[The expression of clock gene CLOCK and its clinical significance in nasopharyngeal carcinoma].

Objective: To explore the relationship between expression levels of CLOCK mRNA and protein and the clinical characteristics of patients with nasopharyngeal carcinoma. Methods: The frozen tissue specimens from 33 patients with nasopharyngeal carcinoma in the Affiliated Tumor Hospital of Guizhou Medical University from 2018 to 2019 were collected. Seventeen cases of tissue specimens from patients with nasopharyngeal chronic inflammation in the Affiliated Hospital of Guizhou Medical University in 2019 were collected. From 2008 to 2014, 68 cases of formalin-fixed paraffin-embedding (FFPE) nasopharyngeal carcinoma tissue and 37 cases of FFPE nasopharyngeal chronic inflammation tissue were collected from the Affiliated Tumor Hospital of Guizhou Medical University. Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot (WB) were used to detect the mRNA and protein expression levels of CLOCK. The nasopharyngeal carcinoma cells including CNE1, CNE2, 5-8F and the normal nasopharyngeal epithelial cell NP69 were cultured. qRT-PCR was used to detect the expression level of CLOCK mRNA in each cell line at the time points of ZT2, ZT6, ZT10, ZT14, ZT18 and ZT22. The cosine method was used to fit the rhythm of CLOCK gene in nasopharyngeal carcinoma. The protein expression of CLOCK protein was detected by using immunohistochemical method in 68 cases of nasopharyngeal carcinoma and 37 cases of nasopharyngeal chronic inflammation tissue. Survival was analyzed by Kaplan-Meier method and Log rank test, and the influencing factors was analyzed by Cox regression model. Results: The expression levels of CLOCK mRNA in CNE1, CNE2 and 5-8F cells (0.63±0.07, 0.91±0.02 and 0.33±0.04, respectively) were lower than that in NP69 cell (1.00±0.00, P<0.05). The expression levels of CLOCK protein in CNE1, CNE2 and 5-8F cells (0.79±0.06, 0.57±0.05 and 0.74±0.10, respectively) were lower than that of NP69 cells (1.00±0.00, P<0.05). The expressions of CLOCK mRNA in nasopharyngeal carcinoma cells including CEN1, CNE2, 5-8F and normal nasopharyngeal epithelial cell NP69 were different at different time points, with temporal fluctuations. The fluctuation periods of CLOCK mRNA in CNE1, CNE2, 5-8F, and NP69 cells were 16, 14, 22 and 24 hours, respectively. The peak and trough times were ZT10: 40 and ZT18: 40, ZT10 and ZT3, ZT14: 30 and ZT3: 30, ZT12: 39 and ZT0: 39, respectively. CLOCK mRNA and protein expression levels in nasopharyngeal carcinoma tissues (0.37±0.20 and 0.20±0.26, respectively) were lower than those in nasopharyngeal chronic inflammation tissues (1.00±0.00 and 0.51±0.41, respectively, P<0.05). The 1, 3, and 5-year survival rates of patients in the CLOCK protein high expression group (CLOCK protein expression level ≥ 0.178) were 96.2%, 92.1%, and 80.1%, respectively, which were higher than those in the low expression group (CLOCK protein expression level <0.178, 92.9% , 78.6% and 57.1%, respectively, P=0.009). The 1, 3, and 5-year progression-free survival (PFS) rates of patients in the CLOCK protein high expression group were 96.2%, 87.8%, and 87.7%, respectively, which were higher than those in the low expression group (92.7%, 82.2%, and 70.8%, respectively, P=0.105). Compared with the low-expression group (100.0%, 96.9%, and 90.0%, respectively), the 1, 3, and 5-year recurrence-free survival rates of patients in the CLOCK protein high expression group (100.0%, 95.7%, and 95.7%, respectively) were not statistically significant (P=0.514). Compared with the low-expression group (92.7%, 82.2%, and 79.3%), the 1, 3, and 5-year survival rates without metastasis in the CLOCK protein high expression group (96.2%, 92.0%, and 92.0%, respectively) were not statistically significant (P=0.136). CLOCK protein expression and T stage were independent prognostic factors of overall survival (P<0.05). Conclusions: The expression of CLCOK is downregulated in the nasopharyngeal carcinoma cell and nasopharyngeal carcinoma tissues. Clock gene CLOCK is rhythmically expressed in the nasopharyngeal carcinoma cells and normal nasopharyngeal epithelial cells. Compared with normal nasopharyngeal epithelial cells, the fluctuation period of CLOCK in nasopharyngeal carcinoma cells is shortened. The overall survival of patients in the CLOCK protein high expression group is better than that of low expression group. The expression of CLOCK protein is an independent influencing factor for overall survival. CLOCK gene may be a potential tumor suppressor gene in the nasopharyngeal carcinoma.

Phosphatase and tensin homolog (PTEN) suppresses triacylglycerol accumulation and monounsaturated fatty acid synthesis in goat mammary epithelial cells.

Phosphatase and tensin homolog (PTEN) is a well-known tumor suppressor in nonruminants and regulates various cellular processes including growth through dephosphorylation of phosphoinositide substrates. Although studies with bovine mammary tissue suggested a role for PTEN during lactation, its potential role in lipid metabolism remains unknown. Objectives of the present study were to determine PTEN abundance in goat mammary tissue at 2 stages of lactation (n = 6 Xinong Saanen dairy goats per stage), and to use gene-silencing and adenoviral transfections in vitro with isolated goat mammary epithelial cells (GMEC) to evaluate the role of PTEN abundance of lipid metabolism-related genes. Abundance of PTEN decreased by 51.5% at peak lactation compared with the dry period. The PTEN was overexpressed in isolated GMEC through adenoviral transfection using an adenovirus system with Ad-GFP (recombinant adenovirus of green fluorescent protein) as control, and silenced via targeted small interfering RNA (siRNA) transfection with a scrambled small interfering RNA as a negative control. Cell culture was performed for 48 h before RNA extraction, triacylglycerol (TAG) analysis, and fatty acid analysis. Overexpression of PTEN downregulated abundance of acetyl-coenzyme A carboxylase α (ACACA), fatty acid synthase (FASN), sterol regulatory element binding transcription factor1 (SREBF1), stearoyl-coenzyme A desaturase 1 (SCD1), diacylglycerol acytransferase 1 (DGAT1), 1-acylglycerol-3-phosphate O-acyltransferase 6 (AGPAT6) coupled with an increase in patatin-like-phospholipase domain containing 2 (PNPLA2), hormone-sensitive lipase (LIPE), and carnitine palmitoyltransferase 1 ß (CPT1B). Furthermore, overexpressing PTEN in vitro resulted in a significant decrease in TAG concentration and concentration of C16:1. In contrast, interference of PTEN led to an opposite effect on lipid metabolism in GMEC. These changes suggested a shift from lipogenesis and esterification to lipolysis and fatty acid oxidation. Collectively, PTEN seems to play a role in monounsaturated fatty acids synthesis and lipid accumulation in GMEC.

[Application of diffusion tensor imaging combined with virtual reality three-dimensional reconstruction in the operation of gliomas involved eloquent regions].

OBJECTIVE: To investigate the values of diffusion tensor imaging (DTI) and virtual reality (VR) techniques in design surgery program of gliomas near eloquent regions. METHODS: In this study, 35 cases were retrospectively analyzed with gliomas involved language areas or rolandic regions operated in Department of Neurosurgery, Peking University Third Hospital from January 2015 to January 2019. Surgery programs were performed by Dextroscope virtual reality system. The pre-operative data, such as the magnetic resonance imaging (MRI), magnetic resonance arteriography (MRA) and DTI was transferred into the VR computer for restitution,Tumors, neural fiber tracts and blood vessels were reconstructed to simulate operation and design individual surgical plan. Neurological function was evaluated 1 week, 1 month and 3 months after operation. RESULTS: Virtual reality three-dimensional images of the 35 cases were successfully achieved, including neural fiber tracts,blood vessels and the lesions. The displacement and destruction of fiber tracts, the anatomic relationship between tumor and important fiber bundle, artery and vein could be shown clearly. Surgical simulation and surgery program of VR of the 35 patients were successfully performed. The 3D images obtained from virtual reality near to the real surgery. Ten of the 35 cases were defined as rolandic regions tumors, 14 of the 35 cases were defined as language areas tumors and 11 of the 35 cases involved both language areas and rolandic regions. Complete resection of enhancing tumor (CRET) was achieved in 30 cases (85.7%), subtotal resection in 5 cases (14.3%), neurological function improved in 34 cases (97.1%) after operation,and 1 case had no improvement compared with that before(2.9%). Thirteen cases without neurological deficit pre-operation, showed transient neurological deficit ,which were recovered about 10 days post-operation, 12 of 22 cases with pre-operative neurologic deficit, improved one week postoperation, 9 of 22 cases with pre-operative neurologic deficit improved one month after operation, the rest 1 case was recurrent with glioblastoma with aggravated hemiplegia symptom after operation, who died of cerebral hernia 2 months later. CONCLUSION: Dextroscope virtual reality system can clearly expose and quantify the 3D anatomic relationship of tumors, neural fiber tracts and blood vessels surrounding gliomas near eloquent regions, which is helpful to design the best individualized surgery program, to improve surgical effect.

[Study on unprotected anal intercourse behavior in HIV-positive men who have sex with men in the context of knowing their HIV infection status in Chengdu].

Objective: To identify related factors associated with unprotected anal intercourse (UAI) among HIV-positive men who have sex with men (MSM) in the context of knowing their HIV infection status. Methods: HIV positive MSM who known that they had been infected with HIV for more than 6 months and accepted follow up services were recruited by convenience sampling method in Chengdu 2015. Semi-structured questionnaire was used to collect their information, such as demographic characteristics, antiviral therapy and sexual behavior characteristics etc. Logistic regression model was used for univariate and multivariate analyses. Results: A total of 330 HIV- positive MSM were recruited, 201 eligible MSM were interviewed. The prevalence of UAI in recent six months was 18.41% (37/201). The results of multivariate logistic regression analysis revealed that the number of anal intercourse with male ≥3 in last month (OR=6.22, 95%CI: 1.88-20.56), low education level (OR=7.29, 95%CI: 1.36-39.16), married, divorced or widowed status (OR=4.65, 95%CI: 1.13-19.17), homosexual cohabitation (OR=3.32, 95%CI: 1.01-10.95) were the risk factors related with UAI among the HIV-positive MSM. Conclusion: Frequent homosexual anal intercourse, low education level, married, divorced or widowed status and homosexual cohabitation might be the risk factors related with UAI in HIV-positive MSM in Chengdu.

Prognostic significance of downregulated BMAL1 and upregulated Ki-67 proteins in nasopharyngeal carcinoma.

This study assessed the prognostic value of BMAL1 and Ki-67 expression in patients with nasopharyngeal carcinoma. Level of BMAL1 mRNA was assessed in tissue specimens from 36 nasopharyngeal carcinomas and 20 nasopharyngeal chronic inflammations using quantitative reverse transcriptase-polymerase chain reaction. Expression of BMAL1 and Ki-67 proteins was analyzed immunohistochemically in 90 paired nasopharyngeal carcinoma and distant normal tissues. The Kaplan-Meier curves and the Log-rank test were used to calculate prognostic significance stratified by BMAL1 and Ki67 protein expression and the COX regression model was to analyze the multivariate prognosis. BMAL1 mRNA was significantly reduced in nasopharyngeal carcinoma (4.67 ± 0.27 versus 6.64 ± 0.51 in chronic inflammation tissues, p = 0.002). Level of BMAL1 mRNA was associated with tumor distant metastasis (3.37 ± 0.66 versus 5.04 ± 0.27 compared with non-metastasis, p = 0.011). Level of BMAL1 protein was also reduced in tumor tissues and BMAL1 expression was associated with better 1-, 3- and 5-year overall survival (OS) of cancer patients (92.6%, 69.2% and 62.3% versus 59.1%, 40.9% and 0% in patients with low BMAL1 expressed tumors; p = 0.000). BMAL1 expression and age were independent prognostic factors for OS (p = 0.032). Furthermore, Ki-67 expression was high in tumor versus normal tissues and associated with poor OS of cancer patients (p = 0.035). The Pearson correlation analysis showed that there was an inverse association between BMAL1 and Ki-67 protein expression (p = 0.021). This study demonstrated that lost BMAL1 and Ki-67 overexpression were associated with poor OS of nasopharyngeal carcinoma patients.

A randomized phase II trial of induction chemotherapy followed by cisplatin chronotherapy versus constant rate delivery combined with radiotherapy.

Chronotherapy is no longer a novel concept in cancer treatment after approximately 20 years of development. Many clinical trials have provided strong supporting evidence that chronomodulated treatment yields better results than a traditional dosage regimen. This study aimed to evaluate the adverse reactions, effect on immune functions, and therapeutic efficacy of chronomodulated infusion versus flat intermittent infusion of cisplatin (DDP) combined with intensity-modulated radiation therapy (IMRT) in patients with locoregionally advanced nasopharyngeal carcinoma (NPC). A total of 148 patients with biopsy-diagnosed untreated stage III-IVb NPC were randomly assigned to undergo two cycles of chronomodulated infusion (study group) or flat intermittent infusion (control group) of DDP (100 mg/m2 on day 1, 21 days/cycle) synchronized with radical radiotherapy. Patients in the study group received chronomodulated infusion, with peak delivery of DDP at 16:00 pm. Patients in the control group received a routine constant rate of infusion. Both groups were treated with the same radiotherapy techniques. Over a median follow-up of 20 months, the study group had better outcomes for adverse effects and immune functions compared with the control group. During the phase of concurrent chemoradiotherapy, the incidence of nausea, vomiting, and oral mucositis in the study and control groups was 66.7% and 79.5% (p < 0.05), 47.9% and 71.2% (p < 0.05), and 73.9% and 87.7% (p < 0.05), respectively. There was no significant difference in 2-year overall survival, progression-free survival, and distant metastasis-free survival between the two groups (p > 0.05). Chronochemotherapy significantly reduced the incidence of adverse reactions and enhanced the tolerance for treatment without affecting survival. It is worth mentioning that reduced destruction of immune function is a novel area of exploration in chronotherapy research.

KCTD12 promotes tumorigenesis by facilitating CDC25B/CDK1/Aurora A-dependent G2/M transition.

Cell cycle dysregulation leads to uncontrolled cell proliferation and tumorigenesis. Understanding the molecular mechanisms underlying cell cycle progression can provide clues leading to the identification of key proteins involved in cancer development. In this study, we performed proteomics analysis to identify novel regulators of the cell cycle. We found that potassium channel tetramerization domain containing 12 (KCTD12) was significantly upregulated in M phase compared with S phase. We also found that KCTD12 overexpression not only facilitated the G2/M transition and induced cancer cell proliferation, but also promoted the growth of subcutaneous tumors and Ki-67 proliferation index in mice. Regarding the mechanism underlying these phenomena, cyclin-dependent kinase 1 (CDK1) was identified as an interacting partner of KCTD12 by immunoprecipitation and mass spectrometry analysis, which showed that KCTD12 activated CDK1 and Aurora kinase A (Aurora A) and that the effects of KCTD12 on CDK1 phosphorylation and cell proliferation were abrogated by cell division cycle 25B (CDC25B) silencing. In addition, Aurora A phosphorylated KCTD12 at serine 243, thereby initiating a positive feedback loop necessary for KCTD12 to exert its cancer-promoting effects. Furthermore, we analyzed the expression levels of various genes and the correlations between the expression of these genes and survival using tumor tissue microarray and Gene Expression Omnibus (GEO) data sets. The data showed that KCTD12 expression was significantly upregulated in cervical and lung cancers. More importantly, high KCTD12 expression was associated with larger tumor sizes, higher pathological stages and poor patient survival. Collectively, our study demonstrate that KCTD12 binds to CDC25B and activates CDK1 and Aurora A to facilitate the G2/M transition and promote tumorigenesis and that Aurora A phosphorylates KCTD12 at serine 243 to trigger a positive feedback loop, thereby potentiating the effects of KCTD12. Thus, the KCTD12-CDC25B-CDK1-Aurora A axis has important implications for cancer diagnoses and prognoses.

MicroRNA-377 suppresses initiation and progression of esophageal cancer by inhibiting CD133 and VEGF.

Esophageal cancer is one of the most lethal cancers worldwide with poor survival and limited therapeutic options. The discovery of microRNAs created a new milestone in cancer research. miR-377 is located in chromosome region 14q32, which is frequently deleted in esophageal squamous cell carcinoma (ESCC), but the biological functions, clinical significance and therapeutic implication of miR-377 in ESCC are largely unknown. In this study, we found that miR-377 expression was significantly downregulated in tumor tissue and serum of patients with ESCC. Both tumor tissue and serum miR-377 expression levels were positively correlated with patient survival. Higher serum miR-377 expression was inversely associated with pathologic tumor stage, distant metastasis, residual tumor status and chemoradiotherapy resistance. The roles of miR-377 in suppressing tumor initiation and progression, and the underlying molecular mechanisms were investigated. Results of in vitro and in vivo experiments showed that miR-377 overexpression inhibited the initiation, growth and angiogenesis of ESCC tumors as well as metastatic colonization of ESCC cells, whereas silencing of miR-377 had opposite effects. Mechanistically, miR-377 regulated CD133 and VEGF by directly binding to their 3' untranslated region. Moreover, systemic delivery of formulated miR-377 mimic not only suppressed tumor growth in nude mice but also blocked tumor angiogenesis and metastasis of ESCC cells to the lungs without overt toxicity to mice. Collectively, our study established that miR-377 plays a functional and significant role in suppressing tumor initiation and progression, and may represent a promising non-invasive diagnostic and prognostic biomarker and therapeutic strategy for patients with ESCC.

Liver X receptor α promotes the synthesis of monounsaturated fatty acids in goat mammary epithelial cells via the control of stearoyl-coenzyme A desaturase 1 in an SREBP-1-dependent manner.

Stearoyl-coenzyme A desaturase 1 (SCD1) is a pivotal enzyme in the biosynthesis of monounsaturated fatty acids (MUFA). It is tightly regulated by transcription factors that control lipogenesis. In nonruminants, liver X receptor α (LXRα) is a nuclear receptor and transcription factor that acts as a key sensor of cholesterol and lipid homeostasis. However, the mechanism whereby LXRα regulates the expression and transcriptional activity of SCD1 in ruminant mammary cells remains unknown. In this study with goat mammary epithelial cells (GMEC), the LXRα agonist T 4506585 (T09) markedly enhanced the mRNA expression of SCD1 and sterol regulatory element binding factor 1 (SREBF1). The concentrations of C16:1 and C18:1 and their desaturation indices also were increased by LXRα activation. However, knockdown of LXRα did not alter the mRNA expression of SCD1. Although SCD1 was repressed by SREBF1 knockdown, T09 significantly increased SCD1 expression. Further analysis revealed that the SCD1 promoter activity was activated by LXRα overexpression. The goat SCD1 promoter contains 2 LXR response elements (LXRE), 1 sterol response element (SRE), and 1 nuclear factor Y (NF-Y) binding site. Site-directed mutagenesis of LXRE1, LXRE2, or SRE alone did not eliminate the upregulation of SCD1 when LXRα was overexpressed. In contrast, when NF-Y alone or in combination with SRE was mutated simultaneously, the basal transcriptional activity of the SCD1 promoter was markedly decreased and did not respond to LXRα overexpression. Furthermore, when SREBF1 was knocked down, overexpression of LXRα did not affect the promoter activity of SCD1. Together, these data suggest that LXRα regulates the expression of SCD1 through increasing SREBP-1 abundance to promote interaction with SRE and NF-Y binding sites. The present study provides evidence that LXRα is involved in the synthesis of MUFA in the goat mammary gland through an indirect mechanism.

Thyroid hormone responsive (THRSP) promotes the synthesis of medium-chain fatty acids in goat mammary epithelial cells.

In nonruminants, thyroid hormone responsive (THRSP) is a crucial protein for cellular de novo lipogenesis. However, the role of THRSP in regulating the synthesis of milk fatty acid composition in goat mammary gland remains unknown. In the present study, we compared gene expression of THRSP among different goat tissues. Results revealed that THRSP had the highest expression in subcutaneous fat, and expression was higher during lactation compared with the dry period. Overexpression of THRSP upregulated the expression of fatty acid synthase (FASN), stearoyl-coenzyme A desaturase 1 (SCD1), diacylglycerol acyltransferase 2 (DGAT2), and glycerol-3-phosphate acyltransferase (GPAM) in goat mammary epithelial cells. In contrast, overexpression of THRSP led to downregulation of thrombospondin receptor (CD36) and had no effect on the expression of acetyl-coenzyme A carboxylase α (ACACA) and sterol regulatory element binding transcription factor1 (SREBF1). In addition, overexpressing THRSP in vitro resulted in a significant increase in triacylglycerol (TAG) concentration and the concentrations of C12:0 and C14:0. Taken together, these results highlight an important role of THRSP in regulating lipogenesis in goat mammary epithelial cells.

Suppression of N-Ras by shRNA-expressing plasmid increases sensitivity of HepG2 cells to vincristine-induced growth inhibition.

Oncogenic ras genes relate to the development of human cancers. In this study, we used a plasmid-mediated short-hairpin RNA (shRNA) targeting N-ras gene to combine with clinical drug vincristine (VCR) for the treatment of human hepatoma cells. Our results showed that anti-N-Ras shRNA expression vector (pCSH1-shNR) knocked down the target mRNA and protein. Higher efficacy on growth inhibition was observed when pCSH1-shNR was combined with VCR. This synergistic effect was associated with abrogation of VCR-induced overexpressions of P-glycoprotein and multidrug resistance-associated protein 1 by pCSH1-shNR through downregulations of N-Ras and total Ras. Western blot analysis showed that pCSH1-shNR-induced downregulations of N-Ras and total Ras were potentiated by VCR. Following Ras downregulation, phosphorylations of ERK1/2 and Akt were dramatically inhibited by combinatory treatment. The data showed that pCSH1-shNR-induced inhibition of nuclear factor-kappaB was enhanced by VCR. In addition, the combination of pCSH1-shNR and VCR synergistically inhibited the growth of human hepatoma HepG2 in vivo. Our findings suggested that combination of gene-specific therapeutics and appropriate chemotherapeutic agents might be a promising approach for the treatment of human hepatocellular carcinoma.

Glucose-regulated protein 78 as a novel effector of BRCA1 for inhibiting stress-induced apoptosis.

The tumor suppressor BRCA1 is mutated in a high percentage of familial breast and ovarian cancer, but our understanding of its mechanisms of action remains incomplete. We report here that glucose-regulated protein (GRP)-78, a critical regulator of the unfolded protein response (UPR), is a novel downstream target of BRCA1. We showed that overexpression of wild-type BRCA1 suppressed the expression of GRP78, whereas expression of mutant BRCA1 gene or targeted inhibition of endogenous BRCA1 using small-interfering RNA (siRNA) enhanced GRP78 expression. Knockdown of BRCA1 also led to induction of other components of UPR, such as GRP94 and CHOP. Consistent with a role of BRCA1 knockdown in mediating cell survival, forced expression of GRP78 stimulated cell proliferation and prevented apoptosis, including that induced by endoplasmic reticulum stress and chemotherapy, in ovarian OVCAR-3 and breast MCF-7 cancer cells. Overexpression of wild-type BRCA1 could increase the apoptosis of GRP78-overexpressing cells. Conversely, knockdown GRP78 by siRNA sensitized ovarian and breast cancer cells to apoptosis. This effect was reduced when the expression of BRCA1 was simultaneously knockdown by siRNA, indicating that BRCA1 also negatively regulates GRP78-mediated cell survival and resistance to apoptosis.

Functional proteomics to identify critical proteins in signal transduction pathways.

Reversible protein phosphorylation plays a crucial role in the regulation of signaling pathways that control various biological responses, such as cell growth, differentiation, invasion, metastasis and apoptosis. Proteomics is a powerful research approach for fully monitoring global molecular responses to the activation of signal transduction pathways. Identification of different phosphoproteins and their phosphorylation sites by functional proteomics provides informational insights into signaling pathways triggered by all kinds of factors. This review summarizes how functional proteomics can be used to answer specific questions related to signal transduction systems of interest. By examining our own example on identifying the novel phosphoproteins in signaling pathways activated by EB virus-encoded latent membrane protein 1 (LMP1), we demonstrated a functional proteomic strategy to elucidate the molecular activity of phosphorylated annexin A2 in LMP1 signaling pathway. Functional profiling of signaling pathways is promising for the identification of novel targets for drug discovery and for the understanding of disease pathogenesis.

Ligand variation in the transferrin family: the crystal structure of the H249Q mutant of the human transferrin N-lobe as a model for iron binding in insect transferrins.

Proteins of the transferrin (Tf) family play a central role in iron homeostasis in vertebrates. In vertebrate Tfs, the four iron-binding ligands, 1 Asp, 2 Tyr, and 1 His, are invariant in both lobes of these bilobal proteins. In contrast, there are striking variations in the Tfs that have been characterized from insect species; in three of them, sequence changes in the C-lobe binding site render it nonfunctional, and in all of them the His ligand in the N-lobe site is changed to Gln. Surprisingly, mutagenesis of the histidine ligand, His249, to glutamine in the N-lobe half-molecule of human Tf (hTf/2N) shows that iron binding is destabilized and suggests that Gln249 does not bind to iron. We have determined the crystal structure of the H249Q mutant of hTf/2N and refined it at 1.85 A resolution (R = 0.221, R(free) = 0.246). The structure reveals that Gln249 does coordinate to iron, albeit with a lengthened Fe-Oepsilon1 bond of 2.34 A. In every other respect, the protein structure is unchanged from wild-type. Examination of insect Tf sequences shows that the K206.K296 dilysine pair, which aids iron release from the N-lobes of vertebrate Tfs, is not present in the insect proteins. We conclude that substitution of Gln for His does destabilize iron binding, but in the insect Tfs this is compensated by the loss of the dilysine interaction. The combination of a His ligand with the dilysine pair in vertebrate Tfs may have been a later evolutionary development that gives more sophisticated pH-mediated control of iron release from the N-lobe of transferrins.

Selective activation of mitomycin A by thiols to form DNA cross-links and monoadducts: biochemical basis for the modulation of mitomycin cytotoxicity by the quinone redox potential.

Mitomycin A (MA) but not mitomycin C (MC) cross-linked linearized (32)P-pBR322 DNA in the presence of dithiothreitol (DTT) or glutathione (GSH), as shown by a sensitive DNA cross-link assay. Incubation of calf-thymus DNA with MA and DTT or mercaptoethanol (MER) resulted in the formation of MA-DNA adducts, which were isolated from nuclease digests of the drug-DNA complexes by HPLC. The adducts were characterized by their UV absorption spectra, electrospray ionization mass spectrometry (ESIMS), and facile conversion from 7-methoxy- to 7-amino-substituted mitosene type adducts upon 10% NH(4)OH treatment, which were identical with known adducts of MC. Both DNA interstrand and intrastrand cross-link adducts, linking two deoxyguanosine residues at N(2), as well as several deoxyguanosine-N(2) monoadducts of MA, were identified. No DNA adducts were formed with MC under the same conditions. A specificity of DNA cross-link formation for the CpG sequence was observed using 12-mer synthetic oligodeoxyribonucleotides as substrates and as DNA sequence models, in analogy to the known CpG sequence specificity of MC-induced DNA cross-links. MA is known to be more cytotoxic by 2-3 orders of magnitude than MC, and this property correlates with redox potentials of MA (-0.19 V) and MA analogues that are higher than those of MC (-0.40 V) and its analogues. It is suggested that the biochemical basis for the higher cytotoxic potency of MA is MA's propensity to be reductively activated by cellular thiols while MC is resistant to thiol activation. This distinction is probably derived from the large difference between the quinone redox potentials of the two drugs.

Crystal structures and iron release properties of mutants (K206A and K296A) that abolish the dilysine interaction in the N-lobe of human transferrin.

Human transferrin (Tf) is responsible for the binding and transport of iron in the bloodstream of vertebrates. Delivery of this bound iron to cells occurs by a process of receptor-mediated endocytosis during which Tf releases its iron at the reduced endosomal pH of approximately 5.6. Iron release from Tf involves a large conformational change in which the two domains that enclose the binding site in each lobe move apart. We have examined the role of two lysines, Lys206 and Lys296, that form a hydrogen-bonded pair close to the N-lobe binding site of human Tf and have been proposed to form a pH-sensitive trigger for iron release. We report high-resolution crystal structures for the K206A and K296A mutants of the N-lobe half-molecule of Tf, hTf/2N, and quantitative iron release data on these mutants and the double mutant K206A/K296A. The refined crystal structures (for K206A, R = 19.6% and R(free) = 23.7%; for K296A, R= 21.2% and R(free) = 29.5%) reveal a highly conserved hydrogen bonding network in the dilysine pair region that appears to be maintained even when individual hydrogen bonding groups change. The iron release data show that the mutants retain iron to a pH 1 unit lower than the pH limit of wild type hTf/2N, and release iron much more slowly as a result of the loss of the dilysine interaction. Added chloride ions are shown to accelerate iron release close to the pH at which iron is naturally lost and the closed structure becomes destabilized, and to retard it at higher pH.

Function of GATA transcription factors in hydroxyurea-induced HEL cells.

HEL cells, a human erythroleukemia cell line, mainly express the fetal (gamma) globin gene and trace amount of the embryonic (epsilon) globin gene, but not adult (beta) globin gene. Here we show that hydroxyurea (HU) can induce HEL cells to express adult (beta) globin gene and lead these cells to terminal differentiation. Results showed in Gel mobility shift assays that GATA factors could specifically bind to the regulatory elements of human beta-globin gene, including the proximal regulatory element (the beta-promoter) and the distal regulatory elements (the DNase I hypersensitive sites in the LCR, HS2-HS4 core sequences). However, the DNA binding patterns of GATA factors were quite different between HU-induced and uninduced HEL cells. Western-blot analysis of nuclear extracts from both the uninduced and HU-induced HEL cells revealed that the level of GATA-2 transcription factor decreased, whereas the level of GATA-1 transcription factor increased following the time of hydroxyurea induction. Furthermore, using RT-PCR analysis the expression of human beta-globin gene in HU-induced HEL cells could be blocked again when HEL cells were incubated in the presence of antisense oligonucleotides for hGATA-1, suggesting that the upregulation of hGATA-1 transcription factor might be critical for the expression of human beta-globin gene in HU-induced HEL cells.

[Characterization of cell death induced by anticancer antibiotic lidamycin in human hepatoma BEL-7402 cells].

AIM: To study the features of cell death induced by the anticancer antibiotic lidamycin (LDM) in human hepatoma BEL-7402 cells. METHODS: Chromatin condensation was observed by co-staining with fluorescent dyes, hoechst 33342 and propidium iodide. "G1 sub-peak" was detected by flow cytometry and DNA ladder was observed using agarose gel electrophoresis. The caspase-3, 6 activities were measured with kits specific for them. RESULTS: Typical apoptotic chromatin condensations appeared when the BEL-7402 cells were treated with the conventional antitumor agent mitomycin C30 mumol.L-1 for 12 h. However, an abnormal type of chromatin condensation occurred when the cells were treated with LDM 1 mumol.L-1 for 6 h, which was characterized with keeping the completeness of nuclear membrane and not forming apoptotic bodies. The DNA ladder patterns were observed using agarose gel electrophoresis. The "G1 sub-peak" occurred only in the cells treated with LDM for 24 h, though chromatin condensation was earlier detected in treatment with LDM for 6 h. The caspase-3, 6 activities were increased about 5 and 4 folds, after the cells were treated with LDM 1 mumol.L-1 for 6 h, as did mitomycin C. The time of initiating chromatin condensation was earlier than that of the high peak activities of caspase-6. CONCLUSION: The characterization of cell death induced by lidamycin in the human hepatoma BEL-7402 cells differs from typical apoptosis. The results make it helpful to explain the molecular mechanism of the highly potent cytotoxicities of lidamycin toward tumor cells.

The chloride effect is related to anion binding in determining the rate of iron release from the human transferrin N-lobe.

The major function of human transferrin is to deliver iron from the bloodstream to actively dividing cells. Upon iron release, the protein changes its conformation from 'closed' to 'open'. Extensive studies in vitro indicate that iron release from transferrin is very complex and involves many factors, including pH, the chelator used, an anion effect, temperature, receptor binding and intra-lobe interactions. Our earlier work [He, Mason and Woodworth (1997) Biochem. J. 328, 439-445] using the isolated transferrin N-lobe (recombinant N-lobe of human transferrin comprising residues 1-337; hTF/2N) has shown that anions and pH modulate iron release from hTF/2N in an interdependent manner: chloride retards iron release at neutral pH, but accelerates the reaction at acidic pH. The present study supports this idea and further details the nature of the dual effect of chloride: the anion effect on iron release is closely related to the strength of anion binding to the apoprotein. The negative effect seems to originate from competition between chloride and the chelator for an anion-binding site(s) near the metal centre. With decreasing pH, the strength of anion binding to hTF/2N increases linearly, decreasing the contribution of competition with the chelator. In the meantime, the 'open' or 'loose' conformation of hTF/2N, induced by the protonation of critical residues such as the Lys-206/Lys-296 pair at low pH, enables chloride to enter the cleft and bind to exposed side chains, thereby promoting cleft opening and synergistically allowing removal of iron by the chelator, leading to a positive anion effect. Disabling one or more of the primary anion-binding residues, namely Arg-124, Lys-206 and Lys-296, substantially decreases the anion-binding ability of the resulting mutant proteins. In these cases, the competition for the remaining binding residue(s) is increased, leading to a negative chloride effect or, at most, a very small positive effect, even at low pH.

Mutations at the histidine 249 ligand profoundly alter the spectral and iron-binding properties of human serum transferrin N-lobe.

Human serum transferrin is an iron-binding and -transport protein which carries iron from the blood stream into various cells. Iron is held in two deep clefts located in the N- and C-lobes by coordinating to four amino acid ligands, Asp 63, Tyr 95, Tyr 188, and His 249 (N-lobe numbering), and to two oxygens from carbonate. We have previously reported the effect on the iron-binding properties of the N-lobe following mutation of the ligands Asp 63, Tyr 95, and Tyr 188. Here we report the profound functional changes which result from mutating His 249 to Ala, Glu, or Gln. The results are consistent with studies done in lactoferrin which showed that the histidine ligand is critical for the stability of the iron-binding site [H. Nicholson, B. F. Anderson, T. Bland, S. C. Shewry, J. W. Tweedie, and E. N. Baker (1997) Biochemistry 36, 341-346]. In the mutant H249A, the histidine ligand is disabled, resulting in a dramatic reduction in the kinetic stability of the protein toward loss of iron. The H249E mutant releases iron three times faster than wild-type protein but shows significant changes in both EPR spectra and the binding of anion. This appears to be the net effect of the metal ligand substitution from a neutral histidine residue to a negative glutamate residue and the disruption of the "dilysine trigger" [MacGillivray, R. T. A., Bewley, M. C., Smith, C. A., He, Q.-Y., Mason, A. B., Woodworth, R. C., and Baker, E. N. (2000) Biochemistry 39, 1211-1216]. In the H249Q mutant, Gln 249 appears not to directly contact the iron, given the similarity in the spectroscopic properties and the lability of iron release of this mutant to the H249A mutant. Further evidence for this idea is provided by the preference of both the H249A and H249Q mutants for nitrilotriacetate rather than carbonate in binding iron, probably because NTA is able to provide a third ligation partner. An intermediate species has been identified during the kinetic interconversion between the NTA and carbonate complexes of the H249A mutant. Thus, mutation of the His 249 residue does not abolish iron binding to the transferrin N-lobe but leads to the appearance of novel iron-binding sites of varying structure and stability.