Hepatitis B and C in pregnancy: a review and recommendations for care.

Our objective was to provide a comprehensive review of the current knowledge regarding pregnancy and hepatitis B virus (HBV) or hepatitis C virus (HCV) infection as well as recent efforts to reduce the rate of mother-to-child transmission (MTCT). Maternal infection with either HBV or HCV has been linked to adverse pregnancy and birth outcomes, including MTCT. MTCT for HBV has been reduced to approximately 5% overall in countries including the US that have instituted postpartum neonatal HBV vaccination and immunoprophylaxis with hepatitis B immune globulin. However, the rate of transmission of HBV to newborns is nearly 30% when maternal HBV levels are greater than 200 000 IU ml(-1) (>6 log10 copies ml(-1)). For these patients, new guidelines from the European Association for the Study of the Liver (EASL) and the Asian Pacific Association for the Study of the Liver (APASL) indicate that, in addition to neonatal vaccination and immunoprophylaxis, treating with antiviral agents such as tenofovir disoproxil fumarate or telbivudine during pregnancy beginning at 32 weeks of gestation is safe and effective in preventing MTCT. In contrast to HBV, no therapeutic agents are yet available or recommended to further decrease the risk of MTCT of HCV, which remains 3 to 10%. HCV MTCT can be minimized by avoiding fetal scalp electrodes and birth trauma whenever possible. Young women with HCV should be referred for treatment post delivery, and neonates should be closely followed to rule out infection. New, better-tolerated treatment regimens for HCV are now available, which should improve outcomes for all infected individuals.

Blocking Epidermal Growth Factor Receptor Signaling in HTR-8/SVneo First Trimester Trophoblast Cells Results in Dephosphorylation of PKBα/AKT and Induces Apoptosis.

We identified a major peptide signaling target of EGF/EGFR pathway and explored the consequences of blocking or activating this pathway in the first trimester extravillous trophoblast cells, HTR-8/SVneo. A global analysis of protein phosphorylation was undertaken using novel technology (Kinexus Kinetworks) that utilizes SDS-polyacrylamide minigel electrophoresis and multi-lane immunoblotting to permit specific and semiquantitative detection of multiple phosphoproteins. Forty-seven protein phosphorylation sites were queried, and the results reported based on relative phosphorylation at each site. EGF- and Iressa-(gefitinib, ZD1839, an inhibitor of EGFR) treated HTR-8/SVneo cells were subjected to immunoblotting and flow cytometry to confirm the phosphoprotein screen and to assess the effects of EGF versus Iressa on cell cycle and apoptosis. EGFR mediates the phosphorylation of important signaling proteins, including PKBα/AKT. This pathway is likely to be central to EGFR-mediated trophoblast survival. Furthermore, EGF treatment induces proliferation and inhibits apoptosis, while Iressa induces apoptosis.

Molecular tools to reestablish progestin control of endometrial cancer cell proliferation.

OBJECTIVE: Endometrial cancers often arise in a setting of estrogen stimulation unopposed by the differentiating effects of progesterone. Our laboratory and others have previously shown that progesterone receptor down-regulation or perturbation of progesterone receptor isoform A or B expression is associated with the development of poorly differentiated endometrial cancers that are not growth inhibited by progestins. The purpose of these studies was to reestablish high progesterone receptor isoform A and B gene expressions in such endometrial cancer cells and to examine the effects of progestin treatment on cell growth and metastatic potential after this transformation. STUDY DESIGN: To induce high levels of expression of the progesterone receptor isoforms in KLE and Hec50 endometrial cancer cells, adenoviral vectors encoding the genes for progesterone receptor isoforms A and B were created. The characteristic ability of cancer cells to grow independently of anchorage to the surrounding solid matrix was measured by counting colony formation on soft agar for 8 to 14 days. Cell proliferation in response to a time course of progestin treatment was tested with flow cytometry. RESULTS: After treatment with a control vector without a progesterone receptor--encoding insert, no effect of progestin treatment on cell proliferation was found; after treatment with vectors encoding progesterone receptor isoform A or B, however, progestin treatment resulted in significant inhibition of cell growth. The anchorage-independent cell growth on soft agar assay showed that by 8 to 14 days the number of cell colonies was reduced by 50% relative to control preparations in the presence of progesterone receptor isoform A plus progestin (P <.0001, both Hec50 and KLE cell lines) and by 90% in the presence of progesterone receptor isoform B plus progestin (P <.0001, both Hec50 and KLE cell lines). Progestin treatment also resulted in a time-dependent reduction in cell proliferation as measured by flow cytometry. Although transfection with both progesterone receptor isoforms A and B reduced cell proliferation according to our assays, progesterone receptor isoform B caused a much more dramatic decrease in cell growth (P =.001, Hec50 cells; P <.0001, KLE cells). CONCLUSION: In poorly differentiated endometrial cancer cells that are resistant to progestin therapy, adenovirus-induced expressions of progesterone receptors A and B reestablish progestin control of endometrial cancer cell proliferation.

A randomized controlled trial of interleukin-1 receptor antagonist in a rabbit model of ascending infection in pregnancy.

OBJECTIVE: To determine whether treatment with interleukin-1 receptor antagonist (IL1-ra) would affect amniotic fluid concentrations of tumor necrosis factor alpha (TNF-alpha) and prostaglandins or clinical or microbiological outcomes in a model of ascending bacterial infection in pregnancy. METHODS: Timed pregnant New Zealand white rabbits at 70% of gestation underwent endoscopic inoculation of the cervices with 10(6) - 10(7) cfu Escherichia coli. Animals were randomly assigned in a blinded manner to a 5-h intravenous infusion of human IL1-ra (10 mg/kg) or placebo beginning 1-2 h after inoculation. Blood was drawn from the does for assay of serum IL1-ra concentration before inoculation, at mid-infusion, after the infusion ended and at necropsy. At necropsy, temperature and cultures were taken, and aspirated amniotic fluid was pooled for assays of TNF-aalpha, prostaglandin E2 (PGE2) and ILI-ra. RESULTS: Serum IL1-ra concentrations rose to a mean of 2 microg/ml at mid-infusion and fell markedly after the infusion to concentrations barely detectable at necropsy. Between the two groups, there were no significant differences in the rates of fever or positive cultures or in amniotic fluid concentrations of PGE2 or TNF-alpha. One unique finding was the demonstration that administration of human IL1-ra to the does resulted in measurable concentrations of human IL1-ra in the amniotic fluid. CONCLUSIONS: Treatment with an intravenous infusion of human IL1-ra after cervical inoculation with E. coli did not affect clinical or microbiological outcomes or amniotic fluid concentrations of TNF-alpha or PGE2. This experiment providesthefirstdemonstration of passage of human IL1-ra from the maternal bloodstream to the amniotic fluid.

Histologic inflammation in the maternal and fetal compartments in a rabbit model of acute intra-amniotic infection.

OBJECTIVE: This study was undertaken to determine the course of acute inflammation in the maternal and fetal compartments during experimentally induced ascending intra-amniotic infection. STUDY DESIGN: Forty pregnant rabbits at 70% gestation were inoculated endocervically with 10(5) colony-forming units of Escherichia coli. Does were killed at 0, 4, 8, 16, 24, and 30 hours after inoculation. At necropsy, blood, peritoneal fluid, amniotic fluid, and uterine tissue were cultured. Fetal brain, lung, heart, gut, and kidney were collected for histologic examination. Necrosis, infiltrates, congestion, and edema were each assessed semiquantitatively, and mean composite histologic-inflammation scores were compared with analysis of variance. Inflammation, mitotic activity, and apoptosis were evaluated in the fetal brain, and groups were compared with analysis of variance. RESULTS: Twenty-six animals were evaluated after 14 were excluded (lack of fever or positive culture results). A significant increase in histologic inflammation score was seen in the uterus (P<.001), placenta(P = .011), and fetal lung (P = .001) but not in other fetal tissues. These changes were seen earlier in the uterus and placenta and later in the fetal lung. Mitotic activity in the fetal brain decreased significantly by 8 hours after cervical inoculation. There was no inflammation in the fetal brain, and apoptosis in the fetal brain did not increase with time. CONCLUSIONS: Histologic inflammation occurs early in both the uterus and the placenta and later in the fetal lung in the rabbit model of acute intra-amniotic infection. This contrasts with the previously reported chronic model of intra-amniotic infection in the rabbit.

Acute intrauterine infection results in an imbalance between pro- and anti-inflammatory cytokines in the pregnant rabbit.

PROBLEM: Intrauterine infection results in an increase in cytokines. This study compared the time courses for the pro- and anti-inflammatory cytokine responses in 33 pregnant rabbits at 70% gestation. Pro-inflammatory markers were activated nuclear factor-kappa B (NF-kappaB) in placenta and tumor necrosis factor-alpha (TNF-alpha) in amniotic fluid. These were compared to the anti-inflammatory cytokine, interleukin-1 receptor antagonist (IL-1ra), in placenta and uterus. METHOD OF STUDY: Does were endoscopically inoculated with Escherichia coli through their cervices and sacrificed at six intervals between 0 and 30 hr post-inoculation. RESULTS: Activated NF-kappaB, determined by electromobility gel shift assay, increased significantly 16 hr after bacterial inoculation (P < or = 0.05). This was directly mirrored by TNF-alpha concentrations, determined by bioassay, in the amniotic fluid. However, IL-1ra levels, determined by enzyme-linked immunosorbent assay, did not increase in response to infection. CONCLUSIONS: Intrauterine infection results in an imbalance between pro- and anti-inflammatory cytokines that may potentiate infection-induced preterm delivery.

Estrogens in intrahepatic cholestasis of pregnancy.

OBJECTIVE: To determine whether estrogen production and excretion are impaired in gravidas with intrahepatic cholestasis. METHODS: Plasma and urine samples were collected from 13 women from the United States and Chile at 35-38 weeks' gestation with mild (n = 9) or severe (n = 4) intrahepatic cholestasis of pregnancy. Urinary and plasma steroid levels from women with cholestasis were compared with levels from 27 normal pregnant women within the same gestational age range. Urinary concentrations of dehydroepiandrosterone (DHEA), estrone (E1), estradiol (E2), estriol (E3), estetrol, progesterone, and 16-hydroxy-pregnenolone were measured by gas chromatography mass spectrometry, and plasma concentrations of DHEA sulfate, progesterone, unconjugated E1, unconjugated E2, unconjugated E3, sulfated E3 derivatives, glucuronidated E3 derivatives, and total E3 were measured by radioimmunoassay. RESULTS: Compared with normal pregnant women, women with cholestasis had significantly lower plasma levels of estrogens and DHEA sulfate, the precursor to placental estrogen production synthesized by the fetal adrenal gland (Hotelling-Lawley trace = 0.81; F4,19 = 3.9; P = .02). The mean plasma DHEA sulfate, unconjugated E2, unconjugated E3, and total E3 concentrations were 0.271, 10.21, 9.80, and 99.53 ng/mL, respectively, in women with cholestasis compared with 0.802, 18.98, 16.28, and 145.07 ng/mL for controls. CONCLUSION: Fetal adrenal production of DHEA sulfate, and in response, downstream placental production of estrogens, was compromised by intrahepatic cholestasis of pregnancy.

Endocrine cancer risks for women during the perimenopause and beyond.

Cancer and its link to reproductive hormones is an area of intense concern for our patients and has been the subject of much speculation. But if estrogen causes breast cancer, for example, most women would eventually develop the disease. We know this is not the case! Actually, estrogen and progesterone have been linked to a decrease as well as an increase in cancer, depending upon the type of tumor under investigation. The purpose of this manuscript is to review the data supporting those relationships.

Selective down-regulation of progesterone receptor isoform B in poorly differentiated human endometrial cancer cells: implications for unopposed estrogen action.

The uterine endometrium responds to unopposed estrogen stimulation with rapid cell proliferation. Progesterone protects the endometrium against the hyperplastic effects of estradiol (E2) through progesterone receptors (PRs), of which two isoforms are expressed: human (h) PRA and PRB. hPRB has a longer NH2 terminus and may function differently from hPRA. Thus, the relative expression of hPRA:hPRB is likely to be important for the action of progesterone. We hypothesized that the hPRA:hPRB ratios may be abnormal in endometrial cancer, leading to a lack of normal progesterone protection against the growth-promoting effects of E2. To test this hypothesis, well-differentiated Ishikawa endometrial cancer cells were compared to poorly differentiated Hec50 and KLE cells. Reverse transcription-PCR was chosen as a sensitive method to detect transcripts for the two forms of PR. The relative expression of PR isoforms under hormonal stimulation was determined by Western blotting. Transient transfections of hPRA and hPRB into endometrial cells allowed the evaluation of the transcriptional activity of each isoform independently on reporter gene transcription under the control of a simple progesterone response element-containing promoter. The effect of coexpressing the estrogen receptor on PR expression was also studied. Ishikawa cells (well-differentiated) express both hPRA and hPRB. Both isoforms, but predominantly hPRB, are up-regulated by E2 and not by tamoxifen or the pure antiestrogen ICI 182,780. Hec50 and KLE cells (poorly differentiated) express only hPRA. No hPRB is present in the poorly differentiated cells, and it is not induced by estrogen receptor expression and/or estrogen treatment. In all cells, hPRB expression, whether endogenous or produced as a result of transfection, acts as a stronger transcription factor than hPRA on a simple progesterone-dependent promoter. We speculate that down-regulation of hPRB may predict for poorly differentiated endometrial cancers that do not respond to progestin therapy.

The role of oestrogen in follicular development.

Most of our knowledge of ovarian physiology is based upon studies that have demonstrated functional oestrogen receptors in the ovaries of lower animal species. The presence of oestrogen receptors in primate granulosa cells has been questioned by some investigators. However, we have found oestrogen receptor messenger RNA in human granulosa cells by reverse transcriptase-PCR assay. Furthermore, using immortalized granulosa cell lines transfected with a plasmid containing an oestrogen response element, a functional oestrogen receptor was confirmed. These experiments strongly support the hypothesis that human granulosa cells express biologically active oestrogen receptor.

Estrogen receptors are present in human granulosa cells.

Recent studies failed to detect estrogen receptors in primate follicles. This study was initiated to determine whether estrogen receptor (ER) messenger ribonucleic acid (mRNA) is present in human granulosa cells and, further, if functional ER proteins are present. To evaluate the presence of ER, RNA from human granulosa cells obtained at the time of oocyte retrieval for assisted reproduction was extracted, and complementary DNA synthesis was performed by the reverse transcriptase-polymerase chain reaction. Oligonucleotide primers were used to amplify basepairs 570-852 in the B- and C- domains of the ER mRNA. Southern blotting was performed and confirmed that the amplified DNA fragment identified in granulosa cells represented ER. By reverse transcriptase-polymerase chain reaction, mRNA for the ER is clearly identified in primary human granulosa cells obtained at the time of oocyte retrieval. To expand these studies and determine whether functional ER were present in human granulosa cells in culture, a simian virus-40-transformed human granulosa cell line was studied. Cells were transfected with a plasmid containing as estrogen response element up-stream from the bacterial reporter gene chloramphenicol acetyltransferase (CAT). In transfected cells, CAT activity is inducible by estradiol if endogenous functional ER are present. In these studies, the transfection analysis confirmed that functional, transcriptionally competent ER are present in a human granulosa cell line, with a 4- to 5-fold enhancement of CAT activity demonstrated after the addition of estradiol compared to that in nonhormone-treated cells. In conclusion, ER mRNA is present in human granulosa cells. Functional ER are also demonstrated in a transformed human granulosa cell line. We hypothesize that low, but biologically significant, amounts of ER protein are present in human granulosa cells, which are not routinely detectable by standard assays.

Low serum estradiol and high serum progesterone concentrations characterize hypertensive pregnancies at high altitude.

OBJECTIVE: Intrauterine growth retardation and preeclampsia are more common at high than at low altitude. Because altered hormonal profiles have been linked with these disorders, we asked whether placental steroid hormone concentrations were altered during pregnancy at high altitude. METHODS: We measured progesterone, unconjugated estradiol, and estriol (by radioimmunoassay) at weeks 20, 30, and 36 of pregnancy in 18 women at low altitude (1600 m) and 40 women at high altitude (3100 m). RESULTS: Women at 3100 m compared with 1600 m had lower serum estradiol concentrations at week 36 of pregnancy, and lower estriol and higher progesterone concentrations throughout pregnancy. As a result, the progesterone/estradiol ratio was greater in the high- versus the low-altitude women. Estradiol fell between weeks 30 and 36 in women who developed transient hypertension or preeclampsia. The fall in estradiol was accompanied by a marked increase in progesterone concentrations among the preeclamptic women. At 3100 m, estradiol correlated negatively (r = -0.37, P < .05) and progesterone positively (r = 0.46, P < .05) with mean arterial pressure at week 36 of pregnancy. CONCLUSIONS: We speculate that reduced placental oxygen pressure (PO2) at high altitude may decrease placental aromatase activity and thereby lower estradiol and estriol concentrations. The factor(s) responsible for the rise in progesterone is unknown. Possibly, high progesterone relative to estradiol concentrations contributes to the development of preeclampsia at high altitude.

Estrogen receptors are identified in the glioblastoma cell line U138MG.

OBJECTIVE: The antiestrogen tamoxifen has been found to be effective in decreasing glioblastoma cell proliferation, but the mechanism underlying this effect and whether it is through the estrogen receptor (ER) is controversial. The objective of this study was to determine whether ERs are present in three human glioblastoma cell lines--HS683, U138MG, and JHN J889H--using the most sensitive techniques available. METHODS: Ligand binding and flow cytometry were employed to identify estrogen and progesterone receptors. The reverse transcriptase-polymerase chain reaction was used to identify ER mRNA, and a novel reporter gene transfection assay demonstrated that the ER was capable of activating gene transcription. RESULTS: U138MG glioblastoma cells contain ERs that are capable of increasing gene transcription in response to estradiol. No ERs were found in HS683 or JHN J889H cells. CONCLUSION: Tamoxifen may be acting through the ER in some glioblastoma cells.

Oestrogen modulation with parturition in the human placenta.

An initial group of term (36-41 6/7 weeks), preterm (less than 36 weeks), and post-term (42 or more weeks) placentae were collected from women at delivery to determine the placental levels of important steroids and steroidogenic enzymes involved in the oestrogen synthesis pathway as a function of gestational age. A second group of placentae were obtained from women delivering at term before and after the onset of labour. Placentae were evaluated individually for cytosolic steroid hormone levels and microsomal steroidogenic enzyme activities. Oestradiol (E2), oestrone (E1), progesterone (P), and delta-4-androstenedione (A) were measured by radioimmunoassay in placental cytosols. Aromatase (AR), sulphatase (S), and 3 beta-hydroxysteroid dehydrogenase/isomerase (3 beta HSD) activities were assayed in placental microsomes. Cytosolic concentrations of E1, E2, P, and A did not differ with respect to gestational age. Correspondingly, the microsomal enzyme activities of 3 beta HSD, S, and AR did not vary as a function of gestational age. However, when patients at term who were in labour prior to delivery were compared to those who were not, the placental cytosolic level of E1 was found to be threefold higher in the non-labouring group (4572 versus 1427 pg/mg cytosolic protein, P < 0.025). Additionally, microsomal aromatase activity was also significantly higher in the non-labouring patients (46 versus 19 pM/min/mg protein, P < 0.025), while the E2 to P ratio in the labouring patients was twice that of the non-labouring group, a difference which was significant at the P < 0.025 level (Wilcoxon rank sum test). These data suggest that at term, prior to labour, the placental production of E1 by AR is high, and that AR activity and E1 levels fall significantly after the onset of labour. Also, the placental cytosolic concentration of the more active oestrogen, E2, demonstrates stable to rising levels with a significant increase in E2/P after the onset of labour. We theorize that in the term pregnancy prior to labour, E1 may represent a large but relatively inactive intracellular oestrogen pool which is maintained by high AR activity, and may function to protect the pregnant local uterine environment from the more oxytocic effects of E2.

Pregnancy specific beta 1 glycoprotein (SP-1) in maternal serum and amniotic fluid; pre-eclampsia, small for gestational age fetus and fetal distress.

Pregnancy specific beta 1 glycoprotein (SP-1) levels have been suggested to correlate with certain obstetrical complications. We compared maternal serum SP-1 levels in normal pregnancies (N = 82), and pregnancies complicated by pre-eclampsia (N = 37), small for gestational age fetuses (N = 8) and fetal distress (N = 13). We also compared levels of this protein in mid-trimester amniotic fluid samples obtained from normal (N = 47) and small for gestational age fetuses (N = 25). Despite trends towards low maternal serum and amniotic fluid SP-1 values in complicated pregnancies, there were no statistically significant differences in their levels as compared with normal gestational age matched controls (P > 0.05). These data do not support the use of SP-1 values in clinical practice.

Differential gene expression in the amnion, chorion, and trophoblast of the human placenta.

Human chorionic gonadotrophin (hCG), placental alkaline phosphatase (PLAP), and pregnancy-specific glycoprotein (PSG) are three major proteins produced by the trophoblast of the human placenta. Immunocytochemical studies suggest that PSG and hCG are also present in the human amnion. In this study, we examined whether amniotic and chorionic membranes were capable of expressing trophoblastic-specific genes. As previously reported, trophoblasts express high levels of hCG beta, hCG alpha, PLAP, and PSG. Both amnion and chorion were found to express PLAP and hCG beta mRNA. However, the hCG alpha transcript was expressed only by the amnion, but not by the chorion in the term placenta. Recent molecular cloning studies indicate that human PSGs are a group of closely related placental proteins that, together with the carcinoembryonic antigen family members, comprise a subfamily within the immunoglobulin superfamily. To demonstrate that amnion and chorion also express PSG transcripts, we employed ribonuclease protection analysis using probes specific to the 5' and 3' region of PSG mRNAs. Our data indicate that while amniotic as well as chorionic membrane expressed low levels of the PSG genes, only a certain subpopulation of PSG transcripts were expressed. Furthermore, the amnion and chorion demonstrated differences in PSG species expression from each other and from trophoblastic tissue. Thus, human amnion, chorion and trophoblast selectively express several placental genes.

Functional analysis of a mutant estrogen receptor isolated from T47Dco breast cancer cells.

OBJECTIVE: Estrogen receptor-positive cancers that initially respond to hormone therapy often progress to a resistant state. The breast cancer cell line T47Dco is a model for such resistance. It is a polymorphic line, composed of multiple cell populations that demonstrate the presence of mutant estrogen receptors by cloning and sequencing techniques. Our objective was to isolate and analyze the structural and functional characteristics of the T47Dco mutant estrogen receptor complementary deoxyribonucleic acid clones. STUDY DESIGN: We constructed two independent T47Dco complementary deoxyribonucleic acid libraries. We isolated and sequenced T47Dco estrogen receptors and have identified a mutant receptor that is truncated near the end of the deoxyribonucleic acid binding domain. This mutant has now been recreated with site-directed mutagenesis and tested for its ability to bind to deoxyribonucleic acid, dimerize with other estrogen receptors, and activate gene transcription by the chloramphenicol acetyltransferase assay and by gel shift assays. RESULTS: The chloramphenicol acetyltransferase assays reveal that in the absence of estradiol low levels of conversion of chloramphenicol to acetylated products occur when the mutant estrogen receptor is used to activate chloramphenicol acetyltransferase gene transcription, supporting that it has some constitutive function. Also, gel mobility shift assays demonstrate low levels of deoxyribonucleic acid binding with the mutant protein. CONCLUSION: This mutant estrogen receptor may contribute to the estrogen receptor-positive, hormone-resistant phenotype of T47Dco cells by constitutively binding to and activating genes that were previously estradiol dependent.

T47DCO cells, genetically unstable and containing estrogen receptor mutations, are a model for the progression of breast cancers to hormone resistance.

We postulate that one mechanism for the progression of breast cancers to hormone resistance involves the acquisition of mutant estrogen receptors (ER)4 by genetically unstable cell subpopulations. The T47D human breast cancer cell line may be a model for such progression, having sublines that are ER positive and estrogen responsive, ER positive and estrogen resistant, or ER negative. Also, T47D cells can be either hyperdiploid (HD) or hypertetraploid (HT) or persistently alternate between these states. T47DCO cells are a HD and ER-positive, but estrogen-resistant, subline of T47D cells that undergoes spontaneous tetraploidization. Such a stable variant, designated T47Dv, is 85% HT (Cancer Res., 49: 3943, 1989). We now show that single-cell clones derived from the mixed HD/HT T47Dv can be either HD or HT, and can be either estrogen responsive or estrogen resistant, for growth and for progesterone receptor regulation. To begin the study of ER in this model system of T47DCO and their derivatives, we have generated complementary DNA libraries from the parental HD T47DCO cells and have isolated three ER complementary DNA mutants. These include two frame-shift/termination mutants that would encode ERs truncated in the DNA-binding domain and in the hormone-binding domain and a third mutant with a large in-frame deletion spanning the hinge region and a part of the hormone-binding domain. If expressed, these mutant ERs would lack hormone-binding capacity and would be undetected by the anti-ER antibodies currently in clinical use. Genetic instability, when associated with mutant ERs in subpopulations of breast tumor cells, may provide the selective pressure leading to hormone resistance. T47DCO cells and their clonal derivatives provide a model for the systematic study of ER mutations and other mechanisms of hormone resistance in Stage IV breast cancer.

Linkage of two human pregnancy-specific beta 1-glycoprotein genes: one is associated with hydatidiform mole.

A genomic clone containing two linked human pregnancy-specific beta 1-glycoprotein (PS beta G) genes has been isolated and characterized. The two genes are arranged in the same 5'----3' orientation; the 3' region (including the A2 and B-C exons) of the upstream gene, PSGGA, is linked to the 5' region (including the 5'/L and L/N exons) of PSGGB, the downstream gene. Depending upon the domains compared, PSGGA and PSGGB share 92-98% nucleotide and 86-95% amino acid sequence identity with PSG93, the most abundant PS beta G transcript. The 3' exon (B-C) of PSGGA contains four alternative splice sites and three polyadenylylation sites, which account for the 3' heterogeneity previously reported in the PS beta G family. Each of the predicted PSGGA-encoded proteins would have a different carboxyl terminus. PSGGB corresponds to the previously identified cDNA PSG6, which encodes proteins containing a 34-amino acid leader peptide and a 108-amino acid N domain, which is one amino acid shorter than the majority of PS beta G N domains. Additionally, the PSGGB-encoded proteins contain the cell-surface recognition tripeptide Arg-Gly-Asp, shared by several previously reported PS beta Gs as deduced from cDNA sequences. Northern blot hybridization performed with a PSGGB-specific oligonucleotide probe to the N domain revealed that PSGGB or a PSGGB-like gene encodes a major 1.7-kilobase mRNA in hydatidiform mole tissues and a major 2.0-kilobase mRNA in term placenta tissues. Moreover, the PSGGB-specific probe hybridized most strongly with mRNA from molar trophoblastic tissue, suggesting that the PSGGB-like species may be the gene preferentially expressed in gestational trophoblastic disease. Additionally, the sequence of a 2315-base-pair PS beta G cDNA (PSG95) that contains an N-A1-A2-B2-C domain arrangement is reported. The coding region of PSG95 is identical to the previously reported cDNA clones PSG1d and FL-NCA, but PSG95 contains an additional 518 and 523 base pairs in the 3' end as compared with PSG1d and FL-NCA, respectively.