Electron spin resonance studies on normal human uterus and cervix and on benign and malignant uterine tumors.

Electron spin resonance (ESR) studies at -130 degrees have been made on frozen samples of normal human cervix and uterus and on frozen samples of various pathological conditions of the cervix and uterus including fibroleiomyoma and carcinoma. Fifty-five samples of normal cervix and endometrium, 40 samples of nonmalignant disturbances, 15 benign tumor samples, and 20 malignant samples were studied. Very strong ESR signals were seen in frozen powders and frozen intact samples of normal cervix and endometrium and in nonmalignant gynecological conditions. In many cases, the ESR signal was greatly decreased or even undetectable in cancer samples. The substance(s) responsible for the ESR signal in frozen intact tissue (g = 2.11 to 2.15) is decreased in concentration when the sample is ground to powder under liquid nitrogen, and an anisotropic signal (g = 2.002 to 2.035) then becomes much more evident. The ESR signals in intact and in powder samples are sensitive to temperature variations; the signals disappear around 0 degrees, and only the intact samples show significant recovery of signal on recooling. The anisotropic g values and temperature sensitivity in the powders may result from an organic peroxy radical that is more strongly associated with a metal ion in intact samples.

Characterization of keratins from rat cervical epithelial cells in vivo and in vitro.

The keratins expressed in cultured rat cervical epithelial cells were analyzed by using two-dimensional gel electrophoresis and monoclonal antikeratin antibodies and were compared with those expressed in the rat cervix in vivo in the presence and absence of estrogen stimulation. The cervical epithelium in vivo responds to estrogen stimulation with alterations in keratin composition. In response to estrogen, the epithelium undergoes proliferation and stratification and begins expressing increased amounts of two basic Mr 57,000 and 58,000 keratins and two acidic Mr 51,000 and 52,000 keratins. Cultured rat cervical epithelial cells were found to express only small amounts of the basic Mr 57,000 and 58,000 keratins and the two acidic Mr 51,000 and 52,000 keratins characteristic of the estrogen-stimulated cervix. In addition to the keratins found in the cervical epithelium in vivo, rat cervical epithelial cells in vitro begin expressing two Mr 55,000 and 56,000 keratins of the basic keratin family (type II) and an acidic component with a molecular weight of 40,000. Although these components are not detected in the rat cervix in vivo, they migrate similarly on two-dimensional electrophoretic gels to proteins expressed by the rat endometrial epithelium in vivo. These findings indicate that alterations in keratin expression could serve as markers for studying the effects of steroid hormones on the differentiation of rat cervical epithelial cells in vitro and during the development of squamous metaplasia.

Characterization of normal human exocervical epithelial cells immortalized in vitro by papillomavirus types 16 and 18 DNA.

An in vitro system for studying the interaction between human papillomavirus (HPV) 16 and 18 recombinant DNA and normal human exocervical epithelial cells is described. Eight HPV-immortalized human exocervical epithelial cell lines were established; all the lines contained either integrated HPV16 or 18 sequences and expressed HPV mRNAs. Thus, integration and expression appear to be required for immortalization. Immortalized cells (greater than 200 population doublings to date) divided rapidly (doubling time of 30 to 46 h) and morphologically resembled primary cultures of normal human exocervical epithelial cells. They expressed a keratin pattern consistent with their origin from exocervical epithelium. When cultured at high density or in the presence of serum they terminally differentiated. Sublines resistant to terminal differentiation were selected by growth in serum-supplemented medium. Keratin pattern changes suggest they have some properties in common with cervical squamous carcinoma cells. However, HPV-immortalized cell lines were not tumorgenic in nude mice. Thus, HPV16/18 is not carcinogenic by itself. These cell lines represent an appropriate model for studying factors that regulate HPV gene expression in normal cervical epithelial cells and examining the influence of cocarcinogens on neoplastic progression.

Keratin subtypes in carcinomas of the uterine cervix: implications for histogenesis and differential diagnosis.

Normal epithelia and carcinomas of the human uterine cervix were studied by monoclonal antibodies chain specific for cytokeratins 4, 8, 10, 13, 14, 18, and 19. Most cells in 13 examined squamous carcinomas revealed a cytokeratin phenotype detected in ectocervical basal cells and endocervical subcolumnar reserve cells: 8+, 14+, 18+, 19+, 4-, 10-, 13-. We propose that these two cell types are closely related or identical and that squamous carcinoma of the cervix originates in this cell type. In more differentiated tumor cells cytokeratins 4, 10, and 13, which are present in suprabasal layers of the normal ectocervical epithelium, were coexpressed with basal cell cytokeratins. Thus, contrary to previous beliefs, all cytokeratins detected in carcinomas were also present in normal epithelium of uterine cervix. The cytokeratin profile of cervical adenocarcinomas corresponded to that of columnar endocervical cells (8+, 18+, 19+), although two of the three adenocarcinomas also expressed cytokeratin 4, which in the normal endocervix was detected in scattered single columnar cells only. The new monoclonal antibody DE-K14, specific for cytokeratin 14, proved a specific marker of subcolumnar reserve cells in the endocervix. It was also the only one that reacted with all cervical squamous carcinomas but with none of the cervical adenocarcinomas and, as such, has a potential value for pathological differential diagnosis of cervical tumors.

Localization of progesterone receptor with monoclonal antibodies to the human progestin receptor.

A series of rat and mouse monoclonal antibodies to the human progesterone receptor (PR) has recently been produced. These antibodies were used for the immunocytochemical identification of PR in several mammalian species including humans. The specificity of the monoclonal antibodies for PR was confirmed by using competition studies with purified PR and by comparison of the immunostained tissues, known from steroid binding assays to be receptor rich, with immunostained tissues known to be receptor-poor. Immunoreactive PR was found in the nuclei of uterine epithelial, stromal, and smooth muscle cells; benign ductal and lobular epithelial cells of the breast; ovarian surface epithelium; ovarian stroma and luteal cells; pulmonary parenchymal cells; and selected pituitary parenchymal cells. A proportion of the following selected human tumors expressed PR: breast carcinomas, endometrial carcinomas, ovarian carcinomas, and meningiomas. PR was localized to the nuclei of all progesterone target tissues even under conditions where the vast majority of the receptor is unoccupied by steroid, suggesting that the unoccupied as well as the steroid-occupied form of PR are predominantly nuclear proteins, as observed previously for estrogen receptor and rabbit PR.

Immunocytochemical localization of estrogen receptors in the macaque reproductive tract with monoclonal antiestrophilins.

Monoclonal antibodies to the estrogen receptor (anti-ER) were used to develop an immunocytochemical method to detect ERs in frozen sections of the macaque reproductive tract. Specific nuclear, but not cytoplasmic, staining occurred with two different methods: direct, in which an antiestrophilin -horseradish peroxidase conjugate was used as the first antibody, and indirect, in which a mixture of antiestrophilins was used in the first incubation step. Nuclear staining was absent when various control antibodies replaced the anti-ER. In uteri from spayed monkeys treated with estradiol (E2) for 14 days, nuclear staining was always present. In uteri from similar animals treated for an additional 14 days with E2 and progesterone, nuclear staining was almost completely absent. Mean endometrial nuclear ER levels, measured by an exchange assay, were 5-fold greater in the E2-treated than in the E2-plus progesterone-treated group. In addition, when samples of estrogenized uterus and oviduct were incubated for 60 min in vitro with 100 nM E2, the intensity of nuclear staining increased in parallel with an increase in the concentration of nuclear ER. The nuclei of stromal, smooth muscle, and epithelial cells of the estrogenized oviduct, cervix, and vagina as well as smooth muscle cells of the estrogenized myometrium were also receptor positive. Nontarget tissues, such as duodenum, colon, esophagus, and skeletal muscle, contained no cells that showed specific nuclear staining. Some staining of cytoplasmic and extracellular components occurred in all preparations. These latter reactions were nonspecific, because they were present in many nontarget tissues or when control antibodies replaced the anti-ER. With current methods, only nuclear ERs can be reliably localized in frozen sections of monkey tissues with monoclonal antiestrophilins .

Changes in nucleic acid and protein content in nuclei of human cervical cells.

Nuclei in five classes of cervical cells observed in Pap smears were studied using quantitative epifluorescence microscopy. The five classes of cells were: parabasal (Pb) cells; intermediate cells with round (I-R), oval (I-O), and rod-pyknotic (I-RP) nuclei; and, pyknotic (P) cells. Six nuclear traits were measured: total nucleic acid, DNA, RNA, total protein, histone, and non-histone protein. The six nuclear indices increased as Pb cells became I-R cells (cell enlargement and maturation), and then decreased as I-R cells degenerated through the following senescence sequence: I-O----I-RP----P. We infer that these changes continue and result in anucleate, superficial cells. Pb cells are probably in early stages of DNA synthesis (S-phase of the cell cycle) since the mean for DNA increased as they became I-R cells. The following types of cells comprised the Pap smears studied: Pb, 7%; I-R, 19%; I-O, 55%; I-RP, 8%; P, 9%; superficial cells with nuclei devoid of nucleic acids, 1%; and, anucleate cells, 1%. We conclude that cervical exfoliative cytology provides a model system for the study of human cell development, maturation, senescence, and death in addition to its use in detecting early through late stages of cervical cancer. The high correlation between the nuclear indices studied suggests that several quantitative nuclear parameters other than DNA may be useful for cancer detection.

Human papillomavirus DNA in genital tract lesions histologically negative for condylomata. Analysis by in situ, Southern blot hybridization and the polymerase chain reaction.

Koilocytotic atypia (nuclear atypia in conjunction with perinuclear halos) is diagnostic of condylomata of the lower female genital tract, over 90% of which contain human papillomavirus (HPV) DNA. Genital tract lesions may be clinically suggestive of condylomata but lack clear-cut koilocytotic atypia. Of 57 vulvar and 60 cervical lesions that lacked clear-cut koilocytotic atypia, four (7%) and two (3%), respectively, had HPV DNA detected by in situ analysis. Using Southern blot analysis, HPV DNA was detected in five of 27 (19%) and 20 of 55 (36%) vulvar and cervical lesions, respectively, that lacked koilocytotic atypia. When analyzed with the polymerase chain reaction (PCR), HPV DNA was detected in six of 22 (27%) and three of 18 (17%) vulvar and cervical lesions, respectively, that lacked koilocytotic atypia. These findings demonstrate that infection by HPV may be found in genital tract lesions that lack koilocytotic atypia. The lower detection rate of HPV in cervical lesions that lacked koilocytotic atypia with PCR as compared with Southern blot analysis may be related to the relatively high proportion of "novel" types (related to, but distinct from, the HPV types in the probe) in such lesions. The increase in the detection rate in vulvar lesions that lacked koilocytotic atypia with PCR compared with in situ hybridization suggests that about one third of such lesions are HPV related, but that in such cases the copy number of the virus is typically below the threshold of the in situ analysis.

Autoradiographic visualization of alpha 1-adrenergic receptors in cervix of early pregnant rat.

alpha 1-Adrenergic receptors were identified, characterized, and localized in rat cervix on Day 6 of pregnancy by autoradiography. Autoradiographic study was performed in slide-mounted rat cervix sections using [3H]-prazosin ([3H]-PRAZ) as ligand. Binding was time dependent and specific. Pharmacological study indicated that specific [3H]-PRAZ binding was inhibited with high affinity by prazosin and phenylephrine and low affinity by yohimbine and clonidine. In cervix, the alpha 1-adrenergic receptors were localized mainly to the inner circular layer of the myometrium. Binding to the outer longitudinal layer of myometrium was moderate, and binding was absent in the endometrium. The regional distribution of alpha 1-adrenergic receptors strongly suggests that the circular layer of myometrium may function as an important modulator of contractile response of the cervix, probably involved in the retention of blastocysts at the utero-cervical end of the horn.

Estrogen receptor immunocytochemistry.

Estrogen receptor activity was preserved in fixed, paraffin-embedded tissue and demonstrated by binding of estrogen which, in turn, was detected immunocytochemically. Estrogen was added to rat endocervial epithelium to protect specifically receptors during fixation. The protective estrogen was apparently lost during embedding and had to be resupplied before staining. Estradiol-mediated immunocytochemical staining was inhibited by diethylstilbestrol and nafoxidine hydrochloride.

S-100 protein in glands within decidua and cervical glands during early pregnancy.

Strong immunoreactivity with polyclonal S-100 protein antisera and monoclonal S-100 alpha subunit antiserum was found in glandular cells of the decidua basalis and cervical polyps during early pregnancy. Immunoreactive S-100 protein was negative in glandular cells of the endometrium and cervix of nonpregnant women. It was also negative in endometrial hyperplasia and endometrial carcinoma. While the function of S-100 protein is not known, a relationship between humoral factors related to pregnancy and expression of S-100 protein gene is suggested by the results of this study.

Immunocytochemical localization of benzo(a)pyrene-DNA adducts in human tissue.

Specimens of human lung, uterine cervix, ovary, and placenta were studied for the presence of benzo(a)pyrene 7,8-diol 9,10 epoxide (BPDE)-DNA adducts by using rabbit anti-BPDE-DNA antibody and light microscopic immunocytochemistry. BPDE-DNA antigenicity was detected in the bronchial epithelial cells, cervical epithelium, oocytes, luteal cells, corpora albicans, and hyalinized media of arteries within the ovaries and trophoblastic cells of the placental villi. In conjunction with immunoassay detection of BPDE-DNA adducts in human peripheral blood lymphocytes, this study demonstrates that a variety of human tissues can metabolize and bind the ubiquitous carcinogen benzo(a)pyrene. The identification and localization of this carcinogen-DNA antigenicity in various tissues and cells may not only help in monitoring exposed persons but also give insight to organ site carcinogenesis, transplacental carcinogenesis, and teratogenesis.

[Possibilities and limits of the quantitative demonstration of protein thiols with the DDD-reagent in cells of cervical epithelium (author's transl)]. / Möglichkeiten und Grenzen der quantitativen Bestimmung von Protein-Thiolen mit dem DDD-Reagens in Cervix-Epithelien.

From six patients, all with invasive carcinoma of the cervix uteri, two groups fo smear preparations, each consisting of eight samples, were taken: The first, group A, before and the second, group B, after mechanical removal of the discharge which was present in abundant quantities. All samples were treated for 24 h with DDD to reach the fast-reaching SH-groups, coupled with Fast Blue B and measured cytophotometrically at 560 nm. Without any exception, the neoplastic cells of group A showed significantly lower extinction values both of the nucleus and of the total cell, if compared to group B. The mean extinction difference amounts to 55% and is highly significant and some evidence is given that the viability of the cells plays an important role. For this reason, the quantitative evaluation of the DDD Fast Blue B does not seem to be useful for cytologic routine investigations.

Localization of human epidermal growth factor receptor in cervical intraepithelial neoplasias.

Immunohistochemical staining of epidermal growth factor receptor (EGF-R) with a monoclonal antibody was performed in 43 biopsies of cervical tissue. The distribution of the receptors in normal cervical tissue differs from that of cervical intraepithelial neoplasia (CIN) and squamous cell carcinoma of the cervix. Whereas the staining reaction in normal squamous epithelium was confined to the basal and deep parabasal cell layer, in all cervical intraepithelial neoplasias, with or without human papilloma virus association, a homogeneous EGF-R staining reaction could be observed throughout the entire lesion. This means that the dysplasia cells of a CIN I-III, like the tumor cells of a squamous cell carcinoma, have a raised EGF-R content, which in the normal squamous epithelium is usually only found in the basal and deep parabasal cells that are capable of dividing. No EGF-R staining reaction could be detected in the higher, differentiated cell layers of the normal squamous epithelium of the cervix.

The histogenesis of mixed cervical carcinomas. The concept of endocervical columnar-cell dysplasia.

In a case of cervical adenocarcinoma in situ, accompanied by relatively minor squamous-cell dysplasia, the neoplastic glandular cells were associated with "abnormal" but not clearly cancerous surface cells of the endocervix. Measurements of the nuclear DNA of these cells revealed an aneuploid pattern similar to that of epidermoid dysplasias. It is suggested that endocervical dysplasia is a valid entity and represents a step in the evolution of cervical adenocarcinoma from reserve cells comparable to the position of epidermoid dysplasia in the genesis of epidermoid carcinoma.

Immunohistochemical localization of carcinoembryonic antigen in microglandular hyperplasia and adenocarcinoma of the endocervix.

Immunoperoxidase localization of carcinoembryonic antigen (CEA) was performed on tissue sections of microglandular hyperplasia and adenocarcinoma of endocervical origin to see if CEA would be a marker of malignancy in this context. Twenty (95%) of 21 of the benign lesions were negative and one was focally positive. Nine (64%) of 14 adenocarcinomas were widely positive, four were focally positive, and one was negative. These results suggest that extensive immunohistochemical positivity for CEA in endocervical glandular lesions is indicative of malignancy.

Simple procedure for assessing relative quantities of neutral and acidic sugars in mucin glycoproteins: its use in assessing cyclical changes in cervical mucins.

A simple histochemical procedure for assessing relative amounts of neutral and acidic sugars in mucin glycoproteins, and its application in the study of cyclical changes of human cervical mucins, is described. This procedure, the saponification/selective periodate oxidation/borohydride reduction/alcian blue pH 2.5/periodic acid Schiff (KOH/PA*/Bh/Ab 2.5/PAS) method, uses a selective oxidation step to remove the PAS positivity of sialic acid; thus only neutral sugars stain positively with PAS, and acidic sugars (O-sulphate esters and carboxyl groups) stain with alcian blue. This differs from the KOH/Ab/PAS technique which stains sialic acid residues with both alcian blue and PAS. Applying the KOH/PA*/Bh/Ab 2.5/PAS technique to the study of cyclical changes of human cervical mucins, a decreased neutral:acidic sugar ratio in the secretory phase mucins compared with those of the proliferative phase was found. This difference was not seen with KOH/Ab/PAS staining in the same cases. The techniques and reagents used in this procedure can be easily applied in a clinical histopathology laboratory.

Immunoreactive alpha interferon in cervical flat koilocytic lesions and intraepithelial neoplasia.

Immunocytochemical staining for alpha-interferon was carried out on cervical biopsy specimens showing non-condylomatous koilocytic atypia (n = 12) and cervical intraepithelial neoplasia (n = 18), both of which are associated with human papilloma virus (HPV) infection. Normal cervical tissue obtained from hysterectomy specimens was also assessed. Koilocytes were not immunoreactive for alpha interferon and keratinocyte staining was observed in only four cases of intraepithelial neoplasia. HPV infection alone does not therefore seem to induce the production of alpha interferon in cervical squamous epithelium. There was variable but, in some cases, prominent staining of cells in the stromal inflammatory infiltrate as well as intraepithelial cells which had morphological and immunocytochemical characteristics of Langerhans' cells. Alpha interferon immunoreactivity in Langerhans' cells is in keeping with derivation from the mononuclear phagocyte system and may be important in the host response to HPV infection.

A new marker of maturation in the cervix: the estrogen-regulated 24K protein.

The presence of an estrogen-regulated protein (24K) in normal, dysplastic, metaplastic, and neoplastic cervical epithelium correlates with histologic criteria for squamous cell maturation. The 24K protein, originally discovered in the MCF-7 breast cancer cell line, was studied in 51 cases by the modified immunoperoxidase Avidin-Biotin Complex method, using an anti-24K mouse monoclonal antibody. Immunostained sections were compared to hematoxylin-and-eosin-stained sections cut from the same tissue block. The 24K protein was observed to be located primarily in the parabasal or "prickle cell layer" of normal cervical tissue (6 of 7 normal cervical tissue specimens tested were positive for 24K protein. Specimens were obtained from surgery for nonneoplastic causes) and in all cases (12 of 12) of dysplasia and carcinoma in situ. Intercellular bridges of these cells showed prominent immunostaining in normal cervix and dysplasia. 24K protein was observed as a granular cytoplasmic stain in all cases of squamous metaplasia (5 of 5) and keratinizing squamous cell carcinoma (9 of 9), and in 8 of 14 cases of nonkeratinizing squamous cell carcinoma. In this latter group, immunostaining was confined to only those cells showing cytoplasmic eosinophilia on H&E sections. In no case was the presence of the 24K protein associated with areas of mature keratin. 24K immunostaining was also observed in the reserve cells of morphologically normal endocervical glands adjacent to areas of dysplasia and carcinoma. We conclude that 24K protein is associated with squamous cell maturation and may be an important marker of reserve cell hyperplasia and squamous metaplasia.

Expression of epidermal growth factor receptor and HER-2/neu in normal and neoplastic cervix, vulva, and vagina.

Monoclonal antibodies were used to localize immunohistochemically epidermal growth factor receptor and HER-2/neu in normal and neoplastic frozen tissue samples from the lower genital tract of women. In squamous epithelia of the cervix, vulva, and vagina, epidermal growth factor receptor and HER-2/neu both were expressed most strongly by basal keratinocytes. Expression of both of these cell surface molecules decreased as cells underwent differentiation toward the mucosal surface. In contrast, both epidermal growth factor receptor and HER-2/neu were expressed throughout the entire thickness of the epithelium by undifferentiated squamous cells in squamous metaplasia, raised condyloma, and carcinoma in situ. In 34 squamous cancers of the cervix, vulva, and vagina, all malignant cells were found to have moderate to heavy staining for epidermal growth factor receptor. Staining of 33 of these cancers for HER-2/neu was light, although one patient who presented with distant metastases had heavy staining for HER-2/neu. These data suggest that although overexpression of HER-2/neu in squamous cancers of the lower genital tract is a rare event, it may be associated with aggressive biologic behavior.