Human Endocervical Epithelial Cells Research Articles

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.

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.

WHI-05, a novel bromo-methoxy substituted phenyl phosphate derivative of zidovudine, is a dual-action spermicide with potent anti-HIV activity

Heterosexual transmission of HIV to women is the fastest-growing mode of transmission. In a systematic effort to develop a microbicide capable of preventing HIV transmission as well as providing fertility control, novel phenyl phosphate derivatives of 3’-azido-3’-deoxythymidine (zidovudine, ZDV) have been identified that exhibit potent anti-HIV and spermicidal activities. This study reports the synthesis, characterization, and preclinical formulation of compound WHI-05, 5-bromo-6-methoxy-5,6-dihydro-3’-azidothymidine-5’-(p-methoxyphenyl) methoxyalaninyl phosphate. The anti-HIV activities of WHI-05 and ZDV were compared by measuring p24 antigen production and reverse transcriptase activity as markers of viral replication using human peripheral blood mononuclear cells (PBMC) infected with both ZDV-sensitive and ZDV-resistant strains of HIV. The sperm immobilizing activity (SIA) of WHI-05 was compared with that of ZDV and nonoxynol - 9 (N - 9) by computer-assisted sperm analysis (CASA). The effect of WHI-05 on sperm membrane integrity was examined by high resolution, low voltage scanning electron microscopy (HR-LVSEM). The in vitro cytotoxicity profile of WHI-05 versus N - 9 were compared using normal human vaginal, ectocervical, and endocervical epithelial cells. The in vivo vaginal tolerance, absorption, and toxicity of a 2% WHI-05 gel-microemulsion was tested in the rabbit. Whereas ZDV displayed potent anti-HIV activity but lacked SIA, WHI-05 elicited both potent anti-HIV activity and SIA. WHI-05 inhibited the replication of ZDV-sensitive as well as ZDV-resistant strains of HIV in PBMC. CASA combined with HR-LVSEM demonstrated that WHI-05-induced SIA was not associated with membrane damage. Unlike, N - 9, the spermicidal activity of WHI-05 was not associated with cytotoxicity to reproductive tract epithelial cells. Repetitive intravaginal application of a 2% WHI-05 gel-microemulsion did not damage the vaginal epithelium or cause local inflammation in the rabbit model. As a potent anti-HIV agent that has spermicidal activity and is devoid of mucosal toxicity, WHI-05 shows a unique clinical potential to become the active ingredient for a vaginal contraceptive for women who are at high risk for acquiring HIV by heterosexual vaginal transmission.

Two distinct human uterine cervical epithelial cell lines established after transfection with human papillomavirus 16 DNA.

We have established two distinct human cervical cell lines, NCC16 and NCE16, after traiisfecting human papillomavirus type 16 (HPV16) DNA into normal human ecto‐cervical and endo‐cervical epithelial cells, respectively. Both lines expressed HPV16 E6 and E7 as detected hy reverse transcriptase‐polymerase chain reaction and northern Wot hybridization. These cells have been passaged for over 100 population doublings and express strong telomerase activity. Neither cell line was tumorigenic in athymic nu/nu mice. However, both NCC16 and NCE16 developed abnormally stratified architectures following implantation with a silicon membrane sheet in the back of athymic nude mice. The former cells were pathohistologically similar to carcinoma, while the latter produced Alcian‐blue positive cells, suggesting the occurrence of metaplastic changes. These distinct cell lines offer a useful model system for the study of cervical carcinogenesis and of its regulatory mechanism after HPV infection in different regions of the uterine cervix.

Escherichia coli expressing a Neisseria gonorrhoeae opacity-associated outer membrane protein invade human cervical and endometrial epithelial cell lines.

Members of the opacity-associated (Opa) outer membrane protein family of Neisseria gonorrhoeae have been proposed to mediate adherence to and invasion of cultured human epithelial cells. We transformed Escherichia coli with a plasmid containing a gonococcal opa gene fused in-frame to the leader sequence of the beta-lactamase gene as described by Palmer et al. [Palmer, L., Brooks, G. F. & Falkow, S. (1989) Mol. Microbiol. 3, 663-671]. These transformed E. coli [E. coli (opa)] expressed the heat-modifiable opa gene product (the Opa protein) in their outer membrane and adhered to and invaded ME-180 human endocervical epithelial cells. In a 2-h adherence assay, an average of 26.7 E. coli (opa) adhered per ME-180 cell, whereas the control E. coli carrying only the expression vector (pKT279) did not adhere at all (less than 0.15 bacterium per cell). We investigated the ability of the adherent E. coli (opa) to invade ME-180 epithelial cells by using a gentamicin selection assay. We recovered up to 1 x 10(6) gentamicin-resistant bacteria per monolayer when ME-180 cells were infected with E. coli (opa) compared to less than 10 bacteria when the epithelial cells were infected with the same number of control E. coli (pKT279). The kinetics and level of invasion by E. coli (opa) were similar to invasion by Opa+ N. gonorrhoeae. Maximum invasion occurred 4 h after infection with 4 x 10(7) bacteria. Transmission electron microscopy studies confirmed that E. coli (opa) invaded ME-180 cells. In comparative studies, the number of E. coli (opa) that invaded HEC-1-B human endometrial epithelial cells was about an order of magnitude less than the number that invaded ME-180 cells, and E. coli (opa) did not invade Chang human conjunctival epithelial cells at all. The observations that early (less than 4 h) invasion by E. coli (opa) was dramatically inhibited, in a dose-responsive manner, by the actin-disrupting reagent cytochalasin D but later invasion (8-24 h) was not suggest that invasion mediated by Opa proteins may occur by two mechanisms, only one of which is dependent upon microfilament function. Transmission electron microscopy also revealed that infected epithelial cells had a dramatically increased amount of cytoplasmic fibrillar material surrounding the nucleus. The function and genesis of this material remain unclear. These studies indicate that at least one gonococcal Opa protein is an invasin.

Polarized expression of major histocompatibility complex class I molecules in human endometrial and endocervical epithelial cells

The localization of major histocompatibility complex (MHC) class I molecules in human endometrial and endocervical epithelial cells was studied with an immunogold technique on ultrathin cryosections. At the cell surface, MHC class I molecules are delivered solely to the basolateral plasma membrane in human epithelia. Therefore, only the basolateral domain of epithelial cells is capable of presenting antigen to MHC class I-restricted cytotoxic T cells.

Growth and characterization of epithelial cells from normal human uterine ectocervix and endocervix

The human uterine cervix consists of an endocervical canal lined with a single layer of columnar mucus-secreting cells and an outer ectocervix covered by a stratified squamous epithelium. We report here the culture of human endocervical epithelial cells (HEnE) and human ectocervical epithelial cells (HEcE) in serum-free medium (KGM). Both HEnE and HEcE cultures were composed of keratinocytelike cells which formed desmosomal contacts and stratified in the presence of high concentrations of calcium ions. Cells with a pleomorphic epithelial morphology were observed in HEnE cultures, but not in HEcE cultures. Keratin 18, which is characteristic of endocervix in vivo, was detected by indirect immunofluorescent staining in all HEnE cells but was never detected in cultured HEcE. HEcE expressed keratin 13 which is characteristic of ectocervix in vivo. Although keratin 13 was never detected in primary HEnE cultures, it was expressed in passaged HEnE cultures grown in medium with high concentrations of calcium and in late passage HEnE cultures. HEnE underwent an average of 15.1 population doublings during serial culture. Mean colony-forming efficiency during Passages 2 to 3 was 14.7% and mean population doubling time was 17.8 h. HEcE cultures underwent significantly more population doublings (29.0) than HEnE cultures, whereas colony-forming efficiencies and doubling times were similar to those determined for HEnE. HEnE and HEcE cells may be useful in developing in vitro models of cervical squamous metaplasia and for exploring the interactions between target cell differentiation, carcinogens, and papillomaviruses in the development of cervical neoplasia.

Human ectocervical and endocervical epithelial cells in culture: A comparative ultrastructural study

Primary and subcultured epithelial cells obtained from the human ectocervix and endocervix were compared by light, immunofluorescent, and electron microscopic techniques. In culture, both cell types were consistently free of surface-associated fibronectin as revealed by immunofluorescent studies. Ectocervical cell colonies exhibited a stratified appearance, where as endocervical epithelial cells grew in a single layer with smooth overlapping of adjacent cells. In addition, the surfaces as well as the cytoplasmic organellar structures of both cell types showed many characteristic features related to the epithelium of their origin. These features were retained with the aging of the cells and were seen also in subcultures. These cultures should permit observation of the effects of steroids, as well as viral and chemical carcinogens, on the ultrastructural integrity of the epithelial cells.