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Antimicrobial Agents and Chemotherapy, June 2003, p. 2022-2026, Vol. 47, No. 6
0066-4804/03/$08.00+0     DOI: 10.1128/AAC.47.6.2022-2026.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Alpha Interferon Augments Cidofovir's Antiviral and Antiproliferative Activities

Jeffrey A. Johnson^* and J. David Gangemi

Department of Microbiology and Molecular Medicine and the Greenville Hospital System Biomedical Cooperative, Clemson University, Clemson, South Carolina 29634

Received 4 October 2002/ Returned for modification 30 December 2002/ Accepted 6 February 2003

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The antiviral and antiproliferative activities of alpha 2a interferon (IFN-2a) and cidofovir in human papillomavirus type 16 (HPV-16)-transformed keratinocytes were evaluated. The compounds in combination were more effective than comparable levels of either drug alone. Evaluation of effective drug ratios revealed a synergistic cooperation between IFN-2a and cidofovir in inhibiting the proliferation of HPV-infected cells.

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Alpha interferons (IFN-) elicit broad activities inhibitory to virus replication including blocking de novo protein synthesis with the induction of the GTPase transducer Mx (30), activating proteolysis of abnormal proteins by inducing the IFN-stimulated gene (ISG15) ubiquitin-like protein (17), and causing disaggregation of polysomes and activation of RNase L by upregulating oligoadenylate 2'-5'-A synthetase (31). IFN- have demonstrated moderate activity against RNA viruses (16, 24); however, their effectiveness against DNA viruses such as herpesviruses (20), hepatitis B virus (36), and poxviruses (11, 23) is nominal.

IFN also has a history of use in the clinical treatment of human papillomavirus (HPV) dermatopathology (21, 32, 35). An intracellular mechanism by which IFN-2a inhibits HPV-transformed-cell proliferation, and presumably HPV-induced papillomas, is through the suppression of viral oncoprotein expression and cytostatic arrest of cell cycling at G₁, by preserving p53 and retinoblastoma tumor suppressor activity (1, 15). Unfortunately, because of the cytostatic nature of IFN, prolonged therapy is required and treatment is problematic (4, 33) due to drug tolerance or to detrimental side effects (e.g., intolerable flu-like symptoms and loss of drug responsiveness).

The activity of cidofovir {CDV; (S)-1-[3-hydroxy-2-(phosphonomethoxy)-propyl]cytosine} against herpesvirus DNA polymerase through inhibition of second-strand synthesis has been reported (13, 34). CDV has also been shown to have some success in the treatment of molluscum contagiosum virus, which also encodes a DNA polymerase (10). CDV applications have achieved modest reductions in the clinical presentation of a variety of HPV-induced tissue proliferations (27-29) and reduced the proliferation of cells transformed by HPV (14), even though this virus lacks a viral DNA polymerase. In contrast to what is found for uninfected cells, CDV is readily phosphorylated to the active triphosphate form in HPV-infected cells and is incorporated into both virus and host cell DNA (14).

When used alone, both IFN-2a and CDV (5, 9, 19) present significant toxicity concerns. Therapeutic regimens may be improved with combinations in which individual drugs work through distinct antiviral mechanisms. We have observed this potential when suboptimal combinations of IFN and CDV produced a more rapid reduction in cell proliferation and at higher concentrations caused a nearly complete inhibition of transformed cells from which cell growth did not recover after treatment was abated (our preliminary data were presented earlier [Abstr. 17th Int. Conf. Papillomavirus Res., abstr. THER4, 1999]). Moreover, therapies that are cytotoxic, and thus potentially lethal, to virus-infected cells may reduce the likelihood of a resurgence of disease and/or a requirement for prolonged treatment by substantially reducing or eliminating the population of infected cells.

This study examined the ability of IFN-2a to augment the activity of CDV at concentrations that would impart minimal host toxicity while still being effective against virus-infected cells. Antiproliferative activities of the compounds were evaluated by colony reduction assays. Briefly, primary normal newborn human foreskin keratinocytes that had undergone less than 10 passages or HPV type 16 (HPV-16)-transfected keratinocytes (HKc/HPV-16-d-2C [d-2C]) (22) with less than 15 passages were plated in triplicate at 500 cells/60-mm-diameter culture dish in keratinocyte-SFM medium (Gibco-Life Technologies, Gaithersburg, Md.) and allowed to adhere for 24 h. Treatment regimens involved addition of antiviral agents three times per week for 2 weeks in a checkerboard combination configuration at the following concentrations: recombinant IFN-2a (Roferon; Hoffman-LaRoche), 12.5, 25, 50, and 100 U/ml; CDV (HPMPC; Gilead Sciences), 0.10, 0.15, 0.20, 0.30, 0.40, 0.50, 0.60, 0.80, 1.00, and 1.20 µM diluted in Dulbecco's phosphate-buffered saline (PBS). These concentrations were all under the maximum tolerance levels of normal cells, defined as the highest concentrations that had no inhibitory effect on normal keratinocytes (100 U/ml for IFN and 1.2 µM for CDV) in vitro. After the 2-week treatment, plates were washed with PBS and fixed and stained with Giemsa stain (Gibco-Life Technologies). The cell colony densities and areas of spread for each dish were determined from densitometric analysis on scanned images of the culture dishes. Mean colony growth in each of the triplicate treatments was compared to that for untreated controls. Statistical analyses of treatment responses were performed by using the Student t test.

Combinations of one-half the maximum doses of both antiviral compounds (50 U/ml for IFN and 0.60 µM for CDV) provided the greatest inhibition of HPV-16-transformed cells (86% inhibition) while exhibiting no inhibition of normal cell growth (data not shown). In combinations where the concentration of IFN was above 50 U/ml and CDV was above 0.6 µM, up to the 1.2 µM level, normal cell proliferation was inhibited 6 to 14% (data not shown). A representation of the results from the transformed-cell colony reduction assays with IFN and CDV alone and in combination is given in Fig. 1, and the data are provided in Table 1. The antiproliferative activity of this half-maximum combination (86% inhibition) was similar to that of the 1.2 µM CDV maximum tolerable dose (89% inhibition). The combination of 25 U of IFN/ml and 0.15 µM CDV yielded the 50% inhibitory concentration for the combination treatments.

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FIG. 1. Inhibition of HPV-transformed cell proliferation after 2-week treatments with CDV and/or IFN. Percent inhibitions are relative to control (untreated cultures). 1, mock control; 2, 0.15 µM CDV; 3, 50 U of IFN/ml; 3, 0.15 µM CDV plus 50 U of IFN/ml; 4, 1.20 µM CDV; 5, 0.60 µM CDV plus 50 U of IFN/ml.

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TABLE 1. Fa values for treatment with IFN and CDV individually or combineda

To evaluate the effects on virus proteins and cell cycle factors, cultures treated for 2 days with 1.0 µM CDV or 0.50 µM CDV plus 50 U of IFN/ml or mock treated were processed for Western blot analysis. These concentrations were selected for their equivalent and substantial levels of inhibition as observed in the 2-week treatments. Cells from the treatment groups were lysed by four freeze-thaws in WCE buffer (10 mM HEPES, 400 mM NaCl, 20% glycerol, 1 mM EDTA) and clarified at 13,000 x g for Western blot analysis. Target proteins in 40 µg of lysates/ml from controls and each treated culture were immunoprecipitated with protein A/G plus agarose beads (Santa Cruz Biotechnology, Santa Cruz, Calif.) bound to a polyclonal goat antibody raised against the cellular regulatory factors p53, pRB, and p21 (CIP1) and the viral E6 and E7 oncogenes. Immunoprecipitates were denatured by boiling them in loading buffer (Bio-Rad, Hercules, Calif.) and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in 12% Tris-glycine Ready gels along with Kaleidescope (molecular sizes: 208, 127, 85, 45, 32.8, 18.1, and 7.4 kDa) and low-range (molecular sizes: 102, 78, 49.5, 34.2, 28.3, and 19.9 kDa) size standards (Bio-Rad). Proteins were transferred to a 0.2-µm-pore-size natural nitrocellulose membrane, and the membranes were probed with 1.5 µg of mouse monoclonal antibodies against p53, pRB, p21, E6, or E7 (Santa Cruz Biotechnology)/ml overnight at 4°C. Membranes were incubated in 0.025 µg of horseradish peroxidase-conjugated goat anti-mouse antibodies/ml for 2 h, and bands were detected by enhanced chemiluminescence and exposure to Hyperfilm-ECL (Amersham Pharmacia, Piscataway, N.J.). Densitometric analysis of the HPV E6 and E7 protein bands in the Western blots revealed a 40% decrease in oncoprotein expression following treatment with the combination of 0.50 µM CDV and 50 U of IFN/ml (Fig. 2A and B). These decreases in the presence of IFN supported our earlier studies of HPV oncoprotein expression inhibition by IFN (15). The reduction in expression could be attributed only to IFN since CDV alone did not appear to suppress oncogene expression nor was IFN-induced suppression enhanced by CDV within the 2 days.

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FIG. 2. Effects of combination treatments on E6, E7, retinoblastoma (Rb), and p21 from lysates of cultures untreated or treated with 1.0 µM CDV or 0.50 µM CDV plus 50 U of IFN/ml.

The levels of p53, p21, and pRB were also quantified after combination treatment to differentiate between the cycle-regulatory effects of the combinations and the IFN (15) and CDV (14) monotherapy effects previously observed by our group. pRB levels increased substantially (39%) after 2 days of treatment with the 50 U of IFN/ml plus 0.50 µM CDV (Fig. 2C), which was similar to the response observed with IFN alone. There was a modest increase in the level of p53 in the combination group (not shown) that had been previously observed with, and is attributable to the effects of, IFN (15). Most strikingly, and unique to CDV, p21 levels decreased remarkably (88% reduction) following 2 days with this combination treatment, a decrease comparable to what was observed with 1.2 µM CDV alone. At 1 µM CDV, twice the concentration that was present in the combination treatment, p21 was reduced 77%, slightly less than what was observed for the combination (Fig. 2D). Since p21 acts to inhibit cycle progression into the DNA synthesis (S) phase, cells do not arrest at G₁ for DNA repair.

The modulatory effects of cell-regulatory factors were measured by flow-cytometric analysis of cell cycling. Cycle analyses were performed on HPV-16-d-2C cultures synchronized by addition of 2 mM thymidine for 24 h (6). Following 2-day treatments with 1 µM CDV, 50 U of IFN/ml, and 50 U of IFN/ml plus 0.50 µM CDV, cells were harvested, trypsinized, and suspended in PBS. Suspended cells were then permeabilized and fixed overnight in ice-cold 70% ethanol, washed, treated with RNase, and incubated with 1 mg of propidium iodide/ml for 1 h prior to analysis of genomic fluorescence (ASICS single-beam cytometer; Coulter). Integrated red fluorescence cell cycle analysis (Multicycle AV; Phoenix Flow Systems) was performed on samples of 20,000 cells from each treatment group. IFN treatment resulted in a 19% reduction in the number of transformed cells transitioning from G₁ into S phase compared to the number of untreated cells (Fig. 3B). In contrast, CDV treatment brought about a 45% increase in the number of HPV-transformed cells in S phase (32%) (Fig. 3C), and the number entering S phase gradually increased as the time span of treatment was extended (not shown). The combination of IFN and CDV further added to the accumulation of cells in G₁ and S, with few cells proceeding through G₂ (Fig. 3D). At higher concentrations, the capacity of CDV to abolish p21 appeared to supersede the cell cycling inhibition of IFN and infected cells transitioned into S phase. Damage to DNA by CDV, along with partial recovery of tumor suppressor function provided by IFN, appears to initiate in the cells given dual treatment a G₁ repair in combination with a priming of DNA synthesis (15). Interestingly, synchronized infected cells in late G₁/S at the time of treatment were detained in late S/pre-G₂ and did not appear to progress into G₂ with time (not shown). Hence, two potential inhibitory mechanisms are illustrated here: (i) when transformed cells, during the G₁ repair arrest, are unable to repair the genomic lesions as a result of CDV incorporation, they become entrapped in G₁/early S and fail to thrive; (ii) CDV incorporation exceeding a critical threshold during DNA synthesis may prevent the completion of the synthesis phase. This condition of DNA stasis is evidenced by a lack of progression to G₂ and mitosis. The stronger push of the cell cycle into S phase following the combined drug treatments correlated with a shorter time to cell death (6 to 8 days versus 7 to 10 days with CDV monotherapy). Though the reduction in time was not remarkable, the trend was reproducible.

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FIG. 3. Effect of IFN and CDV on transformed-cell cycling. Shown are bar graph representations of cell cycle histograms of d-2C cell integrated red fluorescence following 2 days of mock treatment (A) or treatments of 1 µM CDV (B), 50 U of IFN/ml (C), or 50 U of IFN/ml plus 0.50 µM CDV (D).

The combinatorial effectiveness of IFN and CDV was evaluated by using ComboStat software, version 3.03 (ComboStat Corp.) (3, 18), based on the combination index (CI) method of Chou and Taladay (8). Factor effects (fractional inhibition [F_a]), the measure of the reduction in colony growth with each individual compound and combination, were determined from densitometric analysis of the colony assays. Mean colony densities from triplicate plates were analyzed by the program, with the effects of each drug designated mutually exclusive. The program calculated confidence intervals for the cooperation reflected in each F_a. If the CI is >1, the combination is antagonistic, and if the CI for a factor effect is <1, the compounds cooperate synergistically. Colony growth inhibitions for two CDV combination series (series 1 and 2) were analyzed. Series 1 consisted of 100, 50, 25, and 12.5 U of IFN-2a/ml combined with 1.20, 0.60, 0.30, and 0.15 µM CDV, respectively. Series 2 consisted of the same concentrations of IFN-2a combined with 0.80, 0.40, 0.20, and 0.10 µM CDV, respectively. These analyses revealed that CDV concentrations less than 0.15 µM were antagonistic to IFN in inhibiting transformed-cell proliferation (Fig. 4). When the drugs in combination were present at concentrations great enough to produce an F_a of 0.5 (50% inhibition), as was observed with CDV concentrations above 0.15 µM, the CI values fell below 1.0, signifying synergy. Our data indicate that synergy was best achieved when both drugs were combined at concentrations of 25 U of IFN-2a/ml plus 0.15 µM CDV. At concentrations greater than 0.15 µM sufficient analog may become incorporated into the viral DNA and the infected cells to achieve lethal genetic insults within the time period examined.

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FIG. 4. Graphical representation of the median-effect analysis of combinations of IFN-2a and CDV. Combinations of concentrations that elicited CIs <1 (50 U of IFN/ml plus 0.150 µM CDV) were synergistic in their inhibition of HPV-transformed-cell proliferation.

When used in combination, CDV and IFN-2a are more effective in inhibiting early gene expression and cellular proliferation than either drug alone. This observation is similar to what was found when IFN was used in combination with 5-fluorouracil (12), all-trans-retinoic acid (25), and thymosin ₁ (26) to treat proliferating carcinomas. Another potential candidate for combination therapy is the cyclic cogenitor of CDV, 1-[({S}-2-hydroxy-2-oxo-1,4,2-dioxophosphorian-5-yl)-methyl]cytosine dihydrate, which has been shown to be active against herpesvirus infections (7) and HPV (J. A. Johnson and J. D. Gangemi, Abstr. 10th Int. Conf. Antivir. Res., abstr. 184, 1997). That the combinations presented in our study may have clinical relevance was recently illustrated in an unrelated study where IFN and CDV therapy was successfully utilized in the treatment of severe respiratory papillomatosis (2).

Our in vitro assay provides a means with which to study the direct molecular effects of combined-drug treatments on virus-infected cells. It is important that the virus infection model presented in our study was designed to examine antiviral responses and cell toxicities for treatments where longer-term regimens are considered necessary. When attempting to translate this information into clinical practice, it is not unreasonable to predict that these observed molecular responses would not occur in vivo. There are, however, two important factors in vivo that do not exert their influence in our closed culture system. First, fluid is dynamic in the body and, not as in our system, local drug concentrations ebb. As a result, efficacious in vivo treatments would require much higher concentrations of drug than those we use in our system. To avoid complications with toxicity, these higher drug doses in combination must be administered such that intracellular drug levels are not sustained at intolerable concentrations. Second, IFN is a potent immunomodulator, and we have not measured the additional benefit of an immunologic response in controlling the virus-infected cells.

As part of an effort to reduce toxicity, the clinical effectiveness of combination therapy would ultimately require trials that examine suboptimal concentrations of the individual drugs. Our data indicate that drug combinations administered at lower than the current maximum monotherapy doses may prove to be both efficacious and more tolerable to recipients. The same approach of using drugs with different antiviral mechanisms may be effective against otherwise poorly responsive DNA viruses, such as variola major virus, hepatitis B virus, and herpesviruses.

 
We acknowledge Lucia Pirisi at the University of South Carolina School of Medicine for graciously providing the HPV-transformed keratinocytes, John Whitesides at Clemson University for assistance with flow cytometry, Norbert Bischofberger at Gilead Sciences for supplying the HPMPC, and Raymond Schinazi for providing the ComboStat analysis software.

 
* Corresponding author. Present address: Centers for Disease Control and Prevention, NCID/DASTLR/HARB, Bldg. 15, MS G19, 1600 Clifton Rd., Atlanta, GA 30333. Phone: (404) 639-4976. Fax: (404) 639-1174. E-mail: jjohnson1{at}cdc.gov.

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Antimicrobial Agents and Chemotherapy, June 2003, p. 2022-2026, Vol. 47, No. 6
0066-4804/03/$08.00+0     DOI: 10.1128/AAC.47.6.2022-2026.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Johnson, J. A. Articles by Gangemi, J. D. Search for Related Content PubMed PubMed Citation Articles by Johnson, J. A. Articles by Gangemi, J. D.