Direct confirmation of quiescence of CD34+CD38- leukemia stem cell populations using single cell culture, their molecular signature and clinicopathological implications.

BACKGROUND: The proliferating activity of a single leukemia stem cell and the molecular mechanisms for their quiescent property remain unknown, and also their prognostic value remains a matter of debate. Therefore, this study aimed to demonstrate the quiescence property and molecular signature of leukemia stem cell and their clinicopathological implications. METHODS: Single cell sorting and culture were performed in the various sets of hematopoietic stem cells including CD34+CD38- acute myeloid leukemia (AML) cell population (ASCs) from a total of 60 patients with AML, and 11 healthy controls. Their quiescence related-molecular signatures and clinicopathological parameters were evaluated in AML patients. RESULTS: Single cell plating efficiency of ASCs was significantly lower (8.6%) than those of normal hematopoietic stem cells i.e.: cord blood, 79.0%; peripheral blood, 45.3%; and bone marrow stem cell, 31.1%. Members of the TGFß super-family signaling pathway were most significantly decreased; as well as members of the Wnt, Notch, pluripotency maintenance and hedgehog pathways, compared with non ASC populations. mtDNA copy number of ASCs was significantly lower than that of corresponding other cell populations. However, our data couldn't support the prognostic value of the ASCs in AML. CONCLUSIONS: ASCs showed remarkable lower plating efficiency and slower dividing properties at the single cell level. This quiescence is represented as a marked decrease in the mtDNA copy number and also linked with down-regulation of genes in various molecular pathways.

Regulation of cellular RNA nano-particle assembly by splicing factor SRp20.

Cellular RNA nano-particles (RNA granules) such as stress granule (SG) and P-body (PB) are translationally silenced mRNA-protein complexes. Previously, a genome-wide loss-of-function screen using oligomeric siRNAs targeting potential drug target genes was performed to identify genes that are involved in SG and PB assembly. SRp20 (SRSF3), a splicing regulator, was identified as a potential regulator for the RNA granule assembly. Here, we show that SRp20 is a bona-fide RNA granule component using antibody against SRp20 as well as Flag-tagged SRp20 through immunofluorescence microscopy. More importantly, upon knockdown of SRp20 using siRNA, RNA granule formation was potently disrupted indicating that SRp20 is one of the major structural components of SGs and PBs. Interestingly, polysome profiling analyses displayed that SRp20 is distributed in all ribosomal fractions suggesting a potential role of SRp20 as a post-transcriptional mRNA regulator. These results broaden the functional role of SRp20 from the nuclear RNA processing events to the cytoplasmic post-transcriptional mRNA regulatory events through RNA granules that are critical for the regulation of gene expression.

Splicing factor SRSF3 represses translation of p21cip1/waf1 mRNA.

Serine/arginine-rich splicing factor 3 (SRSF3) is an RNA binding protein that most often regulates gene expression at the splicing level. Although the role of SRSF3 in mRNA splicing in the nucleus is well known, its splicing-independent role outside of the nucleus is poorly understood. Here, we found that SRSF3 exerts a translational control of p21 mRNA. Depletion of SRSF3 induces cellular senescence and increases the expression of p21 independent of p53. Consistent with the expression patterns of SRSF3 and p21 mRNA in the TCGA database, SRSF3 knockdown increases the p21 mRNA level and its translation efficiency as well. SRSF3 physically associates with the 3'UTR region of p21 mRNA and the translational initiation factor, eIF4A1. Our study proposes a model in which SRSF3 regulates translation by interacting with eIF4A1 at the 3'UTR region of p21 mRNA. We also found that SRSF3 localizes to the cytoplasmic RNA granule along with eIF4A1, which may assist in translational repression therein. Thus, our results provide a new mode of regulation for p21 expression, a crucial regulator of the cell cycle and senescence, which occurs at the translational level and involves SRSF3.

HOXB13 promotes androgen independent growth of LNCaP prostate cancer cells by the activation of E2F signaling.

BACKGROUND: Androgen signaling plays a critical role in the development of prostate cancer and its progression. However, androgen-independent prostate cancer cells emerge after hormone ablation therapy, resulting in significant clinical problems. We have previously demonstrated that the HOXB13 homeodomain protein functions as a prostate cancer cell growth suppressor by inhibiting androgen-mediated signals. However, the role of the HOXB13 in androgen-independent growth of prostate cancer cells remains unexplained. RESULTS: In this report, we first demonstrated that HOXB13 was highly overexpressed in hormone-refractory tumors compared to tumors without prostate-specific antigen after initial treatment. Functionally, in an androgen-free environment minimal induction of HOXB13 in LNCaP prostate cancer cells, to the level of the normal prostate, markedly promoted cell proliferation while suppression inhibited cell proliferation. The HOXB13-mediated cell growth promotion in the absence of androgen, appears to be mainly accomplished through the activation of RB-E2F signaling by inhibiting the expression of the p21waf tumor suppressor. Indeed, forced expression of HOXB13 dramatically decreased expression of p21waf; this inhibition largely affected HOXB13-mediated promotion of E2F signaling. CONCLUSIONS: Taken together, the results of this study demonstrated the presence of a novel pathway that helps understand androgen-independent survival of prostate cancer cells. These findings suggest that upregulation of HOXB13 is associated with an additive growth advantage of prostate cancer cells in the absence of or low androgen concentrations, by the regulation of p21-mediated E2F signaling.

Differential CARM1 expression in prostate and colorectal cancers.

BACKGROUND: Coactivator-associated arginine methyltransferase 1 (CARM1) functions as a transcriptional coactivator of androgen receptor (AR)-mediated signaling. Correspondingly, overexpression of CARM1 has been associated with the development of prostate cancer (PCa) and its progression to androgen-independent PCa. In our preliminary study, however, the promoting effects of CARM1, with regard to androgen-stimulated AR target gene expression were minimal. These results suggested that the AR target gene expression associated with CARM1 may result primarily from non-hormone dependent activity. The goal of this study was to confirm the pattern of expression of CARM1 in human tumors and determine the mechanism of action in CARM1 overexpressed tumors. METHODS: Tissue microarray was used to determine the pattern of expression of CARM1 in human cancers by immunohistochemistry. CARM1 expression was also evaluated in prostate and colorectal surgical specimens and the clinical records of all cases were reviewed. In addition, a reporter transcription assay using the prostate-specific antigen (PSA) promoter was used to identify the signaling pathways involved in non-hormone-mediated signal activation associated with CARM1. RESULTS: The tissue microarray showed that CARM1 was particularly overexpressed in the colorectal cancers while CARM1 expression was not prevalent in the prostate and breast cancers. Further studies using surgical specimens demonstrated that CARM1 was highly overexpressed in 75% of colorectal cancers (49 out of 65) but not in the androgen-independent PCa. In addition, CARM1's coactivating effect on the entire PSA promoter was very limited in both androgen-dependent and androgen-independent PCa cells. These results suggest that there are other factors associated with CARM1 expression in PSA regulation. Indeed, CARM1 significantly regulated both p53 and NF-kappaB target gene transcription. CONCLUSIONS: The results of this study suggest that, in addition to its role in activation of steroid receptors, CARM1 functions as a transcriptional modulator by altering the activity of many transcriptional factors, especially with regard to androgen independent PCa and colorectal cancers.

Avenanthramide-C Restores Impaired Plasticity and Cognition in Alzheimer's Disease Model Mice.

Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by cognitive decline and dementia with no effective treatment. Here, we investigated a novel compound from oats named avenanthramide-C (Avn-C), on AD-related memory impairment and behavioral deficits in transgenic mouse models. Acute hippocampal slices of wild-type or AD transgenic mice were treated with Avn-C in the presence or absence of oligomeric Aß42. LTP analyses and immunoblotting were performed to assess the effect of Avn-C on Aß-induced memory impairment. To further investigate the effect of Avn-C on impaired memory and Aß pathology, two different AD transgenic mice (Tg2576 and 5XFAD) models were orally treated with either Avn-C or vehicle for 2 weeks. They were then assessed for the effect of the treatment on neuropathologies and behavioral impairments. Avn-C reversed impaired LTP in both ex vivo- and in vivo-treated AD mice hippocampus. Oral administration (6 mg/kg per day) for 2 weeks in AD mice leads to improved recognition and spatial memory, reduced caspase-3 cleavage, reversed neuroinflammation, and to accelerated glycogen synthase kinase-3ß (pS9GSK-3ß) and interleukin (IL-10) levels. Avn-C exerts its beneficial effects by binding to α1A adrenergic receptors to stimulate adenosine monophosphate-activated kinase (AMPK). All of the beneficial effects of Avn-C on LTP retrieval could be blocked by prazosin hydrochloride, a specific inhibitor of α1A adrenergic receptors. Our findings provide evidence, for the first time, that oats' Avn-C reverses the AD-related memory and behavioral impairments, and establish it as a potential candidate for Alzheimer's disease drug development.

Differential expression of osteocalcin during the metastatic progression of prostate cancer.

Osteocalcin expression is restricted to osteoblasts and serum osteocalcin level is elevated in metastatic bone tumors including prostate tumors, which predominantly metastasizes to the bone and causes typical osteoblastic lesions. Previously, we have reported that osteocalcin RNA is widely expressed but incompletely spliced in the prostate including prostate tumors. Considering that many studies using osteocalcin-driven gene therapy have been conducted to treat hormone refractory metastatic tumors, detailed mechanisms controlling osteocalcin expression needs to be clarified. We aim to learn how osteocalcin expression is regulated during the metastatic process of prostate cancer. We applied assays of immunohistochemistry and RNA in situ hybridization in prostate tumors acquired from prostate (15) and metastatic sites, 13 from lymph node and 14 from bone. RT-PCR analysis in various cultured prostate cells was also performed. As predicted, osteocalcin RNA was highly expressed in most prostate epithelial cells of tumors, regardless of metastatic status of the tumor. However, osteocalcin protein was undetectable in tumors acquired from the primary site or lymph nodes whereas protein was highly expressed in the majority of bone-metastasized prostate tumors. RT-PCR analysis demonstrated that there was more completely spliced form of osteocalcin RNA present in bone-derived prostate cancer cells. Our data suggest that osteocalcin RNA was expressed but not completely spliced in non-bone environment, ultimately resulting in improper production of osteocalcin protein. This study explains why serum osteocalcin level is increased in patients with bone-metastasized prostate cancers. Yet, it remains to be clarified what regulates bone-specific osteocalcin RNA splicing in prostate tumors.

Vibrio vulnificus metalloprotease VvpE is essentially required for swarming.

Bacterial swarming constitutes a good in vitro model for surface adherence and colonization, and is accompanied by expressions of virulence factors related to invasiveness. In this study, it was determined that Vibrio vulnificus swarming was abolished by mutation of the vvpE gene encoding a metalloprotease VvpE and this swarming defect was recovered by complementation of the vvpE gene. Expression of the vvpE gene began simultaneously with the beginning of swarming and increased along with expression of the luxS gene encoding the synthase of the precursor of quorum-sensing signal molecule autoinducer 2, and this increased vvpE expression was decreased by mutation of the luxS gene. Moreover, VvpE destroyed IgA and lactoferrins, which are responsible for mucosal immunity. These results suggest that VvpE may play important roles in the surface adherence and colonization of V. vulnificus by facilitating swarming and in the mucosal invasion of V. vulnificus by destroying IgA and lactoferrin.

Effect of the crp mutation on the utilization of transferrin-bound iron by Vibrio vulnificus.

Cyclic AMP-cAMP receptor protein (CRP) complex plays an essential role in the global regulation of Vibrio vulnificus virulence. We found that growth retardation of V. vulnificus caused by mutation of the crp gene encoding CRP was exacerbated under iron-limited conditions. Accordingly, we investigated the effect of crp mutation on the expression of the vulnibactin-mediated iron-uptake system and the ability of V. vulnificus to utilize transferrin-bound iron, and thus to grow in cirrhotic ascites, a human ex vivo system. The production of vulnibactin was suppressed, and the transcription of the vis and vuuA genes, which encode an enzyme required for vulnibactin synthesis and vulnibactin receptor protein, was also suppressed in the crp mutant. Moreover, the crp mutant could not utilize transferrin-bound iron, and its growth was severely suppressed both on transferrin-bound iron and in cirrhotic ascites. All the defects in the crp mutant were recovered by the in trans complementation of the wild-type crp gene. Putative CRP-binding sequences were found in the regulatory regions of the fur, vis and vuuA genes. These results indicate that crp mutation attenuates the ability to grow on transferrin-bound iron and in a human body fluid by down-regulating the vulnibactin-mediated iron-uptake system.

Suppression and inactivation of Vibrio vulnificus hemolysin in cirrhotic ascites, a human ex vivo experimental system.

To elucidate the mechanisms underlying the in vivo suppression and inactivation of Vibrio vulnificus hemolysin (VvhA), we used cirrhotic ascites fluid as a human ex vivo experimental system. VvhA expression was suppressed in proportion to the amount of cirrhotic ascites. The expression of vvhA in undiluted cirrhotic ascites could be suppressed further by the addition of glucose, a constituent of cirrhotic ascites. VvhA was readily inactivated in the presence of cirrhotic ascites by a cholesterol-mediated oligomerization and interaction with an undefined constituent(s) of cirrhotic ascites. These results indicate that the expression of vvhA can be suppressed and that any VvhA produced is inactivated by the constituents of cirrhotic ascites. Our results suggest that only a very small portion of the VvhA that is produced in human body fluids may actually contribute to the pathogenesis of V. vulnificus septicemia. It is suggested that cirrhotic ascites could be used as a human ex vivo experimental system for the studies on the in vivo expression and the significance of V. vulnificus virulence factors.