Structural and biochemical changes underlying a keratoderma-like phenotype in mice lacking suprabasal AP1 transcription factor function.

Epidermal keratinocyte differentiation on the body surface is a carefully choreographed process that leads to assembly of a barrier that is essential for life. Perturbation of keratinocyte differentiation leads to disease. Activator protein 1 (AP1) transcription factors are key controllers of this process. We have shown that inhibiting AP1 transcription factor activity in the suprabasal murine epidermis, by expression of dominant-negative c-jun (TAM67), produces a phenotype type that resembles human keratoderma. However, little is understood regarding the structural and molecular changes that drive this phenotype. In the present study we show that TAM67-positive epidermis displays altered cornified envelope, filaggrin-type keratohyalin granule, keratin filament, desmosome formation and lamellar body secretion leading to reduced barrier integrity. To understand the molecular changes underlying this process, we performed proteomic and RNA array analysis. Proteomic study of the corneocyte cross-linked proteome reveals a reduction in incorporation of cutaneous keratins, filaggrin, filaggrin2, late cornified envelope precursor proteins, hair keratins and hair keratin-associated proteins. This is coupled with increased incorporation of desmosome linker, small proline-rich, S100, transglutaminase and inflammation-associated proteins. Incorporation of most cutaneous keratins (Krt1, Krt5 and Krt10) is reduced, but incorporation of hyperproliferation-associated epidermal keratins (Krt6a, Krt6b and Krt16) is increased. RNA array analysis reveals reduced expression of mRNA encoding differentiation-associated cutaneous keratins, hair keratins and associated proteins, late cornified envelope precursors and filaggrin-related proteins; and increased expression of mRNA encoding small proline-rich proteins, protease inhibitors (serpins), S100 proteins, defensins and hyperproliferation-associated keratins. These findings suggest that AP1 factor inactivation in the suprabasal epidermal layers reduces expression of AP1 factor-responsive genes expressed in late differentiation and is associated with a compensatory increase in expression of early differentiation genes.

AP1 factor inactivation in the suprabasal epidermis causes increased epidermal hyperproliferation and hyperkeratosis but reduced carcinogen-dependent tumor formation.

Activator protein one (AP1) (jun/fos) factors comprise a family of transcriptional regulators (c-jun, junB, junD, c-fos, FosB, Fra-1 and Fra-2) that are key controllers of epidermal keratinocyte survival and differentiation, and are important drivers of cancer development. Understanding the role of these factors in epidermis is complicated by the fact that each member is expressed in defined cell layers during epidermal differentiation, and because AP1 factors regulate competing processes (that is, proliferation, apoptosis and differentiation). We have proposed that AP1 factors function differently in basal versus suprabasal epidermis. To test this, we inactivated suprabasal AP1 factor function in mouse epidermis by targeted expression of dominant-negative c-jun (TAM67), which inactivates function of all AP1 factors. This produces increased basal keratinocyte proliferation, delayed differentiation and extensive hyperkeratosis. These findings contrast with previous studies showing that basal layer AP1 factor inactivation does not perturb resting epidermis. It is interesting that in spite of extensive keratinocyte hyperproliferation, susceptibility to carcinogen-dependent tumor induction is markedly attenuated. These novel observations strongly suggest that AP1 factors have distinct roles in the basal versus suprabasal epidermis, confirm that AP1 factor function is required for normal terminal differentiation, and suggest that AP1 factors have a different role in normal epidermis versus cancer progression.

TGF-beta-mediated cell cycle arrest of HPV16-immortalized human ectocervical cells correlates with decreased E6/E7 mRNA and increased p53 and p21(WAF-1) expression.

Transforming growth factor beta (TGF-beta) suppresses proliferation and potentiates apoptosis of HPV16-immortalized human cervical epithelial cells (ECE16-1). Exposure of ECE16-1 to TGF-beta1 increased expression of p53 and induced cell cycle arrest. We examined, by Western blotting, expression of p53 and related cell cycle regulatory proteins after treatment. p53 levels increased as a function of time and dose. Increased p53 appeared to be active, since TGF-beta1 treatment increased the activity of a p53 transcriptional response element in a luciferase reporter plasmid. Additionally, the proteins of the p53-regulated genes, p21(WAF1), mdm2, and Bax, were increased with similar time and dose responses. We did not observe consistent changes in protein levels of cyclin D, cyclin E, CDK4, CDK6, CDK2, p27(Kip1), p16(INK4a), or RNA levels of p15(INK4b). Activity of CDK4 or 6, measured by phosphorylation of an Rb fragment, remained constant during the response period; however, activity of CDK2 (phosphorylation of histone H1) decreased. Concordantly, increased levels of p21(WAF1) were immunoprecipitated with anti-CDK2 antibodies. During treatment, the phosphorylation state of Rb shifted to a hypophosphorylated form. mRNA for the HPV E6/E7 genes decreased; however, significant changes in the E7 protein were not observed, while increased levels of Rb immunoprecipitated with anti-E7 antibodies were observed. These data are consistent with the following model. In ECE16-1 cells, there exists a fine balance between inhibitory levels of p53 and Rb and the antagonists, E6 and E7. TGF-beta1 treatment decreases steady-state levels of E6/E7 mRNA, which results in a shifted balance (lowered activity of E6) in favor of increased p53 expression, resulting in activation of the cell cycle inhibitory gene, p21(WAF1). This protein binds the cyclin E/CDK2 complex that maintains Rb in a phosphorylated state. Rb shifts to a hypophosphorylated state, resulting in G1 arrest, presumably by binding E2F transcription factors.

Suprabasal expression of the human papillomavirus type 16 oncoproteins in mouse epidermis alters expression of cell cycle regulatory proteins.

Human papillomavirus (HPV) survives by reactivating DNA replication in post-mitotic cells. In the present study, we describe a mouse model of HPV-dependent disease. In these mice, DNA synthesis is activated in suprabasal keratinocytes, leading to acanthosis, parakeratosis and enhanced desquamation. The full-length E6/E7 transcript and two alternately spliced products are produced and in most lines the predominant product is E6*. In the present study, we examine the effects of E6/E7 on cell cycle regulatory protein expression. E6/E7 expression in mouse epidermis is correlated with increased levels of the p53, p21, p27, cdk2, cdk4, cdk6, cyclin D1 and cyclin E regulatory proteins. Hyperproliferation is also observed in the buccal mucosa and the tongue epithelia of E6/E7 mice, and p53 levels are markedly increased in these epithelia. These results suggest that the major changes in cell cycle regulatory protein expression are in response to the presence of E7 and that E6 has a lesser impact.

Transgenic animal models of human papillomavirus-dependent disease (Review).

Human papillomaviruses (HPVs) are DNA tumor viruses that induce hyperproliferative lesions in cutaneous and mucosal epithelia. A wide variety of studies implicate the viral E6 and E7 oncoproteins as cell immortalizing agents, and show that these proteins work, respectively, by interfering with the function of the p53 and pRb tumor suppressor genes. Most of these studies have been performed using cell culture models. However, recently, a variety of in vivo mouse model systems have been developed for the study of HPV-dependent disease. These models use tissue-specific promoters to deliver HPV oncoprotein expression to specific body sites. Using this strategy, mouse models have been designed for the study of cancer progression in epithelia, and additional models have been designed to use E6 and E7, respectively, to probe the role of p53 and pRb on tissue differentiation and function. In the present report, we summarize the literature describing these systems, and highlight some of the important findings derived from these studies.

Vitamin D regulates human ectocervical epithelial cell proliferation and insulin-like growth factor-binding protein-3 level.

The differentiation status of the cervical epithelial cell has an important influence on responsiveness to estrogens and progestins. Several agents, including glucocorticoids and retinoids, are known to influence cervical cell differentiation. However, the effects of vitamin D have not been examined. Vitamin D is known to regulate cell proliferation and gene expression in a variety of epithelial cells. In the present study we investigated the ability of 1alpha25-dihydroxyvitamin D3 (D3) to regulate cell proliferation and expression of insulin-like growth factor-binding protein-3 (IGFBP-3) in human ectocervical epithelial cells. ECE16-1, a non-tumorigenic cervical cell line, was growth inhibited by D3 with maximal inhibition at 1000 nM. IGFBP-3 levels increased in parallel with the growth inhibition. IGFBP-3 levels were half-maximally increased at approximately 10-100 nM and maximally increased (10- to 30-fold) at 1000 nM D3. These studies show that vitamin D regulates cervical epithelial cell gene regulation and cell proliferation and that IGFBP-3 may be an in vivo marker of vitamin D action in the cervix.

Transcriptional regulation of the EGF receptor promoter by HPV16 and retinoic acid in human ectocervical epithelial cells.

We have previously demonstrated that human papillomavirus 16 (HPV16)-immortalized human ectocervical epithelial cells and cells derived from tumors which express HPV16 oncogenes express high levels of epidermal growth factor receptor (EGFR) compared to normal cervical cells. We have also shown that proliferation of these cells is inhibited by retinoid treatment. We have hypothesized that the retinoid inhibition of cell proliferation may be due to the retinoid-dependent reduction in EGFR level. In this study we examine the regulation of EGFR expression in cervical cells with emphasis on two aspects: (1) the mechanism of retinoid-dependent suppression of EGFR levels in HPV16-positive cells and (2) the mechanism of EGFR upregulation by HPV16. EGFR levels were found to be elevated 5-, 3. 7-, and 1.25-fold in the HPV16-immortalized ECE16-1, ECE16-D1, and ECE16-D2 cells, respectively, compared to normal cervical cells. Treatment of ECE16-1 and ECE16-D1 cells with retinoic acid suppresses proliferation, EGFR level, EGFR mRNA level, and EGFR promoter activity. The reduction in EGFR promoter activity appears to account for the reduction in EGFR protein and mRNA levels. In contrast, retinoic acid does not affect cell growth or EGFR level in ECE16-D2 cells or normal cervical cells. To study the mechanisms regulating EGFR expression in HPV16-positive cells, normal ECE cells were cotransfected with an EGFR promoter reporter plasmid and an expression plasmid encoding the HPV16 E6/E7 open reading frames. In the presence of E6/E7, EGFR promoter activity was increased by 2- to 3-fold, suggesting that the E6/E7 proteins are directly or indirectly responsible for the increased EGFR level and that the EGFR promoter contains the DNA elements necessary to mediate this response. Nevertheless expression of E6/E7 proteins did not confer retinoic acid regulation, as EGFR promoter activity remained elevated in normal cells cotransfected with pHPVE6/E7 and treated with retinoic acid. These results suggest that human papillomavirus and retinoic acid regulate EGFR levels by independent effects on the EGFR promoter.

Polycyclic aromatic hydrocarbons enhance terminal cell death of human ectocervical cells.

Polycyclic aromatic hydrocarbons (PAH) are a class of chemical carcinogens whose active metabolites form DNA adducts, resulting in specific mutational events. The tumor suppressor protein p53 is believed to play a pivotal role in the ability of cells to response to DNA damage, resulting in either cell cycle arrest in G1 or apoptosis under conditions of excessive damage. This growth inhibition is associated with the concomitant induction of p53 and enhanced terminal cell differentiation. In this study we evaluated the effects of PAH on cell growth, cell differentiation, xenobiotic metabolism, and DNA adduct levels in normal ectocervical epithelial cells (ECE) and compared them to cervical cells whose p53 have been inactivated either by binding to viral HPV E6 oncogene (ECE16-1) or by mutation (C33A). The PAH 3-methylcholanthrene (3MC) inhibited normal ECE and to a lesser extent ECE16-1 cell proliferation. Not only did the growth inhibition occur at lower concentrations in the normal cells but the extent of inhibition was also greater in normal as compared to immortalized cells. Benzanthracene (BA) had a minor effect on normal ECE cells with no effect on immortalized ECE16-1 cells. C33A cell growth was unaffected by 3MC and BA. Terminal cell death was enhanced only in normal ECE cells as evidenced by increased envelope formation and was paralleled by an increase in the level of p53 following 3MC treatment. The differentiation status of the 3MC-treated cells was similar to untreated cells as indicated by three independent markers of cell differentiation; transglutaminase, involucrin, keratin expression. There was no difference in the pattern or level of DNA adducts formed in normal and immortalized cells following 3MC treatment. In addition the basal level of metabolism of 14C-BaP to phenols, diols and quinnones was unaltered by pretreatment with either 3MC or BA. These results demonstrate that immortalized cervical cells are less sensitive to toxicant damage [i.e. cell proliferation and terminal differentiation], and as a result, immortalized cells proliferate in the presence of genotoxic damage and are at increased risk for mutations and cancer.

Retinoids modulate P2U purinergic receptor-mediated changes in transcervical paracellular permeability.

In human cervical cells, extracellular ATP induces an acute decrease in the resistance of the lateral intercellular space, the phase I response, followed by a delayed increase in tight junctional resistance, the phase II response. These responses depend on vitamin A because incubation of cells in retinoid-free medium (RFM) abolished both responses. Treatment with retinoic acid restored the phase I response in full, but the amplitude of the phase II response was restored only partly. Shorter incubations and lower concentrations of retinoic acid [half-maximal effective concentrations (K 1/2) = 0.1 microM] were required for restoring the phase I response than were required for reversing the phase II response (K 1/2 = 1 microM). The phase I response could be restored by ligands that bind to either retinoic acid receptors (RARs) or retinoid X receptors, but only RAR agonists had an effect on phase II response. RFM had no effect on decreases in resistance induced by ionomycin, but it attenuated phase II-like increases in resistance induced by KCl or by 1,2-dioctanoyl-sn-diglycerol (diC8). Actinomycin D blocked phase II response but not phase I response or the responses to ionomycin, KCl, or diC8. These results suggest that retinoids act on cervical cells via distinct retinoid receptor mechanisms and modulate phase I and phase II changes in resistance by regulating distinct signal mechanisms.

Retinoids regulate tight junctional resistance of cultured human cervical cells.

The objective of the study was to determine the effect of retinoids on paracellular resistance across the cervical epithelium and the mechanisms involved. The experimental model was cultures of human CaSki cells on filters, which retain phenotypic characteristics of the endocervical epithelium. End points for paracellular resistance were measurements of transepithelial electrical resistance and fluxes of pyranine (a trisulfonic acid that traverses the epithelium via the intercellular space). Paracellular resistance was significantly increased in cells grown in retinoid-free medium; the effect could be blocked and reversed with all-trans-retinoic acid (tRA) and with agonists of RAR and RXR receptors but only partially with retinol. The effect of tRA was dose dependent and saturable, with a 50% effective concentration of 0.8 nM. The increases in paracellular resistance induced by vitamin A deficiency required longer incubation in retinoid-free medium than decreases in resistance induced by retinoic acid. tRA had only a minimal effect on paracellular resistance in cells maintained in regular medium. Retinoid-free medium increased and tRA decreased the relative cation mobility across CaSki cultures. Also the effects of tRA were nonadditive to those of cytochalasin D (which decreases tight junctional resistance) and additive to those of ionomycin (which decreases the resistance of the lateral intercellular space), suggesting that tRA modulates tight junctional resistance. It is concluded that vitamin A determines the degree of paracellular resistance across cervical cells by a mechanism that involves modulation of tight junctional resistance.

Role of nitroreductases but not cytochromes P450 in the metabolic activation of 1-nitropyrene in the HepG2 human hepatoblastoma cell line.

1-Nitropyrene is an environmental contaminant that is mutagenic in many prokaryotic and eukaryotic systems, including the hypoxanthine-guanosine phosphoribosyl transferase (HGPRT) locus in the human hepatoma cell line HepG2. Metabolism and DNA adduct formation of [3H]1-nitropyrene in the HepG2 were quantified to understand the role of nitroreduction and/or cytochrome P450-mediated C-oxidation of 1-nitropyrene in DNA adduct formation and mutagenicity. In uninduced HepG2 cells, 10 microM [3H]1-nitropyrene was metabolized principally by nitroreduction to 1-aminopyrene (516 pmol/24 hr/10(6) cells), and by cytochrome P450-mediated C-oxidation to K-region trans-dihydrodiols (37 pmol/24 hr/10(6) cells), 1-nitropyren-3-ol (51 pmol/24 hr/10(6) cells), and 1-nitropyren-6-ol and 1-nitropyren-8-ol (77 pmol/24 hr/10(6) cells). Pretreatment of the HepG2 cells for 24 hr with 5 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) resulted in a complete change in the metabolism of [3H]1-nitropyrene, with 1-nitropyren-6-ol and 1-nitropyren-8-ol formation (449 pmol/24 hr/10(6) cells) being 80-fold greater than 1-aminopyrene formation (6 pmol/24 hr/10(6) cells). This increase in C-oxidation of 1-nitropyrene was consistent with increased levels of cytochrome P450 1A. The only DNA adduct detected using the 32P-postlabeling assay in the HepG2 cells administered 1-nitropyrene was N-(2'-deoxyguanosin-8-yl)-1-aminopyrene (dG-C8-AP). Induction of C-oxidative metabolism through TCDD treatment resulted in a concomitant decrease in dG-C8-AP formation. DNA adducts for oxidized 1-nitropyrene metabolites were not detected in the TCDD-treated HepG2 cells administered 1-nitropyrene, which indicates that cytochrome P450-mediated C-oxidative pathways are detoxification pathways in HepG2 cells.

Relaxin modulates human cervical stromal cell activity.

The cervix undergoes profound changes in size and consistency during pregnancy which are designed to facilitate a normal delivery. These changes are under the control of a number of hormonal factors. Experimental and clinical studies suggest that relaxin, a protein hormone, plays a role in promoting cervical softening. One possible target site for the effects of relaxin on the cervix is the stroma. In the studies reported here, cultures of normal human cervical stromal cells were used to determine what direct relaxin has on cervical cells. Relaxin at concentrations ranging from 0.10-100 ng/mL had no effect on human cervical stromal cell proliferation, but was found to modulate stromal cell activity related to the remodeling of extracellular matrix. When gelatin substrate gels were used, relaxin stimulated the secretion of several cervical stromal proteinase activities, including 36,52, and 116 kDa. Inhibitor studies using ethylenediamine tetraacetate, 1,10-phenanthroline, and L-cysteine identify these gelatinases as metalloproteinases. Neither estradiol nor progesterone augmented the effects of relaxin. A relaxin-induced concentration-dependent increase in collagenase activity was observed when measured with a conventional collagen fibril assay. Finally, relaxin was found to increase glycosaminoglycan synthesis, as indicated by [3H]g-lycocyamine incorporation in human cervical stromal cell cultures. These results indicate the relaxin may regulate cervical ripening in humans, as it does in other species, by direct effects on cervical cells.

AGN193109 is a highly effective antagonist of retinoid action in human ectocervical epithelial cells.

Retinoids are important physiological agents that regulate epithelial cell differentiation and proliferation. The importance of these agents in regulating growth, development, and differentiation has led to a search for new retinoid agonists and antagonists. In the present manuscript we show that AGN193109, a retinoid analog, is an efficient antagonist of retinoid action in human cervical epithelial cells. Treatment of ECE16-1 cells with natural or synthetic retinoids reduces cytokeratin K5, K6, K14, K16, and K17 levels, increases cytokeratin K7, K8, and K19 levels, increases retinoic acid receptor-beta (RAR beta) mRNA levels, suppresses proliferation, and alters cell morphology. Co-treatment with AGN193109 prevents these responses. Half-maximal and maximal antagonism is observed at a molar ratio of AGN193109: retinoid agonist of 1:1 and 10:1, respectively. When administered alone AGN193109 has no agonist activity. Thus, AGN193109, which binds to RAR alpha, RAR beta, and RAR gamma with Kd values = 2,2, and 3 nm, respectively, but is unable to bind to the retinoid X receptors, is a highly active antagonist of retinoid action in ECE16-1 cells.

Differential regulation of human ectocervical epithelial cell line proliferation and differentiation by retinoid X receptor- and retinoic acid receptor-specific retinoids.

Retinoids are important regulators of human papillomavirus (HPV)-immortalized cervical epithelial cell differentiation and have been successfully used in the treatment of HPV-involved cervical cancer. In the present study, we examine the effects of a series of natural and synthetic retinoids on differentiation and proliferation of HPV-16-positive lines, ECE16-1 and CaSki. Retinoic acid receptor alpha (RAR alpha), RAR gamma, and retinoid X receptor alpha (RXR alpha) are the major retinoid receptor subtypes expressed when ECE16-1 cells are grown in retinoid-free medium. Our results indicate that ligands that interact with RARs only or both RARs and RXRs, including all-trans-retinoic acid (all-trans-RA), 9-cis-retinoic acid (9-cis-RA), 13-cis-retinoic acid (13-cis-RA), and several synthetic retinoids, suppress ECE16-1 cell proliferation, regulate expression of the retinoid-responsive differentiation marker cytokeratin K5, and increase RAR beta mRNA levels. In contrast, ligands that specifically interact with RXRs do not suppress proliferation and are less efficient regulators of gene expression. CaSki cells express greatly reduced RAR and RXR levels compared to ECE16-1 cells. However, both RAR- and RXR-specific ligands increase CaSki number by > or = 20%. In addition, RXR-specific ligands suppress cytokeratin K5 mRNA levels slightly, compared to RAR-specific ligands that strongly suppress K5 mRNA levels. We also compare the effects of these agents on the proliferation of other cervical cell lines, including ECE16-D2, ME180, and SiHa cells. ECE16-D2 and ME180 cells are growth suppressed by RAR-specific, but not RXR-specific, retinoids. SiHa cells are not responsive to either class of retinoid. Our results indicate that: (a) the response of different human cervical cell lines varies following treatment with receptor type-specific retinoids; and (b) the relationship between retinoid regulation of proliferation and differentiation can be uncoupled.

Tamoxifen regulation of ectocervical cell differentiation.

OBJECTIVE: To determine the effects of tamoxifen on the growth and differentiation of normal human cervical cells and compare those effects with those of a synthetic estrogen, diethylstilbestrol (DES). In addition, the effects of these compounds on immortalized cervical cells and cervical tumor cells were ascertained. METHODS: Growth curves were used to determine the effects on cell proliferation. The expression of several proteins was used to determine the effects on cell differentiation. Binding assays and Western analysis were used to determine estrogen receptor levels. RESULTS: Both tamoxifen and DES inhibited the proliferation of normal cervical cells. This growth inhibition was coincident with an increase in cell differentiation as determined by cornified envelope formation. The increase in envelope number was not accompanied by an increase in involucrin or cornifin, two protein precursors of the envelope. The activity of transglutaminase, which enzymatically incorporates precursor proteins into the envelope, was not stimulated following treatment. Diethylstilbestrol did not alter the growth or differentiation of the human papillomavirus 16-immortalized cell line ECE16-1 or any of the cervical tumor cell lines. Of all the immortalized or cancer cell lines, only Caski cells were growth inhibited by tamoxifen. Normal ectocervical epithelial cells and Caski cells expressed the high-affinity 56-kDa estrogen receptor, but 3H-estradiol binding was not detected in cell extracts from either ME180 or ECE16-1 cells. Nevertheless, extracts from ME180 cells contained an immunoreactive band at the appropriate molecular weight for the estrogen receptor. CONCLUSIONS: These results suggest that tamoxifen and DES act similarly in normal cervical cells to promote cervical cell differentiation. However, because Caski cell growth was inhibited by tamoxifen and not DES, the effects of tamoxifen in these cells may be mediated by non-estrogen receptor mechanisms.

Transforming growth factor beta regulation of epidermal growth factor receptor in ectocervical epithelial cells.

Transforming growth factor beta (TGF beta) is a pluripotent modulator of cell function and an important suppressor of cervical epithelial cell proliferation. In the present study, we examine the effects of TGF beta 1 on the level and activity of the epidermal growth factor receptor (EGFR) in HPV-16 immortalized cervical epithelial cells. In ECE16-1 cells, increased EGFR levels are observed within 24 h after initiation of TGF beta 1 treatment and levels continue to increase with time. This increase is correlated with a TGF beta 1-dependent decrease in proliferation rate. Scatchard analysis indicates that the population of EGFR sites induced by TGF beta 1 have a low affinity for EGF (Kd = 4.08 nM) compared to the receptors present prior to TGF beta 1 treatment (Kd = 0.3 and 1.6 nM). TGF beta 1 treatment also reduces EGFR kinase autophosphorylation activity. Cell cycle studies indicate that TGF beta 1-treated cells arrest in the G1 phase of the cell cycle and that regulation of EGFR level was independent of cell cycle stage in both TGF beta 1-treated and untreated cells. However, EGFR level was related to the G1 phase time. Parallel studies indicate that a TGF beta 1-dependent increase in p53 level is also correlated with increased time spent in G1. These results suggest that TGF beta 1 inhibition of ECE16-1 cell proliferation may act both by the replacement of high affinity/high kinase activity EGFR sites with low affinity/low kinase activity EGFR sites and a p53-mediated cell cycle arrest.

Differential response of normal and HPV immortalized ectocervical epithelial cells to B[a]P.

Cigarette smoking has been established as a risk factor for the development of cervical cancer. Polycyclic aromatic hydrocarbons such as benzo[a]pyrene (B[a]P), which are present in cigarette smoke, might account for this increased risk. The effects of B[a]P on cell growth, aryl hydrocarbon hydroxylase, DNA adducts and p53 levels was measured in cervical cells. Since 90% of cervical preneoplastic lesions are positive for the human papillomavirus (HPV) we compared the effects of these chemicals in normal ectocervical epithelial cells (ECE) and human papillomavirus 16 (HPV16) immortalized ectocervical epithelial cells (ECE16-1). Exposure of normal ECE and HPV immortalized ECE16-1 cells to B[a]P inhibited cell proliferation. Inhibition occurred at 20-fold lower concentrations in the normal ECE cells compared to ECE16-1 cells. The proliferation of cervical cells which express mutated p53 was unaffected by B[a]P. Neither cervical stromal cells nor endometrial stromal cells were affected by these compounds. The effects of B[a]P on normal ECE cell proliferation correlated with increased terminal differentiation as measured by increased envelope formation. In contrast, B[a]P exposure did not induce envelope formation in immortalized ECE16-1 cells or in cervical tumor cells. Pretreatment of both ECE and ECE16-1 cells with 2,3,7,8-tetrachlorodibenzo-p-dioxin, which induces P450 expression and activity, did not alter B[a]P metabolism in either normal or immortalized cells. Furthermore, equivalent levels of DNA adducts were formed by B[a]P in ECE and ECE16-1 cells. Neither the extent of adduct formation nor the rate of their removal differed in normal and immortalized cervical cells. Therefore, the diminished growth inhibition of the ECE16-1 cells as compared to normal ECE cells by B[a]P is not due to changes in cytochrome P450 of the 1A family metabolism or DNA adduct number. Furthermore, analysis of the p53 levels in both normal and ECE16-1 cells revealed that p53 levels are higher in normal versus immortalized ectocervical cells, and p53 is induced in both cell types following B[a]P treatment. Thus reduced p53 levels in ECE16-1 cells may contribute to a lack of growth suppression following B[a]P treatment. These results demonstrate that HPV16 immortalization diminishes ectocervical epithelial cell responsiveness to toxicant damage (i.e. decreased cell proliferation and increased terminal differentiation). As a result, ECE16-1 cells that sustain genotoxic damage which leads to DNA adduct formation continue to proliferate and may be at increased risk for mutations and further progression towards a fully transformed phenotype.

Biphasic regulation of paracellular permeability in human cervical cells by two distinct nucleotide receptors.

The effects of extracellular ATP (ATPe) were characterized in human cervical cells grown on filters. ATPe changed the transepithelial conductance (GT) in a biphasic manner with an initial acute increase of 13 +/- 3% (phase I), lasting approximately 2 min, followed by a sustained decrease of 49 +/- 17% (phase II). ATPe also effected decreases of permeabilities to pyranine and to sucrose, which correspond to the phase II decrease in GT. Both phase I and II effects appear to be mediated by apical nucleotide receptors. However, the phase I effect differed from the phase II effect as follows: 1) a higher 50% effective concentration for ATPe, 22 vs. 3 microM; 2) different nucleotide specificity; 3) lack of influence of pretreatment with pertussis toxin; 4) independence from time in culture after seeding; and 5) involvement of cytosolic calcium, rather than diacylglycerol, as intracellular messenger. These differences suggest the presence of two different types of nucleotide receptors that use different intracellular signaling systems and have opposite effects on the paracellular permeability and electrical conductance. These results suggest that ATPe could regulate cervical mucus production in vivo by regulating the paracellular permeability. Depending on the specific receptors present, ATPe could either increase or decrease mucus production.

Retinoid regulation of cell differentiation in a series of human papillomavirus type 16-immortalized human cervical epithelial cell lines.

Retinoids are important regulators of cervical epithelial cell differentiation and have been used in the treatment of cervical cancer. In the present study we evaluate the effects of retinoic acid on expression of biochemical markers of differentiation and expression of human papillomavirus reading frames encoding the early gene products E6 and E7 in normal and HPV16-immortalized cervical epithelial cell lines. Our results indicate that the differentiation markers cytokeratins K5 and K16 and transglutaminase type 1 are suppressed by all-trans-retinoic acid (RA). A marked concentration-dependent reduction in the level of of each mRNA is observed with maximal suppression at 1 microM. Each of the HPV16-immortalized cell lines (ECE16-1, ECE16-D1 and ECE16-D2) are more sensitive to the effects of RA than normal cells. The level of HPV16 transcript encoding E6/E7 is not significantly suppressed by 1 microM RA in ECE16-1 cells, but is suppressed in ECE16-D1 and ECE16-D2 cells. In addition, an increase in HPV transcripts encoding E6/E7 is observed at intermediate (10 and 100 nM) retinoic acid concentrations in ECE16-1 and ECE16-D2 cells, but not in ECE16-D1 cells. Our results show that retinoids regulate E6/E7 transcript levels in some cervical cell lines but not in others, suggesting that different cervical tumors may respond to retinoids via different mechanisms.