Effect of the HIV nucleoside reverse transcriptase inhibitor zidovudine on the growth and differentiation of primary gingival epithelium

Abstract

Objectives: Oral complications associated with HIV infection and with the antiretroviral drugs used to treat it are of increasing concern in HIV-infected patients. Protease inhibitors have been shown to change the proliferation and differentiation state of oral tissues but the effect of nucleoside reverse transcriptase inhibitors is currently unknown. This study examined the effect of zidovudine on the growth and differentiation of the gingival epithelium.

Methods: Gingival keratinocyte organotypic (raft) cultures were established. The raft cultures were treated with a range of zidovudine concentrations. Haematoxylin and eosin staining was performed to examine the effect of zidovudine on gingival epithelium growth and stratification. Raft cultures were immunohistochemically analysed to determine the effect of this drug on the expression of key differentiation and proliferation markers, including cytokeratins and proliferating cell nuclear antigen (PCNA).

Results: Zidovudine dramatically changed the proliferation and differentiation state of gingival tissues both when it was present throughout the growth period of the tissue and when it was added to established tissue at day 8. Zidovudine treatment increased the expression of cytokeratin 10, PCNA and cyclin A. Conversely, cytokeratin 5, involucrin and cytokeratin 6 expression was decreased. The tissue exhibited characteristics of increased proliferation in the suprabasal layers as well as an increased fragility and an inability to heal itself.

Conclusions: Zidovudine treatment, even when applied at low concentrations for short periods of time, deregulated the cell cycle/proliferation and differentiation pathways, resulting in abnormal epithelial repair and proliferation. Our system could potentially be developed as a model for studying the effects of HIV and highly active antiretroviral therapy in vitro.

Figure 1

Time line for experiments.

Figure 2. Effect of Zidovudine on gingival epithelium morphology and stratification

Primary gingival keratinocytes were grown in organotypic (raft) cultures and treated with different concentrations of Zidovudine, beginning either at day 0 (Tissue Harvest A) or at day 8 (Tissue Harvest B). (Panels A, G, M, S, Y and EE) untreated rafts; (Panels B, H, N, T, Z, and FF) rafts treated with 0.5 µg/ml Zidovudine; (Panels C, I, O, U, AA, and GG) rafts treated with 1 µg/ml Zidovudine; (Panels D, J, P, V, BB and HH) rafts treated with 2 µg/ml, the cMax of Zidovudine; (Panels E, K, Q, W, CC, and II) rafts treated with 4 µg/ml Zidovudine; (Panels F, L, R, X, DD, and JJ) rafts treated with 6 µg/ml Zidovudine. Rafts were harvested at different points and stained with hematoxylin and eosin. DPT denotes days post treatment (Tissue Harvest B). Images are at 20 X original magnification.

Figure 2. Effect of Zidovudine on gingival epithelium morphology and stratification

Primary gingival keratinocytes were grown in organotypic (raft) cultures and treated with different concentrations of Zidovudine, beginning either at day 0 (Tissue Harvest A) or at day 8 (Tissue Harvest B). (Panels A, G, M, S, Y and EE) untreated rafts; (Panels B, H, N, T, Z, and FF) rafts treated with 0.5 µg/ml Zidovudine; (Panels C, I, O, U, AA, and GG) rafts treated with 1 µg/ml Zidovudine; (Panels D, J, P, V, BB and HH) rafts treated with 2 µg/ml, the cMax of Zidovudine; (Panels E, K, Q, W, CC, and II) rafts treated with 4 µg/ml Zidovudine; (Panels F, L, R, X, DD, and JJ) rafts treated with 6 µg/ml Zidovudine. Rafts were harvested at different points and stained with hematoxylin and eosin. DPT denotes days post treatment (Tissue Harvest B). Images are at 20 X original magnification.

Figure 3. Expression pattern of cytokeratin 5 in untreated and Zidovudine treated gingival raft cultures

Primary gingival keratinocytes were grown in raft cultures and treated with different concentrations of Zidovudine at either day 0 or day 8. (Panels A, G, M, and S) untreated rafts; (Panels B, H, N and T) rafts treated with 0.5 µg/ml Zidovudine; (Panels C, I, O, and U) rafts treated with 1 µg/ml Zidovudine; (Panels D, J, P, and V) rafts treated with 2 µg/ml, the cMax of Zidovudine; (Panels E, K, Q, and W) rafts treated with 4 µg/ml Zidovudine; (Panels F, L, R, and X) rafts treated with 6 µg/ml Zidovudine. Rafts were harvested at the indicated timepoints (DPT denotes days post treatment) and stained with anti-cytokeratin 5. Images are at 20 X original magnification.

Figure 4. Expression pattern of involucrin in untreated and Zidovudine treated gingival raft cultures

Primary gingival keratinocytes were grown in raft cultures and treated with different concentrations of Zidovudine at either day 0 or day 8. (Panels A, G, and M) untreated rafts; (Panels B, H and N) rafts treated with 0.5 µg/ml Zidovudine; (Panels C, I and O) rafts treated with 1 µg/ml Zidovudine; (Panels D, J and P) rafts treated with 2 µg/ml, the cMax of Zidovudine; (Panels E, K, and Q) rafts treated with 4 µg/ml Zidovudine; (Panels F, L, and R) rafts treated with 6 µg/ml Zidovudine. Rafts were harvested at the indicated time points (DPT denotes days post treatment) and stained with anti-cytokeratin involucrin. Images are at 20 X original magnification.

Figure 5. Expression pattern of cytokeratin 10 in untreated and Zidovudine treated gingival raft cultures

Primary gingival keratinocytes were grown in raft cultures and treated with different concentrations of Zidovudine at either day 0 or day 8. ((Panels A, G, M, and S) untreated rafts; (Panels B, H, N and T) rafts treated with 0.5 µg/ml Zidovudine; (Panels C, I, O, and U) rafts treated with 0.1 µg/ml Zidovudine; (Panels D, J, P, and V) rafts treated with 2 µg/ml, the cMax of Zidovudine; (Panels E, K, Q, and W) rafts treated with 4 µg/ml Zidovudine; (Panels F, L, R, and X) rafts treated with 6 µg/ml Zidovudine. Rafts were harvested at the indicated time points (DPT denotes days post treatment) and stained with anti-cytokeratin 5. Images are at 20 X original magnification. Rafts were harvested at the indicated time points (DPT denotes days post treatment) and stained with anti-cytokeratin 10. Images are at 20 X original magnification.

Figure 6. Expression pattern of cytokeratin 6 in untreated and Zidovudine treated gingival raft cultures

Primary gingival keratinocytes were grown in raft cultures and treated with different concentrations of Zidovudine at either day 0 or day 8. (Panels A, G, M, and S) untreated rafts; (Panels B, H, N and T) rafts treated with 0.5 µg/ml Zidovudine; (Panels C, I, O, and U) rafts treated with 1 µg/ml Zidovudine; (Panels D, J, P, and V) rafts treated with 2 µg/ml, the cMax of Zidovudine; (Panels E, K, Q, and W) rafts treated with 4 µg/ml Zidovudine; (Panels F, L, R, and X) rafts treated with 6 µg/ml Zidovudine. Rafts were harvested at the indicated timepoints (DPT denotes days post treatment) and stained with anti-cytokeratin 6. Images are at 20 X original magnification.

Figure 7. Proliferating cell nuclear antigen expression pattern in untreated and Zidovudine treated gingival raft cultures

Primary gingival keratinocytes were grown in raft cultures and treated with different concentrations of Zidovudine at either day 0 (Tissue Harvest A) or day 8 (Tissue Harvest B). (Panels A, G, and M) untreated rafts; (Panels B, H and N) rafts treated with 0.05 µg/ml Zidovudine; (Panels C, I and O) rafts treated with 1 µg/ml Zidovudine; (Panels D, J and P) rafts treated with 2 µg/ml, the c_Max of Zidovudine; (Panels E, K, and Q) rafts treated with 4 µg/ml Zidovudine; (Panels F, L, and R) rafts treated with 6 µg/ml Zidovudine. Rafts were harvested at the indicated timepoints (DPT denotes days post treatment) and stained with anti-cytokeratin pcna or cyclin A. Images are at 20 X original magnification.

Figure 8. Effect of Zidovudine on gingival epithelium morphology, stratification and cytokeratin expression pattern in gingival raft cultures either untreated or treated with Zidovudine for 6, 12 or 24 hours

Primary gingival keratinocytes were grown in raft cultures and treated with different concentrations of Zidovudine at day 8 for either 6, 12 or 24 hours. All rafts were treated with 2 µg/ml, the c_Max of Zidovudine.