The Asian-American E6 Variant Protein of Human Papillomavirus 16 Alone Is Sufficient To Promote Immortalization, Transformation, and Migration of Primary Human Foreskin Keratinocytes

Abstract

We examined how well the human papillomavirus (HPV) E6 oncogene can function in the absence of the E7 oncogene during the carcinogenic process in human keratinocytes using a common HPV variant strongly associated with cervical cancer: the Asian-American E6 variant (AAE6). This E6 variant is 20 times more frequently detected in cervical cancer than the prototype European E6 variant, as evidenced by independent epidemiological data. Using cell culture and cell-based functional assays, we assessed how this variant can perform crucial carcinogenesis steps compared to the prototype E6 variant. The ability to immortalize and transform primary human foreskin keratinocytes (PHFKs) to acquire resilient phenotypes and the ability to promote cell migration were evaluated. The immortalization capability was assayed based on population doublings, number of passages, surpassing mortality stages 1 and 2, human telomerase reverse transcriptase (hTERT) expression, and the ability to overcome G₁ arrest via p53 degradation. Transformation and migration efficiency were analyzed using a combination of functional cell-based assays. We observed that either AAE6 or prototype E6 proteins alone were sufficient to immortalize PHFKs, although AAE6 was more potent in doing so. The AAE6 variant protein alone pushed PHFKs through transformation and significantly increased their migration ability over that of the E6 prototype. Our findings are in line with epidemiological data that the AA variant of HPV16 confers an increased risk over the European prototype for cervical cancer, as evidenced by a superior immortalization, transformation, and metastatic potential.

INTRODUCTION

Human papillomavirus (HPV) is a double-stranded DNA virus that infects epithelial keratinocytes. It is implicated in virtually all cervical cancers worldwide, with HPV16 being the most common high-risk type (76). Within the HPV16 type, variation occurs among the genomes, resulting in variants such as the European prototype (E), Asian-American (AA), African (Af), or Asian (As) (72). Epidemiological evidence suggests that the AA variant is more aggressive than other variants (3, 6, 63, 70, 71, 75), particularly in the presence of the HPV16 E7 oncogene (51).

Over the years, cellular immortalization has been defined in numerous ways. Landmark publications in 1989 defined immortalization as simply an extension of life span (21, 29, 43). On this basis, they showed that both the E6 and E7 oncogenes cooperated to accomplish immortalization of keratinocytes. This established that the E6 and E7 oncogenes were the cause of cellular immortalization in HPV16-positive primary human keratinocytes (PHKs) (21, 29, 43).

Experimental methods in the late 1980s were suboptimal as gene technology was rudimentary, but they have quickly evolved since then. In 1990, amphotropic, high-titer virus technology was developed for the purpose of transducing PHKs (42); this mimicked conditions occurring in vivo through the natural infection process. As more research was conducted over the course of time, the process of cell immortalization was partially resolved.

The discovery of the enzyme telomerase (18) and subsequent demonstration of the link between telomerase expression and immortalization (9) provided the foundation to further expand research in this field. The point when cells become immortalized was later defined by an immortalization crisis, referred to as mortality stage 2, (M2), which takes place after a period of replicative senescence, mortality stage 1 (M1) (4, 55, 56, 58, 67). During M1, cells remain viable for months and then escape from this stage, resulting in extended life span. This could occur when cells are infected with certain viral particles, as in the case of high-risk HPV16 infections (4). This stage can be confused for immortalization. M2 occurs once telomeres reach a critically short length and apoptosis is induced (4, 40, 50). The process of escaping from this stage has been associated with the stabilization of telomere length through reactivation of telomerase (4, 40, 50); this critical stage of immortalization was observed to be promoted by the E6 oncoprotein of HPV16 (36, 41, 47, 58, 62).

In subsequent articles, many researchers accepted the model that E6 and E7 must work together to induce immortalization in human keratinocytes, while it was Halbert et al. (20) who initially observed that E7 alone was sufficient to immortalize keratinocytes (11, 20, 27, 30, 36, 37, 62). However, none of these articles were able to show a complete picture of immortalization, as they relied only on one segment of the complex process as indicators for immortalization, namely, extension of life span (20), inactivation of the retinoblastoma protein (27), or increased telomerase expression (11, 30, 36, 37, 62).

Until now, no long-term study has been performed to highlight the differences in immortalization potential that may be seen among individual, naturally occurring E6 variants without the help of E7. Our laboratory extensively studied the role of E6 and E7 proteins in HPV16 AA and prototype E6 variants (51, 74, 75). We have previously reported that the HPV16 AAE6 variant in the presence of E7 showed a phenotype more reminiscent of transformed cells than the HPV16 prototype E6 due to differentially modulating metabolic enzymes and their associated pathways (51). These differences in the metabolic activities between AAE6/E7 and E6/E7 prototypes suggested that the AAE6 variant alone has a clear advantage over other variants during in vitro immortalization and transformation.

Here, we show through a long-term study that the E6 oncoprotein alone can immortalize host PHFKs and that the AAE6 variant was consistently more efficient than the prototype E6 in doing so. Furthermore, we hypothesized that the AAE6 variant protein alone can push PHFKs through the stages of carcinogenesis, such as transformation, migration, and invasion, and we demonstrate that this variant is more efficient in doing so than the E6 prototype protein. Finally, we confirmed a part of these findings through another independent long-term study using PHFKs procured from a separate donor showing that the observed effects were donor independent.

MATERIALS AND METHODS

Cell culture and propagation.

PHFKs (Cell Applications Inc., San Diego, CA) were cultured in serum-free keratinocyte growth medium (KGM; Cell Applications Inc.). Briefly, at 70 to 80% confluence, cells were incubated with trypsin-EDTA (Invitrogen, Carlsbad, CA), which was then inactivated with trypsin neutralization solution (Cell Applications Inc.). At each passage, cells were counted using a Bio-Rad TC10 cell counter (Bio-Rad, Hercules, CA). Phoenix cells (a kind gift of Garry P. Nolan, Stanford University) were cultured in Dulbecco's modified Eagle medium (DMEM) with 10% fetal bovine serum (FBS) and 1% antibiotics-antimycotics. The cells were grown until 70% confluence, and fresh media were given every 3 days.

Transfection of Phoenix cells.

To generate E6 and E7 viral particles, 5-alpha competent Escherichia coli cells (New England BioLabs, Ipswich, MA) were transformed with pLXSN plasmid DNA carrying either E6 with the hemagglutinin (HA) tag sequence at the C terminus followed by a stop codon (51) or E7 gene inserts. This HA tag approach was chosen to help subsequently detect variant E6 proteins using Western blotting. After amplification, the EndoFree Plasmid Maxi kit (Qiagen, Hilden, Germany) was used to isolate plasmid DNA from E. coli for transfection. A transfection step based on calcium phosphate precipitation was performed on Phoenix cells (42) using the CalPhos kit from Clontech (Mountain View, CA) and 10 μg plasmid DNA as previously described (51, 74).

Retroviral transduction of PHFKs.

PHFKs from two donors were grown to passage 3, after which biological triplicates were transduced with the viral supernatant from the transfected Phoenix cells (42) containing the following constructs: prototype E6, AAE6, and pLXSN. Untransduced parental PHFKs were also grown. In each flask, 2 ml of the respective viral supernatant was added in the presence of Polybrene (a cationic polymer used to increase the transduction efficiency; 1 μg per ml of viral supernatant). A cotransduction was also performed using 1 ml of E7 supernatant and 1 ml of prototype E6 or AAE6 supernatant to create the positive controls prototype E6E7 and AAE6/E7. Total infection time was 3 h, after which KGM was added (1:1) to avoid supernatant serum-induced differentiation from the DMEM of the viral supernatant. The flasks were incubated for another 3 h, after which the cells were maintained in KGM. One day after transduction, selection was performed to isolate transduced cell populations by growing the cells in the presence of 100 μg/ml gentamicin (G418; Roche Applied Science, Laval, Quebec, Canada) for a total of 6 days.

Flow cytometry analysis.

To determine cell cycle stages and anoikis resistance, flow cytometry was performed using a FACSCalibur flow cytometer (Becton Dickson). Briefly, at passages 6, 16, 30, and 60, cell cycle analysis was performed on both E6 cell lines using 0.5 nM actinomycin D (AD; Sigma, St. Louis, MO) as previously described by Richard et al. (51). Also for these passages, apoptosis was induced by resuspending the variant cell lines in semisolid KGM for 24 h as previously described by our group (74). Negative controls were adherent cells overlaid with normal medium. The cells were analyzed using the annexin V-fluorescein isothiocyanate (FITC) assay (Sigma) following 3 washes in phosphate-buffered saline (PBS). G₁:S ratios were calculated by comparing the number of cells in G₁ phase to the number of cells in S phase. The ratios approaching or below 1 were considered to reflect cells overcoming the cell cycle arrest.

PCR and qRT-PCR analysis.

To characterize E6 and E7 oncogene expression in the PHFKs, DNA was first extracted using a DNeasy blood and tissue kit (Qiagen, Hilden, Germany) per the manufacturer's protocol, and PCR analysis was performed. Briefly, 100 ng DNA was placed in the master mix containing 1× PCR buffer, 1 mM MgCl₂, 200 μM deoxynucleoside triphosphates (dNTPs), 0.5 μM forward and reverse primers (E6 primer sequences were from Zehbe et al. [75] and E7 primer sequences were from Lesnikova et al. [34]), and 1 U of Taq polymerase. The thermocycler parameters were 40 cycles of 94°C for 1 min, 55°C for 1 min, 72°C for 2 min, and a final extension of 72°C for 7 min. The resulting amplified DNA was imaged following 1.5% agarose gel electrophoresis. To determine the relative expression of E6 and E7 oncogenes in PHFKs, RNA was extracted using the Arcturus PicoPure RNA isolation kit (Applied Biosystems, Carlsbad, CA), and the quality and purity of the isolated RNA was determined using the Experion automated electrophoresis system using the StdSens analysis kit (Bio-Rad). The RNA was reverse transcribed to cDNA using the high-capacity cDNA archive kit (Applied Biosystems) with random hexamer primers. The parameters for the thermocycler were 25°C for 10 min, 37°C for 2 h, and 85°C for 5 min. For quantitative real-time PCR (qRT-PCR), 150 ng of cDNA was mixed with 45 μl TaqMan master mix, 4.5 μl of the gene assay mixes containing hypoxanthine phosphoribosyltransferase 1 (HPRT1), human telomerase reverse transcriptase (hTERT), E6, or E7 (Applied Biosystems), and nuclease-free water to a final volume of 90 μl. Triplicates of 25 μl each were pipetted into a 96-well plate and analyzed by a 7500 ABI cycler.

Transformation and clonogenic assay.

To determine the transformation and clonogenic capacity of the transduced PHFKs, high-passage-number keratinocyte cultures (passage 65) containing either E6 gene, as well as negative- and positive-control cells (parental PHFKs from passage 3 and HeLa cells, respectively), were assessed for their ability to form colonies in semisoft agar using the CytoSelect 96-well cell transformation kit (Cell Biolabs, Inc., Burlington, Ontario, Canada) as described previously (51). To determine the ability of the cells to form colonies in the presence of basement membrane proteins mimicking in vivo conditions, a modified clonogenic assay described by Franken et al. (15) was performed. Briefly, 6-well plates were immobilized with and without 20 μg/ml Matrigel (BD Biosciences) and blocked with 1% bovine serum albumin (BSA) to prevent nonspecific binding of cells to the substratum. High-passage prototype E6 and AAE6-containing keratinocytes, as well as PHFKs and HeLa cells, were seeded on the plates and grown for 11 days in serum-free DMEM/F12. The cells were replenished with fresh serum-free medium every 3rd day, and on the 11th day they were fixed with cold methanol, stained with 0.1% crystal violet Gram stain (Sigma-Aldrich), and imaged under low magnification. The different types of colonies were distinguished and quantified.

Adhesion and viability assays.

To determine whether the basement membrane proteins promote cell adhesion capacity, an adhesion assay was performed. Briefly, 96-well plates were immobilized with different concentrations of Matrigel (0, 5, 10, 20, and 40 μg/ml) in replicates of 4 and blocked with 1% BSA for at least 10 min. About 100,000 cells were seeded per well, after which plates were incubated at 37°C for 40 min. The cells were washed with serum-free DMEM/F12 until cells in the BSA wells stopped detaching. The cells were fixed using ice-cold methanol and stained with 0.1% crystal violet Gram stain. Filtered 2% SDS was added to all wells, and the plates were incubated with agitation at room temperature for 30 min, after which absorbance readings at 550 nm were taken using the PowerWave XS plate reader (BioTek, VT). To determine if the Matrigel affects cell proliferation, a resazurin-based fluorimetric assay (R & D Systems, Minnesota) was performed. After the 96-well plates were immobilized with Matrigel as described above, 4,000 cells were seeded per well and allowed to grow in their normal growth medium until wells containing BSA reached a confluence of 80%. Resazurin solution was added to each well, as described by the manufacturer's protocol, and plates were incubated at 37°C for 2 h, after which time the plates were incubated with agitation in the dark at room temperature for 20 min. The fluorescence intensities were measured using the FLx800 plate reader at an excitation of 540 nm and emission of 590 nm.

Invasion/migration assay.

To determine the invasiveness and metastatic ability of transduced PHFKs, high-passage-number (passage 70) keratinocyte cultures containing both E6 genes under study (AAE6 and prototype E6), as well as negative (PHFK) and positive controls (HeLa, prototype E6E7, and AAE6/E7), were assessed for their ability to migrate through a porous membrane. A CytoSelect 24-well Cell Haptotaxis assay (Cell Biolabs) coated with collagen I was used for this study. Ten percent FBS in KGM was added to the bottom chamber of three wells per variant. Each variant was seeded at 150,000 cells per insert, and the plate was incubated at 37°C for 16 h based on similar experiments (2, 5, 23, 48, 61). Migratory cells were stained with crystal violet staining solution and imaged using an inverted microscope at ×200 magnification. Three fields per insert were used for imaging, and the stain was then extracted and the absorbance reading at 560 nm taken using a PowerWave XS plate reader (BioTek, VT).

SDS-PAGE gel electrophoresis.

To observe protein expression, SDS-PAGE gels were prepared at different concentrations to appropriately resolve the protein of interest. Protein samples were lysed on ice using 110 μl of lysis buffer (1 ml of nucleus buffer containing 0.25 M sucrose, 0.2 M NaCl, 10 mM Tris-HCl, 2 mM MgCl₂, 1 mM CaCl₂, 1% Triton X-100, and water to 1 liter, 10 μl phenylmethylsulfonyl fluoride [PMSF; Sigma, St. Louis, MO], and 1 μl protease inhibitor cocktail [Sigma, St. Louis, MO]). Prior to addition of SDS to the cell lysates, the total protein in the samples was estimated by a Bradford assay using the Bio-Rad protein assay (Bio-Rad, Hercules, CA). Equal amounts of total protein were loaded into each well based on the known concentrations of each sample. The gel was run at 100 V until the samples ran into the separating gel, and then the voltage was turned up to 120 V.

Western blot analysis.

The SDS-PAGE gel was transferred to a polyvinylidene difluoride (PVDF) membrane for 1 h at 100 V and was then blocked in an appropriate percentage of milk or BSA for 1 h at room temperature. The membrane was put in primary antibody solution. Depending on the experiment, we used either monoclonal mouse E-cadherin (1:200; M3612; Dako), polyclonal rabbit CAIX (1:5,000; ab15086; Abcam), monoclonal rabbit p53 (1:500; M3629; Dako), or rat monoclonal HA (1:500; 11867423001; Roche) dissolved in milk and secondary antibody solutions corresponding to each primary antibody. Chemiluminescence was done with Western Lightning Plus-ECL (PerkinElmer, Inc.) solutions, and pictures were then taken using the BioSpectrum 410 Imaging System (UVP). Densitometry was performed, and the housekeeping gene actin was used as a loading control to normalize the results.

Statistical analysis.

All statistical analyses were performed using open-source statistical software, called R (a language for data analysis and graphics; 1996), or Graphpad Prism. All data were tested for normality and homogeneity of variances using a Shapiro-Wilks test and a Bartlett's test, respectively, before choosing a suitable parametric or nonparametric statistical test. A resulting P value of less than 0.05 was considered significant. Parametric data sets were analyzed using Student's t tests and one- and two-way analyses of variance (ANOVAs), followed by an appropriate post hoc test, such as a Tukey's honestly significant difference. Nonparametric data sets were analyzed using two-way ANOVAs or one-way Kruskal-Wallis ANOVA followed by a Nemenyi's post hoc test and Friedman's tests followed by a pairwise comparison using a Wilcoxon signed rank test with a Bonferroni adjustment. Cell population doublings were calculated based on the method described by Willey et al. (66).

RESULTS

The E6 oncoprotein alone is sufficient for PHFK life span extension and escape from the immortalization crisis.

We confirmed AAE6 and prototype E6 gene presence and expression in transduced PHFKs by conventional and quantitative reverse transcriptase PCR (qRT-PCR), respectively, at passages 6, 16, 30, and 60 (data not shown). All E6 variants were devoid of any E7 expression, and vector pLXSN and untransduced PHFKs (both negative controls) were clear of any E6 or E7 expression. We also completed Western blotting against the HA tag present in the E6 gene constructs used for retroviral transduction to quantify respective E6 protein expression levels (Fig. 1). Both the variant and the prototype had comparable levels of E6 protein (P > 0.05 by Student's t test; n = 3); we can conclude that experimental effects are not a result of differential E6 quantities. To observe immortalization, PHFKs from two separate donors were each transduced with the E6 oncogene from the AAE6 variant or the prototype E6. The E6 prototype together with prototype E7 (positive control), the vector pLXSN, and untransduced PHFKs (negative controls) were also cultured. From PHFK donor 1, both types of E6 as well as E6E7 were grown up to passage 65. Both untransduced and pLXSN-transduced PHFKs senesced at passage 9. From PHFK donor 2, both types of E6 as well as E6E7 were grown up to passage 30. Untransduced PHFKs survived until passage 12, while pLXSN-transduced PHFKs senesced at passage 7. In donor 1, AAE6 and prototype E6 reached passage 65 after 495 and 662 days, respectively, resulting in significantly fewer days for AAE6 to reach the endpoint compared to prototype E6 (P < 0.001) (Fig. 2A and B and Table 1).

Fig 1.

HA tag protein expression as a relative measure of E6 oncoprotein expression. Western blot analysis of transduced PHFKs from donor 2 (passages 6, 16, and 30) and donor 1 (passage 70) was performed. Densitometry was performed relative to the housekeeping gene beta-actin. HA tag protein expression was found in both transduced variants at all passages. Student's t test reveals no significant differences in protein expression levels between variants among passages (P > 0.05; n = 3). (B) Respective densitometry results from triplicate experiments shown in panel A. The mass of beta-actin is 42 kDa.

Fig 2.

Immortalization of primary human keratinocytes (PHFKs). Retrovirally transduced keratinocytes from two separate donors were grown in serum-free KGM and passaged through 30 and 60 passages. Transduced PHFKs from donor 1 (A) and donor 2 (B) were passaged, and the number of passages and time points were plotted. Donor 1 cells that were transduced with the AAE6 variant took significantly fewer days to reach passage 60 (P < 0.001 by Friedman test followed by pair-wise comparison using the Wilcoxon signed rank test with Bonferroni adjustment; n = 3). In donor 2, there was no significance with regard to days to reach passage 30, but the same trend as that for donor 1 was achieved. (C and D) Total population doublings from donor 1- and donor 2-transduced PHFKs were determined. Compared to prototype E6 cells, AAE6 cells had significantly more population doublings (P < 0.00001 by one-way ANOVA; n = 60) with significantly shorter population doubling times (P < 0.000001 by one-way ANOVA; n = 60). In donor 2 there were no statistically significant differences in population doublings, but the same trend as that for donor 1 was achieved.

Table 1.

Population doubling time for transduced PHFKs^a

Donor and variant	Result for passage:
	6–16		17–30		6–30		31–65		6–65
	Doubling time (h)	No. of daysb	Doubling time (h)	No. of days	Doubling time (h)	No. of days	Doubling time (h)	No. of days	Doubling time (h)	No. of days
Donor 1
AAE6	93.22 ± 83.78	128	55.83 ± 15.21	112			48.29 ± 12.72	255	58.28 ± 40.28	495
Prototype E6	91.53 ± 63.50	114	164.77 ± 157.91	191			91.45 ± 63.52	357	131.22 ± 112.32	662
E6E7	84.74 ± 69.00	120	51.23 ± 8.39	113			57.95 ± 65.10	271	61.29 ± 58.29	504
Donor 2
AAE6	73.63 ± 28.82	57	62.76 ± 20.71	69	67.54 ± 24.67	126
Prototype E6	83.26 ± 38.01	60	82.60 ± 32.01	78	82.89 ± 34.02	138
E6E7	61.79 ± 15.64	70	43.27 ± 16.17	60	51.42 ± 18.21	130

^aPHFKs containing the E6 oncogene variants were passaged up to 60 times in donor 1 and up to 30 times in donor 2. The population doubling times were calculated using the standard formula 3.32(log N_t − log N_o) = tf, where N_t is the number of cells counted and N_o is the number of cells plated, t is the time in days between passages, and f is the growth rate constant.

^bShown is the number of days gone by between the passages.

We observed more drastic differences between AAE6 and prototype E6 when we calculated population doubling times (66) in both donor PHFKs. In donor 1, AAE6 had significantly more population doublings than prototype E6 at passage 65 (P < 0.00001), with significantly shorter population doubling times compared to prototype E6 (P < 0.000001) (58.28 ± 40.28 h and 131.22 ± 112.32 h, respectively) (Fig. 2C, Table 1). This trend was also seen in donor 2, with AAE6 reaching passage 30 in 126 days and E6 prototype in 138 days and with a difference in population doubling times of 67.54 ± 24.67 h (AAE6) and 82.89 ± 34.02 h (E6 prototype) (Fig. 2D, Table 1). Together, these results demonstrate a similar trend in the growth and passage-specific pattern between the donors and that AAE6 was significantly faster than prototype E6.

The difference in growth rate between AAE6 and prototype E6 may be caused by variations in metabolic enzyme-mediated pathways (e.g., amino acid metabolism, tricarboxylic acid [TCA] cycle, and glycolytic pathways) between the two variants, as evidenced in our earlier publication (51). In particular, isocitrate dehydrogenase (IDH) isotypes 1 and 2 were found to be reduced in the AAE6 variant compared to prototype E6 (51), which leads to increased hypoxia-inducible factor 1α (HIF-1α) levels. To test this notion further, we examined the expression levels of one downstream effector molecule, the metabolic enzyme carbonic anhydrase IX (CAIX), in high-passage-number PHFKs expressing either E6 protein through Western blotting. CAIX expression was higher in AAE6 than in the prototype E6 and HeLa cells, and this was significant in the presence of E7 (P < 0.001) (Fig. 3), suggesting that CAIX expression is one means by which the oncogenic potential in the AAE6 variant is increased over that of the prototype.

Fig 3.

Carbonic anhydrase IX expression in the presence of different E6 oncogene variants. Western blot analysis on high-passage-transduced PHFKs was performed in triplicate. Densitometry was performed relative to the housekeeping gene beta-actin. (A) CAIX protein expression was found to be higher in the AAE6 variant than the prototype E6; however, no statistical significance was observed (P > 0.05 by one-way ANOVA; n = 3). (B) Respective densitometry results from triplicate experiments shown in panel A. (C) CAIX expression in AAE6 and prototype E6 variants in the presence of E7 were compared. It was observed that AAE6/E7 had significantly higher CAIX expression than prototype E6E7 (P < 0.001 by one-way ANOVA; n = 6). (D) Respective densitometry results from triplicate experiments shown in panel C. The mass of beta-actin is 42 kDa.

Both E6 proteins can overcome p53-induced G₁ arrest.

In the absence of tumor suppressor p53, it has been shown that cells bypass M1 (68), the first stage of senescence. Since E6 is known to abolish p53 activity, its presence in PHFKs should be enough to bypass cell cycle arrest and, consequently, M1. To test this notion, actinomycin D was used to induce DNA damage through strand breaks by interfering with cellular topoisomerase activity (14). This results in increased p53 expression, which leads to G₁ arrest (28). Our PHFKs transduced with E6 variants were expected to block this induced p53 activity. We observed that both E6 proteins equally and significantly overcame G₁ arrest in both donors compared to untransduced PHFKs (P < 0.01) (Table 2). This shows that the E6 oncoprotein is functional and the transduced PHFKs can overcome M1 without the help of E7. Western blotting was performed to ensure that p53 expression was downregulated. We found that p53 expression in passages 6, 16, and 30 for both E6- and E6E7-transduced cell lysates was undetectable, while control PHFKs showed strong p53 expression (Fig. 4), which is in line with our earlier findings (51, 74).

Table 2.

Cell cycle stage analysis of transduced PHFKs^a

Donor and variant	G1:S ratio at passage:
	6	16	30	62
Donor 1
AAE6	1.159523	0.963343394	1.078434835	0.89521312
Prototype E6	1.155732	1.187382696	0.957111242	0.822580843
E6E7	0.727265	0.640061664	0.873832927	1.031583798
PHFK	1.616437	NA	NA	NA
Donor 2
AAE6	0.871189	0.800391187	0.846311
Prototype E6	0.949242	0.922922372	0.824532
E6E7	1.097474687	1.011198634	0.995241
PHFK	2.605856	NA	NA

^aFlow cytometry was performed to observe the effect of the E6 oncogene on induced cell cycle arrest. PHFKs containing the E6 oncogene variants were exposed to actinomycin D, and the G₁:S ratio was calculated. This experiment showed that in both donors, E6 oncogene expression resulted in overcoming G₁ cell cycle arrest, leading to a significantly higher number of cells in S phase compared to PHFKs (P < 0.01 by two-way ANOVA; n = 3). NA, not applicable.

Fig 4.

Expression of p53 protein in PHFKs transduced with and without E6 variants. Western blotting was performed on the cell lysates obtained from passages 6, 16, and 30 in duplicates from donor 1. p53 expression was abolished in every variant containing E6, while normal PHFKs showed p53 protein expression. The mass of beta-actin is 42 kDa. For passage 6, C33a was used as a positive p53 control rather than PHFKs, as there was no sample available. A minus denotes DMSO treatment without actinomycin D, and a plus denotes actinomycin D treatment.

Immortalization status of PHFKs in the presence of AAE6 or prototype E6 is further confirmed by hTERT expression.

Once keratinocytes bypass M1, they are characterized by an extension of life span after which an immortalization crisis, M2, ensues (54, 67). Both E6 PHFK cultures exhibited some level of crisis during the culture period from which the immortalized subpopulation emerged. AAE6 had a shorter major crisis time (for donor 1 it was 350 h and for donor 2 it was 140 h) compared to the prototype E6 (700 h for donor 1 and 175 h for donor 2) (Fig. 5). Overcoming this stage has been attributed to combating telomere shortening, as measured by telomerase reverse transcriptase's catalytic subunit, hTERT.

Fig 5.

Average population doubling time in hours per passage in PHFKs transduced with E6 oncogene variants. Doubling time, in hours, was plotted against the passage number of each variant from both donor 1 and donor 2. An extreme increase in doubling time suggests a point of crisis in culture growth. A, B, and C represent donor 1, while D and E represent donor 2. Major crises have been circled.

To further assess the immortalization capability of the two E6 proteins in our study, qRT-PCR for hTERT was performed on transduced PHFKs, as well as in negative-control PHFKs and positive-control HeLa cells. The expression of hTERT was standardized to hypoxanthine phosphoribosyltransferase 1 (HPRT1) expression in the E6 PHFK cultures, as previous results indicate that HPRT1 expression is unaffected by HPV infection (10). As expected, we observed that as the passage number increased from passage 6 to 30, hTERT expression steadily increased in a time-dependent manner (Fig. 6A). Once at passage 30, hTERT expression was highest in both E6 cultures but significantly higher in AAE6 than in prototype E6 (P < 0.001). However, hTERT expression seemed variable, because this difference was not confirmed in donor 2 (Fig. 6B). This activation and increased expression of hTERT allows for the conclusion to be drawn that the E6 oncogene is sufficient for immortalization without E7.

Fig 6.

Relative expression of hTERT gene in PHFKs transduced with and without E6 variants. A real-time PCR was performed using hTERT and HPRT1 (control housekeeping gene). (A) hTERT expression in donor 1. At passage 30, it was found that the AAE6 variant showed significantly higher expression than the prototype E6 (P < 0.001 by two-way ANOVA; n = 9). Within the AAE6 variant, the passage 30 PHFKs had significantly higher expression of hTERT than passage 6 PHFKs (P < 0.0001 by two-way ANOVA; n = 9). (B) In donor 2, it was observed that at passage 30, the prototype E6 had significantly higher expression than AAE6 (P < 0.00001 by two-way ANOVA; n = 9). An asterisk denotes statistical significance.

AAE6 and prototype E6 oncoprotein expression equally facilitate PHFKs in overcoming anoikis, but only AAE6 is able to induce transformation in vitro.

To assess the ability of both E6 proteins to survive anoikis (detachment-induced apoptosis) through E6 expression, transduced PHFKs were resuspended in media containing methylcellulose, after which annexin V-FITC flow cytometry was performed. Annexin V-FITC allows the cells to incorporate annexin V-FITC agent when the membrane has been compromised, signifying early apoptosis, and also allows cells to incorporate propidium iodide when the cell membrane has been abolished, signifying cell death. Both E6 PHFK cultures from both donors showed similar anchorage-independent survival at a level that was significantly higher than that of PHFKs treated with semisolid medium (P < 0.00001). Both E6 cultures and both donors were able to overcome anoikis, as evidenced by a general trend of decreased cell death with increasing passage number beginning at passage 16, suggesting that they were in the beginning stages of becoming transformed (Fig. 7A and B). This observation was consistent for both donors.

Fig 7.

Transformation of PHFKs transduced with and without E6 variants. (A and B) Annexin V-FITC flow cytometry was performed to determine if the viral protein-transduced cells could overcome anoikis. In both donors, all high-passage-number E6 variants had significantly decreased anoikis compared to normal PHFKs (P < 0.00001 by two-way ANOVA; n = 3). (C) A semisoft agar transformation assay shows that the AAE6 variant was the only E6 variant that was able to form colonies. Cell proliferation was calculated with the MTT assay. The averages from three independent experiments are shown. (D) The representative colonies for each variant from the semisoft agar transformation assay are shown here. A second transformation assay under serum starvation conditions was performed in the absence (E) and presence (F) of anchorage-supporting basement membrane proteins. It was observed that in the presence and absence of basement membrane protein, the AAE6 variant formed significantly more colonies than prototype E6 and HeLa cells and normal PHFKs (P < 0.05 by Student's t test; n = 3). An asterisk denotes statistical significance.

Anchorage-independent growth through colony formation is another method for detecting in vitro transformation of cells. The ability of each E6 culture to form colonies in semisolid medium at passage 65 was assessed through a transformation assay (51). We observed that high-passage-number AAE6 PHFKs (passage 65) were able to form colonies while E6 prototype PHFKs were not, suggesting that AAE6 surpassed the transformation potential of prototype E6 (Fig. 7C and D), as supported by our previous study using both E6 and E7 (51). The ability of the cells to withstand serum-starved conditions in anchorage-dependent environments was also evaluated. The AAE6 PHFKs were still able to form colonies, while the ability of HeLa cells to form colonies was greatly reduced. However, no CFU were observed in the culture plates containing prototype E6-transduced PHFKs (Fig. 7E and F). Together, these results indicate that compared to prototype E6-transduced PHFKs, the AAE6 variant may create a microenvironment more conducive to tumor cell growth even in nutrient-deficient conditions.

We next tested resilience by means of clonogenic assays to observe any attributes of the variants that assist them in growth under unfavorable conditions. Soft agar-based clonogenic assays are used to gather information about the ability of neoplastic cells to grow in a contact-inhibited, exogenous growth factor-depleted, and anchorage-independent environment. Our modified clonogenic assays confer anchorage-dependent conditions that are free of exogenous growth factors and have extremely low seeding dilutions per square area of substratum (33). These conditions promote an environment that is conducive to tumor growth, causing cells to group together to form a microenvironment with defined edges that allow cell survival. The resistance potential and resiliency of the E6 PHFKs were evaluated. It was observed that colonies with definite resistance potential (closed and mixed types) were only present in AAE6 populations (Fig. 8), suggesting that this variant has an increased potential to survive adverse conditions.

Fig 8.

PHFKs transduced with various E6 oncogene variants formed distinct CFU. A modified clonogenic assay showed that the two E6 oncogene variants were able to form different kinds of colonies. (A) Morphology of a closed CFU. Scattered CFU (B) and mixed CFU (C) that comprised both closed and scattered colonies. (D) We observed that in the absence of basement membrane protein, the AAE6 variant preferentially formed closed colonies while HeLa formed scattered colonies, and the prototype E6-transduced PHFKs, along with normal PHFKs, did not form any colonies. (E) In the presence of basement membrane proteins, the AAE6 variant preferentially formed mixed colonies, while HeLa cells and the prototype E6 formed scattered colonies.

AAE6 showed significantly better migration ability than prototype E6.

Once cells gain a malignant phenotype, metastasis can occur. The initial and gateway step in the metastasis cascade is invasion of the basement membrane of blood vessels, which occurs when the primary tumor cells strongly attach to and invade through the epithelial layer and stroma (12). To experimentally mimic these conditions in vitro, we initially performed a cell adhesion assay to determine the E6 PHFKs' adhesive capability, as this is an important first step before invasion can occur. Briefly, we seeded PHFKs transduced with the E6 variants on a gradient concentration of immobilized basement proteins (Matrigel). We observed that both E6 cultures showed a concentration-dependent increase in adhesion, beginning at 10 μg/ml Matrigel (Fig. 9A). Interestingly, we observed that while both variants showed significantly increased adhesion at 40 μg/ml Matrigel compared to control PHFKs (P < 0.05), AAE6 had a significantly higher adhesion capacity than prototype E6 and HeLa cells (P < 0.05). It was also observed that Matrigel did not have a significant effect on cell proliferation or viability, suggesting that the experimental conditions were conducive for cell growth (Fig. 9B).

Fig 9.

Metastatic potential of immortalized PHFKs. (A) Adhesiveness of the transduced PHFKs was tested using different concentrations of Matrigel. We observed that between 10 and 40 μg/ml, AAE6 had significantly increased adhesion capacity compared to prototype E6 and HeLa cells and PHFKs (P < 0.05 by Student's t test; n = 4). Prototype E6 also had significantly higher adhesion capacity than PHFKs (P < 0.01 by Student's t test; n = 4). An asterisk denotes statistical significance. (B) To determine whether the Matrigel used in the analysis had any significant effect on cell viability, the cells were treated with resazurin and incubated for 2 h. We observed that there was no significant effect of Matrigel on the transduced PHFKs. (C) A Boyden chamber assay using a type I collagen-coated Cytoselect 24-well Cell Haptotaxis assay was performed using high-passage-number E6 variants. FBS was added to the bottom chamber of the wells to act as a minor chemoattractant. It was observed that the AAE6 variant had significantly higher migration capacity than prototype E6 with and without E7 (P < 0.05 by one-way ANOVA; n = 3). One and two asterisks denote statistical significance between the two relative variants, while three asterisks denote overall significance.

To determine the migratory capability of these cells, a Boyden chamber assay was completed using the transduced PHFKs (48). It was observed that AAE6 PHFKs migrated significantly more than prototype E6 PHFKs in the Boyden chamber (P < 0.05) (Fig. 9C), which suggests that the AAE6 variant is able to better adhere to and migrate through the basement membrane. This trend was also observed in the presence of E7. HeLa cells had the highest migration capacity of all variants (P < 0.0001). Interestingly, parental nontransduced PHFKs showed a better migratory ability compared to prototype E6 and E6E7 but not to AAE6 with and without E7. This may be attributed to the natural expression of collagenase I by PHFKs (46). This enzyme may become dysregulated after infection by the HPV16 E6 oncogenes during the carcinogenesis process. One potential mechanism of migration in these keratinocytes is the downregulation of E-cadherin, a cell-cell adhesion molecule. To investigate this, Western blotting was performed on high-passage-number keratinocytes. It was found that the AAE6 variant had lower expression of E-cadherin than the prototype E6, as would be expected. HeLa cells had lower E-cadherin expression than both variants, which was significant compared to the prototype E6 (P < 0.009). The PHFKs also had lower E-cadherin expression than both E6 proteins (Fig. 10).

Fig 10.

E-cadherin expression in the presence of different E6 oncogene variants. Western blot analysis on high-passage-transduced PHFKs was performed. Densitometry was performed relative to the housekeeping gene beta-actin. (A) E-cadherin protein expression was found to be lower in the AAE6 variant than the prototype E6, although this was not significant. Control HeLa cells and PHFKs both had completely downregulated E-cadherin, which was significant in HeLa cells compared to prototype E6 (P < 0.009 by one-way Kruskal Wallis; n = 3). (B) The representative densitometry from triplicate experiments shown in panel A. An asterisk denotes significance. The mass of beta-actin is 42 kDa.

DISCUSSION

Our study fills a knowledge gap within the field of keratinocyte carcinogenesis which traces its beginnings to the late 1980s, when high-risk HPV E6 and E7 oncoproteins were first reported to cooperate synergistically to extend primary human keratinocyte life span (21, 29, 43). It is imperative to clarify the role of the HPV16 E6 oncoprotein in the cellular immortalization process, as this is the initial step in the molecular signaling cascade that may promote malignancy. Based on previous literature, we composed a complete definition of immortalization and performed appropriate experiments to validate its definition. Before cells can reach the immortalized state, they must pass through the extension-of-life-span stage (4). Our experimental keratinocyte models demonstrated much beyond this, with the passage 65 endpoint reached significantly faster by the AAE6 variant than prototype E6. This was expected, as earlier epidemiological findings have suggested that non-European variants (such as the AA variant) are more oncogenic than the E6 prototype based on their increased prevalence in cervical cancer (3, 75). This was also seen in our earlier studies which showed that in the presence of E7, the metabolic enzyme IDH was found to be expressed significantly less in the AAE6 variant than in the prototype E6 (51). IDH is necessary for α-ketoglutarate production, which is used for hydroxylation of HIF-1α in normoxic conditions. Therefore, a decrease in IDH would result in an increase of HIF-1 expression (53). As a downstream gene in the HIF-1 pathway, we examined CAIX, which regulates pH and cellular adhesion to nonadhesive supports (45). AAE6 had higher CAIX expression than prototype E6, which was significant in the presence of E7. As CAIX is a known indicator of poor patient prognosis (45) and aggressive tumors (32, 38), this higher expression in the AAE6 variant may correlate with the increased prevalence of this variant in epidemiological studies.

The first stage that the cells must overcome leading to immortalization is the replicative senescence stage, during which cell cycle arrest is induced through elevated p53 following DNA damage (24). After DNA damage was induced in the cultures in this study, the cells overcame G₁ arrest and, consequently, M1 by destroying functional p53. Once cells bypass M1 and an extension of life span, immortalization crises occur caused by telomere shortening (8), during which nonimmortalized cells senesce and an immortalized subpopulation emerges. Cells with this ability overcome telomere shortening through transient expression of telomerase (18). This enzyme is detectable at very low levels in normal cells but is inactive (8, 19, 73). When it is activated, it assists cells past the M2 crisis stage, leading to immortalization (54). E6 has been shown to induce telomerase activity through an increase in hTERT transcription in keratinocytes, a hallmark for immortalization (20, 31, 36, 41, 44, 47, 58, 62, 69). As expected, we observed that both E6 variants reached and surpassed M2, as evidenced by steadily increasing hTERT levels (elevating at least as early as passage 6 and culminating at passage 30). In passage 30, we observed optimal hTERT expression for both E6 proteins, potentially due to the cells completely overcoming M2. After this, hTERT expression levels decreased to a more sustainable level. The E6 oncoprotein also aids in the ubiquitin-dependent proteosomal degradation of p53 protein, which may indirectly result in bypassing the M2 immortalization crisis stage, as described in earlier reports (40, 50). Also, there were variable amounts of hTERT expression between the E6 variants, suggesting E7 is necessary to stabilize the expression levels, as seen in our previous results (51) and as studied by Liu et al. (35).

One aspect of carcinogenesis involves keratinocytes attaining a malignant phenotype through transformation. Transformation of cells into this phenotype allows for formation of colonies that are resistant to immune-mediated clearance and provides an environment conducive to tumor growth (33). Usually, when normal keratinocytes lose their ability to adhere to a substratum and to each other, terminal differentiation is induced (1) and cells commit to detachment-induced apoptosis, which is termed anoikis (16). Transformed cells can overcome anoikis, likely through CFU that adhere strongly to each other. This may be supported by HPV16 infection, where the E6 gene can counteract the proapoptotic effects of p53 (52). Anchorage-independent conditions can be recreated in vitro through the use of semisolid medium (1). One of the main causes of transformation is an upregulation of Notch1 signaling (49), which results in resistance to anoikis and can be induced by E6 (64). Under anoikis-inducing conditions, we found that both E6 proteins from both donors equally showed increased resistance to anoikis as the passage number increased. However, AAE6 showed increased potential for transformation through its ability to form colonies in both anchorage-inhibiting and -promoting conditions. In addition, when AAE6-transduced PHFKs were grown in serum-starved nutrient media in the absence of a basement membrane protein substratum, they resisted apoptosis and produced closed cell colonies. This suggests that AAE6-PHFKs could be capable of surviving inhospitable conditions in the human body, likely through an upregulation of the Akt signaling pathway, which is known to promote antiapoptotic functions (57). Additionally, when serum-starved AAE6 cells were grown in the presence of basement membrane protein substratum, they formed more mixed colonies that had both closed and scattered CFU, suggesting an adaptable survival mechanism. The differences seen in CAIX expression could be one reason behind this. The ability of CAIX to modulate its environment with respect to pH (45) so that it is conducive to cell growth suggests that a higher expression of CAIX would create a more favorable microenvironment around the colonies. Together, these findings suggest that E6 gene expression is necessary for in vitro transformation as well as anchorage to basement membranes, and that the increased transformational potential of the AAE6 variant may augment its invasive potential.

Some groups have completed invasion and migration studies involving HPV16 and human umbilical vein endothelial cells (HUVEC) (48), trophoblast-like cells (5), cervical keratinocytes (61), or mutant keratinocytes (2), but the ability of different E6 proteins alone to cause migration and invasion in keratinocytes has not been examined until now. Consistently in the presence or absence of the E7 protein, the AAE6 variant migrated more than the prototype E6 variant. Although there was this difference between the variants, the presence of the E6 oncoprotein alone did not cause the PHFKs to migrate any more than they would normally. However, the migratory potential was enhanced for AAE6 in the presence of E7, which correlates with results from Charette et al. (7) and suggests that the E7 oncoprotein enhances migration in keratinocytes through the Akt signaling pathway. To further investigate the potential mechanism behind this difference in migration between variants, the role of E-cadherin was examined. During the metastasis stage, the epithelial-mesenchymal transition (EMT) is known to promote an invasive and migratory phenotype, possibly via modulation of the Wnt/beta-catenin signaling pathway (13, 60). During this phase, E-cadherin is downregulated (65), allowing cells to release themselves from each other and prepare for invasion and metastasis (39). In a study by Matthews et al. (39), it was found that E6 downregulates E-cadherin expression, which might partially induce an EMT. However, our results show that E-cadherin expression is not completely downregulated by the E6 oncoprotein alone, which suggests that, in the case of HPV16, E7 is necessary for optimal migration. This correlates with the Boyden chamber assay results, which show that in the presence of E7, the AAE6 variant migrates more than its counterpart containing only E6. Also, for controls, the decreased level of E-cadherin expression directly correlates with an increase in migration. The same trend is seen between the E6 variants, with the AAE6 cells showing less E-cadherin expression than prototype E6 cells and having higher migration ability. This signifies that E-cadherin downregulation is one mechanism to induce migration in PHFKs. The downregulated E-cadherin expression in PHFKs may be because they are primary cells from neonates at early passage (i.e., passage 3) with a mesenchymal phenotype due to retained embryogenesis pathways. Moreover, several reports have suggested that E-cadherin expression patterns in keratinocytes are variable based on the source, culture conditions, and other experimental factors (17, 22, 25, 26, 59). Potential contamination of our PHFKs with fibroblasts was ruled out because they tested negative for fibronectin (a marker for fibroblasts) in Western blotting (data not shown).

In summary, our results reveal the power of the E6 oncoprotein alone during the carcinogenesis process and that naturally occurring HPV16 E6 variants have differential abilities in accomplishing the various stages involved. We demonstrated that either E6 oncoprotein alone is sufficient to immortalize keratinocytes in vitro but that the AAE6 variant is more efficient at doing so. AAE6 was also superior to the prototype E6 at inducing transformational traits in the PHFKs. These data further support epidemiological findings that the incidence of the AA variant in cervical cancers is 20 times higher than other variants or the prototype (3) and highlight AAE6's enhanced carcinogenic properties as a reason behind this.