RESUMO
There have been tens of thousands of RNAs deposited in different databases that contain sequences of two genes and are coined chimeric RNAs, or chimeras. However, "chimeric RNA" has never been lucidly defined, partly because "gene" itself is still ill-defined and because the means of production for many RNAs is unclear. Since the number of putative chimeras is soaring, it is imperative to establish a pellucid definition for it, in order to differentiate chimeras from regular RNAs. Otherwise, not only will chimeric RNA studies be misled but also characterization of fusion genes and unannotated genes will be hindered. We propose that only those RNAs that are formed by joining two RNA transcripts together without a fusion gene as a genomic basis should be regarded as authentic chimeras, whereas those RNAs transcribed as, and cis-spliced from, single transcripts should not be deemed as chimeras. Many RNAs containing sequences of two neighboring genes may be transcribed via a readthrough mechanism, and thus are actually RNAs of unannotated genes or RNA variants of known genes, but not chimeras. In today's chimeric RNA research, there are still several key flaws, technical constraints and understudied tasks, which are also described in this perspective essay.
Assuntos
Bases de Dados de Ácidos Nucleicos , RNA/genética , Terminologia como Assunto , Animais , Sequência de Bases , Humanos , PesquisaRESUMO
Spinal muscular atrophy (SMA) is an autosomal recessive genetic disease and a leading cause of infant mortality. Deletions or mutations of SMN1 cause SMA, a gene that encodes a SMN protein. SMN is important for the assembly of Sm proteins onto UsnRNA to UsnRNP. SMN has also been suggested to direct axonal transport of ß-actin mRNA in neurons. Humans contain a second SMN gene called SMN2 thus SMA patients produce some SMN but not with sufficient levels. The majority of SMN2 mRNA does not include exon 7. Here we show that increased expression of PSF promotes inclusion of exon 7 in the SMN2 whereas reduced expression of PSF promotes exon 7 skipping. In addition, we present evidence showing that PSF interacts with the GAAGGA enhancer in exon 7. We also demonstrate that a mutation in this enhancer abolishes the effects of PSF on exon 7 splicing. Furthermore we show that the RNA target sequences of PSF and tra2ß in exon 7 are partially overlapped. These results lead us to conclude that PSF interacts with an enhancer in exon 7 to promote exon 7 splicing of SMN2 pre-mRNA.
Assuntos
Éxons/genética , Regulação Neoplásica da Expressão Gênica , Precursores de RNA/genética , Proteínas de Ligação a RNA/metabolismo , Western Blotting , Primers do DNA/química , Primers do DNA/genética , Humanos , Luciferases/metabolismo , Neuroblastoma/genética , Neuroblastoma/metabolismo , Fator de Processamento Associado a PTB , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína 2 de Sobrevivência do Neurônio Motor/genética , Células Tumorais CultivadasRESUMO
The product of proto-oncogene Ron is a human receptor for the macrophage-stimulating protein (MSP). Upon activation, Ron is able to induce cell dissociation, migration and matrix invasion. Exon 11 skipping of Ron pre-mRNA produces Ronâ³165 protein that is constitutively active even in the absence of its ligand. Here we show that knockdown of SRSF2 promotes the decrease of exon 11 inclusion, whereas overexpression of SRSF2 promotes exon 11 inclusion. We demonstrate that SRSF2 promotes exon 11 inclusion through splicing and transcription procedure. We also present evidence that reduced expression of SRSF2 induces a decrease in the splicing of both introns 10 and 11; by contrast, overexpression of SRSF2 induces an increase in the splicing of introns 10 and 11. Through mutation analysis, we show that SRSF2 functionally targets and physically interacts with CGAG sequence on exon 11. In addition, we reveal that the weak strength of splice sites of exon 11 is not required for the function of SRSF2 on the splicing of Ron exon 11. Our results indicate that SRSF2 promotes exon 11 inclusion of Ron proto-oncogene through targeting exon 11. Our study provides a novel mechanism by which Ron is expressed.
Assuntos
Proteínas Nucleares/fisiologia , Splicing de RNA , Receptores Proteína Tirosina Quinases/genética , Ribonucleoproteínas/fisiologia , Transcrição Gênica , Células Cultivadas , Éxons/genética , Células HeLa , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Proto-Oncogene Mas , Proto-Oncogenes/genética , Receptores Proteína Tirosina Quinases/metabolismo , Fatores de Processamento de Serina-ArgininaRESUMO
Spinal muscular atrophy (SMA) is a human genetic disease which occurs because of the deletion or mutation of SMN1 gene. SMN1 gene encodes the SMN protein which plays a key role in spliceosome assembly. Although human patients contain SMN2, a duplicate of SMN1, splicing of SMN2 produces predominantly exon 7 skipped isoform. In order to understand the functions of splice site sequences on exon 7 and 8, we analyzed the effects of conserved splice site sequences on exon 7 skipping of SMN2 and SMN1 pre-mRNA. We show here that conserved 5' splice site sequence of exon 7 promoted splicing of nearby exons and subsequently reduced splicing of distant exons. However, to our surprise, conserved 3' splice site sequence of exon 7 and 8 did not promote splicing of nearby exons. By contrast, the mutation inhibited splicing of nearby exons and subsequently promoted splicing of distant exons. Our study shows that 3' splice sites of exon 7 and 8 contain enhancer for their splice site selection, in addition to providing cleavage sites.
Assuntos
Elementos Facilitadores Genéticos , Éxons , Atrofia Muscular Espinal/genética , Precursores de RNA , Sítios de Splice de RNA , Proteína 2 de Sobrevivência do Neurônio Motor/genética , Sequência de Bases , Linhagem Celular , Sequência Conservada , Humanos , Proteína 1 de Sobrevivência do Neurônio Motor/genéticaRESUMO
We established new methods for cloning cDNA ends that start with reverse transcription (RT) and soon proceed with the synthesis of the second cDNA strand, avoiding manipulations of fragile RNA. Our 3'-end cloning method does not involve poly-dT primers and polymerase chain reactions (PCR), is low in efficiency but high in fidelity and can clone those RNAs without a poly-A tail. We also established a cDNA protection assay to supersede RNA protection assay. The protected cDNA can be amplified, cloned and sequenced, enhancing sensitivity and fidelity. We report that RT product using gene-specific primer (GSP) cannot be gene- or strand-specific because RNA sample contains endogenous random primers (ERP). The gene-specificity may be improved by adding a linker sequence at the 5'-end of the GSP to prime RT and using the linker as a primer in the ensuing PCR. The strand-specificity may be improved by using strand-specific DNA oligos in our protection assay. The CDK4 mRNA and TSPAN31 mRNA are transcribed from the opposite DNA strands and overlap at their 3' ends. Using this relationship as a model, we found that the overlapped sequence might serve as a primer with its antisense as the template to create a wrong-template extension in RT or PCR. We infer that two unrelated RNAs or cDNAs overlapping at the 5'- or 3'-end might create a spurious chimera in this way, and many chimeras with a homologous sequence may be such artifacts. The ERP and overlapping antisense together set complex pitfalls, which one should be aware of.
Assuntos
Clonagem Molecular/métodos , RNA Antissenso/análise , RNA Antissenso/genética , RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Sequência de Bases , Células Cultivadas , Primers do DNA , DNA Complementar , Células HeLa , Humanos , Células MCF-7 , Dados de Sequência MolecularRESUMO
Cannabis sativa has long been used as a traditional medicine in China. Among its effective compounds are cannabinoids. This study determined the effect of WIN55,212-2 (WIN), a synthetic cannabinoid, on the BEL-7402 human hepatocellular carcinoma (HCC) cell line. The results showed that WIN could decrease the proliferation of BEL-7402 cells. Moreover, WIN could cause apoptosis of the cells via up-regulation of Bax expression, down-regulation of Bcl-2 expression, induction of the mitochondrial membrane potential, increase of caspase-3, -8 and -9 activities, and induction of the cleavage of caspase-3 and poly-ADP-ribose polymerase (PARP). The WIN-induced apoptosis was accompanied by the up-regulation of PPARγ expression, the activation of PPARγ DNA binding activity, and a down-regulation of PPARγ target oncogene c-myc. Conversely, the effects of WIN could be attenuated by PPARγ antagonist GW9662, and the WIN induced PPARγ expression was partially attenuated by AM630, a cannabinoid receptor-2 antagonist, whereas the WIN-induced reduction of c-myc expression was partially restored by GW9662. Collectively, our results suggest that WIN can decrease the proliferation and cause apoptosis of the BEL-7402 cells via a mitochondrial-caspase pathway and mediated by PPARγ. These results may provide a basis for the application of WIN in HCC treatment.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Benzoxazinas/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Morfolinas/farmacologia , Naftalenos/farmacologia , PPAR gama/metabolismo , Caspases/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , HumanosRESUMO
Activated Kras gene coupled with activation of Akt and nuclear factor-kappa B (NF-κB) triggers the development of pancreatic intraepithelial neoplasia, the precursor lesion for pancreatic ductal adenocarcinoma (PDAC) in humans. Therefore, intervention at premalignant stage of disease is considered as an ideal strategy to delay the tumor development. Pancreatic malignant tumor cell lines are widely used; however, there are not relevant cell-based models representing premalignant stages of PDAC to test intervention agents. By employing a novel Kras-driven cell-based model representing premalignant and malignant stages of PDAC, we investigated the efficacy of ACTICOA-grade cocoa polyphenol (CP) as a potent chemopreventive agent under in vitro and in vivo conditions. It is noteworthy that several human intervention/clinical trials have successfully established the pharmacological benefits of cocoa-based foods. The liquid chromatography (LC)-mass spectrometry (MS)/MS data confirmed epicatechin as the major polyphenol of CP. Normal, nontumorigenic and tumorigenic pancreatic ductal epithelial (PDE) cells (exhibiting varying Kras activity) were treated with CP and epicatechin. CP and epicatechin treatments induced no effect on normal PDE cells, however, caused a decrease in the (i) proliferation, (ii) guanosine triphosphate (GTP)-bound Ras protein, (iii) Akt phosphorylation and (iv) NF-κB transcriptional activity of premalignant and malignant Kras-activated PDE cells. Further, oral administration of CP (25 mg/kg) inhibited the growth of Kras-PDE cell-originated tumors in a xenograft mouse model. LC-MS/MS analysis of the blood showed epicatechin to be bioavailable to mice after CP consumption. We suggest that (i) Kras-driven cell-based model is an excellent model for testing intervention agents and (ii) CP is a promising chemopreventive agent for inhibiting PDAC development.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Cacau/química , Carcinoma Ductal Pancreático/genética , Catequina/química , Genes ras , Neoplasias Pancreáticas/genética , Polifenóis/farmacologia , Animais , Antineoplásicos Fitogênicos/administração & dosagem , Apoptose/efeitos dos fármacos , Carcinoma Ductal Pancreático/tratamento farmacológico , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Proliferação de Células , Transformação Celular Neoplásica/efeitos dos fármacos , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Nus , NF-kappa B/genética , NF-kappa B/metabolismo , Neoplasias Pancreáticas/tratamento farmacológico , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação/efeitos dos fármacos , Polifenóis/administração & dosagem , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Espectrometria de Massas em Tandem , Transcrição GênicaRESUMO
BACKGROUND: CyclinD1 is an essential sensor and activator of cell cycle initiation and progression; overexpression of cyclinD1 is linked to various human cancers, including breast cancer. The elevated cyclinD1 in some types of cancers is believed to be associated with tumor progression and response to systemic treatments. AIMS: In this study, we anticipate to address the questions in human breast cancer; the function of cyclinD1 in mediating chemoresponses; and the signaling pathway cooperating with cyclinD1 to interfere with the drug functions. MATERIALS AND METHODS: Using the cell clones, concurrent ectopic expression of the wild-type or K112E-mutated human cyclinD1 protein in the MCF7 and MDA-MB231 (MB231) breast cancer cells to study the function of cyclinD1 in responses to the chemotherapeutic treatments. Three drugs, cisplatin (CDDP), 5-fluorouracil (5-FU), and Gemzar were used in this study; the 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, cell cycle and cell death analysis, clonogenic survival assay, acridine orange (AO)/ethidium bromide (EB) staining, and Western blot assay were conducted to evaluate the drugs' effects in the cell clones. RESULTS: The cell clones expressing the D1 protein in MCF7 and MB231 cells result in distinct effects on the responses to chemotherapeutic treatments. Particularly with Gemzar, ectopic expression of cyclinD1 protein in MCF7 cells results in a potentiated effect, which is CDK4 kinase activity dependent, whereas in MB231 cells, an opposite effect was observed. Moreover, our results suggested that the distinct chemosensitivities among those cell clones were not resulted from accelerated cell cycle, cell proliferation driven by the cyclinD1CDK4/6-Rb-E2F signaling chain, rather, they were results of the cell cycle-independent functions led by cyclinD1 alone or in complex with CDK4. CONCLUSIONS: Our results suggest that the functions of cyclinD1 protein in modulating chemoresponses in the MCF7 and MB231 cells are independent to its function as cell cycle initiator through activation of CDK4/6. Furthermore, the signals modulated by cyclinD1 upon treatment are determined by the drug and the cellular network.
RESUMO
Transcription of a pre-mRNA in eukaryotic cells elongates from the 5' to the 3' end, but intron removal during a pre-mRNA splicing does not always proceed in this orientation. In this study, we identified eight mouse p53 transcripts that retained one or more of introns 6, 7 and 8. The 5' part of intron 9 was also retained while the 3' part was not studied. These intron-containing transcripts, abbreviated as p53-ICTs, were detected at low abundance in many mouse embryonic fibroblasts (MEF) as well as cancer cell lines and tissues, and the highest ratio of these p53-ICTs to the mature p53 mRNA was seen in the normal pancreas. Serum starvation decreased those p53-ICTs that retained introns 6 and 7 but increased the levels of those lacking these introns while the level of the mature p53 mRNA was unaffected. Treatment of several cancer cell lines with cisplatin increased the mature p53 mRNA level but decreased these p53-ICTs. Transfection of p53(-/-) MEF with the p53 cDNA or several p53-ICT mini-genes slightly increased the cell viability and rendered the cells resistant to cisplatin. These data also suggest that p53 pre-mRNA splicing may have multiple orders of intron removal, some of which may not follow the "first come, first served" principle. It remains possible that these p53-ICTs are splicing intermediates existing as a mechanism for the cell to respond more promptly to a demand for more p53 and that p53 protein may be required for a normal life of MEF.
Assuntos
Cisplatino/farmacologia , Precursores de RNA/genética , Splicing de RNA/efeitos dos fármacos , Splicing de RNA/genética , Proteína Supressora de Tumor p53/genética , Animais , Sequência de Bases , Linhagem Celular , Meios de Cultura Livres de Soro , Regulação da Expressão Gênica/efeitos dos fármacos , Ordem dos Genes , Camundongos , Dados de Sequência Molecular , Precursores de RNA/química , RNA Mensageiro/química , RNA Mensageiro/genética , Transcrição GênicaRESUMO
Human longevity is an interesting and complicated subject, with many associated variations, geographic and genetic, including some known mitochondrial variations. The population of the Bama County of Guangxi Province of China is well known for its longevity and serves as a good model for studying a potential molecular mechanism. In this study, a full sequence analysis of mitochondrial DNA (mtDNA) has been done in ten Bama centenarians using direct sequencing. Polymorphisms of the displacement loop (D-loop) region of mtDNA and several serum parameters were analyzed for a total of 313 Bama individuals with ages between 10 and 110 years. The results showed that there were seven mitochondrial variations, A73G, A263G, A2076G, A8860G, G11719A, C14766T, and A15326G, and four haplogroups, M(*), F1, D* and D(4) in 10 Bama centenarians. In the D-loop region of mtDNA, the mt146T occurred at a significantly lower frequency in those is the older age group (90-110 years) than in the middle (80-89 years) and in the younger (10-79 years) groups (P < 0.05). The mt146T also had lower systolic blood pressure and serum markers such as total cholesterol, triglyceride and low density lipoprotein than did mt146C in the older age group (P < 0.05). No significant differences were observed between the mt146C and the mt146T individuals in the middle and the younger groups (P > 0.05). The mt5178C/A polymorphisms did not show any significant differences among the three age-groups (P > 0.05), but different nationalities in the Bama County did show a significant difference in the mt5178C/A polymorphisms (P < 0.05). These results suggest that the mt146T/C polymorphisms in Guangxi Bama individuals may partly account for the Bama longevity whereas the mt5178C/A polymorphisms are strongly associated with the nationalities in the Guangxi Bama population.
Assuntos
Povo Asiático/etnologia , DNA Mitocondrial/genética , Longevidade/genética , Polimorfismo Genético , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Envelhecimento/genética , Criança , China , Feminino , Ordem dos Genes , Loci Gênicos , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Adulto JovemRESUMO
BACKGROUND: Cisplatin (CDDP) is a drug used for treatment of many types of malignancy but pancreatic cancer is relatively resistant to it. This study aims to determine whether and how cyclin D1 (D1) and c-Myc influence the response of pancreatic cancer cells to CDDP. MATERIALS AND METHODS: Ela-mycPT mouse pancreatic cancer cells were transfected with D1 or c-myc cDNA and treated with CDDP alone or together with NPCD, an inhibitor of cyclin dependent ckinase (CDK) 4 and 6. Reverse transcription followed by polymerase chain reaction (RT-PCR) and western blot assays were used to determine the mRNA and protein levels of interested genes. Cell viability was determined using 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. RESULTS: Treatment of Ela-mycPT1 cells with CDDP caused an increase in c-myc expression but a slightly latent decrease in D1 expression, whereas D1 and c-Myc proteins repressed each other. D1 or c-Myc rendered Ela-mycPT1 cells resistant or sensitive, respectively, to CDDP. D1 induced the expression of several members of the NF-κB family, including RelA, RelB, Nfκb1 and Nfκb2. D1 also induced BIRC5 and several pro-survival members of the Bcl-2 gene family, including Bcl-2 , Mcl-1 and Bad while it decreased the level of the pro-apoptotic Noxa. Inhibition of CDK4 or CDK6 kinase activity by NPCD did not affect these effects of D1. In contrast, c-Myc in Ela-mycPT1 and Ela-mycPT4 cells has the opposite effects to D1 on the expression of most of these apoptosis regulating genes. CONCLUSION: Our results suggest that induction of c-Myc and inhibition of D1 may be mechanisms for CDDP to elicit cytotoxicity. On the other hand, D1 induces whereas c-Myc represses the expression of key NF-κB family members to induce and repress, respectively, the expression of BIRC5 and several Bcl-2 family members, in turn inhibiting or enhancing the response to CDDP.
RESUMO
c-Myc is a transcription factor overexpression of which induces mammary cancer in transgenic mice. To explore whether certain microRNAs (mirRNA) mediate c-Myc induced mammary carcinogenesis, we studied mirRNA expression profile in mammary tumors developed from MMTV-c-myc transgenic mice, and found 50 and 59 mirRNAs showing increased and decreased expression, respectively, compared with lactating mammary glands of wild type mice. Twenty-four of these mirRNAs could be grouped into eight clusters because they had the same chromosomal localizations and might be processed from the same primary RNA transcripts. The increased expression of mir-20a, mir-20b, and mir-9 as well as decreased expression of mir-222 were verified by RT-PCR, real-time RT-PCR, and cDNA sequencing. Moreover, we fortuitously identified a novel non-coding RNA, the level of which was decreased in proliferating mammary glands of MMTV-c-myc mice was further decreased to undetectable level in the mammary tumors. Sequencing of this novel RNA revealed that it was transcribed from a region of mouse chromosome 19 that harbored the metastasis associated lung adenocarcinoma transcript-1 (Malat-1), a non-protein-coding gene. These results suggest that certain mirRNAs and the chromosome 19 derived non-coding RNAs may mediate c-myc induced mammary carcinogenesis.
Assuntos
Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Genes myc , Neoplasias Mamárias Experimentais/genética , MicroRNAs/genética , RNA Neoplásico/genética , Animais , Transformação Celular Viral/genética , Mapeamento Cromossômico , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Lactação/genética , Glândulas Mamárias Animais/metabolismo , Vírus do Tumor Mamário do Camundongo/genética , Camundongos , Camundongos Transgênicos , MicroRNAs/biossíntese , RNA Neoplásico/biossínteseRESUMO
Cyclin D1 plays a key regulatory role during the G1 phase of the cell cycle and its gene is amplified and over-expressed in many cancers. The cyclin D1b mRNA variant was established in human cells and recent functional analyses revealed that its protein product harbors unique activities in human cancer cells. By performing reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) experiments, we identified the cyclin D1b mRNA variant in mouse. Similar to its human counterpart, the mouse cyclin D1b transcript consists of exon 1, 2, 3, 4 and part of intron 4, and contains a long open reading frame (ORF). The predicted peptide from this ORF is 34-amino acid longer than the human cyclin D1b. The expression of this mouse mRNA variant was investigated. It appears to be expressed ubiquitously and differentially in various mouse cell lines and tissues and its level might be proportional to that of the canonical endogenous cyclin D1a mRNA.
Assuntos
Ciclina D1/genética , Sequência de Aminoácidos , Animais , Linhagem Celular , Ciclina D1/química , Ciclina D1/metabolismo , Regulação da Expressão Gênica , Camundongos , Dados de Sequência Molecular , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
PURPOSE: We have previously shown that p90 ribosomal protein S6 kinase 4 (RSK4), an X-linked gene, is highly up-regulated in mammary tumors of MMTV-c-Myc transgenic mice. In this study, we further investigated whether RSK4 inhibits or promotes breast tumor growth and progression. EXPERIMENTAL DESIGN: Stable overexpression or small interfering RNA-mediated knockdown of RSK4 was done in the MDA-MB-231 cell line. Stable clones were tested for cell proliferation, anchorage-independent growth in soft agar, invasive and metastatic ability of these clones in vitro and tumorigenesis, invasive and metastatic ability in vivo in severe combined immunodeficient mice. RESULTS: Here, we show that exogenous expression of RSK4 resulted in decreased cell proliferation and increased accumulation of cells in G(0)-G(1) phase, which paralleled with enhanced expression of tumor suppressor genes: retinoblastoma protein, retinobl astoma-associated 46 kDa protein, and p21 protein. Overexpression of RSK4 resulted in reduced colony formation in soft agar and suppressed invasive and migratory activities of MDA-MB-231 cells both in vitro and in vivo. Importantly, RSK4-overexpressing cells showed up-regulation of claudin-2 and down-regulation of CXCR4, both of these play roles in invasion and chemotaxis. CONCLUSIONS: These results indicate that RSK4 expression may limit the oncogenic, invasive, and metastatic potential of breast cancer cells. Anti-invasive and antimetastatic activities of RSK4 may be, in part, due to its regulation of claudin-2. Increased expression of RSK4 in c-Myc-overexpressing cells and a dose-dependent induction of luciferase reporter gene activity suggest that c-Myc may regulate RSK4 expression.
Assuntos
Neoplasias da Mama/enzimologia , Regulação Neoplásica da Expressão Gênica/fisiologia , Invasividade Neoplásica , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Animais , Western Blotting , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Proliferação de Células , Claudinas , Feminino , Humanos , Imuno-Histoquímica , Proteínas de Membrana/biossíntese , Camundongos , Camundongos SCID , Receptores CXCR4/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Cyclin D1 is one of the most commonly overexpressed oncogenes in breast cancer; yet, it is not clear whether cyclin D1 alone is capable of causing malignant transformation of mammary epithelial cells. Here, we show that ectopic expression of cyclin D1 in benign mouse mammary epithelial cells promotes cell proliferation, anchorage-independent growth in soft agar, and tumorigenesis in severe combined immunodeficient mice. To address the possible interaction of cyclin D1 and c-myc in malignant transformation, we used cyclin D1/c-myc dual-expressing clones, which displayed more aggressive and invasive phenotype than cyclin D1-expressing clones. These data provide evidence that overexpression of cyclin D1 or coexpression with c-myc could cause invasive malignant transformation of benign mouse mammary epithelial cells. Furthermore, microarray analysis of cyclin D1 and cyclin D1/c-myc clones showed that these two tumor-producing clones might use distinct invasive pathways. In summary, overexpression of cyclin D1 may commit mammary epithelia to a tumor-prone phenotype in which cooperation with other genes, such as synergy with c-myc, may lead to a more aggressive phenotype.
Assuntos
Transformação Celular Neoplásica/patologia , Ciclina D1/biossíntese , Neoplasias Mamárias Experimentais/patologia , Proteínas Proto-Oncogênicas c-myc/biossíntese , Animais , Adesão Celular/fisiologia , Processos de Crescimento Celular/fisiologia , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Ciclina D1/genética , Células Epiteliais/patologia , Feminino , Perfilação da Expressão Gênica , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/metabolismo , Camundongos , Camundongos SCID , Proteínas Proto-Oncogênicas c-myc/genética , TransfecçãoRESUMO
Therapy failure and metastasis-associated mortality are stumbling blocks in the management of PDAC in patients. Failure of therapy is associated to intense hypoxic conditions of tumors. To develop effective therapies, a complete understanding of hypoxia-associated changes in genetic landscape of tumors during disease progression is needed. Because artificially immortalized cell lines do not rightly represent the disease progression, studying genetics of tumors in spontaneous models is warranted. In the current study, we generated a spectrum of spontaneous human (UM-PDC1; UM-PDC2) and murine (HI-PanL, HI-PancI, HI-PanM) models representing localized, invasive, and metastatic PDAC from a patient and transgenic mice (K-rasG12D/Pdxcre/Ink4a/p16-/). These spontaneous models grow vigorously under hypoxia and exhibit activated K-ras signaling, progressive loss of PTEN, and tumorigenicity in vivo. Whereas UM-PDC1 form localized tumors, the UM-PDC2 metastasize to lungs in mice. In an order of progression, these models exhibit genomic instability marked by gross chromosomal rearrangements, centrosome-number variations, Aurora-kinase/H2AX colocalization, loss of primary cilia, and α-tubulin acetylation. The RNA sequencing of hypoxic models followed by qRT-PCR validation and gene-set enrichment identified Intestine-Specific Homeobox factor (ISX)-driven molecular pathway as an indicator PDAC aggressivness. TCGA-PAAD clinical data analysis showed high ISX expression correlation to poor survival of PDAC patients, particularly women. The functional studies showed ISX as a regulator of i) invasiveness and migratory potential and ii) VEGF, MMP2, and NFκB activation in PDAC cells. We suggest that ISX is a potential druggable target and newly developed spontaneous cell models are valuable tools for studying mechanism and testing therapies for PDAC.
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Since the early 1990s, multiple human estrogen receptor-α (hER-α) splice variants have been identified, of which the majority contain ≥1 deleted exon, and some are expressed as proteins with modified functions from the wild-type 66 kDa hER-α (ER-α66). In the present study, a novel hER-α splice variant, ER-α30, was identified and cloned from clinical breast cancer tissue. The ER-α30 sequence lacked a ligand-binding domain and a ligand-dependent transcriptional activation domain but retained the N-terminal transcriptional activation domain, the DNA-binding domain and a partial hinge domain, and possesses a unique 10-amino-acid domain. The expression of ER-α30 was associated with ER-α66-negative and progesterone receptor-negative breast cancer. The 30 kDa protein was expressed in stably transfected MDA-MB-231 cells, and the overexpression of ER-α30 in MDA-MB-231 cells enhanced malignant biological behaviors, including cellular proliferation, migration and invasion in vitro. The results of the present study indicated that ER-α30 might represent a potential biomarker for breast cancer.
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There have been enough cell death modes delineated in the biomedical literature to befuddle all cell death researchers. Mulling over cell death from the viewpoints of the host tissue or organ and of the host animal, we construe that there should be only two physiological cell death modes, i.e. apoptosis and senescent death (SD), as well as two pathological modes, i.e. necrosis and stress-induced cell death (SICD). Other death modes described in the literature are ad-hoc variants or coalescences of some of these four basic ones in different physiological or pathological situations. SD, SICD and necrosis kill useful cells and will thus trigger regeneration, wound healing and probably also scar formation. SICD and necrosis will likely instigate inflammation as well. Apoptosis occurs as a mechanism to purge no-longer useful cells from a tissue via phagocytosis by cells with phagocytic ability that are collectively tagged by us as scavengers, including macrophages; therefore apoptosis is not followed by regeneration and inflammation. The answer for the question of "who dies" clearly differentiates apoptosis from SD, SICD and necrosis, despite other similarities and disparities among the four demise modes. Apoptosis cannot occur in cell lines in vitro, because cell lines are immortalized by reprogramming the death program of the parental cells, because in culture there lack scavengers and complex communications among different cell types, and because culture condition is a stress to the cells. Several issues of cell death that remain enigmatic to us are also described for peers to deliberate and debate.
RESUMO
Objectives: While evidence that episodes of mania in bipolar I are associated with changes in bioenergetic and regional cerebral blood flow (rCBF) and cerebral blood flow velocity (rCBFV), both the regions and the extent of these changes have not yet been defined. Therefore, we determined the pattern of regional cerebral perfusion mania patients and using patients with major depressive disorder (MDD) as positive controls and healthy participants as negative controls. Methods: Twenty participants with mania, together with 22 MDD patients and 24 healthy volunteers, were recruited for this study. On all participants, Transcranial Doppler (TCD) was conducted to measure rCBFV parameters, 320-slice CT was conducted to measure rCBF in the different cerebral artery regions, and hematological parameters were assessed. ANOVA and Pearson's tests were used for the statistical analysis. Results: Our data indicated that rCBF in the medial temporal lobe and hippocampus, especially in the left medial temporal lobe and the right hippocampus, was increased in the mania group compared with the control and MDD groups (p < 0.01). In contrast, rCBF in the medial temporal lobe and hippocampus was decreased in the depression group (p < 0.01) compared with healthy controls. In addition, values of rCBFV in the bilateral internal carotid arteries (ICAs) and middle cerebral arteries (MCA) were increased in mania (p < 0.01) in comparison to the MDD group. Whole blood viscosity and hematocrit as well as red blood cell sedimentation rate remained unchanged in all group (p > 0.05). Conclusions: In mania, rCBF is increased in the medial temporal lobe and hippocampus, with a corresponding increase in rCBFV in the same regions.
RESUMO
The prevalence of infection by different genotypes of human papillomavirus (HPV) varies among different geographic areas. We studied the prevalence of infection by 21 HPV genotypes in cervical tissue specimens from 4213 women in the Guiyang district, that is located in the southwest of China and is dominated by minor ethnicities of Chinese, and 2074 cases in our cohort had pathological diagnosis available. The overall infection rate was 36.98%. Most (72.08%) infectors were positive for only one HPV subtype, with the remaining being cases infected by two or more subtypes. Infections by the HPV subtypes 16, 52 and 58 were the most prevalent, having rates of 34.66%, 16.03%% and 15.53%, respectively. The most common cervical lesions in HPV infections were genital warts, cervical cancer (CC) and cervical intraepithelial neoplasia (CIN). Age and age at first sexual activity were independent risk factors for HPV infections that in turn cause certain cervical lesions. Intriguingly, while 94.90% of the CC patients were infected by oncogenically high-risk (HR) HPV subtypes, only 2.75% and 2.29% of these patients were infected by oncogenically low-risk (LR) subtypes or other-subtypes with their oncogenicity unclear. The rates of infection by LR-HPVs and other-HPVs were also low, being 4.63% and 6.76%, respectively, in the patients with CIN that is a precursor lesion of CC, lower than the 8.54% and 18.20%, respectively, in the women without a cervical lesion. Our data provides an important foundation for prevention, diagnosis and treatment of HPV infection in Guiyang district and suggests that development of vaccines for prevention and treatment of CC in this area should first target the HPV subtypes 16, 52 and 58, but not subtype 18 as for many other places. It deserves study whether infections by certain LR-HPVs and other-HPVs may serve as attenuated live vaccines for prevention of CC.