relative to GEM-exposed non-silencing control cells. In support of these observations, depletion of TAp63 attenuated GEM-dependent transactivation of a subset of p53/TAp63-target genes.
Considering that knockdown of RUNX2 significantly enhances ADR sensitivity of U2OS cells,7 we have examined whether silencing of RUNX2 could affect GEM sensitivity of AsPC-1 cells. On the basis of our present results, RUNX2 knockdown enhanced GEM sensitivity of AsPC-1 cells accompanied by further accumulation of TAp63 as well as a subset of its target genes in response to GEM, implying that RUNX2-mediated trans-repression of TAp63 has a pivotal role in the regulation of GEM-resistant phenotype of p53-deficient pancreatic cancer cells (Figure 1). It is worth noting that depletion of TAp63 reduced GEM-mediated accumulation of DNA damage marker γH2AX, whereas the amounts of γH2AX was elevated in GEM-exposed RUNX2 knockdown cells. As DNA damage-mediated phosphorylation of H2AX is mediated by phosphorylated ataxia telangiectasia mutated (ATM),11 we have checked the phosphorylation status of ATM in the presence or absence of GEM. Our immunoprecipitation/immunoblotting experiments clearly demonstrated that GEM-mediated phosphorylation of ATM is abrogated in TAp63-silencing cells, suggesting that TAp63 participates in the regulation of ATM-dependent phosphorylation of H2AX following GEM exposure. However, the precise molecular mechanisms how TAp63 contributes to ATM-dependent phosphorylation of H2AX in response to GEM remain elusive. Further studies should be required to adequately address this issue.
Figure 1
Depletion of RUNX2 enhances gemcitabine sensitivity of p53-deficient human pancreatic cancer AsPC-1 cells through the stimulation of TAp63-mediated cell death pathway
Taken together, our present findings strongly suggest that RUNX2 attenuates TAp63-dependent cell death pathway in p53-deficient pancreatic cancer cells following GEM exposure, and thus the depletion of RUNX2 might be an attractive strategy to enhance the efficacy of the clinically approved GEM, which contributes to save cost to treat patients with advanced pancreatic cancer when compared with the development of novel anticancer drug(s) targeting pancreatic cancer.12
Recently, we have described that siRNA-mediated silencing of runt-related transcription factor 2 (RUNX2) improves anti-cancer drug gemcitabine (GEM) sensitivity of p53-deficient human pancreatic cancer AsPC-1 cells through the augmentation of p53 family TAp63-dependent cell death pathway. In this manuscript, we have extended our study to p53-mutated human pancreatic cancer Panc-1 cells. According to our present results, knockdown of mutant p53 alone had a marginal effect on GEM-mediated cell death of Panc-1 cells. We then sought to deplete RUNX2 using siRNA in Panc-1 cells and examined its effect on GEM sensitivity. Under our experimental conditions, RUNX2 knockdown caused a significant enhancement of GEM sensitivity of Panc-1 cells. Notably, GEM-mediated induction of TAp63 but not of TAp73 was further stimulated in RUNX2-depleted Panc-1 cells, indicating that, like AsPC-1 cells, TAp63 might play a pivotal role in the regulation of GEM sensitivity of Panc-1 cells. Consistent with this notion, forced expression of TAp63α in Panc-1 cells promoted cell cycle arrest and/or cell death, and massively increased luciferase activities driven by TAp63-target gene promoters such as p21WAF1 and NOXA. In addition, immunoprecipitation experiments indicated that RUNX2 forms a complex with TAp63 in Panc-1 cells. Taken together, our current observations strongly suggest that depletion of RUNX2 enhances the cytotoxic effect of GEM on p53-mutated Panc-1 cells through the stimulation of TAp63-dependent cell death pathway even in the presence of a large amount of pro-oncogenic mutant p53, and might provide an attractive strategy to treat pancreatic cancer patients with p53 mutations.
Propolis is a nature resinous mixture produced by honeybees that exhibit a wide range of biological functions.We previously reported that the propolis derived from Philippine endemic stingless bees (Tetragonula biroi, Friese) had anti-gastric cancer and hair growth activities in the JALAS annual meetings.However, the hair growth stimulating activity of the propolis remains poorly understood.In this study, we investigate the effects and the underlying mechanism of Philippine stingless bees propolis on hair growth using mice.Treatment of 99.5% ethanol-crude extract of Philippine stingless bees propolis (EEPP) significantly stimulated hair growth by inducing "Anagen phase" of hair follicle.Additionally, EEPP upregulated expression of Wnt3a, Dvl2, β-catenin, Lef1 and Bmp2.These results indicate that EEPP has the activity of hair growth through the Wnt /β-catenin signaling pathway and may be a potential treatment for hair loss in humans.
Abstract PIK3CA is the most frequently mutated oncogene in cervical cancer, and somatic mutations in the PIK3CA gene result in increased activity of PI3K. In cervical cancer, the E545K mutation in PIK3CA leads to elevated cell proliferation and reduced apoptosis. In the present study, we designed and synthesized a novel pyrrole‐imidazole polyamide‐ seco ‐CBI conjugate, P3AE5K, to target the PIK3CA gene bearing the E545K mutation, rendered possible by nuclear access and the unique sequence specificity of pyrrole‐imidazole polyamides. P3AE5K interacted with double‐stranded DNA of the coding region containing the E545K mutation. When compared with conventional PI3K inhibitors, P3AE5K demonstrated strong cytotoxicity in E545K‐positive cervical cancer cells at lower concentrations. PIK3CA mutant cells exposed to P3AE5K exhibited reduced expression levels of PIK3CA mRNA and protein, and subsequent apoptotic cell death. Moreover, P3AE5K significantly decreased the tumor growth in mouse xenograft models derived from PIK3CA mutant cells. Overall, the present data strongly suggest that the alkylating pyrrole‐imidazole polyamide P3AE5K should be a promising new drug candidate targeting a constitutively activating mutation of PIK3CA in cervical cancer.
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