Human Ectocervical Epithelial Cells Research Articles

Cross talk: trafficking and functional impact of maternal exosomes at the feto-maternal interface under normal and pathologic states†.

Fetal cell-derived exosomes promote inflammation in uterine and cervical cells to promote labor and delivery. However, the effect of maternal exosomes on fetal cells is still not known. We tested the hypothesis that cervical cells exposed to infectious and oxidative stress (OS) signals produce exosomes that can induce inflammation at the feto-maternal interface (FMi). Exosomes isolated from medium samples from human ectocervical epithelial cells (ecto), endocervical epithelial cells (endo), and cervical stromal cells (stroma) in normal cell culture (control) or exposed to infection or OS conditions were characterized based on morphology, size, quantity, expression of tetraspanin markers, and cargo proteins. Human decidual cells, chorion trophoblast cells (CTC), chorion mesenchymal cells (CMC), amnion mesenchymal cells (AMC), and amnion epithelial cells (AEC) were treated with control, LPS-, or OS-treated cervical exosomes. Enzyme-linked immunosorbent assay for pro-inflammatory cytokines and progesterone was done to determine the recipient cells' inflammatory status. Ecto, endo, and stroma released ∼110nm, cup-shaped exosomes. LPS and OS treatments did not affect exosome size; however, OS significantly increased the number of exosomes released by all cervical cells. Cervical exosomes were detected by fluorescence microscopy in each target cell after treatment. Exosomes from LPS- and CSE-treated cervical cells increased the inflammatory cytokine levels in the decidual cells, CMC, AMC, and AEC. LPS-treated stromal cell exosomes increased IL-6, IL-8, and progesterone in CTC. In conclusion, infection and OS can produce inflammatory cargo-enriched cervical exosomes that can destabilize FMi cells. However, the refractoriness of CTC to exosome treatments suggests a barrier function of the chorion at the FMi.

Estradiol alters the immune-responsiveness of cervical epithelial cells stimulated with ligands of Toll-like receptors 2 and 4.

The mucosa of the female reproductive tract plays a pivotal role in host defence. Pregnancy must alter immunological mechanisms at this interface to protect the conceptus. We sought to determine how estradiol (E2) alters the immune-responsiveness of cervical epithelial cells to ligand stimulation of Toll-like receptor (TLR)-2 and -4. Human ectocervical epithelial cells (HECECs) were cultured and co-incubated with two concentrations of E2 and peptidoglycan (PGN) or lipopolysaccharide (LPS) over durations that ranged between 10 minutes and 18 hours. Cytometric Bead Array was performed to quantify eight cytokines in the supernatant fluid. In response to PGN, HECECs co-incubated with E2 released lesser quantities of IL-1ß and IFNγ, higher levels of RANTES, and variable levels of IL-6 and IL-8 than those not exposed to E2. In contrast, HECECs co-incubated with LPS and E2 secreted increased levels of IL-1ß, IL-6, IL-8, and IFNγ at 2 and 18 hours than HECECs not exposed to E2, and reduced levels of RANTES at same study time-points. Estradiol alters the immune-responsiveness of cultured HECECs to TLR2 and TLR4 ligands in a complex fashion that appears to vary with bacterial ligand, TLR subtype, and duration of exposure. Our observations are consistent with the functional complexity that this mucosal interface requires for its immunological roles.

Medroxyprogesterone Acetate Differentially Regulates Interleukin (IL)-12 and IL-10 in a Human Ectocervical Epithelial Cell Line in a Glucocorticoid Receptor (GR)-dependent Manner

Medroxyprogesterone acetate (MPA), designed to mimic the actions of the endogenous hormone progesterone (P4), is extensively used by women as a contraceptive and in hormone replacement therapy. However, little is known about the steroid receptor-mediated molecular mechanisms of action of MPA in the female genital tract. In this study, we investigated the regulation of the pro-inflammatory cytokine, interleukin (IL)-12, and the anti-inflammatory cytokine IL-10, by MPA versus P4, in an in vitro cell culture model of the female ectocervical environment. This study shows that P4 and MPA significantly increase the expression of the IL-12p40 and IL-12p35 genes, whereas IL-10 gene expression is suppressed in a dose-dependent manner. Moreover, these effects were abrogated when reducing the glucocorticoid receptor (GR) levels with siRNA. Using a combination of chromatin immunoprecipitation (ChIP), siRNA, and re-ChIP assays, we show that recruitment of the P4- and MPA-bound GR to the IL-12p40 promoter requires CCAAT enhancer-binding protein (C/EBP)-β and nuclear factor κB (NFκB), although recruitment to the IL-10 promoter requires signal transducer and activator of transcription (STAT)-3. These results suggest that both P4 and MPA may modulate inflammation in the ectocervix via this genomic mechanism.

PL.89 Functional Effects of Oestradiol on Cytokine Responses of Human Ectocervical Epithelial Cells to Toll-Like Receptor 2 and 4 Ligands

We have demonstrated that the pattern recognition toll-like receptors (TLR) 2&4 are upregulated during pregnancy in human cervical epithelium. We sought to determine whether oestradiol (E2) alters the immunological responses...

PL.84 Short-Term Culture of Human Ecto-Cervical Epithelial Cells for Genomic, Proteomic and Functional Studies

Background Understanding cell physiology is limited by reliance on tumour-derived immortalised cell lines. Primary cell culture models may offer more relevant mechanistic insight into cell physiology but are often difficult...

Trichomonas vaginalis lipophosphoglycan triggers a selective upregulation of cytokines by human female reproductive tract epithelial cells.

Trichomonas vaginalis is one of the most common nonviral sexually transmitted human infections and, worldwide, has been linked to increased incidence of human immunodeficiency virus type 1 transmission, preterm delivery, low birth weight, cervical cancer, and vaginitis. The molecular pathways that are important in initiating host inflammatory and immune responses to T. vaginalis are poorly understood. Here we report interactions of human cervicovaginal epithelial cells with the most abundant cell surface glycoconjugate of the parasite, the T. vaginalis lipophosphoglycan (LPG). Purified LPG mediated the adhesion of parasites to human vaginal epithelial cells in a dose-dependent manner. Furthermore, T. vaginalis LPG (but not LPG from Tritrichomonas foetus, the causative agent of bovine trichomoniasis) induced a selective upregulation of chemotactic cytokines by human endocervical, ectocervical, and vaginal epithelial cells, which do not express Toll-like receptor 4/MD2. The T. vaginalis LPG triggered interleukin 8 (IL-8), which promotes the adhesion and transmigration of neutrophils across the endothelium, and macrophage inflammatory protein 3alpha, which is a chemoattractant for immune cells and is essential for dendritic cell maturation. These effects were dose dependent and sustained in the absence of cytotoxicity and IL-1beta release and utilized, at least in part, a signaling pathway independent from the Toll-like/IL-1 receptor adaptor protein MyD88.

Estrogen Attenuates P2X7‐R — Mediated Apoptosis of Uterine Cervical Cells by Blocking Calcium Influx

AbstractFor Abstract see ChemInform Abstract in Full Text.

Estrogen Attenuates P2X7‐R—Mediated Apoptosis of Uterine Cervical Cells by Blocking Calcium Influx

Estrogen blocks apoptosis of human ectocervical epithelial cells by modulating P2X7/Ca2 + influx. The effect involves decreased Ca2 +‐influx and cytosolic‐calcium‐increase via ATP‐activated P2X7 pores. This mechanism may have physiological significance in the human cervix, in‐vivo, and the results suggest a physiological role for estrogen in the cervix as an anti‐apoptotic factor.

Aging and estrogen effects on transcervical-transvaginal epithelial permeability.

The objective of the study was to understand age-related contributions of the resistance of the intercellular tight junctions (R(TJ)) and the resistance of the lateral intercellular space (R(LIS)) to the transcervical-transvaginal permeability. The experiments used normal human ectocervical epithelial cells obtained from women aged 36-65 yr. Twenty-four hours of treatment of cells with 10 nm 17beta-estradiol decreased the R(LIS), whereas longer treatments were required to decrease The R(TJ). Aging had no effect on baseline or estrogen decrease in R(TJ), but it blocked both baseline and the estrogen decrease in R(LIS). Actin assays showed age-related decrease in the fraction of monomeric G-actin and attenuation of sodium-nitroprusside-induced increase in G-actin. These results suggest that the aging-related diminished capacity of human ectocervical epithelial cells to remodel cellular actin from polymerized actin toward monomeric G-actin confers increased cell rigidity and therefore increased R(LIS). Therefore, the hypoestrogenism-related decrease in R(TJ) and the hypoestrogenism- and aging-related increase in R(LIS) could be the cellular mechanisms of decreased permeability that lead to decreased fluid transport and decreased lubrication of the lower genital tract in older postmenopausal women.

P2X7 receptor-mediated apoptosis of human cervical epithelial cells.

Normal human ectocervical epithelial (hECE) cells undergo apoptosis in culture. Baseline apoptosis could be increased by shifting cells to serum-free medium and blocked by lowering extracellular calcium. Treatment with the ATPase apyrase attenuated baseline apoptosis, suggesting that extracellular ATP and purinergic mechanisms control the apoptosis. Treatment with ATP and the P2X7 receptor analog 2'-3'-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate (BzATP) increased apoptosis significantly, in a time- and dose-related manner. The threshold of ATP effect was 0.5 microM in hECE cells and approximately 1 microM in CaSki cancer cells. The apoptotic effect of BzATP was additive in part to that of tumor necrosis factor (TNF)-alpha, and it could be attenuated by lowering extracellular calcium and by treatment with the caspase-9 inhibitor Leu-Glu-His-Asp-O-methyl-fluoromethylketone (LEHD-FMK). Treatment with BzATP activated caspase-9, and, in contrast to TNF-alpha, it had only a mild effect on caspase-8. Both BzATP and TNF-alpha activated caspase-3, suggesting that BzATP activates predominantly the mitochondrial apoptotic pathway. Both hECE and CaSki cells secrete ATP into the extracellular fluid, and mean ATP activity in conditioned medium was approximately 0.5 microM, which is in the range of values that suffice to activate the P2X7 receptor. On the basis of these findings we propose a novel autocrine-paracrine mechanism of cervical cell apoptosis that operates by P2X7 receptor control of cytosolic calcium and utilizes the mitochondrial apoptotic pathway.

Gene expression of telomerase related proteins in human normal oral and ectocervical epithelial cells

We analyzed telomerase activities and gene expressions of telomerase components: hTERT, hTR, hTEP1, telomeric repeat binding factors: TRF1, TRF2, and c-myc, Max and Mad in human normal oral and ectocervical epithelial keratinocytes, comparing with those of squamous carcinoma cells and HPV16- or SV40-immortalized cells. Significant telomerase activity and hTERT expression were detected in primary keratinocytes. However, both were dramatically down-regulated during serial passages. The down-regulation of hTERT mRNA was associated with augmented expression of TRF1. Expression of c-myc was slightly decreased, whereas Mad was expressed in parallel with that of hTERT during passages. We also detected an alternate splicing of hTERT transcript in two of four cancer cells and normal aged epithelial cells. These results suggest that the senescence of normal oral and ectocervical keratinocytes is accompanied with up-regulation of TRF1 and down-regulation of telomerase activity due to transcriptional suppression of active form of hTERT in vitro.

Retinoids Suppress Epidermal Growth Factor-associated Cell Proliferation by Inhibiting Epidermal Growth Factor Receptor-dependent ERK1/2 Activation

Human papillomavirus (HPV) is an important etiological agent in the genesis of cervical cancer. HPV-positive cervical tumors and human papillomavirus-positive cell lines display increased epidermal growth factor receptor (EGFR) expression, which is associated with increased cell proliferation. ECE16-1 cells are an HPV-immortalized human ectocervical epithelial cell line that is a model of HPV-associated cervical neoplasia and displays elevated EGFR levels. In the present study, we evaluated the effects of receptor-selective retinoid ligands on EGFR-associated signal transduction. We show that retinoic acid receptor (RAR)-selective ligands reduce EGFR level and the magnitude and duration of EGFR activation in EGF-stimulated cells. These effects are reversed by cotreatment with an RAR antagonist. To identify the mechanism, we examined the effects of retinoid treatments on EGF-dependent signaling. Stimulation with EGF causes a biphasic activation of the ERK1/2 MAPK. The first peak of activation is present at 20 min, and the second is present at 36 h. This activation subsequently leads to an increase in the cyclin D1 level and increased cell proliferation. Simultaneous treatment with EGF and a RAR-selective retinoid inhibits both phases of ERK1/2 activation, completely eliminates the cyclin D1 induction, and suppresses EGF-dependent cell proliferation. This effect is specific as retinoid treatment does not alter the level or activity of other EGFR-regulated kinases, including AKT and the MAPKs p38 and JNK. Retinoid X receptor-selective ligands, in contrast, did not regulate these responses. These results suggest that RAR ligand-associated down-regulation of EGFR activity reduces cell proliferation by reducing the magnitude and duration of EGF-dependent ERK1/2 activation.

Vaginal-cervical epithelial permeability decreases after menopause

Objective: To determine the effects of menopause (aging and E) on vaginal-cervical epithelial paracellular permeability.Design: Experimental, basic clinical research.Setting: Academic research environment.Patient(s):Premenopausal, perimenopausal, and postmenopausal women, aged 35–65 years.Intervention(s): Primary to tertiary cultures of normal human ectocervical epithelial cells on filters. Cells were outgrown from surgically discarded ectocervical minces.Main Outcome Measurement(s): Changes in paracellular permeability were determined as changes in transepithelial electrical conductance and pyranine permeability.Result(s): [1] Levels of transepithelial electrical conductance and pyranine permeability decreased as women’s age advanced. [2] Removal of E from the culture medium decreased paracellular permeability. Treatment of cells in vitro with 10 nmol/L 17β-E2 increased transepithelial electrical conductance and pyranine permeability, but the effects were additive to the age-related decrease in permeability. [3] Coadministration of 100 nmol/L tamoxifen blocked the E increase in paracellular permeability in cells of both premenopausal and postmenopausal women.Conclusion(s): [1] Aging and E deficiency decrease independently vaginal-cervical epithelial paracellular permeability. [2] The E increase in vaginal-cervical epithelial paracellular permeability in cells of postmenopausal women is mediated by the E receptor. [3] The E increase in vaginal-cervical epithelial paracellular permeability in cells of postmenopausal women is masked by age-related increase in the tight junctional resistance, leading to overall decrease in paracellular permeability.

Estrogen Biphasic Regulation of Paracellular Permeability of Cultured Human Vaginal- Cervical Epithelia

The objective of the study was to understand effects of estrogen and aging on paracellular permeability of human vaginal-cervical epithelia. Assays included determinations of transepithelial electrical conductance across cultures of normal human ectocervical epithelial cells on filters. Baseline transepithelial electrical conductance across steroid-deprived cells from postmenopausal women was lower than across cells of premenopausal women. Short-term (24-48 h) treatment with 10 nM 17beta-estradiol increased transepithelial electrical conductance in both groups of cells. In cells of premenopausal women longer-term treatment with estrogen for up to 14 d had no additional effect on permeability, but in cells of postmenopausal women it caused a secondary increase in transepithelial electrical conductance that continued for the duration of the 2-wk treatment. Binding assays of 17beta-[(3)H]estradiol revealed saturable binding to high affinity (1.2-1.3 nM), low capacity sites (0.2-1.2 pmol/mg DNA) in cells of both premenopausal and postmenopausal women. In both types of cells treatment with 17beta-estradiol increased 17beta-[(3)H]estradiol binding activity in a time- and dose-related manner (EC(50) 1 nM; maximal effect within 9-12 h), and increased estrogen receptor-alpha and -beta mRNA. 8-Br-cGMP, a stable cell-permeant analog of cGMP, could mimic the estrogen first phase increase in transepithelial electrical conductance, but not the secondary increase. Treatment with estrogen augmented the increase in transepithelial electrical conductance that was induced by hydrostatic gradients (modulator of the resistance of the lateral intercellular space), and the effect was independent of woman's age or baseline transepithelial electrical conductance. In contrast, the effect of low extracellular calcium (modulator of the tight junctional resistance) was more potent in cells of premenopausal women than in cells of postmenopausal women and was independent of treatment with estrogen. These results suggest that changes in vaginal-cervical epithelial permeability after menopause are determined by aging-related increase in tight junctional resistance, and by low estrogen-dependent increase in lateral intercellular space that lead to net increase in total paracellular resistance and decreased permeability and result in reduced lubrication of the lower genital canal.

Thymidine kinase-independent intracellular delivery of bioactive nucleotides by aryl phosphate derivatives of bromo-methoxy zidovudine (compounds WHI-05 and WHI-07) in normal human female genital tract epithelial cells and sperm.

The compounds WHI-05 (5-bromo-6-methoxy-5, 6-dihydro-3'-azidothymidine-5'-[p-methoxyphenyl] methoxyalaninyl phosphate) and WHI-07 (5-bromo-6-methoxy-5, 6-dihydro-3'-azidothymidine-5'-[p-bromophenyl] methoxyalaninyl phosphate) are aryl phosphate derivatives of zidovudine (ZDV) with dual-function anti-human immunodeficiency virus and contraceptive activity. These drugs were rationally designed to bypass the thymidine kinase (TK) dependency of ZDV activation as well as to achieve spermicidal activity. We investigated the TK activity and intracellular metabolism of WHI-05 and WHI-07 in normal human vaginal and cervical epithelial cells as well as sperm. The time- and concentration-dependent intracellular formation of ZDV metabolites following addition of WHI-05 and WHI-07 to normal human vaginal, ectocervical, and endocervical epithelial cells as well as motile sperm was studied by analytical HPLC. Thymidine kinase activity in these cells was determined by the flow cytometric method based on intracellular phosphorylation of the fluorescent nucleoside, 5-amino-2-deoxyuridine-dansyl chloride and by the ability of cell-free extracts to convert [(3)H]thymidine to thymidine monophosphate in comparison to NALM-6, a pre-B leukemia cell line. TK activity of genital tract epithelial cells and sperm was found to be relatively low or lacking. Addition of WHI-05 and WHI-07 to vaginal and cervical epithelial cells resulted in their concentration- and time-dependent conversion to alaninyl ZDV monophosphate (Ala-ZDV-MP) and 5'-ZDV monophosphate as the major metabolites. Studies using motile human sperm also demonstrated the conversion of WHI-05 and WHI-07 to Ala-ZDV-MP. These results demonstrate that human female genital tract epithelial cells and sperm efficiently convert WHI-05 and WHI-07 to bioactive ZDV metabolites despite their TK deficiency.

Novel thiourea compounds as dual-function microbicides.

Sexually active women represent the fastest growing human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome risk group. In an effort to develop a vaginal microbicidal contraceptive potentially capable of preventing HIV transmission as well as providing fertility control, we previously reported the synthesis of novel nonnucleoside inhibitors (NNIs) of HIV-1 reverse transcriptase with sperm-immobilizing activity (SIA). To gain further insight into the structure-function relationship controlling these two properties of NNIs, we have rationally designed and synthesized 30 novel thiourea compounds and examined them for dual-function, anti-HIV and spermicidal activity. Twelve of the 30 thiourea compounds exhibited potent anti-HIV activity in the nanomolar range (IC(50) = <1-9 nM). Nine of the 30 thiourea derivatives exhibited both anti-HIV and spermicidal activity. Among the phenyl ring-containing thioureas, the 2-fluoro (HI-240) -substituted and 2-chloro (HI-253) -substituted derivatives exhibited potent anti-HIV activity (IC(50) = <1 nM) with SIA (EC(50) = 70 microM and 147 microM). Among the alicyclic ring-containing thioureas, the 5-bromo (HI-346) and 5-chloro (HI-445) functionalized cyclohexenyl ring-substituted thioureas were the most potent dual-function spermicides (EC(50) = 42 and 57 microM), with anti-HIV activity at nanomolar range (IC(50) = 3 nM). Unlike nonoxynol-9 (N-9), none of the potent dual-function thiourea compounds were cytotoxic to normal human vaginal, ectocervical, and endocervical epithelial cells at spermicidal concentrations. We conclude that as potent anti-HIV agents with SIA and reduced cytotoxicity when compared with N-9, the phenyl-substituted and cyclohexenyl-substituted thiourea derivatives, especially compounds HI-253 (N-[2-(2-chlorophenethyl)]-N'-[2-(5-bromopyridyl)-thiourea), HI-346 (N-[2-(5-bromopyridinyl)]-N'-[2-(1-cyclohexenyl)ethyl-thiourea), and HI-445 (N-[2-(5-chloropyridinyl)]-N'-[2-(1-cyclohexenyl)ethyl-thiourea) show unique clinical potential to become the active ingredients of a vaginal contraceptive for women who are at high risk for acquiring HIV by heterosexual vaginal transmission.

Effects of Menopause and Estrogen on Cervical Epithelial Permeability1

The objective of the study was to characterize the effect and explore the mechanisms by which menopause affects paracellular permeability of cultured human cervical epithelium. The experimental system was cultures of human ectocervical epithelial (hECE) cells on filters. Assays included flux measurements of cell-impermeable molecules and determinations of transepithelial electrical conductance. hECE cells of postmenopausal women formed epithelia with lower paracellular permeability than hECE cells of premenopausal women. Treatment with estrogen increased paracellular permeability, but levels remained lower compared to cultures from premenopausal women. Lowering extracellular calcium or treatment with cytochalasin-D, conditions that decrease the tight junctional resistance (R(TJ)), increased the permeability, and the relative effects were greater in cells of premenopausal women than in postmenopausal women. Treatment of cells with sn-1,2-dioctanoyl diglyceride, an agent that increases the R(TJ), decreased the permeability, and the relative effect was greater in cells of postmenopausal women than in cells of premenopausal women. Estrogen had no significant effect on the responses to low calcium, cytochalasin-D, or sn-1,2-dioctanoyl diglyceride. Hydrostatic and hypertonic gradients in the subluminal to luminal direction, conditions that decrease the resistance of the lateral intercellular space increased the permeability to a similar degree in cultures of cells from premenopausal and from postmenopausal women. Pretreatment with estrogen augmented the increases in permeability in response to hydrostatic and hypertonic gradients. In cells exposed to low extracellular calcium, hydrostatic gradients had an additive increase in permeability. By extrapolation it was determined that in cultures of postmenopausal women R(TJ) contributes 97% to the total paracellular resistance, whereas in hECE cultures of premenopausal women the R(TJ) contributes only 84%. These results indicate that after menopause the transcervical paracellular permeability decreases significantly; this can explain the decrease in lubrication of the cervix and vagina in postmenopausal women. Part of the effect is due to lack of estrogen, and it can be reversed by treatment with the hormone. However, most of the effect is unrelated to estrogen and is caused by an increase in R(TJ).

Structural requirements for potent human spermicidal activity of dual-function aryl phosphate derivative of bromo-methoxy zidovudine (compound WHI-07).

WHI-07, a novel bromo-methoxy-substituted aryl phosphate derivative of zidovudine (ZDV), is a potent dual-function contraceptive agent. Although the bromo-methoxy functional groups in the thymine ring of its ZDV are very important for its sperm-immobilizing activity (SIA), the importance of the esterification of the phosphate group with an amino acid side chain and the identity of the para substituent in the aryl moiety remain unclear. In the present study, we have synthesized 23 new analogues of WHI-07 by replacing the alanine (Ala) side chain with different amino acids containing nonpolar side chains, namely tryptophan (Trp), proline (Pro), phenylalanine (Phe), leucine (Leu), methionine (Met), valine (Val), or glycine (Gly). The para substituents on the aryl moiety included bromo, chloro, fluoro, nitro, or methoxy groups. The SIA of each of the 23 WHI-07 analogues was evaluated by computer-assisted sperm analysis. The potential cytotoxicity of these compounds against normal human ectocervical and endocervical epithelial cells was evaluated using MTT (3-[4, 5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) cell viability assays. The replacement of the Ala side chain of WHI-07 with Val, Leu, or Phe led to a complete loss of SIA (EC(50) values > 500 microM), whereas replacement with Trp reduced the SIA by 4-fold. The presence of para substituents on the phenyl moiety led to significant alterations in SIA. The anti-human immunodeficiency virus (HIV) activity of Trp-containing WHI-07 analogues was also diminished. Our finding highlights the necessity of Ala side chain and the presence of electron-withdrawing para-bromo substituent on the phenyl moiety in addition to bromo-methoxy functionalization groups on the thymine ring in order for the phosphoramidate derivatives of ZDV to be effective dual-function spermicidal agents. Unlike the detergent-type microbicide, nonoxynol-9, which was cytotoxic to normal human ectocervical and endocervical epithelial cells (IC(50) values of 22 microM and 16 microM, respectively) at spermicidal concentrations (EC(50) = 81 microM), WHI-07 and its active analogues were selectively spermicidal without cytotoxicity against female genital tract epithelial cells. WHI-07 and its Trp analogues hold particular clinical promise for the development of novel, nondetergent-type prophylactic contraceptives for the prevention of heterosexual HIV/acquired immunodeficiency syndrome transmission.

Insulin-like growth factor binding protein-3 does not mediate the interferon-dependent suppression of human ectocervical epithelial cell proliferation.

Interferon is a potential therapeutic agent for the treatment of cervical cancer. In the present study we examine the role of IFNgamma as a regulator of proliferation and production of IGFBP-3 expression in ectocervical epithelial cells. ECE16-1 cells are a model for studying early human papillomavirus-dependent cervical disease. IFNgamma produces a concentration-dependent inhibition of ECE16-1 cell proliferation that is associated with an increase in insulin-like growth factor binding protein-3 level. Growth suppression and IGFBP-3 increase is maximal at concentrations of IFNgamma >/=0.75 ng/ml. The increased IGFBP-3 expression is mediated via an increase in IGFBP-3 encoding mRNA. In contrast, IFNgamma inhibits proliferation of CaSki and SiHa cells, but IGFBP-3 is barely detectable and levels are not regulated by IFNgamma. These results suggest that the IFNgamma-dependent suppression of CaSki and SiHa cell proliferation is not mediated by secreted IGFBP-3. This result was confirmed when vector-mediated overexpression of immunoreactive IGFBP-3 in SiHa and CaSki cells did not consistently result in reduced cell proliferation rate.

Synthesis, characterization and preclinical formulation of a dual-action phenyl phosphate derivative of bromo-methoxy zidovudine (compound WHI-07) with potent anti-HIV and spermicidal activities.

In a systematic effort to develop a microbicide contraceptive capable of preventing transmission of human immunodeficiency virus (HIV), as well as providing fertility control, we have previously identified novel phenyl phosphate derivatives of zidovudine (ZDV) with 5-halo 6-alkoxy substitutions in the thymine ring and halo substitution in the phenyl moiety respectively. Here, we describe the synthesis, characterization, and successful preclinical formulation of our lead compound, 5-bromo-6-methoxy-3'-azidothymidine-5'-(p-bromophenyl) methoxyalaninyl phosphate (WHI-07), which exhibits potent anti-HIV and sperm immobilizing activities. The anti-HIV activity of WHI-07 was tested by measuring viral p24 antigen production and reverse transcriptase activity as markers of viral replication in HIV-1 infected human peripheral blood mononuclear cells (PBMC). WHI-07 inhibited replication of HIV in a concentration-dependent fashion with nanomolar IC50 values. The effects of WHI-07 on human sperm motion kinematics were analysed by computer-assisted sperm analysis (CASA), and its effects on sperm membrane integrity were examined by confocal laser scanning microscopy (CLSM), and high-resolution low-voltage scanning electron microscopy (HR-LVSEM). WHI-07 caused cessation of sperm motility in a concentration- and time-dependent fashion. The in-vitro cytotoxicities of WHI-07 and nonoxynol-9 (N-9) were compared using normal human ectocervical and endocervical epithelial cells by the MTT cell viability assay. Unlike N-9, WHI-07 had no effect upon sperm plasma and acrosomal membrane integrity. N-9 was cytotoxic to normal human ectocervical and endocervical cells at spermicidal doses, whereas WHI-07 was selectively spermicidal. The in-vivo vaginal absorption and vaginal toxicity of 2% gel-microemulsion of WHI-07 was studied in the rabbit model. The sperm immobilizing activity of WHI-07 was 18-fold more potent than that of N-9. Over a 10 day period, there was no irritation or local toxicity to the vaginal epithelia or systemic absorption of WHI-07. Therefore, as a potent anti-HIV agent with spermicidal activity, and lack of mucosal toxicity, WHI-07 may have the clinical potential to become the active ingredient of a vaginal contraceptive for women who are at high risk for acquiring HIV by heterosexual vaginal transmission.