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Background:. Nipple-sparing mastectomy (NSM) preserves the natural nipple–areola complex and entire native breast skin, with the goal of better cosmetic outcomes in breast reconstruction. In bilateral TE/implant-based reconstruction requiring unilateral postmastectomy radiotherapy (PMRT), progressive radiation-induced fibrosis can lead to increasing nipple asymmetry with cosmetic dissatisfaction. Thus, PMRT may ultimately negate the intended positive cosmetic value of NSM compared with skin-sparing mastectomy (SSM). This study compares (1) surgical complications, (2) patient satisfaction, and (3) aesthetic outcomes between NSM versus SSM in bilateral implant-based reconstruction with unilateral PMRT. Methods:. This retrospective matched cohort study included consecutive NSM patients with bilateral TE/implant breast reconstruction + unilateral PMRT matched 1:2 to SSM group. Patients completed PMRT and TE exchange to implants. Demographics, oncologic stage, comorbidities, and complications were collected. Patient satisfaction was evaluated by BREAST-Q. Aesthetic outcomes were assessed by blinded reviewers with a five-point Likert scale. Results:. Among 58 patients who underwent bilateral TE/implant reconstruction with unilateral PMRT, 17 NSM patients were matched to 41 SSM patients by age, body mass index, and comorbidities. No significant differences existed in overall surgical complications and individual BREAST-Q questionnaire scores between cohorts. However, aesthetic outcomes scores were higher in SSM compared with NSM. Conclusions:. Although NSM is generally associated with superior cosmetic outcomes compared with SSM, it has far less impact in bilateral implant-based breast reconstruction with unilateral PMRT due to the negative postradiotherapy effect on nipple symmetry.

Diabetes is a global public health problem. Maintaining optimal glycemic control is critical for minimizing associated long-term complications and achieving better quality of life. Effective diabetes self-management education is one key component to enhance diabetes clients' self-management capabilities. The research team established a "Caring for Yourself-Managing Your Diabetes" Web site, which contained 35 video clips about diabetes management. The aim of this study was to evaluate user satisfaction with the Web-based diabetes self-management education program. A convenience sample of 100 diabetes clients (mean age, 61.5 [SD, 10.7] years) was invited to view one of the video clips via a laptop computer. A modified version of the Computer-Aided Learning Evaluation Questionnaire and the End-User Computing Satisfaction Questionnaire was used to evaluate participants' satisfaction with the program. The results indicate that participants were satisfied with the format, content, and accuracy of the Web-based diabetes education program. Some participants suggested adding different types of exercises that are specific to the needs of client groups and more explanation of diabetes medications. The results of this study support the use of computer-assisted learning as a promising method for delivering diabetes self-management education, which is satisfactory to diabetes clients.

Aims: To assess the potential value of chromosome in situ hybridisation (CISH), Ki-67, and telomerase immunocytochemistry in liquid based cervical cytology to help detect carcinoma cells and precursors., Method: Sixty ThinPrep processed cervical cytology samples were studied: 23 cases within the normal limit, 13 low grade squamous intraepithelial lesions (LSILs), 10 high grade squamous intraepithelial lesions (HSILs), six squamous cell carcinomas, three endocervical adenocarcinomas, two cervical adenosquamous cell carcinomas, and three endometrial adenocarcinomas. CISH was performed with DNA probes specific for the pericentromeric regions of chromosome 11 and 16. Hybridisation signals were visualised with the streptavidin-biotin peroxidase technique. The monoclonal MIB1 and polyclonal TRT-H231 antibodies were used to detect Ki-67 and telomerase immunoreactivity, respectively., Results: Non-specific background staining was almost absent in CISH slides. Normal squamous and glandular cells showed a diploid chromosomal pattern. A relative gain in chromosomes 11 and 16 (aneusomy) was seen in HSIL and the carcinomas (p<0.0001). In MIB1 stained smears, normal cells and koilocytes showed inconspicuous immunoreactivity, whereas strongly immunoreactive nuclei were found in cancer cells and HSIL (p<0.0001). Not only carcinoma and HSIL cells, but also some normal cells, showed cytoplasmic staining for telomerase., Conclusions: These preliminary results indicate that ThinPrep processed cervical smears are suitable for CISH and immunocytochemical studies. The neoplastic squamous and glandular cells were easily identified based on nuclear aneusomy and strong Ki-67 immuoreactivity in the context of abnormal nuclear morphology. This is the first study to apply CISH in cervical cytology using an immunoenzymatic approach.

This paper proposes a qualitative assessment methodology that is comprised of a cluster analysis and an autoregression analysis that assess the effects of various road safety strategies implemented in Hong Kong over the last 10 years. The cluster analysis is first used to group over a hundred road safety projects and programs into a smaller set of meaningful road safety policy strategy clusters. These strategies, together with the trend factor, seasonal pattern, car crashworthiness and meteorological data are then used in the autoregression analysis to relate to the fatality and casualty rates of drivers, passengers, motorcyclists, and pedestrians. This method allows the evaluation of the overall effects of the road safety strategies, and the effects and relative significance of each individual strategy. The evaluation method is described, and the main findings of the study are discussed.

Background: This study compared the findings of cervical cytology screening by ThinPrep Papanicolaou (Pap) tests (TP) with the findings of screening by conventional cervical smears (CS) in a screening population involving what to the authors' knowledge is the largest sample of ThinPrep Pap tests published to date., Method: Data from 191,581 CS that were screened in the period from March 1, 1998 to February 28, 2000 were compared with data from 190,667 TP performed from March 1, 2000 to February 28, 2002 and that were obtained from the same sources., Results: With TP, the unsatisfactory rate was reduced from 0.48% to 0.32%. Fewer cases were considered to be suboptimal (19.12% vs. 12.97%). The detection rates of squamous cell carcinomas, adenocarcinomas, and high-grade squamous intraepithelial lesions (HSIL) were essentially unchanged in the TP group (0.005%, 0.003%, and 0.25%, respectively) compared with the rates for the CS group (0.01%, 0.006%, and 0.25%, respectively). There was an increase in the detection of atypical squamous cells of undetermined significance (ASCUS; 3.74% vs. 3.19%) and low-grade SIL (LSIL; 1.67% vs. 1.01%) with a decrease in the ASCUS-to-LSIL ratio from 3.15 for CS to 2.33 for TP. The detection of atypical glandular cells of undetermined significance (AGUS) increased slightly from 0.07% to 0.09%. The proportion of cells reported to have reactive atypia dropped from 2.71% to 1.48%. Limited biopsy correlation (range, 73.2-76.2%) confirmed the increased sensitivity of TP. More actinomyces (1.07% vs. 0.52%) were detected in TP samples despite of a similar portion of intrauterine-device users. The average primary screening and rapid rescreening time of each slide were reduced from 8 minutes to 4 minutes and from 2 minutes to less than 1 minute, respectively., Conclusions: The preliminary experience of the authors of the current study appears to support the use of the ThinPrep Pap test to enhance the efficiency of cervical cytology screening., (Copyright 2003 American Cancer Society.)

Objectives: The aim of this study was to determine cervical cancer cell sensitivity to chemotherapy-induced apoptosis based on human papillomavirus (HPV) status., Methods: CaSki (HPV-positive) and C33A (HPV-negative) cells were treated with camptothecin or cisplatin. Cellular viability was determined by trypan blue exclusion. Apoptotic indexes were determined by flow cytometric analysis of annexin V labeling and morphological changes. Mitochondrial release of cytochrome c was determined by immunofluorescence using confocal microscopy. Caspase 3 activation and bax expression were assessed by immunoblotting., Results: CaSki cells displayed chemoresistance to both camptothecin and cisplatin. Low response to apoptogenic stimuli was evidenced by a marginal increase in the apoptotic cell fraction after camptothecin treatment (22.9 +/- 2.56%) compared with control (17.8 +/- 1.95%). Cisplatin (14.8 +/- 1.01%) caused a slight decrease in apoptosis. Mitochondrial release of cytochrome c and cleavage of caspase 3 could not be demonstrated in CaSki cells after treatment. Despite p53 mutation, C33A cells were sensitive to the antiproliferative effects of camptothecin and cisplatin. Mean apoptotic events were 17.5 +/- 0.33 for control, 42 +/- 1.76 for cisplatin, and 38.1 +/- 0.75 for camptothecin. An intact cytochrome c pathway was demonstrated in C33A cells leading to cleavage of caspase 3 after camptothecin treatment. The constitutive bax expression demonstrated in both cell lines displayed no change after camptothecin treatment., Conclusion: HPV-positive cervical cancer cells have an inherent resistance to chemotherapy-induced apoptosis. HPV-dependant inactivation of apoptotic regulators such as p53 and blockage of downstream events such as cytochrome c release and caspase 3 activation might be elemental to this cellular survival advantage provided by high-risk oncogenic papillomavirus.

Our objective is to test the hypothesis that inhibition of mitogen-activated protein (MAP) kinase kinase (MEK) with PD98059 in human luteinized granulosa cells will block epidermal growth (EGF)-stimulated MAP kinase activity and induce apoptosis. Luteinized granulosa cells from human in vitro fertilization aspirates were cultured and treated with the following: (1) vehicle; (2) PD98059; (3) EGF; (4) PD98059 + EGF. Treatment with PD98059 suppressed MAP kinase activity, inhibited MAP kinase phosphorylation by Western blot analysis, blocked nuclear translocation of phosphorylated MAP kinase by confocal microscopy, and increased percentages of subdiploid apoptotic nuclei by flow cytometry. Our data are the first evidence that a relationship may exist between the MAP kinase pathway and control of apoptosis in human luteinized granulosa cells. These results support the hypothesis that suppression of the MAP kinase pathway may lead to apoptosis in these cells., (Copyright 1999 Academic Press.)

Objective: To assess the expression of messenger RNA (mRNA) for estrogen receptors alpha and beta in the vaginal walls and uterosacral ligaments of premenopausal and postmenopausal women., Design: In vitro experiment., Setting: Academic research environment., Patient(s): Sixteen consecutively seen women who underwent hysterectomy., Intervention(s): Samples of anterior vaginal walls and uterosacral ligaments were obtained during hysterectomy., Main Outcome Measure(s): Reverse transcriptase-polymerase chain reaction analysis for estrogen receptor mRNA., Result(s): Messenger RNA transcripts for estrogen receptor alpha were present in all samples of vaginal walls (16/16) and uterosacral ligaments (16/16). Estrogen receptor beta mRNA was detected in all samples of vaginal walls from premenopausal women (12/12) but in none of those from postmenopausal women (0/4). Estrogen receptor beta mRNA was found in most samples of uterosacral ligaments from premenopausal women (10/12) and in some of those from postmenopausal women (2/4)., Conclusion(s): Estrogen receptors alpha and beta were expressed in the vaginal walls and uterosacral ligaments of premenopausal and postmenopausal women. Estrogen receptor beta was absent from the vaginal walls of postmenopausal women.

There is growing evidence that various isoforms of 17 beta-hydroxysteroid dehydrogenase (17-HSD) are regulated at the level of catalysis in intact cells. A number of investigators have proposed that the NAD(P)/NAD(P)H ratio may control the direction of reaction. In a previous study, we obtained evidence that A431 cells, derived from an epidermoid carcinoma of the vulva, are enriched in 17-HSD type 2, a membrane-bound isoform reactive with C18 and C19 17 beta-hydroxysteroids and 17-ketosteroids. The present investigation was undertaken to confirm the presence of 17-HSD type 2 in A431 cells and to assess intracellular regulation of 17-HSD at the level of catalysis by comparing the activity of homogenates and microsomes with that of cell monolayers. Northern blot analysis confirmed the presence of 17-HSD type 2 mRNA. Exposure of cells to epidermal growth factor resulted in an increase in type 2 mRNA and, for microsomes, increases in maximum velocity (Vmax) with no change in Michaelis constant (Km) for testosterone and androstenedione, resulting in equivalent increases in the Vmax/Km ratio consistent with the presence of a single enzyme. Initial velocity data and inhibition patterns were consistent with a highly ordered reaction sequence in vitro in which testosterone and androstenedione bind only to either an enzyme-NAD or an enzyme-NADH complex respectively. Microsomal dehydrogenase activity with testosterone was 2- to 3-fold higher than reductase activity with androstenedione. In contrast, although cell monolayers rapidly converted testosterone to androstenedione, reductase activity with androstenedione or dehydroepiandrosterone (DHEA) was barely detectable. lactate but not glucose, pyruvate or isocitrate stimulated the conversion of androstenedione to testosterone by monolayers, suggesting that cytoplasmic NADH may be the cofactor for 17-HSD type 2 reductase activity with androstenedione. However, exposure to lactate did not result in a significant change in the NAD/NADH ratio of cell monolayers. It appears that within A431 cells 17-HSD type 2 is regulated at the level of catalysis to function almost exclusively as a dehydrogenase. These findings give further support to the concept that 17-HSD type 2 functions in vivo principally as a dehydrogenase and that its role as a reductase in testosterone formation by either the delta 4 or delta 5 pathway is limited.

Although there is a growing body of evidence that 17 beta-hydroxysteroid oxidoreductase plays a role in the regulation of steroid levels in epithelial tumors of the endometrium and breast, our knowledge of its role in other gynecologic tumors is limited. In this investigation, the 17 beta-hydroxysteroid oxidoreductase activity of cell lines derived from two ovarian tumors (OVCAR-3, CAOV-3) and an epidermoid tumor of the vulva (A431) was assayed under conditions which differentiate between 17 beta-hydroxysteroid oxidoreductase type 1, a cytosolic isoform highly specific for estradiol, and type 2, a membrane bound isoform reactive with both estradiol and testosterone. On the basis of estradiol/testosterone activity ratios, all three cell lines appear to have type 2-like activity, with the specific activity of A431 markedly greater than that of the other cell lines. Estradiol, progesterone, or EGF, alone or in combination, were without effect on the enzymatic activity of OVCAR-3 cells. EGF decreased the activity of CAOV-3 cells slightly. In contrast, EGF stimulated A431 17 beta-hydroxysteroid oxidoreductase activity 7-8-fold over a 5-day exposure. Estradiol or progesterone, singly or in combination, also did not effect the enzymatic activity of A431 cells. However, progesterone inhibited the increase in activity seen in the presence of EGF. With EGF, estradiol, and progesterone together, the increase in enzymatic activity was comparable to that with EGF alone. The effects of estradiol and progesterone appear to result from steroid actions following binding of EGF to low-affinity receptors on A431 cells.

Tamoxifen has been an effective antiestrogen in suppressing breast cancer growth which is estrogen-responsive or dependent. Early studies have provided circumstantial evidence that transforming growth factor-beta (TGF-beta) may be an autocrine mediator of tamoxifen action. Therefore, it is both fundamentally important and clinically relevant to investigate the relationship between tamoxifen and TGF-beta. In this study, we demonstrated that CAMA-1 cells, which are sensitive to tamoxifen inhibition, did not respond to TGF-beta growth inhibition. The type I and II TGF-beta receptors were undetectable by the radio-ligand affinity labeling technique. Despite the presence of a normal TGF-beta type II receptor gene, the mRNA transcript of the gene was undetectable by the extremely sensitive Intron-differential RNA/PCR method. The possibility that the lack of TGF-beta receptors might be intimately linked to the absence of normal retinoblastoma (Rb) gene products, as suggested by previous studies of retinoblastoma cells, was further investigated. The lack of TGF-beta receptor expression was found due to reasons other than the absence, deletion or abnormality of the Rb gene because a normal Rb gene and its hyper- and hypo-phosphorylated protein products were detected in CAMA-1 cells. In conclusion, our results suggest that the TGF-beta system is not obligatory for antiestrogen growth inhibition of CAMA-1 cells.

Regulation of ovarian cancer growth is poorly understood. In this study, the effects of EGF, TGF alpha and TGF beta 1 on two ovarian cancer cell lines (OVCAR-3 and CAOV-3) were investigated. The results showed that EGF/TGF alpha stimulated cell growth and DNA synthesis in OVCAR-3 cells, but inhibited cell proliferation and DNA synthesis in CAOV-3 cells. TGF beta 1 invariably inhibited cell proliferation and DNA synthesis in both cell lines. These effects on growth factors are dose dependent. The interaction of TGF beta 1 and EGF/TGF alpha was antagonistic in OVCAR-3 cells. In contrast, EGF/TGF alpha and TGF beta 1 had an additive inhibitory effect on CAOV-3 cells. Our results demonstrated that mature and functional EGF receptors are present in both cell lines and that they are capable of ligand binding, internalization, processing and ligand-enhanced autophosphorylation. Both high- and low-affinity binding are present in these cell lines, with CAOV-3 cells having about 2-3-fold higher total receptors than OVCAR-3 cells. These results together with those from our previous studies show that these cells express TGF alpha, TGF beta 1 and EGF receptors and that cell growth may be modulated by these growth factors in an autocrine and paracrine manner. This report presents evidence supporting the important roles of growth factors in ovarian cancer growth and provides a foundation for further study into the mechanism of growth regulation by growth factors in these cell lines.

Induction of indoleamine-2,3-dioxygenase (IDO) by interferon-gamma (IFN-gamma) is thought to be one mechanism underlying IFN-gamma's antineoplastic properties. Since clinical trials with IFN-gamma have yielded variable efficacy in treating cancers of gynecological origin, we tested the effects of IFN-gamma on cell growth and IDO activity in cell lines from seven gynecologic and five breast cancers. At a dose of 250 IU/ml, IFN-gamma suppressed cell growth and induced IDO activity in one cervical (C41), one vulva (A431), one breast (HS578T) and two ovarian (OVCAR-3, CAOV-3) cancer cell lines. Differing inhibition of cell growth, but with no induction of IDO activity, was found with IFN-gamma treatment of the other cell lines.

To elucidate the relationship between epidermal growth factor (EGF)/transforming growth factor (TGF-alpha) and estradiol-17 beta (E) in cell proliferation, we examined their effects on the breast cancer cell line, CAMA-1. While E was able to consistently induce cell proliferation under a variety of experimental conditions, EGF/TGF-alpha was without effect. Despite the presence of the receptor (EGFR) gene, mature EGFR protein and mRNA were not detected by radioreceptor assay, 35S Met-labelling, and the Intron Differential RNA/PCR method under conditions in which cells remain responsive to E. Furthermore, TGF-alpha is not an autocrine factor in CAMA-1 cells. We demonstrated unequivocally that EGF/TGF-alpha interaction with EGFR is not an obligatory event in mediating estrogen-stimulated cell proliferation.

Objective: Immature mammalian oocytes cultured in vitro undergo inadequate cytoplasmic maturation and hence have a limited potential for fertilization. Our primary objective was to determine if the addition of epidermal growth factor (EGF) to the in vitro culture system would have a positive effect on oocyte cytoplasmic maturation., Design: We studied the effect of different EGF concentrations on both denuded and cumulus-enclosed mouse oocytes cultured in vitro., Main Outcome Measures: The percentage of oocytes undergoing germinal vesicle breakdown (GVBD) and polar body one formation over time as a function of EGF concentration was determined., Results: A dose-related positive effect of EGF on both GVBD and polar body one formation over time was observed for mouse oocytes. As well, a similar effect of EGF was seen on immature human oocytes that had not been stimulated with exogenous gonadotropins., Conclusions: The use of EGF may allow for the performance of successful in vitro fertilization procedures using immature human oocytes retrieved during unstimulated cycles.

To elucidate the possible roles of proto-oncogenes and growth factors in estrogen-regulated cell proliferation of human breast and gynecologic cancers, we have determined the gene expressions of c-myc, transforming growth factor-alpha and beta 1 (TGF-alpha, beta 1) and epidermal growth factor receptor (EGFR) in a number of these cancer cell lines by using an intron-Differential (ID) RNA/PCR method, which differentially identifies the amplified cDNA from PCR products of genomic DNA contaminants. With this method, we demonstrated the expression of these genes, except EGFR, in an estrogen-dependent breast cancer cell line (CAMA-1). Our results show that TGF-alpha/EGF does not function as an autocrine factor in this cell line. Accordingly, it is unlikely that the TGF-alpha/EGFR system participates as a mediator in the estrogen-induced cell proliferation of CAMA-1 cells. The ID RNA/PCR method is a rapid, sensitive and specific technique for mRNA phenotyping and will have great clinical utility.

A retrospective study of 1,855 fine-needle aspirations of the lymph node (LN) from 1,484 Chinese patients in Queen Mary Hospital over a period of 11.5 yr (January 1, 1976, to June 30, 1987) was reviewed and analyzed. Positive cytologic results diagnosed malignancies in 49.3%. Sensitivity and specificity of cytology reached 95% and 96.5%, respectively. The order of frequency of the metastatic tumors according to primary sites was analyzed. The most common metastatic carcinomas in various groups of LNs were nasopharyngeal carcinoma in the cervical LN, breast carcinoma in the axillary LN, and cervical carcinoma in both the groin and pelvic LNs. Tuberculosis (TB) is an important differential diagnosis; FNAC showed a sensitivity of 76.9% in the detection of TB lymphadenopathy.

Studies on the mechanism of activation of estrogen receptor (ER) have led us to identify two classes of receptors. Effect of molybdate on the activation of ER was determined by their affinity for nuclei or DNA-cellulose. Molybdate caused inhibition of nuclear binding with rat uterine ER, but an increase in the binding was observed with rabbit uterine ER. Similar results were observed when DNA-cellulose was used instead of nuclei. To further test the diversity in ER activation, DNA-cellulose binding of ER from 10 primary breast cancer tissues was determined in the presence or absence of molybdate. ER from 8 tissues showed inhibition of binding, one showed an increase, and one was not affected by molybdate. These results indicate the presence of two classes of ER, one whose activation is inhibited by molybdate and another whose activation is either unaffected or stimulated by molybdate. Other differences between rat and rabbit uterine ER are also discussed.

Growth factors (GFs) and growth inhibitors (GIs) are important regulators of cell proliferation and differentiation in many cell types. Insulin growth factor I and II, epidermal growth factor, transforming growth factor alpha and transforming growth factor beta are produced and secreted by various fetal tissues, and their cognate membrane receptors are present in the fetal-placental unit. These findings suggest that fetal growth and development are regulated by these or other GFs and GIs in an autocrine or paracrine manner. A well-coordinated and integrated action of several GFs/GIs may be operating in promoting the growth and development of different fetal organs during the various stages of gestation. An interruption of the coordinated pathway due to an abnormal expression of GFs/GIs, a structural aberration of their receptors, or an impairment in the postreceptor signaling mechanism may contribute to abnormal fetal growth. The investigation of the unique role of a single GF/GI, the integrated action of several of them in fetal growth, and the points of abnormalities in this mechanism in abnormal fetal growth have both fundamental and clinical relevance.

CAMA-1 cells, isolated from malignant pleural effusion, are grown in long-term cultures as monolayers. The rate of growth is dependent upon fetal bovine serum and estradiol. These cells also exhibit a dose response to added 17 beta-estradiol with respect to the incorporation of radiolabeled thymidine into cells. The uptake is increased at low levels of estradiol and decreased at pharmacological levels of estradiol. The uptake of uridine and leucine is also stimulated by estradiol in a dose-related manner. Induction of precursor uptake is not observable until cells have been exposed to estradiol for approximately 10 hr or longer. Cells plated for different periods in steroid-stripped serum remain sensitive to estrogenic stimulation and show similar lag time in response. Estrogenic effect is not noticeable in the absence of serum. Addition of prolactin can partially restore estrogenic stimulation of thymidine uptake in serum-free medium. Like other estrogen target tissues, these cells contain cytoplasmic and nuclear estrogen receptors. These results demonstrate that the CAMA-1 cell line is estrogen dependent and that these cells may provide a promising model for the in vitro investigation of the mode of estrogen action in human breast cancer.

The mammary cancer cell line CAMA-1 synchronized at the G1/S boundary by thymidine block or at the G1/M boundary by nocodazole was used to evaluate 1) the sensitivity of a specific cell cycle phase or phases to 17 beta-estradiol (E2), 2) the effect of E2 on cell cycle kinetics, and 3) the resultant E2 effect on cell proliferation. In synchronized G1/S cells, E2-induced 3H-thymidine uptake, which indicated a newly formed S population, was observed only when E2 was added during, but not after, thymidine synchronization. Synchronized G2/M cells, enriched by Percoll gradient centrifugation to approximately 90% mitotic cells, responded to E2 added immediately following selection; the total E2-treated population traversed the cycle faster and reached S phase approximately 4 hr earlier than cells not exposed to E2. When E2 was added during the last hour of synchronization (ie, at late G2 or G2/M), or for 1 hr during mitotic cell enrichment, a mixed response occurred: a small portion had an accelerated G1 exit, while the majority of cells behaved the same as controls not incubated with E2. When E2 addition was delayed until 2 hr, 7 hr, or 12 hr following cell selection, to allow many early G1 phase cells to miss E2 exposure, the response to E2 was again mixed. When E2 was added during the 16 hr of nocodazole synchronization, when cells were largely at S or possibly at early G2, it inhibited entry into S phase. The E2-induced increase or decrease of S phase cells in the nocodazole experiments also showed corresponding changes in mitotic index and cell number. These results showed that the early G1 phase and possibly the G2/M phase are sensitive to E2 stimulation, late G1, G1/S, or G2 are refractory; the E2 stimualtion of cell proliferation is due primarily to an increased proportion of G1 cells that traverse the cell cycle and a shortened G1 period, E2 does not facilitate faster cell division; and estrogen-induced cell proliferation or G1/S transition occurs only when very early G1 phase cells are exposed to estrogen. These results are consistent with the constant transition probability hypothesis, that is, E2 alters the probability of cells entering into DNA synthesis without significantly affecting the duration of other cell cycle phases. Results from this study provide new information for further studies aimed at elucidating E2-modulated G1 events related to tumor growth.

In order to test the in vivo effect of prolactin on estrogen receptor (ER) binding capacity in tumors induced by 7,12 dimethylbenz(a)anthrancene (DMBA-tumor), growth of the tumors from changes in prolactin and estrogen levels was compared retrospectively with cytoplasmic ER levels. It was demonstrated that some tumors required prolactin, some needed prolactin-estrogen during their growth period anda small number were not influenced by hormonal milieu. ER was present in hormonally dependent tumors but was low or absent in hormonaly-independent tumors. Deletion of hormones by endocrine ablation in the host rat resulted in tumor regression loss of ER. Replenishment of ER and subsequent tumor growth were accomplished by injection of prolactin or prolactin-estrogen in endocrine ablated rats but were not achieved in rats bearing tumors exposed to prolactin-nafoxidine. Our results demonstrate that both estrogen and prolactin were essential for growth of hormonally dependent DMBA-tumors. Tumor growth was also prevented when cytoplasmic ER was not replenished , indicating that ER may be an indispensable prerequisite for growth. Prolactin, independently of or cooperatively with estrogen, stimulated ER binding capacity. These results support the hypothesis that there may exist a prolactin regulatory mechanism of estrogen action at the tumor site. The interactions of estrogen and prolactin in situ in modulating hormonal receptor binding capacities may contribute to the overall stimulatory effect of these two hormones on DMBA-tumors.

Tamoxifen (NSC-180973), a synthetic antiestrogen, was studied for efficacy and toxicity in patients with metastatic breast adenocarcinoma. Two dose levels were used, 10 mg bid and 15 mg/m2 bid, in separate groups. In the 10-mg bid dosage group, 30 of the 31 patients were considered evaluable for efficacy. Five complete and 11 partial responses were recorded, for an overall response rate of 53%. In the 15-mg/m2 bid dosage group, 44 of the 45 patients were considered evaluable for efficacy. Three complete and 16 partial responses were recorded, for an overall response rate of 43%. All 76 patients were evaluated for toxicity. Side effects were generally mild, consisting mostly of hot flushes, transient leukopenia, transient thrombocytopenia, nausea, and fluid retention. A high degree of correlation between response and positive estrogen-receptor assay suggests the value of the test as a means to select patients for tamoxifen treatment. The conclusion from this study is that tamoxifen used as a single agent is an effective drug with minimal toxicity for treatment of metastatic breast adenocarcinoma.

The cytoplasmic estrogen receptor (ERc) and progesterone receptor (PRc) in mammary tumors have been recognized as useful biochemical markers for predicting the objective response of patients with advanced breast cancers to endocrine therapy. These proteins are also useful in the prognosis of gynecologic carcinoma. This report presents data showing the effect of sodium molybdate in the stabilization of estrogen and progesterone receptors. In rabbit uterine tissue, molybdate (20 mM) increased the binding of progesterone and estrogen to the receptors in several ways: (a) the apparent loss of detectable receptors during lengthy sucrose gradient analysis and at elevated temperature (30 degrees C) was reduced; (b) the instability of receptors due to storage at -70 degrees C was lessened, and (c) the conversion of the 7S PRc to the 3.5S form was minimized. Similarly, molybdate caused a qualitative and/or a statistically significant quantitative difference in receptor values for some human gynecologic tumors presented herein; the molybdate-associated changes vary with tumor specimen. Of the 8 tumors for which receptor values in the presence of molybdate (M+) and its absence (M-) can be compared, detectable ERc of 6 and PRc of 7 tumors increased with molybdate, and ERc of 2 and PRc of 1 tumor showed no change. In addition to the increase in receptor values, a concomitant shift of the 3-4S molecules to the 7-8S moieties was noted for some tumors (1 of 6 for ERc and 3 of 7 for PRc). In 2 receptor-poor tumor samples, ERc was only detected in M+ cytosols. These results show that molybdate is effective in reducing receptor degradation and stabilizes the 7-8S molecules from converting to 4S moieties. The addition of molybdate may be helpful for better quantitation of steroid receptors in clinical specimens.

Hormonal influences on dimethylbenz(a)anthracene-induced tumor growth were investigated in detail by endocrine ablation and replacement of hormones. The majority of tumors regressed following ablation and most of them were reactivated by subsequent administrations of estrogen (0.1 to 5 mug) or prolactin (2 mg). Increasing numbers of tumors, however, were not stimulated by prolactin when administration was delayed, and a basal level of estradiol (0.01 mug) in addition to prolactin was required for reactivation of tumors. Nafoxidine hydrochloride, a competitor of estrogen at the receptor sites, arrested growth of a large portion of dimethylbenz(a)anthracene-induced tumors in intact animals but failed to retard growth of prolactin-stimulated tumors. On withdrawal of prolactin-nafoxidine, rapid regression of tumor occurred and readministration of prolactin failed to activate most of the tumors for as long as 28 days. Our results give good supporting evidence that estrogen plays a primary role in tumor growth. The interactions of prolactin and estrogen at tumor sites are necessary for regulatory events related to tumor growth.

The cytoplasmic content of estrogen and progesterone receptors (ERc and PRc) was measured in the rat uterine and mammary tissues during the period from late pregnancy through lactation. Uterine ERc and PRc, which sedimented at the 8S and 4S regions, respectively, of a 5-20% sucrose gradient, showed similar rise and decline patterns before and after parturition. Following a decline from the 15th day (114 +/- 13.7 fmol/mg protein) to the 20th day of pregnancy (57.7 +/- 10.2 fmol/mg protein), ERc levels increased almost 2.5-fold at the time of parturition. A decrease to the predelivery level by 24 h post-delivery was followed by a steady increase to 270.6 +/- 46 fmol/mg protein by 15 days of lactation. Uterine PRc levels also showed a peak similar to that of ERc at the time of parturition (24.3 +/- 1.1 fmol/mg protein). The level of ERc in mammary gland 6 h postpartum was the lowest (1.5 +/- 0.3 fmol/mg protein) and during the first 10 days of lactation ERc levels were maintained at about 10 fmol per mg of cytosol protein. Results herein suggest that levels of these steroid receptors may be hormonally regulated and that the uterus and mammary gland may have a different sensitivity to these regulatory control mechanisms. The physiological significance in the variation of these receptors in conjunction with the onset of parturition is discussed.

In order to study whether cytosolic progesterone receptor (PRc) is involved in progesterone-induced proliferative arrest of breast cancer cells (CAMA-1N), subunit composition of PRc in these cells was characterized. In the TSK G3000 high-performance liquid chromatography (HPLC) column, molybdate-stabilized PRc eluted as a single sharp peak immediately following the void volume. PRc from this peak had a sedimentation coefficient of 7.6 S and was eluted at 0.22 M salt from a linear gradient (0.1 to 0.4 M NaCl) in a DEAE column. When covalently labeled with [3H]R5020, analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and visualized by fluorography, PRc from crude cytosol exhibited two major protein bands with molecular weights of 105 +/- 2.1 and 83 +/- 3.2 kD, respectively. However, HPLC-purified PRc showed only one major band of 105 kD. These results show that PRc in CAMA-1N cells has steroid-binding components similar to other target tissues and provides a basis for further quantitative and qualitative analyses of PRc during cell proliferation and progesterone-induced growth arrest of CAMA cells.

In an attempt to differentiate between the biochemical characteristics of hormonal-dependent and independent gynecologic tumors, monoclonal antibodies (mABs) MBr1 and MBr3 were used to detect tumor antigens. Steroid receptors and MBr1 and MBr3 determinants were assayed independently in a double-blinded manner. Of the 45 gynecologic tumors analyzed, 16 of 20 (80%) with both progesterone receptors (PR) (greater than 10 fm/mg protein) and estrogen receptors (ER) (greater than 5 fm/mg protein) also contained high levels of MBr1 determinant. However, these receptors were not quantitatively related to MBr1 activity. The 15 of 17 (88%) tumors that contained receptors below the discriminant levels also had low or undetectable levels of MBr1 activity. In tumors where only either PR or ER was present, 2 of 2 PR+/ER- tumors and 2 of 6 PR-/ER+ tumors expressed MBr1 antigen. No relationship of MBr3 antigenic activity to the receptors was established. These results show that MBr1-recognized antigen is expressed largely in steroid receptor-positive tumors and these data suggest that either the antigen itself or its carrier protein may be regulated by estrogen. Like steroid receptors, MBr1 antigens may have important prognostic value in gynecologic tumors.

Nafoxidine hydrochloride can provide additional palliation to patients who responded to previous endocrine ablation and had tumors containing an estrogen receptor. In the present study, 24 patients were evaluated for their response to nafoxidine therapy, 180 to 240 milligrams given orally per day. When tumor response was obtained, doses were reduced to 60 to 120 milligrams every other day or longer without loss of control. In the estrogen receptor positive group, eight of ten patients responded objectively to nafoxidine, while all seven estrogen receptor negative patients progressed with this therapy. These results demonstrated that nafoxidine is effective in the treatment of estrogen receptor positive cutaneous disease. These results demonstrated that nafoxidine is effective in the treatment of estrogen receptor positive cutaneous disease. Patients with lesions to other sites should receive cytoxic agents along with nafoxidine. Alternative therapy to nafoxidine should be considered for patients in whom the tumors contain negligible estrogen receptor.

The phospholipids from subcellular fractions of human intracranial tumors were examined. For comparison, microsomes were isolated from a fetal human brain and from the gray matter of adult human brains. The subcellular membranes of tumors had a higher protein-to-phospholipid ratio than the normal brain membranes. The microsomes from tumors had a lower proportion of diacylglycerophosphorylethanolamine and higher proportions of alkenylacylglycerophosphorylcholine and sphingomyelin (plus diacylglycerophosphorylinositol) than microsomes from the gray matter. Also, the ratios of alkenylacylglycerophosphorylethanolamine to diacylglycerophosphorylethanolamine were higher in the tumors than in the normal controls. The acyl groups of ethanolamine phosphoglycerides in tumor microsomes had relatively more 18:1, 18:2, and 20:4(n - 6) and less 18:0, 22:4(n - 6), and 22:6(n - 3) than the adult brain gray matter. Except for the increase in 18:2, acyl group changes in choline phosphoglycerides between tumors and controls were not as extensive as in the ethanolamine phosphoglycerides. The characteristic features of phospholipids and their constituent acyl groups of tumors were often present in all the subcellular fractions. Although the acyl group profiles of the tumor phosphoglycerides were in closer resemblance to the fetal brain than to the adult brain, other differences were observed. Results indicate that neoplastic brain cells are unique in their cellular composition, and consequently they deviate from the normal neurons and glials in metabolism and functions.

From 1977 to 1981, 39 cases of primary cervical carcinoma were assayed for the presence or absence of cytoplasmic estrogen (ERc) and progesterone (PRc) receptors by a saturation point dextran-coated charcoal assay. The levels of ERc and PRc were compared with clinical stage, histologic type, histologic grade, menstrual status, age, and survival. Carcinomas were divided into positive and negative receptor groups with the use of greater than 5 fmol/mg of cytosol protein for ERc and 10 fmol/mg of cytosol protein for PRc as discrimination points. Survival of the patients studied was computed by means of the product-limit analysis according to Kaplan-Meier. A statistically significant difference in survival in the PRc + group versus the PRc - group in the total group of patients was noted with the Mantel-Cox test (P = 0.049). When patients were stratified according to menopausal status, a statistically significant difference in survival was noted between the PRc + group versus the PRc - group in the premenopausal patients only.

Both serum and estrogen affect cell proliferation in CAMA-1 cells. Their effects on cell cycle kinetics are being investigated with partially synchronized cells following 48 hours of serum deprivation. By comparing the growth kinetics of synchronized (serum-deprived) cells with asynchronized (normal-fed) cells, we observed that there was a delay of cell proliferation for approximately 25 hr for synchronized cells and that estrogen only induced cell proliferation in serum-supplemented culture. A serum protein (RPF), precipitated by ammonium sulfate, dialyzed in 3,500 daltons cut off membrane and reconstituted in culture medium, was shown to stimulate cell proliferation in a dose-related manner. In the absence of RPF, estrogen had no effect on cell growth and S-phase formation in serum-free medium, but significantly induced cell growth in the presence of RPF. Experiments conducted with a synchronized cell population showed that estrogen increased the proportion of G1 phase cells to enter S phase, shortened the G1 phase duration, and ultimately increased the proportion of mitotic cells per cycle. Collectively, these results show that (a) estrogen effects of cell proliferation in vitro requires a serum component, a "G1/S-promoting factor" and (b) estrogen-induced tumor growth is a result of an accelerated rate of G1/S transition and an increased number of dividing cells per cycle. The knowledge of the interaction of estrogen with the G1/S promoting factor on cell growth is of both fundamental and therapeutic importance.

Cytosolic progesterone receptors (PRcs) from the reproductive tract of the female turkey were analyzed by high-performance liquid chromatography using a diethylaminoethyl (DEAE) ion-exchange column. PRcs from oviduct tissue of laying, incubating, photorefractory and short-day turkey hens were compared. In general, three types of PRcs were identified: Receptor I, a partially displaceable species that was eluted at a 0.13 M salt concentration; and Receptors II and III, which were two specific binding species eluting at 0.23 M and 0.26 M, respectively. In the subdivided tissue from the laying hen oviduct, Receptor I was the major PRc species of the isthmus and Receptor III was the only receptor present in the uterus. The infundibulum and magnum each contained a small amount of Receptor II and a substantial amount of Receptor III. The whole oviduct of incubating hens contained a greater proportion of Receptor I than Receptor II or III, and these last two receptor types were present in equal quantity. The whole oviduct of the short-day hens had an equal distribution of the three receptor types. In the presence of sodium molybdate, an inhibitor of phosphatase and protease, only one sharp Receptor II species was seen in the magnum and uterus of the laying hen oviduct and in the whole oviducts of incubating and short-day hens. The transformation of Receptor II to Receptor III in the absence of sodium molybdate was facilitated by the aging of cytosol at 0-4 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Two sublines, CAMA-1R and CAMA-1N, were derived from the original CAMA-1 cells after continuous passages in our laboratory. 1R cells were supplemented in culture with estradiol and retained the properties of the original cell line, whereas 1N cells, not supplemented with estradiol, had a partial loss of estrogenic responsiveness. The doubling times for 1R and 1N cells were 2 and 2.4, respectively. These two sublines showed different effects on thymidine uptake after estradiol stimulation. Cells of 1R had a 2-fold greater thymidine incorporation than did the control without estradiol. In contrast, no difference of thymidine uptake was demonstrated in cells of 1N in the presence or absence of estradiol. Both cell lines contained cytoplasmic progesterone receptors with a Kd of 0.56 nM and a sedimentation coefficient of 6 to 7S on a 5 to 20% sucrose gradient. The progesterone receptor level in 1N was about 8-fold higher than in 1R cells. Both sublines exhibited a dose-dependent increase in progesterone receptor levels in response to estradiol with the maximal stimulation (8-fold increase) reached by using 5 nmol of estradiol. The rate of increase for estradiol-induced progesterone receptor appears to be the same for both sublines; a liner increase was demonstrated between Days 3 and 7 following plating. Our results show that estrogen regulation of thymidine uptake and progesterone receptor induction may be mediated through two separate mechanisms. Thus, following a chronically deficient supply of estradiol to 1N cells, there is apparent loss of responsiveness to estradiol-induced thymidine uptake, but the ability of these cells to respond to estradiol induction of progesterone receptor was retained.

Cytosolic (ERc) and nuclear (ERn) estrogen receptors prepared from rat uteri were characterized by size-exclusion and ion-exchange HPLC. The oligomeric ERc eluted as a single, sharp peak near the exclusion volume of the gel column; ERn eluted as a broad peak. When salt-extracted ERn was partially purified sequentially by Sephadex G-200, DEAE-cellulose chromatography and polyacrylamide gel electrophoresis, the partially purified receptor moieties were not distinguishable by the sucrose gradient method, but showed characteristic retention times in the size-exclusion HPLC column. Further distinction in net surface charges was observed between ERc and ERn moieties by ion-exchange high-pressure liquid chromatography (HPLC). Molybdate-stabilized ERc was eluted as sharp peak at 0.27 M salt gradient. In contrast, fresh extracts of ERn emerged as a broad peak in the region of 0.1-0.2 M salt gradient. In the absence of molybdate, ERc dissociated into several 4-5 S molecules, which were well resolved in the DEAE column. This report, therefore, demonstrates the usefulness of size-exclusion and ion-exchange HPLC for steroid receptor analysis.

The ability of L-dopa to arrest pain can be used to predict objective response of skeletal disease to endocrine ablation or additive therapy. In the present study, 43 patients with painful skeletal metastases were evaluated for the relief of pain by L-dopa, given 250 mg to 500 mg orally every 4 hours for 4 days. Sixteen of the 25 responders to L-dopa had objective response to either previous or later hormonal therapy while all the 18 non-responders did not benefit from endocrine ablation. The results of L-dopa responses also correlated well to the presence of absence of cytoplasmic ER in tumor. These results demonstrate that both tests (L-dopa and ER) are reliable indicators, one complimenting the other, in prognosticating response to endocrine therapy and should be used prior to hormone treatment. Alternative therapy should be considered for patients who are non-responders to the L-dopa test and/or whose tumors contain negligible ER. The long term therapeutic value of L-Dopa, however, is limited.

Tissue levels of cytosolic estrogen receptors (ERc) and progesterone receptors (PRc) have been shown to have prognostic meaning in hormonally responsive cancers arising in the breast and endometrium. Although carcinomas of the uterine cervix rarely respond to hormonal manipulation, the normal cervical stroma and epithelia contain ERc and PRc. To study the prognostic value of these receptors, 65 cervical carcinomas for ERc and PRc were assayed before initiation of surgical or radiation therapy. In premenopausal women, logistic models demonstrated that clinical stage, surgical stage, and receptor level independently predict survival. If confirmed in further studies, ERc and PRc may prove to be useful prognostic factors in the management of cervical carcinomas.

Estrogen (E) stimulatory effects on mammary carcinoma are well documented; however, little is known of the precise mechanism regulating E-induced cell proliferation. This study attempts to investigate the in vitro effects of E and antiestrogen (AE) on promoting cell proliferation and the cell cycle kinetics of an experimental mammary carcinoma model, CAMA-1. Sublines IR and IN, which have been shown to respond to E somewhat differently in 3H-thymidine (3H-dThd) uptake and in the induction of progesterone receptor (Cancer Res 41: 5004-5009, 1981), were evaluated concurrently. The present report showed that E stimulated and AE, as shown by tamoxifen (TAM) and nafoxidine hydrochloride (NAF), inhibited cell growth and 3H-dThd uptake in a dose-related manner for both sublines supplemented with serum. In a chemically defined medium with 1% steroid-stripped serum, the E-stimulatory effect was nullified, and cells were more sensitive to AE. These results suggest that serum component(s) is (are) involved in AE and E action. The TAM inhibition could be partially reversed by E. A partially synchronized G1 population, arrested by serum deprivation for 48 hrs, responded promptly to E stimulation. While E promoted a faster G1 exit, faster for IN cells than for IR subline, AE inhibited cell progression at the G1 phase. Furthermore, E stimulated a higher proportion of S-phase formation during the first cycle of hormone treatment while AE inhibited this process. These results are consistent with the notion that TAM and E act on some common events at the G1 phase in cell proliferation. The net result of E stimulation was the promotion of new cell to traverse the cell cycle, the acceleration of G1 exit, and an increase in the proportion of S-phase and dividing cells per cycle. In contrast, AE inhibited the progression of cells at the G1 phase, resulting in a marked decrease of S-phase and dividing cells per cycle. Our results also demonstrate the importance of careful kinetic studies in evaluating the E and AE responses of mammary carcinoma cells in culture, and this is best conducted with synchronized populations.

Forty-two cases of recurrent and 14 cases of advanced clinical stage (III and IV) endometrial carcinoma are presented, in which progesterone and estrogen receptors from the metastatic sites were measured. Mean survival time (time from recurrence or, in advanced stages, from the time of diagnosis to death or last follow-up), mean total survival time (time from diagnosis to death or last follow-up), and mean time to recurrence (time from diagnosis of primary tumor to the time of recurrence) were positively correlated with positive progesterone and estrogen receptor status and with histologic grade of tumor. No correlation was found with age, clinical stage, depth of myometrial invasion, or site of metastasis. However, when multiple variables were considered with the Cox regression model, the combination most highly correlated with survival included progesterone receptor, grade of tumor, and site of metastasis (pelvis vs. other sites). All differences were statistically significant (p less than 0.05). We conclude that measurement of progesterone and estrogen receptors in metastatic or recurrent endometrial tumors may be used as an additional prognostic variable.