Article Figures & Data
Figures
- Fig. 1.
HPV16 and E6 and E7 expression levels in HeLa cell subclones. (A) Total RNA was isolated from confluent plates of each indicated HeLa clone. Ten micrograms of total RNA was subjected to electrophoresis in a 1.2% formaldehyde gel, transferred to a positively charged nylon membrane (Amersham), and hybridized with a32P-radiolabeled probe specific for HPV16 E6/E7 mRNA (Top). Hybridization with a GAPDH probe was used to normalize the data for loading variability (bottom). (B) The relative levels of HPV16 E6/E7 mRNA were quantitated using a densitometer (Molecular Dynamics) and normalized against the GAPDH RNA levels. The relative levels of normalized mRNA relative to SiHa HPV16 mRNA levels were graphed. RNA from HPV18-positive HeLa cells was used as the negative control. RNA from the HPV16-positive SiHa cell line was used as a positive control. HPV16 and HPV18 probes do not cross-hybridize under the stringent annealing and washing conditions used.
- Fig. 2.
HPV16 E7 protein levels in HeLa cell subclones. Whole-cell lysates from HPV16 E6- and E7-expressing clones were resolved by SDS-PAGE and then analyzed by immunoblotting with monoclonal antibody 8c9, specific for HPV16 E7, as indicated (top). Extracts from normal HeLa cells were used as a negative control. As positive controls, whole-cell extracts from rat kidney fibroblasts cells constitutively expressing HPV16 E7 (RKO-7.2) (27), as well as from the HPV16-positive cell line SiHa, were used. To control for protein loading, the blot was also probed with an antibody specific for actin (bottom).
- Fig. 3.
Colony reduction assay. Normal HeLa cells, retroviral LXSN vector cells, and HPV16 E6- and E7-expressing HeLa cell clones 1, 2, 16, and 17 were maintained in DMEM supplemented with 10% fetal bovine serum. Cells were seeded into six-well dishes at densities that resulted in approximately 50% confluence after 24 h. As described in Materials and Methods, cells were transfected with 1 μg of pBabe-puro along with 9 μg of vector or the E2TA or E2TR plasmid using Fugene (Boehringer Mannheim). After 21 days, the remaining cells were fixed and stained with methylene blue and the colonies were counted. The averaged total numbers of colonies from three independent experiments are shown. E2TA, full-length BPV-1 E2 protein; E2TR, BPV-1 E2 variant lacking the N terminus transactivation domain; vector, empty expression plasmid.
- Fig. 4.
Dose response of transient HPV16 E6 and E7 expression on E2-mediated growth arrest. HeLa cells were cotransfected with 6 μg of empty vector or the E2TA plasmid along with increasing concentrations of HPV16 E6/E7 plasmid as indicated. After 21 days, the total number of puromycin-resistant colonies was determined as described in the legend to Fig. 3. Results shown are representative of a single experiment.
- Fig. 5.
Northern blot analysis of HPV18 E6/E7 mRNA levels. Cytoplasmic total RNA was isolated from the indicated cell lines transfected with empty vector (vector) or the E2TA or E2TR plasmid and electrophoresed on a 1.2% agarose gel containing formaldehyde. The membrane was hybridized with a 32P-radiolabeled probe specific for HPV18 E6/E7 (A), HPV16 E6/E7 (B), or GAPDH (C) mRNA.
- Fig. 6.
E2TR inhibition of reporter gene transcription in HeLa cells. HeLa cells were cotransfected with 20 ng of the E2TA plasmid, 1 μg of a luciferase reporter plasmid containing four E2-binding sites, and increasing amounts of the E2TR plasmid. Cellular extracts were prepared at 48 h posttransfection, and luciferase activity was measured. The basal activity of the luciferase reporter plasmid in the absence of E2TA was 391. The graphed results are the averages of three independent experiments.
Tables
- Table 1.
Colony reduction assaya
Cell line HPV DNA statusb Status of gene forc: No. of puromycin-resistant colonies after transfection with: pRb p53 Vector E2TA plasmid E2TR plasmid HeLa + wt wt 740 70 762 SiHa + wt wt 111 10 117 C33A − mut mut 1,424 1,486 1,392 U20S − wt wt 564 552 452 a The indicated cell lines were transfected with vector alone or E2TA or E2TR plasmids, along with pBabe-puro. Twenty-four hours after transfection, cell cultures were divided between two 10-cm-diameter plates, allowed to attach, and placed under selection in media containing 0.7 μg of the selection antibiotic/ml. The number of drug-resistant colonies was determined after 3 weeks.
b +, positive; −, negative.
c wt, wild type; mut, mutant.
