Iron deficiency: prevalence, mortality risk, and dietary relationships in general and heart failure populations.

Background: Iron deficiency (ID) is the most common nutritional deficiency, with little research on its prevalence and long-term outcomes in the general population and those with heart failure (HF). Both the relationships between dietary iron and ID, as well as dietary folate and ID, are understudied. Methods: We used data from the National Health and Nutrition Examination Survey from 1999 to 2002 to investigate the prevalence, prognosis, and relationship between dietary and ID defined by different criteria in the general population (n = 6,660) and those with HF (n = 182). Results: There was no significant difference in the prevalence of ID between HF patients and the general population after propensity score matching. Transferrin saturation (TSAT) <20% was associated with higher 5-year all-cause mortality (HR: 3.49, CI: 1.40-8.72, P = 0.007), while ferritin <30 ng/ml was associated with higher 10-year (HR: 2.70, CI: 1.10-6.67, P = 0.031) and 15-year all-cause mortality (HR: 2.64, CI: 1.40-5.00, P = 0.003) in HF patients. Higher dietary total folate but dietary iron reduced the risk of ID (defined as ferritin <100 ng/ml) in HF patients (OR: 0.80; 95% CI: 0.65-1.00; P = 0.047). Conclusions: The prevalence of ID was identical in HF and non-HF individuals. Ferritin <30 ng/ml was associated with long-term outcomes whereas TSAT <20% was associated with short-term prognosis in both the general population and HF patients. A diet rich in folate might have the potential for prevention and treatment of ID in HF patients.

MRE11 liberates cGAS from nucleosome sequestration during tumorigenesis.

Oncogene-induced replication stress generates endogenous DNA damage that activates cGAS-STING-mediated signalling and tumour suppression1-3. However, the precise mechanism of cGAS activation by endogenous DNA damage remains enigmatic, particularly given that high-affinity histone acidic patch (AP) binding constitutively inhibits cGAS by sterically hindering its activation by double-stranded DNA (dsDNA)4-10. Here we report that the DNA double-strand break sensor MRE11 suppresses mammary tumorigenesis through a pivotal role in regulating cGAS activation. We demonstrate that binding of the MRE11-RAD50-NBN complex to nucleosome fragments is necessary to displace cGAS from acidic-patch-mediated sequestration, which enables its mobilization and activation by dsDNA. MRE11 is therefore essential for cGAS activation in response to oncogenic stress, cytosolic dsDNA and ionizing radiation. Furthermore, MRE11-dependent cGAS activation promotes ZBP1-RIPK3-MLKL-mediated necroptosis, which is essential to suppress oncogenic proliferation and breast tumorigenesis. Notably, downregulation of ZBP1 in human triple-negative breast cancer is associated with increased genome instability, immune suppression and poor patient prognosis. These findings establish MRE11 as a crucial mediator that links DNA damage and cGAS activation, resulting in tumour suppression through ZBP1-dependent necroptosis.

Discovery of a 53BP1 Small Molecule Antagonist Using a Focused DNA-Encoded Library Screen.

Methyl-lysine reader p53 binding protein 1 (53BP1) is a central mediator of DNA break repair and is associated with various human diseases, including cancer. Thus, high-quality 53BP1 chemical probes can aid in further understanding the role of 53BP1 in genome repair pathways. Herein, we utilized focused DNA-encoded library screening to identify the novel hit compound UNC8531, which binds the 53BP1 tandem Tudor domain (TTD) with an IC50 of 0.47 ± 0.09 µM in a TR-FRET assay and Kd values of 0.85 ± 0.17 and 0.79 ± 0.52 µM in ITC and SPR, respectively. UNC8531 was cocrystallized with the 53BP1 TTD to guide further optimization efforts, leading to UNC9512. NanoBRET and 53BP1-dependent foci formation experiments confirmed cellular target engagement. These results show that UNC9512 is a best-in-class small molecule 53BP1 antagonist that can aid further studies investigating the role of 53BP1 in DNA repair, gene editing, and oncogenesis.

Understanding the scope of intracardiac echocardiography in catheter ablation of ventricular arrhythmia.

Over the last few decades, catheter ablation has emerged as the first-line treatment for ventricular arrhythmias. However, detailed knowledge of cardiac anatomy during the surgery remains the prerequisite for successful ablation. Intracardiac echocardiography (ICE) is a unique imaging technique, which provides real-time visualization of cardiac structures, and is superior to other imaging modalities in terms of precise display of cardiac tissue characteristics as well as the orientation of anatomical landmarks. This article aimed to introduce the various advantages and limitations of ICE in the ablation of ventricular arrhythmias.

Reck-Notch1 Signaling Mediates miR-221/222 Regulation of Lung Cancer Stem Cells in NSCLC.

Cancer stem cells (CSCs) contribute to the cancer initiation, metastasis and drug resistance in non-small cell lung cancer (NSCLC). Herein, we identified a miR-221/222 cluster as a novel regulator of CSCs in NSCLC. Targeted overexpression or knockdown of miR-221/222 in NSCLC cells revealed the essential roles of miR-221/222 in regulation of lung cancer cell proliferation, mammosphere formation, subpopulation of CD133+ CSCs and the expression of stemness genes including OCT4, NANOG and h-TERT. The in vivo animal study showed that overexpression of miR-221/222 significantly enhanced the capacity of lung cancer cells to develop tumor and grow faster, indicating the importance of miR-221/222 in tumorigenesis and tumor growth. Mechanistically, Reck was found to be a key direct target gene of miR-221/222 in NSCLC. Overexpression of miR-221/222 significantly suppressed Reck expression, activated Notch1 signaling and increased the level of NICD. As an activated form of Notch1, NICD leads to enhanced stemness in NSCLC cells. In addition, knockdown of Reck by siRNA not only mimicked miR-221/222 effects, but also demonstrated involvement of Reck in the miR-221/222-induced activation of Notch1 signaling, verifying the essential roles of the miR-221/222-Reck-Notch1 axis in regulating stemness of NSCLC cells. These findings uncover a novel mechanism by which lung CSCs are significantly manipulated by miR-221/222, and provide a potential therapeutic target for the treatment of NSCLC.

Thioredoxin mitigates radiation-induced hematopoietic stem cell injury in mice.

BACKGROUND: Radiation exposure poses a significant threat to public health. Hematopoietic injury is one of the major manifestations of acute radiation sickness. Protection and/or mitigation of hematopoietic stem cells (HSCs) from radiation injury is an important goal in the development of medical countermeasure agents (MCM). We recently identified thioredoxin (TXN) as a novel molecule that has marked protective and proliferative effects on HSCs. In the current study, we investigated the effectiveness of TXN in rescuing mice from a lethal dose of total body radiation (TBI) and in enhancing hematopoietic reconstitution following a lethal dose of irradiation. METHODS: We used in-vivo and in-vitro methods to understand the biological and molecular mechanisms of TXN on radiation mitigation. BABL/c mice were used for the survival study and a flow cytometer was used to quantify the HSC population and cell senescence. A hematology analyzer was used for the peripheral blood cell count, including white blood cells (WBCs), red blood cells (RBCs), hemoglobin, and platelets. Colony forming unit (CFU) assay was used to study the colongenic function of HSCs. Hematoxylin and eosin staining was used to determine the bone marrow cellularity. Senescence-associated ß-galactosidase assay was used for cell senescence. Western blot analysis was used to evaluate the DNA damage and senescence protein expression. Immunofluorescence staining was used to measure the expression of γ-H2AX foci for DNA damage. RESULTS: We found that administration of TXN 24 h following irradiation significantly mitigates BALB/c mice from TBI-induced death: 70% of TXN-treated mice survived, whereas only 25% of saline-treated mice survived. TXN administration led to enhanced recovery of peripheral blood cell counts, bone marrow cellularity, and HSC population as measured by c-Kit+Sca-1+Lin- (KSL) cells, SLAM + KSL cells and CFUs. TXN treatment reduced cell senescence and radiation-induced double-strand DNA breaks in both murine bone marrow lineage-negative (Lin-) cells and primary fibroblasts. Furthermore, TXN decreased the expression of p16 and phosphorylated p38. Our data suggest that TXN modulates diverse cellular processes of HSCs. CONCLUSIONS: Administration of TXN 24 h following irradiation mitigates radiation-induced lethality. To the best of our knowledge, this is the first report demonstrating that TXN reduces radiation-induced lethality. TXN shows potential utility in the mitigation of radiation-induced hematopoietic injury.

MiR-215 Is Induced Post-transcriptionally via HIF-Drosha Complex and Mediates Glioma-Initiating Cell Adaptation to Hypoxia by Targeting KDM1B.

The hypoxic tumor microenvironment serves as a niche for maintaining the glioma-initiating cells (GICs) that are critical for glioblastoma (GBM) occurrence and recurrence. Here, we report that hypoxia-induced miR-215 is vital for reprograming GICs to fit the hypoxic microenvironment via suppressing the expression of an epigenetic regulator KDM1B and modulating activities of multiple pathways. Interestingly, biogenesis of miR-215 and several miRNAs is accelerated post-transcriptionally by hypoxia-inducible factors (HIFs) through HIF-Drosha interaction. Moreover, miR-215 expression correlates inversely with KDM1B while correlating positively with HIF1α and GBM progression in patients. These findings reveal a direct role of HIF in regulating miRNA biogenesis and consequently activating the miR-215-KDM1B-mediated signaling required for GIC adaptation to hypoxia.

Small RNAs Recruit Chromatin-Modifying Enzymes MMSET and Tip60 to Reconfigure Damaged DNA upon Double-Strand Break and Facilitate Repair.

Recent reports have demonstrated that DNA double-strand break (DSB)-induced small RNAs (diRNA) play an important role in the DNA damage response (DDR). However, the molecular mechanism by which diRNAs regulate the DDR remains unclear. Here, we report that Dicer- and Drosha-dependent diRNAs function as guiding molecules to promote the recruitment of the methyltransferase MMSET (WHSC1) and the acetyltransferase Tip60 (KAT5) to the DSB, where local levels of histone H4 di- and tri-methylation at lysine 20 (H4K20me2, 3) and H4 acetylation at lysine 16 (H4K16Ac) were enhanced. These histone modification events resulted in an open, flexible chromatin configuration, as indicated by the increased release of histones γH2AX, H2AX, and H3 from damaged chromatin. Furthermore, we found that diRNA-associated AGO2 interacted with MMSET and Tip60 and that the diRNA binding and catalytic activities of AGO2 were dispensable for the interaction but required for the recruitment of MMSET and Tip60 to DSBs. Consequently, diRNA-mediated chromatin remodeling promoted DSB repair by enhancing the recruitment of Rad51 and BRCA1 to the DSB site. Taken together, our findings reveal an unexpected direct role for diRNAs in regulating chromatin remodeling to facilitate DSB repair, revealing a new layer of DDR regulation involving specialized RNA molecules. Cancer Res; 76(7); 1904-15. ©2016 AACR.

Intraventricular injection of human dental pulp stem cells improves hypoxic-ischemic brain damage in neonatal rats.

OBJECTIVE: To investigate the effect of intraventricular injection of human dental pulp stem cells (DPSCs) on hypoxic-ischemic brain damage (HIBD) in neonatal rats. METHODS: Thirty-six neonatal rats (postnatal day 7) were assigned to control, HIBD, or HIBD+DPSC groups (n = 12 each group). For induction of HIBD, rats underwent left carotid artery ligation and were exposed to 8% to 10% oxygen for 2 h. Hoechst 33324-labeled human DPSCs were injected into the left lateral ventricle 3 days after HIBD. Behavioral assays were performed to assess hypoxic-ischemic encephalopathy (HIE), and on postnatal day 45, DPSC survival was assessed and expression of neural and glial markers was evaluated by immunohistochemistry and Western blot. RESULTS: The HIBD group showed significant deficiencies compared to control on T-maze, radial water maze, and postural reflex tests, and the HIBD+DPSC group showed significant improvement on all behavioral tests. On postnatal day 45, Hoechst 33324-labeled DPSC nuclei were visible in the injected region and left cortex. Subsets of DPSCs showed immunostaining for neuronal (neuron-specific enolase [NSE], Nestin) and glial markers (glial fibrillary acidic protein [GFAP], O4). Significantly decreased staining/expression for NSE, GFAP, and O4 was found in the HBID group compared to control, and this was significantly increased in the HBID+DPSC group. CONCLUSION: Intraventricular injection of human DPSCs improves HIBD in neonatal rats.

Directed evolution of cytochrome P450 for sterol epoxidation.

16,17-Epoxysterol plays an important role in pharmaceutical steroid synthesis. To investigate the potential application of cytochrome P450 for epoxysterol synthesis, an approach to the epoxidation of 16,17-epoxysterol, based on directed evolution of cytochrome P450 BM-3, was developed. This comprised random gene mutagenesis for optimizing the activity of P450 BM-3 for epoxidation of hydrophobic sterol, followed by the 7-ethoxycoumarin de-ethylation assay for general enzyme activity detection and the modified picric acid assay for epoxidation activity screening. By the two-step screening, one mutant from 792 clones showed specific substrate activity of converting progesterone to 16,17-epoxysterol, which validated the possibility to evolve the cytochrome P450 for the synthesis of steroidal epoxides.

Bat3 facilitates H3K79 dimethylation by DOT1L and promotes DNA damage-induced 53BP1 foci at G1/G2 cell-cycle phases.

The methyltransferase DOT1L methylates histone H3 at K79 to facilitate specific biological events. H3K79 dimethylation (H3K79-2Me) by DOT1L influences the DNA damage response by promoting 53BP1 recruitment to DNA damage sites; however, it is unclear if this methylation is required as 53BP1 interacts with dimethylated H4 (H4K20-2Me) with a much higher affinity. We demonstrate that H3K79-2Me, while negligible during S-phase, is required for ionizing radiation (IR)-induced 53BP1 foci formation during G1/G2-phases when H4K20-2Me levels are low. Further, we describe an essential role for HLA-B-associated transcript 3 (Bat3) in regulating this process in U2OS cells. Bat3 co-localizes with DOT1L at histone H3, and Bat3 knockdown results in decreased DOT1L-H3 interaction and H3K79-2Me, leading to a reduction in IR-induced 53BP1 foci formation, defects in DNA repair and increased sensitivity to IR. We demonstrate that a conserved Bat3 ubiquitin-like motif and a conserved DOT1L ubiquitin-interacting motif promote DOT1L-Bat3 interaction to facilitate efficient H3K79-2Me and IR-induced 53BP1 foci formation during G1/G2-phases. Taken together, our findings identify a novel role for Bat3 in regulating DOT1L function, which plays a critical role in DNA damage response.

Mono- or double-site phosphorylation distinctly regulates the proapoptotic function of Bax.

Bax is the major multidomain proapoptotic molecule that is required for apoptosis. It has been reported that phosphorylation of Bax at serine(S) 163 or S184 activates or inactivates its proapoptotic function, respectively. To uncover the mechanism(s) by which phosphorylation regulates the proapoptotic function of Bax, a series of serine (S)→ alanine/glutamate (A/E) Bax mutants, including S163A, S184A, S163E, S184E, S163E/S184A (EA), S163A/S184E (AE), S163A/S184A (AA) and S163E/S184E (EE), were created to abrogate or mimic, respectively, either single or double-site phosphorylation. The compound Bax mutants (i.e. EA and AE) can flesh out the functional contribution of individual phosphorylation site(s). WT and each of these Bax mutants were overexpressed in Bax(-/-) MEF or lung cancer H157 cells and the proapoptotic activities were compared. Intriguingly, expression of any of Bax mutants containing the mutation S→A at S184 (i.e. S184A, EA or AA) represents more potent proapoptotic activity as compared to WT Bax in association with increased 6A7 epitope conformational change, mitochondrial localization/insertion and prolonged half-life. In contrast, all Bax mutants containing the mutation S→E at S184 (i.e. S184E, AE or EE) have a mobility-shift and fail to insert into mitochondrial membranes with decreased protein stability and less apoptotic activity. Unexpectedly, mutation either S→A or S→E at S163 site does not significantly affect the proapoptotic activity of Bax. These findings indicate that S184 but not S163 is the major phosphorylation site for functional regulation of Bax's activity. Therefore, manipulation of the phosphorylation status of Bax at S184 may represent a novel strategy for cancer treatment.

A nonhomologous end-joining pathway is required for protein phosphatase 2A promotion of DNA double-strand break repair.

Protein phosphatase 2A (PP2A) functions as a potent tumor suppressor, but its mechanism(s) remains enigmatic. Specific disruption of PP2A by either expression of SV40 small tumor antigen or depletion of endogenous PP2A/C by RNA interference inhibits Ku DNA binding and DNA-PK activities, which results in suppression of DNA double-strand break (DSB) repair and DNA end-joining in association with increased genetic instability (i.e., chromosomal and chromatid breaks). Overexpression of the PP2A catalytic subunit (PP2A/C) enhances Ku and DNA-PK activities with accelerated DSB repair. Camptothecin-induced DSBs promote PP2A to associate with Ku 70 and Ku 86. PP2A directly dephosphorylates Ku as well as the DNA-PK catalytic subunit (DNA-PKcs) in vitro and in vivo, which enhances the formation of a functional Ku/DNA-PKcs complex. Intriguingly, PP2A promotes DSB repair in wild type mouse embryonic fibroblast (MEF) cells but has no such effect in Ku-deficient MEF cells, suggesting that the Ku 70/86 heterodimer is required for PP2A promotion of DSB repair. Thus, PP2A promotion of DSB repair may occur in a novel mechanism by activating the nonhomologous end-joining pathway through direct dephosphorylation of Ku and DNA-PKcs, which may contribute to maintenance of genetic stability.

Reduced ATR or Chk1 expression leads to chromosome instability and chemosensitization of mismatch repair-deficient colorectal cancer cells.

Genomic instability in colorectal cancer is categorized into two distinct classes: chromosome instability (CIN) and microsatellite instability (MSI). MSI is the result of mutations in the mismatch repair (MMR) machinery, whereas CIN is often thought to be associated with a disruption in the APC gene. Clinical data has recently shown the presence of heterozygous mutations in ATR and Chk1 in human cancers that exhibit MSI, suggesting that those mutations may contribute to tumorigenesis. To determine whether reduced activity in the DNA damage checkpoint pathway would cooperate with MMR deficiency to induce CIN, we used siRNA strategies to partially decrease the expression of ATR or Chk1 in MMR-deficient colorectal cancer cells. The resultant cancer cells display a typical CIN phenotype, as characterized by an increase in the number of chromosomal abnormalities. Importantly, restoration of MMR proficiency completely inhibited induction of the CIN phenotype, indicating that the combination of partial checkpoint blockage and MMR deficiency is necessary to trigger CIN. Moreover, disruption of ATR and Chk1 in MMR-deficient cells enhanced the sensitivity to treatment with the commonly used colorectal chemotherapeutic compound, 5-fluorouracil. These results provide a basis for the development of a combination therapy for those cancer patients.

Bcl2 negatively regulates DNA double-strand-break repair through a nonhomologous end-joining pathway.

Bcl2 can enhance susceptibility to carcinogenesis, but the mechanism(s) remains fragmentary. Here we discovered that Bcl2 suppresses DNA double-strand-break (DSB) repair and V(D)J recombination by downregulating Ku DNA binding activity, which is associated with increased genetic instability. Exposure of cells to ionizing radiation enhances Bcl2 expression in the nucleus, which interacts with both Ku70 and Ku86 via its BH1 and BH4 domains. Removal of the BH1 or BH4 domain abrogates the inhibitory effect of Bcl2 on Ku DNA binding, DNA-PK, and DNA end-joining activities, which results in the failure of Bcl2 to block DSB repair as well as V(D)J recombination. Intriguingly, Bcl2 directly disrupts the Ku/DNA-PKcs complex in vivo and in vitro. Thus, Bcl2 suppression of the general DSB repair and V(D)J recombination may occur in a mechanism by inhibiting the nonhomologous end-joining pathway, which may lead to an accumulation of DNA damage and genetic instability.

Engineering bacteria for production of rhamnolipid as an agent for enhanced oil recovery.

Rhamnolipid as a potent natural biosurfactant has a wide range of potential applications, including enhanced oil recovery (EOR), biodegradation, and bioremediation. Rhamnolipid is composed of rhamnose sugar molecule and beta-hydroxyalkanoic acid. The rhamnosyltransferase 1 complex (RhlAB) is the key enzyme responsible for transferring the rhamnose moiety to the beta-hydroxyalkanoic acid moiety to biosynthesize rhamnolipid. Through transposome-mediated chromosome integration, the RhlAB gene was inserted into the chromosome of the Pseudomonas aeruginosa PAO1-rhlA(-) and Escherichia coli BL21 (DE3), neither of which could produce rhamnolipid. After chromosome integration of the RhlAB gene, the constitute strains P. aeruginosa PEER02 and E. coli TnERAB did produce rhamnolipid. The HPLC/MS spectrum showed that the structure of purified rhamnolipid from P. aeruginosa PEER02 was similar to that from other P. aeruginosa strains, but with different percentage for each of the several congeners. The main congener (near 60%) of purified rhamnolipid from E. coli TnERAB was 3-(3-hydroxydecanoyloxy) decanoate (C(10)-C(10)) with mono-rhamnose. The surfactant performance of rhamnolipid was evaluated by measurement of interfacial tension (IFT) and oil recovery via sand-pack flooding tests. As expected, pH and salt concentration of the rhamnolipid solution significantly affected the IFT properties. With just 250 mg/L rhamnolipid (from P. aeruginosa PEER02 with soybean oil as substrate) in citrate-Na(2)HPO(4), pH 5, 2% NaCl, 42% of oil otherwise trapped was recovered from a sand pack. This result suggests rhamnolipid might be considered for EOR applications.

Bcl2 impedes DNA mismatch repair by directly regulating the hMSH2-hMSH6 heterodimeric complex.

Bcl2 has been reported to suppress DNA mismatch repair (MMR) with promotion of mutagenesis, but the mechanism(s) is not fully understood. MutSalpha is the hMSH2-hMSH6 heterodimer that primarily functions to correct mutations that escape the proofreading activity of DNA polymerase. Here we have discovered that Bcl2 potently suppresses MMR in association with decreased MutSalpha activity and increased mutagenesis. Exposure of cells to nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone results in accumulation of Bcl2 in the nucleus, which interacts with hMSH6 but not hMSH2 via its BH4 domain. Deletion of the BH4 domain from Bcl2 abrogates the ability of Bcl2 to interact with hMSH6 and is associated with enhanced MMR efficiency and decreased mutation frequency. Overexpression of Bcl2 reduces formation of the hMSH2-hMSH6 complex in cells, and purified Bcl2 protein directly disrupts the hMSH2-hMSH6 complex and suppresses MMR in vitro. Importantly, depletion of endogenous Bcl2 by RNA interference enhances formation of the hMSH2-hMSH6 complex in association with increased MMR and decreased mutagenesis. Thus, Bcl2 suppression of MMR may occur in a novel mechanism by directly regulating the heterodimeric hMSH2-hMSH6 complex, which potentially contributes to genetic instability and carcinogenesis.

Effect of adenovirus-mediated p27 gene expression on the proliferation and apoptosis of HL-60 and Raji cell lines.

OBJECTIVE: To explore the possible effect of p27 gene expression on the proliferation and apoptosis of HL-60 and Raji cell lines. METHODS: The infections of HL-60 and Raji cells were performed by the adenovirus-mediated p27 gene transfection approach. The efficiency of Adp27 infection and the expression of p27 mRNA and protein were evaluated by X-gal staining, RT-PCR and flow cytometry. The proliferation and apoptosis of HL-60 and Raji cells were estimated by using trypan blue staining, MTT assay, Annexin V/PI and DNA ladder electrophoresis. RESULTS: The infection efficiency of HL-60 and Raji cells were 40.3 and 32%, respectively; RT-PCR and flow cytometry showed that there were significant expressions of p27 mRNA and protein of HL-60 and Raji cells infected by Adp27, while HL-60 cells themselves showed only faint p27 mRNA and protein, and Raji cells hardly presented p27 mRNA and protein. The strong proliferation inhibitions, which were in a time-dependent manner for HL-60 and Raji cells infected by Adp27, were indicated by cell growth curve and MTT assay. After 72 h infection of HL-60 and Raji cells by Adp27, the Annexin V+/PI- apoptotic cell rates were 46.9 and 35.7% respectively, which were significantly increased compared with control group (4.7 and 5.6% respectively). The typical DNA ladder bands were detectable in HL-60 and Raji cells after 48 h of Adp27 infection. CONCLUSION: The infection of HL-60 and Raji cells by means of adenoviral vector-mediated p27 gene could evidently inhibit cellular proliferation and promote cell apoptosis, which would provide experimental evidence for gene therapy of leukemia/lymphoma using adenovirus-mediated p27 gene approach.

Effect of adenovirus-mediated p27 gene expression on the proliferation and apoptosis of HL-60 and Raji cell lines.

BACKGROUND: p27 is an essential mediator of cell cycle control, which plays a key negative role in the proliferation and tumorigenesis of certain cell types. Here, we designed this study to explore the possible effects of p27 on the proliferation and apoptosis of HL-60 and Raji cell lines. METHODS: HL-60 and Raji cells were transfected with p27 via an adenovirus-mediated approach. The efficiency of Adp27 infection and the expression of p27 mRNA and protein were evaluated by X-gal staining, RT-PCR, and flow cytometry. The proliferation and apoptosis of HL-60 and Raji cells were estimated by means of trypan blue staining, MTT assay, Annexin V/PI, and DNA ladder electrophoresis. RESULTS: The infection efficiencies in HL-60 and Raji cells were 40.3% and 32.0%, respectively. RT-PCR and flow cytometry showed that there was significant expression of p27 mRNA and protein in HL-60 and Raji cells infected with Adp27; on the other hand, uninfected HL-60 cells showed faint traces of p27 mRNA and protein and Raji cells showed nearly no signs of p27 mRNA and protein. As demonstrated by a cell growth curve and by an MTT assay, strong time-dependent proliferation inhibition was apparent in HL-60 and Raji cells infected by Adp27. After 72 hours of infection, the Annexin V+/PI- apoptotic cell rates in HL-60 and Raji cell lines were 46.9% and 35.7%, respectively, significantly higher than in the control groups (4.7% and 5.6%, respectively). Typical DNA ladder bands were detectable in HL-60 and Raji cells after 48 hours of Adp27 infection. CONCLUSIONS: Adenoviral vector-mediated p27 gene transfection of HL-60 and Raji cells leads to the inhibition of cellular proliferation and the promotion of cell apoptosis. This technique may provide an approach to gene therapy for leukemia or lymphoma.