Epigenetic and metabolic regulation of breast cancer stem cells.

Breast cancer has a relatively high mortality rate in women due to recurrence and metastasis. Increasing evidence has identified a rare population of cells with stem cell-like properties in breast cancer. These cells, termed cancer stem cells (CSCs), which have the capacity for self-renewal and differentiation, contribute significantly to tumor progression, recurrence, drug resistance and metastasis. Clarifying the mechanisms regulating breast CSCs has important implications for our understanding of breast cancer progression and therapeutics. A strong connection has been found between breast CSCs and epithelial mesenchymal transition (EMT). In addition, recent studies suggest that the maintenance of the breast CSC phenotype is associated with epigenetic and metabolic regulation. In this review, we focus on recent discoveries about the connection between EMT and CSC, and advances made in understanding the roles and mechanisms of epigenetic and metabolic reprogramming in controlling breast CSC properties.

Cytosolic PrP induces apoptosis of cell by disrupting microtubule assembly.

Prion protein (PrP) is able to bind with tubulin and to interfere with the formation of microtubule. To investigate the influence of accumulation of cytosolic PrP in cytoplasm on microtubule, plasmid pcDNA3.1-PrP23-230 expressing human PrP23-230 was introduced into HeLa cells. Immunoprecipitation assays identified the molecular interaction between cytosolic PrP and cellular tubulin. Confocal microscopy showed the co-localization of the expressed cytosolic PrP with tubulin in cytoplasm. Immunofluorescent assays of tubulin illustrated remarkable disruption of microtubular structures in the cells accumulated with cytosolic PrP. Meanwhile, the expressed cytosolic PrP significantly reduced cell viability and induced cell apoptosis. The amounts of microtubule protein in the cells expressing cytosolic PrP were decreased. Moreover, the levels of endogenous tubulin in the brain tissues of scrapie-infected hamsters were significantly lower than that of normal one. It highlights a close linkage between disruption of microtubule framework and cell death caused by abnormal presence of cellular PrP in cytoplasm. The association of apoptosis with microtubule-disrupting activity caused by cytosolic PrP may further provide insight into the unresolved biological function of PrP in the neurons.

Elevated levels of tau protein in cerebrospinal fluid of patients with probable Creutzfeldt-Jakob disease.

INTRODUCTION: A definitive diagnosis of Creutzfeldt-Jakob disease (CJD) can only be made by neuropathologic examination and demonstration of typical pathologic changes and the pathologic prion protein in central nervous tissues. This study investigated the diagnostic sensitivity and specificity of the microtubule-association protein tau in cerebrospinal fluid (CSF) from Chinese patients with sporadic CJD. METHODS: Two hundred two CSF samples from clinically suspected patients with sporadic CJD were analyzed for tau protein by enzyme-linked immunosorbent assay and for the signal transduction regulatory protein 14-3-3 protein by immunoblot. RESULTS: Remarkably increased levels of tau protein and increased incidence of 14-3-3 positivity were observed in probable CJD, when compared with possible CJD and others. With a threshold of 1400 pg/mL, tau determination showed a sensitivity of 90% and a specificity of 94% for the diagnosis of probable CJD. The combination of raised tau and positive 14-3-3 increased the specificity but slightly reduced the sensitivity. Statistical analysis indicated that the raised level of tau positively correlated with the presence of 14-3-3 in CSF but not with other main clinical features, eg, age, gender, clinical manifestations and sampling time. CONCLUSIONS: These data suggest that Chinese patients with probable CJD have similar increased levels of tau in the CSF as in Caucasian patients. Measurement of CSF tau will be another potential technique for antemortem CJD diagnosis.

Changes of tau profiles in brains of the hamsters infected with scrapie strains 263 K or 139 A possibly associated with the alteration of phosphate kinases.

BACKGROUND: Phospho-tau deposition has been described in a rare genetic human prion disease, Gerstmann-Sträussler-Scheinker syndrome, but is not common neuropathological picture for other human and animal transmissible spongiform encephalopathies (TSEs). This study investigated the possible changes of tau and phosphorylated tau (p-tau, at Ser396, Ser404, and Ser202/Thr205) in scrapie experimental animals. METHODS: The profiles of tau and p-tau (p-tau, at Ser396, Ser404, and Ser202/Thr205) in the brain tissues of agents 263K- or 139A-infected hamsters were evaluated by Western blots and real-time PCR. Meanwhile, the transcriptional and expressive levels of GSK3beta and CDK5 in the brains were tested. RESULTS: The contents of total tau and p-tau at Ser202/Thr205 increased, but p-tau at Ser396 and Ser404 decreased at the terminal stages, regardless of scrapie strains. Transcriptional levels of two tau isoforms were also increased. Additionally, it showed higher CDK5, but lower GSK3beta transcriptional and expressive levels in the brains of scrapie-infected animals. Analysis of brain samples collected from different times after inoculated with agent 263 K revealed that the changes of tau profiles and phosphate kinases were time-relative events. CONCLUSION: These data suggest that changes of profiles of p-tau at Ser396, Ser404 and Ser202/Thr205 are illness-correlative phenomena in TSEs, which may arise of the alteration of phosphate kinases. Alteration of tau, p-tau (Ser396, Ser404, and Ser202/Thr205), GSK3beta and CDK5 were either intermediate or consequent events in TSE pathogenesis and proposed the potential linkage of these bioactive proteins with the pathogenesis of prion diseases.

Cytosolic prion protein induces apoptosis in human neuronal cell SH-SY5Y via mitochondrial disruption pathway.

Different neurodegenerative disorders like prion disease, is caused by protein misfolding conformers. Reverse-transfected cytosolic prion protein (PrP) and PrP expressed in the cytosol have been shown to be neurotoxic. To investigate the possible mechanism of neurotoxicity due to accumulation of PrP in cytosol, a PrP mutant lacking the signal and GPI (CytoPrP) was introduced into the SH-SY5Y cell. MTT and trypan blue assays indicated that the viability of cells expressing CytoPrP was remarkably reduced after treatment of MG-132. Obvious apoptosis phenomena were detected in the cells accumulated with CytoPrP, including loss of mitochondrial transmembrane potential, increase of caspase-3 activity, more annexin V/PI-double positive-stained cells and reduced Bcl-2 level. Moreover, DNA fragmentation and TUNEL assays also revealed clear evidences of late apoptosis in the cells accumulated CytoPrP. These data suggest that the accumulation of CytoPrP in cytoplasm may trigger cell apoptosis, in which mitochondrial relative apoptosis pathway seems to play critical role.

Manganese-induced changes of the biochemical characteristics of the recombinant wild-type and mutant PrPs.

Manganese may play some roles in the pathogenesis of prion diseases. In this study, recombinant human wild-type (WT) PrP and PrP mutants with deleted or inserted octarepeats were exposed to manganese, and their biochemical and biophysical characteristics were evaluated by proteinase K (PK) digestion, sedimentation experiments, transmission electron microscopy and circular dichroism. It demonstrated that incubation of manganese remarkably increased PK-resistances, protein aggregations and beta-sheet contents of the PrPs. Moreover, the PrP mutants of inserted or deleted octarepeats were much vulnerable to the influence of manganese, which showed obviously more aggregation and higher beta-sheet content than that of WT-PrP. It highlights that the effect of manganese on the PrP seems to lie on the incorrectness of the octarepeats numbers. The association of the octarepeats number of PrP with manganese may further provide insight into the unresolved biological function of PrP in the neurons.

[Alternations of tau protein and its phosphorylated profiles in the experimental hamsters infected by scrapie agents 263K and 139A].

In human prion diseases, phosphorylated-tau deposition has been described in a rare genetic form, Gerstmann-Straussler-Scheinker disease, but is not considered part of the neuropathological picture of Creutzfeldt-Jakob disease. To investigate the possible changes of tau and phosphorylated tau (Ser396/Ser404) in transmissible spongiform encephalopathies (TSEs), the expressions and transcriptions of above biological factors in the brain tissues of 263K- and 139A-infected hamsters were evaluated by Western blots and Real Time PCR, respectively, followed by quantitative analyses of immunoblot images and relative transcriptional levels compared with normal animals. The contents of total tau increased, but phosphorylated tau at Ser396 and Ser404 decreased, regardless of the types of scrapie agents and clinical incubations. Transcriptions of two tau isoforms were also markedly increased. These findings suggested that dephosphorylation of tau at Ser396/Ser404 was a illness-correlative phenomenon in TSEs. Alterations of tau and phosphorylated tau (Ser396/Ser404) were either intermediate or consequent events in TSE pathogenesis and proposed the potential linkage of these bioactive proteins with the pathogenesis of prion diseases.

Molecular epidemiological study on prevalence of human papillomaviruses in patients with common warts in Beijing area.

OBJECTIVE: To study the circulation, distribution, and genomic diversity of HPVs in common warts in Beijing area of China. METHODS: Forty eight patients with pathologically diagnosed common warts were screened for the presence of HPV with HPV type-specific PCR and direct sequencing analysis. The genomic diversity of HPVs prevalent in Chinese patients was analyzed based on LCR. RESULTS: Forty one (85.5%) samples were positive for HPV DNA, 13 (31.7%)--HPV-57, 12 (29.3%)--HPV-1a, 7 (17%)--HPV-27 and 5(12.2%)--HPV-2a. Four cases were infected with two different HPV types, two (4.9%) with HPV-1a and HPV-27, one (2.4%) with HPV-1 and HPV-57 and one (2.4%) with HPV-27 and HPV-57. In contrast to the prevalence of single strain of novel HPV-57 variant and HPV-1 prototype, two HPV-2 and three HPV-27 novel variants were found to circulate in Beijing. CONCLUSION: HPV-1, -2, -27 and -57 are predominantly prevalent in patients with common warts in Beijing.

The propagation of hamster-adapted scrapie PrPSc can be enhanced by reduced pyridine nucleotide in vitro.

Transmissible spongiform encephalopathies (TSEs), or prion diseases, are fatal neurodegenerative disorders caused by an infectious agent termed a prion, which can convert normal cellular prion protein (PrP(C)) into a pathologically misfolded isoform (PrP(Sc)). Taking advantage of protein misfolding cyclic amplification (PMCA), a series of experiments was conducted to investigate the possible influences of pyridine nucleotides on the propagation activities of hamster-adapted scrapie agents 263K and 139A in vitro using normal hamster brain homogenates and recombinant hamster PrP as the substrates. The results showed that PrP(Sc) from both scrapie agent 263K- and 139A-infected brains propagated more efficiently in PMCA with the addition of reduced NADPH, showing an obvious dose-dependent enhancement. Reduced NADH also prompted PrP(Sc) propagation, whereas NADP, NAD and vitamin C failed. Moreover, following incubation with NADPH, recombinant hamster PrP could be efficiently converted into the proteinase K-resistant form when exposed to the trace of PrP(Sc) from infected hamsters. Our data provide evidence that the reduced pyridine nucleotide plays an important role in the propagation of prion and this process seems to target PrP(C) molecules.

PrP(Sc) of scrapie 263K propagates efficiently in spleen and muscle tissues with protein misfolding cyclic amplification.

Transmissible spongiform encephalopathies (TSEs), or prion diseases, are transmissible neurodegenerative disorders of protein conformation. This group of diseases is caused by infectious agents, termed prions, which can convert normal conformation (PrP(C)) into misfolded protein (PrP(Sc)). The infectivity of non-neuronal tissues has been wildly addressed, but the propagating features and the biochemical properties of prion generated from these tissues are only partially settled. In this study, utilizing protein misfolding cyclic amplification (PMCA), the in vitro conversion of PrP(C) into PrP(Sc) in spleen and muscle tissues can be induced by PrP(Sc) produced in vivo. The further propagation of newly formed PrP(Sc) in normal brain and some of the biochemical properties of new PrP(Sc) are similar as the brain-derived prions, implying the naturally infectious pathway of prion from peripheral generation to neuro-invasion. However, compared with the brain-derived PrP(Sc), the weaker resistance of new PrP(Sc) to some inactivated agents, i.e. sodium hydroxide and thermal inactivation, are observed. Our data provide the reliable evidence that the brain-derived PrP(Sc) can utilize the PrP(C) from non-neuronal tissues for its propagation. Similarity of the replicative ability in PMCA in vitro and the infectivity in vivo highlights the possibility to use PMCA instead of bioassay to investigate the propagation of prion.

[PrP protein can bind to protein kinase CK2 both in native and recombinant forms in vitro].

To explore the possible molecular interaction between CK2 and PrP, the full length sequences of human CK2alpha and CK2beta genes were amplified with RT-PCR using the mRNA from cell line SH-SY5Y as the template, and then the fusion proteins HIS-CK2alpha and GST-HIS-CK2beta were expressed in E. coli. The interaction between CK2 and PrP was evaluated with immunoprecipitation and pull-down assays. The results demonstrated that recombinant PrP bound specifically with CK2alpha, but not with CK2beta. The native CK2 and PrP in the hamster brains interacted each other, forming protein complexes. The domain responsible for interacting with CK2alpha was located at the C-terminal segment of PrP (residues 90-231). This study proposed reliable experimental data for the molecular interaction between PrP and CK2alpha, both in recombinant and native categories. These results supply scientific clues for further assessing the potential biological significance of the interaction of PrP with CK2 and possible role of CK2 in the pathogenesis of prion diseases.

[Expression of cytosolic PrP and analysis of its cytotoxic activities].

In order to study the physicochemical characteristics of cytosolic PrP (CytoPrP) and evaluate its possible influence on cell viability, a recombinant plasmid expressing human CytoPrP eukaryoticly was constructed and transfected into human neuroblastoma cell line SH-SY5Y transiently. Proteinase-resistant activities of CytoPrP were evaluated by a proteinase K (PK) digestion and cytotoxic effects of CytoPrP were tested by MTT assay and Trypan Blue cell-counting. The presence of CytoPrP in cytoplasm after transfection was controlled by the presence of protease inhibitor. Compared with wild-type PrP, CytoPrP possessed relatively stronger PK-resistant activities. Obvious cytotoxic effects were observed in the cells after inducement of CytoPrP in cytoplasm by protease inhibitor, showing a dose-dependent manner. The results provide useful scientific evidences for further studies of potential role of CytoPrP in pathological mechanism of prion disease.

[Establishment of PrP(Sc) conversion based on serial PMCA in vitro].

In order to establish an amplification system in vitro with which the PrP(Sc) is able to convert PrP(C) into proteinase K-resistant isoform infinitely and whether this system is more efficient than conventional protein misfolding cyclic amplification (PMCA), scrapie strain 263K-infected hamster's brain homogenate and homologous normal brain homogenate were prepared, respectively. A new methodology, namely serial PMCA, was utilized to reveal the continuous propagation ability of PrP(Sc). Totally 8 rounds of serial PMCA were proceeded and each round contained 48 cycles of alternative sonication and incubation. Simultaneously 144 cycles of conventional PMCA was used as a control. The results showed the PrP(Sc) from hamsters' brain tissues of scrapie agent 263K could replicate efficiently and infinitely with serial PMCA compared with finite propagation of PrP(Sc) with conventional PMCA system. The study of PrP(Sc) continuous propagation in brain homogenate with serial PMCA may further provide insight into the unsettled mechanism of prions misfolding and replication and apply to detect trace amount of PrP(Sc).

[Establishment of an assay for PrP(Sc) detection based on streptomycin precipitation].

To establish a new Western blotting assay for PrP(Sc) detection, we optimized the Western blotting assay with a precipitation procedure of streptomycin sulfate. After digestion with PK, 10% scrapie infected hamster brain homogenates were incubated with 60 mmol/L streptomycin and the precipitated PrP(Sc) was recovered by centrifugation. The enrichment of PrP(Sc) by streptomycin sulfate precipitation was evaluated using Western blotting assay. The results showed streptomycin could bind to PK-treated PrP(Sc), forming high molecular masses, but not influence the glycosylated patterns on SDS-PAGE. Western blot assay revealed that the detective sensitivity of the streptomycin-precipitation PrP(Sc) was remarkably improved. As a sensitive, specific, rapid and flexible protocol for PrP(Sc), the protocol in this study has the potential utility, alone or combined with other techniques, for the detection of low level PrP(Sc) in the specimens from central nerve system, or from peripheral organs or body fluids.

Establishment of a sandwich ELISA method for detection of vascular endothelial growth factor in serum samples of hepatocellular carcinoma patients.

OBJECTIVE: To establish a sandwich ELISA method for detecting vascular endothelial growth factor (VEGF) in sera of population and the patients with hepatocellular carcinoma (HCC). METHODS: Full length and two truncated human VEGF cDNA sequences were amplified from a commercial plasmid pBLAST49-hVEGF by PCR and inserted into the prokaryotic-expression plasmid pET-32a or pGEX-2T. Various VEGF proteins were expressed and purified from E. coli in His-Trx or GST fusion forms. The specific VEGF antibodies were elicited in experimental rabbits and mice by immunization of the full length VEGF fusion protein His-Trx-VEGF1-165. After purification of antibodies with chromatograph of Protein G, a sandwich ELISA technique was established. Serum VEGF levels were evaluated in 229 adults and 291 HCC patients. RESULTS: SDS-PAGE displayed that the molecular weights of the expressed full length (His-Trx-VEGF1-165), N-terminal (His-Trx-VEGF1-100) and C-terminal (GST-VEGF100-165) human VEGF fusion proteins were about 38KD, 31KD, and 33KD, respectively. Western blots confirmed that the prepared antisera were able to recognize both prokaryoticly and eukaryoticly expressed recombinant VEGF proteins. Assays of serially diluted His-Trx-VEGF1-100 by the established sandwich ELISA method showed that the linear range of the standard curve was 0.625-320 ng/mL, with the squared correlation coefficient R2 = 0.991. Screening of a serum panel containing 291 serum samples of HCC patients and 229 health adults revealed that the average VEGF level in HCC patients was higher than that in healthy controls, with a statically significant difference. CONCLUSION: The established sandwich ELISA reflects the level of serum VEGF and provide scientific basis for screening metastasis and recurrence of HCC using serum VEGF as an index.

Different expression patterns of CK2 subunits in the brains of experimental animals and patients with transmissible spongiform encephalopathies.

To address the possible alteration of casein kinase 2 (CK2) in transmissible spongiform encephalopathies (TSEs), the levels and patterns of CK2 in the brain tissues of hamsters or C57BL mice inoculated intracerebrally with scrapie agents 263K or 139A were evaluated by Western blots, followed by quantitative analysis. Specific semi-quantitative RT-PCR for evaluating the mRNA transcripts of CK2 subunits was performed in parallel. Compared with normal animals, the levels of CK2alpha and CK2beta in the brains of infected hamsters and mice were significantly decreased, regardless of which scrapie agent was. However, the expression of CK2alpha' or CK2alpha'/CK2alpha'' in the animals infected with agents 263K or 139A was considerably increased. Furthermore, decreases of CK2alpha and CK2beta and increases of CK2alpha'/CK2alpha'' were observed in cerebella homogenates from one familial Creutzfeldt-Jakob disease (fCJD) case and one fatal familial insomnia (FFI) case. These results suggest that alterations of CK2 subunits in brains are illness-correlative phenomena in TSEs and indicate a potential linkage of CK2 changes with the pathogenesis of prion diseases.

Molecular interaction between prion protein and GFAP both in native and recombinant forms in vitro.

Gliosis of glial fibrillary acidic protein (GFAP) associated astrocytes is considered to be one of the hallmarks of transmissible spongiform encephalopathies (TSEs). In the present study, remarkable GFAP-PrP(Sc) or GFAP-PrP(C) complexes were separately detected in the brain homogenates of 263 K (Scrapie)-infected or normal hamsters by co-immunoprecipitation assay. To get more exact molecular evidences for interaction between prion protein (PrP) and GFAP, various recombinant PrP or GFAP proteins were expressed using prokaryotic-expressing and in vitro translation system. Using pull down and co-immunoprecipitation assays, reliable molecular interaction between PrP and GFAP was observed, and proteinase K (PK)-digested PrP(Sc) molecules were confirmed to be able to bind the recombinant GFAP specifically as well. The region within PrP that was responsible for interaction with GFAP was narrowed to PK-resistant core of PrP (i.e. aa 91-230). The study of the association of PrP with GFAP supplies the molecular evidence for the observation of co-localization of PrP(Sc) and GFAP in the brains of TSEs and may further provide insight into a potential role of GFAP in the biological function of PrP and the pathogenesis of prion diseases.

The N-terminus of PrP is responsible for interacting with tubulin and fCJD related PrP mutants possess stronger inhibitive effect on microtubule assembly in vitro.

Microtubule dynamics is essential for many vital cellular processes such as in intracellular transport, metabolism, and cell division. Some evidences demonstrate that PrP may associate with microtubular cytoskeleton and its major component, tubulin. In the present study, the molecular interaction between PrP and tubulin was confirmed using pull-down assays, immunoprecipitation and ELISA. The interacting regions within PrP with tubulin were mapped in the N-terminus of PrP spanning residues 23-50 and 51-91. PrP octapeptide repeats are critical for the binding activity with tubulin, that the binding activity of PrP with tubulin became stronger along with the number of the octapeptide repeats increased. Microtubule assembly assays, sedimental tests and transmission electron microscopy demonstrated that the full-length PrP (aa 23-231) obviously inhibited the microtubule polymerization processes in vitro, whereas the N- (aa 23-91) and C- (aa 91-231) terminal peptides of PrP did not affect microtubule polymerization. Moreover, the familial Cruetzfeldt Jacob disease (fCJD) related PrP mutants with inserted or deleted octapeptide repeats showed much stronger inhibitive capacities on the microtubule dynamics in vitro than wild-type PrP. Our data highlight a potential role of PrP in regulating the microtubule dynamics in neurons.

Human tau protein forms complex with PrP and some GSS- and fCJD-related PrP mutants possess stronger binding activities with tau in vitro.

Microtubule associated protein tau is considered to play roles in some types of human transmissible spongiform encephalopathies (TSE). In this study, the full-length and several truncated human tau proteins were expressed from E. coli and purified. Using GST pull down, co-immunoprecipitation assay and tau-coated ELISA, the molecular interaction between tau protein and PrP was confirmed in the context of the full-length human tau. The N terminus (amino acids 1-91) and tandem repeats region (amino acids 186-283) of tau protein were responsible for the interaction with PrP. The octapeptide repeats within PrP directly affected the binding activity of PrP with tau. GSS-related mutant PrP102L and fCJD- related mutants with two and seven extra octarepeats showed more active binding capacity with tau than wild-type PrP. The molecular interactions between PrP and tau protein highlight a potential role of tau in the biological function of PrP and the pathogenesis of TSE.

[Preliminary analyses for influence of mutant PrPs with different number of octapeptide].

OBJECTIVE: The present study was conducted to understand the effects of PrP in different octapeptide repeats on proliferation of HeLa cells. METHODS AND RESULTS: Mutant PrPs with octapeptide repeat insertion were transiently expressed in HeLa cells and their results of MTT assay showed stronger cytotoxic effect on the proliferation of cells than wild-type PrP. Annexin V/PI assay also demonstrated that the expression of mutant PrPs was much easier to induce apoptosis than wild-type in HeLa cells. The percentage of both early and late stage apoptosis in mutant groups were significantly higher than that of wild type. CONCLUSION: These data suggest that the expression of mutant PrPs associated with familial CJD is much easier to induce apoptosis in cultured cells than expression of wild type PrP.