HPV8 early genes modulate differentiation and cell cycle of primary human adult keratinocytes

Abstract

Human papillomaviruses (HPV) have been associated with the development of non-melanoma skin cancer (NMSC) but the molecular mechanisms of the role of the virus in NMSC development are not clearly understood. Abnormal epithelial differentiation seen in malignant transformation of keratinocytes is associated with changes in keratin expression. The purpose of this study was to investigate the phenotype of primary human adult keratinocytes expressing early genes of HPV8 with specific reference to their differentiation and cell cycle profile to determine whether early genes of HPV8 lead to changes that are consistent with transformation. The expression of HPV8 early genes either individually or simultaneously caused distinct changes in the keratinocyte morphology and induced an abnormal keratin expression pattern, that included simple epithelial (K8, K18, K19), hyperproliferation-specific (K6, K16), basal-specific (K14, K15) and differentiation-specific (K1, K10) keratins. Our results indicate that expression of HPV8 early genes disrupts the normal keratin expression pattern in vitro. Expression of HPV8-E7 alone caused polyploidy that was associated with decreased expression of p21 and pRb. Expression of individual genes or in combination differentially influenced cell morphology and cell cycle distribution which might be important in HPV8-induced keratinocyte transformation.

Figure 1

HPV transcripts expressed in human adult primary keratinocytes and morphology of cells transdused with different HPV8 genes. (a) Detection of HPV8 early gene transcripts in PHAKs infected with recombinant retroviruses. cDNA was amplified with primer for E6 and E7 to verify the presence of transcripts in PHAK-8-E6 (lane 1) and PHAK-8-E7 (lane 2) cells. These and also the primers for E1, E2 and E1∧E4 were used to show the presence of all early genes of HPV8 in PHAK-8-CER except for E1∧E4 (lanes 3–7). (b) Morphology of PHAK cells infected with recombinant retroviruses. Phase contrast photographs of PHAK, PHAK-8E6, PHAK-E7 and PHAK-8-CER at passage 2 are shown. Magnification, ×100.

Figure 2

Keratin immunofluorescence of PHAK, PHAK-8-E6, PHAK-8-E7 and PHAK-8-CER cell lines. Keratin staining, which fluoresces green, was detected using the TSA Fluorescein System. Propidium iodide (PI) staining, which fluoresces red, highlights DNA stain in the nuclei. The apparent yellow colour present in the K6 of the HPV8-CER cells was the result of a very intense fluorescence that sometimes obscures the red staining of the nuclei.

Figure 3

Flow cytometric analysis of PHAKs expressing HPV8 early genes. (a, b) Relative DNA content of cells was determined by staining living cells with Hoechst 33342. Results shown are representative of at least three independent experiments. (c) BrdU incorporation into PHAK expressing HPV8 early genes. Box 1: 2n to 4n cycling cells; box 2: 4n to 8n cycling cells; box 3: octaploid cell population.

Figure 4

Western blot analysis for p53, pRb, p21 and GAPDH with extracts from keratinocytes expressing HPV8 early genes.