Volume 13, Issue 6, Pages 1142-1148 (June 2006) A Protease Inhibitor Specifically Inhibits Growth of HPV-Infected Keratinocytes  David A. Drubin, Margaret E. McLaughlin-Drubin, Gary A. Clawson, Craig Meyers  Molecular Therapy  Volume 13, Issue 6, Pages 1142-1148 (June 2006) DOI: 10.1016/j.ymthe.2006.02.005 Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

FIG. 1 Effects of AAPFcmk, GRcmk, and/or DMSO on AWCA organotypic raft cultures. AWCA cells, which contain integrated HPV18, were grown in organotypic raft culture. Treatments were made every other day with feeding for 10 days, and the raft cultures were harvested, fixed, sectioned, and stained with hematoxylin and eosin. Rafts were either (A) untreated or treated with (B) 0.25% DMSO solvent control, (C) 10 μM AAPFcmk, (D) 25 μM AAPFcmk, (E) 50 μM AAPFcmk, (F) 10 μM GRcmk, (G) 25 μM GRcmk, or (H) 50 μM GRcmk. Of note is the significant attenuation of the raft cultures by the higher concentration treatments (D and E) of AAPFcmk. Images are at 20× original magnification. Molecular Therapy 2006 13, 1142-1148DOI: (10.1016/j.ymthe.2006.02.005) Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

FIG. 2 Effects of AAPFcmk, GRcmk, and DMSO on CIN-612 9E organotypic raft cultures. CIN-612 9E cells, which contain episomal high-risk HPV31b, were grown in organotypic raft culture. Treatments were made every other day with feeding for 10 days, and the rafts were harvested, fixed, sectioned, and stained with hematoxylin and eosin. Rafts were either (A) untreated or treated with (B) 0.25% DMSO solvent control, (C) 10 μM AAPFcmk, (D) 25 μM AAPFcmk, (E) 50 μM AAPFcmk, (F) 10 μM GRcmk, (G) 25 μM GRcmk, or (H) 50 μM GRcmk. Of note is the significant raft thinning by the higher concentration treatments (D and E) of AAPFcmk. Images are at 20× original magnification. Molecular Therapy 2006 13, 1142-1148DOI: (10.1016/j.ymthe.2006.02.005) Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

FIG. 3 Effects of AAPFcmk, GRcmk, and/or DMSO on HFK organotypic raft cultures. Primary HFKs were grown in organotypic raft culture. Treatments were made every other day with feeding for 10 days, and the rafts were harvested, fixed, sectioned, and stained with hematoxylin and eosin. Rafts were either (A) untreated or treated with (B) 0.25% DMSO solvent control, (C) 10 μM AAPFcmk, (D) 25 μM AAPFcmk, (E) 50 μM AAPFcmk, (F) 10 μM GRcmk, (G) 25 μM GRcmk, or (H) 50 μM GRcmk. Notable is the lack of thinning effects in (D) and (E) that is observed in HPV-containing cells (Figs. 1 and 2). Images are at 20× original magnification. Molecular Therapy 2006 13, 1142-1148DOI: (10.1016/j.ymthe.2006.02.005) Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

FIG. 4 C33A organotypic raft cultures treated with AAPFcmk, GRcmk, and DMSO. C33A cells, which are derived from a non-HPV-containing carcinoma, were grown as organotypic raft cultures. Treatments were made every other day with feeding for 10 days, and the rafts were harvested, fixed, sectioned, and stained with hematoxylin and eosin. Rafts were either (A) untreated or treated with (B) 0.25% DMSO solvent control, (C) 25 μM AAPFcmk, (D) 50 μM AAPFcmk, (E) 25 μM GRcmk, or (F) 50 μM GRcmk. Of note is the lack of effect of AAPFcmk treatment on the raft growth (C and D). Images are at 20× original magnification. Molecular Therapy 2006 13, 1142-1148DOI: (10.1016/j.ymthe.2006.02.005) Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

FIG. 5 Effects of AAPFcmk on HPV genome copy number in the CIN-612 9E cell line. CIN-612 9E cells were grown in organotypic raft culture. Treatments of 0.25% DMSO and AAPFcmk and GRcmk as labeled at the top were applied to the raft cultures every other day with feeding over the course of 10 days. DNA was harvested and digested with either a noncutter of the HPV31 genome, BamHI (B), or a single-cutter, HindIII (H), and a Southern blot was performed as described with a 32P-labeled probe for the HPV31 genome; controls with 100, 10, or 1 copy number per cell were included. Form II DNA (nicked supercoiled) and Form I DNA (supercoiled) episomal HPV31 bands are observed in the uncut lanes and Form III DNA (linear) is seen in the single-cut lanes. Note the dose-dependent decrease in episomal HPV DNA; it is reduced by ∼50% with 25 μM AAPFcmk and by ∼75% with 50 μM AAPFcmk. Molecular Therapy 2006 13, 1142-1148DOI: (10.1016/j.ymthe.2006.02.005) Copyright © 2006 The American Society of Gene Therapy Terms and Conditions