cervical cancer cell line c33a  (ATCC)

Journal: International Journal of Molecular Sciences

Article Title: Reactivation of P53 Antiproliferative and Pro-Apoptotic Pathways by Resveratrol in Mutant P53 Cancer Cell Lines

doi: 10.3390/ijms27104481

Figure Lengend Snippet: Dose-dependent effect of resveratrol (RES) on cellular metabolic activity in cancer cell lines with different p53 status. Cells were treated with increasing concentrations of RES (50–250 µM) for 48 h. Cellular metabolic activity was assessed by means of MTT assay and normalized to that of vehicle-treated controls (EtOH 0.3% v / v ). Data represent mean ± SD of at least four independent experiments. IC50 values are indicated for each cell line (presented in box).

Article Snippet: The human cervical cancer cell line C33A ( p53 mutation R273C) was purchased from the American Type Culture Collection (ATCC cat. no. HTB-31 [C-33A]; Manassas, VA, USA).

Techniques: Activity Assay, MTT Assay

Journal: International Journal of Molecular Sciences

Article Title: Reactivation of P53 Antiproliferative and Pro-Apoptotic Pathways by Resveratrol in Mutant P53 Cancer Cell Lines

doi: 10.3390/ijms27104481

Figure Lengend Snippet: Resveratrol induces apoptosis in cancer cell lines with mutant or null p53 . ( A ) C33A, SK-BR-3, and Saos-2 cells were treated with vehicle (ethanol) or resveratrol at their respective IC50s for 48 h. Apoptosis was assessed by means of Annexin V/propidium iodide (PI) double staining followed by flow cytometry analysis. Representative dot plots show viable cells in the lower left quadrant (Annexin V − /PI − ), early apoptotic cells in the lower right quadrant (Annexin V + /PI − ), and late apoptotic cells in the upper right quadrant (Annexin V + /PI + ). ( B ) Quantifications of total apoptotic cells (early + late apoptosis) are shown as the mean ± SD from three independent experiments. ** p < 0.01; *** p < 0.001 versus EtOH. RES, resveratrol; EtOH, control with ethanol.

Article Snippet: The human cervical cancer cell line C33A ( p53 mutation R273C) was purchased from the American Type Culture Collection (ATCC cat. no. HTB-31 [C-33A]; Manassas, VA, USA).

Techniques: Mutagenesis, Double Staining, Flow Cytometry, Control

Journal: International Journal of Molecular Sciences

Article Title: Reactivation of P53 Antiproliferative and Pro-Apoptotic Pathways by Resveratrol in Mutant P53 Cancer Cell Lines

doi: 10.3390/ijms27104481

Figure Lengend Snippet: Resveratrol induces the expression of p53 target genes across different p53 backgrounds. Gene expression analysis was performed in ( A ) C33A ( p53 R273C), ( B ) SK-BR-3 ( p53 R175H), ( C ) MCF-7 ( p53 WT), and ( D ) Saos-2 ( p53 null) cells following treatment with vehicle (EtOH) or resveratrol (RES) at their respective IC50s for 48 h. Relative mRNA levels of p53 target genes were determined by means of RT-qPCR and expressed as fold changes relative to vehicle-treated controls, normalized to the housekeeping gene β2-microglobulin (β2M). Data are presented as the mean ± SD of at least three independent experiments. Statistical significance was evaluated using one-way ANOVA followed by appropriate post hoc multiple comparison tests. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001 versus control.

Article Snippet: The human cervical cancer cell line C33A ( p53 mutation R273C) was purchased from the American Type Culture Collection (ATCC cat. no. HTB-31 [C-33A]; Manassas, VA, USA).

Techniques: Expressing, Gene Expression, Quantitative RT-PCR, Comparison, Control

Journal: International Journal of Molecular Sciences

Article Title: Reactivation of P53 Antiproliferative and Pro-Apoptotic Pathways by Resveratrol in Mutant P53 Cancer Cell Lines

doi: 10.3390/ijms27104481

Figure Lengend Snippet: Resveratrol enhances cisplatin sensitivity in cancer cell lines with mutant or null p53 . ( A ) C33A, ( B ) SK-BR-3, and ( C ) Saos-2 cells were treated with increasing concentrations of cisplatin (CDDP; 2.5–20 μM) in the presence or absence of resveratrol (RES), at the corresponding IC 50 for each cell line, for 48 h. Cellular metabolic activity was assessed via MTT assay and normalized to that of vehicle-treated controls. Data represent the mean ± SD of at least four independent experiments. ( D ) IC50 values for CDDP were calculated using non-linear regression analysis (log [CDDP] vs. normalized response, variable slope model) using GraphPad Prism 6.01. * Previously published results from our working group.

Article Snippet: The human cervical cancer cell line C33A ( p53 mutation R273C) was purchased from the American Type Culture Collection (ATCC cat. no. HTB-31 [C-33A]; Manassas, VA, USA).

Techniques: Mutagenesis, Activity Assay, MTT Assay

Journal: Cell Death & Disease

Article Title: Treating ICB-resistant cancer by inhibiting PD-L1 via DHHC3 degradation induced by cell penetrating peptide-induced chimera conjugates

doi: 10.1038/s41419-024-07073-y

Figure Lengend Snippet: A The expression of ZDHHC3 protein in cancers based on the Human Protein Atlas: ZDHHC3 expression levels are evaluated according to the immunohistochemistry staining on cancer tissue samples of the Human Protein Atlas database. The X axis represents the cancer type. Pan-cancer includes 20 different cancer types. BRCA, breast cancer. CESC, cervical cancer. SKCM, skin cancer and melanoma. HNSC, head and neck cancer. The Y axis represents the percentage of patients with specific expression pattern of ZDHHC3. High, high expression. Low, low expression. None, no expression. B The correlation between PDL1 (CD274) and PD1(PDCD1), ZDHHC1, ZDHHC2, or ZDHHC3 expression in TCGA cancer types. TIMER2.0 Gene_Corr Module is used to evaluate the expression between two genes in different cancer types of TCGA database. The heatmap lists the purity-adjusted partial spearman’s rho (Cor) value as the degree of the correlation between two genes and the number (n) of patient cases in each cancer typer. C Western blot analysis was utilized to examine the expression of PD-L1 protein in MDA-MB-231 and C33A cells following the knockdown of DHHC1, DHHC2, and DHHC3. D A schematic representation of the structural design of cp-PCC. E Western blot analysis was conducted to assess the expression of PD-L1 protein in four cell lines (MDA-MB-231, C33A, FaDu, A375) following a 6-hour incubation with 10 µM PCC16/17/18, each corresponding to CRBN/VHL/IAP-based cp-PCC, respectively. Quantitative data are presented as mean ± standard error. Statistical analysis was performed using one-way ANOVA with Tukey’s post hoc test ( n = 3): * P < 0.05, ** P < 0.01, ***P < 0.001.

Article Snippet: Human cervical cancer cell line C33A, mouse mammary carcinoma cell line 4T1, and mouse cervical cancer cell line U14 were purchased from ATCC.

Techniques: Expressing, Immunohistochemistry, Staining, Western Blot, Knockdown, Incubation

Journal: Cell Death & Disease

Article Title: Treating ICB-resistant cancer by inhibiting PD-L1 via DHHC3 degradation induced by cell penetrating peptide-induced chimera conjugates

doi: 10.1038/s41419-024-07073-y

Figure Lengend Snippet: A – C Western blot analysis was utilized to examine the expression of DHHC3/PD-L1 proteins in C33A cells following 6 h of treatment with varying concentrations of PCC16/17/18. D Comparative analysis of the DC50 values for PCC16/17/18. E – G Western blot analysis to assess the expression of DHHC3/PD-L1 proteins in C33A cells post-incubation with specific concentrations of PCC16/17/18 for different time periods. H Confocal microscopy was employed to observe the fluorescence intensity of PCC16/17/18 entering the cells at various time points. Quantitative data are presented as mean ± standard error. Statistical analysis was performed using one-way ANOVA with Tukey’s post hoc test ( n = 3): * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Human cervical cancer cell line C33A, mouse mammary carcinoma cell line 4T1, and mouse cervical cancer cell line U14 were purchased from ATCC.

Techniques: Western Blot, Expressing, Incubation, Confocal Microscopy, Fluorescence

Journal: Cell Death & Disease

Article Title: Treating ICB-resistant cancer by inhibiting PD-L1 via DHHC3 degradation induced by cell penetrating peptide-induced chimera conjugates

doi: 10.1038/s41419-024-07073-y

Figure Lengend Snippet: A Confocal observation of DHHC3 binding with drug PCC16. Scale bar, 1 µM. B C33A cell lysates treated at different temperatures after incubation with 0.5 µM PCC16, with Western blot analysis of DHHC3 protein expression; C CETSA melting curve plotted. D Flow cytometry analysis of fluorescent cells after treatment with Rhodamine-labeled PCC16 at different concentration gradients; E C33A cells treated with PCC16 over time and concentration gradients, with cell toxicity of PCC16 measured by MTT assay.

Article Snippet: Human cervical cancer cell line C33A, mouse mammary carcinoma cell line 4T1, and mouse cervical cancer cell line U14 were purchased from ATCC.

Techniques: Binding Assay, Incubation, Western Blot, Expressing, Flow Cytometry, Labeling, Concentration Assay, MTT Assay

Journal: Cell Death & Disease

Article Title: Treating ICB-resistant cancer by inhibiting PD-L1 via DHHC3 degradation induced by cell penetrating peptide-induced chimera conjugates

doi: 10.1038/s41419-024-07073-y

Figure Lengend Snippet: A C33A cells pre-treated with/without MG132 for 4 h and then treated with PCC16/17/18, with Western blot analysis to detect PD-L1 protein levels; B Bar graph representing the quantification of grayscale values; C Confocal microscopy observation of the immunofluorescence intensity of PD-L1 protein under PCC16/17/18 ± MG132 conditions. Scale bar, 10 μM. Quantitative data expressed as mean ± standard error; analyzed by one-way ANOVA with Tukey’s post hoc test ( n = 3): * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Human cervical cancer cell line C33A, mouse mammary carcinoma cell line 4T1, and mouse cervical cancer cell line U14 were purchased from ATCC.

Techniques: Western Blot, Confocal Microscopy, Immunofluorescence

Journal: Cell Death & Disease

Article Title: Treating ICB-resistant cancer by inhibiting PD-L1 via DHHC3 degradation induced by cell penetrating peptide-induced chimera conjugates

doi: 10.1038/s41419-024-07073-y

Figure Lengend Snippet: A Western blot analysis of the effects of PCC series drugs and PD-L1 monoclonal antibody on PD-L1 protein levels in C33A cells; B TUNEL assay to assess apoptosis in cervical cancer cells treated with cisplatin and PCC16; C Ki67 assay for evaluating the proliferation of cervical cancer cells treated with cisplatin and PCC16; D Plate cloning experiment analyzing the proliferation of cervical cancer cells treated with cisplatin and PCC16; E In a C33A-T cell co-culture model, ELISA was used to measure IFN-γ and TNF-α expression levels after treatment with different concentrations of PCC16; F Hoechest33528 staining in the co-culture system to observe apoptosis of C33A cells after treatment with different concentrations of PCC16. Quantitative data are expressed as mean ± standard error; analyzed by one-way ANOVA with Tukey’s post hoc test ( n = 3): * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Human cervical cancer cell line C33A, mouse mammary carcinoma cell line 4T1, and mouse cervical cancer cell line U14 were purchased from ATCC.

Techniques: Western Blot, TUNEL Assay, Cloning, Co-Culture Assay, Enzyme-linked Immunosorbent Assay, Expressing, Staining

Journal: mBio

Article Title: HPV oncogenes expressed from only one of multiple integrated HPV DNA copies drive clonal cell expansion in cervical cancer

doi: 10.1128/mbio.00729-24

Figure Lengend Snippet: Transcription and RNA splicing from a single HPV16 DNA integration site identified in CaSki cells. ( A ) Virus-host RNA junctions with ≥2 CJRs that mapped to Chr6. ( B ) The integrated HPV16 DNA on Chr6 in CaSki cells and its expression. The integrated HPV 16 DNA between Chr6 nt 45,691,417 and nt 45,691,384 has a redundant E1 and E2 region on each end with the indicated upstream regulatory region (URR) and tail-to-head junction (nt 7,906/1). The fusion RNA is expressed from the viral early promoter P97 and polyadenylated by using a host PAS on Chr6 nt 45,691,310. Arrows below the genome region are the primers used to validate the expression of the virus-host fusion RNA. RNA-seq reads-coverage (0–8,000 scale) in the integrated virus-host genome region and Sashimi plots for splice junctions were visualized by IGV (middle). Three RNA isoforms, including the pre-mRNA (transcript-1) and the spliced mRNA transcript-2 and -3, are shown (bottom). The number in nt below each isoform RNA indicates the estimated length of the RNA without a pA tail. ( C ) Validation of the virus-host DNA junctions at the virus integration site using the primer sets F5 + F10 and B10 + B4. The PCR products in the red rectangles were gel purified and sequenced, with the sequencing chromatograms on the right. ( D ) The cleavage and polyadenylation sites in the virus-host fusion transcripts were mapped by 3′ RACE using an HPV16 primer F4. The sequencing chromatograms on the right show the virus-host junction, PAS (underlined) site, and cleavage site (CS, vertical arrow). ( E ) Northern blot analysis of total CaSki RNA (10 µg) using an antisense oligo probe B3 ( B ) derived from HPV16 nt 855–836. Total RNA from HPV-negative C33A cells served as a negative control. GAPDH served as an RNA loading control. Major isoforms of the virus-host transcripts detected are shown on the right.

Article Snippet: HPV-negative cervical cancer cell line C33A cells (ATCC) with mutant p53 and mutant pRB were used as an HPV-negative cell control.

Techniques: Virus, Expressing, RNA Sequencing, Biomarker Discovery, Purification, Sequencing, Northern Blot, Derivative Assay, Negative Control, Control

Journal: mBio

Article Title: HPV oncogenes expressed from only one of multiple integrated HPV DNA copies drive clonal cell expansion in cervical cancer

doi: 10.1128/mbio.00729-24

Figure Lengend Snippet: HPV18 transcription and RNA splicing from a single HPV18 DNA integration site in HeLa cells. ( A ) Verification of two integration junction sites in HeLa cells by PCR using two separate primer sets B11 + B9 and F8 + F11 shown in ( E ), with sequencing chromatograms on the gel right. ( B and C ) Mapping of the virus-host RNA CJRs to the HPV18 genome ( B ) and human Chr8 ( C ). ( D ) Distribution of top 11 virus-host CJR species identified by RNA-seq. ( E ) HPV18 DNA integration on Chr8 and its expression in HeLa cells. Diagrams showing the truncated HPV18 genome in size of 4,618 nt in a rearranged Chr8 region with one end of the viral DNA at nt 5,736 in L1 joined to Chr8 DNA at nt 127,218,391 and the other end of the viral DNA at nt 2,497 in E1 joined to Chr8 DNA at nt 127,229,301. Arrows below the chimeric host-virus genome show the primers used for validating the structure and expression of integrated viral DNA. RNA-seq reads-coverage (0–24,620 scale) and Sashimi plots for splice junctions in the Chr8 region with the integrated HPV18 DNA are illustrated by IGV, showing the transcription, splicing, and polyadenylation to produce five RNA isoforms, including the unspliced pre-mRNA transcript-1. The number in nt below each RNA isoform indicates the estimated length of the RNA without a pA tail. ( F ) HPV18 RNA splicing and polyadenylation cleavage sites (CS) were determined by 3′ RACE using HPV18-specific primers F6, F7, or F9. The RACE products were gel-purified and sequenced, with the mapped splice sites, integration junctions, and the PA cleavage site (black arrow) shown on the right chromatograms. ( G ) A viral-host fusion RNA splice junction was identified by RNA-seq and validated by RT-PCR using the primer set B12 + B6, with the sequencing chromatogram on the right. ( H ) Northern blot showing the major viral RNA isoforms transcribed from the identified integration site in HeLa cells. Total RNA (10 µg) and polyA + RNA purified from 100 µg of total RNA were analyzed by Northern blotting using an antisense HPV18 probe ( B7 ). Corresponding RNA samples from an HPV-negative C33A cells served as negative controls. GAPDH RNA served as a sample loading control.

Article Snippet: HPV-negative cervical cancer cell line C33A cells (ATCC) with mutant p53 and mutant pRB were used as an HPV-negative cell control.

Techniques: Sequencing, Virus, RNA Sequencing, Expressing, Purification, Reverse Transcription Polymerase Chain Reaction, Northern Blot, Control

Journal: mBio

Article Title: HPV oncogenes expressed from only one of multiple integrated HPV DNA copies drive clonal cell expansion in cervical cancer

doi: 10.1128/mbio.00729-24

Figure Lengend Snippet: Inhibition of viral E6 and E7 expression and cell proliferation and promotion of cell senescence by specific siRNAs targeting to the host portion of virus-host fusion RNAs from a single HPV DNA integration site in HPV16 + CaSki and HPV18 + HeLa cells. ( A and C ) Diagram showing the specific targeting sites of siRNAs, si-C for CaSki ( A ) and si-H for HeLa RNAs ( C ). ( B and D ) Specific siRNAs targeting CaSki (si-C) or HeLa (si-H) host portion of the virus-host fusion transcripts inhibit the expression of viral E6 and E7 proteins but stabilizing p53 in HPV16 + CaSki ( B ) and HPV18 + HeLa cells ( D ). Cell lysates were prepared 48 h after transfection of si-C for CaSki and si-H for HeLa cells, along with non-specific siRNA control (si-NC). The relative HPV E6/E7 and p53 protein levels in each sample were immunoblotted by corresponding antibodies. The signal intensity of each viral protein band was quantified after normalizing with GAPDH serving as the protein loading control. ( E and F ) The siRNAs specific to CaSki (si-C) or HeLa (si-H) portion of the host-virus fusion RNAs inhibited cell proliferation ( E ) but promoted cell senescence ( F ). HPV16 + SiHa and HPV − cervical cancer cells C33A served as cell line controls and si-NC as a siRNA control. A CCK-8 cell proliferation assay was applied to examine the cell proliferation at the indicated time ( H ) after cell transfection once (time zero, vertical arrow) with 40 nM of individual siRNAs. The viable cell numbers (mean ± SE from three experimental repeats) in each group were measured by absorbance at 450 nm ( E ). Cell senescence was examined by β-Gal staining ( F ) at day 8 for the indicated cells transfected twice with 40 nM of siRNA in a 72-h interval after first siRNA transfection. The senescent cells with perinuclear blue staining beyond the background level from each experimental group were counted from ~100 cells from six random fields and averaged (mean ± SE) from three independent experiments ( F ). ** P < 0.01 by Student’s t -test.

Article Snippet: HPV-negative cervical cancer cell line C33A cells (ATCC) with mutant p53 and mutant pRB were used as an HPV-negative cell control.

Techniques: Inhibition, Expressing, Virus, Transfection, Control, CCK-8 Assay, Proliferation Assay, Staining