Processing and juxtacrine activity of membrane-anchored betacellulin

Betacellulin (BTC) was originally isolated as a secreted growth factor from a mouse pancreatic beta-tumor cell line, whereas the cDNA sequence predicts that BTC is synthesized as a larger transmembrane protein. In the present study, we have characterized the membrane-anchored forms of BTC, using Chinese hamster ovary (CHO) cells, mouse fibroblast A9 cells, and a human breast cancer cell line MCF-7, all of which were stably transfected with human BTC cDNA. A9 and MCF-7 transfectants produced membrane-anchored BTC isoforms of 21, 25, 29, and 40 kDa on the cell surface, as well as a secreted BTC isoform. CHO transfectants secreted little BTC but accumulated the membrane-anchored isoforms. The cleavage of the membrane-anchored forms to release a secreted from of BTC was not enhanced by biological mediators such as a phorbol ester, which stimulates the cleavage of other membrane-anchored growth factors. The membrane-anchored forms of BTC expressed on the transfected cells induced the insulin production and/or promoted the growth in subclones of AR42J rat pancreatic cells. These results suggest that the membrane-anchored BTC can function as a juxtacrine factor in regulating the growth and differentiation of pancreatic endocrine cells.     

Impediments to Enhancement of CPT-11 Anticancer Activity by E. coli Directed Beta-Glucuronidase Therapy

CPT-11 is a camptothecin analog used for the clinical treatment of colorectal adenocarcinoma. CPT-11 is converted into the therapeutic anti-cancer agent SN-38 by liver enzymes and can be further metabolized to a non-toxic glucuronide SN-38G, resulting in low SN-38 but high SN-38G concentrations in the circulation. We previously demonstrated that adenoviral expression of membrane-anchored beta-glucuronidase could promote conversion of SN-38G to SN-38 in tumors and increase the anticancer activity of CPT-11. Here, we identified impediments to effective tumor therapy with E. coli that were engineered to constitutively express highly active E. coli beta-glucuronidase intracellularly to enhance the anticancer activity of CPT-11. The engineered bacteria, E. coli (lux/βG), could hydrolyze SN-38G to SN-38, increased the sensitivity of cultured tumor cells to SN-38G by about 100 fold and selectively accumulated in tumors. However, E. coli (lux/βG) did not more effectively increase CPT-11 anticancer activity in human tumor xenografts as compared to non-engineered E. coli. SN-38G conversion to SN-38 by E. coli (lux/βG) appeared to be limited by slow uptake into bacteria as well as by segregation of E. coli in necrotic regions of tumors that may be relatively inaccessible to systemically-administered drug molecules. Studies using a fluorescent glucuronide probe showed that significantly greater glucuronide hydrolysis could be achieved in mice pretreated with E. coli (lux/βG) by direct intratumoral injection of the glucuronide probe or by intratumoral lysis of bacteria to release intracellular beta-glucuronidase. Our study suggests that the distribution of beta-glucuronidase, and possibly other therapeutic proteins, in the tumor microenvironment might be an important barrier for effective bacterial-based tumor therapy. Expression of secreted therapeutic proteins or induction of therapeutic protein release from bacteria might therefore be a promising strategy to enhance anti-tumor activity.     

Hypoxia-targeted over-expression of carboxylesterase as a means of increasing tumour sensitivity to irinotecan (CPT-11)

The induced expression of carboxylesterase (CE) enzymes, which convert the prodrug irinotecan (CPT-11) into its active cytotoxic metabolite SN-38, constitutes a promising strategy for cancer gene therapy. By incorporating hypoxia-responsive elements (HREs) in conjunction with the transgene, expression can be targeted specifically to hypoxic tissues (such as solid tumours), expressing the hypoxia-inducible factor 1 (HIF-1). We have constructed a recombinant adenoviral vector, AdHRE-rCE, encoding the cDNA for the highly efficient rabbit liver CE (rCE), under the control of a HRE derived from the human phosphoglycerate kinase 1 (PGK-1) gene in conjunction with a minimal SV40 promoter. In vitro, HT1080 fibrosarcoma and SW480 colon carcinoma cells demonstrated an approximately 10-fold hypoxia-dependent induction in CE expression following pre-infection with AdHRE-rCE, which led to a15-30-fold increased sensitivity to CPT-11. Furthermore, in vivo, SW480 tumour xenografts infected with AdHRE-rCE demonstrated a 2-fold decrease in tumour doubling time, when combined with 7 days of CPT-11 treatment, in comparison to mock-infected controls, with rCE expression shown to be limited to hypoxic regions only. As the cytotoxicity of CPT-11 is reduced under hypoxic conditions, over-expression of a highly efficient CE such as rCE under hypoxia control within these hypoxic cells could reverse this effect and, therefore, form the basis for future clinical treatment strategies.     

Molecular modeling of CPT-11 metabolism by carboxylesterases (CEs): use of pnb CE as a model

CPT-11 is a prodrug that is converted in vivo to the topoisomerase I poison SN-38 by carboxylesterases (CEs). Among the CEs studied thus far, a rabbit liver CE (rCE) converts CPT-11 to SN-38 most efficiently. Despite extensive sequence homology, however, the human homologues of this protein, hCE1 and hiCE, metabolize CPT-11 with significantly lower efficiencies. To understand these differences in drug metabolism, we wanted to generate mutations at individual amino acid residues to assess the effects of these mutations on CPT-11 conversion. We identified a Bacillus subtilis protein (pnb CE) that could be used as a model for the mammalian CEs. We demonstrated that pnb CE, when expressed in Escherichia coli, metabolizes both the small esterase substrate o-NPA and the bulky prodrug CPT-11. Furthermore, we found that the pnb CE and rCE crystal structures show an only 2.4 A rmsd variation over 400 residues of the alpha-carbon trace. Using the pnb CE model, we demonstrated that the "side-door" residues, S218 and L362, and the corresponding residues in rCE, L252 and L424, were important in CPT-11 metabolism. Furthermore, we found that at position 218 or 252 the size of the residue, and at position 362 or 424 the hydrophobicity and charge of the residue, were the predominant factors in influencing drug activation. The most significant change in CPT-11 metabolism was observed with the L424R variant rCE that converted 10-fold less CPT-11 than the wild-type protein. As a result, COS-7 cells expressing this mutant were 3-fold less sensitive to CPT-11 than COS-7 cells expressing the wild-type protein.     

ACAT2 stimulates cholesteryl ester secretion in apoB-containing lipoproteins

Previous studies in nonhuman primates revealed a striking positive correlation between liver cholesteryl ester (CE) secretion rate and the development of coronary artery atherosclerosis. CE incorporated into hepatic VLDL is necessarily synthesized by ACAT2, the cholesterol-esterifying enzyme in hepatocytes. We tested the hypothesis that the level of ACAT2 expression, in concert with cellular cholesterol availability, affects the CE content of apolipoprotein B (apoB)-containing lipoproteins. In a model system of lipoprotein secretion using COS cells cotransfected with microsomal triglyceride transfer protein and truncated forms of apoB, ACAT2 expression resulted in a 3-fold increase in microsomal ACAT activity and a 4-fold increase in the radiolabeled CE content of apoB-lipoproteins. After cholesterol-cyclodextrin (Chol-CD) treatment, CE secretion was increased by 27-fold in ACAT2-transfected cells but by only 7-fold in control cells. Chol-CD treatment also caused the percentage of CE in the apoB-lipoproteins to increase from 3% to 33% in control cells and from 16% to 54% in ACAT2-transfected cells. In addition, ACAT2-transfected cells secreted 3-fold more apoB than control cells. These results indicate that under all conditions of cellular cholesterol availability tested, the relative level of ACAT2 expression affects the CE content and, hence, the potential atherogenicity, of nascent apoB-containing lipoproteins.     

Intercellular transfer regulation of the paracrine activity of GPI-anchored Cripto-1 as a Nodal co-receptor

Cripto-1 (CR-1) is a glycosylphosphatidylinositol-anchored glycoprotein which acts as an obligate co-receptor of a TGFβ family ligand, Nodal. Previous studies have demonstrated that CR-1 functions in a paracrine fashion by a cellular mechanism which has not been fully described. This paracrine activity was observed only when CR-1 was expressed as a membrane-bound form and was abolished when CR-1 was expressed in a soluble form. In the current study, we found that there were few biochemical differences in post-translational modifications between membrane-anchored and soluble forms of CR-1. Flow cytometric analysis revealed an intercellular transfer of the membrane-bound form of CR-1 between cells. CR-1-expressing cells formed unique membrane extensions, generated more membrane fragments than control cells, and exhibited enhanced cellular adhesion. Thus, expression of CR-1 may alter the physiochemical properties of the plasma membrane resulting in an enhancement of intercellular transfer of cellular signaling components which may account for the paracrine activity of CR-1.     

Identification and activities of human carboxylesterases for the activation of CPT-11, a clinically approved anticancer drug.

CPT-11 is a clinically approved anticancer drug used for the treatment of advanced colorectal cancer. Upon administration, the carbamate side chain of the drug is hydrolyzed, resulting in the release of SN-38, an agent that has approximately 1000-fold increased cytotoxic activity. Since only a very small percentage of the injected dose of CPT-11 is converted to SN-38, there is a significant opportunity to improve its therapeutic efficacy and to diminish its systemic toxicity by selectively activating the drug within tumor sites. We envisioned that a mAb-human enzyme conjugate for CPT-11 activation would be of interest, particularly since the conjugate would likely be minimally immunogenic, and the prodrug is clinically approved. Toward this end, it was necessary to identify the most active human enzyme that could convert CPT-11 to SN-38. We isolated enzymes from human liver microsomes based on their abilities to effect the conversion and identified human carboxylesterase 2 (hCE-2) as having the greatest specific activity. hCE-2 was 26-fold more active than human carboxylesterase 1 and was 65% as active as rabbit liver carboxylesterase, the most active CPT-11 hydrolyzing enzyme known. The anti-p97 mAb 96.5 was linked to hCE-2, forming a conjugate that could bind to antigen-positive cancer cells and convert CPT-11 to SN-38. Cytotoxicity assays established that the conjugate led to the generation of active drug, but the kinetics of prodrug activation (48 pmol x min(-1) x mg(-1) was insufficient for immunologically specific prodrug activation. These results confirm the importance of hCE-2 for CPT-11 activation and underscore the importance of enzyme kinetics for selective prodrug activation.     

Wise retained in the endoplasmic reticulum inhibits Wnt signaling by reducing cell surface LRP6

The Wnt signaling pathway is tightly regulated by extracellular and intracellular modulators. Wise was isolated as a secreted protein capable of interacting with the Wnt co-receptor LRP6. Studies in Xenopus embryos revealed that Wise either enhances or inhibits the Wnt pathway depending on the cellular context. Here we show that the cellular localization of Wise has distinct effects on the Wnt pathway readout. While secreted Wise either synergizes or inhibits the Wnt signals depending on the partner ligand, ER-retained Wise consistently blocks the Wnt pathway. ER-retained Wise reduces LRP6 on the cell surface, making cells less susceptible to the Wnt signal. This study provides a cellular mechanism for the action of Wise and introduces the modulation of cellular susceptibility to Wnt signals as a novel mechanism of the regulation of the Wnt pathway.     

CXCL2/CXCR2 axis induces cancer stem cell characteristics in CPT-11-resistant LoVo colon cancer cells via Gαi-2 and Gαq/11

Cancer stem cells (CSCs) exist in colon cancer and exhibit characteristics of stem cells which are due to lineages of tissues where they arise. Epithelial to mesenchymal transition (EMT)-undergoing cancer cells display CSC properties and therapeutic resistance. Cancer and stromal cells comprise of a tumor microenvironment. One way the two populations communicate with each other is to secret CXC ligands (CXCLs). CXCLs are capable of causing chemotaxis of specific types of stromal cells and control angiogenesis. Double immunofluorescence, western blot analysis, and colony-formation assay were carried out to compare parental and CPT-11-resistant LoVo cells. CPT-11-R LoVo colon cancer cells showed increased expression of CXCL1, CXCL2, CXCL3, and CXCL8. They displayed significantly increased intracellular protein levels of CXCL2 and CXCR2. CPT-11-R LoVo cells showed significantly elevated expression in aldehyde dehydrogenase 1 (ALDH1), cluster of differentiation 24 (CD24), cluster of differentiation 44 (CD44), and epithelial cell adhesion molecule (EpCAM). CXCL2 knockdown by short hairpin RNA resulted in reduced expression of CSC proteins, cyclins, EMT markers, G proteins, and matrix metalloproteinases (MMPs). Finally, Gαi-2 was found to promote expression of CSC genes and tumorigenesis which were more apparent in the resistant cells. In addition, Gαq/11 showed a similar pattern with exceptions of EpCAM and MMP9. Therefore, CXCL2–CXCR2 axis mediates through Gαi-2 and Gαq/11 to promote tumorigenesis and contributes to CSC properties of CPT-11-R LoVo cells.     

Characterization of the SP11/SCR high-affinity binding site involved in self/nonself recognition in brassica self-incompatibility

In Brassica self-incompatibility, the recognition of self/nonself pollen grains, is controlled by the S-locus, which encodes three highly polymorphic proteins: S-locus receptor kinase (SRK), S-locus protein 11 (SP11; also designated S-locus Cys-rich protein), and S-locus glycoprotein (SLG). SP11, located in the pollen coat, determines pollen S-haplotype specificity, whereas SRK, located on the plasma membrane of stigmatic papilla cells, determines stigmatic S-haplotype specificity. SLG shares significant sequence similarity with the extracellular domain of SRK and is abundant in the stigmatic cell wall, but its function is controversial. We previously showed that SP11 binds directly to its cognate SRK with high affinity (K(d) = 0.7 nM) and induces its autophosphorylation. We also found that an SLG-like, 60-kD protein on the stigmatic membrane forms a high-affinity binding site for SP11. Here, we show that the 60-kD stigmatic membrane protein is a truncated form of SRK containing the extracellular domain, transmembrane domain, and part of the juxtamembrane domain. A transiently expressed, membrane-anchored form of SRK exhibits high-affinity binding to SP11, whereas the soluble SRK (eSRK) lacking the transmembrane domain exhibits no high-affinity binding, as is the case with SLG. The different binding affinities of the membrane-anchored SRK and soluble eSRK or SLG will be significant for the specific perception of SP11 by SRK.     

Enhanced Anticancer Effect of Adding Magnesium to Vitamin C Therapy: Inhibition of Hormetic Response by SVCT-2 Activation

l-Ascorbic acid (vitamin C, AA) is known as an antioxidant, but at high concentrations, AA can kill cancer cells through a prooxidant property. Sodium-dependent vitamin C transporter family-2 (SVCT-2) determines the cellular uptake of AA, and the activity of SVCT-2 is directly related to the anticancer activity of AA. Cancer cells that showed high SVCT-2 expression levels were more sensitive to AA treatment than cancer cells with low SVCT-2 expression levels. Cells with low SVCT-2 expression showed a hormetic response to a low dose of AA. Magnesium ions, which are known to activate SVCT-2, could increase the V_max value of SVCT-2, so we investigated whether providing magnesium supplements to cancer cells with low SVCT-2 expression that had shown a hormetic response to AA would elevate the V_max value of SVCT-2, allowing more AA to accumulate. To evaluate the effects of magnesium on cancer cells, MgSO₄ and MgCl₂ were screened as magnesium supplements; both forms showed synergistic anticancer effects with AA. Taken together, the results of this study suggest that magnesium supplementation enhanced the anticancer effect of AA by inhibiting the hormetic response at a low dose. This study has also demonstrated that AA treatment with magnesium supplementation provided more effective anticancer therapy than AA treatment alone.     

Localizing matrix metalloproteinase activities in the pericellular environment

Matrix metalloproteinases (MMPs) are a group of structurally related proteolytic enzymes containing a zinc ion in the active site. They are secreted from cells or bound to the plasma membrane and hydrolyze extracellular matrix (ECM) and cell surface-bound molecules. They therefore play key roles in morphogenesis, wound healing, tissue repair and remodeling in diseases such as cancer and arthritis. Although the cell anchored membrane-type MMPs (MT-MMPs) function pericellularly, the secreted MMPs have been considered to act within the ECM, away from the cells from which they are synthesized. However, recent studies have shown that secreted MMPs bind to specific cell surface receptors, membrane-anchored proteins or cell-associated ECM molecules and function pericellularly at focussed locations. This minireview describes examples of cell surface and pericellular partners of MMPs, as well as how they alter enzyme function and cellular behaviour.     

Mechanisms and effects of retention of over-expressed aquaporin AtPIP2;1 in the endoplasmic reticulum

Plasma membrane intrinsic proteins (PIPs) are aquaporins that mediate water transport across the plant plasma membrane (PM). The present work addresses, using Arabidopsis AtPIP2;1 as a model, the mechanisms and significance of trafficking of newly synthesized PIPs from the endoplasmic reticulum (ER) to the Golgi apparatus. A functional diacidic export motif (Asp4-Val5-Glu6) was identified in the N-terminal tail of AtPIP2;1, using expression in transgenic Arabidopsis of site-directed mutants tagged with the green fluorescent protein (GFP). Confocal fluorescence imaging and a novel fluorescence recovery after photobleaching application based on the distinct diffusion of PM and intracellular AtPIP2;1-GFP forms revealed a retention in the ER of diacidic mutated forms, but with quantitative differences. Thus, the individual role of the two acidic Asp4 and Glu6 residues was established. In addition, expression in transgenic Arabidopsis of ER-retained AtPIP2;1-GFP constructs reduced the root hydraulic conductivity. Co-expression of AtPIP2;1-GFP and AtPIP1;4-mCherry constructs suggested that ER-retained AtPIP2;1-GFP may interact with other PIPs to hamper their trafficking to the PM, thereby contributing to inhibition of root cell hydraulic conductivity.     

Biosynthesis and function of LFA-3 in human mutant cells deficient in phosphatidylinositol-anchored proteins

Mutants that lack expression of phosphatidylinositol (PI)-anchored proteins were derived from the human B lymphoblastoid JY cell line. It was demonstrated that unlike wild-type cells, which normally express both a transmembrane and a PI-linked form of LFA-3 glycoprotein, the mutant cells expressed only the transmembrane form of LFA-3. [3H]Ethanolamine was not incorporated into LFA-3 of mutant cells, indicating that the anchor moiety was entirely missing. Blockade of normal biosynthesis of the PI-anchored form led to accumulation of two intermediates that may have intact and truncated polypeptide chains. The truncated LFA-3, which was not attached to the cell membrane, was secreted by mutant cells into culture supernatants. A possible division of adhesion function between the two forms of LFA-3 was studied by using the JY cell lines as targets for CTL. Wild-type and mutant JY cells formed conjugates with CTL and were subsequently lysed to a similar extent. In addition, wild-type and mutant JY cells stimulated CTL proliferation to the same extent. Antibody-blocking experiments demonstrated a predominant role for the CD2/LFA-3 pathway in interaction of both wild-type and mutant cells with CTL. Because E exclusively express only the PI-linked LFA-3 form, and this form is known to mediate cell adhesion, the present results indicate that the two distinct membrane-anchored LFA-3 forms are each capable of mediating adhesion. A possible division of signaling functions between the two forms of LFA-3 is under investigation.     

Inhibitory role of TRIP-Br1 oncoprotein in anticancer drug-mediated programmed cell death via mitophagy activation

Chemotherapy has been widely used as a clinical treatment for cancer over the years. However, its effectiveness is limited because of resistance of cancer cells to programmed cell death (PCD) after treatment with anticancer drugs. To elucidate the resistance mechanism, we initially focused on cancer cell-specific mitophagy, an autophagic degradation of damaged mitochondria. This is because mitophagy has been reported to provide cancer cells with high resistance to anticancer drugs. Our data showed that TRIP-Br1 oncoprotein level was greatly increased in the mitochondria of breast cancer cells after treatment with various anticancer drugs including staurosporine (STS), the main focus of this study. STS treatment increased cellular ROS generation in cancer cells, which triggered mitochondrial translocation of TRIP-Br1 from the cytosol via dephosphorylation of TRIP-Br1 by protein phosphatase 2A (PP2A). Up-regulated mitochondrial TRIP-Br1 suppressed cellular ROS levels. In addition, TRIP-Br1 rapidly removed STS-mediated damaged mitochondria by activating mitophagy. It eventually suppressed STS-mediated PCD via degradation of VDACI, TOMM20, and TIMM23 mitochondrial membrane proteins. TRIP-Br1 enhanced mitophagy by increasing expression levels of two crucial lysosomal proteases, cathepsins B and D. In conclusion, TRIP-Br1 can suppress the sensitivity of breast cancer cells to anticancer drugs by activating autophagy/mitophagy, eventually promoting cancer cell survival.     

Simple non-ion-paired high-performance liquid chromatographic method for simultaneous quantitation of carboxylate and lactone forms of 14 new camptothecin derivatives

SN-38 (7-ethyl-10-hydroxycamptothecin) is an active metabolite derived from the semi-synthetic compound camptothecin (CPT) named Irinotecan (CPT-11). The antitumor activity of SN-38 is 1000-fold more potent than the parent CPT-11. Fourteen new derivatives of camptothecin have recently been developed by Yakult Honsha (Tokyo, Japan). Here we describe a simple and cost-effective high-performance liquid chromatography (HPLC) method without an ion-pairing agent, which allows the simultaneous determination of both lactone and carboxylate forms of SN-38 and other camptothecin derivatives. A weak linear relationship between the HPLC retention factors (ln k') and the cellular concentrations of these compounds was observed. These results suggest that low-polarity compounds easily accumulate in cancer cells and may circumvent drug resistance. The HPLC analysis herein described is expected to greatly assist in derivative synthesis and chemical modification of camptothecin-based antitumor drugs.     

Membrane-anchored proteoglycans of mouse macrophages: P388D1 cells express a syndecan-4–like heparan sulfate proteoglycan and a distinct chondroitin sulfate form

Proteoglycan accumulation by thioglycollate-elicited mouse peritoneal macrophages and a panel of murine monocyte-macrophage cell lines has been examined to determine whether these cells express plasma membrane-anchored heparan sulfate proteoglycans. Initially, cells were screened for heparan sulfate and chondroitin sulfate glycosaminoglycans after metabolic labeling with radiosulfate. Chondroitin sulfate is secreted to a variable extent by every cell type examined. In contrast, heparan sulfate is all but absent from immature pre-monocytes and is associated predominantly with the cell layer of mature macrophage-like cells. In the P388D1 cell line, the cell-associated chondroitin sulfate is largely present as a plasma membrane-anchored proteoglycan containing a 55 kD core protein moiety, which appears to be unique. In contrast, the cell-associated heparan sulfate is composed of a proteoglycan fraction and protein-free glycosaminoglycan chains, which accumulate intracellularly. A fraction of the heparan sulfate proteoglycan contains a lipophilic domain and can be released from cells following mild treatment with trypsin, suggesting that it is anchored in the plasma membrane. Isolation of this proteoglycan indicates that it is likely syndecan-4: it is expressed as a heparan sulfate proteoglycan at the cell surface, it is cleaved from the plasma membrane by low concentrations of trypsin, and it consists of a single 37 kD core protein moiety that co-migrates with syndecan-4 isolated from NMuMG mouse mammary epithelial cells. Northern analysis reveals that a panel of macrophage-like cell lines accumulate similar amounts of syndecan-4 mRNA, demonstrating that this proteoglycan is expressed by a variety of mature macrophage-like cells. Syndecan-1 mRNA is present only in a subset of these cells, suggesting that the expression of this heparan sulfate proteoglycan may be more highly regulated by these cells. © 1993 Wiley-Liss, Inc.     

Death receptor Fas/Apo-1/CD95 expressed by human placental cytotrophoblasts does not mediate apoptosis

Trophoblasts, the fetal cells that line the villous placenta and separate maternal blood from fetal tissue, express both Fas antigen and the tumor necrosis factor (TNF) receptor p55 (TNFRp55), two members of the TNF receptor family that contain a cytoplasmic "death domain" that mediates apoptotic signals. We show that Fas mRNA expressed by cultured villous cytotrophoblasts isolated from term placentas encodes transmembrane sequences and that the protein is full-length (approximately 45 kDa), suggesting that the product is an active plasma membrane-anchored receptor. Its location on the cell surface was confirmed by cellular ELISA analysis of live cells. Although cytotrophoblast apoptosis was induced by TNFalpha, and both anti-Fas antibody (CH11) and FasL-expressing T lymphocyte hybridoma (activated A1.1) cells induced HeLa cell apoptosis, neither CH11 antibody nor activated A1.1 cells stimulated apoptosis in term or first-trimester cytotrophoblasts or in term syncytiotrophoblasts. We conclude that Fas- but not TNFRp55-mediated apoptosis is blocked in primary villous trophoblasts. These data suggest that the Fas response is specifically inactivated by unknown mechanisms to avoid autocrine or paracrine killing by Fas ligand constitutively expressed on neighboring cyto- or syncytiotrophoblasts.