Kinetics of cytokine and interferon stimulated gene expression in XSCID canine keratinocytes stimulated with poly(dA:dT).

<p><b>A and B</b> Keratinocytes were seeded into multiple wells and cultured as a monolayer for 24(dA:dT) or medium alone. RNA was extracted after 2, 4, and 6 days post-stimulation. Cytokine (<b>A</b>) and interferon stimulated gene (<b>B</b>) expression was determined by quantitative RT-PCR. Resulting Cq values were normalized to a reference gene and calibrated to mRNA expression in unstimulated keratinocytes (ΔΔCq). Results are expressed as mean +/− SD of three replicate experiments performed in triplicate. Each experiment used keratinocytes derived from a different normal control dog (n = 3) and one of two different XSCID dogs (n = 2).</p

Kinetics of cytokine and interferon stimulated gene expression in XSCID canine keratinocytes infected with CPV-2.

<p><b>A and B</b> Keratinocytes were seeded into multiple wells and cultured as a monolayer for 24-2 (CPV-2) (200 viral particle per cell) or medium alone. RNA was extracted after 2, 4, and 6 days post-infection (DPI). Cytokine (<b>A</b>) and interferon stimulated gene (<b>B</b>) expression was determined by quantitative RT-PCR. Resulting Cq values were normalized to a reference gene and calibrated to mRNA expression in unstimulated keratinocytes (ΔΔCq). Results are expressed as mean +/− SD of three replicate experiments performed in triplicate. Each experiment used keratinocytes derived from a different normal control dog (n = 3) and one of two different XSCID dogs (n = 2). <b>C</b> RT-PCR for the CPV-2 spliced transcripts E1∧E2 and reference gene (GAPDH) in non-infected and infected keratinocytes. Results are shown from one experiment performed in triplicate at 2 DPI. Similar results obtained at 4 and 6 days post infection in all replicate experiments. Data is representative of three independent experiments performed in triplicate.</p

Primer sets, product size, and GenBank accession numbers for RT-PCR.

<p>*Primer pair used to detect 4-bp deletion in XSCID keratinocytes.</p

Confirmation of γ_c-deficient primary keratinocyte cultures from bone marrow transplanted XSCID dogs.

<p><b>A</b> The genetic mutation in XSCID dogs is a 4 base pair deletion in exon 1 of γ_c. <b>B</b> RT-PCR was performed in duplicate on cDNA isolated from primary keratinocyte cultures using primer pairs that span the 4 base pair deletion in γ_c. Results are shown from one experiment performed on one normal dog and one XSCID dog. Similar results were obtained from two other experiments using keratinocytes derived from a second XSCID dog and two normal control dogs. The PCR product size from normal dogs is 114 bps and from XSCID dogs is 110 bps. Positive controls included genomic DNA from normal canine skin and splenic genomic DNA from non-bone marrow transplanted XSCID dog.</p

Expression of γ_c -dependent cytokine receptors in XSCID and normal keratinocyte cultures.

<p>RT-PCR for γ_c and alpha subunits was performed on monolayer keratinocyte cultures 24 hours after seeding. Results shown are from cultures derived from two XSCID dogs and three normal control dogs. Positive control included cDNA from either canine histiocytic or lymphocytic cell lines.</p

Treatment type, date of treatment, and course of papillomavirus infection in XSCID dogs from this study.

<p>* No SCC; SCC =  squamous cell carcinoma.</p

HPV16 E7 Protein and hTERT Proteins Defective for Telomere Maintenance Cooperate to Immortalize Human Keratinocytes

<div><p>Previous studies have shown that wild-type human telomerase reverse transcriptase (hTERT) protein can functionally replace the human papillomavirus type 16 (HPV-16) E6 protein, which cooperates with the viral E7 protein in the immortalization of primary keratinocytes. In the current study, we made the surprising finding that catalytically inactive hTERT (hTERT-D868A), elongation-defective hTERT (hTERT-HA), and telomere recruitment-defective hTERT (hTERT N+T) also cooperate with E7 in mediating bypass of the senescence blockade and effecting cell immortalization. This suggests that hTERT has activities independent of its telomere maintenance functions that mediate transit across this restriction point. Since hTERT has been shown to have a role in gene activation, we performed microarray studies and discovered that E6, hTERT and mutant hTERT proteins altered the expression of highly overlapping sets of cellular genes. Most important, the E6 and hTERT proteins induced mRNA and protein levels of Bmi1, the core subunit of the Polycomb Group (PcG) complex 1. We show further that Bmi1 substitutes for E6 or hTERT in cell immortalization. Finally, tissue array studies demonstrated that expression of Bmi1 increased with the severity of cervical dysplasia, suggesting a potential role in the progression of cervical cancer. Together, these data demonstrate that hTERT has extra-telomeric activities that facilitate cell immortalization and that its induction of Bmi1 is one potential mechanism for mediating this activity.</p> </div

hTERT wt and hTERT-D868A do not activate the hTERT promoter.

<p>Keratinocytes were transfected with either wt hTERT core promoter or the cyclin D1 promoter and either HPV16E6, hTERT wt, or hTERT-D8686A. The pRL-CMV <i>R. reniformis</i> reporter plasmid was also transfected into the cells to standardize for transfection efficiency. Luciferase activity was measured 24 hours after transfection using the Dual luciferase reporter assay system (Promega). Relative fold activation reflects the normalized luciferase activity induced by E6 and hTERT compared to the normalized activity of vector control. The value of pGL3B-hTERT activity with empty was set to 1. Error bars show the standard deviation for at least three independent experiments. Neither hTERT wt nor hTERT-D868A induce hTERT core promoter (A), while they are able to activate cyclin D1 promoter (B). HPV16 E6 was as a positive control for induction of hTERT promoter.</p

A telomere elongation-defective hTERT mutant cooperates with HPV E7 for immortalizing human keratinocytes (HFKs).

<p>Primary HFKs were transduced with pBABE-puro-based retroviruses containing hTERT or hTERT-HA and pLXSN-based retroviruses with HPV E7 or empty vector and selected with puromycin and G418 as previously described. Cultures were passed continuously in vitro, and growth curves were plotted. Cultures that did not proliferate and expand in 20 days were considered senescent and were terminated. This experiment was repeated more than three times with similar results. <u>(A) Telomerase activity.</u> Quantitative TRAP assays were done as described in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003284#s4" target="_blank">Materials and Methods</a>. Similar levels of telomerase activity were observed among cells expressing an hTERT construct. <u>(B) Telomere length.</u> Telomeres lengthened in hTERT/E7 cells, but shortened in hTERT-HA/E7 cells. Both wild-type hTERT and hTERT-HA immortalize HFKs in combination with HPV E7.</p

Telomerase activity does not correlate with telomere length during immortalization of human genital keratinocytes by the HPV E6 and E7 oncoproteins.

<p>Primary human foreskin keratinocytes (HFKs) and human ectocervical keratinocytes (HECs) were transduced with pLXSN-based retroviruses containing HPV E6, E7, E6/E7, or empty vector and selected as previously described. Cultures were passed continuously in vitro as described in the text and the number of cell doublings calculated and plotted versus time in culture. Cultures that did not proliferate and expand in 20 days were considered senescent and were terminated. This experiment was repeated more than five times with similar results. <u>(A) Immortalized cells exhibit similar levels of telomerase activity as in cervical cancer cells.</u> Quantitative TRAP assays as described in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003284#s4" target="_blank">Materials and Methods</a> were used to measure telomerase activity in E6/E7 immortalized HFKs, E6/E7 immortalized HECs, and SiHa (HPV-16 positive), HeLa (HPV-18 positive), and C33A (HPV negative) cervical cancer cell lines. <u>(B) Telomere length stabilizes in E6/E7 immortalized cells and cervical cancer cell lines.</u> Cellular DNAs were isolated from HFKs at indicated passages and cervical cancer cells, and relative telomere length (T/S ratio = telomere/single copy gene) was measured using real-time PCR, as described in the Material and Methods. Immortalized cells and cancer cells have relatively shorter telomeres. <u>(C) Telomere length shortens over cell passages during immortalization.</u> Cellular DNAs were isolated and subjected to real-time PCR-based telomere length measurement.</p