cervical cancer derived cell lines c33a  (ATCC)

Journal: International Journal of Biological Sciences

Article Title: Human Papillomavirus Types 16 and 18 Early-expressed Proteins Differentially Modulate the Cellular Redox State and DNA Damage

doi: 10.7150/ijbs.21547

Figure Lengend Snippet: Human papillomavirus (HPV) E6 oncoproteins increase the production of reactive oxygen species (ROS) and GSH in C33A cells. Representative images (A) and quantitative data (B) of ROS induced by E6 from HPV types 16 and 18. Quantitative data were obtained 48 hours after transfection in cells expressing E6 from each viral type, comparing with p3X transfected cells as control. The mean intensity of ethidium fluorescence was measured in 16 different fields per well per condition using Gen5 TM 3.0 software for image acquisition and quantification. E6 decreased GSH (C) and did not change C33A cell viability in comparison with the control vector (D). The fluorescence intensity, GSH levels and cell viability are expressed as the mean±SD. Tukey's test *p<0.05 and **p<0.005 vs control (p3X), n=3.

Article Snippet: C33A cervical cancer derived cell line negative for HPV, was acquired from American Type Culture Collection (ATCC) and maintained in Dulbecco's Modified Eagle's Medium (DMEM) F12 medium supplemented with 10% foetal bovine serum (FBS).

Techniques: Transfection, Expressing, Control, Fluorescence, Software, Comparison, Plasmid Preparation

Journal: International Journal of Biological Sciences

Article Title: Human Papillomavirus Types 16 and 18 Early-expressed Proteins Differentially Modulate the Cellular Redox State and DNA Damage

doi: 10.7150/ijbs.21547

Figure Lengend Snippet: Human papillomavirus (HPV) E6 oncoproteins decrease catalase protein levels and activity and do not affect SOD1/2 levels or activity. Representative immunoblot (A) and quantitative densitometric (B) showing E6 expression and catalase protein levels in C33A cells after 48 hours of transfection with Flag-tagged HPV16 E6 or HPV18 E6 and p3X vector; glyceraldehyde 3 phosphate dehydrogenase (GAPDH) was used as a loading control. Catalase enzymatic activity in C33A transfected cells expressing HPV16 or 18 E6 oncoproteins (C). Representative immunoblot (D) and densitometric analysis (E) of superoxide dismutase (SOD) 1 in C33A transfected cells transfected; GAPDH was used as a loading control. Representative immunoblot (F) and densitometric analysis (G) of SOD2 expression, in relation to GAPDH. SOD enzymatic activity in C33A transfected cells (H). Data are expressed as the mean±SD. Tukey's test *p<0.05, **p<0.005, and ***p<0.0005 vs p3X control, n=3.

Article Snippet: C33A cervical cancer derived cell line negative for HPV, was acquired from American Type Culture Collection (ATCC) and maintained in Dulbecco's Modified Eagle's Medium (DMEM) F12 medium supplemented with 10% foetal bovine serum (FBS).

Techniques: Activity Assay, Western Blot, Expressing, Transfection, Plasmid Preparation, Control

Journal: International Journal of Biological Sciences

Article Title: Human Papillomavirus Types 16 and 18 Early-expressed Proteins Differentially Modulate the Cellular Redox State and DNA Damage

doi: 10.7150/ijbs.21547

Figure Lengend Snippet: Human papillomavirus (HPV) E6 oncoproteins increase DNA damage in C33A cells. Representative immunoblots (A) showing total H2AX and γH2AX in C33A cells 48 hours after transfection with Flag-tagged HPV16 E6, HPV18 E6 and p3X vector. Glyceraldehyde 3 phosphate dehydrogenase (GAPDH) was used as a loading control. Densitometry of γH2AX and H2AX proteins (B). Data are expressed as the ratio of relative units between γH2AX and H2AX. Data are shown as the mean±SD. Tukey's test *p<0.05 vs p3X control. n=3.

Article Snippet: C33A cervical cancer derived cell line negative for HPV, was acquired from American Type Culture Collection (ATCC) and maintained in Dulbecco's Modified Eagle's Medium (DMEM) F12 medium supplemented with 10% foetal bovine serum (FBS).

Techniques: Western Blot, Transfection, Plasmid Preparation, Control

Journal: International Journal of Biological Sciences

Article Title: Human Papillomavirus Types 16 and 18 Early-expressed Proteins Differentially Modulate the Cellular Redox State and DNA Damage

doi: 10.7150/ijbs.21547

Figure Lengend Snippet: Human papillomavirus (HPV) E7 oncoproteins decrease reactive oxygen species (ROS) and increase GSH in C33A cells. Representative images (A) and quantitative data (B) of ROS levels in HA-tagged HPV16 E7, HIS-tagged HPV18 E7 and control pcDNA3 expressing cells. Quantitative data were obtained 48 hours after transfection. The mean intensity of ethidium fluorescence was measured in 16 different fields per well per condition using Gen5 TM 3.0 software for image acquisition and quantification. HPV 16 and 18 E7 increase GSH (C) without affecting cell viability (D), in comparison with pcDNA3 transfected cells. The fluorescence and GSH quantification and cell viability are expressed as the mean±SD. Tukey's test *p<0.05 and **p<0.005 vs control (p3X), n=3.

Article Snippet: C33A cervical cancer derived cell line negative for HPV, was acquired from American Type Culture Collection (ATCC) and maintained in Dulbecco's Modified Eagle's Medium (DMEM) F12 medium supplemented with 10% foetal bovine serum (FBS).

Techniques: Control, Expressing, Transfection, Fluorescence, Software, Comparison

Journal: International Journal of Biological Sciences

Article Title: Human Papillomavirus Types 16 and 18 Early-expressed Proteins Differentially Modulate the Cellular Redox State and DNA Damage

doi: 10.7150/ijbs.21547

Figure Lengend Snippet: Human papillomavirus (HPV) E7 oncoproteins increase catalase protein levels and activity and decrease SOD1 protein levels and SOD activity. Representative immunoblot (A) and densitometric analysis (B) of catalase levels in C33A cells 48 hours after transfection with HA-tagged HPV16 E7, HIS-tagged HPV18 E7 or control pcDNA3 plasmids. Catalase enzymatic activity in cells harbouring HPV E7 oncoproteins (C). Representative immunoblot of SOD1 and SOD2 (D, F) and respective densitometric analysis (E, G) in C33A transfected cells. Glyceraldehyde 3 phosphate dehydrogenase (GAPDH) was used as a loading control. SOD enzymatic activity in C33A transfected cells is shown (H). Data is expressed as the mean±SD, Tukey's test *p<005, **p<0.005 and ***p<0.005 vs pcDNA3 control (pcDNA3). n=3.

Article Snippet: C33A cervical cancer derived cell line negative for HPV, was acquired from American Type Culture Collection (ATCC) and maintained in Dulbecco's Modified Eagle's Medium (DMEM) F12 medium supplemented with 10% foetal bovine serum (FBS).

Techniques: Activity Assay, Western Blot, Transfection, Control

Journal: International Journal of Biological Sciences

Article Title: Human Papillomavirus Types 16 and 18 Early-expressed Proteins Differentially Modulate the Cellular Redox State and DNA Damage

doi: 10.7150/ijbs.21547

Figure Lengend Snippet: Human papillomavirus (HPV) E7 oncoproteins do not alter phospho-histone 2 AX (γH2AX) protein levels. Representative immunoblot of total H2AX and γH2AX (A) and densitometric analysis (B) of the ratio γH2AX/H2AX in C33A cells 48 h post-transfection. Glyceraldehyde 3 phosphate dehydrogenase (GAPDH) was used as a loading control. Data are expressed as the ratio of relative units between γH2AX and H2AX. Data are presented as the mean±SD, n=3.

Article Snippet: C33A cervical cancer derived cell line negative for HPV, was acquired from American Type Culture Collection (ATCC) and maintained in Dulbecco's Modified Eagle's Medium (DMEM) F12 medium supplemented with 10% foetal bovine serum (FBS).

Techniques: Western Blot, Transfection, Control

Journal: International Journal of Biological Sciences

Article Title: Human Papillomavirus Types 16 and 18 Early-expressed Proteins Differentially Modulate the Cellular Redox State and DNA Damage

doi: 10.7150/ijbs.21547

Figure Lengend Snippet: Human papillomavirus (HPV) E6/E7 co-expressed oncoproteins do not alter the production of reactive oxygen species (ROS) neither GSH levels in C33A cells. Representative images (A) and quantitative data (B) of ROS levels in HPV16 E6/E7, HPV18 E6/E7 and control pcDNA3 transfected cells. GSH levels were not altered in the presence of E6/E7 oncoproteins from HPV16 and 18 (C). C33A viability is similar in cells expressing E6/E7 oncoproteins in comparison with cells transfected with the control vector (D). Data are presented as the mean±SD, n=3.

Article Snippet: C33A cervical cancer derived cell line negative for HPV, was acquired from American Type Culture Collection (ATCC) and maintained in Dulbecco's Modified Eagle's Medium (DMEM) F12 medium supplemented with 10% foetal bovine serum (FBS).

Techniques: Control, Transfection, Expressing, Comparison, Plasmid Preparation

Journal: International Journal of Biological Sciences

Article Title: Human Papillomavirus Types 16 and 18 Early-expressed Proteins Differentially Modulate the Cellular Redox State and DNA Damage

doi: 10.7150/ijbs.21547

Figure Lengend Snippet: Human papillomavirus (HPV) E6/E7 co-expressed oncoproteins decrease SOD1 protein levels and SOD activity. Representative immunoblot (A) and densitometric analysis (B) of catalase in C33A cells. Catalase enzymatic activity in transfected cells (C). Immunoblot and densitometric analysis of SOD1 (D, E) and SOD2 (F, G). SOD enzymatic activity (H) in C33A transfected cells. Glyceraldehyde 3 phosphate dehydrogenase (GAPDH) was used as a loading control. Data are presented as the mean±SD, Tukey's test *p<0.05 and **p<0.005 vs control (p3X), n=3.

Article Snippet: C33A cervical cancer derived cell line negative for HPV, was acquired from American Type Culture Collection (ATCC) and maintained in Dulbecco's Modified Eagle's Medium (DMEM) F12 medium supplemented with 10% foetal bovine serum (FBS).

Techniques: Activity Assay, Western Blot, Transfection, Control

Journal: International Journal of Biological Sciences

Article Title: Human Papillomavirus Types 16 and 18 Early-expressed Proteins Differentially Modulate the Cellular Redox State and DNA Damage

doi: 10.7150/ijbs.21547

Figure Lengend Snippet: Human papillomavirus (HPV) E6/E7 co-expressed oncoproteins do not affect DNA integrity in C33A cells. Immunoblot of γH2AX, H2AX (A) and densitometric analysis (B) of γH2AX in relation to H2AX. Glyceraldehyde 3 phosphate dehydrogenase (GAPDH) was used as a loading control. Data are expressed as the ratio of relative units between γH2AX and H2AX. Data are presented as the mean±SD, n=3.

Article Snippet: C33A cervical cancer derived cell line negative for HPV, was acquired from American Type Culture Collection (ATCC) and maintained in Dulbecco's Modified Eagle's Medium (DMEM) F12 medium supplemented with 10% foetal bovine serum (FBS).

Techniques: Western Blot, Control

Journal: International Journal of Biological Sciences

Article Title: Human Papillomavirus Types 16 and 18 Early-expressed Proteins Differentially Modulate the Cellular Redox State and DNA Damage

doi: 10.7150/ijbs.21547

Figure Lengend Snippet: Human papillomavirus (HPV) E1/E2 proteins increase the production reactive oxygen species (ROS), while GSH is decreased in the presence of E1, E2 or E1/E2 in C33A transfected cells. Representative images (A) and quantitative data (B) of ROS levels in cells transfected with HPV18 E1, HPV18 E2, HPV18 E1/E2 or pcDNA3 control. Quantitative data were obtained at 48 h post-transfection. The mean intensity of ethidium fluorescence was measured in 16 different fields per well per condition using Gen5 TM 3.0 software for image acquisition and quantification. Presence of GSH (C) and viability (D) in C33 transfected cells. Data are expressed as the mean±SD. Tukey's test *p<0.05 and **p<0.005 vs control (pcDNA3). n=3.

Article Snippet: C33A cervical cancer derived cell line negative for HPV, was acquired from American Type Culture Collection (ATCC) and maintained in Dulbecco's Modified Eagle's Medium (DMEM) F12 medium supplemented with 10% foetal bovine serum (FBS).

Techniques: Transfection, Control, Fluorescence, Software

Journal: International Journal of Biological Sciences

Article Title: Human Papillomavirus Types 16 and 18 Early-expressed Proteins Differentially Modulate the Cellular Redox State and DNA Damage

doi: 10.7150/ijbs.21547

Figure Lengend Snippet: Human papillomavirus (HPV) E1, E2 and E1/E2 increase catalase activity, while SOD1 and SOD2 protein levels and SOD activity decrease. Representative immunoblot (A) and densitometric analysis (B) of catalase levels in C33 cells transfected with HA-tagged HPV18 E1, HPV18 E2, HPV 18 E1/E2 or control pcDNA3 plasmids 48 hours after transfection. Catalase enzymatic activity in C33A transfected cells (C). Representative immunoblot of SOD1 (D) and SOD2 (F) and densitometric analysis of SOD1 (E) and SOD2 (G) levels in C33 cells transfected with HA-tagged HPV18 E1, HPV18 E2, HPV 18 E1/E2 or control pcDNA3 plasmids. Glyceraldehyde 3 phosphate dehydrogenase (GAPDH) was used as a loading control. SOD enzymatic activity in C33A transfected cells (H). Data are presented as mean±SD. *p<0.05, **p<0.005, and ***p<0.0005 vs control (pcDNA3). n=3.

Article Snippet: C33A cervical cancer derived cell line negative for HPV, was acquired from American Type Culture Collection (ATCC) and maintained in Dulbecco's Modified Eagle's Medium (DMEM) F12 medium supplemented with 10% foetal bovine serum (FBS).

Techniques: Activity Assay, Western Blot, Transfection, Control

Journal: International Journal of Biological Sciences

Article Title: Human Papillomavirus Types 16 and 18 Early-expressed Proteins Differentially Modulate the Cellular Redox State and DNA Damage

doi: 10.7150/ijbs.21547

Figure Lengend Snippet: Human papillomavirus (HPV) 18 E1/E2 promote DNA damage in C33A transfected cells. Representative immunoblot (A) and densitometric analysis (B) of γH2AX and H2AX proteins. Glyceraldehyde 3 phosphate dehydrogenase (GAPDH) was used as a loading control. Data are expressed as the ratio of relative units between γH2AX and H2AX at 48 hours post transfection and presented as the mean±SD, Tukey's test **p<0.005 vs pcDNA3 control. n=3.

Article Snippet: C33A cervical cancer derived cell line negative for HPV, was acquired from American Type Culture Collection (ATCC) and maintained in Dulbecco's Modified Eagle's Medium (DMEM) F12 medium supplemented with 10% foetal bovine serum (FBS).

Techniques: Transfection, Western Blot, Control

Journal:

Article Title: Methylation Patterns of Papillomavirus DNA, Its Influence on E2 Function, and Implications in Viral Infection

doi: 10.1128/JVI.77.23.12450-12459.2003

Figure Lengend Snippet: Effect of the global CpG methylation on BPV1 E2-dependent transcriptional activation. (A) Schematic of BPV1 LCR, the BamC promoter, and luciferase reporter plasmid pKT260, illustrating the relative positions of the BPV1 sequences, the basic BamC promoter from EBV, and the firefly luciferase translational ORF. Asterisk, relative position of the E2BS. (B) Bar graph summarizing the luciferase activities measured in lysates from cell populations transfected with methylated or unmethylated pKT260. Eight micrograms of the plasmid pKT260 that had or had not been treated in vitro with CpG methylase was transfected into C33A cells by electroporation in the presence or absence of the BPV1 E2 expression vector pCGE2 (10 μg). Luciferase activities were measured 48 h posttransfection. All experiments were repeated at least three times. Bars (error bars, standard deviations): 1, no DNA transfection control; 2, methylated pKT260 alone; 3 methylated pKT260 plus pCGE2; 4, unmethylated pKT260 alone; 5, unmethylated pKT260 plus pCGE2.

Article Snippet: The human cervical cancer derived cell line C33A and adenovirus-transformed human kidney epithelial cell line 293 were obtained from the American Type Culture Collection and grown in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum.

Techniques: CpG Methylation Assay, Activation Assay, Luciferase, Plasmid Preparation, Transfection, Methylation, In Vitro, Electroporation, Expressing

Journal:

Article Title: Methylation Patterns of Papillomavirus DNA, Its Influence on E2 Function, and Implications in Viral Infection

doi: 10.1128/JVI.77.23.12450-12459.2003

Figure Lengend Snippet: Effect of global CpG methylation on HPV16 E2-dependent transcriptional activation. (A) Schematic of HPV16 LCR, BamC promoter, and luciferase reporter plasmid pKT267, illustrating the relative positions of the HPV16 LCR, the basic BamC promoter from EBV, and the firefly luciferase translational ORF. Asterisks, relative positions of E2BSs. (B) Bar graph summarizing the luciferase activities measured in lysates from cell populations transfected with methylated or unmethylated pKT267. Three micrograms of the plasmid pKT267 that had or had not been treated in vitro with CpG methylase was transfected into C33A cells by electroporation in the presence or absence of 9 μg of the HPV16 E2 expression vector pCMV4E2. Luciferase activities were measured 48 h posttransfection. All experiments were repeated at least three times. Bars: 1, no DNA transfection control; 2 methylated pKT267 alone; 3 methylated pKT267 plus pCMV4E2; 4 unmethylated pKT267 alone; 5 unmethylated pKT267 plus pCMV4E2 (error bar, standard deviation).

Article Snippet: The human cervical cancer derived cell line C33A and adenovirus-transformed human kidney epithelial cell line 293 were obtained from the American Type Culture Collection and grown in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum.

Techniques: CpG Methylation Assay, Activation Assay, Luciferase, Plasmid Preparation, Transfection, Methylation, In Vitro, Electroporation, Expressing, Standard Deviation