Southern blots of PA25 treated HPV16 episomes from W12E cells.

<p>A. Southern blot of intact HPV episomes following treatment over 5 µM PA25. Migration of linearized HPV16 is shown (BamH1). Note retardation of migration of HPV16 Form 1 DNA (arrow) over time in the presence of PA25, and the appearance of Form 3 viral DNA (linear) at 5 hours of treatment (arrowhead). Open circle form of HPV is indicated with asterisk. B. Southern blot of episomes following treatment with 10 µM PA25. Migration of linearized HPV16 is shown (BamH1). Note the pattern of migration of HPV16 Form 1 DNA (arrow) over time in presence of PA25 resulting in a step-like appearance of HPV topoisomers. Open circle form of HPV is indicated with asterisk.</p

Genes whose expression is significantly altered by PA25 in W12E cells.

<p><b>DSB:</b> double-strand break; <b>BER:</b> base excision repair; <b>MMR:</b> mismatch repair; <b>HR:</b> homologous recombination; <b>TLS:</b> translesion repair; <b>MMR:</b> mismatch repair; <b>NHEJ</b>: non-homologous end-joining.</p

Southern blots of linearized (left) and intact (right) HPV16 episomes over time following treatment with 1 µM PA25 for 48 hours.

<p>The blots are loaded identically except HPV16 was linearized by BamH1 in one set of samples (left) or digested with HindIII, which does not restrict viral DNA (right). An additional, over-exposed HindIII blot is also provided. OC: open circle; SC: super-coiled.</p

DNA Damage Repair Genes Controlling Human Papillomavirus (HPV) Episome Levels under Conditions of Stability and Extreme Instability

<div><p>DNA damage response (DDR) genes and pathways controlling the stability of HPV episomal DNA are reported here. We set out to understand the mechanism by which a DNA-binding, N-methylpyrrole-imidazole hairpin polyamide (PA25) acts to cause the dramatic loss of HPV DNA from cells. Southern blots revealed that PA25 alters HPV episomes within 5 hours of treatment. Gene expression arrays identified numerous DDR genes that were specifically altered in HPV16 episome-containing cells (W12E) by PA25, but not in HPV-negative (C33A) cells or in cells with integrated HPV16 (SiHa). A siRNA screen of 240 DDR genes was then conducted to identify enhancers and repressors of PA25 activity. Serendipitously, the screen also identified many novel genes, such as TDP1 and TDP2, regulating normal HPV episome stability. MRN and 9-1-1 complexes emerged as important for PA25-mediated episome destruction and were selected for follow-up studies. Mre11, along with other homologous recombination and dsDNA break repair genes, was among the highly significant PA25 repressors. The Mre11 inhibitor Mirin was found to sensitize HPV episomes to PA25 resulting in a ∼5-fold reduction of the PA25 IC50. A novel assay that couples end-labeling of DNA to Q-PCR showed that PA25 causes strand breaks within HPV DNA, and that Mirin greatly enhances this activity. The 9-1-1 complex member Rad9, a representative PA25 enhancer, was transiently phosphorylated in response to PA25 treatment suggesting that it has a role in detecting and signaling episome damage by PA25 to the cell. These results establish that DNA-targeted compounds enter cells and specifically target the HPV episome. This action leads to the activation of numerous DDR pathways and the massive elimination of episomal DNA from cells. Our findings demonstrate that viral episomes can be targeted for elimination from cells by minor groove binding agents, and implicate DDR pathways as important mediators of this process.</p></div

Bar graph of PA25 enhancers and repressors identified in the siRNA screen.

<p>The effects of siRNAs targeting the genes on PA25 activity are shown. Knockdown of enhancers, those genes that are required for full PA25 activity, results in a net gain in viral episomes in the presence of PA25. Knockdown of repressors, those genes that oppose the antiviral activity of PA25, causes an increase in PA25 activity resulting in a greater loss of HPV episomes.</p

Antiviral activity and structure of anti-HPV N-methylpyrrole-imidazole polyamides following 48 hours of treatment in W12E cells.

<p>A. PA1 and PA25 dramatically decrease HPV16 episome levels in W12E cells while the related PA11 has no effect. B. Structure of PA11. C. Structure of PA25.</p

Heat map of matrix examining effects of 22 siRNAs on the expression of the same 22 genes measured by Q-PCR using gene specific primers.

<p>All 22 siRNAs were found to specifically down-regulate the appropriate target gene as seen by the mid-linear diagonal effect in the heat map.</p

Effects of PA11 and PA25 on expression of cell cycle, apoptosis, and DDR genes in W12E cells.

<p>A. PA11, an inactive polyamide, does not significantly alter gene expression in W12E cells. B. PA25 significantly alters the expression of numerous genes in W12E cells. The expression of most genes is decreased in response to PA25 while 3 are significantly increased.</p

Experimental design of siRNA screen.

<p>A total of 4 experiments were conducted on 4 separate days with cells that were treated with either vehicle (Set 1) or vehicle plus PA25 (Set 2). Cells maintaining HPV16 (days 1−3) or HPV31 (day 4) were used in these experiments.</p

The Mre11 inhibitor Mirin acts as a PA25 sensitizer in W12E cells.

<p>A. Mirin has no effect on HPV16 episome levels by itself. PA25 causes ∼90% loss of HPV16 episomes in cells pre-treated with vehicle (0.1% DMSO), while showing a ∼98% loss of episomes in cells pre-treated with 100 µM Mirin. * p  =  0.00001 (two-tailed student’s t-test assuming unequal variance), n = 6, error bars represent standard deviation. B. Mirin (100 µM) causes a leftward shift in the PA25 dose response curve demonstrating the increased sensitivity of HPV episomes under conditions of Mre11 inhibition. The IC50 in this experiment for PA25 was 72 nM without Mirin (solid boxes), and 18 nM in the presence of Mirin (open diamonds). C. Single and double strand DNA breaks were detected by ELCQ. PA25 caused an increase in the number of detectable breaks while Mirin significantly enhanced this effect. The numbers over the bars indicate the fold change in detected HPV DNA from the vehicle (0.1% DMSO) treated control, which is set at 1.</p