Search

The prostaglandin F2alpha derivative, latanoprost (LT), used in glaucoma treatment, can induce pigmentation in irises of patients with hazel or heterochromatic eye colour. The mechanism by which LT induces pigmentation in the iris is not yet established, although it does not appear to induce proliferation of iridial melanocytes. The purpose of this study was to develop an in vitro model in which to investigate this mechanism. The pigmentary responses to LT and prostaglandin F(2alpha) (PGF(2alpha)) were examined in human iridial melanocytes alone or in co-culture with epithelial cells (non-ocular human epidermal keratinocytes and iris pigment epithelial cells) or mesenchymal cells (non-ocular dermal fibroblasts or iridial fibroblasts). Melanogenesis was assessed after 4 days culture with prostanoids, using dopa oxidase activity. Prostaglandin FP expression on human iridial fibroblasts and melanocytes was investigated using an immunofluorescent technique employing antibody to PGF(2alpha) receptor and RT-PCR. Iridial melanocytes did not show a convincing increase in dopa oxidase when cultured alone but in the presence of fibroblasts (ocular or non-ocular) there was a significant increase (25-30%) in dopa oxidase activity in response to 10(-7)-10(-5)m LT and PGF(2alpha). Co-culture of melanocytes with epithelial cells, while leading to increased dopa oxidase activity, did not lead to any melanogenic response to LT or PGF(2alpha). FP receptor expression was detected on fibroblasts but not iridial melanocytes by immunocytochemistry and RT-PCR. The melanocyte/fibroblast co-culture model developed in this study also showed that LT and PGF(2alpha) increased dopa oxidase activity in melanocytes from donors with brown but not blue eyes. These results suggest that LT may be inducing pigmentation in the human iris indirectly through the FP receptor on adjacent fibroblasts.

Niemann-Pick type C disease is characterized by the accumulation of cholesterol and other lipids within the lysosomal compartment, a process that is often accompanied by a reduction in acid sphingomyelinase activity. These studies demonstrate that a CHO cell mutant (CT-60), which accumulates lysosomal cholesterol because of a defective NP-C1 protein, has approximately 5-10% of the acid sphingomyelinase activity of its parental cell line (25-RA) or wild type (CHO-K1) cells. The cholesterol-induced reduction in acid sphingomyelinase activity can be reproduced in CHO-K1 cells by incubation in the presence of low density lipoprotein (LDL) and progesterone, which impairs the normal egress of LDL-derived cholesterol from the lysosomal compartment. Kinetic analysis of sphingomyelin hydrolysis in cell extracts suggests that the CT60 cells have a reduced amount of functional acid sphingomyelinase as indicated by a 10-fold reduction in the apparent V(max). Western blot analysis using antibodies generated to synthetic peptides corresponding to segments within the carboxyl-terminal region of acid sphingomyelinase demonstrate that both the CT60 and the LDL/progesterone-treated CHO-K1 cells possess near normal levels of acid sphingomyelinase protein. Likewise, Niemann-Pick type C fibroblasts also displayed normal acid sphingomyelinase protein but negligible levels of acid sphingomyelinase activity. These data suggest that cholesterol-induced inhibition is a posttranslational event, perhaps involving cofactor mediated modulation of enzymatic activity or alterations in acid sphingomyelinase protein trafficking and maturation.

Previously, modified LDLs were shown to stimulate macropinocytosis in pigeon macrophages. Simultaneous intracellular trafficking of LDL and AcLDL, differentially labeled with colloidal gold, was done to determine whether uptake of LDL, which does not cause foam cell formation, was internalized via a separate route from AcLDL, which stimulates foam cell formation. AcLDL and LDL were followed at either low (12 microg/mL) concentrations near the saturation of high affinity binding sites or high (50 to 150 microg/mL) lipoprotein concentrations used to induce foam cell formation. The colloidal gold distribution and percentage of co-labeling as observed by transmission electron microscopy were determined for organelles involved with coated-pit endocytosis or macropinocytosis. LDL simultaneously incubated with AcLDL on macrophages at the low concentration was predominately internalized via coated-pit endocytosis. AcLDL was internalized via both coated-pit endocytosis and macropinocytosis at low concentration. At higher lipoprotein concentrations (50 to 150 microg/mL), AcLDL continued to be internalized via macropinocytosis. Interestingly, a significant portion of the co-incubated LDL, at high concentrations, also trafficked via macropinocytosis. LDL internalized by macropinosomes at high lipoprotein concentrations suggests that AcLDL-stimulated macropinocytosis might increase uptake of co-incubated lipoproteins. When (125)I-LDL was incubated with cold AcLDL, LDL degradation at 37 degrees C doubled, without a corresponding increase in cell association or total binding of LDL at 4 degrees C. These studies suggest that modified LDL-stimulated macropinocytosis is a mechanism for increased degradation of co-incubated LDL potentially leading to foam cell formation.

Using a stable cell line 25-RA derived from wild-type Chinese hamster ovary (CHO) cells as the parental cell, this laboratory previously reported the isolation and characterization of CHO cell mutants (cholesterol-trafficking or CT) defective in transporting LDL-derived cholesterol out of the acidic compartment(s) (lysosomes/endosomes) to the endoplasmic reticulum (ER) for esterification. In this report, we show that the CT mutation can be complemented by fusion with human cells; however, attempts to complement the CT defect through DNA transfection have resulted in a collection of stable cell lines designated as ST cells. Under cholesterol starvation condition, the ST cells exhibit an elevated rate of cholesterol ester biosynthesis (by 3- to 5-fold) compared to both the parental CHO cells and the CT cells. The phenotypes of the ST cells are stable. ST cells are thus new cell lines arisen from the CT cells. When the plasma membranes of the parental, CT, and ST cells are labelled with [3H]cholesterol, ST cells show rates of [3H]cholesterol esterification much higher than that observed in CT cells but lower than that observed in the parental CHO cells. This result shows that translocation of plasma membrane cholesterol to the ER for esterification is defective in the CT cells. This result also suggests that ST cells acquire increased cholesterol trafficking activity between the lysosome and the ER without mixing the plasma membrane cholesterol pool. The characteristics of CT cells and ST cells reported here suggest that translocation of both lysosomal LDL-derived cholesterol and plasma membrane cholesterol to the ER for esterification may require common cellular factors involved in cholesterol egress from the acidic compartment(s) (lysosomes/endosomes).

Low-density lipoproteins (hLDL) and beta-migrating-very-low-density lipoproteins (beta-VLDL) were isolated from the plasma of cholesterol-fed White Carneau (WC) pigeons and low-density lipoproteins (nLDL) were isolated from the plasma of grain-fed WC pigeons. The lipoproteins were radiolabeled with 125I or 131I and injected into normocholesterolemic or hypercholesterolemic WC pigeons to determine their rate of clearance from the plasma. The fractional catabolic rate (FCR) of nLDL and hLDL in normocholesterolemic pigeons averaged 0.202 and 0.206 pools/h.respectively. beta-VLDL was cleared at a significantly slower rate of 0.155 pools/h. The FCR of the same lipoproteins injected into hypercholesterolemic pigeons was reduced by 17% for nLDL, 50% for hLDL and 57% for beta-VLDL, indicating that the effect of hypercholesterolemia on clearance in vivo was different for the three lipoproteins. The FCR of reductively methylated pigeon LDL (MeLDL), which gives a measure of receptor-independent clearance of LDL, was shown previously to be 0.037 pools/h. These studies suggest therefore that LDL and beta-VLDL are cleared from the plasma of normocholesterolemic and hypercholesterolemic pigeons at a rate substantially greater than that predicted for non-specific processes. Despite the reduction in the clearance rate of hLDL and beta-VLDL due to cholesterol feeding, the absolute amount of cholesterol that was cleared from the plasma by these lipoproteins was increased from approx. 200 mg/kg body weight per day in the normocholesterolemic pigeons to greater than 1000 mg/kg body weight per day in the hypercholesterolemic pigeons. This is due principally to the enrichment in cholesterol relative to protein of the lipoproteins isolated from cholesterol-fed pigeons and the failure of hypercholesterolemia to completely inhibit receptor-dependent clearance of LDL and beta-VLDL. The lower rate of clearance of beta-VLDL relative to LDL is in marked contrast to mammalian beta-VLDL, which is cleared much faster than LDL, but is consistent with the lack of apo E on pigeon lipoproteins. Apo E is the apoprotein that is thought to be responsible for the rapid clearance of beta-VLDL in normocholesterolemic mammals. The low rate of beta-VLDL clearance in pigeons also suggests that pigeons lack an apolipoprotein that function like mammalian apo E.

Treatment failure for intraepithelial neoplasia of the uterine cervix following electrocautery, cryosurgery, or carbon dioxide laser therapy is related primarily to incomplete eradication of the lesion. This may be due to the depth of crypt involvement, the linear extent, or the location of the abnormal epithelium. In view of this, the extent (depth and linear extent) and the location of cervical intraepithelial neoplasia (CIN) in 319 cervical cone specimens were analyzed. There is a significant correlation between the severity and the extent of the change. The mean depths of CIN I, II, and III were 0.42 +/- 0.28, 0.93 +/- 0.71, and 1.35 +/- 1.15 mm, respectively. The mean linear extents for CIN I, II, and III were 4.10 +/- 2.84, 5.84 +/- 4.13, and 7.60 +/- 4.32 mm, respectively. To eradicate 99.7% of CIN III lesions, it is necessary to destroy the tissue up to 4.80 mm in depth and of sufficient linear extent. While most lesions (87.2%) involved the transformation zone, 9.7% were higher in the cervical canal and 3.1% were located in the ectocervix. Appropriate cytologic samples, endocervical curettage, and colposcopic examination should be employed in evaluating the CIN, and strict criteria should be followed in selecting the patients for conservative management.

In this survey of 210 women with a history and/or gross changes of the cervix and vagina characteristic of DES exposure, changes related to vaginal adenosis were correlated with age. The mean age of women with columnar epithelium alone was 18.5 +/- 3.2 years, columnar epithelium with squamous metaplasia was 20.2 +/- 3.4 years, and squamous metaplasia alone was 23.0 +/- 2.7 years. These findings suggest that involution of vaginal adenosis occurs with increasing age. Involution of glands in the superficial stroma in most cases occurred concurrently with that of surface columnar epithelium. However, those deeply seated and complex glands in the stroma might lag in the metaplastic process and might persist. The pitfalls of misinterpretation of squamous cell changes in histologic samples were discussed. The lack of correlation between the colposcopic findings of mosaicism and/or punctuation and squamous cell neoplasia was confirmed in this study.

In this study, 59 vulvar intraepithelial squamous neoplasms (8 atypia and 51 carcinoma in situ) and 33 invasive squamous carcinomas were analyzed for their nuclear DNA content using Feulgen microspectrophotometry. Four cases of atypia had a polyploid DNA distribution. The remaining 4 cases of atypia and all cases of carcinoma in situ had an aneuploid pattern, and nearly two thirds of these had high ploidy stem cells (greater than 3N). This is in contrast to the low ploidy stem cells (less than 3N) seen in 70% of the invasive carcinomas and in 82% of the intraepithelial neoplasms in the vicinity of invasive carcinomas. This observation suggests that not all vulvar intraepithelial neoplasms have the same propensity to become invasive. Invasive carcinomas of comparable size and depth of invasion with low ploidy stem cells had a higher frequency of lymph node metastasis than those having high ploidy stem cells. The significance of nuclear DNA findings related to gynecologic neoplasms is discussed.

Sixteen borderline malignant serous ovarian tumors and seven well-differentiated invasive serous ovarian carcinomas were examined with the technique of Feulgen microspectrophotometry for the determination of nuclear deoxyribonucleic acid (DNA) ploidy patterns (diploid versus aneuploid) and ploidy levels of the stem cell lines. Of the nine stage I-II borderline malignant tumors, only one (11%) was aneuploid. In contrast, four of seven (57%) stage III borderline malignant neoplasms and all stage III carcinomas were aneuploid. The stem cell modal values in all borderline serous tumors were less than triploid (3N) while in five of seven carcinomas stem cell modal values were greater than triploidy. This contrast in ploidy patterns and ploidy levels may explain the differences in biologic behavior between borderline malignant serous tumors and invasive serous carcinomas of the ovary.

In the past few years there have been a number of reports correlating a high frequency of herpes simplex virus type 2(HSV-2) infection with lesions of the uterine cervix. These studies have used a clinical history of herpetic infection or the demonstration of herpetic antibodies in the cancer patients. The present study was performed to evaluate any possible carcinogenic activity of the formalin-inoculated herpes simplex virus type 2 in the reproductive tract of the female mouse. This approach to the study was selected because of previous experience with a model system of carcinogenesis of the cervix uteri using coal tar hydrocarbons. Cytologic and histologic preparations from experimental animals and controls are presented to demonstrate the mucosal alterations and tumors observed in the animals. Noninvasive lesions of the cervix were identified in 76.8% and invasive adenocarcinoma detected in 30.2% of the mice.

Initial evidence suggested that estrogen therapy increases the risk of endometrial carcinoma. It was then suggested that some studies may have exaggerated the hazard of estrogen therapy by including patients with atypical endometrial hyperplasia among those having endometrial carcinoma. Three internationally recognized pathologists reviewed the histology slides available from the Ziel and Finkle study, which originally reported a risk ratio of 7.6 for estrogen users. At least one of the pathologists concurred with the original diagnosis in all but one case. Furthermore, all pathologists aggreed that 74 per cent (66/89) were correctly diagnosed. In the 66 patients with unanimous diagnosis, 61 per cent (40/66) had used conjugated estrogens, versus 57 per cent (54/94) in the original study. On the basis of 66 patients and 132 matched controls, the revised risk-ratio estimate is 8.1 (with a one-sided 95 per cent lower confidence limit of 4.5), validating the original estimate.

Histologic sections from cervical and vaginal biopsies showing dysplasia in 37 women exposed to diethylstilbestrol (DES) in utero were stained for human papillomavirus structural antigens using an immunoperoxidase technique. Forty-three percent of the lesions had detectable papillomavirus antigens. These findings indicate that a significant proportion of cervical dysplasia observed in DES-exposed progeny is associated with papillomavirus infection.

This study has analyzed the nuclear DNA content of intraepithelial glandular lesions in diethylstilbestrol (DES)-exposed offspring. The endocervical cells and endometrial-tubal cells present in the vaginal adenosis and pericervical collars had a euploid distribution. However, greater proliferative activity was found among the endometrial-tubal cells. In the cells of vaginal adenosis of a postpartum woman without a confirmed history of maternal exposure to DES, a polyploid pattern was found, indicating that endometrial-tubal cells underwent hyperplastic changes under the influence of hormonal stimulation. In an atypical adenosis derived from endometrial-tubal cells, an aneuploid pattern was found. Based on these findings, it is suggested that endometrial-tubal cells have a greater proliferative potential than endocervical cells.

Previous studies at this laboratory showed that repeated application of inactivated herpes simplex virus type 2 to the mouse cevix produced premalignant and malignant lesions. In the present study mice were inoculated with inactivated herpes simplex virus type 2 or control solution and Freund's adjuvant by intraperitoneal and subcuaneous routes before exposure of the cervix to inactivated virus. It appears that immunization with inactivated virus conferred a protection against the induction of cervical carcinoma.

Forty-nine cases of ovarian endometrioid carcinoma with squamous foci were reviewed. Of particular interest was the biologic behavior of the neoplasms in relation to the appearance of the epithelium. As previously noted for the analogous uterine endometrial tumors, the ovarian adenosquamous lesions occurred later in life, were more advanced, were associated with less differentiated adenocarcinomatous components, and had a poorer prognosis. The five-year survival rate for patients with adenosquamous endometrioid ovarian carcinoma was 21% in comparison with the 90% survival found in patients with ovarian endometrioid adenocanthoma.

Cellular samples obtained by vaginal scrapings from 204 women exposed to diethylstilbestrol (DES) in utero were evaluated and compared with samples collected from a like number of controls. Several different sampling methods were evaluated. Vaginal scrapings contained columnar cells existing alone or in combination with squamous metaplastic cells and less frequently were characterized only by metaplastic cells. None of the control samples contained columnar cells and only 1 of 200 control samples contained metaplastic cells. The cellular findings in the offspring of women exposed to DES in utero varied in relation to age. Cellular abnormalities were demonstrated in 90.4% of women having scrapings of visible lesions and in 88.1% of women studied by 4-quadrant vaginal scrapings in the absence of clinical disease. Of 58 women with histopathologically proven adenosis, 57 (98.3%) had similar cellular abnormalities. Abnormalities were demonstrated in squamous epithelial cells of the vagina in 8 (3.9%) of the women exposed to DES in utero, and in 5 of these women there were similar changes in the uterine cervix. Only 2 (0.9%) of the women had cellular changes suggesting dysplasia of the vagina.

Cytomegalovirus and Chlamydia trachomatis are prevalent sexually transmissible pathogens. They produce persistent infections of the cervix and have been associated with cervical neoplasia. Cytomegalovirus has also been shown to induce transformation of cells in culture. Because of the high prevalence of genital infections with these pathogens and evidence that they may have oncogenic effects on the cervix, cytomegalovirus (strain AD-169) and C. trachomatis (serovar LGV-2) were tested for oncogenicity in a mouse model in which induction of cervical neoplasia by repeated exposure to inactivated herpes simplex viruses has been demonstrated previously. Cotton tampons, saturated with UV-inactivated cytomegalovirus, C. trachomatis, or corresponding control fluids, were inserted into the vaginas of virgin C57 mice 3 times a week. Smears of vaginal aspirates for cytological examination were obtained every 5 weeks. After 75-90 weeks of exposure, the mice were sacrificed and serial sections of their reproductive tracts were examined. Cervical dysplasia was detected by histological examination in 51% and cervical carcinoma in 10% of mice exposed to cytomegalovirus. In control mice, in contrast, dysplasia developed in 3% and carcinoma in none. The progression from normal cervical epithelium to dysplasia to carcinoma observed with cytomegalovirus exposure was similar to that observed previously in this model after exposure of mice to herpes simplex virus types 1 and 2. The frequencies of cervical abnormalities in mice exposed to C. trachomatis or corresponding control fluid were low, and differences between the two groups were not statistically significant. These data indicate that strain AD-169 of cytomegalovirus is oncogenic for the mouse cervix and suggest that the LGV-2 serovar of C. trachomatis is not.